Bioengineered probiotics derived bacterial extracellular vesicle as bioactive nanocarrier for the local VEGF expression to accelerate wound healing

Bioengineered probiotics derived bacterial extracellular vesicle as bioactive nanocarrier for the local VEGF expression to accelerate wound healing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bioengineered probiotics derived bacterial extracellular vesicle as bioactive nanocarrier for the local VEGF expression to accelerate wound healing Zelin Zheng, Xi Liu, Kailu Guo, Shaojie Wu, Wenchen Cai, Yuting Li, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8711594/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Feb, 2026 Read the published version in Journal of Nanobiotechnology → Version 1 posted 9 You are reading this latest preprint version Abstract The angiogenesis is a pivotal process during wound healing. Its deficiency usually causes diminished oxygen and nutrient conveyance, compromising the cell function and decelerating the wound closure. Since being identified as the potent stimulator of angiogenesis, vascular endothelial growth factor (VEGF) has been explored as the leading therapeutic candidate. To overcome its inherent instability, both recombinant proteins and gene therapies have been proposed. Nonetheless, evidences of their therapeutic benefits for wound healing were limited. Over the past decades, bacterial extracellular vesicles (BEVs) have been recognized as versatile bioactive nanocarriers for the cross-kingdom communication. Herein, BEV derived from the recombinant probiotics Escherichia coli Nissle 1917 (BEV-pVEGF) was bioengineered to deliver the shuttle plasmid encoding VEGF. The BEV-pVEGF was proven could facilitate the intracellular delivery and local expression of the exogenous pVEGF, promoting the proliferation, migration, and angiogenesis of the endothelial HUVEC. Moreover, it was also proven to enable the intracellular delivery of the endogenous miR-21-5p, activating the PI3K-AKT signaling pathway and expediting the proliferation and migration of the epidermal HaCaT. Upon its subcutaneous administration for 7 consecutive days, the vascularized granulation tissue formation and re-epithelialized wound closure were significantly accelerated on mice bearing full-thickness wounds, with no obvious immunogenicity and toxicity being detected. These bioengineered BEV-pVEGF nanocarriers provide a readily-available, mass-producible, and cost-effective approach to developed effective and safe therapeutic modality for the future wound management. bacterial extracellular vesicles wound healing angiogenesis probiotics VEGF Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Wound healing is a complex process that requires close cooperation of different cells to consecutively go through four stages including hemostasis, inflammation, proliferation, as well as remodeling [ 1 , 2 ]. To accommodate enough oxygen and nutrient for the successive progress of the four stages, the locally damaged vasculatures need to be reconstructed, and thus the timely commenced angiogenesis is imperative [ 3 , 4 ]. Previous studies have demonstrated that the rapid network formation of blood vessels could significantly accelerate the healing of wounds [ 5 , 6 ]. While in contrast, the inadequate angiogenesis induced by infection, hyperglycemia, or circulatory disorder always lead to chronic wounds that fail to heal or heal slowly [ 7 – 9 ]. Since being discovered, more and more angiogenic proteins and peptides have been explored as therapeutic agents for wound healing [ 10 – 15 ]. Among them, the vascular endothelial growth factor (VEGF) has been recognized as the most famous and effective regulators [ 10 ]. It can bind to receptors on endothelial cells, triggering the formation of new blood vessels from the existing ones [ 16 ]. As such, the expression level of either VEGF or its receptors can significantly affect the angiogenesis process during wound healing [ 17 ]. Accordingly, the VEGF therapy has been developed as a supplementary wound healing treatment for the conventional ones such as the debridement and dressing [ 18 , 19 ]. To overcome the inherent stability deficiency of VEGF as proteins, several advanced formulations including the recombinant VEGF and VEGF hydrogel such as Regranex® have been made [ 20 , 21 ]. Nonetheless, the frequent dosing and high cost still limited their widespread clinical applications, underscoring the high demand for the optimized formulations. VEGF plasmid (pVEGF) refer to the circular plasmid carrying the DNA sequence encoding VEGF [ 22 , 23 ]. By packaged into nanocarriers, the pVEGF can be transported into target cells, which can then produce and secret VEGF directly around the wound area, stimulating and prolonging the healing effects [ 24 – 26 ]. Among all the nanocarriers for the pVEGF delivery, extracellular vesicles (EVs) became more and more popular [ 27 , 28 ]. These phospholipid bilayer nanostructures secreted by cells are capable of shuttling bioactive substances including nucleic acids, proteins, and metabolites from their patent cells to the recipient cells [ 29 ]. Therefore, the EVs are considered as bioactive nanocarriers. For instance, the EVs derived from human mesenchymal stem cells (MSCs) [ 30 – 33 ], pluripotent stem cells (iPSCs) [ 34 ], as well as endothelial progenitor cells [ 35 ] have been extensively explored for wound healing due to their inherited regenerative functions. The MSCs derived EVs have even entered the Phase I Trial for the wound healing in diabetics (NCT05243368, ClinicalTrials.gov) [ 36 ]. Nonetheless, the cultivation and isolation processes of these mammalian EVs are both time and cost consuming. In comparison, the bacterial extracellular vesicles (BEVs) can be more facilely acquired. Owing to the rapid proliferation of bacteria and standard procedure of culturing, the production of BEVs is scalable [ 37 – 39 ]. Besides, benefited from the diverse genetical, physical, and chemical engineering techniques, the manufacture of BEVs is also customizable [ 40 – 42 ]. Moreover, motivated by the emerging research interests in the “gut-skin axis” [ 43 – 45 ], the wound healing capacities of BEVs, especially those derived from probiotics, have been gradually revealed [ 46 ]. For instance, the Lactobacillus reuteri and Lactobacillus plantarum derived BEVs have been found to be capable of regulating the inflammation and proliferation phases of wound healing [ 47 – 49 ]. While the Lactobacillus rhamnosus GG and Lactobacillus druckerii derived BEVs have been found to be capable of modulating the proliferation and remodeling phases of wound healing [ 50 – 52 ]. Nonetheless, except for these gram-positive probiotics derived BEVs, the wound healing capacities of the gram-negative probiotics derived BEVs have been barely reported. Herein, the gram-negative probiotics Escherichia coli Nissle 1917 (EcN) derived BEVs were bioengineered in the presented work as bioactive nanocarriers for the local expression of VEGF to accelerate wound healing. The pVEGF containing BEV (BEV-pVEGF) was firstly constructed and characterized. Its angiogenesis capacity was then examined both in vitro on the endothelial human umbilical vein endothelial cell (HUVEC) and in vivo on the mice with full-thickness wounds. In light of the in vivo wound healing results, the bioactivities of BEV apart from angiogenesis were elucidated. Meanwhile, the primary miRNA comprised in the BEV-pVEGF that responsible for these bioactivities was identified. The miRNA mediated cellular functions on the epidermal HaCaT were furtherly investigated, together with the targeted signaling pathway being examined. Moreover, the biosafety profile of the BEV-pVEGF was lastly evaluated on mice after its subcutaneous treatment for 7 days. The bioengineered BEV-pVEGF are expected as a promising therapeutic modality for the effective and safe wound management. 2. Materials and methods 2.1. EcN-pVEGF construction The VEGF was synthesized and the pVEGF was recombined by Sangon Biotech (Shanghai, China). Their sequences were provided in Table S1 and Table S2, respectively. The pVEGF was then transferred into EcN to construct EcN-pVEGF, which was cultured to generate BEV-pVEGF. 2.2. Bacteria culture EcN and EcN-pVEGF were cultured in the Luria-Bertani (LB) medium containing 5 g/L yeast extract, 10 g/L tryptone, and 10 g/L NaCl. After primary culture at 220 rpm and 37°C for 12 h, 1 mL of the culture medium was added to 50 mL of the fresh LB medium for the secondary culture at 220 rpm and 37°C for 24 h. The obtained medium was used to isolate BEV-pVEGF. 2.3. BEVs isolation and characterization BEV and BEV-pVEGF were isolated as previous report [ 49 ]. The obtained medium was firstly centrifuged at 10,000 g and 4°C for 15 min to remove bacteria. The supernatant was then filtered through a 0.22 µm syringe filter to remove bacterial debris. The filtrate was ultra-centrifugated at 150,000 g and 4°C for 1.5 h to collect the BEV and BEV-pVEGF pellets. The collected pellets were resuspended in PBS, ultra-centrifugated at 150,000 g and 4°C for 1.5 h again for purification. These isolated BEVs were resuspended in PBS and stored at -80 ℃ before further use. Their morphologies were visualized by transmission electron microscopy (TEM; JEM-1400plus, Japan). Their particle concentrations and size distributions were determined by nanoparticle tracking analysis (NTA; NanoSight NS300, Malvern, UK). Their zeta potentials were analyzed by dynamic light scattering (DLS; Zetasizer Nano ZS90, Malvern, UK). Their membrane proteins were verified by Western Blot (WB). 2.4. pVEGF examination The pVEGF contained in the isolated BEV-pVEGF was firstly extracted with the SanPrep Spin Column & Collection Tube (Sangon, Shanghai) following the manufacture’s instruction. Then both the recombined pVEFG and the extracted pVEGF were incubated with the prepared NheI/EcoRI digestion solution under 37 ℃ for 2 h. The agarose gel electrophoresis was performed to examine the base pair number of VEFG for the verification of the successful construction of pVEGF. 2.5. Cell culture The human umbilical vein endothelial cell (HUVEC) and human skin keratinocytes (HaCaT) were obtained from Shanghai Skin Disease Hospital (Shanghai, China). HaCaT and HUVEC were cultured in high-glucose Dulbecco’s modified eagle medium (DMEM; Gibco, 11995500) supplemented with 10% fetal bovine serum (FBS; Gibco, 10091148) and 1% penicillin-streptomycin (Gibco, 15140-122). Both cells were cultured in incubator under 37°C and 5% carbon dioxide conditions. 2.6. Cell viability HUVECs and HaCaT were seeded in the 96-well plate at a density of 5.0×10 3 cells/well. After overnight culture, BEV and BEV-pVEGF was added to the culture medium with the final concentration ranging from 1×10 6 -1×10 9 particles/mL. After 24 h treatment, the cell viability of both cells was examined by the Cell Counting Kit-8 (CCK-8, Beyotime, C0043) according to the manufacture’s guideline. 2.7. Cell internalization BEV and BEV-pVEGF were firstly labeled with the red fluorescent dye DiI (Beyotime, C1036) and then incubated with HUVEC and HaCaT in confocal dishes at 37 ℃ for 6 h. The treated cells were washed with PBS, fixed with 4% paraformaldehyde (PFA; Servicebio, G1101), and stained with the blue fluorescence nuclei dye 4,6-diamidino-2-phenylindole (DAPI; Servicebio, G1012) as well as the green fluorescence cytoskeleton dye phalloidin (Servicebio, G1248). The cell internalization of BEV and BEV-pVEGF was then visualized by the laser scanning confocal microscope (LSCM; Leica Microsystems, Leica MICA, Germany). 2.8. Cell proliferation HUVEC and HaCaT were seeded in confocal dishes at a density of 5.0×10 4 cells/well and cultured overnight before being treated with BEV or BEV-pVEGF at a concentration of 1.0×10 8 particles/mL. After 24 h culture under the same conditions, the cells were washed with PBS for 3 times, fixed with 4% PFA, permeabilized with Immunostaining Permeabilization Buffer (Beyotime, P0095), blocked with goat serum (Servicebio, G1208), and incubated with Ki67 antibody (Abcam, ab15580, 0.5 µg/mL) sequentially at 4°C. Alexa Fluor™ 488-labeled secondary antibody (Abcam, ab150077, 1:500) was used to stain the Ki67, while DAPI was used to stain the cell nuclei. Their fluorescent signals were then visualized by LSCM and quantified by Image J. 2.9. Scratch assay HUVEC and HaCaT were seeded in the 6-well plate at a density of 5×10 5 cells/well. After overnight culture, the cells were scratched by a 1000 µL sterile pipette and the floating cells were removed by PBS. BEV and BEV-pVEGF was then added to the culture medium with a final concentration of 1.0×10 8 particles/mL. After 24 h treatment, the wounded areas were imaged by inverted microscope (ECLIPSE Ts2, Nikon, Japan) and the migration rates were calculated by Image J. 2.10. Transwell assay HUVEC and HaCaT were seeded in the upper chamber of the 24-well plate with 8 µm pore-sized filter (Corning, USA) at a density of 4×10 4 cells/well. After overnight culture, BEV and BEV-pVEGF was added to the culture medium in the lower chamber with a final concentration of 1.0×10 8 particles/mL. After 24 h treatment, the unmigrated cells on the upper surface of the filters were gently removed by a sterile cotton swab. While the migrated cells on the lower surface of the filters were fixed by 4% PFA for 15 min and stained by crystal violet (Solarbio, G1059) for 20 min. The stained migrated cells were imaged by optical microscope (ECLIPSE Ts2, Nikon, Japan) and the migrated cell numbers were calculated by Image J. 2.11. Tube formation HUVEC was seeded in the 96-well plate with Matrigel coating at a density of 4.0×10 4 cells/well. After overnight culture, BEV and BEV-pVEGF was added to the culture medium with a final concentration of 1.0×10 8 particles/mL. After 6 h treatment, the