An Activatable Nanoenzyme Reactor for Coenhanced Chemodynamic and Starving Therapy Against Tumor Hypoxia and Antioxidant Defense System

Open AccessCCS ChemistryRESEARCH ARTICLE1 May 2021An Activatable Nanoenzyme Reactor for Coenhanced Chemodynamic and Starving Therapy Against Tumor Hypoxia and Antioxidant Defense System Zhihe Qing, Ailing Bai, Lifang Chen, Shuohui Xing, Zhen Zou, Yanli Lei, Junbin Li, Juewen Liu and Ronghua Yang Zhihe Qing *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Hunan Provincial Key Laboratory of Cytochemistry, School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha 410114 Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, ON N2L 3G1 Google Scholar More articles by this author , Ailing Bai Hunan Provincial Key Laboratory of Cytochemistry, School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha 410114 Google Scholar More articles by this author , Lifang Chen Hunan Provincial Key Laboratory of Cytochemistry, School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha 410114 Google Scholar More articles by this author , Shuohui Xing Hunan Provincial Key Laboratory of Cytochemistry, School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha 410114 Google Scholar More articles by this author , Zhen Zou Hunan Provincial Key Laboratory of Cytochemistry, School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha 410114 Google Scholar More articles by this author , Yanli Lei Hunan Provincial Key Laboratory of Cytochemistry, School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha 410114 Google Scholar More articles by this author , Junbin Li Hunan Provincial Key Laboratory of Cytochemistry, School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha 410114 Google Scholar More articles by this author , Juewen Liu Department of Chemistry, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, ON N2L 3G1 Google Scholar More articles by this author and Ronghua Yang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] Hunan Provincial Key Laboratory of Cytochemistry, School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha 410114 Laboratory of Chemical Biology and Traditional Chinese Medicine Research, Ministry of Education, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.020.202000259 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail It is critical to improve the efficiency of cancer therapy with minimized side effects. Chemodynamic therapy (CDT) is a tumor therapeutic strategy designed to generate abundant reactive oxygen species (ROS) at tumor sites through a Fenton or Fenton-like reaction. Recently, this developing scheme has demonstrated an incredible promise for tumor therapy. The process involved could induce cell death without the input of external energy, and this could only occur via the conversion of hydrogen peroxide (H2O2) to hydroxyl radicals (·OH). Although Fenton or Fenton-like reactions are being exploited for CDT, along with an application of oxidation reactions to supplement H2O2, it has been proven that in cancer cells, the high levels of the existing antioxidants could suppress CDT via ·OH depletion, and, unfortunately, tumor hypoxia also inhibits the oxidation reactions. Herein, the authors aimed to fabricate an activatable nanoenzyme reactor (NER) to solve this challenge. Fluorescent reporters (FRs) and bioenzyme glucose oxidase (GOX) were coassembled on nanozyme MnO2 nanosheets, which was enwrapped by the tumor-targeting material, hyaluronic acid (HA). NER was internalized explicitly by cancer cells through ligand/receptor recognition-mediated endocytosis, followed by intracellular hyaluronidase (HAase)-dependent activation. As a result, the oxygen level was improved, and the antioxidants were depleted, leading to the promotion of glucose consumption and an increase in ·OH level. Thus, the NER exhibited multiple effects to induce coenhanced, chemodynamic and starving therapy against tumor hypoxia and antioxidant