Bacteria-derived nanovesicles enhance tumour vaccination by trained immunity.

Trained immunity enhances the responsiveness of immune cells to subsequent infections or vaccinations. Here we demonstrate that pre-vaccination with bacteria-derived outer-membrane vesicles, which contain large amounts of pathogen-associated molecular patterns, can be used to potentiate, and enhance, tumour vaccination by trained immunity. Intraperitoneal administration of these outer-membrane vesicles to mice activates inflammasome signalling pathways and induces interleukin-1ß secretion. The elevated interleukin-1ß increases the generation of antigen-presenting cell progenitors. This results in increased immune response when tumour antigens are delivered, and increases tumour-antigen-specific T-cell activation. This trained immunity increased protection from tumour challenge in two distinct cancer models.

Intelligent Responsive Nanoparticles with Multilevel Triggered Drug Penetration for Tumor Photochemotherapy.

Nanomedicines have contradictory size requirements to overcome systemic barriers and penetrate the tumor extracellular matrix (ECM). Larger-sized nanoparticles (50-200 nm) exhibit prolonged blood circulation half-life and improved tumor enrichment, while small-sized nanoparticles (4-20 nm) easily penetrate deep tumor tissues. Therefore, the development of intelligent responsive nanomedicine systems can not only increase nanodrug tumor accumulation but also improve their penetration into the ECM. Herein, we propose an intelligent responsive nanoparticle triggered by near-infrared light (NIR). The nanoparticle was constructed by a temperature-sensitive liposome (TSL) encapsulating ultrasmall melanin nanoparticles (MNPs) loaded with doxorubicin (MNP/doxorubicin (DOX)@TSL). When exposed to NIR irradiation, the tailor-made nanoparticles not only effectively ablated the tumor cells around blood vessels but also destroyed the structural integrity and released loaded ultrasmall MNP/DOX (<10 nm) to promote deep tumor penetration and enhance interior tumor cell killing. This NIR-triggered intelligent nanoparticle successfully integrated photothermal therapy (PTT) for perivascular tumor cells and chemotherapy for deep tumor cell inhibition. The in vivo results showed remarkable tumor regression in 4T1 breast tumor-bearing mice by 74.2%. This controllable size switchable nanosystem with efficient tumor accumulation and penetration has shown great potential in improving synergistic antitumor effects of photochemotherapy.

Restoration of Sinusoid Fenestrae Followed by Targeted Nanoassembly Delivery of an Anti-Fibrotic Agent Improves Treatment Efficacy in Liver Fibrosis.

During the onset of liver fibrosis, capillarized liver sinusoidal endothelial cells (LSECs) limit substance exchange between the blood and the Disse space, further accelerating hepatic stellate cell (HSCs) activation and fibrosis progression. Limited accessibility of therapeutics to the Disse space is often overlooked and remains a major bottleneck for HSCs-targeted therapy in liver fibrosis. Here, an integrated systemic strategy for liver fibrosis treatment is reported, utilizing pretreatment with the soluble guanylate cyclase stimulator, riociguat, followed by insulin growth factor 2 receptor-mediated targeted delivery of the anti-fibrosis agent, JQ1, via peptide-nanoparticles (IGNP-JQ1). The riociguat reversed the liver sinusoid capillarization to maintain a relatively normal LSECs porosity, thus facilitating the transport of IGNP-JQ1 through the liver sinusoid endothelium wall and enhancing the accumulation of IGNP-JQ1 in the Disse space. IGNP-JQ1 is then selectively taken up by activated HSCs, inhibiting their proliferation and decreasing collagen deposition in the liver. The combined strategy results in significant fibrosis resolution in carbon tetrachloride-induced fibrotic mice as well as methionine-choline-deficient-diet-induced nonalcoholic steatohepatitis (NASH) mice. The work highlights the key role of LSECs in therapeutics transport through the liver sinusoid. The strategy of restoring LSECs fenestrae by riociguat represents a promising approach for liver fibrosis treatment.

Modular-designed engineered bacteria for precision tumor immunotherapy via spatiotemporal manipulation by magnetic field.

Micro-nano biorobots based on bacteria have demonstrated great potential for tumor diagnosis and treatment. The bacterial gene expression and drug release should be spatiotemporally controlled to avoid drug release in healthy tissues and undesired toxicity. Herein, we describe an alternating magnetic field-manipulated tumor-homing bacteria developed by genetically modifying engineered Escherichia coli with Fe3O4@lipid nanocomposites. After accumulating in orthotopic colon tumors in female mice, the paramagnetic Fe3O4 nanoparticles enable the engineered bacteria to receive and convert magnetic signals into heat, thereby initiating expression of lysis proteins under the control of a heat-sensitive promoter. The engineered bacteria then lyse, releasing its anti-CD47 nanobody cargo, that is pre-expressed and within the bacteria. The robust immunogenicity of bacterial lysate cooperates with anti-CD47 nanobody to activate both innate and adaptive immune responses, generating robust antitumor effects against not only orthotopic colon tumors but also distal tumors in female mice. The magnetically engineered bacteria also enable the constant magnetic field-controlled motion for enhanced tumor targeting and increased therapeutic efficacy. Thus, the gene expression and drug release behavior of tumor-homing bacteria can be spatiotemporally manipulated in vivo by a magnetic field, achieving tumor-specific CD47 blockage and precision tumor immunotherapy.

Normalizing the Immune Macroenvironment via Debulking Surgery to Strengthen Tumor Nanovaccine Efficacy and Eliminate Metastasis.

In tumor nanovaccines, nanocarriers enhance the delivery of tumor antigens to antigen-presenting cells (APCs), thereby ensuring the robust activation of tumor antigen-specific effector T-cells to kill tumor cells. Through employment of their high immunogenicity and nanosize, we have developed a "Plug-and-Display" delivery platform on the basis of bacterial outer membrane vesicles (OMVs) for tumor nanovaccines (NanoVac), which can rapidly display different tumor antigens and efficiently eliminate lung metastases of melanoma. In this study, we first upgraded the NanoVac to increase their antigen display efficiency. However, we found that the presence of a subcutaneous xenograft seriously hampered the efficiency of NanoVac to eliminate lung metastases, with the subcutaneous xenograft mimicking the primary tumor burden in clinical practice. The primary tumor secreted significant amounts of granulocyte colony-stimulating factor (G-CSF) and altered the epigenetic features of granulocyte monocyte precursor cells (GMPs) in the bone marrow, thus disrupting systemic immunity, particularly the function of APCs, and ultimately resulting in NanoVac failure to affect metastases. These changes in the systemic immune macroenvironment were plastic, and debulking surgery of primary tumor resection reversed the dysfunction of APCs and failure of NanoVac. These results demonstrate that, in addition to the formulation design of the tumor nanovaccines themselves, the systemic immune macroenvironment incapacitated by tumor development is another key factor that cannot be ignored to affect the efficiency of tumor nanovaccines, and the combination of primary tumor resection with NanoVac is a promising radical treatment for widely metastatic tumors.

Engineered Bacterial Outer Membrane Vesicles as Controllable Two-Way Adaptors to Activate Macrophage Phagocytosis for Improved Tumor Immunotherapy.

The most immune cells infiltrating tumor microenvironment (TME), tumor-associated macrophages (TAMs) closely resemble immunosuppressive M2-polarized macrophages. Moreover, tumor cells exhibit high expression of CD47 "don't eat me" signal, which obstructs macrophage phagocytosis. The precise and efficient activation of TAMs is a promising approach to tumor immunotherapy; however, re-education of macrophages remains a challenge. Bacteria-derived outer membrane vesicles (OMVs) are highly immunogenic nanovesicles that can robustly stimulate macrophages. Here, an OMV-based controllable two-way adaptor is reported, in which a CD47 nanobody (CD47nb) is fused onto OMV surface (OMV-CD47nb), with the outer surface coated with a polyethylene glycol (PEG) layer containing diselenide bonds (PEG/Se) to form PEG/Se@OMV-CD47nb. The PEG/Se layer modification not only mitigates the immunogenicity of OMV-CD47nb, thereby remarkedly increasing the dose that can be administered safely through intravenous injection, but also equips the formulation with radiation-triggered controlled release of OMV-CD47nb. Application of radiation to tumors in mice injected with the nanoformulation results in remodeling of TME. As two-way adaptors, OMV-CD47nb activates TAM phagocytosis of tumor cells via multiple pathways, including induction of M1 polarization and blockade of "don't eat me" signal. Moreover, this activation of TAMs results in the stimulation of T cell-mediated antitumor immunity through effective antigen presentation.

Functional Immune Cell-Derived Exosomes Engineered for the Trilogy of Radiotherapy Sensitization.

The limited efficacy of radiotherapy leads to radio-resistance and high rates of tumor recurrence and metastasis, which is caused by tumor hypoxia, rapid DNA damage repair, and especially the suppressive immune microenvironment of tumor. Lots of immune cell-derived exosomes can regulate antitumor immunity, but their application in enhancing radiotherapy is rarely studied. Herein, as a model of concept, M1 macrophage-derived exosomes (M1Exos) is engineered as effective radiotherapy sensitizers, realizing the trilogy of radiotherapy sensitization: 1) M1Exos is engineered to express catalases on the inside of membrane, which can effectively relieve tumor hypoxia, and enhance DNA damage. 2) The DNA damage repair inhibitor is loaded in M1Exos to effectively inhibit DNA damage repair. 3) M1Exos can polarize M2 macrophages into M1 phenotypes, and the anti-PD-L1 nanobody engineered on the outside of M1Exos can relieve the immunosuppression of T cells, both ultimately leading to the remodeling of the tumor suppressive microenvironment. The trilogy of radiotherapy sensitization achieves excellent antitumor efficacy, exhibiting the good utility of engineering immune cell-derived exosomes as radiotherapy sensitizers, inspiring the future efforts to explore different kinds of immune cell-derived exosomes for enhanced radiotherapy.

