Abstract Pulmonary fibrosis (PF) is a chronic lung disease characterized by progressive fibrosis and scar tissue formation, often leading to respiratory failure. Current treatments are limited to alleviating symptoms and slowing disease progression. Mesenchymal stem cells (MSC) can be applied to treat PF owing to their anti-inflammatory, immunomodulatory, tissue repair, and vascular regeneration properties. However, the short pulmonary retention time of intravenously injected MSC limits therapeutic efficacy. We combined MSC with Fe[3]O[4]-polydopamine (Fe[3]O[4]@PDA) nanoparticles and applied an external magnetic field to guide magnetized MSC to damaged lung areas. This strategy not only enhances MSC localization, targeting, and retention but also amplifies their therapeutic effects by activating critical therapeutic molecules involved in MSC migration, adhesion, homing, and intercellular connections, as well as signaling pathways such as MAPK/AKT. In vivo studies using a PF mouse model demonstrated that Fe[3]O[4]@PDA-magnetized MSC therapy effectively alleviated lung tissue fibrosis and significantly reduced the expression of inflammatory factors. RNA sequencing analysis revealed profound changes in gene expression profiles, particularly in pathways such as ECM-receptor interactions, focal adhesion, and the TGF-β pathway. Compared to non-magnetized MSC therapy, magnetized MSC therapy significantly activated essential signaling pathways in lung tissues, including cell adhesion molecules, leukocyte transendothelial migration, and the Rap1 signaling pathway. Thus, Fe[3]O[4]@PDA-magnetized MSC therapy offers a promising strategy for optimizing MSC distribution and therapeutic molecule expression in PF treatment, paving the way for broader MSC-based applications. Keywords: Pulmonary fibrosis, Mesenchymal stem cells, Magnetite nanoparticles, Polydopamine, Lung reparation Graphical abstract [39]Image 1 [40]Open in a new tab Highlights * • We combined MSC with Fe[3]O[4]-polydopamine (Fe[3]O[4]@PDA) NPs for the treatment of PF. * • We applied external magnetic field to guide magnetized MSC to damaged lung areas. * • This strategy enhances MSC targeting, migration, adhesion and retention. * • It amplified therapeutic effects by activating critical therapeutic molecules. * • This is a promising strategy to optimize the efficacy of MSC therapy for PF. 1. Introduction Pulmonary fibrosis (PF) is a chronic respiratory disease known for its extremely difficult treatment and high mortality rates. PF is characterized by progressive and irreversible lung tissue scarring, compromised gas exchange, organ failure, and respiratory collapse [[41]1,[42]2]. Patients with PF typically have a survival expectancy of only 2–3 years, with a 5-year survival rate of less than 30 % [[43]3]. Early intervention is the key to effectively improving PF. The incidence and mortality rates of PF have increased recently and have been exacerbated by the COVID-19 pandemic [[44]4]. Notably, 4 % of patients with COVID-19 develop fibrosis within one week of acute respiratory distress syndrome (ARDS), 24 % within 1–3 weeks, and 61 % after more than three weeks [[45]5], and several COVID-19 patients die from progressive PF. Currently, effective treatment options for PF remain limited. Lung transplantation is currently the only effective treatment for progressive PF [[46]6,[47]7]. Therapeutic drugs approved by the Food and Drug Administration (FDA) of the United States, nintedanib (NIN) and pirfenidone (PFD), can slow disease progression but do not substantially extend the median survival time of clinical patients. Recently, mesenchymal stem cells (MSC) have demonstrated therapeutic potential in various diseases, including lung injury. Experimental evidence suggests that MSC can repair damaged lung epithelial cells, inhibit fibroblast proliferation, and regulate immune responses, thereby reducing fibrosis and promoting lung tissue repair. MSC exert their therapeutic effects through multiple mechanisms, including homing, immunomodulation, and growth factor secretion. However, the homing behavior of MSC is influenced by various factors such as cell dosage, site of inflammation, and individual patient variability. Therefore, optimizing MSC therapy to enhance targeting specificity is crucial to maximize therapeutic efficacy. Magnetite (Fe[3]O[4]) nanoparticles (NPs), approved by the FDA and European Drug Administration (EMA), are widely utilized as superparamagnetic NPs because of their low toxicity, ease of synthesis, strong magnetism, and unique optical properties [[48]8,[49]9]. Fe[3]O[4]NPs have been shown to enhance the homing ability of MSC to damaged tissues, including cardiovascular tissue [[50]10], thereby promoting tissue repair [[51]11]. CXCR4 receptors present on the surface of MSC play a crucial role in regulating their migration and homing by binding to their natural ligand SDF-1. Therefore, another strategy for enhancing MSC targeting is to increase CXCR4 expression. Fe[3]O[4] NPs not only facilitate magnetic targeting but also significantly increase the expression of CXCR4 and migration-related proteins CCR1 and c-Met in MSC, thereby further improving the efficiency and specificity of cell therapy [[52]12]. MSC can activate relevant signaling pathways under harsh conditions, such as stimulation by tumor necrosis factor (TNF)-α, lipopolysaccharide (LPS), or hypoxia. This activation triggers various cellular responses, increases the secretion of growth factors and cytokines, and regulates inflammatory responses and tissue repair processes [[53][13], [54][14], [55][15]]. Previous studies have shown that treatment of MSC with iron oxide NPs can significantly enhance the expression of anti-inflammatory and tissue repair-related cytokines by activating the c-Jun N-terminal kinase (JNK) signaling pathway [[56]16,[57]17]. Upon cellular uptake, iron oxide NPs in the endosomes are exposed to a low pH environment and might undergo slow ionization. This process results in the release of iron ions into the cytoplasm and production of reactive oxygen species (ROS) [[58]18]. ROS can not only activate JNK and c-Jun but also upregulate the expression of Connexin43 [[59][19], [60][20], [61][21]]. These changes further influence the physiological functions and fates of the cells. In this study, we aimed to enhance the targeting efficiency and improve therapeutic outcomes by applying Fe[3]O[4]@PDA-magnetized MSC and selectively guiding them to lung tissues using an external magnetic field. PDA was used to coat Fe[3]O[4] NPs, which significantly enhanced their stability, biocompatibility, and targeting ability [[62]22]. Notably, the PDA coating effectively mitigates the cytotoxicity typically associated with Fe[3]O[4] NPs and does not introduce new cytotoxic effects owing to its natural abundance in the human body [[63][23], [64][24], [65][25]]. Our results indicate that Fe[3]O[4]@PDA-magnetized MSC can be directed to targeted lung sites using an external magnetic field and can activate genes related to cell migration, adhesion, homing, intercellular connections, and MAPK/AKT signaling pathways, thereby improving the effectiveness of PF treatment. 