defense system to achieve a favorable targeted tumor therapeutic, via these rigorously highly effective, and targeted biochemical reactions both in an in vitro cultured cancer cells system or in an in vivo mice tumor model. Download figure Download PowerPoint Introduction Intracellular oxidative stress responding to reactive oxygen species (ROS) is one of the most common ways to induce cellular apoptosis.1–4 Among the ROS, hydroxyl radical (·OH) is the most harmful to biomolecules, including nucleic acids, proteins, and lipids. Thus, considerable efforts have been devoted to converting the less effective reactive hydrogen peroxide (H2O2) to a more potent reactive ·OH species, able to kill the diseased cells efficiently, a process called chemodynamic therapy (CDT).5–8 CDT is a direct chemical energy conversion strategy, independent of external energies such as photic, phonic, or magnetic input; thus, it avoids the inconveniences of low depth tissue penetration and complex radiation devices. To date, several chemical reactions concerning the conversion of H2O2 for CDT have been developed. Besides the classical Fenton reaction induced by iron-containing materials,9–12 Yang and coworkers7 recently exploited a Fenton-like reaction catalyzed by Mn2+/HCO3− to transform intrinsic H2O2 produced by mitochondria into highly harmful ·OH for CDT in cancer cells. Since the intrinsic H2O2 in cancer cells is generally too low (∼50 µM), compared with the level desired to produce enough ·OH for highly efficient tumor therapy,13,14 extra biotransformations, catalyzed by enzymes or nanozymes have been developed to supplement the H2O2 via oxidation of endogenous substances available in high concentrations, as well as possessing high oxidizing power (e.g., glucose [Glu] and lactic acid).15–18 However, in the H2O2-supplemented CDT, two challenging obstacles cannot be overlooked: (1) Tumor hypoxia: The oxidation reactions of biological substances for H2O2 production are oxygen (O2) dependent, but the tumor microenvironment is anoxic.19–21 (2) The suppression of the CDT reaction by endogenous antioxidants: The cellular antioxidant defense system (ADS), powered by reductive substances such as thiol compounds, inhibits CDT via oxydoreduction reaction with ROS.22–25 Thus, it is desirable to develop smart materials that would improve the O2 level and deplete the antioxidants simultaneously. In this study, we addressed this challenge by constructing successive intracellular biochemical reactions as an activatable nanoenzyme reactor (NER) package, tailored specifically for cancer cells’ targeting and cytotoxicity. Experimental Sections Materials and apparatus Tetramethylammonium hydroxide (TMAH), an aqueous solution of H2O2 (30%), manganese chloride tetrahydrate (MnCl2·4H2O), methanol, Glu, acetone, ammonia (NH3·H2O), titanium sulfate (Ti(SO4)2), sulfuric acid (H2SO4), sodium bicarbonate (NaHCO3), and dimethyl sulfoxide (DMSO) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Glucose oxidase (GOX) was purchased from Shanghai Aladdin Bio-Chem Technology (Shanghai, China). HA, glutathione (GSH), and methylene blue (MB) were purchased from J&K Chemical Co., Ltd. (Shanghai, China). HAase was purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS), trypsin, and fetal bovine serum (FBS) were purchased from Beyotime Biotechnology (Shanghai, China). 