Abstract Background Acute lung injury (ALI) is a critical pulmonary condition characterized by high morbidity and mortality rates. Recent studies have highlighted the therapeutic potential of engineered exosomes derived from mesenchymal stem cell (MSC) in modulating the inflammatory response in ALI. Here, a novel approach was developed to fabricate engineered bone mesenchymal stem cells (BMSCs) derived exosomes by utilizing superparamagnetic iron oxide nanoparticles (SPIONs) and an alternating magnetic field (AMF) to precondition BMSCs. This study evaluated and compared the therapeutic potential of different groups of engineered exosomes in ALI mice model and analyzed their underlying mechanisms using high-throughput sequencing. Methods BMSCs were isolated from SD rats and subjected to treatment with SPIONs and/or an AMF. Following this, we established a lipopolysaccharide (LPS)-induced ALI mice model and evaluated the therapeutic efficacy of exosomes from different groups by administering them via tail vein injection. The expression profiles of microRNAs (miRNAs) in exosomes were compared to explore the mechanism of regulating inflammatory response and ameliorating lung injury. Results 25 µg/mL SPIONs and 3mT AMF were the best conditions for preparing engineered exosomes, which reduced the level of pro-inflammatory factors and had the most significant effect in repaired lung damage in vivo. The transfection of miR-145-5p mimics enhanced Bronchial Epithelium transformed with Ad12-SV40 2B (BEAS-2B) cells viability and reduced relevant inflammation expression in vitro experiments. Conclusion The engineered exosomes obtained by low dose SPIONs combined with AMF can help regulate the level of inflammatory factors and improve lung injury. Targeted regulation of kruppel-like factor 5 (KLF5) by exosomal miR-145-5p and inhibition of the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway play a key role in this vitro process. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-025-04598-7. Keywords: Engineered exosomes, Acute lung injury, Alternating magnetic field, Superparamagnetic iron oxide nanoparticles, Inflammation Background Acute lung injury (ALI) is an acute and severe inflammatory lung injury, which accompanied with the activation of various inflammatory mediators and effector factors within the cells, leading to a cascade of inflammation processes. ALI can progress into a more severe form called Acute Respiratory Distress Syndrome (ARDS), which has a significantly high mortality rate ranging from 35 to 40% [[38]1, [39]2]. Currently, the clinical treatment for ALI and ARDS primarily focuses on providing supportive measures, which can only partially help alleviate the progression of the disease, and the overall mortality rate remains high [[40]3]. There is a critical and urgent need for the development of more effective treatment strategies for ALI. Cell therapies using mesenchymal stem cells (MSCs) have emerged as promising treatments for ALI due to their potential in immune system regulation and tissues repair [[41]4]. Nevertheless, the clinical application of stem cell transplantation faced several challenges, including low survival rates of the transplanted cells, the potential risks of tumor formation, and immune rejection [[42]5]. Recent research suggests that MSC derived exosomes (MSC-Exos) plays a crucial role in mediating the therapeutic effects observed in ALI. Exosomes are small extracellular vesicles that contain a wide range of bioactive molecules, including miRNAs, which have been significantly involved in the therapeutic benefits of MSC-Exos for ALI through regulation of various signaling pathways [[43]6–[44]8]. To enhance the therapeutic potential of MSC-Exos, researchers have been exploring methods to engineer the exosomes miRNA [[45]9–[46]12]. However, current engineering methods of exosomes, such as sonication, extrusion, or electroporation, have limitations in preserving the integrity of exosomes membranes and protecting the activity of bioactive molecules [[47]13–[48]16]. Therefore, developing new engineering techniques that can overcome these limitations is crucial to fully harness the therapeutic potential of MSC-Exos for the treatment of ALI. Electromagnetic fields have garnered significant attention as a potent modality for noninvasive modulating MSC behavior, which is crucial for tissue repair and regeneration. Among the various strategies, the employment of superparamagnetic iron oxide nanoparticles (SPIONs) stands out due to their exceptional magnetic responsiveness and biocompatibility [[49]17], which positions them as an ideal candidate for the precise manipulation of cell behaviors [[50]18]. Empirical evidence has demonstrated that the stimulation of SPIONs-loaded MSCs with electromagnetic fields significantly influences cellular functions, notably augmenting migration, proliferation, and differentiation processes [[51]19, [52]20]. Alternating Magnetic Field (AMF) is a type of dynamic electromagnetic field that formed by the combination of a changing magnetic field and an electric field generated by an alternating current power source. In contrast to static magnetic fields, the AMF’s electromagnetic field is highly malleable and adjustable, which renders AMF exceptionally versatile and advantageous for the noninvasive and precise modulation of stem cell behavior and biological contents [[53]21]. In this study, a versatile strategy for the fabrication of engineered bone mesenchymal stem cell derived exosomes (BMSC-Exos) via magnetic preconditioning of BMSCs, was developed for the therapeutic management of ALI. We found that the AMF treatment of SPION-loaded BMSCs had a significant impact on the miRNAs content of BMSC-Exos, potentially enhancing their anti-inflammatory capabilities. In a systematic approach, we have generated three distinct types of engineered BMSC-Exos: one group derived from BMSCs loaded with SPIONs (BS-Exos), another from BMSCs exposed to a 3mT AMF (BA-Exos) and a third group from BMSCs subjected to a synergistic treatment involving both SPIONs and AMF (BSA-Exos). In vivo experiments validated the profound anti-inflammatory and tissue reparative functions of these engineered BMSC-Exos in the treatment of ALI, with the exosomes derived from BMSCs with the combined treatment of SPIONs and AMF demonstrating superior efficacy in mitigating inflammation and promoting tissue repair. The miRNA sequencing was carried out to further explore the underlying