Nano-engineered probiotic treats atherosclerosis via inhibiting intestinal microbiota-TMA-TMAO axis

Nano-engineered probiotic treats atherosclerosis via inhibiting intestinal microbiota-TMA-TMAO axis | 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 Nano-engineered probiotic treats atherosclerosis via inhibiting intestinal microbiota-TMA-TMAO axis Boxuan Ma, Zhezhe Chen, Qiongjun Zhu, Hong Xu, Yiqing Hu, Yanan Wang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5612717/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Considerable numbers of patient are suffering from atherosclerosis without typical risk factors, which can cause severe cardiovascular complication but is lack of practical treatment. Thereinto, trimethylamine N -oxide (TMAO), originated from enteric microorganism, emerges as an unconventional and crucial factor causing atherosclerosis. Here we demonstrate a strategy to inhibit TMAO through intestinal microbiota-trimethylamine (TMA)-TMAO axis for atherosclerotic treatment. The therapy is performed by an oral-treated nano-engineered probiotic PDMF@LGG, where the probiotic Lacticaseibacillus rhamnosus GG (LGG) is armed with polydopamine coating and conjugated with PMF nanoparticles based on a ROS-responsive polymeric prodrug of fluoromethylcholine (FMC). PDMF@LGG can durably colonize the intestinal canal due to sticky polydopamine coating and the protection of PMF against ROS-induced injury. The ROS trigger the delivery of FMC from nanoparticles, which can inhibit TMA production in enteric microorganisms. Meanwhile, LGG can strengthen the tight junctions of intestinal epithelium and reduce TMA entering the blood. The in vivo study suggests that PDMF@LGG reduces plasma TMAO and suppresses atherosclerotic progression. Furthermore, the microbiomics and metabolomics show that PDMF@LGG also regulates gut microbial composition and various metabolites, assisting in the therapeutic outcome. Together, PDMF@LGG offers a potential candidate for atherosclerotic therapy caused by TMAO and broadens the range of treatable atherosclerosis. Health sciences/Cardiology/Cardiovascular biology/Cardiovascular diseases/Vascular diseases/Atherosclerosis Health sciences/Diseases/Cardiovascular diseases/Vascular diseases/Atherosclerosis Health sciences/Gastroenterology/Gastrointestinal system/Microbiota Biological sciences/Biotechnology/Biomaterials/Biomedical materials Biological sciences/Biological techniques/Nanobiotechnology/Nanoparticles Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Atherosclerotic cardiovascular disease (CVD) is intricately associated with metabolic disorders and presents a substantial threat to human health. 1 Abnormal lipid profiles, insulin resistance, hypertension, and chronic inflammatory status are well-established as pivotal contributors to CVDs, making their management a fundamental cornerstone for effective treatment and prevention strategies. 2 , 3 Nevertheless, recent studies highlighted that a concerning proportion of CVD patients do not exhibit specific risk factors, who encountered with a markedly higher 30-day mortality and a lower prescription rate for guideline-directed therapeutics than that of individuals with identifiable risk factors. 4 , 5 This disparity underscores the pressing need to uncover new intervenable targets and to pioneer novel pharmacotherapy options tailored to the unique needs of these populations. Relevant studies have indicated the tight clinical association between high trimethylamine N -oxide (TMAO) levels and cardiovascular risk, including atherosclerosis, aortic aneurysm, and thrombotic events. 6 – 9 High plasma TMAO additionally increases the susceptibility of mice to atherosclerosis, contributing to accelerated foam cell formation, impaired endothelial cell function, chronic inflammation infiltration, excessive platelet activation, and dysregulation of cholesterol metabolism. 10 – 12 The upstream metabolite of TMAO was further traced and identified as trimethylamine (TMA) produced by gut microbiome, which is derived from choline, L-carnitine, or betaine. Followed by hepatic oxidation through flavin monooxygenases (FMOs), TMA is converted to TMAO. 11 , 13 Western diets can seriously affect intestinal metabolic processes, particularly contributing to an overabundance of TMA through the action of the gut microbial choline TMA lyase CutC and CutD. 14 , 15 Furthermore, this dietary pattern disrupts the intestinal homeostasis by altering the gut microbiome composition, increasing reactive oxygen species (ROS) generation, and compromising the intestinal mucosal barrier. 16 Such synergistic adverse effects thereby exacerbate the susceptibility to atherosclerosis. Accordingly, the regulation of the gut microbiome-derived TMAO metabolic pathway could potentially serve as a promising therapeutic target for atherosclerotic patients without standard modifiable risk factors. Currently, approaches targeting the intestinal microbiota-TMA-TMAO metabolic axis are actively investigated. Low-choline dietary modification and FMO3 inhibition emerge as potent methods for lowering circulating TMAO levels. 17 – 19 However, other diseases resulting from the deficiency of indispensable nutrient choline, the detrimental effects on FMO-mediated exogenous drug metabolism, and the accumulation of unmetabolized TMA also greatly limit the ultimate benefits. 20 – 23 Another alternative is to inhibit the activities of the bacterial enzyme CutC/D. Inhibitors that act on microbial TMA lyase activity to reduce circulating TMAO levels appear to offer a safer therapeutic option for mitigating the progression of atherosclerotic plaques. The fluorinated choline analog, fluoromethylcholine (FMC), was specifically developed for this purpose, which demonstrated a significant ability to lower plasma TMAO levels but suffered from the rapid metabolism due to its hydrophilia. 24 Moreover, oral probiotic adjuvants have shown promise in restoring the intestinal microenvironment, whereas the harsh conditions in the gastrointestinal tract often undermine the viability and retention time of most probiotics. 25 , 26 Following a multi-pronged intervention strategy, artificially armed probiotics are designed to enhance their intestinal viability for better bacteriotherapy performance. Meanwhile, by leveraging the intestinal colonization properties, probiotics can be available as carriers to deliver and gradually release therapeutic drugs in the intestine. 27 In this work, we develop a nano-engineered probiotic to treat the atherosclerosis triggered by TMAO, which lacks typical symptoms and practical solutions in clinic. First, the fluoromethylcholine (FMC) drug is introduced to reduce the production of TMA from enteric microorganisms. In order to overcome the rapid drug loss caused by its good hydrophilicity, FMC has been grafted through an oxalate ester bond to a polymer backbone based on methyl thioethanol ester (MEMA), which can be self-assembled into the nanoparticles (PMF) and offer ROS triggered drug release. 28 – 30 In further, to enhance intestinal colonization, PMF nanoparticles have been engineered onto the surface of the oral probiotic Lacticaseibacillus rhamnosus GG (LGG) through the amidation on a polydopamine coating, resulting in the construction of nano-engineered probiotic PDMF@LGG. Upon oral administration, PDMF@LGG can steadily pass through the stomach and reach the intestinal canal. The MEMA component in PMF nanoparticles can scavenge overexpressed ROS, and the polydopamine coating provides enhanced stickiness to the nano-engineered probiotic, which jointly lead to the protection to the probiotic from oxidative stress and more stable colonization for an extended retention time of PDMF@LGG in intestinal canal. On the other hand, under the trigger of local ROS, the FMC drug in PMF nanoparticles can be accurately released to block the CutC/D enzyme and depress the production of TMA. At the same time, LGG can provide protection to the intestinal endothelium and maintain the tight junctions between the epithelial cells, which helps to the reduction of TMA entering the blood. Therefore, PDMF@LGG is expected to inhibit the intestinal microbiota-TMA-TMAO axis via reducing TMA production and its entry into the bloodstream, which results in a decrease in circulatory TMAO and, consequently, an efficient treatment for atherosclerosis (Fig. 1 ). Furthermore, during the treatment procedure in the intestine, PDMF@LGG may also affect the gut microbiota in terms of altering abundance as well as ameliorating dysbiosis, which can further assist in the management of metabolites related to atherosclerosis. Results and Discussion Preparation of PMF nanoparticles and PDMF@LGG Nano-Engineered Probiotic The PMF nanoparticles integrated on LGG probiotic were prepared with a polymeric prodrug of FMC, which was also endowed with the ROS responsiveness (Fig. 2 a). The FMC drug was first modified into a monomer FAOE with ROS breakable oxalic ester bond, which was used for the copolymerization with MEMA monomer through the RAFT polymerization. The chemical structures of FAOE as well as PMF polymer were confirmed by 1 H NMR ( Fig. S1 and S2 ), where the molecular weight of PMF was calculated to be 4300 g mol − 1 , and the polymer could be defined as poly(MEMA 10 -co-FAOE 9 ). Through self-assembly in PBS solution, the PMF polymer could form nanoparticles with a particle size of around 89 nm and a regular spherical morphology (Fig. 2 b). When exposed to a high level of ROS, the methyl sulfide groups in MEMA could realize a hydrophobic-to-hydrophilic conversion, and the oxalate ester bonds in FAOE could be broken, which offered the PMF nanoparticles the ROS triggered disintegration and drug release capabilities. 28 , 31 , 32 The disruption of nanostructure in ROS was first characterized with the size variation under different concentrations of H 2 O 2 (Fig. 2 c). It was determined that PMF nanoparticles maintained a stable particle size in 0.01 mM H 2 O 2 within 8 h, while higher concentrations of H 2 O 2 , exceeding 0.1 mM, could trigger the disassembly of nanoparticles and an increase in particle size. What’s more, the morphology of PMF nanoparticles after treated with 1 mM H 2 O 2 for 4 h was also observed by TEM (Fig. 2 d), which indicated an obvious decomposition and aggregation of the PMF polymer. Relying on the ROS responsiveness of PMF, the ability of H 2 O 2 scavenging and the stimuli-triggered drug delivery were further investigated (Fig. 2 e). With the increasing concentration of PMF, the amount of H 2 O 2 scavenged gradually increased, and almost 80% of H 2 O 2 was cleared within 1 h at a PMF concentration of 2 mg mL − 1 (Fig. 2 f). As a result, due to the scavenging of H 2 O 2 , the protective effect of PMF on LGG under H 2 O 2 was also evaluated by co-incubation in 1 mM H 2 O 2 (Fig. 2 g), where LGG could normally grow in the presence of H 2 O 2 with the PMF concentration at 1 mg mL − 1 . Thus, PMF was expected to help LGG probiotic overcome the pathologically intestinal microbial environment with high levels of oxidative stress and protect the activity of LGG. 33 Besides, the disassembly of PMF in response to ROS led to the breakage of oxalate ester bonds, which in turn facilitated the controlled release of the FMC drug. As shown in Fig. 2 h, the speed and amount of FMC delivered from PMF nanoparticles indicated a concentration-dependent response to H 2 O 2 . When the H 2 O 2 concentration reached 1 mM, over 50% of FMC was released in 12 h and almost 90% was delivered after 48 h, which provided an ROS-specific delivery of the FMC drug with intensive therapeutic accuracy and reduced drug loss. Additionally, PMF nanoparticles were conjugated with LGG probiotic to construct a nano-engineered probiotic through the amidation based on a polydopamine coating. The carboxyls groups on PMF were activated with EDC/NHS and then participated in a dopamine polymerization on the surface of LGG (Fig. 2 i). The TEM images of free LGG, polydopamine coated LGG (PD@LGG) and nano-engineered probiotic (PDMF@LGG) were shown in Fig. 2 j to determine the conjugation of PMF nanoparticles and LGG probiotics. At the same time, fluorescent DiI-labeled PMF and Hoechst 33342-stained LGG were also used to confirm the preparation of PDMF@LGG (Fig. 2 k). The viability of probiotics after nano-engineering was evaluated. Monitoring the optical density at 600 nm with a 30-min interval for 12 h, both polydopamine coating and nano-engineering showed barely any influence on the growth of LGG (Fig. 2 l). Moreover, the conjugated PMF could also protect LGG from the damage of simulated gastric fluid, simulated intestinal fluid and bile salt environment (Fig. 2 m-o). Therefore, the nano-engineered probiotics PDMF@LGG with the maintained activity as well as enhanced viability in harsh environments were expected to achieve better colonization in the intestinal tract, and further regulate the intestinal microbial environment with accurate delivery of FMC drug. Intestinal Colonization of PDMF@LGG During the oral treatment with nano-engineered probiotics, the colonization and retention of probiotics in the intestinal tract were of great importance to the outcome, which were first evaluated in vitro on HT29 cells. 