A nanosystem targeting tissue inhibitor of metalloproteinase-1 for continuous spatiotemporal idiopathic pulmonary fibrosis therapy

Domain: chemistry
Published: 2026-01-19
Editorial outcome: major_revision

Abstract
Idiopathic pulmonary fibrosis (IPF), a progressive, life-threatening disease marked by excessive collagen deposition, severe tissue injury, and dysregulated oxidative stress, poses a major threat to human health. Despite clinical advances, current therapies have limited anti-fibrotic efficacy. Here we show a reactive oxygen species (ROS)-responsive nanosystem targeting tissue inhibitor of metalloproteinase-1 (TIMP-1) for spatiotemporally precise IPF treatment. Anti-TIMP-1 antibodies (aT) are conjugated to mesenchymal stem cell-derived exosomes (Mexo) via ROS-cleavable phenylboronic acid ester linkers (cl), yielding Mexo-cl-aT. Following intratracheal administration, cl linkers are selectively cleaved by elevated ROS in the IPF microenvironment, enabling ROS scavenging while releasing Mexo and aT to mediate tissue repair and collagen degradation, respectively. We demonstrate that a single dose of Mexo-cl-aT exerts robust therapeutic efficacy against IPF in a bleomycin-induced mouse model of advanced-stage fibrosis, thereby validating this nanosystem as a safe and efficient candidate for next-generation IPF therapies.

Introduction
Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease1–3. The progressive accumulation of extracellular matrix (ECM) leads to increased stiffness of injured tissues, seriously impairing pulmonary function and quality of life in patients4,5.

Approximately 5 million people worldwide suffer from IPF, with a median survival of 3–5 years post-diagnosis6. Pirfenidone and nintedanib are the only two U.S. Food and Drug Administration (FDA)-approved antifibrotic drugs that can slow the progression of IPF by inhibiting fibroblast activation7,8. Despite promising advances in the treatment of early-stage patients, they have limited efficacy for advanced-stage patients with massive collagen accumulation within the lesions. Moreover, both medications have been reported to cause adverse side effects on other organs or tissues, such as significant gastrointestinal symptoms and other complications9. Therefore, a new therapeutic strategy is urgently needed to halt or reverse IPF progression.

Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, play a critical role in the normal turnover of the ECM10. Although MMP levels have been reported to facilitate ECM protein degradation during pulmonary fibrosis, there is still excessive ECM deposition in IPF lesions. Recently, researchers have come to realize the importance of maintaining the balance of ECM metabolism homeostasis in the treatment of IPF, which is dynamically regulated by the protease system of MMPs/tissue inhibitor of metalloproteinases (TIMPs) rather than by MMPs alone11. The increased TIMPs, particularly TIMP-1, bind to various MMPs and inhibit their activity12,13. Therefore, blocking TIMP-1 may promote ECM degradation via MMP catalysis, thus restoring the compromised alveolar space. Nonetheless, to the best of our knowledge, the use of TIMP-1 as a novel therapeutic target for IPF to regulate MMP activity for ECM degradation has rarely been reported. In addition, current views suggest that pulmonary redox imbalance, stimulated by oxidative stress, not only drives the pathogenic activation of fibroblasts but also induces apoptosis of epithelial and endothelial cells during the fibrotic process, leading to the destruction of lung architecture14. Therefore, scavenging excess reactive oxygen species (ROS) in the IPF microenvironment and regenerating the disrupted alveolar epithelium and endothelium are indispensable for the reconstruction of disrupted fibrotic alveoli and the restoration of pulmonary function.

Mesenchymal stem cell-derived exosomes (Mexo) serve as innate nano-carriers, adept at targeting inflammation/injury sites and augmenting drug retention at the lesion site while maintaining a superior biosafety profile15,16. Furthermore, Mexo exhibit a spectrum of therapeutic properties, including anti-inflammatory and anti-fibrotic activities, as well as facilitation of tissue repair and regeneration17. Here, we aimed to verify the role of TIMP-1 in the pathogenesis of IPF and develop a combination therapy that targets both ECM metabolism imbalance and pulmonary redox imbalance by exploiting their synergism to reverse the progression of IPF and ameliorate lung function. We engineered Mexo with TIMP-1 antibodies (aT) via a phenylboronic acid ester bond-based ROS-cleavable linker (cl) (Fig. 1a), which is subject to cleavage by ROS in fibrotic lesions. We rationalized that upon ROS exposure, the smart nanosystem Mexo-cl-aT would rapidly scavenge excess ROS and release aT and Mexo to accelerate the clearance of ECM and promote lung repair in pulmonary fibrosis, respectively (Fig. 1b)18,19. Therefore, the inhaled nanoformulation treatment promoted the clearance of excessive ECM and alveolar re-epithelialization in an advanced-stage pulmonary fibrosis mouse model.

Results
High expression of TIMP-1 in IPF patients and bleomycin-induced pulmonary fibrosis mouse models
To study the correlation between TIMP-1 expression and IPF progression, the TIMP-1-positive area in lung tissues from IPF patients was investigated using immunohistochemical (IHC) staining (Fig. 2a, Table S1). The results showed that TIMP-1 expression, which was mainly localized in the extracellular matrix (Supplementary Fig. S1), increased by 4.7-fold compared with that in the lung tissues of non-IPF controls (Fig. 2b). Furthermore, a positive correlation (r = 0.5831) was observed between TIMP-1 expression and fibrosis progression, as identified by the representative fibrosis marker collagen type I alpha 1 chain (COL1A1) (Fig. 2c). Similarly, the TIMP-1-positive area in lung tissues showed a 4.4-fold increase in the bleomycin-induced pulmonary fibrosis mouse model, and a positive correlation (r = 0.7383) was observed with COL1A1 (Fig. 2d–f). Furthermore, enzyme-linked immunosorbent assay (ELISA) analysis revealed that the level of TIMP-1 in the bronchoalveolar lavage fluid (BALF) of bleomycin-induced pulmonary fibrosis mice increased by 36.9-fold, and that in the lung tissues increased by 2-fold (Supplementary Fig. S2a, b). These results reveal the prominent role of TIMP-1 in pulmonary fibrosis pathology, suggesting that TIMP-1 serves as a potential therapeutic target for pulmonary fibrosis.

Given that cellular oxidative stress and alveolar epithelial damage also contribute significantly to IPF pathogenesis, we assessed ROS levels and expression of the type I alveolar epithelial cell (AECI) marker, aquaporin 5 (AQP5), in lung tissues14,20. As shown in Fig. 2g, h, the levels of ROS increased (2.3- and 16.4-fold, respectively) and AQP5 levels decreased significantly (1.8- and 1.9-fold, respectively) in the lung tissues of both IPF patients and mice. Moreover, we found strong correlations between TIMP-1, COL1A1, and AQP5 in mouse samples through quantitative analysis of IHC images, suggesting that TIMP-1, collagen deposition, and alveolar epithelial damage are synergistic in IPF progression (Supplementary Fig. S2c).

Preparation and characterization of Mexo-cl-aT
The abovementioned results motivated us to develop a system to synergize ECM degradation, ROS scavenging, and tissue repair. For this purpose, we fabricated Mexo-cl-aT by conjugating aT with Mexo via an ROS-cleavable linker (cl). First, N3-modified Mexo (Mexo-N3) was obtained via the reaction between phenylboronic acid (PBA) and the glycosyl moieties on the Mexo surface proteins (Supplementary Fig. S3), which were subsequently conjugated with dibenzocyclooctyne (DBCO)-modified aT (aT-DBCO) (Supplementary Fig. S4) to yield Mexo-cl-aT21,22. Western blotting revealed that Mexo-cl-aT was composed of Mexo and aT, which were characterized by the presence of exosome biomarkers (ALIX, CD63, CD9) and the light chain of aT (Fig. 3a)23,24. The aT of Mexo-cl-aT were also verified by the binding of secondary antibody-linked Au nanoparticles (Supplementary Fig. S5). Moreover, the high colocalization (94.1%) of the aT and Mexo signals further confirmed successful construction of Mexo-cl-aT (Fig. 3b).

When the ratio of Mexo to aT was 5:1 (w/w), the Mexo-cl-aT displayed the greatest antifibrotic effect against transforming growth factor-β1 (TGF-β1)-induced primary mouse lung fibroblasts (MLFs) without causing cytotoxicity (Supplementary Fig. S6, S7), and the loading capacity of aT was 89.9% at this time (Supplementary Fig. S8). Transmission electron microscope (TEM) images of Mexo-cl-aT showed no significant alteration in morphology compared to that of Mexo (Fig. 3c), indicating that aT modification did not influence the morphology of Mexo. After conjugation, the mean hydration particle size of Mexo-cl-aT slightly increased from 103.4 nm to 114.3 nm, while the zeta potential decreased (Fig. 3d). There were no detectable changes in particle size or zeta potential after storage in phosphate-buffered saline (PBS) at 4 °C for 7 days, suggesting the desired stability of the engineered Mexo-cl-aT for therapeutic applications (Fig. 3e).

In our design, the phenylboronic acid ester bond in Mexo-cl-aT is ROS-cleavable, leading to the release of free aT (Fig. 3f). To confirm this, H2O2 was exogenously added to the Mexo-cl-aT solution to mimic the high levels of H2O2 in the IPF pathological environment, and the released aT were quantified. As expected, the amount of free aT in the buffer solution gradually increased, reaching a plateau after 4 h of co-incubation, showing a strong time- and H2O2 concentration-dependent profile (Fig. 3g). Notably, more than 53.5% of the aT were released in the 0.1 mM H2O2 solution, and 83.2% were released in the 1 mM H2O2 solution, both significantly greater than the release observed in PBS (10.8%). Moreover, H2O2 levels decreased by 90.0% after incubation with Mexo-cl-aT under both 0.1 mM and 1 mM H2O2 conditions (Fig. 3h). These results indicate that Mexo-cl-aT not only responds to ROS but also efficiently scavenges ROS.

Anti-fibrotic effects and mechanisms of Mexo-cl-aT in vitro
Fibroblasts are recognized as one of the cellular sources of MMP-9, while activated fibroblasts are identified as the primary source of TIMP-112,25. To explore the in vitro antifibrotic activity of Mexo-cl-aT, TGF-β1-induced primary MLFs were treated with different formulations for 24 h, and the myofibroblast marker alpha-smooth muscle actin (α-SMA) and COL1A1 were detected by IF staining26. As shown in Fig. 4a, the levels of α-SMA and COL1A1 expression were significantly upregulated in the PBS group, while aT or Mexo alone slightly inhibited α-SMA and COL1A1 expression. Better antifibrotic effects were observed when aT and Mexo cooperated. Notably, although Mexo-fl-aT (Mexo conjugated with aT via an ROS-uncleavable fixed linker) and Mexo-cl-aT did not differ in their ability to down-regulate α-SMA expression, the former was less effective than the latter in collagen degradation (Fig. 4b, c, Supplementary Fig. S9).

We further investigated the antifibrotic mechanisms of Mexo-cl-aT. Given that aT can block TIMP-1 and thus restore MMP activity (Fig. 5a), MMP-9 activity in the cell culture supernatant of each group was measured at 4 h post-treatment. As shown in Fig. 5b, c, both the aT and Mexo-cl-aT groups exhibited significantly decreased TIMP-1 levels and correspondingly increased active MMP-9 levels compared to the PBS group. In contrast, Mexo-fl-aT treatment failed to improve MMP-9 activity because the release of aT was prevented by the presence of the fixed linker. Similar results were also obtained in TIMP-1-silenced MLFs, confirming TIMP-1 as a key target of Mexo-cl-aT and demonstrating its ability to shift the TIMP-1/MMP-9 balance toward an anti-fibrotic state (Supplementary Figs. S10–S12). It is important to note that while MMP-9 elevation may occur as an early, transient event in this reparative process, the sustained anti-fibrotic outcomes observed are primarily attributable to the rebalancing of the MMPs/TIMP-1 ratio achieved by aT, rather than prolonged MMP-9 activity alone.

Moreover, the percentage of apoptotic A549 cells in the Mexo-cl-aT group was reduced by 37.3% compared to the PBS group (Fig. 5d, e, Supplementary Fig. S13). Since the alveolar epithelium, which is primarily responsible for gas exchange, is vulnerable in IPF, we monitored the cell viability of alveolar epithelial A549 cells after treatment with different formulations daily for 72 h27,28. Cell Counting Kit-8 (CCK-8) assay showed that bleomycin induced clear time-dependent cytotoxicity to A549 cells, whereas Mexo-cl-aT treatment effectively maintained cell viability (Fig. 5f). Consequently, the Mexo-cl-aT group exhibited increased Ki67 expression, indicating that it promoted cell proliferation (Supplementary Fig. S14). To assess tissue repair capacity, we performed a wound healing assay where a scratch was used to mimic tissue injury. Representative images showed a 53.6% increase in the migration area with Mexo-cl-aT treatment versus PBS (Fig. 5g). Similar results were obtained for human umbilical vein endothelial cells (HUVECs) (Supplementary Fig. S15). Additionally, the ability of Mexo-cl-aT to scavenge ROS was assessed using CLSM and flow cytometry. As shown in Fig. 5h and Supplementary Fig. S16, compared to the PBS group, the Mexo, aT, and Mexo-fl-aT groups exhibited reduced ROS levels attributed to the anti-fibrotic effects of aT and/or the cellular repair capacity of Mexo. Notably, the Mexo-cl-aT group exhibited the weakest ROS signal, which decreased from 15.9% in the PBS group to 8.6%. This significant reduction suggests a substantial contribution of the phenylboronic acid ester bond to ROS scavenging, which was further confirmed by Amplex Red assay (Supplementary Fig. S17). These results collectively support the assertion that the therapeutic effects of Mexo-cl-aT are attributable to the synergistic effects of rapid clearance of excess ROS by the phenylboronic acid ester bond, MMP activation by aT, and cellular repair by Mexo.

ROS-responsive behavior and metabolism of Mexo-cl-aT in vivo
The retention and extracellularly responsive release of aT in IPF lesions are critical for therapeutic efficacy. To this end, we separately labeled Mexo with 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR), and aT with Alexa Fluor 647-conjugated antibodies. Subsequently, fluorescently labeled Mexo+aT (physical mixture) or Mexo-cl-aT was intratracheally injected into a bleomycin-induced pulmonary fibrosis mice, and their biodistribution was monitored using an in vivo imaging system (IVIS). Mexo signals peaked at 2 h and gradually declined by 72 h in both groups (Supplementary Fig. S18), indicating that more than half of the Mexo remained in the lungs for at least 72 h. At this point, the main organs were collected and imaged. The Mexo signals did not significantly differ between the two groups, indicating that the engineered modifications did not affect the normal metabolism of Mexo (Fig. 6a). In parallel, we found that the aT signals in the Mexo-cl-aT group were 2.3-fold stronger than those in the Mexo+aT group (Fig. 6b), indicating that the conjugation of aT with Mexo significantly improves the retention time of aT in the lungs.

To further verify the controllable release of aT from Mexo-cl-aT, we labeled Mexo with DiI and aT with FITC-conjugated secondary antibodies. After injecting fluorescently labeled Mexo-fl-aT or Mexo-cl-aT into bleomycin-induced pulmonary fibrosis mice, lung tissues were collected and imaged using CLSM. Since the fixed linker could not be cleaved by ROS in IPF lesions, aT were well colocalized with Mexo in the Mexo-fl-aT group. In contrast, the high level of H2O2 secreted in mouse lung tissues (Supplementary Fig. S19) led to the release of aT from Mexo in the Mexo-cl-aT group, which resulted in successful separation and poor colocalization (Fig. 6c). The results demonstrate that Mexo-cl-aT persists in IPF tissues and subsequently respond to ROS, releasing aT into the extracellular matrix of IPF lesions. This validates our design strategy and lays the foundation for a highly efficient treatment of IPF.

Therapeutic efficacy and safety of Mexo-cl-aT in vivo
Next, we evaluated the antifibrotic efficacy of Mexo-cl-aT in a bleomycin-induced pulmonary fibrosis mouse model (Fig. 7a). As shown in Fig. 7b, Mexo-cl-aT treatment made the greatest improvement in the macroscopic morphology of the lungs, as evidenced by the healthiest color and texture alongside minimal area of dark lesions. In contrast, all other treatment groups still exhibited varying degrees of impairment. Hematoxylin-eosin (H&E) staining, Masson staining, and Ashcroft score also revealed significant alveolar wall damage, tissue structural collapse, and collagen accumulation after bleomycin exposure. The antifibrotic effect was mild in Mexo or aT group, but it was enhanced when the two combined (Mexo+aT, Mexo-fl-aT and Mexo-cl-aT group). Notably, Mexo-cl-aT was more competent in restoring tissue structure and inhibiting ECM deposition, as evidenced by the recovery of the alveolar cavity and wall, and reduced collagen accumulation (Fig. 7c, d and Supplementary Fig. S20a), and the lowest levels of α-SMA and COL1A1 among all the treatment groups (Fig. 7e). Moreover, the average level of hydroxyproline (HYP, 280 μg/g of wet lung tissue) in the Mexo-cl-aT group approached that of healthy controls (286 μg/g of wet lung tissue) (Fig. 7f). These results strongly suggest the significant antifibrotic effects of Mexo-cl-aT in vivo.

