Pulmonary-Targeted Nanoparticles Interrupt the Malignant Mechanical and Biochemical Signaling Crosstalk for Idiopathic Pulmonary Fibrosis Therapy

Abstract

Idiopathic pulmonary fibrosis (IPF) involves transforming growth factor-beta, a key factor that drives biochemical signaling pathways, inducing cellular transdifferentiation and excessive extracellular matrix (ECM) deposition. Increased ECM stiffness alters the mechanical microenvironment of the lung, exacerbating pulmonary dysfunction through mechanical signaling transduction. Here, persistent malignant mechanical and biochemical signaling crosstalk in IPF is demonstrated that drives the relentless progression of the disease. Therefore, inhalable lung-targeted lipid nanoparticles (VB-RT NPs) are developed for co-delivering verteporfin and berbamine to effectively treat IPF by interrupting pulmonary mechanical-biochemical signaling malignant crosstalk. Specifically, VB-RT NPs are modified with tannic acid to scavenge reactive oxygen species and enhance lung targeting, and with L-arginine to penetrate dense ECM and reach deeper lung regions. After being inhaled in a bleomycin model, VB-RT NPs inhibited fibroblast activation and promoted the transition of endothelial cell (EC)-like myofibroblasts to ECs, reducing endothelial-to-mesenchymal transition and fibrotic progression. Additionally, VB-RT NPs blocked the nuclear translocation of the mechanotransducers Yes-associated protein, interrupting fibrosis-related mechanotransduction pathways. The results demonstrate that VB-RT NPs effectively reversed dysregulated mechanical-biochemical signaling crosstalk in fibrotic lungs and halted fibrosis progression, offering a promising therapeutic approach for IPF.

Keywords: extracellular matrix; idiopathic pulmonary fibrosis; malignant crosstalk; mechanical and biochemical signals; mechanotransduction; transforming growth factor‐beta.

Scheme 1

Illustration of pulmonary‐targeted NPs interrupting the malignant mechanical and biochemical signaling crosstalk during IPF therapy. A) Schematic of the preparation of VB‐RT NPs. B) Schematic representation of the in vivo therapeutic process for IPF treatment via pulmonary nebulization of VB‐RT NPs.

Figure 1

The malignant mechanical‐biochemical signaling crosstalk in samples from healthy donors and IPF patients. A) UMAP distribution of samples from healthy donors and IPF patients. B) UMAP plots of cells from healthy and fibrotic lung datasets colored by cell type. C) GO analyses of differentially expressed genes in fibroblasts from healthy and fibrotic lungs. D) GSEA analysis showed the gene sets of TGF‐β signaling pathway in myofibroblasts. E) GSEA analysis showed the gene sets of regulation of the integrin‐mediated signaling pathway in myofibroblasts. F) Heatmap showing Spearman correlation between integrin and TGF‐β related genes in myofibroblasts. G) H&E staining and Masson's trichrome staining of healthy and fibrotic human lungs. H) Representative IF images of CD31, SPC, and α‐SMA in healthy and fibrotic human lungs. I) Young's modulus of healthy and fibrotic human lungs measured by AFM (n = 3). J) Representative Western blotting (WB) assay of integrin β1, pMLC, and total MLC. K) Representative WB assay of YAP and phosphorylated YAP (S127). L) Representative WB assay of LAP‐TGF‐β1 and Active TGF‐β1. M) Representative WB assay of pSMAD2 and active SMAD2. N) Schematic illustration of malignant mechanical‐biochemical signaling crosstalk in IPF pathogenesis.

Figure 2

Characterization and multifunctional properties of the VB‐RT NPs. A) Particle size of different formulations. B) Zeta potential of different formulations. C‐D) Average size distribution and TEM images of VB NPs and VB‐RT NPs. E) UV–vis spectra of different formulations across the full wavelength range. F) Seven‐day stability of VB NPs, VB‐R NPs, and VB‐RT NPs. G) Comparison of particle size before and after nebulization. H) Heatmap of PDI before and after nebulization. I) Fluorescence imaging of ROS levels in 16HBE cells under different formulations using DCFH DA. J) The NO detection in 16HBE cells under different formulations using DAF‐FM DA. K) Quantification of mean fluorescence intensity (MFI) of NO in 16HBE cells under different formulations (n = 3). L) Uptake of different formulations in PMVECs measured by flow cytometry. M) Quantification of MFI of NO in A549 cells under different formulations using DAF‐FM DA (n = 3). N) Illustration of the in vitro collagen barrier model, showing DiI‐labeled NPs crossing the epithelial cell‐collagen layer to reach myofibroblasts. O) CLSM images of myofibroblasts treated with different formulations in Transwell chambers with collagen barrier for 4 h at 37 °C. P) Quantification of MFI in myofibroblasts analyzed using ImageJ (n = 3). All data are presented as the Mean ± SD (n = 3). ^*** p < 0.001.

Figure 3

VB‐RT NPs interrupt malignant mechanical and biochemical signaling crosstalk in fibroblasts in vitro. A) Schematic illustration of potential TGF‐β release from the ECM. B) The TGF‐β expression levels of fibroblasts and PMVECs were cultured on non‐stretched and stretched membranes (n = 3). C) Representative images of collagen gel contraction mediated by fibroblasts after treatment with different formulations. D) Quantitative analysis of gel area contraction (n = 3). E) IF staining of α‐SMA (white) and DAPI (purple) in fibroblasts subjected to cyclic stretching or static culture using the programmable mechanical cell stretch system. F) Representative WB assay of integrin β1, LAP‐TGF‐β1, and active TGF‐β1. G) IF staining of p‐SMAD (red) and DAPI (blue) in fibroblasts cultured on soft and stiff matrices. H) Schematic of live cell imaging of fibroblasts and PMVECs. I) Imaging of fibroblast‐PMVEC contact (1 frame/10 min). J) Quantification of average force on micropillars by fibroblasts under different conditions (n = 3). ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001. n.s., no significant difference.

Figure 4

VB‐RT NPs interrupt malignant mechanical and biochemical signaling crosstalk in ECs in vitro. A) Representative WB assay of VE‐cadherin and Vimentin. B) Young's modulus of PMVECs measured by AFM indentation after treatment with different formulations (n = 200 measurements per cell). C) Representative images of tube formation on soft and stiff matrices at 6 and 12 h. D) IF staining of integrin β1 (red) and DAPI (blue) in fibroblasts cultured on soft and stiff matrices. E) Quantification of branch points in the tube formation on soft and stiff matrices at 6 and 12 h (n = 3). F) Quantitative PCR analysis of gene expression (ALK5, FoxA2, Kdr, Tie2) under different treatments (n = 6). G) ELISA quantification of cytokine levels (TGF‐β, IL‐1β, IL‐6, VEGF) under non‐stretched, stretched, and VB‐RT NPs conditions (n = 3). H) Quantitative analysis of filopodia length after 24 h in encapsulated EC spheroids cultured on soft and stiff matrices (from left to right n = 6, 6, 8, 8, 8, 8 cells). I) Schematic illustration of 3D cell culture in soft‐collagen (0.5–1.0 mg mL⁻¹) and stiff‐collagen (3.0–3.5 mg mL⁻¹) gels. J) IF staining of pMLC (red), F‐actin (green), and DAPI (blue) in cells cultured in 3D gels with soft and stiff conditions. The top images show 2D views, while the bottom images display 3D projections. ^** p < 0.01, and ^*** p < 0.001. n.s., no significant difference.

Figure 5

VB‐RT NPs biodistribution in the lungs of BLM‐induced fibrosis mice. A) Schematic illustration of in vivo and ex vivo imaging in the BLM‐induced pulmonary fibrosis model. The red and blue lines indicate the start of model induction and drug administration, respectively. B) In vivo images of mice nebulized with Lipo/DiR, Lipo/DiR‐R, and Lipo/DiR‐RT for 24 h. C) Quantification of fluorescence intensity in the lungs following treatment (n = 3). D) Ex vivo fluorescence images of the heart, liver, spleen, lungs, and kidneys of treated mice. E) Quantitative analysis of fluorescence intensity in major organs following treatment (n = 3). F) MFI of DiI⁺ cells in BALF measured by flow cytometry (n = 3). G and H) Colocalization of DiI‐labeled NPs with CD31 (G) and FAP (H) in the lungs of fibrotic mice. I) Quantification of fluorescence co‐localization with CD31 and FAP expression using ImageJ (n = 3). All data are presented as the Mean ± SD (n = 3). ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001. n.s., no significant difference.

Figure 6

VB‐RT NPs modulate biochemical signaling and alleviate pulmonary fibrosis in BLM‐induced fibrotic mice. A) Schematic of experimental design for therapeutic assessment in BLM‐induced mice. The red and green lines indicate the start of model induction and drug administration, respectively. B) Representative histological analysis of lung sections, including H&E, Masson's trichrome, and IHC staining for α‐SMA and Collagen I, and IF analysis for TGF‐β1 LAP‐D (R58). C) Body weight changes following treatment with different formulations (n = 6). D) The levels of HYP content in lung tissue (n = 6). E) Ashcroft scores of lung fibrosis in normal and BLM‐treated mice (n = 3). F‐H) The levels of VEGF (F), TGF‐β (G), and IL‐1β (H) in BALF, measured by ELISA (n = 3). I‐K) Pulmonary function tests: tidal volume (I), minute volume (J), and peak expiratory flow (K) (n = 6). All data are presented as the Mean ± SD. ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001. n.s., no significant difference.

Figure 7

VB‐RT NPs modulate mechanical signaling and improve the mechanical microenvironment in BLM‐induced fibrotic mice. A) Representative heatmap of lung tissue stiffness in healthy, fibrotic, and treated mice, based on cantilever‐based AFM micro‐indentation analysis. B) Representative fluorescence images of collagen fibers in lung sections captured by two‐photon microscopy. C) Young's modulus of mice lungs was estimated by AFM indentation after different treatments (n = 200 measurements per lung tissue). D) Representative WB analysis of Collagen I and α‐SMA in lung tissues. E) Representative WB analysis of integrin β1 and F‐actin in lung tissues. F) Representative IF image of CD31 (red) and pMLC (green) in lung tissues. G) Representative WB analysis of pMLC and MLC in lung tissues. H) Representative WB analysis of YAP and YAP (S127) in cytoplasm and nucleus. I) Quantitative analysis of CD31 and pMLC fluorescence intensity using ImageJ (n = 3). All data are presented as the Mean ± SD. ^*** p < 0.001. n.s., no significant difference.