Biomimetic targeted self-adaptive nanodrug for inflammation optimization and AT2 cell modulation in precise ARDS therapy

Abstract

Acute respiratory distress syndrome (ARDS) is a lethal respiratory condition, while effective pharmacological treatments remain elusive. We identified the decreased mechanical capacity and impaired proliferation of alveolar type 2 (AT2) epithelial cells in the inflammatory environment as the primary contributors to respiratory failure of ARDS. A biomimetic, self-adaptive, 7,8-dihydroxyflavone-loaded hollow mesoporous cerium oxide coated with a platelet membrane (HCeO_x-D@PM) was developed for precise ARDS therapy. HCeO_x-D@PM comprises a platelet membrane (PM) shell for targeted delivery to injured lungs and an HCeO_x core, which enables high drug loading, efficient reactive oxygen species (ROS) scavenging, and penetration of the alveolar-capillary barrier. Initially, HCeO_x-D@PM suppresses the inflammation and mitigates the adverse effects of lesions on AT2 cell by scavenging accumulated ROS. It then adaptively releases 7,8-dihydroxyflavone in response to cysteine-aspartic acid protease 3 activation, facilitating AT2 cell proliferation and notably improving survival rates in vivo, offering a promising advancement in the precise treatment of respiratory diseases.

Fig. 1.. Inflammatory microenvironment–driven AT2 dysfunction in ARDS and self-adaptive nanotherapeutic strategy for pulmonary edema.

(A) Diagrammatic representation of the pathological microenvironment in the lungs of patients with ARDS. The inflammatory milieu leads to a reduction in the mechanical and proliferative capabilities of AT2 cells, accompanied by a notable decrease in their population, ultimately resulting in pulmonary edema, respiratory failure, and death. (B) The schematic diagram illustrating the self-adaptive therapy of core-shell nanodrug HCeO_x-D@PM. The PM shell specifically targets the damaged lung tissue, while the hollow mesoporous cerium oxide (HCeO_x) core facilitates enhanced penetration of the alveolar-capillary barrier and ameliorates the inflammatory microenvironment by eliminating excess ROS, thereby increasing the mechanical and proliferative capabilities of AT2 cells. Subsequently, the adaptive on-demand release of 7,8-dihydroxyflavone (DHF) in response to Casp3 activity rapidly promotes AT2 cell growth, leading to a notably reduction in pulmonary edema and an increase in survival rates.

Fig. 2.. Study design and cellular landscape.

(A) Overview of the study design. t-SNE, t-distributed stochastic neighbor embedding. (B) UMAP plot showing scRNA-seq analysis of 55,541 cells isolated from human lungs (n = 10, with three healthy and seven ARDS samples from each genotype). DCs, dendritic cells; NK, natural killer. (C) Bar plots depicting the composition of all cell clusters in the healthy and ARDS groups. (D) KEGG enrichment analysis of notably different genes compared with the healthy group. Rows represent pathways arranged in descending order of richness or importance (key pathways highlighted in red); the x axis indicates false discovery rate. FcγR, Fcγ receptor; HD, human diseases; CP, cellular processes; OS, organismal systems; EIP, environmental information processing. (E) Protein-protein interaction network of differentially expressed genes (DEGs) involved in key pathways (red frame indicates key genes related to biomechanics, while yellow frame indicates key genes related to cell proliferation). EGFR, epidermal growth factor receptor. (F) UMAP plot of biomechanical and cell proliferation gene set scores for the healthy and ARDS groups. (G) Representative immunofluorescence staining images of cell proliferation–related protein expression, including SDC4 (red fluorescence) and E-Cad (orange fluorescence) in AT2 cells (SFTPC; green fluorescence) from cancer-adjacent lung tissue and ARDS-related COVID-19 lung tissue, along with intensity quantification (n = 3). Scale bar, 20 μm. (H) Representative immunofluorescence staining images of biomechanics-related protein expression, including ITGB6 (red fluorescence) and ITGA3 (orange fluorescence) in AT2 cells (SFTPC; green fluorescence) from cancer-adjacent lung tissue and ARDS-related COVID-19 lung tissue, along with intensity quantification (n = 3). Scale bar, 20 μm. *P < 0.05 and **P < 0.01.

Fig. 3.. Analysis of cell communication and DEGs correlation in AT2 cells with human samples validation.

(A) RankNet analysis highlighting conserved signaling pathways. Each bar represents a signaling pathway, with the rank order indicating its significance in the analyzed dataset. Key pathways, such as CCL, TNF, IL-6, and IL-2, are highlighted red for emphasis. MHC-I, major histocompatibility complex I; EGF, epidermal growth factor; NCAM, neural cell adhesion molecule. (B) Heatmaps showing the relative importance of each cell group based on computed network centrality measures of the CCL and TNF signaling networks. (C) Correlation of DEGs in the inflammation (CCL and TNFα signaling) and proliferation (PI3K-AKT and MAPK signaling) pathways in AT2 cells. The heatmap shows the correlation of DEGs associated with these pathways in AT2 cells. Each cell represents the correlation coefficient, with colors ranging from blue (negative correlation) to red (positive correlation), illustrating the interaction between pathways. (D) qPCR and (E) Western blot analysis of AT2 cells induced by LPS, as well as (F) the quantification of Western blot results, showing a significant reduction in SDC4, CDH1 (E-Cad), ITGB6, and ITGA3 compared to healthy AT2 cells (n = 3). *P < 0.05 and **P < 0.01.

Fig. 4.. Synthetic characterization of the HCeO_x-D@PM nanodrug.

(A) Schematic illustration of the synthesis process for HCeO_x-D@PM (x = 1 to 2), highlighting DL and surface bionic modification. h, hours. (B) Representative high-angle annular dark-field scanning TEM images of HCeO_x-D@PM, along with corresponding TEM-mapping images for Ce, O, P, S, and N and their merged Ce/P/S map. Scale bar, 100 nm. (C) Hydrodynamic diameters of HCeO_x, HCeO_x-D, and HCeO_x-D@PM NPs (n = 3). (D) Surface zeta potentials of HCeO_x, HCeO_x-D, and HCeO_x-D@PM NPs (n = 3). (E) Representative dot blots of denatured PM and nondenatured HCeO_x-D@PM stained with extracellular/intracellular anti-CD47 antibodies, with normalized intensity (n = 3). (F) Western blot analysis of CD42b, CD61, and CD62p in Plt, PM, and HCeO_x-D@PM (n = 3). (G) Quantification of proteins in Plt, PM, and HCeO_x-D@PM using label-free quantitative techniques. The Venn diagram shows the protein overlap among Plt, PM, and HCeO_x-D@PM. (H) Volcano plot exhibiting differentially expressed proteins between PM and HCeO_x-D@PM. FC, fold change. (I) Classification of HCeO_x-D@PM proteins by biological process. GO, Gene Ontology. (J) Classification of HCeO_x-D@PM proteins by molecular function. (K) Expression abundance of proteins associated with Plt adhesion and immune evasion in PM and HCeO_x-D@PM.

Fig. 5.. Analysis and characterization of ROS scavenging, motion, and DL properties of HCeO_x-D@PM.

(A) Schematic illustration of ROS scavenging by HCeO_x-D@PM. (B) H₂O₂ scavenging efficiency of HCeO_x-D@PM and 5.68-nm CeO_x at various Ce concentrations (n = 3). (C) Detection of O₂ generation in different environments over 7 min (n = 3). (D) Scavenging efficiency of ·O₂⁻ and (E) ·OH by HCeO_x-D@PM and 5.68-nm CeO_x at various Ce concentrations (n = 3). (F) Electron spin resonance (ESR) curves showing the scavenging of ·OH by various concentrations of HCeO_x-D@PM using 5,5′-dimethylpyrroline-1-oxide (DMPO) as a spin trap agent. a.u., arbitrary units. (G) Schematic diagram of the renewable H₂O₂ scavenging capacity of HCeO_x-D@PM, along with Raman spectra of HCeO_x-D@PM reacting with 1 mM H₂O₂ over 60 min per cycle. (H) Representative tracking trajectories of HCeO_x-D@PM in the presence of different concentrations of H₂O₂ as fuel (movies S1 to S3). (I) Corresponding mean square displacement (MSD) curves of HCeO_x-D@PM (n = 3). (J) Corresponding diffusion coefficients of HCeO_x-D@PM. (K) Corresponding speeds of HCeO_x-D@PM (n = 3). (L) Schematic diagram of drug-responsive release and diffusion in the ARDS microenvironment. (M) Cumulative drug release curves of HCeO_x-D@PM in the presence of PBS (pH 7.4), 1 mM H₂O₂, and Casp3 (0.1 and 1 μg ml⁻¹) at specific time points (n = 3). (N) Cumulative drug release and corresponding fitting of HCeO_x-D@PM in the presence of different Casp3 concentrations over 24 hours (n = 3).

Fig. 6.. ROS scavenging and cytoprotective activities of HCeO_x-D@PM in vitro.

(A) Experimental procedures for apoptosis/ROS analysis in human primary AT2 cells treated with HCeO_x-D@PM in vitro. (B) Cell viability of human primary AT2 cells under different treatment conditions (n = 5). (C) Fluorescence-activated cell sorting results and (D) statistical analysis of the cell death ratio in human primary AT2 cells under various treatments (n = 3). (E) Representative DCFH-DA staining (green fluorescence) of human primary AT2 cells under different treatment conditions. Scale bar, 50 μm. (F) Statistical analysis of DCFH-DA levels detected using a microplate reader (n = 5). (G) Experimental procedures for EdU/Ki67 analysis in AT2 cells treated with HCeO_x-D@PM in vitro. (H) Representative EdU staining (green fluorescence) of AT2 cells under different treatment conditions. Scale bar, 100 μm. Statistical analysis of EdU levels detected using laser confocal microscopy (n = 3). (I) Representative Ki67 staining (purple fluorescence) of AT2 cells under different treatment conditions. Scale bar, 50 μm. Statistical analysis of Ki67 levels detected using laser confocal microscopy (n = 3). (J) Schematic diagram depicting the establishment of an alveolar-capillary barrier injury cell model, where AT2 and HUVEC cells are seeded on opposite sides of the Transwell filter inserts to form a bilayer. (K) TEER values measured to assess the integrity of the alveolar-capillary barrier (n = 5). *P < 0.05 and **P < 0.01. n.s., not significant.

Fig. 7.. Targeting and distribution of HCeO_x-D@PM in ARDS mice.

(A) Flowchart of the experimental design. (B) CT imaging and (C) quantitative analysis of ARDS or sham mice at different time points following intravenous injection of HCeO_x-D@PM or HCeO_x-D (n = 3). HU, Hounsfield units. (D) Ce content in lung tissue of ARDS or sham mice, determined by ICP-MS, 4 hours after intravenous injection of HCeO_x-D@PM or HCeO_x-D (n = 3). (E) Biodistribution of HCeO_x-D@PM in the blood, heart, liver, spleen, lungs, and kidneys at 4 hours postinjection (n = 3). (F) Schematic representation of the active penetration of the damaged alveolar-capillary barrier by HCeO_x-D@PM, showing relative amounts of HSiO@PM, HCeO_x@PM, and HCeO_x-D@PM collected in the lower chamber (n = 5). (G) Schematic representation of HCeO_x-D@PM penetration through the damaged alveolar-capillary barrier. (H) TEM analysis of NP penetration through the damaged alveolar-capillary barrier. Scale bars, 2 μm. *P < 0.05 and **P < 0.01.

Fig. 8.. Therapeutic efficacy of HCeO_x-D@PM in the ARDS mouse model.

(A) Flowchart of the study design. (B) Percentage change in body weight over 48 hours (n = 3). (C) Survival rate of ARDS mice after different treatments over 5 days (n = 10). (D) Lung wet/dry (W/D) weight ratio (n = 3). (E) Average relative levels of TNF-α, IL-6, and IL-1β in lung tissue and bronchoalveolar lavage fluid (BALF). For each group, three mice were analyzed to calculate the average value. (F) Representative dihydroethidium (DHE) staining (red fluorescence) of lung tissues and intensity quantification (n = 3). Scale bar, 50 μm. (G) Proportion and quantitative analysis of neutrophils in BALF detected using flow cytometry (n = 3). (H) Representative H&E staining of lung tissues. Magnified images of the boxed regions in the top panel are displayed in the bottom panel. Scale bars, 100 μm (overview) and 50 μm (magnified). Lung injury scores were calculated on the basis of H&E staining (n = 3). (I) Representative SFTPC staining (purple fluorescence) of lung tissue and intensity quantification (n = 3). Scale bar, 50 μm. (J) Representative immunofluorescence staining images of proliferation-related proteins, including E-Cad (red fluorescence) and SDC4 (orange fluorescence) in AT2 cells (SFTPC; green fluorescence). Immunofluorescence intensity quantification results are shown as heatmaps (n = 3). Scale bar, 50 μm. (K) Representative immunofluorescence staining images of biomechanics-related proteins, including ITGB6 (red fluorescence) and ITGA3 (orange fluorescence) in AT2 cells (SFTPC; green fluorescence). Immunofluorescence intensity quantification results are shown as heatmaps (n = 3). Scale bar, 50 μm. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 9.. The preparation process of Pep-D.

(A) The synthetic route for Int-1. RT, room temperature. (B) The synthetic route for Pep-D.