In the dose optimization study, we further demonstrated that a single high-dose administration is particularly effective in restoring normal lung structure and function in bleomycin-challenged mice (Supplementary Figs. S21, S22). In a comparative study in which pirfenidone was used as a positive control, Mexo-cl-aT also exhibited pronounced anti-fibrotic efficacy (Supplementary Fig. S23), markedly attenuated leukocyte infiltration, and the decreased secretion of inflammatory cytokines such as IL-6, TNF-α, and IL-1β (Supplementary Fig. S24).

To assess the balance of ECM metabolism, we measured the MMP activities of TIMP-1 and MMP-9 in BALF samples from healthy and fibrotic mice via ELISA. The results showed that TIMP-1 levels in mouse fibrotic lungs (PBS group) were significantly greater than those in healthy controls. Although free aT or Mexo reduced TIMP-1 levels and increased MMP-9 activity to some extent, this effect was enhanced when the two were combined. As expected, TIMP-1 levels in the Mexo-cl-aT group decreased by 90.3% compared to those in the PBS group, approaching levels seen in the healthy control group (Fig. 7g). Accordingly, Mexo-cl-aT treatment resulted in the greatest increase in MMP-9 activity among all treatment groups (Fig. 7h). Furthermore, the transcriptome analysis also revealed that Mexo-cl-aT treatment upregulated the expression of multiple MMPs and restored the balance of the MMPs/TIMPs protease system (Supplementary Fig. S25a).

To examine the ROS scavenging ability of Mexo-cl-aT in vivo, the ROS probes (DCFH-DA) were intratracheally injected into the lungs of bleomycin-challenged mice 4 h after the indicated treatments. Flow cytometry analysis revealed that fibrotic lungs had high levels of ROS, and Mexo-cl-aT treatment achieved an average ROS clearance of 76.8% (Fig. 7i and Supplementary Fig. S26). The transcriptomic analysis also revealed that Mexo-cl-aT treatment downregulated the expression of genes associated with oxidative stress, suggesting that effective alleviation of lung tissue damage (Supplementary Fig. S25b). Moreover, a marked decrease in TdT-mediated dUTP Nick-End Labeling (TUNEL) fluorescence was observed in the Mexo-cl-aT group (Fig. 7j). IHC images also showed increased expression of AQP5, prosurfactant protein C (ProSPC, a biomarker of type II alveolar epithelial cells), and von Willebrand factor (vWF, a biomarker of vascular endothelial cells) in lung sections after Mexo-cl-aT treatment (Supplementary Fig. S20b), suggesting its robust performance in tissue repair.

Finally, we assessed the biosafety of Mexo-cl-aT through hematologic and biochemical analysis, as well as H&E staining. Blood cell counts and hepatorenal functions were all within normal reference ranges (Supplementary Figs. S27, S28a). Additionally, the heart, liver, spleen, and kidneys of these mice showed no microscopic abnormalities in cell and tissue morphology (Supplementary Fig. S28b). All results indicate that Mexo-cl-aT does not cause any toxicity or apparent damage. In summary, our nanosystem exerts favorable synergistic therapeutic effects by rebalancing the protease system, scavenging ROS, and promoting tissue repair, while also providing good safety.

Methods
Materials and agents
Triton X-100, bicinchoninic acid (BCA) protein assay kit, nanogold-conjugated secondary antibody, electrogenerated chemiluminescence (ECL), DCFH-DA, H2O2 assay kit, CCK-8, HYP content assay kit, mouse cytokine ELISA kits were purchased from Solarbio Science & Technology Co. Ltd. (Beijing, China). N-Hydroxysuccinimide-(polyethylene glycol)4-DBCO (NHS-PEG4-DBCO), PBA-PEG4-azide and NHS-PEG4-azide were purchased from Xi’an Qiyue Biotechnology Co., Ltd. (Xi’an, China). DiI, DiR, and Amplex Red were purchased from Beyotime Biotechnology Co. Ltd. (Shanghai, China). Rabbit anti-mouse TIMP-1 antibody, Rabbit anti-mouse vWF antibody, Rabbit anti-human α-SMA antibody were purchased from ABclonal Technology Co. Ltd. (Wuhan, China). Rabbit anti-human CD63/CD9/ALIX antibody, HRP-conjugated goat anti-rabbit IgG (H+L) secondary antibody, Tetramethylrhodamine (TRITC)/FITC-conjugated goat anti-rabbit secondary antibody, rabbit anti-human TIMP-1 antibody, and mouse TIMP-1 ELISA kit were purchased from Proteintech (Wuhan, China). MMP-9 ELISA kit was purchased from Elabscience (E-EL-M3052, Wuhan, China). Bleomycin sulfate was purchased from Yuanye Bio-Technology Co. Ltd. (Shanghai, China). Silica was purchased from Shanghai Macklin Biochemical Co. Ltd. (Shanghai, China). Annexin V-FITC/PI apoptosis detection kit was purchased from Vazyme (Nanjing, China). Pirfenidone and recombinant mouse TGF-β1 was purchased from MedChemexpress (New Jersey, USA). Rabbit anti-mouse COL1A1 antibody, Alexa Fluor 488-conjugated rabbit anti-mouse α-SMA antibody and Alexa Fluor 647-conjugated goat anti-rabbit IgG secondary antibody, Rabbit anti-mouse AQP5 antibody were purchased from Thermo Fisher Scientific (Waltham, USA). Rabbit anti-mouse ProSPC antibody was purchased from Abcam (Cambridge, UK).

Human specimens
Diseased lung tissue was harvested from IPF patients (diagnosed via standard criteria and MDT consensus) at the time of lung transplantation at the First Affiliated Hospital, Zhejiang University School of Medicine. Control lung samples (non-cancerous regions of lung cancer patients, or non-diseased areas of benign nodule patients) were collected for IHC and IF staining.
Patient demographics are summarized in Supplementary Table S1. All specimens were collected at the First Affiliated Hospital, Zhejiang University School of Medicine. Written informed consent was obtained from all donors prior to specimen collection. The study protocol was approved by the Clinical Research Ethics Committee of this hospital (Approval ID: 1047) and Shenzhen Children’s Hospital (Approval ID: 2023159021).

Cell culture
Human umbilical cord mesenchymal stem cells (hUMSCs) were obtained from the Cell Resource Center, Peking Union Medical College, and cultured in complete HMSC medium (SIMPSONLIFE, China). A549 cells and HUVECs were obtained from American Type Culture Collection (ATCC) and cultured in high glucose DMEM containing 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37 °C under 5% CO2 environment. Primary MLFs were obtained from C57BL/6J mouse lung tissues. Briefly, the tissues were harvested and cut into small pieces (1 mm3), digested in type IV collagenase (2 mg/mL) at 37 °C for 15–20 min, terminated and washed in high glucose DMEM containing 20% FBS and antibiotics. Small pieces were spread on the petri dish (diameter 10 cm) and cultured in 2 mL complete medium for 24 h for stable adherence, followed by 4 mL medium replacement. Fibroblasts climbed out from the margin of small tissue on day 3. The fibroblasts were subcultured when the density reached 80%. Primary MLFs used for downstream experiments were between passages 3 and 5.

Mexo isolation
hUMSCs were cultured in exosome-free medium. Culture supernatants were collected and centrifuged at 1000 × g for 10 min and 2000 × g for 20 min to remove cells and cell fragments, respectively. The obtained supernatant was centrifuged at 10,000 × g for 60 min at 4 °C to remove debris and microvesicles. The final supernatant was then ultracentrifuged at 100,000 × g for 70 min to obtain exosome sedimentation. The obtained exosomes were resuspended in PBS. The protein concentration was determined by BCA.

Preparation of Mexo-cl-aT and Mexo-fl-aT
Conjugation of NHS-PEG4-DBCO to the aT. NHS reacted with the amino group of lysine on the antibody. NHS-PEG4-DBCO was added to the aT at a molar ratio of 50:1 (DBCO: aT). After incubation on shaker at RT for 1 h, the solution was filtered (Millipore, amicon ultra-0.5, 10 kDa) using centrifugation at 7000 × g for 30 min to remove the excess NHS-PEG4-DBCO. The purified DBCO-aT were suspended in PBS and analyzed using MALDI-TOF to confirm the purity and composition.
Conjugation of PBA-PEG4-azide or NHS-PEG4-azide to the Mexo. PBA and NHS reacted with saccharide groups and amino groups on the surface of the Mexo, respectively. Excessive PBA-PEG4-azide was added to the Mexo at a concentration ratio of 100 μM: 1 mg/mL (PBA-PEG4-azide: Mexo). After incubation at 4 °C for 2 h, the solution was filtered (Millipore, amicon ultra-0.5, 100 kDa) using centrifugation at 7000 × g for 30 min to remove the excess PBA-PEG4-azide. The purified Mexo-cl-azide was suspended in PBS. Mexo-fl-azide was prepared in the same way.
Conjugation of DBCO-aT to the Mexo-cl/fl-azide. DBCO-aT were conjugated with Mexo-cl/fl-azide by click chemistry reaction. After incubation of DBCO-aT and Mexo-cl/fl-azide at 4 °C for 2 h, the resulting Mexo-cl-aT or Mexo-fl-aT were washed and purified using ultracentrifugation, and suspended in PBS. Free DBCO-aT in the ultracentrifuged supernatant was determined using ELISA. Therefore, the capacity of aT on Mexo-cl-aT and Mexo-fl-aT could be calculated.

Morphology of Mexo and Mexo-cl-aT
TEM images of Mexo and Mexo-cl-aT stained with antibody-linked gold nanoparticles (10 nm) were obtained using TEM (HITACHI, Japan) with an accelerating voltage of 80 kV. The size and zeta potential were determined using Zetasizer (Malvern, UK).

Colocalization of Mexo-cl-aT
Mexo and aT were labeled with DiI and FITC-conjugated secondary antibody, respectively. Both dyes were added to the solution at a final concentration of 5 µM. Double-labeled Mexo-cl-aT were dropped on a glass slide, covered with a coverslip, and visualized using CLSM (Nikon, Japan). The fluorescence was also analyzed by flow nanoanalysis (Beckman, USA). Measurements were taken from distinct samples.

Western blotting
All samples were denatured in boiling water bath for 10 min. Each sample containing 20 μg protein was loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Exosomes were characterized using anti CD63, anti CD9, and anti-ALIX antibodies. The light chain of aT was characterized using HRP-conjugated secondary antibody. Target bands were observed by a gel imager (Thermo Fisher, USA).

Release assay in vitro
Mexo-cl-aT was labeled with FITC-conjugated secondary antibody. The labeled Mexo-cl-aT in 200 μL PBS containing 0 mM, 0.1 mM, or 1 mM H2O2, was added into dialysis bags (1000 kDa). Then, dialysis bags were immersed in 5 mL solution mentioned above, respectively. At predetermined time intervals, 200 μL liquid outside bags was removed for analysis and an equal volume of corresponding solution was supplemented. Finally, the absorbance at 488 nm of samples were measured using a microplate reader (BioTek, USA). Measurements were taken from distinct samples.

H2O2 determination in vitro
The samples were collected at 4 h in the release assay. The content of H2O2 was determined using H2O2 assay kit according to the manufacturer’s instructions. Briefly, H2O2 reacts with titanium sulfate to form a yellow titanium peroxide complex, which has characteristic absorption at 415 nm. Finally, the absorbance at 415 nm of samples was measured using a microplate reader. Measurements were taken from distinct samples.

Determination of H2O2 in vitro (Amplex Red assay)
A549 cells were seeded on confocal dishes (5 × 105 per dish) and incubated overnight. First, the cells were injured by bleomycin and then were co-incubated with various groups (PBS, aT, Mexo, Mexo-fl-aT and Mexo-cl-aT) for 48 h. Normal A549 cells were as the control group. Next, remove the culture medium and add 100 μM Amplex Red and 0.25 U/mL HRP. After 30 min, transfer the supernatant to a 96-well plate and read the absorbance at 570 nm using a fluorescence microplate reader.

ROS detection in vitro
A549 cells were seeded on confocal dishes (5 × 105 per dish) and incubated overnight. First, the cells were injured by bleomycin and then various groups (PBS, aT, Mexo, Mexo-fl-aT and Mexo-cl-aT) were co-incubated for 48 h. Normal A549 cells were as the control group. Next, the ROS probe DCFH-DA (10 μM) was added into the dishes and incubated with cells in FBS-free DMEM at 37 °C under 5% CO2 in the dark for 20 min. Last, the level of ROS was imaged and quantified by CLSM and its analytical system. 3D maps of relative fluorescence intensity were produced by ImageJ software. For flow cytometry (BD FACSAria II, USA), treated cells were digested from the dishes and incubated with DCFH-DA (10 μM) in the same conditions above. Measurements were taken from distinct samples.

Anti-apoptosis effects in vitro
A549 cells were seeded on 6-well plates (2 × 106 per well) and incubated overnight. First, the cells were injured by bleomycin and then various groups (PBS, aT, Mexo, Mexo-fl-aT and Mexo-cl-aT) were co-incubated for 48 h. Normal A549 cells were as the control group. Next, cells were digested from the dishes using EDTA-free trypsin, and Annexin V-FITC/PI staining was performed using apoptosis detection kit. Last, the cell apoptosis was analyzed by flow cytometry. Measurements were taken from distinct samples.

Cell viability in vitro
The proliferation ability and viability of A549 cells and HUVECs treated with various groups (PBS, aT, Mexo, Mexo-fl-aT, Mexo-cl-aT) was evaluated by CCK-8. First, A549 cells and HUVECs were seeded in 96-well plates (2.5 × 103 per well) and injured by bleomycin for 48 h. Next, medium with bleomycin was removed and replaced with fresh DMEM with 2% FBS. Then, various groups (PBS, aT, Mexo, Mexo-fl-aT, Mexo-cl-aT) were added. Last, CCK-8 assays were performed on 96-well plates cultured for 24, 48, and 72 h, respectively. The absorbance at 450 nm was detected using a microplate reader (Thermo Scientific, USA). Measurements were taken from distinct samples.

Cell proliferation in vitro
A549 cells and HUVECs were seeded on confocal dishes (5 × 105 per dish) and incubated overnight. First, the cells were injured by bleomycin for 48 h. Normal cells were as the control group. Next, bleomycin was removed and replaced with fresh DMEM containing 2% FBS, then various groups (PBS, aT, Mexo, Mexo-fl-aT and Mexo-cl-aT) were added and co-incubated for 72 h. Last, Ki67 staining was performed, and images were captured using CLSM. Measurements were taken from distinct samples.

Wound healing assay in vitro
The proliferation and migration ability of A549 cells and HUVECs treated with various groups (PBS, aT, Mexo, Mexo-fl-aT, Mexo-cl-aT) was evaluated by wound healing tests. First, A549 cells and HUVECs were seeded in six-well plates (2 × 106 per well) and incubated until the fusion of cells reached 100%. Next, a scratch line was created in the middle of each well using a 20 μL pipette tip, and then cells were washed three times with PBS to remove suspended cells. The cells were treated with various groups (PBS, aT, Mexo, Mexo-fl-aT, Mexo-cl-aT) and cultured in DMEM with 2% FBS for 24 h. Last, the cells were imaged by inverted fluorescence microscope (Olympus, Japan) to observe the area of the scratch. Measurements were taken from distinct samples.

Determination of MMP-9 activity
For in vitro study, Primary MLFs were seeded on 12-well (5 × 105 per well) and incubated overnight. First, the cells were activated by TGF-β1 (10 ng/mL) for 24 h. Next, various groups (PBS, aT, Mexo, Mexo-fl-aT and Mexo-cl-aT) were added and co-incubated with cells for 4 h. Then, the cultural supernatant was collected and added 1 mM PMSF to prevent protein degradation followed by centrifugation at 2000 × g for 10 min at 4 °C to remove cells and cell debris. For in vivo study, mouse BALF samples were collected and centrifuged to remove cells and cell debris. Finally, MMP-9 activity was determined by ELISA. The absorbance at 450 nm was detected using a microplate reader. Measurements were taken from distinct samples.

Anti-fibrotic effects in vitro
Primary MLFs were seeded on confocal dishes (5 × 105 per dish) and incubated overnight. First, the cells were activated by TGF-β1 (10 ng/mL) and then various groups (PBS, aT, Mexo, Mexo-fl-aT and Mexo-cl-aT) were co-incubated for 24 h. Normal MLFs were as the control group. Next, cells were washed with PBS, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and then blocked with 5% BSA before incubation in proper order with COL1A1 primary antibodies, TRITC-conjugated secondary antibodies, and Alexa Fluor 488-conjugated α-SMA antibodies. The cell nucleus was stained with Hoechst 33342. Last, the images were captured using CLSM. For the quantification of α-SMA+ and COL1A1+ cells in MLFs, treated cells were digested from the dishes, stained with the corresponding fluorescent antibodies, and analyzed by flow cytometry. Measurements were taken from distinct samples.

