Engineered vesicles enhance oral antibiotic absorption in proximal small intestine and mitigate gut dysbiosis

Domain: physics
Published: 2025-12-25
Editorial outcome: major_revision

Abstract
Oral antibiotics are a mainstay for treating bacterial infections, but unabsorbed portions can reach the caecum and colon, leading to gut dysbiosis. Herein, we engineer a biohybrid delivery system through the integration of milk extracellular vesicles with liposomes. The hybrid vesicles employ targeting mechanisms via neonatal Fc receptor and peptide transporter 1, facilitating antibiotic transport across the proximal small intestine. These vesicles exhibit superior drug encapsulation efficiency, stable release behavior, efficient mucus traversal, higher endocytosis, increased basolateral exocytosis, and improved oral absorption, achieving a 3.24-fold increase in oral bioavailability compared to free antibiotics. In lung bacterial infections and bacteremia models, hybrid vesicle-encapsulated cefdinir outperforms free antibiotics in eliminating infections. Notably, this approach also mitigates adverse effects on the intestinal microbiota, safeguarding the animals from dysbiosis-associated metabolic syndromes and opportunistic pathogen infections. This innovative hybrid vesicle system holds promise for the oral delivery of other drugs that suffer from limited absorption or cause gut dysbiosis.

Introduction
Antibiotics are among the most widely prescribed medications globally, with their usage escalating at an alarming rate1. However, the majority of antibiotics are hampered by their low oral bioavailability, necessitating the use of excessive doses to achieve the desired therapeutic outcomes. The frequent administration of high doses of antibiotics not only exerts a significant burden on the body but also fosters the emergence of antibiotic-resistant strains2. Moreover, the unabsorbed fraction of orally administered antibiotics reaches the caecum and colon, potentially causing intestinal microbiota dysbiosis3. The mutualistic microbes residing in the intestine are vital for various physiological functions, including the regulation of immune and metabolic homoeostasis4–6. When this delicate balance is disrupted by antibiotics, it may trigger a cascade of adverse effects, such as diarrhea, allergic reactions, inflammation, and neurological disorders7–11. Thus, it is essential to improve the oral bioavailability of antibiotics and reduce their unintended negative impacts on gut microbiota to optimize their use.

The population and composition of gut microbiota differ greatly in various parts of the intestine. Bacterial counts typically stretch from approximately 105/mL in the upper small intestine to approximately 1012/mL in the colon12. This bacterial density gradient hints that if antibiotics are effectively absorbed in the proximal small intestine, it would restrict contact with the large intestine’s flora, thereby cutting down their presence in feces13. In recent years, extracellular vesicles have emerged as promising drug delivery carriers for targeted delivery and overcoming physiological barriers14–16. Milk extracellular vesicles (mEV) stand out as particularly promising oral delivery candidates due to their ample sources, efficient cargo protection capabilities, minimal immunogenicity, and stable gastrointestinal physicochemical properties17. Notably, research has shown that mEV are transported through neonatal Fc receptor (FcRn) and peptide transporter 1 (PEPT1) on the intestinal epithelial cells18. Since FcRn and PEPT1 are highly expressed in the proximal small intestine, this transport mechanism may promote absorption by the proximal small intestine while minimizing side effects on the microbiota in the colon19,20. Nevertheless, natural mEV still face several limitations as oral carriers. First, mEV have limited ability to traverse the mucus layer21. Second, mEV encounter challenges in encapsulating cargo efficiently22. Third, the phenomenon of rapid release of small molecule drugs encapsulated in mEV is quite pronounced23. To overcome these limitations and construct a more efficient carrier for oral delivery, it is essential to modify natural mEV to enhance their mucus penetration ability, drug loading capacity, and release behavior.

In this study, we design a milk extracellular vesicle-liposome (mEV-Lip) hybrid vesicle with the aim of enhancing absorption in the proximal small intestine and thereby circumventing adverse effects on the colonic microbiota (Fig. 1). The hydrophilic modification of polyethyleneglycol (PEG) enables the mEV-Lip to penetrate the intestinal mucus layer, while the proteins on the surface of mEV facilitate targeting to epithelial cells in the proximal small intestine, thus enhancing the targeted absorption of antibiotics. We select cefdinir (Cef), a β-lactam antibiotic, as the representative antibiotic for our study. The results demonstrate that oral administration of Cef-loaded mEV-Lip (mEV-Lip@Cef) exhibits higher efficacy in Klebsiella pneumoniae infection in both lung and bacteremia models. More importantly, treatment with mEV-Lip@Cef markedly diminishes the negative impact on intestinal microbiota, mitigating the risk of dysbiosis-associated opportunistic pathogen infection and metabolic syndromes.

Results
Hybrid vesicles facilitate enhanced absorption at proximal small intestine
The uptake of mEV at the intestinal epithelium is facilitated by FcRn and PEPT118. To explore the potential of targeted antibiotic delivery, we first investigated the expression of these transporters in the intestine. As depicted in Fig. 2a and Supplementary Fig. 1, the expression levels of FcRn and PEPT1 were notably elevated in small intestine relative to the colon. This disparity in transporter distribution suggests a promising avenue for transporter-mediated targeting of mEV in the proximal small intestine.

Inspired by this insight, we engineered mEV-Lip hybrid vesicles, leveraging the transporter-binding ability to target the proximal small intestine. Initially, mEV was isolated using a classical ultracentrifugation method. To remove casein from milk, ethylenediaminetetraacetic acid disodium salt (EDTA-2Na, 30 mM) was added to chelate calcium ions and disrupt casein micelle integrity, removing casein through micellar disassembly before ultracentrifugation. Dynamic light scattering (Fig. 2b) and nanoparticle-tracking analysis (NTA, Fig. 2c) revealed hydrated particle sizes of 132.9±1.8 nm and 113.5±2.5 nm, respectively, aligning with the typical size range of extracellular vesicles (30–150 nm). The modest polydispersity index (PDI, 0.208±0.016) and substantial yield (1.08×1011±2.62×109 particles/mL) indicated successful isolation of mEV.

Next, we modified liposomes with PEG-functionalized phospholipids utilizing the classical thin-film hydration method. The hybrid vesicles were subsequently fabricated by capitalizing on the flow and fusion characteristics inherent to the mEV membrane’s phospholipid bilayer. Fluorescence scanning patterns of mEV, liposomes (Lip), and mEV-Lip demonstrated the fluorescence resonance energy transfer (FRET) phenomenon in the hybrid vesicles (Fig. 2d). Confocal laser scanning microscope (CLSM) further confirmed the successful hybridization of the mEV-Lip (Supplementary Fig. 2). Cryo-transmission electron microscope (cryo-TEM) images showed that mEV-Lip had spheroidal morphology (Supplementary Fig. 3). Western Blot (WB) analysis corroborate the presence of mEV signature proteins (CD9, CD63, HSP70) in both mEV and mEV-Lip, while calnexin, an endoplasmic reticulum marker, was absent in both groups except the supernatant (Fig. 2e). Particle size and zeta potential measurements indicated that after fusion with Lip, mEV had an increased particle size and a more negative potential (Supplementary Fig. 4). Moreover, stability tests showed that both mEV and mEV-Lip maintained favorable colloidal stability in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF), as evidenced by minimal changes in particle size (Supplementary Fig. 5). These vesicles also exhibited excellent stability over 7 days at 4 °C and −80 °C (Supplementary Fig. 6). In summary, we successfully prepared hybrid vesicles modified with PEG-functionalized phospholipids, which possess stable colloidal properties.

One of the primary objectives of this study was to enhance the mucus penetration ability of natural mEV by introducing hydrophilic PEG. To closely mimic the in vivo environment, fresh porcine small intestinal mucus was used to evaluate mucus penetration. NTA showed that the mobility of mEV was restricted in the mucus, whereas Lip and mEV-Lip had significantly wider mobility ranges (Fig. 2f). Mean squared displacement (MSD) calculations revealed that within a 1 s interval, the MSD values of Lip and mEV-Lip were greater than that of mEV (Fig. 2g). Besides, a transwell permeable scaffold was used to construct a three-dimensional mucus model (Supplementary Fig. 7a), and the apparent permeability coefficient (Papp) was measured to quantify the transport rate from the donor chamber to the recipient chamber24. The hydrophilic modification by PEG significantly enhanced the mucus penetration ability of natural extracellular vesicles, with a 1.6-fold increase (Supplementary Fig. 7b). These findings imply that the PEG layer is instrumental in improving permeability of vesicles in the intestinal mucus.

After penetrating the mucus layer, intestinal epithelial cells pose another barrier to absorption. We investigated the intestinal epithelial cell barrier using Caco-2/HT29 coculture model. Cellular uptake studies showed that mEV-Lip had a significant increase compared to Lip (2.7-fold) and mEV (1.7-fold), indicating that the combination of PEG with mEV is an effective strategy to overcome both the mucus and apical membrane barriers (Supplementary Fig. 8). Additionally, mEV-Lip demonstrated enhanced exocytosis ability compared to Lip and mEV (Fig. 2h). A cell monolayer was established to assess the transcytosis efficiency of various formulations across the intestinal epithelium. The Papp results indicated that mEV-Lip improved the transcytosis efficiency of Lip and mEV in the Caco-2/HT29 coculture cell monolayer model (Fig. 2i). Bidirectional exocytosis profiling revealed that the exocytosis rate of Lip from basolateral side was only 63% of that from the apical side, whereas mEV-Lip raised this fraction to 131% (Supplementary Fig. 9), yielding a 2-fold higher basolateral-to-apical exocytosis ratio. To determine whether vesicles remain structurally intact during transcytosis, double fluorophores-labeled mEV-Lip were applied to the cell monolayer. A robust FRET signal detected in the basolateral compartment (Supplementary Fig. 10) confirmed that mEV-Lip cross the epithelium with intact structure. These findings suggest that mEV-Lip can be more efficiently transported into and out of the epithelial cells, with an “easy entry, easy exit” characteristic.

To visualize the in vivo distribution of vesicles, we encapsulated a fluorescent dye (1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide, DiR) into the vesicles. Mice were orally given DiR-labeled vesicles, and their intestines were subsequently imaged using an in vivo imaging system (IVIS). Compared with free DiR, mEV-Lip had a higher signal intensity in the small intestine and a markedly lower intensity in the large intestine (Fig. 2j–m, and Supplementary Fig. 11). In vitro intestinal absorption experiments also showed that mEV-Lip had significantly higher absorption in the small intestine compared with Lip (Supplementary Fig. 12). We further investigated the micro-distribution of the vesicles within the proximal small intestine. The findings indicated that the uptake of mEV-Lip by intestinal epithelium surpassed that of other vesicles (Supplementary Fig. 13). Immunofluorescent staining of intestinal sections showed that mEV-Lip co-localized with FcRn and PEPT1 in small intestinal epithelial cells (Supplementary Fig. 14). Moreover, linear regression analysis demonstrated that epithelial uptake of mEV-Lip correlated tightly with FcRn and PEPT1 expression levels (Supplementary Fig. 15). The above results collectively suggest that the absorption of mEV-Lip in the small intestine is potentially mediated by specific interactions with FcRn/PEPT1.

