Engineered exosome nanovesicles for delivery of antibodies to treat inflammatory bowel disease

Domain: neuroscience
Published: 2026-02-13
Editorial outcome: major_revision

Abstract
Inflammatory bowel disease (IBD) is characterized by chronic inflammation and impaired immune tolerance, for which current therapies provide only partial and transient relief. Here, we introduce PrEXO-a23, a biomimetic nanotherapeutic engineered by fusing regulatory T cell (Treg)-derived exosomes with platelet membrane vesicles and conjugating interleukin-23 (IL-23) antibodies via a matrix metalloproteinase (MMP)-cleavable linker. This design exploits the inherent homing ability of platelets and Tregs, enabling PrEXO-a23 to preferentially accumulate in inflamed colonic tissues in murine IBD models. At the disease site, elevated MMP activity triggers antibody release to inhibit IL-23-mediated inflammation, while exosomal cargo reprograms dendritic cells and promotes Treg expansion, thereby restoring immune tolerance. This dual-action strategy significantly alleviates IBD, prevents complications like intestinal fibrosis and colitis-associated colorectal cancer, and shows p53-dependent efficacy in carcinogenesis prevention. These findings highlight PrEXO-a23 as a promising nanotherapeutic platform for durable immune reprogramming and long-term IBD management.

Introduction
Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), is a chronic relapsing gastrointestinal disorder characterized by debilitating symptoms such as diarrhea, abdominal pain, and rectal bleeding, which often progress to life-threatening complications1. A key driver of IBD pathogenesis is sustained intestinal inflammation coupled with impaired immune tolerance, leading to a vicious cycle of immune dysregulation2,3. Although current treatments, including probiotics, small-molecule medicines, immunosuppressants, and biologics, can reduce disease symptoms, their efficacy in reversing immune dysregulation remains limited4–6. Critically, unresolved immune imbalance increases the risk of disease relapse and contributes to progressive complications such as intestinal fibrosis and colitis-associated colorectal cancer (CAC). Intestinal fibrosis is characterized by thickened intestinal walls and reduced peristalsis, eventually leading to intestinal stenosis and life-threatening obstruction. The cumulative incidence of intestinal fibrosis is reported as >50% in CD and 2–11% in UC7,8. In parallel, chronic immune dysregulation promotes somatic mutations in intestinal epithelial cells, including key tumor suppressor genes such as TP53, driving the progression to CAC. Patients with IBD have an ~2–3-fold increased risk of developing CAC compared to the general population9–11. Despite this, there are currently no FDA-approved treatments for intestinal fibrosis8, and CAC remains associated with poor clinical outcomes11. Therefore, there is an urgent need for therapeutic strategies that not only suppress inflammation but also restore immune tolerance to correct immune dysregulation, with the goal of reducing disease severity and long-term complications.

The proinflammatory cytokine interleukin-23 (IL-23) plays a central role in the pathogenesis of IBD by promoting the differentiation, expansion, and effector function of T-helper 17 (Th17) cells12. Th17 cells exacerbate IBD by secreting proinflammatory cytokines (e.g., IL-17A), which drive epithelial barrier dysfunction and chronic tissue damage, thereby perpetuating the inflammatory cascade13,14. Therapeutic antibodies targeting IL-23 specifically neutralize these cytokines, effectively disrupting the inflammatory cascade and reducing disease symptoms. Clinical studies have shown that IL-23 antibodies achieve remission rates of ~40%, substantially outperforming conventional therapies, which typically yield remission rates below 20%15,16. However, systemic administration of IL-23 antibodies may lead to off-target immunosuppression, increasing the risk of opportunistic infections17. Consequently, the development of targeted delivery systems is essential to enhance therapeutic safety and improve treatment efficacy.

Regulatory T cells (Tregs) are crucial for maintaining immune tolerance18,19, making Treg-based therapies promising candidates for IBD treatment, as supported by ongoing clinical and preclinical studies20–22. However, the direct application of Tregs is challenged by complex isolation and expansion protocols, as well as their reduced stability within the inflammatory microenvironment14,23. As an alternative, exosomes derived from Tregs have emerged as potent immunomodulatory nanovesicles. These nanoscale vesicles carry diverse functional biomolecules, including cytokines, microRNAs, and proteins, that facilitate immune regulation by targeting and reprogramming recipient immune cells24,25. Importantly, rEXO therapy bypasses the stability issues associated with cell-based Treg therapy, making it a compelling strategy for modulating immune tolerance in IBD.

The pathological features of IBD include an inflammatory environment and damaged intestinal vasculature26,27. High-affinity integrins that bind inflammation-upregulated endothelial adhesion molecules are expressed on the Treg cell surface, enabling the targeting of inflamed lesions28,29. Platelets, which possess unique surface molecules (e.g., GPIbα), can preferentially localize to damaged vasculature through robust adhesion to collagen exposed on the injured vascular endothelium30,31. The platelet-Treg hybrid engineering strategy offers an approach for enabling lesion-specific drug delivery through a dual-targeting mechanism for IBD therapy.

Inspired by these findings, we engineered a platelet-Treg hybrid nanovesicle system (PrEXO-a23) for lesion-specific delivery of anti-inflammatory and immune tolerance agents to address immune dysregulation for IBD treatment. rEXO was fused with platelet membrane vesicles (PMV) to generate hybrid nanovesicles (PrEXO), followed by the conjugation of IL-23 antibodies (a23) via a matrix metalloproteinase (MMP)-cleavable peptide linker. MMPs are overexpressed and released by activated stromal cells and immune cells infiltrating the inflamed intestinal mucosa in IBD32. The introduction of an MMP9-cleavable linker can exploit the pathological microenvironment to control drug release, thereby maximizing local therapeutic efficacy while minimizing systemic exposure and off-target immunosuppression24,33. Thus, after intravenous administration of PrEXO-a23, it selectively accumulated at sites of damaged and inflamed colonic lesions due to platelet-mediated damage adhesion and Treg-derived inflammation homing. Cleavage by MMPs promoted the release of a23 and PrEXO within MMP-enriched IBD lesions. The release of a23 neutralized the cytokine IL-23, which suppressed inflammation, while the PrEXO, enriched with immunomodulatory molecules from Tregs, reprogrammed local immune cells into regulatory phenotypes, such as tolerogenic dendritic cells (tDCs) and Tregs, promoting immune tolerance. As a result, in murine IBD models, PrEXO-a23 treatment effectively modulated colonic immune dysregulation, leading to comprehensive IBD remission, including symptom relief, intestinal barrier repair, and microbiota normalization. Furthermore, PrEXO-a23 significantly decreased the risk of complications, such as intestinal fibrosis and CAC. Notably, the action of PrEXO-a23 to prevent CAC progression is partially dependent on p53 (Fig. 1).

Results
Engineering biomimetic exosome-based nanovesicle (PrEXO-a23) for targeted delivery of the immunomodulatory IL-23 antibody

Naïve CD4+ T cells isolated from mouse spleens successfully differentiated into Tregs, as confirmed by flow cytometry analysis showing CD25 and FOXP3 expression on day 7 (Supplementary Fig. 1). Subsequently, Treg-derived exosomes (rEXO) were isolated from the cellular supernatant by ultracentrifugation, and verified by Western blotting to retain Treg immunosuppressive proteins, such as IL-10 and CTLA-4 (Supplementary Fig. 2). Platelet membrane vesicles (PMV) were prepared from whole blood using repeated freeze-thaw cycles followed by serial extrusion. rEXO and PMV were then fused through additional coextrusion to generate hybrid vesicles (PrEXO). Nanoflow cytometry analysis revealed that a 1:1 mass ratio of PMV to rEXO achieved the highest fusion efficiency (Supplementary Fig. 3), and this ratio was used for all subsequent experiments. To equip these hybrid vesicles for controlled antibody delivery, we employed an MMP-cleavable peptide linker (Arg-Val-Gly-Leu-Pro) bearing N-hydroxysuccinimide (NHS) and maleimide (Mal) termini. The NHS end reacted with primary amines on the antibody to yield linker-a23 (Supplementary Fig. 4). When the molar ratio of a23 to linker was 1:50, the conjugation efficiency of linker to a23 was the highest (Supplementary Fig. 5a). Subsequently, the surface thiols of PrEXO were activated by mild reduction and reacted with linker-a23 to form stable thioether bonds through the maleimide group to generate PrEXO-a23. Enzyme-linked immunosorbent assay (ELISA) determined the conjugation efficiency of a23 to PrEXO, revealing that a PrEXO-to-a23 mass ratio of 1:0.5 achieved the optimal conjugation efficiency (Supplementary Fig. 5b). Therefore, throughout this study, an a23-to-linker molar ratio of 1:50 and a PrEXO-to-a23 mass ratio of 1:0.5 were used to prepare the PrEXO-a23 formulations.

Transmission electron microscopy (TEM) analysis demonstrated that PrEXO-a23 maintained the typical cup-shaped morphology of exosomes (~100 nm), and immunogold labeling confirmed surface conjugation of a23 (Fig. 2a). Compared with PrEXO, PrEXO-a23 exhibited a slight increase in particle size and a reduction in zeta potential (Fig. 2b, c and Supplementary Fig. 6). Confocal laser scanning microscopy (CLSM) results revealed high colocalization of DiI-labeled rEXO, DiD-labeled PMV, and Alexa Fluor 488-labeled a23 signals in PrEXO-a23 (Fig. 2d), confirming successful construction of PrEXO-a23. Western blot analysis verified the presence of exosomal proteins (ALIX, TSG101), chemokine receptor (CCR6), platelet markers (GPIbα and CD62P), and a23 on PrEXO-a23 (Fig. 2e and Supplementary Fig. 7), indicating the retention of functional components. MMP9 expression was markedly upregulated in inflamed colonic tissues of IBD mice compared with healthy controls (Supplementary Fig. 8a, b) and it was significantly higher than that in the serum of the same mice (Supplementary Fig. 8c). This enzyme facilitated responsive release of a23 from PrEXO-a23, which was suppressed by the MMP9 inhibitor GM6001 (Fig. 2f). The released a23 preserved its IL-23 neutralization activity (Fig. 2g). Notably, PrEXO-a23 remained stable over 7 days in PBS at 4 °C (Fig. 2h) and in plasma at 37 °C (Supplementary Fig. 9), with no detectable changes in size or zeta potential, supporting its potential for in vivo application.

Anti-inflammatory and epithelial cell protective effects of PrEXO-a23

To investigate the regulatory effects of PrEXO-a23 on Th17 cell differentiation and its potential to protect intestinal epithelial cells, we established a coculture model. Naïve CD4+ T cells were seeded in the upper chamber and treated with IL-6 and IL-23 to induce Th17 polarization. Cells were then treated with PBS (Th17 group), rEXO (rEXO group), PMV (PMV group), PrEXO (PrEXO group), a23 (a23 group), or PrEXO-a23 (PrEXO-a23 group). Untreated cells served as the negative control (Control group). After 72 h of treatment, colonic epithelial NCM460 cells were seeded into the lower chamber and cocultured with the pretreated T cells for an additional 24 h to evaluate the epithelial response (Fig. 3a).

Flow cytometry analysis revealed that IL-6 and IL-23 successfully induced the differentiation of naïve T cells into Th17 cells in the Th17 group, as evidenced by an increased proportion of CD4+ IL-17A+ cells compared to the control (Fig. 3b and Supplementary Fig. 10a). Compared with the Th17 group, PMV treatment showed no inhibitory effects on Th17 cell differentiation. Treatment with the IL-23 neutralizing antibody a23 significantly reduced Th17 differentiation (P=0.0001), confirming its functional blocking activity. rEXO and PrEXO treatments showed effective inhibitory effects, while PrEXO-a23 exhibited the most pronounced suppression, reducing Th17 cell differentiation to levels comparable to untreated controls. Meanwhile, ELISA analysis of supernatants revealed elevated levels of proinflammatory cytokines IL-17A and TNF-α in the Th17 group, consistent with Th17 activation. There was no significant difference in the secretion of these inflammatory cytokines in the PMV group (Fig. 3c, d). In contrast, rEXO and PrEXO treatments reduced the levels of these cytokines, with PrEXO-a23 displaying a more pronounced reduction and bringing them to levels comparable to the control.

We next examined the effects of Th17 polarization on epithelial cell viability. The results showed that apoptosis and necrosis of NCM460 cells were significantly increased when cocultured with Th17 cells in the Th17 group (P<0.0001), indicating epithelial cell damage. Similarly, PMV alone exerted minimal protective effect against Th17-induced epithelial damage. Notably, PrEXO-a23 treatment significantly alleviated this damage, reducing both apoptosis and necrosis and demonstrating a robust epithelial protective effect (P<0.0001, Fig. 3e and Supplementary Fig. 10b, c). These findings clearly indicate that PrEXO-a23 protects intestinal epithelial cells from damage by inhibiting Th17 cell differentiation and inflammatory cytokine secretion, thereby exerting anti-inflammatory and epithelial protective effects.

PrEXO-a23 reprograms dendritic cells toward a tolerogenic phenotype and promotes the expansion of Tregs

Dendritic cells (DCs) are key regulators of immune responses, shaping T-cell differentiation through their distinct phenotypes34. Mature DCs (mDCs) drive immune effector functions, characterized by elevated expression of the costimulatory markers (CD80, CD86 and MHC II) and secretion of proinflammatory cytokines (e.g., IL-6 and TNF-α). In contrast, tolerogenic DCs (tDCs) display reduced levels of these markers and increased production of immunosuppressive cytokines, including IL-10 and TGF-β, promoting immune tolerance by facilitating Treg induction and suppressing Th17 cell differentiation35–37. Given the role of Treg-derived exosomes in maintaining immune tolerance38, we evaluated whether PrEXO-a23 could reprogram mDCs toward a tolerogenic phenotype. The immature DCs (imDCs) were stimulated with lipopolysaccharide (LPS) and IFN-γ to induce mature mDCs and subsequently treated with various formulations, including PBS (mDC group), rEXO (rEXO group), PMV (PMV group), PrEXO (PrEXO group), a23 (a23 group), or PrEXO-a23 (PrEXO-a23 group) for 24 h. Untreated immature DCs (imDCs group) served as the negative control (Fig. 3f). The expression of surface markers and cytokine profiles was analyzed using flow cytometry and ELISA, respectively.

As expected, mDCs showed increased expression of CD80, CD86, and MHC II compared to imDCs (Fig. 3g and Supplementary Fig. 11a), as well as increased production of IL-6 and TNF-α (Fig. 3h). PMV treatment did not significantly affect these markers or cytokines on mDCs. While treatment with a23 markedly downregulated these maturation markers and reduced the proinflammatory cytokine levels, indicating an anti-inflammatory effect, the levels of IL-10 and TGF-β were comparable to those in mDCs, suggesting that a23 alone was insufficient to fully reprogram DCs into a tolerogenic state. In contrast, both rEXO and PrEXO treatments resulted in downregulation of maturation markers and proinflammatory cytokines while promoting secretion of IL-10 and TGF-β, making a shift toward a tolerogenic phenotype. Notably, PrEXO-a23 exhibited the most potent suppressive effects on inflammatory markers and the strongest promotion of anti-inflammatory cytokine production, suggesting its robust role in inducing DC transformation into tDCs. This enhanced efficacy was likely due to the anti-inflammatory effects of a23, which created a favorable microenvironment for PrEXO to reprogram DCs into tDCs.

To further explore the potential of PrEXO-a23–induced tDCs in promoting Treg differentiation, we cocultured the pretreated mDCs with naïve T cells for 72 h, and T cell phenotypes were determined via flow cytometry (Fig. 3i). Flow cytometry analysis revealed that PBS-treated mDCs from the mDC group did not significantly induce Treg differentiation compared to imDCs (P=0.9998, Fig. 3j and Supplementary Fig. 11b). Likewise, PMV-treated and a23-treated mDCs failed to promote Treg differentiation. However, the proportion of Tregs (CD25+FOXP3+) among naïve T cells was significantly increased in both rEXO- and PrEXO-treated mDCs groups compared to the mDCs group. Remarkably, PrEXO-a23-induced tDCs led to the most significant increase in Treg differentiation (P<0.0001). Additionally, these tDCs effectively reduced the proportion of Th17 cells (CD4+ IL-17A+) compared to other treatments, with rEXO, PrEXO, and a23 showing moderate effects, and PMV alone showing a negligible effect (Fig. 3k and Supplementary Fig. 11c).

Collectively, while a23 monotherapy effectively inhibited Th17 differentiation and proinflammatory cytokine secretion, it showed limited capacity to promote tDC and Treg generation. PMV did not induce immune tolerance under these conditions. In contrast, both rEXO and PrEXO effectively induced Treg differentiation over Th17 cells by promoting DC polarization toward a tolerogenic phenotype, thereby supporting immune tolerance. Notably, PrEXO-a23 demonstrated a substantially greater ability to induce tDCs and expand Tregs. This enhanced efficacy stems from the environmental regulation of DC and T cell differentiation, where an inflammatory microenvironment dominated by cytokines like IL-23, drives polarization toward proinflammatory states. Sustained IL-23 signaling suppresses the expression of FOXP3, a transcription factor critical for Treg function, thereby impairing Treg differentiation and exacerbating immune tolerance loss19,39,40. By neutralizing IL-23, a23 mitigated this inhibitory milieu, enabling the intrinsic tolerogenic cargo of PrEXO to fully reprogram DCs and T cells toward a regulatory state.

PrEXO-a23 enables precise targeted delivery and MMP-responsive release of antibody to inflamed colonic lesions

During IBD, inflammatory injury to the intestinal endothelium exposes subendothelial collagen, to which platelets rapidly adhere via GPIbα-von Willebrand factor (vWF) interactions in the bloodstream41. To determine whether PrEXO-a23 retains this critical adhesive capability, we performed an in vitro blood circulation assay using collagen-coated flow channels that mimic damaged vessels. DiO-labeled formulations, including rEXO, PMV, PrEXO, or PrEXO-a23, were introduced into the flow channel under arterial flow conditions (1000 s−1), and fluorescence imaging was performed using CLSM. While rEXO displayed weak fluorescence in the flow channel, both PrEXO and PrEXO-a23 showed strong fluorescent signals (Supplementary Fig. 12a), indicating strong adherence to the collagen matrix. These results confirmed that platelet membrane hybridization conferred enhanced adhesion under high shear, prolonging retention at lesion sites, and that a23 conjugation did not impair this capability.

Next, to determine its targeting to inflammatory cells, we incubated LPS-activated RAW264.7 macrophages and neutrophils with DiI-labeled formulations. CLSM revealed that all formulations strongly bound to LPS-treated RAW264.7 cells after 2 h, whereas binding to untreated controls was minimal (Supplementary Fig. 12b). Flow cytometry result of LPS-pretreated neutrophils confirmed this trend (Supplementary Fig. 12c). This enhanced targeting ability is likely due to the increased molecular interactions between adhesion receptors overexpressed on inflamed cells and the corresponding ligands on the hybrid membranes. Additionally, in the presence of RAW264.7 cells, PrEXO-a23 efficiently released a23 upon exposure to MMP9 (Supplementary Fig. 12d).

Encouraged by these in vitro findings, we investigated the in vivo targeting performance of PrEXO-a23 in a dextran sulfate sodium (DSS)-induced murine IBD model. DiD-labeled formulations, including rEXO, PMV, rEXO+PMV (physical mixture), PrEXO or PrEXO-a23, were administered intravenously, and biodistribution was monitored via an in vivo imaging system. PrEXO and PrEXO-a23 showed strong accumulation in inflamed intestinal tissues within 2 h after injection, reached a peak at 12 h, and maintained strong retention for up to 48 h. The other formulations showed weaker and less durable intestinal targeting (Fig. 4a and Supplementary Fig. 13a). Pharmacokinetic analysis showed that the blood circulation half-life of PrEXO-a23 was ~1.44 h (Supplementary Fig. 13b). Ex vivo imaging of harvested colons and frozen tissue sections confirmed significantly higher lesion-targeting by PrEXO and PrEXO-a23 compared to controls (P<0.0001, Fig. 4b–d). PrEXO-a23 exhibited the highest colon-to-liver ratios of fluorescence intensity, further indicating its enhanced disease-site selectivity (Supplementary Fig. 13c–e). Additionally, flow cytometry of intestinal immune cells revealed efficient uptake of PrEXO-a23 by dendritic cells, macrophages, T cells, B cells, and neutrophils (Fig. 4e and Supplementary Fig. 13f). Importantly, PrEXO-a23 outperformed rEXO in lesion-targeting, suggesting that membrane fusion synergistically enhances adhesion to damaged vessels (via PMV) and targeting of inflamed cells (via rEXO). Thus, PrEXO-a23 demonstrates dual-targeting capability, efficiently homing to both damaged vasculature and inflamed colonic tissue, offering a promising targeted delivery strategy for IBD therapy.

To further elucidate the mechanism of targeted delivery and MMP-responsive antibody release by PrEXO-a23, we performed dual-label in vivo tracking analysis to independently monitor the antibody (a23) and the carrier (PrEXO). a23 was labeled with Alexa Fluor 647, and the PrEXO was labeled with DiR. Three groups were included for comparison: PrEXO+a23 (a physical mixture of PrEXO and a23), PrEXO-fL-a23 (PrEXO and a23 were conjugated with a non-cleavable peptide, fL), and PrEXO-a23 (PrEXO and a23 were conjugated with the MMP-cleavable peptide). Following intravenous administration, in vivo and ex vivo imaging were performed at predetermined timepoints to assess distribution and retention in DSS-induced IBD mice and isolated colons. In the PrEXO+a23 group, the DiR-PrEXO signal robustly accumulated in colonic lesions, peaked at 12 h, and persisted for up to 48 h, demonstrating that PrEXO effectively targets inflamed colon tissues (Fig. 4f–h), consistent with the targeting observed in Fig. 4a–c. However, the Alexa Fluor 647-a23 signal in this group was weak and rapidly diminished, becoming almost undetectable after 12 h, indicating poor lesion-targeting and rapid clearance of free antibody. In contrast, both PrEXO-fL-a23 and PrEXO-a23 groups displayed PrEXO signals of similar intensity and duration to the physical mixture, but importantly, their a23 signals were markedly stronger and more persistent (higher than in the physical mixture, Fig. 4h). These results demonstrated that both PrEXO and a23 in PrEXO-a23 were effectively delivered to colonic lesions, which was attributed to the lesion-targeting capability of PrEXO, enabling the co-delivery of a23 via linkage.

Next, we performed CLSM analysis on colon tissue sections from IBD mice to confirm the release of PrEXO and a23 following MMP9-responsive cleavage of PrEXO-a23 at lesions. As shown in Fig. 4i, the non-cleavable PrEXO-fL-a23 group displayed strong spatial colocalization of a23 (green) and PrEXO (red), indicating the simultaneous delivery of the antibody and carrier to lesions. In contrast, the clear spatial separation of the two signals was detected in the PrEXO-a23 group, confirming in situ cleavage of the MMP-cleavable linker and subsequent release of a23 and PrEXO at the colonic lesions.

Taken together, these findings demonstrate that PrEXO-a23 not only achieves targeted co-delivery of both carrier and antibody to the site of inflammation but also undergoes efficient MMP-mediated cleavage to release the therapeutic antibody and PrEXO.

PrEXO-a23 corrects immune dysregulation via dual modulation of inflammation and tolerance in IBD

We evaluated the therapeutic efficacy of PrEXO-a23 in a DSS-induced colitis model, focusing on its ability to modulate immune dysregulation by simultaneously suppressing inflammation and promoting immune tolerance. C57BL/6 mice were administered 3% (w/v) DSS in drinking water for 7 days to induce colitis, and randomly divided into five treatment groups receiving intravenous injections every other day: PBS (DSS group), PrEXO (PrEXO group), IL-23 antibody (a23 group), rEXO-a23 (rEXO-a23 group), or PrEXO-a23 (PrEXO-a23 group). Healthy mice without DSS served as controls (Health group). Colonic tissues were collected on day 8 for downstream analyses.

