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

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Herein, we engineer milk exosome-liposome hybrid vesicles that enhance antibiotic absorption in the proximal small intestine via neonatal Fc receptor and peptide transporter 1-mediated transport. 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 while reducing exposure to the large-intestine microbiota. 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. Health sciences/Medical research/Drug development Health sciences/Health care/Therapeutics/Drug therapy/Drug delivery Oral antibiotics milk exosomes hybrid vesicles bioavailability gut dysbiosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Antibiotics are among the most widely prescribed medications globally, with their usage escalating at an alarming rate 1 . 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 strains 2 . Moreover, the unabsorbed fraction of orally administered antibiotics reaches the caecum and colon, potentially causing intestinal microbiota dysbiosis 3 . The mutualistic microbes residing in the intestine are vital for various physiological functions, including the regulation of immune and metabolic homoeostasis 4 – 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 disorders 7 – 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 10 5 /mL in the upper small intestine to approximately 10 12 /mL in the colon 12 . 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 feces 13 . In recent years, exosomes have emerged as promising drug delivery carriers for targeted delivery and overcoming physiological barriers 14 – 16 . Milk-derived exosomes (mEXO) stand out as particularly promising oral delivery candidates due to their ample sources, efficient cargo protection capabilities, minimal immunogenicity, and stable gastrointestinal physicochemical properties 17 . Notably, research has shown that mEXO are transported through neonatal Fc receptor (FcRn) and peptide transporter 1 (PEPT1) on the intestinal epithelial cells 18 . Since FcRn and PEPT1 are highly expressed in the proximal small intestine, this transport mechanism may promote absorption by the proximal small intestine and minimizing side effects on the microbiota in the colon 19 , 20 . Nevertheless, natural mEXO still face several limitations as oral carriers. First, mEXO have limited ability to traverse the mucus layer 21 . Second, mEXO encounter challenges in encapsulating cargo efficiently 22 . Third, the phenomenon of rapid release of small molecule drugs encapsulated in mEXO is quite pronounced 23 . To overcome these limitations and construct a more efficient carrier for oral delivery, it is essential to modify natural mEXO to enhance their mucus penetration ability, drug loading capacity, and release behavior. In this study, we design a milk exosome-liposome (mEXO-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 polyethylene glycol (PEG) enables the mEXO-Lip to penetrate the intestinal mucus layer, while the proteins on the surface of exosomes 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 mEXO-Lip (mEXO-Lip@Cef) exhibits higher efficacy in Klebsiella pneumoniae infection in both lung and bacteremia models. More importantly, treatment with mEXO-Lip@Cef markedly diminishes the negative impact on intestinal microbiota, mitigating the risk of dysbiosis-associated opportunistic pathogen infection and metabolic syndromes. 2. Results 2.1. Hybrid vesicles facilitate enhanced absorption at proximal small intestine The uptake of mEXO at the intestinal epithelium is facilitated by FcRn and PEPT1 18 . To explore the potential of targeted antibiotic delivery, we first investigated the expression of these transporters in the intestine. As depicted in Fig. 2 a, 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 mEXO in the proximal small intestine. Inspired by this insight, we engineered mEXO-Lip hybrid vesicles, leveraging the transporter-binding ability to target the proximal small intestine. Initially, mEXO was isolated using a classical ultracentrifugation method. To remove casein from milk, ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) was added to chelate and precipitate casein before ultracentrifugation. Dynamic light scattering (Fig. 2 b) and nanoparticle-tracking analysis (NTA, Fig. 2 c) revealed hydrated particle sizes of 132.9 ± 1.8 nm and 113.5 ± 2.5 nm, respectively, aligning with the typical size range of exosomes (30–150 nm). The modest polydispersity index (PDI, 0.208 ± 0.016) and substantial yield (1.08 × 10 11 ± 2.62 × 10 9 particles/mL) indicated successful isolation of mEXO. 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 exosomal membrane’s phospholipid bilayer. Fluorescence scanning patterns of exosomes, liposomes (Lip), and mEXO-Lip demonstrated the fluorescence resonance energy transfer (FRET) phenomenon in the hybrid vesicles (Fig. 2 d). Transmission electron microscope (TEM) images showed that mEXO-Lip had distinct morphological features compared to exosomes; while exosomes typically exhibit a teacup-shaped morphology, mEXO-Lip presented irregular vesicle-like structures (Supplementary Fig. 1). Western Blot (WB) analysis corroborate the presence of exosomal signature proteins (CD63, CD9, HSP70) in both mEXO and mEXO-Lip, while calnexin, an endoplasmic reticulum marker, was absent in both groups except the supernatant (Fig. 2 e). Particle size and zeta potential measurements indicated that after fusion with liposomes, exosomes had an increased particle size and a more negative potential (Supplementary Fig. 2). Moreover, stability tests showed that both mEXO and mEXO-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. 3). These vesicles also exhibited excellent stability over 7 days at 4℃ and − 80℃ (Supplementary Fig. 4). 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 mEXO by introducing hydrophilic PEG. To closely mimic the in vivo environment, freshly obtained porcine small intestinal mucus was used to evaluate mucus penetration. NTA showed that the mobility of mEXO was restricted in the mucus, whereas Lip and mEXO-Lip had significantly wider mobility ranges (Fig. 2 f). Mean square displacement (MSD) calculations revealed that within a 1 s interval, the MSD values of Lip and mEXO-Lip were greater than that of mEXO (Fig. 2 g). Besides, a transwell permeable scaffold was used to construct a three-dimensional mucus model (Supplementary Fig. 5a), and the apparent permeability coefficient (Papp) was measured to quantify the transport rate from the donor chamber to the recipient chamber 24 . The hydrophilic modification by PEG significantly enhanced the mucus penetration ability of natural exosomes, with a 1.6-fold increase (Supplementary Fig. 5b). These findings imply that the PEG layer is instrument 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 mEXO-Lip had a significant increase compared to Lip (2.7-fold) and mEXO (1.7-fold), indicating that the combination of PEG with mEXO is an effective strategy to overcome both the mucus and apical membrane barriers (Supplementary Fig. 6). Additionally, mEXO-Lip demonstrated enhanced exocytosis ability compared to Lip and mEXO (Fig. 2 h). A cell monolayer was established to assess the transcytosis efficiency of various formulations across the intestinal epithelium. The Papp results indicated that mEXO-Lip improved the transcytosis efficiency of Lip and mEXO in the Caco-2/HT29 coculture cell monolayer model (Fig. 2 i). These findings suggest that mEXO-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, mEXO-Lip had a higher signal intensity in the small intestine and a markedly lower intensity in the large intestine (Fig. 2 j-m, and Supplementary Fig. 7). In vitro intestinal absorption experiments also showed that mEXO-Lip had significantly higher absorption in the small intestine compared with Lip (Fig. 2 n). We further investigated the micro-distribution of the vesicles within the proximal small intestine. The findings indicated that the uptake of mEXO-Lip by intestinal epithelium surpassed that of other vesicles (Supplementary Fig. 8). Immunofluorescent staining of intestinal sections showed that mEXO-Lip co-localized with FcRn and PEPT1 in small intestinal epithelial cells (Supplementary Fig. 9). Moreover, when FcRn and PEPT1 were overexpressed in Caco-2 cells (Fig. 2 o), FcRn inhibitor (human serum albumin, HSA) and PEPT1 inhibitor (glycylsarcosine, Gly-Sar) significantly inhibited the endocytosis of mEXO and mEXO-Lip, while having a relatively weaker inhibitory effect on Lip (Fig. 2 p). Collectively, these results suggest that the hybrid vesicles promote their absorption in the proximal small intestine through the transporters FcRn and PEPT1 (Fig. 2 q), which holds great promise for oral antibiotic delivery. 2.2. Hybrid vesicles reduce intestinal residues and enhance oral bioavailability To evaluate whether the increased absorption of mEXO-Lip at the proximal small intestine could decrease intestinal residues, the quantity of residual DiR-labeled mEXO-Lip in feces was measured using the IVIS imaging system at various time points post oral administration. Comparative analysis revealed that mice treated with mEXO-Lip showed a marked reduction in fecal residues compared to free DiR and DiR-labeled Lip (Fig. 3 a,b), which may be correlated with enhanced proximal intestinal absorption. We subsequently explored whether mEXO-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, mEXO@Cef, and mEXO-Lip@Cef. Although the encapsulation efficiency of natural mEXO was only 11.29%, mEXO-Lip significantly increased the drug encapsulation efficiency to 56.02% (4.96-fold) (Supplementary Table. 