Bacteria extracellular vesicle as nanopharmaceuticals for versatile biomedical potential.

Bacteria extracellular vesicles (BEVs), characterized as the lipid bilayer membrane-surrounded nanoparticles filled with molecular cargo from parent cells, play fundamental roles in the bacteria growth and pathogenesis, as well as facilitating essential interaction between bacteria and host systems. Notably, benefiting from their unique biological functions, BEVs hold great promise as novel nanopharmaceuticals for diverse biomedical potential, attracting significant interest from both industry and academia. Typically, BEVs are evaluated as promising drug delivery platforms, on account of their intrinsic cell-targeting capability, ease of versatile cargo engineering, and capability to penetrate physiological barriers. Moreover, attributing to considerable intrinsic immunogenicity, BEVs are able to interact with the host immune system to boost immunotherapy as the novel nanovaccine against a wide range of diseases. Towards these significant directions, in this review, we elucidate the nature of BEVs and their role in activating host immune response for a better understanding of BEV-based nanopharmaceuticals' development. Additionally, we also systematically summarize recent advances in BEVs for achieving the target delivery of genetic material, therapeutic agents, and functional materials. Furthermore, vaccination strategies using BEVs are carefully covered, illustrating their flexible therapeutic potential in combating bacterial infections, viral infections, and cancer. Finally, the current hurdles and further outlook of these BEV-based nanopharmaceuticals will also be provided.

Enzymes in Synergy: Bacteria Specific Molecular Probe for Locoregional Imaging of Urinary Tract Infection in vivo.

Uropathogenic Escherichia coli (UPECs) is a leading cause for urinary tract infections (UTI), accounting for 70-90 % of community or hospital-acquired bacterial infections owing to high recurrence, imprecision in diagnosis and management, and increasing prevalence of antibiotic resistance. Current methods for clinical UPECs detection still rely on labor-intensive urine cultures that impede rapid and accurate diagnosis for timely UTI therapeutic management. Herein, we developed a first-in-class near-infrared (NIR) UPECs fluorescent probe (NO-AH) capable of specifically targeting UPECs through its collaborative response to bacterial enzymes, enabling locoregional imaging of UTIs both in vitro and in vivo. Our NO-AH probe incorporates a dual protease activatable moiety, which first reacts with OmpT, an endopeptidase abundantly present on the outer membrane of UPECs, releasing an intermediate amino acid residue conjugated with a NIR hemicyanine fluorophore. Such liberated fragment would be subsequently recognized by aminopeptidase (APN) within the periplasm of UPECs, activating localized fluorescence for precise imaging of UTIs in complex living environments. The peculiar specificity and selectivity of NO-AH, facilitated by the collaborative action of bacterial enzymes, features a timely and accurate identification of UPECs-infected UTIs, which could overcome misdiagnosis in conventional urine tests, thus opening new avenues towards reliable UTI diagnosis and personalized antimicrobial therapy management.

Enzyme-Responsive Fluorescent Labeling Strategy for In Vivo Imaging of Gut Bacteria.

Myrosinase (Myr), as a unique ß-thioglucosidase enzyme capable of converting natural and gut bacterial metabolite glucosinolates into bioactive agents, has recently attracted a great deal of attention because of its essential functions in exerting homeostasis dynamics and promoting human health. Such nutraceutical and biomedical significance demands unique and reliable strategies for specific identification of Myr enzymes of gut bacterial origin in living systems, whereas the dearth of methods for bacterial Myr detection and visualization remains a challenging concern. Herein, we present a series of unique molecular probes for specific identification and imaging of Myr-expressing gut bacterial strains. Typically, an artificial glucosinolate with an azide group in aglycone was synthesized and sequentially linked with the probe moieties of versatile channels through simple click conjugation. Upon gut bacterial enzymatic cleavage, the as-prepared probe molecules could be converted into reactive isothiocyanate forms, which can further act as reactive electrophiles for the covalent labeling of gut bacteria, thus realizing their localized fluorescent imaging within a wide range of wavelength channels in live bacterial strains and animal models. Overall, our proposed method presents a novel technology for selective gut bacterial Myr enzyme labeling in vitro and in vivo. We envision that such a rational probe design would serve as a promising solution for chemoprevention assessment, microflora metabolic mechanistic study, and gut bacterium-mediated physiopathological exploration.