Persistent inflammation in IBD is characterized by excessive infiltration of innate immune cells and dysregulated cytokine production3,42. Immunofluorescence analysis of colon tissues revealed that PrEXO-a23 treatment significantly reduced infiltration of CD86+ dendritic cells (P<0.0001) and CD68+ macrophages (P=0.0010), with greater reductions than those observed for PrEXO or a23 monotherapies (Fig. 5a–c). Further evaluation of colonic cytokine profiles via immunohistochemistry, ELISA, and RT-qPCR demonstrated that DSS treatment markedly upregulated proinflammatory cytokines IL-17A, IL-6, TNF-α, IL-23, and IFN-γ, while anti-inflammatory IL-10 was suppressed compared to healthy controls (Fig. 5d–f and Supplementary Fig. 14a, b). These cytokines are critical mediators of IBD pathogenesis: IL-23 and IL-17 promote pathogenic Th17 differentiation3, IFN-γ and TNF-α exacerbate chronic inflammation and intestinal tissue damage43,44, and IL-6 correlates with disease activity45, whereas IL-10 promotes immune tolerance and mucosal protection46. While free a23 demonstrated some anti-inflammatory activity in vitro, its in vivo efficacy was limited, only moderately reducing IFN-γ, IL-23, and IL-17A, and showing negligible impact on IL-6, TNF-α, or IL-10. This was attributed to poor lesion-targeting and insufficient drug enrichment. Moreover, a23 monotherapy had insufficient efficacy in regulating immune tolerance. In contrast, PrEXO-a23 broadly suppressed all major proinflammatory cytokines to near-healthy levels, while significantly upregulating IL-10 (P<0.0001). These therapeutic effects likely result from the hybrid membrane-mediated lesion-targeting, which delivers a23 to locally inhibit the IL-23/Th17 axis, combined with PrEXO’s role as an immune tolerance modulator to regulate the inflammatory microenvironment.

Previous studies have suggested that promoting Treg differentiation and inhibiting Th1/Th17 responses can significantly alleviate IBD by restoring immune tolerance22,47. Immunofluorescence staining of colon tissues showed that PrEXO-a23 treatment substantially decreased CD4+ T cell infiltration compared to DSS mice (Supplementary Fig. 14c). Flow cytometry analysis was then performed to quantify the proportions of Tregs (CD4+ FOXP3+), Th1 cells (CD4+ IFN-γ+), and Th17 cells (CD4+ IL-17A+) among CD4+ T cells. Compared with healthy mice, DSS mice exhibited reduced Treg frequencies and elevated Th1 and Th17 proportions, confirming immune tolerance breakdown (Fig. 5g, h). Compared to the DSS group, treatment with a23 significantly decreased Th1 (P=0.0061) and Th17 cell (P=0.0090) populations but had minimal effects on Treg proportion, consistent with its in vitro anti-inflammatory profile and limited capacity to promote tolerogenic reprogramming. Although PrEXO showed some potential to induce Tregs and suppress Th17 cells in vitro, its Treg-promoting effect was attenuated in vivo, likely due to antagonistic inflammatory signals such as IL-23-mediated Treg instability19,39,40. In contrast, PrEXO-a23 elicited the strongest immunoregulatory response, with significant Treg induction and concurrent suppression of Th1 and Th17 cells (P<0.0001), restoring an immune profile comparable to healthy mice. This enhanced effect likely results from the synergistic combination of a23, which establishes an anti-inflammatory milieu conducive to tolerance, and PrEXO, which promotes Treg differentiation. Collectively, these data demonstrate that PrEXO-a23 provides a dual therapeutic strategy in the complex IBD microenvironment by: (1) suppressing the IL-23/Th17 inflammatory axis through targeted a23 delivery and (2) promoting immune tolerance via Treg induction and tolerogenic reprogramming.

To further elucidate the molecular basis of PrEXO-a23’s immunoregulatory effects, we performed RNA sequencing (RNA-seq) on colonic tissues, using the DSS group as a control. PrEXO-a23 treatment induced 1,134 differentially expressed genes (DEGs): 199 upregulated and 935 downregulated (Supplementary Fig. 15a). Gene set enrichment analysis (GSEA) showed significant downregulation of IBD-related and Th17-differentiation gene signatures (P<0.05; Fig. 5i and Supplementary Fig. 15b). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of downregulated DEGs highlighted suppression of cytokine–cytokine receptor interaction, TNF, NF-κB, IL-17, JAK/STAT, Toll-like receptor signaling, and Th17 differentiation pathways (Fig. 5j). Gene Ontology (GO) analysis further indicated significant downregulation of processes involved in immune cell infiltration, inflammatory response, proinflammatory cytokine production, and neutrophil chemotaxis (P<0.05, Supplementary Fig. 15c). Importantly, PrEXO-a23 markedly downregulated key inflammatory genes (e.g., Ccr2, Ccr5, Il-17a, and Tnf), and upregulated anti-inflammatory or tolerance-associated genes (e.g., Fem1a) (Fig. 5k). Collectively, these results establish PrEXO-a23 as a dual-function therapeutic platform capable of concurrently extinguishing pathogenic IL-23 signaling and regulating immune tolerance, thereby resolving the fundamental immune dysregulation underlying IBD pathogenesis.

Potent therapeutic efficacy of PrEXO-a23 in treating IBD

To evaluate the therapeutic efficacy of PrEXO-a23, we employed a DSS-induced colitis model characterized by hallmark pathological features such as body weight loss, elevated disease activity index (DAI), colon shortening, and splenomegaly. Mice received 3% (w/v) DSS in drinking water for 7 days. Meanwhile, mice were treated on days 2, 4, and 6 by intravenous administration, as outlined in Fig. 6a. DAI, including weight change, stool consistency, and fecal bleeding, was monitored daily. Colon and spleen tissues were harvested for analysis on day 8. Treatment with PrEXO, a23, or rEXO-a23 partially alleviated the DSS-induced weight loss (Fig. 6b) and DAI elevation (Fig. 6c and Supplementary Fig. 16a–c), with PrEXO-a23 demonstrating the most significant therapeutic benefit. Colon length, severely reduced in the DSS group (~4.2 cm), was partially restored by rEXO-a23 (~5.4 cm) and further extended to near-normal levels by PrEXO-a23 (~6.6 cm; Fig. 6d). Spleen index analysis indicated significant splenomegaly in DSS-treated mice (P<0.0001), which was not observed in the PrEXO-a23 group (Supplementary Fig. 16d), suggesting attenuation of systemic inflammation48. These findings highlight the robust efficacy of PrEXO-a23 compared to monotherapies. Mechanistic analysis suggests that while PrEXO or a23 alone showed limited therapeutic effects due to insufficient immune regulation, PrEXO-a23 combines lesion-targeted delivery with synergistic immunomodulation. rEXO facilitates precise delivery and contributes to immune tolerance through its intrinsic bioactivity, while PMV-fusion-mediated vascular adhesion enhances retention at inflamed sites, amplifying payload accumulation and therapeutic outcomes beyond those of rEXO-a23.

In IBD, intestinal epithelial cell damage and inflammatory cell infiltration disrupt the integrity of the mucosal barrier49. Hematoxylin and eosin (H&E) staining and histological scoring revealed that PrEXO-a23 treatment preserved colonic epithelial integrity, maintaining intact epithelia, regular crypt architecture, and mucosal layers, along with reduced inflammatory cell infiltration compared to DSS group (Fig. 6e and Supplementary Fig. 16e). Immunofluorescence staining demonstrated restored expression of the tight junction proteins zonula occludens-1 (ZO-1) and Occludin, which were downregulated in DSS-treated mice, indicating improved intestinal barrier repair (Fig. 6f and Supplementary Fig. 16f). These results confirm that PrEXO-a23 substantially prevents physiological damage to colonic tissue and preserves intestinal barrier function.

Beyond barrier integrity, gut microbiota composition is a critical indicator of intestinal health. Dysregulated immune microenvironment in IBD favors pathogenic bacteria overgrowth while suppressing beneficial probiotics, leading to dysbiosis50. We next investigated microbiota modulation by PrEXO-a23 in IBD mice via 16S ribosomal RNA (rRNA) gene sequencing of fecal samples. A community circos diagram illustrated that PrEXO-a23 alleviated DSS-induced perturbations in microbiota composition (Fig. 6g). Specifically, PrEXO-a23 mitigated the reduction of beneficial bacteria, such as Lachnospiraceae and Akkermansiaceae, families known for anti-inflammatory and probiotic properties51,52, while suppressing overgrowth of Erysipelotrichaceae (Fig. 6h), a family associated with IBD progression53,54. Linear discriminant analysis (LDA) effect size (LEfSe) analysis further identified dominant microbial taxa influencing the gut environment from family to species levels. Consistent with previous findings, the abundances of IBD-associated pathogenic bacteria, including Erysipelotrichaceae, Turicibacter, and Bacteroides, were increased in DSS-treated mice (Fig. 6i). PrEXO-a23 treatment reduced these pathogenic bacteria while enriching beneficial microbes such as Lachnospiraceae and Akkermansiaceae, suggesting restoration of gut microbial homeostasis. This significant microbiota-regulating effect of PrEXO-a23 is likely attributed to its dual anti-inflammatory and pro-tolerant actions, which together restore immune homeostasis and repair the epithelial barrier, thereby creating a microenvironment favorable for microbiota recovery.

In clinical practice, IBD is associated with various extra-intestinal inflammatory responses, such as skin inflammation. To explore whether PrEXO-a23 can simultaneously treat these inflammatory conditions, we established an IBD-comorbid psoriasis mouse model using DSS and Imiquimod (IMQ). PrEXO-a23 was then administered intravenously to assess its accumulation and therapeutic effects at the site of inflammation. The results showed that PrEXO-a23, through its synergistic “inflammation homing + damage adhesion” strategy, was enriched not only in colonic lesions but also in extraintestinal inflamed skin sites, without accumulating in healthy tissues (Supplementary Fig. 17). In addition, PrEXO-a23 treatment effectively alleviated IBD and psoriasis symptoms, as demonstrated by suppressed body weight loss and colon shortening, and reduced disease activity scores for both diseases (Supplementary Fig. 18).

Finally, the biosafety of PrEXO-a23 was evaluated both in vitro and in vivo. In vitro cytotoxicity assays showed no adverse effects on colonic epithelial cell viability across a range of PrEXO-a23 concentrations (Supplementary Fig. 19a, b). In vivo safety assessment following intravenous administration of PrEXO-a23 in healthy mice revealed no histopathological abnormalities in major organs (Supplementary Fig. 19c). Blood biochemistry (Supplementary Fig. 19d, e) and cytokine analyses confirmed that levels of IFN-γ, IL-6, and TNF-α (Supplementary Fig. 19f) remained within normal physiological ranges, indicating favorable biocompatibility.

PrEXO-a23 effectively decreases the risk of CAC and fibrosis

We further analyzed the RNA-seq data on colonic tissues from DSS-induced IBD mice, comparing gene expression profiles across healthy, DSS-treated, and PrEXO-a23-treated groups. Among the expressed genes, 295 were consistently different in DSS mice compared with healthy controls and the PrEXO-a23 group. Using STRING and Cytoscape, we constructed a protein-protein interaction network and extracted an 89-node, 1044-edge subnetwork. MCODE analysis revealed two highly interconnected modules (module 1, score=23.5; module 2, score=8.0), with module 1 containing 49 targets and 564 interactions. Degree-centrality ranking within this module identified Trp53 as the pivotal hub (Fig. 7a and Supplementary Fig. 20). As a tumor suppressor governing cell cycle, DNA repair, and apoptosis, Trp53 dysfunction is an early event in ulcerative colitis-associated colorectal carcinogenesis10,55. Notably, PrEXO-a23 therapy regulated p53 in DSS-treated colons, suggesting that PrEXO-a23 has the potential to decrease the risk of progression from IBD to CAC.

To evaluate the long-term therapeutic potential of PrEXO-a23 in preventing IBD-associated complications such as intestinal fibrosis and CAC, we employed an azoxymethane (AOM)/DSS-induced CAC mouse model. Mice were administered AOM followed by repeated cycles of DSS to induce chronic colitis and tumorigenesis, with untreated healthy mice serving as controls (Health group). During disease progression, mice received intravenous injections of PBS (AOM/DSS group), PrEXO (PrEXO group), a23 (a23 group), rEXO-a23 (rEXO-a23 group), or PrEXO-a23 (PrEXO-a23 group) (Fig. 7b). As expected, AOM/DSS-treated mice exhibited significantly reduced survival (P=0.0208; Fig. 7c), substantial weight loss (Supplementary Fig. 21a), and significantly elevated DAI scores (Fig. 7d), shortened colon length, and increased tumor burden, both in terms of number and size (Fig. 7e–g and Supplementary Fig. 21b). Among all treatment groups, PrEXO-a23 demonstrated the robust therapeutic efficacy, markedly improving survival, body weight, DAI, and colon morphology to levels comparable with healthy controls. Importantly, while PrEXO, a23, and rEXO-a23 treatments conferred partial protection, none of these monotherapies prevented tumor development. In contrast, PrEXO-a23 treatment completely inhibited tumor formation, highlighting its potent anti-carcinogenic effect and capacity to halt IBD-to-CAC progression.

Given that CAC development involves sustained mucosal immune activation, epithelial barrier disruption, and hyperproliferation11, we further investigated the impact of PrEXO-a23 on these pathological hallmarks. Compared with the AOM/DSS group, PrEXO-a23 significantly decreased proinflammatory cytokine levels (TNF-α and IL-6) in colonic tissues (P=0.0016 and P=0.0112; Fig. 7h, i) and normalized the spleen index (Supplementary Fig. 21c), suggesting effective suppression of both local and systemic inflammation48. Histological analysis by H&E staining confirmed preserved epithelial architecture and reduced inflammatory cell infiltration in the PrEXO-a23 group (Fig. 7j). Furthermore, Ki67 immunofluorescence staining demonstrated suppressed epithelial hyperproliferation, with proliferation indices comparable to those of healthy controls (Fig. 7k and Supplementary Fig. 21d). Together, these data indicate that PrEXO-a23 improves colonic integrity and function, as demonstrated by its ability to modulate immune response, repair colonic barrier and control epithelial proliferation.

To evaluate whether PrEXO-a23 also protects against intestinal fibrosis, a common and severe consequence of chronic IBD, we employed a chronic DSS-induced colitis model with three DSS cycles. Mice in the AOM/DSS group exhibited marked thickening of the colonic wall, muscularis mucosa, and muscularis propria, characteristic features of fibrosis (Fig. 7l–o). Immunohistochemical staining for α-smooth muscle actin (α-SMA), a marker of myofibroblast activation, showed significant upregulation in fibrotic colons (P<0.0001; Fig. 7p, q). While PrEXO, a23, and rEXO-a23 monotherapies did not inhibit fibrotic progression, PrEXO-a23 treatment preserved normal colon thickness and suppressed α-SMA expression to baseline levels, indicating effective prevention of fibrotic changes. PrEXO-a23 mitigated fibrotic progression through a coordinated dual mechanism. By neutralizing IL-23, it could suppress Th17-driven inflammation, while simultaneously delivering Treg-derived exosomes to promote immune tolerance. This combined anti-inflammatory and pro-tolerance strategy not only corrected the underlying immune dysregulation before pathological fibrosis occurs but also reprogrammed the local microenvironment to favor beneficial immunosuppressive and tolerogenic Treg functions, thereby preemptively blocking the pathological progression toward fibrosis.

We previously demonstrated that PrEXO-a23 affected the Trp53 expression change in DSS-treated colons. To evaluate the contribution of p53 to the therapeutic effect of PrEXO-a23, the mice were co-treated with the p53 inhibitor pifithrin-α (ip53). This intervention markedly diminished the therapeutic efficacy of PrEXO-a23 and negated its anti-tumor effects (Fig. 8a–g), suggesting that p53 pathway activity is associated with the nanotherapy’s ability to prevent CAC. We therefore examined key mediators of p53 signaling in colonic tissues using RT-qPCR and Western blot analysis. RT-qPCR analysis revealed that during CAC progression in the AOM/DSS group, expression of Trp53, Cdkn1a, and Bax was significantly downregulated compared to healthy controls, reflecting pathway dysfunction. Treatment with PrEXO-a23 restored their expression to near-normal levels, an effect that was partially blocked by p53 inhibition (PrEXO-a23 + ip53) (Fig. 8h). Western blot analysis confirmed these changes at the protein level for p53, p21, and Bax (Fig. 8i, j). Together, these data demonstrate that the capacity of PrEXO-a23 to prevent the progression from chronic inflammation to cancer is partially dependent on functional p53 signaling. Collectively, PrEXO-a23 not only attenuates active inflammation but also provides durable protection against fibrosis and tumorigenesis, highlighting it as a promising therapeutic strategy for the long-term management of IBD and its complications.

Methods
Reagents and materials
Naïve CD4+ T cell isolation kit was purchased from Miltenyi Biotec. The peptide linker (Arg-Val-Gly-Leu-Pro, RVGLP) was designed and synthesized by China Peptides. In Vivo MAb IL-23 was obtained from BioXcell. Primary antibodies for immunostaining MMP9, ALIX, TSG101, CCR6, CD68, CD4, ZO-1, Occludin, CD62P, CTLA-4, GAPDH, and Ki67 were provided by Abcam. Anti-mouse GPIbα, IFN-γ, IL-10 and α-SMA were supplied by Affinity. Anti-mouse CD86, IL-17A and p53 were purchased from Proteintech. Anti-mouse p21 and Bax were obtained from Cell Signaling Technology. BV421-anti-CD11c, PE-anti-CD86, APC-anti-CD80, FITC-anti-MHCII, BV421-anti-CD45, FITC-anti-CD3, APC/FireTM 750-anti-CD4, APC-anti-CD25, PE-anti-IL-17A, APC/FireTM 750-anti-CD45, PE-anti-CD19, BV421-anti-F4/80, and PE-Ly6G were purchased from BioLegend. PE-anti-FOXP3 and APC-anti-IFN-γ were provided by eBioscience. 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI), 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate (DiD), and tris(2-carboxyethyl)phosphine (TCEP) were obtained from Invitrogen. IL-6, IL-23, IFN-γ, and IL-1β, were provided by PeproTech. IL-2, TGF-β, MMP9, GM6001, NHS-PEG-MAL linker, and pifithrin-α were purchased from MedChemExpress. Dextran sulfate sodium salt (DSS), calcein-AM/propidium iodide (PI) stain kit, PI/Annexin V apoptosis detection kit, and mouse type I collagen were supplied by Yeasen. Azoxymethane (AOM) and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich. 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and ELISA kits for IFN-γ, TNF-α, IL-17A, IL-23, IL-6, and IL-10 were purchased from Solarbio. ELISA kits for TGF-β1 and MMP9 were obtained from Elabscience. X-VIVO medium was purchased from Lonza. Dulbecco’s Modified Eagle Medium (DMEM), RPMI 1640 medium, fetal bovine serum (FBS), and penicillin–streptomycin (P/S) were purchased from Gibco.

Animal and cell lines
All C57BL/6 female mice (6−8 weeks old) were obtained from the SPF Biotechnology Co., Ltd (Beijing, China), and housed in groups of six in individually ventilated cages under a 12 h light/dark cycle at 20–22 °C with ad libitum access to standard rodent chow and water. All animal studies were conducted in accordance with the animal experimental protocols approved by the Institutional Animal Care and Use Committee of the Chinese Academy of Medical Sciences & Peking Union Medical College Institute of Radiation Medicine (Approval number: IRM/2-IACUC-2311-002).

RAW264.7 (murine macrophage cell line) and HT-29 cells (human colorectal carcinoma cell line) were obtained from Procell. NCM460 cells (human normal colonic epithelial cell line) were purchased from SUNNCELL. All cell lines were maintained in DMEM supplemented with 10% FBS and 1% P/S. Bone marrow-derived neutrophils and DCs were isolated from 6-week-old C57BL/6 mice. The bone marrow cells were cultured in RPMI 1640 medium containing 10% FBS, 1% P/S, 20 ng/mL recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF; Sino Biological), and 10 ng/mL IL-4 (Sino Biological) for DC differentiation. Half of the culture medium was replaced with fresh medium on day 3, and cells were used on day 6 as immature DCs. For neutrophil isolation, bone marrow cells were added to a Percoll gradient (55, 65, and 78%; v/v), followed by centrifugation at 500 × g for 30 min. Neutrophils were collected at the interface between the 65 and 78% Percoll layers, washed three times with PBS, and maintained in RPMI 1640 medium containing 10% FBS and 1% P/S for further analysis. All cells were cultured at 37 °C in a humidified 5% CO2 atmosphere.

Preparation of rEXO and PMV
Naïve CD4+ T cells were isolated from the spleen of healthy C57BL/6 mice using a CD4-positive T cell isolation kit and polarized to regulatory T cells in X-VIVO medium supplemented with anti-CD3 (5 μg/mL), anti-CD28 (2 μg/mL), IL-2 (20 ng/mL), and TGF-β (5 ng/mL) for 7 days. Treg culture supernatant was sequentially centrifuged at 300 × g for 10 min, 2000 × g for 20 min, and 10,000 × g for 30 min to remove debris, followed by ultracentrifugation at 100,000 × g for 70 min to obtain rEXO.

Platelets isolated from the whole blood of healthy C57BL/6 mice were subjected to freeze-thaw cycles, and platelet membranes were extracted by centrifugation at 8000 × g for 20 min. The processed membranes were sequentially extruded through polycarbonate porous membrane filters with decreasing pore sizes (0.8, 0.4, 0.2, and 0.1 μm) to obtain PMV. Protein concentrations of rEXO and PMV were quantified using a BCA assay kit (Accurate Biotechnology).

Preparation and characterization of PrEXO-a23
Co-extrusion of PMV and rEXO. PrEXO was prepared by mixing PMV and rEXO at protein mass ratios of 1:1, 1:2, or 1:3, followed by sonication (40 kHz, 5 min) and then repeated extrusion as described for the PMV preparation. Fusion efficiency of PrEXO was assessed by NanoFlow Cytometer (CytoFLEX, Beckman).

Synthesis and conjugation of MMP9-cleavable linker to a23. An MMP-cleavable peptide linker (Arg-Val-Gly-Leu-Pro, RVGLP) with an NHS group at the arginine (Arg) terminus and a Mal group at the proline (Pro) terminus was used to conjugate the IL-23 antibody (a23) to the hybrid vesicles (PrEXO). To determine the optimal molar ratio for linker-a23 conjugation, a23 was reacted with a linker at molar ratios (a23:linker) of 1:1, 1:10, 1:50, 1:75, or 1:100 in PBS at 4 °C for 12–18 h. Unreacted linker was removed by centrifugation at 8000 × g for 30 min using a centrifugal filter (100 kDa; Jet Biofil). The purified linker-a23 was analyzed using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS; AXIMA-Performance MA, Shimadzu).

Preparation of PrEXO-a23. PrEXO was prepared by coextrusion of platelet membrane vesicles and Treg-derived exosomes at a 1:1 protein mass ratio, followed by protein quantification using a BCA assay. PrEXO was treated with 1 mM TCEP at 37 °C for 30 min to expose sulfhydryl (SH) groups on its membrane. Pretreated PrEXO was then added to purified linker-a23 at a mass ratio (PrEXO:a23) of 1:0.1, 1:0.25, 1:0.5, 1:1, or 1:2, and incubated at 25 °C for 1 h. Unreacted linker-a23 was removed by centrifugation at 8000 × g for 30 min using a centrifugal filter (300 kDa). ELISA was used to determine the conjugation efficiency of a23 to PrEXO. Using the same method, PrEXO was conjugated to a23 using a non-MMP-cleavable linker (fL: NHS-PEG-MAL) to generate PrEXO-fL-a23.

Characterization of PrEXO-a23. Morphological analysis was performed using a TEM instrument (JEM-1400 Flash, JEOL, Japan). Particle sizes and zeta potential were measured by a NanoZS Malvern analyzer (Malvern, UK). PrEXO-a23 was stored in PBS (pH 7.4) at 4 °C and in plasma at 37 °C, and particle size and zeta potential were measured daily for 7 days.

Protein analysis of PrEXO-a23
IL-10 (1:50), CTLA-4 (1:50), ALIX (1:50), TSG101 (1:50), CCR6 (1:50), CD62P (1:50), GPIbα (1:50), and a23 were detected using an automated Digital Capillary Electrophoresis (CE)-Western system (JS4167, ProteinSimple, USA) according to the manufacturer’s protocol. Specifically, protein samples were prepared using loading buffer without reducing agents (e.g., DTT) to allow analysis under non-reducing conditions. Protein samples, primary antibody dilution, secondary antibody dilution, and chemiluminescence substrate were sequentially added to the lane plates for protein detection. All analyses were carried out using Compass for Simple Western software (v 6.0.0, ProteinSimple).