therapeutic mechanisms of BSA-Exos. Our findings indicate that the engineered exosomes were enriched with miR-145-5p, a microRNA that plays a crucial role in suppressing the phosphorylation NF-κB by modulating the transcriptional activity of KLF5 at the p65 subunit, thereby exerting a pivotal role in the attenuation of inflammatory signaling pathways. Our study underscores the potential of the magnetic engineered BMSC-Exos as a potent therapeutic modality, and highlighting their promise in the treatment of ALI. The graphical abstract of study is illustrated in Fig. [54]1. Fig. 1. [55]Fig. 1 [56]Open in a new tab Schematic description of BSA-Exos-derived miR-145-5p regulate inflammation levels in acute lung injury. Created by Biorender.com Materials and methods The work has been reported in line with the ARRIVE guidelines 2.0. Cell culture and reagents The cells used in our study were primary bone marrow mesenchymal stem cells isolated from SD rats. Primary BMSCs were cultured in DMEM/F12 containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution. BEAS-2B cells were obtained from the Chinese Academy of Sciences Shanghai Cell Bank (Shanghai, China). They were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin solution. All cells were incubated at 37 °C with 5% CO[2]. Most cell culture media and reagents for cell culture were purchased from Gibco (Carlsbad, CA, USA). Cell culture plates were obtained from NEST Biotechnology Co., Ltd. (Wuxi, China). Isolation and identification of bone marrow mesenchymal stem cells All animal use and research protocols in this study were approved by Animal Care & Welfare Committee of Southeast University, and were in accordance with the Guide for the Care and Use of Laboratory Animals. BMSCs were isolated and cultured according to previous reports [[57]22]. All female SD rats (3–4 weeks old) were purchased from Hangzhou Medical College (license no. SYXK [Su] 2021-0022). Three rats in each cage were kept in a temperature-controlled (24 ± 2℃) specific pathogen-free facility and given free food and water. Six SD rats at a time were anesthetized with isoflurane and were euthanized by inhalation of carbon dioxide. Then soaked in 75% alcohol to sterilize. Cells were collected by flushing the femurs and tibias with phosphate-buffered saline (PBS) and were cultured in complete DMEM/F12. The cells were maintained in a 37 °C humidified incubator with 5% CO[2]. To identify BMSCs, the cells morphology was observed under a microscope, and flow cytometry and differentiation assays were performed. SPIONs and AMF conditions Dextran-coated SPIONs were prepared according to the literature [[58]23, [59]24]. A sterile suspension of SPIONs was first added to the complete medium, and then the concentration of SPIONs in the medium was successively diluted to 25, 50, and 100 µg/mL. Helmholtz coil with length, width, and height of 280 mm*160 mm*330 mm (Hu Nan Pang Sheng Elegance Technology Co., Ltd) was used to connect the constant current power supply, and the AMF environment of BMSCs was set to 0, 1, 3 and 5mT respectively. BMSCs added with different concentrations of SPIONs were stimulated with AMF for one hour a day. At the same time, in order to prove that the interaction between the AMF and the SPIONs caused the change of cell contents, we measured the temperature change of the cell culture medium every 10 min to rule out the thermal effect. Prussian blue iron staining In order to confirm the presence of SPIONs in BMSCs, when BMSCs grew to the logarithmic stage, cells were seeded in 12-well plates with 8 × 10^4 cells per well. After cell adhesion, 25 µg/mL SPIONs (mixed with medium) were added and incubated for 24 h. The control group without SPIONs was added with the same amount of medium. The culture medium was sucked out after 24 h, the cells were fixed with 4% paraformaldehyde after washing with PBS, and fixed at room temperature for 20 min. Prussian blue stain (Solarbio, Beijing, China) was added and incubated at 37℃ for 30 min. Perls redye solution was added into the wells for 1 min and the result was observed under an inverted optical microscope. Isolation and purification of exosomes First, FBS was centrifuged at 110,000×g for 70 min to remove endogenous exosomes before used for cell culture. BMSCs were cultured in DMEM containing 10% Exo-free FBS, and the supernatant was collected and centrifuged sequentially to isolate exosomes. In accordance with the manufacturer’s instructions and ultracentrifuged method described by literatures [[60]25, [61]26]. Briefly, the collected cell supernatant was precentrifuged at 300×g for 10 min, 2000×g for 10 min and 10,000×g for 30 min to remove some cell debris and miscellaneous proteins. Then, the supernatant was ultracentrifuged at 110,000×g for 70 min to obtain pellet exosomes. The exosomes pellets were resuspended in PBS and ultracentrifuged at 110,000×g for 70 min again and final exosomes were subpackaged and then stored at -80 °C. Identification and internalization of exosomes According to the newly updated guidelines on Minimal information for studies of extracellular vesicles in 2023 (MISEV2023) [[62]27], exosomes were characterized as follows: (1) the morphology of exosomes was observed by transmission electron microscopy (TEM; Hitachi, Tokyo, Japan) after staining with 1% uranyl acetate, (2) the particle size distribution were detected by nanoparticle tracking analysis (NTA; Particle Metrix, Germany), and (3) the characteristic proteins of exosomes were detected by western blotting (WB), including CD81, CD9, tumor susceptibility gene 101(TSG101) and Calnexin. Establishment of acute lung injury model in vivo All male C57BL/6 mice (5–6 weeks old) were purchased from Hangzhou Medical College (license no. SYXK [Su] 2021-0022). Five mice in each cage were kept in a temperature-controlled (24 ± 2℃) specific pathogen-free facility and given free food and water. At least 1 week before the experiment, the mice had acclimated to the environment. Sixty mice were anesthetized with isoflurane; the trachea was fully exposed, and LPS( Sigma, L2880, 10 mg/kg) was injected with a microsampler. All mice were randomly assigned to experimental groups (n = 5 per group): (1) LPS + PBS, (2) LPS + B-Exos, (3) LPS + BS-Exos, (4) LPS + BA-Exos, (5) LPS + BSA-Exos, and (6) Control (no treat group). The exosomes suspension (50 µg, top up to 100µL with PBS) was injected through the tail vein after 4 h LPS induction. The same volume of PBS was used as a control. The