1). This substantial improvement highlights the advantage of preparing hybrid vesicles, utilizing the drug-loading capacity of liposomes to address the issue of low encapsulation efficiency of natural exosomes when loading drugs. Besides, in vitro drug release experiments revealed that Cef was released in a slow and sustained manner from mEXO-Lip@Cef when incubated in simulated buffers (Fig. 3 c). This sustained release profile overcomes the problem of rapid drug release from natural mEXO. The serum concentration of Cef in the mEXO-Lip@Cef group was significantly higher than that of free Cef (Fig. 3 d and Supplementary Table. 2). Additionally, the bioavailability of mEXO-Lip@Cef was 3.24 times higher than that of free Cef (Fig. 3 e), indicating that mEXO-Lip@Cef significantly promoted the oral absorption of Cef. These results collectively suggest that the mEXO-Lip packaging enhances oral antibiotics bioavailability and reduces intestinal residues, which underscores the potential of mEXO-Lip as an effective oral delivery system for antibiotics. 2.3. mEXO-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. 4 a) 25 . Compared to free Cef, which achieved modest bacterial reduction in the lung, both Lip@Cef and mEXO@Cef demonstrated superior antibiotic activity (Fig. 4 b-d). Remarkably, mEXO-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. 4 e). 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. 4 f-h). Importantly, lower inflammation and attenuated tissue damage were observed in mEXO-Lip@Cef-treated mice compared with free Cef-treated and other Cef-loaded vesicle-treated mice. In summary, these results suggest that mEXO-Lip@Cef exhibits enhanced efficacy in treating bacterial pneumonia. 2.4. mEXO-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. 5 a). 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. 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. 5 b). Remarkably, the mEXO-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 mEXO-Lip@Cef demonstrated a more efficient clearance of Klebsiella pneumoniae compared to those treated with free Cef, Lip@Cef, and mEXO@Cef (Fig. 5 c-e). In addition, hematoxylin and eosin (H&E) staining was performed on liver and spleen tissue sections (Fig. 5 f). The staining results showed that mEXO-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 mEXO-Lip@Cef possesses superior efficacy in eliminating bacterial pathogens, highlighting its potential as an effective approach for antibiotic therapy. 2.5. 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 aboundance 26 . 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 populations 27 – 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. 6 a). As anticipated, 16S ribosomal RNA sequencing revealed marked depletion of stool α-diversity and substantial dysbiosis in fecal community communities following free Cef administration (Fig. 6 b-e). Strikingly, the mEXO-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 mEXO-Lip@Cef group compared to the pronounced dispersion observed with free Cef (Fig. 6 f). 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. 6 g,h). In contrast, mEXO-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. 2.6. 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 accumulation 30 . Pathological conditions including obesity, diabetes mellitus type 2, and hepatic steatosis demonstrate enhanced dietary energy harvest efficiency, a characteristic feature of metabolic dysregulation 31 . To investigate the potential impact of antibiotic-loaded vesicles on antibiotic-associated metabolic perturbations, we established a dietary obesity model (Fig. 7 a). Treatment with free Cef resulted in a significant increase in HFD-induced body mass accumulation (Fig. 7 b). In contrast, the mEXO-Lip@Cef showed moderated weight alterations, suggesting a reduced impact on weight gain. Similarly, metabolic parameter analysis demonstrated that free Cef treatment exacerbated high-fat diet (HFD)-induced metabolic dysregulation, manifesting as impaired glucose tolerance and hepatic cholesterol accumulation (Fig. 7 c-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 mEXO-Lip@Cef compared to those treated with free Cef (Fig. 7 f). To investigate whether these metabolic alterations were driven by changes in gut microbiota, we conducted fecal microbiota transplantation (FMT) studies (Fig. 7 g). Three donor cohorts were established: free Cef treatment, mEXO-Lip@Cef treatment, and antibiotic-naïve controls. Recipient mice received fecal transplants from donor groups and were subsequently placed on an 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 mEXO-Lip@Cef-treated donors (Fig. 7 h-l). These findings jointly indicate that engineered vesicle-mediated antibiotic delivery preserves metabolic homeostasis by maintaining microbial ecological balance. 2.7. 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 pathobionts 32 – 34 . To evaluate the effects of pretreatment with free Cef and mEXO-Lip@Cef on the severity of Escherichia coli intestinal infection, we conducted an experimental study (Fig. 8 a). 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. 8 b-e). This heightened susceptibility manifested as elevated pathogen burdens across fecal samples, cecal contents, and colonic tissues, accompanied by pronounced mucosal hyperplasia. Of note, mice receiving mEXO-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. 3. Discussion Antibiotics have long been hailed as life-saving agents in the battle against bacterial infections, enjoying near-universal adoption in clinical practice. However, the shadow of their overuse looms large, casting a multitude of risks upon human health. In response to this pressing issue, we have developed an innovative antibiotic delivery technology that enhances oral bioavailability while minimizing the impact on the gut microbiota. Our study demonstrates the potential of milk exosome-liposome hybrid vesicles to achieve targeted and efficient absorption of antibiotics at the proximal small intestine. The mEXO-Lip system significantly enhances the oral absorption of antibiotics, as evidenced by our experiments with Cef. This hybrid vesicle formulation not only improves drug bioavailability but also reduces perturbations to the gut microbiota, thereby mitigating dysbiosis-induced adverse effects. Our findings, although preliminary and based on murine models, suggest that mEXO-Lip-based antibiotic delivery can substantially reduce the severity of antibiotic-induced perturbation to gut microbiota. This is particularly significant given the well-established clinical manifestations of dysbiosis, which can severely endanger patients in hospital settings. The ability to deliver antibiotics orally while minimizing the need for intravenous administration could revolutionize the treatment of bacterial infections. Oral delivery of antibiotic-loaded mEXO-Lip could potentially reduce the duration and necessity of hospitalizations, thereby lowering associated costs and risks. Our study has shown that mEXO-Lip performs efficiently for the delivery of Cef, but the potential applications extend beyond this single antibiotic. Future research should explore the efficacy of this system with other antibiotics and even other drugs that face absorption-related or gut-dysbiosis-related limitations. The strong and selective absorption efficiency enabled by mEXO-Lip suggests its potential use for the oral delivery of a wide range of drugs. This technology could address the challenges associated with low oral bioavailability and the unintended consequences of drug-induced dysbiosis. 4. Materials and Methods 4.1. Materials Egg lecithin 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-mPEG 2000 was purchased from AVT (Shanghai) Pharmaceutical Tech Co., Ltd. (Shanghai, China). Recombinant human serum albumin (HSA) was purchased from Tianjin Xiensi Biochemical Technology Co., Ltd. (Tianjin, 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-FCGRT/FcRn antibody, anti-SLC15A1/PEPT1 antibody, and goat anti-rabbit IgG H&L (HRP) were obtained from Abcam (Shanghai, 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. 4.2. Strains Klebsiella pneumoniae used in this study is a clinical strain isolated from human, preserved in our laboratory; Escherichia coli used in this study was obtained from the American Type Culture Collection and is preserved in our laboratory. 4.3. 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℃, 5% CO 2 , 95% relative humidity). Male Balb/c mice certified by Hunan Slac Jingda 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. All procedures complied with guidelines and received institutional approval (IACUC-20250407-01) from the Laboratory Animal Use and Management Committee of Southwest University. 4.4. Isolation of milk exosomes (mEXO) Pasteurized bovine milk was sourced from retail sources. mEXO isolation employed a multi-step differential centrifugation protocol: primary clarification at 5000 g (30 min, 4℃) 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 (100,000 g for 60 min, 210,000 g 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. mEXO samples with a total protein concentration below 2 mg/mL were cryopreserved at -80℃ until further use. 4.5. 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-mPEG 2000 (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 mEXO solution (4 mg of mEXO 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 mEXO 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. 4.6. 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 mEXO suspensions was performed via nanoparticle-tracking analysis (NTA, Nanosight LM10, Malvern Instruments Ltd., UK). For morphology characterization, negatively-stained specimens (2% phosphotungstic acid) were imaged via transmission electron microscopy (TEM, Hitachi HT-7800, Japan). To confirm the successful extraction of exosomes, Western Blot (WB) analysis was conducted on both mEXO and the hybrid vesicles. The presence of several exosomal markers, including CD9, CD63, and HSP70, was detected, along with the supernatant protein calnexin, following standard protocols. To validate the fusion of mEXO with liposomes, a fluorescence resonance energy transfer (FRET)-based analytical approach was employed. Specifically, mEXO 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℃. 