2. Materials and methods 2.1. Fe[3]O[4]@PDA NP synthesis The Fe[3]O[4]@PDA NPs used in this study were kindly provided by Professor Zhang Hao (Jilin University, Changchun, China) and were synthesized following previously published procedures [[66]23]. The Fe[3]O[4]@PDA NPs were attracted by circular neodymium magnets (Shanghai Jinneodymium Magnet Co., Ltd., Shanghai, China) with a diameter of 15 mm and a thickness of 5 mm. 2.2. Transmission electron microscopy (TEM) TEM images of the Fe[3]O[4]@PDA NPs were obtained using a Hitachi H-800 electron microscope at an acceleration voltage of 200 kV using a charge-coupled device camera. The ultrastructural features of the MSC and MSC + NPs were obtained using a Tecnai Spirit TEM (FEI Company, Hillsboro, OR, USA). 2.3. Human umbilical-cord-derived MSC isolation and identification MSC derived from human umbilical cord were cultured in α-minimum essential medium (MEM) supplemented with 10 % fetal bovine serum (FBS; Hyclone, Australia) in a 5 % CO[2] incubator at 37 °C. The cells were analyzed using flow cytometry for the appropriate positive markers, including CD73, CD90, and CD105, combined with the negative marker CD45, using a Human MSC Analysis Kit (BD Biosciences, San Jose, CA, USA). The differentiation potential of MSC was evaluated according to the manufacturer's protocol for osteogenic or adipogenic differentiation (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). The adipogenic or osteogenic differentiation abilities of cells with or without Fe[3]O[4]@PDA NPs were detected using Alizarin Red or Oil Red O staining kits (Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China) after the cells were cultured for 21 or 28 d, respectively. 2.4. A549 cell culture The human alveolar adenocarcinoma cell line (A549) was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). A549 cells were grown in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) supplemented with 10 % FBS in a 5 % CO[2] incubator at 37 °C. 2.5. Prussian blue staining Fe[3]O[4]@PDA NPs were added to MSC, which were incubated for 24 h. After washing three times with PBS, the cells were fixed with 4 % paraformaldehyde for 10 min and stained using a Prussian blue iron staining kit (Solarbio, Beijing, China), according to the manufacturer's instructions. 2.6. MSC iron determination MSC or MSC + NPs cell pellets were lysed in aqua regia, and their iron content was quantified using an inductively coupled plasma-optical emission spectrometer (ICP-OES) with a Perkin-Elmer Optima 3300DV (Perkin-Elmer, Norwalk, CT, USA). 2.7. Cell viability determination The cytotoxicity of Fe[3]O[4]@PDA NPs was evaluated using the Cell Counting Kit-8 (CCK-8; Dojindo, Kyushu, Japan) assay. Briefly, A549 cells were seeded in 96-well plates at a density of 5 × 10^3 cells/well for 24 h, after which Fe[3]O[4]@PDA NPs (0, 25, 50, 100, and 200 μg/mL) were added to the plates and cultured for 24, 48, or 72 h. The optical density (OD) at 450 nm was determined using a microplate reader (Synergy HT; BioTek Instruments, Winooski, VT, USA), and the cell viability was calculated as (OD test sample/OD control cells) × 100 %. 2.8. Cell cycle determination Fe[3]O[4]@PDA NPs at different concentrations (0, 25, 50, and 100 μg/mL) were added to the MSC and incubated for 24 h. After the incubation, the supernatant was discarded. The cells were washed three times with PBS and then collected. Subsequently, the cells were fixed with cold 70 % ethanol and incubated overnight at 4 °C. The next day, the fixed cells were centrifuged, and the supernatant was discarded. The cell pellet was then washed with PBS to remove any residual fixative. Subsequently, the cells were treated with 100 μg/mL ribonuclease A (RNase A) for 30 min to eliminate intracellular RNA and then stained with 50 μg/mL propidium iodide for 30 min at room temperature in the dark. After the stained cell suspension was filtered through a strainer, a flow cytometer (FC500; Beckman Coulter, Inc., Fullerton, CA, USA) was used to analyze the DNA content histogram to determine the proportions of cells in each cell cycle phase (G[0]/G[1] phase, S phase, and G[2]/M phase). 2.9. Cell adhesion analysis MSC with or without Fe[3]O[4]@PDA NPs were seeded into a 96-well plate at a density of 6 × 10^3/well. The plate was then incubated at 37 °Cfor 3 h. Following incubation, the culture medium was discarded, and the cells were washed three times with PBS to remove non-adherent and loosely adherent cells. After taking images using a microscope, 10 μL of CCK-8 solution was added to each well. The plate was incubated at 37 °C for 3 h, and the OD value was measured, as follows: [MATH: Celladhesionrate=[(ODblank−ODtestcells)/(ODblank−ODcontrolcells)]×100% :MATH] 2.10. Western blots Lysed cell proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blotting was performed using the following antibodies: anti-AKT (S0B0114, STARTER, Hangzhou, China), anti-P-AKT (S0B0363, STARTER, Hangzhou, China), anti-JNK (9252, Cell Signaling Technology, USA), anti-P-JNK (4668, Cell Signaling Technology, USA), anti-ERK (4695, Cell Signaling Technology, USA), anti-P-ERK (4370, Cell Signaling Technology, USA), anti-p38 (9212, Cell Signaling Technology, USA), anti-P-p38 (4511, Cell Signaling Technology, USA), anti-CXCR1 (DF7730; Affinity Biosciences, Beijing, China), anti-CXCR4 (60042-1-Ig; Proteintech, Wuhan, China), anti-CXCR6 (CY7429; Always, Shanghai, China), and anti-GAPDH (14497-1-AP; Proteintech, Wuhan, China). The corresponding IRDye 700 or 800 secondary antibodies were obtained from Li-Cor Biosciences (Lincoln, NE, USA). Bound images were acquired using an Odyssey Infrared Imaging System (Li-Cor Biosciences). 