Application of Engineering Efferocytosis-Mimicking Nanovesicles in Atherosclerosis Treatment via M1 Macrophages and celluar lipid metabolic reprogramming

Application of Engineering Efferocytosis-Mimicking Nanovesicles in Atherosclerosis Treatment via M1 Macrophages and celluar lipid metabolic reprogramming | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Application of Engineering Efferocytosis-Mimicking Nanovesicles in Atherosclerosis Treatment via M1 Macrophages and celluar lipid metabolic reprogramming Jiaxuan Mei, Shanshan Yuan, Xiaoxi Fan, Bozhi Ye, Yixin Zhou, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8705147/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 Background and aims : Atherosclerosis is a chronic inflammatory disease driven by macrophage-mediated inflammation. This study aims to clarify the effect and mechanism of nanovesicles mimicking engineering efferocytosis on atherosclerosis, offering a multi-modal approach for atherosclerosis treatment. Methods : In this study, we investigated the therapeutic potential of Engineering efferocytosis-mimicking nanovesicles (EMNV), specifically S1P-PS-MMV and SIP-PS-MMV@SPD, in targeting and alleviating atherosclerotic lesions. Results : We demonstrated that S1P-PS-MMV could specifically target atherosclerotic plaques in a mouse model. Mechanistically, treatment with S1P-PS-MMV and S1P-PS-MMV@SPD reduced the infiltration of inflammatory cells, particularly pro-inflammatory M1 macrophages, as well as the expression of pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α).Furthermore,EMNV significantly reduced the expression levels of scavenger receptors SR-A1 and CD36 involved in lipid uptake, while increasing the expression of SR-B1 stabilizing plasma cholesterol, eventually led to celluar lipid metabolic reprogramming. Conclusion: This finding demonstrates that S1P-PS-MMV and S1P-PS-MMV@SPD can effectively target atherosclerotic lesions, reduce inflammatory cell infiltration, and modulate scavenger receptor expression in macrophages, thereby alleviating plaque formation. Atherosclerosis Macrophage EMNV Inflammation lipid metabolic reprogramming Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Atherosclerosis, recognized as a systemic inflammatory disorder affecting vascular homeostasis, has been etiologically linked to the pathogenesis of life-threatening circulatory conditions including myocardial infarction and cerebrovascular events 1 . Pathologically, advanced atherosclerotic lesions—known as plaques or atheromas—narrow the arterial lumen, restrict blood flow, and promote thrombosis. These processes can lead to ischemia, resulting in insufficient blood supply to downstream organs and tissues 2 .Emerging evidence underscores that macrophage populations derived from peripheral blood monocytes demonstrate microenvironmental remodeling capabilities within arterial walls 3 . These immunocompetent cells exhibit dual regulatory functions, orchestrating not only the primordial phase of lipid-rich core development but also critically modulating plaque destabilization mechanisms throughout the pathological continuum 4 . Consequently, the development of drugs aimed at targeting macrophage inflammation presents a highly promising therapeutic approach for the treatment of atherosclerosis 5 . Presently, among the various drug delivery systems available, nanocarrier vesicles originating from macrophage membranes exhibit superior targeting capabilities towards macrophages 6 . These multifunctional vesicles not only serve as efficient nanocarriers for targeted delivery but also retain intrinsic biological properties derived from their parent cell membranes, enabling macrophage-mediated tropism toward inflammatory lesions through specific cellular recognition mechanisms 7 . In the field of cardiovascular disease treatment, there have been successful cases utilizing macrophage-biomimetic nanoparticles for the treatment of coronary artery disease 8 . The macrophages within atherosclerotic plaques also promote a positive feedback loop, further attracting more inflammatory cells to gather in these areas 9 . Macrophages can recognize apoptotic cells by identifying surface markers such as sphingosine-1-phosphate (S1P) and phosphatidyl serine (PS), which are pivotal in the regulation of efferocytosis 10 . S1P, a metabolite of sphingolipids, acts as a chemoattractant and signaling molecule, facilitating macrophage recruitment and modulating inflammation via G protein-coupled receptors. PS, typically restricted to the inner leaflet of plasma membranes, externalizes during apoptosis and serves as a universal "eat-me" signal, interacting with phagocyte receptors to enable selective clearance of apoptotic cells through efferocytosis 11 . Spermidine (SPD), a naturally occurring polyamine present in all living cells, plays a crucial role in cellular homeostasis and disease modulation. It regulates key biological processes, including the induction of autophagy, reduction of oxidative stress, and suppression of inflammation by inhibiting pathways such as NLRP3 inflammasome activation 12 . SPD's capacity to both promote cellular repair and reduce pro-inflammatory cytokine production (e.g.,IL-1β and TNF-α ) synergizes with S1P/PS-mediated efferocytosis, enhancing therapeutic efficacy 13 . We strategically selected S1P and PS as dual-functional components for nanovesicle engineering, capitalizing on their complementary biological roles and therapeutic potential. In our previous studies, efferocytosis-mimicking nanovesicles (S1P-PS-MMV@SPD) could effectively target inflammatory site and inhibit the progression of rheumatoid arthritis 14 . This S1P/PS strategy offers significant potential for treating chronic inflammatory disorders marked by aberrant efferocytosis, such as rheumatoid arthritis or atherosclerosis. Herein, we employ this approach to treat atherosclerosis with the goal of developing innovative methodologies for its management. Concurrently, this endeavor expands the scope of application for our nanovesicle. Previous research 14 has demonstrated that S1P-PS-MMV and S1P-PS-MMV@SPD exhibit exceptional recruitment and rapid recognition capabilities, thus we selected these entities for subsequent experimental investigations. The objective was to ascertain the precise role and mechanism of the constructed EMNV within the context of atherosclerosis. 2. Results 2.1 EMNV tended towards AS inflammatory plaque location As shown in Fig. 1 A the preparation of EMNV, including S1P-PS-MMV and S1P-PS-MMV@SPD, have been established. Previous works have elaborated dynamic-light-scattering size distributions, zeta potentials, transmission-electron-microscopy images, polydispersity indices, drug-loading efficiencies or stability curves in serum. EMNV could target inflammation site due to the macrophage membrane 14 . Atherosclerosis is also an inflammation-driven disease mediated by macrophages. To evaluate the effects of EMNV on atherosclerosis, we first injected S1P-PS-MMV into the tail veins of atherosclerotic mice. Concurrently, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DID) was administered as a fluorescent indicator. After 8 hours, we observed the fluorescence intensity of DID in the mice's vasculature (Fig. 1 B) and found that the fluorescence intensity in the DID/S1P-PS-MMV group was significantly greater than that of the control group (only DID in vehicle) (Fig. 1 C), and it was notably concentrated in the atherosclerotic lesions (Fig. 1 B). Dynamic light scattering analysis revealed that the average diameter of S1P-PS-MMV@Spd was ∼412.8 ± 1.6 nm, which falls within the size range of natural apoptotic bodies (100–5000 nm) (Fig. 1 D). Meanwhile, the zeta potential of S1P-PS-MMV@Spd (− 8.2 ± 0.6 mV) was higher than that of S1P-PS-MMV(Fig. 1 E), indicating the successful loading of positively charged SPD into S1P-PS-MMV. 2.2 S1P-PS-MMV@SPD Alleviated Atherosclerotic Plaque Formation Since S1P-PS-MMV could target plaques in atherosclerosis, we further explored the functions of S1P-PS-MMV and S1P-PS-MMV@SPD in mouse atherosclerosis. After injecting S1P-PS-MMV and S1P-PS-MMV@SPD into HFD-fed ApoE −/− mice, compared with the saline group, the atherosclerotic plaques in the mice were significantly alleviated (Fig. 2 A-B). In the heart aortic root, the area of pathological (Fig. 2 C), the area of oil red staining (Fig. 2 D-E), and the area of fibrosis (Fig. 2 F-G) were significantly reduced. Moreover, the effects of S1P-PS-MMV@SPD were more pronounced than those of S1P-PS-MMV alone. This indicated that S1P-PS-MMV could effectively treat atherosclerosis in mice. The S1P-PS-MMV@SPD vesicles (Fig. 2 A-D), after being loaded with SPD, exhibited even more pronounced therapeutic effects.In addition, serum lipid levels were measured in mice. The lipid profile results ( Supplementary Fig. 1A )were consistent with the trends from earlier experiments. This trend was also reflected in the body weight ( Supplementary Fig. 1B )changes of the mice. 2.3 S1P-PS-MMV@SPD inhibited the aggregation of inflammatory cells in plaques EMNV primarily alleviated rheumatoid arthritis by modulating the content of inflammatory macrophages. To explore the mechanism by which EMNV alleviates the development of atherosclerosis, we first verified the effect of EMNV on inflammatory cells. By performing immunohistochemical staining for macrophage (F4/80, Fig. 3 A), neutrophil (Ly6G, Fig. 3 B), and monocyte (Ly6C, Fig. 3 C) markers on pathological tissue sections in the aortic root of HFD-fed ApoE −/− mice, we clearly observed an increase in the three major inflammatory cells in the saline group, with the most significant increase in macrophages (Fig. 3 A-D). Additionally, compared with the saline group, SPD and S1P-PS-MMV treatments resulted in a reduction in inflammatory cells, and the S1P-PS-MMV@SPD treatment showed the least positive area of inflammatory cells (Fig. 3 A-D). Meanwhile, the inflammatory factors IL-1β (Fig. 3 E) and TNF-α (Fig. 3 F) at the atherosclerotic plaque sites under S1P-PS-MMV and S1P-PS-MMV@SPD treatments also exhibited a similar decreasing trend (Fig. 3 E-G). These results indicated that EMNV treatment could reduce inflammatory infiltration in atherosclerosis in HFD-fed ApoE −/− mice, with the most significant changes observed in macrophages. 