Development of Enzyme Loaded Polyion Complex Vesicle (PICsome): Thermal Stability of Enzyme in PICsome Compartment and Effect of Coencapsulation of Dextran on Enzyme Activity

Applications of enzymes are intensively studied, particularly for biomedical applications. However, encapsulation or immobilization of enzymes without deactivation and long‐term use of enzymes are still at issue. This study focuses on the polymeric vesicles “PICsomes” for encapsulation of enzymes to develop a hecto‐nanometer‐scaled enzyme‐loaded reactor. The catalytic activity of a PICsome‐based enzyme nanoreactor is carefully examined to clarify the effect of compartmentalization by PICsome. Encapsulation by PICsome provides a stability enhancement of enzymes after 24 h incubation at 37 °C, which is particularly helpful for maintaining the high effective concentration of β‐galactosidase. Moreover, to control the microenvironment inside the nanoreactor, a large amount of dextran, a neutral macromolecule, is encapsulated together with β‐galactosidase in the PICsome. The resulting dextran‐coloaded nanoreactor contributes to the enhancement of enzyme stability, even after exposure to 24 h incubation at −20 °C, mainly due to the antifreezing effect.

     

Direct Real‐Time Monitoring of Prodrug Activation by Chemiluminescence

The majority of theranostic prodrugs reported so far relay information through a fluorogenic response generated upon release of the active chemotherapeutic agent. A chemiluminescence detection mode offers significant advantages over fluorescence, mainly due to the superior signal‐to‐noise ratio of chemiluminescence. Here we report the design and synthesis of the first theranostic prodrug monitored by a chemiluminescence diagnostic mode. As a representative model, we prepared a prodrug from the chemotherapeutic monomethyl auristatin E, which was modified for activation by β‐galactosidase. The activation of the prodrug in the presence of β‐galactosidase is accompanied by emission of a green photon. Light emission intensities, which increase with increasing concentration of the prodrug, were linearly correlated with a decrease in the viability of a human cell line that stably expresses β‐galactosidase. We obtained sharp intravital chemiluminescent images of endogenous enzymatic activity in β‐galactosidase‐overexpressing tumor‐bearing mice. The exceptional sensitivity achieved with the chemiluminescence diagnostic mode should allow the exploitation of theranostic prodrugs for personalized cancer treatment.     

Enzyme‐MOF Nanoreactor Activates Nontoxic Paracetamol for Cancer Therapy

Prodrug activation, by exogenously administered enzymes, for cancer therapy is an approach to achieve better selectivity and less systemic toxicity than conventional chemotherapy. However, the short half‐lives of the activating enzymes in the bloodstream has limited its success. Demonstrated here is that a tyrosinase‐MOF nanoreactor activates the prodrug paracetamol in cancer cells in a long‐lasting manner. By generating reactive oxygen species (ROS) and depleting glutathione (GSH), the product of the enzymatic conversion of paracetamol is toxic to drug‐resistant cancer cells. Tyrosinase‐MOF nanoreactors cause significant cell death in the presence of paracetamol for up to three days after being internalized by cells, while free enzymes totally lose activity in a few hours. Thus, enzyme‐MOF nanocomposites are envisioned to be novel persistent platforms for various biomedical applications.     

Imino‐ and Azasugar Protonation Inside Human Acid β‐Glucosidase,the Enzyme that is Defective in Gaucher Disease

Gaucher disease is caused by mutations in human acid β‐glucosidase or glucocerebrosidase (GCase), the enzyme responsible for hydrolysis of glucosyl ceramide in the lysosomes. Imino‐ and azasugars such as 1‐deoxynojirimycin and isofagomine are strong inhibitors of the enzyme and are of interest in pharmacological chaperone therapy of the disease. Despite several crystal structures of the enzyme with the imino‐ and azasugars bound in the active site having been resolved, the actual acid–base chemistry of the binding is not known. In this study we show, using photoinduced electron transfer (PET), that 1‐deoxynojirimycin and isofagomine derivatives are protonated by human acid β‐glucosidase when bound, even if they are completely unprotonated outside the enzyme. While isofagomine derivative protonation to some degree was foreshadowed by earlier crystal structures, 1‐deoxynojirimycin derivatives were not believed to act as basic amines in the enzyme.     