2.11. Mouse model and treatments Male 6–8 weeks old C57BL/6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animal experiments were approved by the Ethics Committee of Animal Experiments of Jilin University and were performed in accordance with the U.K. Animals (Scientific Procedures) Act, 1986. Mice were randomly divided into four groups: sham control, PF model, MSC-treatment, and MSC + NPs treatment. To induce PF, mice were anesthetized with [67]isoflurane using an R520 Portable Veterinary Anesthesia Machine (RWD Life Science, Shenzhen, China) and then administered 5 mg/kg bleomycin (BLM; Selleck, USA) or the same volume of saline as a control via intratracheal injection. We added 0 or 50 μg/mL of Fe[3]O[4]@PDA NPs to the MSC and incubated them for 24 h. After the incubation, the cells were washed three times with PBS and then collected. Twenty-four hours after model establishment, BLM-treated mice were intravenously injected with 100 μL PBS or 100 μL PBS containing 3 × 10^5 MSC or 3 × 10^5 MSC + NPs cells. Simultaneously, for the MSC + NPs group, a magnet was placed on the external surface of the lungs of the mice for 2 h to induce magnetic targeting. After 10 d, the cells were injected repeatedly, and magnetic targeting was induced once. After 28 d, all mice were euthanized, and lung tissues and serum were collected for histological and immunological analyses. 2.12. In vivo imaging Mice were injected with DiO (Beyotime Biotechnology, Shanghai, China)-labeled MSC or MSC + NPs through the tail vein and attracted to a magnetic field in the lungs for 2 h prior to imaging. Subsequently, the animals were euthanized, and DiO-labeled MSC or MSC + NPs in the heart, liver, spleen, lungs, and kidneys were visualized using live animal imaging (In-Vivo FX PRO, Bruker, USA) and probe-based confocal laser endoscopy (pCLE; Cellvizio, Mauna Kea Technologies, Paris, France). 2.13. Histological analyses Lung tissues were fixed with 4 % paraformaldehyde, embedded in paraffin, and sectioned. The sections were stained with hematoxylin and eosin (H&E) for histopathological analysis. Collagen accumulation was evaluated using Masson, picrosirius red, and periodic acid–Schiff (PAS) staining. Sections were stained using a Prussian blue iron stain kit (Servicebio, Wuhan, China), according to the manufacturer's instructions. 2.14. Immunohistochemistry (IHC) staining Lung tissues were fixed with 4 % paraformaldehyde overnight and sliced into 4-μm paraffin-embedded sections. Lung tissue protein levels were assessed using the following primary antibodies: anti-α-SMA (19245, Cell Signaling Technology, USA), anti-CDH1 (D160656, Sangon Biotech, Shanghai, China), anti-CDH2 (D199282, Sangon Biotech, Shanghai, China), and anti-vimentin (5741S, Cell Signaling Technology, USA). An immunohistochemical staining kit (ZSGB-BIO, Beijing, China) was used according to the manufacturer's instructions. Images were captured using a confocal laser scanning microscope (FluoviewFV3000; Olympus, Tokyo, Japan). 2.15. Immunofluorescence (IF) analysis Cells were seeded in a 24-well Transwell plate (bottom), washed thrice with PBS, and fixed for 10 min in 4 % paraformaldehyde (w/v). Subsequently, cells were pre-blocked with 5 % BSA (A1933; Sigma–Aldrich), incubated with anti-CDH1 (D160656, Sangon Biotech, Shanghai, China), anti-CDH2 (D199282, Sangon Biotech, Shanghai, China), and anti-vimentin (5741S, Cell Signaling Technology, USA) antibodies for 1 h at room temperature and then incubated with the fluorophore-conjugated secondary antibody (SA00003-1 and SA00003-2, Proteintech, Wuhan, China) for 30 min at room temperature. Nuclei were stained with DAPI (Beyotime Biotechnology, Shanghai, China). Images were acquired using a High-Content Imaging System (Perkin-Elmer Operetta, USA). 2.16. Hydroxyproline content determination The concentration of hydroxyproline in the lung tissue was determined using a hydroxyproline detection kit (Jiancheng, Nanjing, China) following the manufacturer's instructions. The OD was measured at 550 nm using an ultraviolet spectrophotometer. 2.17. RNA isolation and real-time quantitative polymerase chain reaction (qRT-PCR) RNA from lung tissues was extracted using TRIzol reagent (Invitrogen, USA). Total RNA was reverse-transcribed into cDNA using Reverse Transcriptase (Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions. qRT-PCR was performed using the SYBR Green PCR Master Mix (Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions. The primer pairs used for qRT-PCR are listed in [68]Table S1. 2.18. Luminex analysis-serum cytokine expression The serum cytokine concentrations were determined using a Luminex assay with mouse multiplex-cytokine kits (12002798; Bio-Rad, USA) according to the manufacturer's instructions. 2.19. Serum analysis The mouse blood sample was coagulated at room temperature for 1 h, and centrifuged at 3000 rpm for 15 min to obtain serum. According to the manufacturer's instructions, commercial testing kits (Mindray, Shenzhen, China) were used to measure liver function parameters (alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total protein (TP)) and kidney function related indicators (creatine (Crea), blood urea nitrogen (BUN), and uric acid (UA)). 2.20. Transcriptome sequencing analysis Total RNA was extracted from mouse lung tissue using MagZol Reagent (Magen, Shanghai, China). The absorbance and RIN values of the A260/A280 ratios were measured using a NanoDrop spectrophotometer. A PE library of qualified RNA was prepared using the ABclonal mRNA Seq Lib Prep Kit (ABclonal, China) according to the manufacturer's instructions. The NovaSeq 6000 sequencing platform, PE150, was used to determine the reading length. Bioinformatic analysis was performed on the Illumina platform using the generated experimental data. All transcriptome analyses were conducted by Shanghai Zhongke New Life Biotechnology Co. Ltd. 2.21. Statistical analyses Data are expressed as the mean ± standard deviation of three independent experiments. Differences between groups were assessed using one-way analysis of variance (ANOVA). Values were considered statistically significant at P < 0.05. 