To further demonstrate this, we employed the FcRn inhibitor human serum albumin (HSA) and the PEPT1 inhibitor glycylsarcosine (Gly-Sar) to block each pathway in vivo, which resulted in a marked reduction in the absorption of mEV-Lip in the small intestine (Supplementary Fig. 16). In contrast, the absorption of Lip remained unchanged upon treatment with FcRn or PEPT1 inhibitor. Moreover, when FcRn and PEPT1 were overexpressed in Caco-2 cells (Supplementary Fig. 17), HSA and Gly-Sar significantly inhibited the endocytosis of mEV and mEV-Lip, while having a relatively weaker inhibitory effect on Lip (Supplementary Fig. 18). The transcytosis assays revealed similar pathway dependency, with mEV and mEV-Lip transport across epithelial barriers being significantly compromised by both inhibitors (Supplementary Fig. 19). Collectively, these results suggest that the hybrid vesicles promote their absorption in the proximal small intestine through the transporters FcRn and PEPT1 (Fig. 2n), which holds great promise for oral antibiotic delivery.

Hybrid vesicles reduce intestinal residues and enhance oral bioavailability
To evaluate whether the increased absorption of mEV-Lip at the proximal small intestine could decrease intestinal residues, the quantity of residual DiR-labeled mEV-Lip in feces was measured using the IVIS imaging system at various time points post oral administration. Comparative analysis revealed that mice treated with mEV-Lip showed a marked reduction in fecal residues compared to free DiR and DiR-labeled Lip (Fig. 3a, b), which may be correlated with enhanced proximal intestinal absorption.

We subsequently explored whether mEV-Lip could serve as carriers to boost the bioavailability of encapsulated antibiotics. For this purpose, Cef was employed as a model antibiotic, and the corresponding Cef-loaded vesicles were designated as Lip@Cef, mEV@Cef, and mEV-Lip@Cef. Although the encapsulation efficiency of natural mEV was only 11.29%, mEV-Lip significantly increased the drug encapsulation efficiency to 56.02% (4.96-fold, P=0.0002) (Supplementary Table. 1). The same trend was observed for seven additional antibiotics representing distinct chemical classes: amoxicillin (Amo), cefalexin (Cax), rifampicin (Rif), ciprofloxacin hydrochloride (Cfh), ceftriaxone sodium (Cxs), ampicillin sodium (Aps), sulfamethoxazole (Sul) (Supplementary Table. 2). This substantial improvement highlights the advantage of preparing hybrid vesicles, where the enhanced membrane fluidity imparted by liposomal components facilitates superior drug-loading capacity compared to natural extracellular vesicles. Besides, in vitro drug release experiments revealed that Cef was released in a slow and sustained manner from mEV-Lip@Cef when incubated in simulated buffers (Fig. 3c). This sustained release profile overcomes the problem of rapid drug release from natural mEV. Furthermore, Lip@Cef and mEV-Lip@Cef had a certain sustained release effect and played important roles in blood circulation (Supplementary Fig. 20). The serum concentration of Cef in the mEV-Lip@Cef group was significantly higher than that of free Cef (Fig. 3d and Supplementary Table. 3). Additionally, the bioavailability of mEV-Lip@Cef was 3.24 times higher than that of free Cef (Fig. 3e), indicating that mEV-Lip@Cef significantly promoted the oral absorption of Cef. These results collectively suggest that the mEV-Lip packaging enhances oral antibiotics bioavailability and reduces intestinal residues, which underscores the potential of mEV-Lip as an effective oral delivery system for antibiotics.

mEV-Lip@Cef effectively eliminates Klebsiella pneumoniae infection in the lung
To evaluate the therapeutic potential of hybrid vesicles, we established a murine model of pulmonary infection induced by Klebsiella pneumoniae, a predominant nosocomial pathogen causing pneumonia (Fig. 4a)25. Whereas only 50% of mice receiving free Cef survived the 4 d observation period, 88% of animals treated with mEV-Lip@Cef remained alive (Supplementary Fig. 21). Compared to free Cef, which achieved modest bacterial reduction in the lung, both Lip@Cef and mEV@Cef demonstrated superior antibiotic activity (Fig. 4b–d). Remarkably, mEV-Lip@Cef exhibited the highest therapeutic efficacy, achieving the lowest bacterial load in the lung. Besides, the measurement of inflammatory cytokines in lungs revealed substantially reduced inflammation in the Cef-loaded vesicles groups compared with mice treated with free Cef (Fig. 4e). Pulmonary pathological and histological analyses further showed that both free Cef and other Cef-loaded vesicle groups exhibited diminished inflammation and pathology compared with the phosphate buffer solution (PBS) group (Fig. 4f–h). Importantly, lower inflammation and attenuated tissue damage were observed in mEV-Lip@Cef-treated mice compared with free Cef-treated and other Cef-loaded vesicle-treated mice. In summary, these results suggest that mEV-Lip@Cef exhibits enhanced efficacy in treating bacterial pneumonia.

mEV-Lip@Cef effectively eradicates Klebsiella pneumoniae infection in a bacteremia model
To further evaluate the antibacterial efficacy of antibiotic-loaded vesicles, we employed a bacteremia model (Fig. 5a). Mice were intravenously inoculated with a standardized dose of Klebsiella pneumoniae. Subsequently, at pre-determined time points, the mice were treated with free Cef and Cef-loaded vesicles. Only 12.5% of mice receiving free Cef survived the 8 d monitoring period, while 62.5% of animals administered mEV-Lip@Cef remained alive (Supplementary Fig. 22). The appearance of liver and spleen revealed a notable reduction in inflammation in free Cef and Cef-loaded vesicle groups compared to PBS group (Fig. 5b). Remarkably, the mEV-Lip@Cef group exhibited a significant attenuation of inflammation. To quantify the pathogen burden, we measured the colony-forming units (CFU) in the peripheral blood, liver, and spleen 24 h post-infection. Consistent with our findings in the lung infection model, mice treated with mEV-Lip@Cef demonstrated a more efficient clearance of Klebsiella pneumoniae compared to those treated with free Cef, Lip@Cef, and mEV@Cef (Fig. 5c–e). In addition, hematoxylin and eosin (H&E) staining was performed on liver and spleen tissue sections (Fig. 5f). The staining results showed that mEV-Lip@Cef-treated mice had less inflammation and reduced tissue damage in the liver and spleen compared to mice treated with free Cef and other Cef-loaded vesicles. Collectively, these results suggest that mEV-Lip@Cef possesses superior efficacy in eliminating bacterial pathogens, highlighting its potential as an effective approach for antibiotic therapy.

Hybrid vesicles mitigate antibiotic-driven dysbiosis
The enhanced proximal small intestine absorption profile of oral hybrid vesicles may confer ecological advantages by minimizing gut microbiota disruption. Notably, pharmacological alteration of enteric flora through conventional antibiotic formulations can induce substantial community restricting, affecting approximately 30% of bacterial taxa abundance26. This dysbiotic transformation is manifested through abrupt declines in α-diversity metrics including species richness and evenness, with incomplete microbiome restoration post-treatment potentially potentiating susceptibility to dysbiosis-associated pathologies, particularly in pediatric populations27–29. To evaluate these differential effects, we conducted longitudinal comparative analyses of gut microbiota ecology following administration of free Cef versus hybrid vesicle-encapsulated Cef formulations at multiple intervals post-administration (Fig. 6a). As anticipated, 16S ribosomal RNA sequencing revealed marked depletion of stool α-diversity and substantial dysbiosis in fecal communities following free Cef administration (Fig. 6b–e). Strikingly, the mEV-Lip@Cef demonstrated superior preservation of gut microbiota homeostasis, facilitating accelerated recovery of both microbial richness and ecological architecture compared to free Cef. The protective effect was further corroborated by principal components analysis (PCA) of microbial communities, which showed tighter clustering patterns in the mEV-Lip@Cef group compared to the pronounced dispersion observed with free Cef (Fig. 6f). Taxonomic profiling at the phylum level identified significant perturbations induced by free Cef, characterized by decrease of Bacteroidota and Bacillota populations alongside increase of Pseudomonadota (Fig. 6g, h). In contrast, mEV-Lip@Cef administration maintained near-physiological phylum distributions. Taken together, these findings suggest that the engineered hybrid vesicle system effectively mitigates antibiotic-associated microbiota perturbations, preserving critical ecological parameters including bacterial richness, diversity, and composition.

Hybrid vesicle-mediated antibiotic delivery ameliorates dysbiosis-associated obesity
Emerging evidence underscores the critical involvement of intestinal microbial communities in regulating systemic metabolic process, particularly energy balance regulation and lipid accumulation30. Pathological conditions including obesity, diabetes mellitus type 2, and hepatic steatosis demonstrate enhanced dietary energy harvest efficiency, a characteristic feature of metabolic dysregulation31. To investigate the potential impact of antibiotic-loaded vesicles on antibiotic-associated metabolic perturbations, we established a dietary obesity model (Fig. 7a). Treatment with free Cef resulted in a significant increase in high-fat diet (HFD)-induced body mass accumulation (Fig. 7b). In contrast, the mEV-Lip@Cef showed moderated weight alterations, suggesting a reduced impact on weight gain. Similarly, metabolic parameter analysis demonstrated that free Cef treatment exacerbated HFD-induced metabolic dysregulation, manifesting as impaired glucose tolerance and hepatic cholesterol accumulation (Fig. 7c–e). In comparison, the engineered nanocarrier system maintained near-physiological glycemic responses and effectively constrained hepatic cholesterol levels. Furthermore, histological analysis revealed milder liver steatosis in mice treated with mEV-Lip@Cef compared to those treated with free Cef (Fig. 7f).

To investigate whether these metabolic alterations were driven by changes in gut microbiota, we conducted fecal microbiota transplantation (FMT) studies (Fig. 7g). Three donor cohorts were established: free Cef treatment, mEV-Lip@Cef treatment, and antibiotic-naïve controls. Recipient mice received fecal transplants from donor groups and were subsequently placed on a HFD regimen. Notably, animals receiving microbiota from free Cef-treated donors developed exacerbated weight gain, impaired glucose resistance, elevated hepatic cholesterol accumulation, and pronounced steatotic changes compared to those that received microbiota from mEV-Lip@Cef-treated donors (Fig. 7h–l). These findings jointly indicate that engineered vesicle-mediated antibiotic delivery preserves metabolic homeostasis by maintaining microbial ecological balance.

Hybrid vesicle delivery of antibiotics decreases dysbiosis-associated Escherichia coli infection
Antibiotic-mediated disruption of gut microbiota homeostasis has been widely documented to enhance vulnerability to enteric infections, primarily through two mechanisms: facilitating colonization by exogenous pathogens or promoting proliferation of resident opportunistic pathobionts32–34. To evaluate the effects of pretreatment with free Cef and mEV-Lip@Cef on the severity of Escherichia coli intestinal infection, we conducted an experimental study (Fig. 8a). Mice were pretreated with antibiotics to simulate the initial treatment and associated perturbation of the gut microbiota, followed by an intestinal challenge with pathogenic Escherichia coli at day 5. Quantitative analysis revealed that free Cef-pretreated mice exhibited significantly compromised resistance to Escherichia coli colonization compared to antibiotic-naïve controls (Fig. 8b–e). This heightened susceptibility manifested as elevated pathogen burdens across fecal samples, cecal contents, and colonic tissues, accompanied by pronounced mucosal hyperplasia. Notably, mice receiving mEV-Lip@Cef pretreatment demonstrated infection resistance comparable to untreated controls, with no statistically significant differences in pathogen colonization levels and histopathological manifestations. These findings collectively suggest that engineered vesicle-based antibiotic delivery effectively mitigates dysbiosis-associated pathogen infections (Fig. 8f).

