Research ArticleHEALTH AND MEDICINE

Nanoparticle-induced neutrophil apoptosis increases survival in sepsis and alleviates neurological damage in stroke

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Science Advances  06 Nov 2019:
Vol. 5, no. 11, eaax7964
DOI: 10.1126/sciadv.aax7964
  • Fig. 1 Scheme shows NP targeting of proinflammatory neutrophils to induce their apoptosis for treatment of inflammatory diseases.

    (A) The therapeutic process of DOX-conjugated BSA NPs includes neutrophil uptake of NPs in situ and cell apoptosis to prevent neutrophil transmigration and inflammatory responses. (B) DOX is conjugated to BSA via a hydrazone bond, followed by aggregating BSA conjugates to form DOX-hyd-BSA NPs, and DOX release from NPs triggered by low pH in neutrophils to promote neutrophil apoptosis.

  • Fig. 2 Characterization of DOX-conjugated BSA NPs and DOX release from NPs at acidic condition to cause cell death.

    (A) Particle size of BSA NPs, DOX-ab-BSA NPs, and DOX-hyd-BSA NPs measured by DLS in PBS at pH 7.4. (B) TEM image of DOX-hyd-BSA NPs. Scale bars, 500 and 100 nm for inset. (C) Zeta potential of BSA NPs, DOX-ab-BSA NPs, and DOX-hyd-BSA NPs in PBS at different pH. (D) UV-vis spectra of their supernatants after NPs were incubated in PBS at different pH for 2 hours. a.u., arbitrary unit. (E) Cumulative DOX release from DOX-ab-BSA NPs or DOX-hyd-BSA NPs in PBS at pH 7.4, 6.5, or 5.0. (F) Cell death of differentiated HL-60 cells (neutrophil-like cells) induced by DOX. Cells were treated for 24 hours at different concentrations of DOX. Data are shown as means ± SD (n = 6 independent experiments).

  • Fig. 3 Neutrophil activation up-regulates Fcγ receptors to mediate NP uptake.

    (A) Intravital microscopy of mouse cremaster muscle venules shows that neutrophil activation was associated with up-regulation of Fcγ receptors. The resting condition of neutrophils was established by no intrascrotal injection of TNF-α (0.5 μg per mouse) and the tail vein injection of Fcγ antibodies 3 hours before performing intravital microscopy (top). Intrascrotal injection of TNF-α (0.5 μg per mouse) activated neutrophils. Alexa Fluor 647–labeled anti-mouse CD16/32 (red) and Alexa Fluor 488–labeled anti-mouse LY-6G (green) antibodies were intravenously injected to stain Fcγ receptors and neutrophils, respectively (bottom). The images were taken using a Nikon A1R+ resonant-scanning confocal microscope at 488 and 640 nm. Scale bars, 10 μm. (B) Percentage of costaining between anti-mouse CD16/32 and anti-mouse LY-6G based on intravital images of (A). (C) Confocal laser scanning microscopy (CLSM) images of blood neutrophils from healthy mice or LPS-challenged mice. Four hours after intraperitoneal LPS injection, DOX-hyd-BSA NPs were intravenously administered to a mouse. Three hours later, the mouse blood was collected, and neutrophils were isolated using anti-mouse LY-6G beads. Alexa Fluor 488–labeled anti-mouse LY- 6G antibody was used to label neutrophils. Scale bars, 10 μm. (D) Uptake of BSA NPs by blood leukocytes analyzed by flow cytometry. Neutrophils, T cells, monocytes, and natural killer (NK) cells were isolated from blood and stained by Alexa Fluor 647–labeled anti-mouse LY-6G, CD3, CD115, and CD335 antibodies, respectively. All data are expressed as means ± SD (three mice per group).

  • Fig. 4 DOX-hyd-BSA NPs promote neutrophil apoptosis to inhibit inflammatory responses.

    (A) CLSM images show the apoptosis of differentiated HL-60 cells. Annexin V–FITC is an apoptosis marker, and 7AAD (emission at 650 nm) is a fluorescent dye to mark dead cells. Cells were treated with PBS, free DOX, DOX-ab-BSA NPs, or DOX-hyd-BSA NPs for 24 hours, respectively. Scale bar, 20 μm. (B) Percentage of apoptotic cells analyzed by flow cytometry after HL-60 cells treated with various DOX formulations. (C) CLSM images of HL-60 cells stained with TUNEL after treatment with several DOX formulations for 24 hours. TUNEL and DOX were excited at 488 and 560 nm. Scale bar, 20 μm. (D) Percentage of costaining between DOX and TUNEL based on CLSM images of (C). N.S., not significant. (E) Diagram shows the experimental protocol in acute lung inflammation mouse model. Intratracheal (i.t.) administration of LPS causes local lung acute inflammation, and subsequently, neutrophils transmigrate from blood to airspace in the lung. Twenty-four hours after intravenous injection of several DOX formulations, BALF was collected to assess neutrophil number and cytokines. Number of neutrophils (F), inflammatory factors TNF-α (G), IL-1β (H), and IL-6 (I) in mouse BALF after i.t. LPS challenge (10 mg/kg). All data are expressed as means ± SD (three mice per group). *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 5 DOX-hyd-BSA NPs protect mouse death from sepsis and does not impair the neutrophil generation in the bone marrow.