2′,7′-Dichlorofluorescein diacetate (DCFH-DA) was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Cyanine-5.5 NS acid (Cy5.5) and 5-carboxyfluorescein (FAM) were purchased from AAT Bioquest Inc. Annexin V-FITC/propidium iodide (PI) cell apoptosis kit was purchased from Fuyuanbio (Shanghai, China). Hematoxylin–Eosin (H&E) staining kit was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). A Milli-Q system (Millipore Corporation, Bedford, MA, USA) was used to produce the ultrapure water used in this study. All experiments involving the use of live mice were performed in compliance with the guidelines set up by the National Institutes of Health (NIH) for the care and use of laboratory animals. pH was tested by pH meter (PHB-4; LEICI, Shanghai, China). Malvern Zetasizer NanoZS90 (Malvern, United Kingdom) was used to measure the hydrodynamic size and zeta potential. The morphology of the nanoparticles was imaged by Tecnai G2 F20S-TWIN (Hillsborough, Oregon, USA) transmission electron microscopy (TEM) and Bruker AXS Dimension Icon (Bluker, Germany) atomic force microscopy (AFM). UV–Vis spectrophotometer (UV-2700; Shimadzu, Tokyo, Japan) was used to characterize the UV–Vis absorption spectra. Cytotoxicity assays were performed on a Versa Max Microplate reader (Sunnyvale, CA, USA). The fluorescence imaging studies were carried out on an FV-3000 confocal laser scanning microscopy (CLSM; Olympus Corporation, Japan). Flow cytometric assay was carried out by flow cytometry (BD AccuriTM C6, Lake Franklin, New Jersey, USA). In vivo imaging experiments were performed by in vivo imaging system (IVIS) (PerkinElmer IVIS Lumina III, PerkinElmer, USA). Preparation of manganese dioxide nanosheets Manganese dioxide nanosheets (MDN) were prepared, following previous reports.26 Briefly, 20 mL of H2O2 (3 wt %) and TMAH (0.6 M) were first mixed. Then 10 mL MnCl2·4H2O (0.3 M) was quickly added to the above solution within 15 s. The color of the mixed solution changed immediately to dark brown. The solution was stirred vigorously for 24 h at room temperature. The synthesized MDN was centrifugated at 2000 rpm for 20 min and washed with ultrapure water first and then methanol for three times, respectively. Finally, the above purified bulk MDN was dried in a vacuum drying oven at 60 °C and stored at 4 °C for further experiments. If needed, 10 mg MDN was added in 20 mL ultrapure water and ultrasonicated for 12 h for further use. Preparation of NER The mixture of 500 μL MDN (0.25 mg/mL) and 10 μL GOX (2.5 mg/mL) was added to 1.5 mL of HA solution (2 mg/mL) and then stirred for 12 h at room temperature. Then the suspension was centrifugated at 6000 rpm for 20 min and washed with ultrapure water for three times. After that, the NER prepared nanoparticles were dried in a freeze dryer and then dispersed in a 2 mL of ultrapure water for further use. As a control, [email protected] was synthesized in a parallel reaction, without the addition of GOX, and Mn2+[email protected] was obtained using MnCl2 instead of MDN while keeping the other conditions the same. Besides, green fluorescein amidites (FAM)-loaded NER (FAM-NER) and far-red Cy5.5-loaded NER (Cy5.5-NER) were prepared by adding the fluorescent reporters (FRs) before HA encapsulation. Investigation of NER stability Drug stability is an important factor that alters therapeutic efficiency. Thus, the stability of the prepared NER was investigated by adding it to different solutions, including deionized (DI) water, saline, PBS, and 10% FBS medium, and then kept at room temperature for 30 days. Subsequently, their size change was measured by dynamic light scattering (DLS) apparatus (Malvern Zetasizer NanoZS90, Worcestershire, United Kingdom), and photographs were taken to monitor the precipitate visually. In addition, MDN and a mixture of MDN and GOX were also investigated under the same conditions as controls. Verification of MDN degradation, ·OH generation, and O2 supplement To verify MDN degradation by GSH, 100 μL MDN solution (0.25 mg/mL) was added into 300 μL PBS buffers (pH 6.5) containing GSH of different concentrations (0, 0.1, 0.2, 0.4, 0.8, and 1 mM), respectively, and slightly shaken at 37 °C for 5 min. Then photographs of the resulted solutions were taken to show the color change of MDN. Then UV–Vis absorption of the resultant solutions was measured. To verify ·OH generation resulted from MDN, MDN (0.25 mg/mL) was first mixed with GSH (10 mM) at 37 °C for 5 min. Then the mixture was incubated in 25 mM NaHCO3/5% CO2 buffer containing MB (10 