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, following established protocols 18 . 4.7. 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 (100 µL) were homogenously dispersed in 900 µL 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 SGF, 37℃, 2 h) followed by SIF (50 mL, 37℃, 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. 4.8. Mucus permeability evaluation The mobility of the vesicles within mucus was assessed using NTA. A volume of 0.1 mL vesicles was introduced into 1 mL porcine intestinal mucus and incubated for 30 min. The movement of the vesicles within the mucus was then recorded. The averaged mean square displacement (MSD) of the particles was calculated based on established methodologies described in the literature 35 . The mucus permeability of the vesicles was quantitatively assessed using a Transwell system. Initially, the donor chamber received 100 µL of porcine intestinal mucus, while the acceptor compartment was filled with 800 µL of PBS. To achieve uniform mucus layer formulation, the assembly underwent equilibration at 37℃ with shaking for 30 min. Subsequently, 150 µL of C6-labeled vesicles was carefully administered to the mucus-containing donor chamber, followed by continuous incubation at 37℃. 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 as described in the literature 24 . 4.9. 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 mmol/L) 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. 4.10. 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 experimental protocol involved administration of C6-labeled vesicles (C6 concentration: 0.5 µg/mL) with subsequent 3 h cellular internalization. Post-uptake processing included triple-rinsing with cold PBS to remove extracellular particulates. To monitor secretory trafficking, preloaded cultures were maintained in for 1 h under standard culture conditions. Finally, the fluorescence intensity was determined using a microplate reader. 4.11. 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 approximately300 Ω·cm 2 was achieved. Prior to transport experiments, the differentiated monolayers were pre-incubated with blank medium for 30 min at 37℃. The apical compartment then received 200 µL of medium containing C6-labeled vesicles (C6 concentration: 1.0 µg/mL), while the basolateral chamber contained 200 µL 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. 4.12. Biodistribution of vesicles Balb/c mice 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). 4.13. Intestinal absorption of Cef-loaded vesicles After fasting the Balb/c mice 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% O 2 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 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 counterstaining was performed with DAPI prior to fluorescence imaging analysis using an Olympus confocal laser scanning microscope (CLSM, Japan). 4.14. Pharmacokinetics of Cef-loaded vesicles in vivo Balb/c mice were fasted overnight and orally administered free Cef, Lip@Cef, mEXO@Cef or mEXO-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℃. 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. 4.15. In vivo pharmacodynamics studies in lung infection model with Klebsiella pneumoniae A murine pneumonia model was established in Balb/c mice by intranasal inoculation with 1 × 10 7 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℃ 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. 4.16. In vivo pharmacodynamics studies in bacteremia model with Klebsiella pneumoniae Bacteremia was induced in Balb/c mice by intravenous injection with 1 × 10 7 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℃. Colonies were counted to calculate the CFU. The liver and spleen were harvested for H&E staining histological examination. 4.17. High-fat diet (HFD) mouse model Balb/c mice were divided into three groups receiving PBS, free Cef or mEXO-Lip@Cef via oral gavage for 5 d. Subsequently, all animals were transitioned to HFD 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 week17 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. 4.18. Fecal microbiota transplantation (FMT) Donor Balb/c mice received daily oral gavage of PBS, free Cef, or mEXO-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. 4.19. Intestinal infection model with Escherichia coli Balb/c mice were pretreated with free Cef or mEXO-Lip@Cef via oral administration daily for 5 d, while control animals were administered equivalent volumes of PBS via the sane route. Subsequently, all experimental groups were subjected to intraperitoneal challenge with Escherichia coli (1 × 10 8 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℃ overnight before enumeration of CFU, with final counts normalized to tissue mass. The colons were harvested for histological examination via H&E staining. 4.20. 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 values < 0.05 were considered statistically significant. Declarations Declaration of competing interest The authors declare no competing interest. Acknowledgments This research was financially supported by National Key Research and Development Program of China (2021YFD1800900), Chongqing Science and Technology Commission (CSTB2023NSCQ-JQX0002), Special Fund for Youth Team of Southwest University (SWU-XJLJ202306), Chongqing Natural Science Foundation (CSTB2024NSCQ-MSX0547), Science and Technology Innovation Key R&D Program of Chongqing (CSTB2024TIAD-STX0038, CSTB2022TIAD-KPX0094). Data availability All data are available within the article and supplementary information. References Dickey SW, Cheung GYC, Otto M (2017) Different drugs for bad bugs: antivirulence strategies in the age of antibiotic resistance. Nat Rev Drug Discov 16:457–471 Sommer MOA, Dantas G, Church GM (2009) Functional characterization of the antibiotic resistance reservoir in the human microflora. Science 325:1128–1131 Levison ME, Levison JH (2009) Pharmacokinetics and pharmacodynamics of antibacterial agents. 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Mater. 20, 539–547 ( (2023)) Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryInformation.docx Supplementary Information Cite Share Download PDF Status: Published Journal Publication published 25 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7033003","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":481747313,"identity":"da98df66-b068-4a49-85ab-49851dc5b774","order_by":0,"name":"Lei 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1","display":"","copyAsset":false,"role":"figure","size":499312,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration of the milk exosome-liposome hybrid vesicle (mEXO-Lip) for oral delivery of antibiotics.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mEXO-Lip system is designed to enhance the absorption of antibiotics in the proximal small intestine, thereby reducing the contact between antibiotics and the microbiota residing in colon, thus mitigating antibiotic-associated dysbiosis.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7033003/v1/aa5e3f5b49090d0ea62dcf75.png"},{"id":86409698,"identity":"6fc91d39-e446-4d65-b26c-443a8f3c8c39","added_by":"auto","created_at":"2025-07-10 10:33:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":643790,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHybrid vesicles enhance absorption in the proximal small intestine.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Immunofluorescence analysis of FcRn and PEPT1 expression in mouse small intestines and colon.\u003cstrong\u003e \u003c/strong\u003eScale bars, 100 μm. \u003cstrong\u003eb\u003c/strong\u003e, Dynamic light scattering-measured mEXO particle size. \u003cstrong\u003ec\u003c/strong\u003e, Nanoparticle-tracking analysis measurement of mEXOs. \u003cstrong\u003ed\u003c/strong\u003e, FRET phenomenon of mEXO-Lip encapsulating C6 and RhB. \u003cstrong\u003ee\u003c/strong\u003e, WB results of exosomal characteristic proteins. \u003cstrong\u003ef\u003c/strong\u003e, Representative trajectories of different vesicles in fresh porcine mucus. Scale bars, 50 pixels.\u003cstrong\u003e g\u003c/strong\u003e, The mean square displacement (MSD) of vesicles over time. \u003cstrong\u003eh\u003c/strong\u003e, Quantitative exocytosis of various vesicles. \u003cstrong\u003ei\u003c/strong\u003e, Apparent permeability coefficient (Papp) values of vesicles across Caco-2/HT29 cell monolayers in Transwell inserts. \u003cstrong\u003ej\u003c/strong\u003e, 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. \u003cstrong\u003ek-m\u003c/strong\u003e, 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 (\u003cstrong\u003ek\u003c/strong\u003e), SI-D (\u003cstrong\u003el\u003c/strong\u003e), or LI (\u003cstrong\u003em\u003c/strong\u003e). \u003cstrong\u003en\u003c/strong\u003e, Papp values of vesicles transported across the intestine. \u003cstrong\u003eo\u003c/strong\u003e, Immunofluorescence analysis of FcRn and PEPT1 expression in Caco-2 cells. Scale bars, 5 μm. \u003cstrong\u003ep\u003c/strong\u003e, Endocytosis efficiency of vesicles under different endocytic inhibitors. \u003cstrong\u003eq\u003c/strong\u003e, Schematic representation showing that mEXO-Lip, with PEG modification, penetrating the mucus layer and being taken up and transported by intestinal epithelial cells (IECs) \u003cem\u003evia\u003c/em\u003e FcRn/PEPT1-mediated transport after oral administration. Data are mean ± SD (\u003cem\u003en \u003c/em\u003e= 3). One-way ANOVA with a Tukey post hoc test was used for statistical significance. * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, **** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001. NS, not significant.