Preparation And Immunological Effects Of Newcastle Disease DNA Vaccine Nanoparticles Using Chitosan As Carrier

Newcastle disease(ND)is a highly contagious,pathogenic disease caused by Newcastle disease virus(NDV),which causes great harm to animal husbandry.vaccines are usually used to prevent ND,such as attenuated vaccine and inactivated vaccine,but they have more or less shortcomings in practice.Attenuated vaccine may be atavistic because of mutation;while,inactivated ND vaccine has slow effect.It has been focused on applicating DNA vaccine to control ND.Although DNA vaccine is superior than traditional vaccines in some respects,but it is easy to degrade.nanopartciles mucosa immunity delivery system using biodegradable materials,which lowers degradation,enhances targeting,and decreases immunization.In this study,the research is focused on establishing a model with ND DNA vaccine pVAXl-optiF encapsulated into pDNA-CS-NPs and pDNA-CS-PLGA-NPs,which was prepared using complex coacervation method and coated method.The influential factor,preparation procedure,physicochemical characteris,drug stability,cell toxicity were evaluated.The results demonstrated that:1)ND DNA vaccine encapsulated in chitosan nanoparticles prepared by complex coacervation showed suitable size,morphous regulation and well-distributed observed by transmission electron microscopy.The average particle size of the pDNA-CS-NPs produced by the optimal formulation was 199.5nm with low polydispersity index 0.336 and proper Zata Potential +11.2mV.The entrapment efficiency was(98.59±0.03)%(n=5),in addition,the drug loading was(36.12 ± 0.19)%(n=5);2)pDNA-PLGA-NPs coated with chitosan by coated methond showed suitable size,morphous regulation,smooth surface,extremely spherical shape observed by transmission electron microscopy.The average particle size of the pDNA-CS-PLGA-NPs produced by the optimal formulation was 699,1nm with low polydispersity index 0.005 and proper Zata Potential +6.35mV.The entrapment efficiency was(98.12 ± 0.35)%(n=5);3)DNase I digestion test showed that pDNA-CS-NPs had the ability to protect ND DNA from DNase I degradation,plasmid DNA loaded in pDNA-CS-NPs not only retain its structure but also keep its biological activity;4)The release of both pDNA-CS-NPs and pDNA-CS-PLGA-NPs test in vitro demonstrated that the release of pDNA-CS-NPs was a continuous process,whereas the release of pDNA-CS-PLGA-NPs possesses preferable slow-release result,avoiding initial burst release phenomenon;5)The indirect immunofluorescence and Western blotting of both pDNA-CS-NPs and pDNA-CS-PLGA-NPs test in vitro test showed that the preparation method of both pDNA-CS-NPs and pDNA-CS-PLGA-NPs didn't destroy the biological activity of plasmid DNA,which had expressed in vitro;6)Both pDNA-CS-NPs and pDNA-CS-PLGA-NPs was low toxicity and excellent safety,which could be proved by a cytotoxicity test;7)The SPF chicken vaccination results illustrated that mucosal immunity by both pDNA-CS-NPs and pDNA-CS-PLGA-NPs used,the IgG and IgA contents of mucosal immunity response positions were higher and possessed better protection effect than intramuscular injection.The result proved that the chitosan-based nanodimensional mucosal delivery system not only induced immune responses in mucosal location but also may stimulus organism to produce strong humoral and cellular immune responses.ND DNA vaccine encapsulated in the chitosan-based nanodimensional imucosal delivery system had implemented a mechanism of the long-term release of the plasmid DNA successfully,which could decrease the expense of the vaccine production,vaccination times would be reduced and the vaccination methods would be increased.At the same time,the results also provided theoretical reference for the clinical application of nanodimensional mucosal delivery system.