NIR triggered polydopamine coated cerium dioxide nanozyme for ameliorating acute lung injury via enhanced ROS scavenging

Abstract

Acute lung injury (ALI) is a life threatening disease in critically ill patients, and characterized by excessive reactive oxygen species (ROS) and inflammatory factors levels in the lung. Multiple evidences suggest that nanozyme with diversified catalytic capabilities plays a vital role in this fatal lung injury. At present, we developed a novel class of polydopamine (PDA) coated cerium dioxide (CeO₂) nanozyme (Ce@P) that acts as the potent ROS scavenger for scavenging intracellular ROS and suppressing inflammatory responses against ALI. Herein, we aimed to identify that Ce@P combining with NIR irradiation could further strengthen its ROS scavenging capacity. Specifically, NIR triggered Ce@P exhibited the most potent antioxidant and anti-inflammatory behaviors in lipopolysaccharide (LPS) induced macrophages through decreasing the intracellular ROS levels, down-regulating the levels of TNF-α, IL-1β and IL-6, up-regulating the level of antioxidant cytokine (SOD-2), inducing M2 directional polarization (CD206 up-regulation), and increasing the expression level of HSP70. Besides, we performed intravenous (IV) injection of Ce@P in LPS induced ALI rat model, and found that it significantly accumulated in the lung tissue for 6 h after injection. It was also observed that Ce@P + NIR presented the superior behaviors of decreasing lung inflammation, alleviating diffuse alveolar damage, as well as promoting lung tissue repair. All in all, it has developed the strategy of using Ce@P combining with NIR irradiation for the synergistic enhanced treatment of ALI, which can serve as a promising therapeutic strategy for the clinical treatment of ROS derived diseases as well.

Keywords: Acute lung injury; M2 directional polarization; Nanozyme; ROS scavenging; Synergistic enhanced therapy.

Fig. 1

Schematic illustration of synthesis of Ce@P and in vivo ALI therapy by intravenous (IV) injection of Ce@P. The synergistic enhanced ALI therapy was achieved by the strategy of Ce@P combining with NIR irradiation via antioxidant, anti-inflammation and M2 directional polarization

Fig. 2

Preparation and basic characterization of NPs. A) Synthesis procedure of Ce@P. B) Zeta potential of CeO₂ and Ce@P. C) XRD results of CeO₂ and Ce@P. D) TGA results of CeO₂ and Ce@P. E) TEM-mapping results of Ce@P and the corresponding element composition (HAADF, C, N, O and Ce images). F) XPS results of CeO₂ and Ce@P (Full spectrum, C, N, O and Ce detailed spectrum)

Fig. 3

Physicochemical properties of NPs. A) Dispersion and stability of CeO₂ and Ce@P at predetermined time points (0, 1, 2, 6, 12, 24 and 48 h), and the corresponding solutions were PBS, H₂O₂ (5 mM), DMEM and FBS respectively (from left to right). B) Photothermal images of different NPs (CeO₂ and Ce@P) with the same concentration of 100 µg/mL under NIR irradiation (1.5 W/cm²) versus time. C) Temperature changes of PBS, CeO₂ and Ce@P with the same concentration of 100 µg/mL under NIR irradiation (1.5 W/cm²) (i), 100 µg/mL Ce@P under different power intensity of NIR irradiation (0.5, 1, 1.5 and 2 W/cm²) (ii), different concentrations (0, 50, 100 and 200 µg/mL) of Ce@P under NIR irradiation (1.5 W/cm²) (iii) versus time, and photothermal stability of 100 µg/mL Ce@P under NIR irradiation (1.5 W/cm²) for 4 “on” and “off” cycles (iv). D) ROS scavenging capacity of different NPs by ROS testing kits: H₂O₂ (i), ·OH (ii) and ·O₂⁻ (iii). E) ESR results of ROS scavenging ability of different NPs: ·OH (i), ·O₂⁻ (ii) and ¹O₂ (iii)