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7033003/v1/d32c670ab0597c6f6e6291a3.png"},{"id":86408367,"identity":"d64bef44-029a-4b1c-8863-92844dadf4b1","added_by":"auto","created_at":"2025-07-10 10:17:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":588354,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHybrid vesicles mitigate intestinal residues and enhance oral antibiotics absorption.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Representative images of residual vesicles in murine fecal samples at different time points. \u003cstrong\u003eb\u003c/strong\u003e, Quantification of vesicle fluorescence intensity in fecal matter. \u003cem\u003en\u003c/em\u003e = 3 mice. \u003cstrong\u003ec\u003c/strong\u003e, Drug release profile in 2 h simulated gastric fluid (SGF) followed by 6 h simulated intestinal fluid (SIF). \u003cem\u003en \u003c/em\u003e= 3. \u003cstrong\u003ed\u003c/strong\u003e, Plasma concentration-time profiles of free Cef and Cef-loaded vesicles in mice. \u003cem\u003en\u003c/em\u003e = 5 mice. \u003cstrong\u003ee\u003c/strong\u003e, Relative bioavailability calculation by comparing the area under the curve (AUC) of the vesicle formulation to that of free Cef. All data presented as mean ± SD. Statistical significance determined \u003cem\u003evia\u003c/em\u003eone-way ANOVA with a Tukey post hoc test.* \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7033003/v1/ec578fb2fa745241a3d885c4.png"},{"id":86409407,"identity":"70f56e18-0492-40ff-a6a3-1512130bbdf6","added_by":"auto","created_at":"2025-07-10 10:25:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":749309,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCef-loaded hybrid vesicles effectively eliminate \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eKlebsiella pneumoniae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection in the lung.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Schematic illustration of the experimental protocol for therapy in the mouse model of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e infection. \u003cstrong\u003eb\u003c/strong\u003e, Representative \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e colonies formed on plates from the lung homogenates of mice with pneumonia after different treatments. \u003cstrong\u003ec\u003c/strong\u003e, Quantitative analysis of bacterial loads in the lungs. \u003cstrong\u003ed\u003c/strong\u003e, Representative images of lung tissue from different groups after Gram staining. Scale bars, 100 μm. Arrows indicate \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. \u003cstrong\u003ee\u003c/strong\u003e, Levels of inflammatory cytokines in the lung tissue. \u003cstrong\u003ef\u003c/strong\u003e, The appearance of lungs from different treatment groups. \u003cstrong\u003eg\u003c/strong\u003e, Hematoxylin and eosin (H\u0026amp;E) staining of lung sections. Scale bars, 100 μm. \u003cstrong\u003eh\u003c/strong\u003e, Lung index (lung/body weight ratio) quantification. Data are presented as the mean ± SD. \u003cem\u003en\u003c/em\u003e = 5 mice. Statistical significance was determined using one-way ANOVA with a Tukey post hoc test. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7033003/v1/c54df7b065c8fb36b31777e8.png"},{"id":86408372,"identity":"8f85a0f5-5043-4521-92b0-b3d6f4b55801","added_by":"auto","created_at":"2025-07-10 10:17:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":793365,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCef-loaded hybrid vesicles effectively eradicate \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eKlebsiella pneumoniae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection in a bacteremia model.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Experimental timeline schematic of the bacteremia infection model. \u003cstrong\u003eb\u003c/strong\u003e, Macroscopic pathological changes observed in hepatic and splenic tissues. \u003cstrong\u003ec-e\u003c/strong\u003e, Comparative analysis of microbial burden in circulatory (\u003cstrong\u003ec\u003c/strong\u003e), hepatic (\u003cstrong\u003ed\u003c/strong\u003e), and splenic (\u003cstrong\u003ee\u003c/strong\u003e). \u003cstrong\u003ef\u003c/strong\u003e, Histopathological evaluation of liver and spleen sections through H\u0026amp;E staining. Scale bars, 100 μm. Data presented mean ± SD. \u003cem\u003en\u003c/em\u003e = 5 mice. Statistical comparisons were performed using one-way ANOVA with a Tukey post hoc test.* \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7033003/v1/cbf2569884bcdc7d57c4529f.png"},{"id":86408389,"identity":"63ed710f-1bfe-4381-8b96-b6e89efcaf05","added_by":"auto","created_at":"2025-07-10 10:17:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":795065,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtective effects of hybrid vesicle delivery against antibiotic-induced microbial imbalance.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Schematic design workflow. \u003cstrong\u003eb\u003c/strong\u003e, Temporal changes in gut microbiota α-diversity indices across different groups. The pink band indicates therapeutic intervention periods with either free Cef or Cef-loaded vesicles. \u003cstrong\u003ec\u003c/strong\u003e, Comparative analysis of α-diversity through AUC. \u003cstrong\u003ed\u003c/strong\u003e, Longitudinal variations in fecal microbial β-diversity between modalities, assessed by Unweighted UniFrac metric relative to baseline. \u003cstrong\u003ee\u003c/strong\u003e, Cumulative β-diversity alterations quantified by AUC. \u003cstrong\u003ef\u003c/strong\u003e, Multivariate separation of intestinal bacteria profiles visualized through principal components analysis (PCA). The circles represent the confidence ellipses, illustrating the distribution of samples within each group. \u003cstrong\u003eg\u003c/strong\u003e, Taxonomic composition profiling of gut microbiota. \u003cstrong\u003eh\u003c/strong\u003e, Hierarchical clustering analysis of microbial abundance patterns. Data represent mean ± SD. \u003cem\u003en\u003c/em\u003e = 3 mice. Statistical comparisons performed using one-way ANOVA with a Tukey post hoc test. ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001, **** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7033003/v1/3d51308ca30d85bd2ba2a5f4.png"},{"id":86408375,"identity":"e097ef63-67a4-4a84-acaa-9a21b7a08e76","added_by":"auto","created_at":"2025-07-10 10:17:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":777346,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHybrid vesicle-mediated delivery of antibiotics ameliorates dysbiosis-associated obesity.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Graphical overview of the experimental design. \u003cstrong\u003eb\u003c/strong\u003e, Body weight changesacross intervention group. \u003cstrong\u003ec\u003c/strong\u003e, Intraperitoneal glucose tolerance test (IPGTT) results for mice. \u003cstrong\u003ed\u003c/strong\u003e, AUC analysis of blood glucose levels during IPGTT. \u003cstrong\u003ee\u003c/strong\u003e, Hepatic cholesterolaccumulation after prolonged high-fat diet (HFD) exposure. \u003cstrong\u003ef\u003c/strong\u003e, Representative H\u0026amp;E-stained sections of liver tissue. Scale bars: 100 μm (overview), 50 μm (magnified regions indicated by dashed squares). \u003cstrong\u003eg\u003c/strong\u003e, Schematic showing the experimental protocol. \u003cstrong\u003eh\u003c/strong\u003e, Body weight changes in recipient mice. \u003cstrong\u003ei\u003c/strong\u003e, IPGTT results for recipient mice. \u003cstrong\u003ej\u003c/strong\u003e, AUC analysis of blood glucose during IPGTT in recipient mice. \u003cstrong\u003ek\u003c/strong\u003e, Hepatic cholesterol deposition. \u003cstrong\u003el\u003c/strong\u003e, Parenchymal morphology assessment in recipient mice. Scale bars, 100 μm (overview), 50 μm (magnified regions indicated by dashed squares). Data are expressed as the mean ± SD. \u003cem\u003en\u003c/em\u003e = 5 mice. Statistical comparisons employed one-way ANOVA with a Tukey post hoc test. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. NS, not significant.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7033003/v1/e0e69fe20997f00dced4f3eb.png"},{"id":86409408,"identity":"f39c313e-1da7-4351-8906-2d9c23eb71b7","added_by":"auto","created_at":"2025-07-10 10:25:46","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":439525,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe hybrid vesicle-mediated delivery of antibiotics decreases dysbiosis-associated infection by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEscherichia coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Experimental design workflow. \u003cstrong\u003eb-d\u003c/strong\u003e, Microbial burden assessment in feces (\u003cstrong\u003eb\u003c/strong\u003e), caecal contents (\u003cstrong\u003ec\u003c/strong\u003e), and colon (\u003cstrong\u003ed\u003c/strong\u003e). \u003cstrong\u003ee\u003c/strong\u003e, Histopathological characterization of colon sections by H\u0026amp;E staining. Scale bars, 100 μm. \u003cstrong\u003ef\u003c/strong\u003e, Schematic diagram illustrating the mechanism by which treatment with free Cef decreases microbiota diversity and leads to the expansion of the \u003cem\u003eEscherichia coli\u003c/em\u003e population. In contrast, treatment with mEXO-Lip@Cef has minimal impact on microbiota, maintaining diversity and resistance to pathogens colonization. Data expressed as mean ± SD. \u003cem\u003en\u003c/em\u003e = 5 mice. Statistical analyses employed one-way ANOVA with a Tukey post hoc test.* \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. NS, not significant.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7033003/v1/90cf13749b7fffe3619ff510.png"},{"id":101933176,"identity":"e07fc594-23a7-4f69-8d66-f1ee71cce7d1","added_by":"auto","created_at":"2026-02-05 08:06:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6612288,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7033003/v1/91cef3c7-0555-4a1c-b0db-1a0ae25e583f.pdf"},{"id":86409405,"identity":"23a4c114-5958-4079-9db1-036b063131ae","added_by":"auto","created_at":"2025-07-10 10:25:45","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":866497,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7033003/v1/68e85d3d0ce8f9a832af7061.