lung tissue or BALF was harvested after 24 h exosomes administration. According to previous experiments, the ALI mice showed a deterioration in mental state, including wheezing and nose scratching, reduced eating and movement, and crouching in the corners of their cages. Mice that died during the experiment or did not develop symptoms after LPS induction were excluded from the criteria. The sample size is determined on the basis of previous experiments, and the success probability of the ALI model is maintained at more than 80%. To avoid any influence of the BALF collection procedure on histology features, separate animal experiments were implemented for BALF analysis. Sample sizes for each experiment are included in the figure legends. The researchers involved in experiments were blinded to group allocations for the duration of the study. Measurement of inflammatory cytokines and protein concentration in BALF The lungs of mice were lavaged three times with 500µL ice-cold PBS to collect BALF. Approximately 1 mL of net BALF was recovered and centrifuged in a centrifugal tube at 3000 rpm and 4℃ for 10 min. The supernatant was subpackaged and stored at -80℃ for cytokine detection. The proinflammatory cytokine TNF-α, IL-12p70, IL-1β, and IL-10 levels were then measured in BALF by Enzyme-linked immunosorbent assay (ELISA) kit (Proteintech Group, Inc). The concentration of BALF protein was detected by using the bicinchoninic acid (BCA) protein assay kit (NCM Biotech, Suzhou, China) according to the manufacturer’s instructions. Evaluation of the pulmonary aggregation of exosomes in vivo Most of the current studies used exosomes labeling to facilitate tracing in vivo. The exosomes are labeled with 1µL 1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindotricarbocyanine Iodide (DIR) (Beyotime, Shanghai, China) and incubated at 37℃ for 20 min. Excess dye was removed by ultracentrifugation (110000×g, 70 min) after incubation. Twenty C57BL/6 mice were randomly divided into control group and LPS group. The labeled exosomes (100µL, 1 × 10^6 cells) were injected into the tail vein, and then the pulmonary retention of exosomes was observed using the Maestro in vivo optical imaging system (Cambridge Research & Instrumentation, Inc.) at 2 h, 4 h, 8 h, 12 h, and 24 h. The heart, liver, spleen, lung and kidney were taken for in vitro fluorescence imaging to observe the distribution of exosomes after the sacrificed mice were dissected. Lung wet/dry (W/D) weight measurement The severity of pulmonary edema was assessed by W/D ratio. In short, after 24 h of caudal intravenous administration, mice anesthetized with isoflurane were euthanized by inhalation of carbon dioxide. The lung tissue was removed and immediately weighed wet weight (W). The wet lung tissue was then placed in an oven at 70℃ for 48 h before being reweighed to obtain dry weight (D). Finally, the W/D ratio was calculated. The higher the W/D ratio, the more serious the lung injury was. Hematoxylin and Eosin (H&E) staining The right lower lungs of mice were fixed in 4% paraformaldehyde for 24 h at 4℃. Then the lung tissues were rinsed, dehydrated, embedded in paraffin, and sliced ona rotary microtome with a thickness of 5 μm. Pathological changes of the lung tissues were analyzed by H&E staining. The degree of pulmonary injury was graded from 0 (normal) to 4 (severe) based on the following histopathological parameters: interstitial inflammation, neutrophil infiltration, congestion, and edema. The scoring criteria were defined as follows: Score 0: no damage; Score 1: <25% damage; Score 2: 25–50% damage; Score 3: 50–75% damage; Score 4: >75% damage. The total lung injury score was calculated as the sum of all four parameter scores [[63]28]. Tissue sections were examined under an inverted fluorescence microscope (Olympus, Japan) by evaluating five randomly selected high-power fields with the average score being calculated for statistical analysis. Biosafety of the engineered exosomes in vivo The biosafety of the engineered exosomes in vivo was evaluated. To evaluate the biosafety in vivo, fifteen healthy C57BL/6 mice were randomly divided into three groups: (1) PBS; (2) B-Exos; (3) BSA-Exos. The dose of exosomes is consistent with the amount injected for in vivo treatment. Different groups of exosomes were injected into the mice respectively. One month after administration, the mice were sacrificed to collect serum for biochemical examination, and each organ was isolated for histological analysis. Real-time PCR analysis The expression of different miRNAs and inflammatory factors associated with acute lung injury were assessed using real-time quantitative polymerase chain reaction. Total RNA was extracted from cells using Trizol and then reverse-transcribed into cDNA using a reagent kit according to the instructions. The mRNA expression was evaluated by qRT-PCR using SYBR Green mRNA analysis kit and ABI StepOneTM Real-time PCR system. We normalize mRNA expression levels using the GAPDH method and perform relative quantification using Inline graphic method. The qRT-PCR primer sequences are provided in Table [64]S1and [65]S2 of the attachment. RNA sequencing and bioinformatics analysis The miRNA expression profiles beween B-Exos and BSA- Exos were compared after they were determined by small RNA sequencing. The total RNA of exosomes was extracted by Trizol method, and the RNA was intact by NanoDrop One and Aglient 2100 Degree of quality evaluation. The Library was constructed using the NEBNext^®Multiplex Small RNA Library Prep Set for Illumina kit. Finally, the second generation sequencing technology was applied to the library based on Illumina NovaSeq 6000 sequencing platform. The splicer sequences were excised with the Cutadapt v3.4 tool, the excised sequences ≥ 16 bp were retained, and then compared with the non-coding RNA sequences in miRBase Release 22.1. The Ensembl database with bowtie 1.3.0. The DESeq2 toolkit was used to analyze the difference in miRNA expression data by statistical model, and the statistical significance of the difference P < 0.05 was statistically different in miRNA expression. The absolute value of FC ≥ 2 and P < 0.05 were used as criteria to screen differentially expressed miRNAs, and the results were presented in the form of volcanic maps. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis will show the top 20 relevant entries. The RNA sequence data has been added in supplement information. Dual-luciferase reporter assay The wild-type(wt) and mutant(mut) 3’-UTR of KLF5 were amplified by PCR and were inserted into the pGL3 plasmid (RiboBio, Guangzhou, China). HEK293T cells (5 × 10^4 cells/well) were seeded in a 24-well plate and co-transfected with wt or mut luciferase reporter plasmid (1 µg) and miR-145-5p mimics (50nM) or negative controls (NCs) using Lipofectamine 3000 (L3000001, Invitrogen). Relative luciferase activity was measured using the luciferase reporter assay after 48 h (Promega, Madison, WI, USA). Transwell assay BMSCs (1 × 10^4 cells/well) were suspended in serum-free medium and seeded in the upper chamber of a 24-well transwell plate with an 8-µm pore filter (Corning, NewYork, USA), with BEAS-2B cells containing 10% serum medium in the lower chamber. Three experimental settings were as follows: (1) The negative control was transfected (BMSCs-NC + BEAS-2B); (2) miR-145-5p mimics was transfected (BMSCs-mimics + BEAS-2B); (3) The overexpressed BMSCs were pretreated with GW4869 (10µM, MedChemExpress, New Jersey, USA) for 24 h (BMSCs-mimics + GW4869 + BEAS-2B). The relative expression of miR-145-5p was detected by qRT-PCR after incubation with BEAS-2B cells in Transwell plates at 37 °C for 24 h. Cell counting Kit-8 assay In order to establish a cellular inflammation model, we stimulated BEAS-2B cells with TNF-α [[66]29]. BEAS-2B cells (1 × 10^3 cells/well) were seeded in 96-well plates. After the cells entered the logarithmic growth phase, the culture medium was discarded and 100µL medium containing different concentrations of TNF-α (0, 10, 15, and 20ng/mL; GenScript, Nanjing, China) was added when equal amount of normal medium was added to the cells in the control group. In a follow-up experiment, cells were transfected with different concentrations of miR-145-5p mimics (0, 5, 10, 15, 20, 25, and 30 nM) for 24 h after TNF-α treatment. Cell Counting Kit-8 (Beyotime, Shanghai, China) was used to detect cell viability. Cell transfection BEAS-2B cells (1 × 10^5 cells/well) were seeded in 6-well plates. After cell attachment, an inflammatory model was induced by stimulation with TNF-α. Subsequently, cells were transfected with miR-145-5p mimics (15nM) for 24 h using Lipofectamine 3000 (L3000001, Invitrogen). Similarly, miR-145-5p and KLF5 overexpression plasmid were co-transfected into the cells. The relative expression levels of inflammatory markers were measured by quantitative real-time PCR (qRT-PCR) after 24 h. Western blotting RIPA Lysis Buffer (NCM Biotech, Suzhou, China) was used to lyse cells or exosomes. A BCA protein assay kit was used to measure the concentration of total proteins in each sample that were separated by electrophoresis on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes. After blocking with the 5% bovine serum albumin solution for 1 h, the membranes were incubated with appropriate primary antibodies against CD81, CD9, TSG101, Calnexin, GAPDH, KLF5 (Proteintech Group, Inc, Chicago, USA), Phospho-NF-κB p65 (Ser536, #3033, Cell Signaling Technology (CST), Beverly, MA, USA), NF-κB p65 (#8424, CST) at 4 °C overnight and with horseradish peroxidase-coupled secondary antibody at ambient temperature for 1 h. Immunoreactive bands were displayed in chemiluminescent systems (VILBER LOURMAT, Paris, France) with chemiluminescent reagents (Vazyme, Nanjing, China). Statistical analysis The data are shown as the means ± standard deviation (SD) of at least three independent experiments and were analyzed using GraphPad Prism version 8.0 software (GraphPad Software, Inc., San Diego, CA, USA). The differences in two groups with normally distributed data were compared by Student’s t-test. For more than two groups, one-way analysis of variance (ANOVA) with Tukey’s multiple comparison post hoc test was used. Data are presented as mean ± SD. The p < 0.05 was considered statistically significant. Results Magnetic spions and AMF conditions To establish the optimal conditions for BMSCs stimulation, we initially evaluated the effects of SPIONs and AMF on BMSCs viability. Prussian blue staining confirmed the uptake and internalization of SPIONs by BMSCs, with the nanoparticles localized to the cytoplasm (Fig. [67]2A). The viability of BMSCs was significantly increased with the treatment with 25 µg/mL SPIONs, suggesting that low concentrations of SPIONs promoted BMSCs proliferation (Fig. [68]2B). Importantly, AMF treatment also enhanced BMSCs viability in a field strength-dependent manner within the range of 1-5mT (Fig. [69]2C). Based on these findings, the viabilities of SPIONs-loaded BMSCs under different AMF strengths were further investigated. The results indicated that a combination of 25 µg/mL SPIONs and a 3mT AMF was optimal for promoting the growth and proliferation of BMSCs (Fig. [70]2D). Therefore, we selected 25 µg/mL SPIONs and 3mT AMF as the ideal conditions for our subsequent experiments. Additionally, we observed no significant changes in the temperature of the culture medium during the stimulation process (Fig. [71]2E), which avoids any confounding effects of magnetocaloric heating on BMSCs. Fig. 2. [72]Fig. 2 [73]Open in a new tab (A) Prussian blue staining of BMSCs exposed to 25 µg/mL SPIONs. Untreated cells were used as a control. (B) CCK8 assay for the proliferation of BMSCs cultured in the presence of various concentrations of SPIONs. (C) CCK8 assay for the proliferation of BMSCs cultured in the presence of different AMF strengths. (D) The best proliferation of BMSCs treated with the optimal working concentration (25 µg/mL) of SPIONs and exposed to 3mT AMF. (E) The temperature change of the medium containing SPIONs with magnetic stimulation at different time points. (F) Schematic illustration of magnetic stimulation using SPIONs and AMF. (1) AMF generator; (2) a pair of Helmhotz coil; (3) culture flasks or plates Isolation and characterization of the exosomes To generate the engineered BMSC-Exos, primary BMSCs were isolated from SD rats and characterized [[74]22]. The cells exhibited a typical elongated and spindle-shaped appearance, with a regular radial and swirling arrangement (Fig. [75]S1A). The multi-direction differentiation potential of the prepared BMSCs was evaluated. Upon induction of adipogenesis and osteogenesis, the cells successfully produced lipid droplets and calcium nodules (Fig. [76]S1B). Flow cytometric analysis also showed high expression levels of the BMSCs specific surface antigens CD29 (99.3%) and CD90 (98.2%), and a low expression level of the hematopoietic stem cell