2.4 S1P-PS-MMV@SPD mainly inhibited the aggregation of M1 macrophages within the plaque Monocyte-derived macrophages are key inflammatory cells involved in the process of atherosclerosis, differentiating into various subtypes during the progression of atherosclerosis. Given the significant role that macrophages played in the treatment of mouse atherosclerosis with EMNV, we performed cellular immunofluorescence assays to assess the expression levels of macrophage subtypes M2 (CD206, Fig. 4 A-B) and M1 (CD86 Fig. 4 A and C ) within the aortic root plaques of HFD -fed mice treated with S1P-PS-MMV and S1P-PS-MMV@SPD. We found that the number of macrophages M1 significantly decreased due to the use of S1P-PS-MMV and S1P-PS-MMV@SPD treatments, with the S1P-PS-MMV@SPD group showing a more pronounced effect (Fig. 3 D). Macrophages M2 tend to decrease after treatment with EMNV, but there is no significant statistical significance (Fig. 3 E). This might be the EMNV had a good targeting and inhibitory effect on M1-type inflammatory macrophages. 2.5 S1P-PS-MMV@SPD Inhibited Scavenger Receptors in M1 Macrophages Stimulated by ox-LDL To elucidate the molecular mechanism of EMNV in atherosclerotic M1 macrophages, we obtained mouse peritoneal primary macrophages and stimulated using ox-LDL (50 µg/ml, 24h). By culturing cells with PBS, S1P-PS-MMV, and S1P-PS-MMV@SPD respectively, we performed oil red staining on the cells. The results indicated a significant reduction in lipid accumulation within the cells due to the application of S1P-PS-MMV and S1P-PS-MMV@SPD, with a more pronounced effect observed in S1P-PS-MMV@SPD group (Fig. 5 A). Given the intimate relationship between macrophage lipids and scavenger receptors, we conducted an assessment of the scavenger receptor content within the cells. The findings revealed a significant reduction in the expression levels of SRA1 (Fig. 5 B-C) and CD36 (Fig. 5 B and D ), proteins responsible for regulating lipid uptake by macrophages. Conversely, there was a marked increase in the expression of SRB1(Fig. 5 B and E ), which stabilize plasma cholesterol through the reverse cholesterol transport pathway 15 (Fig. 5 B-E). Additionally, the ratio of cholesteryl esters to total cholesterol diminished, indicating a mitigation in foam cell formation within macrophages 15 (Fig. 5 F). The impact of S1P-PS-MMV@SPD was more pronounced than that of S1P-PS-MMV alone (Fig. 5 A-F). These results suggested that EMNV might suppress the expression of scavenger receptors in inflammatory macrophages, thereby decreasing cholesterol accumulation and alleviating foam cell formation in these cells. 3. Discussion The present study revealed a multi-layered mechanism involving plaque targeting, inflammatory cells regulation, and lipid metabolic reprogramming of macrophages by effecting the expression of scavenger receptors, thereby reducing macrophage foam cell formation. We demonstrated that the therapeutic potential of EMNV in atherosclerosis by targeting modulation of inflammatory macrophages. Currently, emerging therapeutic strategies aimed at macrophage inflammation in atherosclerosis encompass a variety of approaches. These include targeting specific receptors on the surface of macrophages, utilizing nanocarriers to deliver drugs that inhibit the inflammatory response of these cells, and also involve modulating the macrophage phenotype. For instance, efforts are made to shift the macrophage phenotype from a pro-inflammatory M1 type to an anti-inflammatory M2 type 17 . Furthermore, therapeutic interventions focus on altering macrophage metabolic pathways, such as cholesterol metabolism and inflammatory signaling pathways, to influence plaque stability 17 – 19 . Additionally, the treatment of atherosclerosis can be facilitated by regulating the interaction between macrophages and other cell types, including CD8 + T cells 20 . These pathways also have good therapeutic effects, and the related mechanisms and targets may be combined with nanomaterials in the future to create more targeted treatment methods. In this study, we firstly found that the enhanced DID/S1P-PS-MMV accumulation in atherosclerotic lesions validates the design rationale of macrophage-derived vesicles for inflammatory targeting. This phenomenon may be attributed to the natural tropism of macrophage-derived vesicles toward inflammatory sites 21 , 22 and the attraction of their loaded apoptotic signals SIP and PS on anti-inflammatory macrophages surrounding the plaque. This targeting efficiency establishes a critical foundation for subsequent therapeutic effects. Secondly, we observed that S1P-PS-MMV@SPD has superior efficacy in reducing plaque burden than S1P-PS-MMV, suggesting synergistic effects between the vesicle platform and SPD payload. For potential reasons, SPD can further improve nanovesicle's efficacy because of its multi anti-inflammatory effects. As reported, SPD plays a critical role in anti-inflammatory by suppressing oxidative stress, and disrupts inflammatory pathways such as NLRP3 inflammasome activation 12 , 23 , and exihibiting excellent anti-rejection with inflammation-regulating functions in drug-loading applications with other materials 24 . Meanwhile, these differential results also indicated that the vesicle itself possesses inherent anti-inflammatory properties, SPD loading amplifies therapeutic effects through additional pathways. This is consistent with previous report on rheumatoid arthritis therapy 14 , now extended to atherosclerotic treatment. Thirdly, our cellular analysis provides a potential mechanism of action through macrophage polarization modulation. As we known, the M1 macrophages-specific inhibition is particularly relevant given the central role of M1-derived inflammatory cytokines in plaque instability 25 . Here, our findings indicate that EMNV predominantly influences the population of M1 macrophages in atherosclerosis, with no significant statistical difference observed in the quantity of M2 macrophages. This disparity may be attributed to the predominance of M1 macrophages in atherosclerotic conditions 26 , as well as the possible inhibition of SPD on the inflammatory response from M1 macrophages. Nowadays, many related targeted therapeutics and drug development tend to focus more on distinguishing between M1 and M2 for precise treatment of atherosclerosis 18 , 27 , 28 . The selective reduction of M1 macrophages without significant M2 population alteration suggests a precision modulation strategy distinct from conventional anti-inflammatory approaches. Moreover, these M1 macrophages are known to engulf lipids and subsequently differentiate into foam cells, secreting pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, thereby exacerbating the inflammatory response 5 . The observed TNF-α and IL-1β reduction induced by EMNV treatment may also involved in this macrophage’s polarization shift, potentially creating an anti-inflammatory microenvironment conducive to plaque stabilization. In atherosclerotic pathogenesis, dysregulated lipid metabolism synergizes with inflammatory mechanisms to drive plaque formation. Emerging studies highlight inflammation-induced lipid metabolic reprogramming as a pivotal pathobiological process 29 – 31 . Scavenger receptors onstitute a diverse family of receptor molecules that are ubiquitously expressed on the plasma membrane of phagocytic cells. This family encompasses several members, such as SR-A1, SR-B1, CD36 32,33 . These receptors are pivotal in lipid metabolism and cardiovascular health, primarily through their interaction with lipoproteins and their role in modulating intracellular lipid transport 32 , 34 . Previous studies have demonstrated that nucleic acid nanovesicles predominantly gain cellular entry via endocytosis that is mediated by scavenger receptors and dependent on caveolae 35 . This underscores the association between nanovesicles and scavenger receptors. Our investigation indicates that the dual regulation of scavenger receptors in macrophages represents a novel therapeutic mechanism of EMNV. The differential modulation of a decrease in SRA1/CD36 versus an increase in SRB1 suggests EMNV's ability to reprogram macrophage lipid metabolism, rather than simple inhibition. This switching of scavenger receptors may explain the reduced cholesteryl ester accumulation in macrophages, effectively disrupting the vicious cycle of ox-LDL uptake and foam cell formation. The precise mechanisms underlying this phenomenon could be modulated by the structural component among nanovesicles and the pathological microenvironment. Furthermore, scavenger receptors are recognized to exert a substantial regulatory influence on macrophage inflammation in atherosclerosis, extending beyond their role in lipid transport 36 , 37 . For example, SR-A1 and CD36 facilitate the activation of downstream signaling pathways, including NF-κB and JNK, by internalizing oxidized lipoproteins, thereby promoting inflammatory responses 38 . Conversely, the activation of SRB1 can attenuate the secretion of pro-inflammatory cytokines, such as TNF-α and IL-6, while enhancing the expression of anti-inflammatory cytokines, such as IL-10, to mitigate inflammation 39 . Studies indicate that pyroptosis effector protein, can induce IL-1β release and amplify inflammatory cascades in M1 macrophages 18 . We speculate that EMNV's anti-pyroptotic components, like SPD, may indirectly alter gene expression by affecting macrophages' inflammatory responses, thereby influencing cellular lipid synthesis and transport 29 . Additionally, existing research indicates that celluar lipid metabolism may be hindered by oxidative stress 40 . The suppression of oxidative stress in inflammatory cells by SPD may promote a positive cycle of lipids, thereby regulating the normal distribution of lipid transport receptors. Therefore, the direct mechanism of action of EMNV on macrophage scavenger receptors in atherosclerosis necessitates further investigation. Besides, another limitation warrant consideration. While our in vivo models demonstrate therapeutic efficacy of EMNV, the long-term safety profile of repeated EMNV administration needs systematic evaluation. 