Surface Mineralization of Engineered Bacterial Outer Membrane Vesicles to Enhance Tumor Photothermal/Immunotherapy.

Gram-negative bacteria can naturally produce nanosized spherical outer membrane vesicles (OMVs) with a lipid bilayer membrane, possessing immunostimulatory capabilities to be potentially applied in tumor therapy. However, the systemic toxicity induced by pathogen-associated molecular patterns (PAMPs) of OMVs is the main obstacle for their clinical translation. Herein, melanin-loaded OMVs were produced with a genetic engineering strategy and further coated with calcium phosphate (CaP) to reduce their toxicity to enhance tumor treatment effects. Wild-type bacterium Escherichia coli Nissle 1917 (EcN) was genetically engineered to highly express tyrosinase to catalyze the intracellular synthesis of melanin, giving melanin-loaded OMVs (OMVMel). To reduce the systemic toxicity in tumor therapy, OMVMel was coated with CaP by surface mineralization to obtain OMVMel@CaP. In comparison with OMVMel, OMVMel@CaP showed lower systemic inflammatory responses in healthy mice and less damage to the liver, spleen, lung, and kidney, so the administration dose could be increased to enhance the antitumor effect. In the acidic tumor microenvironment, the CaP shell disintegrated to release OMVMel to trigger antitumor immune responses. Under costimulation of OMVMel acting as immunoadjuvants and the damage-associated molecular patterns (DAMPs) released by the photothermal effect, the efficiency of tumor photothermal/immunotherapy was largely boosted through promoting the infiltration of matured DCs, M1 macrophages, and activated CD8+ T cells, decreasing the ratio of MDSCs in tumors.

Genetically engineered probiotics for an optical imaging-guided tumor photothermal therapy/immunotherapy.

Bacteria-based cancer therapy (BCT) has been extensively investigated because of the tumor targeting and antitumor immunity activating abilities of bacteria over traditional nanodrugs, but their potential systemic toxicity poses a challenge. Therefore, it is important to visualize the precise localization and real-time distribution of bacteria in vivo to guide the treatment. Herein, biogenetically engineered Escherichia coli Nissle 1917 (EcN) were constructed to highly express tyrosinase to intracellularly generate cyanine 5-labeled melanin-like polymers (Cy5-Mel), thus endowing them with a bright fluorescence and an excellent photothermal performance upon NIR laser irradiation, thereby inducing the intense immunogenic death of tumor cells and release of tumor-associated antigens. Acting as adjuvants, bacteria can greatly stimulate the maturation of dendritic (DC) cells. The in vivo behaviors of these bacteria was monitored via noninvasive optical imaging when they were intravenously administrated to tumor-bearing mice. From this, NIR exposure on tumor sites was carried out at an appropriate time point to induce the damage to tumor cells and for the modulation of tumor immune microenvironments. Thus, via a simple bioengineering strategy, a promising bacteria-based theranostic platform was constructed for tumor treatment.

Engineered Extracellular Vesicles to Enhance Antigen Presentation for Boosting Light-Driven Tumor Immunotherapy.

Extracellular vesicles (EVs) have emerged as potential tools for tumor-target therapy accompanied with activating anticancer immune responses by serving as an integrated platform, but usually suffered from the limited cross presentation of tumor-associated antigen by dendritic cells (DCs). Here, a straightforward engineering strategy to construct heat shock proteins 70 (HSP70) highly expressed EVs incapsulated with Te nanoparticles (Te@EVsHSP70 ) for tumor photothermal therapy triggering improved immunotherapy is proposed. Tumor cells are firstly used as bioreactors for intracellular synthesis of Te nanoparticles, and NIR irradiation is subsequently introduced to upregulate the expression of HSP70 to give engineered Te@EVsHSP70 through exocytosis. Te@EVsHSP70 exhibits excellent photothermal performance and enhanced tumor antigen capture capability, which induces significant immunogenic death of tumor cells and improves DCs maturation both in vitro and in vivo. Thus, the engineered EVs demonstrate superior antitumor efficacy through photothermal effect and following provoked antitumor immune responses. This work provides a facile method to fabricate multifunctional EVs-based drug delivery system for improving photothermal-triggered tumor immunotherapy.

