Enzymatic catalysis in microreactors has attracted growing scientific interest because of high specific surface enabling heat and mass transfer and easier control of reaction parameters in microreactors. However, two major challenges that limit their application are fast inactivation and the inability to the biocatalysts in microchannel reactors. A fluid and unsinkable immobilized enzyme were firstly applied in a microchannel reactor for biocatalysis in this study. Functionalized forms of graphene-immobilized naringinase flowing in microchannels have yielded excellent results for isoquercitrin production. A maximum yield of 92.24 ± 3.26% was obtained after 20 min in a microchannel reactor. Ten cycles of enzymatic hydrolysis reaction were successively completed and an enzyme activity above 85.51 ± 2.76% was maintained. The kinetic parameter V m/K m increased to 1.9-fold and reaction time was decreased to 1/3 compared with that in a batch reactor. These results indicated that the moving and unsinkable graphene sheets immobilized enzyme with a high persistent specificity and a mild catalytic characteristic enabled the repetitive use of enzyme and significant cost saving for the application of enzyme catalysis. Thus, the developed method has provided an efficient and simple approach for the productive and repeatable microfluidic biocatalysis.
Here we described nano-polyplexes (NPs) made of oleoyl-carboxymethy-chitosan (OCMCS)/hyaluronic acid (HA) as novel potential carriers for oral gene vaccines delivery. Aerolysin gene (aerA) of Aeromonas hydrophila as microbial antigen was efficiently loaded to form OCMCS-HA/aerA (OHA) NPs. OHA NPs performed the optimal parameters, i.e. smallest (154.5±9.4nm), positive charged (+7.9±0.5mV) and monodispersed system with the N/P ratio of 5 and OCMCS/HA weight ratio of 4. Upon the introduction of HA, OHA NPs was beneficial for the DNA release in intestinal environments in comparison to OA NPs. The mean fluorescence intensity detected in Caco-2 cells incubated with OHA NPs was about 2.5-fold higher than that of OA NPs; however, it decreased significantly in the presence of excess free HA. The OHA NPs and OA NPs decreased the transepithelial electric resistance (TEER) of Caco-2 monolayers obviously and induced increasing the apparent permeability coefficient (Papp) of DNA by 5.45-6.09 folds compared with free DNA. Significantly higher (P<0.05) antigen-specific antibodies were detected in serum after orally immunized with OHA NPs than that immunized with OA NPs and DNA alone in carps. These results enable the OHA NPs might resolve challenges arising from gastrointestinal damage to gene antigens, and offer an approach applicable for oral vaccination.
Caffeic acid (CA) can be considered as an important natural antioxidant. However, the low solubility and stability of CA in various solvent systems is a major limiting factor governing the overall application in the lipid industry, so the synthesis of methyl caffeate (MC)using CA and methanol is a feasible way to improve its lipophilicity. Here, the reaction conditions and kinetic parameters for the synthesis of MC using p-toluenesulfonic acid (PTSA) as a catalyst were firstly investigated, and the product was confirmed byliquid chromatography-mass spectrometry (LC-MS),Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and melting point analysis. The highest yield of MC catalyzed by PTSA reached 84.0% under the optimum conditions as follows: molar ratio of methanol to CA of 20:1, reaction temperature of 65°C, mass ratio of catalyst to substrate of 8 %, and reaction time of 4 h. The esterification kinetics of CA and methanol is described by the pseudo-homogeneous second order reversible model, the relationship between temperature and the forward rate constant is k1 = exp (358.7 - 2111/T), and the activation energy is 17.5 kJ mol-1. These results indicated that the PTSA possesses high catalytic activity in the synthesis of MC, which is an efficient catalyst suitable for MC production in the chemical industry
A series of deoxycholate-chitosan-hydroxybutyl (DAHBCs) with different degrees of substitution (DS) of hydrophobic deoxycholate (DOCA) were successfully synthesized. The lower critical solution temperature (LCST) of various DAHBCs could be adjusted from 35.4°C to 42.1°C by controlling the graft density of DOCA. DAHBCs could self-assemble into nanoparticles (NPs) which gradually evolved from irregular aggregates to spherical particles with the decrease of the DS of DOCA groups. The size of DAHBCs NPs ranged from 100nm to 250nm and their zeta potential varied between 3.85 and 12.37mV. Hemolysis tests and protein adsorption assay exhibited DAHBCs NPs had few adverse effects on the blood components even at a concentration as high as 1mg/mL. DAHBCs NPs showed high curcumin (CUR) encapsulation efficiency up to 80%. CUR-loaded DAHBCs NPs displayed thermal-dependent drug release profiles, and the release rate of CUR (∼75%) was significantly (p<0.05) accelerated at a temperature above the LCST compared with that (∼40%) below the LCST. Cytotoxicity analysis identified no toxicity associated with DAHBCs NPs at a concentration up to 0.5mg/mL. However, when the cells were incubated with the CUR-loaded NPs, their growth was significantly inhibited at 43°C (>LCST), demonstrating the thermal-responsive release of encapsulated cargoes from the NPs. With the capacity to control the LCST of DAHBCs NPs at specific temperatures, it could be speculated that DAHBCs NPs might serve as a promising thermo-responsive nanoplatform for the delivery of antitumor drugs.
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