tube formations were imaged by optical microscope (ECLIPSE Ts2, Nikon, Japan) and the tube lengths, mesh numbers, as well as branching points were calculated by Image J. 2.12. Western blot The BEV and BEV-pVEGF treated cells were lysed by the radioimmunoprecipitation (RIPA) buffer containing PMSF (Servicebio, G2008) to extract their total cellular proteins. After being quantified by the BCA protein quantification kit (Servicebio, G2026), the total cellular proteins were separated by SDS-PAGE and transferred to PVDF membrane. The membranes were firstly blocked with 5% skimmed milk and then successively incubated with the primary antibody, the secondary antibody, as well as the chemiluminescent substrate. The primary antibodies and secondary antibodies purchased from Servicebio included the GAPDH antibody (GB15004, 1: 5000), VEGFA antibody (GB11034B, 1:1000), AKT antibody (GB15689, 1:1000), pAKT antibody (GB150002, 1:1000), HIF-1α antibody (GB111339, 1:500), and horseradish peroxidase-conjugated goat anti-rabbit IgG (H + L) (GB23303, 1:10000). The bands on the membrane were images by ChemiDoc MP imaging system (BIO-RAD, USA) and analyzed by Image J. 2.13. Wound model and BEV treatment All animal experiments were approved by the Institutional Animal Care and Use Committee of Shanghai University (ECSHU 2024-074) in accordance with the National Institutes of Health guidelines. The 8-week-old male C57BL/6J mice were purchased from Huachuang Sino (JiangSu, China). All mice were anesthetized and shaved before the four full-thickness wounds with a diameter of 6 mm being excised on their backs by a biopsy punch [ 53 ]. The mice were then randomly divided into 3 groups and subcutaneously treated with 100 µL PBS or 1×10 9 particles/mL BEV and BEV-pVEGF. The treatments were performed every other day. On the day 3 and 7, the wound areas were images by smartphone to directly visualize the wound closure, and examined by PeriCam PSI-ZR (PERIMED Ltd, Sweden) to evaluate the blood perfusion. The wound tissues were collected after the mice being sacrificed, the underside of the skin was images by stereomicroscope (Leica Germany) to evaluate the new blood vessel formation. Then the whole tissues were fixed, embedded, sectioned, and stained for the further histological staining and immunostaining analysis. 2.14. Blood perfusion evaluation Laser speckle contrast imaging (LSCI) was performed to evaluate the new blood vessel formation and the blood perfusion around the wounds by PeriCam PSI-ZR (PERIMED Ltd, Sweden) after the 7-day treatment. Under the same scan site dimension, the blood perfusions were imaged at a fixed distance. The images of blood flux were processed by the PIMSoft (Moor Instruments Ltd, UK) to calculate the mean perfusion units (MPUs). 2.15. Histological staining and immunostaining analysis The collected tissues were successively fixed with 4% PFA, embedded in paraffin, and sectioned for staining analysis. For the immunofluorescence analysis, the sectioned tissues were firstly stained with antibodies of VEGF-A (GB11034B, 1:1000) and then stained with Alexa Fluor™ 488-conjugated secondary antibodies (Abcam, ab150080, 1:1000). For immunohistochemistry analysis, the sectioned tissues were firstly stained with antibodies of CD31 (Servicebio, GB11063, 1:1000) and Ki67 (Servicebio, GB111141, 1:1000) then stained with DAPI. For histological analysis, the sectioned tissues were H&E stained to evaluate the epidermal regeneration and Masson stained to evaluate the collogen deposition. All the stained tissue sections were images under SLIDEVIEW VS200 scanner (Olympus). All the fluorescence intensities were quantified by Image J. 2.16. miRNA sequencing The total RNAs of BEV and BEV-pVEGF were extracted by TRIzol Reagent (Invitrogen, USA), quantified by Nanodrop ND-100 (Thermo Fisher, USA), and high-throughput sequenced by OBiO (Shanghai, China). The miRNA sequencing library was constructed by TruSeq Small RNA Sample Prep Kits (Illumina, USA) before the sequencing by Illumina Hiseq 2000/ 2500 (Illumina, USA). The ACGF101-miR (V4.2) was used for data analysis. 2.17. RT-qPCR analysis The total RNAs of HUVEC and HaCaT were extracted by TRIzol (Invitrogen, 15596026). The extracted RNA was reverse-transcribed into cDNA by Mir-X miRNA 1st-Strand Synthesis Kit (Takara Bio, CN638313). The cDNA was quantified by CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA). The primers used were synthesized by Sangon (China) and listed in Table S3. U6 was used to normalize the gene expression level. The relative expression levels of miRNA were quantified using the 2 −ΔΔCT method. 2.18. Inhibitor transfection HUVEC and HaCaT were transfected with 100 nmol/L miR-21a-5p inhibitor (RiboBio, China) by the Lipofectanube 3000 (Invitrogen, L3000008) according to the manufacture’s guideline. After 8 h transfection, the culture medium was replaced and the cells were cultured for another 16 h before the following experiments [ 52 ]. 2.19. ELISA The total proteins of the collected wound tissues were extracted by RIPA buffer containing PMSF and quantified by BCA protein quantification kit. The expression levels of IL-1β and IL-10 were then determined by the ELISA kits (MultiSciences, EK210, EK206, EK201B, and EK282) according to the manufacture’s guidelines. 2.20. Hemolysis assay 200 uL 4% rabbit erythrocytes (Sbjbio, SBJ-RBC-RAB003) were mixed with 200 µL ddH 2 O, PBS, BEV and BEV-pVEGF with the final concentrations ranging from 1×10 5 -1×10 9 particles/mL, respectively. After co-incubation for 2 h at 37 ℃, the blood samples were centrifugated at 800 g for 5 min and photographed. Their absorbances at 540 nm were then measured by the microplate reader (Cytation, Biotek, USA) to calculate the hemolysis ratios. 2.21. Safety profile After the 7-day treatments with PBS, BEV, and BEV-pVEGF, the blood of all mice was collected by removing their eyeballs. Subsequently, the blood was centrifugated for extracting the serum. The expression level of key biochemical indicators in serum including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine (CREA) was evaluated. Besides, the major organs of all mice including heart, liver, spleen, lung, and kidney were also collected after sacrifice and sectioned for the further H&E staining analyses. 2.22. Statistical analysis All experiments were performed for at least 3 times. All data were displayed as mean ± standard deviation (SD). GraphPad Prism 8.0 (La Jolla, USA) was used to analysis the experimental data. Image J was used to analysis the experimental images. One-way ANOVA was used for the significance assessments. p value of less than 0.05 was considered as significant difference with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. 3. Results and discussions 3.1. Construction and characterization of BEV-pVEGF To obtain the pVEGF containing BEVs (BEV-pVEGF), the bioengineering approach was employed. As illustrated in Fig. 2 A, the VEGF gene element was firstly synthesized and used to generate the shuttle plasmid pVEGF (Table S1 , S2). After being successfully validated by the agarose gel electrophoresis (Fig. 2 B, C), the pVEGF was then transfected into the Escherichia coli Nissle 1917 (EcN) to obtain the recombinant probiotics EcN-pVEGF, which were further cultured to generate the bioengineered BEV-pVEFG. Through the ultracentrifugation based process as previously reported [ 49 ], both the EcN secreted BEV and the EcN-pVEGF secreted BEV-pVEGF were effectively isolated and purified. Despite of their different contents, the BEV and BEV-pVEGF possess the same surface components, similarly to their parental EcN. Thereby, almost the same zeta potentials at approximate − 20 mV were determined for the BEV and BEV-pVEGF, respectively (Fig. 2 D). While compared to EcN, these values were slightly lower, in accordance with the previous founding that the repulsion caused by the local enrichment of negative charges on the outer membrane of bacteria contributed to their secretion of BEVs [ 54 , 55 ]. Besides, the successful loading of pVEGF in the BEV-pVEGF was also confirmed by the agarose gel electrophoresis as demonstrated in Fig. 2 E. According to the transmission electron microscopy (TEM) visualization, both of the BEV and BEV-pVEGF exhibited the featured spherical bilayer nanostructures with average diameter of approximately 125.6 nm (Fig. 2 F). These diameters were further verified by the dynamic light scattering (DLS) (Figure S1 ) and nanoparticle tracking analysis (NTA) (Fig. 2 G), which also shown that the concentration of the obtained BEV and BEV-pVEGF were around 2.34×10 9 particles/mL and 1.69×10 9 particles/mL, respectively. All these characterization results indicated that the rationally designed BEV-pVEGF were successfully engineered and effectively isolated, facilitating the following evaluations on their therapeutic efficacies for wound healing. 3.2. BEV-pVEGF delivering pVEGF to promote the proliferation, migration, and angiogenesis of endothelial cell To validate the angiogenesis performance of BEV-pVEGF, their cellular functions mediated by the delivered pVEGF were examined. Since the VEGF mainly participate in the vascularizing process during the proliferative stage of wound healing [ 49 , 56 ], the angiogenic endothelial cell HUVEC was selected for the examination. According to the CCK-8 assay, the BEV-pVEGF displayed no cytotoxicity against HUVEC up to concentration as high as 1×10 9 particles/mL (Figure S2). After incubation with the HUVEC for 6 h, the red fluorescence Dil labelled BEV-pVEGF could be successfully uptake by the blue fluorescence DAPI and green fluorescence Phalloidin labelled HUVEC, simultaneously delivering their loaded pVEGF into the HUVEC (Fig. 3 A). The successfully internalized BEV-pVEGF were then demonstrated could effectively facilitate the proliferation of HUVEC as the mostly increased population of the Ki67 positive cells were observed (Fig. 3 B, C). Meanwhile, a significantly higher expression of the VEGF was detected for the HUVEC with the treatment of BEV-pVEGF than PBS or BEV (Fig. 3 D), indicating the effective intracellular delivery and expression of pVEGF by the BEV-pVEGF. Subsequently, the accelerated migration of HUVEC with the treatment of BEV-pVEGF was observed. The migration rate of HUVEC increased approximately 2.1 times with the treatment of BEV-pVEGF than PBS based on the scratch assay (Fig. 3 E, F). While the migration number of HUVEC increased approximately 2.9 times with the treatment of BEV-pVEGF than PBS based on the transwell assay (Fig. 3 G, H). Most notably, more than 2.1 times increased branching points were formed by HUVEC with the treatment of BEV-pVEGF (Fig. 3 I, J), confirming the BEV-pVEGF enhanced angiogenesis of HUVEC. All these cellular function results constituted compelling validation that the rationally engineered BEV-pVEGF could effectively deliver the pVEGF into HUVEC, simultaneously upregulating its expression of VEGF-A. As a consequence, the significantly expressed VEGF-A could mediate and promote the proliferation, migration, and angiogenesis of HUVEC, holding great potential in expediting the wound healing. 3.3. BEV-pVEGF Expedited the Angiogenesis and Closure of Cutaneous Wounds To assess the in vivo angiogenesis and healing efficiency of BEV-pVEGF, full-thickness wounds were bilaterally created on the dorsal of the C57BL/6J mice. As demonstrated in Fig. 4 A, the mice with 6 mm diameter wounds were randomly divided into 3 groups and subcutaneously administrated with PBS, BEV, and BEV-pVEGF around the edge of wounds every other day. According to the macroscopic images (Fig. 4 B) and tracing analyses (Fig. 4 C) of the wounds, the BEV-pVEGF treatment significantly accelerated the cutaneous wound closure. With the BEV-pVEGF treatment, more than 64.7% and 80.4% wound cloresures were restores at day 3 and day 7, respectively (Fig. 4 D). While although not as effective as the BEV-pVEGF treatment, the BEV treatment still achieved 50.8% and 64.7% wound cloresures, which were approaximately 1.8 and 1.4 times higher than the control group with the PBS treatment at day 3 and day 7, respectively. To confirm the accelerated wound closure is attributed to the promoted angiogenesis, the blood flow perfusion around the wounds were firstly imaged after the treatment for 7 days. As demonstrated in Fig. 4 E and Figure S3, compared to the PBS and BEV treatment, the BEV-pVEGF significantly enhanced the blood flow perfusion around the wounds. The intensity of blood flow perfusion increased 1.97 and 1.34 times, respectively, suggesting the improved vascularization. Besides, to further verify the improved vasculatization, all mice were euthanatized at day 7 for the tissue colllection and analyses. According to the dermoscope images displayed in Fig. 4 F, the maximum blood vessles were formed subcutaneously with the BEV-pVEGF treatment. Morevoer, based on the immunohistochemistry analyses, both the BEV and BEV-pVEGF treatments up-regulated the expression of Ki67 from 20.4% to about 50.0%, suggesting their promoted cell proliferation (Fig. 4 G). Under such circumstances, only the BEV-pVEGF treatments achieved the highest expression of VEGF, which is 1.7 and 4.1 times higher than the BEV and PBS treatment, respectively (Fig. 4 H). The BEV-pVEGF facilitated expression of VEGF ultimately stimulated the vasculatization, with 2.0 and 3.5 times increased expression of the angiogenesis marker CD31 being observed than BEV and PBS (Fig. 4 I). All these in vivo evidences togetherly confirmed that the BEV-pVEGF could significantly increase the expression of VEGF and consequently stimulate the angiogenesis of wounds. The enhanced vascularization could restore the oxygen supply and nutriention delivery to wounds, accelerating the healing process and thus expediting the wound closure. 