34 After confirming the cytotoxicity of PMF nanoparticles ( Fig. S3 ), different formulas of PDMF@LGG were used to indicate the adhesion on HT29 cells with in 2 h ( Fig. S4 ), where PMF was labeled with DiI (red) and LGG was stained with MycoLight Green JJ98 (green). Both PD@LGG and PDMF@LGG exhibited markedly enhanced adhesion capacity to the cells, which was owing to the viscous polydopamine coating. Furthermore, the colonization of probiotics was also estimated in C57BL/6 mice (Fig. 3 a). According to the fluorescent signals of PMF and LGG (Fig. 3 b), free LGG and PMF nanoparticles were found in the intestinal tract of mice after 6 h from the oral administration. The decrease in fluorescent intensity over extended time indicated a loss of probiotics or nanoparticles (Fig. 3 c,d), while free PMF showed a faster reduction because of its nanoscale. After being coated with polydopamine, PD@LGG presented better reservation in the intestinal tract due to the stickiness of dopamine. Moreover, it was notable that PDMF@LGG exhibited the best colonization ability compared to other formulas in the first 12 h, which resulted from the combined effect of the viscosity provided by dopamine and the protective role of PMF to the LGG in the intestine. Furthermore, the retention of PD@LGG and PDMF@LGG in the intestinal tract were observed to be nearly identical after a 24-hour period, suggesting that PMF might provide early-time protection to LGG. To delve deeper into this phenomenon, the short-time colonization was investigated by ex vivo imaging of the separated intestinal tract after 2 h since oral treatment (Fig. 3 e). In accordance with the in vivo results, PDMF@LGG revealed the most reservation in the intestine (Fig. 3 f), and the specific imaging of the cecum also confirmed the consistent results (Fig. 3 g,h). Moreover, the sections of the cecum were observed under CLSM (Fig. 3 i), where more and stronger fluorescent signals from PMF and LGG were found in PDMF@LGG treated group (Fig. 3 j). Therefore, the synergistic effects of the dopamine-given stickiness and the PMF-mediated protection could enhance the viability and promote extended colonization of PDMF@LGG for better therapeutic outcomes. In Vivo Regulation on Intestinal Microbiota-TMA-TMAO axis The nano-engineered probiotics, PDMF@LGG, were designed to combat atherosclerosis through reducing the circulating TMAO, which was regulated by the intestinal microbiota-TMA-TMAO axis. 35 , 36 Thus, FMC drug encapsulated in PMF nanoparticles was utilized to inhibit TMA production in the intestinal flora, while LGG probiotics were expected to protect the integrity of intestinal barriers and prevent TMA invasion into the bloodstream. 7 , 37 To evaluate the regulation of these processes, mice fed a hypercholine diet were prepared, exhibiting elevated plasma TMAO levels (Fig. 4 a). After confirming the biosafety towards mean organs ( Fig. S5 ), the regulation of PDMF@LGG on intestinal microbiota-TMA-TMAO axis was illustrated in Fig. 4 b. Following 6 weeks of high choline diet supplemented with oral administration of various treatments, the choline-related metabolites including TMA and TMAO were quantified (Fig. 4 c-e, Fig. S6 ). LGG could inhibit circulating TMA (Fig. 4 c) and TMAO (Fig. 4 d) to a certain extent, while PMF nanoparticles revealed a better outcome, which suggested that the suppression on TMA production contributed more to the control of plasma TMA and TMAO, rather than blocking TMA from entering bloodstream. On the other hand, combining the advantages of probiotics and nanomedicine, PDMF@LGG indicated the most remarkable inhibition on TMA and TMAO, where circulating choline was also nearly restored to the levels under a normal diet by these formulas (Fig. 4 e). Furthermore, the expression of key metabolic enzyme FMO3 in the liver was investigated, which undertook the conversion of TMA to TMAO. The immunofluorescence exhibited that FMO3 was clearly suppressed after treatment with PMF and PDMF@LGG (Fig. 4 f,g), where the quantitative western blot analysis also confirmed similar results (Fig. 4 h,i). Although previous studies have shown that the increase in plasma TMAO following a high-choline diet was not accompanied by an increase in hepatic FMO3 expression, there was also solid evidences pointing out that insulin resistance caused by high TMAO may increase the expression of FMO3, which supported our findings. 13 , 19 , 38 On the other side, typical proteins involved in the tight junctions of the intestinal epithelium, which affected TMA absorption from the the intestinal environment, such as ZO-1, Occludin, and Claudin, were detected to investigate the integrity of intestinal barrier. 39 As shown in Fig. 4 j and 4 k, the LGG-treated group revealed a more complete mechanical intestinal barrier, while PDMF@LGG indicated a better barrier integrity owing to the protective benefits of PMF for LGG. Therefore, during the regulation of PDMF@LGG to intestinal microbiota-TMA-TMAO axis, the FMC drug delivered from PMF nanoparticles could inhibit TMA production and thereby reduce circulating TMA/TMAO levels. At the same time, LGG probiotics offered protection to the intestinal endothelium by maintaining the tight junction proteins, which restrained the TMA produced by intestinal flora entering into the blood. As a result, the inhibition of TMA production and the preservation of intestinal barrier collaboratively led to a significant decrease in plasma TMAO, which could contribute to the treatment of atherosclerosis. In Vivo Treatment to Atherosclerosis According to the clinical results, a number of patients suffered from atherosclerosis without typical risk factors, where a traceable culprit was identified to be TMAO from choline metabolism by enteric microorganism. 40 As a result, the therapeutic strategy based on PDMF@LGG inhibiting intestinal microbiota-TMA-TMAO axis to overcome TMAO-triggered atherosclerosis was investigated in ApoE −/− mice with atherosclerosis by feeding high-choline diet (Fig. 5 a). First, the in vivo regulation of PDMF@LGG on intestinal microbiota-TMA-TMAO axis was evaluated (Fig. 5 b,c and Fig. S7 ). The typical choline-related metabolites showed a similar trend to the results observed in C57BL/6 mice, where TMA (Fig. 5 b) and TMAO (Fig. 5 c) were discovered reduced by PDMF@LGG. In addition, the immunofluorescence signal (Fig. 5 d,e) as well as the western blot result (Fig. 5 f,g) also suggested that the expression of FMO3 in liver was suppressed by PDMF@LGG. Furthermore, the tight junctions of the intestinal barrier were also protected by PDMF@LGG to cut down the entry of TMA into the blood, where the expression of ZO-1, Occludin, and Claudin were maintained by PDMF@LGG (Fig. 5 h,i). Therefore, the inhibition of PDMF@LGG on the intestinal microbiota-TMA-TMAO axis, which was based on the decline of TMA production and obstruction of TMA entering the blood, was expected to suppress atherosclerotic progression through reduced circulating TMAO (Fig. 5 j). As a crucial trigger for atherosclerosis, plasma TMAO could facilitate the progression of atherosclerotic plaques. 10 After treatment with PDMF@LGG and its different formulas, plaques in the aortas were stained with ORO and the en face views of the aortas were shown in Fig. 6 a. It was suggested that both LGG and PMF could deliver significant inhibition on formation of plaques (Fig. 6 b). Compared with LGG, PMF indicated a better effect on inhibiting the plaques, which was in accordance with the circulating TMAO levels of these groups. Moreover, PDMF@LGG showed the best suppression on atherosclerotic plaque progression, where the plaques in the aortas were similar to the control group without high-choline diet. On the other hand, the various compositions in the plaques of aortic roots were also stained and quantified (Fig. 6 c). While both LGG and PMF exhibited distinct limitations to the plaques’ progression, PDMF@LGG indicated the most beneficial anti-atherosclerotic effect. After the treatment of PDMF@LGG, the plaques showed a significant reduction in lipid accumulation (Fig. 6 d), collagen degradation (Fig. 6 e ) , necrocytosis (Fig. 6 f), macrophage infiltration (Fig. 6 g), and MMP-9 expression (Fig. 6 h), which suggested a delayed development and a stable plaque structure. Therefore, depending on the inhibition to intestinal microbiota-TMA-TMAO axis, PDMF@LGG could realize the suppression of circulating TMAO which led to the restraint of atherosclerotic progression. Regulation on Enteric Microorganism and Metabolism Considering the critical role of PDMF@LGG in modulating intestinal microbial homeostasis and other potential additional benefits for atherosclerosis beyond inhibiting the intestinal microbiota-TMA-TMAO axis, the composition changes in gut microbiome and serum metabolites were detected in high-choline induced atherosclerotic mice treated with saline, LGG, PMF, and PDMF@LGG using 16S ribosomal RNA gene sequencing and targeted LC-MS/MS metabolomics assay (Fig. 7 a). Conspicuously, the increased biodiversity in gut microbiome presented by inverse-Simpson diversity index could be observed in all treatment groups compared to saline group, indicating the protective and restorative capacity of LGG and PMF on intestine bacterial richness (Fig. 7 b). The β-diversity analysis performed by non-metric multidimensional scaling (NMDS) and principal coordinates analysis (PCoA) plots revealed favorable clustering trends within the same treatment group, while isolated clusters occurred between various groups, suggesting segregated microbiome compositions (Fig. 7 c,d). Further analyses at the family- (Fig. 7 e) and genus-level (Fig. 7 f) taxonomy were conducted to explore the relative abundance of gut microbiome after various treatments. Apparently, the bacterial composition of atherosclerotic mice with PDMF@LGG treatment was significantly different compared with other groups, and the relative abundance of characterized microbiota was investigated in detail at different levels. Benefiting from the treatment of PMF and PDMF@LGG, an increase in the abundance of Clostridia at the class-level taxonomy was observed, which was verified to prevent metabolic syndrome through constraining lipid absorption (Fig. 7 g). 41 This may be due to the fact that the ROS-scavenging capacity of PMF improved the living environment for Clostridia , thus enhancing the survival. Meanwhile, PDMF@LGG treatment dramatically increased the relative abundance of Lachnospiraceae family (Fig. 7 h) and Oscillospiraceae family (Fig. 7 i). Lachnospiraceae family, as a promising probiotic candidate, was reported to be correlated inversely with atherosclerotic cardiovascular diseases, possibly due to its role in intestinal barrier repair, microbiota homeostasis, and production of secondary metabolites. 42 , 43 The abundance of Oscillospiraceae family was markedly related to the regulation of lipid metabolism and body weight loss, and exerted anti-atherosclerotic effects. 44 , 45 After relative abundance screening at genus-level taxonomy, Oscillibacter (Fig. 7 j) and Roseburia (Fig. 7 k) were disclosed to be effectively increased in the PDMF@LGG treated group. Convincing evidence was provided that the cholesterol metabolizing ability of Oscillibacter genus contributed to the maintenance of lipid homeostasis and ultimately benefited cardiovascular health. 46 Multi-omic analysis of atherosclerotic mice and patients showed a strong negative correlation between Roseburia abundance and cardiovascular diseases, owing to its behaviors on ameliorating atherosclerosis by influencing intestinal gene expression, regulating glycolysis-fatty acid metabolism, and lowering inflammatory responses. 47 , 48 LDA effect size (LEfSe) analysis was further performed to explore the differential gut microbiome composition among the groups ( Fig. S8 ). The histogram of LDA discriminant showed the gut microbiome with significant differences in each group, and the cladogram visually presented the multi-taxonomy circle from inside out which represented the taxonomy level from phylum to genus. Consistent with the above results, LefSe clearly exhibited the enrichment of the aforementioned microbiome after PDMF@LGG treatment. These findings confirmed that PDMF@LGG was beneficial in regulating gut microbiome abundance and ameliorating dysbiosis. Atherosclerosis is accompanied with metabolic remodeling especially involving glucose abnormality, lipid metabolism disorder, and other metabolites disturbance. 