surface-specific antigen CD45 (1.57%), which further validated the identity of the isolated cells as BMSCs (Fig. [77]S1C). Together, these results confirm the successful extraction and purification of BMSCs with the expected phenotypic and functional characteristics. As depicted in Fig. [78]2F, BMSCs were subjected to distinct treatments: incubation with SPIONs, exposure to an AMF, a synergistic treatment involving both SPIONs and AMF, and a control group without any treatment. Exosomes were subsequently isolated from the culture medium of each group using differential ultracentrifugation. TEM images revealed that exosomes from all four groups displayed a characteristic saucer-like concave vesicle morphology with a double-layer membrane structure (Fig. [79]3A). No significant differences in size or shape were observed among the exosomes derived from the different treatment groups. NTA results indicated that the particle size distribution of the exosomes across all groups ranged from 100 nm to 150 nm. The concentration of the B-Exos was 22 ± 1.2 × 10^10 particles/mL, and the size distribution peak was 128.0 ± 2.5 nm. The concentration of the BS-Exos was 22 ± 1.6 × 10^10 particles/mL, and the size distribution peak was 127.2 ± 3.8 nm. The concentration of the BA-Exos was 22 ± 2.1 × 10^10 particles/mL, and the size distribution peak was 114.5 ± 3.2 nm. The concentration of the BSA-Exos was 23 ± 1.3 × 10^10 particles/mL, and the size distribution peak was 117.4 ± 2.6 nm (Fig. [80]3B). Western blotting confirmed the presence of exosomes-specific markers CD9, CD81, and TSG101 in all exosomes samples, and the negative exosomes marker Calnexin was absent (Fig. [81]3C). These findings suggest that the treatments of BMSCs with SPIONs and AMF did not significantly affect the morphology, size, or specific biomolecular markers of the engineered BMSC-Exos. Fig. 3. [82]Fig. 3 [83]Open in a new tab (A) Morphology of four types of exosomes under TEM, the red arrows indicate exosomes. Scale bars are 200 Inline graphic . (B) NTA analysis of four types of exosomes. (C) Western blotting analysis of exosomal proteins CD81, CD9, TSG101 and the negative marker Calnexin. Full-length blots/gels are presented in Supplementary Figure [84]S2. (D) Uptake of red fluorescent dye (DiI)-labeled BSA-Exos by BEAS-2B cells To evaluate the cellular uptake of the engineered BMSC-Exos by BEAS-2B cells, the 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorat (DiI)-stained exosomes were co-cultured with BEAS-2B cells for 4 h. As shown in Fig. [85]3D, the red fluorescence emitted by the DiI-labeled exosomes was found to match well with the blue fluorescence from the 4’,6-Diamidino-2-Phenylindole (DAPI)-stained nuclei of BEAS-2B cells. The co-localization of fluorescence indicates that the exosomes could be efficiently internalized by BEAS-2B cells. In vivo distribution and retention of the engineered BMSC-Exos The in vivo distribution and retention of BMSC-Exos were examined in an LPS-induced ALI model. DIR-labeled BSA-Exos were administered intravenously through the tail vein, with sham-operated mice serving as controls. In vivo NIR fluorescence imaging showed that the fluorescence intensity in the lungs reached its peak within the first 2 h post-injection and was still detectable for at least 24 h (Fig. [86]4A). Notably, the fluorescence intensity in the lungs of ALI mice was markedly elevated compared to that of the control group, suggesting a specific accumulation of BSA-Exos in the injured lungs. This observation was further supported by ex vivo imaging, which revealed a pronounced accumulation of BSA-Exos in the lungs of mice with lung injury, in contrast to the control group. The preferential accumulation of BSA-Exos in LPS-induced ALI is likely due to the damage-targeting properties of BMSC-Exos [[87]30–[88]32]. Among the organs assessed, the liver displayed the most significant fluorescence intensity, potentially due to the nonspecific uptake of exosomes by the mononuclear phagocyte system [[89]33]. Collectively, these findings suggest that BSA-Exos could specifically accumulate in injured lung tissue. Fig. 4. [90]Fig. 4 [91]Open in a new tab (A) The result of fluorescence imaging at different time points and important organs 24 h later. (B) Schematic diagram of in vivo animal experiment procedure. (C-F). Relative expression of inflammatory factors TNF-α、IL-1β、IL-12p70、IL-10 in BALF of mice was detected by ELISA. (mean ± SD, n = 5, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). G. The total protein contenst of BALF in control and LPS mice were measured by BCA assay. (H). The W/D ratio of mice lung sections after each group exosomes treatment Enhanced therapeutic efficacy of engineered BMSC-Exos in a LPS-induced ALI model The therapeutic efficacy of the engineered BMSC-Exos was assessed in an LPS-induced ALI model, with the animal experiments following the schematic diagram shown in Fig. [92]4B. The ELISA results showed that all the exosomes formulations significantly reduced the expression of inflammatory factors such as TNF-α, IL-1β, and IL-12p70 as shown in Fig. [93]4C, D and E, which are known to exacerbate inflammation and tissue damage. Furthermore, the exosomes formulations bolstered the expression of the anti-inflammatory factor IL-10, which is critical for maintaining tissue homeostasis and preventing additional tissue damage during inflammation responses (Fig. [94]4F) [[95]34]. Notably, BSA-Exos demonstrated superior performance in cytokine modulation compared to other formulations, suggesting that the synergistic treatment of BMSCs with SPIONs and AMF effectively enhance the anti-inflammation capabilities of the BMSC-Exos. To evaluate the efficacy of the engineered BMSC-Exos in mitigating alveolar-capillary barrier dysfunction in ALI, the total protein content in BALF supernatant was evaluated using a BCA assay. The results indicated that the group treated with BSA-Exos exhibited the lowest total protein content, indicating that BSA-Exos administration effectively preserves the integrity of the pulmonary capillary barrier and prevents further protein leakage (Fig. [96]4G). Additionally, the W/D ratio of mice lung tissues were measured, revealing a significant decrease in the BSA-Exos treated group compared to other exosomes-treated groups (Fig. [97]4H). Moreover, histopathological examination of lung tissues stained with H&E showed reduced congestion in the alveolar walls, decreased interstitial thickness, and reduced