Synthesis of Chemically Asymmetric Silica Nanobottles and Their Application for Cargo Loading and as Nanoreactors and Nanomotors

We report the synthesis of chemically asymmetric silica nanobottles (NBs) with a hydrophobic exterior surface (capped with 3‐chloropropyl groups) and a hydrophilic interior surface for spatially selective cargo loading, and for application as nanoreactors and nanomotors. The silica NBs, which have a “flask bottle” shape with an average diameter of 350 nm and an opening of ca. 100 nm, are prepared by anisotropic sol–gel growth in a water/n‐pentanol emulsion. Due to their chemically asymmetric properties, nanoparticles (NPs) with hydrophilic or hydrophobic surface properties can be selectively loaded inside the NBs or on the outside of the NBs, respectively. A high‐performance nanomotor is constructed by selectively loading catalytically active hydrophilic Pt NPs inside the NBs. It is also demonstrated that these NBs can be used as vessels for various reactions, such as the in situ synthesis of Au NPs, and using Au NP‐loaded NBs as nanoreactors for catalytic reactions.     

Synthesis and preliminary cytotoxicity study of a cephalosporin-CC-1065 analogue prodrug

Background  

Antibody-directed enzyme prodrug therapy (ADEPT) is a promising new approach to deliver anticancer drugs selectively to tumor cells. In this approach, an enzyme is conjugated to a tumor-specific antibody. The antibody selectively localizes the enzyme to the tumor cell surface. Subsequent administration of a prodrug substrate of the enzyme leads to the enzyme-catalyzed release of the free drug at the tumor site. The free drug will destroy the tumor cells selectively, thus, reducing side effects.     

Therapeutic Vesicular Nanoreactors with Tumor‐Specific Activation and Self‐Destruction for Synergistic Tumor Ablation

Polymeric nanoreactors (NRs) have distinct advantages to improve chemical reaction efficiency, but the in vivo applications are limited by lack of tissue‐specificity. Herein, novel glucose oxidase (GOD)‐loaded therapeutic vesicular NRs (thera NR) are constructed based on a diblock copolymer containing poly(ethylene glycol) (PEG) and copolymerized phenylboronic ester or piperidine‐functionalized methacrylate (P(PBEM‐co ‐PEM)). Upon systemic injection, thera NR are inactive in normal tissues. At a tumor site, thera NR are specifically activated by the tumor acidity via improved permeability of the membranes. Hydrogen peroxide (H₂O₂) production by the catalysis of GOD in thera NR increases tumor oxidative stress significantly. Meanwhile, high levels of H₂O₂ induce self‐destruction of thera NR releasing quinone methide (QM) to deplete glutathione and suppress the antioxidant ability of cancer cells. Finally, thera NR efficiently kill cancer cells and ablate tumors via the synergistic effect.     

Core-Shell Reactor Partitioning Enzyme and Prodrug by ZIF-8 for NADPH-Sensitive In Situ Prodrug Activation