Animals
C57BL/6J male mice (8–10 weeks) were purchased from Laboratory Animal Center, Peking University. Mice were anesthetized and received a single intratracheal injection of bleomycin sulfate at a dose of 2 units/kg. Mice challenged with bleomycin for 10 days were used for all subsequent animal experiments in vivo. For in vivo therapeutic studies, PBS, aT, Mexo, Mexo+aT, Mexo-fl-aT and Mexo-cl-aT (200 μg/mL Mexo, 40 μg/mL aT) in 50 μL solution were given one dose intratracheal injection in pulmonary fibrosis mouse lungs. Pirfenidone was as the positive control (p.o. 300 mg/kg, 100 μL, once daily from day 10–17). Mouse lungs were harvested and analyzed on day 18. For silica-induced pulmonary fibrosis model, mice challenged with silica for 2 weeks were used for all subsequent animal experiments in vivo. PBS, aT, Mexo, Mexo+aT, Mexo-fl-aT and Mexo-cl-aT (200 μg/mL Mexo, 40 μg/mL aT) in 50 μL solution were given one dose intratracheal injection in pulmonary fibrosis mouse lungs. Mouse lungs were harvested and analyzed on week 4. All the procedures were approved by the Animal Ethics Association and the Ethics Committee of Beijing Institute of Technology (approval ID: 2022-0009-167).

Determination of cytokines in mouse lung tissues
First, the tissues were washed with pre-cooled PBS, weighed, and chopped up. Next, chopped tissues were added to PBS (1 mL PBS for 0.1 g tissues), and PMSF was added to prevent protein degradation. The tissue samples were transferred to a homogenizer and ground thoroughly on ice. Finally, the homogenate was centrifuged at 2–8 °C, 5000 × g for 10 min. The cytokines (TIMP-1, TGF-β, IL-6, IL-1β, TNF-α) in the supernatant were determined by ELISA.

Transcriptomics
Total RNA was extracted from the mouse lung tissues using TRIzol® Reagent according the manufacturer’s instructions (Magen). RNA samples were detected based on the A260/A280 absorbance ratio with a Nanodrop ND-2000 system (Thermo Scientific, USA), and the RIN of RNA was determined by an Agilent Bioanalyzer 4150 system (Agilent Technologies, CA, USA). Only qualified samples will be used for library construction. Paired-end libraries were prepared using a ABclonal mRNA-seq Lib Prep Kit (ABclonal, China) following the manufacturer’s instructions. The mRNA was purified from 1 μg total RNA using oligo (dT) magnetic beads followed by fragmentation carried out using divalent cations at elevated temperatures in ABclonal First Strand Synthesis Reaction Buffer. Subsequently, first-strand cDNAs were synthesized with random hexamer primers and Reverse Transcriptase (RNase H) using mRNA fragments as templates, followed by second-strand cDNA synthesis using DNA polymerase I, RNAse H, buffer, and dNTPs. The synthesized double stranded cDNA fragments were then adapter-ligated for preparation of the paired-end library. Adaptor-ligated cDNA was used for PCR amplification. PCR products were purified (AMPure XP system) and library quality was assessed on an Agilent Bioanalyzer 4150 system. Finally, sequencing was performed with an Illumina Novaseq 6000/MGISEQ-T7 instrument. The data generated from Illumina/BGI platform were used for bioinformatics analysis. Differential expression analysis was performed using the DESeq2, DEGs with |log2FC| > 1 and Padj < 0.05 were considered to be significantly different expressed genes.

H2O2 determination in vivo
Mice were intratracheally injected with 50 μL PBS or bleomycin sulfate (2 U/kg). After 10 days, the mouse lung tissues were collected and immediately homogenized in pre-cooled acetone (tissue: acetone = 0.1 g/mL). The process was operated on ice. The samples were centrifuged at 8000 rpm for 10 min at 4 °C, and the supernatants were obtained. The content of H2O2 was determined using H2O2 assay kit according to the manufacturer’s instructions. Briefly, H2O2 reacts with titanium sulfate to form a yellow titanium peroxide complex, which has characteristic absorption at 415 nm. Finally, the absorbance at 415 nm of samples was measured using a microplate reader. Measurements were taken from distinct samples.

Imaging of drug metabolism
For in vivo imaging, Mexo were labeled with DiR and aT were labeled with Alexa Fluor 647-conjugated secondary antibody. Bleomycin-induced pulmonary fibrosis mice were given intratracheal injection of Mexo+aT (simple mixed) and Mexo-cl-aT. Images were captured at timepoint 0, 2, 8, 24, 48, 72 h after injection by animal in vivo imaging system (PerkinElmer, USA). The major organs were dissected and subjected for ex vivo imaging. Average radiant efficiency of DiR and Alexa Fluor 647-conjugated secondary antibody was quantified by Living Image analysis software. For confocal microscopy, Mexo were labeled with DiI and aT were labeled with FITC-conjugated secondary antibody. Bleomycin-induced pulmonary fibrosis mice were given intratracheal injection of Mexo-fl-aT and Mexo-cl-aT. After 4 h, the lungs of mice were collected, fixed in 4% paraformaldehyde for 24 h, dehydrated in 30% sucrose solution for 1–3 days and stored in the optimum cutting temperature (OCT) compound at −20 °C. The frozen lungs were further cut into 10 µm slices using freezing microtome (RWD Life Science, China). These sections were imaged using CLSM to analyze the controlled release of drugs.

Histology analysis
Mouse lung tissues were collected, washed with PBS, and fixed in 4% paraformaldehyde for 24 to 48 h. For H&E, Masson’s trichrome and picrosirius red staining, lungs were dehydrated and embedded in paraffin followed by sectioning and staining. Ashcroft scores were evaluated based on the results above. For IF and IHC staining, lung sections were stained with anti α-SMA, anti COL1A1, anti ProSPC, anti AQP5, anti vWF antibodies and anti-TIMP-1 antibodies. Measurements were taken from distinct samples.

Determination of hydroxyproline
The HYP content in mouse lungs was determined using a hydrochloric acid hydrolysis kit according to the manufacturer’s instructions. In brief, ~200 mg lung tissues were accurately weighed and hydrolyzed in hydrochloric acid solution at 100 °C for 2–6 h. After cooling, the hydrolysate was adjusted to a pH of 6.0–8.0 with NaOH and centrifuged at 16,000 rpm for 20 min. The collected supernatant was incubated with detection agents, and absorbance at 560 nm was measured to determine the HYP content using a microplate reader (Thermo Scientific, USA). Measurements were taken from distinct samples.

Analysis of ROS in lung tissues
For flow cytometry, mice were intratracheally injected with 50 μL DCFH-DA (10 μM). After 20 min, the mouse lung tissues were collected and immediately ground into a single-cell suspension through a 40 μm filter. The process was operated on ice. Then, the level of ROS was quantified by flow cytometry. Measurements were taken from distinct samples.

Determination of TIMP-1 and MMP-9 in mouse BALF
Mouse BALF was collected and centrifuged at 1000 × g for 10 min at 4 °C to remove cells. The level of TIMP-1 and MMP-9 in mouse BALF of various groups were assayed using ELISA method as instructed by the manufacturer. Measurements were taken from distinct samples.