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Engineered vesicles enhance oral antibiotic absorption in proximal small intestine and mitigate gut dysbiosis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAntibiotics are among the most widely prescribed medications globally, with their usage escalating at an alarming rate\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. 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 strains\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Moreover, the unabsorbed fraction of orally administered antibiotics reaches the caecum and colon, potentially causing intestinal microbiota dysbiosis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The mutualistic microbes residing in the intestine are vital for various physiological functions, including the regulation of immune and metabolic homoeostasis\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. When this delicate balance is disrupted by antibiotics, it may trigger a cascade of adverse effects, such as diarrhea, allergic reactions, inflammation, and neurological disorders\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9 CR10\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Thus, it is essential to improve the oral bioavailability of antibiotics and reduce their unintended negative impacts on gut microbiota to optimize their use.\u003c/p\u003e \u003cp\u003eThe population and composition of gut microbiota differ greatly in various parts of the intestine. Bacterial counts typically stretch from approximately 10\u003csup\u003e5\u003c/sup\u003e /mL in the upper small intestine to approximately 10\u003csup\u003e12\u003c/sup\u003e /mL in the colon\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. This bacterial density gradient hints that if antibiotics are effectively absorbed in the proximal small intestine, it would restrict contact with the large intestine\u0026rsquo;s flora, thereby cutting down their presence in feces\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In recent years, exosomes have emerged as promising drug delivery carriers for targeted delivery and overcoming physiological barriers\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Milk-derived exosomes (mEXO) stand out as particularly promising oral delivery candidates due to their ample sources, efficient cargo protection capabilities, minimal immunogenicity, and stable gastrointestinal physicochemical properties\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Notably, research has shown that mEXO are transported through neonatal Fc receptor (FcRn) and peptide transporter 1 (PEPT1) on the intestinal epithelial cells\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Since FcRn and PEPT1 are highly expressed in the proximal small intestine, this transport mechanism may promote absorption by the proximal small intestine and minimizing side effects on the microbiota in the colon\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Nevertheless, natural mEXO still face several limitations as oral carriers. First, mEXO have limited ability to traverse the mucus layer\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Second, mEXO encounter challenges in encapsulating cargo efficiently\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Third, the phenomenon of rapid release of small molecule drugs encapsulated in mEXO is quite pronounced\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. To overcome these limitations and construct a more efficient carrier for oral delivery, it is essential to modify natural mEXO to enhance their mucus penetration ability, drug loading capacity, and release behavior.\u003c/p\u003e \u003cp\u003eIn this study, we design a milk exosome-liposome (mEXO-Lip) hybrid vesicle with the aim of enhancing absorption in the proximal small intestine and thereby circumventing adverse effects on the colonic microbiota (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The hydrophilic modification of polyethylene glycol (PEG) enables the mEXO-Lip to penetrate the intestinal mucus layer, while the proteins on the surface of exosomes 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 mEXO-Lip (mEXO-Lip@Cef) exhibits higher efficacy in \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e infection in both lung and bacteremia models. More importantly, treatment with mEXO-Lip@Cef markedly diminishes the negative impact on intestinal microbiota, mitigating the risk of dysbiosis-associated opportunistic pathogen infection and metabolic syndromes.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Hybrid vesicles facilitate enhanced absorption at proximal small intestine\u003c/h2\u003e \u003cp\u003eThe uptake of mEXO at the intestinal epithelium is facilitated by FcRn and PEPT1\u003csup\u003e18\u003c/sup\u003e. To explore the potential of targeted antibiotic delivery, we first investigated the expression of these transporters in the intestine. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, 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 mEXO in the proximal small intestine.\u003c/p\u003e \u003cp\u003eInspired by this insight, we engineered mEXO-Lip hybrid vesicles, leveraging the transporter-binding ability to target the proximal small intestine. Initially, mEXO was isolated using a classical ultracentrifugation method. To remove casein from milk, ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) was added to chelate and precipitate casein before ultracentrifugation. Dynamic light scattering (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) and nanoparticle-tracking analysis (NTA, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) revealed hydrated particle sizes of 132.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 nm and 113.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 nm, respectively, aligning with the typical size range of exosomes (30\u0026ndash;150 nm). The modest polydispersity index (PDI, 0.208\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016) and substantial yield (1.08 \u0026times; 10\u003csup\u003e11\u003c/sup\u003e \u0026plusmn; 2.62 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e particles/mL) indicated successful isolation of mEXO.\u003c/p\u003e \u003cp\u003eNext, 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 exosomal membrane\u0026rsquo;s phospholipid bilayer. Fluorescence scanning patterns of exosomes, liposomes (Lip), and mEXO-Lip demonstrated the fluorescence resonance energy transfer (FRET) phenomenon in the hybrid vesicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Transmission electron microscope (TEM) images showed that mEXO-Lip had distinct morphological features compared to exosomes; while exosomes typically exhibit a teacup-shaped morphology, mEXO-Lip presented irregular vesicle-like structures (Supplementary Fig.\u0026nbsp;1). Western Blot (WB) analysis corroborate the presence of exosomal signature proteins (CD63, CD9, HSP70) in both mEXO and mEXO-Lip, while calnexin, an endoplasmic reticulum marker, was absent in both groups except the supernatant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Particle size and zeta potential measurements indicated that after fusion with liposomes, exosomes had an increased particle size and a more negative potential (Supplementary Fig.\u0026nbsp;2). Moreover, stability tests showed that both mEXO and mEXO-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.\u0026nbsp;3). These vesicles also exhibited excellent stability over 7 days at 4℃ and \u0026minus;\u0026thinsp;80℃ (Supplementary Fig.\u0026nbsp;4). In summary, we successfully prepared hybrid vesicles modified with PEG-functionalized phospholipids, which possess stable colloidal properties.\u003c/p\u003e \u003cp\u003eOne of the primary objectives of this study was to enhance the mucus penetration ability of natural mEXO by introducing hydrophilic PEG. To closely mimic the \u003cem\u003ein vivo\u003c/em\u003e environment, freshly obtained porcine small intestinal mucus was used to evaluate mucus penetration. NTA showed that the mobility of mEXO was restricted in the mucus, whereas Lip and mEXO-Lip had significantly wider mobility ranges (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Mean square displacement (MSD) calculations revealed that within a 1 s interval, the MSD values of Lip and mEXO-Lip were greater than that of mEXO (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). Besides, a transwell permeable scaffold was used to construct a three-dimensional mucus model (Supplementary Fig.\u0026nbsp;5a), and the apparent permeability coefficient (Papp) was measured to quantify the transport rate from the donor chamber to the recipient chamber\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The hydrophilic modification by PEG significantly enhanced the mucus penetration ability of natural exosomes, with a 1.6-fold increase (Supplementary Fig.\u0026nbsp;5b). These findings imply that the PEG layer is instrument in improving permeability of vesicles in the intestinal mucus.\u003c/p\u003e \u003cp\u003eAfter 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 mEXO-Lip had a significant increase compared to Lip (2.7-fold) and mEXO (1.7-fold), indicating that the combination of PEG with mEXO is an effective strategy to overcome both the mucus and apical membrane barriers (Supplementary Fig.