Upconversion dual-photosensitizer-expressing bacteria for near-infrared monochromatically excitable synergistic phototherapy.

Synergistic phototherapy stands for superior treatment prospects than a single phototherapeutic modality. However, the combined photosensitizers often suffer from incompatible excitation mode, limited irradiation penetration depth, and lack of specificity. We describe the development of upconversion dual-photosensitizer-expressing bacteria (UDPB) for near-infrared monochromatically excitable combination phototherapy. UDPB are prepared by integrating genetic engineering and surface modification, in which bacteria are encoded to simultaneously express photothermal melanin and phototoxic KillerRed protein and the surface primary amino groups are derived to free thiols for biorthogonal conjugation of upconversion nanoparticles. UDPB exhibit a near-infrared monochromatic irradiation-mediated dual-activation characteristic as the photothermal conversion of melanin can be initiated directly, while the photodynamic effect of KillerRed can be stimulated indirectly by upconverted visible light emission. UDPB also show living features to colonize hypoxic lesion sites and inhibit pathogens via bacterial community competition. In two murine models of solid tumor and skin wound infection, UDPB separately induce robust antitumor response and a rapid wound healing effect.

Bacteria-based drug delivery for treating non-oncological diseases.

Bacteria inhabit all over the human body, especially the skin, gastrointestinal tract, respiratory tract, urogenital tract, as well as specific lesion sites, such as wound and tumor. By leveraging their distinctive attributes including rapid proliferation, inherent abilities to colonize various biointerfaces in vivo and produce diverse biomolecules, and the flexibility to be functionalized via genetic engineering or surface modification, bacteria have been widely developed as living therapeutic agents, showing promising potential to make a great impact on the exploration of advanced drug delivery systems. In this review, we present an overview of bacteria-based drug delivery and its applications in treating non-oncological diseases. We systematically summarize the physiological positions where living bacterial therapeutic agents can be delivered to, including the skin, gastrointestinal tract, respiratory tract, and female genital tract. We discuss the success of using bacteria-based drug delivery systems in the treatment of diseases that occur in specific locations, such as skin wound healing/infection, inflammatory bowel disease, respiratory diseases, and vaginitis. We also discuss the advantages as well as the limitations of these living therapeutics and bacteria-based drug delivery, highlighting the key points that need to be considered for further translation. This review article may provide unique insights for designing next-generation bacteria-based therapeutics and developing advanced drug delivery systems.

Generating dual structurally and functionally skin-mimicking hydrogels by crosslinking cell-membrane compartments.

The skin is intrinsically a cell-membrane-compartmentalized hydrogel with high mechanical strength, potent antimicrobial ability, and robust immunological competence, which provide multiple protective effects to the body. Methods capable of preparing hydrogels that can simultaneously mimic the structure and function of the skin are highly desirable but have been proven to be a challenge. Here, dual structurally and functionally skin-mimicking hydrogels are generated by crosslinking cell-membrane compartments. The crosslinked network is formed via free radical polymerization using olefinic double bond-functionalized extracellular vesicles as a crosslinker. Due to the dissipation of stretching energy mediated by vesicular deformation, the obtained compartment-crosslinked network shows enhanced mechanical strength compared to hydrogels crosslinked by regular divinyl monomers. Biomimetic hydrogels also exhibit specific antibacterial activity and adequate ability to promote the maturation and activation of dendritic cells given the existence of numerous extracellular vesicle-associated bioactive substances. In addition, the versatility of this approach to tune both the structure and function of the resulting hydrogels is demonstrated through introducing a second network by catalyst-free click reaction-mediated crosslinking between alkyne-double-ended polymers and azido-decorated extracellular vesicles. This study provides a platform to develop dual structure- and function-controllable skin-inspired biomaterials.

Bacteria-Based Living Probes: Preparation and the Applications in Bioimaging and Diagnosis.

Bacteria can colonize a variety of in vivo biointerfaces, particularly the skin, nasal, and oral mucosa, the gastrointestinal tract, and the reproductive tract, but also target specific lesion sites, such as tumor and wound. By virtue of their prominent characteristics in motility, editability, and targeting ability, bacteria carrying imageable agents are widely developed as living probes for bioimaging and diagnosis of different diseases. This review first introduces the strategies used for preparing bacteria-based living probes, including biological engineering, chemical modification, intracellular loading, and optical manipulation. It then summarizes the recent progress of these living probes for fluorescence imaging, near-infrared imaging, ultrasonic imaging, photoacoustic imaging, magnetic resonance imaging, and positron emission tomography imaging. The biomedical applications of bacteria-based living probes are also reviewed particularly in the bioimaging and diagnosis of bacterial infections, cancers, and intestine-associated diseases. In addition, the advantages and challenges of bacteria-based living probes are discussed and future perspectives are also proposed. This review provides an updated overview of bacteria-based living probes, highlighting their great potential as a unique yet versatile platform for developing next-generation imageable agents for intelligent bioimaging, diagnosis, and even therapy.

Recent advances in targeted antibacterial therapy basing on nanomaterials.

Bacterial infection has become one of the leading causes of death worldwide, particularly in low-income countries. Despite the fact that antibiotics have provided successful management in bacterial infections, the long-term overconsumption and abuse of antibiotics has contributed to the emergence of multidrug resistant bacteria. To address this challenge, nanomaterials with intrinsic antibacterial properties or that serve as drug carriers have been substantially developed as an alternative to fight against bacterial infection. Systematically and deeply understanding the antibacterial mechanisms of nanomaterials is extremely important for designing new therapeutics. Recently, nanomaterials-mediated targeted bacteria depletion in either a passive or active manner is one of the most promising approaches for antibacterial treatment by increasing local concentration around bacterial cells to enhance inhibitory activity and reduce side effects. Passive targeting approach is widely explored by searching nanomaterial-based alternatives to antibiotics, while active targeting strategy relies on biomimetic or biomolecular surface feature that can selectively recognize targeted bacteria. In this review article, we summarize the recent developments in the field of targeted antibacterial therapy based on nanomaterials, which will promote more innovative thinking focusing on the treatment of multidrug-resistant bacteria.

Artificially engineered bacteria to treat gastrointestinal disease and cancer.

Therapeutics based on living organisms provide a roadmap for next-generation biomedicine. Bacteria have an essential role in the development, regulation, and treatment of gastrointestinal disease and cancer through similar mechanisms. However, primitive bacteria lack the stability to overcome complex drug delivery barriers, and their multifunctionality in reinforcing both conventional and emerging therapeutics is limited. Artificially engineered bacteria (ArtBac) with modified surfaces and genetic functions show promise for tackling these problems. Herein, we discuss recent applications of ArtBac as living biomedicine for the treatment of gastrointestinal diseases and tumors. Future perspectives are given to guide the rational design of ArtBac toward safe multifunctional medicine.

Biointerface mineralization generates ultraresistant gut microbes as oral biotherapeutics.

Despite the fact that oral microecologics are effective in modulating the gut microbiome, they always suffer from multiple insults during the journey from manufacture to arrival at the intestine. Inspired by the protective mechanism of mineralization, we describe a cytocompatible approach of biointerface mineralization that can generate an ultraresistant and self-removable coating on bacterial surface to solve these challenges. Mineral coating endows bacteria with robust resistances against manufacture-associated oxygen exposure, ultraviolet irradiation, and 75% ethanol. Following oral ingestion, the coating is able to actively neutralize gastric acid and release encapsulated bacteria through spontaneous yet rapid double-decomposition reaction. In addition to acid neutralization, the generated calcium ions can trigger micellar aggregation of bile acid, enabling dual exemptions from the insults of gastric acid and bile acid to achieve uncompromised bacterial viability. Further supported by the therapeutic efficacy of coated bacteria toward colitis mice, biointerface mineralization provides a versatile platform for developing next-generation living oral biotherapeutics.

Versatility of bacterial outer membrane vesicles in regulating intestinal homeostasis.

Outer membrane vesicles (OMVs) play vital roles in bacterial communication both intraspecifically and interspecifically. However, extracellular mechanisms of gut microbiota-derived OMVs in the intestine remain poorly understood. Here, we report that OMVs released from Akkermansia muciniphila are able to (i) restore disturbed balance of the gut microbiota by selectively promoting the proliferation of beneficial bacteria through membrane fusion, (ii) elicit mucosal immunoglobulin A response by translocating into Peyer's patches and subsequently activating B cells and dendritic cells, and (iii) maintain the integrity of the intestinal barrier by entering intestinal epithelial cells to stimulate the expressions of tight junctions and mucus. We demonstrate that transplantation of gut microbiota-associated OMVs to the intestine can alleviate colitis and enhance anti-programmed cell death protein 1 therapy against colorectal cancer by regulating intestinal homeostasis. This work discloses the importance of gut microbiota-derived OMVs in intestinal ecology, providing an alternative target for disease intervention and treatment.

Integrating Bacteria with a Ternary Combination of Photosensitizers for Monochromatic Irradiation-Mediated Photoacoustic Imaging-Guided Synergistic Photothermal Therapy.

Photosensitizer-based therapy often suffers from unitary and easily attenuated photosensitive effects, limited tumor penetration and retention, and requirement of multiple irradiation for combination therapy, which largely restrict its application. Here, bacteria are integrated with a monochromatic irradiation-mediated ternary combination of photosensitizers for photoacoustic imaging-guided synergistic photothermal therapy. Bacteria that are bioengineered to express natural melanin are decorated with dual synthetic photosensitizers by nanodeposition with indocyanine green and polydopamine under a cytocompatible condition. The combined photosensitizers, which share an adequate excitation at 808 nm, endow integrated bacteria with a stable triple photoacoustic and photothermal effect under a monochromatic irradiation. Due to their living characteristics, these bacteria preferentially colonize hypoxic tumor tissue with homogeneous distribution and durable retention and generate uniform imaging signals and a sufficient heating of tumor upon laser irradiation. Supported by significantly inhibited tumor growth and extended survival of animals in different tumor-bearing murine models, our work proposes the development of bacteria-based innovative photosensitizers for imaging-guided therapy.

Fluorescence-Activating and Absorption-Shifting Nanoprobes for Anaerobic Tracking of Gut Microbiota Derived Vesicles.

Outer membrane vesicles (OMVs) are crucial for bacterial intercellular communication and the crosstalk between the gut microbiota and its host. Methods capable of visualizing gut microbiota derived OMVs would be of great significance but have been rarely reported. Here, nanoprobes carrying a fluorescence-activating and absorption-shifting tag are prepared by combining genetic engineering and antibiotic-boosted vesicle formation and release. Benefiting from their natural structure and molecular oxygen-independent emission, the resulting nanovesicles can be applied as endogenous fluorescence probes to anaerobically track gut microbiota associated OMVs. These nanoprobes show flexibility in on-demand fluorescence turn-on/off and reversibly switchable emission bands for intelligent and dual-color imaging. With these special characteristics, the behaviors of microbiota OMVs to not only inhibit specific pathogenic strains through membrane fusion but also repair the intestinal barrier via entering intestinal epithelia and promoting the expressions of tight junctions are tracked and identified in the gut. Based on these discoveries, OMVs are disclosed to be able to remit inflammation in a murine model of colitis following transplantation to the intestine by oral delivery. This work provides an approach to visualize the dynamics of the gut microbiota and disclose potential targets for disease intervention.

Dressing Bacteria With a Hybrid Immunoactive Nanosurface to Elicit Dual Anticancer and Antiviral Immunity.

Approaches capable of simultaneously treating cancer and protecting susceptible patients from lethal infections such as coronavirus disease 2019, are highly desirable but prove to be difficult. Here, dressing bacteria with a hybrid immunoactive nanosurface is reported to elicit dual anticancer and antiviral immunity. A combination of a checkpoint blocking antibody and a virus-specific antigen is covalently conjugated to polydopamine nanoparticles, which can be anchored onto bacterial surface, by a one-step in situ polymerization of dopamine under a cell-friendly condition. By virtue of the ability to colonize and penetrate deep tumor tissue, dressed bacteria enable sustained release and expanded exposure of carried immunoactivators to stimulate immune cells. In addition to a carrier role, bacteria are able to further provoke innate immunity due to the native immunogenicity of the pathogen-associated molecular patterns. Immunization with dressed bacteria promotes the maturation, and activation of antigen-presenting cells, which induces robust humoral and cellular immune responses in tumor-bearing mice. As evidenced by efficient production of viral-antigen-specific immunoglobulin G antibody in serum and significantly suppressed tumor growth in different models, dressing bacteria with a hybrid immunoactive nanosurface paves an avenue to prepare next-generation therapeutics for synergistic treatment and prevention.

Chiral-Selective Antigen-Presentation by Supramolecular Chiral Polymer Micelles.

Chirality is ubiquitous in biological systems, which is closely related to biological functions, life processes, and even the pathogenesis of diseases. However, the interface between the chirality of synthetic materials and organisms, particularly the immune system, remains poorly understood. Here, supramolecular chiral polymer micelles (SCPMs) are prepared by complexing antigenic proteins with chiral amino acid-modified polyethyleneimine. The introduction of chirality not only reduces the toxicity of cationic polymer, but also benefits cell uptake and antigen presentation. Especially, D-chirality presents the lowest cytotoxicity, while promoting the highest expression level of costimulatory molecules on dendritic cells compared to L-chirality and achirality. The superiority of D-chirality to stimulate dendritic cell maturation is supported by immunization with D-SCPMs, which achieves significant antigen-specific proliferation of T cells in the spleen, lymph nodes, and tumor of mice. Chirality-mediated antigen processing and presentation are demonstrated by D-SCPMs self-assembled from chiral alkaline histidine or neutral phenylalanine modified polyethyleneimine and tumor associated ovalbumin or severe acute respiratory syndrome coronavirus 2 spike 1 antigenic protein. Immunoactivation enabled by D-chirality opens a window to prepare potent nanotherapeutics for disease prevention and treatment.

Chemical reaction-mediated covalent localization of bacteria.

Methods capable of manipulating bacterial colonization are of great significance for modulating host-microbiota relationships. Here, we describe a strategy of in-situ chemical reaction-mediated covalent localization of bacteria. Through a simple one-step imidoester reaction, primary amino groups on bacterial surface can be converted to free thiols under cytocompatible conditions. Surface thiolation is applicable to modify diverse strains and the number of introduced thiols per bacterium can be easily tuned by varying feed ratios. These chemically reactive bacteria are able to spontaneously bond with mucous layer by catalyst-free thiol-disulfide exchange between mucin-associated disulfides and newly converted thiols on bacterial surface and show thiolation level-dependent attachment. Bacteria optimized with 9.3 × 107 thiols per cell achieve 170-fold higher attachment in mucin-enriched jejunum, a challenging location for gut microbiota to colonize. As a proof-of-concept application for microbiota transplantation, covalent bonding-assisted localization of an oral probiotic in the jejunum generates an improved remission of jejunal mucositis. Our findings demonstrate that transforming bacteria with a reactive surface provides an approach to chemically control bacterial localization, which is highly desirable for developing next-generation bacterial living bioagents.

Nanocapping-enabled charge reversal generates cell-enterable endosomal-escapable bacteriophages for intracellular pathogen inhibition.

Bacteriophages (phages) are widely explored as antimicrobials for treating infectious diseases due to their specificity and potency to infect and inhibit host bacteria. However, the application of phages to inhibit intracellular pathogens has been greatly restricted by inadequacy in cell entry and endosomal escape. Here, we describe the use of cationic polymers to selectively cap negatively charged phage head rather than positively charged tail by electrostatic interaction, resulting in charge-reversed phages with uninfluenced vitality. Given the positive surface charge and proton sponge effect of the nanocapping, capped phages are able to enter intestinal epithelial cells and subsequently escape from endosomes to lyse harbored pathogens. In a murine model of intestinal infection, oral ingestion of capped phages significantly reduces the translocation of pathogens to major organs, showing a remarkable inhibition efficacy. Our work proposes that simple synthetic nanocapping can manipulate phage bioactivity, offering a facile platform for preparing next-generation antimicrobials.

Encoding with a fluorescence-activating and absorption-shifting tag generates living bacterial probes for mammalian microbiota imaging.