3.4. BEV-pVEGF Facilitated the Granulation Tissue Formation and Re-epithelialization via the PI3K-AKT Signaling Pathway To elucidate the other biofunctions of the BEV-pVEGF during wound healing, the collected tissues at day 3 and day 7 were sectioned for further Hematoxylin and Eosin (H&E), Masson’s Trichrome, as well as immunofluorescence staining. According to the H&E staining images (Fig. 5 A), both of the BEV-pVEGF and BEV treatments decreased the wound width (Fig. 5 B) while increased the granulation tissue and epidermal thickness (Fig. 5 C, D). Even though not as efficient as BEV-pVEGF, the BEV still exerted significantly improved wound healing efficiency than the control. After the 7 days administration, the BEV-pVEGF and BEV stimulated the denser collagen deposition as well based on the Masson staining images (Fig. 5 E, F). The deposited collagen fibers formed a scaffold that favoring cell proliferation and migration, furtherly accelerated the epidermal coverage and wound closure. Since both of the BEV-pVEGF and BEV facilitated the granulation tissue formation and re-epithelialization, the bioactivities of BEV during wound healing were also investigated. Derived from the probiotics EcN, the BEV comprising diverse bioactive molecules that inherited from EcN has been considered as bioactive nanocarrier. For instance, previous study has found that the miR-21-5p was comprised in the BEV [ 57 ]. Most recently, the miR-21-5p has been furtherly proven to activate the PI3K/AKT signaling pathway, facilitating the wound healing activities of both endothelial and epidermal cells.[ 49 , 52 ] In consistent with the previous studies, after the treatment by BEV-pVEGF and BEV, the up-regulated expressions of p-AKT (Fig. 5 G) and HIF-1α (Fig. 5 H) were both detected, confirming the activated PI3K/AKT signaling pathway as expected. All these tissue staining analyses revealed that apart from the exogenous pVEGF expedited angiogenesis, the endogenous bioactive molecules that comprised in the BEV-pVEGF could simultaneously facilitated the granulation tissue formation and re-epithelialization via the PI3K-AKT signaling pathway. All the exogenous and endogenous bioactive molecules inside BEV-pVEGF can work together, synergistically contributing to their accelerated wound closure and healing. 3.5. BEV-pVEGF Preserved the Endogenous Wound Healing miRNA To identify the key endogenous bioactive molecule inside the BEV-pVEGF that facilitate wound healing, the next-generation miRNA sequencing was performed. As demonstrated in Fig. 6 A, 241 miRNAs were detected inside the BEV generated by EcN. According to the miRWalk database, 129 of these miRNAs were associated with wound healing.[ 58 ] Subsequently, the top 20 highly expressed miRNAs inside the BEV were listed in the heatmap (Fig. 6 B). Among them, 5 miRNAs including the hsa-miR-21-5p,[ 52 ] hsa-miR-423-5p,[ 59 ] hsa-miR-148a-5p,[ 60 ] hsa-miR-221-3p,[ 61 ] and hsa-miR-92a-3p[ 62 ] are related to wound healing. Besides, the BEV-pVEGF generated by the bioengineered EcN preserved the miRNA content. There were no significantly differences being examined between the BEV-pVEGF and the BEV in terms of the expression on the top 5 wound healing miRNAs (Fig. 6 C). Based on the top 5 highly expressed wound healing miRNA, the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed as well to elucidate the potential signaling pathways facilitating wound healing. As displayed in Fig. 6 D, the 3 closely relevant signaling pathways were metabolic, PI3K-AKT, and HIF-1. Further Gene Ontology (GO) analyses revealed that these miRNAs predominately interfered with the protein binding in molecular function (Figure S4A), membrane formation in cellular component (Figure S4B), as well as signal transduction in biological process (Figure S4C). In addition, the network on the interactions between the top 5 highly expressed wound healing miRNA and their targeted genes were also visualized by Cytoscape (Fig. 6 E). Multiple targeted genes, such as RPS6KA5, MTOR, SKP2, TGFB1, and FAM168, are correlated with the cell proliferation and migration. All these miRNA sequencing analyses demonstrated that the BEV-pVEGF preserved the endogenous wound healing miRNA from the EcN even after bioengineering. The top 5 highly expressed wound healing miRNA inside the BEV-pVEGF, including the hsa-miR-21-5p, hsa-miR-423-5p, has-miR-148a-5p, has-miR-221-3p, and has-miR-92a-3p, predominately relevant to 3 signaling pathways. Among them, the PI3K-AKT signaling pathway coincided with the one detected during the tissue staining analyses. Thereby, it was supposed to be activated by the endogenous miRNA inside the BEV-pVEGF to facilitate the wound healing. 3.6. BEV-pVEGF delivering mir-21-5p to prompt the proliferation and migration of epidermal cells via activating the PI3K-AKT signaling pathway To further clarify the underlying relationship between the endogenous bioactive substances and wound healing capacities of BEV-pVEGF, their cellular functions mediated by the most highly loaded miR-21-5p were examined on the epidermal HaCaT. Similarly to the HUVEC, no obvious cytotoxicity was detected against the HaCaT with the BEV and BEV-pVEGF treatment up to the concentration of 1×10 9 particles/mL (Figure S5). After culturing with the HaCaT for 6 h, the Dil-labeled BEV-pVEGF could successfully penetrate the cell membrane thus effectively deliver the intrinsic miR-21-5p into the Phalloidin and DAPI stained HaCaT (Figure S6 and Fig. 7 B). Subsequently, an increased population of the Ki67 positive cells was observed with the prolonged culturing by BEV-pVEGF (Fig. 7 A), indicating the significantly induced proliferation of HaCaT. While upon the transfection of the miR-21-5p inhibitor, the cell proliferation was severely inhibited to an extent similarly to the control group that treated with only the PBS. Besides, based on the scratch assay (Fig. 7 C) and transwell assay (Fig. 7 D, the migration rate and number of HaCaT that cultured by BEV-pVEGF also increased more than 1.5 times, but barely changed in the presence of the miR-21-5p inhibitor (Fig. 7 D). Altogether, these results demonstrated that the BEV-pVEGF could promote the proliferation and migration of the epidermal HaCaT. These effects were achieved mainly through the BEV-pVEGF facilitated intracellular delivery of their endogenous miR-21-5p. Furthermore, the increased expression of the phosphorylated AKT (p-AKT) and the hypoxia-inducible factor-1 alpha (HIF-1α), which are the indicators of the PI3K-AKT signaling pathway activation,[ 52 ] were detected in HaCaT according to the western blot (WB) analyses (Fig. 7 E). While once again, the p-AKT and HIF-1α expressions were suppressed in the presence of the miR-21-5p inhibitor, confirming the PI3K-AKT signaling pathway was activated by the endogenous miR-21-5p of BEV-pVEGF. In combined with the observed PI3K-AKT pathway activation in the closured wound tissues after the treatment of BEV-pVEGF, these results provided compelling evidence that apart from the exogenous pVEGF, the BEV-pVEGF could also acted as bioactive nanocarrier to delivery their endogenous miR-21-5p into the epidermal cells. Thereby, the proliferation and migration of epidermal HaCaT were promoted. The endogenous miR-21-5p worked together with the exogenous pVEGF of BEV-pVEGF, facilitating the vascularized granulation tissue formation and re-epithelialization thus accelerating the effective wound closure on mice. 3.7. BEV-pVEGF Displayed Low Immunogenicity and Toxicity During the 7-Day Wound Treatment To guarantee the safe BEV-pVEGF treatment for wound healing, its safety profile in terms of toxicity was furtherly examined on mice after the different treatments for 7 days. Due to the defect in its LPS biosynthesis, the non-pathogenic probiotics EcN and its derived BEV bear a truncated LPS chain and exhibit a semi-rough phenotype.[ 63 ] Thereby, the immunogenicity of EcN and its BEV were found significantly reduced.[ 63 ] In consistent with previous founding, both of the BEV and BEV-pVEGF displayed low immunogenicity. After the treatment with BEV and BEV-pVEGF for 7 days, there were no significant differences on the expression of the pro-inflammatory cytokine interleukin 1β (IL-1β) (Fig. 8 A). Most notably, after the treatment with the BEV-pVEGF for 7 days, the expression of the anti-inflammatory cytokine interleukin-10 (IL-10) was even slightly increased compared to the mice with the PBS and BEV treatments (Fig. 8 A). Attributed to the pro-angiogenesis capacity of BEV-pVEGF, the anti-inflammatory phenomenon should come up simultaneously with the expedited vascularized granulation tissue formation.[ 64 , 65 ] Besides, both of the BEV and BEV-pVEGF exhibited excellent hemocompatibility (Fig. 8 B), with less than 1% hemolysis being detected up to concentrations of 1×10 9 particles/mL. After the treatment with PBS, BEV, and BEV-pVEGF for 7 days, no significant differences were detected on the expression level of the key biochemical indicators in the serum of mice (Fig. 8 C), including the alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), and creatinine (CREA). These results indicated that the subcutaneous administrations of neither the BEV nor the BEV-pVEGF for 7 consecutive days had adverse effects on the liver and kidney functions of mice. Moreover, there were no noticeable pathological changes and inflammatory cell infiltrations being observed in the major organs of mice, including the heart, liver, spleen, lung, as well as kidney, after all the treatments based on the H&E staining images displayed in Fig. 8 D. All these results substantiated the low immunogenicity and toxicity of both BEV and BEV-pVEGF, illustrating their distinct in vivo safety profiles for the safe wound treatment. 4. Conclusion Featured by their scalable, customizable, and bioactive properties, the probiotics EcN derived BEVs were bioengineered as bioactive nanocarriers in the presented work for the local expression of VEGF to accelerate wound healing. Based on the bioengineering approach and ultracentrifugation procedure, the 125.6 nm lipid bilayer BEVs comprising shuttle plasmid that encoded VEGF-A were obtained. The BEV-pVEGF facilitated the cell internalization of pVEGF, up-regulated the cell expression of VEGF-A, and thus promoted the angiogenesis of HUVEC. Besides, in addition to the exogeneous pVEGF, the BEV-pVEGF also intracellularly delivered their endogenous miR-21-5p, subsequently activated the PI3K-AKT signaling pathway, and thus promoted the proliferation and migration of the epithelial HaCaT. Upon the BEV-pVEGF treatment, expedited vascularization and enhanced blood flow perfusion were observed around the wound on mice. Sequentially, the accelerated granulation tissue formation and wound re-epithelialization were observed as well within 7 days. Moreover, no significant immunogenicity and toxicity were detected on mice after the BEV-pVEGF treatment. With more and more researches on the gut microbiota and their cross-kingdom regulation in wound repair and regeneration, these results filled the existing gaps between the gut microbiota and wound homeostatic, contributing to the future development of innovative nanotherapeutics for the wound treatment. Meanwhile, the bioengineering of BEV-pVEGF provides a readily-available, mass-producible, and cost-effective approach to drive the clinical translation of the effective and safe nanotherapeutic modality of BEVs for the future wound management. Abbreviations BEV Bacterial extracellular vesicle VEGF Vascular endothelial growth factor EcN Escherichia coli Nissle 1917 TEM Transmission electron microscope DLS Dynamic light scattering NTA Nanoparticles tracking analysis HUVEC Human umbilical vein endothelial cell HaCaT Human skin keratinocytes LCSM Laser confocal scanning microscopy WB Western blot RT-qPCR Real-time quantitative polymerase chain reaction ELISA Enzyme linked immunosorbent assay miRNA Micro-RNA DiI 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate Gt Granulation tissue H&E Hematoxylin and eosin staining AKT Protein kinase B p-AKT Phospho-protein kinase B HIF-1α Hypoxia-inducible factor 1-alpha GAPDH Glyceraldehyde-3-phosphate dehydrogenase ALT Alanine aminotransferase AST Aspartate aminotransferase BUN Blood urea nitrogen CREA Creatinine Declarations Ethics approval and consent to participate All animal experiments were approved by the Institutional Animal Care and Use Committee of Shanghai University (ECSHU 2024-074) in accordance with the National Institutes of Health guidelines. Consent for publication No applicable. Competing interests The authors declare no competing financial interest. Supplementary Information The online version contains supplementary material available at… Funding The work was supported by the Shanghai Pujiang Program (21PJ1404100), the Young Elite Scientists Sponsorship Program by CAST (2023QNRC001), and the National Natural Science Foundation of China (No. 22477075, No.22077078, and No. 22205260). Author Contribution Zelin Zheng, Xi Liu, and Kailu Guo contributed equally to the work. Zelin Zheng: Data curation, methodology, visualization, writing–original draft; Xi Liu: Conceptualization, methodology, writing–original draft; Kailu Guo: Data curation, methodology, visualization; Yuting Li: Data curation, validation, formal analysis; Yirong Wang: Data curation, validation, formal analysis; Cuiping Zhang: Funding acquisition, resources, supervision, writing–review and editing; Honggang Hu: Funding acquisition, resources, supervision, writing–review and editing; Yejiao Shi: Conceptualization, funding acquisition, project administration, writing–review and editing. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Acknowledgements The authors gratefully thank Assoc Prof Han Liu from Shanghai University for his valuable help and guidance on bioengineering of the EcN-pVEGF. 