Preparation And Performance Study Of Carboxymethyl Cellulose-lysozyme Nanogel Loaded With Tea Polyphenols

Natural polymers are attracting more and more attention due to their good biocompatibility,biodegradability and the ability of their groups to be modified,in particular,they have become a research hotspot in recent years as a bioactive substance and transport carrier for anticancer drugs.Among them,the nano-compelx carrier systems based on the natural polysaccharide-protein interaction have many characteristics such as natural non-toxic,easy self-assembly,mild reaction conditions,no toxic cross-linking agent in the reaction process.Therefore,it has extensive application value in the food and health fields.Lysozyme(Ly)is a positively charged protein under neutral conditions,carboxymethyl cellulose(CMC)is a polysaccharide that is stable at pH 2.0-10.0 and has strong negative charges.Both of them can self-assemble to form nano-complex/nanogels by electrostatic interaction in this pH range.In our paper,Ly and CMC were used as research objects,and the interaction behavior and mechanism of the two were systematically investigated.The application prospects of tea polyphenols(TP)as a functional nutrient carrier were discussed.The main contents and results are list as follows:(1)The interaction between Ly and CMC was studied.The effects of the degree of substitution of CMC,the ratio of Ly to CMC,temperature,pH and salt on the complex formed by Ly and CMC were studied by measuring the turbidity,particle size,PDI andζ-potential of the nano-complex system.The results showed that when the degree of substitution of CMC was 0.7 and the ratio of Ly:CMC was 5:2.the particle size of the nano-complex was the smallest,which was 369.5 nm.Heating could cause the denaturation of Ly to expose its hydrophobic groups,accelerate the combination of polymers,and form a microgel structure.Therefore,after heating 30 min at 80℃,the particle size of Ly-CMC nanogels(NGs)was smaller than that of Ly-CMC nano-complex(COAs),and all the ratios were less than 400 nm.At the ratio of 5:2,NGs particle size and PDI value were the smallest at 261.1 nm and 0.25,respectively.The change of ζ-potential was not obvious after heating,but the turbidity increased slightly.By adding urea and NaCl,SDS in salt interference test,it was found that urea had no effect on turbidity of NGs and COAs,SDS could clarify both solutions,and NaCl could only clarify COAs solution.It was indicated that the main force of maintaining COAs is electrostatic interaction,while the action of maintaining NGs may have both electrostatic interaction and hydrophobic interaction at the same time.Under the condition of adjusting pH value of pH 2.0-10.0,the results showed that the size of NGs obtained under the condition of pH 2.0 was the smallest,and the particle size of NGs was as low at 108 nm.By adding a certain concentration of NaCl to the prepared NGs,it was found that the salt ion shielded the charge and weakened the electrostatic interaction between the Ly-CMC,which was not conducive to the formation of NGs.(2)The physicochemical properties of Ly-CMC nanogels and its loading effect on functional nutrient TP.The morphology and assembly mechanism of nanogels were characterized by SEM,fluorescence spectrum and UV spectrophotometer.The results showed that the nanogels prepared under the optimal parameters were regular sphericity.In the presence of chitosan,the nanogels were agglomerated and dissociated and lose their 3D structure.The results of endogenous fluorescence and ultraviolet spectrometric analysis corresponded to the previous turbidity results.The results showed that the hydrophobic groups of the nanocomposites are exposed due to Ly denaturation after heating,and the interaction force of the nanogels is in addition to the electrostatic interaction.Hydrophobic interaction is also produced,which makes the structure of nanogels more stable.The stability,entrapment efficiency,drug loading,in vitro sustained release of Ly-CMC nanogels on TP and the scavenging ability of TP loaded nanogels on DPPH and ABTS free radicals were determined by spectrophotometry and HPLC.The results showed that:The encapsulation efficiency of TP was 89.05 13.14%,and the stability of TP was significantly improved by Ly-CMC nanogel.The in vitro sustained release time of TP was prolonged from 5 hours to 24 hours,and the scavenging rates of DPPH and ABTS free radicals were 76.5%and 86.1%,respectively.The results showed that TP-loaded nanogels have good drug release properties and free radical scavenging ability.(3)The effect of TP-loaded Ly-CMC nanogels on HepG2 cells.The cytotoxicity of TP loaded Ly-CMC nanogels to HepG2 cells was studied by MTT assay,DAPI and AO/EB staining and Annexin V-FITC/PI double staining.The results of MTT assay showed that free TP and TP-loaded Ly-CMC nanogels could significantly inhibit the growth of HepG2 cells in a time-and concentration-dependent manner,and TP inhibited the growth of HepG2 cells for a longer time.When the concentration of TP was 100 μg/mL,the survival rate of HepG2 cells treated with TP for 12 hours was less than 10%after 24 and 48 hours.The results of HepG2 cells stained by DAPI and AO/EB showed that compared with the normal control cells,the morphology of the treated cells changed significantly,and the shape became round,irregular and fragmented,and there was a gap between the cells.The characteristics of cell apoptosis,such as cell rupture and shrinkage,were significant.The results of Annexin V-FITC/PI double staining further confirmed that TP-loaded Ly-CMC nanogels could induce apoptosis of HepG2 cells more significantly than free TP.TP-loaded Ly-CMC nanogels could induce 37.62%early apoptosis and 6.89%late apoptosis of HepG2 cells.However,free TP could only induce early apoptosis in 15.61%and late apoptosis in 6.71%.Therefore,as the carrier of TP,Ly-CMC nanogels can prevent the degradation of TP,increase the content of TP in HepG2 cells,promote the apoptosis of HepG2 cells and enhance the anti-cancer activity of TP.