Statistical analysis
All results are analyzed using GraphPad Prism software (version 8.0.2) and expressed as means ± SEM. Significant differences between two groups were analyzed by two-tailed unpaired Student’s t test. Significant differences between three or more groups were analyzed by one-way ANOVA. ns, no significance, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Differences were considered statistically significant at P < 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript utilizes a ROS-responsive nanosystem targeting TIMP-1 to regulate ECM metabolic balance in fibrosis, presenting an innovative therapeutic strategy. By simultaneously scavenging ROS and releasing therapeutic exosomes, it offers a dual therapeutic mechanism for IPF. The in vivo and in vitro experimental data are abundant, and the significant antifibrotic effects of the nanosystem have been fully validated in a mouse model.\nHowever, there are some issues that should be addressed:\n1. Line 141-“When the ratio of Mexo to aT was 5:1 (w/w), the Mexo-cl-aT displayed the greatest antifibrotic effect on transforming growth factor-β1 (TGF-β1)-induced primary mouse lung fibroblasts (MLFs)”\nIn this case, what is the cytotoxic effect of Mexo-cl-aT on cells? Was the CCK8 assay used for detection? Is this screening ratio method reasonable? Should the effect on cells be prioritized? Additionally, how was the concentration of TGF-β determined?\n2. How does Mexo-cl-aT achieve better collagen degradation? Is it through an extracellular or intracellular degradation pathway?\n3. In in vivo studies, only the effect of a single dose administration was reported, but the effect of different doses of Mexo-cl-aT on IPF was not explored in depth. Dose optimisation experiments are recommended to determine the most effective and safest dose range. There are also fewer time-dependent studies, and research could add monitoring of drug effects at different time points to determine the optimal timing of therapeutic effects.\nIn addition, IPF is a chronic progressive disease and only one time point (10 days) was observed in the manuscript, it is recommended to extend the observation period to determine the presence of delayed side effects and cumulative organ toxicity.\n4. The current experimental control groups, which mainly consist of PBS, aT, Mexo and Mexo-fl-aT group, lack a direct comparison with other standard antifibrotic treatments such as pirfenidone and nidaneb. The addition of a positive control group for these drugs could provide a clearer picture of the advantages and disadvantages of Mexo-cl-aT over existing treatments.\n5. Figure 6C-Most models of fibrosis are quite patchy in nature, thus just choosing one relatively high powered microscope field is not sufficient to quantify the levels of fibrosis. The addition of mRNA and western blotting results is recommended to more fully quantify the therapeutic effect of Mexo-cl-aT.", "reviewer_2": "The manuscript by Li et al. presents an antibody-based therapeutic approach for advanced lung fibrosis. While the study touches on an important issue, the work needs further experimental evidence before can be consider for publication in NC:\n1. Choice of TIMP-1 as a therapeutic target. The justification for selecting TIMP-1 as the target is not sufficiently supported. Although increased expression of TIMP-1 is observed in fibrotic areas, this alone does not establish it as a viable therapeutic target. Moreover, the efficacy of inhibiting TIMP-1—or any single-factor inhibition, particularly in advanced lung fibrosis—remains controversial in the previous studies.\n2. Boronic acid-based ROS-responsive materials. The materials described are not broadly responsive to ROS but are, in fact, solely responsive to hydrogen peroxide at relatively high concentrations (micromolar to millimolar range). Achieving such concentrations in the human body is challenging, which often necessitates the use of GOx to induce H2O2 production in previous studies. Furthermore, based on the authors’ results, the nanosystem did not demonstrate significantly enhanced sensitivity to hydrogen peroxide (100 μM), raising concerns about whether it will function as designed in vivo or whether the observed effects are actually obtained from a different mechanism.\n3. Rationale in the use of exosomes. The inclusion of exosomes in this study needs further consideration. Given that polymer conjugation can yield similar outcomes in terms of lung accumulation and hydrogen peroxide scavenging when administered via inhalation, the added value of exosomes in this context is not clear.\n4. Mechanistic study. The mechanistic investigation needs further depth experiments to get to certain mechanistic claim. For example, the authors suggest that TIMP-1 inhibition restores MMP activity, but MMP is a broad family with diverse roles depending on the subtype. Moreover, only MMP-9 was evaluated in the study, whose role in lung fibrosis is both controversial and largely unknown, therefore it would be even beneficial to explore whether MMP-9 can be adopted as a robust pathogenesis for lung fibrosis. Additionally, other experiments, such as measuring ROS content and cell viability, seem more indicative of particle functionality rather than offering insight into the underlying mechanisms.", "reviewer_3": "In this manuscript, Li et al. developed a nano system targeting tissue inhibitor of metalloproteinase-1 (TIMP-1) for continuous IPF therapy. It seems that the anti-fibrotic effects were good, but more data or evidence are needed to confirm the anti-fibrotic effects without adverse effects. I have several concerns with the claimed mechanism of anti-fibrosis.\n1. I would concern about the mechanism of the anti-fibrotic effects. The authors claimed that the nano-system with anti-TIMP-1 could rebalance TIMP-1 and MMP9 further ameliorated BLM-induced fibrosis. Based on the published articles, the claim was still controversial. From their claim, the anti-TIMP-1 treatment would upregulate MMP9 and dissolve the fibrosis. However, the increased MMP9 would enhance inflammatory cells infiltration and led to increased inflammation. Besides, the increased MMP9 would lead to increased TGF-beta activation that further aggravate the fibrosis. I highly recommended the authors provided more information about the phenotype of in vivo anti-fibrosis effects.\n2. Figure 1. The author correlated TIMP-1 with fibrosis in patients and fibrotic mice. However, I am much curious about the relation of MMP9 and Fibrosis. If there is an imbalance with TIMP-1 and MMP9, whether MMP9 would decrease within the fibrotic lung?\n3. Authors focused on fibroblasts and epithelial with in vitro experiments. While the in vivo experiments were much more complicated than the in vitro system. The author should provide more information about the cellular source of TIMP1 and MMP9, which would smooth the results to fibroblasts and epithelial. Besides, whether the anti-TIMP-1 treatment would increase the inflammatory cell infiltration into the lung?\n4. Figure 6. I am quite curious about the effects when the nano system applied to the mice in vivo. Though the authors provide information about the anti-fibrotic effect, I will ask that what the effects that would be about the inflammation. Some related cytokines IL-1b, TNF-a, IL-6 and the most critical TGF-b experession should be detected to fulfill its anti-fibrotic effects.\n5. Figure 6. As for the Masson’s and H&E staining, authors are expected to provide full lung lobe images with zoomed area for the readers better to evaluate the anti-fibrotic effects. The fibrosis in the lung were very heterogeneity. It would be better for the readers to evaluated the anti-fibrosis effects. Besides, IF staining to a-SMA and COL-1 were bad quality, confocal images would be better if the author could do that.\n6. Figure 6. The levels of TIMP1 and MMP9 in the BALF were not that convincing, since majority of the cytokines were in the lung interstitial. Also, there were huge variation when BAL was prepared. Western blot detecting the two important proteins with the lung tissue are needed to better illuminate the protein levels in the lung. The results would better illuminate the balance between the two proteins.\n7. BLM induced pulmonary fibrosis would dissolve within 3-5 weeks after BML treated. If the author could provide more information about the Nano system against Crystalline silica induced fibrosis would be more attracting. Since CS particle would induced persist and progressive pulmonary fibrosis.\n8. How many mice were used in the experiment of each group. HE Masson staining would be evaluated with semi quantification.\n9. The anti-fibrotic effects would rely more on the anti-ROS system or the anti-TIMP-1 effects?", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "Overall:\nThis manuscript describes a study deploying umbilical cord-derived mesenchymal exosomes decorated with ROS-cleavable antibodies to TIMP-1 and delivered intratracheally as a potential therapeutic nanosystem for idiopathic pulmonary fibrosis. While many aspects of the study are intriguing, the exact cellular and molecular targets of this complex nanosystem are poorly defined, and the data as presented do not clearly reveal therapeutic targets and mechanism of action. The use of a single model which does not recapitulate many aspects of the human disease limits the broad applicability of the findings.\nMajor Points:\n- The rationale for the focus on TIMP-1 and MMP9 specifically is poorly defined. Additional discussion is needed about which MMPs are implicated in IPF pathogenesis and why TIMP-1 was chosen (ie does it have higher affinity binding for these specific MMPs?).\n- Figure 1 - TIMP staining – Figures are low resolution and lack use of additional cell markers (Krt5, aSMA etc) to determine location of TIMP expression. In human IPF images shown the staining is rather diffuse. Is it intracellular or extracellular? Their BAL ELISA would suggest extracellular, but their staining shows it in tissue - is it being produced / secreted by specific cell types in the IPF lung? Can the authors use mouse lung tissue from their bleomycin experiment to look for TIMP levels via ELISA or Western blot?\n- Figure 1g/h – the method used to stain for “ROS” is not specified in the methods section. What was used? Very few reagents will react with all forms of ROS and are not stoichiometric. DCFH-DA was used later in the paper and may have been used here? If so, DCFH-DA is not a reliable measure of all ROS and has several limitations (e.g., PMID: 22027063). Additionally, staining fixed tissue sections for ROS is not ideal – reactive species are produced and reduced dynamically and fixation will not preserve these species. The authors should state these limitations clearly and use a complementary assay to assess redox dysregulation in these samples, such as the formation of oxidative byproducts or modifications (see PMID: 29988126). It is also unclear if human lung tissue was formalin fixed or frozen, as well as any demographic information or source of IPF and healthy control lung tissues - these should also be specified in the methods.\n- Rationale for choice of mesenchymal exosomes (e.g., vs. an engineered nanoparticle) could be better developed. Is the goal to protect the antibody, or to only release it when ROS levels are high? Or is exosome delivery meant to reach intracellular space? Also, what is the rationale for sourcing exosomes from umbilical cord mesenchymal stem cells, and what additional molecular do these exosome preparations contain?\n- Figure 3 – The authors treat MLF stimulated with TGFB with the exosome/TIMP antibody, but do not confirm that TIMP and/or MMP activity/levels were affected by TGFB treatment in these fibroblasts. Staining for TIMP/MMP or an activity assay in the TGFB treated fibroblasts would be helpful here. Also, in Fig3a, there is no information on number of fields of view assessed or replicates, or any quantification for these samples. It is hard to get an idea of the scale of the change from isolated images. These are cells in culture, so it should be possible to also perform western blotting and/or ELISAs of the targets investigated in this panel (aSMA, col1a1, and add TIMP)?\n- Figure 3 – when MLF are treated with the exosome alone, there is significant reduction of aSMA and it looks like collagen (although it is not clear if this comparison was not significant or just not reported). What is the proposed explanation for this? It looks like the exosome has equal effect to the antibody alone? In the scheme in Figure 1 it shows the proposed effect of Mexo alone acting on vascular endothelial cells and epithelial cells, but this point is not really discussed in the introduction or results.\n- Figure 4 in general – it seems like the Mexo-fl-aT and the Mexo-cl-aT both have the same effect on viability, apoptosis, and wound healing, with the only difference being that Mexo-fl-aT is not able to increase MMP9 activity. Would this indicate that these phenotypic effects on wound healing, apoptosis, cell viability, are not mediated through MMP9 activity? For some of the endpoints, like wound healing, it looks like the exosome alone increases the response.\n- Figure 4f - Again, DCFH-DA is not a reliable probe for ROS generation. Something like Amplex Red might be a better assay here, which also focuses on extracellular H2O2 levels, and would better reflect the extracellular oxidative environment in which the Mexo-cl-aT would be cleaved.\n- Figure 6g – TIMP1 levels decrease in response to these treatments, but even the Mexo alone condition causes this. What is the proposed mechanism for this? Does binding of TIMP-1 with the aT antibody cause it to be undetected by the ELISA? Also, this is in BAL- do tissue levels of TIMP-1 decrease as well? Can ELISA/staining/WB be performed on these?\n- Can the authors demonstrate that when aT is released from Mexo-cl-aT, that the antibody then binds TIMP? Can this complex be identified and measured, ie via immunoprecipitation from in vivo samples? This would also help determine non-specific binding of aT to other targets (other TIMPs?) occurs in vivo.\n- The discussion is not very detailed and the first two paragraphs merely summarize the results of the study, while only the last one discusses broader implications. What are the limitations of this study?\n- Overall, the main goal of the treatment is unclear – is the Mexo-cl-aT meant to deliver the anti-TIMP antibody and scavenge ROS through cleavage of the linker? Or is the Mexo component serving some value in wound repair? I do not think this distinction is well described in the introduction/results sections. In lines 341 – 351 of the discussion this is mentioned, although the statement “Mexo have a natural ability to home to injury sites” is not cited. A more thorough introduction about why Mexo was chosen and what is known about exosomes ability to promote wound repair is needed in the introduction.\nMinor Points:\n- Citations appear to be after the period for each sentence.\n- Methods – “Determination of ROS in lung tissues” and “Determination of TIMP-1 in BALF” have the same description, which is for the TIMP measurement. ROS method section should be updated.\n- Figure 2h – How was H2O2 quantified in these samples? This is not stated in the methods section.\n- The MMP9 activity assay in Figure 4B makes more sense to be included in Figure 3 – it is from the fibroblast experiment and involves the same treatments. The y-axis of this figure is confusing – is this activity or abundance (the ng/mL suggests abundance). This is also an issue in Figure 6C.\n- Figure 4c – it is unclear what the treatment is. I’m assuming it is the bleomycin treatment, but no label is provided on the figure and no information on dose is given in the legend. The significance notation here is also unclear – is everything significant, or is it referring to a specific comparison?\n- Figure 4d – I think having the graph shown in Figure S7a would be helpful here to compare the apoptotic cells in the same format as other assays\n- Mouse model is referred to as “IPF” – these mice don’t have IPF, they’ve been injured with bleomycin", "reviewer_6": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to Referees\nManuscript entitled“A Nanosystem Targeting Tissue Inhibitor of Metalloproteinase-1\nfor Continuous Spatiotemporal Idiopathic Pulmonary Fibrosis Therapy ”\n(NCOMMS-24-53302).\nResponses to the comments from the reviewers (Note: All the responses here are in\nblue. All the changes have been highlighted in red in the revised manuscript).\nReviewer #1 (Remarks to the Author):\nThe manuscript utilizes a ROS-responsive nanosystem targeting TIMP-1 to regulate\nECM metabolic balance in fibrosis, presenting an innovative therapeutic strategy. By\nsimultaneously scavenging ROS and releasing therapeutic exosomes, it offers a dual\ntherapeutic mechanism for IPF. The in vivo and in vitro experimental data are\nabundant, and the significant antifibrotic effects of the nanosystem have been fully\nvalidated in a mouse model.\nHowever, there are some issues that should be addressed:\n⚫ Response: We thank the reviewer for the positive comments and for recognizing\nour work as an “innovative therapeutic strategy”.\n1. Line 141-“When the ratio of Mexo to aT was 5:1 (w/w), the Mexo-cl-aT displayed\nthe greatest antifibrotic effect on transforming growth factor-β1 (TGF-β1)-induced\nprimary mouse lung fibroblasts (MLFs)” In this case, what is the cytotoxic effect of\nMexo-cl-aT on cells? Was the CCK8 assay used for detection? Is this screening\nratio method reasonable? Should the effect on cells be prioritized? Additionally, how was\nthe concentration of TGF-β determined?\n⚫ Response: Thanks for the reviewer’s kind reminder. we evaluated the cytotoxic\neffect of Mexo-cl-aT on cells by CCK8 assay. The results showed that 5 μg/mL\nMexo + 1 μg/mL aT (Mexo:aT=5:1 (w/w)) displayed no significant toxicity or\nside effects on A549, HUVEC, and MLFs. We have added the data as Fig.S7 and\ncorresponding description (line 149-150) in the revised manuscript.\nFig.S7 The cytotoxic effect of Mexo-cl-aT on A549, HUVEC, and MLF cells by\nCCK8 assay. The cells were treated with different ratio of Mexo and aT for 24 hours\nbefore measurement (N=3). Data are means ± SEM; ns, not significant (one-way\nANOVA with Sidak's multiple comparisons test).\n⚫ Response: The concentration of 10 ng/mL TGF-β1 employed in our study was\nselected based on well-established protocols from previous studies (PMID:\n36402779; PMID: 35731866; PMID: 39791575) demonstrating its efficacy in\nactivating fibroblasts in vitro.\n2. How does Mexo-cl-aT achieve better collagen degradation? Is it through an\nextracellular or intracellular degradation pathway?\n⚫ Response: We are sorry for the confusion. When Mexo-cl-aT enters a fibrotic\nmicroenvironment characterized by high levels of ROS, its phenylboronic acid\nester bond breaks, releasing aT. Since MMP-9\n(https://www.uniprot.org/uniprotkb/P41245/entry) and TIMP-1\n(https://www.uniprot.org/uniprotkb/P12032/entry) are secreted proteins that\nreside in the extracellular matrix, where aT can effectively exert its function.\nSpecifically, aT neutralize free TIMP-1, leading to increased activity of MMP-9.\nAs shown in Fig. 4b-c, Mexo-cl-aT treatment resulted in significant decrease of\nfree TIMP-1 concentration and increase of MMP-9 activity in the culture\nsupernatant of TGF-β1-stimulated mouse lung fibroblast (MLF) cells. We have\nadded the data as Fig. 4b-c and corresponding description (line 200-204) in the\nrevised manuscript.