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