μg/mL) and H2O2 of varying concentrations at 37 °C for 30 min. Next, photographs of the resultant solutions were taken to show the color change of MB, and the absorption changes of MB induced by ·OH were measured at 665 nm using UV–Vis spectrometry. To verify O2 reproduction catalyzed by MDN, MDN (0.25 mg/mL) was added into PBS buffer (pH 6.5) with or without 10 mM H2O2. The production of O2 was determined through the bubbles occurring in the solutions. Verification of O2 dependence and H + generation in GOX-catalyzed Glu oxidation A hypoxia condition was controlled by nitrogen (N2)-saturated with Glu (10 mM) in phosphate-buffered saline (PBS; pH 6.5). Either MDN (62.5 μg/mL), GOX (12.5 μg/mL), and H2O2 (2.5 mM) were added into the Glu solutions, or a parallel experiment was set up with the saturated Glu solutions, but without the latter reagents. After reacting for 4 h, the residue of Glu was measured by a portable blood sugar meter. For comparison, the GOX-catalyzed Glu oxidation was also carried out under normoxia condition. To monitor the pH change in the process, Glu (10 mM) was mixed in PBS buffer (pH 7.4), with or without the addition of GOX (12.5 μg/mL), along with the reaction at 37 °C under mild shock, and the pH value of each solution was tested by a pH meter at different time points of the reaction. HAase-dependent activation of NER for H2O2 generation The activation of GOX in NER was verified by H2O2 generation. Typically, NER (2 mg/mL) was added to PBS buffers (containing 10 mM Glu) with or without HAase (150 U/mL). The mixtures were gently vibrated for 4 h at 37 °C. Then these resulted solutions were centrifuged, and their supernatants were obtained. About 200 μL of 10% acetone and 200 μL of 10% NH3·H2O were mixed and added into each supernatant, and 200 μL Ti(SO4)2 (0.3 M) was subsequently added. The yellow precipitate was acquired by centrifugation at 3000 rpm for 15 min and dissolved with 1 mL H2SO4 (1 M). Finally, the UV–Vis absorbance of each solution was recorded at 405 nm. ·OH elimination by GSH and NER-induced GSH depletion for improving ·OH level To verify ·OH elimination by GSH, MnCl2 (0.5 mM) was added into 25 mM NaHCO3/5% CO2 buffer solution with H2O2 (20 mM) under the 37 °C shocking for 1 h, and then the different concentrations of GSH and MB (10 μg/mL) were mixed thoroughly with the above mixture solution. Following that, photographs of the resultant solutions were taken to show the color change of MB, and the UV–Vis absorption changes of MB induced by ·OH were determined at 665 nm. To verify NER-induced GSH depletion for improving ·OH level, NER (2 mg/mL) was added into 25 mM NaHCO3/5% CO2 buffer solutions (containing 10 mM GSH and 10 mM Glu) with or without HAase (150 U/mL), following shocking for 4 h at 37 °C. For comparison, Mn2+[email protected] was also used in a parallel experiment. There was an equivalent Mn concentration (0.5 mM) in two cases. The supernatant was obtained by centrifugation at 3000 rpm for 15 min. Then, MB (10 μg/mL) and H2O2 (50 μM) were added to the above supernatants and incubated at 37 °C for 30 min. Furthermore, an MB solution without nanoreagents was used as a blank under the same conditions. Photographs of the resulted solutions were taken to show the color change of MB, and the UV–Vis absorption changes of MB induced by ·OH were determined at 665 nm. Cell culture Cervical cancer cell line (Hela) with highly expressed CD44 receptors and normal human embryonic kidney cells (HEK-293) without CD44 receptors were purchased from the cell bank of the Central Laboratory of Xiangya Hospital (Changsha, China). Hela and HEK-293 cells were separately cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin (PS, 10,000 IU penicillin and 10,000 μg/mL streptomycin, multicell) at 37 °C in a humidified incubator with 5% CO2. Cytotoxicity assay Cytotoxicity experiments of the NER nanosystem were estimated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. Hela