Fig. 4

Cell biocompability and cellular uptake capacity. A) Cell viability of CeO₂ and Ce@P with different concentrations ranging from 0 to 500 µg/mL. B) Live/dead staining images of treated cells and their corresponding quantified results (C). The corresponding groups were: cells without treatment (normal group), cells pre-treated with LPS followed by incubating with PBS buffer (LPS group), cells pre-treated with LPS followed by incubating with 100 µg/mL CeO₂ (CeO₂), cells pre-treated with LPS followed by incubating with 100 µg/mL Ce@P (Ce@P), and cells pre-treated with LPS followed by incubating with 100 µg/mL Ce@P and NIR irradiation (1.5 W/cm²) (Ce@P + NIR). D) Cellular uptake images of cells incubated with Cy5-CeO₂ and Cy5-Ce@P for 3 h by confocal microscope and the corresponding quantified results (E). (“*” symbol compared with CeO₂, **p < 0.01)

Fig. 5

Antioxidant and anti-inflammation capacity in cellular levels. A) Intracellular ROS (DCFA, DHE and HPF) levels of treated cells by fluorescent microscope and the corresponding quantified results (DCFA (i), DHE (ii) and HPF (iii)) (B). C) Inflammatory factors (TNF-α (i), IL-6 (ii) and IL-1β (iii)) expression levels of the supernatent of treated cells by ELISA. D) Quantified results of IL-6 (i), iNOS (ii), CD206 (iii) and HSP70 (iv) expression level of treated cells by fluorescent microscope. F) Inflammatory and anti-inflammatory genes (TNF-α (i), IL-6 (ii), iNOS (iii), SOD-2 (iv) and CD206 (v)), and HSP70 (vi) gene expression levels of treated cells by qRT-PCR. The corresponding groups were: cells without treatment (normal group), cells pre-treated with LPS followed by incubating with PBS buffer (LPS group), cells pre-treated with LPS followed by incubating with 100 µg/mL CeO₂ (CeO₂), cells pre-treated with LPS followed by incubating with 100 µg/mL Ce@P (Ce@P), and cells pre-treated with LPS followed by incubating with 100 µg/mL Ce@P and NIR irradiation (1.5 W/cm²) (Ce@P + NIR). (“*” symbol compared with normal group, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001)

Fig. 6

In vivo bio-distribution, photothermal effect and biosafety. A) Fluorescent images of major isolated organs (heart (H), liver (Li), spleen (S), lung (Lu) and kidney (K)) at predetermined time points (0, 0.5, 1, 2, 6 and 24 h) by IVIS and the corresponding quantified results of lung tissue (B). The corresponding groups were: rats injected with Ce@P (Ce@P), rats injected with Cy5 (Cy5), and rats injected with Cy5-Ce@P (Cy5-Ce@P). C) In vivo photothermal images of treated rats versus time under NIR irradiation (1.5 W/cm²) and the corresponding quantified results (D). The corresponding groups were: rat with PBS injection (sham group) and rat with Ce@P injection (Ce@P). E) H&E staining results of major organs of treated rats. The corresponding groups were: rats without treatment (sham group) and rats with Ce@P injection (Ce@P)

Fig. 7

In vivo ALI therapy evaluation. A) Macroscopic observation of lung tissue of treated rats. B) The wet/dry ratio of lung tissue of treated rats. C) Inflammatory factors (TNF-α (i), IL-6 (ii) and IL-1β (iii)) expression levels of lung homogenate of treated rats by ELISA. D) ROS staining images of lung tissue of treated rats. E) SOD (i) and MDA (ii) levels of lung homogenate of treated rats by the corresponding testing kits. F) H&E staining images of lung tissue of treated rats and the corresponding Smith score (G). H) HSP70 expression level of lung tissue of treated rats by immunohistochemical staining and the corresponding average optical density (AOD) (I). The corresponding groups were: rats without treatment (sham group), LPS induced rats with PBS injection (ALI group), LPS induced rats with Ce@P injection (Ce@P) and LPS induced rats with Ce@P injection and NIR irradiation (Ce@P + NIR). (“*” symbol compared with normal group, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001)