Beyond the TPP+ "gold standard": a new generation mitochondrial delivery vector based on extended PN frameworks.

Mitochondrial targeting represents an attractive strategy for treating metabolic, degenerative and hyperproliferative diseases, since this organelle plays key roles in essential cellular functions. Triphenylphosphonium (TPP+) moieties - the current "gold standard" - have been widely used as mitochondrial targeting vectors for a wide range of molecular cargo. Recently, further optimisation of the TPP+ platform drew considerable interest as a way to enhance mitochondrial therapies. However, although the modification of this system appears promising, the core structure of the TPP+ moiety remains largely unchanged. Thus, this study explored the use of aminophosphonium (PN+) and phosphazenylphosphonium (PPN+) main group frameworks as novel mitochondrial delivery vectors. The PPN+ moiety was found to be a highly promising platform for this purpose, owing to its unique electronic properties and high lipophilicity. This has been demonstrated by the high mitochondrial accumulation of a PPN+-conjugated fluorophore relative to its TPP+-conjugated counterpart, and has been further supported by density functional theory and molecular dynamics calculations, highlighting the PPN+ moiety's unusual electronic properties. These results demonstrate the potential of novel phosphorus-nitrogen based frameworks as highly effective mitochondrial delivery vectors over traditional TPP+ vectors.

Antimicrobial Photodynamic Therapy for the Remote Eradication of Bacteria.

The emergence of multi-drug resistant bacteria strains has been an uphill battle in modern healthcare worldwide, due to the increasing difficulty of killing them. The evolving pathogenicity of bacteria has led to researchers searching for more effective antimicrobial therapeutics to successfully eliminate them without undesirable consequences to the human body. In recent years, antimicrobial photodynamic therapy (APDT), an obsolete technique for cancer treatments, has been reported to eradicate bacteria and biofilm-related infections. The principle of antimicrobial photodynamic therapy solely relies on the photosensitizers (PSs) generating reactive oxygen species, in the presence of oxygen and light, to destroy pathogens. Thus, it can target a broad spectrum of microorganisms, owing to the indirect interaction between PSs and the bacteria, resulting in the less likelihood for the development of drug resistant bacteria strains. This review will focus on the recent progress of APDT in the last five years and some future perspectives of APDT. The mechanism of APDT against bacteria and biofilms, various PSs used for APDT, and some common multidrug-resistant bacteria strains will be briefly introduced. The reported in vivo applications of APDT in the several types of bacterial infections that includes periodontitis, wound infections, keratitis, endophthalmitis and tuberculosis in the last five years will be summarized in detail.

Near Infrared-II Photothermal and Colorimetric Synergistic Sensing for Intelligent Onsite Dietary Myrosinase Profiling.

Myrosinase (Myr) is a type of critical ß-thioglucosidase enzyme activator essential for sustaining many functional foods to perform their health-promoting functions. An accurate and reliable Myr test is meaningful for food quality and dietary nutrition assessments, whereas the currently reported methods do not guarantee specificity and have high reliance on instrumentation, which are not suitable for rapid and onsite Myr screening especially in complex systems from various sources. Herein, we present a unique NIR-II absorption-based photothermal-responsive colorimetric biosensor for anti-interference onsite Myr determination and realization of rapid visualized outputs with the aid of smartphone calculation. Typically, assisted by glucose oxidase (GOx), Myr specifically converts the sinigrin substrate into hydrogen peroxide (H2O2) that can oxidize 3,3',5,5'-tetramethylbenzidine (TMB) catalyzed by AuNPs to form a charge transfer complex (CTC) with NIR-II absorption and photothermal characters. Delightfully, such a proposed method is able to determine Myr within a wide range of 0 to 172.5 mU/mL with a detection limit down to 2.96 mU/mL. Moreover, simple, rapid, and real-time visual Myr identification in actual food-sourced samples could also be readily achieved by smartphone readout processing, with the promising advantages of anti-interference, high accuracy, and low cost as well as labor-saving and intelligence engagement, thus providing great feasibility for precise measurement in complex and dynamic dietary sample analysis. Overall, our proposed method presents a novel technology for onsite dietary Myr enzyme profiling, which is promising to be applied in the food industry for nutritional composition profiles, freshness evaluation, and quality assessment.

Nanoparticles-mediated ion channels manipulation: From their membrane interactions to bioapplications.

Ion channels are transmembrane proteins ubiquitously expressed in all cells that control various ions (e.g. Na+, K+, Ca2+ and Cl- etc) crossing cellular plasma membrane, which play critical roles in physiological processes including regulating signal transduction, cell proliferation as well as excitatory cell excitation and conduction. Abnormal ion channel function is usually associated with dysfunctions and many diseases, such as neurodegenerative disorders, ophthalmic diseases, pulmonary diseases and even cancers. The precise regulation of ion channels not only helps to decipher physiological and pathological processes, but also is expected to become cutting-edge means for disease treatment. Recently, nanoparticles-mediated ion channel manipulation emerges as a highly promising way to meet the increasing requirements with respect to their simple, efficient, precise, spatiotemporally controllable and non-invasive regulation in biomedicine and other research frontiers. Thanks the advantages of their unique properties, nanoparticles can not only directly block the pore sites or kinetics of ion channels through their tiny size effect, and perturb active voltage-gated ion channel by their charged surface, but they can also act as antennas to conduct or enhance external physical stimuli to achieve spatiotemporal, precise and efficient regulation of various ion channel activities (e.g. light-, mechanical-, and temperature-gated ion channels etc). So far, nanoparticles-mediated ion channel regulation has shown potential prospects in many biomedical fields at the interfaces of neuro- and cardiovascular modulation, physiological function regeneration and tumor therapy et al. Towards such important fields, in this typical review, we specifically outline the latest studies of different types of ion channels and their activities relevant to the diseases. In addition, the different types of stimulation responsive nanoparticles, their interaction modes and targeting strategies towards the plasma membrane ion channels will be systematically summarized. More importantly, the ion channel regulatory methods mediated by functional nanoparticles and their bioapplications associated with physiological modulation and therapeutic development will be discussed. Last but not least, current challenges and future perspectives in this field will be covered as well.

Highly Stretchable, Swelling-Resistant, Self-Healed, and Biocompatible Dual-Reinforced Double Polymer Network Hydrogels.

Superior flexibility and toughness can be achieved in bioactive hydrogels by the use of a double polymer network with complementary properties. Inspired by this design principle, we here combine polyacrylic acid (PAA) and sodium alginate (SA) to obtain a dual-reinforced double interpenetrating network (d-DIPN) hydrogel. The dual reinforcement involves ionic cross-linking and introduction of SiO2 nanoparticles, which leads to extraordinary improvements in strength and toughness. Compared with the standard PAA hydrogel that offers an elongation of 240% and a breakage stress of 0.03 MPa, the prepared SA(Ca2+)-PAA-SiO2 hydrogel shows an elongation above 1000% and a breakage stress of 1.62 MPa. Moreover, the combination of strong covalent cross-links and weak reversible interactions provides the d-DIPN hydrogel with swelling resistance and self-healing behavior, adhesive abilities, and shape memory performance. Furthermore, we show that the biocompatibility and bone cell proliferation ability of the hydrogels can be improved through a mineralization process despite an observed reduction in breakage strain and stress. Taken as a whole, our work paves the way for the design of strong and tough hydrogels, with potential applications within biomedicine and particularly tissue engineering.

Hypoxia deactivates epigenetic feedbacks via enzyme-derived clicking proteolysis-targeting chimeras.

Epigenetic mediation through bromodomain and extraterminal (BET) proteins have progressively translated protein imbalance into effective cancer treatment. Perturbation of druggable BET proteins through proteolysis-targeting chimeras (PROTACs) has recently contributed to the discovery of effective therapeutics. Unfortunately, precise and microenvironment-activatable BET protein degradation content with promising tumor selectivity and pharmacological suitability remains elusive. Here, we present an enzyme-derived clicking PROTACs (ENCTACs) capable of orthogonally cross-linking two disparate small-molecule warhead ligands that recognize BET bromodomain-containing protein 4 (BRD4) protein and E3 ligase within tumors only upon hypoxia-induced activation of nitroreductase enzyme. This localized formation of heterobifunctional degraders promotes specific down-regulation of BRD4, which subsequently alters expression of epigenetic targets and, therefore, allows precise modulation of hypoxic signaling in live cells, zebrafish, and living mice with solid tumors. Our activation-feedback system demonstrates compelling superiorities and may enable the PROTAC technology with more flexible practicality and druggable potency for precision medicine in the near future.

Screening, Expression, and Identification of Nanobody against SARS-CoV-2 Spike Protein.

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an infectious disease that has become a serious burden on global public health. This study screened and yielded specific nanobodies (Nbs) against SARS-CoV-2 spike protein receptor binding domain (RBD), following testing its basic characteristics. A nanobody phage library was established by immunizing a camel with RBD protein. After three rounds of panning, the positive colonies were screened by enzyme-linked immunosorbent assay (ELISA). By sequencing, four different sequences of nanobody gene fragments were selected. The four nanobody fusion proteins were expressed and purified, respectively. The specificity and affinity of the four nanobodies were identified by ELISA. Our results showed that an immune phage display library against SARS-CoV-2 has been successfully constructed with a library capacity of which was 4.7 × 108 CFU. The four purified nanobodies showed specific high-affinity binding SARS-CoV-2 S-RBD. Among these, the antigen binding affinity of Nb61 was more comparable to that of commercial rabbit anti-SARS-CoV-2 S-RBD antibodies. In sum, our study has obtained four nanobody strains against SARS-CoV-2 S-RBD with significant affinity and specificity, therefore laying an essential foundation for further research as well as the applications of diagnostic and therapeutic tools of SARS-CoV-2.

Detection of Reactive Oxygen and Nitrogen Species by Upconversion Nanoparticle-Based Near-Infrared Nanoprobes: Recent Progress and Perspectives.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are essential oxidative metabolites of organisms, which are closely related to physiological, pathological and pharmacological processes. The accurate detection of ROS/RNS is important for the understanding of biological processes, monitoring of pharmacological effects, and predicting the course of disease. The recently developed NIR nanoprobes based on upconversion nanoparticles (UCNPs) hold great prospects in sensitive and deep-tissue detection of ROS/RNS, and considerable progress has been achieved so far. In this review, we systematically summarize the up-to-date advances of UCNPs-based near-infrared (NIR) probes for ROS/RNS sensing, and the potential challenges and perspectives for further research are also highlighted. We envision that such a research field will have a bright future for modern biomedical applications.

Resolving the Conflict between Strength and Toughness in Bioactive Silica-Polymer Hybrid Materials.

Simultaneously improving the strength and toughness of materials is a major challenge. Inorganic-polymer hybrids offer the potential to combine mechanical properties of a stiff inorganic glass with a flexible organic polymer. However, the toughening mechanism at the atomic scale remains largely unknown. Based on combined experimental and molecular dynamics simulation results, we find that the deformation and fracture behavior of hybrids are governed by noncovalent intermolecular interactions between polymer and silica networks rather than the breakage of covalent bonds. We then attempt three methods to improve the balance between strength and toughness of hybrids, namely the total inorganic/organic (I/O) weight ratio, the size of silica nanoparticles, and the ratio of -C-O vs -C-C bonds in the polymer chains. Specifically, for a hybrid with matched silica size and I/O ratio, we demonstrate optimized mechanical properties in terms of strength (1.75 MPa at breakage), degree of elongation at the fracture point (31%), toughness (219 kPa), hardness (1.08 MPa), as well as Young's modulus (3.0 MPa). We also demonstrate that this hybrid material shows excellent biocompatibility and ability to support cell attachment as well as proliferation. This supports the possible application of this material as a strong yet tough bone scaffold material.

Screening of multifunctional fruit carbon dots for fluorescent labeling and sensing in living immune cells and zebrafishes.

Nine kinds of carbon dots (CDs) were synthesized by using fruits with different varieties as carbon sources; meanwhile, the fluorescence characteristics, quantum yield, and response ability to different metal ions and free radicals were systematically studied. These CDs showed similar excitation and emission spectral ranges (λex ≈ 345 nm, λem ≈ 435 nm), but very different fluorescence quantum yield (QY), in which orange and cantaloupe CDs have the highest QY around 0.25 and green plum CDs showed the lowest quantum yield around 0.1. Interestingly, the fluorescence of all of these CDs can be significantly quenched by hydroxyl radical (•OH) and iron ion (Fe3+); however, these CDs showed very different response characteristics to other metal ions (e.g., Co2+, Ni2+, Cu2+, Ce3+, Mn2+, Ag+, and Fe2+). Through in-depth analysis, we found some interesting patterns of the influence of carbon sources on the fluorescence characteristics of CDs. Finally, by using white pitaya CDs as fluorescence probe, we realized sensing of Fe3+ and •OH with limits of detection (LOD) of 19.4 µM and 0.7 µM, respectively. Moreover, the CDs were also capable for sensitive detection in immune cells and even in zebrafishes. Our work can provide valuable guidance for the rational design of functional CDs for biological applications.

Tumour enzyme affinity mediated peptide molecular crowding for targeted disruption of hyperactivated glucose uptake.

An unconventional environment-responsive molecular crowding via specific binding between small molecule peptide inhibitor derivatives and an overexpressed tumour enzyme has been developed. Assemblies of such short peptides selectively localize on tumour surfaces and exhibited unique functions in disrupting hyperactivated glucose uptake, providing novel insights towards strategic tumour treatment.

Protein-Mediated Fluorescence Resonance Energy Transfer (P-FRET) Probe: Fabrication and Hydroxyl Radical Detection.

Fluorescent probes based on fluorescence resonance energy transfer (FRET) are highly promising for diverse bioapplications. The key to constructing FRET probes is to confine the donor and acceptor within a sufficiently close distance. However, the commonly used covalent linkage often requires elaborate design and complex organic synthesis, and sometimes causes changes in the fluorescence properties of the donor and acceptor. Inspired by the binding between small molecules and protein in nature, herein, we propose a protein-mediated strategy to fabricate FRET probe. In such protein-mediated FRET (P-FRET) probe, protein acts as a carrier to simultaneously confine donor and acceptor in its cavity. As a proof of concept, we use bovine serum albumin (BSA) as a model protein, coumarin derivative as a donor and hydroxyl radical (·OH)-responsive dye fluorescein as an acceptor. Through a series of investigations, including binding parameters, fluorescence properties and detection performance, we prove that the construction of P-FRET probe is simple and feasible and the detection is sensitive. Our P-FRET strategy will provide new insights for the design of FRET probes.

A tumor microenvironment-responsive Co/ZIF-8/ICG/Pt nanoplatform for chemodynamic and enhanced photodynamic antitumor therapy.