\u0026nbsp;6). Additionally, mEXO-Lip demonstrated enhanced exocytosis ability compared to Lip and mEXO (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). A cell monolayer was established to assess the transcytosis efficiency of various formulations across the intestinal epithelium. The Papp results indicated that mEXO-Lip improved the transcytosis efficiency of Lip and mEXO in the Caco-2/HT29 coculture cell monolayer model (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). These findings suggest that mEXO-Lip can be more efficiently transported into and out of the epithelial cells, with an \u0026ldquo;easy entry, easy exit\u0026rdquo; characteristic.\u003c/p\u003e \u003cp\u003eTo visualize the \u003cem\u003ein vivo\u003c/em\u003e 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 \u003cem\u003ein vivo\u003c/em\u003e imaging system (IVIS). Compared with free DiR, mEXO-Lip had a higher signal intensity in the small intestine and a markedly lower intensity in the large intestine (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej-m, and Supplementary Fig.\u0026nbsp;7). \u003cem\u003eIn vitro\u003c/em\u003e intestinal absorption experiments also showed that mEXO-Lip had significantly higher absorption in the small intestine compared with Lip (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003en). We further investigated the micro-distribution of the vesicles within the proximal small intestine. The findings indicated that the uptake of mEXO-Lip by intestinal epithelium surpassed that of other vesicles (Supplementary Fig.\u0026nbsp;8). Immunofluorescent staining of intestinal sections showed that mEXO-Lip co-localized with FcRn and PEPT1 in small intestinal epithelial cells (Supplementary Fig.\u0026nbsp;9). Moreover, when FcRn and PEPT1 were overexpressed in Caco-2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eo), FcRn inhibitor (human serum albumin, HSA) and PEPT1 inhibitor (glycylsarcosine, Gly-Sar) significantly inhibited the endocytosis of mEXO and mEXO-Lip, while having a relatively weaker inhibitory effect on Lip (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ep). Collectively, these results suggest that the hybrid vesicles promote their absorption in the proximal small intestine through the transporters FcRn and PEPT1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eq), which holds great promise for oral antibiotic delivery.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Hybrid vesicles reduce intestinal residues and enhance oral bioavailability\u003c/h2\u003e \u003cp\u003eTo evaluate whether the increased absorption of mEXO-Lip at the proximal small intestine could decrease intestinal residues, the quantity of residual DiR-labeled mEXO-Lip in feces was measured using the IVIS imaging system at various time points post oral administration. Comparative analysis revealed that mice treated with mEXO-Lip showed a marked reduction in fecal residues compared to free DiR and DiR-labeled Lip (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,b), which may be correlated with enhanced proximal intestinal absorption.\u003c/p\u003e \u003cp\u003eWe subsequently explored whether mEXO-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, mEXO@Cef, and mEXO-Lip@Cef. Although the encapsulation efficiency of natural mEXO was only 11.29%, mEXO-Lip significantly increased the drug encapsulation efficiency to 56.02% (4.96-fold) (Supplementary Table. 1). This substantial improvement highlights the advantage of preparing hybrid vesicles, utilizing the drug-loading capacity of liposomes to address the issue of low encapsulation efficiency of natural exosomes when loading drugs. Besides, \u003cem\u003ein vitro\u003c/em\u003e drug release experiments revealed that Cef was released in a slow and sustained manner from mEXO-Lip@Cef when incubated in simulated buffers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). This sustained release profile overcomes the problem of rapid drug release from natural mEXO. The serum concentration of Cef in the mEXO-Lip@Cef group was significantly higher than that of free Cef (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed and Supplementary Table. 2). Additionally, the bioavailability of mEXO-Lip@Cef was 3.24 times higher than that of free Cef (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee), indicating that mEXO-Lip@Cef significantly promoted the oral absorption of Cef. These results collectively suggest that the mEXO-Lip packaging enhances oral antibiotics bioavailability and reduces intestinal residues, which underscores the potential of mEXO-Lip as an effective oral delivery system for antibiotics.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. mEXO-Lip@Cef effectively eliminates \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e infection in the lung\u003c/h2\u003e \u003cp\u003eTo evaluate the therapeutic potential of hybrid vesicles, we established a murine model of pulmonary infection induced by \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, a predominant nosocomial pathogen causing pneumonia (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea)\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Compared to free Cef, which achieved modest bacterial reduction in the lung, both Lip@Cef and mEXO@Cef demonstrated superior antibiotic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-d). Remarkably, mEXO-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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef-h). Importantly, lower inflammation and attenuated tissue damage were observed in mEXO-Lip@Cef-treated mice compared with free Cef-treated and other Cef-loaded vesicle-treated mice. In summary, these results suggest that mEXO-Lip@Cef exhibits enhanced efficacy in treating bacterial pneumonia.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. mEXO-Lip@Cef effectively eradicates \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e infection in a bacteremia model\u003c/h2\u003e \u003cp\u003eTo further evaluate the antibacterial efficacy of antibiotic-loaded vesicles, we employed a bacteremia model (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Mice were intravenously inoculated with a standardized dose of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. Subsequently, at pre-determined time points, the mice were treated with free Cef and Cef-loaded vesicles. 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Remarkably, the mEXO-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 mEXO-Lip@Cef demonstrated a more efficient clearance of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e compared to those treated with free Cef, Lip@Cef, and mEXO@Cef (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-e). In addition, hematoxylin and eosin (H\u0026amp;E) staining was performed on liver and spleen tissue sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). The staining results showed that mEXO-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 mEXO-Lip@Cef possesses superior efficacy in eliminating bacterial pathogens, highlighting its potential as an effective approach for antibiotic therapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Hybrid vesicles mitigate antibiotic-driven dysbiosis\u003c/h2\u003e \u003cp\u003eThe 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 aboundance\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. 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 populations\u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eAs anticipated, 16S ribosomal RNA sequencing revealed marked depletion of stool α-diversity and substantial dysbiosis in fecal community communities following free Cef administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb-e). Strikingly, the mEXO-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 mEXO-Lip@Cef group compared to the pronounced dispersion observed with free Cef (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg,h). In contrast, mEXO-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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Hybrid vesicle-mediated antibiotic delivery ameliorates dysbiosis-associated obesity\u003c/h2\u003e \u003cp\u003eEmerging evidence underscores the critical involvement of intestinal microbial communities in regulating systemic metabolic process, particularly energy balance regulation and lipid accumulation\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Pathological conditions including obesity, diabetes mellitus type 2, and hepatic steatosis demonstrate enhanced dietary energy harvest efficiency, a characteristic feature of metabolic dysregulation\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. To investigate the potential impact of antibiotic-loaded vesicles on antibiotic-associated metabolic perturbations, we established a dietary obesity model (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eTreatment with free Cef resulted in a significant increase in HFD-induced body mass accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). In contrast, the mEXO-Lip@Cef showed moderated weight alterations, suggesting a reduced impact on weight gain. Similarly, metabolic parameter analysis demonstrated that free Cef treatment exacerbated high-fat diet (HFD)-induced metabolic dysregulation, manifesting as impaired glucose tolerance and hepatic cholesterol accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec-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 mEXO-Lip@Cef compared to those treated with free Cef (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eTo investigate whether these metabolic alterations were driven by changes in gut microbiota, we conducted fecal microbiota transplantation (FMT) studies (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg). Three donor cohorts were established: free Cef treatment, mEXO-Lip@Cef treatment, and antibiotic-na\u0026iuml;ve controls. Recipient mice received fecal transplants from donor groups and were subsequently placed on an 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 mEXO-Lip@Cef-treated donors (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh-l). These findings jointly indicate that engineered vesicle-mediated antibiotic