Preparation Of Chitosan Nanoparticle Loaded With Helicobacter Pylori Lpp20 And Primary Study Of Its Mucosal Vaccination In Mice

Objectives:(1) To construct chitosan nanoparticles containing the lipoprotein Lpp20 gene of Helicobacter pylori (H. pylori).(2) To observe the humoral and cellular immune responses in BALB/c mice induced by chitosan-pcDNA3.1(+)/Lpp20 nanoparticles(CS-pcD/Lpp20 NPs) following mucosal immunization, and to provide theoretical and experimental basis for H.pylori DNA vaccine using chitosan nanoparticles vector.Methods:The chitosan nanoparticles containing plasmid DNA encoding H.pylori lipoprotein Lpp20 gene were prepared using a complex coacervation method.The combination manner of CS-pcD/Lpp20 NPs was analyzed by gel retardation test,and the morphology and size were observed by transmission electronic microscopy.The diametric distribution and Zeta potential were determined using laser nanoparticle analyzer,and the efficiency of the encapsulation was measured with a spectrophotometer.The stability of CS-pcD/Lpp20 NPs was assessed by agarose gel electrophoresis analysis,and the effect of protection to plasmid DNA(pDNA) from DNase degradation was examined using DNase I.Then in vitro cytotoxicit of CS-pcD/Lpp20 NPs was evaluated by MTT assay. In order to observe the humoral and cellular immune responses of naked plasmid DNA and CS-pcD/Lpp20 NPs, we administered them to 6-week-old female BALB/c mice by intranasal or oral mucosal routes.The murine serum and intestinal mucus were collected at different time after immunization and the specific IgG and sIgA antibodies against Lpp20 were detected by ELISA.ELISA were also used for the quantitative determination of the concentrations of IFN-γand IL-4 in the supernatants of mice spleen lymphocyte culture medium after stimulating by Lpp20. The proliferation response of spleen cells was detected by MTT assay. H. pylori Lpp20 expression in nasal mucous tissue was detected by immunohistochemical method.Results:(1) The results of gel retardation showed chitosan could completely combine the Lpp20 by electrostactic interaction.The encapsulation rates for these NPs all exceeded 90%.(2) The morphology of the CS-pcD/Lpp20 NPs was most uniform, spherical and well distributed with a mean diameter of about l50-300nm. Its Zeta potential was about 13.4mV.(3) The DNase I protection test confirmed that pDNA could be protected from DNase I degradation. The stability test showed that at 4℃the recombinant Lpp20 plasmid could be parcelled well for 60 days.MTT assay proved that CS/DNA nanoparticles had very low cytotoxicity under high test concentration.(4) Naked plasmid pcDNA3.1(+)/Lpp20 and CS-pcD/Lpp20 NPs could induce effective immune response in mice through intranasal or oral vaccination.Specific IgG and sIgA antibodies of CS-pcD/Lpp20 NPs groups were higher than that of naked pcDNA3.1(+)/Lpp20 group. The concentration of cytokines IFN-γand IL-4 in cultural supernatant of T lymphocytes from CS-pcD/Lpp20 NPs immunized mice increased greatly than that of control groups(P<0.01).After stimulated by corresponding antigen, the stimulation index of intranasal or oral delivery of CS-pcD/Lpp20 NPs group were 1.466±0.29 and 1.169±0.17, respectively, which were higher than that of pcDNA3.1(+)/Lpp20 group(0.788±0.11),CS group(0.473±0.09) and PBS control group(0.442±0.12).Conclusions:(1) The chitosan nanoparticles containing the lipoprotein Lpp20 gene of H. pylori were constructed successfully.(2) Chitosan nanoparticles enhanced the immune response of pcDNA3.1(+)/Lpp20 DNA vaccine by intranasal or oral administration in BALB/c mice.(3) Compared to oral administration,intranasally delivery of chitosan- pcDNA3.1(+)/Lpp20 DNA nanoparticles could induce stronger cellular and humoral immune responses in BALB/c mice.