\nFig. 4b-c. The levels of TIMP-1 (PBS vs. Mexo-cl-aT, P < 0.0001) and active MMP-9\n(PBS vs. Mexo-cl-aT, P = 0.0077) in MLF culture supernatants (N=3). Data are means\n± SEM; ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001\n(one-way ANOVA with Dunnett's multiple comparisons test).\n3. In in vivo studies, only the effect of a single dose administration was reported, but\nthe effect of different doses of Mexo-cl-aT on IPF was not explored in depth. Dose\noptimisation experiments are recommended to determine the most effective and safest\ndose range. There are also fewer time-dependent studies, and research could add\nmonitoring of drug effects at different time points to determine the optimal timing of\ntherapeutic effects. In addition, IPF is a chronic progressive disease and only one time\npoint (10 days) was observed in the manuscript, it is recommended to extend the\nobservation period to determine the presence of delayed side effects and cumulative\norgan toxicity.\n⚫ Response: Much thanks to the reviewers for the in-depth review of our\nmanuscript. According to the reviewer’s suggestion, we performed the dose\noptimization experiments to determine the most effective and safest dose range.\nSimultaneously, we extended the observation period from 18 days to 28 days to\ndetermine the presence of delayed side effects and cumulative organ toxicity. In\naddition, we monitored the drug effects at different time points via lung function\ntesting to determine the optimal timing of therapeutic effects.\n⚫ The dosage and frequency of Mexo-cl-aT administration. Dose 1, high-dose\nsingle administration of Mexo-cl-aT, contains 10 μg Mexo and 2 μg aT; Dose 2,\nlow-dose multiple administrations. The total dose is the same as Dose 1; Dose 3,\nhigh-dose multiple administrations. The total dose is three times that of Dose 1\nand Dose 2.\n⚫ According to Fig. S16a, three different doses of Mexo-cl-aT were individually\nadministered to a bleomycin-induced fibrosis mouse model via inhalation, and\nlung function was measured at 10, 18, and 28 days post-bleomycin administration.\nThe results showed that compared to the control group, the breathing frequency\nof the mice in the PBS group was significantly enhanced, while the levels of peak\nexpiratory flow rate and tidal volume were reduced significantly. Compared with\nthe PBS group, all the lung function indexes were restored considerably in three\ndifferent doses of Mexo-cl-aT groups (Fig. S16b-d). Obviously, Dose 3\n(high-dose multiple administrations) is less effective than Dose 1 (high-dose\nsingle administration) and Dose 2 (low-dose multiple administrations) in\nrestoring respiratory frequency and tidal volume in mice with pulmonary fibrosis\n(Fig. S16b-d). Moreover, multiple administrations may impose a burden on the\nrespiratory function of the mice. Therefore, Dose 1 is most beneficial for\nrestoring normal lung function in mice with pulmonary fibrosis. We have added\nthe data as Fig.S16 and corresponding description (line 301-303) in the revised\nmanuscript.\nFig. S16 Inhalation of Mexo-cl-aT restores the pulmonary functions of mice with\nbleomycin-induced pulmonary fibrosis. a. Illustration of animal experiment\nprocedure. b-d. Measurements of pulmonary function parameters including frequency,\n(b), peak expiratory flow rate (c), and tidal volume (d) at day 10, 18, 28 treatment (N\n= 6). Data are means ± SEM. Dose 1, high-dose single administration of Mexo-cl-aT\n(total equivalent: 10 μg Mexo and 2 μg aT). Dose 2, low-dose multiple\nadministrations of Mexo-cl-aT (total equivalent: 10 μg Mexo and 2 μg aT). Dose 3,\nhigh-dose multiple administrations of Mexo-cl-aT (total equivalent: 30 μg Mexo and\n6 μg aT). ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001\n(one-way ANOVA with Dunnett's multiple comparisons test).\n⚫ To further assess the therapeutic efficacy of three different doses of Mexo-cl-aT,\nwe used HE and Masson staining to observe the histopathological changes of\nlung tissues in the different groups. The control group showed normal lung\nstructure without inflammation, while the PBS group exhibited severe lung\ndamage and inflammation. Mexo-cl-aT treatment significantly improved lung\nhistopathology, reduced alveolar wall damage, and had antifibrotic effects\ncompared to the PBS group (Fig. S15). Particularly noteworthy was the superior\nantifibrotic performance of the Dose 1 group, evidenced by a cleared and intact\nalveolar structure and diminished collagen deposition (Fig. S15). Taken together,\nDose 1 group could effectively improve lung function, and reduce tissue fibrosis\nin mice with pulmonary fibrosis. We have added the data as Fig. S15 and\ncorresponding description (line 301-303) in the revised manuscript.\nFig. S15 Anti-fibrotic effects of Mexo-cl-aT inhalation treatment in mice of\nbleomycin-induced pulmonary fibrosis. a. Illustration of animal experiment\nprocedure. b. H&E and Masson staining of mouse lungs after different treatments\n(N=6). Dose 1, high-dose single administration of Mexo-cl-aT (total equivalent: 10 μg\nMexo and 2 μg aT). Dose 2, low-dose multiple administrations of Mexo-cl-aT (total\nequivalent: 10 μg Mexo and 2 μg aT). Dose 3, high-dose multiple administrations of\nMexo-cl-aT (total equivalent: 30 μg Mexo and 6 μg aT). Scale bar = 200 μm.\n⚫ Finally, we assessed the biosafety of Mexo-cl-aT via hematologic and\nbiochemical analysis. The hepatic and renal function parameters remained within\nnormal reference ranges; however, treatment with Dose 3 resulted in a significant\ndecrease in BUN (Fig. S20). In summary, The Dose 1 employed in our study is\nthe most effective and safest dose range. We have added the data as Fig. S20 and\ncorresponding description (line 334-336) in the revised manuscript.\nFig. S20 Biosafety of Mexo-cl-aT inhalation treatment in a bleomycin-induced\npulmonary fibrosis mouse model. a. Illustration of animal experiment procedure. b.\nEvaluation of liver and kidney functions in mice treated with different doses of\nMexo-cl-aT (N=6). Dose 1, high-dose single administration of Mexo-cl-aT (total\nequivalent: 10 μg Mexo and 2 μg aT). Dose 2, low-dose multiple administrations of\nMexo-cl-aT (total equivalent: 10 μg Mexo and 2 μg aT). Dose 3, high-dose multiple\nadministrations of Mexo-cl-aT (total equivalent: 30 μg Mexo and 6 μg aT). ns, not\nsignificant; *P < 0.05 (one-way ANOVA with Dunnett's multiple comparisons test).\n4. The current experimental control groups, which mainly consist of PBS, aT, Mexo\nand Mexo-fl-aT group, lack a direct comparison with other standard antifibrotic\ntreatments such as pirfenidone and nidaneb. The addition of a positive control\ngroup for these drugs could provide a clearer picture of the advantages and disadvantages of\nMexo-cl-aT over existing treatments.\n⚫ Response: Thank the reviewer for the suggestion. We added pirfenidone, a\nstandard antifibrotic treatment, as a positive control and evaluated its therapeutic\nefficacy using HE and Masson staining. The control group showed normal lung\nstructure, while the PBS group exhibited severe lung damage. Mexo-cl-aT and\npirfenidone treatment significantly improved lung histopathology, reduced\nalveolar wall damage. Remarkably, the Mexo-cl-aT group demonstrated better\nantifibrotic effects compared to the pirfenidone group, as shown by a\nwell-preserved alveolar structure and reduced collagen accumulation (Fig. S17a).\nWe have added the data as Fig. S17a and corresponding description (line\n304-305) in the revised manuscript.\nFig. S17 Anti-fibrosis effect of Mexo-cl-aT vs. pirfenidone in a bleomycin-induced\npulmonary fibrosis mouse model. a. HE and masson staining of the lung sections of\nmice treated with different formulations. Scale bar = 200 μm.\n5. Figure 6C-Most models of fibrosis are quite patchy in nature, thus just choosing\none relatively high powered microscope field is not sufficient to quantify the levels of\nfibrosis. The addition of mRNA and western blotting results is recommended to more\nfully quantify the therapeutic effect of Mexo-cl-aT.\n⚫ Response: Thank the reviewer for the good suggestion. We have added full lung\nlobe images with zoomed area in Fig. 6c of the revised manuscript.\n⚫ Response: We further evaluated the therapeutic effect of Mexo-cl-aT using RNA\nsequencing. The results revealed upregulated expression of MMP-related genes,\nincluding MMP1b, MMP3, MMP7 MMP8 MMP9 and MMP12, in lungs treated\nwith Mexo-cl-aT. Furthermore, the upregulation of MMPs was significantly\nhigher than that of TIMPs, indicating a shift towards extracellular matrix\ndegradation. Additionally, the expression of genes associated with oxidative\nstress decreased, suggesting effective alleviation of lung tissue damage. We have\nadded the data as Fig. S19 and corresponding description (line 317-320 and line\n324-327) in the revised manuscript.\nFig. 6c. Masson staining of mouse lung sections with different treatments. Scale bar =\n200 μm.\nFig. S19 Transcriptome analysis in the lung of mice with bleomycin-induced\npulmonary fibrosis. a. Heatmap of MMP- and TIMP-related genes. b. Heatmap of\noxidative stress-related genes. N=3. M-c-a: Mexo-cl-aT.\nReviewer #2 (Remarks to the Author):\nThe manuscript by Li et al. presents an antibody-based therapeutic approach for\nadvanced lung fibrosis. While the study touches on an important issue, the work needs\nfurther experimental evidence before can be consider for publication in NC:\n⚫ Response: We appreciate the reviewer’s insightful comments. In response, we\nhave conducted additional relevant experiments and implemented appropriate\nrevisions to enhance the manuscript.\n1. Choice of TIMP-1 as a therapeutic target. The justification for selecting TIMP-1 as\nthe target is not sufficiently supported. Although increased expression of TIMP-1 is\nobserved in fibrotic areas, this alone does not establish it as a viable therapeutic target.\nMoreover, the efficacy of inhibiting TIMP-1—or any single-factor inhibition,\nparticularly in advanced lung fibrosis—remains controversial in the previous studies.\n⚫ Response: Thanks for the reviewer’s kind reminder. Previous studies have\nreported that TIMP-1 is overexpressed by activated fibroblasts and promotes\nfibrosis by inhibiting MMP-mediated ECM degradation (PMID: 30243515;\nPMID35715541), and the impaired proteases and anti-proteases balance was\nfound in IPF (PMID: 29518524). We have preliminarily verified the effectiveness\nof anti-TIMP-1 therapy to enhance collagen degradation in vitro, as shown in Fig.\n3a. COL1A1 was upregulated in MLFs after TGF-β1 stimulation. Inhibiting\nTIMP-1 by aT promoted collagen degradation, and finally decreased COL1A1\n(weakened red fluorescence signal).\nFig. 3a IF staining of α-SMA and COL1A1 in MLFs.\n⚫ Response: Mexo-cl-aT does more than just single-factor inhibition to combat\nfibrosis. It contains three active ingredients: Mexo inhibits fibroblast activation\nand repairs epithelium and endothelium; phenylboronic acid ester-based\nROS-cleavable linker (cl) removes excess H O at the lesion; aT restores MMP\n2 2\nactivity to promote collagen degradation. They synergize to achieve superior\nanti-fibrotic treatment.\n2. Boronic acid-based ROS-responsive materials. The materials described are not\nbroadly responsive to ROS but are, in fact, solely responsive to hydrogen peroxide at\nrelatively high concentrations (micromolar to millimolar range). Achieving such\nconcentrations in the human body is challenging, which often necessitates the use of\nGOx to induce H2O2 production in previous studies. Furthermore, based on the\nauthors’ results, the nanosystem did not demonstrate significantly enhanced sensitivity\nto hydrogen peroxide (100 μM), raising concerns about whether it will function as\ndesigned in vivo or whether the observed effects are actually obtained from a different\nmechanism.\n⚫ Response: We thank the reviewer for drawing this point to our attention. We\nmeasured the concentration of hydrogen peroxide in lung tissues of\nbleomycin-induced pulmonary fibrosis mouse model, and the concentration\nreached 0.9 mM (Fig. S13). It is feasible that our nanosystem respond to the\npulmonary fibrosis microenvironment. Furthermore, extensive research has\ndemonstrated that the lesion site of pulmonary fibrosis is characterized by a\npersistent and aberrant production of reactive oxygen species, including hydrogen\nperoxide, which leads to the pathophysiology of the disease (PMID: 33201251;\nPMID: 37707699). We have added the data as Fig. S13 and corresponding\ndescription (line 268) and experimental procedure (line 597-605) in the revised\nmanuscript.\no\nntr\nol\nmy\nci\nn\nC o\ne\nBl\nFig. S13 The level of H O in lung tissues of bleomycin-induced pulmonary\n2 2\nfibrosis mouse model. P = 0.0181, N=3. Data are means ± SEM. *P < 0.05\n(two-tailed unpaired Student’s t test).\n3. Rationale in the use of exosomes. The inclusion of exosomes in this study needs\nfurther consideration. Given that polymer conjugation can yield similar outcomes in\nterms of lung accumulation and hydrogen peroxide scavenging when administered via\ninhalation, the added value of exosomes in this context is not clear.\n⚫ Response: Thanks to the reviewer’s suggestion. MSC-derived exosomes (Mexo)\nare ideal nanocarriers for lung-targeted drug delivery via inhalation, given their\noptimal size (30–150 nm), biocompatibility, low immunogenicity, and inherent\ntropism for injured or inflamed tissues, offering advantages over polymer\nnanoparticles. Furthermore, they can transfer bioactive molecules (miRNAs,\nproteins, lipids) to recipient cells, exerting diverse therapeutic effects such as\nenhancing angiogenesis, reducing fibrosis, and stimulating cell proliferation and\n)Mm(\nO\nH\n2\n2\n1\n1\n0\n0\n.5\n.0\n.5\n.0\n✱\ndifferentiation. Additionally, Mexo exhibit potent immunomodulatory properties\nby suppressing pro-inflammatory cytokines and promoting anti-inflammatory\nresponses, emerging as a promising cell-free therapy for regenerative medicine,\ninflammation-related diseases, and organ injury (PMID: 32855385;\nPMID: 32939235). In our manuscript, we also demonstrate that Mexo serves not\nonly as a delivery system but also as an active therapeutic ingredient, inhibiting\nfibroblast activation (Fig. 3a) and repairing the epithelium (Figs. 4d-g, S9) and\nendothelium (Fig. S10) in the treatment of pulmonary fibrosis. In the introduction\nof the revised manuscript (line 67-72), we have provided a comprehensive\nexplanation for the selection of Mexo.\n4. Mechanistic study. The mechanistic investigation needs further depth experiments\nto get to certain mechanistic claim. For example, the authors suggest that TIMP-1\ninhibition restores MMP activity, but MMP is a broad family with diverse roles\ndepending on the subtype. Moreover, only MMP-9 was evaluated in the study, whose\nrole in lung fibrosis is both controversial and largely unknown, therefore it would be\neven beneficial to explore whether MMP-9 can be adopted as a robust pathogenesis\nfor lung fibrosis. Additionally, other experiments, such as measuring ROS content and\ncell viability, seem more indicative of particle functionality rather than offering\ninsight into the underlying mechanisms.\n⚫ Response: We sincerely thank the reviewers for the in-depth review of our\nmanuscript. We further evaluated the antifibrotic effect of Mexo-cl-aT through\ntranscriptome analysis. The results showed that mice treated with Mexo-cl-aT\nshowed the upregulated expression of MMP-related genes, including MMP-1,\nMMP-2, MMP-3, MMP-7, MMP-8, MMP-9 and MMP-12. Furthermore, the\nupregulation of MMPs was significantly higher than that of TIMPs, indicating a\nshift towards extracellular matrix degradation. Additionally, the expression of\ngenes associated with oxidative stress decreased, suggesting effective alleviation\nof lung tissue damage. We have added the data as Fig. S19 and corresponding\ndescription (line 317-320 and line 324-327) in the revised manuscript.\nFig. S19 Transcriptome analysis in the lung of mice with bleomycin-induced\npulmonary fibrosis. a. Heatmap of MMP- and TIMP-related genes. b. Heatmap of\noxidative stress-related genes. N=3. M-c-a: Mexo-cl-aT.\nReviewer #3 (Remarks to the Author):\nIn this manuscript, Li et al. developed a nano system targeting tissue inhibitor of\nmetalloproteinase-1 (TIMP-1) for continuous IPF therapy. It seems that the\nanti-fibrotic effects were good, but more data or evidence are needed to confirm the\nanti-fibrotic effects without adverse effects. I have several concerns with the claimed\nmechanism of anti-fibrosis.\n⚫ Response: We sincerely appreciate the reviewer’s insightful comments. Below\nwe have conducted additional relevant experiments and implemented appropriate\nrevisions to enhance the manuscript.\n1. I would concern about the mechanism of the anti-fibrotic effects. The authors\nclaimed that the nano-system with anti-TIMP-1 could rebalance TIMP-1 and MMP9\nfurther ameliorated BLM-induced fibrosis. Based on the published articles, the claim\nwas still controversial. From their claim, the anti-TIMP-1 treatment would upregulate\nMMP9 and dissolve the fibrosis. However, the increased MMP9 would enhance\ninflammatory cells infiltration and led to increased inflammation. Besides, the\nincreased MMP9 would lead to increased TGF-beta activation that further aggravate\nthe fibrosis. I highly recommended the authors provided more information about the\nphenotype of in vivo anti-fibrosis effects.\n⚫ Response: We appreciate the reviewer’s insightful comments. The role of\nMMP-9 in the occurrence and development of fibrosis is indeed a subject of\ndebate. This study (PMID: 35804363) has reported that MMP-2 and MMP-9 are\nnot the main driving factors for pulmonary fibrosis. For confirmation, we\nexamined leukocyte infiltration at the lesion site and the secretion of\ninflammatory factors in bleomycin-induced mouse lung tissues using IF and\nELISA assay after administering Mexo-cl-aT via inhalation. As shown in Fig.\nS18a, the fluorescence intensity of CD45+ cells (a biomarker of leukocytes) was\nsignificantly higher in lung sections from the PBS group compared to a\nsubstantial decline observed in the Mexo-cl-aT group. In line with these\nobservations, Mexo-cl-aT treatment resulted in a marked reduction in the\nsecretion of inflammatory cytokines, such as IL-6, TNF-α, and IL-1β,\ndemonstrating its robust capacity to inhibit leukocyte infiltration and the\nsecretion of inflammatory cytokines in vivo. We have added the Fig. S18 and\ncorresponding description (line 305-307) in the revised manuscript.\nFig. S18 Anti-inflammation effect of Mexo-cl-aT. a. Immunofluorescence staining\nof CD45+ cells in mouse lung sections. Scale bar = 200 μm. b. The levels of IL-6\n(PBS vs. Mexo-cl-aT, P < 0.0001; N=6), TNF-α (PBS vs. Mexo-cl-aT, P < 0.0001; N=6),\nand IL-1β (PBS vs. Mexo-cl-aT, P = 0.2009; N=6) in mouse lung tissues\ndetermined by ELISA. Data are means ± SEM. ns, not significant; *P < 0.05; **P <\n0.01; ****P < 0.0001 (one-way ANOVA with Dunnett's multiple comparisons test).