4. Conclusion In our investigation, we found the application of EMNV to alleviate the development of atherosclerosisHere the combination of targeted delivery, macrophage polarization control, and celluar lipid metabolic reprogramming addresses multiple pathological aspects simultaneously, followed by the reduction of foam cell formation and inflammatory responses, eventually led to mitigate plaque development in the context of atherosclerosis. These findings highlight the potential of EMNV as a novel therapeutic strategy for atherosclerosis by targeting macrophage-mediated inflammation and affecting lipid metabolism, and position EMNV as a promising multi-modal therapeutic platform for atherosclerosis. 5. Methods 5.1 Reagents Ox-LDL was purchased from Yiyuan Biotechnology (YB-002, Guangzhou, China). Antibodies against F4/80 (sc-377009) and TNF-α (sc-52746) were obtained from Santa Cruz (CA, USA). Antibodies against SRA1 (24655-1-AP), SRB1 (ET1602-32), CD36 (ET1701-24), AEBP1 (ER61507), Ly6C (HA500088), Ly6G (0809 − 11), CD206 (ET1702-04), and CD86 (ET1606-50) were sourced from HUABIO (Hangzhou, China). Antibodies against Il-1β (ab9722) was obtained from Abcam (Cambridge, UK). And antibodies against GAPDH (60004-1-Ig) from Proteintech (Wuhan, China) were also included in the study. The kit of HE staining, Oil Red O staining, Masson’s trichrome staining were all purchasd from Solarbio (Beijing, China), Amplex red cholesterol and cholesteryl ester assay kit were obtained from Beyotime Biotechnology (shanghai, China) and the manufacturer's instructions were followed during use. 5.2 Preparation of EMNV Following our previous research 14 , macrophage membranes were first extracted, then sonicated, and subsequently passed twice through a 5 µm polycarbonate membrane to yield MMV. EMNV were prepared by introducing PS and S1P onto the MMV surface and encapsulating SPD within the MMV using a co-extrusion technique. Specifically, EMNV, with 16.8 µg of membrane protein, was capable of co-encapsulating approximately 1µg of S1P and 10 µg of SPD. 5.3 Animals ApoE -/- mice on C57BL/6J background were purchased from GemPharmatech (Nanjing, China). We randomly divided 8-week-old male mice into four groups. All studies utilized double-blind randomized controlled trials.Mice in the atherosclerotic group were fed with high-fat diet (HFD), while mice in the control group were fed with Normal diet. Mice in HFD were intravenously injected with saline, S1P-PS-MMV, or S1P-PS-MMV@SPD respectively. The mice were raised at Animal Experimental Center of the First Affiliated Hospital of Wenzhou Medical University, following ethical guidelines approved by the Laboratory Animal Ethics Committee (document no. WYYY-IACUC-AEC-2024-055). All experiments were complied with Directive 2010/63/EU of the European Parliament on animal protection for scientific research. After raising, mice were euthanized with sodium Pentobarbital (100 mg/kg, once, ip.), and their blood vessels and hearts were collected. 5.4 Inflorescence Select age-appropriate ApoE −/− mice on a C57BL/6J background that have been fed a high-fat diet. The experimental group of mice receive tail vein injections of S1P-PS-MMV@SPD-DID, while the control group receive tail vein injections of the DID alone. The fluorescence intensity of various organs in the mice will be detected using a live animal imaging system. 5.5 Atherosclerotic lesion analysis After removing the fat from the mouse's blood vessels, the aortic arch was cut open starting from the aortic arch to expose the intima, which was then stained with Oil Red O staining solution. Photos were taken to calculate the area percentage of lipid plaques. For the vascular valve slices, a cryostat microtome was used to make cross-sectional cuts through the frozen heart. After initially displaying the valve tissue at 8–10 mm, the following sections was transferred onto adhesive slides. Subsequently, the slides were then subjected to HE staining, Oil Red O staining, Masson’s trichrome staining, and immunohistochemistry. 3 to 6 microscope fields were randomly selected by blinded experimenters for analysis. 5.6 Immunohistochemistry After washing the frozen sections, they were blocked with 5% BSA, incubated with the primary antibody overnight, and then observed under a confocal microscope after incubation with Alexa-488/647 (Abcam, 1:5000) and DAPI. 5.7 Immunofluorescence For immunohistochemical staining, primary antibodies were used. Subsequently, the sections underwent incubation with secondary antibodies, diaminobenzidine, and hematoxylin. 5.8 Cell culture Mouse primary peritoneal macrophages (MPMs) were harvested from the peritoneal cavity of ApoE −/− mice as previously described (Han et al., 2022b). After pretreatment with PBS, S1P-PS-MMV, or S1P-PS-MMV@SPD, ox-LDL (50 µg/ml) was added to induce the atherosclerotic process in macrophages according to the manufacturer's instructions, followed by Oil Red O staining and Western Blot experiments. 5.9 Cytology Oil Red O staining According to the Oil Red O Staining Kit (Solarbio, Beijing, China), the manufacturer's instructions were followed during use. 5.10 Westernblots. Separate protein samples using SDS-PAGE gel, then transfer proteins onto an NC membrane. After blocking with 5% BSA at room temperature for two hours, incubate with primary antibody overnight. The next day, incubate with secondary antibody (catalogue nos. A0216 and A0208, Beyotime Biotechnology, Shanghai, China) and expose using an exposure machine (Tanon-5200, Serial Number 14T12NPFLI6-901). 5.11 Statistical Analysis. The experimental evaluation and data analysis were performed by personnel who did not participate in sampling. Statistical analysis was performed using GraphPad Prism version 10.1.2. First, conduct a normality test (Shapiro-Wilk test) on the data. For data that do not conform to a normal distribution, perform non-parametric analysis (Mann-Whitney test for 2 groups; Kruskal-Wallis test with Dunn's multiple comparisons for more than 2 groups). For normally distributed data, use the Student's t-test or One-Way ANOVA with Bonferroni correction. P value < 0.05 was considered to indicate statistical significance Abbreviations EMNV Efferocytosis-Mimicking Nanovesicles MMV macrophage membrane vesicles PS phosphatidyl serine S1P sphingosine-1-phosphate H&E Hematoxylin and eosin HFD high-fat diet ND norml diet Tnf-α tumor necrosis factor-α IL-1β Interleukin-1β ox-LDL oxidized low-density lipoprotein. Declarations Acknowledgment The authors are grateful to the Scientific Research Center of Wenzhou Medical University for their assistance with the immunofluorescence experiments. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author contributions Zhouqing Huang,Junjie Deng and Jianmin li contributed to the literature search and study design.Jiaxuan Mei,Shanshan Yuan, Xiaoxi Fan, Bozhi Ye,Yixin Zhou ,Zhentong Yang and Zhuoqun Wang performed the experiments and analyzed the data. Zhouqing Huang and Junjie Deng approve the version to be published. Jiaxuan Mei and Xiaoxi Fan participated in the drafting of the article. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy. Sources of Funding This work was supported by the Key project of Zhejiang Provincial Health Commission (WKJ-ZJ-2540) and S Discipline Plan of the Affiliated First Hospital of Wenzhou Medical University(A), and Natural Science Foundation of Zhejiang Province (Grant No.LMS25H020006), and Zhejiang Province Key Laboratory of Intelligent Cancer Biomarker Discovery and Translation (G2023006 and G2023014). Data availability The data underlying this article will be shared on reasonable request to the corresponding author. 