infiltration of inflammatory cells in the alveoli across all exosomes-treated groups, signifying a substantial improvement in pneumonia injury. Notably, the BSA-Exos treated group displayed the most pronounced histological improvement (Fig. [98]5A), highlighting the superior protective effects of BSA-Exos. The pathological scoring data of lung injury was in line with this outcome from another perspective (Fig. [99]5B). Collectively, these results demonstrate the exceptional protective effects of BSA-Exos in maintaining the integrity of alveolar-capillary barrier and alleviating ALI. Fig. 5. [100]Fig. 5 [101]Open in a new tab (A) H&E staining of mice lung sections after each group exosomes treatment. (B) The lung injury score was calculated by histological analysis in different groups (n = 5). (C) H&E staining analysis of main organs in mice after injection of each group. (D) ALT, AST and ALB levels in serum of mice in each group (n = 5). (E) BUN, CREA and UA levels in serum of mice in each group (n = 5) Biosafety of the engineered exosomes in vivo We evaluated the toxicity induced by engineered exosomes in vivo. H&E staining of various organs revealed no pathological changes in any group (Fig. [102]5C). Furthermore, Serum of mice collected in the in vivo toxicity assay was tested for hepatic biochemicals (alanine aminotransferase, aspartate aminotransferase and albumin) and renal biochemicals (urea, creatinine, and uric acid). The results showed all the indexes in each group were within the normal reference ranges in Fig. [103]5D and E. These results indicated that engineered exosomes were safe biomaterials for in vivo application. RNA sequencing and bioinformatics analysis Based on previous studies, exosomes-carried miRNAs are crucial for intercellular communication among cells implicated in acute lung inflammation and injury [[104]3, [105]35, [106]36]. This has promoted our hypothesis that the enhanced inhibitory effects of BSA-Exos might be ascribed to specific miRNAs induced by AMF stimulation of SPIONs-loaded BMSCs. To validate this hypothesis, a rigorous analysis of the miRNA profiles of BSA-Exos was conducted using the RNA sequencing technique. Compared to B-Exos, BSA-Exos exhibited significant differences in the expression levels of 76 miRNAs, with 44 being upregulated and 32 downregulated (Fig. [107]6A). Fig. 6. [108]Fig. 6 [109]Open in a new tab (A) The number of miRNA differences between B-Exos and BSA-Exos. (B) Volcano map of miRNA sequencing analysis of mRNAs with ≥ 2-fold and P < 0.05 difference in expression between B-Exos and BSA-Exos. Red and green indicate up and down, respectively. (C) KEGG enrichment analysis of the first 20 entries. (D) Comparison of the top four elevated miRNAs (miR-145-5p, miR-99a-5p, miR-186a-5p and miR-140-5p) by using qRT-PCR. (E) The miR-145-5p binding sequence with the 3′-UTR of KLF5. (F) Luciferase reporter assay from wild-type or mutant KLF5 3′-UTR co-transfected in HEK293T with control mimics or miR-145-5p mimics The differential expression patterns of these miRNAs were graphically represented using volcanic maps (Fig. [110]6B). Subsequent pathway enrichment analysis revealed that the miRNAs with differentially expressed were predominantly involved in critical signaling pathways, including the NF-κB, TNF, and MAPK pathways (Fig. [111]6C), which are well-established for their roles in mediating inflammation and immune responses. To elucidate the functional roles of the differentially expressed miRNAs, we focused on the four most significantly upregulated candidates: miR-145-5p, miR-99a-5p, miR-186-5p, and miR-140-5p, for subsequent analysis and validation. qRT-PCR analysis confirmed a significant upregulation of miR-145-5p in BSA-Exos relative to exosomes from untreated BMSCs (Fig. [112]6D). Exsomal miR-145-5p regulates KLF5 by targeting the 3′-UTR Following a thorough bioinformatics analysis, KLF5 was identified as a candidate target gene of miR-145-5p. To experimentally validate the interaction between miR-145-5p and the 3’-untranslated region (3’-UTR) of KLF5, a dual-luciferase reporter assay was conducted using luciferase reporter special plasmids that contained either the wild-type (wt) or mutant (mut) KLF5 3’-UTR with the miR-145-5p binding sites (Fig. [113]6E). The transfection of HEK293T cells with miR-145-5p mimics resulted in a significant reduction in luciferase activity when the cells were co-transfected with the plasmid containing the wild-type KLF5 3’-UTR, as compared to those co-transfected with the control mimics. In contrast, the luciferase activity in cells co-transfected with the mutant KLF5 3’-UTR plasmid remained unchanged in the presence of miR-145-5p mimics (Fig. [114]6F). This differential response between the wild-type and mutant constructs strongly suggests that miR-145-5p directly binds to and regulates the expression of KLF5. Treatment with miR-145-5p mimics alleviated TNF-α-induced BEAS-2B inflammation cytokines To verify whether miR-145-5p could be transferred to recipient cells via exosomes, we conducted in vitro experiments using a Transwell system. As depicted in Fig. [115]7A, BMSCs overexpressing miR-145-5p effectively delivered miR-145-5p to BEAS-2B cells. However, treatment with GW4869, a specific inhibitor of extracellular vesicles, abrogated the miR-145-5p transfer, indicating that exosome-mediated communication is essential for miR-145-5p delivery from BMSCs to BEAS-2B cells. Fig. 7. [116]Fig. 7 [117]Open in a new tab (A) miR-145-5p is transferred from BMSCs to BEAS-2B cells by an EV-dependent pathway. (B) Different concentrations and time point of TNF-α treatment decreased cell viability. (C) The release of IL-1β, IL-6, and CXCL10 of BEAS-2B cells were increase after TNF-α treatment by qRT-PCR detection. (D) Transfection of mimics increased cell viability after TNF-α treatment. (E) The release of IL-1β, IL-6, and CXCL10 of BEAS-2B cells after different transfection. (F) The protein expression of KLF5 after transfection of BEAS-2B cells with miR-145-5p mimics, miR-145-5p inhibitor and their NCs. (G) pNF-κB p65 level in BEAS-2B cells was determined by western blotting. Full-length blots/gels are presented in Supplementary Figure S3 and S4 We treated BEAS-2B cells with TNF-α over a time course to examine its cytotoxic effects. As shown in Fig. [118]7B, TNF-α treatment induced a time-dependent reduction in cell viability, with the most significant effect observed at 24 h post-treatment. Therefore, we selected cells treated