Antigen-bearing outer membrane vesicles as tumour vaccines produced in situ by ingested genetically engineered bacteria.

The complex gastrointestinal environment and the intestinal epithelial barrier constrain the design and effectiveness of orally administered tumour vaccines. Here we show that outer membrane vesicles (OMVs) fused to a tumour antigen and produced in the intestine by ingested genetically engineered bacteria function as effective tumour vaccines in mice. We modified Escherichia coli to express, under the control of a promoter induced by the monosaccharide arabinose, a specific tumour antigen fused with the protein cytolysin A on the surface of OMVs released by the commensal bacteria. In mice, oral administration of arabinose and the genetically engineered E. coli led to the production of OMVs that crossed the intestinal epithelium into the lamina propria, where they stimulated dendritic cell maturation. In a mouse model of pulmonary metastatic melanoma and in mice bearing subcutaneous colon tumours, the antigen-bearing OMVs inhibited tumour growth and protected the animals against tumour re-challenge. The in situ production of OMVs by genetically modified commensal bacteria for the delivery of stimulatory molecules could be leveraged for the development of other oral vaccines and therapeutics.

Rapid Surface Display of mRNA Antigens by Bacteria-Derived Outer Membrane Vesicles for a Personalized Tumor Vaccine.

Therapeutic mRNA vaccination is an attractive approach to trigger antitumor immunity. However, the mRNA delivery technology for customized tumor vaccine is still limited. In this work, bacteria-derived outer membrane vesicles (OMVs) are employed as an mRNA delivery platform by genetically engineering with surface decoration of RNA binding protein, L7Ae, and lysosomal escape protein, listeriolysin O (OMV-LL). OMV-LL can rapidly adsorb box C/D sequence-labelled mRNA antigens through L7Ae binding (OMV-LL-mRNA) and deliver them into dendritic cells (DCs), following by the cross-presentation via listeriolysin O-mediated endosomal escape. OMV-LL-mRNA significantly inhibits melanoma progression and elicits 37.5% complete regression in a colon cancer model. OMV-LL-mRNA induces a long-term immune memory and protects the mice from tumor challenge after 60 days. In summary, this platform provides a delivery technology distinct from lipid nanoparticles (LNPs) for personalized mRNA tumor vaccination, and with a "Plug-and-Display" strategy that enables its versatile application in mRNA vaccines.

Antigen Capture and Immune Modulation by Bacterial Outer Membrane Vesicles as In Situ Vaccine for Cancer Immunotherapy Post-Photothermal Therapy.

Tumor antigens released from tumor cells after local photothermal therapy (PTT) can activate the tumor-specific immune responses, which are critical for eliminating the residual lesions and distant metastases. However, the limited recognition efficiency of released tumor antigens by the immune system and the immunosuppressive microenvironment lead to ineffective antitumor immunity. Here, an in situ multifunctional vaccine based on bacterial outer membrane vesicles (OMVs, 1-MT@OMV-Mal) is developed by surface conjunction of maleimide groups (Mal) and interior loading with inhibitor of indoleamine 2, 3-dioxygenase (IDO), 1-methyl-tryptophan (1-MT). 1-MT@OMV-Mal can bind to the released tumor antigens after PTT, and be efficiently recognized and taken up by dendritic cells. Furthermore, in situ injection of 1-MT@OMV-Mal simultaneously overcomes the immune inhibition of IDO on tumor-infiltrating effector T cells, leading to remarkable inhibition on both primary and distant tumors. Together, a promising in situ vaccine based on OMVs to facilitate immune-mediated tumor clearance after PTT through orchestrating antigen capture and immune modulation is presented.

Personalized cancer vaccines from bacteria-derived outer membrane vesicles with antibody-mediated persistent uptake by dendritic cells.

Nanocarriers with intrinsic immune adjuvant properties can activate the innate immune system while delivering tumor antigen, thus efficiently facilitating antitumor adaptive immunity. Bacteria-derived outer membrane vesicles (OMVs) are an excellent candidate due to their abundance of pathogen associated molecular patterns. However, during the uptake of OMVs by dendritic cells (DCs), the interaction between lipopolysaccharide and toll-like receptor 4 induces rapid DC maturation and uptake blockage, a phenomenon we refer to as "maturation-induced uptake obstruction" (MUO). Herein we decorated OMV with the DC-targeting αDEC205 antibody (OMV-DEC), which endowed the nanovaccine with an uptake mechanism termed as "not restricted to maturation via antibody modifying" (Normandy), thereby overcoming the MUO phenomenon. We also proved the applicability of this nanovaccine in identifying the human tumor neoantigens through rapid antigen display. In summary, this engineered OMV represents a powerful nanocarrier for personalized cancer vaccines, and this antibody modification strategy provides a reference to remodel the DC uptake pattern in nanocarrier design.

Biomimetic Nanoparticles Carrying a Repolarization Agent of Tumor-Associated Macrophages for Remodeling of the Inflammatory Microenvironment Following Photothermal Therapy.

The complete regression of residual tumors after photothermal therapy (PTT) depends on the activation and recognition of the immune system. However, the inevitable local inflammation after PTT in residual tumor recruits abundant abnormal immune cells, especially the tumor-associated macrophages (TAMs) which further promote immune escape and survival of the remaining tumor cells, resulting in the tumor recurrence and progression. To solve this problem, herein we explored biomimetic nanoparticles carrying repolarization agent of TAMs to remodel the post-PTT inflammatory microenvironment. The polydopamine nanoparticles were used simultaneously as photothermal transduction agents to ablate tumor cells and the delivery vehicles for TMP195 which can repolarize the M2-like TAMs into an antitumor phenotype. In addition, a biomimetic decoration of macrophage membrane coating was designed to endow nanoparticles the ability to actively target the tumor site after PTT mediated by inflammation-mediated chemotaxis. In the breast tumor model, these biomimetic nanoparticles with immune-modulating ability significantly elevated the levels of M1-like TAMs, ultimately resulting in a tumor-elimination rate of 60%, increased from 10% after PTT. This synergistic treatment strategy of PTT and TAMs repolarization provides a promising approach to address the deteriorated tumor microenvironment after PTT and proposes a more effective way for combinational treatment option in clinic.

Melanin-Like Nanomedicine in Photothermal Therapy Applications.

Photothermal therapy (PTT) mediated by nanomaterial has become an attractive tumor treatment method due to its obvious advantages. Among various nanomaterials, melanin-like nanoparticles with nature biocompatibility and photothermal conversion properties have attracted more and more attention. Melanin is a natural biological macromolecule widely distributed in the body and displays many fascinating physicochemical properties such as excellent biocompatibility and prominent photothermal conversion ability. Due to the similar properties, Melanin-like nanoparticles have been extensively studied and become promising candidates for clinical application. In this review, we give a comprehensive introduction to the recent advancements of melanin-like nanoparticles in the field of photothermal therapy in the past decade. In this review, the synthesis pathway, internal mechanism and basic physical and chemical properties of melanin-like nanomaterials are systematically classified and evaluated. It also summarizes the application of melanin-like nanoparticles in bioimaging and tumor photothermal therapy (PTT)in detail and discussed the challenges they faced in clinical translation rationally. Overall, melanin-like nanoparticles still have significant room for development in the field of biomedicine and are expected to applied in clinical PTT in the future.

Synthesis of new tetracyclic benzodiazepine-fused isoindolinones using recyclable mesoporous silica nanoparticles.

Pseudo natural products (NPs) feature structural novelty and diversity and thus are a new source of lead compounds for drug discovery. We first report the mesoporous silica nanoparticles (MSNs)-catalyzed de novo combination of benzodiazepine and isoindolinone, giving tetracyclic benzodiazepine-fused isoindolinone pseudo natural products (21 examples, 55-91% yields). The work also demonstrates that MSNs are efficient acidic catalysts for multi-component reactions.

Rapid Capture and Nondestructive Release of Extracellular Vesicles Using Aptamer-Based Magnetic Isolation.

Extracellular vesicles (EVs) play important roles in cell-cell communication by transferring cargo proteins and nucleic acids between cells. Due to their small size (50-150 nm) and low density, rapid capture and nondestructive release of EVs remains a technical challenge which significantly hinders study of their biofunction and biomedical application. To address this issue, we designed a DNA aptamer-based system that enabled rapid capture and nondestructive release of EVs in 90 min with similar isolation efficiency to ultracentrifugation (around 78%). Moreover, because we designed a DNA structure-switch process to release the exosomes, the isolated EVs maintained high bioactivity in cell-uptake assay and wound-healing assays. Using this method, we can isolate EVs from clinical samples and found that the amount of MUC1 positive EVs in breast cancer patient plasma sample is significantly higher than that in healthy donors. This DNA aptamer-based magnetic isolation strategy can be potentially applied for the biofunction study of EVs and EV-based point-of-care clinical tests.