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Supplementary Files ZhengetalSIJNB20260123.docx floatimage1.png Graphical abstract Cite Share Download PDF Status: Published Journal Publication published 22 Feb, 2026 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Revision requested 04 Feb, 2026 Reviews received at journal 03 Feb, 2026 Reviews received at journal 02 Feb, 2026 Reviewers agreed at journal 02 Feb, 2026 Reviewers agreed at journal 02 Feb, 2026 Reviewers invited by journal 02 Feb, 2026 Editor assigned by journal 28 Jan, 2026 Submission checks completed at journal 28 Jan, 2026 First submitted to journal 27 Jan, 2026 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. 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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-8711594","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":584727690,"identity":"6ab32724-a636-4874-898a-8b0c85ea5876","order_by":0,"name":"Zelin Zheng","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"prefix":"","firstName":"Zelin","middleName":"","lastName":"Zheng","suffix":""},{"id":584727691,"identity":"3e32738d-d006-475a-a48a-7a479bd4b4a9","order_by":1,"name":"Xi Liu","email":"","orcid":"","institution":"PLA General Hospital and PLA Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xi","middleName":"","lastName":"Liu","suffix":""},{"id":584727692,"identity":"e0da7815-26e1-4af2-919c-1c30094ddd23","order_by":2,"name":"Kailu Guo","email":"","orcid":"","institution":"PLA General Hospital and PLA Medical College","correspondingAuthor":false,"prefix":"","firstName":"Kailu","middleName":"","lastName":"Guo","suffix":""},{"id":584727693,"identity":"a1bae60f-e5f8-4e24-a908-6773e5eb7ec0","order_by":3,"name":"Shaojie Wu","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"prefix":"","firstName":"Shaojie","middleName":"","lastName":"Wu","suffix":""},{"id":584727694,"identity":"74abfbe5-cb64-4ea6-b2c0-5afa1d936cc4","order_by":4,"name":"Wenchen Cai","email":"","orcid":"","institution":"Macau University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Wenchen","middleName":"","lastName":"Cai","suffix":""},{"id":584727695,"identity":"03f95480-c7e6-4916-947f-ed4496bd942b","order_by":5,"name":"Yuting Li","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"prefix":"","firstName":"Yuting","middleName":"","lastName":"Li","suffix":""},{"id":584727696,"identity":"799ec739-0f06-416c-9dbd-4acfda61fb8e","order_by":6,"name":"Yirong Wang","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"prefix":"","firstName":"Yirong","middleName":"","lastName":"Wang","suffix":""},{"id":584727697,"identity":"a9cfa147-a73e-4023-8fbd-e3ba8c924bdc","order_by":7,"name":"Cuiping Zhang","email":"","orcid":"","institution":"PLA General Hospital and PLA Medical College","correspondingAuthor":false,"prefix":"","firstName":"Cuiping","middleName":"","lastName":"Zhang","suffix":""},{"id":584727698,"identity":"b2912706-4fba-4017-823a-25eac684507a","order_by":8,"name":"Honggang Hu","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"prefix":"","firstName":"Honggang","middleName":"","lastName":"Hu","suffix":""},{"id":584727699,"identity":"8b9480c3-d3b0-4fa0-9122-a87f287b6b4c","order_by":9,"name":"Yejiao Shi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYDACCcaGAww8NgwGIA4PcVqYGx8w8KSRpIW9Gaj6MAla5Gc3tknzyJy3N5dIYHzwto1B3pyQFoM7B4FaeG4n7pyRwGw4t43BcGcDIS0SiWAtCQY3EtikedsYEgwOEHLYDLCWc/ZALey/idLCcCOx2ZiH5wDjBqAtzERpMbiR2PhwDk9y4oYzD5sl55yTMNxA2GHpDw687bGzNziefPDDmzIbecIOAwIm3h4QxdgAJCSIUA9S++MHcQpHwSgYBaNghAIAQs9ALAhTSNMAAAAASUVORK5CYII=","orcid":"","institution":"Shanghai University","correspondingAuthor":true,"prefix":"","firstName":"Yejiao","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2026-01-27 14:25:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8711594/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8711594/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12951-026-04217-4","type":"published","date":"2026-02-22T15:57:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":101795225,"identity":"4aae9598-ca95-4343-b6e9-016c95136d28","added_by":"auto","created_at":"2026-02-03 16:41:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":548781,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe construction procedures and wound healing mechanisms of BEV-pVEGF. \u003c/strong\u003eShuttle plasmid pVEGF was firstly synthesized and then transfected into \u003cem\u003eEscherichia coli\u003c/em\u003e Nissle 1917 to construct the recombinant EcN-pVEGF, which could be cultured to generate the pVEGF containing extracellular vesicles BEV-pVEGF. The BEV-pVEGF could not only deliver the exogenous pVEGF to HUVEC, promoting its proliferation, migration, and angiogenesis; but could also deliver the endogenous miR-21-5p to HaCaT, activating its PI3K-AKT signaling pathway and promoting its proliferation and migration. The subcutaneous administration of BEV-pVEGF on mouse with full thickness wounds expedited the vascularized granulation tissue formation and re-epithelialization, facilitating the timely wound closure without any obvious immunogenicity and toxicity.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8711594/v1/06e7ce0c5199c42ab3dce90b.png"},{"id":101795218,"identity":"20ba5912-87ef-4ab7-ad51-e517bb693fcd","added_by":"auto","created_at":"2026-02-03 16:41:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":550203,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction and characterization of BEV-pVEGF. \u003c/strong\u003e(A) Schematic illustration on the construction of BEV-pVEGF. (B) Schematic map on the rationally designed shuttle plasmid pVEGF. (C) Agarose gel electrophoresis validation on the shuttle plasmid pVEGF following the NheI/EcoRI double digestion. pV: pVEGF, M: marker 10000. (D) Zeta potential of EcN, BEV, and BEV-pVEGF in the pH 7.4 PBS. (E) Agarose gel electrophoresis validation on the VEGF element in BEV-pVEGF. (F) Representative TEM images of BEV and BEV-pVEGF. (G) NTA particle concentrations and size distributions of BEV and BEV-pVEGF.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8711594/v1/104f2fd064a3d055e6133992.png"},{"id":101795220,"identity":"b8b06cf3-197d-4574-94ea-036553a5df68","added_by":"auto","created_at":"2026-02-03 16:41:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1346131,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBEV-pVEGF delivering pVEGF to promote the proliferation, migration, and angiogenesis of endothelial cell\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e(A)\u003cstrong\u003e \u003c/strong\u003eRepresentative internalization images of the DiI labeled BEV-pVEGF by the DAPI and Phalloidin stained HUVEC for 6 h. (B) Representative immunofluorescent images of the Ki67 that expressed on the HUVEC treated with PBS, BEV, or BEV-pVEGF for 24 h. (C) Quantification on the Ki67 positive ratio based on (B). (D) WB analyses on the VEGF-A that expressed in HUVEC treated with PBS, BEV, or BEV-pVEGF for 24 h. (E) Representative scratch based migration images of the HUVEC treated with PBS, BEV, or BEV-pVEGF for 24 h. (F) Quantification on the migration rate of HUVEC based on (E). (G) Representative transwell based migration images of the HUVEC treated with PBS, BEV, or BEV-pVEGF for 24 h. (H) Quantification on the migrated cell number of HUVEC based on (G). (I) Representative tube formation images of the HUVEC treated with PBS, BEV, or BEV-pVEGF for 6 h. (J) Quantification on the total branching points based on (I). Data were presented as the mean ± SD, \u003cem\u003en\u003c/em\u003e = 3. \u003cem\u003ens\u003c/em\u003e, no significant, *\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":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8711594/v1/689315fbf82c8da7a94837a3.png"},{"id":101795224,"identity":"7d15a3f8-1e75-443b-9f8b-bb6456551d70","added_by":"auto","created_at":"2026-02-03 16:41:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1116436,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBEV-pVEGF expedited the angiogenesis and closure of cutaneous wounds. \u003c/strong\u003e(A) Schematic illustration on the wound construction and treatment procedures. (B) Representative images of the full-thickness wounds after the treatment with PBS, BEV, or BEV-pVEGF for 0, 3, and 7 days. (C) Simulation analyses on the wound healing progress based on (B). (D) Quantification on the wound closure rate based on (B). (E) Representative blood flow perfusion images of the wounds after the treatment with PBS, BEV, or BEV-pVEGF for 7 days. (F) Representative subcutaneous vessel formation images of the wound tissues after the treatment with PBS, BEV, or BEV-pVEGF for 7 days. Representative immunohistochemical images and quantification of the Ki67 (G), VEGF-A (H), and CD31 (I) that expressed in the wound tissues after the treatment with PBS, BEV, or BEV-pVEGF for 7 days.Data were presented as the mean ± SD, \u003cem\u003en\u003c/em\u003e = 3. \u003cem\u003ens\u003c/em\u003e, no significant, *\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":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8711594/v1/ddfbabdd37b19755eee12ec1.png"},{"id":101795223,"identity":"ff4ae5ee-88cb-4387-bdbf-211c180c02a3","added_by":"auto","created_at":"2026-02-03 16:41:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1460674,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBEV-pVEGF facilitated the vascularized granulation tissue formation and re-epithelialization via the PI3K-AKT signaling pathway. \u003c/strong\u003e(A) Representative H\u0026amp;E staining images with the histological analyses on the wound tissues after the treatment with PBS, BEV, or BEV-pVEGF for 7 days. The green arrowed lines indicate the wound width, the purple arrowed lines indicate the epidermal thickness, the blue arrowed lines indicate the granulation tissue (Gt). (B) Quantification on the wound width. (C) Quantification on the Gt thickness. (D) Quantification on the epidermal thickness. (E) Representative Masson’s trichrome staining images with the histological analyses on the wound tissues after the treatment with PBS, BEV, or BEV-pVEGF for 7 days. (F) Quantification on the collagen deposition. Representative immunofluorescent images and quantification of the p-AKT (G) and HIF-1α (I) that expressed in the wound tissues after the treatment with PBS, BEV, or BEV-pVEGF for 7 days. \u0026nbsp;Data were presented as the mean ± SD, \u003cem\u003en\u003c/em\u003e= 3. \u003cem\u003ens\u003c/em\u003e, no significant, *\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":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8711594/v1/1b175db5b82348824cfbb633.png"},{"id":101795228,"identity":"3d1eef35-ee4a-4913-9bdc-3c92226cae6d","added_by":"auto","created_at":"2026-02-03 16:41:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":584074,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBEV-pVEGF preserved the endogenous wound healing miRNA.\u003c/strong\u003e (A) Venn diagram for the screened miRNAs inside the BEV. (B) Heatmap analysis on the top 20 highly expressed miRNAs inside the BEV. (C) Quantification analysis on the top 5 highly expressed wound healing miRNAs inside the BEV-pVEGF and BEV. (D) KEGG pathway enrichment analysis on the top 5 highly expressed wound healing miRNAs inside the BEV-pVEGF and BEV. (E) Mapping on the network between the predicted key target genes and the top 5 significantly expressed wound healing miRNAs inside the BEV-pVEGF and BEV. Data were presented as the mean ± SD, \u003cem\u003en\u003c/em\u003e = 3. \u003cem\u003ens\u003c/em\u003e, no significant, *\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":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8711594/v1/bc5b20b84ad9d0771a9f6cc6.png"},{"id":101795222,"identity":"8b1eccfd-6a5e-4c71-8ab8-b88a7d60e286","added_by":"auto","created_at":"2026-02-03 16:41:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":957031,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBEV-pVEGF delivering miR-21-5p to prompt the proliferation and migration of epidermal cells via activating the PI3K-AKT signaling pathway. \u003c/strong\u003e(A) Representative immunofluorescent images and quantification of the Ki67 that expressed on the HaCaT treated with PBS, BEV-pVEGF, or BEV-pVEGF in combined with the miR-21-5p inhibitor for 24 h. (B) Quantification on the relative miR-21-5p content of the HaCaT. (C) Representative scratch based migration images and quantification of the HaCaT treated with PBS, BEV-pVEGF, or BEV-pVEGF in combined with the miR-21-5p inhibitor for 24 h. (D) Representative transwell based migration images and quantification of the HaCaT treated with PBS, BEV-pVEGF, or BEV-pVEGF in combined with the miR-21-5p inhibitor for 24 h. (E) WB analyses and quantifications on the p-AKT and HIF-1α that expressed in HaCaT treated with PBS, BEV-pVEGF, or BEV-pVEGF in combined with the miR-21-5p inhibitor for 24 h. Data were presented as the mean ± SD, \u003cem\u003en\u003c/em\u003e = 3. \u003cem\u003ens\u003c/em\u003e, no significant, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8711594/v1/32c81049557393ba5d43a1fd.png"},{"id":101795219,"identity":"b841b8cf-43ad-493a-a343-e20aa2f76a37","added_by":"auto","created_at":"2026-02-03 16:41:17","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1084773,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e safety profile of BEV-pVEGF. \u003c/strong\u003e(A) Quantifications on the inflammatory cytokines IL-1β and anti-inflammatory cytokine IL-10 expressed in the wound tissues of mice after the treatment with PBS, BEV, and BEV-pVEGF for 7 days. (B) The hemolysis of rabbit blood cells with the treatment of BEV and BEV-pVEGF at varied concentrations ranging from 1×10\u003csup\u003e6\u003c/sup\u003e-1×10\u003csup\u003e9\u003c/sup\u003e particles/mL. (C) Quantifications on the key biochemical indicator in the serum of mice after the treatment with PBS, BEV, and BEV-pVEGF for 7 days. (D Representative H\u0026amp;E staining images of the heart, liver, spleen, lung, and kidney of mice after the treatment with PBS, BEV, and BEV-pVEGF for 7 days. Data were presented as mean ± SD, \u003cem\u003en\u003c/em\u003e = 3. \u003cem\u003ens\u003c/em\u003e, no 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.