3. Results 3.1. Construction and characterization of magnetized MSC A schematic illustration of the preparation of the magnetized MSC is shown in [69]Fig. 1A. Dopamine and Fe[3]O[4] particles were co-incubated at pH 8.5 to form NPs containing Fe[3]O[4] cores encapsulated within PDA shells. Examination under an electron microscope revealed that the Fe[3]O[4]@PDA NPs exhibited a spherical structure with a PDA shell layer ([70]Fig. 1B and C). Dynamic light scattering experiments revealed that the diameter of these NPs was approximately 119.71 nm ([71]Fig. 1D). Next, Fe[3]O[4]@PDA NPs were added to the MSC culture environment. TEM images confirmed that these NPs had successfully entered MSC and were predominantly localized within the lysosomes ([72]Fig. 1E). Quantification using inductively coupled plasma (ICP) revealed that an increase in the NPs concentration led to a gradual elevation in the iron content of the MSC ([73]Fig. 1F). Similar findings were observed in the Prussian blue staining experiments, which visually confirmed the presence of iron within the MSC ([74]Fig. 1G). Fig. 1. [75]Fig. 1 [76]Open in a new tab Characterization and uptake of Fe[3]O[4]@PDA NPs by MSC. A. Schematic diagram illustrating the synthesis process of Fe[3]O[4]@PDA NPs. B. Transmission electron microscopy (TEM) image depicting the morphology of Fe[3]O[4]@PDA NPs. C. Scanning electron microscopy (SEM) image depicting the morphology of Fe[3]O[4]@PDA NPs. D. DLS size distribution analysis of Fe[3]O[4]@PDA NPs. E. TEM image illustrating the internalization of Fe[3]O[4]@PDA NPs by MSC. F. Quantification of iron concentration in MSC using inductively coupled plasma-optical emission spectroscopy (ICP-OES). G. Prussian blue staining of MSC treated with varying concentrations of Fe[3]O[4]@PDA NPs. Next, the potential cytotoxic effects of Fe[3]O[4]@PDA NPs on MSC were examined. The CCK-8 results indicated that Fe[3]O[4]@PDA NPs did not significantly affect cell proliferation at 24, 48, or 72 h ([77]Fig. 2A). Furthermore, cell cycle analysis showed that, compared with the control group, no notable alterations were observed in the cell cycle upon the introduction of Fe[3]O[4]@PDA NPs ([78]Fig. 2B). Subsequently, the effect of Fe[3]O[4]@PDA NPs on MSC stemness was investigated. The results showed that Fe[3]O[4]@PDA NPs did not alter MSC characteristics, and the expression of surface markers CD90, CD105, CD73, and CD45 remained unchanged compared to those in the control cells ([79]Fig. 2C). Induction experiments further confirmed that the magnetized MSC retained their capacity for directional differentiation into osteoblasts and adipocytes, similar to the control MSC ([80]Fig. 2D). Taken together, these results demonstrate that the utilization of an appropriate concentration of Fe[3]O[4]@PDA NPs did not impair the viability or stemness of MSC after 72 h of exposure. Fig. 2. [81]Fig. 2 [82]Open in a new tab Effect of Fe[3]O[4]@PDA NPs on MSC characterization. A. Cytotoxicity assessment using the CCK-8 assay after 24, 48, and 72 h of incubation. B. Cell cycle analysis using flow cytometry. C. Flow cytometry analysis showing the absence of CD45 and CD34 expression and strong expression of CD44 and CD90 surface markers on MSC and MSC + NPs. D. Evaluation of the adipogenic and osteogenic differentiation potential in MSC and MSC + NPs. E. Detection of the cell migration ability of MSC and MSC + NPs. Left: Schematic illustration of the transwell assay. Middle: Crystal violet staining of cells that migrated through the inserts. Right: Histogram for statistical analysis of the number of migrating cells. F. Cell adhesion capability analysis of MSC and MSC + NPs. Top: Microscopic image of adherent cells; Bottom: Column chart of cell adhesion analysis of MSC treated with Fe[3]O[4]@PDA NPs at different concentrations. G. Detection of mRNA expression levels of Fn1, PECAM1, VCAM1, MMP-3, MMP-8, CDH2, CDH1, and OCLN genes. H. Western blot to analyze Cx43, CXCR1, CXCR4, and CXCR6 expression in MSC and MSC + NPs. GAPDH was used as the loading control. I. Detection of the expression levels of the phosphorylated and nonphosphorylated forms of AKT (P-AKT), p38 (P-p38), ERK (P-ERK), and JNK (P-JNK) in MSC and MSC + NPs using western blotting. GAPDH was used as the loading control. Data are presented as the mean ± standard deviation. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Moreover, Transwell chamber assays were employed to compare the migration behavior of MSC in the presence and absence of Fe[3]O[4]@PDA NPs. The results showed that when MSC were exposed to Fe[3]O[4]@PDA NPs, the number of migrating MSC increased significantly, indicating that Fe[3]O[4]@PDA NPs enhanced the migration ability of MSC ([83]Fig. 2E). In addition, the effects of Fe[3]O[4]@PDA NPs on the adhesion ability of MSC were also examined. The results revealed that compared with the 0 μg/mL group, the groups with the addition of 25 μg/mL or 50 μg/mL of Fe[3]O[4]@PDA NPs had significantly more remaining cells after the washing treatment. This implies that Fe[3]O[4]@PDA NPs can effectively enhance the adhesion ability of MSC ([84]Fig. 2F). To further explore the molecular mechanisms underlying the enhanced migration and adhesion, the gene expression levels related to the extracellular matrix and cell adhesion were explored. The results revealed an upregulation of transcripts for Fn1, PECAM1, VCAM1, MMP-3, MMP-8, and CDH2, whereas the expression of CDH1 and OCLN was downregulated post-treatment with Fe[3]O[4]@PDA NPs ([85]Fig. 2G). Interestingly, the proteins CXCR1, CXCR4, and CXCR6, which play crucial roles in MSC homing, were also upregulated. Moreover, the gap junction protein Cx43 exhibited increased expression levels after Fe[3]O[4]@PDA NPs treatment. These findings suggest that Fe[3]O[4]@PDA NPs not only enhance the expression of genes linked to migration and adhesion but also modulate those involved in MSC homing and intercellular connections ([86]Fig. 2H). Further exploration of the signaling pathways related to cell adhesion and migration revealed that Fe[3]O[4]@PDA NPs treatment activated the MAPK and PI3K-AKT signaling pathways in MSC. Compared to the non-phosphorylated forms, the phosphorylated forms of p38 (P-p38), ERK (P-ERK), JNK (P-JNK), PI3K (P-PI3K), and AKT (P-AKT) were significantly upregulated in the MSC treated with Fe[3]O[4]@PDA NPs ([87]Fig. 2I). These results indicate that Fe[3]O[4]@PDA NPs regulate MSC adhesion, migration, and intercellular communication by activating the MAPK and PI3K-AKT signaling pathways. 3.2. Magnetized MSC reverse TGF-β-induced epithelial-mesenchymal transition (EMT) in A549 cells Several studies have highlighted the crucial role of the EMT in the progression and development of PF. To elucidate the potential therapeutic effects on PF, A549 cells exposed to TGF-β were co-cultured with MSC or MSC + NPs. The methodology is illustrated in [88]Fig. 3A. A549 cells were seeded in a 24-well plate, while MSC were placed in another transwell upper chamber. The following day, the A549 cells were switched to serum-free medium, whereas Fe[3]O[4]@PDA NPs was added to the MSC. On the third day, TGF-β was added to the A549 cells to induce EMT, and the upper chamber containing MSC or MSC + NPs was placed into the 24-well plate with A549 cells for 48 h of co-culture. Finally, IF staining was performed to observe the expression of EMT marker proteins in A549 cells. The results showed that in the A549 cells treated with MSC or MSC + NPs, expression of the epithelial marker CDH1 was upregulated, whereas the mesenchymal markers CDH2 and vimentin were downregulated ([89]Fig. 3B–D). These results suggest that MSC and MSC + NPs may offer promising therapeutic avenues for reversing EMT. Fig. 3. [90]Fig. 3 [91]Open in a new tab Impact of MSC and MSC + NPs treatment on TGF-β-induced EMT in A549 cells. A. Schematic representation of the assay. B, C, D. Immunofluorescence assays showing the expression levels of the epithelial marker B: CDH1 and mesenchymal markers (C: CDH2 and D: vimentin) in A549 cells following different treatments. 