Research ArticleHEALTH AND MEDICINE

Self-regulated hirudin delivery for anticoagulant therapy

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Science Advances  09 Oct 2020:
Vol. 6, no. 41, eabc0382
DOI: 10.1126/sciadv.abc0382
  • Fig. 1 Schematic of self-regulated hirudin delivery for anticoagulant therapy.

    (A) Schematic of synthesis of HV/ctNGs and adaptive release of HV in response to thrombin. (B) Schematic of mechanism of HV/ctNGs for closed-loop HV delivery and anticoagulant regulation.

  • Fig. 2 Thrombin-responsive self-regulated HV release.

    (A) Representative histogram of the hydrodynamic diameter distribution and TEM image of HV/ctNGs. Scale bar, 100 nm. (B) Representative HPLC profiles of the TCP cross-linker after incubation with thrombin for different time. a.u., arbitrary unit. (C) Change in the conversion ratio of the TCP cross-linker after incubation under different conditions over time. Data are shown as means ± SD (n = 3). (D) Release profiles of Rho-HV from Rho-HV/ctNGs after incubation with different concentrations of thrombin. Data are shown as means ± SD (n = 3). (E) Representative HPLC profiles of the TCP cross-linker after incubation with thrombin in the presence of HV for different time. (F) Release profiles of Rho-HV from Rho-HV/ctNGs after repeated incubation with and without thrombin. Data are shown as means ± SD (n = 3).

  • Fig. 3 In vitro and in vivo clot-targeting evaluation.

    (A) Fluorescent images of the blood clots after different treatments: (1) saline, (2) Cy7-BSA/tNGs, (3) Cy7-BSA/ctNGs, and (4) the clot-targeted peptide + Cy7-BSA/ctNGs. (B) Fluorescent intensities of Cy7 in the blood clots after different treatments. Data are shown as means ± SD (n = 3). ***P < 0.001. (C) Representative fluorescent images of the lung tissue harvested from the thromboplastin-induced pulmonary embolism mouse models after intravenous injection of Cy7-BSA/ctNGs and Cy7-BSA/tNGs at 15 min after induction. The pulmonary emboli were labeled with Cy5.5. (D) Relative fluorescent intensity ratio of Cy7 to Cy5.5 in the lung tissues. Data are shown as means ± SD (n = 3). ***P < 0.001. (E) Representative fluorescent images of the lung tissues harvested from the thromboplastin-induced pulmonary embolism mouse models after intravenous injection of FITC-BSA/ctNGs and FITC-BSA/tNGs at 15 min after induction. Scale bar, 100 μm. (F) Representative fluorescent images of the carotid arteries of the FeCl3-induced thrombosis mouse models after intravenous injection of FITC-BSA/ctNGs for different time. Scale bar, 500 μm. (G) Changes in the relative fluorescent intensity ratio of FITC to Rho in the carotid arteries over time after intravenous injection of FITC-BSA/ctNGs and FITC-BSA/tNGs. Data are shown as means ± SD (n = 3).

  • Fig. 4 In vivo therapeutic efficacy study on the thromboplastin-induced pulmonary embolism mouse model.

    (A) Fluorescent images of the lung tissues harvested from the mice after different treatments at 15 min after induction. The pulmonary emboli were labeled with Cy5.5. (B) Fluorescent intensity of Cy5.5 in the lung tissues. Data are shown as means ± SD (n = 3). P > 0.05 (no significance, n.s.), *P < 0.05, **P < 0.01, and ***P < 0.001. (C) Representative H&E- and Masson’s trichrome–stained images of the lung sections after different treatments at 15 min after induction. Scale bar, 100 μm. (D) Relative clot areas in the lung tissues after different treatments at 15 min after induction. Data are shown as means ± SD (n = 3). P > 0.05 (no significance, n.s.) and ***P < 0.001. (E) Survival rates of the mice after different treatments within 2 hours (n = 6).

  • Fig. 5 In vivo therapeutic efficacy study on the FeCl3-induced carotid artery thrombosis mouse model.

    (A) Representative fluorescent images of the carotid arteries of the mice after intravenous injection of different HV formulations for different time. The blood clots were labeled with Rho 6G. Scale bars, 500 μm. (B) Representative H&E- and Masson’s trichrome–stained images of the carotid arterial transverse sections after intravenous injection of different HV formulations for 25 min. Scale bar, 100 μm. (C) Changes in the relative fluorescent intensity of Rho after different HV formulations over time. Data are shown as means ± SD (n = 3). (D) Embolization rates in the H&E-stained carotid arterial transverse sections after different treatments. Data are shown as means ± SD (n = 3). P > 0.05 (no significance, n.s.) and ***P < 0.001. (E) VTI of the carotid arteries of the mice before and after intravenous injection of HV/ctNGs for 25 min. (F) Representative fluorescent images of the carotid arteries of the mice after intravenous injection of HV and HV/ctNGs for different time upon sequential induction. The blood clots were labeled with Rho 6G. Scale bar, 500 μm. (G) Relative fluorescent intensity ratios of Rho after intravenous injection of HV and HV/ctNGs upon sequential induction. Data are shown as means ± SD (n = 3). P > 0.05 (no significance, n.s.) and ***P < 0.001. (H) Pharmacokinetic profiles of Rho-HV and Rho-HV/ctNGs after intravenous injection into the rats. Data are shown as means ± SD (n = 3).

Supplementary Materials

  • Supplementary Materials

    Self-regulated hirudin delivery for anticoagulant therapy

    Xiao Xu, Xuechao Huang, Ying Zhang, Shiyang Shen, Zhizi Feng, He Dong, Can Zhang, Ran Mo

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