PrEXO-a23 colocalization assay
rEXO, PMV, and a23 were labeled with DiI, DiD, and Alexa Fluor 488, respectively. All samples were incubated with 5 μg/mL dye at 37 °C for 30 min. Triple-labeled PrEXO-a23 was observed using confocal laser scanning microscopy (Eclipse Ti2, Nikon, Japan) with a 100× oil-immersion objective.

In vitro assessment of a23 release from PrEXO-a23
To evaluate MMP9-responsive properties of the peptide linker, PrEXO-a23 was incubated with MMP9 with or without the MMP9 inhibitor GM6001 at predetermined time intervals. Released a23 was quantified using an ELISA kit.

Coculture model of CD4+ T cells and intestinal epithelial cells
Naïve CD4+ T cells were polarized to Th17 cells by culturing in RPMI 1640 medium containing TGF-β (1 ng/mL), IL-6 (50 ng/mL), IL-23 (5 ng/mL), IL-1β (10 ng/mL), anti-IFN-γ (10 μg/mL), and anti-IL-4 (10 μg/mL) for 72 h. Cells were treated with rEXO (25 μg/mL), PMV (25 μg/mL), PrEXO (25 μg/mL rEXO and 25 μg/mL PMV), a23 (7.5 μg/mL), or PrEXO-a23 (25 μg/mL rEXO, 25 μg/mL PMV, and 7.5 μg/mL a23), respectively. After 72 h, T cells were collected and stained with APC/FireTM 750-CD4 antibody and PE-IL-17A antibody to detect the percentage of Th17 cells using a flow cytometer (CytoFLEX, Beckman).

For coculture, the pretreated T cells (2 × 105 per well) were seeded in the upper chamber of 12-well Transwell inserts (NEST Biotechnology), while NCM460 cells (5 × 105 cells per well) were plated in the lower chamber. After 24 h, NCM460 cells were collected and stained with PI/Calcein-AM or Annexin V/PI for 15 min according to the manufacturer’s protocol. The stained cells were then analyzed using fluorescence microscopy or flow cytometry.

Induction of tDCs and Tregs
On day 6, immature DCs were stimulated with LPS (1 μg/mL) and IFN-γ (20 ng/mL) to generate mature DCs for 24 h. For the induction of tDCs, the cells were co-treated with rEXO, PMV, PrEXO, a23, or PrEXO-a23, respectively. After 24 h, cells were incubated with CD16/CD32 (BioLegend) for 15 min to block Fcγ receptors, then stained with BV421-CD11c, APC-CD80, PE-CD86, and FITC-MHCII antibodies, and analyzed by flow cytometry. Culture supernatants were collected for cytokine analysis (IL-10, TGF-β, IL-6, and TNF-α) using ELISA kits.

To activate lymphocytes, 5 × 105 CD4+ T cells were cocultured with pretreated 1 × 105 DCs for 72 h. After incubation, CD4+ T cells were stained with antibodies, including APC/FireTM 750-CD4, PE-IL-17A, APC-CD25, and PE-FOXP3, to assess the proportion of Th17 cells and Tregs.

In vitro flow assay
A flow channel was coated with 100 μg/mL Type I collagen by incubating overnight at 37 °C. DiO-labeled rEXO, PMV, PrEXO, or PrEXO-a23 were loaded into a 1 mL syringe for perfusion over the collagen-coated flow channel at a shear rate of 500 s−1 using a syringe pump (Harvard, USA). Then, the flow channel was perfused with PBS at the same shear rate before imaging by CLSM.

In vitro inflammatory targeting assay
RAW264.7 cells or neutrophils were incubated with DiI-labeled rEXO, PMV, PrEXO, or PrEXO-a23 for 2 h, with or without the pre-stimulation of LPS (1 μg/mL). After incubation, cells were washed twice using PBS, then the percentage of DiI-positive cells or mean fluorescence intensity was measured using a flow cytometer or CLSM. PrEXO-a23 was incubated with or without LPS-activated RAW264.7 at 37 °C in the MMP9 condition. The cumulative release of a23 was measured by ELISA at the indicated time points.

In vivo biodistribution of PrEXO-a23 in the IBD model
To generate the IBD mouse model, C57BL/6 mice were fed with 3% (w/v) DSS for 7 days after being pretreated with a fluorescence-free diet. Mice were intravenously injected with DiD-labeled rEXO, PMV, rEXO+PMV, PrEXO, or PrEXO-a23, respectively. At 0, 2, 6, 12, 24, 36, and 48 h after injection, the mice were imaged using an in vivo imaging system (ABL-X5, Tanon, China) to analyze the fluorescence biodistribution of DiD. Mice from each group were euthanized, and their major organs (heart, liver, spleen, lung, kidney, and colon) were collected for further analysis. For localization analysis, frozen colon sections were stained with DAPI, and images were captured using CLSM. For cellular targeting analysis, colon tissues were processed into single-cell suspensions for flow cytometry analysis. The relative DiD fluorescence in DCs (APC/FireTM 750-CD45 and BV421-CD11c), macrophages (APC/FireTM 750-CD45 and BV421-F4/80), T cells (APC/FireTM 750-CD45 and FITC-CD3), B cells (APC/FireTM 750-CD45 and PE-CD19) and neutrophils (APC/FireTM 750-CD45 and PE-Ly6G) were analyzed to determine immune cell uptake.

In vivo pharmacokinetics study
DiD-labeled PrEXO-a23 was intravenously administered to C57BL/6 mice, and blood samples were collected at predetermined time points (0.5, 1, 2, 4, 24, 48, and 72 h). Blood samples were placed in 96-well plates, and fluorescence intensity was measured at an excitation wavelength of 650 nm using an in vivo imaging system.

Dual-label tracking analysis in vivo
a23 was labeled with Alexa Fluor 647, and PrEXO was labeled with DiR. DSS-induced IBD mice were intravenously injected with PrEXO+a23 (a physical mixture of PrEXO and a23), PrEXO-fL-a23 (PrEXO and a23 were conjugated with a non-cleavable peptide, fL), or PrEXO-a23 (PrEXO and a23 were conjugated with the MMP-cleavable peptide), respectively. The biodistribution of PrEXO and a23 was monitored using an in vivo imaging system at 0, 2, 6, 12, 24, 36, and 48 h after injection. Colon tissues were harvested at 12 h after injection for ex vivo imaging analysis. Cryosections were processed for CLSM imaging to examine the colocalization of PrEXO (DiI-labeled) and a23 (Alexa Fluor 647-labeled) signals.

Evaluation of clinical signs in DSS-induced IBD model
Following a 7-day acclimatization period, female C57BL/6 mice (8 weeks old) were given 3% (w/v) DSS in drinking water for 7 days to establish an IBD model. Healthy mice in the normal group were not treated, while DSS-induced mice in the DSS, PrEXO, a23, rEXO-a23, and PrEXO-a23 groups were injected intravenously with PBS, PrEXO, a23, rEXO-a23, and PrEXO-a23 on days 2, 4, and 6 of DSS feeding, respectively. The dose was administered at 5 mg/kg for rEXO and 1.5 mg/kg for a23. Mice were observed daily, and the body weight and disease activity index (DAI) were evaluated. DAI scores were calculated daily based on weight loss (0 to 4), stool consistency (0 to 4), and fecal bleeding (0 to 4) scores. On day 8, mice were euthanized, and their colons were collected for length measurement and subsequent analysis. Spleen index was calculated as follows: spleen index = (spleen weight/body weight) × 100%.

Evaluation of the inhibition effect in the AOM/DSS-induced CAC model
To induce the CAC mouse model, six-week-old female C57BL/6 mice were intraperitoneally injected with AOM (10 mg/kg). After 7 days, mice were subjected to three cycles of DSS treatment, each consisting of 2.5% DSS for 7 days followed by a 14-day recovery period with regular water. During each 7-day DSS treatment period, PBS, PrEXO, a23, rEXO-a23, and PrEXO-a23 were administered intravenously every 2 days, as described for the IBD model. At the end of the 10-week experiment, mice were euthanized, and colons were excised for tumor enumeration and length measurement.

Histological analysis
Colonic tissues were fixed in 10% formalin for 24 h, embedded in paraffin, and sliced into 4 μm sections with a microtome (Thermo Fisher). Subsequently, hematoxylin and eosin (H&E) staining was performed on the tissue sections. Colonic histological damage was blindly scored for epithelial damage (0–6) and inflammatory infiltration (mucosa: 0–3; submucosa: 0–2; muscle/serosa: 0–1) according to established criteria60.

For immunofluorescence, the fixed colon slides were permeabilized with 0.3% Triton X-100 for 10 min, blocked with 5% goat serum, and incubated overnight at 4 °C with primary antibodies against ZO-1 (1:100), Occludin (1:100), CD86 (1:400), CD68 (1:1000), CD4 (1:500), or Ki67 (1:1000). Alexa Fluor 488 or Alexa Fluor 647 conjugated secondary antibodies (1:500) were applied for 1 h, and cell nuclei were stained with DAPI (1:1000).

For immunohistochemistry, preprocessed colonic sections were labeled with primary antibodies of anti-MMP9 (1:500), anti-IL-17A (1:900), anti-IFN-γ (1:200), or anti-α-SMA (1:900) overnight, followed by secondary antibodies and a color reaction using a DAB Kit (Maxim). Cell nuclei were counterstained with hematoxylin. All images were acquired using a Pannoramic digital slide scanner (3DHISTECH), and analyzed with ImageJ.

Flow cytometry analysis of colonic cell subgroups
Colonic lamina propria lymphocytes were isolated by digesting tissue with Liberase (31.25 μg/mL, Roche) and DNase I (50 μg/mL, Roche) at 37 °C for 1 h with rotation. Single-cell suspensions were obtained through a 70-μm cell strainer (Biosharp), and 1 × 106 cells were separated from each group for staining. After Fc receptor blocking, the cells were incubated with cell surface fluorescent antibodies containing BV421-CD45, FITC-CD3, and APC/FireTM 750-CD4 antibodies. For transcription factor staining, cells were fixed and permeabilized using a commercial FOXP3 staining kit (eBioscience) according to the manufacturer’s protocol, followed by incubation with PE-FOXP3 antibody.

For intracellular cytokine staining, isolated lymphocytes were stimulated with a Cell Activation Cocktail (BioLegend) for 6 h at 37 °C before cell surface staining. Subsequently, PE-anti-IL-17 antibody and APC-anti-IFN-γ antibody were used. The proportions of Th1, Th17, and Treg cells in each sample were analyzed using a flow cytometer. Finally, all data were processed using FlowJo V10.8.1.

ELISA assay
Serum and colonic tissues were collected from IBD mice. Colon segments were homogenized in PBS using a homogenizer at 4 °C. Homogenates were centrifuged at 10,000 × g for 10 min, and supernatants were analyzed for cytokines and MMP9 using ELISA kits following manufacturer protocols.

Reverse transcription quantitative real-time PCR (RT-qPCR)
Total RNA was extracted from frozen colon tissues using TRIzol, quantified on a NanoDrop 2000 spectrophotometer (Thermo Fisher, USA), and reverse-transcribed using HiScript III RT SuperMix (Vazyme). Then, qPCR was performed on the CFX Connect Real-Time PCR Detection System with SYBR Green Master Mix. Primer pairs for target mouse genes are shown in Supplementary Table 1. Relative gene expression was calculated using the 2−ΔΔCt method, normalized to GAPDH.

RNA sequencing analysis
On day 8, mice were euthanized, and their colon tissues were excised (n=5). Total RNA from colon tissues was extracted using TRIzol. After total RNA was extracted, eukaryotic mRNA was enriched using Oligo (dT) beads. Following mRNA enrichment, RNA was processed for cDNA synthesis and library construction with the MGIEasy RNA Library Prep Kit. Libraries were sequenced on the MGISEQ-2000 sequencing platform (Beijing Genomic Institution, BGI). Raw reads were filtered with SOAPnuke 1.5.6, and clean reads were aligned to the mouse genome (GRCm38.p6) using HISAT2. Gene expression counts were obtained with feature counts, and differential expression analysis was performed using DESeq2 (v1.4.5) between two different groups. The genes with the parameter of |log2 (fold change)| ≥ 1 and adjusted P ≤ 0.05 were considered as differentially expressed genes (DEGs). Functional enrichment analyses, including heatmap clustering, GSEA, GO, and KEGG analysis, were performed on the BGI Dr. Tom platform (https://biosys.bgi.com). GSEA was conducted using the GSEA software v4.0.3 to determine whether predefined gene sets from the KEGG pathways were significantly enriched between two groups.

Western blot analysis
Protein expression was quantified using an automated Digital Capillary CE-Western system (JS4167, ProteinSimple, USA) according to the manufacturer’s protocol. Briefly, colon segments from mice were homogenized in RIPA buffer to extract protein, and protein concentration was determined using a BCA assay kit. Protein samples were added to each well, followed by the corresponding primary antibodies (p53 at 1:50, p21 at 1:50, Bax at 1:50, and GAPDH at 1:50) and HRP-conjugated secondary antibody. Relative protein levels were calculated by measuring the area under the peak of a chemiluminescent chromatogram and normalized to GAPDH. All analyses were carried out using Compass for Simple Western software (v 6.0.0, ProteinSimple). Uncropped scans are available in the Source Data file.

16S rRNA microbiota sequencing assay
On day 7, mouse feces were collected for gut microbiota analysis via 16S rRNA gene sequencing at Genesky Biotechnologies Inc. (Shanghai, China). Total DNA was extracted from fecal content using a fecal DNA extraction kit. The hypervariable V3-V4 regions of the bacterial 16S rRNA gene were amplified by PCR, and products were purified with the Agencourt AMPure XP Kit (Beckman Coulter, CA, USA). Libraries were constructed with Illumina adapters, quantified via Qubit 3.0 Fluorometer, and validated with an Agilent Bioanalyzer 2100 system. Sequencing was performed on an Illumina NovaSeq platform to generate 250 bp paired-end reads. Data analysis was conducted on the Genesky Cloud Platform (http://cloud.geneskybiotech.com), with microbial composition analyzed at the family level.