49 Although our study suggested that the therapeutic strategy of modulating microorganism-TMA-TMAO axis relied on PDMF@LGG was advantageous in reducing atherosclerotic plaques, it remained unclear whether other gut microboime-associated metabolites exerted anti-atherosclerosis effects. Therefore, LC-MS/MS metabolomics assay was used to identify the differential metabolites in the serum. Orthogonal partial least squares discriminant analysis (OPLS-DA) plot showed the distinguished tendency of metabolites among the groups (Fig. 7 l). Additionally, heatmap of serum metabolic profiles also intuitively visualized the metabolite differences, including fatty acids, amino acid and its metabolites, glycoprotein, etc ( Fig. S9 ). To further understand the classification and functional properties of these metabolites, KEGG pathway annotation was conducted and key metabolic pathways were noted. Compared with the saline group, LGG and PMF treated groups were both involved in carbohydrate digestion and absorption, ABC transporters, and starch and sucrose metabolism, whereas LGG treatment significantly regulated the pathways of unsaturated fatty acids biosynthesis, arginine biosynthesis, and metabolism of alanine, aspartate and glutamate, and PMF treatment mainly participated in the vascular smooth muscle contraction and arachidonic acid metabolism (Fig. 7 m,n). The metabolic pathways of primary bile acid biosynthesis, cholesterol metabolism, and choline metabolism in cancer, which were found to be tightly associated with the onset and progression of atherosclerosis, were significantly enriched after PDMF@LGG treatment (Fig. 7 o). Interactions between the gut microbiota and serum metabolites serve a vital function in the pathogenesis of atherosclerosis. Consequently, we investigated the association between differential microbes and metabolites under PDMF@LGG treatment in comparison to the saline group. The cluster heatmap based on genus-level taxonomy was drawn ( Fig. S10 ). Detailed information on the top 20 differential metabolites and microbes was also provided (Fig. 7 p,q). The results showed that Roseburia and unidentified Clostridia were positively correlated with Trigonelline (beneficial for cardiovascular diseases because of its strong antioxidant and anti-inflammatory activity and ability to modulate lipid metabolism) 50 and Ectoine (helpful to treat various inflammatory diseases and known as protein protectant, membrane modulator, and DNA protectant) 51 , 52 , and negatively correlated to 13(R)-HODE (reported to exacerbate atherosclerosis through facilitating foam cell formation, inducing apoptosis, and promoting oxidative stress) 53 , 54 . Roseburia also kept negative association with 12,13-EpOME which affected the production of pro-inflammatory mediators and the activation of immune cells, and 13-HOTrE which was recognized as a compound involving in lipid-mediated inflammation response and oxidative stress. 55 , 56 Overall, in addition to regulating the intestinal microbiota-TMA-TMAO axis, PDMF@LGG treatment also modulated the composition of gut microbiome and caused changes in key metabolites in the serum, thereby driving the enhanced therapeutic efficacy in hypercholine diet-induced atherosclerotic mice. Conclusion Aiming to overcome the atherosclerosis caused by high-level TMAO, which was not considered as a typical clinical risk factor, we developed a nano-engineered probiotic PDMF@LGG to realize the inhibition on intestinal microbiota-TMA-TMAO axis for the suppression of circulating TMAO and atherosclerotic progression. To construct PDMF@LGG, the prodrug nanoparticles were prepared with ROS scavenging and stimuli-responsive drug delivery abilities for inhibition on TMA production, which were further engineered onto the surface of LGG probiotic via a polydopamine coating. Through oral administration, PDMF@LGG could sustainably colonize and maintain activity in the intestinal tract, owing to the stickiness provided by polydopamine coating as well as the protection from oxidative stress injury offered by the PMF nanoparticles. Under conditions of locally overexpressed ROS, PMF nanoparticles could responsively deliver the FMC drug to reduce the production of TMA. On the other hand, it could be found that LGG probiotics maintained the integrity of the tight intestinal epithelial junctions and decreased TMA entering the blood. As a result, PDMF@LGG suppressed the circulating TMA, while the plasma TMAO levels and the expression of hepatic FMO3 were also observed reduced, thereby efficiently restraining the progression of atherosclerosis. In further, by combining microbiomic and metabolomic results, it was noted that PDMF@LGG treatment not only regulated the gut microbiome composition but also modulated a series of serum metabolites, thereby assisting to the therapeutic outcome. In conclusion, the nano-engineered PDMF@LGG probiotic indicated an anti-atherosclerosis activity through inhibiting the intestinal microbiota-TMA-TMAO axis, which provided a promising clinical strategy to address atherosclerosis caused by atypical TMAO. Methods Preparation and Characterization of PDMF@LGG In order to arm the LGG probiotic with PMF nanoparticles, a coating based on polydopamine and EDC/NHS reaction was utilized. Briefly, PMF nanoparticles (2 mg mL − 1 ) in PBS solution were first modified with EDC (0.16 mg mL − 1 ) and NHS (0.08 mg mL − 1 ) for 1 h. At the same time, PBS solution of LGG (at an absorbance of 1.0 at 600 nm) was stirred for 2 h with the addition of dopamine (0.5 mg mL − 1 ). Afterwards, the polydopamine coated LGG (PD@LGG) was washed with PBS three times and further co-incubated with modified PMF nanoparticles (2 mg mL − 1 ). After another 2 h, the nano-engineered LGG (PDMF@LGG) was purified by washing with PBS and stored at 4 o C. To evaluate the integration of PMF nanoparticles on LGG probiotic, transmission electron microscope (TEM, Thermo Scientific) was used to observe the morphology of LGG, PD@LGG and PDMF@LGG. In further, DiI labeled PMF was also used to arm LGG, which was stained with Hoechst 33342, and the fluorescent signals of PMF and LGG were detected under a CLSM (Nikon A1 Ti). On the other hand, the growth of LGG, PD@LGG, and PDMF@LGG was confirmed by measuring the absorbance at 600 nm every 30 min for 12 h by a microplate reader. Moreover, the survivability of PDMF@LGG in external environment under SGF (pH 1.5) supplemented with pepsin (0.32%), SIF (pH 6.8) supplemented with trypsin (10 mg/ml), and bile salt (0.4%) solutions was determined by calculating the proliferation with CCK-8 assay, with un-coated LGG as a control. In Vivo and Ex Vivo Colonization of PDMF@LGG in Intestinal Canal C57BL/6 mice were used to investigate the colonization of PDMF@LGG as well as its different formulations. Briefly, the mice were fed with LGG, PMF, PD@LGG and PDMF@LGG, where PMF were labeled with DiI and LGG were stained with MycoLight Green JJ98. At 6, 12, and 24 h post-administration, the mice were anesthetized and the fluorescent signals of PMF and LGG were detected in vivo by a IVIS spectrum system (Lumina LT). Moreover, the intestinal tracts were separated from the mice after 2 h since oral administration, and the ex vivo fluorescence of the entire intestinal tracts and cecum was measured. On the other hand, the cecum from mice after 24 h treatment was prepared into sections and stained with DAPI. The distribution of fluorescent signals from PMF and LGG in the cecal tissue was observed under a CLSM (Nikon A1 Ti). In Vivo Regulation on TMAO Metabolism The regulation of PDMF@LGG on TMAO was first investigated in C57BL/6 mice. Along with a high-choline diet, the mice were orally treated with saline, PMF (0.2 mg every time), LGG (10 8 CFU every time), and PDMF@LGG (10 8 CFU every time) every two days. After 6 weeks, the mice were sacrificed and the serum was collected, which was used to calculate the choline related metabolites such as choline, TMA, TMAO, betaine, creatinine, and L-carnitine. In further, the liver tissue was isolated for immunofluorescence as well as western blot to determine the expression of FMO3, and the colonic tissue was used for immunofluorescence staining of intestinal barrier proteins such as ZO-1, Occludin, and Claudin. In Vivo Treatment to Atherosclerosis The ApoE −/− mice were fed with high-choline diet to construct a high-TMAO induced atherosclerosis model, which were also orally administrated with saline, LGG (10 8 CFU each time), PMF (0.2 mg each time), and PDMF@LGG (10 8 CFU each time) every two days. After 16 weeks, the mice were sacrificed and the choline related metabolites in serum were measured. Concurrently, the expression of hepatic FMO3 was determined by immunofluorescence and western blot, while the intestinal barrier proteins in colonic tissue were evaluated by immunofluorescence. Furthermore, to investigate the atherosclerotic progression, the aortas of mice were separated and lengthwise opened, where the en face was stained with oil red O (ORO). On the other hand, the sections of aortic roots were prepared for various histological staining such as ORO, H&E, Masson, CD68, and MMP-9. Microbiome and Metabolomic Analysis After four months of different treatments in high-choline diet induced atherosclerotic mice, three pellets of fecal samples were collected and serum were extracted, which were frozen in liquid nitrogen immediately and sent to Metware Metabolic Biotechnology Co., LTD (Wuhan, China) for microbiome and metabolomics analysis. The data were analyzed using a free online platform called Metware Cloud ( https://cloud.metware.cn ). Briefly, total genome DNA from feces was extracted using CTAB method, and the purity and concentration were monitored. Genes of 16S V4 region were amplified used specific primers (515F: GTGCCAGCMGCCGCGGTAA; 806R: GGACTACHVGGGTWTCTAAT) with the barcode. PCR products were mixed in equal density ratios and purified with Qiagen Gel Extraction Kit (Qiagen, Germany). Sequencing libraries were generated using TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, USA) and the quantified libraties were sequenced on an Illumina NovaSeq6000 platform and 250 bp paired-end reads were generated. Effective tags were obtained after data split, filtration, sequence assembly and chimera removal. ASV denoising was performed by Deblur (Version 1.1.1) in the QIIME2 software (Version 2023.2). The Silva Database was utilized to annotate taxonomic information. The inverse-Simpson diversity index was calculated to assess the alpha diversity, while NMDS and PCoA were performed to assess beta diversity. Relative abundance of gut microbiome at family and genus levels were visualized and quantitative data of representative microbiome at different levels were shown. The pre-treated serum samples were transferred for LC-MS analysis using an LC-ESI-MS/MS system (UPLC, ExionLC AD; MS, QTRAP® System). The original data were qualitatively analyzed based on the Metware Database and the quantification was performed using multiple reaction monitoring (MRM) by triple quadrupole scans. Differential metabolites were determined according to VIP (VIP > 1) and P value (P value < 0.05, Student’s t test). VIP values were extracted from OPLS-DA models, which also contained score plots to show the inter-group differences. Identified metabolites were annotated using KEGG Compound database ( http://www.kegg.jp/kegg/compound/ ) and annotated metabolites were then mapped to KEGG Pathway database ( http://www.kegg.jp/kegg/pathway.html ). Statistical analysis All experiments were performed at least three times (n = 3–6) and expressed as means ± standard deviation (s.d.). The contrast between two groups was analyzed by SPSS 20.0 software using the Student’s t test and the non-parametric test to assess statistical significance. Statistical significance was assessed at P < 0.05. Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Data availability The relevant data are available from the corresponding authors upon reasonable request. Declarations All animal experiments were approved by the Laboratory Animal Research Center of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. The animal protocols were assessed and approved by the Animal Laboratory Ethics Committee of Zhejiang University (SYXK2017-0006). Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Data availability The relevant data are available from the corresponding authors upon reasonable request. Acknowledgements This research was financially supported by Noncommunicable Chronic Diseases-National Science and Technology Major Project (NO. 2023ZD0503904), National Natural Science Foundation of China (No. 32201128, No. 82270262, No. 32301100), Zhejiang TCM Science and Technology Program TCM modernization Special project, China (No. 2022ZX012) and Natural Science Funds of Zhejiang Province (No. ZCLY24H1801). The schematic figures were created with BioRender.com. We thank Xiaoli Hong from the Core Facilities, Zhejiang University School of Medicine for their technical support. Author contributions All authors have read and approved the manuscript. Z.C. and B.M. conceived and designed the study. Z.C., Q.Z., H.X., and B.M. performed the experiments. H.X. and B.M. synthesized and prepared the nanoparticles. Z.C., Y.H., and X.H. analyzed the microbiome and metabolomics data. Y.W., Z.C., and Y,W assisted in the experiments and drew the figures. Z.C. and B.M. wrote the manuscript. G.F., B.M., and W.Z. revised the manuscript and supervised this study. Competing interests The authors declare no competing interests. Additional information Supplementary information The online version contains supplementary materials available at Correspondence and requests for materials should be addressed to Guosheng Fu, Boxuan Ma or Wenbin Zhang. Peer review information Reprints and permissions information Publisher’s note References Libby P (2021) The changing landscape of atherosclerosis. Nature 592:524–533 Libby P et al (2019) Atherosclerosis Nat Rev Dis Primers 5:56 Kong P et al (2022) Inflammation and atherosclerosis: signaling pathways and therapeutic intervention. 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Nat Microbiol 4:1851–1861 Kumar N et al (2016) 15-Lipoxygenase metabolites of α-linolenic acid, [13-(S)-HPOTrE and 13-(S)-HOTrE], mediate anti-inflammatory effects by inactivating NLRP3 inflammasome. Sci Rep 6:31649 Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaryinformation.docx Supplementary information Cite Share Download PDF Status: Published Journal Publication published 13 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-5612717","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":398238475,"identity":"7eb05221-7cd9-436a-b5b7-efa4d6f0c9e0","order_by":0,"name":"Boxuan 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Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wenbin","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-12-10 03:25:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5612717/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5612717/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-66448-7","type":"published","date":"2025-12-13T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":73267636,"identity":"3da4aeb5-a886-431e-a44f-abe1bc67a084","added_by":"auto","created_at":"2025-01-08 10:37:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":126395379,"visible":true,"origin":"","legend":"\u003cp\u003eIllustration of the intestinal microbiota-TMA-TMAO axis resulting in atherosclerosis and the therapeutic strategy based on nano-engineered probiotic PDMF@LGG.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5612717/v1/43509c51aefde9a35734bfc7.png"},{"id":73267639,"identity":"39758ba4-08de-4634-99fd-9de65234994a","added_by":"auto","created_at":"2025-01-08 10:37:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":57281569,"visible":true,"origin":"","legend":"\u003cp\u003ePreparation and characterization of PMF nanoparticles and PDMF@LGG nano-engineered probiotic. \u003cstrong\u003ea\u003c/strong\u003e The synthetic route of PMF polymer. \u003cstrong\u003eb\u003c/strong\u003e The size distribution and TEM image of PMF nanoparticles. The scale bar was 100 nm. \u003cstrong\u003ec\u003c/strong\u003e The size variation of PMF under different H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations. \u003cstrong\u003ed\u003c/strong\u003e TEM image of PMF after incubated in 1 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003efor 4 h. The scale bar was 100 nm. \u003cstrong\u003ee\u003c/strong\u003e Illustration of PMF responding to ROS and realizing the ROS scavenging as well as the drug delivery. \u003cstrong\u003ef\u003c/strong\u003e H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003escavenged\u003csub\u003e \u003c/sub\u003eby various concentrations of PMF in 1 h. \u003cstrong\u003eg\u003c/strong\u003e Proliferation of LGG in 1 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003etreated with different concentrations of PMF in 12 h. The viability of LGG was measured by CCK-8 assay. \u003cstrong\u003eh\u003c/strong\u003e Accumulative release of FMC from PMF nanoparticles under different H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003econcentrations. \u003cstrong\u003ei\u003c/strong\u003e Illustration of preparing PDMF@LGG by conjugating PMF and LGG through amidation and polydopamine coating. \u003cstrong\u003ej\u003c/strong\u003e TEM images of LGG, PD@LGG and PDMF@LGG. The scale bars were 1 μm. \u003cstrong\u003ek\u003c/strong\u003e Fluorescent images of PDMF@LGG prepared with DiI (red) labeled PMF and Hoechst 33342 (blue) stained LGG. The scale bars were 5 μm. \u003cstrong\u003el\u003c/strong\u003e The growth curves of LGG, PD@LGG, and PDMF@LGG monitored by the absorption at 600 nm in 30-min intervals. Survivals of LGG and PDMF@LGG after exposure to SGF (pH 1.5) supplemented with pepsin (0.32%) (\u003cstrong\u003em\u003c/strong\u003e), SIF (pH 6.8) supplemented with trypsin (10 mg/ml) (\u003cstrong\u003en\u003c/strong\u003e), and bile salt (0.4%) solutions (\u003cstrong\u003eo\u003c/strong\u003e). Data in (c, f-h, l-o) were expressed as mean ± s.d. (n = 3).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5612717/v1/c9b02b47430fcac8edb21677.png"},{"id":73267632,"identity":"f9d15726-9a6c-4784-abc6-2ca4794fba95","added_by":"auto","created_at":"2025-01-08 10:37:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":100746018,"visible":true,"origin":"","legend":"\u003cp\u003eIntestinal colonization of PDMF@LGG and its different formulas. \u003cstrong\u003ea\u003c/strong\u003e Illustration of mice orally treated with various formulas and used for \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e imaging. \u003cem\u003eIn vivo\u003c/em\u003e imaging of mice after different treatments for 6, 12 and 24 h (\u003cstrong\u003eb\u003c/strong\u003e). PMF was labeled with DiI and LGG was stained with MycoLight Green JJ98. The fluorescent intensities of LGG (\u003cstrong\u003ec\u003c/strong\u003e) and PMF (\u003cstrong\u003ed\u003c/strong\u003e) were calculated, respectively. \u003cem\u003eEx vivo\u003c/em\u003e imaging of the intestinal tract (\u003cstrong\u003ee\u003c/strong\u003e) and the cecum (\u003cstrong\u003eg\u003c/strong\u003e) separated from the mice after various treatments for 2 h, and the quantitative results of fluorescent intensity in the intestinal tract (\u003cstrong\u003ef\u003c/strong\u003e) and the cecum (\u003cstrong\u003eh\u003c/strong\u003e) were measured. \u003cstrong\u003ei\u003c/strong\u003eSections of the cecum with different treatments for 2 h.\u003cstrong\u003e j\u003c/strong\u003e The fluorescent intensities of LGG and PMF were quantified. The scale bars were 100 μm. Data in (c, d, f, h, j) were expressed as mean ± s.d. (n = 3).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5612717/v1/8a0bacbb6413deb2e757eb58.png"},{"id":73267637,"identity":"cf993a0b-333c-463f-87df-2b7dc6347ef8","added_by":"auto","created_at":"2025-01-08 10:37:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":100096462,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e regulation of PDMF@LGG to the intestinal microbiota-TMA-TMAO axis in C57BL/6 mice. \u003cstrong\u003ea\u003c/strong\u003eTreatment protocol of C57BL/6 mice with high-choline diet and PDMF@LGG treatment. \u003cstrong\u003eb\u003c/strong\u003e Illustration of the PDMF@LGG regulating the intestinal microbiota-TMA-TMAO axis. Choline related metabolites in the plasma of C57BL/6 mice treated with saline, LGG, PMF and PDMF@LGG, including TMA (\u003cstrong\u003ec\u003c/strong\u003e), TMAO (\u003cstrong\u003ed\u003c/strong\u003e) and choline (\u003cstrong\u003ee\u003c/strong\u003e). Immunofluorescent images (\u003cstrong\u003ef\u003c/strong\u003e) and quantitative data (\u003cstrong\u003eg\u003c/strong\u003e) of FMO3 in liver of mice with various treatments. The scale bar was 100 μm. Western blot (\u003cstrong\u003eh\u003c/strong\u003e) and quantitative data (\u003cstrong\u003ei\u003c/strong\u003e) of hepatic FMO3 expression in mice treated with different formulas. Immunofluorescent images (\u003cstrong\u003ej\u003c/strong\u003e) and quantitative data (\u003cstrong\u003ek\u003c/strong\u003e) of tight junction proteins, including ZO-1, Occludin, and Claudin, in the intestinal endothelium with different treatments. The scale bars were 100 μm. Data in (c-e, g, i, k) were expressed as mean ± s.d. (c-e: n = 5; g, i, k: n = 6).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5612717/v1/2841642a6da9f96a9247366f.png"},{"id":73267647,"identity":"fde1b6d9-dc12-45c2-a35a-6eb5c86c224e","added_by":"auto","created_at":"2025-01-08 10:37:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":123349331,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e regulation of PDMF@LGG to the intestinal microbiota-TMA-TMAO axis in ApoE\u003csup\u003e-/-\u003c/sup\u003e mice. \u003cstrong\u003ea\u003c/strong\u003e Therapeutic protocol of PDMF@LGG treating ApoE\u003csup\u003e-/-\u003c/sup\u003e mice with high-choline diet. Plasma TMA (\u003cstrong\u003eb\u003c/strong\u003e) and TMAO (\u003cstrong\u003ec\u003c/strong\u003e) levels after different treatments. Immunofluorescence image (\u003cstrong\u003ed\u003c/strong\u003e) and quantitative data (\u003cstrong\u003ee\u003c/strong\u003e) of FMO3 in sections of liver from the ApoE\u003csup\u003e-/-\u003c/sup\u003e mice after various administrations. The scale bar was 100 μm. Western blot (\u003cstrong\u003ef\u003c/strong\u003e) and quantitative data (\u003cstrong\u003eg\u003c/strong\u003e) of FMO3 protein in liver tissue treated with different formulas. Immunofluorescence image (\u003cstrong\u003eh\u003c/strong\u003e) and quantitative data (\u003cstrong\u003ei\u003c/strong\u003e) of typical tight junction proteins expressed in intestine, including ZO-1, Occludin, and Claudin. The scale bars were 100 μm. \u003cstrong\u003ej\u003c/strong\u003e Illustration of PDMF@LGG regulating intestinal microbiota-TMA-TMAO axis for atherosclerosis treatment. Data in (b, c, e, g, i) were expressed as mean ± s.d. (b,c : n = 5; e, g, i: n = 6).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5612717/v1/6448b208bc762004ce8d7e1c.png"},{"id":73267654,"identity":"8e84f288-54b1-4cc2-8195-b62222314b73","added_by":"auto","created_at":"2025-01-08 10:37:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":299708139,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e treatment of PDMF@LGG to atherosclerosis. Photographs (\u003cstrong\u003ea\u003c/strong\u003e) and relative areas (\u003cstrong\u003eb\u003c/strong\u003e) of plaques on the en face of aortas, which were stained with ORO. \u003cstrong\u003ec\u003c/strong\u003eSections of aortic roots after treatment with different formulas, which were stained with ORO, H\u0026amp;E, Masson’s trichrome, as well as antibodies to CD68 and MMP-9. The scale bars were 500 μm. Quantitative results of lipid area (\u003cstrong\u003ed\u003c/strong\u003e), collagen (\u003cstrong\u003ee\u003c/strong\u003e), necrotic core (\u003cstrong\u003ef\u003c/strong\u003e), macrophages (\u003cstrong\u003eg\u003c/strong\u003e), and MMP-9 (\u003cstrong\u003eh\u003c/strong\u003e) in the plaques. Data in (b, d-h) were expressed as mean ± s.d. (n = 6).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5612717/v1/2e96470d26f97c7731125ace.png"},{"id":73270875,"identity":"6694e3af-592a-4029-a253-644175444eb9","added_by":"auto","created_at":"2025-01-08 10:53:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":94454274,"visible":true,"origin":"","legend":"\u003cp\u003eRegulation of PDMF@LGG to the gut microbiome homeostasis and gut microbiome-related metabolites. \u003cstrong\u003ea\u003c/strong\u003e Illustration of gut microbiome and serum metabolomic analyses in ApoE\u003csup\u003e-/-\u003c/sup\u003e mice with high-choline diet under different treatments. \u003cstrong\u003eb\u003c/strong\u003e The gut microbiome α-diversity analysis \u003cem\u003evia\u003c/em\u003e inverse-Simpson diversity index. The gut microbiome β-diversity analysis demonstrated by NMDS (\u003cstrong\u003ec\u003c/strong\u003e) and PCoA plots (\u003cstrong\u003ed\u003c/strong\u003e). \u003cstrong\u003ee\u003c/strong\u003e Relative abundance of gut microbiome at family-level taxonomy after different treatments. \u003cstrong\u003ef\u003c/strong\u003e Heatmap of relative abundance of gut microbiome at genus-level taxonomy after various treatments. Relative abundance of \u003cem\u003eClostridia \u003c/em\u003e(\u003cstrong\u003eg\u003c/strong\u003e) at class-level taxonomy, \u003cem\u003eLachnospiraceae \u003c/em\u003e(\u003cstrong\u003eh\u003c/strong\u003e) and \u003cem\u003eOscillospiraceae \u003c/em\u003e(\u003cstrong\u003ei\u003c/strong\u003e) at family-level taxonomy, \u003cem\u003eOscillibacter \u003c/em\u003e(\u003cstrong\u003ej\u003c/strong\u003e) and \u003cem\u003eRoseburia \u003c/em\u003e(\u003cstrong\u003ek\u003c/strong\u003e) at genus-level taxonomy after different treatments. \u003cstrong\u003el\u003c/strong\u003e Differences in serum metabolic profiles illustrated by OPLS-DA score plot. KEGG enrichment analyses revealing main metabolism pathways between LGG (\u003cstrong\u003em\u003c/strong\u003e), PMF (\u003cstrong\u003en\u003c/strong\u003e), PDMF@LGG (\u003cstrong\u003eo\u003c/strong\u003e) with Saline group. \u003cstrong\u003ep\u003c/strong\u003e Spearman correlation heatmaps of differential serum metabolites and gut microbiome at genus-level taxonomy. The blank grid indicated a P-value greater than 0.05. \u003cstrong\u003eq\u003c/strong\u003e Spearman chord diagram of differential serum metabolites and gut microbiome at genus-level taxonomy. Pink curves represented positive correlations, while blue curves represented negative correlations. Data in (b, g-k) were expressed as mean ± s.d. (n=3).