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8711594/v1/0c82e8d6190fa1bc85d7b36a.png"},{"id":103252228,"identity":"2ad976f4-23b8-4d72-82fa-ad77babaa274","added_by":"auto","created_at":"2026-02-23 16:13:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9024844,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8711594/v1/5d167b16-edc4-4325-a8f0-843036f0f549.pdf"},{"id":101795226,"identity":"04fc8f21-9d48-4f64-9453-c0e40c3de182","added_by":"auto","created_at":"2026-02-03 16:41:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3791352,"visible":true,"origin":"","legend":"","description":"","filename":"ZhengetalSIJNB20260123.docx","url":"https://assets-eu.researchsquare.com/files/rs-8711594/v1/5fbeeda5b8a24ab8f8f723e8.docx"},{"id":101795227,"identity":"b772da63-da83-41b8-bdfe-5b68d9dcdda2","added_by":"auto","created_at":"2026-02-03 16:41:17","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":283585,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8711594/v1/8430fbda93068ce0327f40a5.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bioengineered probiotics derived bacterial extracellular vesicle as bioactive nanocarrier for the local VEGF expression to accelerate wound healing","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eWound healing is a complex process that requires close cooperation of different cells to consecutively go through four stages including hemostasis, inflammation, proliferation, as well as remodeling [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. To accommodate enough oxygen and nutrient for the successive progress of the four stages, the locally damaged vasculatures need to be reconstructed, and thus the timely commenced angiogenesis is imperative [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Previous studies have demonstrated that the rapid network formation of blood vessels could significantly accelerate the healing of wounds [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. While in contrast, the inadequate angiogenesis induced by infection, hyperglycemia, or circulatory disorder always lead to chronic wounds that fail to heal or heal slowly [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSince being discovered, more and more angiogenic proteins and peptides have been explored as therapeutic agents for wound healing [\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Among them, the vascular endothelial growth factor (VEGF) has been recognized as the most famous and effective regulators [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It can bind to receptors on endothelial cells, triggering the formation of new blood vessels from the existing ones [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. As such, the expression level of either VEGF or its receptors can significantly affect the angiogenesis process during wound healing [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Accordingly, the VEGF therapy has been developed as a supplementary wound healing treatment for the conventional ones such as the debridement and dressing [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. To overcome the inherent stability deficiency of VEGF as proteins, several advanced formulations including the recombinant VEGF and VEGF hydrogel such as Regranex\u0026reg; have been made [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Nonetheless, the frequent dosing and high cost still limited their widespread clinical applications, underscoring the high demand for the optimized formulations.\u003c/p\u003e \u003cp\u003eVEGF plasmid (pVEGF) refer to the circular plasmid carrying the DNA sequence encoding VEGF [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. By packaged into nanocarriers, the pVEGF can be transported into target cells, which can then produce and secret VEGF directly around the wound area, stimulating and prolonging the healing effects [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Among all the nanocarriers for the pVEGF delivery, extracellular vesicles (EVs) became more and more popular [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These phospholipid bilayer nanostructures secreted by cells are capable of shuttling bioactive substances including nucleic acids, proteins, and metabolites from their patent cells to the recipient cells [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, the EVs are considered as bioactive nanocarriers. For instance, the EVs derived from human mesenchymal stem cells (MSCs) [\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], pluripotent stem cells (iPSCs) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], as well as endothelial progenitor cells [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] have been extensively explored for wound healing due to their inherited regenerative functions. The MSCs derived EVs have even entered the Phase I Trial for the wound healing in diabetics (NCT05243368, ClinicalTrials.gov) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Nonetheless, the cultivation and isolation processes of these mammalian EVs are both time and cost consuming.\u003c/p\u003e \u003cp\u003eIn comparison, the bacterial extracellular vesicles (BEVs) can be more facilely acquired. Owing to the rapid proliferation of bacteria and standard procedure of culturing, the production of BEVs is scalable [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Besides, benefited from the diverse genetical, physical, and chemical engineering techniques, the manufacture of BEVs is also customizable [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Moreover, motivated by the emerging research interests in the \u0026ldquo;gut-skin axis\u0026rdquo; [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], the wound healing capacities of BEVs, especially those derived from probiotics, have been gradually revealed [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. For instance, the \u003cem\u003eLactobacillus reuteri\u003c/em\u003e and \u003cem\u003eLactobacillus plantarum\u003c/em\u003e derived BEVs have been found to be capable of regulating the inflammation and proliferation phases of wound healing [\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. While the \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e GG and \u003cem\u003eLactobacillus druckerii\u003c/em\u003e derived BEVs have been found to be capable of modulating the proliferation and remodeling phases of wound healing [\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Nonetheless, except for these gram-positive probiotics derived BEVs, the wound healing capacities of the gram-negative probiotics derived BEVs have been barely reported.\u003c/p\u003e \u003cp\u003eHerein, the gram-negative probiotics \u003cem\u003eEscherichia coli Nissle\u003c/em\u003e 1917 (EcN) derived BEVs were bioengineered in the presented work as bioactive nanocarriers for the local expression of VEGF to accelerate wound healing. The pVEGF containing BEV (BEV-pVEGF) was firstly constructed and characterized. Its angiogenesis capacity was then examined both \u003cem\u003ein vitro\u003c/em\u003e on the endothelial human umbilical vein endothelial cell (HUVEC) and \u003cem\u003ein vivo\u003c/em\u003e on the mice with full-thickness wounds. In light of the \u003cem\u003ein vivo\u003c/em\u003e wound healing results, the bioactivities of BEV apart from angiogenesis were elucidated. Meanwhile, the primary miRNA comprised in the BEV-pVEGF that responsible for these bioactivities was identified. The miRNA mediated cellular functions on the epidermal HaCaT were furtherly investigated, together with the targeted signaling pathway being examined. Moreover, the biosafety profile of the BEV-pVEGF was lastly evaluated on mice after its subcutaneous treatment for 7 days. The bioengineered BEV-pVEGF are expected as a promising therapeutic modality for the effective and safe wound management.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. EcN-pVEGF construction\u003c/h2\u003e \u003cp\u003eThe VEGF was synthesized and the pVEGF was recombined by Sangon Biotech (Shanghai, China). Their sequences were provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Table S2, respectively. The pVEGF was then transferred into EcN to construct EcN-pVEGF, which was cultured to generate BEV-pVEGF.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Bacteria culture\u003c/h2\u003e \u003cp\u003eEcN and EcN-pVEGF were cultured in the Luria-Bertani (LB) medium containing 5 g/L yeast extract, 10 g/L tryptone, and 10 g/L NaCl. After primary culture at 220 rpm and 37\u0026deg;C for 12 h, 1 mL of the culture medium was added to 50 mL of the fresh LB medium for the secondary culture at 220 rpm and 37\u0026deg;C for 24 h. The obtained medium was used to isolate BEV-pVEGF.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. BEVs isolation and characterization\u003c/h2\u003e \u003cp\u003eBEV and BEV-pVEGF were isolated as previous report [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The obtained medium was firstly centrifuged at 10,000 g and 4\u0026deg;C for 15 min to remove bacteria. The supernatant was then filtered through a 0.22 \u0026micro;m syringe filter to remove bacterial debris. The filtrate was ultra-centrifugated at 150,000 g and 4\u0026deg;C for 1.5 h to collect the BEV and BEV-pVEGF pellets. The collected pellets were resuspended in PBS, ultra-centrifugated at 150,000 g and 4\u0026deg;C for 1.5 h again for purification. These isolated BEVs were resuspended in PBS and stored at -80 ℃ before further use. Their morphologies were visualized by transmission electron microscopy (TEM; JEM-1400plus, Japan). Their particle concentrations and size distributions were determined by nanoparticle tracking analysis (NTA; NanoSight NS300, Malvern, UK). Their zeta potentials were analyzed by dynamic light scattering (DLS; Zetasizer Nano ZS90, Malvern, UK). Their membrane proteins were verified by Western Blot (WB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. pVEGF examination\u003c/h2\u003e \u003cp\u003eThe pVEGF contained in the isolated BEV-pVEGF was firstly extracted with the SanPrep Spin Column \u0026amp; Collection Tube (Sangon, Shanghai) following the manufacture\u0026rsquo;s instruction. Then both the recombined pVEFG and the extracted pVEGF were incubated with the prepared NheI/EcoRI digestion solution under 37 ℃ for 2 h. The agarose gel electrophoresis was performed to examine the base pair number of VEFG for the verification of the successful construction of pVEGF.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Cell culture\u003c/h2\u003e \u003cp\u003eThe human umbilical vein endothelial cell (HUVEC) and human skin keratinocytes (HaCaT) were obtained from Shanghai Skin Disease Hospital (Shanghai, China). HaCaT and HUVEC were cultured in high-glucose Dulbecco\u0026rsquo;s modified eagle medium (DMEM; Gibco, 11995500) supplemented with 10% fetal bovine serum (FBS; Gibco, 10091148) and 1% penicillin-streptomycin (Gibco, 15140-122). Both cells were cultured in incubator under 37\u0026deg;C and 5% carbon dioxide conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Cell viability\u003c/h2\u003e \u003cp\u003eHUVECs and HaCaT were seeded in the 96-well plate at a density of 5.0\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well. After overnight culture, BEV and BEV-pVEGF was added to the culture medium with the final concentration ranging from 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e-1\u0026times;10\u003csup\u003e9\u003c/sup\u003e particles/mL. After 24 h treatment, the cell viability of both cells was examined by the Cell Counting Kit-8 (CCK-8, Beyotime, C0043) according to the manufacture\u0026rsquo;s guideline.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Cell internalization\u003c/h2\u003e \u003cp\u003eBEV and BEV-pVEGF were firstly labeled with the red fluorescent dye DiI (Beyotime, C1036) and then incubated with HUVEC and HaCaT in confocal dishes at 37 ℃ for 6 h. The treated cells were washed with PBS, fixed with 4% paraformaldehyde (PFA; Servicebio, G1101), and stained with the blue fluorescence nuclei dye 4,6-diamidino-2-phenylindole (DAPI; Servicebio, G1012) as well as the green fluorescence cytoskeleton dye phalloidin (Servicebio, G1248). The cell internalization of BEV and BEV-pVEGF was then visualized by the laser scanning confocal microscope (LSCM; Leica Microsystems, Leica MICA, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Cell proliferation\u003c/h2\u003e \u003cp\u003eHUVEC and HaCaT were seeded in confocal dishes at a density of 5.0\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well and cultured overnight before being treated with BEV or BEV-pVEGF at a concentration of 1.0\u0026times;10\u003csup\u003e8\u003c/sup\u003e particles/mL. After 24 h culture under the same conditions, the cells were washed with PBS for 3 times, fixed with 4% PFA, permeabilized with Immunostaining Permeabilization Buffer (Beyotime, P0095), blocked with goat serum (Servicebio, G1208), and incubated with Ki67 antibody (Abcam, ab15580, 0.5 \u0026micro;g/mL) sequentially at 4\u0026deg;C. Alexa Fluor\u0026trade; 488-labeled secondary antibody (Abcam, ab150077, 1:500) was used to stain the Ki67, while DAPI was used to stain the cell nuclei. Their fluorescent signals were then visualized by LSCM and quantified by Image J.