Statistical analysis
Data are presented as mean ± SD (standard deviations). For comparisons between two groups, a two-tailed Student’s t-test was applied. For multiple comparisons, one-way ANOVA followed by Tukey’s post hoc test was used. Survival curves were analyzed by the log-rank test. All statistical analyses were performed using GraphPad Prism (v9.0). P < 0.05 was considered significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Recommendation: Major revision\nGeneral Comments:\nZhang et al. present an intriguing study entitled \"Biomimetic Exosome-Based Nanotherapy for Targeted Immunomodulation\nin Inflammatory Bowel Disease\", wherein they propose PrEXO-a23—a dual-targeting nanoplatform combining Treg-derived\nexosomes and platelet membranes conjugated with IL-23 antibodies. The rationale of leveraging both platelet-mediated\nhoming and Treg immunomodulation is compelling, and the therapeutic potential is conceptually promising. However, the\nmanuscript requires significant revision before it can be considered for publication in a high-impact journal like Nature\nCommunications. Key mechanistic claims are currently underdeveloped or insufficiently supported by experimental\nevidence, and several data presentation issues limit clarity and reproducibility.\nMajor Comments:\n1. The MMP9 levels reported in colitic tissues (~50 ng/mL, Supplementary Fig. 5) appear substantially lower than the\nconcentrations used for in vitro cleavage studies (Figure 2f). The authors should explain this discrepancy, and ideally justify\nthe physiological relevance of the in vitro model.\n2. Is the dosage of PrEXO-a23 based on lyophilized vesicle weight? After co-extrusion at a 1:1 ratio, the concentration of\neach component should theoretically be halved. The authors need to clarify how PrEXO-a23 retains comparable bioactivity\nto single-component vesicles in Figure 3, despite this dilution effect.\n3. Figure 4a suggests that platelet-membrane-derived proteins contribute to adhesion with collagen. Yet PMVs alone do not\nexhibit equivalent performance. Could this discrepancy be explained by a protein corona effect, or by synergistic effects with\nexosomal components? Furthermore, the authors should examine the vesicle stability in whole blood and assess potential\nopsonization or clearance. Relevant literature, such as Nat Commun 2024, 15, 4876, may offer guidance. Additionally,\norgan-level biodistribution data should be provided.\n4. Figure 4b shows increased uptake by macrophages and neutrophils. The authors should consider whether intracellular\ndegradation or trafficking might affect IL-23 antibody release kinetics or impair bioavailability.\n5. The utility of the MMP9-cleavable linker hinges on differential biodistribution and release of the antibody vs. carrier.\nSeparate tracking of antibody and carrier at the inflammatory site is essential to support this claim. Without this mechanistic\nevidence, the cleavable linker lacks justification. Moreover, line 563 asserts successful separation of antibody and carrier\npost-cleavage—this must be experimentally validated.\n6. Splenomegaly is a known indicator of systemic inflammation. The authors should discuss whether elevated systemic\nMMP9 levels could lead to premature antibody release in circulation, compromising targeted delivery.\nMinor Comments:\n1. All flow cytometry data should be accompanied by clearly defined gating strategies to enhance reproducibility and\ntransparency.\n2. In Figures 3b and 3g, the x-axis labels are unclear—does the labeling include the control group? Similar ambiguities may\nexist in other figures; consistent and precise labeling should be ensured throughout.\n3. For datasets with high variability (e.g., Figure 4d), the number of biological replicates should be increased to ensure\nstatistical robustness.\n4. The manuscript briefly mentions maintenance of immune tolerance (e.g., Line 171). A more nuanced discussion is\nwarranted, particularly regarding the dual role of Tregs in both suppressing inflammation and promoting fibrosis in chronic\nconditions.\n5. The rationale for using MMP9-responsive linkers should be clearly introduced in the opening section. The biological\nsignificance and therapeutic relevance of such linkers in IBD should be explicitly stated.\n6. In Lines 166 and 170, the text refers to statistical comparisons with control groups, yet no corresponding statistical\nindicators (e.g., asterisks or p-values) are shown in the figures. This inconsistency should be rectified.\n7. Quantification data in Figure 4h would be more convincing if accompanied by representative flow cytometry histograms or\ndot plots.\n8. The manuscript frequently refers to significant differences without specifying the exact p-values. It is strongly\nrecommended that the authors include exact p-values wherever possible to enhance scientific rigor.\n9. The authors should carefully proofread the manuscript to eliminate typographical errors and redundancies. For example,\nline 376 reads: “Although PrEXO showed Although PrEXO showed some potential to...”.", "reviewer_2": "The article introduces PrEXO-a23, a novel nanotherapeutic for inflammatory bowel disease (IBD). It combines regulatory T\ncell (Treg)-derived exosomes (rEXO) with platelet membrane vesicles (PMV) and uses an MMP-cleavable linker to attach IL-\n23 antibodies (a23). This design leverages the homing abilities of platelets and Treg cells to target inflamed colonic tissues\nin IBD mice. At the inflamed site, MMPs release a23, which neutralizes IL-23 and inhibits Th17-mediated inflammation.\nMeanwhile, rEXO reprograms dendritic cells and promotes Treg expansion to restore immune tolerance. Tests show\nPrEXO-a23 reduces colitis severity, enhances epithelial barrier integrity, rebalances the gut microbiota, and prevents\ncomplications like intestinal fibrosis and colitis-associated colorectal cancer. Overall, PrEXO-a23 shows potential for\ndurable immune reprogramming and long-term management of IBD and its complications. However, there are still a few\nissues that need to be addressed:\n1. The material preparation section is too concise and doesn't elaborate on determining the optimal concentrations of the\nbinder and a23.\n2. It's recommended that the role of Th17 cells in IBD be highlighted in the introduction.\n3. It's suggested that a discussion on rEXO's immune-regulating effects, such as inhibiting Th17 cell differentiation, be\nincluded or that further experiments on its related mechanisms be conducted.\n4. The enrichment mechanism of this study deserves discussion. The material developed in this study can be enriched in\nIBD lesions through the vWF factor highly expressed in the inflamed intestine of IBD, and the targeting effectiveness has\nbeen verified in animal models. However, in clinical settings, IBD is associated with various extraintestinal inflammatory\nresponses, such as skin inflammation, eye lesions, and joint involvement. How to prevent the material from accumulating in\nextraintestinal IBD lesions and whether it still has a therapeutic effect in these locations need to be explored. Also,\nconsidering that vWF is highly expressed in many other diseases like coronary heart disease and diabetes, how can we\nprevent this biomaterial from aggregating in sites related to comorbidities with high vWF expression in patients, or will it\nhave a therapeutic effect after accumulation?\n5. There seem to be some unexplained characters in Supplementary Fig. 12c. The author should replace the image.", "reviewer_3": "In this study, Cao et al. made a nanotherapeutic material, PrEXO-a23, to treat inflammatory bowel disease (IBD), by fusing\nregulatory T cell (Treg)-derived exosomes (rEXO) with platelet membrane vesicles, and conjugating IL-23 antibodies (a23)\nvia a matrix metalloproteinase (MMP)-cleavable linker. Authors present in vitro and in vivo data to show PrEXO-a23 can\nreduce colitis severity, rebalance gut microbiota, and even prevent chronic complications including intestinal fibrosis and\ncolitis-associated colorectal cancer. However, its design has obvious defects, the key data are not convincing and cannot\nsupport conclusions. These flaws prohibit its publication. The following are my comments.\nMajor concerns:\n1. The design of this study highly depends on MMP activity to cleave the linker and release anti-IL-23 antibody in\ninflammatory site. However, PrEXO-a23 was injected intravenously into mice in the animal study. There are MMPs in the\nserum. The anti-IL-23 antibodies could be released from PrEXO-a23 in the serum before it arrives the inflammatory site. In\naddition, IL-23 level is high in the serum when the body undergoes inflammation. The serum IL-23 will bind to anti-IL-23\nantibody on PrEXO-a23 before it arrives the inflammatory site. This study didn’t take into account MMP and IL-23 in serum.\nIts design has obvious defects.\n2. Figure 5g. PrEXO treated mice showed very higher IFN-gamma level in CD4 T cell compared with Dextran Sulfate\nSodium Salt (DSS) treated mice and healthy control mice. This phenotype suggests that PrEXO actually promote\ninflammation in IBD model. In addition, a23, anti-IL-23 antibody treated mice also showed quite higher IFN-gamma level in\nCD4 T cell compared with DSS treated mice and healthy control mice. This result also suggests that anti-IL-23 antibody can\nactually enhance inflammation. These results actually are against authors’ claims. Authors proposed that, at inflammation\nsites, MMP can cleavage PrEXO-a23 to release a23 and PrEXO to inhibit inflammation. Moreover, authors showed PrEXO-\na23 treated mice had almost no IFN-gamma expression in CD4 T cell. How could this happen? Both PrEXO and a23\ninduced high levels of IFN-gamma. How could PrEXO-a23, which combines PrEXO and a23, wipe out IFN-gamma\nexpression?\n3. Figure 2e, authors said that “The rEXO and PMV were then fused through additional coextrusion to generate hybrid\nvesicles (PrEXO).”Therefore, PrEXO should contain both exosomal and platelet markers. However, Figure 2e show that\nPrEXO has no exosomal proteins (ALIX, TSG101, and CCR6), platelet markers (GPIbα and CD62P). In contrast, the so\ncalled PrEXO-a23, originated from PrEXO, has ALIX, TSG101, and CCR6, as well as GPIbα and CD62P. Weirdly, PrEXO,\nwithout the conjugation of anti-IL-23 antibody, shows the presence of anti-IL-23 antibody (a23). One more problem is the\nmolecular weight of a23. a23 is a monoclonal rat antibody. It should have light and heavy chain. Light chain is around\n24kDa. Heavy chain is around 55kDa. The whole IgG molecule is around 150kDa. When performing western blot, IgG\nantibody will disassemble. Only light and heavy chain appear in the gel at around 25kDa and 55kDa. The 150kDa antibody\ncannot be detected. The 150kDa band in Figure 2e is not a23. Overall, this western blot demonstrates that PrEXO is wrong\nand the presence of anti-IL-23 antibody in PrEXO-a23 is suspicious.\n4. The quality of Flow cytometer data is poor. Supplementary Figure 7b, the staining of FOXP3 is very poor and not\nconvincing. Foxp3 is very specific for Treg cells. There is no clear FOXP3 positive cell population. How can authors tell\nthese cells are Tregs? In addition, it is CD4+FOXP3+ cell, not CD25+FOXP3+ cell mentioned in the manuscript (Line 274).\nThe staining of IL17A has the similar problem in Figure 7c and 6a.\n5. Authors claimed that “PrEXO-a23 treatment upregulated the expression of the tumor suppressor gene p53, which further\ninhibited CAC development (Fig.1).” (Line113-115). However, they fail to provide convincing evidence. The expression of\np53 was not confirmed by RT-PCR and western blot in the whole study. Figure 7b-n, the expression of p53 and its target\ngenes in the AOM/DSS-induced colon cancer model is missing. In Figure Extended Data Fig. 3, no data showed that the\nfunction of p53 was suppressed.\nMinor concerns:\n1. Figure 1. The most left of the Figure. There are some problems. Naïve T cell cannot inhibit Treg. Treg cannot inhibit IL-10\nand TGF-beta.\n2. Figure 2a，the size distribution of PMV, rEXO, PrEXO, PrEXO-a23 particles should be provided.\n3. Typing error: Line 376, “Although PrEXO showed Although PrEXO showed some potential to”.\n4. Supplementary Figure 12. There are seven Chinese words in c panel.", "reviewer_4": "This is an interesting paper by Cao et al. reporting a potential approach for management of IBD-related inflammation.\nThe setting is nicely explained, so that even with relatively superficial knowledge the reader can understand the rationale of\nthe experiment.\nI have a few comments, listed below, some touching areas where I am not an expert. While it is great that excess CAC risk\nwas reduced (actually eliminated), it is stretched to credit restored Trp53 expression for it.\n“Notably, p53 mRNA was suppressed in DSS-treated colons but restored to near-normal levels following PrEXOa23\ntherapy,”\nThe number of mice in each group was small. The exact numbers utilized for RNA-seq should be clearly stated. In addition\nto the volcano plot in the supplement, please give a supplementary table presenting the detailed expression results per\nanimal & gene, plus groups compiled, including statistics. Highlight Trp53 results.\nSo, short term effect of PrEXO-a23 is said to be restoration of Trp53 levels that had been reduced after DSS. Would one not\nexpect Trp53 to be upregulated after such a stress and insult? Please carefully look into the literature re TP53 expression in\nnormal epithelium of human IBD patients and murine models.\nSee eg from the abstract of Hartl et al. Science Advances 2024 doi: 10.1126/sciadv.adp8783 We find that p53 signaling is\nsilent and dispensable during homeostasis but strongly up-regulated in the epithelium upon DSS-induced colitis.\n(and references therein).\nOther comments:\nThroughout, please use correct nomenclature for murine genes and proteins.\nPage 6\n“Cells were treated with PBS, rEXO, PMV, PrEXO, a23, or PrEXO-a23, with untreated cells serving as controls”\nIt is not mentioned whether PMV produced any effect. This would serve as additional control.\n“We next examined the downstream effects of Th17 polarization on epithelial viability.”\nThe experiments are on cells, not epithelium.\nPage 7\n“The immature DCs (imDCs) were activated with lipopolysaccharide (LPS) and IFN-γ to induce maturation and\nsubsequently treated with various formulations, including PBS, rEXO, PMV, PrEXO, a23 or PrEXO-a23 for 24 h.”\nAlso here nothing is said re results with PMV.\nPage 10\n“These therapeutic effects likely result from the hybrid membrane-mediated lesion targeting, which delivers a23 to locally\ninhibit the IL-23/Th17 axis, combined with PrEXO’s role as an immune tolerance modulator to regulate the inflammatory\nmicroenvironment”\nThe anti-inflammatory agents within rEXO’s should be described. Any measurements that they contained the expected\nmolecules?\nPage 11\n“Although PrEXO showed Although PrEXO showed some potential to induce Tregs and suppress Th17 cells in vitro,”\nPlease delete extra words\nPage 12\n“Mice received 3% (w/v) DSS in drinking water for 7 days, followed by daily intravenous treatments as outlined in Fig. 6a.”\nConfusing timeline in text.\nPage 13\n“We next investigated microbiota modulation by PrEXO-a23 in IBD mice via 16S ribosomal RNA (rRNA) gene sequencing of\nfecal samples.”\nIs it unexpected to see these changes after eight days?"}, "author_response": "Point-by-point responses to Reviewers\nReviewer #1: Zhang et al. present an intriguing study entitled “Biomimetic Exosome-\nBased Nanotherapy for Targeted Immunomodulation in Inflammatory Bowel Disease”,\nwherein they propose PrEXO-a23—a dual-targeting nanoplatform combining Treg-\nderived exosomes and platelet membranes conjugated with IL-23 antibodies.\nThe rationale of leveraging both platelet-mediated homing and Treg immunomodulation is\ncompelling, and the therapeutic potential is conceptually promising. However, the\nmanuscript requires significant revision before it can be considered for publication in a\nhigh-impact journal like Nature Communications. Key mechanistic claims are currently\nunderdeveloped or insufficiently supported by experimental evidence, and several data\npresentation issues limit clarity and reproducibility.\nMajor Comments:\nQuestion 1. The MMP9 levels reported in colitic tissues (~50 ng/mL, Supplementary\nFig. 5) appear substantially lower than the concentrations used for in vitro cleavage\nstudies (Figure 2f). The authors should explain this discrepancy, and ideally justify\nthe physiological relevance of the in vitro model.\nResponse: We sincerely thank the reviewer for the positive comments and for pointing\nout this discrepancy. After careful review, we found an error in the description of\ncolonic MMP9 concentrations in the DSS-induced IBD model. The original unit of the\nMMP9 concentration value in Supplementary Fig. 5 (~35 ng/mL) should actually be\nng/mg. We sincerely apologize for this error and have corrected accordingly. We have\nalso provided the detailed ELISA assay procedure as below.\n1 mg of colon tissue was homogenized in 90 µL of PBS. Therefore, the original value\nof ~35 ng/mg corresponds to a corrected concentration of ~389 ng/mL (35 ng/mg × 1\nmg / 0.09 mL ≈ 389 ng/mL) in colon homogenates from IBD mice (Supplementary Fig.\n8b), which is more consistent with physiologically reported levels in inflamed IBD\ntissues (Gastroenterology 2011, 141, 1283-1292).\nAdditionally, we repeated the release experiments using 389 ng/mL MMP9 to mimic\nthe colonic concentration in vivo. The results confirmed that PrEXO-a23 showed\nresponsive a23 release under this condition, while the MMP9 inhibitor GM6001\nblocked this release (Fig. 2f), consistent with our initial findings.\nSupplementary Fig. 8b | MMP9 concentrations in colonic tissue of mice detected by ELISA. n =\n3 mice. Data are presented as mean ± s.d. Statistical significance was determined by Student's t-test\n(two-tailed).\nFig. 2f | ELISA quantification of a23 release under different treatments (PBS, MMP9 and MMP9 +\nMMP9 inhibitor). n = 3. Data are presented as mean ± s.d. Statistical significance was determined\nusing one-way ANOVA with Tukey's multiple comparisons test.\nQuestion 2. Is the dosage of PrEXO-a23 based on lyophilized vesicle weight? After\nco-extrusion at a 1:1 ratio, the concentration of each component should theoretically be\nhalved. The authors need to clarify how PrEXO-a23 retains comparable bioactivity to\nsingle-component vesicles in Figure 3, despite this dilution effect.\nResponse: We thank the reviewer for the comments. The dosage of PrEXO-a23 is based\non the amount of total loaded proteins, which were quantified by the bicinchoninic acid\nassay (BCA), not the weight of the lyophilized vesicles.\nIn this study, rEXO and a23 are the core components responsible for the key\nbioactivities. All experimental samples were carefully normalized to ensure equal\namounts of these active components across groups. Specifically, Treg-derived\nexosomes (rEXO) and platelet membrane vesicles (PMV) were first measured by BCA\nassay, and then fused at a 1:1 protein ratio to form PrEXO. PrEXO was then mixed with\na23 at a mass ratio of 1: 0.5 (e.g., 50 μg PrEXO: 25 μg a23), and unconjugated a23 was\nremoved by ultrafiltration to yield PrEXO-a23. Based on ELISA quantification, the\nfinal PrEXO-a23 contained 25 μg/mL PMV, 25 μg/mL rEXO, and 7.5 μg/mL a23.\nTherefore, in all in vitro experiments (e.g., Fig. 3), the single-component treatment\ngroups (PMV, rEXO, and a23) were administered at the same protein concentrations as\nthose in PrEXO-a23. PMV: 25 μg/mL; rEXO: 25 μg/mL; a23: 7.5 μg/mL; PrEXO-a23:\n25 μg/mL PMV + 25 μg/mL rEXO + 7.5 μg/mL a23.\nWe have added the descriptions of the dosing measurement in the Methods section.\nQuestion 3. Figure 4a suggests that platelet-membrane-derived proteins contribute to\nadhesion with collagen. Yet PMVs alone do not exhibit equivalent performance. Could\nthis discrepancy be explained by a protein corona effect, or by synergistic effects with\nexosomal components? Furthermore, the authors should examine the vesicle stability\nin whole blood and assess potential opsonization or clearance. Relevant literature, such\nas Nat Commun 2024, 15, 4876, may offer guidance. Additionally, organ-level\nbiodistribution data should be provided.\nResponse: We appreciate these important questions. Please find below our point-by-\npoint responses to each of them.\n(1) Regarding the issue that PMV alone cannot show the equivalent ability to adhere to\ncollagen. We would like to clarify that both PMV and rEXO alone exhibit moderate\nadhesion to collagen under flow, while PrEXO-a23 demonstrates significantly stronger\nbinding (Supplementary Fig. 12a). This enhancement was not primarily attributable to\nprotein corona effects, as the adhesion assay was performed in serum-free PBS buffer.\nInstead, we believe it was due to synergistic interactions between platelet and exosome\nmembrane components. Specifically, platelet-derived adhesion receptors (e.g., GPIbα,\nGPVI) provide high-affinity binding to collagen/exposed subendothelial matrix, while\nTreg exosomal tetraspanins (e.g., CD9, CD81) and integrins contribute to additional\navidity and membrane reorganization, promoting stable adhesion under shear stress\n(Blood 2017, 130, 2819-2828; Int. J. Mol. Sci 2021, 21, 7568).\n(2) Regarding your suggestion to measure the stability of vesicles in whole blood, we\nhave carefully reviewed the paper entitled “Exploiting histidine kinase-phosphatase\nduality to shape two-component signaling” (Nat Commun 2024, 15, 4876). This study\nfocused on regulating two-component signaling by modulating the kinase phosphatase\nbalance of sensing histidine kinases, and we did not find any techniques related to\nassessing vesicle stability or clearance. Therefore, we used the following methods to\nassess the stability of PrEXO-a23 in whole blood.\nPrEXO-a23 was incubated in mouse plasma at 37 °C, and its size and zeta potential\nwere monitored over 7 days.\nAs shown in Supplementary Fig. 9, no significant changes were observed, indicating\ngood stability in plasma. To measure blood clearance kinetics, DiD-labeled PrEXO-a23\nwas intravenously injected into mice, and blood was collected at predetermined time\npoints for quantitative fluorescence analysis. As shown in Supplementary Fig. 13c, the\nblood circulation half-life of PrEXO-a23 was ~1.44 h, and it was essentially cleared\nwithin 72 h.\nSupplementary Fig. 9 | Size and zeta potential of PrEXO-a23 in plasma over 7 days. n = 3. Data\nare presented as mean ± s.d.\nSupplementary Fig. 13c | Fluorescence images and normalized fluorescence analysis of DiD-\nPrEXO-a23 in blood collected from mice at the predetermined time points after intravenous\ninjection. n = 3 mice. Data are presented as mean ± s.d.\n(3) Regarding organ biodistribution, we performed ex vivo fluorescence imaging of\nmajor organs (heart, liver, spleen, lung, and kidney) in IBD mice receiving DiD-labeled\nPrEXO-a23. PrEXO-a23 primarily accumulated in the liver, which may be due to\nhepatic clearance (Supplementary Fig. 13b). Notably, PrEXO-a23 still demonstrated\nsignificant enrichment in inflammatory colon lesions (Fig. 4a-d in revision), supporting\nthat it preferentially accumulated at sites of inflammation.\nThese new data have been added as Supplementary Fig. 9 and Supplementary Fig. 13b\nand 13c, respectively. The corresponding descriptions have been revised in the\nmanuscript.\nSupplementary Fig. 13b | Ex vivo fluorescence images and the quantification of fluorescent radiant\nefficiency of different organs (heart, liver, spleen, lung, and kidney) of IBD mice 12 h after injection\nof DiD-labeled formulations. n = 3 mice. Data are presented as mean ± s.d.\nQuestion 4. Figure 4b shows increased uptake by macrophages and neutrophils. The\nauthors should consider whether intracellular degradation or trafficking might affect\nIL-23 antibody release kinetics or impair bioavailability.\nResponse: We sincerely appreciate the reviewer for raising this important point.\nAccording to your suggestion, we performed experiments to determine the IL-23\nantibody release in the presence or absence of RAW 264.7 macrophages under\nphysiologically relevant MMP9 conditions (389 ng/mL). As shown in Supplementary\nFig. 12d, although PrEXO-a23 showed strong adhesion to macrophages, it was still able\nto effectively release a23 at 389 ng/mL MMP9, which may be attributed to the MMP-\nresponsive linker connecting PrEXO and a23 by MMP9 (highly expressed in IBD\nlesions).\nThe data have been added as Supplementary Fig. 12d in the revised manuscript, and the\ncorresponding descriptions have been revised in the Results section.\nSupplementary Fig. 12d | a23 release from PrEXO-a23 in the presence or absence of RAW 264.7\ncells. n = 3. Data are presented as mean ± s.d.\nQuestion 5. The utility of the MMP9-cleavable linker hinges on differential\nbiodistribution and release of the antibody vs. carrier. Separate tracking of antibody and\ncarrier at the inflammatory site is essential to support this claim. Without this\nmechanistic evidence, the cleavable linker lacks justification. Moreover, line 563\nasserts successful separation of antibody and carrier post-cleavage—this must be\nexperimentally validated.\nResponse: We thank the reviewer for this constructive comment. To address the\nseparate tracking of the antibody and carrier at the inflammatory site, we performed\ndual-label tracking analysis in DSS-induced IBD mice to independently monitor the\nantibody and the carrier. Specifically, the antibody (a23) was labeled with Alexa Fluor\n647, and the carrier (PrEXO) was labeled with DiR. Three groups were included for\ncomparison: PrEXO + a23 (a physical mixture of PrEXO and a23), PrEXO-fL-a23\n(PrEXO and a23 were conjugated with a non-cleavable peptide, fL), and PrEXO-a23\n(PrEXO and a23 were conjugated with the MMP- cleavable peptide). Following\nintravenous administration, in vivo and ex vivo imaging was performed at\npredetermined time points to assess distribution and retention in DSS-induced IBD\nmice and isolated colons. As shown in Fig. 4f-h, in the PrEXO + a23 group, the DiR-\nPrEXO signal robustly accumulated in colonic lesions, peaked at 12 h and persisted for\nup to 48 h, demonstrating that PrEXO effectively targeted to inflamed colon tissues,\nconsistent with the targeting observed in Fig. 4a-c. However, the Alexa Fluor 647-a23\nsignal in this group was weak and rapidly diminished, becoming almost undetectable\nafter 12 h, indicating poor lesion-targeting and fast clearance of the free antibody. In\ncontrast, both the PrEXO-fL-a23 and PrEXO-a23 groups displayed PrEXO signals of\nsimilar intensity and duration to the physical mixture, but importantly, their a23 signals\nwere markedly stronger and more persistent (~11-fold higher than in the physical\nmixture, Fig. 4h). These results demonstrate that both PrEXO and a23 in PrEXO-a23\nwere effectively delivered to colonic lesions, which was attributed to the lesion-\ntargeting capability of PrEXO, enabling the co-delivery of a23 through linkage.