Methods
Materials
Egglecithin was procured from Shanghai Taiwei Pharmaceutical Co., Ltd. (Shanghai, China). Cholesterol, Rhodamine B (RhB), and glycylsarcosine (Gly-Sar) were obtained from Shanghai Maclean Biochemical Technology Co., Ltd. (Shanghai, China). DSPE-mPEG2000 was purchased from AVT (Shanghai) Pharmaceutical Tech Co., Ltd. (Shanghai, China). Recombinant human serum albumin (HSA) was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Cefdinir (Cef) and coumarin 6 (C6) were obtained from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) was acquired from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). Enhanced BCA protein assay kit, 4’,6-diamidino-2-phenylindole (DAPI), and goat anti-rabbit IgG Alexa Fluor 647 were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Anti-CD9 antibody, anti-CD63 antibody, anti-HSP70 antibody, and anti-calnexin antibody were purchased from Bioss Biotechnology (Beijing, China). Anti-FCGRT/FcRn antibody was obtained from ABclonal Technology Co., Ltd. (Wuhan, China). Anti-SLC15A1/PEPT1 antibody, β-actin antibody, and goat anti-rabbit IgG H&L (HRP) were obtained from Proteintech Group (Wuhan, China). Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) enzyme-linked immunosorbent assay (ELISA) kits were supplied by Shanghai Jianglai Biotechnology Co., Ltd. (Shanghai, China). Total cholesterol detection kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Methanol and acetonitrile, both HPLC grade, were purchased from Aladdin Biotechnology Co., Ltd. (Shanghai, China). All other chemicals and solvents used for this study were of analytical grade.

Strains
Klebsiella pneumoniae (KP2237, serotype K173, resistance gene: aac(3)-IIa, aph(3”)-Ib, aph(6)-Id, blaCTX-M-15, blaKPC-2, blaSHV-106, blaTEM-1B, dfrA14, fosA, oqxA, oqxB, qnrB1, sul2) used in this study is a clinical strain isolated from human, preserved in our laboratory; Escherichia coli (ATCC 25922) used in this study was obtained from the American Type Culture Collection and is preserved in our laboratory.

Cells and animals
Human intestinal cell lines, Caco-2 and HT29 cells, were procured from Beijing Dingguo Biotechnology Co., Ltd. (Beijing, China). Cellular maintenance was conducted in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin, 1% streptomycin, and 1% of non-essential amino acids. All cultures were maintained under standardized conditions (37 °C, 5% CO2, 95% relative humidity).

Male Balb/c mice certified by Hunan SJA Laboratory Animal Co., Ltd. (SCXK Xiang 2021-0002) were housed at the School of Pharmacy, Southwest University, under the experimental animal use license number SYXK (Yu) 2020-0006. Animals were housed under a 12 h light/12 h dark cycle, with the ambient temperature at approximately 25 °C and relative humidity at 50 ± 10%. All procedures complied with guidelines and received institutional approval (IACUC-20250407-01) from the Laboratory Animal Use and Management Committee of Southwest University.

Isolation of milk extracellular vesicles (mEV)
Pasteurized bovine milk was sourced from retail sources. mEV isolation employed a multi-step differential centrifugation protocol: primary clarification at 5000g (30 min, 4 °C) removed somatic cells and debris, followed by casein dissociation using ethylenediaminetetraacetic acid disodium salt (EDTA-2Na, 30 mM). The supernatant underwent filtration (0.22 μm polyethersulfone membrane) prior to sequential ultracentrifugation (100,000g for 60 min, 210,000g for 60 min, Optima L-90K, Beckman, USA) for vesicle subfractionation. Pelleted vesicles were suspended in phosphate-buffered saline (PBS) and quantified via a BCA protein assay kit. mEV samples with a total protein concentration below 2 mg/mL were cryopreserved at −80 °C until further use.

Preparation of hybrid vesicles
The hybrid vesicles were fabricated using the dried film hydration-extrusion technique. Primary constituents including phospholipids (10 mg), cholesterol (2 mg), and DSPE-mPEG2000 (1.67 mg) were initially solubilized in chloroform to form the liposome component. Following complete solvent removal under vacuum conditions, the resultant dry lipid films were hydrated with an mEV suspension (4 mg of mEV diluted to 4 mL with PBS). Subsequently, the mixture was extruded through a liposome extruder fitted with a 200 nm polycarbonate membrane filter. For the preparation of Cef, C6, RhB or DiR-loaded hybrid vesicles, the following procedure was employed. A mixture of mEV and the respective drug solution (Cef: 5 mg, C6: 0.1 mg, RhB: 0.4 mg, or DiR: 0.4 mg) was sonicated (120 W, 1 min, with a 2 s on/2 s off cycle). This sonicate mixture was then processed using the same hydration and extrusion steps as described for the unloaded hybrid vesicles.

Characterization of vesicles
Colloidal properties of the engineered vesicles were assessed using dynamic light scattering (3000+ Ultra, Malvern Instruments Ltd., UK). The particle concentration of mEV suspensions was performed via nanoparticle-tracking analysis (NTA, Nanosight LM10, Malvern Instruments Ltd., UK). For morphology characterization, vesicles were observed by cryo-TEM (TF20, FEI, USA) with an acceleration voltage of 200 kV. To confirm the successful extraction of mEV, Western Blot (WB) analysis was conducted on both mEV and the hybrid vesicles. The presence of several mEV markers, including CD9, CD63, and HSP70, was detected, along with the supernatant protein calnexin, following standard protocols.

To validate the fusion of mEV with liposomes, a fluorescence resonance energy transfer (FRET)-based analytical approach was employed. Specifically, mEV was labeled by incubation with a RhB solution, while liposomes were labeled by incorporating a chloroform solution of C6 during the film preparation process. Subsequently, RhB/C6 co-labeled hybrid vesicles were fabricated using the dried film hydration-extrusion method. Spectral characterization was performed using a fluorescence spectrophotometer with 445 nm excitation, capturing emission profiles across a 475–700 nm wavelength range to quantify energy transfer dynamics.

The encapsulation efficiency was evaluated by eliminating unencapsulated drugs through 100 kDa molecular weight cutoff (MWCO) ultrafiltration membranes. Quantitative analysis of Cef was performed using an optimized high performance liquid chromatography (HPLC, LC-20AD, Shimadzu, Japan) protocol utilizing C18 column (150 mm × 4.6 mm, 5 μm). Chromatographic separation was achieved with a mobile phase consisting 0.25% tetramethylammonium hydroxide (pH-adjusted to 5.5 using phosphoric acid), acetonitrile, and methanol (900:60:40, v/v/v), supplemented with 0.4 mL/L of 0.1 mol/L EDTA-2Na solution. Operational parameters include isocratic elution at 1.0 mL/min, UV detection at 254 nm, 20 μL injection volume, and column thermostating at 25 °C. Encapsulation efficiency was derived from the percentage ratio between the amount of Cef detected in the vesicles to the total amount of Cef used in the preparation.

Stability and in vitro release profile
The colloidal stability of the various vesicles was evaluated under physiologically relevant conditions, including simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Specifically, vesicle suspensions (0.1 mL) were homogenously dispersed in 0.9 mL of either SGF or SIF and incubated. The particle size of the vesicles was measured at predetermined time intervals.

The in vitro release profiles of Cef-loaded vesicles were evaluated in SGF and SIF. A volume of 2 mL of Cef-loaded vesicles was placed in a dialysis bag with a MWCO of 10 kDa. The sealed bag underwent phased incubation: primary SGF (50 mL, 37 °C, 2 h) followed by SIF (50 mL, 37 °C, 6 h) under continuous agitation (90 rpm). At each sampling time point, aliquots (0.5 mL) were periodically collected and immediately replaced with thermo-equilibrated fresh medium. The amount of Cef released was subsequently quantified using HPLC analysis.

Mucus permeability evaluation
The mobility of the vesicles within mucus was assessed using NTA. A volume of 0.1 mL vesicles was introduced into 0.9 mL porcine intestinal mucus and incubated for 30 min. The movement of the vesicles within the mucus was then recorded. The averaged mean squared displacement (MSD) of the particles was calculated based on their trajectories38.

The mucus permeability of the vesicles was quantitatively assessed using a Transwell system. Initially, the donor chamber received 0.1 mL of porcine intestinal mucus, while the acceptor compartment was filled with 0.8 mL of PBS. To achieve uniform mucus layer formulation, the assembly underwent equilibration at 37 °C with shaking for 30 min. Subsequently, 0.15 mL of C6-labeled vesicles was carefully administered to the mucus-containing donor chamber, followed by continuous incubation at 37 °C. Aliquots (80 μL) were periodically harvested from the acceptor compartment at designed intervals, with immediate replenishment of preheated PBS. The vesicles that traversed the mucus layer were quantified using a microplate reader (Synergy H1, American Berton Instrument Company, USA). The apparent permeability coefficient (Papp) values were calculated.

Cellular uptake and endocytic mechanisms
Cellular internalization studies of diverse vesicles were performed using the Caco-2/HT29 coculture model. The intestinal epithelial models were co-seeded in 96-well plates at a 7:3 ratio (Caco-2:HT29) and maintained under standard culture condition for 48 h. Cellular confluence was quantitatively assessed through Alamar Blue assay. Subsequently, the culture medium was exchanged with serum-free medium containing C6-labeled vesicles (C6 concentration: 0.5 μg/mL). After continuous incubation for 3 h, the cells were lysed with dimethyl sulfoxide (DMSO), and intracellular fluorescence accumulation was determined using a microplate reader.

Endocytic pathway analysis was conducted employing the Caco-2 cell model. Following 48 h period in 96-well plates, cellular density was assessed using the Alamar Blue assay. To investigate the specific pathways of endocytosis, HSA (40 μg/mL) and Gly-Sar (100 mM) were added to separate wells and incubated for 1 h. Thereafter, C6-labeled vesicles (C6 concentration: 0.5 μg/mL) were added and incubated for an additional 3 h. The cells were then lysed with DMSO, followed by fluorometric analysis of intracellular accumulation using a microplate reader.

Exocytosis studies
Exocytosis experiments of various vesicles were conducted using the Caco-2/HT29 coculture model (7:3). Following 48 h culture in 96-well plates, cellular density was quantified through Alamar Blue assay. The cells were then exposed to C6-labeled vesicles (C6 concentration: 0.5 μg/mL) for 3 h, washed three times with cold PBS, and incubated with DMEM for 1 h. After lysis with DMSO, the fluorescence intensity was quantified on a microplate reader.

Transcellular transport studies
A co-culture model of intestinal epithelial cells was established using Caco-2 and HT29 cells at a 7:3 ratio in Transwell inserts. Following 21 days of differentiation under standard culture condition, the transepithelial electrical resistance (TEER) of approximately 300 Ω cm2 was achieved. Prior to transport experiments, the differentiated monolayers were pre-incubated with blank medium for 30 min at 37 °C. The apical compartment then received 0.2 mL of medium containing C6-labeled vesicles (C6 concentration: 1.0 μg/mL), while the basolateral chamber contained 0.2 mL fresh medium. Aliquots (80 μL) were collected from the basolateral side at designated time intervals with immediate volume replacement using pre-warmed medium. The collected samples were subjected to disruption using DMSO, followed by fluorescence quantification employing a microplate reader. The Papp values were calculated.