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5612717/v1/af0348ac53b716b15e203ac0.png"},{"id":73269837,"identity":"b9621e5a-2c7f-469d-9a53-85259f7859bc","added_by":"auto","created_at":"2025-01-08 10:45:42","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2618104,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5612717/v1/3f504b3b41a04988c25b1837.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Nano-engineered probiotic treats atherosclerosis via inhibiting intestinal microbiota-TMA-TMAO axis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAtherosclerotic cardiovascular disease (CVD) is intricately associated with metabolic disorders and presents a substantial threat to human health.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Abnormal lipid profiles, insulin resistance, hypertension, and chronic inflammatory status are well-established as pivotal contributors to CVDs, making their management a fundamental cornerstone for effective treatment and prevention strategies.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Nevertheless, recent studies highlighted that a concerning proportion of CVD patients do not exhibit specific risk factors, who encountered with a markedly higher 30-day mortality and a lower prescription rate for guideline-directed therapeutics than that of individuals with identifiable risk factors.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e This disparity underscores the pressing need to uncover new intervenable targets and to pioneer novel pharmacotherapy options tailored to the unique needs of these populations.\u003c/p\u003e \u003cp\u003eRelevant studies have indicated the tight clinical association between high trimethylamine \u003cem\u003eN\u003c/em\u003e-oxide (TMAO) levels and cardiovascular risk, including atherosclerosis, aortic aneurysm, and thrombotic events.\u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e High plasma TMAO additionally increases the susceptibility of mice to atherosclerosis, contributing to accelerated foam cell formation, impaired endothelial cell function, chronic inflammation infiltration, excessive platelet activation, and dysregulation of cholesterol metabolism.\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e The upstream metabolite of TMAO was further traced and identified as trimethylamine (TMA) produced by gut microbiome, which is derived from choline, L-carnitine, or betaine. Followed by hepatic oxidation through flavin monooxygenases (FMOs), TMA is converted to TMAO.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Western diets can seriously affect intestinal metabolic processes, particularly contributing to an overabundance of TMA through the action of the gut microbial choline TMA lyase CutC and CutD.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Furthermore, this dietary pattern disrupts the intestinal homeostasis by altering the gut microbiome composition, increasing reactive oxygen species (ROS) generation, and compromising the intestinal mucosal barrier.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Such synergistic adverse effects thereby exacerbate the susceptibility to atherosclerosis. Accordingly, the regulation of the gut microbiome-derived TMAO metabolic pathway could potentially serve as a promising therapeutic target for atherosclerotic patients without standard modifiable risk factors.\u003c/p\u003e \u003cp\u003eCurrently, approaches targeting the intestinal microbiota-TMA-TMAO metabolic axis are actively investigated. Low-choline dietary modification and FMO3 inhibition emerge as potent methods for lowering circulating TMAO levels.\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 However, other diseases resulting from the deficiency of indispensable nutrient choline, the detrimental effects on FMO-mediated exogenous drug metabolism, and the accumulation of unmetabolized TMA also greatly limit the ultimate benefits.\u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Another alternative is to inhibit the activities of the bacterial enzyme CutC/D. Inhibitors that act on microbial TMA lyase activity to reduce circulating TMAO levels appear to offer a safer therapeutic option for mitigating the progression of atherosclerotic plaques. The fluorinated choline analog, fluoromethylcholine (FMC), was specifically developed for this purpose, which demonstrated a significant ability to lower plasma TMAO levels but suffered from the rapid metabolism due to its hydrophilia.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Moreover, oral probiotic adjuvants have shown promise in restoring the intestinal microenvironment, whereas the harsh conditions in the gastrointestinal tract often undermine the viability and retention time of most probiotics.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Following a multi-pronged intervention strategy, artificially armed probiotics are designed to enhance their intestinal viability for better bacteriotherapy performance. Meanwhile, by leveraging the intestinal colonization properties, probiotics can be available as carriers to deliver and gradually release therapeutic drugs in the intestine.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn this work, we develop a nano-engineered probiotic to treat the atherosclerosis triggered by TMAO, which lacks typical symptoms and practical solutions in clinic. First, the fluoromethylcholine (FMC) drug is introduced to reduce the production of TMA from enteric microorganisms. In order to overcome the rapid drug loss caused by its good hydrophilicity, FMC has been grafted through an oxalate ester bond to a polymer backbone based on methyl thioethanol ester (MEMA), which can be self-assembled into the nanoparticles (PMF) and offer ROS triggered drug release.\u003csup\u003e\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e In further, to enhance intestinal colonization, PMF nanoparticles have been engineered onto the surface of the oral probiotic \u003cem\u003eLacticaseibacillus rhamnosus GG\u003c/em\u003e (LGG) through the amidation on a polydopamine coating, resulting in the construction of nano-engineered probiotic PDMF@LGG. Upon oral administration, PDMF@LGG can steadily pass through the stomach and reach the intestinal canal. The MEMA component in PMF nanoparticles can scavenge overexpressed ROS, and the polydopamine coating provides enhanced stickiness to the nano-engineered probiotic, which jointly lead to the protection to the probiotic from oxidative stress and more stable colonization for an extended retention time of PDMF@LGG in intestinal canal. On the other hand, under the trigger of local ROS, the FMC drug in PMF nanoparticles can be accurately released to block the CutC/D enzyme and depress the production of TMA. At the same time, LGG can provide protection to the intestinal endothelium and maintain the tight junctions between the epithelial cells, which helps to the reduction of TMA entering the blood. Therefore, PDMF@LGG is expected to inhibit the intestinal microbiota-TMA-TMAO axis \u003cem\u003evia\u003c/em\u003e reducing TMA production and its entry into the bloodstream, which results in a decrease in circulatory TMAO and, consequently, an efficient treatment for atherosclerosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Furthermore, during the treatment procedure in the intestine, PDMF@LGG may also affect the gut microbiota in terms of altering abundance as well as ameliorating dysbiosis, which can further assist in the management of metabolites related to atherosclerosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of PMF nanoparticles and PDMF@LGG Nano-Engineered Probiotic\u003c/h2\u003e \u003cp\u003eThe PMF nanoparticles integrated on LGG probiotic were prepared with a polymeric prodrug of FMC, which was also endowed with the ROS responsiveness (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The FMC drug was first modified into a monomer FAOE with ROS breakable oxalic ester bond, which was used for the copolymerization with MEMA monomer through the RAFT polymerization. The chemical structures of FAOE as well as PMF polymer were confirmed by \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2\u003c/b\u003e), where the molecular weight of PMF was calculated to be 4300 g mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and the polymer could be defined as poly(MEMA\u003csub\u003e10\u003c/sub\u003e-co-FAOE\u003csub\u003e9\u003c/sub\u003e). Through self-assembly in PBS solution, the PMF polymer could form nanoparticles with a particle size of around 89 nm and a regular spherical morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). When exposed to a high level of ROS, the methyl sulfide groups in MEMA could realize a hydrophobic-to-hydrophilic conversion, and the oxalate ester bonds in FAOE could be broken, which offered the PMF nanoparticles the ROS triggered disintegration and drug release capabilities.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e The disruption of nanostructure in ROS was first characterized with the size variation under different concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). It was determined that PMF nanoparticles maintained a stable particle size in 0.01 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e within 8 h, while higher concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, exceeding 0.1 mM, could trigger the disassembly of nanoparticles and an increase in particle size. What\u0026rsquo;s more, the morphology of PMF nanoparticles after treated with 1 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 4 h was also observed by TEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), which indicated an obvious decomposition and aggregation of the PMF polymer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRelying on the ROS responsiveness of PMF, the ability of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging and the stimuli-triggered drug delivery were further investigated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). With the increasing concentration of PMF, the amount of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenged gradually increased, and almost 80% of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was cleared within 1 h at a PMF concentration of 2 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). As a result, due to the scavenging of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, the protective effect of PMF on LGG under H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was also evaluated by co-incubation in 1 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg), where LGG could normally grow in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e with the PMF concentration at 1 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Thus, PMF was expected to help LGG probiotic overcome the pathologically intestinal microbial environment with high levels of oxidative stress and protect the activity of LGG.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Besides, the disassembly of PMF in response to ROS led to the breakage of oxalate ester bonds, which in turn facilitated the controlled release of the FMC drug. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh, the speed and amount of FMC delivered from PMF nanoparticles indicated a concentration-dependent response to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. When the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration reached 1 mM, over 50% of FMC was released in 12 h and almost 90% was delivered after 48 h, which provided an ROS-specific delivery of the FMC drug with intensive therapeutic accuracy and reduced drug loss.