with TNF-α (20 ng/mL) for 24 h for subsequent assays. The protective mechanism of miR-145-5p mimics on the inflammatory cells was further evaluated using a TNF-α-induced inflammation model in BEAS-2B cells. As shown in Fig. [119]7C, exposure to TNF-α resulted in a dose-dependent upregulation of the inflammatory cytokines IL-1β, IL-6, and CXCL10 within BEAS-2B cells. Notably, transfection with 15nM miR-145-5p mimics significantly increased cell viability and attenuated the expression of these pro-inflammatory cytokines. Meanwhile, the experimental results demonstrated that KLF5 overexpression partially counteracted miR-145-5p’s therapeutic effects, with corresponding increases in inflammatory cytokine levels (Fig. [120]7D, E). Additionally, we observed that transfection with miR-145-5p mimics downregulated KLF5 protein levels in BEAS-2B cells, whereas transfection with miR-145-5p inhibitors had the opposite effect (Fig. [121]7F). These findings further substantiate the regulatory role of miR-145-5p on KLF5 expression. Subsequently, the protein levels of phosphorylated NF-κB (pNF-κB) were evaluated by western blotting analysis. The results revealed that the expression of pNF-κB was diminished following the transfection of miR-145-5p mimics (Fig. [122]7G). Collectively, these results suggest that miR-145-5p mimics exert a protective effect against TNF-α-induced damage in BEAS-2B cells through the KLF5/NF-κB signaling pathway. Discussion ALI is an acute inflammatory reaction caused by a variety of internal and external pulmonary pathogenic factors, and is a clinical syndrome with high morbidity and mortality [[123]37]. Currently, clinical treatment consists mainly of supportive ventilation and limited fluid management and there is no specific treatment for ALI. MSCs are one of the most widely studied pluripotent stem cells with low heterogeneity, immunomodulatory properties, and self-healing capabilities. MSC-Exos, as a new therapeutic tool of “cell-free therapy”, are used instead of stem cells in the treatment of various diseases to promote tissue regeneration and repair [[124]6, [125]7]. Engineered exosomes take advantage of the versatility of Exos structure to modify their original configuration using various methods, including genetic engineering, chemical modification, physical technology and microfluidic technology, in order to regulate the active substances loaded by exosomes and expand biomedical applications [[126]12]. According to literature reports, low-dose magnetic Iron nanoparticles are safe. Lee et al. injected iron oxide nanoparticles (IONPs) into the infarcted heart. Magnetic guidance can modulate inflammatory response and reduce the degree of apoptosis and fibrosis. It also enhances angiogenesis and cardiac function recovery [[127]38]. Kim et al. showed that magnetic nanovesicles (MNV) derived from BMSCs treated with IONPs can effectively ameliorate ischemic injury because MNV contains more therapeutic molecules than natural BMSC-Exos [[128]39]. The results of this study demonstrate that the treatment of BMSCs with SPIONs, AMF, or their combination does not significantly alter the morphology, size distribution, or specific biomolecular markers of the derived exosomes. This finding is crucial for the potential application of SPIONs and AMF in exosome-based therapies, as it suggests that these treatments do not compromise the fundamental characteristics and biological function of exosomes. SPIONs and AMF are widely used in biomedical applications, such as magnetic targeting, imaging, and hyperthermia therapy [[129]40, [130]41]. The ability to combine these technologies with exosome engineering without compromising exosome integrity opens new possibilities for targeted drug delivery, regenerative medicine, and diagnostic applications. Similarly, the efficient uptake of BMSC-Exos by BEAS-2B cells suggests that the exosomes retain their functional properties, such as membrane fusion or endocytosis-mediated internalization, even after engineering and labeling with DiI (Fig. [131]3D). This is an important observation, as it confirms that the labeling process did not impair the exosomes’ ability to interact with target cells. The rapid internalization within 4 h further underscores the potential of exosomes as efficient delivery vehicles for therapeutic molecules, such as proteins, nucleic acids, or drugs, to bronchial epithelial cells. We aimed to validate the therapeutic efficacy of engineered exosomes in vivo, and established a LPS-induced ALI mice model. Administration of LPS led to severe lung injury, characterized by an acute pulmonary inflammatory response and the secretion of multiple inflammatory cytokines. The excessive cytokine release led to the infiltration and proliferation of various inflammatory cells, which in turn caused damage to pulmonary capillary endothelial cells and alveolar epithelial cells [[132]42, [133]43]. Therefore, inhibition of cytokine release is pivotal for effectively alleviating the progression of ALI. The ELISA results revealed that all exosome formulations significantly reduced the expression of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-12p70, which are known to exacerbate inflammation and tissue damage in ALI. Concurrently, the exosomes enhanced the expression of the anti-inflammatory cytokine IL-10, which plays a critical role in maintaining tissue homeostasis and resolving inflammation. These findings suggest that BMSC-Exos possess potent immunomodulatory properties. Notably, the BSA-Exos, derived from BMSCs treated with both SPIONs and AMF, exhibited superior performance in cytokine modulation compared to other exosomes formulations. BSA-Exos administration effectively preserved the integrity of the alveolar-capillary barrier, as evidenced by the reduced protein content in BALF and the lower W/D ratio in lung tissues. These results indicate that BSA-Exos can prevent vascular leakage and pulmonary edema, which are central to the pathophysiology of ALI. Histopathological analysis revealed that BSA-Exos significantly reduced alveolar congestion, interstitial thickness and inflammatory cell infiltration. These improvements align with the observed cytokine modulation and barrier protection, demonstrating the comprehensive therapeutic effects of BSA-Exos in alleviating lung injury, which indicates that the synergistic treatment of BMSCs with SPIONs and AMF enhances the anti-inflammatory capabilities of the derived exosomes, potentially through the