and HEK-293 cells were seeded (5 × 103 cells/well) separately into 96-well plates, the number of cells in each well was kept the same, and cultured for 24 h at 37 °C in a humidified incubator set at 5% CO2. The plated cells were divided into three main groups, based on the following nanosystem treatments: Group 1: NER, Group 2: [email protected], or Group 3: MDN/GOX, at varying serially diluted concentrations and incubated for 24 h at 37 °C in a humidified incubator (5% CO2). Then, the cells were washed three times with PBS. Subsequently, 10 μL MTT reagent (0.5 mg/mL) was added into each well and incubated for another 4 h. Finally, the medium was removed and 100 μL DMSO was added into each well, and the absorbance was recorded at 490 nm using a multimode microplate reader (Varioskan LUX, Thermo Fisher Scientific, Guangzhou, China). Monitoring in situ generations of ·OH in cells Hela and HEK-293 cells were seeded in confocal dishes (for confocal microscopy) and 6-well plates (for flow cytometry) at 5 × 105 cells/well for 24 h at 37 °C in a humidified incubator with 5% CO2. The plates were divided into three main groups, as described earlier for the NER, [email protected] or Mn2+[email protected] nanoreagents treatments at concentrations of Mn (10 μg/mL), and GOX (2 μg/mL), and the treated cells were incubated at different time points (0–8 h). After that, the cells were washed three times with PBS and stained with the ·OH indicator DCFH-DA (10 μM) at 37 °C for 20 min. Then the cells were analyzed by FV-3000 CLSM and flow cytometry. Monitoring cell death induced by the nanoreagents Hela and HEK-293 cells were seeded in confocal dishes for 24 h at 37 °C in a humidified incubator with 5% CO2. NER, [email protected] or Mn2+[email protected] nanoreagents with an equivalent amount of Mn (10 μg/mL), and GOX (2 μg/mL) were added to the dishes and incubated at different time intervals. After that, the cells were washed three times with PBS and stained simultaneously with Calcein-acetoxymethyl ester (Calcein AM; live cell indicator) and propidium iodide (PI; death cell indicator) at 37 °C for 20 min. Finally, the cells were imaged by FV-3000 CLSM. Apoptosis mechanism assay We investigated the NER killing mechanism after treating cells with NER and analyzed by Annexin-FITC/PI two-color flow cytometry. Hela and HEK-293 cells were seeded in 6-well plate 5 × 105 cells/well for 24 h at 37 °C in a humidified incubator with 5% CO2. The cells were incubated with NER or [email protected] nanoreagents with an equivalent amount of Mn (10 μg/mL) for 4 h. Then the cells were washed three times with PBS, digested by trypsin, and collected in centrifuge tubes, followed by staining for 20 min with the Annexin V-FITC/PI cellular apoptosis fluorescent dye kit, according to the manufacturer’s instructions. Finally, the cells were tested by flow cytometry. Hemolysis assay Mice whole blood was collected from the heart, adding ethylenediaminetetraacetic acid (EDTA; 1.5 mg/mL) as a stabilizer. The red blood cells (RBC) were collected by centrifugation at 1300 rpm for 5 min and washed 6 times with PBS, following by resuspension in PBS (pH 7.4). Varying concentrations NER (serial dilution ranging from 800–30 μg/mL) were added into the RBC-contained PBS buffer solutions, and coincubated at 37 °C for 1 h. All solutions were centrifuged to examine the hemolysis. For comparison, the RBC suspensions in PBS and DI water without NER were used as control groups. In vivo targeting imaging of tumor by NER Hela cells (4 × 106 cells/plate) were injected subcutaneously into the right leg of female BALB/C nude mice aged for 5–6 weeks. When the growing tumor volume had reached ∼50 mm3, the mice were divided randomly into 2 groups for the following treatments: (1) Cy5.5-NER and (2) Cy5.5 were injected intravenously into Hela tumor-bearing BALB/C mice, respectively. The equivalent dose of MDN was 1 mg/kg, GOX was 0.2 mg/kg, and Cy5.5 