Chemotaxis-driven hybrid liposomes trilogically recover intestinal homeostasis for targeted therapy of ulcerative colitis

Chemotaxis-driven hybrid liposomes trilogically recover intestinal homeostasis for targeted therapy of ulcerative colitis | 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 Article Chemotaxis-driven hybrid liposomes trilogically recover intestinal homeostasis for targeted therapy of ulcerative colitis Kaili Hu, Xier Pan, Yushu Li, Xiao Zhao, Jiaxin Zhang, Peng Xian, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4710969/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Ulcerative colitis (UC) is tightly linked to the dysbiosis of intestinal homeostasis, accompanied by the intestinal epithelial barrier destruction, subsequent inflammatory factor infiltration, and excessive oxidative stress. However, conventional therapeutics only focus on suppressing inflammation and often suffer from metabolic instability as well as limited targeting, thereby leading to suboptimal remission rates and severe side effects. Here, we initially fabricated bacterial outer membrane vesicle (OMV, from Stenotrophomonas maltophilia )-fused and borneol-modified liposomes (BO/OMV-lipo@LU) to deliver luteolin for targeted therapy of UC by recovering intestinal homeostasis. Benefitting from the chemotaxis-driven colon-targeting ability of OMVs and intestinal epithelial uptake promotion of borneol, the hybrid liposomes exhibited excellent targeting capability towards the inflamed colon. Moreover, under the combined effects of OMVs and borneol, interestingly, the hybrid liposomes achieved deep penetration into the mucosa for better uptake of luteolin by the intestinal epithelium. Notably, BO/OMV-lipo@LU with favorable biosafety exerted promising therapeutic efficacy and trilogically recovered intestinal homeostasis in UC by alleviating intestinal inflammation, modulating redox balance, and restoring intestinal epithelial barriers. These results demonstrated that such a biomimetic nanoplatform could be exploited as a safe and effective gut-targeted delivery system for UC intervention. Health sciences/Diseases/Gastrointestinal diseases/Intestinal diseases/Inflammatory bowel disease/Ulcerative colitis Health sciences/Medical research/Drug development Bacterial outer membrane vesicle Borneol Liposome Luteolin Ulcerative colitis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Ulcerative colitis (UC), a chronic and relapsing inflammatory disease, has an estimated prevalence of 5 million cases around the world in 2023 and a rapidly increasing incidence worldwide, tremendously reducing the quality of patients’ life 1–3 . Emerging evidence has indicated that UC flare is closely associated with the dysbiosis of intestinal homeostasis, accompanied by the intestinal epithelial barrier destruction 4–6 , subsequent inflammatory factor infiltration and excessive oxidative stress 7–10 . The clinical therapeutics of UC include anti-inflammatory molecules (e.g., 5-aminosalicylates and corticosteroids) and immunosuppressants (e.g., infliximab and azathioprine), which primarily focus on ameliorating disease-related symptoms by reducing inflammatory burden 11 . However, they generally do not address other underlying pathogenic factors, such as the destruction of epithelial barriers and redox imbalance, potentially resulting in suboptimal remission rates among patients 9,12,13 . Besides, the clinical outcomes are also hampered by their inherent hydrophobicity, ease of metabolization, and lack of targeting ability 14 . Moreover, severe side effects are observed during the use of these traditional drugs. For example, the long-term use of corticosteroids may lead to osteoporosis, and the infliximab (anti-tumor necrosis factor antibodies) therapy comes with an increased risk of opportunistic infections and drug-induced lupus 15–19 . Therefore, there exists an unmet demand for a more effective strategy that can achieve multipronged intervention against UC and inflamed colon-targeted delivery. Previous studies attempted to achieve inflamed colon-targeted delivery of therapeutics by enhancing adhesion to mucus or equipping them with targeting moieties (e.g., hyaluronic acid and anti-CD98 antibodies) to target inflammatory epithelial cells and macrophages 20–22 . Despite their high in vitro uptake efficiency, the in vivo penetration depth into mucosa remains limited. Biomimetic nanotechnology offers new opportunities for the development of targeted delivery by integrating nanosystems’ functional versatility with biomimetic materials’ features. Bacterial membrane-originated components, such as outer membrane vesicles (OMVs) and cytoplasmic membranes, have been widely applied to targeted drug delivery 23–25 . Interestingly, we surprisingly found that OMVs derived from Stenotrophomonas maltophilia ( S.mal. ) exhibited excellent biological chemotaxis to the inflamed colon tissue. S.mal. is a Gram-negative and low-virulent bacterium that is found ubiquitously in water, foods, plants, animal tissues, and the human body 26 . The chemotaxis of OMVs from S.mal. may be attributed to the adhesion to inflamed colon mucosa via functional components inherited from parent bacteria, such as outer membrane protein A, which is associated with the adhesion of bacteria[23]. Thereby, biomimetic OMV-fused hybrid liposomes may faithfully replicate the adhesion property that can enhance the retention of liposomes in the inflamed colon to achieve chemotaxis-driven UC-targeted delivery. Moreover, according to previous studies, borneol, a bicyclic monoterpene, showed the potential to improve the uptake of nanoparticles by intestinal epithelial cells 29,30 . Accordingly, we used borneol to further engineer OMV-fused hybrid liposome surface for improving inflamed epithelial uptake in UC. Luteolin, a natural flavonoid compound, is widely found in fruits, vegetables, and medicinal herbs such as Dendranthema morifolium (Ramat.) Tzvel. and Lonicera japonica Thunb. 31,32 . Currently, luteolin has been widely exploited as a dietary supplement for inflammation-related diseases in clinical trials, such as COVID-19 (NCT05311852) and chronic periodontitis (CTRI/2024/05/067436). Increasing evidence indicates that luteolin is a promising candidate for UC intervention due to its multiple pharmacological actions that can restore intestinal homeostasis, such as anti-inflammatory, anti-oxidant, and intestinal epithelial barrier protection effects 33–36 . However, its anti-UC efficacy is limited by poor solubility and the lack of targeting ability towards inflamed colon 35,37 . Therefore, in this study, chemotaxis-driven hybrid liposomes (BO/OMV-lipo@LU) were designed by functionalizing liposomes with borneol and OMVs, aiming to achieve UC-targeted delivery of luteolin, as shown in Scheme 1 . Our results demonstrated that OMV presence contributed to the retention of liposomes in the inflamed colon. Interestingly, under combined effects of OMVs and borneol, BO/OMV-lipo@LU achieved deep mucosa penetration and improved epithelial uptake. In addition, the formulation could sustainably release luteolin in the inflamed colon and exhibit prominent therapeutic potentials in a dextran sulfate sodium (DSS)-induced UC mice model. Notably, BO/OMV-lipo@LU with favorable biosafety trilogically recovered intestinal homeostasis in UC by alleviating intestinal inflammation, modulating redox balance, and restoring intestinal epithelial barriers. The novel chemotaxis-driven hybrid liposomes proposed in this study may serve as a safe and efficient targeted delivery system for UC therapy. 2. Results and discussion 2.1 Characterization of bacterial OMVs and distearyl phosphatidylethanolamine-polyethylene glycol-borneol (DSPE-PEG-BO) Bacterial OMVs and DSPE-PEG-BO required for BO/OMV-lipo@LU formulation were prepared and characterized. Bacterial OMVs were isolated from S.mal. via the ultracentrifugation method. Dynamic light scattering (DLS) analysis results suggested that the particle size peak of OMVs was 100.8 ± 16.5 nm (Fig. 1 a), and the zeta potential was − 12.4 ± 1.11 mV. The transmission electron microscopy (TEM) image showed that OMVs presented a typical phospholipid bilayer structure as described in previous studies (Fig. 1 b) 38,39 . The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed protein profiles of isolated bacterial OMVs (Fig. 1 i). The synthetic route of borneol-modified lipid materials (DSPE-PEG-BO) was shown in Fig. 1 c. The amino groups of (R)-(+)-bornylamine (the amino derivative of borneol) reacted with NHS ester groups of DSPE-PEG-NHS to synthesize DSPE-PEG-BO via amide bonds. Figure 1 E showed the 1 H nuclear magnetic resonance (NMR) spectra of (R)-(+)-bornylamine, DSPE-PEG-NHS and DSPE-PEG-BO. It could be seen that a new proton peak at 8.2 ppm (Peak e) appeared in the NMR spectra of DSPE-PEG-BO compared with (R)-(+)-bornylamine and DSPE-PEG-NHS, suggesting the formation of amide bonds. In addition, the characteristic proton peaks (Peak a, b, and c) of DSPE-PEG-NHS, and the methyl proton peak (Peak 3) of (R)-(+)-bornylamine were also found in the NMR spectra of DSPE-PEG-BO. Moreover, compared with DSPE-PEG-NHS, the carbodiimides proton signal (Peak d) of DSPE-PEG-BO disappeared, further demonstrating the successful conjugation of borneol to DSPE-PEG. These collective results confirmed the successful synthesis of DSPE-PEG-BO through the NHS ester reaction. 2.2 Preparation and characterization of BO/OMV-lipo@LU Four kinds of luteolin-loaded liposome formulations were constructed, including liposomes without any modification (Lipo@LU), borneol-modified liposomes (BO-lipo@LU), OMV-mimetic hybrid liposomes (OMV-lipo@LU), and borneol-modified OMV-mimetic hybrid liposomes (BO/OMV-lipo@LU), aiming to investigate benefits that OMVs and DSPE-PEG-BO brought to liposomes in targeting ability. Initially, Lipo@LU were prepared using the thin-film method, and the formulation optimization was carried out by the single-factor test using the particle size and encapsulation efficiency (EE) as evaluation indexes. As illustrated in Fig. 1 d, the organic solvent type and the ratio of soybean lecithin to cholesterol markedly affected the particle size and EE, respectively. The significant effects of luteolin dosage could also be observed on the particle size and EE of Lipo@LU, while the hydration temperature had little effect on them. The results showed that the optimum formulation of Lipo@LU was confirmed as follows: the organic solvent was ethanol, the ratio of soybean lecithin to cholesterol was 3:1, the hydration temperature was 25℃, and the luteolin dosage was 1.2 mg. Additionally, it could be seen from the TEM image that Lipo@LU had a typical spherical vesicle structure (Fig. 1 e), which is in keeping with other studies of liposomes 40,41 . Subsequently, Lipo@LU was further modified with DSPE-PEG-BO and fused with OMVs to fabricate BO/OMV-lipo@LU. BO-lipo@LU and OMV-lipo@LU, used as control liposome formulations, were prepared by adding DSPE-PEG-BO and OMVs, respectively. Similar to Lipo@LU, BO/OMV-lipo@LU exhibited a vesicle structure (Fig. 1 e). DLS analysis results suggested that all liposome formulations (Lipo@LU, BO-lipo@LU, OMV-lipo@LU, and BO/OMV-lipo@LU) possessed a unimodal distribution of particle sizes (Fig. 1 f). The particle size and polydispersity index (PDI) of BO/OMV-lipo@LU were 156.5 ± 0.9 nm and 0.16 ± 0.02, respectively. As could be seen in Fig. 1 g, the zeta potentials (about − 24 mV) of those OMV-fused liposomes (including OMV-lipo@LU and BO/OMV-lipo@LU) were between that of native OMVs (about − 12 mV) and Lipo@LU (about − 30 mV), which indicated the successful hybridization of OMVs with liposomes as reported previously 42–44 . Additionally, previous studies demonstrated that those nanoparticles with less than 200 nm in diameter and a negative surface charge, exhibited better tissue-penetrating ability for UC therapy 45,46 . Hence, the size and charge properties might contribute to the targeting delivery of BO/OMV-lipo@LU to inflamed colon sites. Moreover, the drug loading efficiency (DLC) and EE of BO/OMV-lipo@LU were 2.16 ± 0.20% and 62.71 ± 2.73%, respectively (Fig. 1 h). SDS-PAGE analysis revealed BO/OMV-lipo@LU and OMV samples showed similar protein bands (Fig. 1 i), indicating that BO/OMV-lipo@LU inherited OMV proteins after hybridization and might possess corresponding biological functions. Next, the stability of BO/OMV-lipo@LU in PBS and gastrointestinal tract (GIT) was tested by monitoring the change of liposomes in the particle size and PDI. As demonstrated in Fig. 1 j, BO/OMV-lipo@LU exhibited excellent storage stability in PBS, as evidenced by the almost stable particle size and PDI within one week at 4 ℃. Apart from storage ability, the stability in the GIT was also important for BO/OMV-lipo@LU to reach the inflamed colon safely and exert curative effects. BO/OMV-lipo@LU presented a slightly increased size after incubation in the simulated gastric fluid (SGF) and the simulated small intestine fluid (SIF), but the change did not exceed 40 nm (Fig. 1 k). No significant size change could be observed in BO/OMV-lipo@LU incubated in the simulated colon fluid (SCF). Meanwhile, during incubation in three simulated gastrointestinal fluids, the PDI of BO/OMV-lipo@LU showed a minor increase and was still within an acceptable range (< 0.3). These results demonstrated that BO/OMV-lipo@LU exhibited favorable stability under gastrointestinal conditions, which might protect luteolin from degradation during their passage through the GIT. To further explore the drug release behavior of BO/OMV-lipo@LU in the GIT, the in vitro release study was conducted in SGF, SIF, and SCF sequentially. As depicted in Fig. 1 l, free luteolin (Free-LU) exhibited rapid release in the SGF and SIF, and the cumulative release rate had already reached over 90% before the release medium was replaced with SCF. Compared with Free-LU, BO/OMV-lipo@LU dramatically slowed down the luteolin release rate under the simulated conditions of the stomach and small intestine. It suggested that the liposome coating could prevent early luteolin release before reaching the inflamed colon. When transferred to SCF, BO/OMV-lipo@LU presented sustained drug release, and the cumulative release rate could reach 70% over 12 h, which might enhance the colonic release of luteolin from the liposomes. 2.3 BO/OMV-lipo@DiD showed promoted epithelial cell uptake in vitro In this study, The DiD fluorescent dye was used to track liposomes in the assay of cellular uptake and in vivo bio-distribution by fabricating DiD-loaded liposome formulations, including Lipo@DiD, OMV-lipo@DiD, BO-lipo@DiD and BO/OMV-lipo@DiD. These DiD-loaded liposomes were characterized by particle size, zeta potential, DLC, and EE. As demonstrated in Fig. 2 a, they possessed similar particle size (~ 120 nm) with a narrow distribution (PDI < 0.3). DLS analysis showed that the surface charge of hybrid liposomes (OMV-lipo@DiD and BO/OMV-lipo@DiD) was closer to that of OMVs in comparison with other non-hybrid liposomes, confirming the successful membrane fusion (Fig. 2 b). DLC and EE of BO/OMV-lipo@DiD were 0.25 ± 0.02% and 53.23 ± 4.28%, respectively (Fig. 2 c). Next, effects of the OMV proportion and DSPE-PEG-BO proportion on the intestinal epithelial cellular uptake abilities of BO/OMV-lipo@DiD were investigated by fluorescence imaging. Different ratios of OMV proteins to lipids (1:100, 1:50, and 1:20) were applied to fabricate OMV-lipo@DiD. As illustrated in Fig. 2 d, e, with the increase of the OMV proportion, the efficiency of liposome uptake by Caco-2 cells presented an increased trend. Amongst, OMV-lipo@DiD with the 1:20 OMV ratio dramatically enhanced the uptake efficiency of Lipo@DiD by 2.5 folds and showed the best cell internalization effects compared with those with other ratios. It revealed that the fusion of OMVs contributed to improving the ability of liposomes to target intestinal epithelial cells. Besides, the effects of the DSPE-PEG-BO proportion on the uptake efficiency of liposomes were also investigated, by applying different DSPE-PEG-BO proportions (3.3%, 6.7%, and 10%) but maintaining the fixed OMV proportion (1:20 ratio) to fabricate BO/OMV-lipo@DiD. It could be seen in Fig. 2 f, g, that there was no significant difference in relative fluorescence intensity between OMV-lipo@DiD and 3.3%-BO/OMV-lipo@DiD, revealing that the presence of DSPE-PEG-BO in a low proportion (3.3%) could not affect Caco-2 cell uptake of liposomes. Whereas, the fluorescence intensity of 10%-BO/OMV-lipo@DiD was 4 folds higher than that of OMV-lipo@DiD. It indicated that the high counterpart (10%) dramatically promoted intestinal epithelial cell uptake efficiency. Taken together, the 1:20 ratio of OMV proteins to lipids and 10% proportion of DSPE-PEG-BO were determined as the optimal ratios to formulate BO/OMV-lipo@DiD that exhibited excellent epithelial targeting ability. 