Construction And Properties Of Catalase-coated Gliadin Particles

Gliadin is usually used as nano-carrier to load bioactive molecules because of its unique properties in self-assembly,film-forming,adhesion and biological compatibility.The antisolvent precipitation method of fabricating Gliadin Nanoparticles(GNPs)has the advantages of simplicity,energy-saving and rapidity compared to the others,but the produced GNPs were thermo-instable,particularly at human body temperature,which greatly limited their potentials in nutraceutical or pharmaceutical applications.In this study,an attempt was made to improve the stability of GNPs by coating with catalase(CAT),the properties and cellular biological activities of the obtained catalase coated GNPs(CAT-GNPs)were subsequently investigated,including their fabrication approaches,colloidal properties and enzymatic properties,interactions between GNPs and CAT,furthermore,the intracellular antioxidant activities of CAT-GNPs on primary cultured peritoneal macrophages and macrophage RAW 264.7 were also evaluated.(1)By adjusting pH,CAT was combined to GNPs surface by electrostatic attraction to form CAT-GNPs complex.The zeta potential of CAT-GNPsbecame stable while increasing the concentration of CAT over 1.4 ?M,which might be due to saturation adsorption of CAT on GNPs surface caused by steric hindrance effect.The average particle size of the obtained CAT-GNPs is about 239 nm with the zeta potential of approximately-12 mV.Furthermore,the surface net charge is zero when the pH is near to 6.2.Compared with GNPs,the storage stability of CAT-GNPs at 37 °C was significantly improved,and the stability at high temperatures was also significantly enhanced,while the stability to pH and the tolerance to salt ions were not improved.(2)The optimum temperature of CAT enzyme activity in CAT-GNPs particles changed slightly compared to that of CAT.The range of optimum temperature for enzyme activity and the apparent optimum pH range became wider.The thermal and low pH(pH3-5.5)resistance of composite particles were also enhanced compared to those of CAT.(3)The addition of other negatively charged proteins,e.g.bovine serum albumin or superoxide dismutase didn't improve the storage stability of GNPs at the same pH in the pH4.CAT significantly enhanced the storage stability at 37? and thermal resistance of GNPs in pH4,in which both CAT and GNPs are positive charged resulting in electrostatic repulsion interaction instead of electrostatic attraction.The results implied that the improvement of the storage stability of GNPs at 37 °C could not totally be explained by the formation of CAT-GNPs via electrostatic attractions,the mechanism behind remains unknown yet.(4)The results of Fourier spectroscopy showed the amide group of GNPs changed significantly after coating with in surface,indicating electrostatic interaction occurred between CAT and GNPs.According to the results obtained from,peak at 346 nm in fluorescence spectrogram of GNPs underwent blue shift with a certain degree of fluorescence quenching after coating CAT to form CAT-GNPs,suggesting that the interaction between them caused the tryptophan microenvironment changes.The results of circular dichroism show that the coexistence of GNPs and CAT,no matter what interaction,either electrostatic attraction or repulsion,led the changes in secondary structure.(5)Cytotoxicity results showed that samples of GNPs,CAT-GNPs and gliadin did not exhibited significant toxicity to primary peritoneal macrophages and RAW264.7 cells.Some samples could promote the growth of both cells at certain concentrations.(6)The results of intracellular antioxidant activity assay showed that CAT did show intracellular antioxidant activity in a higher concentration range but slightly aggravated intracellular oxidative stress.CAT-GNPs didn't exhibit higher values in intracellular antioxidant activity than GNPs within this concentration range.However,at certain range of CAT concentration,CAT showed significant intracellular antioxidant activity.The CAT-GNPs showed higher intracellular antioxidant activity than GNPs in both cell lines after the sample dilution reached 64 times.Its intracellular antioxidant activity value was even significantly greater than those of both CAT and GNPs alone.In summary,this study mainly explored the technical feasibility of catalase-coated gliadin nanoparticles,and investigated the properties,structural characteristics and biological activities of the obtained composite CAT-GNPs particles.Our research findings provide reference value for the stability study of gliadin nanoparticles and the improvement of catalase by encapsulation.