3.3. Magnetized MSC have magnetic targeting movement abilities The magnetic targeting movement ability of magnetized cells was experimentally verified. As shown in [92]Fig. 4A, a round magnet was affixed to the bottom of a cell culture bottle, in which MSC were cultured and magnetized for 24 h. Subsequently, the culture medium was removed and cell movement was observed using crystal violet staining. Under the influence of an external magnetic field, the magnetized MSC aggregated at the magnet's location, whereas the control MSC did not exhibit such behavior ([93]Fig. 4B). Fig. 4. [94]Fig. 4 [95]Open in a new tab Monitoring magnetized MSC movement. A. Schematic illustration outlining the method for monitoring magnetized MSC movement. B. Visualization of magnetized cell accumulation at the magnetic field source. C. Ex vivo imaging of major organs post-injection of MSC or MSC + NPs with or without a magnetic field using an in vivo imaging system FX. D, E. Tissue imaging (D) and quantification of the fluorescence area (E) of the heart, liver, spleen, lungs, and kidneys using a Cellvizio Dual Band System to show the distribution of MSC and MSC + NPs. Data are presented as the mean ± standard deviation. ns, no significant difference; ∗∗∗P < 0.001. Next, MSC or magnetized MSC labeled with the fluorescent dye DiO were injected into mice via the tail vein. A magnetic field was applied to the lungs of the mice to induce cell targeting. After 2 h, fluorescence signals were observed in the lungs and other organs. In vivo imaging results demonstrated that, compared to the control group, both the MSC and MSC + NPs groups exhibited significant enrichment of red fluorescence signals in the lungs. Notably, the fluorescence intensity in the MSC + NPs group was substantially higher than that in the MSC group ([96]Fig. 4C). Similarly, confocal laser endoscopy revealed that DiO-labeled cells in the MSC + NPs group were more extensively distributed in the mouse lung tissue than in the MSC group ([97]Fig. 4D). These findings indicate that magnetic attraction significantly enhanced the accumulation of magnetized MSC in the mouse lungs. 3.4. Magnetized MSC exhibit an enhanced therapeutic effect on PF A PF mouse model was established by intratracheal instillation of BLM followed by treatment with MSC or MSC + NPs. Magnetic targeting was induced by placing magnets near the lungs ([98]Fig. 5A). Compared to the control group, lung tissues in the model groups showed evident hemorrhagic necrosis, whereas the necrosis was markedly reduced in both the MSC and MSC + NPs treatment groups ([99]Fig. 5B). Prussian blue staining of lung tissue revealed a pronounced blue coloration in the MSC + NPs group, indicating successful magnetic targeting and enrichment of magnetized MSC within the lungs ([100]Fig. 5C). Fig. 5. [101]Fig. 5 [102]Open in a new tab Therapeutic effects of MSC and MSC + NPs in the BLM-induced PF mouse model. A. Schematic representation of the BLM-induced PF mouse model used in this study. B. Representative photograph showing lung morphology at the study endpoint. C. Prussian blue staining illustrating iron deposition in lung tissues. D. Histological assessment of lung tissues, including hematoxylin and eosin (H&E), Masson's trichrome, Picrosirius red, and periodic acid–Schiff (PAS) staining. E. Histogram showing the percentage of the positive area of Masson, Picrosirius red, and PAS staining. F. Hydroxyproline content in lung tissues. Data are presented as the mean ± standard deviation. Data are presented as the mean ± standard deviation. ns, no significant difference; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Next, the progression of PF in the mice was assessed. H&E and collagen staining revealed that the alveolar epithelial structure in the model group was destroyed, with severe collagen deposition. However, treatment with the MSC and MSC + NPs effectively preserved the lung tissue structure and reduced collagen deposition. Notably, the MSC + NPs group exhibited superior therapeutic efficacy, showing a more intact alveolar structure and less collagen deposition compared to the MSC group alone ([103]Fig. 5D and E). Additionally, both the MSC and MSC + NPs treatments normalized the hydroxyproline content, a marker for collagen levels ([104]Fig. 5F). Moreover, the treatment with MSC + NPs was more effective than the treatment with MSC alone, showing a lower hydroxyproline content. Furthermore, the expression of epithelial and mesenchymal markers was detected in the lung tissues of mice. RT-PCR, immunohistochemistry, and IF results showed that BLM induction led to a significant increase in the expression of mesenchymal markers CDH2, vimentin, and α-SMA and a decrease in the expression of the epithelial marker CDH1. However, upon treatment with MSC and MSC + NPs, the expression of CDH2, vimentin, and α-SMA was reduced, and the expression of CDH1 partially recovered ([105]Fig. 6A–G). Further comparison of the therapeutic effects between MSC and MSC + NPs revealed that at the RNA level, there were significant differences in the expression of α-SMA and CDH1 between the two groups, while the expression of the other two markers did not show evident differences. At the protein level, the immunohistochemical results indicated that compared with the MSC treatment group alone, the MSC + NPs treatment could more significantly reduce the expression of vimentin and α-SMA. However, the IF results only showed a difference in α-SMA expression between the two groups. The above results suggest that α-SMA may be a more crucial indicator of the therapeutic advantages of MSC + NPs. Fig. 6. [106]Fig. 6 [107]Open in a new tab Expression of epithelial and mesenchymal markers in the lung tissues of BLM-induced PF mice. A. RT-PCR analysis of marker expression. B. Immunohistochemistry depicting the localization of markers in lung tissues. C. Histogram showing the percentage of the positive area of immunohistochemistry staining. D, E, F. Immunofluorescence images showing marker expression patterns. G. Quantification