The mammalian microbiota plays essential roles in health. A primary determinant to understand the interaction with the host is the distribution and viability of its key microorganisms. Here, a strategy of encoding with a fluorescence-activating and absorption-shifting tag (FAST) is reported to prepare living bacterial probes for real-time dynamic, dual-modal, and molecular oxygen-independent imaging of the host microbiota. Carrying FAST endows bacteria with rapid on-demand turn on-off fluorescence by adding or removal of corresponding fluorogens. Encoded bacteria are able to reversibly switch emission bands for dual-color fluorescence imaging via fluorogen exchange. Due to molecular oxygen-independent emission of FAST, encoded bacteria can emit fluorescence under anaerobic environments including the gut and tumor. These living probes demonstrate the applicability to quantify the vitality of bacteria transplanted to the gut microbiota. This work proposes a unique fluorescence probe for investigating the dynamics of the host microbiota.

Spatiotemporally Controllable Distribution of Combination Therapeutics in Solid Tumors by Dually Modified Bacteria.

Methods capable of distributing antitumor therapeutics uniformly and durably throughout an entire tumor would be of great significance in maximizing their treatment efficacy, but they have proven to be extremely challenging. Here, bacteria-mediated spatiotemporally controllable distribution of combination therapeutics in solid tumors is reported to reprogram the immune microenvironment for optimizing antitumor efficacy. By combining synthetic biology and interfacial chemistry, bacteria are inside and outside concurrently modified to express photothermal melanin and to attach immune checkpoint inhibitors on their surface. Due to the nature of bacteria to colonize the hypoxia intratumoral environment, both therapeutic agents can be distributed homogenously and lastingly in tumors during ex vivo human and in vivo mouse studies. Spatiotemporally controllable localization of melanin can repeatedly generate a moderate yet uniform heating of the tumor upon light exposure in a broad treatment window. Combination with similarly localized inhibitors elicits a dual photothermally stimulated and checkpoint-blockade-mediated immune activation effect, synergistically reprogramming the immunosuppressive tumor microenvironment. Therapeutic values are demonstrated by significantly inhibited tumor growth and prolonged survival of mice in both subcutaneous and orthotopic murine models. Colonization of dually modified bacteria paves an avenue for spatiotemporally controllable distribution of therapeutic drugs in solid tumors.

Aptamer-assisted tumor localization of bacteria for enhanced biotherapy.

Despite bacterial-mediated biotherapies have been widely explored for treating different types of cancer, their implementation has been restricted by low treatment efficacy, due largely to the absence of tumor-specific accumulation following administration. Here, the conjugation of aptamers to bacterial surface is described by a simple and cytocompatible amidation procedure, which can significantly promote the localization of bacteria in tumor site after systemic administration. The surface density of aptamers can be easily adjusted by varying feed ratio and the conjugation is able to increase the stability of anchored aptamers. Optimal bacteria conjugated with an average of 2.8 × 105 aptamers per cell present the highest specificity to tumor cells in vitro, separately generating near 2- and 4-times higher accumulation in tumor tissue at 12 and 60 hours compared to unmodified bacteria. In both 4T1 and H22 tumor-bearing mouse models, aptamer-conjugated attenuated Salmonella show enhanced antitumor efficacy, along with highly activated immune responses inside the tumor. This work demonstrates how bacterial behaviors can be tuned by surface conjugation and supports the potential of aptamer-conjugated bacteria for both targeted intratumoral localization and enhanced tumor biotherapy.

Bacteria-Based Microdevices for the Oral Delivery of Macromolecules.

The oral delivery of macromolecules is quite challenging due to environmental insults and biological barriers encountered along the gastrointestinal (GI) tract. Benefiting from their living characteristics, diverse bacterial species have been engineered as intelligent platforms to deliver various therapeutics. To tackle difficulties in oral delivery, innovative bacteria-based microdevices have been developed by virtue of advancements in synthetic biology and nanotechnology, with aims to overcome the instability and short half-life of macromolecules in the GI tract. In this review, we summarize the main classes of macromolecules that are produced and delivered through the oral ingestion of bacteria and bacterial derivatives. Furtherly, we discuss the engineering strategies and biomedical applications of these living microdevices in disease diagnosis, bioimaging, and treatment. Finally, we highlight the advantages as well as the limitations of these engineered bacteria used as platforms for the oral delivery of macromolecules and also propose their potential for clinical translation. The results summarized in this review article would contribute to the invention of next-generation bacteria-based systems for the oral delivery of macromolecules.