enrichment of specific bioactive molecules or the activation of signaling pathways that amplify their therapeutic effects. The observed preferential accumulation of BSA-Exos in the lungs of LPS-induced ALI mice, as demonstrated by in vivo and ex vivo imaging, highlights the potential of exosomes as targeted therapeutic carriers for ALI. The enhanced fluorescence intensity in the lungs of ALI mice, compared to the control group, suggests that the damaged lung tissue may create a microenvironment that facilitates the homing and retention of BSA-Exos. This phenomenon could be attributed to the inflammatory signals and increased vascular permeability associated with ALI, which may enhance the extravasation and uptake of exosomes by the injured tissue. The damage-targeting properties of BMSC-exos, as referenced in previous studies [[134]30–[135]32], further support this hypothesis, indicating that exosomes may inherently possess mechanisms to recognize and respond to injured tissues. The significant fluorescence intensity observed in the liver, likely due to nonspecific uptake by the mononuclear phagocyte system, underscores a common challenge in exosome-based therapeutics: systemic distribution and off-target accumulation. While the liver’s role in clearing exosomes from circulation is well-documented [[136]33], strategies to minimize nonspecific uptake and enhance targeted delivery to the lungs will be crucial for optimizing the therapeutic efficacy of BSA-Exos in ALI. These findings provide valuable insights into the biological distribution and retention dynamics of BSA-Exos in ALI. Future studies could explore surface modifications of exosomes, such as the incorporation of lung-specific targeting ligands, to further improve their specificity and reduce off-target effects. The results of our miRNA analysis provide compelling evidence that the enhanced anti-inflammatory and tissue repair effects of BSA-Exos may be mediated by specific miRNAs induced by AMF stimulation of SPIONs-loaded BMSCs. The significant differences in miRNA expression profiles between B-Exos and BSA-Exos suggest that SPIONs and AMF stimulation induces a unique molecular signature in exosomes, which may be the key to their therapeutic effect. Among them, miR-145-5p emerged as a particularly promising candidate which has been reported to play a critical role in fibrosis and inflammation within cardiovascular diseases, and was identified as a significant modulator in LPS-induced cardiomyocyte injury [[137]44, [138]45]. Therefore, miR-145-5p was selected as a potential key factor in deciphering the superior performance of BSA-Exos in regulating inflammation and promoting tissue repair during ALI. The upregulation of miR-145-5p in BSA-Exos, as confirmed by qRT-PCR, aligns with its known functions in suppressing inflammatory pathways and promoting tissue repair. KLF5, a member of the Krüppel-like family of transcription factors, has been implicated in various cellular processes, including inflammation, proliferation, and fibrosis. It has been reported that the suppression of KLF5 was closely associated with the inhibition of NF-κB phosphorylation [[139]46, [140]47], which serves as a pivotal driver in cellular and tissue injury induced by a variety of inflammatory cytokines [[141]48–[142]50]. By downregulating KLF5, miR-145-5p may modulate key inflammatory and fibrotic pathways, thereby contributing to the attenuation of lung injury and promotion of tissue repair. The suppression of pNF-κB by miR-145-5p mimics suggests that miR-145-5p not only targets KLF5 but also exerts downstream effects on the NF-κB pathway, thereby attenuating the inflammatory cascade. The KLF5/NF-κB axis thus emerges as a critical pathway through which miR-145-5p mediates its anti-inflammatory and tissue-protective functions. By modulating this pathway, miR-145-5p could potentially attenuate the excessive inflammation observed in ALI, thereby contributing to the protective effects of BSA-Exos. In addition to miR-145-5p, the upregulation of other miRNAs such as miR-99a-5p, miR-186-5p, and miR-140-5p warrants further investigation. These miRNAs may also contribute to the observed therapeutic effects, either independently or in concert with miR-145-5p. Future studies should aim to elucidate the specific roles of these miRNAs in the context of ALI and explore their potential synergistic effects. This study highlights the therapeutic potential of BSA-Exos, which not only significantly reduces the expression of inflammatory factors and improves histopathological changes, but also represents an effective and promising protocol for the therapeutic effects of lung injury. However, the current study has some limitations. First, how SPIONs and AMF induce BMSCs to release more exosomes of miR-145-5p remains to be studied to determine the specific molecular mechanisms involved. In addition, the design of transfecting miRNA mimics into Exos to further validate the therapeutic effect in vivo has yet to be perfected. Conclusion In summary, a straightforward methodology has been developed for the fabrication of engineered BMSC-Exos through the preconditioning of BMSCs with SPIONs and AMF. These engineered exosomes exhibited superior therapeutic efficacy in vivo, markedly attenuating inflammation and improving lung tissue pathology in an ALI model when compared to untreated BMSC-Exos. High-throughput sequencing analysis of miRNAs revealed that the upregulated miR-145-5p acted as a key mechanistic driver in the outstanding protective effects of the engineered BMSC-Exos. Luciferase reporter assays further validated KLF5 as a potential target gene of miR-145-5p. Western blotting confirmed that miR-145-5p mimics suppressed the expression of KLF5 and reduced the phosphorylation level of NF-κB, thus preventing the overactivation of inflammation. This study not only presents an efficient and promising therapeutic strategy for the treatment of ALI, but also possesses considerable potential for clinical application. Supplementary Information Below is the link to the electronic supplementary material. [143]13287_2025_4598_MOESM1_ESM.pdf^ (136.9KB, pdf) Supplementary Material 1: Additional experiments about identification of BMSCs (Figure [144]S1); Full-length blots of four group exosomes (Figure [145]S2); Full-length blots of KLF5(Figure [146]S3); Full-length blots of pNF-κB p65 (Figure [147]S4). Primer sequences (Table [148]S1, [149]S2). [150]Supplementary Material 2^ (10.3MB, docx) Acknowledgements