Biodistribution of vesicles
Balb/c mice (8 weeks old) were fasted overnight. Subsequently, they were orally administered DiR-labeled vesicles (DiR: 0.5 mg/kg). At predetermined time points, the mice were sacrificed, and their digestive tract and fecal samples were collected. The distribution of DiR fluorescence in the intestines and fecal samples was collected and determined using an in vivo imaging system (IVIS, Vilber Lourmat).

Intestinal absorption of Cef-loaded vesicles
After fasting the Balb/c mice (8 weeks old) overnight, they were anesthetized and the segments of the proximal small intestine, distal small intestine, and colon were carefully extracted. Each intestinal segment was washed thoroughly, and one end was ligated. At the other end, 0.1 mL of C6-labeled vesicles (C6 concentration: 1.0 μg/mL) was injected into each segment. The intestinal segments were then immersed in fresh Krebs-Ringer (K-R) solution at 37 °C in a water bath, with a continuous supply of 95% O2 to maintain tissue viability. At predetermined time intervals, 0.1 mL of the extracellular fluid was collected from the K-R solution, and an equal volume of fresh extracellular fluid was immediately replenished. The collected samples were mixed with DMSO to disrupt the vesicles, and the fluorescence intensity was measured using a microplate reader. After the experiment, the intestinal segments were removed, and the surface area of each segment was recorded. The Papp values were calculated.

Balb/c mice (8 weeks old) were fasted overnight. Subsequently, the mice were anesthetized, and a midline laparotomy was performed to expose the proximal small intestine. Approximately 2 cm intestinal segments were carefully isolated. C6-labeled vesicles (C6 concentration: 1.0 μg/mL) were injected into these loops, which were then ligated to form closed systems. After a 3 h incubation period, the intestinal segments were excised and subjected to sequential processing: initial rinsing with K-R solution, fixation in 4% paraformaldehyde, and dehydration through 30% sucrose immersion. Tissue specimens were embedded in optimum cutting temperature medium and sectioned at 10 μm thickness using a Leica cryostat (Germany). Nuclear counter-staining was performed with DAPI prior to fluorescence imaging analysis using an Olympus confocal laser scanning microscope (CLSM, Japan).

Pharmacokinetics of Cef-loaded vesicles in vivo
Balb/c mice (8 weeks old) were fasted overnight and orally administered free Cef, Lip@Cef, mEV@Cef or mEV-Lip@Cef (Cef: 35 mg/kg). Serial blood samples (0.2 mL) were obtained via retro-orbital puncture at specified intervals and immediately transferred to heparinized tubes. The quantification of Cef content in the plasma was performed utilizing HPLC. The chromatographic separation employed a C18 column (250 mm × 4.6 mm, 5 μm) maintained at 40 °C. An isocratic mobile phase containing water-methanol-formic acid (100:10:0.1, v/v/v) and methanol (82:18, v/v) was delivered at 1.0 mL/min. Drug detection was achieved through ultraviolet absorbance monitoring at 286 nm.

In vivo pharmacodynamics studies in lung infection model with Klebsiella pneumoniae
A murine pneumonia model was established in Balb/c mice (8 weeks old) by intranasal inoculation with 1×107 CFU of Klebsiella pneumoniae suspended in 50 μL of medium. Therapeutic interventions were initiated 24 h post-infection, with three oral administrations (24, 36, and 48 h) of either free Cef (20 mg/kg) or Cef-loaded vesicles at equivalent dosage. Lung tissue processing and analysis were conducted 72 h post-infection. Collected pulmonary tissues were homogenized in sterile PBS and subjected to serial dilutions. Quantitative bacteriological analysis was performed by culturing homogenate on selective MacConkey agar, followed by 12 h incubation at 37 °C for CFU enumeration. Proinflammatory cytokine concentrations (IL-6 and TNF-α) were determined in lung homogenates using Elisa kits. For morphological analysis, tissue specimens were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned for histological staining. Sections underwent hematoxylin and eosin (H&E) and Gram staining, with microscopic examination conducted using light-field optics.

In vivo pharmacodynamics studies in bacteremia model with Klebsiella pneumoniae
Bacteremia was induced in Balb/c mice (8 weeks old) by intravenous injection with 1×107 CFU Klebsiella pneumoniae. Therapeutic interventions were initiated at two time points (2 and 8 h) post-infection, with experimental groups receiving either free Cef (20 mg/kg) or equivalent Cef-loaded vesicles via oral gavage. To quantify bacterial dissemination, peripheral blood specimens were obtained at 24 h post-infection. Each 0.1 mL blood aliquot underwent initial tenfold dilution with sterile PBS, followed by serial decimal dilutions for plating on selective MacConkey agar. Concurrently, hepatic and splenic tissues were aseptically excised, weighed and homogenized in 1 mL PBS. Tissue homogenates were processed through successive tenfold dilutions before culturing on MacConkey agar. All culture plates underwent overnight incubation at 37 °C. Colonies were counted to calculate the CFU. The liver and spleen were harvested for H&E staining histological examination.

High-fat diet (HFD) mouse model
Balb/c mice (3 weeks old) were divided into three groups receiving PBS, free Cef or mEV-Lip@Cef via oral gavage for 5 d. Subsequently, all animals were transitioned to HFD (XTHF60-1, Jiangsu Collaborative Biotechnology Engineering Co., Ltd., Nanjing, China) from week 7 to week 10 of age. Longitudinal body weight measurements were recorded at weekly intervals during the HFD phase. Metabolic characterization was performed at week 17 through an overnight-fasted intraperitoneal glucose tolerance test (IPGTT), where animals received 1 g/kg glucose solution via intraperitoneally injection. Blood glucose concentrations were quantified using a glucometer at predetermined time points after glucose injection. Terminal procedures were conducted at week 19, with subsequent organ collection for biochemical and histopathological evaluation. Excised hepatic tissues were immediately weighed and processed into homogeneous suspensions for quantitative analysis of total cholesterol content using a commercial cholesterol assay kit following manufacturer specifications. The livers were harvested for histological examination via H&E staining.

Fecal microbiota transplantation (FMT)
Donor Balb/c mice received daily oral gavage of PBS, free Cef, or mEV-Lip@Cef formulations for 5 d. Fecal specimens were collected from the donor mice under sterile conditions for FMT. Individual fecal samples underwent sterile processing involving homogenization in PBS followed by gravitational sedimentation. The supernatants were immediately aliquoted under controlled conditions to recipient mice. Following FMT administration, recipient mice were subjected to HFD for 13 weeks and analyzed as described above.

Intestinal infection model with Escherichia coli
Balb/c mice (8 weeks old) were pretreated with free Cef or mEV-Lip@Cef via oral administration daily for 5 d, while control animals were administered equivalent volumes of PBS via the same route. Subsequently, all experimental groups were subjected to intraperitoneal challenge with Escherichia coli (1×108 CFU per injection) on days 5 and 9, with analysis on day 13. Fecal, caecum, and colon specimens were aseptically collected, weighed and immediately homogenized in sterile PBS. The resulting homogenates underwent serial dilutions in PBS followed by plating on selective MacConkey agar medium for bacterial quantification. Cultured plates were maintained at 37 °C overnight before enumeration of CFU, with final counts normalized to tissue mass. The colons were harvested for histological examination via H&E staining.