\u003c/p\u003e \u003cp\u003eAdditionally, PMF nanoparticles were conjugated with LGG probiotic to construct a nano-engineered probiotic through the amidation based on a polydopamine coating. The carboxyls groups on PMF were activated with EDC/NHS and then participated in a dopamine polymerization on the surface of LGG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). The TEM images of free LGG, polydopamine coated LGG (PD@LGG) and nano-engineered probiotic (PDMF@LGG) were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej to determine the conjugation of PMF nanoparticles and LGG probiotics. At the same time, fluorescent DiI-labeled PMF and Hoechst 33342-stained LGG were also used to confirm the preparation of PDMF@LGG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek). The viability of probiotics after nano-engineering was evaluated. Monitoring the optical density at 600 nm with a 30-min interval for 12 h, both polydopamine coating and nano-engineering showed barely any influence on the growth of LGG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003el). Moreover, the conjugated PMF could also protect LGG from the damage of simulated gastric fluid, simulated intestinal fluid and bile salt environment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003em-o). Therefore, the nano-engineered probiotics PDMF@LGG with the maintained activity as well as enhanced viability in harsh environments were expected to achieve better colonization in the intestinal tract, and further regulate the intestinal microbial environment with accurate delivery of FMC drug.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIntestinal Colonization of PDMF@LGG\u003c/h3\u003e\n\u003cp\u003eDuring the oral treatment with nano-engineered probiotics, the colonization and retention of probiotics in the intestinal tract were of great importance to the outcome, which were first evaluated \u003cem\u003ein vitro\u003c/em\u003e on HT29 cells.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e After confirming the cytotoxicity of PMF nanoparticles (\u003cb\u003eFig. S3\u003c/b\u003e), different formulas of PDMF@LGG were used to indicate the adhesion on HT29 cells with in 2 h (\u003cb\u003eFig. S4\u003c/b\u003e), where PMF was labeled with DiI (red) and LGG was stained with MycoLight Green JJ98 (green). Both PD@LGG and PDMF@LGG exhibited markedly enhanced adhesion capacity to the cells, which was owing to the viscous polydopamine coating. Furthermore, the colonization of probiotics was also estimated in C57BL/6 mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). According to the fluorescent signals of PMF and LGG (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), free LGG and PMF nanoparticles were found in the intestinal tract of mice after 6 h from the oral administration. The decrease in fluorescent intensity over extended time indicated a loss of probiotics or nanoparticles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec,d), while free PMF showed a faster reduction because of its nanoscale. After being coated with polydopamine, PD@LGG presented better reservation in the intestinal tract due to the stickiness of dopamine. Moreover, it was notable that PDMF@LGG exhibited the best colonization ability compared to other formulas in the first 12 h, which resulted from the combined effect of the viscosity provided by dopamine and the protective role of PMF to the LGG in the intestine. Furthermore, the retention of PD@LGG and PDMF@LGG in the intestinal tract were observed to be nearly identical after a 24-hour period, suggesting that PMF might provide early-time protection to LGG. To delve deeper into this phenomenon, the short-time colonization was investigated by \u003cem\u003eex vivo\u003c/em\u003e imaging of the separated intestinal tract after 2 h since oral treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). In accordance with the \u003cem\u003ein vivo\u003c/em\u003e results, PDMF@LGG revealed the most reservation in the intestine (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef), and the specific imaging of the cecum also confirmed the consistent results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg,h). Moreover, the sections of the cecum were observed under CLSM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei), where more and stronger fluorescent signals from PMF and LGG were found in PDMF@LGG treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej). Therefore, the synergistic effects of the dopamine-given stickiness and the PMF-mediated protection could enhance the viability and promote extended colonization of PDMF@LGG for better therapeutic outcomes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn Vivo\u003c/b\u003e \u003cb\u003eRegulation on Intestinal Microbiota-TMA-TMAO axis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe nano-engineered probiotics, PDMF@LGG, were designed to combat atherosclerosis through reducing the circulating TMAO, which was regulated by the intestinal microbiota-TMA-TMAO axis.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Thus, FMC drug encapsulated in PMF nanoparticles was utilized to inhibit TMA production in the intestinal flora, while LGG probiotics were expected to protect the integrity of intestinal barriers and prevent TMA invasion into the bloodstream.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e To evaluate the regulation of these processes, mice fed a hypercholine diet were prepared, exhibiting elevated plasma TMAO levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). After confirming the biosafety towards mean organs (\u003cb\u003eFig. S5\u003c/b\u003e), the regulation of PDMF@LGG on intestinal microbiota-TMA-TMAO axis was illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb. Following 6 weeks of high choline diet supplemented with oral administration of various treatments, the choline-related metabolites including TMA and TMAO were quantified (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec-e, \u003cb\u003eFig. S6\u003c/b\u003e). LGG could inhibit circulating TMA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) and TMAO (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed) to a certain extent, while PMF nanoparticles revealed a better outcome, which suggested that the suppression on TMA production contributed more to the control of plasma TMA and TMAO, rather than blocking TMA from entering bloodstream. On the other hand, combining the advantages of probiotics and nanomedicine, PDMF@LGG indicated the most remarkable inhibition on TMA and TMAO, where circulating choline was also nearly restored to the levels under a normal diet by these formulas (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, the expression of key metabolic enzyme FMO3 in the liver was investigated, which undertook the conversion of TMA to TMAO. The immunofluorescence exhibited that FMO3 was clearly suppressed after treatment with PMF and PDMF@LGG (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef,g), where the quantitative western blot analysis also confirmed similar results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh,i). Although previous studies have shown that the increase in plasma TMAO following a high-choline diet was not accompanied by an increase in hepatic FMO3 expression, there was also solid evidences pointing out that insulin resistance caused by high TMAO may increase the expression of FMO3, which supported our findings. \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e On the other side, typical proteins involved in the tight junctions of the intestinal epithelium, which affected TMA absorption from the the intestinal environment, such as ZO-1, Occludin, and Claudin, were detected to investigate the integrity of intestinal barrier.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek, the LGG-treated group revealed a more complete mechanical intestinal barrier, while PDMF@LGG indicated a better barrier integrity owing to the protective benefits of PMF for LGG. Therefore, during the regulation of PDMF@LGG to intestinal microbiota-TMA-TMAO axis, the FMC drug delivered from PMF nanoparticles could inhibit TMA production and thereby reduce circulating TMA/TMAO levels. At the same time, LGG probiotics offered protection to the intestinal endothelium by maintaining the tight junction proteins, which restrained the TMA produced by intestinal flora entering into the blood. As a result, the inhibition of TMA production and the preservation of intestinal barrier collaboratively led to a significant decrease in plasma TMAO, which could contribute to the treatment of atherosclerosis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn Vivo\u003c/b\u003e \u003cb\u003eTreatment to Atherosclerosis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAccording to the clinical results, a number of patients suffered from atherosclerosis without typical risk factors, where a traceable culprit was identified to be TMAO from choline metabolism by enteric microorganism.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e As a result, the therapeutic strategy based on PDMF@LGG inhibiting intestinal microbiota-TMA-TMAO axis to overcome TMAO-triggered atherosclerosis was investigated in ApoE\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice with atherosclerosis by feeding high-choline diet (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). First, the \u003cem\u003ein vivo\u003c/em\u003e regulation of PDMF@LGG on intestinal microbiota-TMA-TMAO axis was evaluated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb,c and \u003cb\u003eFig. S7\u003c/b\u003e). The typical choline-related metabolites showed a similar trend to the results observed in C57BL/6 mice, where TMA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) and TMAO (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) were discovered reduced by PDMF@LGG. In addition, the immunofluorescence signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed,e) as well as the western blot result (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef,g) also suggested that the expression of FMO3 in liver was suppressed by PDMF@LGG. Furthermore, the tight junctions of the intestinal barrier were also protected by PDMF@LGG to cut down the entry of TMA into the blood, where the expression of ZO-1, Occludin, and Claudin were maintained by PDMF@LGG (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh,i). Therefore, the inhibition of PDMF@LGG on the intestinal microbiota-TMA-TMAO axis, which was based on the decline of TMA production and obstruction of TMA entering the blood, was expected to suppress atherosclerotic progression through reduced circulating TMAO (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs a crucial trigger for atherosclerosis, plasma TMAO could facilitate the progression of atherosclerotic plaques.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e After treatment with PDMF@LGG and its different formulas, plaques in the aortas were stained with ORO and the en face views of the aortas were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea. It was suggested that both LGG and PMF could deliver significant inhibition on formation of plaques (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Compared with LGG, PMF indicated a better effect on inhibiting the plaques, which was in accordance with the circulating TMAO levels of these groups. Moreover, PDMF@LGG showed the best suppression on atherosclerotic plaque progression, where the plaques in the aortas were similar to the control group without high-choline diet. On the other hand, the various compositions in the plaques of aortic roots were also stained and quantified (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). While both LGG and PMF exhibited distinct limitations to the plaques\u0026rsquo; progression, PDMF@LGG indicated the most beneficial anti-atherosclerotic effect. After the treatment of PDMF@LGG, the plaques showed a significant reduction in lipid accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed), collagen degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e, necrocytosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef), macrophage infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg), and MMP-9 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh), which suggested a delayed development and a stable plaque structure. Therefore, depending on the inhibition to intestinal microbiota-TMA-TMAO axis, PDMF@LGG could realize the suppression of circulating TMAO which led to the restraint of atherosclerotic progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eRegulation on Enteric Microorganism and Metabolism\u003c/h3\u003e\n\u003cp\u003eConsidering the critical role of PDMF@LGG in modulating intestinal microbial homeostasis and other potential additional benefits for atherosclerosis beyond inhibiting the intestinal microbiota-TMA-TMAO axis, the composition changes in gut microbiome and serum metabolites were detected in high-choline induced atherosclerotic mice treated with saline, LGG, PMF, and PDMF@LGG using 16S ribosomal RNA gene sequencing and targeted LC-MS/MS metabolomics assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Conspicuously, the increased biodiversity in gut microbiome presented by inverse-Simpson diversity index could be observed in all treatment groups compared to saline group, indicating the protective and restorative capacity of LGG and PMF on intestine bacterial richness (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). The β-diversity analysis performed by non-metric multidimensional scaling (NMDS) and principal coordinates analysis (PCoA) plots revealed favorable clustering trends within the same treatment group, while isolated clusters occurred between various groups, suggesting segregated microbiome compositions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec,d). Further analyses at the family- (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee) and genus-level (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef) taxonomy were conducted to explore the relative abundance of gut microbiome after various treatments. Apparently, the bacterial composition of atherosclerotic mice with PDMF@LGG treatment was significantly different compared with other groups, and the relative abundance of characterized microbiota was investigated in detail at different levels. Benefiting from the treatment of PMF and PDMF@LGG, an increase in the abundance of \u003cem\u003eClostridia\u003c/em\u003e at the class-level taxonomy was observed, which was verified to prevent metabolic syndrome through constraining lipid absorption (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg).\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e This may be due to the fact that the ROS-scavenging capacity of PMF improved the living environment for \u003cem\u003eClostridia\u003c/em\u003e, thus enhancing the survival. Meanwhile, PDMF@LGG treatment dramatically increased the relative abundance of \u003cem\u003eLachnospiraceae\u003c/em\u003e family (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh) and \u003cem\u003eOscillospiraceae\u003c/em\u003e family (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ei). \u003cem\u003eLachnospiraceae\u003c/em\u003e family, as a promising probiotic candidate, was reported to be correlated inversely with atherosclerotic cardiovascular diseases, possibly due to its role in intestinal barrier repair, microbiota homeostasis, and production of secondary metabolites.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e The abundance of \u003cem\u003eOscillospiraceae\u003c/em\u003e family was markedly related to the regulation of lipid metabolism and body weight loss, and exerted anti-atherosclerotic effects.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e After relative abundance screening at genus-level taxonomy, \u003cem\u003eOscillibacter\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ej) and \u003cem\u003eRoseburia\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ek) were disclosed to be effectively increased in the PDMF@LGG treated group. Convincing evidence was provided that the cholesterol metabolizing ability of \u003cem\u003eOscillibacter\u003c/em\u003e genus contributed to the maintenance of lipid homeostasis and ultimately benefited cardiovascular health.\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e Multi-omic analysis of atherosclerotic mice and patients showed a strong negative correlation between \u003cem\u003eRoseburia\u003c/em\u003e abundance and cardiovascular diseases, owing to its behaviors on ameliorating atherosclerosis by influencing intestinal gene expression, regulating glycolysis-fatty acid metabolism, and lowering inflammatory responses.\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e LDA effect size (LEfSe) analysis was further performed to explore the differential gut microbiome composition among the groups (\u003cb\u003eFig. S8\u003c/b\u003e). The histogram of LDA discriminant showed the gut microbiome with significant differences in each group, and the cladogram visually presented the multi-taxonomy circle from inside out which represented the taxonomy level from phylum to genus. Consistent with the above results, LefSe clearly exhibited the enrichment of the aforementioned microbiome after PDMF@LGG treatment. These findings confirmed that PDMF@LGG was beneficial in regulating gut microbiome abundance and ameliorating dysbiosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAtherosclerosis is accompanied with metabolic remodeling especially involving glucose abnormality, lipid metabolism disorder, and other metabolites disturbance.\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e Although our study suggested that the therapeutic strategy of modulating microorganism-TMA-TMAO axis relied on PDMF@LGG was advantageous in reducing atherosclerotic plaques, it remained unclear whether other gut microboime-associated metabolites exerted anti-atherosclerosis effects. Therefore, LC-MS/MS metabolomics assay was used to identify the differential metabolites in the serum. Orthogonal partial least squares discriminant analysis (OPLS-DA) plot showed the distinguished tendency of metabolites among the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003el). Additionally, heatmap of serum metabolic profiles also intuitively visualized the metabolite differences, including fatty acids, amino acid and its metabolites, glycoprotein, etc (\u003cb\u003eFig. S9\u003c/b\u003e). To further understand the classification and functional properties of these metabolites, KEGG pathway annotation was conducted and key metabolic pathways were noted. Compared with the saline group, LGG and PMF treated groups were both involved in carbohydrate digestion and absorption, ABC transporters, and starch and sucrose metabolism, whereas LGG treatment significantly regulated the pathways of unsaturated fatty acids biosynthesis, arginine biosynthesis, and metabolism of alanine, aspartate and glutamate, and PMF treatment mainly participated in the vascular smooth muscle contraction and arachidonic acid metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003em,n). The metabolic pathways of primary bile acid biosynthesis, cholesterol metabolism, and choline metabolism in cancer, which were found to be tightly associated with the onset and progression of atherosclerosis, were significantly enriched after PDMF@LGG treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eo).\u003c/p\u003e \u003cp\u003eInteractions between the gut microbiota and serum metabolites serve a vital function in the pathogenesis of atherosclerosis. Consequently, we investigated the association between differential microbes and metabolites under PDMF@LGG treatment in comparison to the saline group. The cluster heatmap based on genus-level taxonomy was drawn (\u003cb\u003eFig. S10\u003c/b\u003e). Detailed information on the top 20 differential metabolites and microbes was also provided (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ep,q). The results showed that \u003cem\u003eRoseburia\u003c/em\u003e and \u003cem\u003eunidentified Clostridia\u003c/em\u003e were positively correlated with Trigonelline (beneficial for cardiovascular diseases because of its strong antioxidant and anti-inflammatory activity and ability to modulate lipid metabolism) \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e and Ectoine (helpful to treat various inflammatory diseases and known as protein protectant, membrane modulator, and DNA protectant) \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, and negatively correlated to 13(R)-HODE (reported to exacerbate atherosclerosis through facilitating foam cell formation, inducing apoptosis, and promoting oxidative stress) \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eRoseburia\u003c/em\u003e also kept negative association with 12,13-EpOME which affected the production of pro-inflammatory mediators and the activation of immune cells, and 13-HOTrE which was recognized as a compound involving in lipid-mediated inflammation response and oxidative stress.\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e Overall, in addition to regulating the intestinal microbiota-TMA-TMAO axis, PDMF@LGG treatment also modulated the composition of gut microbiome and caused changes in key metabolites in the serum, thereby driving the enhanced therapeutic efficacy in hypercholine diet-induced atherosclerotic mice.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAiming to overcome the atherosclerosis caused by high-level TMAO, which was not considered as a typical clinical risk factor, we developed a nano-engineered probiotic PDMF@LGG to realize the inhibition on intestinal microbiota-TMA-TMAO axis for the suppression of circulating TMAO and atherosclerotic progression. To construct PDMF@LGG, the prodrug nanoparticles were prepared with ROS scavenging and stimuli-responsive drug delivery abilities for inhibition on TMA production, which were further engineered onto the surface of LGG probiotic \u003cem\u003evia\u003c/em\u003e a polydopamine coating. Through oral administration, PDMF@LGG could sustainably colonize and maintain activity in the intestinal tract, owing to the stickiness provided by polydopamine coating as well as the protection from oxidative stress injury offered by the PMF nanoparticles. Under conditions of locally overexpressed ROS, PMF nanoparticles could responsively deliver the FMC drug to reduce the production of TMA. On the other hand, it could be found that LGG probiotics maintained the integrity of the tight intestinal epithelial junctions and decreased TMA entering the blood. As a result, PDMF@LGG suppressed the circulating TMA, while the plasma TMAO levels and the expression of hepatic FMO3 were also observed reduced, thereby efficiently restraining the progression of atherosclerosis. In further, by combining microbiomic and metabolomic results, it was noted that PDMF@LGG treatment not only regulated the gut microbiome composition but also modulated a series of serum metabolites, thereby assisting to the therapeutic outcome. In conclusion, the nano-engineered PDMF@LGG probiotic indicated an anti-atherosclerosis activity through inhibiting the intestinal microbiota-TMA-TMAO axis, which provided a promising clinical strategy to address atherosclerosis caused by atypical TMAO.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePreparation and Characterization of PDMF@LGG\u003c/h2\u003e \u003cp\u003eIn order to arm the LGG probiotic with PMF nanoparticles, a coating based on polydopamine and EDC/NHS reaction was utilized. Briefly, PMF nanoparticles (2 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in PBS solution were first modified with EDC (0.16 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and NHS (0.08 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for 1 h. At the same time, PBS solution of LGG (at an absorbance of 1.0 at 600 nm) was stirred for 2 h with the addition of dopamine (0.5 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Afterwards, the polydopamine coated LGG (PD@LGG) was washed with PBS three times and further co-incubated with modified PMF nanoparticles (2 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). After another 2 h, the nano-engineered LGG (PDMF@LGG) was purified by washing with PBS and stored at 4 \u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e \u003cp\u003eTo evaluate the integration of PMF nanoparticles on LGG probiotic, transmission electron microscope (TEM, Thermo Scientific) was used to observe the morphology of LGG, PD@LGG and PDMF@LGG. In further, DiI labeled PMF was also used to arm LGG, which was stained with Hoechst 33342, and the fluorescent signals of PMF and LGG were detected under a CLSM (Nikon A1 Ti). On the other hand, the growth of LGG, PD@LGG, and PDMF@LGG was confirmed by measuring the absorbance at 600 nm every 30 min for 12 h by a microplate reader. Moreover, the survivability of PDMF@LGG in external environment under SGF (pH 1.5) supplemented with pepsin (0.32%), SIF (pH 6.8) supplemented with trypsin (10 mg/ml), and bile salt (0.4%) solutions was determined by calculating the proliferation with CCK-8 assay, with un-coated LGG as a control.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn Vivo\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eEx Vivo\u003c/b\u003e \u003cb\u003eColonization of PDMF@LGG in Intestinal Canal\u003c/b\u003e\u003c/p\u003e \u003cp\u003eC57BL/6 mice were used to investigate the colonization of PDMF@LGG as well as its different formulations. Briefly, the mice were fed with LGG, PMF, PD@LGG and PDMF@LGG, where PMF were labeled with DiI and LGG were stained with MycoLight Green JJ98. At 6, 12, and 24 h post-administration, the mice were anesthetized and the fluorescent signals of PMF and LGG were detected \u003cem\u003ein vivo\u003c/em\u003e by a IVIS spectrum system (Lumina LT). Moreover, the intestinal tracts were separated from the mice after 2 h since oral administration, and the \u003cem\u003eex vivo\u003c/em\u003e fluorescence of the entire intestinal tracts and cecum was measured. On the other hand, the cecum from mice after 24 h treatment was prepared into sections and stained with DAPI. The distribution of fluorescent signals from PMF and LGG in the cecal tissue was observed under a CLSM (Nikon A1 Ti).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn Vivo\u003c/b\u003e \u003cb\u003eRegulation on TMAO Metabolism\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe regulation of PDMF@LGG on TMAO was first investigated in C57BL/6 mice. Along with a high-choline diet, the mice were orally treated with saline, PMF (0.2 mg every time), LGG (10\u003csup\u003e8\u003c/sup\u003e CFU every time), and PDMF@LGG (10\u003csup\u003e8\u003c/sup\u003e CFU every time) every two days. After 6 weeks, the mice were sacrificed and the serum was collected, which was used to calculate the choline related metabolites such as choline, TMA, TMAO, betaine, creatinine, and L-carnitine. In further, the liver tissue was isolated for immunofluorescence as well as western blot to determine the expression of FMO3, and the colonic tissue was used for immunofluorescence staining of intestinal barrier proteins such as ZO-1, Occludin, and Claudin.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn Vivo\u003c/b\u003e \u003cb\u003eTreatment to Atherosclerosis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe ApoE\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were fed with high-choline diet to construct a high-TMAO induced atherosclerosis model, which were also orally administrated with saline, LGG (10\u003csup\u003e8\u003c/sup\u003e CFU each time), PMF (0.2 mg each time), and PDMF@LGG (10\u003csup\u003e8\u003c/sup\u003e CFU each time) every two days. After 16 weeks, the mice were sacrificed and the choline related metabolites in serum were measured. Concurrently, the expression of hepatic FMO3 was determined by immunofluorescence and western blot, while the intestinal barrier proteins in colonic tissue were evaluated by immunofluorescence. Furthermore, to investigate the atherosclerotic progression, the aortas of mice were separated and lengthwise opened, where the \u003cem\u003een face\u003c/em\u003e was stained with oil red O (ORO). On the other hand, the sections of aortic roots were prepared for various histological staining such as ORO, H\u0026amp;E, Masson, CD68, and MMP-9.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMicrobiome and Metabolomic Analysis\u003c/h3\u003e\n\u003cp\u003eAfter four months of different treatments in high-choline diet induced atherosclerotic mice, three pellets of fecal samples were collected and serum were extracted, which were frozen in liquid nitrogen immediately and sent to Metware Metabolic Biotechnology Co., LTD (Wuhan, China) for microbiome and metabolomics analysis. The data were analyzed using a free online platform called Metware Cloud (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cloud.metware.cn\u003c/span\u003e\u003cspan address=\"https://cloud.metware.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Briefly, total genome DNA from feces was extracted using CTAB method, and the purity and concentration were monitored. Genes of 16S V4 region were amplified used specific primers (515F: GTGCCAGCMGCCGCGGTAA; 806R: GGACTACHVGGGTWTCTAAT) with the barcode. PCR products were mixed in equal density ratios and purified with Qiagen Gel Extraction Kit (Qiagen, Germany). Sequencing libraries were generated using TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, USA) and the quantified libraties were sequenced on an Illumina NovaSeq6000 platform and 250 bp paired-end reads were generated. Effective tags were obtained after data split, filtration, sequence assembly and chimera removal. ASV denoising was performed by Deblur (Version 1.1.1) in the QIIME2 software (Version 2023.2). The Silva Database was utilized to annotate taxonomic information. The inverse-Simpson diversity index was calculated to assess the alpha diversity, while NMDS and PCoA were performed to assess beta diversity. Relative abundance of gut microbiome at family and genus levels were visualized and quantitative data of representative microbiome at different levels were shown.\u003c/p\u003e \u003cp\u003eThe pre-treated serum samples were transferred for LC-MS analysis using an LC-ESI-MS/MS system (UPLC, ExionLC AD; MS, QTRAP\u0026reg; System). The original data were qualitatively analyzed based on the Metware Database and the quantification was performed using multiple reaction monitoring (MRM) by triple quadrupole scans. Differential metabolites were determined according to VIP (VIP\u0026thinsp;\u0026gt;\u0026thinsp;1) and P value (P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test). VIP values were extracted from OPLS-DA models, which also contained score plots to show the inter-group differences. Identified metabolites were annotated using KEGG Compound database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.kegg.jp/kegg/compound/\u003c/span\u003e\u003cspan address=\"http://www.kegg.jp/kegg/compound/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and annotated metabolites were then mapped to KEGG Pathway database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.kegg.jp/kegg/pathway.html\u003c/span\u003e\u003cspan address=\"http://www.kegg.jp/kegg/pathway.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed at least three times (n\u0026thinsp;=\u0026thinsp;3\u0026ndash;6) and expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (s.d.). The contrast between two groups was analyzed by SPSS 20.0 software using the Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test and the non-parametric test to assess statistical significance. Statistical significance was assessed at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eReporting summary\u003c/h2\u003e \u003cp\u003eFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe relevant data are available from the corresponding authors upon reasonable request.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAll animal experiments were approved by the Laboratory Animal Research Center of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. The animal protocols were assessed and approved by the Animal Laboratory Ethics Committee of Zhejiang University (SYXK2017-0006).\u003c/p\u003e\u003ch2\u003eReporting summary\u003c/h2\u003e\n\u003cp\u003eFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.\u003c/p\u003e\n\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eThe relevant data are available from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThis research was financially supported by Noncommunicable Chronic Diseases-National Science and Technology Major Project (NO. 2023ZD0503904), National Natural Science Foundation of China (No. 32201128, No. 82270262, No. 32301100), Zhejiang TCM Science and Technology Program TCM modernization Special project, China (No. 2022ZX012) and Natural Science Funds of Zhejiang Province (No. ZCLY24H1801). The schematic figures were created with BioRender.com. We thank Xiaoli Hong from the Core Facilities, Zhejiang University School of Medicine for their technical support.\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eAll authors have read and approved the manuscript. Z.C. and B.M. conceived and designed the study. Z.C., Q.Z., H.X., and B.M. performed the experiments. H.X. and B.M. synthesized and prepared the nanoparticles. Z.C., Y.H., and X.H. analyzed the microbiome and\u0026nbsp;metabolomics data. Y.W., Z.C., and Y,W assisted in the experiments and drew the figures.\u0026nbsp;Z.C. and B.M. wrote the manuscript. G.F., B.M., and W.Z. revised the manuscript and supervised this study.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eAdditional information\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e The online version contains supplementary materials available at\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u003c/strong\u003e and requests for materials should be addressed to Guosheng Fu, Boxuan Ma or Wenbin Zhang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePeer review information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReprints and permissions information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublisher\u0026rsquo;s note\u003c/strong\u003e\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLibby P (2021) The changing landscape of atherosclerosis. 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Nat Commun 14:8151\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLevan SR et al (2019) Elevated faecal 12,13-diHOME concentration in neonates at high risk for asthma is produced by gut bacteria and impedes immune tolerance. Nat Microbiol 4:1851\u0026ndash;1861\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar N et al (2016) 15-Lipoxygenase metabolites of α-linolenic acid, [13-(S)-HPOTrE and 13-(S)-HOTrE], mediate anti-inflammatory effects by inactivating NLRP3 inflammasome. Sci Rep 6:31649\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5612717/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5612717/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eConsiderable numbers of patient are suffering from atherosclerosis without typical risk factors, which can cause severe cardiovascular complication but is lack of practical treatment. Thereinto, trimethylamine \u003cem\u003eN\u003c/em\u003e-oxide (TMAO), originated from enteric microorganism, emerges as an unconventional and crucial factor causing atherosclerosis. Here we demonstrate a strategy to inhibit TMAO through intestinal microbiota-trimethylamine (TMA)-TMAO axis for atherosclerotic treatment. The therapy is performed by an oral-treated nano-engineered probiotic PDMF@LGG, where the probiotic \u003cem\u003eLacticaseibacillus rhamnosus GG\u003c/em\u003e (LGG) is armed with polydopamine coating and conjugated with PMF nanoparticles based on a ROS-responsive polymeric prodrug of fluoromethylcholine (FMC). PDMF@LGG can durably colonize the intestinal canal due to sticky polydopamine coating and the protection of PMF against ROS-induced injury. The ROS trigger the delivery of FMC from nanoparticles, which can inhibit TMA production in enteric microorganisms. Meanwhile, LGG can strengthen the tight junctions of intestinal epithelium and reduce TMA entering the blood. The \u003cem\u003ein vivo\u003c/em\u003e study suggests that PDMF@LGG reduces plasma TMAO and suppresses atherosclerotic progression. Furthermore, the microbiomics and metabolomics show that PDMF@LGG also regulates gut microbial composition and various metabolites, assisting in the therapeutic outcome. Together, PDMF@LGG offers a potential candidate for atherosclerotic therapy caused by TMAO and broadens the range of treatable atherosclerosis.\u003c/p\u003e","manuscriptTitle":"Nano-engineered probiotic treats atherosclerosis via inhibiting intestinal microbiota-TMA-TMAO axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-08 10:37:35","doi":"10.21203/rs.3.rs-5612717/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"17bb3c52-15b9-412e-bb34-d87460ebb3da","owner":[],"postedDate":"January 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":42421153,"name":"Health sciences/Cardiology/Cardiovascular biology/Cardiovascular diseases/Vascular diseases/Atherosclerosis"},{"id":42421154,"name":"Health sciences/Diseases/Cardiovascular diseases/Vascular diseases/Atherosclerosis"},{"id":42421155,"name":"Health sciences/Gastroenterology/Gastrointestinal system/Microbiota"},{"id":42421156,"name":"Biological sciences/Biotechnology/Biomaterials/Biomedical materials"},{"id":42421157,"name":"Biological sciences/Biological techniques/Nanobiotechnology/Nanoparticles"}],"tags":[],"updatedAt":"2025-12-23T08:05:41+00:00","versionOfRecord":{"articleIdentity":"rs-5612717","link":"https://doi.org/10.1038/s41467-025-66448-7","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-12-13 05:00:00","publishedOnDateReadable":"December 13th, 2025"},"versionCreatedAt":"2025-01-08 10:37:35","video":"","vorDoi":"10.1038/s41467-025-66448-7","vorDoiUrl":"https://doi.org/10.1038/s41467-025-66448-7","workflowStages":[]},"version":"v1","identity":"rs-5612717","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5612717","identity":"rs-5612717","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}