\n⚫ To further investigate the in vivo anti-fibrosis effects of Mexo-cl-aT, we added\npirfenidone, a standard antifibrotic treatment, as a positive control and evaluated\nits therapeutic efficacy through HE and Masson staining. The healthy mice\n(control group) showed normal lung structure, whereas the bleomycin-induced\npulmonary fibrosis mice (PBS group) exhibited severe lung damage. Both\nMexo-cl-aT and pirfenidone treatments significantly ameliorated lung\nhistopathology and reduced alveolar wall damage. Remarkably, the Mexo-cl-aT\ngroup demonstrated superior antifibrotic effects compared to the pirfenidone\ngroup, as evidenced by a well-preserved alveolar structure and diminished\ncollagen accumulation (Fig. S17). Correspondingly, Mexo-cl-aT treatment led\nto a marked decrease in the secretion of TGF-β1 and TIMP-1, highlighting its\nrobust anti-fibrotic capability. We have added the data as Fig. S17 and\ncorresponding description (line 305) in the revised manuscript.\nFig. S17 Anti-fibrosis effect of Mexo-cl-aT vs. pirfenidone in a bleomycin-induced\npulmonary fibrosis mouse model. a. HE and masson staining of the lung sections of\nmice treated with different formulations. Scale bar = 200 μm. b. The levels of TGF-β1\n(PBS vs. Mexo-cl-aT, P = 0.0076; N=6) and TIMP-1 (PBS vs. Mexo-cl-aT, P <\n0.0001; N=6) in mouse lung tissues determined by ELISA. Data are means ± SEM. ns,\nnot significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (one-way\nANOVA with Dunnett's multiple comparisons test).\n2. Figure 1. The author correlated TIMP-1 with fibrosis in patients and fibrotic mice.\nHowever, I am much curious about the relation of MMP9 and Fibrosis. If there is an\nimbalance with TIMP-1 and MMP9, whether MMP9 would decrease within the\nfibrotic lung?\n⚫ Response: We thank the reviewer for drawing this point to our attention.\nExtensive research has demonstrated that the expression of MMP-9 is\nupregulated in pulmonary fibrosis (PMID: 29625182; PMID: 11983918; PMID:\n10956632). Although the abundance of MMP-9 increases, with the increase of\nTIMP-1 expression, its activity is inhibited thereby it cannot fully exert the\nfunction of degrading collagen (PMID: 29518524; PMID: 32641389).\n⚫ To further assess the balance of ECM metabolism, BALF samples from healthy\nand fibrotic mice were collected, and the activities of TIMP-1 and MMP-9 were\nmeasured by ELISA. The results showed that TIMP-1 levels in mouse fibrotic\nlungs (PBS group) were significantly greater than those in healthy controls.\nAlthough free aT or Mexo reduced TIMP-1 levels and increased MMP-9 activity\nto some extent, this effect was enhanced when the two were combined (Mexo+aT,\nMexo-fl-aT, and Mexo-cl-aT groups). As expected, TIMP-1 levels in the\nMexo-cl-aT group decreased by 90.3% compared to those in the PBS group,\napproaching levels seen in the healthy control group (Fig. 6g). Accordingly,\nMexo-cl-aT treatment resulted in the greatest increase in MMP-9 activity among\nall treatment groups (Fig. 6h). We have added the data as Fig. 6g-h and\ncorresponding description (line 314-317) and experimental procedure (line\n647-650) in the revised manuscript.\nFig.6g-h The level of TIMP-1 (g) and active MMP-9 (h) in mouse BALF (PBS vs.\nMexo-cl-aT, P < 0.0001, N=4). Data are means ± SEM; ns, not significant; *P < 0.05;\n**P < 0.01; ***P < 0.001; ****P < 0.0001 (one-way ANOVA with Dunnett's multiple\ncomparisons test).\n3. Authors focused on fibroblasts and epithelial with in vitro experiments. While the\nin vivo experiments were much more complicated than the in vitro system. The author\nshould provide more information about the cellular source of TIMP1 and MMP9,\nwhich would smooth the results to fibroblasts and epithelial. Besides, whether the\nanti-TIMP-1 treatment would increase the inflammatory cell infiltration into the lung?\n⚫ Response: Thanks for the reviewer’s kind reminder. The cellular source of\nTIMP1 and MMP-9 has been extensively documented by other researchers.\nFibroblasts are recognized as one of the cellular sources of MMP-9 (PMID:\n36352225), while activated fibroblasts are identified as the primary source of\nTIMP-1 (PMID: 30243515). We have added the references as ref.12,25 in the\nrevised manuscript.\n⚫ To investigate whether the Mexo-cl-aT treatment would increase the\ninflammatory response, we examined leukocyte infiltration at the lesion site and\nthe secretion of inflammatory factors in bleomycin-induced mouse lung tissues\nusing IF and ELISA assay after administering Mexo-cl-aT via inhalation. As\nshown in Fig. S18a, the fluorescence intensity of CD45+ cells (a biomarker of\nleukocytes) was significantly higher in lung sections from the PBS group\ncompared to a substantial decline observed in the Mexo-cl-aT group. In line with\nthese observations, treatment with Mexo-cl-aT resulted in a marked reduction in\nthe secretion of inflammatory cytokines, such as IL-6, TNF-α, and IL-1β,\ndemonstrating its robust capacity to inhibit leukocyte infiltration and the\nsecretion of inflammatory cytokines in vivo. We have added the Fig. S18 and\ncorresponding description (line 305-307) in the revised manuscript.\nFig. S18 Anti-inflammation effect of Mexo-cl-aT. a. Immunofluorescence staining of\nCD45+ cells in mouse lung sections. Scale bar = 200 μm. b. The levels of IL-6 (PBS\nvs. Mexo-cl-aT, P < 0.0001; N=6), TNF-α (PBS vs. Mexo-cl-aT, P < 0.0001; N=6),\nand IL-1β (PBS vs. Mexo-cl-aT, P = 0.2009; N=6) in mouse lung tissues determined\nby ELISA. Data are means ± SEM. ns, not significant; *P < 0.05; **P < 0.01; ****P <\n0.0001 (one-way ANOVA with Dunnett's multiple comparisons test).\n4. Figure 6. I am quite curious about the effects when the nano system applied to the\nmice in vivo. Though the authors provide information about the anti-fibrotic effect, I\nwill ask that what the effects that would be about the inflammation. Some related\ncytokines IL-1b, TNF-a, IL-6 and the most critical TGF-b expression should be\ndetected to fulfill its anti-fibrotic effects.\n⚫ Response: Thanks for the reviewer’s kind reminder. We assessed the levels of\ninflammatory cytokines (e.g. IL-1β, TNF-a, IL-6) and TGF-β in mouse lung\ntissues by ELISA. The results demonstrated that treatment with Mexo-cl-aT\nsignificantly reduced the secretion of inflammatory cytokines (Fig. S18).\nFurthermore, the expression of TGF-β in mouse lung tissues also showed a\nmarked decrease (Fig. S17), indicating the robust anti-fibrotic capability of\nMexo-cl-aT in vivo. We have added the data as Fig. S17 and Fig. S18 and\nFig. S17b. The levels of TGF-β1 (PBS vs. Mexo-cl-aT, P = 0.0076; N=6) and TIMP-1\n(PBS vs. Mexo-cl-aT, P < 0.0001; N=6) in mouse lung tissues determined by ELISA.\nData are means ± SEM. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001;\n****P < 0.0001 (one-way ANOVA with Dunnett's multiple comparisons test).\nFig. S18b. The levels of IL-6 (PBS vs. Mexo-cl-aT, P < 0.0001; N=6), TNF-α (PBS\nvs. Mexo-cl-aT, P < 0.0001; N=6), and IL-1β (PBS vs. Mexo-cl-aT, P = 0.2009; N=6)\nin mouse lung tissues determined by ELISA. N=6. Data are means ± SEM. ns, not\nsignificant; *P < 0.05; **P < 0.01; ****P < 0.0001 (one-way ANOVA with Dunnett's\nmultiple comparisons test).\n5. Figure 6. As for the Masson’s and H&E staining, authors are expected to provide\nfull lung lobe images with zoomed area for the readers better to evaluate the\nanti-fibrotic effects. The fibrosis in the lung were very heterogeneity. It would be\nbetter for the readers to evaluated the anti-fibrosis effects. Besides, IF staining to\na-SMA and COL-1 were bad quality, confocal images would be better if the author\ncould do that.\n⚫ Response: Thank the reviewer for the good suggestion. We have added full lung\nlobe images with zoomed area in Fig. S14a and Fig. 6c, and provided over-300\nppi IF images of a-SMA and COL-1 IF staining in Fig. 6e of the revised\nmanuscript.\nFig. S14a H&E staining of mouse lungs with different treatments. Scale bar = 200\nμm.\nFig. 6c. Masson staining of mouse lungs with different treatments. Scale bar = 200\nμm.\nFig. 6e. IF staining of α-SMA and COL1A1 in mouse lung sections. Scale bar = 200\nμm.\n6. Figure 6. The levels of TIMP1 and MMP9 in the BALF were not that convincing,\nsince majority of the cytokines were in the lung interstitial. Also, there were huge\nvariation when BAL was prepared. Western blot detecting the two important proteins\nwith the lung tissue are needed to better illuminate the protein levels in the lung. The\nresults would better illuminate the balance between the two proteins.\n⚫ Response: We thank the reviewer for the critical suggestions. To ensure the\ncredibility of the data, we had recalibrated the protein concentrations of the\nsamples by BCA assay before detecting the levels of TIMP-1 and MMP-9 in the\nBALF. The ELISA assay revealed a substantial reduction in TIMP-1 levels in the\nMexo-cl-aT group, decreasing by 90.3% compared to the PBS group, and\napproaching the levels observed in the healthy control group (Fig. 6g).\nCorrespondingly, Mexo-cl-aT treatment led to the most significant increase in\nMMP-9 activity across all treatment groups (Fig. 6h).\nFig. 6g-h. The level of TIMP-1 (g) and active MMP-9 (h) in mouse BALF (PBS\nvs. Mexo-cl-aT, P < 0.0001, N=4).\n⚫ Response: To elucidate the expression changes of various MMPs and provide a\nmore comprehensive understanding of the MMP-TIMP balance, we performed\ntranscriptomic analysis. The results revealed a dominance of TIMPs in the PBS\ngroup. Treatment with Mexo-cl-aT significantly upregulated the expression of\nmultiple MMPs, including MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9,\nand MMP-12. Moreover, the upregulation of MMPs surpassed that of TIMPs,\nindicating a shift towards extracellular matrix degradation. Additionally, there\nwas a decrease in the expression of genes related to oxidative stress, suggesting\neffective alleviation of lung tissue damage. We have added the data as Fig. S19\nand corresponding description (line 317-320 and line 324-327) in the revised\nmanuscript.\nFig. S19 Transcriptome analysis in the lung of mice with bleomycin-induced\npulmonary fibrosis. a. Heatmap of MMP- and TIMP-related genes. b. Heatmap of\noxidative stress-related genes. N=3. M-c-a: Mexo-cl-aT.\n7. BLM induced pulmonary fibrosis would dissolve within 3-5 weeks after BML\ntreated. If the author could provide more information about the Nano system against\nCrystalline silica induced fibrosis would be more attracting. Since CS particle would\ninduced persist and progressive pulmonary fibrosis.\n⚫ Response: Thank the reviewers for their constructive comments. We further\ninvestigated the antifibrotic potential of Mexo-cl-aT in a mouse model of\ncrystalline silica-induced fibrosis. However, the results indicated that its efficacy\nwas limited, as evidenced by the persistence of large, fused fibrotic nodules in\nMexo-cl-aT group (Fig. S22). This phenomenon may be attributed to the\nchallenges in metabolizing CS particles in vivo, which could lead to sustained\nand progressive pulmonary fibrosis.\nFig. S22 Anti-fibrosis effect of Mexo-cl-aT in a silica-induced pulmonary fibrosis\nmouse model. a. Illustration of animal experiment procedure. b. Representative\nimage, HE and masson staining of the lung sections of mice treated with different\nformulations. Arrows indicate fibrotic lesions. Scale bar = 200 μm.\n8. How many mice were used in the experiment of each group. HE Masson staining\nwould be evaluated with semi quantification.\n⚫ Response: We are sorry for the confusion. Six mice were included in the\nexperiment of each group. Ashcroft score is a semi-quantitative method used to\nassess the degree of pulmonary fibrosis (PMID: 18476815), based on HE and\nMasson staining. The results are presented in Fig. 6d. The ashcroft score of\nhealthy lungs is within the range of 0-1. The score of bleomycin-challenged mice\nsignificantly increased, averaging 4.5. Different treatment groups showed varying\ndegrees of alleviation of fibrosis. Among them, the Mexo-cl-aT group had the\nbest effect, with a score of approximately 1.2.\nFig. 6d. Ashcroft score of mouse lungs after different treatments (PBS vs. Mexo-cl-aT,\nP < 0.0001; N=6). Data are means ± SEM; ns, not significant; *P < 0.05; **P < 0.01;\n***P < 0.001; ****P < 0.0001 (one-way ANOVA with Dunnett's multiple comparisons test).\n9. The anti-fibrotic effects would rely more on the anti-ROS system or the\nanti-TIMP-1 effects?\n⚫ Response: Thank the reviewers for their constructive comments. In fact, the\nsynergistic interplay between anti-ROS and anti-TIMP-1 mechanisms underpins\nthe efficacy of our anti-fibrosis therapy. Specifically, aT exhibits a targeted effect\non restoring the functional capacity of MMPs for collagen degradation, while the\nanti-ROS system plays a fundamental role in improving the microenvironment\nfor tissue repair. Our findings revealed a substantial reduction in TIMP-1 levels\nin the Mexo-cl-aT group, decreasing by 90.3% compared to the PBS group, and\napproaching the levels observed in the healthy control group (Fig. 6g).\nCorrespondingly, Mexo-cl-aT treatment led to the most significant increase in\nMMP-9 activity across all treatment groups (Fig. 6h). Furthermore, flow\ncytometry analysis demonstrated elevated ROS levels in fibrotic lungs, which\nwere markedly reduced by an average of 76.8% following Mexo-cl-aT treatment\n(Fig. 6i).\nFig. 6 Anti-fibrotic effects of Mexo-cl-aT in vivo. g-h. The level of TIMP-1 (g) and\nactive MMP-9 (h) in mouse BALF (PBS vs. Mexo-cl-aT, P < 0.0001, N=4). i. Flow\ncytometry analysis of the ROS levels in mouse lung tissues (PBS vs. Mexo-cl-aT, P <\n0.0001, N=5). Data are means ± SEM; ns, not significant; *P < 0.05; **P < 0.01; ***P\n< 0.001; ****P < 0.0001 (one-way ANOVA with Dunnett's multiple comparisons test).\nReviewer #5 (Remarks to the Author):\nOverall:\nThis manuscript describes a study deploying umbilical cord-derived mesenchymal\nexosomes decorated with ROS-cleavable antibodies to TIMP-1 and delivered\nintratracheally as a potential therapeutic nanosystem for idiopathic pulmonary fibrosis.\nWhile many aspects of the study are intriguing, the exact cellular and molecular\ntargets of this complex nanosystem are poorly defined, and the data as presented do\nnot clearly reveal therapeutic targets and mechanism of action. The use of a single\nmodel which does not recapitulate many aspects of the human disease limits the broad\napplicability of the findings.\n⚫ Response: We thank the reviewer for the positive comments and considering our\nwork “intriguing”.\nMajor Points:\n- The rationale for the focus on TIMP-1 and MMP9 specifically is poorly defined.\nAdditional discussion is needed about which MMPs are implicated in IPF\npathogenesis and why TIMP-1 was chosen (ie does it have higher affinity binding for\nthese specific MMPs?\n⚫ Response: Much thanks to the reviewers for the in-depth review of our\nmanuscript. Previous studies and our study have shown that TIMP-1 is one of the\nkey factors leading to collagen deposition in pulmonary fibrosis. Relative\nresearch (PMID: 28585723) demonstrated that TIMP-1 inhibit broad-spectrum\nMMPs (MMP-1, -2, -3, -7, -9, etc.), among which MMP-9 is most efficiently\ninhibited, K value < 50 pM. Therefore, our study focuses on the MMP-9/TIMP-1\ni\nrebalance. We have added the references as ref.13 in the revised manuscript.\n- Figure 1 - TIMP staining – Figures are low resolution and lack use of additional cell\nmarkers (Krt5, aSMA etc) to determine location of TIMP expression. In human IPF\nimages shown the staining is rather diffuse. Is it intracellular or extracellular?\nTheir BAL ELISA would suggest extracellular, but their staining shows it in tissue - is it\nbeing produced / secreted by specific cell types in the IPF lung? Can the authors use\nmouse lung tissue from their bleomycin experiment to look for TIMP levels via\nELISA or Western blot?\n⚫ Response: Thanks for the reviewer’s kind reminder. The cellular source of\nTIMP1 has been extensively documented by other researchers, and the activated\nfibroblasts are identified as the primary source of TIMP-1 (PMID: 30243515).\nWe have added the references as ref.12 in the revised manuscript.\n⚫ Since TIMP-1 (https://www.uniprot.org/uniprotkb/P12032/entry) are secreted\nproteins that reside in the extracellular matrix, where aT can effectively exert its\nfunction. Specifically, aT neutralize free TIMP-1, leading to increased activity of\nMMP-9. For further confirmation, we examined performed IF staining for\nα-SMA and TIMP-1 in bleomycin-induced mouse lung tissues after administering\nMexo-cl-aT via inhalation. As shown in Fig. S1, the fluorescence signals for\nα-SMA (red) did not colocalize with those for TIMP-1 (green), suggesting an\nextracellular localization of TIMP-1. Additionally, we assessed the levels of\nTIMP-1 in mouse lung tissues by ELISA. The results showed that TIMP-1 levels\nin the lung tissues of mice were approximately twice as high as those in the\nnormal control group at day 10 post-bleomycin injury (Fig. S2b). We have added\nthe Fig. S1, Fig. S2b and corresponding description (line 95-96 and line 102-105)\nin the revised manuscript.\nFig. S1 Immunofluorescence staining of α-SMA and TIMP-1 in human lung sections.\nScale bar = 100 μm.\no\nntr\nol\nm\ny\nci\nn\nC o\ne\nBl\nFig. S2b The level of TIMP-1 in mouse lung tissues (P = 0.0154, N=3). Control,\nhealthy mouse; Bleomycin, bleomycin-induced pulmonary fibrosis mouse model.\nData are means ± SEM; ns, not significant; *P < 0.05 (one-way ANOVA with\nDunnett's multiple comparisons test).\n- Figure 1g/h – the method used to stain for “ROS” is not specified in the methods\nsection. What was used? Very few reagents will react with all forms of ROS and are\nnot stoichiometric. DCFH-DA was used later in the paper and may have been used\nhere? If so, DCFH-DA is not a reliable measure of all ROS and has several limitations\n(e.g., PMID: 22027063). Additionally, staining fixed tissue sections for ROS is not\nideal – reactive species are produced and reduced dynamically and fixation will not\npreserve these species. The authors should state these limitations clearly and use a\ncomplementary assay to assess redox dysregulation in these samples, such as the\nformation of oxidative byproducts or modifications (see PMID: 29988126). It is also\nunclear if human lung tissue was formalin fixed or frozen, as well as any demographic\ninformation or source of IPF and healthy control lung tissues - these should also be\nspecified in the methods.\n⚫ Response: Much thanks to the reviewers for the in-depth review of our\nmanuscript. DCFH-DA is a widely used ROS probe in many studies (e.g. PMID:\n32457361; PMID: 37933888). According to the reviewer’s suggestion, we have a\nfurther understanding of the limitations of DCFH-DA in ROS detection. We\nrepeated the relative experiments with a more reasonable ROS probe, Amplex\nRed. The results showed that Mexo-cl-aT significantly reduced H O in the\n2 2\nsupernatant of bleomycin-treated MLFs. We have added the Fig. S11,\nexperimental procedure in the supplementary information, and corresponding\ndescription (line 222-224) in the revised manuscript.\nFig. S11 Quantitative analysis of the H O levels in the supernatant of\n2 2\nbleomycin-treated MLFs (PBS vs. Mexo-cl-aT, P < 0.0001; N=3). Data are means ±\nSEM; ns, not significant; ****P < 0.0001 (one-way ANOVA with Dunnett's multiple\ncomparisons test).\n⚫ Given that the reactive species are produced and reduced dynamically and lung\ntissue fixation may not preserve these species. we performed transcriptomic\nanalysis to further assess redox dysregulation. The results revealed a dominance\nof oxidative stress in the PBS group. Treatment with Mexo-cl-aT significantly\ndecreased the expression of multiple genes related to oxidative stress (e.g. GST,\nSOD, GPx), suggesting effective alleviation of lung tissue damage. We have\nadded the data as Fig. S19b and corresponding description (line 325-327) in the\nrevised manuscript.