of the relative expression of CDH2, vimentin and α-SMA. Data are presented as the mean ± standard deviation. ns, no significant difference; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Decreased inflammation is an important indicator of successful PF treatment. Therefore, changes in serum inflammatory factors were detected using a Luminex chip. As shown in [108]Fig. 7A, the levels of pro-inflammatory factors IL-1β, TNF-α, IFN-γ, IL-2 and IL-12p70 in the model group increased significantly compared with those in the control group. Treatment with MSC and MSC + NPs effectively reduced the expression of the majority of these pro-inflammatory factors in the serum, although MSC treatment did not appear to exert a significant effect on the level of IL-12p70. The anti-inflammatory effect was further validated through RT-PCR analysis in lung tissue. In PF, several important pro-inflammatory factors, including IFN-γ, IL-1β, IL-6 and IL-17A, significantly increased in the model group but decreased significantly after treatment with MSC and MSC + NPs, indicating that inflammation was effectively controlled in PF mice ([109]Fig. 7B). Fig. 7. [110]Fig. 7 [111]Open in a new tab Expression levels of inflammatory factors. A. Luminex chip analysis demonstrating changes in serum inflammatory factors. B. RT-PCR analysis of inflammatory factor expression in lung tissues. Data are presented as the mean ± standard deviation. ns, no significant difference; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. In addition, we further conducted multiplex immunofluorescence assays on the lung tissue sections of mice. The results showed that in the MSC + NPs treatment group, MSC (fluorescing red) and macrophages (fluorescing cyan) were found to be either overlapping or have adjacent localization patterns ([112]Fig. S1). This observation implied the existence of an interaction between MSC and macrophages. 3.5. In vivo safety analysis of magnetized MSC To determine the systemic safety of administration, PBS or 3 × 10^5 magnetized MSC were injected into mice, and after 28 days, changes in blood and major organs were observed. By detecting various serum biochemical indicators in mice, it was found that compared with the PBS group, MSC + NPs treatment did not change liver function parameters (ALT, AST, and TP) or the indicators related to renal function (Crea, BUN, and UA) ([113]Fig. S2A). H&E staining analysis showed that there was no damage to the heart, liver, spleen, lungs, and kidneys ([114]Fig. S2B). These results indicate that the treatment with Fe[3]O[4]@PDA-magnetized MSC is safe. 3.6. Global transcriptomic response to MSC + NPs treatment in BLM-induced PF mice To study the global transcriptomic changes in gene expression in BLM-induced PF mice, RNA-seq analysis was performed. The similarity and heterogeneity of samples from the BLM and control groups in terms of the transcriptome are shown using heatmaps ([115]Fig. 8A). A total of 35701 genes were identified in the lung tissue samples from the two groups. Among these, 1631 significantly differentially expressed genes (DEGs; 520 downregulated and 1111 upregulated) were identified in the BLM-induced PF model. The distribution and up/downregulation of these DEGs are further illustrated in a volcano plot ([116]Fig. 8B). Fig. 8. [117]Fig. 8 [118]Open in a new tab RNA-Seq analysis of the control and BLM models. A. Heatmaps illustrating differentially expressed features in the transcriptome between control and BLM models. B. Volcano plot highlighting the differences in mRNA expression between the control and BLM models. C. Top 20 enriched pathways identified through KEGG analysis between the control and BLM groups. D. Protein–protein interaction (PPI) network prediction. The left diagram shows the top 25 proteins with the highest connectivity in the PPI network. Circles represent differentially expressed proteins/genes, with red indicating upregulation, blue indicating downregulation, and circle size representing connectivity; larger circles denote higher connectivity. The right diagram presents the expression bar chart of the top 25 high-connectivity proteins. To further identify the relevant biological pathways, KEGG enrichment analysis of the DEGs was performed. The most significantly enriched pathways included “ECM-receptor interaction,” “metabolism of xenobiotics by cytochrome P450,” “glutathione metabolism,” “phagosomes,” “malaria,” “human papillomavirus infection,” and “focal adhesion.” The top 20 KEGG pathways are shown in [119]Fig. 8C. In the STRING database, we selected species/closely related species (BLAST e-value: 1e-05) to analyze differentially expressed proteins and obtain their interaction relationships. We then selected the top 25 proteins with the highest connectivity (default setting), recalculated their connectivity, and drew an interaction network diagram ([120]Fig. 8D, left). [121]Fig. 8D (right) shows the expression bar chart of these proteins. To elucidate the underlying mechanism of the therapeutic effects of MSC + NPs on BLM-induced PF mice, the DEGs in each group were compared. [122]Fig. 9A depicts the numbers of unique and shared genes in each comparison group. A statistical analysis was then performed on the number of DEGs among the comparison groups, as presented in [123]Fig. 9B. Trend clustering analysis showed that the DEGs in the distinct groups exhibited a roughly similar trend ([124]Fig. 9C). We further explored the alterations in the signaling pathways triggered by treating PF mice with MSC alone and MSC + NPs. The most significantly enriched pathway analysis revealed that the gene changes induced by treating PF mice with MSC and MSC + NPs involved multiple pathways, including the ECM-receptor interaction, focal adhesion, human papillomavirus infection, malaria, osteoclast differentiation, phagosome, and Toll-like receptor signaling pathways ([125]Fig. 9D). Within these pathways, a notable disparity in gene expression was observed between the model and control groups. This substantial variation strongly suggests that these pathways might all play a role in PF development. After treatment with MSC and MSC + NPs, the gene expression patterns underwent a more advantageous change, indicating that MSC and MSC + NPs treatment may exert a common regulatory effect by modulating these pathways. Notably, in the majority of these pathways, the changes in the MSC + NPs treatment group were even more pronounced. To verify the accuracy of the results, we performed qPCR validation on selected genes within the enriched pathways, as illustrated in [126]Fig. 9E. Fig. 9. [127]Fig. 9 [128]Open in a new tab Gene expression changes in the lung tissues of BLM-induced PF mice following treatment with MSC + NPs. A. Venn diagrams displaying DEGs between various comparison groups. B. Bar chart depicting DEGs content. C. Gene expression trend chart illustrating changes in expression patterns. D. Heatmap depicting enriched pathways affected by MSC and MSC + NPs treatments. E. RT-PCR analysis of gene expression in enriched pathways. Data are presented as the mean ± standard deviation. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Next, to explore the molecular mechanism underlying the more favorable therapeutic effect of the MSC + NPs group compared with the MSC group, the DEGs in lung tissues treated with MSC or MSC + NPs were compared. A total of 3424 significant DEGs were identified through transcriptome sequencing, of which 1397 were downregulated and 2027 were upregulated. A volcano plot displays the distribution and the patterns of down/upregulation of DEGs in the two sets of samples ([129]Fig. 10A). Furthermore, we performed a KEGG pathway analysis on all the genes that changed in the MSC + NPs group compared with the MSC group. [130]Fig. 10B shows the top 20 KEGG pathways, including cell adhesion molecules, complement and coagulation cascades, leukocyte transendothelial migration, the Rap1 signaling pathway, vascular smooth muscle contraction, and Th1 and Th2 cell differentiation ([131]Fig. 10C). This suggests that these pathways may contribute to the improved therapeutic outcome of MSC + NPs in treating PF. The GO pathway enrichment analysis of DEGs indicated a significant enrichment in GO molecular function (GO-MF) terms such as dynein intermediate chain binding and ATP-dependent microtubule motor activity. For the GO cellular component (GO-CC) terms, the changed genes were predominantly located in the cilium, ciliary plasma, and axoneme. In the GO biological process (GO-BP) analysis, the altered genes were enriched in processes related to cilium organization, assembly, and movement ([132]Fig. 10D). Fig. 10. [133]Fig. 10 [134]Open in a new tab RNA-seq analysis of MSC and MSC + NPs. A. Volcano plot highlighting mRNA expression differences between MSC and MSC + NPs. B. Top 20 enriched pathways identified through KEGG analysis between MSC and MSC + NPs. C. Heatmaps illustrating differentially expressed features in the transcriptome of MSC and MSC + NPs. D. Bar chart showing GO enrichment of the differential gene expression levels between MSC and MSC + NPs. The vertical axis is -log[10] (P-value), and the entries are arranged in ascending order of P-value from the smallest to largest. The three major GO categories are represented by different colored columns (green for biological processes, blue for molecular functions, and red for cellular components), with the horizontal axis representing specific functional description information. 4. Discussion PF is an irreversible and a chronic and progressive disease characterized by fibroblast foci, alveolar honeycomb-like structures, and continuous fibrosis, ultimately leading to irreversible destruction of the lung architecture and respiratory failure [[135]1]. MSC possess multi-differentiation potential and self-renewal capabilities. Studies have shown that MSC can differentiate into alveolar epithelial cells (AECs) and fibroblasts in the pulmonary microenvironment, directly participating in tissue repair [[136]26,[137]27]. Additionally, MSC exert strong paracrine effects by secreting various bioactive factors, promoting inflammation resolution, fibrosis reduction, and tissue function recovery. Moreover, MSC have a long survival time in the body, enabling sustained therapeutic effects, which gives them greater potential for long-term treatment compared to the short-term effects of exosomes [[138]28,[139]29]. However, challenges remain in optimizing MSC delivery to ensure their effective localization and sustained presence at the injury site. Direct infusion of MSC may be hindered due to their large diameter, which affects their ability to pass through capillaries and target damaged tissues. Magnetic targeting technology offers a potential solution by using magnetic fields to guide magnetized MSC to the damaged areas. This approach aims to enhance therapeutic efficacy by concentrating MSC at the repair site without increasing the total number of administered cells, thereby reducing the risk of side effects on other organs by limiting cell distribution to the specific injury site. Among various magnetic materials, Fe[3]O[4]@PDA NPs were chosen in this study due to their high magnetic moment, adjustability, biocompatibility, and low toxicity. Here, we observed that MSC treated with Fe[3]O[4]@PDA NPs exhibited a higher tendency to migrate toward injured sites. This enhanced migration capacity was not only attributed to external magnetic guidance but also to the upregulated expression of genes related to migration, homing, and intercellular connections. Fundamentally, the internalization of Fe[3]O[4]@PDA NPs by MSC triggers a series of intracellular changes, including the activation of signaling pathways such as MAPK/AKT and alterations in the expression of specific proteins. These changes lead to an enhanced response of MSC, as demonstrated by their increased migration toward injury sites. AECs play a crucial role in lung tissue repair and regeneration following injury [[140]30,[141]31]. Type II AECs serve as stem cells within the alveolar epithelium and are capable of self-renewal and differentiation into type I AECs [[142]32]. Under conditions such as inflammation and injury, the differentiation of type II into type I AECs is inhibited, leading to their transformation into interstitial cells via EMT, thereby promoting PF formation [[143]6]. In the TGF-β-induced A549 cell EMT model, MSC and magnetized MSC effectively reduced the expression of mesenchymal markers, such as CDH2 and vimentin while increasing the expression of epithelial markers such as CDH1, thereby reversing the TGF-β-induced EMT process. This reversal highlights the potential of MSC-based therapies—particularly when enhanced by magnetization—to mitigate fibrotic transformation and support epithelial recovery in PF. In the in vivo BLM-induced PF mouse model, the results of H&E, Masson's and Picrosirius red and PAS staining, and hydroxyproline content analysis, revealed that MSC and magnetized MSC improved collagen deposition, alleviated PF, and restored lung structure at both the histopathological and imaging levels. Additionally, RT-PCR, IHC, and IF staining indicated that MSC and magnetized MSC influenced the expression of