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Mol Med 2011; 17 :1056-1064. doi: 10.2119/molmed.2011.00141 Hu S, Zhu Y, Zhao X, Li R, Shao G, Gong D, Hu C, Liu H, Xu K, Liu C, Xu M, Zhao Z, Li T, Hu Z, Shao M, Liu J, Li X, Wu H, Li J, Xu Y. Hepatocytic lipocalin-2 controls HDL metabolism and atherosclerosis via Nedd4-1-SR-BI axis in mice. Dev Cell 2023; 58 :2326-2337. doi: 10.1016/j.devcel.2023.09.007 Govaere O, Petersen SK, Martinez-Lopez N, Wouters J, Van Haele M, Mancina RM, Jamialahmadi O, Bilkei-Gorzo O, Lassen PB, Darlay R, Peltier J, Palmer JM, Younes R, Tiniakos D, Aithal GP, Allison M, Vacca M, Göransson M, Berlinguer-Palmini R, Clark JE, Drinnan MJ, Yki-Järvinen H, Dufour JF, Ekstedt M, Francque S, Petta S, Bugianesi E, Schattenberg JM, Day CP, Cordell HJ, Topal B, Clément K, Romeo S, Ratziu V, Roskams T, Daly AK, Anstee QM, Trost M, Härtlova A. Macrophage scavenger receptor 1 mediates lipid-induced inflammation in non-alcoholic fatty liver disease. J Hepatol 2022; 76 :1001-1012. doi: 10.1016/j.jhep.2021.12.012 Trujillo J, Calvert AE, Rink JS, Perez White BE, Sepulveda F, Biyashev D, Lu KQ, Lavker RM, Peng H, Thaxton CS. Keratinocyte SR-B1 expression and targeting in cytokine-driven skin inflammation. Commun Med (Lond) 2025; 5 :100. doi: 10.1038/s43856-025-00804-y Zhang Q, Shen X, Yuan X, Huang J, Zhu Y, Zhu T, Zhang T, Wu H, Wu Q, Fan Y, Ni J, Meng L, He A, Shi C, Li H, Hu Q, Wang J, Chang C, Huang F, Li F, Chen M, Liu A, Ye S, Zheng M, Fang H. Lipopolysaccharide binding protein resists hepatic oxidative stress by regulating lipid droplet homeostasis. Nat Commun 2024; 15 :3213. doi: 10.1038/s41467-024-47553-5 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8705147","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":586455099,"identity":"08a12c1c-7bd1-435a-b09d-770258484b5e","order_by":0,"name":"Jiaxuan Mei","email":"","orcid":"","institution":"First Affiliated Hospital of Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiaxuan","middleName":"","lastName":"Mei","suffix":""},{"id":586455100,"identity":"1d97de1b-d094-400c-a5cb-eec378bc9ac2","order_by":1,"name":"Shanshan Yuan","email":"","orcid":"","institution":"Joint centre of Translational Medicine,Wenzhou institute,University of Chinese Academy of Sciences,Wenzhou,Zhejian","correspondingAuthor":false,"prefix":"","firstName":"Shanshan","middleName":"","lastName":"Yuan","suffix":""},{"id":586455101,"identity":"e98161ed-7289-4cb4-8be7-3b03f91616b6","order_by":2,"name":"Xiaoxi Fan","email":"","orcid":"","institution":"First Affiliated Hospital of Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoxi","middleName":"","lastName":"Fan","suffix":""},{"id":586455102,"identity":"b35bc9e5-7cac-4f0e-865a-24f263cc4cfe","order_by":3,"name":"Bozhi Ye","email":"","orcid":"","institution":"First Affiliated Hospital of Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bozhi","middleName":"","lastName":"Ye","suffix":""},{"id":586455103,"identity":"9a0146d8-6a85-4b7e-af76-e33776784433","order_by":4,"name":"Yixin Zhou","email":"","orcid":"","institution":"First Affiliated Hospital of Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yixin","middleName":"","lastName":"Zhou","suffix":""},{"id":586455104,"identity":"7d4f0244-5156-4fc6-8e73-83962aec7816","order_by":5,"name":"Zhentong Yang","email":"","orcid":"","institution":"First Affiliated Hospital of Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhentong","middleName":"","lastName":"Yang","suffix":""},{"id":586455105,"identity":"33cf5df0-5349-43b2-bf65-3cd2c140495e","order_by":6,"name":"Zhuoqun Wang","email":"","orcid":"","institution":"First Affiliated Hospital of Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhuoqun","middleName":"","lastName":"Wang","suffix":""},{"id":586455106,"identity":"a722da0d-c89e-4348-bb26-c5ee3516dafd","order_by":7,"name":"Jianmin Li","email":"","orcid":"","institution":"First Affiliated Hospital of Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jianmin","middleName":"","lastName":"Li","suffix":""},{"id":586455107,"identity":"b547da68-73e1-4194-a87e-d25eb5b3b978","order_by":8,"name":"Junjie Deng","email":"","orcid":"","institution":"Joint centre of Translational Medicine,Wenzhou institute,University of Chinese Academy of Sciences,Wenzhou,Zhejian","correspondingAuthor":false,"prefix":"","firstName":"Junjie","middleName":"","lastName":"Deng","suffix":""},{"id":586455108,"identity":"00721501-153e-4b33-8a01-2dfdaa1b5d2b","order_by":9,"name":"Zhouqing Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYJCCA4wNDAz8zMwHH5CmRbKdLdmAeGtAWgzO85gJEKVafkb6wwMfd9TJbj7MYMbAUGMTTVCLwY0cg4Mzz7AZbzvMkPaA4VhabgNBLRI5DId523gSgVqOGzA2HCasBeiwB4f/tkkkbm5mbJMgSgvDjQSDw4xtBokbmJnZiNNicOaNwcHetgTjGYfZmA0SiPGLfHv64w8/2+pk+/vPf3zwocaGCIdBASNYZQKxyhFaRsEoGAWjYBRgAwADDEM/i6jBiwAAAABJRU5ErkJggg==","orcid":"","institution":"First Affiliated Hospital of Wenzhou Medical University","correspondingAuthor":true,"prefix":"","firstName":"Zhouqing","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2026-01-27 03:08:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8705147/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8705147/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102236142,"identity":"c3391efe-39ab-4c84-882c-43638ef1dc60","added_by":"auto","created_at":"2026-02-09 16:19:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":327261,"visible":true,"origin":"","legend":"\u003cp\u003ePreparation of S1P-PS-MMV and S1P-PS-MMV@SPD as well as the validation of their targeting effect. \u003cstrong\u003eA\u003c/strong\u003e. Schematic illustration of SIP-PS-MMV and S1P-PS-MMV@SPD fabrication. \u003cstrong\u003eB\u003c/strong\u003e. Fluorescence imaging map of the en face aorta of atherosclerotic mice injected with DID and DID/S1P-PS-MMV. \u003cstrong\u003eC\u003c/strong\u003e. Quantification of the fluorescence indensity of Fig1 B . \u003cstrong\u003eD\u003c/strong\u003e. Representative TEM image of S1P-PS-MMV@SPD. \u003cstrong\u003eE\u003c/strong\u003e. The zeta potentials of S1P-PS-MMV@SPD.(n=3)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8705147/v1/a7184a7bbe0eaf7eade63d92.png"},{"id":102236134,"identity":"6d3fb350-e4ac-402d-bf32-dbb3853a1596","added_by":"auto","created_at":"2026-02-09 16:19:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":438026,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS1P-PS-MMV@SPD Alleviates Atherosclerotic Plaque Formation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApoE\u003csup\u003e-/-\u003c/sup\u003e mice were fed a HFD for 8 weeks, followed by administration of SIP-PS-MMV and SIP-PS-MMV@SPD for an additional 4 weeks. \u003cstrong\u003eA-B\u003c/strong\u003e. Oil Red O staining of the en face aorta (A) and quantification (B). \u003cstrong\u003eC-D\u003c/strong\u003e Oil Red O staining (C) and quantification of positive areas (D) of the aortic root.(scale bar, 200 μm). \u003cstrong\u003eE-F\u003c/strong\u003e Masson's trichrome staining (E) and quantification of fibrotic areas (F) in the aortic root.(scale bar, 200 μm). \u003cstrong\u003eG\u003c/strong\u003e. HE staining of the aortic root. (N=6).(scale bar, 200 μm).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8705147/v1/af3d59a06cf5e938a6564b06.png"},{"id":102236137,"identity":"df462340-d961-4165-b18d-b75a84fca070","added_by":"auto","created_at":"2026-02-09 16:19:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":537860,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSIP-PS-MMV@SPD inhibits the aggregation of inflammatory cells in plaques\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApoE\u003csup\u003e-/-\u003c/sup\u003e mice were fed a HFD for 8 weeks, followed by administration of SIP-PS-MMV and SIP-PS-MMV@SPD for an additional 4 weeks. \u003cstrong\u003eA-B \u003c/strong\u003eImmunohistochemical staining of F4/80 (\u003cstrong\u003eA\u003c/strong\u003e) was performed on the aortic root, and the positive areas were quantified (\u003cstrong\u003eB\u003c/strong\u003e).(scale bar, 200 μm). \u003cstrong\u003eC-D \u003c/strong\u003eImmunohistochemical staining of Ly6G (\u003cstrong\u003eC\u003c/strong\u003e) in the aortic root and quantification of Ly6G-positive areas (\u003cstrong\u003eD\u003c/strong\u003e).(scale bar, 200 μm). \u003cstrong\u003eE-F \u003c/strong\u003eImmunohistochemical staining of Ly6C (\u003cstrong\u003eE\u003c/strong\u003e) was performed on the aortic root, and the positive areas were quantified (\u003cstrong\u003eF\u003c/strong\u003e).(scale bar, 200 μm) \u003cstrong\u003eG-H \u003c/strong\u003eImmunohistochemical staining of IL-1β (G) performed locally on the aortic root and quantification of IL-1β positive areas (H).(scale bar, 100 μm) \u003cstrong\u003eI-J \u003c/strong\u003eImmunohistochemical staining of TNF-α (\u003cstrong\u003eI\u003c/strong\u003e) was performed locally on the aortic root, and the positive areas were quantified (\u003cstrong\u003eJ\u003c/strong\u003e). (n=6).(scale bar, 100 μm).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8705147/v1/c8441734f6f2b910ed85bdf8.png"},{"id":102236139,"identity":"b5ef807e-e2f6-47f8-aa0f-d7aa3dc741f1","added_by":"auto","created_at":"2026-02-09 16:19:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":268249,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS1P-PS-MMV@SPD mainly inhibits the aggregation of M1 macrophages within the plaque\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApoE\u003csup\u003e-/-\u003c/sup\u003e mice were fed a HFD for 8 weeks, followed by administration of SIP-PS-MMV and SIP-PS-MMV@SPD for an additional 4 weeks. \u003cstrong\u003eA-B \u003c/strong\u003eImmunofluorescence images showing the localization of CD206 (A) and CD86 (B) in aortic sections. \u003cstrong\u003eC\u003c/strong\u003e Immunofluorescence images showing the colocalization of CD206 and CD86 in aortic sections. \u003cstrong\u003eD-E\u003c/strong\u003e Quantification of CD206 (D) and CD86 (E) positive fluorescence staining. (N=3).(scale bar, 50 μm).