Data analysis
Experimental data are presented as mean ± standard deviation values (Mean ± SD). Statistical analyses were performed using IBM SPSS Statistics 23.0. Comparisons between two experimental groups were conducted using the two-tailed Student’s t test. For multiple comparisons, one-way analysis of variance (ANOVA) was employed, followed by post hoc analysis with Tukey’s honest significant difference (HSD) test. P < 0.05 were considered statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript presents an innovative oral drug delivery system using a milk exosome-liposome hybrid vesicle (mEXO-Lip) to enhance the absorption of antibiotics in the proximal small intestine while minimizing gut microbiota disruption. The authors combine biologically derived milk exosomes with PEGylated liposomes to improve drug loading, mucus penetration, and cellular uptake. Mechanistically, the hybrid vesicles leverage FcRn and PEPT1 transporters for targeted uptake in the upper small intestine, thus avoiding unnecessary drug exposure to the distal gut and preserving microbiota composition. The platform demonstrates the increased bioavailability and therapeutic efficacy in several murine models, including lung infection, bacteremia, obesity, and E. coli gut infection. Microbiome and metabolomic analyses further show that mEXO-Lip reduces antibiotic-induced dysbiosis and metabolic imbalance. Although substantial work has been conducted, particularly with multiple in vivo model validations, the use of mEXO as oral delivery vehicles, including for small molecules, macromolecules, and peptide-based drugs, has been extensively reported. This also includes hybrid milk exosome vesicles. Moreover, the intestinal absorption mechanisms mediated by the neonatal Fc receptor (FcRn) and peptide transporter 1 (PepT1) are well-established. In fact, the unique surface characteristics of EXOs enable efficient internalization through active multi-targeting to the epithelium, beyond just these two proteins. Overall, this study is well-designed and sheds light on the development of alternative antibacterial strategies.\n1. The authors state that Klebsiella pneumoniae and E. coli were used, but it remains unclear which strain ID, serotype, or resistance profile was employed. This is critical for reproducibility, particularly in the context of antibiotic efficacy.\n2. A control is required to clarify the role of FcRn and PEPT1 in enhancing the absorption of mEXO-Lip@Cb/RhB in the proximal small intestine. Moreover, it is not clear whether the promotion of absorption in the proximal small intestine is caused by FcRn and PEPT1 or by the unique design of mEXO-Lip, and the functional distinction between the two is not clear.\n3. In line 167-68, The encapsulation efficiency of natural mEXO (11.3%) vs. mEXO-Lip (56.0%) shows a major difference. Was this result consistent across replicates or formulations with different drugs? Variability across batches should be reported if applicable.\n4. From Fig. 2i, it is concluded that the high transport efficiency of mEXO-Lip, does it affect drug release? Is there an explanation for the direct correlation between transport proteins and this efficient transport effect? Is this migration unidirectional or bidirectional, and does it have a negative impact on drug uptake?\n5. In Fig. 2j, mEXO-Lip stays and accumulates in the middle segment of the intestine, and the intestinal length of the mEXO-Lip group is quite different from that of the first two groups. Add a scale bar in the figure.\n6. In Fig. 3c, Cef is released very little in SIF from 2-8 h. Does this affect the normal antibacterial effect of the drug? Can the evaluation index of drug bioavailability be used for intestinal antibacterial evaluation? Is there an intestinal bacterial infection treatment model designed to test the therapeutic effect of mEXO-Lip@CEF on the intestine?\n7. Line 377, The cell culture medium contained 1% penicillin/streptomycin. Dose the continued presence of antibiotics affect vesicle uptake or transporter activity?\n8. The study reports AUC and bioavailability data but lacks detailed pharmacokinetic parameters (Cmax, Tmax, half-life). Including these metrics would strengthen claims about absorption efficiency and systemic exposure.\n9. Fig. 2q, the manuscript describes PEGylation as a strategy to enhance mucus penetration, but does not address potential immunogenicity or safety concerns associated with repeated PEG exposure. A brief discussion of PEG biocompatibility in the context of chronic oral use is recommended.\n10. The manuscript lacks data on survival rates in the infection model. Monitoring survival is a crucial parameter for evaluating disease severity and model validity. If no mortality was observed, how was the success of the infection model confirmed? Without this evidence, the robustness of the model and the therapeutic efficacy claims remain unsubstantiated.\n11. Some data points in the picture are abnormal and do not match the values of n. For example, in Fig. 5E, the figure legend states ‘n=5’, but certain columns display only three data points. Please clarify whether this reflects missing data, an error in plotting, or an inconsistency in sample size reporting. If data points are excluded, justification for their removal (e.g., outliers with statistical criteria) must be provided to ensure reproducibility.", "reviewer_2": "What are the noteworthy results?\nThe study by Luo et al is comprehensive. The authors engineered milk extracellular vesicles and showed convincingly that these systems enhance oral antibiotic absorption in proximal small intestine and mitigate gut dysbiosis. The use of milk extracellular vesicles for enhance the intestinal absorption itself is not novel. Additionally, it is well established in the field that the small intestine has evolved to participate in absorption process (hence the vast surface area) so it is not surprising that the absorption of formulations takes place from that region. The mitigation against gut dysbiosis is therefore a result of these phenomena. Although to be expected, the finding is overall noteworthy.\nWill the work be of significance to the field and related fields? How does it compare to the established literature? If the work is not original, please provide relevant references.\nSee above. The work adds to the literature on milk extracellular vesicles in that it details another potential application (antibiotic delivery). This is of sufficient significance to the field.\nDoes the work support the conclusions and claims, or is additional evidence needed?\nThe dysbiosis aspects of the work in my opinion are strong but the work suffers from the lack of detailed characterisation of the vesicles, as highlighted below.\nAre there any flaws in the data analysis, interpretation and conclusions? Do these prohibit publication or require revision?\n- TEM does not convincingly reveal the identity of the vesicles. Cryo TEM or super resolution imaging.\n- Characterisation of particles is limited. Are all the vesicles in the sample hybrids or do you expect to have a proportion of EVs, liposomes and hybrids? If yes, this should be quantified (FRET does not help with this). Consider using nano flow cytometry and / or RAMAN and / or super resolution microscopy. A mixed particle pullulation unfortunately makes some of the analysis (eg western blotting and mucus diffusion) difficult to interpret and potentially invalid since the samples will contain a mixed population of unfused and fused vesicles not allowing for discrimination of findings related to each particle species.\n- There is too much reliance on literature reports of phenomena that are not convincingly established / confirmed, including involvement of FcRn and mucus diffusivity of EVs. The conclusions should be toned down to potentially rather than convincingly suggest FcRn and PEPT1 involvement in EV trafficking. Own data in Fig2p highlight only a 10% decrease in endocytosis upon blocked of these receptors - this does not convincingly evidence that the uptake of EVs is mediated by these receptors.\n- EVs should be called EVs or small EVs rather than exosomes\nIs the methodology sound? Does the work meet the expected standards in your field?\nMostly yes, but I have some reservations:\n- Fig2a: missing secondary antibody only control\n- Supplementary figure 6 and Fig 2h and i: why were HT29 cells used? It is indeed a subclone of these (HT29-MTX) which is capable of producing mucus and is commonly used in co-culture as a goblet-like cell type to render the model as mucus-producing\n- Colocalisation studies in Supplementary figure 9 are not convincing, primarily due to the very low expression of FcRn and PEPT1.\n- Fig 2o: controls lacking\n- How were the antibiotic doses standardised when the loading efficiency in liposomes, exos and hybrids were different?\nIs there enough detail provided in the methods for the work to be reproduced?\n- Methods related to exocytosis studies unclear\nOverall, considering the points raised above on data analysis, interpretation and conclusions, in my view the manuscript currently does not meet the requirements of the current journal regarding the soundness / rigour of the methodology, although with major revisions (additional experiments), this may be addressed.", "reviewer_3": "The article is excellent and with strong data and novel information.\nHowever some suggestions need to be improved as below.\n1. Language and Grammar Improvements\n• Line 16 – Break the long sentence into two shorter ones to improve readability.\n• Line 50 – Change “and minimizing” to “while minimizing” for correct parallel structure.\n• Line 113 – Correct typo: change “instrument” to “instrumental”.\n• Line 246 – Remove redundancy: change “fecal community communities” to “fecal communities”.\n• Line 314 – Replace colloquial “Of note” with “Notably” for a more formal academic tone.\n• Line 563 – Correct typo: change “sane route” to “same route”.\n2. Structural and Content Refinements\n• Line 335 – Condense the first sentence of the Discussion to remove repetitive background information.\n• Line 82 – In the Methods section, specify EDTA concentration units (e.g., mM) and provide a brief rationale for casein removal.\n• Line 167 – Include statistical analysis to confirm the significance of the encapsulation efficiency results.\n• Also address why entrapment efficiency is much lower without liposome incorporation (11.29% vs. 56%).\n3. Formatting and Style Consistency\n• Line 493 – Ensure temperature is written consistently as “37°C” throughout the manuscript.\n• Keep units consistent and in correct SI format.\n• Define all abbreviations upon first appearance in the main text, even if defined in the Abstract (e.g., Papp, HFD).\n• Follow gene/protein naming conventions (e.g., italicize FcRn, PEPT1 only if referring to genes).\n• Ensure statistical notation is consistent, e.g., P < 0.05 (with a space after P).\n4. Figures, Abstract, and Overall Presentation\n• Consider reducing the number of figures; some sections are visually overloaded, which may distract from key findings.\n• Shorten and revise the Abstract to improve clarity and focus, avoiding redundancy while highlighting key quantitative results."}, "author_response": "Response to Reviewers\nWe express our gratitude to the Editorial Office and the reviewers for providing us with constructive comments and suggestions. We have diligently revised the paper in accordance with the reviewers’ feedback and have highlighted all the changes made in revised version.\nReviewer #1:\nThis manuscript presents an innovative oral drug delivery system using a milk exosome-liposome hybrid vesicle (mEXO-Lip) to enhance the absorption of antibiotics in the proximal small intestine while minimizing gut microbiota disruption. The authors combine biologically derived milk exosomes with PEGylated liposomes to improve drug loading, mucus penetration, and cellular uptake. Mechanistically, the hybrid vesicles leverage FcRn and PEPT1 transporters for targeted uptake in the upper small intestine, thus avoiding unnecessary drug exposure to the distal gut and preserving microbiota composition. The platform demonstrates the increased bioavailability and therapeutic efficacy in several murine models, including lung infection, bacteremia, obesity, and E. coli gut infection. Microbiome and metabolomic analyses further show that mEXO-Lip reduces antibiotic-induced dysbiosis and metabolic imbalance. Although substantial work has been conducted, particularly with multiple in vivo model validations, the use of mEXO as oral delivery vehicles, including for small molecules, macromolecules, and peptide-based drugs, has been extensively reported. This also includes hybrid milk exosome vesicles. Moreover, the intestinal absorption mechanisms mediated by the neonatal Fc receptor (FcRn) and peptide transporter 1 (PepT1) are well-established. In fact, the unique surface characteristics of EXOs enable efficient internalization through active multi-targeting to the epithelium, beyond just these two proteins. Overall, this study is well-designed and sheds light on the development of alternative antibacterial strategies.