Research On Chitosan Nanomedicine Targeting Mulberry Ralstonia Pseudosolanacearum

Ralstonia pseudosolanacearum is a bacterial soil-borne disease caused by R.pseudosolanacearum,which is difficult to control.Although traditional pesticides have a certain control effect,problems such as environmental pollution and increased drug resistance are gradually exposed.The previous research of our group found that caffeic acid phenethyl ester（CAPE）and other caffeic acid ester compounds,as a green natural antibacterial compound,have good antibacterial activity against R.pseudosolanacearum.Therefore,in this study,chitosan was used as the functional carrier,phenethyl caffeate was used as the pharmacodynamic molecule,and the anti-R.pseudosolanacearum monoclonal antibody was coupled to prepare a nano-drug against R.pseudosolanacearum to achieve the prevention and treatment of bacterial wilt.control.The main research contents are as follows:（1）Preparation of novel chitosan nanoparticles targeting R.pseudosolanacearum.Chitosan nanoparticles（CS-NPs）loaded with caffeic acid phenethyl ester were prepared by ion gel method using chitosan as functional carrier.The preparation process was optimized according to the encapsulation efficiency and drug loading rate,and the encapsulation efficiency and drug loading rate of CS-NPs were detected,and the sustained-release kinetics was analyzed by the Fickian model.The results showed that when the ratio of CAPE to methyl caffeate（MC）was 8:0,the encapsulation efficiency and drug loading efficiency of CS-NPs were 94.10%and 97.30%,respectively.The particles have no obvious burst release period in phosphate buffer at p H 6.5,7.4 and 9.5,and the theoretical half-life is 7.3-11.5 d,which is1.5-2.4 d longer than the nanoparticles prepared in the previous stage.On this basis,using EDC/NHS chemical coupling method,the monoclonal antibody of R.pseudosolanacearum was modified on the surface of chitosan nanoparticles to prepare chitosan-targeted nanoparticles（CS-TNPs）.It was verified by immuno-PCR and SEM that R.pseudosolanacearum could be specifically captured by antibodies.After CS-TNPs treatment,the binding and adsorption of drug particles could be observed on the surface of R.pseudosolanacearum.Therefore,this study successfully prepared a chitosan-targeted nanomedicine with specific binding ability to R.pseudosolanacearum.（2）Antibacterial properties of CAPE-loaded chitosan-targeted nanomedicines.The median inhibitory concentration and minimum inhibitory concentration（MIC）of CS-TNPs were determined by microplate reader.The results showed that when the concentration of CAPE was 4 mg/m L,the inhibition rate of CS-TNPs was 93.02%,and the EC50 value was1.94 times lower than that of the original drug.In addition,the fractional inhibitory concentration index of CS-TNPs was 0.375,indicating for the first time that chitosan and CAPE had a synergistic inhibitory effect on R.pseudosolanacearum.At the same dose,CS-TNPs significantly inhibited the biofilm formation of R.pseudosolanacearum,which was 5.3times and 4 times that of chitosan and CAPE alone,respectively,and the antibacterial diameter of the plate（13 mm）was 1.5 mm higher than that of R.pseudosolanacearum.drug molecules,confirmed to have a strong inhibitory effect on R.pseudosolanacearum.Scanning electron microscope results showed that the strains of R.pseudosolanacearum treated with CS-TNPs were more severely deformed and damaged,and obvious holes and depressions appeared on the cell membrane surface.The FTIR results of CS-TNPs showed that the main peaks of CAPE decreased at 3199 cm^-1,2883 cm^-1 and 1602 cm^-1,and chitosan was successfully loaded.The results of Zeta potential,TEM and particle size analysis showed that the CS-TNPs had an average particle size of 175.13 nm and a porous structure.After coupling with the antibody,the Zeta potential of the nanoparticles decreased from 49.5 m V to 2.71 m V,which proved that the antibody was successfully coupled.Therefore,this study verifies that CS-TNPs can destroy the surface structure of the cell membrane by specifically binding to R.pseudosolanacearum,and have stronger antibacterial activity.（3）Effects of chitosan-targeted nanoparticles on the generation of reactive oxygen species in R.pseudosolanacearum and analysis of the antibacterial mechanism.Production of reactive oxygen species and changes in pathogenicity-related gene expression in R.pseudosolanacearum.The results showed that CS-TNPs could disrupt bacterial biofilm by increasing the production of bacterial reactive oxygen species and significantly down-regulated the expression of the pathogenicity-related genes egl,phc A,hrp B,pil T,pol A and peh C of R.pseudosolanacearum,compared with the API group.Down-regulated by 1.33,2.11,1.30,1.53,1.61 and 1.32 times,respectively;the expression levels of R.pseudosolanacearum III secretion system-related effectors RS5-1997,RS5-2036,RS5-4055,RS5-4374 and RS5-4819 were compared with API,decreased by 1.22,1.43,1.43,1.28 and1.59 times respectively,all of which were significantly down-regulated.Therefore,CS-TNPs can reduce pathogenicity by enhancing bacterial oxidative stress and inhibiting R.pseudosolanacearum motility and environmental adaptation.The immune response of host plants and the proliferation and spread after colonization have led to a new understanding of the bacteriostatic process of CS-TNPs at the gene level.In summary,this research used cheap chitosan instead of PLGA as the drug carrier,and successfully developed chitosan nanoparticles with the targeting effect of R.pseudosolanacearum,which has potential application value in the prevention and control of bacterial wilt.