2.4 BO/OMV-lipo@DiD targeted the inflamed colon and penetrated into mucosa for improved epithelial uptake in vivo The bio-distribution of BO/OMV-lipo@DiD in UC mice after oral administration was investigated by an in vivo imaging system (IVIS). At 2 h and 6 h post administration of Lipo@DiD, BO-lipo@DiD, OMV-lipo@DiD, and BO/OMV-lipo@DiD, mice were euthanized to obtain the IVIS images. Results presented that the UC mice treated with the OMV-lipo@DiD or BO/OMV-lipo@DiD group exhibited about 2-fold abdominal fluorescence intensity of those treated with Lipo@DiD after 6 h of administration. It indicated that OMV fusion significantly prolonged the retention time of liposomes in the intestinal tract, thereby retarding the intestinal clearance (Fig. 3 a, b). After administration of liposomes for 8 h, the mice were sacrificed. The visceral organs and colon tissues were collected for further fluorescence analysis. As shown in Fig. 3 c, d, visceral organs had negligible fluorescence intensity in all groups compared with colon tissues. Importantly, OMV-lipo@DiD displayed significantly greater accumulation in the inflamed colon tissue than Lipo@DiD, suggesting that the presence of the biomimetic OMVs contributed to the colon-targeting capability of liposomes in UC mice. It could be conjectured that OMVs might faithfully replicate the adhesion property of parent bacteria, thus providing excellent chemotaxis-driven UC-targeted delivery performance. Notably, the sole modification of borneol did not improve the liposome accumulation in the colon. In contrast, mice treated with BO/OMV-lipo@DiD (involving OMV fusion and borneol modification) exhibited higher colonic fluorescence intensity than those treated with OMV-lipo@DiD. The possible explanation was that OMV-induced colon retention of liposomes increased the contact time between intestinal epithelial cells and liposomes, allowing surface-modified borneol to mediate enhanced inflamed epithelial uptake. To further verify whether BO/OMV-lipo@DiD could target colon epithelial cells in vivo , frozen sections of the colon tissue were stained with anti-Epcam mouse antibodies, followed by Alexa Fluor (AF) 555-labeled anti-mouse IgG to indicate epithelial cells. The Pearson correlation coefficient (PCC) was analyzed to quantify colocalization between DiD fluorescence (indicating liposomes) and AF555 fluorescence (indicating epithelial cells). Results showed that BO-lipo@DiD and Lipo@DiD had no difference in PCC. In contrast, the PCC of OMV-lipo@DiD was higher than Lipo@DiD, signifying that OMVs promoted epithelial cell uptake of liposomes to some extent (Fig. 3 e, f). More importantly, among all groups, BO/OMV-lipo@DiD exhibited the most apparent colocalized signals with epithelial cells, demonstrating that OMVs and borneol jointly enhanced colon epithelial uptake efficiency. The excellent targeting capability of BO/OMV-lipo@DiD towards inflamed colon might be attributed to the chemotaxis-driven colon-targeting ability of OMVs and the borneol-mediated further epithelial uptake. Interestingly, under combined effects of OMVs and borneol, BO/OMV-lipo@LU achieved the most robust penetration ability into mucosa for better infiltration of liposomes in the inflamed colon. The other studies employed targeting molecules (such as anti-CD98 antibodies and hyaluronic acid) to engineer the nanosystem surface for targeting inflammatory intestinal epithelial cells and macrophages 21,22 . Notwithstanding their improved targeting ability of the inflamed colon, the penetration depth of these nanosystems was limited (only about 1/3 or less of the colonic mucosa layer). However, BO/OMV-lipo@DiD could penetrate the mucosal layer entirely (reaching the white lines as indicated in Fig. 3 e) after oral administration, indicating the good colonic mucosal permeability of BO/OMV-lipo@DiD. Previous studies reported that membrane-originated components of some specific bacteria inherited chemotaxis towards the intestine from parent bacteria, which might be achieved by specific adhesion protein-mediated adhesion to the intestinal epithelium and/or mucus layer 23,24,47–49 . Nevertheless, the inflamed colon-targeting mechanisms of OMVs derived from S.mal remained to be explored in the future. 2.5 BO/OMV-lipo@LU improved efficacy in the DSS-induced UC model The therapeutic efficacy of BO/OMV-lipo@LU on the DSS-induced UC mice model was evaluated. First, we investigated whether the ratios of OMV proteins to lipids influenced the in vivo therapeutic outcomes of BO/OMV-lipo@LU by improving the colon-targeting ability of hybrid liposomes. Mice received the drinking water containing 3% DSS for 7 days to induce the UC model and were orally administered BO/OMV-lipo@LU with various ratios of OMV proteins to lipids (including 1:20, 1:50, and 1:100), conforming to the regimen shown in Fig. 4 a. The anti-UC efficacy of BO/OMV-lipo@LU was evaluated by detecting body weight changes, disease activity index (DAI) scores, colon length and colonic pathological damage. As shown in Fig. 4 b, c, with either OMV proportion, BO/OMV-lipo@LU treatment distinctly reduced DAI scores and recovered the body weight of UC mice. More importantly, the UC mice treated with 1:20-BO/OMV-lipo@LU showed an increased colon length (Fig. 4 d, e) and less weight loss (Fig. 4 c) than those treated with 1:50-BO/OMV-lipo@LU or 1:100-BO/OMV-lipo@LU. Moreover, the 1:20-BO/OMV-lipo@LU group exhibited a lower DAI score than the 1:100-BO/OMV-lipo@LU group (Fig. 4 b). These results were consistent with the findings of the in vitro uptake assay, demonstrating that the 1:20 ratio of OMV proteins to lipids endowed BO/OMV-lipo@LU with excellent targeting ability and anti-UC efficacy. Based on the promising therapeutic results of 1:20-BO/OMV-lipo@LU, its efficacy was directly compared against Free-LU, Lipo@LU, BO-lipo@LU, and OMV-lipo@LU (with an equivalent OMV proportion). The administration regimen was shown in Fig. 5 a, and an equivalent luteolin dose (17 mg/kg) was given. As presented in Fig. 5 b, c, the colon in the BO/OMV-lipo@LU group was markedly longer than that in other treatment groups, and there existed no difference in colon length between the control group and the BO/OMV-lipo@LU group. Moreover, BO/OMV-lipo@LU treatment significantly reduced the DAI score of UC mice (Fig. 5 d). At the end of the experiment, mice treated with BO/OMV-lipo@LU and OMV-lipo@LU recovered the body weight basically, while other control treatment groups failed to achieve full body weight recovery (Fig. 5 e). Thereafter, to evaluate the colonic pathological change, the colon tissue was stained by hematoxylin and eosin (H&E), followed by the histological scoring. The results showed that the BO/OMV-lipo@LU group had significantly lower colonic histological damage scores compared with the DSS group (P < 0.01, Fig. 5 g). BO/OMV-lipo@LU tremendously alleviated DSS-induced colonic damage characterized by inflammatory cell infiltration, damaged crypt structure, and decreased goblet cell number (Fig. 5 f). In contrast, apparent colonic damage could still be observed in other groups treated with Free-LU, Lipo@LU or BO-lipo@LU. These results indicated that the OMV hybridization and borneol modification were necessary to achieve effective anti-UC treatment. Notably, it was previously reported that the treatment with a high oral dose of free luteolin (100 mg/kg) could exhibit marked therapeutic effects on UC, while the low counterpart (50 mg/kg) had little effects on it 50 . However, our results demonstrated that luteolin in low-dose (17 mg/kg) could be delivered effectively by the biomimetic hybrid liposomes to inflamed colon sites, thereby enough to exert significant anti-UC efficacy. It would seem that BO/OMV-lipo@LU treatment with 17 mg/kg luteolin dose even exerted a more robust efficacy than 100 mg/kg free luteolin treatment. 2.6 BO/OMV-lipo@LU alleviated inflammation, modulated redox balance, and recovered dysregulated intestinal barriers Intrigued by these promising therapeutic results, the underlying action mechanisms of BO/OMV-lipo@LU on UC were further studied. The UC flare tends to be accompanied by dysbiosis of intestinal homeostasis involving multiple pathological mechanisms, including inflammation factor infiltration, excessive oxidative stress, and disrupted epithelial barrier function. Previous experimental evidence indicated that luteolin could affect various pathological aspects of UC based on its action mode of multichannels and multitargets, thereby turning the intestinal microenvironment towards a beneficial state 33–36 . Accordingly, the role of BO/OMV-lipo@LU in regulating intestinal homeostasis was investigated by detecting its ability to suppress inflammation, alleviate oxidative stress, and repair the intestinal epithelial barrier. The local and systemic infiltration of inflammatory factors is one of the primary pathological elements controlling UC initiation and progression. Thus, the levels of inflammatory factors in the serum and colon tissues were determined by enzyme-linked immunosorbent assay (ELISA). BO/OMV-lipo@LU treatment not only significantly inhibited the local level of inflammatory factors in the colon tissues, including tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6), but reduced the systemic content of pro-inflammatory interferon-γ (IFN-γ) and IL-6 (Fig. 6 a-d). The level of these inflammatory factors in the mice treated with BO/OMV-lipo@LU nearly returned to the normal range, whereas other treatment groups did not efficiently suppress inflammatory factor infiltration. Besides, as shown in Fig. 6 e, reactive oxygen species (ROS), such as superoxide anion radicals (O 2 − ), hydrogen peroxide (H 2 O 2 ), and hypochlorous acid (HOCl), rapidly increase in the intestinal microenvironment of UC mice. It leads to excessive oxidative stress, which is recognized as a vital pathological mechanism of UC, followed by severe intestinal lesions. Therefore, reducing ROS levels is necessary to retard UC progression 51 . In this study, the redox-related indicators were detected to examine the ability of BO/OMV-lipo@LU to regulate redox balance in the intestinal microenvironment. The protective effects of BO/OMV-lipo@LU in UC mice were reflected in the elevated activity of superoxide dismutase (SOD) (Fig. 6 f), which was an anti-oxidant metalloenzyme that catalyzed the disproportionation of O 2 − to O 2 and H 2 O 2 . Additionally, BO/OMV-lipo@LU treatment markedly suppressed the activity of myeloperoxidase (MPO) (Fig. 6 g), which generated HOCl from H 2 O 2 in the presence of Cl − and consequently contributed to inflammation and intestinal oxidative damage. Furthermore, glutathione (GSH) was also an anti-oxidant indicator since GSH could reduce H 2 O 2 to water, and BO/OMV-lipo@LU treatment significantly induced an upregulated GSH level in the colon tissue (Fig. 6 h). However, other control treatment groups did not exhibit apparent anti-oxidant performance. These results indicated that BO/OMV-lipo@LU dramatically decreased ROS levels and alleviated intestinal oxidative stress in the colon tissues of UC mice. Given the protection role of the intestinal epithelial barrier in intestinal homeostasis, the expression of tight junction proteins, including ZO-1 and occludin, was assessed to explore the effects of BO/OMV-lipo@LU on colonic epithelial restoration. As shown in Fig. 6 i-k, Free-LU, Lipo@LU, and BO-lipo@LU treatments had minimal impact on ZO-1 and occludin expression. However, the UC mice treated with OMV-lipo@LU or BO/OMV-lipo@LU significantly recovered the expression of tight junction proteins to different extents. These two groups presented similar ZO-1 levels, while the BO/OMV-lipo@LU group had a higher level of occludin, indicating BO/OMV-lipo@LU exerted the most excellent effects on restoring the intestinal epithelial barrier among all treatment groups. Taken together, BO/OMV-lipo@LU trilogically recovered intestinal homeostasis in UC by reducing inflammatory factor levels, alleviating oxidative stress, and repairing the intestinal epithelial barrier, which suggested BO/OMV-lipo@LU could protect animals against UC in an effective manner. 2.7 BO/OMV-lipo@LU showed favorable biosafety The toxicity of BO/OMV-lipo@LU against five major organs was assessed by H&E staining. Results showed that no apparent histological abnormality was observed in the H&E staining sections following the oral administration of BO/OMV-lipo@LU (Fig. 6 l). In addition, the organ indexes of major organs (including heart, liver, spleen, lung, and kidney) were examined for systemic toxicity evaluation. As shown in Fig. 6 m, DSS-induced UC mice were observed with obviously increased organ indexes of the liver and spleen. However, the abnormal hepatosplenomegaly could be alleviated by the BO/OMV-lipo@LU treatment, and the other organ indexes in the BO/OMV-lipo@LU group were close to that in the healthy control group. These results indicated favorable systemic biosafety of BO/OMV-lipo@LU under the current treatment conditions. 3. Conclusions In this study, we constructed a novel chemotaxis-driven biomimetic delivery system for UC-targeted therapy by fusing liposomes with OMVs and functionalizing liposome surfaces with borneol. Benefitting from the chemotaxis-driven colon-targeting ability of OMVs and intestinal epithelial uptake promotion of borneol, BO/OMV-lipo@DiD exhibited excellent targeting capability towards the inflamed colon. Interestingly, under combined effects of OMVs and borneol, BO/OMV-lipo@LU achieved the most robust penetration ability into mucosa for better infiltration of liposomes in the inflamed colon. Thus, the liposomes were designed to achieve UC-targeted delivery of luteolin, a natural flavonoid compound that could exert multiple effects on UC. We found BO/OMV-lipo@LU distinctly alleviated the pathological symptoms of DSS-induced UC mice. Significantly, BO/OMV-lipo@LU trilogically restored intestinal homeostasis in UC by alleviating intestinal inflammation, modulating redox balance, and restoring intestinal epithelial barriers. During the treatment, no apparent toxicity was observed in mice, demonstrating the favorable biosafety of BO/OMV-lipo@LU. This study suggests BO/OMV-lipo@LU are promising nanoplatforms for safe and effective gut-targeted delivery in UC intervention. 4. Experimental section 4.1 Materials, cell lines, and animals Soybean lecithin, cholesterol, and DSPE-mPEG(2000) were purchased from AVT Pharmaceutical Tech Co., Ltd. (China). (R)-(+)-bornylamine was purchased from Sigma-Aldrich Co. (USA). DSPE-PEG(2000)-NHS was purchased from Shanghai Ponsure Biotech, Inc. (China). Pierce BCA Protein Assay Kit was purchased from Thermo Fisher Scientific (USA). Luteolin (98% purity) was purchased from Chengdu Herbpurify Co., Ltd. (China). RIPA lysis buffer, protease inhibitor cocktail, phosphatase inhibitor cocktail, and phenylmethanesulfonyl fluoride (PMSF) were purchased from Beyotime (China). 4% Paraformaldehyde was purchased from Shanghai Biotend Biotech Co., Ltd. (China). Difco™ Nutrient Broth ( Cat. No. 234000 ) was purchased from BD Bioscience (USA). DiD dye was purchased from Dalian MeiLun Biotechnology Co., Ltd. (China). DAPI was purchased from MedChemExpress (USA). DSS was purchased from Yeasen Biotechnology (China). The fecal occult blood detection kit and the GSH assay kit were purchased from Nanjing Jiancheng (China). The MPO activity assay kit and the SOD activity assay kit were purchased from Beijing Solarbio Science & Technology Co., Ltd (China). The mouse IFN-γ ELISA kit, the TNF-α ELISA kit, and the IL-6 ELISA kit were purchased from Shanghai Jianglai Industrial Limited By Share Ltd. (China). The anti-Epcam mouse antibody was purchased from Hangzhou HuaAn Biotechnology Co., Ltd. (China). The anti-mouse IgG conjugated with AF555 was purchased from Cell Signaling Technology (USA). The anti-ZO-1 rat monoclonal antibody (sc-33725) was purchased from Santa Cruz (USA). The Horseradish peroxidase (HRP)-labeled goat anti-rat IgG (GB23302) was purchased from Wuhan Servicebio Technology Co., Ltd. (China). The anti-occludin rabbit monoclonal antibody (ab216327) was purchased from Abcam (UK). The HRP-labeled goat anti-rabbit IgG (RCA054), the TYR-570Plus red dye, and the TYR-520Plus green dye were purchased from Refinebio (China). Caco-2 cells (Human epithelial colorectal adenocarcinoma cells) were purchased from Cell Bank of Chinese Academy of Sciences (China). They were cultured with Dulbecco’s modified Eagle’s medium (DMEM; Meilunbio, China) containing 10% (v/v) fetal bovine serum (FBS; ExCell Bio., China) and 1% penicillin/streptomycin (Biosharp, Beijing Lanjieke Technology Co., Ltd., China) at 37°C in 5% CO 2 . Female C57BL/6J mice (18 ~ 20 g) were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. (China). All animal work complied with the protocols approved by Institutional Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine (IACUC, No: PZSHUTCM2303240001). All animal experiments were conducted following the “3R” principle (reduction, replacement, refinement). 4.2 Isolation and characterization of bacterial OMVs The isolation of bacterial OMVs from S.mal. was conducted using the ultracentrifugation method reported previously with minor modifications 52,53 . S.mal. was inoculated into Nutrient Broth medium and cultured in a rotary shaker (220 rpm) at 32℃ for 12 h. Then, the bacterial suspension was diluted 1:100 with fresh medium and cultured for another 12 h. Next, the bacterial medium was centrifuged at 4℃, 4000 g for 10 min to remove S.mal. , followed by filtration with a 0.22-µm vacuum filter (Beyotime, China). Thereafter, the filtrate was concentrated using an ultrafiltration tube (MWCO 100 kDa, Millipore). The concentrated solution was ultracentrifuged at 20,0000 g, 4℃ for 3 h with SW 70 Ti rotor (Beckman coulter, USA) to obtain OMV pellets, which were suspended finally in PBS and stored at -80℃ for further use. The size distribution and zeta potential of OMVs were detected by a NICOMPTM 380 ZLS Zeta Potential/Particle Sizer (PSS, USA) using DLS. The morphology of OMVs was characterized by 120 kV TEM (FEI, USA). Briefly, the OMV sample was dropped onto 300-mesh carbon-coated copper grids. Following incubation for 1 min, the sample was removed from the grid, which was then stained with uranyl acetate for another 1 min and dried before TEM analysis. In addition, the OMV protein content was measured by a BCA protein assay kit, following the lysis of OMV samples in RIPA buffer supplemented with protease and phosphatase inhibitors. 4.3 Synthesis and characterization of DSPE-PEG-BO DSPE-PEG-BO was synthesized through NHS easter reaction by conjugating the amino group of (R)-(+)-bornylamine (an amino derivative of borneol) and DSPE-PEG-NHS easter. Briefly, DSPE-PEG-NHS (100 mg, 1 equiv.) was dissolved in chloroform and stirred at 300 rpm, followed by rapidly adding (R)-(+)-bornylamine (5.6 mg, 1.1 equiv.) and N, N-Diisopropylethylamine (DIPEA). The mixture was stirred under a nitrogen atmosphere for 24 h at room temperature. Then, the product was purified by immersing the mixture in the ether at -20℃ for 3 ~ 4 h and collecting the precipitate by centrifuging at 5000 g, 4℃ for 30 min. The purification procedure was conducted three times, and the final powder product of DSPE-PEG-BO was obtained through further lyophilization. The structure of DSPE-PEG-BO was characterized by the 600 MHz 1 H-NMR spectrometry (Bruker, Germany). 4.4 Preparation of luteolin-loaded liposomes In this study, four kinds of luteolin-loaded liposome formulations were constructed, including liposomes without any modification (Lipo@LU), single borneol-modified liposomes (BO-lipo@LU), single OMV-mimetic hybrid liposomes (OMV-lipo@LU), and borneol-modified OMV-mimetic hybrid liposomes (BO/OMV-lipo@LU). Lipo@LU were initially fabricated using the thin film hydration method 43,54 . Firstly, a mixture of soybean lecithin, cholesterol, DSPE-mPEG, and luteolin with different weights was placed in a round-bottom flask and ultrasonically dissolved in the organic solvent. Subsequently, the solvent was evaporated entirely under reduced pressure using a rotary evaporator at 45℃ and 125 rpm for 20 min until a thin-lipid film was formed. Then, PBS was added to hydrate the film for 10 min to obtain multilamellar liposomes, followed by probe-sonicating (Ningbo Scientz Biotechnology Co., Ltd., China) at 25% power (2 s pulse on/4 s pulse off) for 5 min to acquire unilamellar liposomes. Next, the suspension was filtered through a 0.45-µm filter to remove large particles, and Lipo@LU was ultimately obtained by extrusion through 400 and 200 nm polycarbonate membranes 9 times using a mini extruder (Avestin, Canada), respectively. Any unentrapped luteolin was removed by ultrafiltration (MWCO 10 kDa, Millipore). To optimize the formulation of Lipo@LU, the single-factor test was performed to investigate effects of the organic solvent type (a 9:1 chloroform-methanol mixture or ethanol), the ratio of soybean lecithin to cholesterol (3:1, 4:1 or 6:1), hydration temperature (25℃ or 37℃) and luteolin dosage (1.2 mg, 2 mg or 3 mg) on the particle size and encapsulation efficiency of Lipo@LU. Based on the formulation mentioned above of Lipo@LU, BO/OMV-lipo@LU were prepared by replacement of DSPE-mPEG with DSPE-PEG-BO and membrane fusion with OMVs. Briefly, DSPE-PEG-BO was added to lipid materials to form liposomes with targeting moieties of borneol (BO-lipo@LU). Additionally, OMVs were mixed with hydration suspension containing liposomes and then were extruded to induce membrane fusion and generate OMV-mimetic hybrid liposomes (OMV-lipo@LU). BO/OMV-lipo@LU was synthesized by combining these two processes. 4.5 Characterization of luteolin-loaded liposomes 4.5.1 Particle size, zeta potential, morphology, and storage stability The size distribution and zeta potential of luteolin-loaded liposomes were detected by a NICOMPTM 380 ZLS Zeta Potential/Particle Sizer. The morphology of these liposomes was observed by 120 kV TEM. To evaluate the storage stability at 4 ℃ within one week, their particle size and PDI were detected and recorded daily. 4.5.2 Encapsulation efficiency and drug loading capability The EE and DLC of luteolin-loaded liposomes were calculated using Eqs. ( 1 ) and ( 2 ). The content of luteolin entrapped in the liposomes was determined with high performance liquid chromatography (HPLC, Shimadzu, Japan). The HPLC analysis for determining luteolin was conducted with an Agilent ZORBAX SB-C18 column (250 mm × 4.6 mm), mobile phase of the mixture of acetonitrile and 0.2% phosphoric acid (33:67, v/v), flow rate of 1 mL/min, column temperature of 25℃, injection volume of 20 µL, and ultraviolet (UV) detection wavelength of 350 nm. Prior to HPLC analysis, liposomes were mixed with methanol by vigorous vortexing (1 min) and sonication in a bath sonicator (10 min, Shanghai Titan, China), followed by centrifuging at 10,012 g for 10 min to collect the supernate. Additionally, the total dry weight of liposomes was measured to calculate DLC after liposomes were freeze-dried for 24 h. $$\:\text{E}\text{E}（\text{%}）=\text{M}\text{o}\text{u}\text{n}\text{t}\:\text{o}\text{f}\:\text{e}\text{n}\text{c}\text{a}\text{p}\text{s}\text{u}\text{l}\text{a}\text{t}\text{e}\text{d}\:\text{l}\text{u}\text{t}\text{e}\text{o}\text{l}\text{i}\text{n}/\text{M}\text{o}\text{u}\text{n}\text{t}\:\text{o}\text{f}\:\text{a}\text{d}\text{d}\text{e}\text{d}\:\text{l}\text{u}\text{t}\text{e}\text{o}\text{l}\text{i}\text{n}\:\times\:100\%$$ 1 $$\:\text{D}\text{L}\text{C}（\text{%}）=\text{M}\text{o}\text{u}\text{n}\text{t}\:\text{o}\text{f}\:\text{e}\text{n}\text{c}\text{a}\text{p}\text{s}\text{u}\text{l}\text{a}\text{t}\text{e}\text{d}\:\text{l}\text{u}\text{t}\text{e}\text{o}\text{l}\text{i}\text{n}/\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\:\text{o}\text{f}\:\text{l}\text{i}\text{p}\text{o}\text{s}\text{o}\text{m}\text{e}\text{s}\times\:100\%$$ 2 4.5.3 SDS-PAGE protein analysis SDS-PAGE analysis was employed to characterize proteins. The OMVs, BO/OMV-lipo@LU, and Lipo@LU samples were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors, mixed with loading buffer, and then incubated at 95℃ for 10 min. Each sample (10 µg) was loaded in the wells of a Bio-Rad electrophoresis system (Bio-Rad Laboratories, USA). To visualize the protein profile, the gel was stained with Coomassie Brilliant Blue G-250 for 15 min and destained for 1 h, and imaged using a Bio-Rad gel imaging system. 4.5.4 Stability and in vitro drug release in simulated gastrointestinal fluids Simulated gastrointestinal fluids included SGF (pH 1.2, consisting of HCl and pepsin), SIF (pH 6.8, consisting of KH 2 PO 4 , NaOH, and trypsin), and SCF (pH 7.8, comprising KH 2 PO 4 and K 2 HPO 4 ), which were used to simulate the physiological conditions in the stomach, small intestine and colon regions, respectively. To investigate the stability of luteolin-loaded liposomes in the gastrointestinal fluids, their particle size and PDI were determined after the liposome samples were incubated in SGF, SIF, and SCF, respectively, at 37℃ for 2 h. The in vitro release properties of luteolin-loaded liposomes were investigated via dialysis. To mimic the environment of liposomes during gastrointestinal passage in vivo , the selection of release medium applied a three-stage method according to previous studies 55,56 . Briefly, Free-LU, Lipo@LU, BO-lipo@LU, OMV-lipo@LU, and BO/OMV-lipo@LU were put in dialysis bags (MWCO 8 ~ 14 kDa, Shanghai Yuanye Bio-Technology, China), respectively. Then, the dialysis bags were fastened and immersed in the release medium in an order of SGF, SIF, and SCF, which contained 0.5% Tween-80. The experiment was performed by a shaker at 37 ℃ and 150 rpm. The samples of release medium were collected at predetermined time points, and the same volume of fresh medium was immediately replenished. Afterward, the luteolin content in the samples diluted with methanol was determined by HPLC analysis to calculate the cumulative release of luteolin. 4.6 Preparation and characterization of DiD-loaded liposomes Aiming to investigate the cell internation and in vivo bio-distribution of liposomes, DiD dye was encapsulated into liposomes as a fluorescent marker. The preparation process of DiD-loaded liposomes was consistent with that of luteolin-loaded liposomes using the thin-film hydration method, except that those lipid materials and DiD dye were dissolved in chloroform rather than ethanol. The particle size, zeta potential, DLC, and EE of Lipo@DiD, OMV-lipo@DiD, BO-lipo@DiD, and BO/OMV-lipo@DiD were determined as previously mentioned in 4.5.1 and 4.5.2. The DiD concentration was qualified by determining the fluorescence intensity with a microplate reader (Tecan, Switzerland). 4.7 In vitro epithelial uptake assay The cellular uptake ability of BO/OMV-lipo@LU was investigated in the Caco-2 cell monolayer model. To observe effects of the OMV proportion on the uptake efficiency of liposomes, three ratios of OMV protein to lipids (1:100, 1:50, and 1:20) were applied to prepare hybrid liposomes, respectively, for subsequent cellular uptake experiments. Caco-2 cells were seeded on 24-well plates at a density of 10 4 cells/well and incubated with Lipo@DiD and hybrid liposomes with various OMV proportions (including 1:100-OMV-lipo@DiD, 1:50-OMV-lipo@DiD, and 1:20-OMV-lipo@DiD) at 37 ℃ for 1 h. Then, the cells were washed with PBS three times and fixed for 10 min using paraformaldehyde, followed by DAPI (10 µg/mL) staining for 10 min. Cellular uptake imaging was acquired using a fluorescence microscope (Leica, Germany), and the fluorescence intensity was semiquantified using Image J 1.53t (USA). In addition, to investigate the effects of the DSPE-PEG-BO proportion on uptake efficiency, Caco-2 cells were incubated with liposomes modified with DSPE-PEG-BO of different proportions (of lipids, 3.3%, 6.7%, 10%). Briefly, Caco-2 cells were treated with OMV-lipo@DiD and BO/OMV-lipo@DiD with various DSPE-PEG-BO proportions (including 3.3%-BO/OMV-lipo@DiD, 6.7%-BO/OMV-lipo@DiD, 10%-BO/OMV-lipo@DiD). Then, the cells were washed, fixed, stained with DAPI, and observed by the fluorescence microscope to visualize the epithelial uptake. 4.8 DSS-induced UC model Female C57BL/6J mice (18 ~ 20 g) were cohoused in cages under specific pathogen-free (SPF) conditions of a 12-h light/dark cycle and acclimatized for a week before random allocation to experimental groups. To establish a DSS-induced UC model, the mice received 3% DSS (w/v) supplemented in drinking water for 7 days 23,57 . 4.9 In vivo imaging To investigate the targeting ability of BO/OMV-lipo@LU to the inflamed colon, UC mice were divided into 4 groups and treated orally with Lipo@DiD, BO-lipo@DiD, OMV-lipo@DiD and BO/OMV-lipo@DiD, respectively, at an equivalent DiD dose of 1.25 mg/kg). At the predetermined time points, in vivo fluorescent images were observed with the IVIS (Perkinelmer, USA) after the mice were anesthetized using isoflurane. After 8 h of administration, the mice were euthanized. The main organs (including the heart, liver, spleen, lung, kidney, and colon) were excised, followed by the collection of fluorescence images via the IVIS. The fluorescence intensity analysis was performed by the IVIS and living image software (version 4.4). 4.10 In vivo immunofluorescence imaging The colon tissue was prepared into frozen sections for immunofluorescence staining to further analyze the colocalization of BO/OMV-lipo@DiD and colon epithelial cells. Specifically, the tissues were fixed in 4% paraformaldehyde overnight and dehydrated in 30% sucrose for 24 h. Then, the tissues were embedded in OCT (optimal cutting temperature), stored at -80 ℃, and cut into slices (slice thickness 10 µm) by a freezing microtome (Leica, Germany). Next, the colon frozen sections were stained with Anti-Epcam mouse antibodies (1:100) and subsequent anti-mouse IgG conjugated with AF555 (1:1000), followed by counter staining with DAPI. The fluorescence images were observed with a laser scanning confocal microscope (SP8, Leica, Germany), and the Pearson correlation coefficient (PCC) was analyzed using Image J. 4.11 Treatment of DSS-induced UC model For exploring effects of the OMV proportion on the anti-UC efficacy of BO/OMV-lipo@LU, the UC mice model was induced by 3% DSS for 7 days and orally treated with 1:20-BO/OMV-lipo@LU, 1:50-BO/OMV-lipo@LU or 1:100-BO/OMV-lipo@LU. Amongst, the proportion referred to the ratio of OMV proteins to lipids in BO/OMV-lipo@LU. All formulation administration (luteolin dose of 17 mg/kg) was performed on predetermined days complying with the treatment regimen as depicted in Fig. 5 a. The healthy mice were provided with normal water as a control. In the therapeutic experiments, C57BL/6J mice were randomly divided into 7 groups, including i) the healthy control group, ii) the DSS control group, iii) the Free-LU-treated DSS group, iv) the Lipo@LU-treated DSS group, v) the BO-lipo@LU-treated DSS group, vi) the OMV-lipo@LU-treated DSS group, and vii) the BO/OMV-lipo@LU-treated DSS group. According to the treatment regimen in Fig. 6 a, all formulations (equivalent luteolin dose of 17 mg/kg) were administrated once daily for 5 days, starting on the third day of DSS treatment. After 7 days of DSS treatment, mice received normal water without DSS for 3 days and were euthanized. During the experimental periods, the body weight, stool consistency, and fecal blood of mice were monitored daily. The DAI score was calculated based on the summation of body weight loss (0: 0%, 1: 1–5%,2: 6–10%, 3: 11–15%, 4: 15%), stool consistency state (0: hard, 1: soft, 3: diarrhea), and fecal occult blood (0: negative, 1: positive, 3: macroscopic) 56 . The fecal occult blood detection kit was used to test the degree of occult blood by the benzidine method. At the end of the experiments, the colon tissue was collected and measured for length. Then, the colon was washed with PBS to remove feces, and a segment from each group was fixed with 4% paraformaldehyde for histological analysis and immunofluorescence staining assays. Additionally, the remaining samples were stored at -80℃ for further use. H&E staining sections of colon tissue were prepared for histological assessment through further hydration and paraffin embedding. The colonic damage severity was scored conforming to a previously described scoring criterion as follows: inflammation cell infiltration (0: few inflammatory cells in the lamina propria, 1: enhanced granulocyte infiltration into the lamina propria, 2: extending into submucosa, 3: extending into muscular and serosal layer), crypts lesion (0: intact crypts, 1: loss of the basal one-third, 2: loss of the basal two-thirds, 3: entire crypt loss, 4: alternation of epithelial surface with erosion, 5: confluent erosion) and ulceration (0: absence of ulceration, 1: 1 ~ 2 foci of ulcerations, 2: 3 ~ 4 foci of ulcerations, 3: confluent ulceration) 58 . 4.12 In vivo ELISA analysis of inflammatory factors To determine the concentration of cytokines in serum, mice blood was collected in the procoagulant tubes by eyeball extraction and centrifuged at 1000 g for 20 min to isolate serum. In addition, the cytokine concentration in colon tissues was also detected using the tissue homogenate sample. The colon segments were cryogenically homogenized (60 Hz, 4 min) in PBS containing 1% PMSF by a tissue grinder (Shanghai Jingxin Industrial Development Co., Ltd., China). Subsequently, the homogenized solution was centrifuged at 10,000 g for 10 min to obtain supernatant for further detection. Following the preparation of serum and colon tissue samples, the levels of cytokines, including IL-6, TNF-α, and IFN-γ, were quantified by ELISA assay kits according to the manufacturer's instructions. 4.13 In vivo MPO, GSH, and SOD activity assay For testing the ability of BO/OMV-lipo@LU to alleviate intestinal oxidative stress, the colon tissue homogenate of mice was prepared for MPO, SOD, and GSH activity analysis. According to the manufacturer’s instructions, the activity of MPO, SOD, and GSH was tested using an MPO activity assay kit, a SOD activity assay kit, and a GSH assay kit, respectively. 4.14 In vivo immunofluorescence imaging of ZO-1 and occludin The immunofluorescence staining method was employed to detect the expression of tight junction proteins (ZO-1 and occludin) in the colon epithelium. Specifically, the paraffin sections of colon tissue were prepared for multiplex immunofluorescence staining based on the method of tyramide signal amplification (TSA). The sections were dewaxed and placed in EDTA alkaline buffer for antigen repair, followed by 3% H 2 O 2 to block endogenous peroxidase. Then, the tissue sections were blocked using blocking buffer to avoid nonspecific binding and stained with anti-ZO-1 rat monoclonal antibodies (1:50), followed by HRP-labeled goat anti-rat IgG (1:200) and TSA buffer containing the TYR-570Plus red dye. Next, the antibodies were eluted in the citrate antigen repair buffer (pH 6.0). The sections were subsequently reblocked and restained with anti-occludin rabbit monoclonal antibodies (1:200), HRP-labeled goat anti-rabbit IgG, and TSA buffer containing the TYR-520Plus green dye. Nuclei were counter-stained with DAPI. Immunofluorescence images were acquired by confocal laser-scanning microscopy. 4.15 In vivo safety test Once mice were sacrificed, the main organs (including heart, liver, spleen, lung, and kidney) were separated and weighed to calculate the organ index using the following formula: $$\:\text{O}\text{r}\text{g}\text{a}\text{n}\:\text{i}\text{n}\text{d}\text{e}\text{x}\:\left(\text{%}\right)=\text{O}\text{r}\text{g}\text{a}\text{n}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}/\text{b}\text{o}\text{d}\text{y}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\:\text{o}\text{f}\:\text{m}\text{i}\text{c}\text{e}\times\:100\text{\%}$$ 3 Then, these organs were fixed in 4% paraformaldehyde and embedded in paraffin. The tissues were sliced, stained with H&E, and observed under a microscope for histological assessments. 4.16 Statistical analysis All data were expressed as Mean ± SD. Statistical analysis was performed using GraphPad Prism software (Version 9.0). The two-tailed T-test and one-way ANOVA were applied to statistically analyze all data. The data were collected from at least three independent measurements. Statistical significance was defined as * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. ns indicated that there is no significant difference. Declarations Competing interests The authors declare no competing interests. Author contributions Xier Pan conceptualized, designed, and performed the experiments, analyzed and interpreted the data, wrote the original draft, and revised the manuscript. Yushu Li, Xiao Zhao, Jiaxin Zhang, Peng Xian, Yangjie Song, Yunrong Nan performed some experiments. Shuting Ni revised the manuscript. Kaili Hu conceptualized and designed the research, provided the resources and funding, supervised the experiments, and revised the manuscript. All authors have approved the final version of the manuscript for publication. Acknowledgments This work was supported by “Shuguang Program” supported by Shanghai Education Development Foundation and Shanghai Municipal Education Commission (22SG41), the National Natural Science Foundation of China (Grant No. 81773909), Shanghai Municipal Commission of Health and Family Planning (Grant No. 2017YQ060, China), and the combination of the medical care and health project of the Shanghai University of Traditional Chinese Medicine (YYKC-2021-01-008). We thank the staff members of the Integrated Laser Microscopy System at the National Facility for Protein Science in Shanghai (NFPS), Shanghai Advanced Research Institute, Chinese Academy of Sciences, China for instrument guidance. Data availability The data related to this study are available from the corresponding author upon reasonable request. References Ng SC et al (2017) Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century: a systematic review of population-based studies. 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Proc Natl Acad Sci U S A 115:E2706–E2715 Vukelić I, Detel D, Batičić L, Potočnjak I, Domitrović R (2020) Luteolin ameliorates experimental colitis in mice through ERK-mediated suppression of inflammation, apoptosis and autophagy. Food Chem Toxicol 145:111680 E N, Vm D (2011) Mitochondrial reactive oxygen species drive proinflammatory cytokine production. J Exp Med 208 Noh I et al (2022) Cellular Nanodiscs Made from Bacterial Outer Membrane as a Platform for Antibacterial Vaccination. ACS Nano. 10.1021/acsnano.2c08360 Li M et al (2020) Chemotaxis-driven delivery of nano-pathogenoids for complete eradication of tumors post-phototherapy. Nat Commun 11:1126 Li L et al (2022) Exosome-liposome hybrid nanoparticle codelivery of TP and miR497 conspicuously overcomes chemoresistant ovarian cancer. J Nanobiotechnol 20:50 Sun L, Ouyang J, Zeng F, Wu S (2022) An AIEgen-based oral-administration nanosystem for detection and therapy of ulcerative colitis via 3D-MSOT/NIR-II fluorescent imaging and inhibiting NLRP3 inflammasome. Biomaterials 283:121468 Zhang J et al (2022) Two-birds-one-stone’ colon-targeted nanomedicine treats ulcerative colitis via remodeling immune microenvironment and anti-fibrosis. J Nanobiotechnol 20:389 Luo R et al (2020) Genipin-crosslinked human serum albumin coating using a tannic acid layer for enhanced oral administration of curcumin in the treatment of ulcerative colitis. Food Chem 330:127241 Laroui H et al (2014) Targeting intestinal inflammation with CD98 siRNA/PEI-loaded nanoparticles. Mol Ther 22:69–80 Schemes Scheme 1 is available in the Supplementary Files section Additional Declarations There is NO Competing Interest. Supplementary Files Scheme1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4710969","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":333103284,"identity":"9fa7b048-3ddf-49e8-85b0-81ecd8b0e50e","order_by":0,"name":"Kaili Hu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYBACCQbGxgMJDAcY+JmZDz8gVksDWItkO1uaAZFaGIDqgcjgPI+CBFFaJNsPNxx4wHAncfNhHgYDhhqbaIJapHkSQQ57lrjtMC9Q67G03AZCWuQYgFoS/x0GauFLMGBsOEyEFv6HIFsOJ25u5jGQIEqLtEQiRMsGZmK1SM6A2GI84zAwkBOI8YvE+fSHD38wHJbt7z98+MGHGhvCWlBBAmnKR8EoGAWjYBTgAgD5q0WWeRCpHgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-0269-909X","institution":"Shanghai University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Kaili","middleName":"","lastName":"Hu","suffix":""},{"id":333103285,"identity":"63e63157-3bae-4041-b17d-4b0bc063309a","order_by":1,"name":"Xier Pan","email":"","orcid":"https://orcid.org/0009-0003-5147-8485","institution":"Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xier","middleName":"","lastName":"Pan","suffix":""},{"id":333103286,"identity":"1069b9af-bc27-41bd-be90-a7351bbd239e","order_by":2,"name":"Yushu Li","email":"","orcid":"","institution":"Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yushu","middleName":"","lastName":"Li","suffix":""},{"id":333103287,"identity":"4578fa4e-ac08-45d6-86cb-56066b8d73a8","order_by":3,"name":"Xiao Zhao","email":"","orcid":"","institution":"Shanghai University of Traditional Chinese Medicine; Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Zhao","suffix":""},{"id":333103288,"identity":"bf4c703c-0901-4f70-a6bd-33aac9a3b2f9","order_by":4,"name":"Jiaxin Zhang","email":"","orcid":"","institution":"Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jiaxin","middleName":"","lastName":"Zhang","suffix":""},{"id":333103289,"identity":"7e15e543-a180-4916-822b-0ca88a7aaa9b","order_by":5,"name":"Peng Xian","email":"","orcid":"","institution":"Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Xian","suffix":""},{"id":333103290,"identity":"727b9fdc-498f-4198-b5ff-4751d308f6c6","order_by":6,"name":"Yangjie Song","email":"","orcid":"","institution":"Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yangjie","middleName":"","lastName":"Song","suffix":""},{"id":333103291,"identity":"868f851b-4c5a-409d-8fd9-67efc6b9a724","order_by":7,"name":"Yunrong Nan","email":"","orcid":"","institution":"Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yunrong","middleName":"","lastName":"Nan","suffix":""},{"id":333103292,"identity":"f8d4e665-7234-46c5-ba49-0ff01a008ecd","order_by":8,"name":"Shuting Ni","email":"","orcid":"","institution":"Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shuting","middleName":"","lastName":"Ni","suffix":""}],"badges":[],"createdAt":"2024-07-09 09:45:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4710969/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4710969/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61381970,"identity":"1d622e5b-4d5b-4dae-8c9c-454c4b3a44c0","added_by":"auto","created_at":"2024-07-30 06:03:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":558719,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePreparation and characterization of BO/OMV-lipo@LU.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003e The synthetic route of DSPE-PEG-BO and the \u003csup\u003e1\u003c/sup\u003eH nuclear magnetic resonance spectra of (R)-(+)-bornylamine, DSPE-PEG-NHS and DSPE-PEG-BO. \u003cstrong\u003e(b)\u003c/strong\u003e Size distribution of OMVs from \u003cem\u003eStenotrophomonas maltophilia\u003c/em\u003e by analysis of dynamic light scattering. \u003cstrong\u003e(c)\u003c/strong\u003e The transmission electron microscopy (TEM) image of OMVs (scale bar = 25 nm). White arrows indicated the phospholipid bilayer. \u003cstrong\u003e(d) \u003c/strong\u003eFormulation optimization of Lipo@LU prepared with the thin-film dispersion method. The effects of the organic solvent type, the ratio of soybean lecithin to cholesterol, hydration temperature, and luteolin dosage on the particle size (\u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) and encapsulation efficiency (EE) (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) of Lipo@LU were investigated. n = 3, \u003csup\u003e#\u003c/sup\u003eas compared to the particle size and \u003csup\u003e*\u003c/sup\u003eas compared to EE. ns indicated there is no significant difference in both the particle size and EE. \u003cstrong\u003e(e)\u003c/strong\u003e The TEM image of Lipo@LU and BO/OMV-lipo@LU (scale bar = 30 nm). \u003cstrong\u003e(f-h)\u003c/strong\u003e Particle size distribution (f), zeta potentials (g), drug loading capacity (DLC), and EE (h) of Lipo@LU, BO-lipo@LU, OMV-lipo@LU and BO/OMV-lipo@LU. n = 3. \u003cstrong\u003e(i)\u003c/strong\u003e SDS-PAGE analysis of total membrane proteins isolated from OMVs, BO/OMV-lipo@LU, Lipo@LU. \u003cstrong\u003e(j)\u003c/strong\u003e Variations of the particle size and the polydispersity index (PDI) of BO/OMV-lipo@LU stored in PBS within one week. n = 3. \u003cstrong\u003e(k)\u003c/strong\u003e Variations of the particle size and PDI of BO/OMV-lipo@LU during 2-h incubation at 37℃ in three simulated gastrointestinal fluids. The simulated fluids included the simulated gastric fluid (SGF), the simulated small intestine fluid (SIF), and the simulated colon fluid (SCF). n = 3. \u003cstrong\u003e(l)\u003c/strong\u003e The\u003cem\u003e in vitro\u003c/em\u003e drug release profile of luteolin from BO/OMV-lipo@LU in simulated gastrointestinal fluids. n = 3.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4710969/v1/49e9541f91727d88d158af90.png"},{"id":61381973,"identity":"2b7033ca-bc4b-4e6f-9644-71e2e23b663c","added_by":"auto","created_at":"2024-07-30 06:03:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":717175,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBO/OMV-lipo@DiD showed promoted epithelial cell uptake\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e in vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. (a-c) \u003c/strong\u003eParticle size distribution (a), zeta potentials (b), DLC and EE (c) of Lipo@DiD, BO-lipo@DiD, OMV-lipo@DiD and BO/OMV-lipo@DiD. n = 3. \u003cstrong\u003e(d-e)\u003c/strong\u003e Fluorescence images of cell internalization profiles of Lipo@DiD (as a control group) and OMV-lipo@DiD with different ratios of OMV proteins to lipids (1:100, 1:50, and 1:20) in Caco-2 cells (scale bar = 100 μm) and semiquantification of fluorescence intensity (n = 3). \u003cstrong\u003e(f-g)\u003c/strong\u003e Fluorescence images of cell internalization profiles of OMV-lipo@DiD (as a control group) and BO/OMV-lipo@DiD with different DSPE-PEG-BO proportions (3.3%, 6.7%, and 10%) in Caco-2 cells and semiquantitative analysis of fluorescence intensity (n = 3).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4710969/v1/40e64cc3008451ddab09e705.png"},{"id":61381974,"identity":"d6c8b851-641e-45ac-8b6c-852128271ae5","added_by":"auto","created_at":"2024-07-30 06:03:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":885637,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBO/OMV-lipo@DiD targeted the inflamed colon and penetrated into mucosa for improved epithelial uptake\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e in vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. (a-b) \u003c/strong\u003eThe\u003cem\u003e \u003c/em\u003efluorescence images of UC mice and semiquantification of abdominal fluorescence intensity by an \u003cem\u003ein vivo\u003c/em\u003e imaging system (IVIS) at 2 h and 6 h post-oral administration of different DiD-loaded liposomes (DiD dose of 1.25 mg/kg).\u003cstrong\u003e \u003c/strong\u003eBO/OMV-lipo@DiD group, n = 4; other groups, n = 3.\u003cstrong\u003e (c-d)\u003c/strong\u003e\u003cem\u003e Ex vivo\u003c/em\u003efluorescence images of main organs (including heart, liver, spleen, lung, kidney, and colon tissues) from mice and semiquantified for DiD fluorescence signal after 8 h of treatment with different DiD-loaded liposomes. BO/OMV-lipo@DiD group, n = 4; other groups, n = 3. \u003cstrong\u003e(e)\u003c/strong\u003e Immunofluorescence staining images of colon tissues after 8 h of administration (scale bar = 100 μm).Red, DiD-loaded liposomes; yellow, intestinal epithelial cells (anti-Epcam antibody and AF555-labeled IgG); blue, nucleus (DAPI). White lines were between the mucosa layer (downward side) and the submucosa layer (upward side). \u003cstrong\u003e(f) \u003c/strong\u003eColocalization analysis of DiD-loaded liposomes and intestinal epithelial cells using the Pearson correlation coefficient. n = 3.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4710969/v1/c7a1a52d6a8ee15fbcae453c.png"},{"id":61382375,"identity":"82f23613-f1b4-401b-a267-d26406bd9342","added_by":"auto","created_at":"2024-07-30 06:11:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":546767,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of the OMV proportion on therapeutic outcomes of BO/OMV-lipo@LU in the DSS-induced UC mice model. (a) \u003c/strong\u003eExperimental timeline for UC therapy. C57BL/6J mice were provided with 3% DSS for 7 days to establish the UC model. On days 3, 4, 5, 6, and 7, UC mice were orally administered with BO/OMV-lipo@LU with different ratios of OMV proteins to lipids, including 1:20-BO/OMV-lipo@LU, 1:50-BO/OMV-lipo@LU or 1:100-BO/OMV-lipo@LU (luteolin dose of 17 mg/kg). \u003cstrong\u003e(b) \u003c/strong\u003eDisease activity index (DAI) scores in each group for 10 days, summarizing the weight loss index, stool consistency index, and fecal bleeding index.\u003cstrong\u003e (c) \u003c/strong\u003eDaily body weight changes in each group for 10 days. \u003cstrong\u003e(d-e)\u003c/strong\u003e Colon images and colon length of mice on day 10. The control group and the 1:20-BO/OMV-lipo@LU group, n = 5; other groups, n = 4.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4710969/v1/212cf10d5c9b6a9ded5375bb.png"},{"id":61381971,"identity":"6d8965af-160f-45cf-add4-556ea4dabe4a","added_by":"auto","created_at":"2024-07-30 06:03:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":856801,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBO/OMV-lipo@LU improved efficacy in the DSS-induced UC model. (a) \u003c/strong\u003eExperimental timeline for UC therapy. C57BL/6J mice were provided with 3% DSS for 7 days to establish the UC model. On days 3, 4, 5, 6, and 7, UC mice were orally administered with Free-LU, Lipo@LU, BO-lipo@LU, OMV-lipo@LU or BO/OMV-lipo@LU (equivalent luteolin dose of 17 mg/kg). BO/OMV-lipo@LU had an equivalent ratio of OMV proteins to lipids (1:20) as OMV-lipo@LU. \u003cstrong\u003e(b-c)\u003c/strong\u003e Colon images and colon length of mice on day 10. \u003cstrong\u003e(d-e) \u003c/strong\u003eDisease activity index (DAI) scores and daily body weight changes in each group for 10 days. The control group, BO-lipo@LU group, and the 1:20-BO/OMV-lipo@LU group, n = 5; other groups, n = 4. \u003cstrong\u003e(f-g) \u003c/strong\u003eRepresentative H\u0026amp;E staining images of colonic sections of mice on day 10 (scale bar = 100 μm) and colonic histological damage scores. n = 3.