\nNext, we performed CLSM analysis on colon tissue sections from IBD mice to confirm\nthe release of PrEXO and a23 following MMP-responsive cleavage of PrEXO-a23 at\nlesions. As shown in Fig. 4i, the non-cleavable PrEXO-fL-a23 group displayed strong\nspatial co-localization of a23 (green) and PrEXO (red), indicating the simultaneous\ndelivery of the antibody and carrier to lesions. In contrast, the clear separation of the\ntwo signals was detected in the PrEXO-a23 group, confirming that in situ cleavage of\nthe MMP-cleavable linker and subsequent release of a23 and PrEXO at the colonic\nlesions.\nTaken together, these findings demonstrate that PrEXO-a23 not only achieves targeted\nco-delivery of both the carrier and antibody to the site of inflammation but also\nundergoes efficient MMP-mediated cleavage to release the therapeutic antibody and\nPrEXO.\nThe data have been added as Fig. 4f-i in the revised manuscript, and the corresponding\ndescriptions have been revised in the Results section.\nFig. 4f-h | f, Representative in vivo fluorescence images of the whole body and isolated colon of\nIBD mice at the indicated time points after injection of PrEXO + a23, PrEXO-fL-a23 or PrEXO-\na23 (a23 labeled with Alexa Fluor 647; PrEXO labeled with DiR). g, Quantification of fluorescence\nradiant efficiency in the whole body of PrEXO (top) and a23 (bottom) in f. h, Quantification of\nfluorescence radiant efficiency in colonic tissue at 12 h after injection for PrEXO and a23 in f. n =\n5 mice. Data are presented as mean ± s.d. Statistical significance was determined using one-way\nANOVA with Tukey's multiple comparisons test.\nFig. 4i | Representative CLSM images (left) and quantitative analysis (right) of colonic tissue\nsections. Red: PrEXO; Green: a23. Scale bar, 20 μm.\nQuestion 6. Splenomegaly is a known indicator of systemic inflammation. The authors\nshould discuss whether elevated systemic MMP9 levels could lead to premature\nantibody release in circulation, compromising targeted delivery.\nResponse: We thank the reviewer for the valuable suggestion. According to the\nsuggestion, we performed ELISA to determine MMP9 levels in both the serum and the\ninflamed colonic tissue of IBD mice. As shown in Supplementary Fig. 8c, the serum\nMMP9 level was ~11 ng/mL, which is markedly lower than the level of ~378 ng/mL\ndetected in the inflamed colonic tissue (P < 0.0001). This significant difference in\nMMP9 concentration favors the MMP9-responsive cleavage of PrEXO-a23 to release\na23 specifically within the inflamed colonic tissue rather than in the circulation.\nWe further assessed the release kinetics of PrEXO-a23 at the serum MMP9\nconcentration of 11 ng/mL, and the results showed that less than 3.0% of a23 was\nreleased within 2 h (Fig. R1). Given that the circulatory half-life of PrEXO-a23 is ~1.44\nh, the low level of MMP9 in the blood results in negligible a23 release in the\nbloodstream. We have added the new data as Supplementary Fig. 8c in the revised\nmanuscript.\nSupplementary Fig. 8c | MMP9 concentrations in serum and colonic lesion of IBD mice measured\nby ELISA. n = 5 mice. Data are presented as mean ± s.d. Statistical significance was determined by\nStudent's t-test (two-tailed).\nFig. R1 | a23 release from PrEXO-a23 under 11 ng/mL of MMP9 in vitro. n = 3. Data are presented\nas mean ± s.d.\nMinor Comments:\nQuestion 7. All flow cytometry data should be accompanied by clearly defined gating\nstrategies to enhance reproducibility and transparency.\nResponse: We appreciate the reviewer’s suggestion. We have added detailed gating\nmethods for all flow cytometry experiments in the revised supplementary information.\nThese plots illustrate the sequential gating, including singlet selection, live cell gating,\nand marker-based population identification.\nSupplementary Fig. 22 | Flow cytometry gating strategy for dead NCM460 cells in vitro. The\ngating strategy was applied for Fig. 3e and Supplementary Fig. 10b.\nSupplementary Fig. 23 | Flow cytometry gating strategy for analysis of immune cells after\ntreatment with PrEXO-a23 in vitro. a, The gating strategy for CD4+IL-17A+ cells (Th17) of CD4+\nT cells, which was applied for Fig. 3b and Supplementary Fig. 10a. b, The gating strategy for\nCD80+CD86+ cells and MHC II+ cells of CD11c+ cells, which was applied for Fig. 3g and\nSupplementary Fig. 11a. c, The gating strategy for CD25+FOXP3+ cells (Tregs) of CD4+ T cells,\nwhich was applied for Fig. 3j and Supplementary Fig. 11b. d, The gating strategy for CD4+IL-17A+\ncells (Th17) of CD4+ T cells, which was applied for Fig. 3k and Supplementary Fig. 11c.\nSupplementary Fig. 24 | Flow cytometry gating strategy for DiI-positive Neutrophils in vitro. The\ngating strategy was applied for Supplementary Fig. 12c.\nSupplementary Fig. 25 | Flow cytometry gating strategy for DiD-positive immune cells in colonic\nlamina propria cells of IBD mice. The gating strategy was applied for Fig. 4e and Supplementary\nFig. 13d.\nSupplementary Fig. 26 | Flow cytometry gating strategy for analysis of immune cells in colonic\nlamina propria cells of IBD mice. a, The gating strategy for CD4+ FOXP3+ cells (Tregs) of CD3+ T\ncells. b, The gating strategy for CD4+ IFN-γ+ cells (Th1) and CD4+ IL-17A+ cells (Th17) of CD3+\nT cells. The gating strategy was applied for Fig. 5g, h.\nQuestion 8. In Figures 3b and 3g, the x-axis labels are unclear—does the labeling\ninclude the control group? Similar ambiguities may exist in other figures; consistent\nand precise labeling should be ensured throughout.\nResponse: We appreciate this suggestion. In the revised Fig. 3b (Th17 differentiation\nvia flow cytometry analysis), naïve T cells without any treatment served as the “Control”\ngroup. The treated groups were stimulated with IL-6 + IL-23, and then co-treated with\nPBS (Th17 group), rEXO (rEXO group), PMV (PMV group), PrEXO (PrEXO group),\na23 (a23 group), or PrEXO-a23 (PrEXO-a23 group), respectively. Similarly, in the\nrevised Fig. 3g (DC maturation via flow cytometry analysis), untreated immature DCs\n(imDCs) served as the negative control (imDCs group), while the other groups were\nstimulated with LPS + IFN-γ and then co-treated with PBS (mDC group), rEXO (rEXO\ngroup), PMV (PMV group), PrEXO (PrEXO group), a23 (a23 group), or PrEXO-a23\n(PrEXO-a23 group), respectively.\nWe have also revised the labeling in the affected panels (Fig. 3b-e, 3g, 3h, 3j and 3k) in\nthe revised manuscript, to clarify the experimental groups.\nFig. 3b | Flow cytometric quantification of Th17 cells (CD4+ IL-17A+) in CD4+ T cells. n = 4. Data\nare presented as mean ± s.d. Statistical significance was determined using one-way ANOVA with\nTukey's multiple comparisons test.\nFig. 3g | Flow cytometry analysis of the expression of CD80/CD86 and MHC II in CD11c+ cells for\nDCs. n = 5. Data are presented as mean ± s.d. Statistical significance was determined using one-\nway ANOVA with Tukey's multiple comparisons test.\nQuestion 9. For datasets with high variability (e.g., Figure 4d), the number of biological\nreplicates should be increased to ensure statistical robustness.\nResponse: We appreciate the reviewer for this kind suggestion. Accordingly, we have\nperformed in vivo targeting experiments in the DSS-induced IBD mouse model with 5\nmice per group. These data have been updated as Fig. 4a-c and Supplementary Fig. 13a\nin the revised manuscript.\nFig. 4a-c | a, Quantification of fluorescent radiant efficiency of the whole body in IBD mice at the\nindicated time points after injection of DiD-labeled formulations. b, c, Colon tissues were isolated\nand imaged at 12 h. Ex vivo fluorescence images (b) and quantification of fluorescent radiant\nefficiency (c) in isolated colonic tissues. n = 5 mice. Data are presented as mean ± s.d. Statistical\nsignificance was determined using one-way ANOVA with Tukey's multiple comparisons test.\nSupplementary Fig. 13a | In vivo biodistribution images of the whole body in IBD mice at the\nindicated time points after injection of DiD-labeled formulations. n = 5 mice.\nQuestion 10. The manuscript briefly mentions maintenance of immune tolerance (e.g.,\nLine 171). A more nuanced discussion is warranted, particularly regarding the dual role\nof Tregs in both suppressing inflammation and promoting fibrosis in chronic conditions.\nResponse: We thank the reviewer for raising this insightful and important comment. In\nthe revised manuscript, we have incorporated a more nuanced discussion to clarify the\ndual roles of Tregs in inflammation and fibrosis, with particular emphasis on how our\ntherapeutic strategy, PrEXO-a23, is designed to harness their beneficial functions while\nmitigating potential risks. The key points are summarized as follows.\n(1) The function of Tregs is stage-dependent: their dominant role is protective prior to\nthe establishment of fibrosis.\nPathological fibrosis represents a maladaptive repair outcome following sustained\nchronic inflammatory injury. Throughout this process, the role of Tregs evolves\ndynamically. During early disease stages and active inflammation, the core function of\nTregs is to suppress excessive immune responses and limit tissue damage, thereby\ncreating conditions conducive to proper repair. At this stage, expanding Tregs and\nenhancing their function constitute a key protective mechanism to prevent the\nprogression from inflammation to fibrosis (Sci. Transl. Med. 2025, 17, eadq3516; JCI\nInsight 2020, 5, e130651). Our therapy, PrEXO-a23, is designed to intervene in\ninflammatory bowel disease before intestinal fibrosis develops. By simultaneously\nneutralizing IL-23 (to inhibit Th17-driven inflammation) and delivering Treg-derived\nexosomes (to induce immune tolerance), it fundamentally corrects the immune\ndysregulation that drives fibrosis. As demonstrated in our results, PrEXO-a23 treatment\nsignificantly upregulated the proportion of Tregs in the colon and effectively prevented\nintestinal fibrosis in the fibrosis mouse model (Fig. 7l-q). This indicates that Tregs\ninduced at this stage primarily exert beneficial effects by suppressing inflammation and\npreventing the onset of fibrosis.\n(2) Treg function is tightly regulated by the local microenvironment: PrEXO-a23 shapes\na tolerogenic microenvironment that suppresses fibrosis.\nTreg functionality is not fixed or cell-intrinsic but is profoundly influenced by the\nsurrounding microenvironment. In a milieu dominated by strong pro-inflammatory\ncytokines such as TNF-α, IL-6, and IL-17, Tregs may be reprogrammed or lose stability,\nand may even indirectly promote fibroblast activation and extracellular matrix\ndeposition, thereby exacerbating fibrosis. In contrast, within an immunologically\nbalanced or tolerogenic microenvironment, Tregs predominantly exert their classic\nanti-inflammatory and immunoregulatory functions (Sci. Immunol. 2022, 7, eabi4613;\nCirculation 2019, 139, 206-221; J. Leukoc. Biol. 2020, 107, 573-587). The novelty of\nour nanotherapeutic strategy, PrEXO-a23, lies in its ability not merely to increase the\nproportion of Tregs, but to synergistically reshape the local immune microenvironment:\nthe a23 component effectively clears key pro-inflammatory signals such as IL-23, while\nthe rEXO component actively induces tolerogenic dendritic cells (tDCs) and anti-\ninflammatory cytokines including IL-10. Thus, PrEXO-a23 establishes a\nmicroenvironment that favors the anti-inflammatory and immunoregulatory function of\nTregs, rather than their pro-fibrotic potential. This resembles the physiological state in\nhealthy individuals, where abundant Tregs are present without triggering fibrosis.\nIn summary, we agree that Tregs may exhibit pro-fibrotic potential in advanced disease\nstages or under specific microenvironmental conditions, which is a limitation of many\ncurrent mono-modal therapies. PrEXO-a23, however, is a multi-targeted and stage-\nspecific combination therapy specifically designed to guide Tregs toward exerting\nbeneficial immunosuppressive and tolerogenic functions during the inflammatory phase,\nbefore fibrosis occurs, thereby preemptively blocking the pathological progression to\nfibrosis. Our experimental results robustly demonstrate the efficacy of this strategy.\nWe have incorporated a more nuanced discussion in the revised manuscript as follows.\n“PrEXO-a23 mitigated fibrotic progression through a coordinated dual mechanism. By\nneutralizing IL-23, it could suppress Th17-driven inflammation, while simultaneously\ndelivering Treg-derived exosomes to promote immune tolerance. This combined anti-\ninflammatory and pro-tolerance strategy not only corrected the underlying immune\ndysregulation before pathological fibrosis occurs but also reprogrammed the local\nmicroenvironment to favor beneficial immunosuppressive and tolerogenic Treg\nfunctions, thereby preemptively blocking the pathological progression toward fibrosis.”\nQuestion 11. The rationale for using MMP9-responsive linkers should be clearly\nintroduced in the opening section. The biological significance and therapeutic relevance\nof such linkers in IBD should be explicitly stated.\nResponse: We thank the reviewer for the thoughtful suggestion. We have revised the\nintroduction to clearly highlight the biological significance and therapeutic relevance\nof such MMP9-responsive linkers.\nSpecifically, matrix metalloproteinases (MMPs), particularly MMP9, are highly\noverexpressed and secreted by activated stromal and immune cells at inflamed\nintestinal lesions in IBD (J. Crohns Colitis 2018, 12, 1011-1013). Incorporating an\nMMP9-cleavable linker leverages the pathological microenvironment for controlled\ndrug release, thereby maximizing local therapeutic efficacy while minimizing systemic\nexposure and off-target immunosuppression. This strategy has been validated by prior\nworks. For instance, MMP-mediated cleavage enabled the inflammation site–selective\nrelease of a VEGF antibody, markedly improving its bioavailability and inhibitory\neffect on neovascularization (Nat. Biomed. Eng. 2021, 5, 968-982). Similarly, a\nmultifunctional PROTAC nanoplatform employing an MMP-cleavable peptide was\nactivated by tumor-overexpressed MMP-2, leading to responsive drug release that\nselectively eliminated tumor cells while reducing adverse effects (Nat. Commun. 2024,\n15, 10382). In this study, we therefore employed an MMP-cleavable linker to conjugate\nTreg–platelet hybrid vesicles (PrEXO) with an anti-IL-23 antibody (a23), producing\nPrEXO-a23. Thus, after intravenous administration of PrEXO-a23, it selectively\naccumulated at sites of damaged and inflamed colonic lesions due to platelet-mediated\ndamage adhesion and Treg-derived inflammation homing. Cleavage by MMPs\npromoted the release of a23 and PrEXO within MMP-enriched IBD lesions, thereby\nincreasing local antibody bioavailability and immunomodulatory activity while\nminimizing systemic exposure and off-target immunosuppression.\nAccordingly, we have revised the manuscript description with the following highlighted\ncontent:\n“The introduction of an MMP9-cleavable linker can exploit the pathological\nmicroenvironment to control drug release, thereby maximizing local therapeutic\nefficacy while minimizing systemic exposure and off-target immunosuppression. Thus,\nafter intravenous administration of PrEXO-a23, it selectively accumulated at sites of\ndamaged and inflamed colonic lesions due to platelet-mediated damage adhesion and\nTreg-derived inflammation homing. Cleavage by MMPs promoted the release of a23\nand PrEXO within MMP-enriched IBD lesions.”\nQuestion 12. In Lines 166 and 170, the text refers to statistical comparisons with\ncontrol groups, yet no corresponding statistical indicators (e.g., asterisks or p-values)\nare shown in the figures. This inconsistency should be rectified.\nResponse: We thank the reviewer for this comment. We have added the P-values in the\nFig. 3b-e and other figures in the revised manuscript.\nFig. 3b-e | b, Flow cytometric quantification of Th17 cells (CD4+ IL-17A+) in CD4+ T cells. c, d,\nELISA analysis of IL-17A (c) and TNF-α (d) levels in CD4+ T cell culture supernatants. e, Flow\ncytometric quantification of apoptotic rate in NCM460 cells. n = 4. Data are presented as mean ±\ns.d. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test.\nQuestion 13. Quantification data in Figure 4h would be more convincing if\naccompanied by representative flow cytometry histograms or dot plots.\nResponse: We appreciate the reviewer’s suggestion. We have added representative flow\ncytometry histograms corresponding to the quantification as shown in Supplementary\nFig. 13d.\nSupplementary Fig. 13d | Representative flow cytometry histograms of DiD-positive immune cells\nin colonic lamina propria including neutrophils, DCs, macrophages, T cells, and B cells.\nQuestion 14. The manuscript frequently refers to significant differences without\nspecifying the exact p-values. It is strongly recommended that the authors include exact\np-values wherever possible to enhance scientific rigor.\nResponse: Thanks for this kind suggestion. We have added the corresponding P-values\nfor all instances of reported significant differences.\nQuestion 15. The authors should carefully proofread the manuscript to eliminate\ntypographical errors and redundancies. For example, line 376 reads: “Although PrEXO\nshowed Although PrEXO showed some potential to...”.\nResponse: We thank the reviewer for pointing out this typographical error. We have\ncarefully reviewed the manuscript and corrected it as well as other minor errors\nthroughout the main text.\nReviewer #2: The article introduces PrEXO-a23, a novel nanotherapeutic for\ninflammatory bowel disease (IBD). It combines regulatory T cell (Treg)-derived\nexosomes (rEXO) with platelet membrane vesicles (PMV) and uses an MMP-cleavable\nlinker to attach IL-23 antibodies (a23). This design leverages the homing abilities of\nplatelets and Treg cells to target inflamed colonic tissues in IBD mice. At the inflamed\nsite, MMPs release a23, which neutralizes IL-23 and inhibits Th17-mediated\ninflammation. Meanwhile, rEXO reprograms dendritic cells and promotes Treg\nexpansion to restore immune tolerance. Tests show PrEXO-a23 reduces colitis severity,\nenhances epithelial barrier integrity, rebalances the gut microbiota, and prevents\ncomplications like intestinal fibrosis and colitis-associated colorectal cancer. Overall,\nPrEXO-a23 shows potential for durable immune reprogramming and long-term\nmanagement of IBD and its complications. However, there are still a few issues that\nneed to be addressed:\nQuestion 1. The material preparation section is too concise and doesn't elaborate on\ndetermining the optimal concentrations of the binder and a23.\nResponse: We appreciate the reviewer’s insightful suggestions. We have added a more\ndetailed description of the material preparation, including the optimization of the ratios\nbetween the linker and a23, as well as between linker-a23 and PrEXO. The specific\nprocedures are described below.\nAn MMP-cleavable peptide linker (Arg-Val-Gly-Leu-Pro, RVGLP) with an NHS ester\nat the Arg terminus and a 6-maleimidocaproyl (6-Mal) group at the Pro terminus was\nused to conjugate an antibody (a23) to the hybrid vesicles (PrEXO). To determine the\noptimal molar ratio for linker-a23 conjugation, a23 (1 mg/mL) was reacted with the\nlinker at molar ratios (a23: linker) of 1:1, 1:10, 1:50, 1:75, or 1:100 in PBS buffer at\n4 °C for 12-18 h, forming linker-a23 via NHS-amine condensation. Unreacted linker\nwas removed by centrifugation at 8,000 g for 30 min using a centrifugal filter (100 kDa).\nThe purified linker-a23 was analyzed using matrix-assisted laser desorption ionization\ntime of flight mass spectrometry (MALDI-TOF MS, AXIMA-Performance MA,\nShimadzu). As shown in Supplementary Fig. 5a, the molar ratio of 1:50 (corresponding\nto 1:4 w/w in the original manuscript) resulted in the highest conjugation efficiency\n(∼60%).\nWe next optimized the mass ratio for conjugating linker-a23 to PrEXO. PrEXO was\nprepared by co-extrusion of platelet membrane vesicles (PMV) and Treg-derived\nexosomes (rEXO) at a 1:1 protein mass ratio, followed by protein quantification using\na BCA assay. PrEXO was treated with 1 mM tris (2-carboxyethyl) phosphine (TCEP)\nsolution at 37 °C for 30 min to expose sulfhydryl (SH) groups on its membrane.\nPretreated PrEXO was then added to purified linker-a23 at a mass ratio (PrEXO: a23)\nof 1:0.1 ,1:0.25, 1:0.5, 1:1, or 1:2, and incubated at 25 °C for 1 h to induce the reaction\nbetween the SH groups of PrEXO and the 6-Mal groups of linker. Unreacted linker-a23\nwas removed by centrifugation at 8,000 g for 30 min using a centrifugal filter (300 kDa).\nELISA was used to determine the conjugation efficiency of a23 to PrEXO, which\nshowed that a PrEXO-to-a23 mass ratio of 1:0.5 provided the highest a23 conjugation\nefficiency of ~30% (Supplementary Fig. 5b).\nTherefore, throughout this study, a a23-to-linker molar ratio of 1:50 and a PrEXO-to-\na23 mass ratio of 1:0.5 were used to prepare the PrEXO-a23 formulations.\nSupplementary Fig. 5 | Optimization of the conjugation ratios for PrEXO-a23. a, The conjugation\nefficiency of linker to a23 at different molar ratio (a23: linker). b, The conjugation efficiency of a23\nto PrEXO at different mass ratio (PrEXO: a23). n = 3. Data are presented as mean ± s.d.\nQuestion 2. It's recommended that the role of Th17 cells in IBD be highlighted in the\nintroduction.\nResponse: We thank the reviewer for the valuable suggestion. We have revised the\nIntroduction section to highlight the pathogenic role of Th17 cells and their relevance\nto our therapeutic strategy.\nSpecifically, Th17 cells differentiated and expanded by the cytokine IL-23, produce\nproinflammatory cytokines such as IL-17A, which act on epithelial, stromal, and\nimmune cells to amplify inflammatory responses, disrupt epithelial barrier function,\nand recruit additional immune cells to the intestinal mucosa (Nat. Rev. Gastroenterol.\nHepatol. 2023, 20, 433-446; Cell 2020, 180, 79-91). Dysregulated Th17 responses also\ncontribute to chronic inflammation and promote tissue remodeling, fibrosis, and colitis-\nassociated carcinogenesis (Nature 2006, 441, 235-238). Therefore, targeting the Th17\naxis represents an important therapeutic approach in IBD. We have added the highlight\nin the Introduction sections as follows.\n“Th17 cells exacerbate IBD by secreting proinflammatory cytokines (e.g., IL-17A),\nwhich drive epithelial barrier dysfunction and chronic tissue damage, thereby\nperpetuating the inflammatory cascade.”\nQuestion 3. It's suggested that a discussion on rEXO's immune-regulating effects, such\nas inhibiting Th17 cell differentiation, be included or that further experiments on its\nrelated mechanisms be conducted.\nResponse: We thank the reviewer for this constructive suggestion. Treg-derived\nexosomes (rEXO) are enriched in Treg-derived immunomodulatory molecules,\nincluding proteins and miRNAs, which contribute to their function for immune\nmodulation (Nat. Biomed. Eng. 2021, 5, 968-982; Immunity 2014, 41, 89-103). We have\nadded more discussion on the immune-regulating effects of rEXO in the revised\nmanuscript.\nIn addition, we have employed proteomic analysis to confirmed that rEXO are enriched\nwith immunosuppressive proteins such as CTLA-4, LAG-3, IL-10, PD-L1, and TGF-\nβ1 (Fig. R2), suggesting its potential to suppress excessive immune activation (Acta\nBiomater. 2025, 198, 452-466). We have also performed additional Western blot\nvalidation, which indicated the abundant presence of key immunomodulatory proteins\nincluding IL-10 and CTLA-4 in rEXO (Supplementary Fig. 2).\nFig. R2 | The relative abundance of CTLA-4, LAG-3, IL-10, PD-L1, and TGF-β1 in rExo and IL-\n6-treated rExo using proteomic analysis. n = 4. Data are presented as mean ± s.d. (Acta Biomater.\n2025, 198, 452-466).\nSupplementary Fig. 2 | Western blot analysis of IL-10 and CTLA-4 in rEXO using an automated\nDigital CE-Western system.\nTo investigate the regulatory effects of rEXO on Th17 cell differentiation, we\nestablished a coculture model. Naïve CD4+ T cells were seeded in the upper chamber\nand treated with IL-6 and IL-23 to induce Th17 polarization. Cells were treated with\nPBS (Th17 group), rEXO (rEXO group), PMV (PMV group), PrEXO (PrEXO group),\na23 (a23 group), or PrEXO-a23 (PrEXO-a23 group), with untreated cells serving as\nthe negative control (Control group). Flow cytometry analysis revealed that IL-6 and\nIL-23 successfully induced the differentiation of naïve T cells into Th17 cells in the Th17\ngroup, as evidenced by an increased proportion of CD4+IL-17A+ cells compared to the\ncontrol (Fig. 3b). rEXO treatment showed effective inhibitory effects on reducing Th17\ncell differentiation (P = 0.0349). Meanwhile, ELISA analysis of supernatants revealed\nelevated levels of the proinflammatory cytokines IL-17A and TNF-α in the Th17 group,\nconsistent with Th17 activation. In contrast, rEXO treatment reduced the levels of these\ncytokines (Fig. 3c, d). These results suggest that rEXO can suppress the Th17-driven\ninflammation.\nFig. 3b-d | b, Flow cytometric quantification of Th17 cells (CD4+ IL-17A+) in CD4+ T cells. c, d,\nELISA analysis of IL-17A (c) and TNF-α (d) levels in CD4+ T cell culture supernatants. n = 4. Data\nare presented as mean ± s.d. Statistical significance was determined using one-way ANOVA with\nTukey's multiple comparisons test.\nGiven the role of Treg-derived exosomes in maintaining immune tolerance, we\nevaluated whether rEXO could reprogram DCs toward a tolerogenic phenotype.\nImmature DCs (imDCs) were stimulated with lipopolysaccharide (LPS) and IFN-γ to\ninduce mature DCs (mDCs) and subsequently treated with various formulations,\nincluding PBS, rEXO, PMV, PrEXO, a23, or PrEXO-a23 for 24 h. Untreated immature\nDCs (imDCs group) served as the negative control (Fig. 3f). The expression of surface\nmarkers and cytokine profiles was analyzed using flow cytometry and ELISA,\nrespectively. As expected, mDCs showed increased expression of CD80, CD86, and\nMHC II compared to imDCs (Fig. 3g), as well as increased production of IL-6 and\nTNF-α (Fig. 3h). In contrast, rEXO treatment resulted in downregulation of maturation\nmarkers and proinflammatory cytokines while promoting secretion of IL-10 and TGF-\nβ, indicating a shift toward a tolerogenic phenotype.