was 0.2 mg/kg. After injection the mice were anesthetized at different times (0, 1, 2, 4, 6, 12, 24, and 48 h), and vivo imaging of the tumor-bearing BALB/C mice was performed in Perkin Elmer IVISlumina III imaging system (PerkinElmer, USA). Finally, the mice were dissected, and the organs, including the heart, liver, spleen, lung, kidney, as well as the tumor, were imaged using the IVIS imaging system. In vivo antitumor therapy Hela tumor-bearing BALB/C mice were randomly divided into three groups (n = 3) when the tumor had reached about 50 mm3. Group 1 received an intravenous injection of PBS, Groups 2 and 3 received nanoreagents ([email protected] and NER) with the equivalent amount of MDN (1 mg/kg) and GOX (0.2 mg/kg) every 24 h for 16 days. Tumor length (L), tumor width (W), and mice body weight were measured and recorded every 2 days for 16 days. The formula used for the calculation of the tumor volume (V) is V = (L × W2)/2. The relative tumor volume was calculated via the formula of V/V0 (V was the tumor volume at each tumor culture time, and V0 was the initial volume before treatment with reagents). The weight of each mouse was real-time recorded simultaneously. Finally, all mice were sacrificed and dissected to remove the main organs and tumors after 16 days of treatment. Their tumors were collected, tumor slices were prepared after fixation (5 μm) and stained with ·OH indicator DCFH-DA (10 μM) at 37 °C for 20 min. The ·OH level in the different tumor was imaged by an FV-3000 CLSM. Results and Discussion Mechanism and characterization of the activatable NER We sought to develop an activatable NER for promoting Glu oxidation in order to avoid the antioxidants impediment to overcome the challenges in CDT. NER was fabricated by a simple programmed packaging (Figure 1a). FRs and GOX were absorbed on a synthesized MDN, which was enwrapped by a compact HA. In this state, the bioenzyme GOX and the nanozyme MnO2 remained at an “inactive” state. The underlying functional principle was that when NER encountered the overexpressed CD44 receptor on the cancer cytomembrane,27 it would be specifically internalized via HA/CD44 recognition-mediated endocytosis (Figure 1b). Inside the cell, the HA shell is degraded by hyaluronidase (HAase), which is also overexpressed in cancer cells.28 Then the released MDN catalyzes the hydrolysis of endogenous H2O2 into O2 to improve the O2 level in the anoxic microenvironment and, in turn, facilitate Glu oxidation to achieve cell starving therapy. GSH is depleted by MDN to form manganese ion (Mn2+) and glutathione disulfide (GSSG),7,26,29 in which the FRs are released with a corresponding fluorescence recovery, thus, enabling the detection of the activatable NER.30,31 Subsequently, the released GOX from the NER package catalyzes the conversion of Glu into gluconic acid and H2O2. Notably, plenty of carbon dioxide (CO2) is produced by aerobic respiration in cells, and HCO3−/CO2 is a critical intracellular buffer that could generate sufficient HCO3−. After disrupting ADS and in the presence of HCO3− and Mn2+, H2O2-catalyzed fragmentation yielded ·OH for CDT. Thus, a tumor-specific biochemical route was established artificially in cancer cells, and the simultaneously enhanced starving, along with the CDT process against tumor hypoxia and ADS, was achieved. Figure 1 | (a) The preparation route for the nanoenzyme reactor (NER) with manganese dioxide nanosheets (MDN), fluorescent reporter (FR), glucose oxidase (GOX), and hyaluronic acid (HA). (b) Schematic illustration of specific uptake of NER via HA/CD44 binding-mediated endocytosis, and hyaluronidase (HAase)-dependent intracellular activation of NER to work against hypoxia and antioxidant defense system via an artificially biochemical route: (1) MDN + H2O2 + H+ → Mn2+ + H2O + O2↑, (2) MDN + GSH → Mn2+ + GSSG, (3) Glu +O2 → H+ + H2O2, and (4) Mn2+ + H2O2 → ·OH. (c) TEM images and (d) zeta potential of MDN, NER, and NER after