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4710969/v1/3545b9c6157d51aa81e76c08.png"},{"id":61381976,"identity":"b3beb228-9921-435c-a479-75dbb8a581d0","added_by":"auto","created_at":"2024-07-30 06:03:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1233139,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBO/OMV-lipo@LU with favorable biosafety alleviated inflammation, modulated redox homeostasis, and recovered dysregulated intestinal barriers. \u003c/strong\u003eThe samples of colon tissue and serum were collected from mice on day 10 after the indicated treatments for subsequent analysis. \u003cstrong\u003e(a-d)\u003c/strong\u003e Inflammatory cytokines in serum (a-b) and the colon tissue (c-d), as determined by ELISA. n = 3. \u003cstrong\u003e(e) \u003c/strong\u003eSchematic of BO/OMV-lipo@LU reducing ROS in the colon. Created with BioRender.com. \u003cstrong\u003e(f-h)\u003c/strong\u003eRedox-related indicators: superoxide dismutase (SOD), myeloperoxidase (MPO) activity, and glutathione (GSH) in the colon tissue. n = 3. \u003cstrong\u003e(i) \u003c/strong\u003eImmunofluorescence staining images of tight junction proteins (including ZO-1 and occludin) in the colon tissue (scale bar = 30 μm for the amplification panels, and 150 μm for all the other panels). Red, ZO-1; green, occludin; blue, nucleus (DAPI). \u003cstrong\u003e(j-k) \u003c/strong\u003eSemiquantitative analysis of the fluorescence intensity of ZO-1 and occludin. n = 3.\u003cstrong\u003e (l-m) \u003c/strong\u003eH\u0026amp;E staining imaging (scale bar = 150 μm) and organ indexes (n = 3) of major organs (heart, liver, spleen, lung, and kidney) were analyzed for systemic biosafety evaluation after the indicated treatments.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4710969/v1/8235a8b55a61ffefd9c45d3f.png"},{"id":64048551,"identity":"4bc14f52-24a3-44ba-afe5-7f88102ffe6a","added_by":"auto","created_at":"2024-09-05 15:13:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5949722,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4710969/v1/8e94f77e-62f5-4d15-9241-5afd2cccadcf.pdf"},{"id":61381972,"identity":"c76ecfbb-83bb-4980-ab1a-7049e5fb705e","added_by":"auto","created_at":"2024-07-30 06:03:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":484038,"visible":true,"origin":"","legend":"","description":"","filename":"Scheme1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4710969/v1/8de424618bcd5e1efd9d5ec8.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Chemotaxis-driven hybrid liposomes trilogically recover intestinal homeostasis for targeted therapy of ulcerative colitis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eUlcerative colitis (UC), a chronic and relapsing inflammatory disease, has an estimated prevalence of 5\u0026nbsp;million cases around the world in 2023 and a rapidly increasing incidence worldwide, tremendously reducing the quality of patients\u0026rsquo; life\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e. Emerging evidence has indicated that UC flare is closely associated with the dysbiosis of intestinal homeostasis, accompanied by the intestinal epithelial barrier destruction\u003csup\u003e4\u0026ndash;6\u003c/sup\u003e, subsequent inflammatory factor infiltration and excessive oxidative stress\u003csup\u003e7\u0026ndash;10\u003c/sup\u003e. The clinical therapeutics of UC include anti-inflammatory molecules (e.g., 5-aminosalicylates and corticosteroids) and immunosuppressants (e.g., infliximab and azathioprine), which primarily focus on ameliorating disease-related symptoms by reducing inflammatory burden\u003csup\u003e11\u003c/sup\u003e. However, they generally do not address other underlying pathogenic factors, such as the destruction of epithelial barriers and redox imbalance, potentially resulting in suboptimal remission rates among patients\u003csup\u003e9,12,13\u003c/sup\u003e. Besides, the clinical outcomes are also hampered by their inherent hydrophobicity, ease of metabolization, and lack of targeting ability\u003csup\u003e14\u003c/sup\u003e. Moreover, severe side effects are observed during the use of these traditional drugs. For example, the long-term use of corticosteroids may lead to osteoporosis, and the infliximab (anti-tumor necrosis factor antibodies) therapy comes with an increased risk of opportunistic infections and drug-induced lupus\u003csup\u003e15\u0026ndash;19\u003c/sup\u003e. Therefore, there exists an unmet demand for a more effective strategy that can achieve multipronged intervention against UC and inflamed colon-targeted delivery.\u003c/p\u003e \u003cp\u003ePrevious studies attempted to achieve inflamed colon-targeted delivery of therapeutics by enhancing adhesion to mucus or equipping them with targeting moieties (e.g., hyaluronic acid and anti-CD98 antibodies) to target inflammatory epithelial cells and macrophages\u003csup\u003e20\u0026ndash;22\u003c/sup\u003e. Despite their high \u003cem\u003ein vitro\u003c/em\u003e uptake efficiency, the \u003cem\u003ein vivo\u003c/em\u003e penetration depth into mucosa remains limited. Biomimetic nanotechnology offers new opportunities for the development of targeted delivery by integrating nanosystems\u0026rsquo; functional versatility with biomimetic materials\u0026rsquo; features. Bacterial membrane-originated components, such as outer membrane vesicles (OMVs) and cytoplasmic membranes, have been widely applied to targeted drug delivery\u003csup\u003e23\u0026ndash;25\u003c/sup\u003e. Interestingly, we surprisingly found that OMVs derived from \u003cem\u003eStenotrophomonas maltophilia\u003c/em\u003e (\u003cem\u003eS.mal.\u003c/em\u003e) exhibited excellent biological chemotaxis to the inflamed colon tissue. \u003cem\u003eS.mal.\u003c/em\u003e is a Gram-negative and low-virulent bacterium that is found ubiquitously in water, foods, plants, animal tissues, and the human body\u003csup\u003e26\u003c/sup\u003e. The chemotaxis of OMVs from \u003cem\u003eS.mal.\u003c/em\u003e may be attributed to the adhesion to inflamed colon mucosa via functional components inherited from parent bacteria, such as outer membrane protein A, which is associated with the adhesion of bacteria[23]. Thereby, biomimetic OMV-fused hybrid liposomes may faithfully replicate the adhesion property that can enhance the retention of liposomes in the inflamed colon to achieve chemotaxis-driven UC-targeted delivery. Moreover, according to previous studies, borneol, a bicyclic monoterpene, showed the potential to improve the uptake of nanoparticles by intestinal epithelial cells\u003csup\u003e29,30\u003c/sup\u003e. Accordingly, we used borneol to further engineer OMV-fused hybrid liposome surface for improving inflamed epithelial uptake in UC.\u003c/p\u003e \u003cp\u003eLuteolin, a natural flavonoid compound, is widely found in fruits, vegetables, and medicinal herbs such as \u003cem\u003eDendranthema morifolium\u003c/em\u003e (Ramat.) Tzvel. and \u003cem\u003eLonicera japonica\u003c/em\u003e Thunb.\u003csup\u003e31,32\u003c/sup\u003e. Currently, luteolin has been widely exploited as a dietary supplement for inflammation-related diseases in clinical trials, such as COVID-19 (NCT05311852) and chronic periodontitis (CTRI/2024/05/067436). Increasing evidence indicates that luteolin is a promising candidate for UC intervention due to its multiple pharmacological actions that can restore intestinal homeostasis, such as anti-inflammatory, anti-oxidant, and intestinal epithelial barrier protection effects\u003csup\u003e33\u0026ndash;36\u003c/sup\u003e. However, its anti-UC efficacy is limited by poor solubility and the lack of targeting ability towards inflamed colon\u003csup\u003e35,37\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTherefore, in this study, chemotaxis-driven hybrid liposomes (BO/OMV-lipo@LU) were designed by functionalizing liposomes with borneol and OMVs, aiming to achieve UC-targeted delivery of luteolin, as shown in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Our results demonstrated that OMV presence contributed to the retention of liposomes in the inflamed colon. Interestingly, under combined effects of OMVs and borneol, BO/OMV-lipo@LU achieved deep mucosa penetration and improved epithelial uptake. In addition, the formulation could sustainably release luteolin in the inflamed colon and exhibit prominent therapeutic potentials in a dextran sulfate sodium (DSS)-induced UC mice model. Notably, BO/OMV-lipo@LU with favorable biosafety trilogically recovered intestinal homeostasis in UC by alleviating intestinal inflammation, modulating redox balance, and restoring intestinal epithelial barriers. The novel chemotaxis-driven hybrid liposomes proposed in this study may serve as a safe and efficient targeted delivery system for UC therapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Results and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Characterization of bacterial OMVs and distearyl phosphatidylethanolamine-polyethylene glycol-borneol (DSPE-PEG-BO)\u003c/h2\u003e \u003cp\u003eBacterial OMVs and DSPE-PEG-BO required for BO/OMV-lipo@LU formulation were prepared and characterized. Bacterial OMVs were isolated from \u003cem\u003eS.mal.\u003c/em\u003e via the ultracentrifugation method. Dynamic light scattering (DLS) analysis results suggested that the particle size peak of OMVs was 100.8\u0026thinsp;\u0026plusmn;\u0026thinsp;16.5 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), and the zeta potential was \u0026minus;\u0026thinsp;12.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11 mV. The transmission electron microscopy (TEM) image showed that OMVs presented a typical phospholipid bilayer structure as described in previous studies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb)\u003csup\u003e38,39\u003c/sup\u003e. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed protein profiles of isolated bacterial OMVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei).\u003c/p\u003e\u003cp\u003eThe synthetic route of borneol-modified lipid materials (DSPE-PEG-BO) was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec. The amino groups of (R)-(+)-bornylamine (the amino derivative of borneol) reacted with NHS ester groups of DSPE-PEG-NHS to synthesize DSPE-PEG-BO via amide bonds. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE showed the \u003csup\u003e1\u003c/sup\u003eH nuclear magnetic resonance (NMR) spectra of (R)-(+)-bornylamine, DSPE-PEG-NHS and DSPE-PEG-BO. It could be seen that a new proton peak at 8.2 ppm (Peak e) appeared in the NMR spectra of DSPE-PEG-BO compared with (R)-(+)-bornylamine and DSPE-PEG-NHS, suggesting the formation of amide bonds. In addition, the characteristic proton peaks (Peak a, b, and c) of DSPE-PEG-NHS, and the methyl proton peak (Peak 3) of (R)-(+)-bornylamine were also found in the NMR spectra of DSPE-PEG-BO. Moreover, compared with DSPE-PEG-NHS, the carbodiimides proton signal (Peak d) of DSPE-PEG-BO disappeared, further demonstrating the successful conjugation of borneol to DSPE-PEG. These collective results confirmed the successful synthesis of DSPE-PEG-BO through the NHS ester reaction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation and characterization of BO/OMV-lipo@LU\u003c/h2\u003e \u003cp\u003eFour kinds of luteolin-loaded liposome formulations were constructed, including liposomes without any modification (Lipo@LU), borneol-modified liposomes (BO-lipo@LU), OMV-mimetic hybrid liposomes (OMV-lipo@LU), and borneol-modified OMV-mimetic hybrid liposomes (BO/OMV-lipo@LU), aiming to investigate benefits that OMVs and DSPE-PEG-BO brought to liposomes in targeting ability.\u003c/p\u003e \u003cp\u003eInitially, Lipo@LU were prepared using the thin-film method, and the formulation optimization was carried out by the single-factor test using the particle size and encapsulation efficiency (EE) as evaluation indexes. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, the organic solvent type and the ratio of soybean lecithin to cholesterol markedly affected the particle size and EE, respectively. The significant effects of luteolin dosage could also be observed on the particle size and EE of Lipo@LU, while the hydration temperature had little effect on them. The results showed that the optimum formulation of Lipo@LU was confirmed as follows: the organic solvent was ethanol, the ratio of soybean lecithin to cholesterol was 3:1, the hydration temperature was 25℃, and the luteolin dosage was 1.2 mg. Additionally, it could be seen from the TEM image that Lipo@LU had a typical spherical vesicle structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee), which is in keeping with other studies of liposomes\u003csup\u003e40,41\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSubsequently, Lipo@LU was further modified with DSPE-PEG-BO and fused with OMVs to fabricate BO/OMV-lipo@LU. BO-lipo@LU and OMV-lipo@LU, used as control liposome formulations, were prepared by adding DSPE-PEG-BO and OMVs, respectively. Similar to Lipo@LU, BO/OMV-lipo@LU exhibited a vesicle structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). DLS analysis results suggested that all liposome formulations (Lipo@LU, BO-lipo@LU, OMV-lipo@LU, and BO/OMV-lipo@LU) possessed a unimodal distribution of particle sizes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). The particle size and polydispersity index (PDI) of BO/OMV-lipo@LU were 156.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 nm and 0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, respectively. As could be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, the zeta potentials (about \u0026minus;\u0026thinsp;24 mV) of those OMV-fused liposomes (including OMV-lipo@LU and BO/OMV-lipo@LU) were between that of native OMVs (about \u0026minus;\u0026thinsp;12 mV) and Lipo@LU (about \u0026minus;\u0026thinsp;30 mV), which indicated the successful hybridization of OMVs with liposomes as reported previously\u003csup\u003e42\u0026ndash;44\u003c/sup\u003e. Additionally, previous studies demonstrated that those nanoparticles with less than 200 nm in diameter and a negative surface charge, exhibited better tissue-penetrating ability for UC therapy\u003csup\u003e45,46\u003c/sup\u003e. Hence, the size and charge properties might contribute to the targeting delivery of BO/OMV-lipo@LU to inflamed colon sites. Moreover, the drug loading efficiency (DLC) and EE of BO/OMV-lipo@LU were 2.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20% and 62.71\u0026thinsp;\u0026plusmn;\u0026thinsp;2.73%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). SDS-PAGE analysis revealed BO/OMV-lipo@LU and OMV samples showed similar protein bands (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei), indicating that BO/OMV-lipo@LU inherited OMV proteins after hybridization and might possess corresponding biological functions.\u003c/p\u003e \u003cp\u003eNext, the stability of BO/OMV-lipo@LU in PBS and gastrointestinal tract (GIT) was tested by monitoring the change of liposomes in the particle size and PDI. As demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej, BO/OMV-lipo@LU exhibited excellent storage stability in PBS, as evidenced by the almost stable particle size and PDI within one week at 4 ℃. Apart from storage ability, the stability in the GIT was also important for BO/OMV-lipo@LU to reach the inflamed colon safely and exert curative effects. BO/OMV-lipo@LU presented a slightly increased size after incubation in the simulated gastric fluid (SGF) and the simulated small intestine fluid (SIF), but the change did not exceed 40 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek). No significant size change could be observed in BO/OMV-lipo@LU incubated in the simulated colon fluid (SCF). Meanwhile, during incubation in three simulated gastrointestinal fluids, the PDI of BO/OMV-lipo@LU showed a minor increase and was still within an acceptable range (\u0026lt;\u0026thinsp;0.3). These results demonstrated that BO/OMV-lipo@LU exhibited favorable stability under gastrointestinal conditions, which might protect luteolin from degradation during their passage through the GIT.\u003c/p\u003e \u003cp\u003eTo further explore the drug release behavior of BO/OMV-lipo@LU in the GIT, the \u003cem\u003ein vitro\u003c/em\u003e release study was conducted in SGF, SIF, and SCF sequentially. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003el, free luteolin (Free-LU) exhibited rapid release in the SGF and SIF, and the cumulative release rate had already reached over 90% before the release medium was replaced with SCF. Compared with Free-LU, BO/OMV-lipo@LU dramatically slowed down the luteolin release rate under the simulated conditions of the stomach and small intestine. It suggested that the liposome coating could prevent early luteolin release before reaching the inflamed colon. When transferred to SCF, BO/OMV-lipo@LU presented sustained drug release, and the cumulative release rate could reach 70% over 12 h, which might enhance the colonic release of luteolin from the liposomes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 BO/OMV-lipo@DiD showed promoted epithelial cell uptake \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eIn this study, The DiD fluorescent dye was used to track liposomes in the assay of cellular uptake and \u003cem\u003ein vivo\u003c/em\u003e bio-distribution by fabricating DiD-loaded liposome formulations, including Lipo@DiD, OMV-lipo@DiD, BO-lipo@DiD and BO/OMV-lipo@DiD. These DiD-loaded liposomes were characterized by particle size, zeta potential, DLC, and EE. As demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, they possessed similar particle size (~\u0026thinsp;120 nm) with a narrow distribution (PDI\u0026thinsp;\u0026lt;\u0026thinsp;0.3). DLS analysis showed that the surface charge of hybrid liposomes (OMV-lipo@DiD and BO/OMV-lipo@DiD) was closer to that of OMVs in comparison with other non-hybrid liposomes, confirming the successful membrane fusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). DLC and EE of BO/OMV-lipo@DiD were 0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02% and 53.23\u0026thinsp;\u0026plusmn;\u0026thinsp;4.28%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, effects of the OMV proportion and DSPE-PEG-BO proportion on the intestinal epithelial cellular uptake abilities of BO/OMV-lipo@DiD were investigated by fluorescence imaging. Different ratios of OMV proteins to lipids (1:100, 1:50, and 1:20) were applied to fabricate OMV-lipo@DiD. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e, with the increase of the OMV proportion, the efficiency of liposome uptake by Caco-2 cells presented an increased trend. Amongst, OMV-lipo@DiD with the 1:20 OMV ratio dramatically enhanced the uptake efficiency of Lipo@DiD by 2.5 folds and showed the best cell internalization effects compared with those with other ratios. It revealed that the fusion of OMVs contributed to improving the ability of liposomes to target intestinal epithelial cells. Besides, the effects of the DSPE-PEG-BO proportion on the uptake efficiency of liposomes were also investigated, by applying different DSPE-PEG-BO proportions (3.3%, 6.7%, and 10%) but maintaining the fixed OMV proportion (1:20 ratio) to fabricate BO/OMV-lipo@DiD. It could be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, g, that there was no significant difference in relative fluorescence intensity between OMV-lipo@DiD and 3.3%-BO/OMV-lipo@DiD, revealing that the presence of DSPE-PEG-BO in a low proportion (3.3%) could not affect Caco-2 cell uptake of liposomes. Whereas, the fluorescence intensity of 10%-BO/OMV-lipo@DiD was 4 folds higher than that of OMV-lipo@DiD. It indicated that the high counterpart (10%) dramatically promoted intestinal epithelial cell uptake efficiency.\u003c/p\u003e \u003cp\u003eTaken together, the 1:20 ratio of OMV proteins to lipids and 10% proportion of DSPE-PEG-BO were determined as the optimal ratios to formulate BO/OMV-lipo@DiD that exhibited excellent epithelial targeting ability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 BO/OMV-lipo@DiD targeted the inflamed colon and penetrated into mucosa for improved epithelial uptake \u003cem\u003ein vivo\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe bio-distribution of BO/OMV-lipo@DiD in UC mice after oral administration was investigated by an \u003cem\u003ein vivo\u003c/em\u003e imaging system (IVIS). At 2 h and 6 h post administration of Lipo@DiD, BO-lipo@DiD, OMV-lipo@DiD, and BO/OMV-lipo@DiD, mice were euthanized to obtain the IVIS images. Results presented that the UC mice treated with the OMV-lipo@DiD or BO/OMV-lipo@DiD group exhibited about 2-fold abdominal fluorescence intensity of those treated with Lipo@DiD after 6 h of administration. It indicated that OMV fusion significantly prolonged the retention time of liposomes in the intestinal tract, thereby retarding the intestinal clearance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter administration of liposomes for 8 h, the mice were sacrificed. The visceral organs and colon tissues were collected for further fluorescence analysis. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d, visceral organs had negligible fluorescence intensity in all groups compared with colon tissues. Importantly, OMV-lipo@DiD displayed significantly greater accumulation in the inflamed colon tissue than Lipo@DiD, suggesting that the presence of the biomimetic OMVs contributed to the colon-targeting capability of liposomes in UC mice. It could be conjectured that OMVs might faithfully replicate the adhesion property of parent bacteria, thus providing excellent chemotaxis-driven UC-targeted delivery performance. Notably, the sole modification of borneol did not improve the liposome accumulation in the colon. In contrast, mice treated with BO/OMV-lipo@DiD (involving OMV fusion and borneol modification) exhibited higher colonic fluorescence intensity than those treated with OMV-lipo@DiD. The possible explanation was that OMV-induced colon retention of liposomes increased the contact time between intestinal epithelial cells and liposomes, allowing surface-modified borneol to mediate enhanced inflamed epithelial uptake.\u003c/p\u003e \u003cp\u003eTo further verify whether BO/OMV-lipo@DiD could target colon epithelial cells \u003cem\u003ein vivo\u003c/em\u003e, frozen sections of the colon tissue were stained with anti-Epcam mouse antibodies, followed by Alexa Fluor (AF) 555-labeled anti-mouse IgG to indicate epithelial cells. The Pearson correlation coefficient (PCC) was analyzed to quantify colocalization between DiD fluorescence (indicating liposomes) and AF555 fluorescence (indicating epithelial cells). Results showed that BO-lipo@DiD and Lipo@DiD had no difference in PCC. In contrast, the PCC of OMV-lipo@DiD was higher than Lipo@DiD, signifying that OMVs promoted epithelial cell uptake of liposomes to some extent (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, f). More importantly, among all groups, BO/OMV-lipo@DiD exhibited the most apparent colocalized signals with epithelial cells, demonstrating that OMVs and borneol jointly enhanced colon epithelial uptake efficiency. The excellent targeting capability of BO/OMV-lipo@DiD towards inflamed colon might be attributed to the chemotaxis-driven colon-targeting ability of OMVs and the borneol-mediated further epithelial uptake.\u003c/p\u003e \u003cp\u003eInterestingly, under combined effects of OMVs and borneol, BO/OMV-lipo@LU achieved the most robust penetration ability into mucosa for better infiltration of liposomes in the inflamed colon. The other studies employed targeting molecules (such as anti-CD98 antibodies and hyaluronic acid) to engineer the nanosystem surface for targeting inflammatory intestinal epithelial cells and macrophages\u003csup\u003e21,22\u003c/sup\u003e. Notwithstanding their improved targeting ability of the inflamed colon, the penetration depth of these nanosystems was limited (only about 1/3 or less of the colonic mucosa layer). However, BO/OMV-lipo@DiD could penetrate the mucosal layer entirely (reaching the white lines as indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee) after oral administration, indicating the good colonic mucosal permeability of BO/OMV-lipo@DiD. Previous studies reported that membrane-originated components of some specific bacteria inherited chemotaxis towards the intestine from parent bacteria, which might be achieved by specific adhesion protein-mediated adhesion to the intestinal epithelium and/or mucus layer\u003csup\u003e23,24,47\u0026ndash;49\u003c/sup\u003e. Nevertheless, the inflamed colon-targeting mechanisms of OMVs derived from \u003cem\u003eS.mal\u003c/em\u003e remained to be explored in the future.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 BO/OMV-lipo@LU improved efficacy in the DSS-induced UC model\u003c/h2\u003e \u003cp\u003eThe therapeutic efficacy of BO/OMV-lipo@LU on the DSS-induced UC mice model was evaluated. First, we investigated whether the ratios of OMV proteins to lipids influenced the \u003cem\u003ein vivo\u003c/em\u003e therapeutic outcomes of BO/OMV-lipo@LU by improving the colon-targeting ability of hybrid liposomes. Mice received the drinking water containing 3% DSS for 7 days to induce the UC model and were orally administered BO/OMV-lipo@LU with various ratios of OMV proteins to lipids (including 1:20, 1:50, and 1:100), conforming to the regimen shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea. The anti-UC efficacy of BO/OMV-lipo@LU was evaluated by detecting body weight changes, disease activity index (DAI) scores, colon length and colonic pathological damage. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, c, with either OMV proportion, BO/OMV-lipo@LU treatment distinctly reduced DAI scores and recovered the body weight of UC mice. More importantly, the UC mice treated with 1:20-BO/OMV-lipo@LU showed an increased colon length (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, e) and less weight loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) than those treated with 1:50-BO/OMV-lipo@LU or 1:100-BO/OMV-lipo@LU. Moreover, the 1:20-BO/OMV-lipo@LU group exhibited a lower DAI score than the 1:100-BO/OMV-lipo@LU group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). These results were consistent with the findings of the \u003cem\u003ein vitro\u003c/em\u003e uptake assay, demonstrating that the 1:20 ratio of OMV proteins to lipids endowed BO/OMV-lipo@LU with excellent targeting ability and anti-UC efficacy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the promising therapeutic results of 1:20-BO/OMV-lipo@LU, its efficacy was directly compared against Free-LU, Lipo@LU, BO-lipo@LU, and OMV-lipo@LU (with an equivalent OMV proportion). The administration regimen was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, and an equivalent luteolin dose (17 mg/kg) was given. As presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, c, the colon in the BO/OMV-lipo@LU group was markedly longer than that in other treatment groups, and there existed no difference in colon length between the control group and the BO/OMV-lipo@LU group. Moreover, BO/OMV-lipo@LU treatment significantly reduced the DAI score of UC mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). At the end of the experiment, mice treated with BO/OMV-lipo@LU and OMV-lipo@LU recovered the body weight basically, while other control treatment groups failed to achieve full body weight recovery (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Thereafter, to evaluate the colonic pathological change, the colon tissue was stained by hematoxylin and eosin (H\u0026amp;E), followed by the histological scoring. The results showed that the BO/OMV-lipo@LU group had significantly lower colonic histological damage scores compared with the DSS group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). BO/OMV-lipo@LU tremendously alleviated DSS-induced colonic damage characterized by inflammatory cell infiltration, damaged crypt structure, and decreased goblet cell number (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). In contrast, apparent colonic damage could still be observed in other groups treated with Free-LU, Lipo@LU or BO-lipo@LU. These results indicated that the OMV hybridization and borneol modification were necessary to achieve effective anti-UC treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNotably, it was previously reported that the treatment with a high oral dose of free luteolin (100 mg/kg) could exhibit marked therapeutic effects on UC, while the low counterpart (50 mg/kg) had little effects on it\u003csup\u003e50\u003c/sup\u003e. However, our results demonstrated that luteolin in low-dose (17 mg/kg) could be delivered effectively by the biomimetic hybrid liposomes to inflamed colon sites, thereby enough to exert significant anti-UC efficacy. It would seem that BO/OMV-lipo@LU treatment with 17 mg/kg luteolin dose even exerted a more robust efficacy than 100 mg/kg free luteolin treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 BO/OMV-lipo@LU alleviated inflammation, modulated redox balance, and recovered dysregulated intestinal barriers\u003c/h2\u003e \u003cp\u003eIntrigued by these promising therapeutic results, the underlying action mechanisms of BO/OMV-lipo@LU on UC were further studied. The UC flare tends to be accompanied by dysbiosis of intestinal homeostasis involving multiple pathological mechanisms, including inflammation factor infiltration, excessive oxidative stress, and disrupted epithelial barrier function. Previous experimental evidence indicated that luteolin could affect various pathological aspects of UC based on its action mode of multichannels and multitargets, thereby turning the intestinal microenvironment towards a beneficial state\u003csup\u003e33\u0026ndash;36\u003c/sup\u003e. Accordingly, the role of BO/OMV-lipo@LU in regulating intestinal homeostasis was investigated by detecting its ability to suppress inflammation, alleviate oxidative stress, and repair the intestinal epithelial barrier.\u003c/p\u003e \u003cp\u003eThe local and systemic infiltration of inflammatory factors is one of the primary pathological elements controlling UC initiation and progression. Thus, the levels of inflammatory factors in the serum and colon tissues were determined by enzyme-linked immunosorbent assay (ELISA). BO/OMV-lipo@LU treatment not only significantly inhibited the local level of inflammatory factors in the colon tissues, including tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6), but reduced the systemic content of pro-inflammatory interferon-γ (IFN-γ) and IL-6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-d). The level of these inflammatory factors in the mice treated with BO/OMV-lipo@LU nearly returned to the normal range, whereas other treatment groups did not efficiently suppress inflammatory factor infiltration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBesides, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, reactive oxygen species (ROS), such as superoxide anion radicals (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and hypochlorous acid (HOCl), rapidly increase in the intestinal microenvironment of UC mice. It leads to excessive oxidative stress, which is recognized as a vital pathological mechanism of UC, followed by severe intestinal lesions. Therefore, reducing ROS levels is necessary to retard UC progression\u003csup\u003e51\u003c/sup\u003e. In this study, the redox-related indicators were detected to examine the ability of BO/OMV-lipo@LU to regulate redox balance in the intestinal microenvironment. The protective effects of BO/OMV-lipo@LU in UC mice were reflected in the elevated activity of superoxide dismutase (SOD) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef), which was an anti-oxidant metalloenzyme that catalyzed the disproportionation of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e to O\u003csub\u003e2\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Additionally, BO/OMV-lipo@LU treatment markedly suppressed the activity of myeloperoxidase (MPO) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg), which generated HOCl from H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the presence of Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e and consequently contributed to inflammation and intestinal oxidative damage. Furthermore, glutathione (GSH) was also an anti-oxidant indicator since GSH could reduce H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to water, and BO/OMV-lipo@LU treatment significantly induced an upregulated GSH level in the colon tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh). However, other control treatment groups did not exhibit apparent anti-oxidant performance. These results indicated that BO/OMV-lipo@LU dramatically decreased ROS levels and alleviated intestinal oxidative stress in the colon tissues of UC mice.\u003c/p\u003e \u003cp\u003eGiven the protection role of the intestinal epithelial barrier in intestinal homeostasis, the expression of tight junction proteins, including ZO-1 and occludin, was assessed to explore the effects of BO/OMV-lipo@LU on colonic epithelial restoration. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei-k, Free-LU, Lipo@LU, and BO-lipo@LU treatments had minimal impact on ZO-1 and occludin expression. However, the UC mice treated with OMV-lipo@LU or BO/OMV-lipo@LU significantly recovered the expression of tight junction proteins to different extents. These two groups presented similar ZO-1 levels, while the BO/OMV-lipo@LU group had a higher level of occludin, indicating BO/OMV-lipo@LU exerted the most excellent effects on restoring the intestinal epithelial barrier among all treatment groups.\u003c/p\u003e \u003cp\u003eTaken together, BO/OMV-lipo@LU trilogically recovered intestinal homeostasis in UC by reducing inflammatory factor levels, alleviating oxidative stress, and repairing the intestinal epithelial barrier, which suggested BO/OMV-lipo@LU could protect animals against UC in an effective manner.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 BO/OMV-lipo@LU showed favorable biosafety\u003c/h2\u003e \u003cp\u003eThe toxicity of BO/OMV-lipo@LU against five major organs was assessed by H\u0026amp;E staining. Results showed that no apparent histological abnormality was observed in the H\u0026amp;E staining sections following the oral administration of BO/OMV-lipo@LU (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003el). In addition, the organ indexes of major organs (including heart, liver, spleen, lung, and kidney) were examined for systemic toxicity evaluation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003em, DSS-induced UC mice were observed with obviously increased organ indexes of the liver and spleen. However, the abnormal hepatosplenomegaly could be alleviated by the BO/OMV-lipo@LU treatment, and the other organ indexes in the BO/OMV-lipo@LU group were close to that in the healthy control group. These results indicated favorable systemic biosafety of BO/OMV-lipo@LU under the current treatment conditions.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Conclusions","content":"\u003cp\u003eIn this study, we constructed a novel chemotaxis-driven biomimetic delivery system for UC-targeted therapy by fusing liposomes with OMVs and functionalizing liposome surfaces with borneol. Benefitting from the chemotaxis-driven colon-targeting ability of OMVs and intestinal epithelial uptake promotion of borneol, BO/OMV-lipo@DiD exhibited excellent targeting capability towards the inflamed colon. Interestingly, under combined effects of OMVs and borneol, BO/OMV-lipo@LU achieved the most robust penetration ability into mucosa for better infiltration of liposomes in the inflamed colon. Thus, the liposomes were designed to achieve UC-targeted delivery of luteolin, a natural flavonoid compound that could exert multiple effects on UC. We found BO/OMV-lipo@LU distinctly alleviated the pathological symptoms of DSS-induced UC mice. Significantly, BO/OMV-lipo@LU trilogically restored intestinal homeostasis in UC by alleviating intestinal inflammation, modulating redox balance, and restoring intestinal epithelial barriers. During the treatment, no apparent toxicity was observed in mice, demonstrating the favorable biosafety of BO/OMV-lipo@LU. This study suggests BO/OMV-lipo@LU are promising nanoplatforms for safe and effective gut-targeted delivery in UC intervention.\u003c/p\u003e"},{"header":"4. Experimental section","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Materials, cell lines, and animals\u003c/h2\u003e \u003cp\u003eSoybean lecithin, cholesterol, and DSPE-mPEG(2000) were purchased from AVT Pharmaceutical Tech Co., Ltd. (China). (R)-(+)-bornylamine was purchased from Sigma-Aldrich Co. (USA). DSPE-PEG(2000)-NHS was purchased from Shanghai Ponsure Biotech, Inc. (China). Pierce BCA Protein Assay Kit was purchased from Thermo Fisher Scientific (USA). Luteolin (98% purity) was purchased from Chengdu Herbpurify Co., Ltd. (China). RIPA lysis buffer, protease inhibitor cocktail, phosphatase inhibitor cocktail, and phenylmethanesulfonyl fluoride (PMSF) were purchased from Beyotime (China). 4% Paraformaldehyde was purchased from Shanghai Biotend Biotech Co., Ltd. (China). Difco\u0026trade; Nutrient Broth \u003cb\u003e(\u003c/b\u003eCat. No. 234000\u003cb\u003e)\u003c/b\u003e was purchased from BD Bioscience (USA). DiD dye was purchased from Dalian MeiLun Biotechnology Co., Ltd. (China). DAPI was purchased from MedChemExpress (USA). DSS was purchased from Yeasen Biotechnology (China). The fecal occult blood detection kit and the GSH assay kit were purchased from Nanjing Jiancheng (China). The MPO activity assay kit and the SOD activity assay kit were purchased from Beijing Solarbio Science \u0026amp; Technology Co., Ltd (China). The mouse IFN-γ ELISA kit, the TNF-α ELISA kit, and the IL-6 ELISA kit were purchased from Shanghai Jianglai Industrial Limited By Share Ltd. (China). The anti-Epcam mouse antibody was purchased from Hangzhou HuaAn Biotechnology Co., Ltd. (China). The anti-mouse IgG conjugated with AF555 was purchased from Cell Signaling Technology (USA). The anti-ZO-1 rat monoclonal antibody (sc-33725) was purchased from Santa Cruz (USA). The Horseradish peroxidase (HRP)-labeled goat anti-rat IgG (GB23302) was purchased from Wuhan Servicebio Technology Co., Ltd. (China). The anti-occludin rabbit monoclonal antibody (ab216327) was purchased from Abcam (UK). The HRP-labeled goat anti-rabbit IgG (RCA054), the TYR-570Plus red dye, and the TYR-520Plus green dye were purchased from Refinebio (China).\u003c/p\u003e \u003cp\u003eCaco-2 cells (Human epithelial colorectal adenocarcinoma cells) were purchased from Cell Bank of Chinese Academy of Sciences (China). They were cultured with Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM; Meilunbio, China) containing 10% (v/v) fetal bovine serum (FBS; ExCell Bio., China) and 1% penicillin/streptomycin (Biosharp, Beijing Lanjieke Technology Co., Ltd., China) at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eFemale C57BL/6J mice (18\u0026thinsp;~\u0026thinsp;20 g) were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. (China). All animal work complied with the protocols approved by Institutional Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine (IACUC, No: PZSHUTCM2303240001). All animal experiments were conducted following the \u0026ldquo;3R\u0026rdquo; principle (reduction, replacement, refinement).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Isolation and characterization of bacterial OMVs\u003c/h2\u003e \u003cp\u003eThe isolation of bacterial OMVs from \u003cem\u003eS.mal.\u003c/em\u003e was conducted using the ultracentrifugation method reported previously with minor modifications\u003csup\u003e52,53\u003c/sup\u003e. \u003cem\u003eS.mal.\u003c/em\u003e was inoculated into Nutrient Broth medium and cultured in a rotary shaker (220 rpm) at 32℃ for 12 h. Then, the bacterial suspension was diluted 1:100 with fresh medium and cultured for another 12 h. Next, the bacterial medium was centrifuged at 4℃, 4000 g for 10 min to remove \u003cem\u003eS.mal.\u003c/em\u003e, followed by filtration with a 0.22-\u0026micro;m vacuum filter (Beyotime, China). Thereafter, the filtrate was concentrated using an ultrafiltration tube (MWCO 100 kDa, Millipore). The concentrated solution was ultracentrifuged at 20,0000 g, 4℃ for 3 h with SW 70 Ti rotor (Beckman coulter, USA) to obtain OMV pellets, which were suspended finally in PBS and stored at -80℃ for further use.\u003c/p\u003e \u003cp\u003eThe size distribution and zeta potential of OMVs were detected by a NICOMPTM 380 ZLS Zeta Potential/Particle Sizer (PSS, USA) using DLS. The morphology of OMVs was characterized by 120 kV TEM (FEI, USA). Briefly, the OMV sample was dropped onto 300-mesh carbon-coated copper grids. Following incubation for 1 min, the sample was removed from the grid, which was then stained with uranyl acetate for another 1 min and dried before TEM analysis. In addition, the OMV protein content was measured by a BCA protein assay kit, following the lysis of OMV samples in RIPA buffer supplemented with protease and phosphatase inhibitors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Synthesis and characterization of DSPE-PEG-BO\u003c/h2\u003e \u003cp\u003eDSPE-PEG-BO was synthesized through NHS easter reaction by conjugating the amino group of (R)-(+)-bornylamine (an amino derivative of borneol) and DSPE-PEG-NHS easter. Briefly, DSPE-PEG-NHS (100 mg, 1 equiv.) was dissolved in chloroform and stirred at 300 rpm, followed by rapidly adding (R)-(+)-bornylamine (5.6 mg, 1.1 equiv.) and N, N-Diisopropylethylamine (DIPEA). The mixture was stirred under a nitrogen atmosphere for 24 h at room temperature. Then, the product was purified by immersing the mixture in the ether at -20℃ for 3\u0026thinsp;~\u0026thinsp;4 h and collecting the precipitate by centrifuging at 5000 g, 4℃ for 30 min. The purification procedure was conducted three times, and the final powder product of DSPE-PEG-BO was obtained through further lyophilization. The structure of DSPE-PEG-BO was characterized by the 600 MHz \u003csup\u003e1\u003c/sup\u003eH-NMR spectrometry (Bruker, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Preparation of luteolin-loaded liposomes\u003c/h2\u003e \u003cp\u003eIn this study, four kinds of luteolin-loaded liposome formulations were constructed, including liposomes without any modification (Lipo@LU), single borneol-modified liposomes (BO-lipo@LU), single OMV-mimetic hybrid liposomes (OMV-lipo@LU), and borneol-modified OMV-mimetic hybrid liposomes (BO/OMV-lipo@LU).\u003c/p\u003e \u003cp\u003eLipo@LU were initially fabricated using the thin film hydration method\u003csup\u003e43,54\u003c/sup\u003e. Firstly, a mixture of soybean lecithin, cholesterol, DSPE-mPEG, and luteolin with different weights was placed in a round-bottom flask and ultrasonically dissolved in the organic solvent. Subsequently, the solvent was evaporated entirely under reduced pressure using a rotary evaporator at 45℃ and 125 rpm for 20 min until a thin-lipid film was formed. Then, PBS was added to hydrate the film for 10 min to obtain multilamellar liposomes, followed by probe-sonicating (Ningbo Scientz Biotechnology Co., Ltd., China) at 25% power (2 s pulse on/4 s pulse off) for 5 min to acquire unilamellar liposomes. Next, the suspension was filtered through a 0.45-\u0026micro;m filter to remove large particles, and Lipo@LU was ultimately obtained by extrusion through 400 and 200 nm polycarbonate membranes 9 times using a mini extruder (Avestin, Canada), respectively. Any unentrapped luteolin was removed by ultrafiltration (MWCO 10 kDa, Millipore). To optimize the formulation of Lipo@LU, the single-factor test was performed to investigate effects of the organic solvent type (a 9:1 chloroform-methanol mixture or ethanol), the ratio of soybean lecithin to cholesterol (3:1, 4:1 or 6:1), hydration temperature (25℃ or 37℃) and luteolin dosage (1.2 mg, 2 mg or 3 mg) on the particle size and encapsulation efficiency of Lipo@LU.\u003c/p\u003e \u003cp\u003eBased on the formulation mentioned above of Lipo@LU, BO/OMV-lipo@LU were prepared by replacement of DSPE-mPEG with DSPE-PEG-BO and membrane fusion with OMVs. Briefly, DSPE-PEG-BO was added to lipid materials to form liposomes with targeting moieties of borneol (BO-lipo@LU). Additionally, OMVs were mixed with hydration suspension containing liposomes and then were extruded to induce membrane fusion and generate OMV-mimetic hybrid liposomes (OMV-lipo@LU). BO/OMV-lipo@LU was synthesized by combining these two processes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Characterization of luteolin-loaded liposomes\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e4.5.1 Particle size, zeta potential, morphology, and storage stability\u003c/h2\u003e \u003cp\u003eThe size distribution and zeta potential of luteolin-loaded liposomes were detected by a NICOMPTM 380 ZLS Zeta Potential/Particle Sizer. The morphology of these liposomes was observed by 120 kV TEM. To evaluate the storage stability at 4 ℃ within one week, their particle size and PDI were detected and recorded daily.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e4.5.2 Encapsulation efficiency and drug loading capability\u003c/h2\u003e \u003cp\u003eThe EE and DLC of luteolin-loaded liposomes were calculated using Eqs.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and (\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The content of luteolin entrapped in the liposomes was determined with high performance liquid chromatography (HPLC, Shimadzu, Japan). The HPLC analysis for determining luteolin was conducted with an Agilent ZORBAX SB-C18 column (250 mm \u0026times; 4.6 mm), mobile phase of the mixture of acetonitrile and 0.2% phosphoric acid (33:67, v/v), flow rate of 1 mL/min, column temperature of 25℃, injection volume of 20 \u0026micro;L, and ultraviolet (UV) detection wavelength of 350 nm. Prior to HPLC analysis, liposomes were mixed with methanol by vigorous vortexing (1 min) and sonication in a bath sonicator (10 min, Shanghai Titan, China), followed by centrifuging at 10,012 g for 10 min to collect the supernate. Additionally, the total dry weight of liposomes was measured to calculate DLC after liposomes were freeze-dried for 24 h.\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\text{E}\\text{E}（\\text{%}）=\\text{M}\\text{o}\\text{u}\\text{n}\\text{t}\\:\\text{o}\\text{f}\\:\\text{e}\\text{n}\\text{c}\\text{a}\\text{p}\\text{s}\\text{u}\\text{l}\\text{a}\\text{t}\\text{e}\\text{d}\\:\\text{l}\\text{u}\\text{t}\\text{e}\\text{o}\\text{l}\\text{i}\\text{n}/\\text{M}\\text{o}\\text{u}\\text{n}\\text{t}\\:\\text{o}\\text{f}\\:\\text{a}\\text{d}\\text{d}\\text{e}\\text{d}\\:\\text{l}\\text{u}\\text{t}\\text{e}\\text{o}\\text{l}\\text{i}\\text{n}\\:\\times\\:100\\%$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:\\text{D}\\text{L}\\text{C}（\\text{%}）=\\text{M}\\text{o}\\text{u}\\text{n}\\text{t}\\:\\text{o}\\text{f}\\:\\text{e}\\text{n}\\text{c}\\text{a}\\text{p}\\text{s}\\text{u}\\text{l}\\text{a}\\text{t}\\text{e}\\text{d}\\:\\text{l}\\text{u}\\text{t}\\text{e}\\text{o}\\text{l}\\text{i}\\text{n}/\\text{T}\\text{o}\\text{t}\\text{a}\\text{l}\\:\\text{w}\\text{e}\\text{i}\\text{g}\\text{h}\\text{t}\\:\\text{o}\\text{f}\\:\\text{l}\\text{i}\\text{p}\\text{o}\\text{s}\\text{o}\\text{m}\\text{e}\\text{s}\\times\\:100\\%$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e4.5.3 SDS-PAGE protein analysis\u003c/h2\u003e \u003cp\u003eSDS-PAGE analysis was employed to characterize proteins. The OMVs, BO/OMV-lipo@LU, and Lipo@LU samples were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors, mixed with loading buffer, and then incubated at 95℃ for 10 min. Each sample (10 \u0026micro;g) was loaded in the wells of a Bio-Rad electrophoresis system (Bio-Rad Laboratories, USA). To visualize the protein profile, the gel was stained with Coomassie Brilliant Blue G-250 for 15 min and destained for 1 h, and imaged using a Bio-Rad gel imaging system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e4.5.4 Stability and \u003cem\u003ein vitro\u003c/em\u003e drug release in simulated gastrointestinal fluids\u003c/h2\u003e \u003cp\u003eSimulated gastrointestinal fluids included SGF (pH 1.2, consisting of HCl and pepsin), SIF (pH 6.8, consisting of KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, NaOH, and trypsin), and SCF (pH 7.8, comprising KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e and K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e), which were used to simulate the physiological conditions in the stomach, small intestine and colon regions, respectively. To investigate the stability of luteolin-loaded liposomes in the gastrointestinal fluids, their particle size and PDI were determined after the liposome samples were incubated in SGF, SIF, and SCF, respectively, at 37℃ for 2 h.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e release properties of luteolin-loaded liposomes were investigated via dialysis. To mimic the environment of liposomes during gastrointestinal passage \u003cem\u003ein vivo\u003c/em\u003e, the selection of release medium applied a three-stage method according to previous studies\u003csup\u003e55,56\u003c/sup\u003e. Briefly, Free-LU, Lipo@LU, BO-lipo@LU, OMV-lipo@LU, and BO/OMV-lipo@LU were put in dialysis bags (MWCO 8\u0026thinsp;~\u0026thinsp;14 kDa, Shanghai Yuanye Bio-Technology, China), respectively. Then, the dialysis bags were fastened and immersed in the release medium in an order of SGF, SIF, and SCF, which contained 0.5% Tween-80. The experiment was performed by a shaker at 37 ℃ and 150 rpm. The samples of release medium were collected at predetermined time points, and the same volume of fresh medium was immediately replenished. Afterward, the luteolin content in the samples diluted with methanol was determined by HPLC analysis to calculate the cumulative release of luteolin.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.6 Preparation and characterization of DiD-loaded liposomes\u003c/h2\u003e \u003cp\u003eAiming to investigate the cell internation and \u003cem\u003ein vivo\u003c/em\u003e bio-distribution of liposomes, DiD dye was encapsulated into liposomes as a fluorescent marker. The preparation process of DiD-loaded liposomes was consistent with that of luteolin-loaded liposomes using the thin-film hydration method, except that those lipid materials and DiD dye were dissolved in chloroform rather than ethanol. The particle size, zeta potential, DLC, and EE of Lipo@DiD, OMV-lipo@DiD, BO-lipo@DiD, and BO/OMV-lipo@DiD were determined as previously mentioned in 4.5.1 and 4.5.2. The DiD concentration was qualified by determining the fluorescence intensity with a microplate reader (Tecan, Switzerland).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4.7 \u003cem\u003eIn vitro\u003c/em\u003e epithelial uptake assay\u003c/h2\u003e \u003cp\u003eThe cellular uptake ability of BO/OMV-lipo@LU was investigated in the Caco-2 cell monolayer model. To observe effects of the OMV proportion on the uptake efficiency of liposomes, three ratios of OMV protein to lipids (1:100, 1:50, and 1:20) were applied to prepare hybrid liposomes, respectively, for subsequent cellular uptake experiments. Caco-2 cells were seeded on 24-well plates at a density of 10\u003csup\u003e4\u003c/sup\u003e cells/well and incubated with Lipo@DiD and hybrid liposomes with various OMV proportions (including 1:100-OMV-lipo@DiD, 1:50-OMV-lipo@DiD, and 1:20-OMV-lipo@DiD) at 37 ℃ for 1 h. Then, the cells were washed with PBS three times and fixed for 10 min using paraformaldehyde, followed by DAPI (10 \u0026micro;g/mL) staining for 10 min. Cellular uptake imaging was acquired using a fluorescence microscope (Leica, Germany), and the fluorescence intensity was semiquantified using Image J 1.53t (USA).\u003c/p\u003e \u003cp\u003eIn addition, to investigate the effects of the DSPE-PEG-BO proportion on uptake efficiency, Caco-2 cells were incubated with liposomes modified with DSPE-PEG-BO of different proportions (of lipids, 3.3%, 6.7%, 10%). Briefly, Caco-2 cells were treated with OMV-lipo@DiD and BO/OMV-lipo@DiD with various DSPE-PEG-BO proportions (including 3.3%-BO/OMV-lipo@DiD, 6.7%-BO/OMV-lipo@DiD, 10%-BO/OMV-lipo@DiD). Then, the cells were washed, fixed, stained with DAPI, and observed by the fluorescence microscope to visualize the epithelial uptake.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e4.8 DSS-induced UC model\u003c/h2\u003e \u003cp\u003eFemale C57BL/6J mice (18\u0026thinsp;~\u0026thinsp;20 g) were cohoused in cages under specific pathogen-free (SPF) conditions of a 12-h light/dark cycle and acclimatized for a week before random allocation to experimental groups. To establish a DSS-induced UC model, the mice received 3% DSS (w/v) supplemented in drinking water for 7 days\u003csup\u003e23,57\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e4.9 \u003cem\u003eIn vivo\u003c/em\u003e imaging\u003c/h2\u003e \u003cp\u003eTo investigate the targeting ability of BO/OMV-lipo@LU to the inflamed colon, UC mice were divided into 4 groups and treated orally with Lipo@DiD, BO-lipo@DiD, OMV-lipo@DiD and BO/OMV-lipo@DiD, respectively, at an equivalent DiD dose of 1.25 mg/kg). At the predetermined time points, \u003cem\u003ein vivo\u003c/em\u003e fluorescent images were observed with the IVIS (Perkinelmer, USA) after the mice were anesthetized using isoflurane. After 8 h of administration, the mice were euthanized. The main organs (including the heart, liver, spleen, lung, kidney, and colon) were excised, followed by the collection of fluorescence images via the IVIS. The fluorescence intensity analysis was performed by the IVIS and living image software (version 4.4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e4.10 \u003cem\u003eIn vivo\u003c/em\u003e immunofluorescence imaging\u003c/h2\u003e \u003cp\u003eThe colon tissue was prepared into frozen sections for immunofluorescence staining to further analyze the colocalization of BO/OMV-lipo@DiD and colon epithelial cells. Specifically, the tissues were fixed in 4% paraformaldehyde overnight and dehydrated in 30% sucrose for 24 h. Then, the tissues were embedded in OCT (optimal cutting temperature), stored at -80 ℃, and cut into slices (slice thickness 10 \u0026micro;m) by a freezing microtome (Leica, Germany). Next, the colon frozen sections were stained with Anti-Epcam mouse antibodies (1:100) and subsequent anti-mouse IgG conjugated with AF555 (1:1000), followed by counter staining with DAPI. The fluorescence images were observed with a laser scanning confocal microscope (SP8, Leica, Germany), and the Pearson correlation coefficient (PCC) was analyzed using Image J.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e4.11 Treatment of DSS-induced UC model\u003c/h2\u003e \u003cp\u003eFor exploring effects of the OMV proportion on the anti-UC efficacy of BO/OMV-lipo@LU, the UC mice model was induced by 3% DSS for 7 days and orally treated with 1:20-BO/OMV-lipo@LU, 1:50-BO/OMV-lipo@LU or 1:100-BO/OMV-lipo@LU. Amongst, the proportion referred to the ratio of OMV proteins to lipids in BO/OMV-lipo@LU. All formulation administration (luteolin dose of 17 mg/kg) was performed on predetermined days complying with the treatment regimen as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea. The healthy mice were provided with normal water as a control.\u003c/p\u003e \u003cp\u003eIn the therapeutic experiments, C57BL/6J mice were randomly divided into 7 groups, including i) the healthy control group, ii) the DSS control group, iii) the Free-LU-treated DSS group, iv) the Lipo@LU-treated DSS group, v) the BO-lipo@LU-treated DSS group, vi) the OMV-lipo@LU-treated DSS group, and vii) the BO/OMV-lipo@LU-treated DSS group. According to the treatment regimen in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, all formulations (equivalent luteolin dose of 17 mg/kg) were administrated once daily for 5 days, starting on the third day of DSS treatment. After 7 days of DSS treatment, mice received normal water without DSS for 3 days and were euthanized.\u003c/p\u003e \u003cp\u003eDuring the experimental periods, the body weight, stool consistency, and fecal blood of mice were monitored daily. The DAI score was calculated based on the summation of body weight loss (0: 0%, 1: 1\u0026ndash;5%,2: 6\u0026ndash;10%, 3: 11\u0026ndash;15%, 4: 15%), stool consistency state (0: hard, 1: soft, 3: diarrhea), and fecal occult blood (0: negative, 1: positive, 3: macroscopic)\u003csup\u003e56\u003c/sup\u003e. The fecal occult blood detection kit was used to test the degree of occult blood by the benzidine method.\u003c/p\u003e \u003cp\u003eAt the end of the experiments, the colon tissue was collected and measured for length. Then, the colon was washed with PBS to remove feces, and a segment from each group was fixed with 4% paraformaldehyde for histological analysis and immunofluorescence staining assays. Additionally, the remaining samples were stored at -80℃ for further use. H\u0026amp;E staining sections of colon tissue were prepared for histological assessment through further hydration and paraffin embedding. The colonic damage severity was scored conforming to a previously described scoring criterion as follows: inflammation cell infiltration (0: few inflammatory cells in the lamina propria, 1: enhanced granulocyte infiltration into the lamina propria, 2: extending into submucosa, 3: extending into muscular and serosal layer), crypts lesion (0: intact crypts, 1: loss of the basal one-third, 2: loss of the basal two-thirds, 3: entire crypt loss, 4: alternation of epithelial surface with erosion, 5: confluent erosion) and ulceration (0: absence of ulceration, 1: 1\u0026thinsp;~\u0026thinsp;2 foci of ulcerations, 2: 3\u0026thinsp;~\u0026thinsp;4 foci of ulcerations, 3: confluent ulceration)\u003csup\u003e58\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e4.12 \u003cem\u003eIn vivo\u003c/em\u003e ELISA analysis of inflammatory factors\u003c/h2\u003e \u003cp\u003eTo determine the concentration of cytokines in serum, mice blood was collected in the procoagulant tubes by eyeball extraction and centrifuged at 1000 g for 20 min to isolate serum. In addition, the cytokine concentration in colon tissues was also detected using the tissue homogenate sample. The colon segments were cryogenically homogenized (60 Hz, 4 min) in PBS containing 1% PMSF by a tissue grinder (Shanghai Jingxin Industrial Development Co., Ltd., China). Subsequently, the homogenized solution was centrifuged at 10,000 g for 10 min to obtain supernatant for further detection. Following the preparation of serum and colon tissue samples, the levels of cytokines, including IL-6, TNF-α, and IFN-γ, were quantified by ELISA assay kits according to the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e4.13 \u003cem\u003eIn vivo\u003c/em\u003e MPO, GSH, and SOD activity assay\u003c/h2\u003e \u003cp\u003eFor testing the ability of BO/OMV-lipo@LU to alleviate intestinal oxidative stress, the colon tissue homogenate of mice was prepared for MPO, SOD, and GSH activity analysis. According to the manufacturer\u0026rsquo;s instructions, the activity of MPO, SOD, and GSH was tested using an MPO activity assay kit, a SOD activity assay kit, and a GSH assay kit, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e4.14 \u003cem\u003eIn vivo\u003c/em\u003e immunofluorescence imaging of ZO-1 and occludin\u003c/h2\u003e \u003cp\u003eThe immunofluorescence staining method was employed to detect the expression of tight junction proteins (ZO-1 and occludin) in the colon epithelium. Specifically, the paraffin sections of colon tissue were prepared for multiplex immunofluorescence staining based on the method of tyramide signal amplification (TSA). The sections were dewaxed and placed in EDTA alkaline buffer for antigen repair, followed by 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to block endogenous peroxidase. Then, the tissue sections were blocked using blocking buffer to avoid nonspecific binding and stained with anti-ZO-1 rat monoclonal antibodies (1:50), followed by HRP-labeled goat anti-rat IgG (1:200) and TSA buffer containing the TYR-570Plus red dye. Next, the antibodies were eluted in the citrate antigen repair buffer (pH 6.0). The sections were subsequently reblocked and restained with anti-occludin rabbit monoclonal antibodies (1:200), HRP-labeled goat anti-rabbit IgG, and TSA buffer containing the TYR-520Plus green dye. Nuclei were counter-stained with DAPI. Immunofluorescence images were acquired by confocal laser-scanning microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e4.15 \u003cem\u003eIn vivo\u003c/em\u003e safety test\u003c/h2\u003e \u003cp\u003eOnce mice were sacrificed, the main organs (including heart, liver, spleen, lung, and kidney) were separated and weighed to calculate the organ index using the following formula:\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:\\text{O}\\text{r}\\text{g}\\text{a}\\text{n}\\:\\text{i}\\text{n}\\text{d}\\text{e}\\text{x}\\:\\left(\\text{%}\\right)=\\text{O}\\text{r}\\text{g}\\text{a}\\text{n}\\:\\text{w}\\text{e}\\text{i}\\text{g}\\text{h}\\text{t}/\\text{b}\\text{o}\\text{d}\\text{y}\\:\\text{w}\\text{e}\\text{i}\\text{g}\\text{h}\\text{t}\\:\\text{o}\\text{f}\\:\\text{m}\\text{i}\\text{c}\\text{e}\\times\\:100\\text{\\%}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThen, these organs were fixed in 4% paraformaldehyde and embedded in paraffin. The tissues were sliced, stained with H\u0026amp;E, and observed under a microscope for histological assessments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e4.16 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data were expressed as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical analysis was performed using GraphPad Prism software (Version 9.0). The two-tailed T-test and one-way ANOVA were applied to statistically analyze all data. The data were collected from at least three independent measurements. Statistical significance was defined as *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ****\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. ns indicated that there is no significant difference.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eXier Pan conceptualized, designed, and performed the experiments, analyzed and interpreted the data, wrote the original draft, and revised the manuscript. Yushu Li, Xiao Zhao, Jiaxin Zhang, Peng Xian, Yangjie Song, Yunrong Nan performed some experiments. Shuting Ni revised the manuscript. Kaili Hu conceptualized and designed the research, provided the resources and funding, supervised the experiments, and revised the manuscript. All authors have approved the final version of the manuscript for publication.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was supported by \u0026ldquo;Shuguang Program\u0026rdquo; supported by Shanghai Education Development Foundation and Shanghai Municipal Education Commission (22SG41), the National Natural Science Foundation of China (Grant No. 81773909), Shanghai Municipal Commission of Health and Family Planning (Grant No. 2017YQ060, China), and the combination of the medical care and health project of the Shanghai University of Traditional Chinese Medicine (YYKC-2021-01-008). We thank the staff members of the Integrated Laser Microscopy System at the National Facility for Protein Science in Shanghai (NFPS), Shanghai Advanced Research Institute, Chinese Academy of Sciences, China for instrument guidance.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe data related to this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNg SC et al (2017) Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century: a systematic review of population-based studies. Lancet 390:2769\u0026ndash;2778\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRol \u0026Aacute; et al (2021) Structure-based design of a Cortistatin analogue with immunomodulatory activity in models of inflammatory bowel disease. 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Mol Ther 22:69\u0026ndash;80\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bacterial outer membrane vesicle, Borneol, Liposome, Luteolin, Ulcerative colitis","lastPublishedDoi":"10.21203/rs.3.rs-4710969/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4710969/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUlcerative colitis (UC) is tightly linked to the dysbiosis of intestinal homeostasis, accompanied by the intestinal epithelial barrier destruction, subsequent inflammatory factor infiltration, and excessive oxidative stress. However, conventional therapeutics only focus on suppressing inflammation and often suffer from metabolic instability as well as limited targeting, thereby leading to suboptimal remission rates and severe side effects. Here, we initially fabricated bacterial outer membrane vesicle (OMV, from \u003cem\u003eStenotrophomonas maltophilia\u003c/em\u003e)-fused and borneol-modified liposomes (BO/OMV-lipo@LU) to deliver luteolin for targeted therapy of UC by recovering intestinal homeostasis. Benefitting from the chemotaxis-driven colon-targeting ability of OMVs and intestinal epithelial uptake promotion of borneol, the hybrid liposomes exhibited excellent targeting capability towards the inflamed colon. Moreover, under the combined effects of OMVs and borneol, interestingly, the hybrid liposomes achieved deep penetration into the mucosa for better uptake of luteolin by the intestinal epithelium. Notably, BO/OMV-lipo@LU with favorable biosafety exerted promising therapeutic efficacy and trilogically recovered intestinal homeostasis in UC by alleviating intestinal inflammation, modulating redox balance, and restoring intestinal epithelial barriers. These results demonstrated that such a biomimetic nanoplatform could be exploited as a safe and effective gut-targeted delivery system for UC intervention.\u003c/p\u003e","manuscriptTitle":"Chemotaxis-driven hybrid liposomes trilogically recover intestinal homeostasis for targeted therapy of ulcerative colitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-30 06:03:12","doi":"10.21203/rs.3.rs-4710969/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a0a74282-b1f2-42ef-bb06-8f3669c29f60","owner":[],"postedDate":"July 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":35266808,"name":"Health sciences/Diseases/Gastrointestinal diseases/Intestinal diseases/Inflammatory bowel disease/Ulcerative colitis"},{"id":35266809,"name":"Health sciences/Medical research/Drug development"}],"tags":[],"updatedAt":"2024-09-05T15:05:30+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-30 06:03:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4710969","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4710969","identity":"rs-4710969","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}