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Scratch assay\u003c/h2\u003e \u003cp\u003eHUVEC and HaCaT were seeded in the 6-well plate at a density of 5\u0026times;10\u003csup\u003e5\u003c/sup\u003ecells/well. After overnight culture, the cells were scratched by a 1000 \u0026micro;L sterile pipette and the floating cells were removed by PBS. BEV and BEV-pVEGF was then added to the culture medium with a final concentration of 1.0\u0026times;10\u003csup\u003e8\u003c/sup\u003e particles/mL. After 24 h treatment, the wounded areas were imaged by inverted microscope (ECLIPSE Ts2, Nikon, Japan) and the migration rates were calculated by Image J.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Transwell assay\u003c/h2\u003e \u003cp\u003eHUVEC and HaCaT were seeded in the upper chamber of the 24-well plate with 8 \u0026micro;m pore-sized filter (Corning, USA) at a density of 4\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well. After overnight culture, BEV and BEV-pVEGF was added to the culture medium in the lower chamber with a final concentration of 1.0\u0026times;10\u003csup\u003e8\u003c/sup\u003e particles/mL. After 24 h treatment, the unmigrated cells on the upper surface of the filters were gently removed by a sterile cotton swab. While the migrated cells on the lower surface of the filters were fixed by 4% PFA for 15 min and stained by crystal violet (Solarbio, G1059) for 20 min. The stained migrated cells were imaged by optical microscope (ECLIPSE Ts2, Nikon, Japan) and the migrated cell numbers were calculated by Image J.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Tube formation\u003c/h2\u003e \u003cp\u003eHUVEC was seeded in the 96-well plate with Matrigel coating at a density of 4.0\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well. After overnight culture, BEV and BEV-pVEGF was added to the culture medium with a final concentration of 1.0\u0026times;10\u003csup\u003e8\u003c/sup\u003e particles/mL. After 6 h treatment, the tube formations were imaged by optical microscope (ECLIPSE Ts2, Nikon, Japan) and the tube lengths, mesh numbers, as well as branching points were calculated by Image J.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Western blot\u003c/h2\u003e \u003cp\u003eThe BEV and BEV-pVEGF treated cells were lysed by the radioimmunoprecipitation (RIPA) buffer containing PMSF (Servicebio, G2008) to extract their total cellular proteins. After being quantified by the BCA protein quantification kit (Servicebio, G2026), the total cellular proteins were separated by SDS-PAGE and transferred to PVDF membrane. The membranes were firstly blocked with 5% skimmed milk and then successively incubated with the primary antibody, the secondary antibody, as well as the chemiluminescent substrate. The primary antibodies and secondary antibodies purchased from Servicebio included the GAPDH antibody (GB15004, 1: 5000), VEGFA antibody (GB11034B, 1:1000), AKT antibody (GB15689, 1:1000), pAKT antibody (GB150002, 1:1000), HIF-1α antibody (GB111339, 1:500), and horseradish peroxidase-conjugated goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) (GB23303, 1:10000). The bands on the membrane were images by ChemiDoc MP imaging system (BIO-RAD, USA) and analyzed by Image J.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Wound model and BEV treatment\u003c/h2\u003e \u003cp\u003e All animal experiments were approved by the Institutional Animal Care and Use Committee of Shanghai University (ECSHU 2024-074) in accordance with the National Institutes of Health guidelines. The 8-week-old male C57BL/6J mice were purchased from Huachuang Sino (JiangSu, China). All mice were anesthetized and shaved before the four full-thickness wounds with a diameter of 6 mm being excised on their backs by a biopsy punch [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The mice were then randomly divided into 3 groups and subcutaneously treated with 100 \u0026micro;L PBS or 1\u0026times;10\u003csup\u003e9\u003c/sup\u003e particles/mL BEV and BEV-pVEGF. The treatments were performed every other day. On the day 3 and 7, the wound areas were images by smartphone to directly visualize the wound closure, and examined by PeriCam PSI-ZR (PERIMED Ltd, Sweden) to evaluate the blood perfusion. The wound tissues were collected after the mice being sacrificed, the underside of the skin was images by stereomicroscope (Leica Germany) to evaluate the new blood vessel formation. Then the whole tissues were fixed, embedded, sectioned, and stained for the further histological staining and immunostaining analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14. Blood perfusion evaluation\u003c/h2\u003e \u003cp\u003eLaser speckle contrast imaging (LSCI) was performed to evaluate the new blood vessel formation and the blood perfusion around the wounds by PeriCam PSI-ZR (PERIMED Ltd, Sweden) after the 7-day treatment. Under the same scan site dimension, the blood perfusions were imaged at a fixed distance. The images of blood flux were processed by the PIMSoft (Moor Instruments Ltd, UK) to calculate the mean perfusion units (MPUs).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15. Histological staining and immunostaining analysis\u003c/h2\u003e \u003cp\u003eThe collected tissues were successively fixed with 4% PFA, embedded in paraffin, and sectioned for staining analysis. For the immunofluorescence analysis, the sectioned tissues were firstly stained with antibodies of VEGF-A (GB11034B, 1:1000) and then stained with Alexa Fluor\u0026trade; 488-conjugated secondary antibodies (Abcam, ab150080, 1:1000). For immunohistochemistry analysis, the sectioned tissues were firstly stained with antibodies of CD31 (Servicebio, GB11063, 1:1000) and Ki67 (Servicebio, GB111141, 1:1000) then stained with DAPI. For histological analysis, the sectioned tissues were H\u0026amp;E stained to evaluate the epidermal regeneration and Masson stained to evaluate the collogen deposition. All the stained tissue sections were images under SLIDEVIEW VS200 scanner (Olympus). All the fluorescence intensities were quantified by Image J.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16. miRNA sequencing\u003c/h2\u003e \u003cp\u003eThe total RNAs of BEV and BEV-pVEGF were extracted by TRIzol Reagent (Invitrogen, USA), quantified by Nanodrop ND-100 (Thermo Fisher, USA), and high-throughput sequenced by OBiO (Shanghai, China). The miRNA sequencing library was constructed by TruSeq Small RNA Sample Prep Kits (Illumina, USA) before the sequencing by Illumina Hiseq 2000/ 2500 (Illumina, USA). The ACGF101-miR (V4.2) was used for data analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17. RT-qPCR analysis\u003c/h2\u003e \u003cp\u003eThe total RNAs of HUVEC and HaCaT were extracted by TRIzol (Invitrogen, 15596026). The extracted RNA was reverse-transcribed into cDNA by Mir-X miRNA 1st-Strand Synthesis Kit (Takara Bio, CN638313). The cDNA was quantified by CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA). The primers used were synthesized by Sangon (China) and listed in Table S3. \u003cem\u003eU6\u003c/em\u003e was used to normalize the gene expression level. The relative expression levels of miRNA were quantified using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.18. Inhibitor transfection\u003c/h2\u003e \u003cp\u003e HUVEC and HaCaT were transfected with 100 nmol/L miR-21a-5p inhibitor (RiboBio, China) by the Lipofectanube 3000 (Invitrogen, L3000008) according to the manufacture\u0026rsquo;s guideline. After 8 h transfection, the culture medium was replaced and the cells were cultured for another 16 h before the following experiments [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e2.19. ELISA\u003c/h2\u003e \u003cp\u003eThe total proteins of the collected wound tissues were extracted by RIPA buffer containing PMSF and quantified by BCA protein quantification kit. The expression levels of IL-1β and IL-10 were then determined by the ELISA kits (MultiSciences, EK210, EK206, EK201B, and EK282) according to the manufacture\u0026rsquo;s guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e2.20. Hemolysis assay\u003c/h2\u003e \u003cp\u003e200 uL 4% rabbit erythrocytes (Sbjbio, SBJ-RBC-RAB003) were mixed with 200 \u0026micro;L ddH\u003csub\u003e2\u003c/sub\u003eO, PBS, BEV and BEV-pVEGF with the final concentrations ranging from 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e-1\u0026times;10\u003csup\u003e9\u003c/sup\u003e particles/mL, respectively. After co-incubation for 2 h at 37 ℃, the blood samples were centrifugated at 800 g for 5 min and photographed. Their absorbances at 540 nm were then measured by the microplate reader (Cytation, Biotek, USA) to calculate the hemolysis ratios.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e2.21. Safety profile\u003c/h2\u003e \u003cp\u003eAfter the 7-day treatments with PBS, BEV, and BEV-pVEGF, the blood of all mice was collected by removing their eyeballs. Subsequently, the blood was centrifugated for extracting the serum. The expression level of key biochemical indicators in serum including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine (CREA) was evaluated. Besides, the major organs of all mice including heart, liver, spleen, lung, and kidney were also collected after sacrifice and sectioned for the further H\u0026amp;E staining analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e2.22. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed for at least 3 times. All data were displayed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). GraphPad Prism 8.0 (La Jolla, USA) was used to analysis the experimental data. Image J was used to analysis the experimental images. One-way ANOVA was used for the significance assessments. \u003cem\u003ep\u003c/em\u003e value of less than 0.05 was considered as significant difference with \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and \u003csup\u003e****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussions","content":"\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Construction and characterization of BEV-pVEGF\u003c/h2\u003e \u003cp\u003eTo obtain the pVEGF containing BEVs (BEV-pVEGF), the bioengineering approach was employed. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, the VEGF gene element was firstly synthesized and used to generate the shuttle plasmid pVEGF (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, S2). After being successfully validated by the agarose gel electrophoresis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C), the pVEGF was then transfected into the \u003cem\u003eEscherichia coli\u003c/em\u003e Nissle 1917 (EcN) to obtain the recombinant probiotics EcN-pVEGF, which were further cultured to generate the bioengineered BEV-pVEFG.\u003c/p\u003e \u003cp\u003eThrough the ultracentrifugation based process as previously reported [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], both the EcN secreted BEV and the EcN-pVEGF secreted BEV-pVEGF were effectively isolated and purified. Despite of their different contents, the BEV and BEV-pVEGF possess the same surface components, similarly to their parental EcN. Thereby, almost the same zeta potentials at approximate\u0026thinsp;\u0026minus;\u0026thinsp;20 mV were determined for the BEV and BEV-pVEGF, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). While compared to EcN, these values were slightly lower, in accordance with the previous founding that the repulsion caused by the local enrichment of negative charges on the outer membrane of bacteria contributed to their secretion of BEVs [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Besides, the successful loading of pVEGF in the BEV-pVEGF was also confirmed by the agarose gel electrophoresis as demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE.\u003c/p\u003e \u003cp\u003eAccording to the transmission electron microscopy (TEM) visualization, both of the BEV and BEV-pVEGF exhibited the featured spherical bilayer nanostructures with average diameter of approximately 125.6 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These diameters were further verified by the dynamic light scattering (DLS) (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and nanoparticle tracking analysis (NTA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG), which also shown that the concentration of the obtained BEV and BEV-pVEGF were around 2.34\u0026times;10\u003csup\u003e9\u003c/sup\u003e particles/mL and 1.69\u0026times;10\u003csup\u003e9\u003c/sup\u003e particles/mL, respectively. All these characterization results indicated that the rationally designed BEV-pVEGF were successfully engineered and effectively isolated, facilitating the following evaluations on their therapeutic efficacies for wound healing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.2. BEV-pVEGF delivering pVEGF to promote the proliferation, migration, and angiogenesis of endothelial cell\u003c/h2\u003e \u003cp\u003eTo validate the angiogenesis performance of BEV-pVEGF, their cellular functions mediated by the delivered pVEGF were examined. Since the VEGF mainly participate in the vascularizing process during the proliferative stage of wound healing [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], the angiogenic endothelial cell HUVEC was selected for the examination. According to the CCK-8 assay, the BEV-pVEGF displayed no cytotoxicity against HUVEC up to concentration as high as 1\u0026times;10\u003csup\u003e9\u003c/sup\u003e particles/mL (Figure S2). After incubation with the HUVEC for 6 h, the red fluorescence Dil labelled BEV-pVEGF could be successfully uptake by the blue fluorescence DAPI and green fluorescence Phalloidin labelled HUVEC, simultaneously delivering their loaded pVEGF into the HUVEC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eThe successfully internalized BEV-pVEGF were then demonstrated could effectively facilitate the proliferation of HUVEC as the mostly increased population of the Ki67 positive cells were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). Meanwhile, a significantly higher expression of the VEGF was detected for the HUVEC with the treatment of BEV-pVEGF than PBS or BEV (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), indicating the effective intracellular delivery and expression of pVEGF by the BEV-pVEGF. Subsequently, the accelerated migration of HUVEC with the treatment of BEV-pVEGF was observed. The migration rate of HUVEC increased approximately 2.1 times with the treatment of BEV-pVEGF than PBS based on the scratch assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F). While the migration number of HUVEC increased approximately 2.9 times with the treatment of BEV-pVEGF than PBS based on the transwell assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, H). Most notably, more than 2.1 times increased branching points were formed by HUVEC with the treatment of BEV-pVEGF (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, J), confirming the BEV-pVEGF enhanced angiogenesis of HUVEC.