    (A) Mouse survival rates in sepsis after treatments of NPs. Four hours after intraperitoneal LPS (50 mg/kg) challenge to mice, mice were treated with PBS, free DOX, and prodrug DOX-hyd-BSA NPs at 0.2 mg/kg of DOX, respectively. All data expressed are as means ± SD (10 mice per group). Statistical analysis was done using the Kaplan-Meier method. ***P < 0.001 compared to controls (PBS and free DOX). Mouse body weights were measured after treatments of PBS (B), free DOX (C), and DOX-hyd-BSA NPs (D) (equal to 0.2 mg/kg free DOX). Number of neutrophils (E), TNF-α (F), IL-1β (G), and IL-6 (H) in blood, and number of neutrophils (I), TNF-α (J), IL-1β (K), and IL-6 (L) in BALF at 16 and 72 hours after LPS challenge, respectively. N.D. (not detected) represents the mouse death. All data expressed as mean ± SD (five mice per group). (M) Diagram shows the experimental protocol to address whether DOX-conjugated BSA NPs impair neutrophil immune sentinel to the secondary infection. The mice were challenged with LPS (intraperitoneal, 50 mg/kg) or PBS (control). Four hours later, the LPS-challenged mice were intravenously treated with DOX-hyd-BSA NPs at 0.2 mg/kg of DOX. The control mice were not treated with LPS and NPs. Seventy-two hours later, all survival and control (healthy) mice were challenged with LPS [i.t. (intratracheal), 10 mg/kg)]. At 84 hours, BALF was collected to assess neutrophil number (N), TNF-α (O), IL-1β (P), and IL-6 (Q). All data are expressed as means ± SD [seven survival mice for the DOX-hyd-BSA NPs-treatment group (equal to 0.2 mg/kg free DOX), and three healthy mice for the control group]. Statistics were performed by a two-sample Student’s t test. Statistics were performed by a two-sample Student’s t test (*P < 0.05, **P < 0.01, and ***P < 0.001).

  • Fig. 6 DOX-hyd-BSA NPs mitigate neutrophil-induced neuroinflammation in cerebral I/R mouse model to restore neurological functions.

    (A) Experimental design to examine the benefit of DOX-hyd-BSA NPs in cerebral I/R. OD, optical density. (B) MPO activity and (C) TNF-α, (D) IL-1β, and (E) IL-6 in brain damage tissues at 22 hours after administration of PBS, free DOX, and DOX-hyd-BSA NPs. (F) Mouse neurological behavior scores after treatments with PBS, free DOX, and DOX-hyd-BSA NPs at 0.2 mg/kg of DOX. All data are expressed as means ± SD (three mice per group). Statistics was performed by a two-sample Student’s t test (*P < 0.05, **P < 0.01, and ***P < 0.001). Photo Credit: Xinyue Dong, Washington State University.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/11/eaax7964/DC1

    Fig. S1. Synthetic pathways of DOX-hyd-BSA.

    Fig. S2. 1H NMR analysis.

    Fig. S3. TEM images of BSA NPs and DOX-ab-BSA NPs.

    Fig. S4. The stability of BSA NPs in serum.

    Fig. S5. Quantitative analysis of cellular uptake of DOX by differentiated HL-60 cells.

    Fig. S6. Flow cytometric analysis on apoptosis of differentiated HL-60 cells 24 hours after treatments with free DOX (0.2 mg/kg), DOX-ab-BSA, or DOX-hyd-BSA NPs (equal to DOX of 0.2 mg/kg), respectively.

    Fig. S7. Flow cytometric analysis of neutrophils in BALF.

    Fig. S8. DOX-hyd-BSA NPs decrease systemic inflammation in the mouse sepsis model.

    Fig. S9. Mouse body weights were monitored following the experiment as shown in Fig. 5M.

    Fig. S10. Toxicity of DOX-conjugated BSA NPs evaluated by histological analysis.

    Movie S1. Intravital microscopy of a cremaster venule shows resting neutrophils in circulation.

    Movie S2. Intravital microscopy of a cremaster venule shows activated neutrophils in circulation.

    Movie S3. Movement of a sham mouse in the cerebral I/R model.

    Movie S4. Movement of a mouse with cerebral I/R after PBS treatment.

    Movie S5. Movement of a mouse with cerebral I/R after DOX treatment.

    Movie S6. Movement of a mouse with cerebral I/R after treatment of DOX-hyd-BSA NPs.

  • Supplementary Materials

    The PDFset includes:

    • Fig. S1. Synthetic pathways of DOX-hyd-BSA.
    • Fig. S2. 1H NMR analysis.
    • Fig. S3. TEM images of BSA NPs and DOX-ab-BSA NPs.
    • Fig. S4. The stability of BSA NPs in serum.
    • Fig. S5. Quantitative analysis of cellular uptake of DOX by differentiated HL-60 cells.
    • Fig. S6. Flow cytometric analysis on apoptosis of differentiated HL-60 cells 24 hours after treatments with free DOX (0.2 mg/kg), DOX-ab-BSA, or DOX-hyd-BSA NPs (equal to DOX of 0.2 mg/kg), respectively.
    • Fig. S7. Flow cytometric analysis of neutrophils in BALF.
    • Fig. S8. DOX-hyd-BSA NPs decrease systemic inflammation in the mouse sepsis model.
    • Fig. S9. Mouse body weights were monitored following the experiment as shown in Fig. 5M.
    • Fig. S10. Toxicity of DOX-conjugated BSA NPs evaluated by histological analysis.

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Intravital microscopy of a cremaster venule shows resting neutrophils in circulation.
    • Movie S2 (.mp4 format). Intravital microscopy of a cremaster venule shows activated neutrophils in circulation.
    • Movie S3 (.mp4 format). Movement of a sham mouse in the cerebral I/R model.
    • Movie S4 (.mp4 format). Movement of a mouse with cerebral I/R after PBS treatment.
    • Movie S5 (.mp4 format). Movement of a mouse with cerebral I/R after DOX treatment.
    • Movie S6 (.mp4 format). Movement of a mouse with cerebral I/R after treatment of DOX-hyd-BSA NPs.

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