\nResponse: Thank you for your thoughtful review and insightful comments on our manuscript. Your constructive feedback has significantly strengthened the scientific robustness of our work. We have worked diligently to address your concerns, and we sincerely hope that our revisions will meet your expectations and further enhance the quality of our manuscript.\n1. The authors state that Klebsiella pneumoniae and E. coli were used, but it remains unclear which strain ID, serotype, or resistance profile was employed. This is critical for reproducibility, particularly in the context of antibiotic efficacy.\nResponse: Thanks for the comments. We have supplemented the strain ID, serotype, or resistance profile as below:\nKlebsiella pneumoniae (KP2237, serotype K173, resistance gene: aac(3)-IIa, aph(3'')-Ib, aph(6)-Id, blaCTX-M-15, blaKPC-2, blaSHV-106, blaTEM-1B, dfrA14, fosA, oqxA, oqxB, qnrB1, sul2) used in this study is a clinical strain isolated from human, preserved in our laboratory; Escherichia coli (ATCC25922) used in this study was obtained from the American Type Culture Collection and is preserved in our laboratory.\n2. A control is required to clarify the role of FcRn and PEPT1 in enhancing the absorption of mEXO-Lip@Cb/RhB in the proximal small intestine. Moreover, it is not clear whether the promotion of absorption in the proximal small intestine is caused by FcRn and PEPT1 or by the unique design of mEXO-Lip, and the functional distinction between the two is not clear.\nResponse: Thanks for the comments. mEV-Lip leverages the intrinsic targeting properties of milk extracellular vesicles toward FcRn and PEPT1 to facilitate intestinal absorption. Administration of transporter inhibitors resulted in a significant reduction in small intestinal absorption. Further analysis demonstrated a positive correlation between the extent of intestinal absorption and the expression levels of both transporters. We have supplemented transporter inhibitor group as the control and the linear regression analysis between mEV-Lip and transporters expression levels as below:\nMoreover, linear regression analysis demonstrated that epithelial uptake of mEV-Lip correlated tightly with FcRn and PEPT1 expression levels (Supplementary Fig. 15). The above results collectively suggest that the absorption of mEV-Lip in the small intestine is potentially mediated by specific interactions with FcRn/PEPT1.\nTo further demonstrate this, we employed the FcRn inhibitor human serum albumin (HSA) and the PEPT1 inhibitor glycylsarcosine (Gly-Sar) to block each pathway in vivo, which resulted in a marked reduction in the absorption of mEV-Lip in the small intestine (Supplementary Fig. 16). In contrast, the absorption of Lip remained unchanged upon treatment with FcRn or PEPT1 inhibitor.\nSupplementary Fig. 15 The uptake efficiency of mEV-Lip was correlated with the expression levels of FcRn (a) and PEPT1 (b). n = 5 mice.\nSupplementary Fig. 16 a, Representative images of intestine from mice after oral gavage of DiR-loaded vesicles with inhibitor of FcRn HSA or PEPT1 Gly-Sar. Scale bars, 2.5 cm. b-d, Quantification of the fluorescence intensity in SI-P (b), SI-D (c), and LI (d) at 4 h after oral administration of Lip or mEV-Lip. Data are mean ± SD (n = 3). One-way ANOVA with a Tukey post hoc test was used for statistical significance. * P < 0.05, *** P < 0.001. NS, not significant.\n3. In line 167-68, The encapsulation efficiency of natural mEXO (11.3%) vs. mEXO-Lip (56.0%) shows a major difference. Was this result consistent across replicates or formulations with different drugs? Variability across batches should be reported if applicable.\nResponse: Thanks for the comments. This result consistent across replicates and formulations with different drugs. We have supplemented the different batches and different drugs as below:\nThe same trend was observed for seven additional antibiotics representing distinct chemical classes: amoxicillin (Amo), cefalexin (Cax), rifampicin (Rif), ciprofloxacin hydrochloride (Cfh), ceftriaxone sodium (Cxs), ampicillin sodium (Aps), sulfamethoxazole (Sul) (Supplementary Table. 2).\nSupplementary Table 1. Characterization of Cef-loaded vesicles. Mean ± SD (n = 3).\nBatches Samples Size (nm) PDI Zeta potential Encapsulation\n(mV) efficiency (%)\nmEV@Cef 128.30 ± 3.12 0.14 ± 0.02 -9.95 ± 1.13 11.29 ± 3.26\nmEV-Lip@Cef 168.92 ± 2.64 0.23 ± 0.02 -19.78 ± 2.06 56.02 ± 3.16***P=0.0002\nmEV@Cef 126.53 ± 11.3 0.23 ± 0.03 -10.82 ± 1.67 7.66 ± 1.13\nmEV-Lip@Cef 168.93 ± 6.10 0.22 ± 0.02 -19.12 ± 1.11 59.61 ± 1.39\nmEV@Cef 127.34 ± 8.92 0.24 ± 0.06 -9.35 ± 1.65 10.89 ± 1.26\nmEV-Lip@Cef 167.98 ± 5.74 0.22 ± 0.03 -19.65 ± 3.23 60.28 ± 2.97\nStatistical significance was determined using one-way ANOVA with a Tukey post hoc test. *** P＜0.001 versus mEV@Cef.\nSupplementary Table 2. Characterization of drug-loaded vesicles. Mean ± SD (n = 3).\nSamples Size (nm) PDI Zeta potential Encapsulation\n(mV) efficiency (%)\nLip@Amo 161.77 ± 11.1 0.29 ± 0.04 -17.02 ± 1.39 69.04 ± 2.39\nmEV@Amo 129.63 ± 15.9 0.26 ± 0.03 -11.47 ± 1.58 13.95 ± 2.41\nmEV-Lip@Amo 158.53 ± 6.64 0.26 ± 0.04 -17.38 ± 1.54 66.02 ± 2.60\nLip@Cax 183.80 ± 14.7 0.24 ± 0.02 -17.28 ± 1.20 83.62 ± 3.90\nmEV@Cax 135.43 ± 13.0 0.25 ± 0.01 -16.10 ± 1.57 21.80 ± 2.45\nmEV-Lip@Cax 176.97 ± 20.6 0.26 ± 0.02 -18.56 ± 1.88 73.12 ± 2.43\nLip@Rif 169.47 ± 6.62 0.22 ± 0.01 -18.20 ± 1.61 96.04 ± 2.12\nmEV@Rif 127.87 ± 15.4 0.17 ± 0.03 -14.00 ± 1.23 8.39 ± 1.11\nmEV-Lip@Rif 178.80 ± 4.16 0.24 ± 0.04 -17.10 ± 1.62 92.99 ± 2.16\nLip@Cfh 157.40 ± 26.1 0.26 ± 0.07 -17.72 ± 1.22 93.44 ± 2.11\nmEV@Cfh 138.90 ± 0.98 0.25 ± 0.01 -14.70 ± 1.95 7.29 ± 1.01\nmEV-Lip@Cfh 166.80 ± 12.2 0.23 ± 0.01 -18.63 ± 2.50 89.41 ± 2.06\nLip@Cxs 160.60 ± 28.6 0.21 ± 0.06 -17.93 ± 1.47 67.78 ± 2.73\nmEV@Cxs 132.13 ± 58.3 0.20 ± 0.05 -12.70 ± 1.79 8.77 ± 1.11\nmEV-Lip@Cxs 166.63 ± 10.6 0.27 ± 0.04 -19.04 ± 1.48 67.40 ± 2.35\nLip@Aps 150.10 ± 9.46 0.29 ± 0.04 -17.38 ± 1.46 69.39 ± 2.06\nmEV@Aps 123.67 ± 5.92 0.24 ± 0.01 -13.23 ± 3.33 10.63 ± 1.01\nmEV-Lip@Aps 161.97 ± 9.80 0.26 ± 0.03 -18.78 ± 1.88 66.09 ± 3.03\nLip@Sul 170.47 ± 6.93 0.27 ± 0.05 -16.09 ± 1.02 88.14 ± 3.10\nmEV@Sul 134.43 ± 7.97 0.24 ± 0.01 -16.07 ± 3.41 8.62 ± 1.21\nmEV-Lip@Sul 186.90 ± 13.9 0.26 ± 0.06 -16.99 ± 2.72 76.33 ± 6.04\n4. From Fig. 2i, it is concluded that the high transport efficiency of mEXO-Lip, does it affect drug release? Is there an explanation for the direct correlation between transport proteins and this efficient transport effect? Is this migration unidirectional or bidirectional, and does it have a negative impact on drug uptake?\nResponse: Thanks for the comments. mEV-Lip retained structurally integrity throughout transcytosis without compromising cargo release. Blocking FcRn with human serum albumin (HSA) or PEPT1 with glycylsarcosine (Gly-Sar) reduced transcytosis, confirming transportor-mediated transport. The intestinal epithelial cells are polar cells. The nanoparticles taken into the cells may exit the cells from the basolateral side or the apical side. The ratio of Lip exiting from the basolateral side to the apical side was 63%, while the ratio for mEXO-Lip was 131%. This indicates that mEV-Lip could more readily exit from the basolateral side. We have supplemented the intact transcytosis, inhibitor-treated transcytosis and bidirectional exocytosis experiments as below:\nBidirectional exocytosis profiling revealed that the exocytosis rate of Lip from basolateral side was only 63% of that from the apical side, whereas mEV-Lip raised this fraction to 131% (Supplementary Fig. 9), yielding a 2-fold higher basolateral-to apical exocytosis ratio. To determine whether vesicles remain structurally intact during transcytosis, double fluorophores-labeled mEV-Lip were applied to the cell monolayer. A robust FRET signal detected in the basolateral compartment (Supplementary Fig. 10) confirmed that mEV-Lip cross the epithelium with intact structure.\nThe transcytosis assays revealed similar pathway dependency, with mEV and mEV-Lip transport across epithelial barriers being significantly compromised by both inhibitors (Supplementary Fig. 19).\nTo apical side\nTo basolateral side\n150 * P = 0.0102*** P = 0.0009\n)%\n(\ns\n* P = 0.0118\nis\no100\nty\nc\no\nx\ne\ne 50\nv\\ita\\le\nR\nSupplementary Fig. 9 Relative rate of apical/basolateral exocytosis of vesicles. All values are presented as the mean ± SD (n = 3). Statistical significance was determined using one-way ANOVA with a Tukey post hoc test. * P < 0.05. NS, not significant.\nSupplementary Fig. 10 Emission spectra and CLSM images of the basolateral medium after the cell monolayer was incubated with C6/RhB-loaded mEV-Lip. Scale bar: 5 μm.\nControl HSA Gly-Sar\nNS ** P = 0.0020 ** P = 0.0065\nNS ** P = 0.0022 ** P = 0.0063\n120\n)%\n( s100\nis\no ty 80\nc\ns n 60\na\rt\ne\n40\nv\\ita\n20\nle\nR\nSupplementary Fig. 19 Transcytosis efficiency of vesicles under different transporter inhibitors. All values are presented as the mean ± SD (n = 3). Statistical significance was determined using one-way ANOVA with a Tukey post hoc test. ** P < 0.01. NS, not significant.\n5. In Fig. 2j, mEXO-Lip stays and accumulates in the middle segment of the intestine, and the intestinal length of the mEXO-Lip group is quite different from that of the first two groups. Add a scale bar in the figure.\nResponse: Thanks for the comments. In Fig. 2j, the strongest fluorescence signal in the mEV-Lip group is observed in the proximal small intestine (predominantly red), whereas the middle segment displays a weaker signal (predominantly blue). Due to the inherent inter-individual variability, intestinal length differed markedly among mice. We have added the scale bar in the figure as below:\nFig. 2j IVIS images 4 h post-oral administration of free DiR or DiR-loaded vesicles in intestine. SI-P: proximal small intestine; SI-D: distal small intestine; LI: large intestine. Scale bars, 2.5 cm.\n6. In Fig. 3c, Cef is released very little in SIF from 2-8 h. Does this affect the normal antibacterial effect of the drug? Can the evaluation index of drug bioavailability be used for intestinal antibacterial evaluation? Is there an intestinal bacterial infection treatment model designed to test the therapeutic effect of mEXO-Lip@CEF on the intestine?\nResponse: Thanks for the comments. The orally administrated formulation remains largely intact in the gastrointestinal (GI) lumen; ＜40% of the Cef is released during GI transit. Subsequent passage across the intestinal mucosa is followed by a sustained release in the systemic circulation, attaining 80% cumulative release within 12 h without compromising antibacterial potency. This pharmacokinetic profile was deliberately engineered to maximise systemic exposure for the treatment of invasive infections. So, there was no intestinal bacterial infection treatment model designed to test the therapeutic effect of mEV-Lip@Cef on the intestine. Exploration of intestinal infection would require an alternative architecture that either (1) triggers site-specific release within the intestine or (2) extends intestinal residence time, objectives that lie outside the scope of the present study. We have supplemented in in vitro release in PBS (pH = 7.4) as below:\nFurthermore, Lip@Cef and mEV-Lip@Cef had a certain sustained release effect and played important roles in blood circulation (Supplementary Fig. 20).\n)%100\n(\ne\ns\n80\na\ne\nle\nr\nfe\ne\nv\\ita\nmEV@Cef\nlu\n20\nm mEV-Lip@Cef\nu\nC\n0\n0 3 6 9 12\nTime (h)\nSupplementary Fig. 20 In vitro release profiles of vesicles in PBS (pH = 7.4). All values are presented as the mean ± SD (n = 3).\n7. Line 377, The cell culture medium contained 1% penicillin/streptomycin. Dose the continued presence of antibiotics affect vesicle uptake or transporter activity?\nResponse: Thanks for the comments. We additionally profiled transporter expression and cellular uptake in cells cultured without 1% penicillin/streptomycin. The continuous presence of antibiotics did not alter vesicle uptake or transporter activity as below:\nSupplementary Fig. 8b, Relative cellular uptake in cells cultured with or without 1% penicillin/streptomycin. All values are presented as the mean ± SD (n = 3). Statistical significance was determined using one-way ANOVA with a Tukey post hoc test. NS, not significant.