Bimetallic Ions Functionalized Metal-Organic-Framework Nanozyme for Tumor Microenvironment Regulating and Enhanced Photodynamic Therapy for Hypoxic Tumor.

Photodynamic therapy (PDT), as a light irradiation inducing reactive oxygen species (ROS) generation for cancer treatment, offers facile and promising solutions with respect to spatiotemporal control of ROS generation, and minimizes the systemic toxicity and side effects for highly precise tumor therapy. However, the PDT efficiency is often severely compromised by the complex tumor microenvironment (TME), such as the hypoxic condition and overexpressed antioxidants. Here, for the first time, a bimetallic ion-modified metal-organic framework nanozyme (Zr4+ -MOF-Ru3+ /Pt4+ -Ce6@HA, ZMRPC@HA) is designed. ZMRPC@HA with catalase (CAT) and glutathione oxidase (GSHOx) mimetic activities, can efficiently regulate TME by generation of O2 and deplete the GSH synergistically for enhancing the long-term PDT efficacy toward the hypoxic tumor. The in vitro cell inhibition and in vivo on tumor xenograft evaluations demonstrate the PDT strategy by using ZMRPC@HA can successfully inhibit the differentiation and proliferation of tumor cells under a 660 nm laser irradiation in deep tissues. These findings open a new avenue for the design of multimetallic ions functionalized MOF-based nanozymes with multienzyme mimetic activities toward the antitumor and various other biological applications.

The protective role of microbiota in the prevention of MPTP/P-induced Parkinson's disease by resveratrol.

Parkinson's disease (PD) is a tricky neurodegenerative disease characterized with motor deficits and gastrointestinal (GI) dysfunction. Gut microbiota disturbance is reported to be involved in the clinical phenotypes of PD and its pathogenesis through the brain-gut-microbiota axis. Resveratrol is a natural polyphenol that possesses various biological activities in alleviating many diseases, including PD. The present study was aimed to investigate the role of gut microbiota in resveratrol-treated PD mice. A chronic mouse model of PD was generated via the injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probenecid (MPTP/P) for 5 consecutive weeks. Resveratrol was orally administered once a day (30 mg kg-1 d-1) for a total of 8 weeks. From the 6th week to the 8th week, fecal microbiota transplantation (FMT) was performed from resveratrol-treated PD mice to PD mice to evaluate the contribution of resveratrol-shaped microbiota in the alleviation of PD. The results showed that FMT from resveratrol-shaped microbiota remarkably alleviated the mice phenotype from PD progression, including increased latency in the rotarod, shortened beam walking time, increased the number of tyrosine hydroxylase (TH)-positive cells in the substantia nigra pars compacta (SNpc) and enriched TH-positive fiber density in the striatum. Further experiments revealed that FMT could ameliorate the GI dysfunction by increasing the small intestinal transport rate and the colon length, decreasing the relative abundances of inflammatory cytokines (TNF-α, IL-6 and IL-1ß) in colon epithelial tissue. The 16S rDNA sequencing indicated that FMT attenuated the gut microbial dysbiosis in PD mice by increasing the abundances of Prevotellaceae, Rikenellaceae, Erysipelotrichaceae, Blautia and Alistipes, lowering the ratio of Fimicutes/Bacteroidetes, and decreasing the abundances of Lachnospiraceae and Akkermansia. Therefore, results in this study demonstrated that gut microbiota played a vital role in the prevention of PD progression, and the shaping of the gut microbiota was the pharmacological mechanism of resveratrol in alleviating the phenotype of Parkinson's disease in PD mice.

The role of imaging in targeted delivery of nanomedicine for cancer therapy.

Nanomedicines overcome the pharmacokinetic limitations of traditional drug formulations and have promising prospect in cancer treatment. However, nanomedicine delivery in vivo is still facing challenges from the complex physiological environment. For the purpose of effective tumor therapy, they should be designed to guarantee the five features principle, including long blood circulation, efficient tumor accumulation, deep matrix penetration, enhanced cell internalization and accurate drug release. To ensure the excellent performance of the designed nanomedicine, it would be better to monitor the drug delivery process as well as the therapeutic effects by real-time imaging. In this review, we summarize strategies in developing nanomedicines for efficiently meeting the five features of drug delivery, and the role of several imaging modalities (fluorescent imaging (FL), magnetic resonance imaging (MRI), computed tomography (CT), photoacoustic imaging (PAI), positron emission tomography (PET), and electron microscopy) in tracing drug delivery and therapeutic effect in vivo based on five features principle.

Bacterially Synthesized Tellurium Nanorods for Elimination of Advanced Malignant Tumor by Photothermal Immunotherapy.

Probiotic Escherichia coli Nissle 1917 (EcN) are employed as a bioreactor for intracellularly synthesizing tellurium nanorods (TeNRs) providing a biohybrid therapeutic platform (Te@EcN) for the elimination of advanced malignant tumor by photothermal immunotherapy. Te@EcN is found to possess superior photothermal property upon near-infrared irradiation, and can efficiently accumulate and retain in tumors, although EcN loses proliferation ability after the synthesis of TeNRs, thus inducing considerable immunogenic tumor cell death. Under co-stimulation by EcN acting as immunoadjuvants, maturation of dendritic cells and priming of cytotoxic T cells are largely promoted. In addition, Te@EcN can reprogram tumor-associated macrophages to ameliorate the immunosuppressive tumor microenvironment. Thus, tumor metastasis and recurrence can be efficiently suppressed. Most importantly, owing to the non-pathogenicity of probiotic EcN and their non-proliferative characteristics after TeNRs synthesis, Te@EcN is found to be rapidly metabolized and cleared from the normal tissues, showing very slight acute side effects in healthy mice even at a relatively high administration dose. Therefore, the proposed combined therapeutic strategy based on bacteria-synthesized TeNRs may find great potential in improving bacteria-mediated tumor therapy with increased antitumor efficacy and reduced toxicity.

Inhibitor of Differentiation-2 Protein Ameliorates DSS-Induced Ulcerative Colitis by Inhibiting NF-κB Activation in Neutrophils.

The loss of inhibitor of differentiation-2 (ID2) could lead to the development of colitis in mice, supplementation with exogenous ID2 protein might be a potential strategy to ameliorate colitis. In this study, the effects of ID2 protein supplementation on Dextran sodium sulfate (DSS)-induced colitis were investigated. Firstly, we confirmed that the expression of ID2 was reduced in the colon tissues of DSS-induced colitis mice and patients with ulcerative colitis (UC). Then, we constructed a recombinant plasmid containing the human Id2 gene and expressed it in Escherichia coli (E. coli) successfully. After purification and identification, purified hID2 could ameliorate DSS-induced colitis efficiently in mice by improving disease symptoms, decreasing the levels of proinflammatory cytokines in colon tissues, maintaining the integrity of intestinal barrier and reducing the infiltration of neutrophils and macrophages in the colon. Further study showed that hID2 could be endocytosed efficiently by neutrophils and macrophages, and hID2 lost its protection function against colitis when neutrophils were depleted with an anti-Gr-1 antibody. hID2 decreased the mRNA levels of IL-6, IL-1ß and TNF-α in lipopolysaccharides (LPS)-stimulated neutrophils and efficiently inhibited the activation of NF-κB signalling pathway in neutrophils. Interestingly, hID2 showed a synergistic role in inhibition of NF-κB activation with pyrrolidine dithiocarbamic acid (PDTC), an inhibitor of NF-κB activation. Therefore, this study demonstrated the potential use of hID2 to treat UC, and hID2 protein might be a promising anti-inflammatory agent that targets the NF-κB signalling pathway in neutrophils.

Interplay between the Gut Microbiome and Metabolism in Ulcerative Colitis Mice Treated with the Dietary Ingredient Phloretin.

A growing number of healthy dietary ingredients in fruits and vegetables have been shown to exhibit diverse biological activities. Phloretin, a dihydrochalcone flavonoid that is abundant in apples and pears, has anti-inflammatory effects on ulcerative colitis (UC) mice. The gut microbiota and metabolism are closely related to each other due to the existence of the food-gut axis in the human colon. To investigate the interplay of faecal metabolites and the microbiota in UC mice after phloretin treatment, phloretin (60 mg/kg) was administered by gavage to ameliorate dextran sulfate sodium (DSS)-induced UC in mice. Gut microbes and faecal metabolite profiles were detected by high-throughput sequencing and liquid chromatography mass spectrometry (LC-MS) analysis, respectively. The correlations between gut microbes and their metabolites were evaluated by Spearman correlation coefficients. The results indicated that phloretin reshaped the disturbed faecal metabolite profile in UC mice and improved the metabolic pathways by balancing the composition of faecal metabolites such as norepinephrine, mesalazine, tyrosine, 5-acetyl-2,4-dimethyloxazole, and 6-acetyl-2,3-dihydro-2-(hydroxymethyl)-4(1H)-pyridinone. Correlation analysis identified the relations between the gut microbes and their metabolites. Proteus was negatively related to many faecal metabolites, such as norepinephrine, L-tyrosine, laccarin, dopamine glucuronide, and 5-acetyl-2,4-dimethyloxazole. The abundance of unidentified Bacteriodales_S24-7_group was positively related to ecgonine, 15-KETE and 6-acetyl-2,3-dihydro-2-(hydroxymethyl)-4(1H)-pyridinone. The abundance of Christensenellaceae_R-7_group was negatively related to the levels of 15-KETE and netilmicin. Stenotrophomonas and 15-KETE were negatively related, while Intestinimonas and alanyl-serine were positively related. In conclusion, phloretin treatment had positive impacts on faecal metabolites in UC mice, and the changes in faecal metabolites were closely related to the gut microbiota.

Albumin-Templated Bi2Se3-MnO2 Nanocomposites with Promoted Catalase-Like Activity for Enhanced Radiotherapy of Cancer.

Novel and effective radiosensitizers that can enhance radiosensitivity of tumor tissues and increase the local radiation dose are highly desirable. In this work, templated by bovine serum albumin (BSA), Bi2Se3-MnO2 nanocomposites (Bi2Se3-MnO2@BSA) were fabricated via biomineralization, while Bi2Se3 nanodots act as radiosensitizers to increase the local radiation dosage because of their strong X-ray attenuation ability, and MnO2 with catalase-like activity can increase the oxygen concentration in tumors by triggering the decomposition of tumor endogenous H2O2 so as to improve the hypoxia-associated radioresistance of tumors. Owing to the interaction of the two components in the interface, Bi2Se3-MnO2@BSA showed promoted catalytic activity compared to MnO2@BSA, favoring tumor radiotherapy (RT) sensitization. BSA templating enabled the nanocomposites with high colloidal stability and biocompatibility as well as satisfactory tumor targeting both in vitro and in vivo; thus, an enhanced RT efficacy was obtained. Moreover, the proposed Bi2Se3-MnO2@BSA exhibited excellent performances in computerized tomography and magnetic resonance imaging. Thus, this work provides a tumor microenvironment-responsive multifunctional theranostic nanoagent with an improved performance for imaging-guided tumor RT sensitization.

Influence of the densities and nutritional components of bacterial colonies on the culture-enriched gut bacterial community structure.

Isolating relevant microorganisms is still a substantial challenge that limits the use of bacteria in the maintenance of human health. To confirm which media and which bacterial colony densities can enrich certain kinds of bacteria, we selected eight common media and used them to enrich the gut microorganisms on agar plates. Then, we calculated the numbers of bacterial colonies and collected the bacterial culture mixtures from each kind of medium. Using the Illumina HiSeq platform, we analyzed the composition and diversity of the culture-enriched gut bacterial community. Our data suggested that medium supplemented with blood could increase the diversity of the bacterial community. In addition, beef powder and peptone could significantly change the culture-enriched bacterial community. A moderate density (100-150 colony-forming units per plate) was optimal for obtaining the highest diversity on the agar. Similarly, membrane transport was significantly enriched in the moderate-density group, which indicated a more active metabolism in this density range. Overall, these results reveal the optimal culture conditions, including the densities of colonies and nutritional components for various gut bacteria, that provide a novel strategy for isolating bacteria in a way that is targeted and avoids blinded and repetitive work.

The responses of the gut microbiota to MBL deficiency.

Mannose-binding lectin (MBL) deficiency is a common innate immune system deficiency, and is associated with exacerbations and increased colonization of some pathogens. However, the response of the gut microbiota, a pivotal factor in host health, to MBL deficiency is not clear. In this study, MBL-/- and wild-type (WT) mice were generated by backcrossing from MBL-A and MBL-C knockout (KO) mice, and fecal samples were collected at different ages (4th, 8th, 12th, 19th and 27th weeks). The gut microbiota was analyzed by high-throughput sequencing with universal 16S rDNA primers (V3-V5 region). The results showed that structural segregation of the gut microbiota occurred at the 8th, 12th, 19th and 27th weeks of age, although there were no significant differences in alpha diversities between MBL-/- and WT mice at different ages. Impressively, in MBL-/- mice, Akkermansia (from the family Verrucomicrobiaceae) were decreased significantly, Lactobacillus (from the family Lactobacillaceae) abundances, Alistipes and Rikenella (both from the family of Rikenellaceae) were always enriched. Network analysis showed that more interactions existed in the gut microbiota from WT mice (33 nodes and 70 edges) than in the gut microbiota from MBL-/- mice (23 nodes and 40 edges). The 16S rDNA function prediction results indicated that the abundances of predicted genes in the "immune system disease", "metabolic disease" and "nucleotide metabolism" pathways were significantly increased in the MBL-/- mice. In conclusion, this study revealed that the gut microbiota changed in MBL deficient mice, especially at ages older than 4 weeks.

Saikosaponin-d ameliorates dextran sulfate sodium-induced colitis by suppressing NF-κB activation and modulating the gut microbiota in mice.

Saikosaponin-d (SSd), extracts from Bupleurum falcatum L, exhibits anti-inflammatory and anti-infectious activities. However, the effect of SSd on intestinal inflammation has not been investigated. The aim of this study was to evaluate the effect of SSd on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) mice, and to elucidate the underlying mechanisms. UC was induced in mice by administrating 3% DSS in drinking water for 7 days. SSd (4 mg/kg and 8 mg/kg) was administered by gavage every day during the experimental process. The results showed that SSd treatment (8 mg/kg) significantly ameliorated UC mice by decreasing disease activity index (DAI), increasing colon length and improving pathological characteristics. SSd treatment (8 mg/kg) significantly suppressed the mRNA levels of pro-inflammatory cytokines including TNF-α, IL-6 and IL-1ß, increased that of anti-inflammatory cytokine IL-10. Furthermore, SSd (8 mg/kg) suppressed the activation of NF-κB by decreasing the degradation and phosphorylation of IκB. SSd (8 mg/kg) also protected the intestinal barrier by increasing the mRNA levels of mucin (Muc1 and Muc2) and the protein levels of zonula occludens-1 (ZO-1) and Claudin-1. The 16S rDNA gene high-throughput sequencing revealed that SSd treatment (8 mg/kg) increased the alpha diversity and regulated the structure of gut microbiota in UC mice. Taken together, our findings demonstrated that SSd (8 mg/kg) improved DSS-induced intestinal inflammation by inhibiting NF-κB activation and regulated the gut microbiota.

The Differences between Luminal Microbiota and Mucosal Microbiota in Mice.

The differences between luminal microbiota (LM) and mucosal microbiota (MAM) were little known, especially in duodenum. In this study, LM and MAM in colon and duodenum of mice were investigated through 16S rRNA high-throughput sequencing. The lowest bacterial diversity and evenness were observed in duodenal LM (D_LM), followed by duodenal MAM (D_MAM). Meanwhile, the bacterial diversity and evenness were obviously increased in D_MAM than these in D_LM, while no significant difference was observed between colonic MAM (C_MAM) and colonic LM (C_LM). PCoA analysis also showed that bacterial communities of LM and MAM in duodenum were completely separated, while these in colon overlapped partly. The ratio of Firmicutes to Bacteroidetes (F/B) in D_MAM was significantly higher than that in D_LM. Lactobacillus was largely enriched and was the characteristic bacteria in D_LM. The characteristic bacteria in D_MAM were Turicibacter, Parasutterella, Marvinbryantia and Bifidobacterium, while in C_LM they were Ruminiclostridium_6, Ruminiclostridium_9, Ruminococcaceae_UCG_007 and Lachnospiraceae_UCG_010, and in C_MAM they were Lachnospiraceae_NK4A136, Mucispirillum, Alistipes, Ruminiclostridium and Odoribacter. The networks showed that more interactions existed in colonic microbiota (24 nodes and 74 edges) than in duodenal microbiota (17 nodes and 29 edges). The 16S rDNA function prediction results indicated that bigger differences of function exist between LM and MAM in duodenum than these in colon. In conclusion, microbiota from intestinal luminal content and mucosa were different both in colon and in duodenum, and bacteria in colon interacted with each other much more closely than those in duodenum.

