固体脂质纳米粒佐剂效应研究

Study on adjuvant effect of solid lipid nanoparticles

为了研究固体脂质纳米粒通过表面吸附抗原蛋白所发挥的免疫佐剂效应,试验用热融超声乳化法制备固体脂质纳米粒悬液(SLN1和SLN2),用激光纳米粒度仪对纳米粒进行了平均粒径(mean diameter,MD)、多分散性系数(polydispersity index,PDI)、表面电位(zeta potential,ZP)的检测。以BSA蛋白为标志抗原,将36只ICR雌性小鼠随机分为6组,即BSA溶液组(500μg/mL BSA溶液1 mL)、BSA-SLN1组(BSA-SLN1950μL+10 mg/mL BSA 50μL)、BSA-SLN2组(BSA-SLN2950μL+10 mg/mL BSA 50μL)、BSA-CFA组(1 mg/mL BSA溶液500μL+CFA 500μL)组、空白SLN组(不加BSA的SLN悬液1 mL)和生理盐水阴性对照组(灭菌生理盐水1 mL),各组小鼠通过腿部肌肉注射相应溶液100μL免疫,免疫后2,4,6周采血,采用间接ELISA法检测血清抗体效价。结果表明:制得的固体脂质纳米粒悬液外观呈乳白色、无味、无沉淀。光学显微镜观察大小比较均匀、分散性好。SLN1悬液的MD、PDI、ZP与SLN2悬液相比均差异显著(P<0.05)。BSA-SLN组抗体效价约为14 lb,显著高于BSA溶液组(10 lb,P<0.05),但显著低于BSA-CFA组(18 lb,P<0.05),而空白SLN组与阴性对照组检测结果都为阴性。第6周BSA溶液组抗体效价大幅降低,而BSA-SLN组与BSA-CFA组继续维持原有水平。比较不同粒径的纳米粒佐剂效应结果显示,SLN1[(237±2)nm]与SLN2[(95±5)nm]固体脂质纳米粒具有非常相似的佐剂作用,二者抗体效价无显著差异(P>0.05)。说明SLN能够通过吸附抗原蛋白显著地提高免疫反应,具有良好的佐剂作用。

In order to study the immune adjuvant effect of solid lipid nanoparticles through surface adsorption of antigen proteins,the experiment was conducted to prepare solid lipid nanoparticle suspensions(SLN1 and SLN2)by hot-melt phacoemulsification,and the mean diameter(MD),polydispersity index(PDI),and zeta potential(ZP)of the nanoparticles were measured with a laser nanoparticle sizer.Using BSA protein as the marker antigen,36 ICR female mice were randomly divided into 6 groups,namely BSA solution group(500μg/mL BSA solution 1 mL),BSA-SLN1 group(BSA-SLN1950μL+10 mg/mL BSA 50μL),BSA-SLN2 group(BSA-SLN2950μL+10 mg/mL BSA 50μL),BSA-CFA group(1 mg/mL BSA solution 500μL+CFA 500μL),blank SLN group(SLN suspension without BSA 1 mL)and normal saline negative control group(sterilized normal saline 1 mL).Mice in each group were immunized by injecting 100μL of the corresponding solution into the leg muscles;blood was collected at 2,4,and 6 weeks after immunization,and the serum antibody titer was detected by indirect ELISA.The results showed that the prepared solid lipid nanoparticle suspension was milky white in appearance,tasteless,and had no precipitation.Observed by the optical microscope,the size was relatively uniform and the dispersion was good.Compared with the SLN2 suspension,the MD,PDI and ZP of the SLN1 suspension were significantly different(P<0.05).The antibody titer of the BSA-SLN group was about 14 lb,which was significantly higher than the BSA solution group(10 lb,P<0.05),but significantly lower than the BSA-CFA group(18 lb,P<0.05),while the blank SLN group had the same test results as the negative control group,which were negative.In the 6 th week,the antibody titer of the BSA solution group was greatly reduced,while the BSA-SLN group and BSA-CFA group continued to maintain the original level.Comparing the adjuvant effects of nanoparticles of different particle sizes,the results showed that SLN1([237±2]nm)and SLN2([95±5]nm)of solid lipid nanoparticles had very similar adjuvant effects,and there was no significant difference in antibody titer between SLN1 and SLN2(P>0.05).The results suggested that SLN can significantly improve the immune response by adsorbing antigen proteins,and has a good adjuvant effect.