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8705147/v1/ec46066fc64fe461dd5dd167.png"},{"id":102297484,"identity":"75212b8d-f4f4-4b24-87c3-aac33f658bb0","added_by":"auto","created_at":"2026-02-10 10:27:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":190872,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS1P-PS-MMV@SPD Inhibits Scavenger Receptors in M1 Macrophages Stimulated by ox-LDL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMPMs were pretreated with S1P-PS-MMV and S1P-PS-MMV@SPD or PBS (1 h) and then stimulated with ox-LDL (50 μg/ml, 24 h). \u003cstrong\u003eA\u003c/strong\u003e Oil Red O staining of ox-LDL-induced MPMs pretreated with S1P-PS-MMV and SIP-PS-MMV@SPD. \u003cstrong\u003eB\u003c/strong\u003e Immunoblot analysis of protein SRA1, SRB1, and CD36. \u003cstrong\u003eC-E\u003c/strong\u003e. Densitometric quantification of immunoblot detection of protein SRA1 (\u003cstrong\u003eC\u003c/strong\u003e), SRB1 (\u003cstrong\u003eD\u003c/strong\u003e), and CD36 (\u003cstrong\u003eE\u003c/strong\u003e). \u003cstrong\u003eF\u003c/strong\u003e. Ratios of cholesterol ester (CE) and total cholesterol (TC) in MPMs. (n=3).(scale bar, 20 μm).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8705147/v1/233eb9f559f4fc48e6a2e9d8.png"},{"id":103224151,"identity":"0430e7c5-0cd0-4f08-8bf3-e0187a1e2583","added_by":"auto","created_at":"2026-02-23 10:42:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2676213,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8705147/v1/7a513927-bc37-4ee4-8c2f-3781539d60dd.pdf"},{"id":102297396,"identity":"b351f95f-aa8e-4c38-bd0c-f3200fc6c2c6","added_by":"auto","created_at":"2026-02-10 10:27:17","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":106941,"visible":true,"origin":"","legend":"","description":"","filename":"originaluncroppedanduncutimagesofthemembranes.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8705147/v1/1704a1057d260c4f0dbfcf18.pdf"},{"id":102236140,"identity":"3083b195-dfe8-4330-bad3-614ddf0b6e4f","added_by":"auto","created_at":"2026-02-09 16:19:13","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":56254,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8705147/v1/8694476ce25af2e27aece9d5.tif"},{"id":102236141,"identity":"9819ccf8-5913-4328-8799-5b87a8219cbf","added_by":"auto","created_at":"2026-02-09 16:19:13","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2378597,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicabstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-8705147/v1/351b0daecc1badb4e9e7a84f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Application of Engineering Efferocytosis-Mimicking Nanovesicles in Atherosclerosis Treatment via M1 Macrophages and celluar lipid metabolic reprogramming","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAtherosclerosis, recognized as a systemic inflammatory disorder affecting vascular homeostasis, has been etiologically linked to the pathogenesis of life-threatening circulatory conditions including myocardial infarction and cerebrovascular events\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Pathologically, advanced atherosclerotic lesions\u0026mdash;known as plaques or atheromas\u0026mdash;narrow the arterial lumen, restrict blood flow, and promote thrombosis. These processes can lead to ischemia, resulting in insufficient blood supply to downstream organs and tissues\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.Emerging evidence underscores that macrophage populations derived from peripheral blood monocytes demonstrate microenvironmental remodeling capabilities within arterial walls\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. These immunocompetent cells exhibit dual regulatory functions, orchestrating not only the primordial phase of lipid-rich core development but also critically modulating plaque destabilization mechanisms throughout the pathological continuum\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eConsequently, the development of drugs aimed at targeting macrophage inflammation presents a highly promising therapeutic approach for the treatment of atherosclerosis\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Presently, among the various drug delivery systems available, nanocarrier vesicles originating from macrophage membranes exhibit superior targeting capabilities towards macrophages\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. These multifunctional vesicles not only serve as efficient nanocarriers for targeted delivery but also retain intrinsic biological properties derived from their parent cell membranes, enabling macrophage-mediated tropism toward inflammatory lesions through specific cellular recognition mechanisms\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In the field of cardiovascular disease treatment, there have been successful cases utilizing macrophage-biomimetic nanoparticles for the treatment of coronary artery disease\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The macrophages within atherosclerotic plaques also promote a positive feedback loop, further attracting more inflammatory cells to gather in these areas\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMacrophages can recognize apoptotic cells by identifying surface markers such as sphingosine-1-phosphate (S1P) and phosphatidyl serine (PS), which are pivotal in the regulation of efferocytosis\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. S1P, a metabolite of sphingolipids, acts as a chemoattractant and signaling molecule, facilitating macrophage recruitment and modulating inflammation via G protein-coupled receptors. PS, typically restricted to the inner leaflet of plasma membranes, externalizes during apoptosis and serves as a universal \"eat-me\" signal, interacting with phagocyte receptors to enable selective clearance of apoptotic cells through efferocytosis\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Spermidine (SPD), a naturally occurring polyamine present in all living cells, plays a crucial role in cellular homeostasis and disease modulation. It regulates key biological processes, including the induction of autophagy, reduction of oxidative stress, and suppression of inflammation by inhibiting pathways such as NLRP3 inflammasome activation\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. SPD's capacity to both promote cellular repair and reduce pro-inflammatory cytokine production (e.g.,IL-1β and TNF-α ) synergizes with S1P/PS-mediated efferocytosis, enhancing therapeutic efficacy\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. We strategically selected S1P and PS as dual-functional components for nanovesicle engineering, capitalizing on their complementary biological roles and therapeutic potential. In our previous studies, efferocytosis-mimicking nanovesicles (S1P-PS-MMV@SPD) could effectively target inflammatory site and inhibit the progression of rheumatoid arthritis\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. This S1P/PS strategy offers significant potential for treating chronic inflammatory disorders marked by aberrant efferocytosis, such as rheumatoid arthritis or atherosclerosis.\u003c/p\u003e \u003cp\u003eHerein, we employ this approach to treat atherosclerosis with the goal of developing innovative methodologies for its management. Concurrently, this endeavor expands the scope of application for our nanovesicle. Previous research\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e has demonstrated that S1P-PS-MMV and S1P-PS-MMV@SPD exhibit exceptional recruitment and rapid recognition capabilities, thus we selected these entities for subsequent experimental investigations. The objective was to ascertain the precise role and mechanism of the constructed EMNV within the context of atherosclerosis.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 EMNV tended towards AS inflammatory plaque location\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA the preparation of EMNV, including S1P-PS-MMV and S1P-PS-MMV@SPD, have been established. Previous works have elaborated dynamic-light-scattering size distributions, zeta potentials, transmission-electron-microscopy images, polydispersity indices, drug-loading efficiencies or stability curves in serum. EMNV could target inflammation site due to the macrophage membrane\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Atherosclerosis is also an inflammation-driven disease mediated by macrophages. To evaluate the effects of EMNV on atherosclerosis, we first injected S1P-PS-MMV into the tail veins of atherosclerotic mice. Concurrently, 1,1\u0026prime;-dioctadecyl-3,3,3\u0026prime;,3\u0026prime;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DID) was administered as a fluorescent indicator. After 8 hours, we observed the fluorescence intensity of DID in the mice's vasculature (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and found that the fluorescence intensity in the DID/S1P-PS-MMV group was significantly greater than that of the control group (only DID in vehicle) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), and it was notably concentrated in the atherosclerotic lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Dynamic light scattering analysis revealed that the average diameter of S1P-PS-MMV@Spd was \u0026sim;412.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 nm, which falls within the size range of natural apoptotic bodies (100\u0026ndash;5000 nm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Meanwhile, the zeta potential of S1P-PS-MMV@Spd (\u0026minus;\u0026thinsp;8.