HAase treatment. (e) Photographs of MDN, MDN/GOX, and NER in various aqueous media incubated for different days. Download figure Download PowerPoint The MDN was prepared by a chemical reduction method,26 and its sheet structure was verified by TEM (Figure 1c, left). It was also characterized by UV–Vis absorption and energy dispersive spectrometer (EDS) spectrum ( Supporting Information Figure S1); its size and zeta potential were about 125 nm ( Supporting Information Figure S2, black curve) and −27.9 mV (Figure 1d, black curve), as measured by DLS. Then the NER was prepared by the programmed assembly of MDN, GOX, and HA. TEM imaging showed that NERs were approximatively spherical nanoparticles (Figure 1c, middle), the transformation from MDN to NER was also characterized by AFM ( Supporting Information Figure S3). The characteristic absorption peak of the precipitation redispersion of NER indicated the successful coencapsulation of GOX and MDN ( Supporting Information Figure S4). Furthermore, the hydration particle size and zeta potential after assembly were enlarged to 294.8 nm ( Supporting Information Figure S2, red curve) and −59.4 mV (Figure 1d, red curve). Attractively, NER stability was sustained after dispersion in different solutions, including water, saline, PBS, and 10% FBS medium for up to 30 days, and its size barely changed, while the controls precipitated in only 1 day under the same experimental conditions (Figure 1e and Supporting Information S5). In addition, when the NER was added into the solution containing a high level of FBS, negligible change was noted in the transmittance and size, indicating good stability under a physiological condition ( Supporting Information Figure S6).32 Thus, we envisioned that these good physicochemical properties of the NER could well be applicable in vivo. Verification of the biochemical route and activation of NER First, the biochemical route of NER was investigated step by step. The O2-dependent oxidation and MDN-catalyzed supplement of O2 were investigated under normoxia and N2-saturated hypoxia. We could observe the oxidation of Glu entirely under normoxia (Figure 2a, red), but unfavorable under hypoxia (Figure 2a, green); nevertheless, the reaction was promoted by the introduction of MDN (Figure 2a, yellow). This was due to the supplement of O2 catalyzed by MDN ( Supporting Information Figure S7). The production of H2O2 through oxidation of Glu catalyzed by GOX was verified using an H2O2 indicator Ti(SO4)2 that could be transformed to a peroxide titanium complex with yellow color and absorption at 405 nm ( Supporting Information Figure S8).33 The supplement of hydrion (H+) through the oxidation reaction was demonstrated by the decrease in pH ( Supporting Information Figure S9). The transformation from MDN to Mn2+ by GSH depletion was verified by the decrease in absorption and fading of MDN solution ( Supporting Information Figure S10). The transformation from H2O2 to ·OH in the presence of Mn2+ and HCO3− was proved by the absorption and color decrease of MB which could be oxidized to colorlessness by ·OH ( Supporting Information Figure S11).34 Notably, reductive GSH depleted ·OH, resulting in only a little decrease of MB sorption (red curve in Figure 2b and Supporting Information Figure S12), but MDN improved ·OH level due to depletion of GSH (green curve in Figure 2b). Figure 2 | (a) O2 dependence of GOX-catalyzed Glu oxidation, and O2 supplement by the reaction of H2O2 and MDN. (b) UV–Vis absorption spectra of MB solutions after treatment with Mn2+ or MDN in the presence of GSH, H2O2 (10 mM), and NaHCO3, to demonstrate the suppressing effect on ·OH production from GSH and the depletion of GSH by MDN. (c) The UV–Vis absorption spectra and photo of solution colors after incubating NER without or with HAase in the presence of Glu, finally adding Ti(SO4)2 as the H2O2 indicator, to demonstrate HAase-dependent activation of NER. (d) UV–Vis abs