Preparation,in Vitro Safety Evaluation Of Magnetic Chitosan Nanoparticles And Their Food Application

Magnetic nanoparticles as an effective deliver carrier have been widely studies in food fields,such as enzyme immobilization,food fast inspection and waste water treatment.However,the agglomeration of magnetic nanoparticles in aqueous solutions limits their practical applications.Chitosan,as protective layer,coated on the surface of magnetic nanoparticle could effectively avoid aggregate of magnetic dipole-dipole attractions between particles,and functional groups for various application.Meanwhile,chitosan functionalized magnetic nanoparticles also ensure as-prepared magnetic chitosan nanoparticles have a strong magnetic properties and high biocompatible.Currently,in order to expanding application range of magnetic chitosan nanoparticles,the preparation method for nano-scaled magnetic chitosan particles with a narrow size distribution is needed.Meanwhile,the unclear potential safety risk of magnetic chitosan nanoparticles also affected their widely application in food or other fields.Therefore,the research objective of this dissertation is develop a new method to prepare magnetic chitosan nanoparticles using two-step route and water-in-oil microemulsion encapsulation method;evaluate their safety issues through in vitro evaluation of hemolysis,cytotoxicity and cellular uptake on this new developed magnetic chitosan nanoparticles;and develop their practical applications on food dye adsorption and extracellular ice nucleators(ECINs)immobilization.The main results are as following:(1)A new two-step route for the preparation of nano-scaled magnetic chitosan particles has been developed,different from reported one-step in situ preparation and two-step preparation method of reversed-phase suspension,Triton X-100 reversed-phase water-in-oil microemulsion encapsulation method was employed in coating the pre-prepared Fe3O4 nanoparticles with chitosan.The resultant magnetic chitosan particles owned a narrow size distribution ranging from 50 nm to 92 nm.X-ray diffraction patterns(XRD)indicated that the chitosan coating procedure did not change the spinal structure of Fe3O4 magnetic nanoparticles.The results of Fourier transform infrared(FTIR)analysis and thermogravimetric analysis(TGA)demonstrated that the chitosan was coated on Fe3O4 nanoparticles and its average mass content was～50%.The saturated magnetization of the magnetic Fe3O4/chitosan nanoparticles reached 18.62 emu/g,meanwhile,the nanoparticles showed the characteristics of superparamagnetism.The magnetic chitosan nanoparticles showed a high recoverability of 99.99%in 10 min when pH exceeded 4.The results suggested that the as-prepared magnetic chitosan particles were nano-scaled with a narrow size distribution and a high recoverability.(2)In vitro evaluation of hemolysis,cytotoxicity and cellular uptake on bare and chitosan coated magnetic nanoparticles were achieved.Gastric digestion of magnetic chitosan nanoparticles was evaluated at the beginning,and chitosan coated mangetic nanoparticles(MCNs,Fe3O4CS NPs)could significantly increase digestion rate of Fe3O4 in gastric juice compare with bare magnetic nanoparticles(Fe3O4 NPs).The results of hemolysis indicated that there is no harmful effect of magnetic nanoparticles with or without chitosan on erythrocytes,but the hemolysis rate of Fe3O4CS NPs is slight higher than Fe3O4 NPs.Fe3O4CS NPs showed low cytotoxicity and good biocompatible in HepG2 and Caco2 cell lines.Fe3O4CS NPs have a higher cumulative fraction transported and apparent permeability coefficients in Caco-2 cell monolayers,but they still belonged to poor permeability,meanwhile,Fe3O4CS NPs with positive charge could help them have a higher cellular uptake amount in HepG2 cells,indicated chitosan coated magnetic nanoparticles could effectively overcome the cell membrane barrier and transferred into cell.Finnally,Caco2 cell monolayers were used to evaluate the intestinal recovery treated by Fe3O4CS NPs,and TEER values of Caco2 cell monolayers could complete recovery after a period of time,indicated Fe3O4CS NPs are no harm to Caco2 cell monolayers.Systematic in vitro safety evaluation results indicated that the magnetic chitosan nanoparticles are safety on in vitro evaluation level.(3)Glutaraldehyde cross-linked magnetic chitosan nanoparticles(GMCNs)were prepared through cross-linking modification of magnetic chitosan nanoparticles(MCNs)using glutaraldehyde as crosslinker,that not only exhibited excellent food dyes adsorption performance,but also showed low cytotoxicity.Adsorption characteristics of GMCNs in FD&C Blue 1 and D&C Yellow 5 aqueous solutions have been studied and results indicated that the adsorption capacities were affected by initial pH values,initial dye concentrations and temperatures.Food dyes adsorption followed with the pseudo-second-order reaction,and equilibrium experiments were well fitted the Langmuir isothem model.Maximum adsorption capacities of GMCNs displayed at pH 3.0 and at 25 ℃,being up to 475.61 and 292.07 mg/g,for FD&C Blue 1 and D&C Yellow 5 respectively.Thermodynamic results demonstrated that the adsorption processes were spontaneous and exothermic.Furthermore,it was found that the GMCNs can be regenerated and reused through dye desorption in alkaline solution.It is a safety,effective and economical way for Food dye adsorption by GMCNs.(4)A magnetically separable extracellular ice nucleators(ECINs)immobilized system was constructed,ECINs from Erwinia herbicola were covalently immobilized on magnetic Fe3O4/chitosan nanoparticles by coupling via glutaraldehyde(GA)or epichlorohydrin(ECH).The immobilization process parameters were optimized,and under optimized conditions,ice nucleation activity(INA)of GA and ECH immobilized ECINs reached at 1.56 × 106 Units/mg and 1.62 × 106 Units/mg,respectively.The stability,reusability and cytotoxicity of GA and ECH immobilized ECINs were characterized and compared;both GA and ECH immobilized ECINs demonstrated good reusability performance after fifteen freeze-thaw cycles and low cytotoxicity,while GA immobilized ECINs were recovered more easily and more stable at different pH compared to ECH immobilized ECINs..Application of the immobilized ECINs in freeze concentration was investigated,and an increase of ice nucleation temperature for water,seawater and apple juice(6.83 ℃,8.08 ℃ and 9.38 ℃)was achieved by using GA immobilized ECINs.The results collected so far show that there are extensively promising applications for GA immobilized ECINs in freeze concentration industry fields.