\nTo further explore the potential of rEXO-induced tolerogenic DCs (tDCs) in promoting\nTreg differentiation, we cocultured the pretreated mDCs with naïve T cells for 72 h, and\nthe phenotypes of the T cells were determined via flow cytometry (Fig. 3i). Flow\ncytometry analysis revealed that PBS-treated mDCs from the mDC group did not\nsignificantly induce Treg differentiation compared to imDCs (P = 0.9998, Fig. 3j).\nHowever, the proportion of Tregs (CD25+FOXP3+) among naïve T cells was\nsignificantly increased in the rEXO-treated mDCs group compared to the mDCs group.\nAdditionally, these tDCs effectively reduced the proportion of (CD4+IL-17A+) Th17\ncells (Fig. 3k).\nCollectively, rEXO effectively induced Treg differentiation over Th17 cells by\npromoting DC polarization toward a tolerogenic phenotype, thereby supporting\nimmune tolerance.\nWe have added these data into the revised manuscript and expanded the discussion on\nthe immune-regulating effect of rEXO in the Results section.\nFig. 3f-h | f, Experimental schema of the shift of mDCs into tDCs. g, Flow cytometry analysis of\nthe expression of CD80/CD86 and MHC II in CD11c+ cells for DCs. h, ELISA analysis of IL-6,\nTNF-α, IL-10, and TGF-β in supernatant of DCs. n = 5. Data are presented as mean ± s.d. Statistical\nsignificance was determined using one-way ANOVA with Tukey's multiple comparisons test.\nFig. 3i-k | i, Experimental schema of T cell differentiation assay in naïve T cells co-cultured with\npretreated DCs, analyzed using flow cytometry. j, k, Polarization of Tregs (CD25+ FOXP3+) (j) and\nTh17 (CD4+ IL-17A+) cells (k) in CD4+ T cells. n = 5. Data are presented as mean ± s.d. Statistical\nsignificance was determined using one-way ANOVA with Tukey's multiple comparisons test.\nQuestion 4. The enrichment mechanism of this study deserves discussion. The material\ndeveloped in this study can be enriched in IBD lesions through the vWF factor highly\nexpressed in the inflamed intestine of IBD, and the targeting effectiveness has been\nverified in animal models. However, in clinical settings, IBD is associated with various\nextraintestinal inflammatory responses, such as skin inflammation, eye lesions, and\njoint involvement. How to prevent the material from accumulating in extraintestinal\nIBD lesions and whether it still has a therapeutic effect in these locations need to be\nexplored. Also, considering that vWF is highly expressed in many other diseases like\ncoronary heart disease and diabetes, how can we prevent this biomaterial from\naggregating in sites related to comorbidities with high vWF expression in patients, or\nwill it have a therapeutic effect after accumulation?\nResponse: We sincerely thank the reviewer for these insightful questions. To address\nthe enrichment mechanism of PrEXO-a23, we have performed additional experiments\nin relevant mouse models. Below, we outline the targeting mechanism and respond\npoint by point.\nPrEXO-a23 achieves site-specific enrichment in IBD lesions via a synergistic dual-\ntargeting strategy: Treg-derived exosomes (rEXO) express high-affinity integrins that\nbind to inflammation-upregulated endothelial adhesion molecules (e.g., ICAM-1,\nVCAM-1) for homing to inflamed sites (Gut 2019, 68, 1688-1700; Gut 2016, 65, 1642-\n1664), while platelet membrane vesicles (PMV) enable adhesion to exposed collagen\nin damaged vasculature through GPIbα-von Willebrand factor (vWF) interactions\n(Nature 2015, 526, 118-121; Nat. Rev. Immunol. 2023, 23, 495-510). This combined\nstrategy of “inflammatory homing + damage adhesion” allows PrEXO-a23 to\npreferentially accumulate in inflamed intestinal tissues, as demonstrated in our IBD\nmodels (Fig. 4).\n(1) Regarding potential accumulation and therapeutic effects in extraintestinal IBD\nmanifestations (e.g., skin inflammation): We developed an IBD-comorbid psoriasis\nmodel by administering 3% DSS in drinking water and applying topical imiquimod\n(IMQ) on the back skin for 7 days. DiD-labeled PrEXO-a23 was injected intravenously\ninto healthy (Health group) and comorbid (IBD + psoriasis group) mice, with\nbiodistribution monitored via an in vivo imaging. As shown in Supplementary Fig. 17,\nstrong fluorescence signals were observed not only in colonic lesions but also in\npsoriatic skin sites in the IBD + psoriasis group mice, whereas the signals were\nnegligible in healthy tissues. These results confirmed that PrEXO-a23 targets\ninflammation in sites beyond the colon, likely due to shared endothelial adhesion\nmolecules and vWF/collagen exposure in extraintestinal inflamed areas. This finding\nsuggests it could offer benefits for IBD comorbidities without undesired off-target\neffects in non-inflamed tissues.\nTo assess therapeutic efficacy, IBD + psoriasis mice were grouped into PBS and\nPrEXO-a23, with treatments administered intravenously every other day. As shown in\nSupplementary Fig. 18, compared to the PBS group (15% body weight loss, IBD\ndisease activity index (DAI) score ~10.2, colon length ~5.2 cm, Psoriasis DAI score\n~8.0), PrEXO-a23 treatment significantly alleviated symptoms, as evidenced by 5%\nweight loss, IBD score ~3.8, psoriasis score ~3.2, and colon length ~6.7 cm. These\nresults indicate that PrEXO-a23 not only accumulated in extraintestinal sites but also\nshowed therapeutic effects, possibly by modulating local inflammation and tolerance,\nconsistent with its dual-action design.\n(2) Concerning accumulation and effects in IBD comorbidities with high vWF\nexpression (e.g., acute wounds): We modeled IBD-comorbid acute wounds by 3% DSS\nadministration and dorsal skin excision. In vivo imaging of DiD-PrEXO-a23 showed\naccumulation in both colonic and wound sites in the IBD + wound group mice, with\nminimal signals in healthy controls (Fig. R3), indicating that its targeting is not organ-\nrestricted but extends to peripheral vWF-rich or damaged vasculature, which is\nattributable to PMV's collagen-binding affinity at injury sites.\nWhile PrEXO-a23 itself is designed as an immunomodulatory agent for IBD and may\nnot directly match the pathophysiology of non-inflammatory conditions like acute\nwounds, its strong lesion-homing capability suggests its potential utility as a targeted\ndelivery vehicle for combination therapies, for instance, co-loading with wound-\nhealing or disease-specific agents. To support this, recent studies (e.g., by Wang et al.)\nhave demonstrated that platelet-derived extracellular vesicles can serve as universal\ncarriers for inflammatory diseases including acute lung injury, atherosclerosis, and\nrheumatoid arthritis, highlighting the broad applicability of inflammation-targeting\nbiomaterials (Matter 2020, 3, 287-301). Such studies affirm the broad utility of\ninflammation/vWF-directed biomaterials, implying PrEXO-a23 can offer benefits in\nIBD comorbidities without undesired off-target effects in non-inflamed tissues.\nIn summary, the inflammation/damage dual-targeting strategy embodied by PrEXO-\na23-based on Treg exosome and platelet membrane hybridization—enables precise\ndrug delivery not only to inflamed intestines but also to extraintestinal inflammatory\nsites and other vWF-high pathological environments. This broader targeting capability\nmay offer a therapeutic advantage for IBD patients with systemic comorbidities.\nWe have added the new data as Supplementary Fig. 17 and 18 in the revised manuscript.\nSupplementary Fig. 17 | In vivo distribution of PrEXO-a23 in DSS- and IMQ-induced IBD-\ncomorbid psoriasis mice. a, Representative in vivo and ex vivo fluorescence imaging of intestine\nand dorsal skin in IBD-comorbid psoriasis mice at the indicated time points after injection of DiD-\nlabeled PrEXO-a23. b-e, Quantification of fluorescence radiant efficiency for PrEXO-a23 in vivo\nintestine (b), in vivo dorsal skin (c), ex vivo colon (d), and ex vivo dorsal skin (e). n = 4 mice. Data\nare presented as mean ± s.d. Statistical significance was determined by Student's t-test (two-tailed).\nSupplementary Fig. 18 | PrEXO-a23 effectively alleviates IBD and its comorbid psoriasis in vivo.\nIBD-comorbid psoriasis mice were induced using DSS and IMQ. a, b, Daily body weight changes\n(a) and DAI scores (b) of IBD-comorbid psoriasis mice obtained after PBS or PrEXO-a23 treatment.\nc, Photographs of colon tissues and the corresponding colon length on day 7. d, Representative\nimages of dorsal skins from day 0 to day 7. e, DAI scores for psoriasis measured from day 0 to day\n7. n = 5 mice. Data are presented as mean ± s.d. Statistical significance was determined by Student's\nt-test (two-tailed).\nFig. R3 | a, Representative in vivo and ex vivo fluorescence imaging of intestine and dorsal skin in\nIBD-comorbid acute wounds mice at the indicated time points after injection of DiD-labeled\nPrEXO-a23. b, c, Quantification of fluorescence radiant efficiency for PrEXO-a23 in vivo intestine\n(b), in vivo dorsal skin (c). n = 4 mice. Data are presented as mean ± s.d.\nQuestion 5. There seem to be some unexplained characters in Supplementary Fig. 12c.\nThe author should replace the image.\nResponse: We thank the reviewer for pointing out this comment. We have replaced the\nimage with a corrected version (Supplementary Fig. 21c).\nSupplementary Fig. 21c | Spleen index analysis of CAC mice after indicated treatment. n = 3 mice.\nData are presented as mean ± s.d. Statistical significance was determined by one-way ANOVA with\nTukey's multiple comparisons test.\nReviewer #3: In this study, Cao et al. made a nanotherapeutic material, PrEXO-a23, to\ntreat inflammatory bowel disease (IBD), by fusing regulatory T cell (Treg)-derived\nexosomes (rEXO) with platelet membrane vesicles, and conjugating IL-23 antibodies\n(a23) via a matrix metalloproteinase (MMP)-cleavable linker. Authors present in vitro\nand in vivo data to show PrEXO-a23 can reduce colitis severity, rebalance gut\nmicrobiota, and even prevent chronic complications including intestinal fibrosis and\ncolitis-associated colorectal cancer. However, its design has obvious defects, the key\ndata are not convincing and cannot support conclusions. These flaws prohibit its\npublication. The following are my comments.\nMajor concerns:\nQuestion 1. The design of this study highly depends on MMP activity to cleave the\nlinker and release anti-IL-23 antibody in inflammatory site. However, PrEXO-a23 was\ninjected intravenously into mice in the animal study. There are MMPs in the serum. The\nanti-IL-23 antibodies could be released from PrEXO-a23 in the serum before it arrives\nthe inflammatory site. In addition, IL-23 level is high in the serum when the body\nundergoes inflammation. The serum IL-23 will bind to anti-IL-23 antibody on PrEXO-\na23 before it arrives the inflammatory site. This study didn’t take into account MMP\nand IL-23 in serum. Its design has obvious defects.\nResponse: We thank the reviewer for these insightful questions.\n(1) To address whether the IL-23 antibody could be released from PrEXO-a23 in the\nserum before it arrives inflammatory sites, we first measured MMP9 levels in the serum\nand inflamed colonic tissues of IBD mice using ELISA. As shown in Supplementary\nFig. 8c, the serum MMP9 level was ~11 ng/mL, significantly lower than that in the\ninflamed colon tissue (~378 ng/mL; P < 0.0001). We further assessed the release\nkinetics of PrEXO-a23 at the serum MMP9 concentration (11 ng/mL), and the results\nshowed that less than 3.0% of a23 was released within 2 h (Fig. R4). Given the short\ncirculatory half-life of PrEXO-a23 (1.44 h; Supplementary Fig. 13c), the low MMP9 in\nthe blood results in negligible a23 release.\nAdditionally, we performed a dual-label in vivo tracking study to independently\nmonitor the distribution of the antibody (a23) and the carrier (PrEXO). As demonstrated\nin Fig. 4h, PrEXO-a23 showed strong and sustained accumulation of a23 signals in the\ncolonic lesions of IBD mice. These results further confirmed that the IL-23 antibody\nwas not prematurely released before reaching the inflammatory site.\nSupplementary Fig. 8c | MMP9 concentrations in serum and colonic lesion of IBD mice measured\nby ELISA. n = 5 mice. Data are presented as mean ± s.d. Statistical significance was determined by\nStudent's t-test (two-tailed).\nFig. R4 | a23 release from PrEXO-a23 under 11 ng/mL of MMP9 in vitro. n = 3. Data are presented\nas mean ± s.d.\nSupplementary Fig. 13c | Fluorescence images and normalized fluorescence analysis of DiD-\nPrEXO-a23 in blood collected from mice at predetermined time points after intravenous injection.\nn = 3 mice. Data are presented as mean ± s.d.\nFig. 4h | Quantification of fluorescence radiant efficiency in colonic tissue of IBD mice at 12 h after\ninjection. n = 5 mice. Data are presented as mean ± s.d. Statistical significance was determined by\none-way ANOVA with Tukey's multiple comparisons test.\n(2) Regarding whether systemic IL-23 during inflammation would bind to the IL-23\nantibody on PrEXO-a23 in the bloodstream, we first quantified IL-23 levels in both\nserum and inflamed colonic tissues of IBD mice. As shown in Fig. R5, the concentration\nof IL-23 in serum (~3 pg/mL) was significantly lower than that in the inflamed colon\ntissue (~22 pg/mL; P < 0.0001). We also measured IL-23 cytokine levels in the colonic\nlesions of IBD mice that received different treatments (Health, DSS, and PrEXO-a23\ngroups) to evaluate the biological effect of the delivered antibody. As shown in Fig. 5f,\nIL-23 levels were significantly elevated in the DSS group compared to the Health group\ndue to inflammation. In contrast, the PrEXO-a23 group showed a substantial reduction\nin IL-23, reaching levels comparable to those in the Health group, confirming that the\nIL-23 antibody delivered and released by PrEXO-a23 remained highly biologically\neffective.\nWe have incorporated these data in the revised manuscript, and the Methods section has\nbeen updated.\nFig. R5 | IL-23 concentrations at serum and colonic lesions of IBD mice measured by ELISA. n =\n5 mice. Data are presented as mean ± s.d. Statistical significance was determined by Student's t-test\n(two-tailed).\nFig. 5f | Cytokine IL-23 levels in colonic homogenates of IBD mice with indicated treatment. n = 5\nmice. Data are presented as mean ± s.d. Statistical significance was determined by one-way ANOVA\nwith Tukey's multiple comparisons test.\nQuestion 2. Figure 5g. PrEXO treated mice showed very higher IFN-gamma level in\nCD4 T cell compared with Dextran Sulfate Sodium Salt (DSS) treated mice and healthy\ncontrol mice. This phenotype suggests that PrEXO actually promote inflammation in\nIBD model. In addition, a23, anti-IL-23 antibody treated mice also showed quite higher\nIFN-gamma level in CD4 T cell compared with DSS treated mice and healthy control\nmice. This result also suggests that anti-IL-23 antibody can actually enhance\ninflammation. These results actually are against authors’ claims. Authors proposed that,\nat inflammation sites, MMP can cleavage PrEXO-a23 to release a23 and PrEXO to\ninhibit inflammation. Moreover, authors showed PrEXO-a23 treated mice had almost\nno IFN-gamma expression in CD4 T cell. How could this happen? Both PrEXO and\na23 induced high levels of IFN-gamma. How could PrEXO-a23, which combines\nPrEXO and a23, wipe out IFN-gamma expression?\nResponse: We sincerely appreciate the reviewer’s comments. We apologize for any\nmisunderstanding caused by the lack of clarity in our original figure labels. To address\nthis concern, we have revised the figure legends and re-performed flow cytometry\nanalysis to avoid potential misinterpretation.\nAs shown in Fig. 5g, the representative flow cytometry plots from left to right\ncorrespond to the following groups with their respective colors in the graph: Health\n(blue), DSS (red), PrEXO (purple), a23 (yellow), rEXO-a23 (cyan), and PrEXO-a23\n(green). The percentage of CD4+IFN-γ+ T cells was 0.43% in the Health group,\nsignificantly increasing to 20.80% in the DSS group (P < 0.0001), indicating elevated\ninflammation in IBD mice. PrEXO reduced this percentage to 13.00%, which was\nsignificantly lower than that in the DSS group (P = 0.0174), demonstrating its anti-\ninflammatory effect rather than promotion of inflammation, consistent with our\nprevious findings. Similarly, a23 treatment decreased it to 13.60% compared to the DSS\ngroup (P = 0.0061), aligning with known anti-inflammatory effects of a23 (Blood 2007,\n109, 1156-1164). Notably, PrEXO-a23 achieved the most substantial reduction in\nCD4⁺IFN-γ⁺ cells (1.45%), which was significantly lower than that in the DSS group\n(P < 0.0001). These results demonstrate a potent synergistic anti-inflammatory\nresponse, robustly supporting our conclusion.\nWe have added more detailed captions to Fig. 5g and 5h and updated Supplementary\nFig. 26b to illustrate the gating strategy for improved clarity.\nFig. 5g, h | Flow cytometric plots (g) and statistical analysis (h) of CD4+ IFN-γ+ T cells in colonic\nlamina propria. n = 5 mice. Data are presented as mean ± s.d. Statistical significance was determined\nby one-way ANOVA with Tukey's multiple comparisons test.\nSupplementary Fig. 26b | The gating strategy for CD4+ IFN-γ+ cells of CD3+ T cells. The gating\nstrategy was applied for Fig. 5g, h.\nQuestion 3. Figure 2e, authors said that “The rEXO and PMV were then fused through\nadditional coextrusion to generate hybrid vesicles (PrEXO).” Therefore, PrEXO should\ncontain both exosomal and platelet markers. However, Figure 2e show that PrEXO has\nno exosomal proteins (ALIX, TSG101, and CCR6), platelet markers (GPIbα and\nCD62P). In contrast, the so called PrEXO-a23, originated from PrEXO, has ALIX,\nTSG101, and CCR6, as well as GPIbα and CD62P. Weirdly, PrEXO, without the\nconjugation of anti-IL-23 antibody, shows the presence of anti-IL-23 antibody (a23).\nOne more problem is the molecular weight of a23. a23 is a monoclonal rat antibody. It\nshould have light and heavy chain. Light chain is around 24kDa. Heavy chain is around\n55kDa. The whole IgG molecule is around 150 kDa. When performing western blot,\nIgG antibody will disassemble. Only light and heavy chain appear in the gel at around\n25kDa and 55kDa. The 150kDa antibody cannot be detected. The 150kDa band in\nFigure 2e is not a23. Overall, this western blot demonstrates that PrEXO is wrong and\nthe presence of anti-IL-23 antibody in PrEXO-a23 is suspicious.\nResponse: We sincerely thank the reviewer for these professional comments.\n(1) Regarding the original Fig. 2e, the lane originally labeled as “PrEXO” did not show\nbands corresponding to exosomal proteins (ALIX, TSG101, and CCR6) or platelet\nmarkers (GPIbα and CD62P) but instead showed the presence of the IL-23 antibody\n(~150 kDa). After careful review, the lane labeled as “PrEXO” should be “IL-23\nantibody (a23)”, we apologize for this error and have corrected them in the revised\nfigure as shown in Fig. 2e and Supplementary Fig. 7.\n(2) Regarding the concern about the molecular weight of the IL-23 antibody (a23), we\nagree with the reviewer that under general Western blot conditions, which utilize\nreducing agents such as DTT and β-mercaptoethanol, IgG antibodies undergo disulfide\nbond reduction, resulting in dissociation into light (~25 kDa) and heavy chains (~50-55\nkDa), thereby preventing detection of the full-length ~150 kDa immunoglobulin. In this\nstudy, we employed an alternative immunodetection system, a fully automated Digital\nCapillary Electrophoresis-Western (CE-Western) platform for protein detection. This\nautomated CE technology uses proprietary-coated capillaries to perform size-based\nseparation, immobilization, immunoprobing, and detection in a single, integrated\nprocess. Critically, samples were prepared without reducing agents, enabling analysis\nunder non-reducing conditions. This preserves the native quaternary structure of\nantibodies, allowing the a23 antibody (IgG monoclonal antibody) to migrate at its intact\nmolecular weight of ~150 kDa. The CE-Western method is well established and has\nbeen extensively validated in peer-reviewed studies for accurate analysis of proteins in\nnative or semi-native states (Nat. Biomed. Eng. 2021, 5, 968-982; Nature 2025, 638,\n1095-1103; Nat. Commun. 2025, 16, 7375).\nWe sincerely apologize for the lack of detailed methods in our original manuscript. We\nhave now added a detailed description of the Digital CE-Western system to the Methods\nsection and, crucially, have explicitly stated that the analysis was performed under non-\nreducing conditions.\nFig. 2e | Western blot analysis of protein markers of rEXO, PMV, a23, and PrEXO-a23. Full scans\nare provided in Supplementary Fig. 7.\nSupplementary Fig. 7 | The full scans of Fig. 2e. Western blot analysis of rEXO, PMV, a23, and\nPrEXO-a23 using an automated Digital CE-Western system.\nQuestion 4. The quality of Flow cytometer data is poor. Supplementary Figure 7b, the\nstaining of FOXP3 is very poor and not convincing. Foxp3 is very specific for Treg\ncells. There is no clear FOXP3 positive cell population. How can authors tell these cells\nare Tregs? In addition, it is CD4+FOXP3+ cell, not CD25+FOXP3+ cell mentioned in\nthe manuscript (Line 274). The staining of IL17A has the similar problem in Figure 7c\nand 6a.\nResponse: We sincerely thank the reviewer for pointing out these questions.\nAs noted by the reviewer, CD4⁺CD25⁺FOXP3⁺ constitutes the specific markers for\nTregs, and we fully adhere to this definition in our gating strategy as mentioned in our\nmanuscript. We apologize for the error in the original Supplementary Fig. 7b where\n“CD25” was mistakenly labeled as “CD4”, and this has been corrected in the revised\nmanuscript. As shown in Supplementary Fig. 11b, the data presented in this panel in\nfact represent CD25⁺FOXP3⁺ Tregs, not CD4⁺FOXP3⁺ cells as previously misstated,\nand a clear population of CD25⁺FOXP3⁺ Tregs is observable.\nRegarding the concern about the FOXP3 staining, we have now added a representative\nsingle-positive control for FOXP3 as shown in Fig. R6, which confirms the presence of\na distinct FOXP3⁺ population and validates the specificity of our staining. We have also\nprovided the corresponding complete gating strategy for CD25⁺FOXP3⁺ T cells, as\nshown in Supplementary Fig. 23c.\nRegarding the concern about IL-17A staining, we have repeated the flow cytometry\nexperiments and the results have been added as Supplementary Fig. 10a and\nSupplementary Fig. 11c. These results demonstrated clear and distinct CD4+IL-17A⁺\ncell populations. To further ensure the rigor and reproducibility of our analysis, we have\nalso provided the complete gating strategy for these assays (Supplementary Fig. 23a\nand 23d).\nSupplementary Fig. 11b | Flow cytometric plots of Tregs (CD25+ FOXP3+) in CD4+ T cells that\nwere coculture with DCs pretreated with indicated formulations.\nFig. R6 | Flow cytometric plots of FOXP3+ cells in CD4+CD25+T cells that were coculture with\nDCs pretreated with indicated formulations.\nSupplementary Fig. 23c | The gating strategy for CD25+ FOXP3+cells (Tregs) of CD4+ T cells,\nwhich was applied for Fig. 3j and Supplementary Fig. 11b.\nSupplementary Fig. 10a | Flow cytometric plots of Th17 (CD4+ IL-17A+) cells in CD4+ T cells\ntreated with indicated formulations under stimulation with IL-6 and IL-23.\nSupplementary Fig. 11c | Flow cytometric plots of Th17 (CD4+ IL-17A+) cells in CD4+ T cells\nthat were coculture with DCs pretreated with indicated formulations.\nSupplementary Fig. 23a | The gating strategy for CD4+ IL-17A+ cells (Th17) of T cells, which\nwas applied for Fig. 3b and Supplementary Fig. 10a.\nSupplementary Fig. 23d | The gating strategy for CD4+ IL-17A+ cells (Th17) of T cells, which\nwas applied for Fig. 3k and Supplementary Fig. 11c.\n5. Authors claimed that “PrEXO-a23 treatment upregulated the expression of the tumor\nsuppressor gene p53, which further inhibited CAC development (Fig.1).” (Line113-\n115). However, they fail to provide convincing evidence. The expression of p53 was\nnot confirmed by RT-PCR and western blot in the whole study. Figure 7b-n, the\nexpression of p53 and its target genes in the AOM/DSS-induced colon cancer model\nis missing. In Figure Extended Data Fig. 3, no data showed that the function of p53 was\nsuppressed.\nResponse: We thank the reviewer for these insightful suggestions. As suggested, we\nperformed additional experiments using RT-qPCR and Western blot to examine the\nexpression of p53 and its downstream target genes, to assess both the transcriptional\nactivity and functional status of the p53 pathway. The RT-qPCR results revealed that,\ncompared to the healthy control group, the AOM/DSS group exhibited significant\ndownregulation of Trp53 mRNA, along with its target genes Cdkn1a (which promotes\ncell cycle arrest) and Bax (which promotes apoptosis). Notably, treatment with PrEXO-\na23 led to the upregulation of these genes compared to the AOM/DSS group (Fig. 8h).\nHowever, this recovery was abolished upon p53 inhibition (PrEXO‑a23 + ip53).\nWestern blot analysis revealed that PrEXO-a23 treatment upregulated the expression of\np53, p21, and Bax compared to the AOM/DSS group, restoring their levels to those\ncomparable to the healthy controls (Fig. 8i, j). These findings indicate that PrEXO-a23\nexerts inhibitory effects on colitis-associated colorectal cancer through modulation of\nthe p53 signaling pathway.\nFig. 8h | h, Relative mRNA expression levels of Trp53, Cdkn1a, and Bax in colonic tissues of CAC\nmice with indicated treatment measured by RT-qPCR. n = 5 mice. Data are presented as mean ± s.d.\nStatistical significance was determined by one-way ANOVA with Tukey's multiple comparisons test.\nFig. 8i, j | Western blot images (i) and relative protein level analysis (j) of p53, p21, and Bax\nexpressed in colonic tissues. n = 3 mice. Data are presented as mean ± s.d. Statistical significance\nwas determined by one-way ANOVA with Tukey's multiple comparisons test.\nMinor concerns:\nQuestion 5. Figure 1. The most left of the Figure. There are some problems. Naïve T\ncell cannot inhibit Treg. Treg cannot inhibit IL-10 and TGF-beta.\nResponse: We apologize for the error in the schematic of Figure 1. We have corrected\nthem, and in the revised Fig. 1, these arrows have been corrected to directional arrows\n(→) marked with an “×”.\nFig. 1 | Schematic illustration of the engineered platelet-Treg hybrid nanovesicles system (PrEXO-\na23) for targeted immune modulation and tolerance induction in inflammatory bowel disease.