delivery preserves metabolic homeostasis by maintaining microbial ecological balance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Hybrid vesicle delivery of antibiotics decreases dysbiosis-associated \u003cem\u003eEscherichia coli\u003c/em\u003e infection\u003c/h2\u003e \u003cp\u003eAntibiotic-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 pathobionts\u003csup\u003e\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. To evaluate the effects of pretreatment with free Cef and mEXO-Lip@Cef on the severity of \u003cem\u003eEscherichia coli\u003c/em\u003e intestinal infection, we conducted an experimental study (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Mice were pretreated with antibiotics to simulate the initial treatment and associated perturbation of the gut microbiota, followed by an intestinal challenge with pathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e at day 5. Quantitative analysis revealed that free Cef-pretreated mice exhibited significantly compromised resistance to \u003cem\u003eEscherichia coli\u003c/em\u003e colonization compared to antibiotic-na\u0026iuml;ve controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb-e). This heightened susceptibility manifested as elevated pathogen burdens across fecal samples, cecal contents, and colonic tissues, accompanied by pronounced mucosal hyperplasia. Of note, mice receiving mEXO-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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eAntibiotics have long been hailed as life-saving agents in the battle against bacterial infections, enjoying\u003c/p\u003e \u003cp\u003enear-universal adoption in clinical practice. However, the shadow of their overuse looms large, casting a multitude of risks upon human health. In response to this pressing issue, we have developed an innovative antibiotic delivery technology that enhances oral bioavailability while minimizing the impact on the gut microbiota. Our study demonstrates the potential of milk exosome-liposome hybrid vesicles to achieve targeted and efficient absorption of antibiotics at the proximal small intestine.\u003c/p\u003e \u003cp\u003eThe mEXO-Lip system significantly enhances the oral absorption of antibiotics, as evidenced by our experiments with Cef. This hybrid vesicle formulation not only improves drug bioavailability but also reduces perturbations to the gut microbiota, thereby mitigating dysbiosis-induced adverse effects. Our findings, although preliminary and based on murine models, suggest that mEXO-Lip-based antibiotic delivery can substantially reduce the severity of antibiotic-induced perturbation to gut microbiota. This is particularly significant given the well-established clinical manifestations of dysbiosis, which can severely endanger patients in hospital settings.\u003c/p\u003e \u003cp\u003eThe ability to deliver antibiotics orally while minimizing the need for intravenous administration could revolutionize the treatment of bacterial infections. Oral delivery of antibiotic-loaded mEXO-Lip could potentially reduce the duration and necessity of hospitalizations, thereby lowering associated costs and risks. Our study has shown that mEXO-Lip performs efficiently for the delivery of Cef, but the potential applications extend beyond this single antibiotic. Future research should explore the efficacy of this system with other antibiotics and even other drugs that face absorption-related or gut-dysbiosis-related limitations. The strong and selective absorption efficiency enabled by mEXO-Lip suggests its potential use for the oral delivery of a wide range of drugs. This technology could address the challenges associated with low oral bioavailability and the unintended consequences of drug-induced dysbiosis.\u003c/p\u003e"},{"header":"4. Materials and Methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Materials\u003c/h2\u003e \u003cp\u003eEgg lecithin 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-mPEG\u003csub\u003e2000\u003c/sub\u003e was purchased from AVT (Shanghai) Pharmaceutical Tech Co., Ltd. (Shanghai, China). Recombinant human serum albumin (HSA) was purchased from Tianjin Xiensi Biochemical Technology Co., Ltd. (Tianjin, 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-FCGRT/FcRn antibody, anti-SLC15A1/PEPT1 antibody, and goat anti-rabbit IgG H\u0026amp;L (HRP) were obtained from Abcam (Shanghai, 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Strains\u003c/h2\u003e \u003cp\u003e \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e used in this study is a clinical strain isolated from human, preserved in our laboratory; \u003cem\u003eEscherichia coli\u003c/em\u003e used in this study was obtained from the American Type Culture Collection and is preserved in our laboratory.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Cells and Animals\u003c/h2\u003e \u003cp\u003eHuman intestinal cell lines, Caco-2 and HT29 cells, were procured from Beijing Dingguo Biotechnology Co., Ltd. (Beijing, China). Cellular maintenance was conducted in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;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℃, 5% CO\u003csub\u003e2\u003c/sub\u003e, 95% relative humidity).\u003c/p\u003e \u003cp\u003eMale Balb/c mice certified by Hunan Slac Jingda 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. All procedures complied with guidelines and received institutional approval (IACUC-20250407-01) from the Laboratory Animal Use and Management Committee of Southwest University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.4. Isolation of milk exosomes (mEXO)\u003c/h2\u003e \u003cp\u003ePasteurized bovine milk was sourced from retail sources. mEXO isolation employed a multi-step differential centrifugation protocol: primary clarification at 5000 g (30 min, 4℃) removed somatic cells and debris, followed by casein dissociation using ethylenediaminetetraacetic acid disodium salt (EDTA-2Na, 30 mM). The supernatant underwent filtration (0.22 \u0026micro;m polyethersulfone membrane) prior to sequential ultracentrifugation (100,000 g for 60 min, 210,000 g for 60 min, Optima L-90K, Beckman, USA) for vesicle subfractionation. Pelleted vesicles were suspended in phosphate-buffered saline (PBS) and quantified \u003cem\u003evia\u003c/em\u003e a BCA protein assay kit. mEXO samples with a total protein concentration below 2 mg/mL were cryopreserved at -80℃ until further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.5. Preparation of hybrid vesicles\u003c/h2\u003e \u003cp\u003eThe hybrid vesicles were fabricated using the dried film hydration-extrusion technique. Primary constituents including phospholipids (10 mg), cholesterol (2 mg), and DSPE-mPEG\u003csub\u003e2000\u003c/sub\u003e (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 mEXO solution (4 mg of mEXO 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 mEXO 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.6. Characterization of vesicles\u003c/h2\u003e \u003cp\u003eColloidal properties of the engineered vesicles were assessed using dynamic light scattering (3000\u0026thinsp;+\u0026thinsp;Ultra, Malvern Instruments Ltd., UK). The particle concentration of mEXO suspensions was performed \u003cem\u003evia\u003c/em\u003e nanoparticle-tracking analysis (NTA, Nanosight LM10, Malvern Instruments Ltd., UK). For morphology characterization, negatively-stained specimens (2% phosphotungstic acid) were imaged \u003cem\u003evia\u003c/em\u003e transmission electron microscopy (TEM, Hitachi HT-7800, Japan). To confirm the successful extraction of exosomes, Western Blot (WB) analysis was conducted on both mEXO and the hybrid vesicles. The presence of several exosomal markers, including CD9, CD63, and HSP70, was detected, along with the supernatant protein calnexin, following standard protocols.\u003c/p\u003e \u003cp\u003eTo validate the fusion of mEXO with liposomes, a fluorescence resonance energy transfer (FRET)-based analytical approach was employed. Specifically, mEXO 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\u0026ndash;700 nm wavelength range to quantify energy transfer dynamics.\u003c/p\u003e \u003cp\u003eThe 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 \u0026times; 4.6 mm, 5 \u0026micro;m). 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 \u0026micro;L injection volume, and column thermostating at 25℃. 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, following established protocols\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.7. Stability and \u003cem\u003ein vitro\u003c/em\u003e release profile\u003c/h2\u003e \u003cp\u003eThe 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 (100 \u0026micro;L) were homogenously dispersed in 900 \u0026micro;L of either SGF or SIF and incubated. The particle size of the vesicles was measured at predetermined time intervals.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e 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 SGF, 37℃, 2 h) followed by SIF (50 mL, 37℃, 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.8. Mucus permeability evaluation\u003c/h2\u003e \u003cp\u003eThe mobility of the vesicles within mucus was assessed using NTA. A volume of 0.1 mL vesicles was introduced into 1 mL porcine intestinal mucus and incubated for 30 min. The movement of the vesicles within the mucus was then recorded. The averaged mean square displacement (MSD) of the particles was calculated based on established methodologies described in the literature\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe mucus permeability of the vesicles was quantitatively assessed using a Transwell system. Initially, the donor chamber received 100 \u0026micro;L of porcine intestinal mucus, while the acceptor compartment was filled with 800 \u0026micro;L of PBS. To achieve uniform mucus layer formulation, the assembly underwent equilibration at 37℃ with shaking for 30 min. Subsequently, 150 \u0026micro;L of C6-labeled vesicles was carefully administered to the mucus-containing donor chamber, followed by continuous incubation at 37℃. Aliquots (80 \u0026micro;L) 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 as described in the literature\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.9. Cellular uptake and endocytic mechanisms\u003c/h2\u003e \u003cp\u003eCellular 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 \u0026micro;g/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.