\u003c/p\u003e \u003cp\u003eAll these cellular function results constituted compelling validation that the rationally engineered BEV-pVEGF could effectively deliver the pVEGF into HUVEC, simultaneously upregulating its expression of VEGF-A. As a consequence, the significantly expressed VEGF-A could mediate and promote the proliferation, migration, and angiogenesis of HUVEC, holding great potential in expediting the wound healing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.3. BEV-pVEGF Expedited the Angiogenesis and Closure of Cutaneous Wounds\u003c/h2\u003e \u003cp\u003eTo assess the \u003cem\u003ein vivo\u003c/em\u003e angiogenesis and healing efficiency of BEV-pVEGF, full-thickness wounds were bilaterally created on the dorsal of the C57BL/6J mice. As demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, the mice with 6 mm diameter wounds were randomly divided into 3 groups and subcutaneously administrated with PBS, BEV, and BEV-pVEGF around the edge of wounds every other day. According to the macroscopic images (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) and tracing analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) of the wounds, the BEV-pVEGF treatment significantly accelerated the cutaneous wound closure. With the BEV-pVEGF treatment, more than 64.7% and 80.4% wound cloresures were restores at day 3 and day 7, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). While although not as effective as the BEV-pVEGF treatment, the BEV treatment still achieved 50.8% and 64.7% wound cloresures, which were approaximately 1.8 and 1.4 times higher than the control group with the PBS treatment at day 3 and day 7, respectively.\u003c/p\u003e \u003cp\u003eTo confirm the accelerated wound closure is attributed to the promoted angiogenesis, the blood flow perfusion around the wounds were firstly imaged after the treatment for 7 days. As demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and Figure S3, compared to the PBS and BEV treatment, the BEV-pVEGF significantly enhanced the blood flow perfusion around the wounds. The intensity of blood flow perfusion increased 1.97 and 1.34 times, respectively, suggesting the improved vascularization. Besides, to further verify the improved vasculatization, all mice were euthanatized at day 7 for the tissue colllection and analyses. According to the dermoscope images displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, the maximum blood vessles were formed subcutaneously with the BEV-pVEGF treatment. Morevoer, based on the immunohistochemistry analyses, both the BEV and BEV-pVEGF treatments up-regulated the expression of Ki67 from 20.4% to about 50.0%, suggesting their promoted cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Under such circumstances, only the BEV-pVEGF treatments achieved the highest expression of VEGF, which is 1.7 and 4.1 times higher than the BEV and PBS treatment, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). The BEV-pVEGF facilitated expression of VEGF ultimately stimulated the vasculatization, with 2.0 and 3.5 times increased expression of the angiogenesis marker CD31 being observed than BEV and PBS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003eAll these \u003cem\u003ein vivo\u003c/em\u003e evidences togetherly confirmed that the BEV-pVEGF could significantly increase the expression of VEGF and consequently stimulate the angiogenesis of wounds. The enhanced vascularization could restore the oxygen supply and nutriention delivery to wounds, accelerating the healing process and thus expediting the wound closure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.4. BEV-pVEGF Facilitated the Granulation Tissue Formation and Re-epithelialization via the PI3K-AKT Signaling Pathway\u003c/h2\u003e \u003cp\u003eTo elucidate the other biofunctions of the BEV-pVEGF during wound healing, the collected tissues at day 3 and day 7 were sectioned for further Hematoxylin and Eosin (H\u0026amp;E), Masson\u0026rsquo;s Trichrome, as well as immunofluorescence staining. According to the H\u0026amp;E staining images (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), both of the BEV-pVEGF and BEV treatments decreased the wound width (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) while increased the granulation tissue and epidermal thickness (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). Even though not as efficient as BEV-pVEGF, the BEV still exerted significantly improved wound healing efficiency than the control. After the 7 days administration, the BEV-pVEGF and BEV stimulated the denser collagen deposition as well based on the Masson staining images (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F). The deposited collagen fibers formed a scaffold that favoring cell proliferation and migration, furtherly accelerated the epidermal coverage and wound closure.\u003c/p\u003e \u003cp\u003eSince both of the BEV-pVEGF and BEV facilitated the granulation tissue formation and re-epithelialization, the bioactivities of BEV during wound healing were also investigated. Derived from the probiotics EcN, the BEV comprising diverse bioactive molecules that inherited from EcN has been considered as bioactive nanocarrier. For instance, previous study has found that the miR-21-5p was comprised in the BEV [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Most recently, the miR-21-5p has been furtherly proven to activate the PI3K/AKT signaling pathway, facilitating the wound healing activities of both endothelial and epidermal cells.[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] In consistent with the previous studies, after the treatment by BEV-pVEGF and BEV, the up-regulated expressions of p-AKT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG) and HIF-1α (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH) were both detected, confirming the activated PI3K/AKT signaling pathway as expected.\u003c/p\u003e \u003cp\u003eAll these tissue staining analyses revealed that apart from the exogenous pVEGF expedited angiogenesis, the endogenous bioactive molecules that comprised in the BEV-pVEGF could simultaneously facilitated the granulation tissue formation and re-epithelialization via the PI3K-AKT signaling pathway. All the exogenous and endogenous bioactive molecules inside BEV-pVEGF can work together, synergistically contributing to their accelerated wound closure and healing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.5. BEV-pVEGF Preserved the Endogenous Wound Healing miRNA\u003c/h2\u003e \u003cp\u003eTo identify the key endogenous bioactive molecule inside the BEV-pVEGF that facilitate wound healing, the next-generation miRNA sequencing was performed. As demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, 241 miRNAs were detected inside the BEV generated by EcN. According to the miRWalk database, 129 of these miRNAs were associated with wound healing.[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] Subsequently, the top 20 highly expressed miRNAs inside the BEV were listed in the heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Among them, 5 miRNAs including the hsa-miR-21-5p,[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] hsa-miR-423-5p,[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] hsa-miR-148a-5p,[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] hsa-miR-221-3p,[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] and hsa-miR-92a-3p[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] are related to wound healing. Besides, the BEV-pVEGF generated by the bioengineered EcN preserved the miRNA content. There were no significantly differences being examined between the BEV-pVEGF and the BEV in terms of the expression on the top 5 wound healing miRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eBased on the top 5 highly expressed wound healing miRNA, the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed as well to elucidate the potential signaling pathways facilitating wound healing. As displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, the 3 closely relevant signaling pathways were metabolic, PI3K-AKT, and HIF-1. Further Gene Ontology (GO) analyses revealed that these miRNAs predominately interfered with the protein binding in molecular function (Figure S4A), membrane formation in cellular component (Figure S4B), as well as signal transduction in biological process (Figure S4C). In addition, the network on the interactions between the top 5 highly expressed wound healing miRNA and their targeted genes were also visualized by Cytoscape (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Multiple targeted genes, such as RPS6KA5, MTOR, SKP2, TGFB1, and FAM168, are correlated with the cell proliferation and migration.\u003c/p\u003e \u003cp\u003eAll these miRNA sequencing analyses demonstrated that the BEV-pVEGF preserved the endogenous wound healing miRNA from the EcN even after bioengineering. The top 5 highly expressed wound healing miRNA inside the BEV-pVEGF, including the hsa-miR-21-5p, hsa-miR-423-5p, has-miR-148a-5p, has-miR-221-3p, and has-miR-92a-3p, predominately relevant to 3 signaling pathways. Among them, the PI3K-AKT signaling pathway coincided with the one detected during the tissue staining analyses. Thereby, it was supposed to be activated by the endogenous miRNA inside the BEV-pVEGF to facilitate the wound healing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3.6. BEV-pVEGF delivering mir-21-5p to prompt the proliferation and migration of epidermal cells via activating the PI3K-AKT signaling pathway\u003c/p\u003e \u003cp\u003eTo further clarify the underlying relationship between the endogenous bioactive substances and wound healing capacities of BEV-pVEGF, their cellular functions mediated by the most highly loaded miR-21-5p were examined on the epidermal HaCaT. Similarly to the HUVEC, no obvious cytotoxicity was detected against the HaCaT with the BEV and BEV-pVEGF treatment up to the concentration of 1\u0026times;10\u003csup\u003e9\u003c/sup\u003e particles/mL (Figure S5). After culturing with the HaCaT for 6 h, the Dil-labeled BEV-pVEGF could successfully penetrate the cell membrane thus effectively deliver the intrinsic miR-21-5p into the Phalloidin and DAPI stained HaCaT (Figure S6 and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eSubsequently, an increased population of the Ki67 positive cells was observed with the prolonged culturing by BEV-pVEGF (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), indicating the significantly induced proliferation of HaCaT. While upon the transfection of the miR-21-5p inhibitor, the cell proliferation was severely inhibited to an extent similarly to the control group that treated with only the PBS. Besides, based on the scratch assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC) and transwell assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, the migration rate and number of HaCaT that cultured by BEV-pVEGF also increased more than 1.5 times, but barely changed in the presence of the miR-21-5p inhibitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Altogether, these results demonstrated that the BEV-pVEGF could promote the proliferation and migration of the epidermal HaCaT. These effects were achieved mainly through the BEV-pVEGF facilitated intracellular delivery of their endogenous miR-21-5p.\u003c/p\u003e \u003cp\u003eFurthermore, the increased expression of the phosphorylated AKT (p-AKT) and the hypoxia-inducible factor-1 alpha (HIF-1α), which are the indicators of the PI3K-AKT signaling pathway activation,[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] were detected in HaCaT according to the western blot (WB) analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). While once again, the p-AKT and HIF-1α expressions were suppressed in the presence of the miR-21-5p inhibitor, confirming the PI3K-AKT signaling pathway was activated by the endogenous miR-21-5p of BEV-pVEGF. In combined with the observed PI3K-AKT pathway activation in the closured wound tissues after the treatment of BEV-pVEGF, these results provided compelling evidence that apart from the exogenous pVEGF, the BEV-pVEGF could also acted as bioactive nanocarrier to delivery their endogenous miR-21-5p into the epidermal cells. Thereby, the proliferation and migration of epidermal HaCaT were promoted. The endogenous miR-21-5p worked together with the exogenous pVEGF of BEV-pVEGF, facilitating the vascularized granulation tissue formation and re-epithelialization thus accelerating the effective wound closure on mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e3.7. BEV-pVEGF Displayed Low Immunogenicity and Toxicity During the 7-Day Wound Treatment\u003c/h2\u003e \u003cp\u003eTo guarantee the safe BEV-pVEGF treatment for wound healing, its safety profile in terms of toxicity was furtherly examined on mice after the different treatments for 7 days. Due to the defect in its LPS biosynthesis, the non-pathogenic probiotics EcN and its derived BEV bear a truncated LPS chain and exhibit a semi-rough phenotype.