\nQuestion 6. Figure 2a，the size distribution of PMV, rEXO, PrEXO, PrEXO-a23\nparticles should be provided.\nResponse: According to the reviewer’s suggestion, we have added the size distribution\nof rEXO, PMV, PrEXO, and PrEXO-a23 as shown in Supplementary Fig. 6 in the\nrevised manuscript.\nSupplementary Fig.6 | Size distribution of rEXO, PMV, PrEXO, and PrEXO-a23.\nQuestion 7. Typing error: Line 376, “Although PrEXO showed Although PrEXO\nshowed some potential to”.\nResponse: We apologize for this error. We have corrected it and have carefully\nproofread the manuscript to ensure such errors are eliminated throughout.\nQuestion 8. Supplementary Figure 12. There are seven Chinese words in c panel.\nResponse: We apologize for this error. We have corrected them in the revised\nmanuscript as follows.\nSupplementary Fig. 21c | Spleen index analysis of CAC mice after indicated treatment. n = 3 mice.\nData are presented as mean ± s.d. Statistical significance was determined by one-way ANOVA with\nTukey's multiple comparisons test.\nReviewer #4: This is an interesting paper by Cao et al. reporting a potential approach\nfor management of IBD-related inflammation. The setting is nicely explained, so that\neven with relatively superficial knowledge the reader can understand the rationale of\nthe experiment. I have a few comments, listed below, some touching areas where I am\nnot an expert.\nQuestion 1: While it is great that excess CAC risk was reduced (actually eliminated),\nit is stretched to credit restored Trp53 expression for it. “Notably, p53 mRNA was\nsuppressed in DSS-treated colons but restored to near-normal levels following\nPrEXOa23 therapy,” The number of mice in each group was small. The exact numbers\nutilized for RNA-seq should be clearly stated. In addition to the volcano plot in the\nsupplement, please give a supplementary table presenting the detailed expression\nresults per animal & gene, plus groups compiled, including statistics. Highlight Trp53\nresults. So, short term effect of PrEXO-a23 is said to be restoration of Trp53 levels that\nhad been reduced after DSS. Would one not expect Trp53 to be upregulated after such\na stress and insult? Please carefully look into the literature re TP53 expression in normal\nepithelium of human IBD patients and murine models.See eg from the abstract of Hartl\net al. Science Advances 2024 doi: 10.1126/sciadv.adp8783 We find that p53 signaling\nis silent and dispensable during homeostasis but strongly up-regulated in the epithelium\nupon DSS-induced colitis.\n(and references therein).\nResponse: We thank the reviewer for these insightful comments. Please find below our\npoint-by-point responses to each of them.\n(1) Regarding sample size and RNA-seq data presentation. We performed RNA-seq\nanalysis using 5 mice per group (n = 5), which we have clearly stated in the Methods\nsection and figure legends for clarity (Fig. 7a and Supplementary Fig. 20). In response\nto your suggestion to present the detailed expression results per animal & gene, we will\nsubmit the data as an Excel file, but it cannot be organized as a supplementary table in\nthe Supporting Information due to too many gene data points. The Excel file contains\ndetailed gene expression data for each animal (e.g., normalized TPM values), group\nmeans, log2 fold changes, adjusted P values, and statistics for all differentially\nexpressed genes (DEGs) in the Health, DSS, and PrEXO-a23 groups.\nThrough mouse experiments and bioinformatics analysis based on RNA-seq results,\nTrp53 is an important regulatory gene for CAC risk, and restoration of Trp53 is of great\nsignificance for preventing the occurrence of CAC. We have emphasized this important\nconclusion in the revised manuscript, and added a new bar graph (Fig. R7) to show\nTrp53 mRNA levels and their statistical comparisons (e.g., DSS vs. Health: P = 0.0034;\nPrEXO-a23 vs. DSS: P = 0.0024).\nFig. R7 | Trp53 mRNA expression levels in colonic tissues analyzed by RNAseq. n = 5 mice. Data\nare presented as mean ± s.d. Statistical significance was determined by one-way ANOVA with\nTukey's multiple comparisons test.\n(2) Regarding the apparent discrepancy between the upregulation of Trp53 in DSS-\ninduced colitis described by you and Hartl et al. (Sci. Adv 2024, adp8783), and the\ndecrease in Trp53 mRNA levels observed in this study, we have performed a\ncomprehensive and careful review and discussion of Trp53 expression in IBD patients\nand mouse models in the published references and the study of this paper. The published\ndata showed that p53 protein was generally upregulated in DSS-induced IBD.\nInterestingly, Trp53 mRNA levels showed different trends with disease progression,\nmodel and sampling time, highlighting the complexity of Trp53 regulation (Science\n2012, 336, 1440-1444; Nat Cell Biol 2025, 27, 1342-1356; Nat Commun 2025, 16, 3264;\nImmunity 2025, 58, 381-396).\nBecause we initially focused only on mRNA changes and did not assess protein\nexpression, we could not directly compare mRNA levels with protein expression results\nreported in the published paper. Therefore, we performed additional Western blot\nanalysis on colon tissues from DSS-induced IBD mice. The results showed that p53\nprotein was significantly elevated in the DSS group compared with healthy controls (P\n= 0.0273), along with elevated expression of its downstream targets p21 and Bax (Fig.\nR8), consistent with the report by Hartl et al. Interestingly, after PrEXO-a23 treatment,\nthese elevated proteins returned to levels comparable to those in healthy mice, which\nmay be attributed to its anti-inflammatory and immunomodulatory effects, thereby\neffectively protecting intestinal cells and alleviating acute cellular damage.\nFurthermore, we evaluated the expression of Trp53 and its downstream target genes\n(Cdkn1a and Bax) using RT-qPCR. The results showed that Trp53, Cdkn1a, and Bax\nmRNA were significantly downregulated in the DSS group compared with healthy mice.\nNotably, PrEXO-a23 treatment also upregulated the expression of these genes to levels\nsimilar to those in the healthy group (Fig. R9). Our results, as well as those from the\nreferences confirm that the p53 signaling pathway plays a key role in IBD, which is\nconsistent with our core conclusion.\nTo further demonstrate whether PrEXO-a23 can suppress CAC by regulating the p53\nsignaling pathway, we collected colonic tissue from mice with AOM/DSS-induced\nCAC and analyzed the expression of p53 and its downstream target factors using RT-\nqPCR and Western blot. RT-qPCR results showed that compared with healthy mice,\nmRNA levels of Trp53 and its target genes, Cdkn1a (which promotes cell cycle arrest)\nand Bax (which promotes apoptosis) were significantly downregulated in the\nAOM/DSS group. However, PrEXO-a23 treatment upregulated their expression (Fig.\n8h). However, this restorative effect was abolished by p53 inhibition (PrEXO-a23 +\nip53). We further examined the protein expression of p53, p21, and Bax. As shown in\nFig. 8i, j, compared to the AOM/DSS group, PrEXO-a23 treatment upregulated the\nexpression of p53, p21, and Bax, restoring them to levels comparable to the healthy\ngroup. These results indicate that PrEXO-a23 inhibits CAC by regulating the p53\nsignaling pathway.\nFig. R8 | Western blot images and relative protein level analysis of p53, p21, and Bax expressed in\ncolonic tissues of IBD mice with indicated treatment. n = 5 mice. Data are presented as mean ± s.d.\nStatistical significance was determined by one-way ANOVA with Tukey's multiple comparisons test.\nFig. R9 | Relative mRNA expression levels of Trp53, Cdkn1a, and Bax in colonic tissues of IBD\nmice with indicated treatment measured by RT-qPCR assay. n = 5 mice. Data are presented as mean\n± s.d. Statistical significance was determined by one-way ANOVA with Tukey's multiple\ncomparisons test.\nFig. 8h | Relative mRNA expression levels of Trp53, Cdkn1a, and Bax in colonic tissues of CAC\nmice with indicated treatment measured by RT-qPCR assay. n = 5 mice. Data are presented as mean\n± s.d. Statistical significance was determined by one-way ANOVA with Tukey's multiple\ncomparisons test.\nFig. 8i, j | Western blot images (i) and relative protein level analysis (j) of p53, p21, and Bax\nexpressed in colonic tissues. n = 3 mice. Data are presented as mean ± s.d. Statistical significance\nwas determined by one-way ANOVA with Tukey's multiple comparisons test.\nQuestion 2. Throughout, please use correct nomenclature for murine genes and proteins.\nResponse: We thank the reviewer for this kind suggestion. We have corrected them in\nthe revised manuscript.\nQuestion 3 Page 6. “Cells were treated with PBS, rEXO, PMV, PrEXO, a23, or PrEXO-\na23, with untreated cells serving as controls” It is not mentioned whether PMV\nproduced any effect. This would serve as additional control.\nResponse: We appreciate the reviewer's thoughtful suggestion. We have revised the\nrelevant paragraphs in the Results section according to your suggestions.\nTo investigate the regulatory effects of PrEXO-a23 on Th17 cell differentiation and its\npotential to protect intestinal epithelial cells, we established a coculture model. Naïve\nCD4+ T cells were seeded in the upper chamber and treated with IL-6 and IL-23 to\ninduce Th17 polarization. Cells were treated with PBS (Th17 group), rEXO (rEXO\ngroup), PMV (PMV group), PrEXO (PrEXO group), a23 (a23 group), or PrEXO-a23\n(PrEXO-a23 group), with untreated cells serving as the negative control (Control\ngroup). After 72 h of treatment, colonic epithelial NCM460 cells were seeded into the\nlower chamber and cocultured with the pretreated T cells for an additional 24 h to\nevaluate the epithelial response (Fig. 3a).\nFlow cytometry analysis revealed that IL-6 and IL-23 successfully induced the\ndifferentiation of naïve T cells into Th17 cells in the Th17 group, as evidenced by an\nincreased proportion of CD4+IL-17A+ cells compared to the Control group (Fig. 3b).\nCompared with the Th17 group, PMV treatment had no inhibitory effects on Th17 cell\ndifferentiation. Meanwhile, ELISA analysis of supernatants revealed elevated levels of\nthe proinflammatory cytokines IL-17A and TNF-α in the Th17 group, consistent with\nTh17 activation. There was no significant difference in the secretion of these\ninflammatory cytokines in the PMV group (Fig. 3c, d).\nWe next examined the effects of Th17 polarization on epithelial cell viability. The results\nshowed that apoptosis and necrosis of NCM460 cells were significantly increased when\nco-cultured with Th17 cells in the Th17 group (P < 0.0001), indicating epithelial cell\ndamage. Similarly, PMV alone had minimal protective effect against Th17-induced\nepithelial damage (Fig. 3e). These findings collectively demonstrate that PMV has no\nsignificant regulatory effect on Th17-driven inflammatory responses.\nFig. 3a-e | a, Experimental schema of the co-culture system between naïve T cells and NCM460\ncells. b, Flow cytometric quantification of Th17 cells (CD4+ IL-17A+) in CD4+ T cells. c, d, ELISA\nanalysis of IL-17A (c) and TNF-α (d) levels in CD4+ T cell culture supernatants. e, Flow cytometric\nquantification of apoptotic rate in NCM460 cells. n = 4. Data are presented as mean ± s.d. Statistical\nsignificance was determined by one-way ANOVA with Tukey's multiple comparisons test.\nQuestion 4 “We next examined the effects of Th17 polarization on epithelial viability.”\nThe experiments are on cells, not epithelium.\nResponse: Thanks for the reviewer’s suggestion. We have revised the text as follows.\n“We next examined the effects of Th17 polarization on epithelial cell viability.”\nQuestion 5 Page 7. “The immature DCs (imDCs) were activated with\nlipopolysaccharide (LPS) and IFN-γ to induce maturation and subsequently treated with\nvarious formulations, including PBS, rEXO, PMV, PrEXO, a23 or PrEXO-a23 for 24\nh.” Also here nothing is said re results with PMV.\nResponse: We thank the reviewer for this valuable suggestion.\nGiven the role of Treg-derived exosomes in maintaining immune tolerance, we\nevaluated whether PrEXO-a23 could reprogram mature DCs (mDCs) toward a\ntolerogenic phenotype. The immature DCs (imDCs) were stimulated with\nlipopolysaccharide (LPS) and IFN-γ to induce mDCs and subsequently treated with\nvarious formulations, including PBS, rEXO, PMV, PrEXO, a23 or PrEXO-a23 for 24\nh. Untreated immature DCs (imDCs group) served as the negative control (Fig. 3f). The\nexpression of surface markers and cytokine profiles were analyzed using flow cytometry\nand ELISA, respectively. As expected, mDCs showed increased expression of CD80,\nCD86, and MHC II compared to imDCs (Fig. 3g), as well as increased production of\nIL-6 and TNF-α (Fig. 3h). PMV treatment did not significantly affect these markers or\ncytokines on mDCs.\nTo further explore the potential of PrEXO-a23-induecd DCs in promoting Treg\ndifferentiation, we cocultured the pretreated DCs with naïve T cells for 72 h, and the\nphenotypes of the T cells were determined via flow cytometry (Fig. 3i). Flow cytometry\nanalysis revealed that mDCs from the mDCs group did not significantly induce Treg\ndifferentiation compared to imDCs (P = 0.9998; Fig. 3j). Likewise, PMV-treated mDCs\nfailed to promote Treg differentiation (Fig. 3j), and showed a negligible effect on the\nproportion of Th17 (CD4+ IL-17A+) cells (Fig. 3k). Collectively, these results indicate\nthat PMV does not induce immune tolerance under these conditions.\nFig. 3f-h | f, Experimental schema of the shift of mDCs into tDCs. g, Flow cytometry analysis of\nthe expression of CD80/CD86 and MHC II in CD11c+ cells for DCs. h, ELISA analysis of IL-6,\nTNF-α, IL-10, and TGF-β in supernatant of DCs. n = 5. Data are presented as mean ± s.d. Statistical\nsignificance was determined by one-way ANOVA with Tukey's multiple comparisons test.\nFig. 3i-k | i, Experimental schema of T cell differentiation assay in naïve T cells co-cultured with\npretreated DCs, analyzed using flow cytometry. j, k, Polarization of Tregs (CD25+ FOXP3+) (j) and\nTh17 (CD4+ IL-17A+) cells (k) in CD4+ T cells. n = 5. Data are presented as mean ± s.d. Statistical\nsignificance was determined by one-way ANOVA with Tukey's multiple comparisons test.\nQuestion 6 Page 10 “These therapeutic effects likely result from the hybrid membrane-\nmediated lesion targeting, which delivers a23 to locally inhibit the IL-23/Th17 axis,\ncombined with PrEXO’s role as an immune tolerance modulator to regulate the\ninflammatory microenvironment” The anti-inflammatory agents within rEXO’s should\nbe described. Any measurements that they contained the expected molecules?\nResponse: We thank the reviewer for the constructive suggestion. Treg-derived\nexosomes (rEXO) are indeed enriched in a variety of immunomodulatory molecules,\nincluding proteins and miRNAs, which contribute to their immunomodulatory\nfunctions (Nat. Biomed. Eng. 2021, 5, 968-982; Immunity 2014, 41, 89-103).\nTo address this point, we have performed proteomic analysis to confirm that rEXO\ncarried multiple immunosuppressive proteins, such as CTLA-4, LAG-3, IL-10, PD-L1,\nand TGF-β1, suggesting their potential to suppress excessive immune activation (Fig.\nR10; Acta Biomater. 2025, 198, 452-466).\nWe also performed additional Western blot validation, and the results clearly showed\nthat rEXO were enriched in key immunoregulatory proteins such as IL-10 and CTLA-\n4 (Supplementary Fig. 2). These results directly demonstrate that rEXO contain the\nexpected immunosuppressive molecules, likely contributing to their therapeutic effects.\nFig. R10 | The relative abundance of CTLA-4, LAG-3, IL-10, PD-L1, and TGF-β1 in rEXO and\nIL-6-treated rEXO using proteomic analysis. n = 4. Data are presented as mean ± s.d. (Acta Biomater.\n2025, 198, 452-466).\nSupplementary Fig. 2 | Western blot analysis of IL-10 and CTLA-4 in rEXO using automated\nDigital CE-Western system.\nQuestion 7 Page 11. “Although PrEXO showed Although PrEXO showed some\npotential to induce Tregs and suppress Th17 cells in vitro,”\nPlease delete extra words\nResponse: We thank the reviewer for pointing out the typographical error. We have\ncorrected them in the revised manuscript.\nQuestion 8 Page 12. “Mice received 3% (w/v) DSS in drinking water for 7 days,\nfollowed by daily intravenous treatments as outlined in Fig. 6a.” Confusing timeline in\ntext.\nResponse: We apologize for this confusion. We have revised the description in the\nmanuscript as follows.\n“Mice received 3% (w/v) DSS in drinking water for 7 days. Meanwhile, mice were\ntreated on days 2, 4, and 6 by intravenous administration, as outlined in Fig. 6a.”\nFig. 6a | Experimental design for evaluating the therapeutic effect of PrEXO-a23 in the IBD mouse\nmodel.\nQuestion 9 Page 13. “We next investigated microbiota modulation by PrEXO-a23 in\nIBD mice via 16S ribosomal RNA (rRNA) gene sequencing of fecal samples.” Is it\nunexpected to see these changes after eight days?\nResponse: We thank the reviewer for raising this important point.\nInflammation rapidly alters the intestinal physicochemical environment (e.g., oxidative\nstate and nitrate levels) and compromises epithelial integrity, thereby imposing\nselective pressures that favor the expansion of inflammation-adapted pathobionts and\nsuppress beneficial commensals. Such changes ultimately lead to functional disruption\nof the microbiota in IBD (Science, 2013, 339, 708-711; Gut Microbes. 2024, 16,\n2361493).\nOur results demonstrated that PrEXO-a23 functioned synergistically through two\nmechanisms. First, it alleviated intestinal inflammation by targeting and neutralizing\nIL-23, inhibiting Th17 cell differentiation, and reducing pro-inflammatory cytokines.\nSecond, it actively promoted immune tolerance by inducing tolerogenic dendritic cells\nand regulatory T cells. Restoration of immune balance was accompanied by significant\nimprovement in epithelial barrier function. With reduced inflammation and restored\nbarrier integrity, the pathological colonic microenvironment was effectively reversed,\ninhibiting pathogenic bacterial overgrowth and promoting overall microbiome\nrestoration. Thus, the changes we observed after 8 days of treatment are appropriate\nand highlight the effectiveness of PrEXO-a23 in rapidly reshaping the intestinal\necosystem through integrated immune and barrier regulation. These effects are\nconsistent with previous findings in DSS-induced IBD models, in which interventions\ntargeting inflammation or barrier repair have been shown to induce detectable\nmicrobiota changes within 7 to 10 days (Nat. Mater. 2020, 19, 118-126; Nat. Commun.\n2025, 16, 3365; Nat. Commun. 2024, 15, 1333; Nat. Rev. Immunol. 2016, 16, 295-309).\nPoint-by-point responses to Reviewers\nReviewer #1 (Remarks to the Author): Comments: The revised manuscript has\nsatisfactorily addressed the majority of the concerns previously raised by this reviewer.\nOnly a few minor points remain for further consideration:\nQuestion 1. Supplementary Fig. 13b: It would be valuable to include a more intuitive\ncomparison of fluorescence intensity specifically at the colon site. As the\nbiodistribution profiles across the four groups show negligible differences in the liver,\npresenting a higher target-to-non-target ratio (e.g., colon/liver) could better emphasize\nthe targeting efficacy of the system.\nResponse: We thank the reviewer for this valuable suggestion. According to your\nsuggestion, we have supplemented the ex vivo colon fluorescence data and calculated\nthe per-animal target-to-non-target ratio (colon/liver radiant efficiency). As shown in\nSupplementary Fig. 13c-e, although the fluorescence intensity of liver was comparable\nacross groups, the colon/liver ratios for both PrEXO and PrEXO-a23 were significantly\nhigher than for rEXO, PMV, and the physical mixture (P < 0.05). These data support\nour conclusion that membrane fusion synergistically enhances the targeting efficacy of\nPrEXO-a23 toward colonic lesions.\nSupplementary Fig. 13c-e | c, Representative ex vivo fluorescence images of major organs\n(heart, liver, spleen, lung, kidney, and colon) collected from IBD mice following injection of\nDiD-labeled formulations. d, Quantitative analysis of fluorescence radiant efficiency for each\nharvested organ. e, Colon-to-liver ratio of fluorescence radiant efficiency. n = 3 mice. Data are\npresented as mean ± s.d. Statistical significance was determined by one-way ANOVA with\nTukey's multiple comparisons test.\nQuestion 2. Supplementary Fig. 8c: Direct comparison between the concentration\nobtained from colon tissue homogenates and that from plasma is not recommended.\nThe concentration in tissue homogenates inherently depends on the dilution factor\nintroduced by the PBS volume used during homogenization; thus, it is not directly\ncomparable to the concentration measured in serum.\nResponse: We sincerely appreciate the reviewer for raising this important point. To\naddress this concern, we have reanalyzed the data using protein concentration\nnormalization. Specifically, we determined the total protein concentrations in colon\ntissue homogenates and plasma samples using the BCA assay, which were ~1.12\nmg/mL (for colon tissue homogenates prepared with 90 μL PBS per 1 mg tissue) and\n~5.01 mg/mL (for plasma), respectively. The MMP9 concentrations were then\nnormalized to nanograms per milligram of total protein (ng/mg). After normalization,\nthe MMP9 concentration in colon tissue was 338 ng/mg (378 ng/mL ÷ 1.12 mg/mL =\n338 ng/mL), while that in plasma was 2.2 ng/mg (11 ng/mL ÷ 5.01 mg/mL = 2.2 ng/mL).\nThe updated results are presented in Supplementary Fig. 8c. This normalized analysis\ndemonstrates that the MMP9 concentration in plasma is significantly lower than that in\ncolon tissue (P < 0.0001), thereby more reliably confirming our original findings.\nSupplementary Fig. 8c | MMP9 concentrations in serum and colonic lesion of IBD mice\nmeasured by ELISA and normalized to total protein. n = 5 mice. Data are presented as mean ±\ns.d. Statistical significance was determined by Student's t-test (two-tailed).\nReviewer #2 (Remarks to the Author): Thank you for submitting the revised manuscript\nand for your detailed responses to the reviewers’ comments. I have carefully reviewed\nthe revised version and the authors’ point-by-point replies. The authors have addressed\nall reviewers’ previous concerns thoroughly and made substantial improvements to the\nmanuscript. I believe this work is suitable for publication in its current form.\nResponse: We sincerely thank the reviewer for the kind words and thoughtful\nevaluation of our revised manuscript. Your positive feedback on the manuscript is\nhighly valued. Thank you again for your time and insightful feedback.\nReviewer #4 (Remarks to the Author): This is a revised version of the study by Cao et\nal \"Engineered exosome nanovesicles efficiently deliver antibodies in vivo to treat\ninflammatory bowel disease\". Much of this work is outside my field and I was pleased\nto see three other expert reviewers tackling the task. I can see that the authors have gone\nthrough significant effort in order to address all comments. My main concern, however,\nwas the claimed mechanism, originally described as “Notably, PrEXO-a23 treatment\nalso upregulates the expression of the tumor suppressor gene p53, further enhancing\nprotection against inflammation-driven carcinogenesis.” The additional data is not that\nhelpful in view of this claim. As might have been expected through previous literature,\nthe inflammation itself increases Trp53 protein expression, and probably the respective\npathway. This effect is then reduced after the treatment. It is difficult to say what causes\nthis reduction; whether e.g. it is just a bystander effect of easing inflammation.\nNevertheless, it is of course interesting that the protein level decreases. But then, Trp53\nmRNA levels increase after PrEXO-a23 treatment. What would be the purpose of that?\nTaken together, the evidence and story could be more robust. It might be wise to leave\nthe mechanism open.\nResponse: We sincerely thank the reviewer for these insightful comments, which have\nbeen instrumental in refining our mechanistic interpretation.\nAs is known, p53 is a crucial tumor suppressor that maintains cellular homeostasis by\nregulating cell cycle arrest, apoptosis, and DNA repair (Gastroenterology 2022, 162,\n715–730; Nat Commun 2023, 14, 110). In IBD, excessive p53 activation can drive\nepithelial cell death and exacerbate tissue injury (Sci Adv 2024, adp8783), while loss or\ndysfunction of p53 serves as an established early driver of colitis-associated colorectal\ncarcinogenesis (Cancer Cell 2013, 23, 634-646; Nat Cancer 2024, 5, 983-995; Nat\nGenet 2023, 55, 1531–1541). Therefore, therapeutic benefit derives not from simply\nincreasing or decreasing p53 expression, but from maintaining homeostasis of the p53\nsignaling pathway. Both chronic loss of p53 and uncontrolled acute hyperactivation are\ndetrimental, whereas homeostasis of p53 signaling confers protection (Cell 2009, 140:\n384-396).\nThe mRNA-protein discordance is frequently observed for p53, which can be attributed\nto the fact that p53 expression is a highly dynamic process governed by multiple\ncomplex regulatory factors (Cancer Cell 2024, 42:946-967): (i) Post-translational\nmodifications: Inflammation-induced phosphorylation or acetylation can stabilize p53\nprotein, leading to its accumulation despite unchanged or decreased mRNA levels\n(Signal Transduct Target Ther 2020, 5, 90; Cancer Cell 2024, 42:946-967). (ii)\nNegative feedback mechanisms: Immune interventions can modulate the MDM2-\nmediated ubiquitin-proteasome pathway, leading to rapid clearance in p53 protein\nlevels that are independent of its mRNA abundance (Nat Rev Cancer 2020, 20, 471-\n480.). These mechanisms may collectively account for the frequent discordance\nbetween p53 mRNA and protein levels.\nTo investigate the effect of PrEXO-a23 on p53 pathway in IBD, we performed Western\nblot analysis on colon tissues from a DSS-induced IBD model. Western blot analysis\nrevealed that compared with healthy controls, the IBD mice exhibited a significant\nincrease in p53 protein levels, along with elevated expression of its downstream targets\np21 and Bax (Fig. R1), indicating aberrant p53 activation in IBD. Following PrEXO-\na23 treatment, these elevated protein levels returned to levels comparable to those in\nhealthy mice, demonstrating that PrEXO-a23 helps maintain homeostasis of the p53\nsignaling pathway. Additionally, RT‑qPCR analysis showed downregulation of Trp53,\nCdkn1a, and Bax mRNA in IBD mice relative to healthy controls (Fig. R2). Notably,\nPrEXO-a23 treatment maintained the expression levels of these genes to those observed\nin the healthy group. Collectively, these data indicate that PrEXO-a23 contributes to\nthe maintenance of p53 signaling homeostasis in IBD.