\u003c/p\u003e \u003cp\u003eEndocytic 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 \u0026micro;g/mL) and Gly-Sar (100 mmol/L) were added to separate wells and incubated for 1 h. Thereafter, C6-labeled vesicles (C6 concentration: 0.5 \u0026micro;g/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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.10. Exocytosis studies\u003c/h2\u003e \u003cp\u003eExocytosis 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 experimental protocol involved administration of C6-labeled vesicles (C6 concentration: 0.5 \u0026micro;g/mL) with subsequent 3 h cellular internalization. Post-uptake processing included triple-rinsing with cold PBS to remove extracellular particulates. To monitor secretory trafficking, preloaded cultures were maintained in for 1 h under standard culture conditions. Finally, the fluorescence intensity was determined using a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4.11. Transcellular transport studies\u003c/h2\u003e \u003cp\u003eA 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 approximately300 Ω\u0026middot;cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e was achieved. Prior to transport experiments, the differentiated monolayers were pre-incubated with blank medium for 30 min at 37℃. The apical compartment then received 200 \u0026micro;L of medium containing C6-labeled vesicles (C6 concentration: 1.0 \u0026micro;g/mL), while the basolateral chamber contained 200 \u0026micro;L fresh medium. Aliquots (80 \u0026micro;L) 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e4.12. Biodistribution of vesicles\u003c/h2\u003e \u003cp\u003eBalb/c mice 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 \u003cem\u003ein vivo\u003c/em\u003e imaging system (IVIS, Vilber Lourmat).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e4.13. Intestinal absorption of Cef-loaded vesicles\u003c/h2\u003e \u003cp\u003eAfter fasting the Balb/c mice 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 \u0026micro;g/mL) was injected into each segment. The intestinal segments were then immersed in fresh Krebs-Ringer (K-R) solution at 37\u0026deg;C in a water bath, with a continuous supply of 95% O\u003csub\u003e2\u003c/sub\u003e 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.\u003c/p\u003e \u003cp\u003eBalb/c mice 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 \u0026micro;g/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 \u0026micro;m thickness using a Leica cryostat (Germany). Nuclear counterstaining was performed with DAPI prior to fluorescence imaging analysis using an Olympus confocal laser scanning microscope (CLSM, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e4.14. Pharmacokinetics of Cef-loaded vesicles \u003cem\u003ein vivo\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eBalb/c mice were fasted overnight and orally administered free Cef, Lip@Cef, mEXO@Cef or mEXO-Lip@Cef (Cef: 35 mg/kg). Serial blood samples (0.2 mL) were obtained \u003cem\u003evia\u003c/em\u003e 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 \u0026times; 4.6 mm, 5 \u0026micro;m) maintained at 40℃. 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e4.15. \u003cem\u003eIn vivo\u003c/em\u003e pharmacodynamics studies in lung infection model with \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eA murine pneumonia model was established in Balb/c mice by intranasal inoculation with 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e suspended in 50 \u0026micro;L 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℃ 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\u0026amp;E) and Gram staining, with microscopic examination conducted using light-field optics.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e4.16. \u003cem\u003eIn vivo\u003c/em\u003e pharmacodynamics studies in bacteremia model with \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eBacteremia was induced in Balb/c mice by intravenous injection with 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e CFU \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. 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 \u003cem\u003evia\u003c/em\u003e 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℃. Colonies were counted to calculate the CFU. The liver and spleen were harvested for H\u0026amp;E staining histological examination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e4.17. High-fat diet (HFD) mouse model\u003c/h2\u003e \u003cp\u003eBalb/c mice were divided into three groups receiving PBS, free Cef or mEXO-Lip@Cef \u003cem\u003evia\u003c/em\u003e oral gavage for 5 d. Subsequently, all animals were transitioned to HFD 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 week17 through an overnight-fasted intraperitoneal glucose tolerance test (IPGTT), where animals received 1 g/kg glucose solution \u003cem\u003evia\u003c/em\u003e 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 \u003cem\u003evia\u003c/em\u003e H\u0026amp;E staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e4.18. Fecal microbiota transplantation (FMT)\u003c/h2\u003e \u003cp\u003eDonor Balb/c mice received daily oral gavage of PBS, free Cef, or mEXO-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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e4.19. Intestinal infection model with \u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eBalb/c mice were pretreated with free Cef or mEXO-Lip@Cef \u003cem\u003evia\u003c/em\u003e oral administration daily for 5 d, while control animals were administered equivalent volumes of PBS \u003cem\u003evia\u003c/em\u003e the sane route. Subsequently, all experimental groups were subjected to intraperitoneal challenge with \u003cem\u003eEscherichia coli\u003c/em\u003e (1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e 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℃ overnight before enumeration of CFU, with final counts normalized to tissue mass. The colons were harvested for histological examination \u003cem\u003evia\u003c/em\u003e H\u0026amp;E staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e4.20. Data analysis\u003c/h2\u003e \u003cp\u003eExperimental data are presented as mean \u0026plusmn; standard deviation values (Mean \u0026plusmn; SD). Statistical analyses were performed using IBM SPSS Statistics 23.0. Comparisons between two experimental groups were conducted using the two-tailed Student\u0026rsquo;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. \u003cem\u003eP\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis research was financially supported by National Key Research and Development Program of China (2021YFD1800900), Chongqing Science and Technology Commission (CSTB2023NSCQ-JQX0002), Special Fund for Youth Team of Southwest University (SWU-XJLJ202306), Chongqing Natural Science Foundation (CSTB2024NSCQ-MSX0547), Science and Technology Innovation Key R\u0026amp;D Program of Chongqing (CSTB2024TIAD-STX0038, CSTB2022TIAD-KPX0094).\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eAll data are available within the article and supplementary information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDickey SW, Cheung GYC, Otto M (2017) Different drugs for bad bugs: antivirulence strategies in the age of antibiotic resistance. 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Mater. 20, 539\u0026ndash;547 (\u003c/em\u003e(2023))\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Oral antibiotics, milk exosomes, hybrid vesicles, bioavailability, gut dysbiosis","lastPublishedDoi":"10.21203/rs.3.rs-7033003/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7033003/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOral antibiotics are a mainstay for treating bacterial infections, but unabsorbed portions can reach the caecum and colon, leading to gut dysbiosis. Herein, we engineer milk exosome-liposome hybrid vesicles that enhance antibiotic absorption in the proximal small intestine \u003cem\u003evia\u003c/em\u003e neonatal Fc receptor and peptide transporter 1-mediated transport. 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 while reducing exposure to the large-intestine microbiota. 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.\u003c/p\u003e","manuscriptTitle":"Engineered vesicles enhance oral antibiotic absorption in proximal small intestine and mitigate gut dysbiosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-10 10:17:40","doi":"10.21203/rs.3.rs-7033003/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8a43cfe2-5517-4e12-9bdf-8d79c39658c8","owner":[],"postedDate":"July 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51149176,"name":"Health sciences/Medical research/Drug development"},{"id":51149177,"name":"Health sciences/Health care/Therapeutics/Drug therapy/Drug delivery"}],"tags":[],"updatedAt":"2026-02-05T08:06:41+00:00","versionOfRecord":{"articleIdentity":"rs-7033003","link":"https://doi.org/10.1038/s41467-025-68082-9","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-12-25 05:00:00","publishedOnDateReadable":"December 25th, 2025"},"versionCreatedAt":"2025-07-10 10:17:40","video":"","vorDoi":"10.1038/s41467-025-68082-9","vorDoiUrl":"https://doi.org/10.1038/s41467-025-68082-9","workflowStages":[]},"version":"v1","identity":"rs-7033003","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7033003","identity":"rs-7033003","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}