单磷酸脂质A(MPLA)pH敏感脂质体制备及免疫增强作用研究

Preparation and immune-enhancing effects of monophosphoryl lipid A pH-sensitive liposome

目的制备pH敏感脂质体,避免溶酶体对单磷酸脂质A(monophosphoryl lipid A,MPLA)的降解,提高MPLA的利用率。方法以DOPE、CHEMS为载体材料,采用薄膜分散法制备pH敏感脂质体。应用动态光散射仪测定脂质体的粒径及Zeta电位,透射电镜观察不同pH值条件下pH敏感脂质体的外观形态,应用流式细胞术评价THP-1、DC2.4细胞对脂质体的吞噬效果,激光共聚焦显微镜下观察脂质体与溶酶体的共定位情况。以乙型肝炎表面抗原(hepatitis B surface antigen,HBsAg)为模式抗原,配伍MPLA pH敏感脂质体免疫BALB/c小鼠,ELISA定量检测血清乙型肝炎表面抗体(anti-HBs)水平,ELISPOT检测小鼠脾淋巴细胞IFN-γ、IL-2斑点形成细胞数(spot forming cells,SFCs)。结果制备的pH敏感脂质体的平均粒径为(90.90±1.13)nm,多分散指数(polydispersity index,PDI)为0.076±0.013,Zeta电位为(-27.900±0.666)mV,随着溶液的pH值减小粒径明显增大,呈现不规则形态,具备显著的pH敏感性。THP-1细胞吞噬pH敏感荧光脂质体的吞噬率为10.40%,DC2.4细胞的吞噬率为12.40%,吞噬荧光脂质体的吞噬率分别为1.09%和0.28%。激光共聚焦显微镜观察到pH敏感荧光脂质体被THP-1细胞吞噬后,存在于细胞质中,而荧光脂质体存在于溶酶体中,MPLA pH敏感脂质体与MPLA脂质体比较可以显著提高小鼠细胞免疫应答水平,MPLA pH敏感脂质体组的IFN-γ和IL-2 SFCs水平均显著高于MPLA脂质体组(P<0.01)和无佐剂组(P<0.001)。结论pH敏感脂质体递送系统可以提高MPLA的利用率,使MPLA更好地发挥佐剂作用。

Objective To prepare pH-sensitive liposomes to avoid the degradation of monophosphoryl lipid A(MPLA)by lysosomes.Methods Using DOPE and CHEMS as carrier materials,pH-sensitive liposomes were prepared by thin-film dispersion method.Particle sizes and Zeta potential of the liposomes were detected by dynamic light scattering.The morphological features of pH-sensitive liposomes under different pH conditions were observed by transmission electron microscopy.Flow cytometry was performed to detect the phagocytosis of liposomes by THP-1 and DC2.4 cells.Confocal laser microscopy was used to observed the colocalization of liposomes and lysosomes.BALB/c mice were immunized with hepatitis B surface antigen(HBsAg)using MPLA pH-sensitive liposome as an adjuvant.The levels of serum anti-HBs were quantitatively detected by ELISA.IFN-γand IL-2 spot forming cells(SFCs)in mouse splenic lymphocytes were detected by ELISPOT.Results The pH-sensitive liposomes were constructed with an average particle size of(90.90±1.13)nm,polydispersity index(PDI)of 0.076±0.013 and Zeta potential of(-27.900±0.666)mV.As the pH value of the solution decreased,the particle size increased significantly and the liposomes presented irregular shapes,indicating the pH-sensitive features.The phagocytosis rates by THP-1 cells and DC2.4 cells were 10.40%and 12.40%for pH-sensitive fluorescent liposomes,and 1.09%and 0.28%for fluorescent liposomes.Confocal laser microscopy revealed that pH-sensitive fluorescent liposomes were phagocytosed by THP-1 cells and existed in the cytoplasm,while fluorescent liposomes existed in lysosomes.Compared with MPLA liposomes,MPLA pH-sensitive liposomes could significantly improve the cellular immune response in mice.The levels of IFN-γand IL-2 SFCs in the MPLA pH-sensitive liposome group were significantly higher than those in the MPLA liposome group(P<0.01)and the non-adjuvant group(P<0.001).Conclusions The pH-sensitive liposome delivery system could improve the utilization of MPLA as an adjuvant.