Research ArticleHEALTH AND MEDICINE

An artificial metalloenzyme for catalytic cancer-specific DNA cleavage and operando imaging

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Science Advances  15 Jul 2020:
Vol. 6, no. 29, eabb1421
DOI: 10.1126/sciadv.abb1421
  • Fig. 1 Schematic diagram of protein-cloaked copper cluster as an artificial metalloenzyme with persistent catalytic activity for high-efficient DNA cleavage and operando chemiluminescent imaging in tumor microenvironment.

    Copper clusters preliminarily recognize αVβ3 overexpressed on vascular endothelial cells and lung tumor cells, then they in situ activate endogenous H2O2 (50 to 100 μM) to give rise to persistent OH and O2 generation in both extracellular (pH 6.5) and intracellular (pH 7.4) tumor microenviroment. The energetically favored, regenerated copper clusters as artificial metalloenzyme maintain long-term satisfactory catalytic performance and lead to significant DNA breakage and genotoxicity in tumor cells and tumor xenograft mice models, achieving high-efficient anticancer effect. The metalloenzyme also facilitates the sensitive tracking of tumor therapeutic intervention in vivo through catalyzing persistent chemiluminescence imaging.

  • Fig. 2 Molecular constitution of synthetic copper clusters and energy diagram of RGD-BSA-CuCs to the redox potential of H2O2/·OH and O2/H2O2.

    MALDI-TOF MS of (A) BSA and BSA-CuCs as well as (B) RGD-BSA and RGD-BSA-CuCs. a.u., arbitrary units. (C) Excitation and emission spectra of BSA-CuCs and RGD-BSA-CuCs. Inset is digital photograph of BSA-CuCs (left) and RGD-BSA-CuCs (right) under 365-nm hand-held ultraviolet lamp excitation. (D) Ultraviolet-visible (UV-Vis) absorption spectra of BSA, BSA-CuCs, and RGD-BSA-CuCs. (E) Cascade image illustrating the representative conformation of RGD-BSA-CuCs (left) and the orientation of Cu13 core stuck in BSA calculated by molecular docking (middle), as well as the optimized Cu13 core configuration with all copper atomic identities calculated by density functional theory (DFT) (right). Cu13 core is stably stuck in BSA through forming coordination bond with His residues (green arrows) or electrostatic interaction with Glu and Asp (black dotted lines). (F) Molar absorption coefficient of Cu13 core with D2h symmetry from time-dependent DFT (TDDFT) calculation. (G) Proposed energy diagram illustrating the CB and VB position of RGD-BSA-CuCs to the redox potential of H2O2/OH and O2/H2O2, which explains the recycled catalytic property of biomimetic catalyst. Clusters’ bandgap energy is determined by Tauc’s plot while the Fermi energy is calculated according to an empirical formula. The cluster bandgap and Fermi level determine the position of the VB and CB energies. The electronic level of clusters and redox couples are ranked by standard hydrogen electrode (SHE) level (left vertical line) and vacuum level (right vertical line).

  • Fig. 3 RGD-BSA-CuCs catalyze hydrogen peroxide decomposition to continually produce hydroxyl radical and oxygen under mimetic physiologic condition.

    Time-dependent catalytic •OH generation of mixture containing 100 μM H2O2 and 12 μM RGD-BSA-CuCs within 24 hours under (A) pH 6.5 and (E) pH 7.4 condition at 37°C. Time-dependent fluorescence change of oxidized Amplex UltraRed at 585 nm in mixture containing 25 μM Amplex UltraRed, 100 μM H2O2, and 12 μM RGD-BSA-CuCs under (B) pH 6.5 and (F) pH 7.4 condition at 37°C. Representative steady-state kinetic analyses of mimetic enzyme by varying the concentrations of H2O2 (0 to 500 μM) with fixed 25 μM Amplex UltraRed and 12 μM RGD-BSA-CuCs under (C) pH 6.5 and (G) pH 7.4 condition at 37°C. Fitted curves (D and H) are relevant double-reciprocal plots reflecting POD activity of RGD-BSA-CuCs using Michaelis-Menten and Lineweaver-Burk models at double pH conditions. Oxygen production traced by mixing 12 μM RGD-BSA-CuCs and 100 μM H2O2 immediately (I) and 24 hours after initiating the catalysis (J) under pH 6.5 condition at 37°C, measured by a Clark-type oxygen electrode. The similar assays (K and L) were conducted under pH 7.4 condition.

  • Fig. 4 Intrinsic geometrical and electronic properties of biomimetic catalyst remain unaltered during the catalytic process studied by the experimental and DFT methods.

