壳聚糖纳米粒在乙型肝炎疫苗中的应用

Application of chitosan nanoparticles as adjuvant of hepatitis B vaccine

目的比较包裹乙型肝炎(简称乙肝)的壳聚糖纳米粒与常规铝佐剂乙肝疫苗的免疫效果差异,并对壳聚糖佐剂的应用效果进行评价。方法采用离子交联法制备20、30及40μg/ml的包裹乙肝疫苗的壳聚糖纳米粒溶液,激光粒度分析仪测定纳米粒粒径和多分散系数,BCA法测定包封率;将BALB/c小鼠随机分为4组:铝佐剂对照组、空白纳米粒对照组、常规疫苗组和壳聚糖疫苗组,肌肉注射免疫小鼠,2μg/只,隔周免疫1次,共2次。采用全自动免疫发光分析仪对小鼠血清中乙肝表面抗体进行定量分析。结果制备的载乙肝疫苗壳聚糖纳米颗粒粒径为(262.7±10.8)nm,与空白壳聚糖纳米粒粒径相比,略有减小,多分散系数为(0.228±0.016);随着抗原浓度升高、4℃放置3个月及反复冻融,纳米粒的包封率均未发生显著变化,可达80%左右;壳聚糖疫苗组在小鼠体内产生的表面抗体滴度最高可达1∶80 000以上,并从第8周起,与常规疫苗组间差异有统计学意义(P<0.01)。结论壳聚糖纳米粒作为新型疫苗佐剂,具有一定的应用价值和进一步研究的意义。

Objective To compare the immune effects of chitosan nanoparticles loaded with bulk of hepatitis B（HB） vaccine and conventional HB vaccine containing alum adjuvant,and evaluate the application of chitosan as an adjuvant.Methods Nanoparticles loaded with bulk of HB vaccine at concentrations of 20,30 and 40 μg / ml were prepared by ionic cross-linking method,measured for size and polydispersity coefficient,and determined for loading efficiency by BCA method.BALB / c mice were randomly divided into four groups,and injected i.m.with alum adjuvant,empty nanoparticles,routine HB vaccine and chitosan adjuvant-containing HB vaccine respectively for 2 times at an interval of one week,2 μg for each,and determined for anti-HBs in sera by full-automatic immune luminescence analyzer.Results The sizes of prepared HB vaccine-loiaded chitosan nanoparticles were（262.7 ± 10.8） nm,which was slightly smaller than those of empty nanoparticles,while the polydispersity coefficient was（0.228 ± 0.016）.The loading efficiency was about 80%,which was not influenced significantly by increased antigen content,storage at 4℃ for 3 months and repeat freezing and thawing.The anti-HBs titers induced by the prepared chitosan nanoparticles reached more than 1︰80 000,which showed significast difference with that induced by routine HB vaccine starting from week 8（P 0.01）.Conclusion Chitosan nanoparticles is a promising adjuvant which is worthy of further research and application.