[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] Thereby, the immunogenicity of EcN and its BEV were found significantly reduced.[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] In consistent with previous founding, both of the BEV and BEV-pVEGF displayed low immunogenicity. After the treatment with BEV and BEV-pVEGF for 7 days, there were no significant differences on the expression of the pro-inflammatory cytokine interleukin 1β (IL-1β) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Most notably, after the treatment with the BEV-pVEGF for 7 days, the expression of the anti-inflammatory cytokine interleukin-10 (IL-10) was even slightly increased compared to the mice with the PBS and BEV treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Attributed to the pro-angiogenesis capacity of BEV-pVEGF, the anti-inflammatory phenomenon should come up simultaneously with the expedited vascularized granulation tissue formation.[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eBesides, both of the BEV and BEV-pVEGF exhibited excellent hemocompatibility (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), with less than 1% hemolysis being detected up to concentrations of 1\u0026times;10\u003csup\u003e9\u003c/sup\u003e particles/mL. After the treatment with PBS, BEV, and BEV-pVEGF for 7 days, no significant differences were detected on the expression level of the key biochemical indicators in the serum of mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC), including the alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), and creatinine (CREA). These results indicated that the subcutaneous administrations of neither the BEV nor the BEV-pVEGF for 7 consecutive days had adverse effects on the liver and kidney functions of mice. Moreover, there were no noticeable pathological changes and inflammatory cell infiltrations being observed in the major organs of mice, including the heart, liver, spleen, lung, as well as kidney, after all the treatments based on the H\u0026amp;E staining images displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD. All these results substantiated the low immunogenicity and toxicity of both BEV and BEV-pVEGF, illustrating their distinct \u003cem\u003ein vivo\u003c/em\u003e safety profiles for the safe wound treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003e Featured by their scalable, customizable, and bioactive properties, the probiotics EcN derived BEVs were bioengineered as bioactive nanocarriers in the presented work for the local expression of VEGF to accelerate wound healing. Based on the bioengineering approach and ultracentrifugation procedure, the 125.6 nm lipid bilayer BEVs comprising shuttle plasmid that encoded VEGF-A were obtained. The BEV-pVEGF facilitated the cell internalization of pVEGF, up-regulated the cell expression of VEGF-A, and thus promoted the angiogenesis of HUVEC. Besides, in addition to the exogeneous pVEGF, the BEV-pVEGF also intracellularly delivered their endogenous miR-21-5p, subsequently activated the PI3K-AKT signaling pathway, and thus promoted the proliferation and migration of the epithelial HaCaT. Upon the BEV-pVEGF treatment, expedited vascularization and enhanced blood flow perfusion were observed around the wound on mice. Sequentially, the accelerated granulation tissue formation and wound re-epithelialization were observed as well within 7 days. Moreover, no significant immunogenicity and toxicity were detected on mice after the BEV-pVEGF treatment. With more and more researches on the gut microbiota and their cross-kingdom regulation in wound repair and regeneration, these results filled the existing gaps between the gut microbiota and wound homeostatic, contributing to the future development of innovative nanotherapeutics for the wound treatment. Meanwhile, the bioengineering of BEV-pVEGF provides a readily-available, mass-producible, and cost-effective approach to drive the clinical translation of the effective and safe nanotherapeutic modality of BEVs for the future wound management.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBEV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBacterial extracellular vesicle\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVEGF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVascular endothelial growth factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEcN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e Nissle 1917\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTransmission electron microscope\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDLS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDynamic light scattering\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNTA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNanoparticles tracking analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHUVEC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman umbilical vein endothelial cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHaCaT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman skin keratinocytes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLCSM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLaser confocal scanning microscopy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWestern blot\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRT-qPCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReal-time quantitative polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eELISA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnzyme linked immunosorbent assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emiRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMicro-RNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDiI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGt\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGranulation tissue\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eH\u0026amp;E\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHematoxylin and eosin staining\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAKT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProtein kinase B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ep-AKT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhospho-protein kinase B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHIF-1α\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHypoxia-inducible factor 1-alpha\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGAPDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlyceraldehyde-3-phosphate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eALT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlanine aminotransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAspartate aminotransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBUN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBlood urea nitrogen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCREA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCreatinine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eAll animal experiments were approved by the Institutional Animal Care and Use Committee of Shanghai University (ECSHU 2024-074) in accordance with the National Institutes of Health guidelines.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNo applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eSupplementary Information\u003c/h2\u003e \u003cp\u003eThe online version contains supplementary material available at\u0026hellip;\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe work was supported by the Shanghai Pujiang Program (21PJ1404100), the Young Elite Scientists Sponsorship Program by CAST (2023QNRC001), and the National Natural Science Foundation of China (No. 22477075, No.22077078, and No. 22205260).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eZelin Zheng, Xi Liu, and Kailu Guo contributed equally to the work. Zelin Zheng: Data curation, methodology, visualization, writing\u0026ndash;original draft; Xi Liu: Conceptualization, methodology, writing\u0026ndash;original draft; Kailu Guo: Data curation, methodology, visualization; Yuting Li: Data curation, validation, formal analysis; Yirong Wang: Data curation, validation, formal analysis; Cuiping Zhang: Funding acquisition, resources, supervision, writing\u0026ndash;review and editing; Honggang Hu: Funding acquisition, resources, supervision, writing\u0026ndash;review and editing; Yejiao Shi: Conceptualization, funding acquisition, project administration, writing\u0026ndash;review and editing. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors gratefully thank Assoc Prof Han Liu from Shanghai University for his valuable help and guidance on bioengineering of the EcN-pVEGF.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGurtner GC, Werner S, Barrandon Y, Longaker MT. Wound repair and regeneration. Nature. 2008;453:314\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePe\u0026ntilde;a OA, Martin P. Cellular and molecular mechanisms of skin wound healing. 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A miRNA cocktail orchestrates coordinated cellular responses to promote diabetic wound healing. Burns Trauma 2025:tkaf060.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrozdanov L, Z\u0026auml;hringer U, Blum-Oehler G, Brade L, Henne A, Knirel YA, Schombel U, Schulze J, Sonnenborn U, Gottschalk G, et al. A single nucleotide exchange in the wzy gene is responsible for the semirough O6 lipopolysaccharide phenotype and serum sensitivity of Escherichia coli strain Nissle 1917. J Bacteriol. 2002;184:5912\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWerner S, Grose R. Regulation of wound healing by growth factors and cytokines. Physiol Rev. 2003;83:835\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu M, He S, Tang H, Hu H, Shi Y. Molecular Engineering of Polymyxin B for Imaging and Treatment of Bacterial Infections. Front Chem. 2021;9:809584.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"bacterial extracellular vesicles, wound healing, angiogenesis, probiotics, VEGF","lastPublishedDoi":"10.21203/rs.3.rs-8711594/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8711594/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The angiogenesis is a pivotal process during wound healing. Its deficiency usually causes diminished oxygen and nutrient conveyance, compromising the cell function and decelerating the wound closure. Since being identified as the potent stimulator of angiogenesis, vascular endothelial growth factor (VEGF) has been explored as the leading therapeutic candidate. To overcome its inherent instability, both recombinant proteins and gene therapies have been proposed. Nonetheless, evidences of their therapeutic benefits for wound healing were limited. Over the past decades, bacterial extracellular vesicles (BEVs) have been recognized as versatile bioactive nanocarriers\nfor the cross-kingdom communication. Herein, BEV derived from the recombinant probiotics Escherichia coli Nissle 1917 (BEV-pVEGF) was bioengineered to deliver the shuttle plasmid encoding VEGF. The BEV-pVEGF was proven could facilitate the intracellular delivery and local expression of the exogenous pVEGF, promoting the proliferation, migration, and angiogenesis of the endothelial HUVEC. Moreover, it was also proven to enable the intracellular delivery of the endogenous miR-21-5p, activating the PI3K-AKT signaling pathway and expediting the proliferation and migration of the epidermal HaCaT. Upon its subcutaneous administration for 7 consecutive days, the vascularized granulation tissue formation and re-epithelialized wound closure were significantly accelerated on mice bearing full-thickness wounds, with no obvious immunogenicity and toxicity being detected. These bioengineered BEV-pVEGF nanocarriers provide a readily-available, mass-producible, and cost-effective approach to developed effective and safe therapeutic modality for the future wound management.","manuscriptTitle":"Bioengineered probiotics derived bacterial extracellular vesicle as bioactive nanocarrier for the local VEGF expression to accelerate wound healing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-03 16:41:03","doi":"10.21203/rs.3.rs-8711594/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-04T07:11:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-04T01:54:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-03T03:50:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"237466522542517259693412576684022619592","date":"2026-02-02T10:30:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"77771585771160613649944223900957436828","date":"2026-02-02T10:20:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-02T09:22:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-28T07:44:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-28T07:41:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2026-01-27T14:10:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e8f1f8b3-2df3-483a-8459-84151ec58df5","owner":[],"postedDate":"February 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T16:09:27+00:00","versionOfRecord":{"articleIdentity":"rs-8711594","link":"https://doi.org/10.1186/s12951-026-04217-4","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2026-02-22 15:57:19","publishedOnDateReadable":"February 22nd, 2026"},"versionCreatedAt":"2026-02-03 16:41:03","video":"","vorDoi":"10.1186/s12951-026-04217-4","vorDoiUrl":"https://doi.org/10.1186/s12951-026-04217-4","workflowStages":[]},"version":"v1","identity":"rs-8711594","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8711594","identity":"rs-8711594","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}