\nSupplementary Fig. 17 Immunofluorescence analysis of FcRn and PEPT1 expression in Caco-2 cells. Scale bars, 5 μm.\n8. The study reports AUC and bioavailability data but lacks detailed pharmacokinetic parameters (Cmax, Tmax, half-life). Including these metrics would strengthen claims about absorption efficiency and systemic exposure.\nResponse: Thanks for the comments. We have supplemented the detailed pharmacokinetic parameters as below:\nThe serum concentration of Cef in the mEV-Lip@Cef group was significantly higher than that of free Cef (Fig. 3d and Supplementary Table. 2).\nSupplementary Table 3. Pharmacokinetic parameters of Cef in mice. Mean ± SD (n = 5).\nPharmacokinetic\nFree Cef Lip@Cef mEXO@Cef mEXO-Lip@Cef\nparameters\nT (h) 89.94 ± 49.15 81.74 ± 25.39 80.52 ± 31.45 67.71 ± 33.73\n1/2\nT (h) 36.00 ± 12.00 36.00 ± 12.00 54.00 ± 12.00 36.00 ± 12.00\nmax\nC (μg/mL) 3.31 ± 0.95 6.25 ± 1.65*P =0.0153 7.95 ± 0.68****P<0.0001 11.52 ± 1.73****P<0.0001\nmax\nAUC 408.68 ± 136.13 716.40 ± 54.46*P=0.0460 1000.69 ± 1323.68 ±\n0-t\n(μg/mL*min) 105.38***P=0.0001 242.44***P=0.0002\nStatistical significance was determined using one-way ANOVA with a Tukey post hoc test. * P < 0.05, *** P < 0.001, **** P < 0.0001 versus free Cef.\n9. Fig. 2q, the manuscript describes PEGylation as a strategy to enhance mucus penetration, but does not address potential immunogenicity or safety concerns associated with repeated PEG exposure. A brief discussion of PEG biocompatibility in the context of chronic oral use is recommended.\nResponse: Thanks for the comments. We have added the discussion of PEG biocompatibility as below:\nTo facilitate penetration of the intestinal mucus layer, we incorporated the PEG corona. While repeated dosing may induce the production of PEG-specific antibodies, which potentially cause a higher incidence of side effects and reduce the therapeutic efficacy35-37.\nReferences:\n35. Armstrong, J.K. et al. Antibody against poly(ethylene glycol) adversely affects PEGasparaginase therapy in acute lymphoblastic leukemia patients. Cancer 110, 103-111 (2007).\n36. Hershfield, M.S. et al. Induced and pre-existing anti-polyethylene glycol antibody in a trial of every 3-week dosing of pegloticase for refractory gout, including in organ transplant recipients. Arthritis Res. Ther. 16, 1-11 (2014).\n37. Miao, G. et al. Accelerated blood clearance of PEGylated nanoparticles induced by PEG-based pharmaceutical excipients. J. Control. Release 363, 12-26 (2023).\n10. The manuscript lacks data on survival rates in the infection model. Monitoring survival is a crucial parameter for evaluating disease severity and model validity. If no mortality was observed, how was the success of the infection model confirmed? Without this evidence, the robustness of the model and the therapeutic efficacy claims remain unsubstantiated.\nResponse: Thanks for the comments. We have supplemented the survival rates in the infection model as below:\nWhereas only 50% of mice receiving free Cef survived the 4 d observation period, 88% of animals treated with mEV-Lip@Cef remained alive (Supplementary Fig. 21).\nOnly 12.5% of mice receiving free Cef survived the 8 d monitoring period, while 62.5% of animals administered mEV-Lip@Cef remained alive (Supplementary Fig. 22).\n)%\n( e 80 PBS\nta\nr 60 Free Cef\nla\nv iv 40 Lip@Cef\nr u mEV@Cef\nS 20 mEV-Lip@Cef\n0\n0 1 2 3 4\nTime (d)\nSupplementary Fig. 21 Survival rates of mice with Klebsiella pneumoniae infection in the lung. n = 8.\n)%\n( e 80 PBS\nta\nr\nla\nv iv 40 Lip@Cef\nru mEV@Cef\nS 20 mEV-Lip@Cef\n0\n0 2 4 6 8\nTime (d)\nSupplementary Fig. 22 Survival rates of mice with Klebsiella pneumoniae infection in a bacteremia model. n = 8.\n11. Some data points in the picture are abnormal and do not match the values of n. For example, in Fig. 5E, the figure legend states ‘n=5’, but certain columns display only three data points. Please clarify whether this reflects missing data, an error in plotting, or an inconsistency in sample size reporting. If data points are excluded, justification for their removal (e.g., outliers with statistical criteria) must be provided to ensure reproducibility.\nResponse: Thanks for the comments. Since some samples did not detect any bacteria, the bacterial load was 0 and was not shown in the bar chart. The original data as below:\nThe microbial burden in spleen (n = 5).\nNormal PBS Free Cef Lip@Cef mEV@Cef mEV-Lip@Cef\n0 188888888.9 9574468.085 12820512.82 3846153.846 0\n0 40322580.65 77777777.78 6986301.37 2564102.564 1830.985915\n0 165137614.7 106666666.7 9278350.515 2173913.043 27500\n0 1038461538 9574468.085 9278350.515 182608.6957 0\n0 228915662.7 35064935.06 6265306.122 5291666.667 1318.681319\nReviewer #2:\n1. What are the noteworthy results?\nThe study Luo et al is comprehensive. The authors engineered milk extracellular vesicles and showed convincingly that these systems enhance oral antibiotic absorption in proximal small intestine and mitigate gut dysbiosis. The use of milk extracellular vesicles for enhance the intestinal absorption itself is not novel. Additionally, it is well established in the field that the small intestine has evolved to participate in absorption process (hence the vast surface area) so it is not surprising that the absorption of formulations takes place from that region. The mitigation against gut dysbiosis is therefore a result of these phenomena. Although to be expected, the finding is overall noteworthy.\nResponse: We are grateful for your thoughtful review. Your constructive comments have been both inspiring and invaluable.\n2. Will the work be of significance to the field and related fields? How does it compare to the established literature? If the work is not original, please provide relevant references.\nSee above. The work adds to the literature on milk extracellular vesicles in that it details another potential application (antibiotic delivery). This is of sufficient significance to the field.\nResponse: Thank you for your incisive review. Your constructive suggestions have given us great encouragement.\n3. Does the work support the conclusions and claims, or is additional evidence needed?\nThe dysbiosis aspects of the work in my opinion are strong but the work suffers from the lack of detailed characterisation of the vesicles, as highlighted below.\nResponse: We sincerely appreciate your meticulous review and the insightful critiques that have substantially elevated the rigor of our study. Every point has been carefully addressed in the revised manuscript, and we sincerely hope that the amendments will meet your expectations.\nAre there any flaws in the data analysis, interpretation and conclusions? Do these prohibit publication or require revision?\n(1) TEM does not convincingly reveal the identity of the vesicles. Cryo TEM or super resolution imaging.\nResponse: Thanks for the suggestions. We have added the Cryo TEM results as below:\nCryo-transmission electron microscope (cryo-TEM) images showed that mEV-Lip had spheroidal morphology (Supplementary Fig. 3).\nSupplementary Fig. 3 The cryo-transmission electron microscope (cryo-TEM) images of Lip, mEV, and mEV-Lip. Scale bars, 25 nm.\n(2) Characterisation of particles is limited. Are all the vesicles in the sample hybrids or do you expect to have a proportion of EVs, liposomes and hybrids? If yes, this should be quantified (FRET does not help with this). Consider using nano flow cytometry and / or RAMAN and / or super resolution microscopy. A mixed particle pullulation unfortunately makes some of the analysis (eg western blotting and mucus diffusion) difficult to interpret and potentially invalid since the samples will contain a mixed population of unfused and fused vesicles not allowing for discrimination of findings related to each particle species.\nResponse: Thanks for the suggestions. Our initial prescription optimization resulted in exclusively hybrid vesicles, as confirmed by the supplementary confocal laser scanning microscope results below:\nConfocal laser scanning microscope (CLSM) further confirmed the successful hybridization of the mEV-Lip (Supplementary Fig. 2).\nSupplementary Fig. 2 Confocal laser scanning microscope images of vesicles. Green: C6; red: RhB. Scale bars, 1 μm.\n(3) There is too much reliance on literature reports of phenomena that are not convincingly established / confirmed, including involvement of FcRn and mucus diffusivity of EVs. The conclusions should be toned down to potentially rather than convincingly suggest FcRn and PEPT1 involvement in EV trafficking. Own data in Fig2p highlight only a 10% decrease in endocytosis upon blocked of these receptors - this does not convincingly evidence that the uptake of EVs is mediated by these receptors.\nResponse: Thanks for the suggestions. We have toned down the conclusions to potentially rather than convincingly suggest FcRn and PEPT1 involvement in EV trafficking. To further explore the relationship between mEV-Lip and these transporters, we have supplemented the study with inhibitors experiments targeting FcRn and PEPT1 in both transcytosis assays and in vivo models. Additionally, linear regression analysis was performed to evaluate the correlation between mEV-Lip and FcRn/PEPT1 expression. We have supplemented these contents as below:\nMoreover, linear regression analysis demonstrated that epithelial uptake of mEV-Lip correlated tightly with FcRn and PEPT1 expression levels (Supplementary Fig. 15). The above results collectively suggest that the absorption of mEV-Lip in the small intestine is potentially mediated by specific interactions with FcRn/PEPT1.\nTo further demonstrate this, we employed the FcRn inhibitor human serum albumin (HSA) and the PEPT1 inhibitor glycylsarcosine (Gly-Sar) to block each pathway in vivo, which resulted in a marked reduction in the absorption of mEV-Lip in the small intestine (Supplementary Fig. 16). In contrast, the absorption of Lip remained unchanged upon treatment with FcRn or PEPT1 inhibitor.\nThe transcytosis assays revealed similar pathway dependency, with mEV and mEV-Lip transport across epithelial barriers being significantly compromised by both inhibitors (Supplementary Fig. 19).\nSupplementary Fig. 15 The uptake efficiency of mEV-Lip was correlated with the expression levels of FcRn (a) and PEPT1 (b). n = 5 mice.\nSupplementary Fig. 16 a, Representative images of intestine from mice after oral gavage of DiR-loaded vesicles with inhibitor of FcRn HSA or PEPT1 Gly-Sar. Scale bars, 2.5 cm. b-d, Quantification of the fluorescence intensity in SI-P (b), SI-D (c), and LI (d) at 4 h after oral administration of Lip or mEV-Lip. Data are mean ± SD (n = 3). One-way ANOVA with a Tukey post hoc test was used for statistical significance. * P < 0.05, *** P < 0.001. NS, not significant.\nControl HSA Gly-Sar\nNS ** P = 0.0020 ** P = 0.0065\nNS ** P = 0.0022 ** P = 0.0063\n120\n)%\n( s100\nis\no ty 80\nc\ns n 60\na\rt\ne\n40\nv\\ita\n20\nle\nR\nSupplementary Fig. 19 Transcytosis efficiency of vesicles under different transporter inhibitors. All values are presented as the mean ± SD (n = 3). Statistical significance was determined using one-way ANOVA with a Tukey post hoc test. ** P < 0.01. NS, not significant.\n(4) EVs should be called EVs or small EVs rather than exosomes.\nResponse: Thanks for the suggestions. We have modified exosomes to extracellular vesicles.\n4. Is the methodology sound? Does the work meet the expected standards in your field?\nMostly yes, but I have some reservations:\n(1) Fig2a: missing secondary antibody only control.\nResponse: Thanks for the suggestions. We have added the control as below:\nFig. 2a, Immunofluorescence analysis of FcRn and PEPT1 expression in mouse small intestines and colon. Scale bars, 100 μm.\n(2) Supplementary figure 6 and Fig 2h and i: why were HT29 cells used? It is indeed a subclone of these (HT29-MTX) which is capable of producing mucus and is commonly used in co-culture as a goblet-like cell type to render the model as mucus-producing.\nResponse: Thanks for the comments. In Supplementary figure 6 and Fig 2h and I, Caco-2/HT29 coculture model was used. HT29 cells were used to simulate the production of mucus by goblet cells, while Caco-2 cells were used to simulate intestinal epithelial cells.\n(3) Colocalisation studies in Supplementary figure 9 are not convincing, primarily due to the very low expression of FcRn and PEPT1.