Enzyme-prodrug therapies have shown unique advantages in efficiency, selectivity, and specificity of in vivo prodrug activation. However, precise spatiotemporal control of both the enzyme and its substrate at the target site, preservation of enzyme activity, and in situ substrate depletion due to low prodrug delivery efficiency continue to be great challenges. Here, we propose a novel core–shell reactor partitioning enzyme and prodrug by ZIF-8, which integrates an enzyme with its substrate and increases the drug loading capacity (DLC) using a prodrug as the building ligand to form a Zn-prodrug shell. Cytochrome P450 (CYP450) is immobilized in ZIF-8, and the antitumor drug dacarbazine (DTIC) is coordinated and deposited in its outer layer with a high DLC of 43.6±0.8 %. With this configuration, a much higher prodrug conversion efficiency of CYP450 (36.5±1.5 %) and lower IC₅₀ value (26.3±2.6 μg/mL) are measured for B16-F10 cells with a higher NADPH concentration than those of L02 cells and HUVECs. With the tumor targeting ability of hyaluronic acid, this core–shell enzyme reactor shows a high tumor suppression rate of 96.6±1.9 % and provides a simple and versatile strategy for enabling in vivo biocatalysis to be more efficient, selective, and safer.     

β‐Actin as a loading control: Less than 2 μg of total protein should be loaded

For the purpose of data normalization in Western blot analysis, journal editors and reviewers usually require authors to reprobe the Western blot membrane with a β‐actin‐specific antibody after detecting the target protein. In most cases, however, β‐actin is overloaded, which results in a failure to detect differences in protein loading. In this study, we attempted to optimize the amount of protein loaded for β‐actin detection to permit suitable Western blot analysis data normalization. Our data suggest that less than 2 μg of total protein should be loaded when β‐actin is used as a loading control. We also suggest avoiding reprobing the membrane with a β‐actin‐specific antibody.     

Functional Polyion Complex Vesicles Enabled by Supramolecular Reversible Coordination Polyelectrolytes

Reported here is a new class of PICsomes (vesicles formed by polyelectrolyte complexation) in which the anionic/neutral diblock copolymer is replaced by an anionic, reversible, supramolecular polyelectrolyte based on metal–ligand coordination. This supramolecular polyelectrolyte forms exclusively inside the wall of the assembly, and therefore self‐adjusts its length to that of the cationic block provided. Moreover, the supramolecular coordination polyelectrolytes introduce new and tunable properties and functions associated with the specific metal. As a proof‐of‐concept Mn‐based PICsomes were prepared and display high magnetic relaxivity, as well as enhanced contrast in in vitro magnetic resonance imaging tests. The simplicity of our approach, together with the new functions derived from the metal ions, demonstrates a robust strategy for the preparation of a variety of PICsomes with well‐defined and tunable structures and properties.     

SpyTag/SpyCatcher Cyclization Confers Resilience to Boiling on a Mesophilic Enzyme

SpyTag is a peptide that spontaneously forms an amide bond with its protein partner SpyCatcher. SpyTag was fused at the N terminus of β‐lactamase and SpyCatcher at the C terminus so that the partners could react to lock together the termini of the enzyme. The wild‐type enzyme aggregates above 37 °C, with irreversible loss of activity. Cyclized β‐lactamase was soluble even after heating at 100 °C; after cooling, the catalytic activity was restored. SpyTag/SpyCatcher cyclization led to a much larger increase in stability than that achieved through point mutation or alternative approaches to cyclization. Cyclized dihydrofolate reductase was similarly resilient. Analyzing unfolding through calorimetry indicated that cyclization did not increase the unfolding temperature but rather facilitated refolding after thermal stress. SpyTag/SpyCatcher sandwiching represents a simple and efficient route to enzyme cyclization, with potential to greatly enhance the robustness of biocatalysts.     

Piece by Piece—Electrochemical Synthesis of Individual Nanoparticles and their Performance in ORR Electrocatalysis

The impact of individual HAuCl₄ nanoreactors is measured electrochemically, which provides operando insights and precise control over the modification of electrodes with functional nanoparticles of well‐defined size. Uniformly sized micelles are loaded with a dissolved metal salt. These solution‐phase precursor entities are then reduced electrochemically—one by one—to form nanoparticles (NPs). The charge transferred during the reduction of each micelle is measured individually and allows operando sizing of each of the formed nanoparticles. Thus, particles of known number and sizes can be deposited homogenously even on nonplanar electrodes. This is demonstrated for the decoration of cylindrical carbon fibre electrodes with 25±7 nm sized Au particles from HAuCl₄‐filled micelles. These Au NP‐decorated electrodes show great catalyst performance for ORR (oxygen reduction reaction) already at low catalyst loadings. Hence, collisions of individual precursor‐filled nanocontainers are presented as a new route to nanoparticle‐modified electrodes with high catalyst utilization.     