\n⚫ Fig. S19 Transcriptome analysis in the lung of mice with bleomycin-induced\npulmonary fibrosis. b. Heatmap of oxidative stress-related genes. N=3. M-c-a: Mexo-cl-aT.\n⚫ Human lung tissue was formalin fixed. Demographic information of IPF and\nnon-IPF lung tissues has been added as Table S1 in the revised manuscript.\nTable. S1 Demographic information of non-IPF and IPF lung tissues.\n- Rationale for choice of mesenchymal exosomes (e.g., vs. an engineered nanoparticle)\ncould be better developed. Is the goal to protect the antibody, or to only release it\nwhen ROS levels are high? Or is exosome delivery meant to reach intracellular space?\nAlso, what is the rationale for sourcing exosomes from umbilical cord mesenchymal\nstem cells, and what additional molecular do these exosome preparations contain?\n⚫ Response: Thanks for the reviewer’s kind reminder. MSC-derived exosomes\n(Mexo) are ideal nanocarriers for lung-targeted drug delivery via inhalation,\ngiven their optimal size (30–150 nm), biocompatibility, low immunogenicity, and\ninherent tropism for injured or inflamed tissues. Furthermore, they can transfer\nbioactive molecules (miRNAs, proteins, lipids) to recipient cells, exerting diverse\ntherapeutic effects such as enhancing angiogenesis, reducing fibrosis, and\nstimulating cell proliferation and differentiation. Additionally, Mexo exhibit\npotent immunomodulatory properties by suppressing pro-inflammatory cytokines\nand promoting anti-inflammatory responses, emerging as a promising cell-free\ntherapy for regenerative medicine, inflammation-related diseases, and organ\ninjury (PMID: 32855385; PMID: 32939235). In our manuscript, we also\ndemonstrate that Mexo serves not only as a delivery system but also as an active\ntherapeutic ingredient, inhibiting fibroblast activation (Fig. 3a) and repairing the\nepithelium (Figs. 4d-g, S9) and endothelium (Fig. S10) in the treatment of\npulmonary fibrosis. In the introduction of the revised manuscript (line 67-72), we\nhave provided a comprehensive explanation for the selection of Mexo.\n⚫ Response: Exosomes from human umbilical cord mesenchymal stem cells\n(hUC-MSCs) are superior to those from other sources. First, they are extracted\nfrom the placenta, generally a discarded tissue, enabling an easy, non-invasive\nacquisition process with minimal ethical concerns (PMID: 26679929). Second,\nhUC-MSCs, originating from neonatal tissue, demonstrate a significantly faster\nproliferation rate than bone marrow mesenchymal stem cells (BM-MSCs) or\nadipose mesenchymal stem cells (AD-MSCs). The proliferation capacity of\nBM-MSCs diminishes with the age of the donor, while AD-MSCs are vulnerable\nto influences such as obesity (PMID: 24428376). Finally, research has indicated\nthat exosomes from hUC-MSCs possess enhanced therapeutic efficacy for lung\ninjury treatment compared to exosomes from other sources (PMID: 39468662).\n- Figure 3 – The authors treat MLF stimulated with TGFB with the exosome/TIMP\nantibody, but do not confirm that TIMP and/or MMP activity/levels were affected by\nTGFB treatment in these fibroblasts. Staining for TIMP/MMP or an activity assay in\nthe TGFB treated fibroblasts would be helpful here. Also, in Fig3a, there is no\ninformation on number of fields of view assessed or replicates, or any quantification\nfor these samples. It is hard to get an idea of the scale of the change from isolated\nimages. These are cells in culture, so it should be possible to also perform western\nblotting and/or ELISAs of the targets investigated in this panel (aSMA, col1a1, and\nadd TIMP)?\n⚫ Response: Thanks for the reviewer’s good suggestion. We determined the\nactivity of MMP-9 and TIMP-1 in TGF-β1-treated MLFs by ELISA. As shown in\nthe Fig. 4b-c, the level of MMP-9 increased by two times, and the level of\nTIMP-1 increased by three times in TGF-β1-activated MLFs compared to the\ncontrol.\nFig. 4b-c The levels of TIMP-1 (PBS vs. Mexo-cl-aT, P < 0.0001; N=3) and active\nMMP-9 (PBS vs. Mexo-cl-aT, P = 0.0077; N=3) in MLF culture supernatants.\n⚫ In Fig.3a, we selected three or more fields of view for each sample.\nRepresentative images are shown in Fig.3a. We also conducted quantitative\nexperiments by flow cytometry (Fig.3b-c), which is more objective and\npersuasive than confocal microscopy.\nFig. 3 Anti-fibrotic effects of Mexo-cl-aT in vitro. a. IF staining of α-SMA and\nCOL1A1 in MLFs. b-c. Flow cytometry analysis and quantification of α-SMA+ (PBS\nvs. Mexo-cl-aT, P < 0.0001) (b) and COL1A1+ cells (PBS vs. Mexo-cl-aT, P < 0.0001)\n(c) in MLFs (N=3). Data are means ± SEM; ns, not significant; *P < 0.05; ***P <\n0.001; ****P < 0.0001 (one-way ANOVA with Dunnett's multiple comparisons test).\n- Figure 3 – when MLF are treated with the exosome alone, there is significant\nreduction of aSMA and it looks like collagen (although it is not clear if this\ncomparison was not significant or just not reported). What is the proposed explanation\nfor this? It looks like the exosome has equal effect to the antibody alone? In the\nscheme in Figure 1 it shows the proposed effect of Mexo alone acting on vascular\nendothelial cells and epithelial cells, but this point is not really discussed in the\nintroduction or results.\n⚫ Response: Thanks for the reviewer’s kind reminder. Numerous studies,\nincluding those with PMID: 33845892 and PMID: 34344478, have documented that\nMSC-derived exosomes can inhibit fibroblast activation, thereby reducing\nα-SMA levels. Our findings align with these reports, as evidenced by the\ndownregulation of α-SMA following Mexo treatment, as shown in Fig. 3a-b.\nMoreover, aT can neutralize free TIMP-1, restoring the functional capacity of\nMMPs for collagen degradation. Therefore, aT treatment also lead to the\ndownregulation of α-SMA (Fig. 3a-b).\nFig. 3 Anti-fibrotic effects of Mexo-cl-aT in vitro. a. IF staining of α-SMA and\nCOL1A1 in MLFs. b-c. Flow cytometry analysis and quantification of α-SMA+ (PBS\nvs. Mexo-cl-aT, P < 0.0001) (b) and COL1A1+ cells (PBS vs. Mexo-cl-aT, P < 0.0001)\n(c) in MLFs (N=3). Data are means ± SEM; ns, not significant; *P < 0.05; ***P <\n0.001; ****P < 0.0001 (one-way ANOVA with Dunnett's multiple comparisons test).\n⚫ Mesenchymal stem cell-derived exosomes (Mexo) serve as innate nano-carriers,\nadept at targeting inflammation/injury sites and augmenting drug retention at the\nlesion site while maintaining a superior biosafety profile. Furthermore, Mexo\nexhibits a spectrum of therapeutic properties, including anti-inflammatory and\nanti-fibrotic activities, as well as facilitation of tissue repair and regeneration. In\nthe introduction of the revised manuscript (line 67-72), we have provided a\ncomprehensive explanation for the selection of Mexo.\n- Figure 4 in general – it seems like the Mexo-fl-aT and the Mexo-cl-aT both have the\nsame effect on viability, apoptosis, and wound healing, with the only difference being\nthat Mexo-fl-aT is not able to increase MMP9 activity. Would this indicate that these\nphenotypic effects on wound healing, apoptosis, cell viability, are not mediated\nthrough MMP9 activity? For some of the endpoints, like wound healing, it looks like\nthe exosome alone increases the response.\n⚫ Response: We are grateful for the reviewer’s insightful feedback. Indeed, Mexo\nis the primary mediator of anti-apoptotic and tissue repair effects. Consequently,\nboth Mexo-fl-aT and Mexo-cl-aT exhibit identical impacts on cell viability,\napoptosis, and wound healing. The primary function of MMP-9 activity is to\nfacilitate collagen degradation, whereas its contribution to anti-apoptosis and\ntissue repair is relatively limited. Therefore, for some of the endpoints, it is the\nexosome play the main role in wound healing.\n- Figure 4f - Again, DCFH-DA is not a reliable probe for ROS generation. Something\nlike Amplex Red might be a better assay here, which also focuses on extracellular\nH2O2 levels, and would better reflect the extracellular oxidative environment in\nwhich the Mexo-cl-aT would be cleaved.\n⚫ Response: According to the reviewer’s suggestion, we repeated the relative\nexperiments with a more reasonable assay, Amplex Red. The results showed that\nMexo-cl-aT significantly reduced H O in the supernatant of bleomycin-treated\n2 2\nMLFs. We have added the Fig. S11, experimental procedure, and corresponding\ndescription (line 222-224) in the revised manuscript.\nFig. S11 Quantitative analysis of the H O levels in the supernatant of\n2 2\nbleomycin-treated MLFs. PBS vs. Mexo-cl-aT, P < 0.0001; N=3. Data are means ±\nSEM; ns, not significant; ****P < 0.0001 (one-way ANOVA with Dunnett's multiple\ncomparisons test).\n- Figure 6g – TIMP1 levels decrease in response to these treatments, but even the\nMexo alone condition causes this. What is the proposed mechanism for this? Does\nbinding of TIMP-1 with the aT antibody cause it to be undetected by the ELISA?\nAlso, this is in BAL- do tissue levels of TIMP-1 decrease as well? Can ELISA/staining/WB\nbe performed on these?\n- Can the authors demonstrate that when aT is released from Mexo-cl-aT, that the\nantibody then binds TIMP? Can this complex be identified and measured, ie via\nimmunoprecipitation from in vivo samples? This would also help determine\nnon-specific binding of aT to other targets (other TIMPs?) occurs in vivo.\n⚫ Response: We thank the reviewer for drawing this point to our attention. Several\nstudies reported that exosomes derived from stem cells could inhibit fibroblast\nactivation (PMID: 33845892; PMID: 34344478), which may lead to decreased\nTIMP-1 secretion. Therefore, we can reasonably speculate that this explains why\nMexo alone can reduce TIMP-1 levels.\n⚫ The molecular weight of TIMP-1 is about 23 kDa, and that of aT is about 150\nkDa. When TIMP-1 is combined with aT, it has almost no excess binding site for\nanother anti-TIMP-1 antibody, thereby undetected by the ELISA.\n⚫ We further quantified the levels of TIMP-1 in mouse lung tissues by ELISA. As\nshown in the Fig. S17b, the level of TIMP-1 in mouse lung tissues increased by\n1.5 times following bleomycin inducement. Mexo-cl-aT treatment led to a\nsignificant decrease in TIMP-1 levels. We have added the Fig. S17b and\ncorresponding description (line 304-305) in the revised manuscript.\nFig. S17b The levels of TIMP-1 in mouse lung tissues determined by ELISA. PBS vs.\nMexo-cl-aT, P < 0.0001; N=6. Data are means ± SEM. ns, not significant; **P < 0.01;\n***P < 0.001; ****P < 0.0001 (one-way ANOVA with Dunnett's multiple comparisons test).\n⚫ We appreciate your insightful question. However, due to the limitations of our\ncurrent experimental setup, we are unable to directly demonstrate the binding of\nthe antibody to TIMP upon the release of aT from Mexo-cl-aT. In the\nco-immunoprecipitation experiment, the magnetic beads coated with protein A/G\nnon-discriminately captured both aT and mouse immunoglobulins, leading to\nimpure co-precipitated samples and significant interference in the detection of the\nTIMP-1-aT complex.\n- The discussion is not very detailed and the first two paragraphs merely summarize\nthe results of the study, while only the last one discusses broader implications. What\nare the limitations of this study?\n⚫ Response: Thanks for the reviewer’s kind reminder. Our research highlights the\nsignificant potential of Mexo-cl-aT as a novel treatment for IPF. We also\nrecognize several challenges that remain for the clinical application of\nMexo-cl-aT. These challenges encompass the limited large-scale production and\nhigh variability of exosomes—persistent issues in bio-optimization,\nindustrialization, and standardization. Additionally, the antifibrotic potential of\nMexo-cl-aT in a mouse model of crystalline silica (CS)-induced fibrosis was\nlimited, with large, fused fibrotic nodules still persisting in the Mexo-cl-aT\ngroup (Fig. S22). This limitation is likely due to the difficulties in metabolizing CS\nparticles in vivo, potentially leading to sustained and progressive pulmonary\nfibrosis. We have added the corresponding description (line 401-403) in the\nrevised manuscript.\nFig. S22 Anti-fibrosis effect of Mexo-cl-aT in a silica-induced pulmonary fibrosis\nmouse model. a. Illustration of animal experiment procedure. b. Representative\nimage, HE and masson staining of the lung sections of mice treated with different\nformulations. Arrows indicate fibrotic lesions. Scale bar = 200 μm.\n- Overall, the main goal of the treatment is unclear – is the Mexo-cl-aT meant to\ndeliver the anti-TIMP antibody and scavenge ROS through cleavage of the linker? Or\nis the Mexo component serving some value in wound repair? I do not think this\ndistinction is well described in the introduction/results sections. In line 341 – 351 of\nthe discussion this is mentioned, although the statement “Mexo have a natural ability\nto home to injury sites” is not cited. A more thorough introduction about why Mexo\nwas chosen and what is known about exosomes ability to promote wound repair is\nneeded in the introduction.\n⚫ Response: We are sorry for the confusion. In the revised manuscript (line 67-72),\nwe have provided a comprehensive introduction explaining the rationale behind\nthe selection of Mexo and the current understanding of exosomes’ capacity to\nfacilitate wound repair. The reasons for our choice of exosomes are outlined as\nfollows: Firstly, exosomes serve as natural nano-carriers. Secondly, they possess\nthe ability to target inflammation/injury sites and enhance the retention time of\ndrugs at the lesion. Thirdly, MSC-derived exosomes exhibit therapeutic\nproperties, including anti-inflammatory and anti-fibrotic activities. Lastly,\nexosomes offer superior biosafety due to their low immunogenicity.\nMinor Points:\n- Citations appear to be after the period for each sentence.\n⚫ Response: We are sorry for this mistake. We replaced citations after the period\nfor each sentence in the revised manuscript.\n- Methods – “Determination of ROS in lung tissues” and “Determination of TIMP-1\nin BALF” have the same description, which is for the TIMP measurement. ROS\nmethod section should be updated.\n⚫ Response: We are sorry for this mistake. We have updated the methods (line\n641-645) in the revised manuscript.\n- Figure 2h – How was H2O2 quantified in these samples? This is not stated in the\nmethods section.\n⚫ Response: We are sorry for the neglect, and have added related content in the\nmethods section of the revised manuscript. H O was quantified by H O assay\n2 2 2 2\nkit according to the manufacturer’s instructions. Briefly, H O reacts with\n2 2\ntitanium sulfate to form a yellow titanium peroxide complex, which has\ncharacteristic absorption at 415 nm. Then, the absorbance at 415 nm of samples\nwas measured using a microplate reader. We have added the methods (line\n506-511) in the revised manuscript.\n- The MMP9 activity assay in Figure 4B makes more sense to be included in Figure 3\n– it is from the fibroblast experiment and involves the same treatments. The y-axis of\nthis figure is confusing – is this activity or abundance (the ng/mL suggests abundance).\nThis is also an issue in Figure 6C.\n⚫ Response: Thanks for the reviewer’s good suggestion. Our research methodology\nis outlined as follows: Figure 3 illustrates the overall anti-fibrotic effect of\nMexo-cl-aT, whereas Figure 4 provides a detailed explanation of the functions of\neach component—aT activates MMP, Mexo facilitates tissue repair, and the\nphenylboronic acid ester bond is responsible for scavenging excess H O .\n2 2\n⚫ The y-axis of Figure 6C (now Figure 6h) represents “activity of MMP-9”. This\nis because only active MMP-9 can be detected by ELISA, whereas MMP-9\ninhibited by TIMP-1 remains undetectable. Accordingly, we revised the sentence\n“The level of MMP-9 in mouse BALF” to “The level of active MMP-9 in mouse\nBALF” in the revised manuscript (line 237, 349).\n- Figure 4c – it is unclear what the treatment is. I’m assuming it is the bleomycin\ntreatment, but no label is provided on the figure and no information on dose is given\nin the legend. The significance notation here is also unclear – is everything significant,\nor is it referring to a specific comparison?\n⚫ Response: We are sorry for this mistake. We have added the related information\nin Fig.4f legend in the revised manuscript. The significance notation here refers\nto a specific comparison (PBS vs. Mexo-cl-aT).\nFig. 4f Relative cell viability of bleomycin-challenged A549 cells after various\ntreatments for 24 (P = 0.0014), 48 (P = 0.0001), and 72 (P = 0.0002) hours (N=3).\n- Figure 4d – I think having the graph shown in Figure S7a would be helpful here to\ncompare the apoptotic cells in the same format as other assays\n⚫ Response: Thanks for the reviewer’s good suggestion. We have added the\noriginal Fig. S7a as new Fig. 4e to compare the apoptotic cells in the same\nformat as other assays. we also added the corresponding description (line\n205-206) in the revised manuscript.\n- Mouse model is referred to as “IPF” – these mice don’t have IPF, they’ve been\ninjured with bleomycin\n⚫ Response: We are sorry for this mistake. We have revised it to “a\nbleomycin-induced mouse model” in the revised manuscript.\nResponses to the comments from the reviewers (Note: All the responses here are in blue.\nAll the changes have been highlighted in red in the revised manuscript).\nReviewer #1 (Remarks to the Author):\nEven though the author added RNA sequencing to evaluate the therapeutic effect of\nMexo-cl-aT on pulmonary fibrosis, this only reflects changes at the genetic level, and\nthe alterations in fibrosis marker proteins remain unknown. Additionally, could you\nprovide the uncropped original membrane for Fig. 2A?\n Response: We are grateful for the reviewers' suggestions. As this study focused on\nTIMP-1, the balance of TIMP-1/MMP-9 was mainly evaluated. The other MMPs,\nTIMPs and redox-related proteins have not been explored in depth. We will\nimprove this part in future studies. In Fig.6, the results showed that TIMP-1 levels\nin mouse fibrotic lungs (PBS group) were significantly greater than those in\nhealthy controls. Although free aT or Mexo reduced TIMP-1 levels and increased\nMMP-9 activity to some extent, this effect was enhanced when the two were\ncombined (Mexo+aT, Mexo-fl-aT, and Mexo-cl-aT groups). As expected, TIMP-1\nlevels in the Mexo-cl-aT group decreased by 90.3% compared to those in the PBS\ngroup, approaching levels seen in the healthy control group (Fig. 6g). Accordingly,\nMexo-cl-aT treatment resulted in the greatest increase in MMP-9 activity among\nall treatment groups (Fig. 6h).\nFig. 6 Anti-fibrotic effects of Mexo-cl-aT in vivo. a. Illustration of animal experiment procedure.\nb-d. Representative images (b), Masson staining (c), and Ashcroft score (d) of mouse lungs after\ndifferent treatments (PBS vs. Mexo-cl-aT, P < 0.0001; N=6). e. IF staining of α-SMA and COL1A1\nin mouse lung sections. Scale bar = 200 μm. f. Hydroxyproline content in mouse lung tissues (PBS\nvs. Mexo-cl-aT, P < 0.0001, N=6). g-h. The level of TIMP-1 (g) and active MMP-9 (h) in mouse\nBALF (PBS vs. Mexo-cl-aT, P < 0.0001, N=4). i. Flow cytometry analysis of the ROS levels in\nmouse lung tissues (PBS vs. Mexo-cl-aT, P < 0.0001, N=5). j. TUNEL staining of mouse lung\nsections. Data are means ± SEM; ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P <\n0.0001 (one-way ANOVA with Dunnett's multiple comparisons test).\n The original membrane for Fig. 2a is as follows. The data was provided in a single\nExcel file.\nFig. 2a Original membrane of western blotting analysis.