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 mV) was higher than that of S1P-PS-MMV(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), indicating the successful loading of positively charged SPD into S1P-PS-MMV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 S1P-PS-MMV@SPD Alleviated Atherosclerotic Plaque Formation\u003c/h2\u003e \u003cp\u003eSince S1P-PS-MMV could target plaques in atherosclerosis, we further explored the functions of S1P-PS-MMV and S1P-PS-MMV@SPD in mouse atherosclerosis. After injecting S1P-PS-MMV and S1P-PS-MMV@SPD into HFD-fed \u003cem\u003eApoE\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, compared with the saline group, the atherosclerotic plaques in the mice were significantly alleviated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). In the heart aortic root, the area of pathological (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), the area of oil red staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E), and the area of fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-G) were significantly reduced. Moreover, the effects of S1P-PS-MMV@SPD were more pronounced than those of S1P-PS-MMV alone. This indicated that S1P-PS-MMV could effectively treat atherosclerosis in mice. The S1P-PS-MMV@SPD vesicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D), after being loaded with SPD, exhibited even more pronounced therapeutic effects.In addition, serum lipid levels were measured in mice. The lipid profile results (\u003cb\u003eSupplementary Fig.\u0026nbsp;1A\u003c/b\u003e)were consistent with the trends from earlier experiments. This trend was also reflected in the body weight (\u003cb\u003eSupplementary Fig.\u0026nbsp;1B\u003c/b\u003e)changes of the mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 S1P-PS-MMV@SPD inhibited the aggregation of inflammatory cells in plaques\u003c/h2\u003e \u003cp\u003eEMNV primarily alleviated rheumatoid arthritis by modulating the content of inflammatory macrophages. To explore the mechanism by which EMNV alleviates the development of atherosclerosis, we first verified the effect of EMNV on inflammatory cells. By performing immunohistochemical staining for macrophage (F4/80, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), neutrophil (Ly6G, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), and monocyte (Ly6C, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) markers on pathological tissue sections in the aortic root of HFD-fed \u003cem\u003eApoE\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice, we clearly observed an increase in the three major inflammatory cells in the saline group, with the most significant increase in macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D). Additionally, compared with the saline group, SPD and S1P-PS-MMV treatments resulted in a reduction in inflammatory cells, and the S1P-PS-MMV@SPD treatment showed the least positive area of inflammatory cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D). Meanwhile, the inflammatory factors IL-1β (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) and TNF-α (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) at the atherosclerotic plaque sites under S1P-PS-MMV and S1P-PS-MMV@SPD treatments also exhibited a similar decreasing trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-G). These results indicated that EMNV treatment could reduce inflammatory infiltration in atherosclerosis in HFD-fed ApoE\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, with the most significant changes observed in macrophages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 S1P-PS-MMV@SPD mainly inhibited the aggregation of M1 macrophages within the plaque\u003c/h2\u003e \u003cp\u003eMonocyte-derived macrophages are key inflammatory cells involved in the process of atherosclerosis, differentiating into various subtypes during the progression of atherosclerosis. Given the significant role that macrophages played in the treatment of mouse atherosclerosis with EMNV, we performed cellular immunofluorescence assays to assess the expression levels of macrophage subtypes M2 (CD206, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B) and M1 (CD86 Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eA \u003cb\u003eand C\u003c/b\u003e) within the aortic root plaques of HFD -fed mice treated with S1P-PS-MMV and S1P-PS-MMV@SPD. We found that the number of macrophages M1 significantly decreased due to the use of S1P-PS-MMV and S1P-PS-MMV@SPD treatments, with the S1P-PS-MMV@SPD group showing a more pronounced effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Macrophages M2 tend to decrease after treatment with EMNV, but there is no significant statistical significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). This might be the EMNV had a good targeting and inhibitory effect on M1-type inflammatory macrophages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 S1P-PS-MMV@SPD Inhibited Scavenger Receptors in M1 Macrophages Stimulated by ox-LDL\u003c/h2\u003e \u003cp\u003eTo elucidate the molecular mechanism of EMNV in atherosclerotic M1 macrophages, we obtained mouse peritoneal primary macrophages and stimulated using ox-LDL (50 \u0026micro;g/ml, 24h). By culturing cells with PBS, S1P-PS-MMV, and S1P-PS-MMV@SPD respectively, we performed oil red staining on the cells. The results indicated a significant reduction in lipid accumulation within the cells due to the application of S1P-PS-MMV and S1P-PS-MMV@SPD, with a more pronounced effect observed in S1P-PS-MMV@SPD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Given the intimate relationship between macrophage lipids and scavenger receptors, we conducted an assessment of the scavenger receptor content within the cells. The findings revealed a significant reduction in the expression levels of SRA1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C) and CD36 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eB \u003cb\u003eand D\u003c/b\u003e), proteins responsible for regulating lipid uptake by macrophages. Conversely, there was a marked increase in the expression of SRB1(Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eB \u003cb\u003eand E\u003c/b\u003e), which stabilize plasma cholesterol through the reverse cholesterol transport pathway\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-E). Additionally, the ratio of cholesteryl esters to total cholesterol diminished, indicating a mitigation in foam cell formation within macrophages\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). The impact of S1P-PS-MMV@SPD was more pronounced than that of S1P-PS-MMV alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-F). These results suggested that EMNV might suppress the expression of scavenger receptors in inflammatory macrophages, thereby decreasing cholesterol accumulation and alleviating foam cell formation in these cells.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eThe present study revealed a multi-layered mechanism involving plaque targeting, inflammatory cells regulation, and lipid metabolic reprogramming of macrophages by effecting the expression of scavenger receptors, thereby reducing macrophage foam cell formation. We demonstrated that the therapeutic potential of EMNV in atherosclerosis by targeting modulation of inflammatory macrophages.\u003c/p\u003e \u003cp\u003eCurrently, emerging therapeutic strategies aimed at macrophage inflammation in atherosclerosis encompass a variety of approaches. These include targeting specific receptors on the surface of macrophages, utilizing nanocarriers to deliver drugs that inhibit the inflammatory response of these cells, and also involve modulating the macrophage phenotype. For instance, efforts are made to shift the macrophage phenotype from a pro-inflammatory M1 type to an anti-inflammatory M2 type\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Furthermore, therapeutic interventions focus on altering macrophage metabolic pathways, such as cholesterol metabolism and inflammatory signaling pathways, to influence plaque stability\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Additionally, the treatment of atherosclerosis can be facilitated by regulating the interaction between macrophages and other cell types, including CD8\u003csup\u003e+\u003c/sup\u003e T cells\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. These pathways also have good therapeutic effects, and the related mechanisms and targets may be combined with nanomaterials in the future to create more targeted treatment methods.