\nWe further evaluated the anti-CAC efficacy of PrEXO-a23 in an AOM/DSS-induced\nCAC mouse model. PrEXO-a23 treatment completely inhibited tumor formation,\ndemonstrating its potent anti-carcinogenic effect and capacity to halt IBD-to-CAC\nprogression. Mice were then co-treated with the p53 inhibitor. This intervention\nmarkedly diminished the therapeutic efficacy of PrEXO-a23 and negated its anti-tumor\neffects (Fig. 8a-g), supporting that the anti-tumor action of PrEXO-a23 is at least\npartially p53-dependent. Furthermore, we analyzed the expression of p53 and its\ndownstream targets in colon tissues from CAC mice using RT‑qPCR and Western blot.\nCompared with the healthy control group, Trp53 mRNAs were decreased, and Cdkn1a\nand Bax were downregulated in the CAC mice, suggesting dysfunction of the p53\npathway during CAC progression. Notably, treatment with PrEXO‑a23 maintained\ntheir expression (Trp53/Cdkn1a/Bax) to near-normal levels (Fig. 8h). However, this\nmaintenance was abolished upon p53 inhibition. Western blot results confirmed these\nchanges in the levels of p53, p21, and Bax proteins (Fig. 8i, j). These findings suggest\nthat PrEXO‑a23 treatment contributes to the maintenance of p53 signaling homeostasis,\nwith the potential to reduce the risk of progression from IBD to CAC.\nBased on your feedback and our experimental data, we think that the action of PrEXO-\na23 cannot be simply characterized as “up-regulating” or “down-regulating” p53, but\ncontributes to the maintenance of p53 pathway homeostasis. p53 pathway homeostasis\nplays a critical role in anti-tumor efficacy. Accordingly, we have revised the initial\nstatement, “Notably, PrEXO-a23 treatment also upregulates the expression of the tumor\nsuppressor gene p53, further enhancing protection against inflammation-driven\ncarcinogenesis.” to the more scientifically rigorous conclusion: “Notably, PrEXO-a23\ntreatment also contributes to the maintenance of p53 signaling homeostasis, with the\npotential to reduce the risk of progression from IBD to CAC.”\nAccordingly, we have revised the manuscript as follows.\nAbstract section: “Notably, PrEXO-a23 treatment also contributes to the maintenance\nof p53 signaling homeostasis, with the potential to reduce the risk of progression from\nIBD to CAC.”\nIntroduction section: “Notably, PrEXO-a23 contributes to the maintenance of p53\nsignaling homeostasis, which potentially further inhibited CAC development (Fig. 1).”\nResult section: “To evaluate the long-term therapeutic potential of PrEXO-a23 in\npreventing IBD-associated complications such as intestinal fibrosis and CAC, we\nemployed an AOM/DSS-induced CAC mouse model. Mice were administered AOM\nfollowed by repeated cycles of DSS to induce chronic colitis and tumorigenesis, with\nuntreated healthy mice serving as controls (Health group). During disease progression,\nmice received intravenous injections of PBS (AOM/DSS group), PrEXO (PrEXO group),\na23 (a23 group), rEXO-a23 (rEXO-a23 group) or PrEXO-a23 (PrEXO-a23 group)\n(Fig. 7b). As expected, AOM/DSS-treated mice exhibited significantly reduced survival\n(P = 0.0208; Fig. 7c), substantial weight loss (Supplementary Fig. 21a), and\nsignificantly elevated DAI scores (Fig. 7d), shortened colon length, and increased\ntumor burden, both in terms of number and size (Fig. 7e-g and Supplementary Fig. 21b).\nAmong all treatment groups, PrEXO-a23 demonstrated the robust therapeutic efficacy,\nmarkedly improving survival, body weight, DAI, and colon morphology to levels\ncomparable with healthy controls. Importantly, while PrEXO, a23, and rEXO-a23\ntreatments conferred partial protection, none of these monotherapies prevented tumor\ndevelopment. In contrast, PrEXO-a23 treatment completely inhibited tumor formation,\nhighlighting its potent anti-carcinogenic effect and capacity to halt IBD-to-CAC\nprogression.\nWe previously demonstrated that PrEXO-a23 affected the Trp53 expression change in\nDSS-treated colons. Mice were co-treated with the p53 inhibitor pifithrin-α. This\nintervention markedly diminished the therapeutic efficacy of PrEXO-a23 and negated\nits anti-tumor effects (Fig. 8a-g), further supporting that the action of PrEXO-a23 in\npreventing CAC progression is at least partially dependent on p53. Notably, the p53\npathway plays a crucial role in tumor suppression, with a mechanism involving various\nfactors such as Cdkn1a/p21 and Bax. Therefore, we investigated the expression of p53,\nCdkn1a/p21, and Bax in colonic tissues using RT-qPCR and Western blot analysis.\nCompared with the health control group, Trp53 mRNAs were decreased, and Cdkn1a\nand Bax were downregulated in the AOM/DSS group, suggesting dysfunction of the p53\npathway during CAC progression. Notably, treatment with PrEXO‑a23 maintained\ntheir expression (Trp53/Cdkn1a/Bax) to near-normal levels (Fig. 8h). However, this\nmaintenance was abolished upon p53 inhibition (PrEXO‑a23 + ip53). Western blot\nresults confirmed these changes in the levels of p53, p21, and Bax proteins (Fig. 8i, j).\nThese findings suggest that PrEXO‑a23 treatment contributes to the maintenance of\np53 signaling homeostasis, with the potential to reduce the risk of progression from\nIBD to CAC. Collectively, PrEXO-a23 not only attenuates active inflammation but also\nprovides durable protection against fibrosis and tumorigenesis, highlighting it as a\npromising therapeutic strategy for the long-term management of IBD and its\ncomplications.”\nConclusion section: “Additionally, it contributes to the maintenance of p53 signaling\nhomeostasis, potentially reducing the risk of progression from IBD to CAC.”\nFig. R1 | Western blot images and relative protein level analysis of p53, p21, and Bax expressed\nin colonic tissues of IBD mice with indicated treatment. n = 5 mice. Data are presented as mean\n± s.d. Statistical significance was determined by one-way ANOVA with Tukey's multiple\ncomparisons test.\nFig. R2 | Relative mRNA expression levels of Trp53, Cdkn1a, and Bax in colonic tissues of\nIBD mice with indicated treatment measured by RT-qPCR assay. n = 5 mice. Data are presented\nas mean ± s.d. Statistical significance was determined by one-way ANOVA with Tukey's\nmultiple comparisons test.\nFig. 8h | Relative mRNA expression levels of Trp53, Cdkn1a, and Bax in colonic tissues of\nCAC mice with indicated treatment measured by RT-qPCR assay. n = 5 mice. Data are\npresented as mean ± s.d. Statistical significance was determined by one-way ANOVA with\nTukey's multiple comparisons test.\nFig. 8i, j | Western blot images (i) and relative protein level analysis (j) of p53, p21, and Bax\nexpressed in colonic tissues. n = 3 mice. Data are presented as mean ± s.d. Statistical\nsignificance was determined by one-way ANOVA with Tukey's multiple comparisons test."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Recommendation: Minor Revision\nComments:\nThe revised manuscript has satisfactorily addressed the majority of the concerns previously raised by this reviewer. Only a\nfew minor points remain for further consideration:\nSupplementary Fig. 13b:\nIt would be valuable to include a more intuitive comparison of fluorescence intensity specifically at the colon site. As the\nbiodistribution profiles across the four groups show negligible differences in the liver, presenting a higher target-to-non-target\nratio (e.g., colon/liver) could better emphasize the targeting efficacy of the system.\nSupplementary Fig. 8c:\nDirect comparison between the concentration obtained from colon tissue homogenates and that from plasma is not\nrecommended. The concentration in tissue homogenates inherently depends on the dilution factor introduced by the PBS\nvolume used during homogenization; thus, it is not directly comparable to the concentration measured in serum.", "reviewer_2": "Thank you for submitting the revised manuscript and for your detailed responses to the reviewers’ comments. I have\ncarefully reviewed the revised version and the authors’ point-by-point replies. The authors have addressed all reviewers\nprevious concerns thoroughly and made substantial improvements to the manuscript. I believe this work is suitable for\npublication in its current form.", "reviewer_4": "This is a revised version of the study by Cao et al \"Engineered exosome nanovesicles efficiently deliver antibodies in\nvivo to treat inflammatory bowel disease\". Much of this work is outside my field and I was pleased to see three other expert\nreviewers tackling the task. I can see that the authors have gone through significant effort in order to address all comments.\nMy main concern, however, was the claimed mechanism, originally described as \"Notably, PrEXO-a23 treatment also\nupregulates the expression of the tumor suppressor gene p53, further enhancing protection against inflammation-driven\ncarcinogenesis.\" The additional data is not that helpful in view of this claim. As might have been expected through previous\nliterature, the inflammation itself increases Trp53 protein expression, and probably the respective pathway. This effect is\nthen reduced after the treatment. It is difficult to say what causes this reduction; whether e.g. it is just a bystander effect of\neasing inflammation. Nevertheless it is of course interesting that the protein level decreases. But then, Trp53 mRNA levels\nincrease after PrEXO-a23 treatment. What would be the purpose of that? Taken together, the evidence and story could be\nmore robust.\nIt might be wise to leave the mechanism open."}, "author_response": "Point-by-point responses to Reviewers\nReviewer #1 (Remarks to the Author): Comments: The revised manuscript has\nsatisfactorily addressed the majority of the concerns previously raised by this reviewer.\nOnly a few minor points remain for further consideration:\nQuestion 1. Supplementary Fig. 13b: It would be valuable to include a more intuitive\ncomparison of fluorescence intensity specifically at the colon site. As the\nbiodistribution profiles across the four groups show negligible differences in the liver,\npresenting a higher target-to-non-target ratio (e.g., colon/liver) could better emphasize\nthe targeting efficacy of the system.\nResponse: We thank the reviewer for this valuable suggestion. According to your\nsuggestion, we have supplemented the ex vivo colon fluorescence data and calculated\nthe per-animal target-to-non-target ratio (colon/liver radiant efficiency). As shown in\nSupplementary Fig. 13c-e, although the fluorescence intensity of liver was comparable\nacross groups, the colon/liver ratios for both PrEXO and PrEXO-a23 were significantly\nhigher than for rEXO, PMV, and the physical mixture (P < 0.05). These data support\nour conclusion that membrane fusion synergistically enhances the targeting efficacy of\nPrEXO-a23 toward colonic lesions.\nSupplementary Fig. 13c-e | c, Representative ex vivo fluorescence images of major organs\n(heart, liver, spleen, lung, kidney, and colon) collected from IBD mice following injection of\nDiD-labeled formulations. d, Quantitative analysis of fluorescence radiant efficiency for each\nharvested organ. e, Colon-to-liver ratio of fluorescence radiant efficiency. n = 3 mice. Data are\npresented as mean ± s.d. Statistical significance was determined by one-way ANOVA with\nTukey's multiple comparisons test.\nQuestion 2. Supplementary Fig. 8c: Direct comparison between the concentration\nobtained from colon tissue homogenates and that from plasma is not recommended.\nThe concentration in tissue homogenates inherently depends on the dilution factor\nintroduced by the PBS volume used during homogenization; thus, it is not directly\ncomparable to the concentration measured in serum.\nResponse: We sincerely appreciate the reviewer for raising this important point. To\naddress this concern, we have reanalyzed the data using protein concentration\nnormalization. Specifically, we determined the total protein concentrations in colon\ntissue homogenates and plasma samples using the BCA assay, which were ~1.12\nmg/mL (for colon tissue homogenates prepared with 90 μL PBS per 1 mg tissue) and\n~5.01 mg/mL (for plasma), respectively. The MMP9 concentrations were then\nnormalized to nanograms per milligram of total protein (ng/mg). After normalization,\nthe MMP9 concentration in colon tissue was 338 ng/mg (378 ng/mL ÷ 1.12 mg/mL =\n338 ng/mL), while that in plasma was 2.2 ng/mg (11 ng/mL ÷ 5.01 mg/mL = 2.2 ng/mL).\nThe updated results are presented in Supplementary Fig. 8c. This normalized analysis\ndemonstrates that the MMP9 concentration in plasma is significantly lower than that in\ncolon tissue (P < 0.0001), thereby more reliably confirming our original findings.\nSupplementary Fig. 8c | MMP9 concentrations in serum and colonic lesion of IBD mice\nmeasured by ELISA and normalized to total protein. n = 5 mice. Data are presented as mean ±\ns.d. Statistical significance was determined by Student's t-test (two-tailed).\nReviewer #2 (Remarks to the Author): Thank you for submitting the revised manuscript\nand for your detailed responses to the reviewers’ comments. I have carefully reviewed\nthe revised version and the authors’ point-by-point replies. The authors have addressed\nall reviewers’ previous concerns thoroughly and made substantial improvements to the\nmanuscript. I believe this work is suitable for publication in its current form.\nResponse: We sincerely thank the reviewer for the kind words and thoughtful\nevaluation of our revised manuscript. Your positive feedback on the manuscript is\nhighly valued. Thank you again for your time and insightful feedback.\nReviewer #4 (Remarks to the Author): This is a revised version of the study by Cao et\nal \"Engineered exosome nanovesicles efficiently deliver antibodies in vivo to treat\ninflammatory bowel disease\". Much of this work is outside my field and I was pleased\nto see three other expert reviewers tackling the task. I can see that the authors have gone\nthrough significant effort in order to address all comments. My main concern, however,\nwas the claimed mechanism, originally described as “Notably, PrEXO-a23 treatment\nalso upregulates the expression of the tumor suppressor gene p53, further enhancing\nprotection against inflammation-driven carcinogenesis.” The additional data is not that\nhelpful in view of this claim. As might have been expected through previous literature,\nthe inflammation itself increases Trp53 protein expression, and probably the respective\npathway. This effect is then reduced after the treatment. It is difficult to say what causes\nthis reduction; whether e.g. it is just a bystander effect of easing inflammation.\nNevertheless, it is of course interesting that the protein level decreases. But then, Trp53\nmRNA levels increase after PrEXO-a23 treatment. What would be the purpose of that?\nTaken together, the evidence and story could be more robust. It might be wise to leave\nthe mechanism open.\nResponse: We sincerely thank the reviewer for these insightful comments, which have\nbeen instrumental in refining our mechanistic interpretation.\nAs is known, p53 is a crucial tumor suppressor that maintains cellular homeostasis by\nregulating cell cycle arrest, apoptosis, and DNA repair (Gastroenterology 2022, 162,\n715–730; Nat Commun 2023, 14, 110). In IBD, excessive p53 activation can drive\nepithelial cell death and exacerbate tissue injury (Sci Adv 2024, adp8783), while loss or\ndysfunction of p53 serves as an established early driver of colitis-associated colorectal\ncarcinogenesis (Cancer Cell 2013, 23, 634-646; Nat Cancer 2024, 5, 983-995; Nat\nGenet 2023, 55, 1531–1541). Therefore, therapeutic benefit derives not from simply\nincreasing or decreasing p53 expression, but from maintaining homeostasis of the p53\nsignaling pathway. Both chronic loss of p53 and uncontrolled acute hyperactivation are\ndetrimental, whereas homeostasis of p53 signaling confers protection (Cell 2009, 140:\n384-396).\nThe mRNA-protein discordance is frequently observed for p53, which can be attributed\nto the fact that p53 expression is a highly dynamic process governed by multiple\ncomplex regulatory factors (Cancer Cell 2024, 42:946-967): (i) Post-translational\nmodifications: Inflammation-induced phosphorylation or acetylation can stabilize p53\nprotein, leading to its accumulation despite unchanged or decreased mRNA levels\n(Signal Transduct Target Ther 2020, 5, 90; Cancer Cell 2024, 42:946-967). (ii)\nNegative feedback mechanisms: Immune interventions can modulate the MDM2-\nmediated ubiquitin-proteasome pathway, leading to rapid clearance in p53 protein\nlevels that are independent of its mRNA abundance (Nat Rev Cancer 2020, 20, 471-\n480.). These mechanisms may collectively account for the frequent discordance\nbetween p53 mRNA and protein levels.\nTo investigate the effect of PrEXO-a23 on p53 pathway in IBD, we performed Western\nblot analysis on colon tissues from a DSS-induced IBD model. Western blot analysis\nrevealed that compared with healthy controls, the IBD mice exhibited a significant\nincrease in p53 protein levels, along with elevated expression of its downstream targets\np21 and Bax (Fig. R1), indicating aberrant p53 activation in IBD. Following PrEXO-\na23 treatment, these elevated protein levels returned to levels comparable to those in\nhealthy mice, demonstrating that PrEXO-a23 helps maintain homeostasis of the p53\nsignaling pathway. Additionally, RT‑qPCR analysis showed downregulation of Trp53,\nCdkn1a, and Bax mRNA in IBD mice relative to healthy controls (Fig. R2). Notably,\nPrEXO-a23 treatment maintained the expression levels of these genes to those observed\nin the healthy group. Collectively, these data indicate that PrEXO-a23 contributes to\nthe maintenance of p53 signaling homeostasis in IBD.\nWe further evaluated the anti-CAC efficacy of PrEXO-a23 in an AOM/DSS-induced\nCAC mouse model. PrEXO-a23 treatment completely inhibited tumor formation,\ndemonstrating its potent anti-carcinogenic effect and capacity to halt IBD-to-CAC\nprogression. Mice were then co-treated with the p53 inhibitor. This intervention\nmarkedly diminished the therapeutic efficacy of PrEXO-a23 and negated its anti-tumor\neffects (Fig. 8a-g), supporting that the anti-tumor action of PrEXO-a23 is at least\npartially p53-dependent. Furthermore, we analyzed the expression of p53 and its\ndownstream targets in colon tissues from CAC mice using RT‑qPCR and Western blot.\nCompared with the healthy control group, Trp53 mRNAs were decreased, and Cdkn1a\nand Bax were downregulated in the CAC mice, suggesting dysfunction of the p53\npathway during CAC progression. Notably, treatment with PrEXO‑a23 maintained\ntheir expression (Trp53/Cdkn1a/Bax) to near-normal levels (Fig. 8h). However, this\nmaintenance was abolished upon p53 inhibition. Western blot results confirmed these\nchanges in the levels of p53, p21, and Bax proteins (Fig. 8i, j). These findings suggest\nthat PrEXO‑a23 treatment contributes to the maintenance of p53 signaling homeostasis,\nwith the potential to reduce the risk of progression from IBD to CAC.\nBased on your feedback and our experimental data, we think that the action of PrEXO-\na23 cannot be simply characterized as “up-regulating” or “down-regulating” p53, but\ncontributes to the maintenance of p53 pathway homeostasis. p53 pathway homeostasis\nplays a critical role in anti-tumor efficacy. Accordingly, we have revised the initial\nstatement, “Notably, PrEXO-a23 treatment also upregulates the expression of the tumor\nsuppressor gene p53, further enhancing protection against inflammation-driven\ncarcinogenesis.” to the more scientifically rigorous conclusion: “Notably, PrEXO-a23\ntreatment also contributes to the maintenance of p53 signaling homeostasis, with the\npotential to reduce the risk of progression from IBD to CAC.”\nAccordingly, we have revised the manuscript as follows.\nAbstract section: “Notably, PrEXO-a23 treatment also contributes to the maintenance\nof p53 signaling homeostasis, with the potential to reduce the risk of progression from\nIBD to CAC.”\nIntroduction section: “Notably, PrEXO-a23 contributes to the maintenance of p53\nsignaling homeostasis, which potentially further inhibited CAC development (Fig. 1).”\nResult section: “To evaluate the long-term therapeutic potential of PrEXO-a23 in\npreventing IBD-associated complications such as intestinal fibrosis and CAC, we\nemployed an AOM/DSS-induced CAC mouse model. Mice were administered AOM\nfollowed by repeated cycles of DSS to induce chronic colitis and tumorigenesis, with\nuntreated healthy mice serving as controls (Health group). During disease progression,\nmice received intravenous injections of PBS (AOM/DSS group), PrEXO (PrEXO group),\na23 (a23 group), rEXO-a23 (rEXO-a23 group) or PrEXO-a23 (PrEXO-a23 group)\n(Fig. 7b). As expected, AOM/DSS-treated mice exhibited significantly reduced survival\n(P = 0.0208; Fig. 7c), substantial weight loss (Supplementary Fig. 21a), and\nsignificantly elevated DAI scores (Fig. 7d), shortened colon length, and increased\ntumor burden, both in terms of number and size (Fig. 7e-g and Supplementary Fig. 21b).\nAmong all treatment groups, PrEXO-a23 demonstrated the robust therapeutic efficacy,\nmarkedly improving survival, body weight, DAI, and colon morphology to levels\ncomparable with healthy controls. Importantly, while PrEXO, a23, and rEXO-a23\ntreatments conferred partial protection, none of these monotherapies prevented tumor\ndevelopment. In contrast, PrEXO-a23 treatment completely inhibited tumor formation,\nhighlighting its potent anti-carcinogenic effect and capacity to halt IBD-to-CAC\nprogression.\nWe previously demonstrated that PrEXO-a23 affected the Trp53 expression change in\nDSS-treated colons. Mice were co-treated with the p53 inhibitor pifithrin-α. This\nintervention markedly diminished the therapeutic efficacy of PrEXO-a23 and negated\nits anti-tumor effects (Fig. 8a-g), further supporting that the action of PrEXO-a23 in\npreventing CAC progression is at least partially dependent on p53. Notably, the p53\npathway plays a crucial role in tumor suppression, with a mechanism involving various\nfactors such as Cdkn1a/p21 and Bax. Therefore, we investigated the expression of p53,\nCdkn1a/p21, and Bax in colonic tissues using RT-qPCR and Western blot analysis.\nCompared with the health control group, Trp53 mRNAs were decreased, and Cdkn1a\nand Bax were downregulated in the AOM/DSS group, suggesting dysfunction of the p53\npathway during CAC progression. Notably, treatment with PrEXO‑a23 maintained\ntheir expression (Trp53/Cdkn1a/Bax) to near-normal levels (Fig. 8h). However, this\nmaintenance was abolished upon p53 inhibition (PrEXO‑a23 + ip53). Western blot\nresults confirmed these changes in the levels of p53, p21, and Bax proteins (Fig. 8i, j).\nThese findings suggest that PrEXO‑a23 treatment contributes to the maintenance of\np53 signaling homeostasis, with the potential to reduce the risk of progression from\nIBD to CAC. Collectively, PrEXO-a23 not only attenuates active inflammation but also\nprovides durable protection against fibrosis and tumorigenesis, highlighting it as a\npromising therapeutic strategy for the long-term management of IBD and its\ncomplications.”\nConclusion section: “Additionally, it contributes to the maintenance of p53 signaling\nhomeostasis, potentially reducing the risk of progression from IBD to CAC.”\nFig. R1 | Western blot images and relative protein level analysis of p53, p21, and Bax expressed\nin colonic tissues of IBD mice with indicated treatment. n = 5 mice. Data are presented as mean\n± s.d. Statistical significance was determined by one-way ANOVA with Tukey's multiple\ncomparisons test.\nFig. R2 | Relative mRNA expression levels of Trp53, Cdkn1a, and Bax in colonic tissues of\nIBD mice with indicated treatment measured by RT-qPCR assay. n = 5 mice. Data are presented\nas mean ± s.d. Statistical significance was determined by one-way ANOVA with Tukey's\nmultiple comparisons test.\nFig. 8h | Relative mRNA expression levels of Trp53, Cdkn1a, and Bax in colonic tissues of\nCAC mice with indicated treatment measured by RT-qPCR assay. n = 5 mice. Data are\npresented as mean ± s.d. Statistical significance was determined by one-way ANOVA with\nTukey's multiple comparisons test.\nFig. 8i, j | Western blot images (i) and relative protein level analysis (j) of p53, p21, and Bax\nexpressed in colonic tissues. n = 3 mice. Data are presented as mean ± s.d. Statistical\nsignificance was determined by one-way ANOVA with Tukey's multiple comparisons test."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have been well addressed all concerns raised by the Reviewer. Thus, the Reviewer recommends to publish the current version of the manuscript.", "reviewer_4": "Thank you again for the revisions. The authors claim that\nthe action of PrEXO-a23 in preventing CAC progression is at least partially dependent on p53.\nand\nit contributes to the maintenance of p53 signaling homeostasis\nI think that the first claim is reasonable; without input from p53 the attenuation of inflammation might not work as it otherwise\nwould.\nHowever, saying that PrEXO-a23 contributes to the maintenance of p53 signaling homeaostasis is a claim that I would be\ncareful about. It is difficult to say if PrEXO-a23 has any specific influence on p53 signaling. If the treatment manages to\ncontrol inflammation, surely many cellular pathways become altered. I would thus stick with my original advice that the\nauthors should discuss with caution what is the specific mechanism underlying the effects of PrEXO-a23.\nI was sorry to see that a new set of comments from ref #3 were not available; it would have been reassuring to see that s/he\nwas happy with those revisions that I am no expert to judge."}, "author_response": "Response: We sincerely thank the reviewer for this insightful comment. Based on your suggestion and our experimental data, we think that the anti‑tumor action of PrEXO‑a23\nis at least partially dependent on p53 (please see Fig. 8a-g). However, due to the multiful\nfunctions of p53 signaling pathway, it is challenging to determine whether PrEXO-a23\nhas a direct, specific effect on maintening the homeostasis of the p53 signaling pathway.\nTherefore, we have revised the manuscript, taking a more cautious approch to\ndescribing the mechanism of PrEXO-a23 “The capacity of PrEXO-a23 to prevent the\nprogression from chronic inflammation to cancer partially depends on functional p53\nsignaling”."}]

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