Phloretin ameliorates dextran sulfate sodium-induced ulcerative colitis in mice by regulating the gut microbiota.

Phloretin, extracted from the pericarp and velamen of apples or pears, is a dihydrochalcone flavonoid with anti-bacterial and anti-inflammatory activities. It has been reported that phloretin has anti-inflammatory effects in ulcerative colitis (UC) mice. However, the role of the gut microbiota in the phloretin anti-UC process remains unclear. In this study, we observed that the anti-UC effect of phloretin was affected by co-housing, probably because of the transmissible nature of the gut micobiota. Through fecal micobiota transplantation (FMT), the effects of the gut microbiota on the anti-UC of phloretin were further confirmed. UC was induced in mice by administrating 3% dextran sulfate sodium (DSS) in drinking water for 7 days. Phloretin (60 mg/kg) was administered by gavage every day during the experiment. Fecal microbes (109 CFU/mL) from phloretin-treated UC mice were administered by gavage to non-phloretin-treated UC mice for 7 days. The results showed that FMT, like phloretin, ameliorated UC by improving disease symptoms and colon inflammation, balancing inflammatory cytokines, maintaining intestinal barrier integrity, restoring systemic immune function, inhibiting NF-κB and NLRP3 inflammasome activation and ameliorating the oxidant stress. Both FMT and phloretin treatment increased the levels of Bacteroidetes, Alistipes and Lactobacillus and decreased those of Firmicutes, Oscillibacter and Ruminiclostridium_6. Correlation analysis between gut microbes and micro-environmental factors revealed that Alistipes abundance was negatively correlated with DAI, pathological score, and TNF-α, IL-6 and IL-1ß levels, and Alistipes was more abundant in phloretin or FMT treated UC mice. Oscillibacter abundance was significantly positively correlated with IL-6 and IL-1ß levels and pathological score, and Oscillibacter was increased in UC mice. Furthermore, network analysis of the dominant genera revealed that Alistipes abundance was negatively related to Oscillibacter abundance. In conclusion, this study suggests that the anti-UC effects of phloretin are achieved through regulation of the gut microbiota and phloretin has the potential to be developed as a promising agent for the treatment of UC.

Chitooligosaccharides Prevents the Development of Colitis-Associated Colorectal Cancer by Modulating the Intestinal Microbiota and Mycobiota.

Gut microbes play a crucial role in the development of colorectal cancer. Chitooligosaccharides (COS), are oligomer that are depolymerized from chitosan and possess a wide range of biological activities. In this study, the effects of COS on colorectal cancer (CRC) development were evaluated using azoxymethane and dextran sulfate sodium (AOM/DSS) induced mouse model of CRC (CACM). In the COS-treated CRC group (CMCOS), COS protected mice from CRC by decreasing the disease activity index, tumor incidences and multiplicity, and the mRNA levels of COX-2, IL-6, TNF-α, IL-1ß, IL-10, and IKK-ß mRNA in colonic epithelial cells. The results of a cage-exchanged experiment, in which mice from the CACMe and CMCOSe treatments exchanged cages every day to interact with microbes, showed that gut microbes play an important role in preventing CAC by COS. The abundances of fecal bacteria (total bacteria, Lactobacillus, Enterococcus, Fusobacterium nucleatum and butyrate-producing bacteria) were detected by qPCR on the 0th, 1st, 3rd, 6th, 9th, and 10th weekends. Furthermore, microbiota and mycobiota were analyzed by high-throughput sequencing on an Illumina MiSeq PE300 system. COS protected mice from CRC by reversing the imbalance of bacteria and fungi, especially by reducing the abundance of Escherichia-Shigella, Enterococcus, and Turicibacter, and increasing the levels of Akkermansia, butyrate-producing bacteria and Cladosporium.