\nReviewer #3 (Remarks to the Author):\nThe authors conducted substantial experiments to prove their conclusions. However, I\nstill have several major concerns.\n Response: We appreciate the reviewer’s insightful comments. In response, we\nhave conducted additional relevant experiments and implemented appropriate\nrevisions to enhance the manuscript.\n1. The first concern is about the cellular source of MMP9. Though there was report\nindicated that Fibroblasts are recognized as one of the cellular sources of MMP-9\n(PMID:36352225), it was not in the fibrotic lung micro-environment. Still, the major\ncellular source of MMP9 is leucocytes, especially Macrophages. The author claimed\nthat delivering Mexo-cl-aT would decrease CD45+ cells, as well as other pro-\ninflammatory cytokines. In that case, how the MMPs increased, what is the cellular\nsource. We suppose that the MMPs came from fibroblast, the alleviated fibrosis did not\nsupport the hypothesis that it came from fibroblasts. The cellular source of increased\nMMPs should be explored in the circumstance. Extend experimental data is needed to\nsupport authors conclusion and for self-justification.\nOverall, the decreased cytokine and CD45+ cells and fibroblast make me confused\nabout how MMP9 increased in the lung microenvironment.\n Response: We sincerely thank the reviewer for raising this important point\nregarding the cellular source of MMP-9 and the apparent paradox between its\nelevation and reduced inflammation/fibrosis. We agree this requires clarification.\n Our data suggest a temporal sequence resolves this observation. In vitro: Active\nMMP-9 was detected early (4 hours post-Mexo-cl-aT treatment), while significant\nanti-fibrotic effects (quantification of α-SMA+ and COL1A1+ cells in MLFs)\nemerged later (24 hours). In vivo: Transcriptome analysis (MMP9/TIMP1) was\nperformed at 24 hours post-intratracheal injection of Mexo-cl-aT, whereas key\nanti-fibrotic and anti-inflammatory endpoints (α-SMA/COL1A1 staining, Masson\ntrichrome, cytokine levels, CD45+ cell counts) were assessed at 8 days.\n This staged timeline indicates that MMP-9 elevation is an early, transient event,\npotentially initiating ECM remodeling. In contrast, the sustained anti-fibrotic and\nanti-inflammatory effects arise from three complementary mechanisms: (1) the\nMMPs/TIMP-1 rebalancing by aT, (2) the potent tissue repair capabilities of Mexo,\nand (3) the ROS scavenging properties of the phenylboronic acid ester bond within\nMexo-cl-aT. Therefore, the early MMP-9 increase is not contradictory to\nsubsequent fibrosis and inflammatory alleviation; rather, it may be a prerequisite\nfor beneficial ECM restructuring.\n We clarified this temporal framework in the Results section (lines 208-211) to\nemphasize that MMP-9 elevation represents an early, self-limiting event within a\nbroader reparative cascade, directly addressing the reviewer’s concern regarding\nits relationship with late-stage anti-fibrotic and anti-inflammatory outcomes.\n2. The auther believed the effects of the Mexo-cl-aT therapy depends on the anti-TIMP1\nactivaty. However, the author did not intervene the TIMP1 in BLM-treated mice. In the\nTimp1 knockout mice, whether it could alleviate BLM-indeced pulmonary fibrosis.\nIf so, as reported in the previous reports, the most important issue is that whether Mexo-\ncl-at treatment is still effective in the Timp1 knockout mice. This experiment can\nanswer whether the treatment depends on the effect of TIMP1.\n Response: We thank the reviewers for their comments. Our key finding is that\nTIMP-1 knockdown significantly reduces fibrotic effects in mouse lung fibroblasts\n(MLFs). As demonstrated in Figure S9, transfection with different siRNA\nformulations targeting TIMP-1 resulted in a significant reduction in TIMP-1\nexpression, confirming successful gene silencing. Subsequently,\nimmunofluorescence (IF) staining for the myofibroblast marker COL1A1 (Figure\nS10) revealed that TGF-β1 treatment induced intense red fluorescence, indicating\nits pro-fibrotic effect. Critically, in TIMP-1-silenced MLFs (si-TIMP1), COL1A1\nfluorescence was markedly reduced, irrespective of TGF-β1 treatment. This result\ndirectly demonstrates that TIMP-1 knockdown attenuates fibrosis in this cellular\nmodel. We regret that we were unable to obtain TIMP-1 knockout mice for the in\nvivo experiments. As an alternative approach, we focused on establishing the\ncellular mechanism using the robust siRNA knockdown model presented here. We\nhave added the data as Fig. S9-S10 and corresponding description (lines 205-208)\nin the revised manuscript.\nFigure S9. Western blotting of TIMP-1 in MLFs after knockdown with different\nsiRNAs.\nFigure S10. Immunofluorescence staining of COL1A1 in control and TIMP-1-\nsilenced MLFs, with or without TGF-β1 treatment. Scale bar = 100 μm.\n3. Why is there was significant differences in the results between the positive control\nPirfenidone and the treatment with Mexo-cl-AT? (FigS18) Why there were 8 groups in\nthe elisa experiments, but 7 groups in the Immunofluorescence staining of CD45+ cells\nin mouse lung sections.\n Response: We are grateful for the reviewer’s attention to the experimental design\ndetails. These assays were specifically requested and added during the first revision\nas independent validation experiments. Notably, all essential comparison groups\nrequired for the core analysis were consistently included in both the original and\nthe supplementary experiments. Specifically, in Figure S18 (now Figure S21), the\nadditional group represents the Pirfenidone treatment group. We incorporated\nPirfenidone, a standard antifibrotic treatment, as a positive control in this\nexperiment to evaluate its therapeutic efficacy and further strengthen the validation\nof our findings.\n4. ELISA analysis of TIMP-1 and MMP-9 would not evaluate the activity of TIMP1\nand MMP9. It can only evaluate the protein contents. Gelatin enzyme spectrum\nexperiment would demonstrate the enzyme activity.\n Response: Thank you for raising this important point regarding the assessment of\nTIMP-1 and MMP-9 activity. We appreciate your suggestion that gelatin\nzymography is the gold standard for directly evaluating MMP-9 enzymatic activity.\n Regarding our use of ELISA, we carefully consulted the manufacturer’s\ninstructions for the Mouse MMP-9 ELISA Kit (Elabscience, E-EL-M3052). These\ninstructions specify that the assay is designed to detect the active form of MMP-9.\nMMP-9 molecules inhibited by TIMP-1 (or other inhibitors) or existing in the pro-\nform are not recognized by the antibodies in this kit. Therefore, while we\nacknowledge that ELISA primarily measures protein content, the specific design\nof this kit allows it to indirectly indicate active MMP-9 levels in the sample.\n We explicitly detailed the kit’s brand and model (Elabscience, E-EL-M3052) in the\nMethods section of the revised manuscript. Additionally, we plan to incorporate\ngelatin zymography in future studies to complement these findings.\n5. In FigS1, it seems that TIMP1 did not came from a-SMA+ fibroblasts. If the authors\nstained macrophages markers F4/80, it may be more co-localization.\n Response: We are very grateful to the reviewer for this exceptional insight and\nconstructive suggestion. We have performed additional immunofluorescence co-\nstaining experiments for TIMP1 and the macrophage marker CD68 on lung\nsections from IPF patients. It is worth noting that we selected CD68 instead of\nF4/80 as the macrophage marker, since F4/80 is specifically expressed on murine\nmacrophages and is not suitable for labeling macrophages in human IPF tissues.\n As shown in Figure S1b, the new results clearly show a substantial degree of co-\nlocalization of TIMP1 with CD68+ macrophages (highlighted by white arrows),\nindicating that macrophages are one of the cellular sources of TIMP1 in this\ncontext. At the same time, we also observed distinct TIMP1 signals located outside\nof CD68-positive cells, suggesting that other cell types, in addition to macrophages,\nmay contribute to TIMP1 production during pulmonary fibrosis. These findings\nalign with existing literature reports indicating that multiple cell types—with\nmacrophages being particularly prominent—can express TIMP1 in IPF [1-3]. We\napologize for not clearly stating this point in our original manuscript and thank the\nreviewer for prompting this clarification. The new image has been incorporated\ninto the Figure. S1 in the revised supplementary materials.\nFigure S1b. Immunofluorescence imaging of TIMP1 and CD68 expression in lung\nsection from IPF patient.\nRef:\n[1] Almuntashiri, S; Alhumaid, A; Zhu, Y; et al. TIMP-1 and its potential diagnostic and\nprognostic value in pulmonary diseases. Chinese Medical Journal Pulmonary and\nCritical Care Medicine, 2023, 1, 67-76.\n[2] Selman, M; Ruiz, V; Cabrera, S; et al. TIMP-1,-2,-3, and-4 in idiopathic pulmonary\nfibrosis. A prevailing nondegradative lung microenvironment? American Journal of\nPhysiology-Lung Cellular and Molecular Physiology, 2000, 279, 562-574.\n[3] Kulshrestha, R; Pandey, A; Jaggi, A; et al. Beneficial effects of N-acetylcysteine on\nprotease-antiprotease balance in attenuating bleomycin-induced pulmonary fibrosis in\nrats. Iranian Journal of Basic Medical Sciences, 2024, 366, 1-17.\n6. The time points of Mexo-cl-aT intervention in vivo is still lack experimental evidence\nin Fig 6a. Confirmed in Fig S18, Mexo-cl-aT could exert anti-inflammatory efficacy\nduring inflammatory stage. In addition, the ROS generation could also be observed in\ninflammatory stage. Please provide experiment data about Mexo-cl-aT intervention\nincluding inflammatory stage and fibrotic stage in IPF model, which could make the\n‘10 day’ time point rational.\n Response: We appreciate the reviewer’s insightful query regarding the selection\nof Day 10 for Mexo-cl-aT intervention. Although fibrotic status of the lung tissues\nwas significantly enhanced during 7-day bleomycin induction [1-3], our data\ndemonstrate significantly higher collagen deposition and larger fibrotic areas at\nDay 10 versus Day 7, justifying this time point for modeling advanced disease\n(Figure R1).\nFigure R1. Histological assessment of bleomycin-induced pulmonary fibrosis\nprogression. Representative H&E-stained lung sections from mice at day 0, 7, and 10\npost-intratracheal bleomycin instillation. Scale bar = 200 μm.\n Our study specifically targets mid-to-late stage fibrosis—a critical unmet need in\nIPF treatment. The core innovation of Mexo-cl-aT lies in disrupting established\nfibrotic networks, and intervention at Day 10 directly addresses the therapeutic\nchallenge of reversing advanced fibrosis. This is evidenced by significant\nreduction in collagen deposition (Fig. 6c-d), downregulation of fibrotic (α-SMA,\nCOL1A1; Fig. 6e), and restoration of MMP9/TIMP1 balance (Fig. 6g-h),\nindicating effective ECM remodeling. These findings demonstrate statistically\nsignificant improvements across fibrosis endpoints, validating the robustness of\nour experimental design.\nRef:\n[1] Zhang, X., Dong, Y., Li, Wc. et al. Roxithromycin attenuates bleomycin-induced\npulmonary fibrosis by targeting senescent cells. Acta Pharmaco.l Sin. 2021, 42, 2058-\n2068.\n[2] Pan, L., Cheng, Y., Yang, W. et al. Nintedanib Ameliorates Bleomycin-Induced\nPulmonary Fibrosis, Inflammation, Apoptosis, and Oxidative Stress by Modulating\nPI3K/Akt/mTOR Pathway in Mice. Inflammation 2023, 46, 1531-1542.\n[3] Liu T, De Los Santos FG, Phan SH. The Bleomycin Model of Pulmonary Fibrosis.\nMethods Mol Biol. 2017, 1627, 27-42.\n7. According to the Graphical Abstract, the core anti-fibrosis target of Mexo-cl-aT was\nregulating the balance between TIMP1 and active MMP9. However, the co-relation of\nMexo-cl-aT and TIMP1 should be confirmed by TIMP1 encoding gene knockout mice,\nand the relationship between active MMP9 and TIMP1 also need demonstration.\nFollowing the Transcriptome analysis in Fig S19, Mexo-cl-aT treatment increased the\nexpression of massive MMP family genes, which was contradictory against TIMP1-\nMMP9 balance. Thus, the ‘Mexo-cl-aT-TIMP1-MMP9-anti-fibrosis’ pathway could\nnot be established with compelling evidence.\n Response: We sincerely thank the reviewer for this insightful critique regarding\nthe establishment of the Mexo-cl-aT–TIMP-1–MMP-9–anti-fibrosis pathway. We\nacknowledge the limitation of not using TIMP-1 knockout mice in this study and\ndeeply appreciate the suggestion to strengthen the causal relationship. To address\nthis within our experimental constraints, we employed TIMP-1-silenced mouse\nlung fibroblasts (MLFs) as an alternative model.\n Our data clearly demonstrate efficient TIMP-1 knockdown (Figure S9), which\nsignificantly reduced the expression of the key myofibroblast marker COL1A1\n(Figure S10), directly linking TIMP-1 suppression to anti-fibrotic effects.\nMoreover, TIMP-1 silencing also resulted in a significant increase in active MMP-\n9 secreted by MLFs (Figure S11). These findings provide direct evidence\nsupporting TIMP-1 as a key target for Mexo-cl-aT and its modulation of the TIMP-\n1/MMP-9 balance towards anti-fibrosis. We have added the data as Fig. S9-S11\nand corresponding description (lines 205-208) in the revised manuscript.\nFigure S9. Western blotting of TIMP-1 in MLFs after knockdown with different\nsiRNAs.\nFigure S10. Immunofluorescence staining of COL1A1 in control and TIMP-1-\nsilenced MLFs, with or without TGF-β1 treatment. Scale bar = 100 μm.\nFig. S11 Quantitative analysis of the active MMP-9 levels in cell lysates and\nsupernatant of bleomycin-treated MLFs. PBS vs. TIMP-1-silenced MLFs; N=3.\nData are means ± SEM; ns, not significant; ***P < 0.001 (one-way ANOVA with\nDunnett's multiple comparisons test).\n Regarding the transcriptome data (Fig. S19, now Fig. S22) showing upregulation\nof multiple MMP genes, previous studies (PMID: 28585723) have demonstrated\nthat TIMP-1 inhibits a broad spectrum of MMPs (including MMP-1, -2, -3, -7, -9,\netc.), with MMP-9 being the most efficiently inhibited (Ki < 50 pM). Therefore,\nour study focuses on the MMP-9/TIMP-1 rebalance. The transcriptome data\nrevealing upregulation of multiple MMP genes after Mexo-cl-aT treatment does\nnot contradict the TIMP-1/MMP-9 balance hypothesis. Instead, it highlights\nthe complexity of MMP regulation: (1) TIMP-1 broadly inhibits MMPs, it is most\npotent against MMP-9. Thus, even if other MMPs are transcriptionally upregulated,\ntheir activity may remain suppressed by TIMP-1. (2) MMP activity is determined\nby the TIMPs/MMPs ratio, not MMPs expression alone. (3) The reduction in\nfibrosis markers (e.g., COL1A1 in TIMP-1-silenced MLFs) confirms that the net\neffect of TIMP-1 knockdown is anti-fibrotic, consistent with MMPs-mediated\nECM degradation.\n8. Although the authors weakened the content about MMP9 in the revised manuscript,\nthe relationship between active MMP9 and reduced fibrosis in IPF should not be\nexplained so simple, which was mentioned in my previous Q2. Please add some test\nindex into in vivo experiment, which can better reflect upregulated collagen\ndegradation specific governed by active MMP9.\n Response: We acknowledge the reviewer’s valid concern regarding the complexity\nof active MMP9’s role in collagen degradation during IPF fibrosis resolution.\nWhile our transcriptomics data confirm broad upregulation of MMPs, we\nspecifically focused on MMP9 in ELISA analyses because TIMP-1 inhibition most\nsignificantly enhanced MMP9 activity (as indicated by preliminary mechanistic\nstudies, PMID: 28585723). This led us to propose MMP-9 as a potential key\nmediator in this context, though we fully recognize that the actual dynamics\ninvolve multifaceted interactions.\n To address your constructive suggestion for more specific indices of MMP-9-\ndriven collagen degradation, we propose to incorporate the following approaches\nin future studies: (1) Direct MMP-9 activity assays (e.g., FRET-based probes,\ngelatin zymography) to quantify enzymatic function. (2) Collagen degradation\nfragment analysis (e.g., C1M for type I collagen, ICTP for crosslinked fragments).\nThese measures would help establish causality between MMP-9 activity and\nfibrosis reduction, if feasible within our experimental framework. We are grateful\nfor your guidance in strengthening the mechanistic rigor of our work and will\ncarefully consider these additions in our revised discussion and future research\nplans.\n9. In Fig S17, the pirfenidone treatment could not effectively hamper lung fibrosis in\nlarge field view, but TGF-β and TIMP1 reduced collectively. What’s more, the level of\nIL-6 and TNF-α should be noticed in pirfenidone treated group in Fig S18.\n Response: We thank the reviewer for highlighting the importance of IL-6/TNF-α\nmodulation in pirfenidone-treated groups. Our data show that pirfenidone reduced\nTGF-β/TIMP1 but failed to ameliorate fibrosis in large-field views (Fig S17, now\nFig S20) or suppress IL-6/TNF-α (Fig S18b, now Fig S21b). This dissociation is\nconsistent with pirfenidone’s mechanism: it inhibits TGF-β-driven collagen\nsynthesis but does not degrade mature ECM due to limited MMP activation (PMID:\n35652284; PMID: 30611717), or block NF-κB-mediated IL-6/TNF-α production\n(PMID: 35332068; PMID: 27498142). These limitations explain its partial clinical\nefficacy in IPF and underscore the advantage of Mexo-cl-aT. Our data demonstrate\nthat Mexo-cl-aT surpasses pirfenidone by three complementary mechanisms: (1)\nthe MMPs/TIMP-1 rebalancing by aT, (2) the potent tissue repair capabilities of\nMexo, and (3) the ROS scavenging properties of the phenylboronic acid ester bond\nwithin Mexo-cl-aT, thereby enabling superior suppression of inflammation,\ninhibition of TGF-β/TIMP1, and MMPs/TIMPs rebalance—leading to significant\nfibrosis resolution. The pirfenidone group thus serves as a valuable control\nhighlighting the multi-targeted advantage of Mexo-cl-aT.\nReviewer #5 (Remarks to the Author):\nOverall: The authors have responded to the reviewer concerns, and have provided\nextensive additional data and figures which have provided additional context and\ngreatly improved the impact of the work.\n Response: We thank the reviewer for the positive comments.\nMajor Comments: none\nMinor Comments:\n1. Lines 67-72: Thank you for adding this helpful text. It is added in a paragraph\ndiscussing ROS, and it seems from the data that the phenylboronic ester bond, not\nthe Mexo themselves, are what is scavenging ROS in this system. Please make the\nadded text a separate paragraph.\n Response: Revised.\n2. Fig. 4.g.: it would be helpful to add reference lines for “no migration” (PBS\ncondition) to all the panels so that the comparable effects of treatments can be more\neasily seen. Also for Fig. S10c\n Response: Revised.\n3. Line 307: “In a comparative study that pirfenidone was as the positive control”;\nsuggest change to “In a comparative study in which pirfenidone was used as a\npositive control” to improve clarity.\n Response: Revised.\n4. Line 403: “of exosomes, which are persistent issues in bio-optimization”;\nsuggest change to “of exosomes-persistent issues in bio-optimization” for clarity.\n Response: Revised.\n5. Line 404: “Additionally, the antifibrotic potential”; suggest change to “Furthermore,\nthe approach may not be optimized for all forms of fibrosis. For example, the\nantifibrotic potential” for clarity.\n Response: Revised."}, {"round": 2, "reviewer_comments": {"reviewer_3": "Thanks for the authors response. I have no further questions. The manuscript could be accepted for publication.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter