Controlled Dendrimersome Nanoreactor System for Localized Hypochlorite-Induced Killing of Bacteria.
ABSTRACT: Antibiotic resistance is a serious global health problem necessitating new bactericidal approaches such as nanomedicines. Dendrimersomes (DSs) have recently become a valuable alternative nanocarrier to polymersomes and liposomes due to their molecular definition and synthetic versatility. Despite this, their biomedical application is still in its infancy. Inspired by the localized antimicrobial function of neutrophil phagosomes and the versatility of DSs, a simple three-component DS-based nanoreactor with broad-spectrum bactericidal activity is presented. This was achieved by encapsulation of glucose oxidase (GOX) and myeloperoxidase (MPO) within DSs (GOX-MPO-DSs), self-assembled from an amphiphilic Janus dendrimer, that possesses a semipermeable membrane. By external addition of glucose to GOX-MPO-DS, the production of hypochlorite (-OCl), a highly potent antimicrobial, by the enzymatic cascade was demonstrated. This cascade nanoreactor yielded a potent bactericidal effect against two important multidrug resistant pathogens, Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa), not observed for H2O2 producing nanoreactors, GOX-DS. The production of highly reactive species such as -OCl represents a harsh bactericidal approach that could also be cytotoxic to mammalian cells. This necessitates the development of strategies for activating -OCl production in a localized manner in response to a bacterial stimulus. One option of locally releasing sufficient amounts of substrate using a bacterial trigger (released toxins) was demonstrated with lipidic glucose-loaded giant unilamellar vesicles (GUVs), envisioning, e.g., implant surface modification with nanoreactors and GUVs for localized production of bactericidal agents in the presence of bacterial growth.
Project description:A hierarchical porous metal-organic framework (MOF), PCN-888, containing three types of cavities was utilized to couple two enzymes into a tandem nanoreactor. The largest cavity (6.2 nm) can only accommodate one molecule of glucose oxidase (GOx). The intermediate cavity (5.0 nm) can accommodate one and only one molecule of horseradish peroxidase (HRP). The small cavity (2.0 nm) has sufficient size for neither GOx nor HRP, and remains open as a substrate diffusion pathway. The coupling of the two enzymes can only be achieved through a unique stepwise encapsulation with a specific order (GOx first, followed by HRP). The tandem nanoreactor shows excellent catalytic performances and negligible enzyme leaching. Its catalytic activity is well maintained within several catalytic cycles. Moreover, PCN-888 can provide protection to the encapsulated enzymes against trypsin digestion, indicating the <i>in vitro</i> and <i>in vivo</i> stability of the nanoreactor.
Project description:Integration of disease diagnosis and therapy in vivo by nanotechnology is a challenge in the design of multifunctional nanocarriers. Herein, we report an intelligent and degradable nanoreactor, an assembly of the 4-mercaptobenzonitrile-decorated silver nanoparticles (AgNPs@MBN) and the glucose oxidase (GOx)-loaded metal-organic-framework (ZIF-8@GOx), which can be activated by tumor microenvironment to start the catalytic cascade-enhanced chemo-starvation synergistic therapy and simultaneous self-sense of cellular glucose level. Under the mild acidic microenvironment of tumor, the nanoreactor will collapse to release GOx that triggers a catalytic cascade reaction in vivo, depleting glucose, etching AgNPs@MBN, and producing toxic H2O2, Ag+, and Zn2+ ions, all of which work together to inhibit tumor growth. The AgNPs@MBN as SERS nanoprobe reads out glucose concentration noninvasively in tumor to achieve instant feedback of therapeutic progression. This work proposes a promising example of using enzyme-encapsulated biomineralized MOFs as an effective anticarcinogen for clinical applications.
Project description:Performing reactions in the presence of self-assembled hierarchical structures of amphiphilic macromolecules can accelerate reactions while using water as the bulk solvent due to the hydrophobic effect. We leveraged non-covalent interactions to self-assemble filled-polymer micelle nanoreactors (NR) incorporating gold nanoparticle catalysts into various amphiphilic polymer nanostructures with comparable hydrodynamic nanoreactor size and gold concentration in the nanoreactor dispersion. We systematically studied the effect of the hydrophobic co-precipitant on self-assembly and catalytic performance. We observed that co-precipitants that interact with gold are beneficial for improving incorporation efficiency of the gold nanoparticles into the nanocomposite nanoreactor during self-assembly but decrease catalytic performance. Hierarchical assemblies with co-precipitants that leverage noncovalent interactions could enhance catalytic performance. For the co-precipitants that do not interact strongly with gold, the catalytic performance was strongly affected by the hydrophobic microenvironment of the co-precipitant. Specifically, the apparent reaction rate per surface area using castor oil (CO) was over 8-fold greater than polystyrene (750 g/mol, PS 750); the turnover frequency was higher than previously reported self-assembled polymer systems. The increase in apparent catalytic performance could be attributed to differences in reactant solubility rather than differences in mass transfer or intrinsic kinetics; higher reactant solubility enhances apparent reaction rates. Full conversion of 4-nitrophenol was achieved within three minutes for at least 10 sequential reactions demonstrating that the nanoreactors could be used for multiple reactions.
Project description:Reliable monitoring of metabolites in biofluids is critical for diagnosis, treatment, and long-term management of various diseases. Although widely used, existing enzymatic metabolite assays face challenges in clinical practice primarily due to the susceptibility of enzyme activity to external conditions and the low sensitivity of sensing strategies. Inspired by the micro/nanoscale confined catalytic environment in living cells, the coencapsulation of oxidoreductase and metal nanoparticles within the nanopores of macroporous silica foams to fabricate all-in-one bio-nanoreactors is reported herein for use in surface-enhanced Raman scattering (SERS)-based metabolic assays. The enhancement of catalytical activity and stability of enzyme against high temperatures, long-time storage or proteolytic agents are demonstrated. The nanoreactors recognize and catalyze oxidation of the metabolite, and provide ratiometric SERS response in the presence of the enzymatic by-product H2O2, enabling sensitive metabolite quantification in a "sample in and answer out" manner. The nanoreactor makes any oxidoreductase-responsible metabolite a candidate for quantitative SERS sensing, as shown for glucose and lactate. Glucose levels of patients with bacterial infection are accurately analyzed with only 20 µL of cerebrospinal fluids, indicating the potential application of the nanoreactor in vitro clinical testing.
Project description:When using the microbial cell factories for green manufacturing, several important issues need to be addressed such as how to maintain the stability of biocatalysts used in the bioprocess and how to improve the synthetic efficiency of the biological system. One strategy widely used during natural evolution is the creation of organelles which can be used for regional control. This kind of compartmentalization strategy has inspired the design of artificial organelle-like nanodevice for synthetic biology and "green chemistry".Mimicking the natural concept of functional compartments, here we show that the engineered thermostable ketohydroxyglutarate aldolase from Thermotoga maritima could be developed as a general platform for nanoreactor design via supramolecular self-assembly. An industrial biocatalyst-(+)-?-lactamase was selected as a model catalyst and successful encapsulated in the nanoreactor with high copies. These nanomaterials could easily be synthesized by Escherichia coli by heterologous expression and subsequently self-assembles into the target organelle-like nanoreactors both in vivo and in vitro. By probing their structural characteristics via transmission electronic microscopy and their catalytic activity under diverse conditions, we proved that these nanoreactors could confer a significant benefit to the cargo proteins. The encapsulated protein exhibits significantly improved stability under conditions such as in the presence of organic solvent or proteases, and shows better substrate tolerance than free enzyme.Our biodesign strategy provides new methods to develop new catalytically active protein-nanoreactors and could easily be applied into other biocatalysts. These artificial organelles could have widely application in sustainable catalysis, synthetic biology and could significantly improve the performance of microbial cell factories.
Project description:Fe<sup>III</sup>-hypohalite complexes have been implicated in a wide range of important enzyme-catalyzed halogenation reactions including the biosynthesis of natural products and antibiotics and post-translational modification of proteins. The absence of spectroscopic data on such species precludes their identification. Herein, we report the generation and spectroscopic characterization of nonheme Fe<sup>III</sup>-hypohalite intermediates of possible relevance to iron halogenases. We show that Fe<sup>III</sup>-OCl polypyridylamine complexes can be sufficiently stable at room temperature to be characterized by UV/Vis absorption, resonance Raman and EPR spectroscopies, and cryo-ESIMS. DFT?methods rationalize the pathways to the formation of the Fe<sup>III</sup>-OCl, and ultimately Fe<sup>IV</sup>=O, species and provide indirect evidence for a short-lived Fe<sup>II</sup>-OCl intermediate. The species observed and the pathways involved offer insight into and, importantly, a spectroscopic database for the investigation of iron halogenases.
Project description:Organelles of eukaryotic cells are structures made up of membranes, which carry out a majority of functions necessary for the surviving of the cell itself. Organelles also differentiate the prokaryotic and eukaryotic cells, and are arranged to form different compartments guaranteeing the activities for which eukaryotic cells are programmed. Cell membranes, containing organelles, are isolated from cancer cells and erythrocytes and used to form biocompatible and long-circulating ghost nanoparticles delivering payloads or catalyzing enzymatic reactions as nanoreactors. In this attempt, red blood cell membranes were isolated from erythrocytes, and engineered to form nanoerythrosomes (NERs) of 150 nm. The horseradish peroxidase, used as an enzyme model, was loaded inside the aqueous compartment of NERs, and its catalytic reaction with Resorufin was monitored. The resulting nanoreactor protected the enzyme from proteolytic degradation, and potentiated the enzymatic reaction in situ as demonstrated by maximal velocity (Vmax) and Michaelis constant (Km), thus suggesting the high catalytic activity of nanoreactors compared to the pure enzymes.
Project description:In this work, we explore the ability to generate well-defined poly(butyl methacrylate) (PBMA) nanostructures by "in situ" polymerization of butyl methacrylate monomer (BMA). PBMA nanostructures of high and low aspect ratios have been successfully obtained through the free radical polymerization (FRP) of a BMA monomer in anodic aluminum oxide (AAO) nanoreactors of suitable size. A polymerization kinetics process has been followed by differential scanning calorimetry (DSC) and proton Nuclear Magnetic Resonance spectroscopy (<sup>1</sup>H-NMR).The determination of the kinetics of polymerization through DSC is based on a quick and direct analysis of the exothermic polymerization process, whereas the analysis through <sup>1</sup>H-NMR also allows the unambiguous chemical analysis of the resulting polymer. When compared to bulk polymerization, both techniques demonstrate confinement effects. Moreover, DSC and <sup>1</sup>H-NMR analysis give the same kinetics results and show a gel-effect in all the cases. The number average molecular weight (Mn) of the PBMA obtained in AAO of 60-300 nm are between 30·10<sup>3</sup>-175·10<sup>3</sup> g/mol. Even if the Mn value is lower with respect to that obtained in bulk polymerization, it is high enough to maintain the polymer properties. As determined by SEM morphological characterization, once extracted from the AAO nanoreactor, the polymer nanostructures show controlled homogeneous aspect/size all throughout the length of nanopillar over a surface area of few cm<sup>2</sup>. The Young's modulus of low aspect ratio PBMA nanopillars determined by AFM gives a value of 3.1 ± 1.1 MPa. In this work, a 100% of PBMA polymer nanostructures are obtained from a BMA monomer in AAO templates through a quick double process: 30 min of monomer immersion at room temperature and 90 min of polymerization reaction at 60 °C. While the same nanostructures are obtained by polymer infiltration of PBMA at 200 °C in about 6 h, polymerization conditions are much softer than those corresponding to the polymer infiltration process. Furthermore, the <sup>1</sup>H-NMR technique has been consolidated as a tool for studying the kinetics of the copolymerization reactions in confinement and the determination of monomer reactivity ratios.
Project description:The covalent chemistry of individual reactants bound within a protein pore can be monitored by observing the ionic current flow through the pore, which acts as a nanoreactor responding to bond-making and bond-breaking events. In the present work, we incorporated an unnatural amino acid into the ?-hemolysin (?HL) pore by using solid-phase peptide synthesis to make the central segment of the polypeptide chain, which forms the transmembrane ?-barrel of the assembled heptamer. The full-length ?HL monomer was obtained by native chemical ligation of the central synthetic peptide to flanking recombinant polypeptides. ?HL pores with one semisynthetic subunit were then used as nanoreactors for single-molecule chemistry. By introducing an amino acid with a terminal alkyne group, we were able to visualize click chemistry at the single-molecule level, which revealed a long-lived (4.5-s) reaction intermediate. Additional side chains might be introduced in a similar fashion, thereby greatly expanding the range of single-molecule covalent chemistry that can be investigated by the nanoreactor approach.
Project description:Observations in patients with ANCA-associated vasculitis suggest that CD8<sup>+</sup> T cells participate in disease, but there is no experimental functional evidence of pathologic involvement for these cells. Myeloperoxidase (MPO) is a well defined autoantigen in ANCA-associated vasculitis. Studies in experimental models of anti-MPO GN suggest that, after ANCA-induced neutrophil localization, deposited MPO within glomeruli is recognized by autoreactive T cells that contribute to injury. We tested the hypothesis that CD8<sup>+</sup> T cells mediate disease in experimental ANCA-associated vasculitis. CD8<sup>+</sup> T cell depletion in the effector phase of disease attenuated injury in murine anti-MPO GN. This protection associated with decreased levels of intrarenal IFN-?, TNF, and inflammatory chemokines and fewer glomerular macrophages. Moreover, we identified a pathogenic CD8<sup>+</sup> T cell MPO epitope (MPO<sub>431-439</sub>) and found that cotransfer of MPO<sub>431-439</sub>-specific CD8<sup>+</sup> T cell clones exacerbated disease mediated by MPO-specific CD4<sup>+</sup> cells in Rag1<sup>-/-</sup> mice. Transfer of MPO<sub>431-439</sub>-specific CD8<sup>+</sup> cells without CD4<sup>+</sup> cells mediated glomerular injury when MPO was planted in glomeruli. These results show a pathogenic role for MPO-specific CD8<sup>+</sup> T cells, provide evidence that CD8<sup>+</sup> cells are a therapeutic target in ANCA-associated vasculitis, and suggest that a molecular hotspot within the MPO molecule contains important CD8<sup>+</sup>, CD4<sup>+</sup>, and B cell epitopes.