\nResponse: Thanks for the suggestions. This variability may be attributed to individual differences among mice, which can lead to variations in receptor expression levels. To address this, we repeated the immunofluorescence experiments and performed Western Blot analysis to investigate intestinal receptor expression. Furthermore, we explored the correlation between receptor expression levels and the intestinal absorption of mEV-Lip. The corresponding results have included in the revised manuscript as follows:\nMoreover, linear regression analysis demonstrated that epithelial uptake of mEV-Lip correlated tightly with FcRn and PEPT1 expression levels (Supplementary Fig. 15).\nSupplementary Fig. 1 Western blot analysis of FcRn and PEPT1 expression in mouse small intestines and colon.\nSupplementary Fig. 14 Representative images of C6-labeled vesicles, FcRn, and PEPT1 in intestine. Blue: nucleus; Green: C6-labeled vesicles; Red: FcRn/PEPT1. Scale bars, 50 μm.\nSupplementary Fig. 15 The uptake efficiency of mEV-Lip was correlated with the expression levels of FcRn (a) and PEPT1 (b). n = 5 mice.\n(4) Fig 2o: controls lacking.\nResponse: Thanks for the comments. We have added the control as below:\nSupplementary Fig. 17 Immunofluorescence analysis of FcRn and PEPT1 expression in Caco-2 cells. Scale bars, 5 μm.\n(5) How were the antibiotic doses standardised when the loading efficiency in liposomes, exos and hybrids were different?\nResponse: Thanks for the comments. Antibiotic loading was first quantified in each vesicle formulation; thereafter, equivalent doses of the encapsulated antibiotics were administered.\n5. Is there enough detail provided in the methods for the work to be reproduced?\nMethods related to exocytosis studies unclear.\nResponse: Thanks for the comments. We have added the method of exocytosis studies as below:\nExocytosis experiments of various vesicles were conducted using the Caco-2/HT29 coculture model (7:3). Following 48 h culture in 96-well plates, cellular density was quantified through Alamar Blue assay. The cells were then exposed to C6-labeled vesicles (C6 concentration: 0.5 μg/mL) for 3 h, washed three times with cold PBS, and incubated with DMEM for 1 h. After lysis with DMSO, the fluorescence intensity was quantified on a microplate reader.\n6. Overall, considering the points raised above on data analysis, interpretation and conclusions, in my view the manuscript currently does not meet the requirements of the current journal regarding the soundness / rigour of the methodology, although with major revisions (additional experiments), this may be addressed.\nResponse: We are grateful for your review and the insightful comments that have materially strengthened the scientific rigor of our manuscript. All concerns have been carefully addressed in the revised version, and we sincerely hope that the amendments meet your expectations.\nReviewer #3:\nThe article is excellent and with strong data and novel information.\nHowever some suggestions need to be improved as below.\nResponse: Thank you for your incisive review and for the constructive suggestions that have markedly improved the scientific rigor of our work. Every point has been carefully addressed in the revised manuscript, and we sincerely hope that the changes will satisfy your expectations.\n1. Language and Grammar Improvements.\n• Line 16 – Break the long sentence into two shorter ones to improve readability.\n• Line 50 – Change “and minimizing” to “while minimizing” for correct parallel structure.\n• Line 113 – Correct typo: change “instrument” to “instrumental”.\n• Line 246 – Remove redundancy: change “fecal community communities” to “fecal communities”.\n• Line 314 – Replace colloquial “Of note” with “Notably” for a more formal academic tone.\n• Line 563 – Correct typo: change “sane route” to “same route”.\nResponse: Thanks for the suggestions. We have rectified these grammatical errors in our revised manuscript as below:\nHerein, we engineer a biohybrid delivery system through the integration of milk extracellular vesicles with liposomes.\nThe hybrid vesicles employ targeting mechanisms via neonatal Fc receptor and peptide transporter 1, facilitating antibiotic transport across the proximal small intestine.\nSince FcRn and PEPT1 are highly expressed in the proximal small intestine, this transport mechanism may promote absorption by the proximal small intestine while minimizing side effects on the microbiota in the colon.\nThese findings imply that the PEG layer is instrumental in improving permeability of vesicles in the intestinal mucus.\nAs anticipated, 16S ribosomal RNA sequencing revealed marked depletion of stool α-diversity and substantial dysbiosis in fecal communities following free Cef administration.\nNotably, mice receiving mEV-Lip@Cef pretreatment demonstrated infection resistance comparable to untreated controls, with no statistically significant differences in pathogen colonization levels and histopathological manifestations.\nBalb/c mice were pretreated with free Cef or mEV-Lip@Cef via oral administration daily for 5 d, while control animals were administered equivalent volumes of PBS via the same route.\n2. Structural and Content Refinements.\n• Line 335 – Condense the first sentence of the Discussion to remove repetitive background information.\n• Line 82 – In the Methods section, specify EDTA concentration units (e.g., mM) and provide a brief rationale for casein removal.\n• Line 167 – Include statistical analysis to confirm the significance of the encapsulation efficiency results.\n• Also address why entrapment efficiency is much lower without liposome incorporation (11.29% vs. 56%).\nResponse: Thanks for the comments. We have refined the structural and content as below:\nAntibiotics have long been hailed as life-saving agents in the battle against bacterial infections.\nTo remove casein from milk, ethylenediaminetetraacetic acid disodium salt (EDTA-2Na, 30 mM) was added to chelate calcium ions and disrupt casein micelle integrity, removing casein through micellar disassembly before ultracentrifugation.\nAlthough the encapsulation efficiency of natural mEV was only 11.29%, mEV-Lip significantly increased the drug encapsulation efficiency to 56.02% (4.96-fold, P = 0.0002) (Supplementary Table. 1).\nThis substantial improvement highlights the advantage of preparing hybrid vesicles, where the enhanced membrane fluidity imparted by liposomal components facilitates superior drug-loading capacity compared to natural extracellular vesicles.\n3. Formatting and Style Consistency.\n• Line 493 – Ensure temperature is written consistently as “37°C” throughout the manuscript.\n• Keep units consistent and in correct SI format.\n• Define all abbreviations upon first appearance in the main text, even if defined in the Abstract (e.g., Papp, HFD).\n• Follow gene/protein naming conventions (e.g., italicize FcRn, PEPT1 only if referring to genes).\n• Ensure statistical notation is consistent, e.g., P < 0.05 (with a space after P).\nResponse: We really appreciate the reviewer for these helpful suggestions. We have corrected these formatting and stylistic inconsistencies in the revised manuscript.\n4. Figures, Abstract, and Overall Presentation.\n• Consider reducing the number of figures; some sections are visually overloaded, which may distract from key findings.\n• Shorten and revise the Abstract to improve clarity and focus, avoiding redundancy while highlighting key quantitative results.\nResponse: Thanks for the suggestions. We have reduced the number of figures and revised the Abstract as below:\nOral antibiotics are a mainstay for treating bacterial infections, but unabsorbed portions can reach the caecum and colon, leading to gut dysbiosis. Herein, we engineer a biohybrid delivery system through the integration of milk extracellular vesicles with liposomes. The hybrid vesicles employ targeting mechanisms via neonatal Fc receptor and peptide transporter 1, facilitating antibiotic transport across the proximal small intestine. These vesicles exhibit superior drug encapsulation efficiency, stable release behavior, efficient mucus traversal, higher endocytosis, increased basolateral exocytosis, and improved oral absorption, achieving a 3.24-fold increase in oral bioavailability compared to free antibiotics. In lung bacterial infections and bacteremia models, hybrid vesicle-encapsulated cefdinir outperforms free antibiotics in eliminating infections. Notably, this approach also mitigates adverse effects on the intestinal microbiota, safeguarding the animals from dysbiosis-associated metabolic syndromes and opportunistic pathogen infections. This innovative hybrid vesicle system holds great promise for the oral delivery of other drugs that suffer from limited absorption or cause gut dysbiosis.\nFig. 2 Hybrid vesicles enhance absorption in the proximal small intestine. a, Immunofluorescence analysis of FcRn and PEPT1 expression in mouse small intestines and colon. Scale bars, 100 μm. b, Dynamic light scattering-measured mEV particle size. c, Nanoparticle-tracking analysis measurement of mEV. d, FRET phenomenon of mEV-Lip encapsulating C6 and RhB. e, WB results of mEV characteristic proteins. f, Representative trajectories of different vesicles in fresh porcine mucus. Scale bars, 50 pixels. g, The mean square displacement (MSD) of vesicles over time. h, Quantitative exocytosis of various vesicles. i, Apparent permeability coefficient (Papp) values of vesicles across Caco-2/HT29 cell monolayers in Transwell inserts. j, IVIS images 4 h post-oral administration of free DiR or DiR-loaded vesicles in intestine. SI-P: proximal small intestine; SI-D: distal small intestine; LI: large intestine. k-m, Absorption dynamics for various vesicles. IVIS image quantification at 1, 4, 8, and 12 hours post-oral administration of DiR-loaded vesicles in SI-P (k), SI-D (l), or LI (m). n, Schematic representation showing that mEV-Lip, with PEG modification, penetrating the mucus layer and being taken up and transported by intestinal epithelial cells (IECs) via FcRn/PEPT1-mediated transport after oral administration. Data are mean ± SD (n = 3). One-way ANOVA with a Tukey post hoc test was used for statistical significance. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. NS, not significant."}, {"round": 2, "reviewer_comments": {"reviewer_1": "All my comments have been well addressed, therefore I have no further comemnts.", "reviewer_2": "The authors have on the whole addressed the comments in a satisfactory manner. However, my point on the use of HT29 instead of a subclone with an increased capacity to produce mucus has not been addressed appropriately. It is well evidenced in the field that the parental HT29 cell line produces little mucus while HT29-MTX secretes significantly more. It would have been more appropriate to use the latter, but since the study uses a separate mucus assay in any case, this is not a substantive point that affects the conclusions. But it should be acknowledged somewhere.\nAnother small point that I missed previously: Line 427 - 'mEV solution' should be replaced with 'mEV suspension'. If this appears elsewhere, then it should be corrected accordingly."}, "author_response": "Response to Reviewers\nWe express our gratitude to the Editorial Office and the reviewers for providing us with constructive comments and suggestions. We have revised the paper in accordance with the reviewers’ feedback.\nReviewer #1:\nAll my comments have been well addressed, therefore I have no further comments.\nResponse: Thank you very much for your recognition.\nReviewer #2:\n1. The authors have on the whole addressed the comments in a satisfactory manner. However, my point on the use of HT29 instead of a subclone with an increased capacity to produce mucus has not been addressed appropriately. It is well evidenced in the field that the parental HT29 cell line produces little mucus while HT29-MTX secretes significantly more. It would have been more appropriate to use the latter, but since the study uses a separate mucus assay in any case, this is not a substantive point that affects the conclusions. But it should be acknowledged somewhere.\nResponse: We appreciate your valuable suggestion. Due to the current constraints of our cell line inventory, only HT29 cells were available for this study. We fully acknowledge the potential benefits of using HT29-MTX cells as you mentioned. We will certainly keep your advice in mind for our future work and will endeavor to procure the HT29-MTX cell line for subsequent experiments.\n2. Another small point that I missed previously: Line 427 - 'mEV solution' should be replaced with 'mEV suspension'. If this appears elsewhere, then it should be corrected accordingly.\nResponse: Thanks for the suggestion. We have replaced the “mEV solution” to “mEV suspension”."}]

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