Hypoxia and the overexpression of hydrogen peroxide (H2O2) in the tumor microenvironment (TME) are conducive to cancer cell proliferation, which greatly hinders cancer treatment. Here, we design a novel TME-responsive therapeutic nanoplatform Co/ZIF-8/ICG/Pt (CZIP) to achieve chemodynamic therapy (CDT) and enhanced photodynamic therapy (PDT). In this nanoplatform, under near-infrared light (NIR) irradiation, the photosensitizer indocyanine green (ICG) can generate singlet oxygen (1O2) for cancer cell apoptosis. Meanwhile, overexpressed H2O2 in the TME could be catalyzed to generate O2 by the loaded Pt to relieve tumor hypoxia and promote the PDT-induced 1O2 production. In addition, the doped Co2+ could react with H2O2 to produce hydroxyl radicals (˙OH) for CDT. The multifunctional nanoplatform CZIP showed high biosafety and a good antitumor effect, which would provide a new route for cancer therapy.

Diazapentabenzocorannulenium: A Hydrophilic/Biophilic Cationic Buckybowl.

Polycyclic aromatic molecules are promising functional materials for a wide range of applications, especially in organic electronics. However, their largely hydrophobic nature has impeded further applications. As such, imparting high solubility/hydrophilicity to polycyclic aromatic molecules leads to a breakthrough in this research field. Herein, we report the synthesis of diazapentabenzocorannulenium, a cationic nitrogen-embedded buckybowl bearing a central imidazolium core, by a bottom-up strategy from polycyclic aromatic azomethine ylide. X-ray crystallography analyses have revealed a bowl-shaped molecular structure that is capable of forming charge-segregated one-dimensional columns by bowl-in-bowl packing. In addition to its fluorescence capabilities and high dispersibility in water, the molecule was found to selectively localize in the mitochondria of various tumor cells, showing potential as viable mitochondria-selective fluorescent probes.

Tumor-Microenvironment-Activated Reactive Oxygen Species Amplifier for Enzymatic Cascade Cancer Starvation/Chemodynamic /Immunotherapy.

At present, some progress has been made in the field of cancer theranostics based on nanocatalysts (NCs), but achieving precise theranostics in response to the specific tumor microenvironment (TME) remains a major challenge. Herein, a TME-responsive upconversion nanoparticles (UCNPs)-based smart UCNPs@Cu-Cys-GOx (UCCG) nanosystem is engineered, which combines natural enzymes and nanozymes so as to amplify reactive oxygen species (ROS) generation in situ for cancer starvation/chemodynamic/immunotherapy. One of the biggest merits of this material is that it can be preserved inert (off) in normal tissues, and only in the TME can it be specifically activated (on) through a series of enzymatic cascades to boost ROS production via a strategy of open source (H2 O2 self-supplying ability) and reduce expenditure (glutathione (GSH) consuming ability). More importantly, the enhanced oxidative stress by UCCG NCs reverses the immunosuppressive TME, and facilitates antitumor immune responses. Meanwhile, the starvation/chemodynamic synergistic therapy triggered by UCCG combined with PD-L1 antibody effectively inhibits the growth of primary tumors and cancer metastasis. In addition, the UCNPs in UCCG present upconversion luminescence enhancement, which can be exploited to visualize the reinforced ROS generation in real time. Collectively, this work provides an original method for the devising and exploitation of UCNPs-based catalytic immunotherapy.

Small-molecule fluorescent probes: big future for specific bacterial labeling and infection detection.

Bacterial infections remain a global healthcare problem that is particularly attributed to the spread of antibiotic resistance and the evolving pathogenicity. Accurate and swift approaches for infection diagnosis are urgently needed to facilitate antibiotic stewardship and effective medical treatment. Direct optical imaging for specific bacterial labeling and infection detection offers an attractive prospect of precisely monitoring the infectious disease status and therapeutic response in real time. This feature article focuses on the recent advances of small-molecule probes developed for fluorescent imaging of bacteria and infection, which covers the probe design, responsive mechanisms and representative applications. In addition, the perspective and challenges to advance small-molecule fluorescent probes in the field of rapid drug-resistant bacterial detection and clinical diagnosis of bacterial infections are discussed. We envision that the continuous advancement and clinical translations of such a technique will have a strong impact on future anti-infective medicine.