epithelial and mesenchymal markers at both the transcriptional and protein levels. Furthermore, treatment with MSC and magnetized MSC inhibited serum inflammatory cytokines, effectively attenuating the inflammatory response. Importantly, magnetized MSC exhibited superior antifibrotic effects compared to MSC alone. RNA-seq analysis of global transcriptomics revealed complex molecular mechanisms of MSC and MSC + NPs in treating PF. These mechanisms include the regulation of the TGF-β and Wnt signaling pathways, which are involved in the EMT; the modulation of ECM-receptor interaction; effects on focal adhesion; and the regulation of multiple signaling pathways such as PI3K and Hippo. These findings are consistent with those of existing research that highlights the immunomodulatory role of MSC, which can secrete various growth factors, cytokines, and chemokines to promote angiogenesis and cellular proliferation, improve tissue function, alleviate oxidative stress, reduce fibrosis, and thus, slow down or treat various diseases [[144][33], [145][34], [146][35], [147][36], [148][37]]. When Fe[3]O[4]@PDA NPs are introduced, MSC exhibit a series of significant changes, including enhanced cell adhesion, improved Th1/Th2 cell differentiation, increased leukocyte transendothelial migration, and activated the Rap1 signaling pathway. These changes are likely closely associated with the enhanced therapeutic efficacy of MSC in the treatment of PF, although the specific molecular mechanisms remain to be elucidated through further in-depth research. Recent reports have indicated that iron oxide NPs can significantly enhance mitochondrial transfer from MSC to damaged mouse AECs, thereby markedly mitigating PF progression [[149]38,[150]39]. Overall, the combination of MSC with Fe[3]O[4]@PDA NPs, complemented by magnetic targeting technology, holds great potential for optimizing MSC-based therapy and improving tissue repair. Fn1 serves as a crucial constituent of the extracellular matrix, playing a pivotal role in governing cell adhesion, migration, proliferation, and differentiation. Studies have demonstrated that Fn1 is closely associated with various pathological processes, including tumor metastasis and invasion, tissue repair following injury, and the progression of fibrotic diseases [[151]40,[152]41]. In our study, we observed a significant upregulation of Fn1 transcription levels in a BLM-induced mouse PF model. Conversely, in both the MSC and MSC + NPs treatment groups, the expression levels of Fn1 exhibited a significant decline. This observation is consistent with previous research results, further confirming the promoting role of Fn1 in the progression of PF, with its expression level being positively correlated with the extent of PF. Tnc, another vital component of the extracellular matrix, participates in several critical biological processes, such as embryonic development, wound healing, tumor development, chronic inflammation, and fibrosis. Research has indicated that the overexpression of Tnc is closely associated with tissue repair and renal fibrosis, while elevated levels are frequently detected in the serum of patients with PF [[153]34,[154]42,[155]43]. In our BLM-induced mouse PF model, Tnc was similarly found to be highly expressed. Following treatment with MSC and MSC + NPs, the transcription levels of Tnc demonstrated a noteworthy reduction. Thbs2 acts as a regulatory factor in collagen fiber formation and angiogenesis; its dysregulation is implicated in various fibrotic conditions, including hepatic and cardiac fibrosis [[156]44,[157]45]. Previous studies have indicated that suppression of Thbs2 can effectively mitigate fibrosis in the context of heart damage induced by pulmonary hypertension, thereby underscoring its potential as an antifibrotic therapeutic agent [[158]34,[159]46,[160]47]. In our experimental model of PF, treatment with MSC and MSC + NPs led to a significant decrease in Thbs2 transcription levels. This finding reinforces the substantial role of Thbs2 in the progression of PF and suggests its potential as a promising therapeutic target for PF. 5. Conclusion Treatment of MSC with Fe[3]O[4]@PDA NPs activates the expression of genes involved in migration, adhesion, homing and intercellular connections as well as the MAPK/AKT signaling pathway. Applying Fe[3]O[4]@PDA-magnetized MSC to treat PF mice significantly repairs fibrosis and reduces the expression of inflammatory factors. This method, which utilizes an external magnetic field to guide magnetized MSC treatment, not only enhances MSC engraftment in the lungs but also activates pathways such as cell adhesion molecules, leukocyte transendothelial migration, and Rap1 signaling pathways. Additionally, changes were induced in the gene expression pathways of ECM-receptor interactions, focal adhesion, and the TGF-β signaling pathway in the lung tissues of PF mice. In summary, Fe[3]O[4]@PDA-magnetized MSC therapy provides an efficient treatment approach for PF. CRediT authorship contribution statement Xiaoming Zhao: Writing – original draft, Data curation. Chenfei Kong: Writing – original draft, Data curation. Xue Xia: Investigation, Data curation. Kelin Zhao: Data curation. Miao Hao: Data curation. Yiyao Gao: Investigation. Yuqian Wang: Formal analysis. Zhiying Chen: Formal analysis, Conceptualization. Jinlan Jiang: Funding acquisition, Conceptualization. Statement of significance Pulmonary fibrosis (PF) is a chronic lung disease characterized by progressive scarring, often leading to severe respiratory issues. Current treatments mainly manage symptoms and slow disease progression. Therapy using mesenchymal stem cells (MSC) therapy shows promise in modulating inflammation, repairing tissue, and regenerating blood vessels, but its effectiveness is limited by the short retention time in the lungs after intravenous administration. In this study, we used Fe[3]O[4]@PDA nanoparticles to magnetize MSC and an external magnetic field to guide them to lung injury sites. This approach significantly enhanced the local concentration, targeting, and retention of MSC, and activated therapeutic molecules involved in migration, adhesion, homing, intercellular connections, and key signaling pathways such as MAPK/AKT. This resulted in notable tissue repair and a substantial reduction in inflammatory markers in PF mice. Transcriptome sequencing further revealed the impact of magnetized MSC on various signaling pathways and gene expression. These findings suggest that Fe[3]O[4]@PDA-magnetized MSC therapy could be a more effective treatment for PF. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments