Bacterial Outer Membrane Vesicles Research Articles

323 Advances in using membrane vesicles for studying intestinal functions

Abstract Enterocytes are the major cell lineage of the small intestinal epithelia along the crypt-villus axis in gut mucosa. Enterocytes participate in the gut terminal nutrient digestion and absorption; and have essential roles in nutrient and metabolite recycling and efflux, nutrient and hormonal sensing, interactions with symbiotic microbiome and binding to infectious viruses, thus influencing the host homeostasis and whole-body physiological status. Intestinal membrane vesicles have been widely used for studying various gut functions in the past several decades. Villus enterocytic brush border membrane and the apical membrane vesicles along the crypt-villus axis in gut mucosa can be prepared and isolated by Mg2+- and Ca2+-precipitation and differential centrifugation procedures. Whereas, gut mucosal basolateral membrane vesicles can be prepared, fractionated and differentiated from other intracellular organelles such as the mitochondrial inner membrane by the Percoll gradient ultracentrifugation. Nutrient transport and exchange activities in the membrane vesicles in vitro are conducted often by rapid hand filtration, and in excess, in an automated fast sampling apparatus. Nutrient transport and exchange activity kinetics are derived according to the well-established classic Michaelis-Menten and the tracer inhibitory kinetic models with a simple linear diffusional component. Physiological and biochemical properties associated with various nutrient uptake systems such as pH optimum, requirements for co-transported ions (e.g., Na+, K+, Cl-), transporter affinity and substrate specificity can be effectively investigated in vitro with the membrane vesicles by altering the compositions of uptake buffers and intra-vesicular pre-loading buffers. Some of the fundamental gut functions are established via using the membrane vesicles in different mammalian species. And these include H+-peptide cotransport, Na+-cotransport and Na+-independent systems of hexose uptake across the apical membrane and the low-affinity Na+-independent hexose bidirectional uptake across the basolateral membrane that were characterized in rodents; Na+-dependent cotransport and Na+-independent systems of amino acid (AA) transport across the apical membrane that were revealed in rodents and pigs; as well as the Na+-hexose and AA cotransport and the co-expression of the coronavirus SARS-CoV-2 host epithelial apical membrane binding receptor, i.e., the angiotensin-converting enzyme-2 (ACE2), along the gut crypt-villus axis that were identified in liquid formula-fed piglets. In the post genomic and metagenomic sequencing era, further research efforts need to pursue understanding of expression and various functionality of the gut terminal hydrolases, nutrient transporters, nutrient exchangers, nutrient sensors, substrate and particle receptors, taste receptors, viral binding receptors such as ACE2 and hormonal receptors and their interactions with luminal factors such as nutrient, metabolites, symbiotic bacterial outer membrane vesicles etc., along the longitudinal and crypt-villus axes, through using the mammalian primary enterocytic apical and the basolateral membrane vesicles under various research settings.

Pleiotropy of biomineralized bacterial outer membrane vesicles in modulating immune systems for liver cancer therapy

The immune landscape of late-stage liver cancer is featured by severe immunosuppression that is characterized by poor immunogenicity, T-cell exhaustion, and infiltration of a large number of immunosuppressive cells, leading to compromised therapeutic efficacy of mainstream immunotherapies. Herein, we developed a pleiotropic immune cell mobilization strategy to modulate immune systems for immunosuppressive liver cancer therapy. In this study, immunogenic bacteria-derived outer membrane vesicles (OMVs) were exploited as a vector to deposit Cu and Mn with mixed valence states via one-step biomineralization, followed by platelet membrane camouflage, which are denoted as OPCM. Mechanistically, the pleiotropic OPCM possesses POD, CAT, and GPX-like activities, thereby achieving cGAS-STING activation, immunogenic ferroptosis, and tumor hypoxia alleviation, ultimately leading to initiation of the cancer immunity cycle, modulation of both innate and adaptive immune system, and reversion of immunosuppression. Notably, the combination with typical αPD-L1 treatment augmented the tumor suppression effect by amplifying the intensity of ferroptosis due to IFN-γ secretion by activated T cells. Overall, the metal biomineralized OPCMs in combination with αPD-L1 formed a closed-loop therapy that cycles from immunotherapy and ferroptosis therapy, providing new insights for treating immunosuppressive liver cancer.

Bacterial outer membrane vesicles increase polymyxin resistance in Pseudomonas aeruginosa while inhibiting its quorum sensing

The persistent emergence of multidrug-resistant bacterial pathogens is leading to a decline in the therapeutic efficacy of antibiotics, with Pseudomonas aeruginosa (P. aeruginosa) emerging as a notable threat. We investigated the antibiotic resistance and quorum sensing (QS) system of P. aeruginosa, with a particular focused on outer membrane vesicles (OMVs) and polymyxin B as the last line of antibiotic defense. Our findings indicate that OMVs increase the resistance of P. aeruginosa to polymyxin B. The overall gene transcription levels within P. aeruginosa also reveal that OMVs can reduce the efficacy of polymyxin B. However, both OMVs and sublethal concentrations of polymyxin B suppressed the transcription levels of genes associated with the QS system. Furthermore, OMVs and polymyxin B acted in concert on the QS system of P. aeruginosa to produce a more potent inhibitory effect. This suppression was evidenced by a decrease in the secretion of virulence factors, impaired bacterial motility, and a notable decline in the ability to form biofilms. These results reveal that OMVs enhance the resistance of P. aeruginosa to polymyxin B, yet they collaborate with polymyxin B to inhibit the QS system. Our research contribute to a deeper understanding of the resistance mechanisms of P. aeruginosa in the environment, and provide new insights into the reduction of bacterial infections caused by P. aeruginosa through the QS system.

Engineering Planar Gram-Negative Outer Membrane Mimics Using Bacterial Outer Membrane Vesicles.

Antibiotic resistance is a major challenge in modern medicine. The unique double membrane structure of gram-negative bacteria limits the efficacy of many existing antibiotics and adds complexity to antibiotic development by limiting transport of antibiotics to the bacterial cytosol. New methods to mimic this barrier would enable high-throughput studies for antibiotic development. In this study, we introduce an innovative approach to modify outer membrane vesicles (OMVs) from Aggregatibacter actinomycetemcomitans, to generate planar supported lipid bilayer membranes. Our method first involves the incorporation of synthetic lipids into OMVs using a rapid freeze-thaw technique to form outer membrane hybrid vesicles (OM-Hybrids). Subsequently, these OM-Hybrids can spontaneously rupture when in contact with SiO 2 surfaces to form a planar outer membrane supported bilayer (OM-SB). We assessed the formation of OM-Hybrids using dynamic light scattering and a fluorescence quenching assay. To analyze the formation of OM-SBs from OM-Hybrids we used quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence recovery after photobleaching (FRAP). Additionally, we conducted assays to detect surface-associated DNA and proteins on OM-SBs. The interaction of an antimicrobial peptide, polymyxin B, with the OM-SBs was also assessed. These findings emphasize the capability of our platform to produce planar surfaces of bacterial outer membranes, which in turn, could function as a valuable tool for streamlining the development of antibiotics.

Enhancement of the anticancer potential and biosafety of BSA-modified, bacterial membrane-coated curcumin nanoparticles

Bacteria and bacterial components have been widely used as bionanocarriers to deliver drugs to treat tumors. In this study, we isolated bacterial outer membrane vesicles (OMVs) with good stability and high yield for macrophage polarization and cell recruitment. Using ultrasound baths, these bacterial OMVs were combined with curcumin nanoparticles (OMV CUR NPs), following which these nanoparticles were modified with bovine serum albumin (BSA) to achieve high biosafety and tumor-targeting effects. The particle size, PDI, and zeta potential of the BSA-OMV CUR NPs were 157.9 nm, 0.233, and −15.1 mV, respectively. The BSA-OMV CUR NPs exhibited high storage stability, low cytotoxicity, sustained release, enhanced cellular uptake of CUR, induction of tumor cell apoptosis, and inhibition of tumor cell proliferation and migration. By determining the survival rate, body length, heart rate, head size, eye size, and pericardium size of the zebrafish, we found that the BSA-OMV CUR NPs were safe for application in vivo. Moreover, an increase in antiproliferation, antiangiogenic and antimetastatic effects of BSA-OMV CUR NPs was demonstrated in wild-type and transgenic tumor-transplanted zebrafish embryos.

Outer membrane vesicles from commensal microbes contribute to the sponge Tedania sp. development by regulating the expression level of apoptosis-inducing factor (AIF)

The transition from the swimming larval stage to the settlement stage represents a significant node in the marine sponge developmental process. Previous research has shown that the outer membrane vesicles (OMVs) from the bacterial species Tenacibaculum mesophilum associated with the sponge Tedania sp. influence larval settlement: low concentrations of OMVs increase the attachment rate, whereas high concentrations decrease the attachment rate. Here, by comparing the transcriptomes of sponge larvae in filtered seawater (FSW group) and in FSW supplemented with OMVs (FSW-OMV group), the results indicated that bacterial OMVs affected larval settlement by modulating the expression levels of apoptosis-inducing factor (AIF) in the host. Subsequently, quantitative real-time PCR revealed a decrease in aif expression near the time of settlement (SE) compared to that in the control group. RNA interference (RNAi) was used to target the aif gene, and the rate of larval settlement was significantly reduced, confirming the inhibitory effect of high concentrations of OMVs. Moreover, small RNA (sRNA) sequencing of OMVs revealed the existence of abundant AIF-sRNAs of 30 nt, further suggesting that one pathway for the involvement of sponge-associated bacteria in host development is the transport of OMVs and the direct function of cargo loading.

Preparation of bacterial outer membrane vesicles with anti-GPC3 single-chain antibody and identification of their targeting effects on hepatocellular carcinoma

This study aims to prepare bacterial outer membrane vesicles (OMVs) with anti-glypican-3 (GPC3) single-chain antibody and analyze their targeting effects on Hep G2 hepatocellular carcinoma (HCC) cells and tissue. The recombinant plasmid pET28a-Hbp-hGC 33-scFv was constructed by ligating Hbp-hGC 33-scFv to pET28a. Western blotting was employed to determine the prokaryotic expression of the fusion protein Hbp-hGC 33-scFv, on the basis of which the optimal induction conditions were determined. Hbp-hGC 33-OMVs secreted from the recombinant expressing strains were collected by ultrafiltration concentration and then characterized. The localization of Hbp-hGC 33-scFv in bacteria and Hbp-hGC 33-OMVs was analyzed by immune electron microscopy. The binding of Hbp-hGC 33-scFv to Hep G2 cells was observed by immunofluorescence. The Hep G2 tumor-bearing mouse model was established, and the targeted retention of Hbp-hGC 33-OMVs in the tumor site of mice was observed by a fluorescence imaging system in vivo. The results showed that the actual molecular weight of the fusion protein was 175.3 kDa, and the optimal induction conditions were as follows: OD600=0.5, IPTG added at a final concentration of 0.5 mmol/L, and overnight induction at 16 ℃. The prepared Hbp-hGC 33-OMVs were irregular spherical structures with an average particle size of (112.3±4.6) nm, expressing OmpC, OmpA, and the fusion protein Hbp-hGC 33-scFv. The Hbp-hGC 33-OMVs prepared in this study demonstrated stronger ability of binding to Hep G2 cells than the wild-type OMVs (P=0.008). All the data indicated that Hbp-hGC 33-OMVs with anti-GPC3 single-chain antibody were successfully prepared and could be used for research on the targeted therapy of hepatocellular carcinoma.

Dual‐Targeted Engineered Bacterial Outer Membrane Vesicles for Hepatocellular Carcinoma Immunotherapy

AbstractCluster of differentiation 47 (CD47) is an important innate immune checkpoint that empowers tumors to send “don't eat me” signals, leading to immune escape. CD47 monoclonal antibodies have achieved remarkable therapeutic success in hematological malignancies. However, their efficacy in solid tumors is limited, mainly because of immunosuppression in the tumor microenvironment. In addition, hematotoxicity severely hinders the clinical translation of CD47 antibodies. Here, a novel genetically programmed nanovesicle, OMV‐CD47/GPC3, is generated for hepatocellular carcinoma (HCC) immunotherapy by fusing anti‐CD47 nanobodies and anti‐GPC3 scFv onto bacterial outer membrane vesicles (OMVs). Following intravenous administration, dual‐targeted OMV‐CD47/GPC3 specifically recognized tumor cells expressing both CD47 and GPC3, thereby actively targeting and accumulating at the orthotopic HCC tumor site without hematotoxicity. OMV‐CD47/GPC3 potently blocked tumor CD47 and promoted macrophage phagocytosis. More importantly, immunogenic OMV‐CD47/GPC3 can effectively remodel the tumor immune microenvironment of orthotopic HCC, especially reprogram tumor‐associated macrophages (TAMs) to the M1‐phenotype and promote dendritic cell maturation, synergistically inducing a robust CD8+ T cell‐mediated anti‐tumor immune response. With the two‐pronged approach of immune checkpoint inhibition and immune activation, engineered OMV‐CD47/GPC3 significantly suppressed orthotopic HCC growth and prevented tumor recurrence and metastasis, thus providing a promising strategy for HCC immunotherapy.

Chitosan oligosaccharides alleviate macrophage pyroptosis and protect sepsis mice via activating the Nrf2/GPX4 pathway

In the process of sepsis, excessive occurrence of pyroptosis, a form of programmed cell death acting as a defense mechanism against pathogens, can disrupt immune responses, thus leading to tissue damage and organ dysfunction. Chitosan oligosaccharide (COS), derived from chitosan degradation, has demonstrated diverse beneficial effects. However, its impact on sepsis-induced pyroptosis remains unexplored. In the present study, ATP/LPS was utilized to induce canonical-pyroptosis in THP-1 cells, while bacterial outer membrane vesicles (OMV) were employed to trigger non-canonical pyroptosis in RAW264.7 cells. Our results revealed a dose-dependent effect of COS on both types of pyroptosis. This was evidenced by a reduction in the expression of pro-inflammatory cytokines, as well as crucial regulatory proteins involved in pyroptosis. In addition, COS inhibited the cleavage of caspase-1 and GSDMD, and reduced ASC oligomerization. The underlying mechanism revealed that COS acts an antioxidant, reducing the release of pyroptosis-induced ROS and malondialdehyde (MDA) by upregulation the expression and promoting the nuclear translocation of nuclear factor erythroid-2-related factor 2 (Nrf2), which led to an elevation of glutathione peroxidase 4 (GPX4) and superoxide dismutase (SOD). Notably, the actions of COS were completely reversed by the Nrf2 inhibitor. Consequently, COS intervention increased the survival rate of sepsis.

Bacterial outer membrane vesicle nanorobot

Autonomous nanorobots represent an advanced tool for precision therapy to improve therapeutic efficacy. However, current nanorobotic designs primarily rely on inorganic materials with compromised biocompatibility and limited biological functions. Here, we introduce enzyme-powered bacterial outer membrane vesicle (OMV) nanorobots. The immobilized urease on the OMV membrane catalyzes the decomposition of bioavailable urea, generating effective propulsion for nanorobots. This OMV nanorobot preserves the unique features of OMVs, including intrinsic biocompatibility, immunogenicity, versatile surface bioengineering for desired biofunctionalities, capability of cargo loading and protection. We present OMV-based nanorobots designed for effective tumor therapy by leveraging the membrane properties of OMVs. These involve surface bioengineering of robotic body with cell-penetrating peptide for tumor targeting and penetration, which is further enhanced by active propulsion of nanorobots. Additionally, OMV nanorobots can effectively safeguard the loaded gene silencing tool, small interfering RNA (siRNA), from enzymatic degradation. Through systematic in vitro and in vivo studies using a rodent model, we demonstrate that these OMV nanorobots substantially enhanced siRNA delivery and immune stimulation, resulting in the utmost effectiveness in tumor suppression when juxtaposed with static groups, particularly evident in the orthotopic bladder tumor model. This OMV nanorobot opens an inspiring avenue to design advanced medical robots with expanded versatility and adaptability, broadening their operation scope in practical biomedical domains.

Biomimetic nanomedicine cocktail enables selective cell targeting to enhance ovarian Cancer chemo- and immunotherapy

Ovarian cancer is one of the deadliest cancers, and combined chemo- and immunotherapies are potential strategies to combat it. However, the anti-cancer efficacy of the combined therapies may be limited by the non-selective co-delivery of chemotherapy and immunotherapy. Herein, a combined chemo- and immunotherapy is designed to selectively target ovarian tumor (ID8) cells and dendritic cells (DCs) using ID8 cell membrane (IM) and bacterial outer membrane vesicles (OMVs), respectively. Doxorubicin (DOX) and Ovalbumin (OVA) peptide (OVA257–264) are chosen as model chemotherapy and immunotherapy agents, respectively. A DNA nanocube capable of easily loading DOX or OVA257–264 is chosen as the carrier. Firstly, the DNA nanocube is used to load DOX or OVA257–264 to prepare cube-DOX or cube-OVA. This nanocube was then encapsulated with IM to form IM@Cube-DOX and with OMV to form OMV@Cube-OVA. IM@Cube-DOX can be selectively taken up by ID8 cells, leading to effective cell killing, while OMV@Cube-OVA targets and activates DC2.4 cells in vitro. Both IM@Cube-DOX and OMV@Cube-OVA show increased accumulation at ID8 tumors in C57BL/6 mice. Combined IM@Cube-DOX + OMV@Cube-OVA therapy demonstrates better anti-tumor efficacy than non-selective delivery methods such as OMV@(Cube-DOX + Cube-OVA) or IM@(Cube-DOX + Cube-OVA) in ID8-OVA tumor-bearing mice. In conclusion, this study demonstrates a biomimetic delivery strategy that enables selective drug delivery to tumor cells and DCs, thereby enhancing the anti-tumor efficacy of combined chemo- and immunotherapy through the selective delivery strategy.

Brain-targeting biomimetic disguised manganese dioxide nanoparticles via hybridization of tumor cell membrane and bacteria vesicles for synergistic chemotherapy/chemodynamic therapy of glioma

Glioma is a prevalent brain malignancy associated with poor prognosis. Although chemotherapy serves as the primary treatment for brain tumors, its effectiveness is hindered by the limited ability of drugs to traverse the blood–brain barrier (BBB) and the development of drug resistance linked to tumor hypoxia. Herein, we report the creation of hybrid camouflaged multifunctional nanovesicles comprising membranes of tumor C6 cells (mT) and bacterial outer membrane vesicles (OMVs) and co-loaded with manganese dioxide nanoparticles (MnO2 NPs) and doxorubicin (DOX) to synergistically enhance the chemotherapy/chemodynamic therapy (CDT) of glioma. Owing to OMV-mediated BBB penetration and mT-inherited tumor-homing properties, MnO2-DOX@mT/OMVs can penetrate the BBB and enhance the tumor cell-specific uptake of DOX via “proton sponge effect”-mediated lysosomal escape. This enhances the apoptotic effect induced by DOX and minimizing DOX-associated cardiotoxicity by facilitating the accumulation of DOX at the tumor site. Furthermore, the MnO2 NPs in MnO2-DOX@mT/OMVs can generate potent CDT by accelerating the Fenton-like reaction with DOX-generated H2O2 and achieving glutathione (GSH)-depletion-induced glutathione peroxidase 4 (GPX4) inactivation. These results showed that MnO2-DOX@mT/OMVs, designed for brain tumor targeting, significantly inhibited tumor growth and exhibited favorable biological safety. This innovative approach offers the augmentation of anticancer treatment efficacy via a potential combination of chemotherapy and CDT.

Gut Pathobiont-Derived Outer Membrane Vesicles Drive Liver Inflammation and Fibrosis in PSC-IBD

Background & AimsPrimary sclerosing cholangitis (PSC), often associated with inflammatory bowel disease (IBD), presents a multifactorial etiology involving genetic, immunological, and environmental factors. Gut dysbiosis and bacterial translocation have been implicated in PSC-IBD, yet the precise mechanisms underlying their pathogenesis remain elusive. Here, we describe the role of gut pathobionts in promoting liver inflammation and fibrosis due to the release of bacterial outer membrane vesicles (OMVs). MethodsPreclinical mouse models in addition to ductal organoids were used to acquire mechanistic data. A proof-of-concept study including serum and liver biopsies of a patient cohort of PSC (n=22), PSC-IBD (n=45) and control individuals (n=27) was performed to detect OMVs in the systemic circulation and liver. ResultsIn both, preclinical model systems and in human PSC-IBD patients, the translocation of OMVs to the liver correlated with enhanced bacterial sensing and accumulation of the NLRP3 inflammasome. Using ductal organoids, we were able to precisely attribute the pro-inflammatory and pro-fibrogenic properties of OMVs to signaling pathways dependent on TLR4 and NLRP3-GSDMD. The immunostimulatory potential of OMVs could be confirmed in macrophages and hepatic stellate cells. Furthermore, when we administered gut pathobiont-derived OMVs to Mdr2-/- mice, we observed a significant enhancement in liver inflammation and fibrosis. In a translational approach, we substantiated the presence of OMVs in the systemic circulation and hepatic regions of severe fibrosis using a PSC-IBD patient cohort. ConclusionThis study demonstrates the contribution of gut pathobionts in releasing OMVs that traverse the mucosal barrier, and thus, promote liver inflammation and fibrosis in PSC-IBD. OMVs might represent a critical new environmental factor that interacts with other disease factors to cause inflammation and thus define potential new targets for fibrosis therapy.

Harnessing Bacterial Membrane Components for Tumor Vaccines: Strategies and Perspectives.

Tumor vaccines stand at the vanguard of tumor immunotherapy, demonstrating significant potential and promise in recent years. While tumor vaccines have achieved breakthroughs in the treatment of cancer, they still encounter numerous challenges, including improving the immunogenicity of vaccines and expanding the scope of vaccine application. As natural immune activators, bacterial components offer inherent advantages in tumor vaccines. Bacterial membrane components, with their safer profile, easy extraction, purification, and engineering, along with their diverse array of immune components, activate the immune system and improve tumor vaccine efficacy. This review systematically summarizes the mechanism of action and therapeutic effects of bacterial membranes and its derivatives (including bacterial membrane vesicles and hybrid membrane biomaterials) in tumor vaccines. Subsequently, the authors delve into the preparation and advantages of tumor vaccines based on bacterial membranes and hybrid membrane biomaterials. Following this, the immune effects of tumor vaccines based on bacterial outer membrane vesicles are elucidated, and their mechanisms are explained. Moreover, their advantages in tumor combination therapy are analyzed. Last, the challenges and trends in this field are discussed. This comprehensive analysis aims to offer a more informed reference and scientific foundation for the design and implementation of bacterial membrane-based tumor vaccines.

Lysosome-Targeting Bacterial Outer Membrane Vesicles for Tumor Specific Degradation of PD-L1.

Increased expression of immune check point genes, such as PD-L1, is one of the main reasons for immunosuppression, especially for colon cancer. Development of novel therapeutic strategies is of great importance to improve the prognosis. In this study, outer membrane vesicles (OMV) derived from Gram-negative bacteria are engineered to immune checkpoint blockade nanosystem for efficient elicitation of anti-tumor immunity. Briefly, the OMVs are engineered with Lyp1-Traptavidin (S52G, R53D mutant of streptavidin) fusion protein displayed on the surface. The Lyp-1 endows the OMV with the capacity to target tumor tissues, while the Traptavidin ensures easy decoration of biotinylated anti-PD-L1 and biotinylated M6P (mannose 6-phosphate). The simultaneously anchored anti-PD-L1 and M6P (ligand for cation-independent mannose 6-phosphate receptor) on the engineered OMVs coordinately direct the membrane PD-L1 to lysosome for degradation, and thus unleash the anti-tumor immunity. With syngeneic tumor model, the engineered OMVs are confirmed to boost immunity, inhibit cancer growth, and thus prolong survival. Together, A proposed OMV-based modular nanosystem that enables assembly of biotinylated anti-PD-L1 and M6P on the surface for tumor-targeted immune checkpoint blockade.

Structural stability for surface display of antigen 43 and application to bacterial outer membrane vesicles production

Structural stability for surface display of antigen 43 and application to bacterial outer membrane vesicles production

Bacterial outer membrane vesicle-cancer cell hybrid membrane-coated nanoparticles for sonodynamic therapy in the treatment of breast cancer bone metastasis

Breast cancer bone metastasis is a terminal-stage disease and is typically treated with radiotherapy and chemotherapy, which causes severe side effects and limited effectiveness. To improve this, Sonodynamic therapy may be a more safe and effective approach in the future. Bacterial outer membrane vesicles (OMV) have excellent immune-regulating properties, including modulating macrophage polarization, promoting DC cell maturation, and enhancing anti-tumor effects. Combining OMV with Sonodynamic therapy can result in synergetic anti-tumor effects. Therefore, we constructed multifunctional nanoparticles for treating breast cancer bone metastasis. We fused breast cancer cell membranes and bacterial outer membrane vesicles to form a hybrid membrane (HM) and then encapsulated IR780-loaded PLGA with HM to produce the nanoparticles, IR780@PLGA@HM, which had tumor targeting, immune regulating, and Sonodynamic abilities. Experiments showed that the IR780@PLGA@HM nanoparticles had good biocompatibility, effectively targeted to 4T1 tumors, promoted macrophage type I polarization and DC cells activation, strengthened anti-tumor inflammatory factors expression, and presented the ability to effectively kill tumors both in vitro and in vivo, which showed a promising therapeutic effect on breast cancer bone metastasis. Therefore, the nanoparticles we constructed provided a new strategy for effectively treating breast cancer bone metastasis.Graphical

The Balance between Protealysin and Its Substrate, the Outer Membrane Protein OmpX, Regulates Serratia proteamaculans Invasion.

Serratia are opportunistic bacteria, causing infections in plants, insects, animals and humans under certain conditions. The development of bacterial infection in the human body involves several stages of host-pathogen interaction, including entry into non-phagocytic cells to evade host immune cells. The facultative pathogen Serratia proteamaculans is capable of penetrating eukaryotic cells. These bacteria synthesize an actin-specific metalloprotease named protealysin. After transformation with a plasmid carrying the protealysin gene, noninvasive E. coli penetrate eukaryotic cells. This suggests that protealysin may play a key role in S. proteamaculans invasion. This review addresses the mechanisms underlying protealysin's involvement in bacterial invasion, highlighting the main findings as follows. Protealysin can be delivered into the eukaryotic cell by the type VI secretion system and/or by bacterial outer membrane vesicles. By cleaving actin in the host cell, protealysin can mediate the reversible actin rearrangements required for bacterial invasion. However, inactivation of the protealysin gene leads to an increase, rather than decrease, in the intensity of S. proteamaculans invasion. This indicates the presence of virulence factors among bacterial protealysin substrates. Indeed, protealysin cleaves the virulence factors, including the bacterial surface protein OmpX. OmpX increases the expression of the EGFR and β1 integrin, which are involved in S. proteamaculans invasion. It has been shown that an increase in the invasion of genetically modified S. proteamaculans may be the result of the accumulation of full-length OmpX on the bacterial surface, which is not cleaved by protealysin. Thus, the intensity of the S. proteamaculans invasion is determined by the balance between the active protealysin and its substrate OmpX.

Engineered Microrobots for Targeted Delivery of Bacterial Outer Membrane Vesicles (OMV) in Thrombus Therapy.

In the realm of thrombosis treatment, bioengineered outer membrane vesicles (OMVs) offer a novel and promising approach, as they have rich content of bacterial-derived components. This study centers on OMVs derived from Escherichia coli BL21 cells, innovatively engineered to encapsulate the staphylokinase-hirudin fusion protein (SFH). SFH synergizes the properties of staphylokinase (SAK) and hirudin (HV) to enhance thrombolytic efficiency while reducing the risks associated with re-embolization and bleeding. Building on this foundation, this study introduces two cutting-edge microrobotic platforms: SFH-OMV@H for venous thromboembolism (VTE) treatment, and SFH-OMV@MΦ, designed specifically for cerebral venous sinus thrombosis (CVST) therapy. These platforms have demonstrated significant efficacy in dissolving thrombi, with SFH-OMV@H showcasing precise vascular navigation and SFH-OMV@MΦ effectively targeting cerebral thrombi. The study shows that the integration of these bioengineered OMVs and microrobotic systems marks a significant advancement in thrombosis treatment, underlining their potential to revolutionize personalized medical approaches to complex health conditions.

Multivariate Analysis of Individual Bacterial Outer Membrane Vesicles Using Fluorescence Microscopy.

Gram-negative bacteria produce outer membrane vesicles (OMVs) that play a critical role in cell-cell communication and virulence. OMVs have emerged as promising therapeutic agents for various biological applications such as vaccines and targeted drug delivery. However, the full potential of OMVs is currently constrained by inherent heterogeneities, such as size and cargo differences, and traditional ensemble assays are limited in their ability to reveal OMV heterogeneity. To overcome this issue, we devised an innovative approach enabling the identification of various characteristics of individual OMVs. This method, employing fluorescence microscopy, facilitates the detection of variations in size and surface markers. To demonstrate our method, we utilize the oral bacterium Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) which produces OMVs with a bimodal size distribution. As part of its virulence, A. actinomycetemcomitans secretes leukotoxin (LtxA) in two forms: soluble and surface associated with the OMVs. We observed a correlation between the size and toxin presence where larger OMVs were much more likely to possess LtxA compared to the smaller OMVs. In addition, we noted that, among the smallest OMVs (<100 nm diameter), the fractions that are toxin positive range from 0 to 30%, while the largest OMVs (>200 nm diameter) are between 70 and 100% toxin positive.