    (A) HRTEM images of RGD-BSA-CuCs before and after catalysis for 24 hours under pH 6.5 (top) and pH 7.4 (bottom) conditions at 37°C. (B) Statistical size analysis of the representative 50 crystalline nanoparticles characterized by HRTEM at pH 6.5 (top) and pH 7.4 (bottom) before and after the catalysis. (C) The relative photoluminescence intensity (Ft/F0) evolution of RGD-BSA-CuCs at both pH conditions at 37°C. F0 and Ft represent the fluorescent intensity of the catalyst before and after initiation of the catalysis, respectively. Error bars represent variation between three measurements. (D) Representative normalized x-ray absorption near-edge structure (XANES) spectra at the Cu K edge of RGD-BSA-CuCs before and after catalysis for 24 hours under two pH conditions at 37°C. The spectra of Cu foil, Cu2O, and CuSO4 compounds are nominated as standards. (E) The reaction pathway of the recycled catalytic reaction from the initial Cu13 core to the intermediate compound Cu13═O. Int1, Int2, and Int3 are the intermediate products, while TS1 and TS2 are the transition states. The reaction rate determining step is highlighted in the blue dash frame. (F) The reaction pathway of the recycled catalytic reaction from the intermediate compound Cu13═O to the initial Cu13 core. Int4, Int5, and Int6 are the intermediate products, while TS3 and TS4 are the transition states. The reaction rate determining step is highlighted in the blue dash frame. Copper, oxygen, and hydrogen atoms are depicted in pink, red, and white, respectively.

  • Fig. 5 Copper clusters as anticancer biomimetic catalysts exert therapeutic effect in vitro and in vivo.

    (A) In vitro viability of A549 lung tumor cells after incubation of RGD-BSA-CuCs or BSA-CuCs with the identical clusters concentration for 48 hours. Viability of A549 cells pretreated by RGD-BSA is set as control group (means ± SD, n = 3). DIC, differential interference contrast. (B) Fluorescence microscopy images showing ROS burst through fluorogenic reaction with CM-H2DCFDA in cells pretreated with a series concentration of RGD-BSA-CuCs for 48 hours. Cells treated with 39 μM RGD-BSA-CuCs and 250 μM ROS quencher mannitol are set as control. (C) Quantification the proportion of apoptotic cells in series dosages of RGD-BSA-CuCs measured by flow cytometry. DIC, differential interference contrast. (D) Agarose gel electrophoretic patterns of supercoiled pUC19 plasmid DNA (20 ng ml−1) in the presence of a series concentration of RGD-BSA-CuCs and 100 μM H2O2 (left). The mixture was preincubated in pH 7.4 buffer solution at 37°C for 12 hours. The persistent DNA cleavage property of 9.6 μM RGD-BSA-CuCs in the presence of 100 μM H2O2 (right). The mixture was preincubated in pH 7.4 buffer solution at 37°C for 0, 3, 6, and 12 hours. DNA treated by 100 μM H2O2 for 12 hours is set as control. (E) DNA lesions induced by the increased concentration of RGD-BSA-CuCs measured by the basic comet assay. DNA content in the comet tail represents the extent of DNA cleavage, analyzed by 50 cells selected randomly by CASP software. ***P < 0.001. (F) Scheme of establishing subcutaneous (s.c.) xenografted A549 cells tumor model and catalytic mediated therapeutic protocol. i.p., intraperitoneal. (G) The relative tumor volumes of mice treated with control group and therapeutic groups (means ± SD, n = 5). *P < 0.05 and ***P < 0.001. (H) Tumor tissues were dissected and compared visually showing inhibition of tumor growth after 21 days of therapy (n = 5). (I) Representative hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL) staining images of dissected tumor tissues. Tumor sections present obvious apoptosis and prominent necrosis. In TUNEL assay, positive apoptotic cells are observed to emit green luminescence [(fluorescein isothiocyanate (FITC)–labeled], while the cell nuclei are stained with 4, 6-diamidino-2-phenylindole (DAPI, blue) (Photo credit: X.G., Beijing University of Technology).

  • Fig. 6 Chemiluminescence imaging in vivo to trace the process of therapeutic intervention.

    (A) Chemiluminescence images (left) and quantitative signal data (right) of 1.0 mM L-012 in the presence of different concentrations of RGD-BSA-CuCs with fixed 100 μM H2O2 under pH 6.5. (B) Chemiluminescence luminescence images (left) and quantitative signal data (right) of 1.0 mM L-012 in the presence of different concentrations of H2O2 with fixed 12 μM RGD-BSA-CuCs under pH 6.5. (C) Time-dependent luminescence (left) and signal intensity (right) of 1.0 mM L-012 upon incubation with 100 μM H2O2 and 9.6 μM RGD-BSA-CuCs. (D) Chemiluminescence images present a higher intensity signal in the tumor region of mice treated with CuCs and L-012 (the right mice in each group) compared with that mice treated with L-012 only (the middle mice) or saline treated mice (the left mice). (E) The corresponding luminescence intensity of tumor region at indicated time (means ± SD, n = 3). The pseudo-colors represent photons per second per square centimeter per steradian. *P < 0.05 and **P < 0.01. (F) Chemiluminescence images of tumor region of mice treated with CuCs and L-012 (the right mice in each group) compared with that mice treated with L-012 only (the middle mice) or saline treated mice (the left mice) for 7, 14, and 21 days of therapeutic intervention. (G) The corresponding luminescence intensity of tumor region at indicated time (means ± SD, n = 3). The pseudo-colors represent photons per second per square centimeter per steradian. *P < 0.05 and **P < 0.01. n.s., no significance. (Photo credit: X.G., Beijing University of Technology).

Supplementary Materials

  • Supplementary Materials

    An artificial metalloenzyme for catalytic cancer-specific DNA cleavage and operando imaging

    Liang Gao, Ya Zhang, Lina Zhao, Wenchao Niu, Yuhua Tang, Fuping Gao, Pengju Cai, Qing Yuan, Xiayan Wang, Huaidong Jiang, Xueyun Gao

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