\u003c/p\u003e \u003cp\u003eIn this study, we firstly found that the enhanced DID/S1P-PS-MMV accumulation in atherosclerotic lesions validates the design rationale of macrophage-derived vesicles for inflammatory targeting. This phenomenon may be attributed to the natural tropism of macrophage-derived vesicles toward inflammatory sites\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and the attraction of their loaded apoptotic signals SIP and PS on anti-inflammatory macrophages surrounding the plaque. This targeting efficiency establishes a critical foundation for subsequent therapeutic effects. Secondly, we observed that S1P-PS-MMV@SPD has superior efficacy in reducing plaque burden than S1P-PS-MMV, suggesting synergistic effects between the vesicle platform and SPD payload. For potential reasons, SPD can further improve nanovesicle's efficacy because of its multi anti-inflammatory effects. As reported, SPD plays a critical role in anti-inflammatory by suppressing oxidative stress, and disrupts inflammatory pathways such as NLRP3 inflammasome activation\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, and exihibiting excellent anti-rejection with inflammation-regulating functions in drug-loading applications with other materials\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Meanwhile, these differential results also indicated that the vesicle itself possesses inherent anti-inflammatory properties, SPD loading amplifies therapeutic effects through additional pathways. This is consistent with previous report on rheumatoid arthritis therapy\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, now extended to atherosclerotic treatment. Thirdly, our cellular analysis provides a potential mechanism of action through macrophage polarization modulation. As we known, the M1 macrophages-specific inhibition is particularly relevant given the central role of M1-derived inflammatory cytokines in plaque instability\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Here, our findings indicate that EMNV predominantly influences the population of M1 macrophages in atherosclerosis, with no significant statistical difference observed in the quantity of M2 macrophages. This disparity may be attributed to the predominance of M1 macrophages in atherosclerotic conditions\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, as well as the possible inhibition of SPD on the inflammatory response from M1 macrophages. Nowadays, many related targeted therapeutics and drug development tend to focus more on distinguishing between M1 and M2 for precise treatment of atherosclerosis\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The selective reduction of M1 macrophages without significant M2 population alteration suggests a precision modulation strategy distinct from conventional anti-inflammatory approaches. Moreover, these M1 macrophages are known to engulf lipids and subsequently differentiate into foam cells, secreting pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, thereby exacerbating the inflammatory response\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The observed TNF-α and IL-1β reduction induced by EMNV treatment may also involved in this macrophage\u0026rsquo;s polarization shift, potentially creating an anti-inflammatory microenvironment conducive to plaque stabilization.\u003c/p\u003e \u003cp\u003eIn atherosclerotic pathogenesis, dysregulated lipid metabolism synergizes with inflammatory mechanisms to drive plaque formation. Emerging studies highlight inflammation-induced lipid metabolic reprogramming as a pivotal pathobiological process\u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Scavenger receptors onstitute a diverse family of receptor molecules that are ubiquitously expressed on the plasma membrane of phagocytic cells. This family encompasses several members, such as SR-A1, SR-B1, CD36\u003csup\u003e32,33\u003c/sup\u003e. These receptors are pivotal in lipid metabolism and cardiovascular health, primarily through their interaction with lipoproteins and their role in modulating intracellular lipid transport\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Previous studies have demonstrated that nucleic acid nanovesicles predominantly gain cellular entry via endocytosis that is mediated by scavenger receptors and dependent on caveolae\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. This underscores the association between nanovesicles and scavenger receptors. Our investigation indicates that the dual regulation of scavenger receptors in macrophages represents a novel therapeutic mechanism of EMNV. The differential modulation of a decrease in SRA1/CD36 versus an increase in SRB1 suggests EMNV's ability to reprogram macrophage lipid metabolism, rather than simple inhibition. This switching of scavenger receptors may explain the reduced cholesteryl ester accumulation in macrophages, effectively disrupting the vicious cycle of ox-LDL uptake and foam cell formation. The precise mechanisms underlying this phenomenon could be modulated by the structural component among nanovesicles and the pathological microenvironment. Furthermore, scavenger receptors are recognized to exert a substantial regulatory influence on macrophage inflammation in atherosclerosis, extending beyond their role in lipid transport\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. For example, SR-A1 and CD36 facilitate the activation of downstream signaling pathways, including NF-κB and JNK, by internalizing oxidized lipoproteins, thereby promoting inflammatory responses\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Conversely, the activation of SRB1 can attenuate the secretion of pro-inflammatory cytokines, such as TNF-α and IL-6, while enhancing the expression of anti-inflammatory cytokines, such as IL-10, to mitigate inflammation\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eStudies indicate that pyroptosis effector protein, can induce IL-1β release and amplify inflammatory cascades in M1 macrophages\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. We speculate that EMNV's anti-pyroptotic components, like SPD, may indirectly alter gene expression by affecting macrophages' inflammatory responses, thereby influencing cellular lipid synthesis and transport\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Additionally, existing research indicates that celluar lipid metabolism may be hindered by oxidative stress\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The suppression of oxidative stress in inflammatory cells by SPD may promote a positive cycle of lipids, thereby regulating the normal distribution of lipid transport receptors. Therefore, the direct mechanism of action of EMNV on macrophage scavenger receptors in atherosclerosis necessitates further investigation. Besides, another limitation warrant consideration. While our in vivo models demonstrate therapeutic efficacy of EMNV, the long-term safety profile of repeated EMNV administration needs systematic evaluation.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn our investigation, we found the application of EMNV to alleviate the development of atherosclerosisHere the combination of targeted delivery, macrophage polarization control, and celluar lipid metabolic reprogramming addresses multiple pathological aspects simultaneously, followed by the reduction of foam cell formation and inflammatory responses, eventually led to mitigate plaque development in the context of atherosclerosis. These findings highlight the potential of EMNV as a novel therapeutic strategy for atherosclerosis by targeting macrophage-mediated inflammation and affecting lipid metabolism, and position EMNV as a promising multi-modal therapeutic platform for atherosclerosis.\u003c/p\u003e"},{"header":"5. Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e5.1 Reagents\u003c/h2\u003e \u003cp\u003eOx-LDL was purchased from Yiyuan Biotechnology (YB-002, Guangzhou, China). Antibodies against F4/80 (sc-377009) and TNF-α (sc-52746) were obtained from Santa Cruz (CA, USA). Antibodies against SRA1 (24655-1-AP), SRB1 (ET1602-32), CD36 (ET1701-24), AEBP1 (ER61507), Ly6C (HA500088), Ly6G (0809\u0026thinsp;\u0026minus;\u0026thinsp;11), CD206 (ET1702-04), and CD86 (ET1606-50) were sourced from HUABIO (Hangzhou, China). Antibodies against Il-1β (ab9722) was obtained from Abcam (Cambridge, UK). And antibodies against GAPDH (60004-1-Ig) from Proteintech (Wuhan, China) were also included in the study. The kit of HE staining, Oil Red O staining, Masson\u0026rsquo;s trichrome staining were all purchasd from Solarbio (Beijing, China), Amplex red cholesterol and cholesteryl ester assay kit were obtained from Beyotime Biotechnology (shanghai, China) and the manufacturer's instructions were followed during use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e5.2 Preparation of EMNV\u003c/h2\u003e \u003cp\u003eFollowing our previous research\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, macrophage membranes were first extracted, then sonicated, and subsequently passed twice through a 5 \u0026micro;m polycarbonate membrane to yield MMV. EMNV were prepared by introducing PS and S1P onto the MMV surface and encapsulating SPD within the MMV using a co-extrusion technique. Specifically, EMNV, with 16.8 \u0026micro;g of membrane protein, was capable of co-encapsulating approximately 1\u0026micro;g of S1P and 10 \u0026micro;g of SPD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e5.3 Animals\u003c/h2\u003e \u003cp\u003eApoE\u003csup\u003e-/-\u003c/sup\u003e mice on C57BL/6J background were purchased from GemPharmatech (Nanjing, China). We randomly divided 8-week-old male mice into four groups. All studies utilized double-blind randomized controlled trials.Mice in the atherosclerotic group were fed with high-fat diet (HFD), while mice in the control group were fed with Normal diet. Mice in HFD were intravenously injected with saline, S1P-PS-MMV, or S1P-PS-MMV@SPD respectively. The mice were raised at Animal Experimental Center of the First Affiliated Hospital of Wenzhou Medical University, following ethical guidelines approved by the Laboratory Animal Ethics Committee (document no. WYYY-IACUC-AEC-2024-055). All experiments were complied with Directive 2010/63/EU of the European Parliament on animal protection for scientific research. After raising, mice were euthanized with sodium Pentobarbital (100 mg/kg, once, ip.), and their blood vessels and hearts were collected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e5.4 Inflorescence\u003c/h2\u003e \u003cp\u003eSelect age-appropriate ApoE\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice on a C57BL/6J background that have been fed a high-fat diet. The experimental group of mice receive tail vein injections of S1P-PS-MMV@SPD-DID, while the control group receive tail vein injections of the DID alone. The fluorescence intensity of various organs in the mice will be detected using a live animal imaging system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e5.5 Atherosclerotic lesion analysis\u003c/h2\u003e \u003cp\u003eAfter removing the fat from the mouse's blood vessels, the aortic arch was cut open starting from the aortic arch to expose the intima, which was then stained with Oil Red O staining solution. Photos were taken to calculate the area percentage of lipid plaques. For the vascular valve slices, a cryostat microtome was used to make cross-sectional cuts through the frozen heart. After initially displaying the valve tissue at 8\u0026ndash;10 mm, the following sections was transferred onto adhesive slides. Subsequently, the slides were then subjected to HE staining, Oil Red O staining, Masson\u0026rsquo;s trichrome staining, and immunohistochemistry. 3 to 6 microscope fields were randomly selected by blinded experimenters for analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e5.6 Immunohistochemistry\u003c/h2\u003e \u003cp\u003eAfter washing the frozen sections, they were blocked with 5% BSA, incubated with the primary antibody overnight, and then observed under a confocal microscope after incubation with Alexa-488/647 (Abcam, 1:5000) and DAPI.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e5.7 Immunofluorescence\u003c/h2\u003e \u003cp\u003eFor immunohistochemical staining, primary antibodies were used. Subsequently, the sections underwent incubation with secondary antibodies, diaminobenzidine, and hematoxylin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e5.8 Cell culture\u003c/h2\u003e \u003cp\u003eMouse primary peritoneal macrophages (MPMs) were harvested from the peritoneal cavity of ApoE\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice as previously described (Han et al., 2022b). After pretreatment with PBS, S1P-PS-MMV, or S1P-PS-MMV@SPD, ox-LDL (50 \u0026micro;g/ml) was added to induce the atherosclerotic process in macrophages according to the manufacturer's instructions, followed by Oil Red O staining and Western Blot experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e5.9 Cytology Oil Red O staining\u003c/h2\u003e \u003cp\u003eAccording to the Oil Red O Staining Kit (Solarbio, Beijing, China), the manufacturer's instructions were followed during use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e5.10 Westernblots.\u003c/h2\u003e \u003cp\u003eSeparate protein samples using SDS-PAGE gel, then transfer proteins onto an NC membrane. After blocking with 5% BSA at room temperature for two hours, incubate with primary antibody overnight. The next day, incubate with secondary antibody (catalogue nos. A0216 and A0208, Beyotime Biotechnology, Shanghai, China) and expose using an exposure machine (Tanon-5200, Serial Number 14T12NPFLI6-901).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e5.11 Statistical Analysis.\u003c/h2\u003e \u003cp\u003eThe experimental evaluation and data analysis were performed by personnel who did not participate in sampling. Statistical analysis was performed using GraphPad Prism version 10.1.2. First, conduct a normality test (Shapiro-Wilk test) on the data. For data that do not conform to a normal distribution, perform non-parametric analysis (Mann-Whitney test for 2 groups; Kruskal-Wallis test with Dunn's multiple comparisons for more than 2 groups). For normally distributed data, use the Student's t-test or One-Way ANOVA with Bonferroni correction. P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate statistical significance\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEMNV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEfferocytosis-Mimicking Nanovesicles\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMMV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emacrophage membrane vesicles\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphatidyl serine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eS1P\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esphingosine-1-phosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eH\u0026amp;E\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHematoxylin and eosin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHFD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehigh-fat diet\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eND\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enorml diet\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTnf-α\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etumor necrosis factor-α\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIL-1β\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterleukin-1β\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eox-LDL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoxidized low-density lipoprotein.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to the Scientific Research Center of Wenzhou Medical University for their assistance with the immunofluorescence experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZhouqing Huang,Junjie Deng\u0026nbsp;and Jianmin li contributed to the literature search and study design.Jiaxuan Mei,Shanshan Yuan, Xiaoxi Fan,\u0026nbsp;Bozhi Ye,Yixin Zhou\u0026nbsp;,Zhentong Yang and Zhuoqun Wang performed the experiments and analyzed the data. Zhouqing Huang and\u0026nbsp;Junjie Deng approve the version to be published. Jiaxuan Mei and\u0026nbsp;Xiaoxi Fan\u0026nbsp;participated in the drafting of the article. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSources of Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Key project of Zhejiang Provincial Health Commission (WKJ-ZJ-2540) and S Discipline Plan of the Affiliated First Hospital of Wenzhou Medical University(A), and Natural Science Foundation of Zhejiang Province (Grant No.LMS25H020006), and Zhejiang Province Key Laboratory of Intelligent Cancer Biomarker Discovery and Translation (G2023006 and G2023014).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data underlying this article will be shared on reasonable request to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eTsao CW, Aday AW, Almarzooq ZI, Anderson CAM, Arora P, Avery CL, Baker-Smith CM, Beaton AZ, Boehme AK, Buxton AE, Commodore-Mensah Y, Elkind MSV, Evenson KR, Eze-Nliam C, Fugar S, Generoso G, Heard DG, Hiremath S, Ho JE, Kalani R, Kazi DS, Ko D, Levine DA, Liu J, Ma J, Magnani JW, Michos ED, Mussolino ME, Navaneethan SD, Parikh NI, Poudel R, Rezk-Hanna M, Roth GA, Shah NS, St-Onge MP, Thacker EL, Virani SS, Voeks JH, Wang NY, Wong ND, Wong SS, Yaffe K, Martin SS. 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Lipopolysaccharide binding protein resists hepatic oxidative stress by regulating lipid droplet homeostasis. \u003cem\u003eNat Commun\u0026nbsp;\u003c/em\u003e2024;\u003cstrong\u003e15\u003c/strong\u003e:3213. doi: 10.1038/s41467-024-47553-5\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Atherosclerosis, Macrophage, EMNV, Inflammation, lipid metabolic reprogramming","lastPublishedDoi":"10.21203/rs.3.rs-8705147/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8705147/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground and aims\u003c/strong\u003e: Atherosclerosis is a chronic inflammatory disease driven by macrophage-mediated inflammation. This study aims to clarify the effect and mechanism of nanovesicles mimicking engineering efferocytosis on atherosclerosis, offering a multi-modal approach for atherosclerosis treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: In this study, we investigated the therapeutic potential of Engineering efferocytosis-mimicking nanovesicles (EMNV), specifically S1P-PS-MMV and SIP-PS-MMV@SPD, in targeting and alleviating atherosclerotic lesions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: We demonstrated that S1P-PS-MMV could specifically target atherosclerotic plaques in a mouse model. Mechanistically, treatment with S1P-PS-MMV and S1P-PS-MMV@SPD reduced the infiltration of inflammatory cells, particularly pro-inflammatory M1 macrophages, as well as the expression of pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α).Furthermore,EMNV significantly reduced the expression levels of scavenger receptors SR-A1 and CD36 involved in lipid uptake, while increasing the expression of SR-B1 stabilizing plasma cholesterol, eventually led to celluar lipid metabolic\u003cstrong\u003e \u003c/strong\u003ereprogramming.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e This finding demonstrates that S1P-PS-MMV and S1P-PS-MMV@SPD can effectively target atherosclerotic lesions, reduce inflammatory cell infiltration, and modulate scavenger receptor expression in macrophages, thereby alleviating plaque formation.\u003c/p\u003e","manuscriptTitle":"Application of Engineering Efferocytosis-Mimicking Nanovesicles in Atherosclerosis Treatment via M1 Macrophages and celluar lipid metabolic reprogramming","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-09 16:19:08","doi":"10.21203/rs.3.rs-8705147/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":"084ff715-3984-4afb-bbf6-35d36f3dfb93","owner":[],"postedDate":"February 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T10:41:47+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-09 16:19:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8705147","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8705147","identity":"rs-8705147","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}