Novel dTDP-l-Rhamnose Synthetic Enzymes (RmlABCD) From Saccharothrix syringae CGMCC 4.1716 for One-Pot Four-Enzyme Synthesis of dTDP-l-Rhamnose.

Deoxythymidine diphospho-l-rhamnose (dTDP-l-rhamnose) is used by prokaryotic rhamnosyltransferases as the glycosyl donor for the synthesis of rhamnose-containing polysaccharides and compounds that have potential in pharmaceutical development, so its efficient synthesis has attracted much attention. In this study, we successfully cloned four putative dTDP-l-rhamnose synthesis genes Ss-rmlABCD from Saccharothrix syringae CGMCC 4.1716 and expressed them in Escherichia coli. The recombinant enzymes, Ss-RmlA (glucose-1-phosphate thymidylyltransferase), Ss-RmlB (dTDP-d-glucose 4,6-dehydratase), Ss-RmlC (dTDP-4-keto-6-deoxy-glucose 3,5-epimerase), and Ss-RmlD (dTDP-4-keto-rhamnose reductase), were confirmed to catalyze the sequential formation of dTDP-l-rhamnose from deoxythymidine triphosphate (dTTP) and glucose-1-phosphate (Glc-1-P). Ss-RmlA showed maximal enzyme activity at 37°C and pH 9.0 with 2.5mMMg2+, and the K m and k cat values for dTTP and Glc-1-P were 49.56µM and 5.39s-1, and 117.30µM and 3.46s-1, respectively. Ss-RmlA was promiscuous in the substrate choice and it could use three nucleoside triphosphates (dTTP, dUTP, and UTP) and three sugar-1-Ps (Glc-1-P, GlcNH2-1-P, and GlcN3-1-P) to form nine sugar nucleotides (dTDP-GlcNH2, dTDP-GlcN3, UDP-Glc, UDP-GlcNH2, UDP-GlcN3, dUDP-Glc, dUDP-GlcNH2, and dUDP-GlcN3). Ss-RmlB showed maximal enzyme activity at 50°C and pH 7.5 with 0.02mM NAD+, and the K m and k cat values for dTDP-glucose were 98.60µM and 11.2s-1, respectively. A one-pot four-enzyme reaction system was developed by simultaneously mixing all of the substrates, reagents, and four enzymes Ss-RmlABCD in one pot for the synthesis of dTDP-l-rhamnose and dUDP-l-rhamnose with the maximal yield of 65% and 46%, respectively, under the optimal conditions. dUDP-l-rhamnose was a novel nucleotide-activated rhamnose reported for the first time.

Rational Design of Multi-Stimuli-Responsive Nanoparticles for Precise Cancer Therapy.

Stimuli-responsive nanoparticles with target capacity are of great interest in drug delivery for cancer therapy. However, the challenge is to achieve highly smart release with precise spatiotemporal control for cancer therapy. Herein, we report the preparation and properties of multi-stimuli-responsive nanoparticles through the co-assembly of a 3-arm star quaterpolymer with a near-infrared (NIR) photothermal agent and chemotherapeutic compound. The nanoparticles can exhibit NIR light/pH/reduction-responsive drug release and intracellular drug translocation in cancer cells, which further integrate photoinduced hyperthermia for synergistic anticancer efficiency, thereby leading to tumor ablation without tumor regrowth. Thus, this rational design of nanoparticles with multiple responsiveness represents a versatile strategy to provide smart drug delivery paradigms for cancer therapy.

Synthesis, Thermal Properties, and Thermoresponsive Behaviors of Cyclic Poly(2-(dimethylamino)ethyl Methacrylate)s.

This study aims at physicochemical properties of thermo- and pH/CO2 -responsive cyclic homopolymers. Three examples of cyclic poly(2-(dimethylamino)ethyl methacrylate)s (PDMAs) are synthesized by combining the reversible addition-fragmentation chain transfer process and the Diels-Alder ring-closure reaction. After cyclization, the glass transition temperature significantly increases (ΔTg = 51.8-59.7 °C) due to the different configurational entropy and end groups, and the maximum decomposition temperature to lose the pendent groups is drastically decreased from 309 to 278 °C. Effects of polymerization degree, polymer concentration, additive of NaCl, and pH/CO2 on lower critical solution temperature behaviors of PDMA aqueous solutions are investigated. The cloud points (Tc ) of ring PDMAs are usually higher than their linear precursors, and the ΔTc values obtained under a fixed condition can reach up to 20.7 °C, revealing the crucial role of the topology effect. This study paves the way for unique properties and applications of smart cyclic polymers and their derivatives.

Multi-responsive graft copolymer micelles comprising acetal and disulfide linkages for stimuli-triggered drug delivery.

Thermo-, pH and reduction triggered drug delivery vehicles based on dual-cleavable polymeric micelles were investigated. A comblike copolymer (G3) comprising one disulfide linkage and PEG, PCL and acetal-bridged PCL-b-PNIPAM grafts was controllably synthesized by successive RAFT copolymerization, ring-opening polymerization and adductive reaction. G3 was liable to self-assemble into spherical micelles at 25 °C and toroidal micelles at 37 °C, and the aggregates formed at 37 °C could be further converted into multicompartment micelles (pH 5.3), spherical micelles (DTT) and hyperbranched or necklace-like cylinders (pH 5.3 + DTT) upon external stimuli due to the stimuli-triggered topological transformation and reaggregation of copolymer aggregates. Upon external stimuli, doxorubicin (DOX) loaded G3 and G3/ß-CD (co)aggregates could exhibit accelerated drug release kinetics. The apparent release rates varied in the range 0.072-0.403 h-1 (for G3 aggregates) and 0.142-0.458 h-1 (for G3/ß-CD coaggregates), revealing that the drug release system bearing host-guest interactions could further extend the ranges of the release rate and cumulative release. Although ß-CD and G3 micelles lacked notable cytotoxicity, the cytotoxicity of DOX-loaded (co)aggregates to 4T1 cells was higher than free DOX. CLSM images revealed that DOX-loaded copolymer aggregates may enter cells via endocytosis in a manner of nanocomplexes. Our study can not only extend the potential of stimuli-cleavable copolymers toward biomedical applications but also enrich the family of multi-responsive copolymer aggregates.