Development of Luminescent Coelenterazine Derivatives Activatable by β‐Galactosidase for Monitoring Dual Gene Expression

Two bioluminogenic caged coelenterazine derivatives (bGalCoel and bGalNoCoel) were designed and synthesized to detect β‐galactosidase activity and expression by means of bioluminescence imaging. Our approach addresses the instability of coelenterazine by introducing β‐galactose caging groups to block the auto‐oxidation of coelenterazine. Both probes contain β‐galactosidase cleavable caging groups at the carbonyl group of the imidazo–pyrazinone moiety. One of the probes in particular, bGalNoCoel, displayed a fast cleavage profile, high stability, and high specificity for β‐galactosidase over other glycoside hydrolases. bGalN‐oCoel could detect β‐galactosidase activity in living HEK‐293T cell cultures that expressed a mutant Gaussia luciferase. It was determined that coelenterazine readily diffuses in and out of cells after uncaging by β‐galactosidase. We showed that this new caged coelenterazine derivative, bGalNoCoel, could function as a dual‐enzyme substrate and detect enzyme activity across two separate cell populations.     

Weak‐Acid Sites Catalyze the Hydrolysis of Crystalline Cellulose to Glucose in Water: Importance of Post‐Synthetic Functionalization of the Carbon Surface

The direct hydrolysis of crystalline cellulose to glucose in water without prior pretreatment enables the transformation of biomass into fuels and chemicals. To understand which features of a solid catalyst are most important for this transformation, the nanoporous carbon material MSC‐30 was post‐synthetically functionalized by oxidation. The most active catalyst depolymerized crystalline cellulose without prior pretreatment in water, providing glucose in an unprecedented 70 % yield. In comparison, virtually no reaction was observed with MSC‐30, even when the reaction was conducted in aqueous solution at pH 2. As no direct correlations between the activity of this solid–solid reaction and internal‐site characteristics, such as the β‐glu adsorption capacity and the rate of catalytic hydrolysis of adsorbed β‐glu strands, were observed, contacts of the external surface with the cellulose crystal are thought to be key for the overall efficiency.     

Self‐Boosting Catalytic Nanoreactors Integrated with Triggerable Crosslinking Membrane Networks for Initiation of Immunogenic Cell Death by Pyroptosis

Synthetic polymer vesicles spur novel strategies for producing intelligent nanodevices with precise and specific functions. Engineering vesicular nanodevices with tunable permeability by a general platform without involving trade‐offs between structural integrity, flexibility, and functionality remains challenging. Herein, we present a general strategy to construct responsive nanoreactors based on polyion complex vesicles by integrating stimuli‐responsive linkers into a crosslinking membrane network. The formulated ROS‐responsive nanoreactor with self‐boosting catalytic glucose oxidation could protect glucose oxidase (GOD) to achieve cytocidal function by oxidative stress induction and glucose starvation, which is ascribed to stimuli‐responsive vesicle expansion without fracture and size‐selective cargo release behavior. The GOD‐loaded therapeutic nanoreactor induced an immunostimulatory form of cell death by pyroptosis, which has the great potential to prime anti‐tumor immune responses.     

Prediction of membrane spanning segments and topology in β‐barrel membrane proteins at better accuracy

Prediction of membrane spanning segments in β‐barrel outer membrane proteins (OMP) and their topology is an important problem in structural and functional genomics. In this work, we propose a method based on radial basis networks for predicting the number of β‐strands in OMPs and identifying their membrane spanning segments. Our method showed a leave‐one‐out cross validation accuracy of 96% in a set of 28 OMPs, which have the range of 8–22 β‐strand segments. The β‐strand segments in OMPs and the residues in membrane spanning segments are correctly predicted with the accuracy of 96% and 87%, respectively. We have developed a web server, TMBETAPRED‐RBF for predicting the transmembrane β‐strands from amino acid sequence and it is available at http://rbf.bioinfo.tw/～sachen/tmrbf.html . We suggest that our method could be an effective tool for predicting the membrane spanning regions and topology of β‐barrel membrane proteins. © 2009 Wiley Periodicals, Inc. J Comput Chem 2010     

Silicon Carbide Nanostructures from Reactions between Vapors of Organochlorosilanes and Liquid of Sodium‐Factors Affecting Morphology and Composition

Cubic shells and spherical nanoparticles of β‐SiC were produced at 1273 K by processing the ceramic precursors formed from the reactions between vapors of organochlorosilanes, Me₂SiCl₂, MeSiCl₃, MeSiHCl₂, and PhSiCl₃, and liquid Na at 523‐723 K. From Me₂SiCl₂, a flexible linear polycarbosilane precursor was synthesized and covered the NaCl byproduct surface to form a cubic shape. Hollow cubic β‐SiC shells were produced after the NaCl templates were removed. From MeSiCl₃, a rigid cross‐linked polycarbosilane was produced and phase segregated from the NaCl byproduct. The precursor was transformed into nanoparticles without special morphology. MeSiHCl₂ produced a cross‐linked polysilane precursor at low temperatures, which can be converted into a mixture of β‐SiC and Si nanoparticles. At high temperatures, the polysilane converted to polycarbosilane and produced hollow cubic β‐SiC shells. The carbon‐rich PhSiCl₃ generated cube‐like particles as the final product, which contained β‐SiC and carbon.     

Palladium nanoparticles in carbon thin film-lined SBA-15 nanoreactors: efficient heterogeneous catalysts for Suzuki-Miyaura cross coupling reaction in aqueous media

Embedding Pd nanoparticles in carbon thin film-lined SBA-15 nanoreactors provides highly efficient catalysts for heterogeneous cross coupling reactions in aqueous media. No leaching or aggregation of Pd nanoparticles was found in these nanoreactors after reusing them several times. The carbon thin film lining of these nanoreactors was further confirmed with small molecular arene probing experiments.     

Hybrid Nanoreactors: Enabling an Off‐the‐Shelf Strategy for Concurrently Enhanced Chemo‐immunotherapy

Immunosuppressive tumors generally exhibit poor response to immune checkpoint blockade based cancer immunotherapy. Rationally designed hybrid nanoreactors are now presented that have integrated functions as Fenton catalysts and glutathione depletion agents for amplifying the immunogenic cell death and activating immune cells. A simple physical mixture of nanoreactors and chemodrugs in combination with immune checkpoint blockades show synergistically and concurrently enhanced chemo‐immunotherapy efficacy, inhibiting the growth of both treated primary immunosuppressive tumors and untreated distant tumors. The off‐the‐shelf strategy uses tumor antigens generated in situ and avoids cargo loading, and is thus a substantial advance in personalized nanomedicine for clinical translation.     

HandaPhos: A General Ligand Enabling Sustainable ppm Levels of Palladium‐Catalyzed Cross‐Couplings in Water at Room Temperature

The new monophosphine ligand HandaPhos has been identified such that when complexed in a 1:1 ratio with Pd(OAc)₂, enables Pd‐catalyzed cross‐couplings to be run using ≤1000 ppm of this pre‐catalyst. Applications to Suzuki–Miyaura reactions involving highly funtionalized reaction partners are demonstrated, all run using environmentally benign nanoreactors in water at ambient temperatures. Comparisons with existing state‐of‐the‐art ligands and catalysts are discussed herein.