地榆皂苷脂质纳米疫苗制备及其诱导特异性免疫应答的作用

Preparation and immunological evaluation of Ziyudisaponin-based lipid nanoparticles for enhancing antigen-specific immune responses

摘要:

目的 制备一种负载模型抗原鸡卵清白蛋白（OVA）的地榆皂苷（Ziyu）脂质纳米疫苗（PZONs），评价 其理化特性、体外免疫活性及诱导特异性免疫应答的能力，并探讨其作为抗肿瘤纳米疫苗的潜力。方法 以Ziyu、 聚乳酸-羟基乙酸共聚物（PLGA）、卵磷脂和OVA为主要成分，二氯甲烷和注射用水为溶剂，采用双乳化-溶剂挥发 法制备PZONs。通过马尔文激光粒度仪测定其粒径、多分散指数和Zeta电位，透射电镜观察纳米疫苗的形貌，并考 察其在磷酸盐缓冲液和血清中的稳定性。以小鼠骨髓来源树突状细胞（BMDCs）为模型，利用流式细胞术和共聚焦 显微镜评价PZONs的细胞摄取效率与入胞机制，以及评估PZONs对BMDCs成熟及抗原交叉呈递的促进作用。建 立 C57BL/6 小鼠免疫模型，经皮下免疫后，采用酶联免疫吸附法检测血清中 OVA 特异性 IgG 及其亚类（IgG1、 IgG2a）的抗体滴度，综合评价PZONs诱导的系统性免疫应答类型。结果 成功制备出形态均一、呈球形或类球形 的 PZONs，平均粒径为 140 nm，多分散指数为 0.24，Zeta 电位为-11 mV。PZONs 在 PBS 和血清中稳定性良好。 PZONs 的细胞摄取效果显著，主要依赖于网格蛋白途径介导内吞入胞。与游离 OVA 相比，PZONs 能显著上调 BMDCs表面共刺激分子 CD40、CD80、MHC-II的表达，并促进抗原通过 MHC I类途径交叉呈递。同时，PZONs免 疫组小鼠血清中OVA特异性总IgG、IgG1和IgG2a抗体滴度均显著高于游离OVA组（P < 0.01），且IgG2a/IgG1比值 升高，表明免疫应答偏向Th1型。结论 PZONs能有效递送抗原并激活DCs，诱导强烈的抗原特异性免疫应答，且 应答类型偏向Th1型，展现出良好的抗肿瘤应用潜力。

Abstract:

Objective To develop a Ziyudisaponin-based lipid nanovaccine (PZONs) loaded with the model antigen ovalbumin (OVA), and to evaluate its physicochemical properties, in vitro immunostimulatory activity , in vivo ability to induce specific immune responses , and its potential as an anti-tumor vaccine platform. Methods PZONs were prepared using a double emulsion-solvent evaporation method with Ziyudisaponin, OVA, lecithin and PLGA. The morphology of the nanovaccine was characterized by transmission electron microscopy (TEM), while particle size, polydispersity index (PDI), and zeta potential were determined using a Malvern Zetasizer. The stability was evaluated in phosphate-buffered saline (PBS) and serum. Bone marrow-derived dendritic cells (BMDCs) were used to assess cellular uptake, internalization mechanisms, maturation, and antigen cross-presentation via flow cytometry and confocal microscopy. Furthermore, C57BL/6 mice were immunized subcutaneously, and serum levels of OVA-specific IgG, IgG1 and IgG2a were measured by ELISA to evaluate the systemic immune response. Results PZONs were successfully prepared with uniform morphology, exhibiting spherical or sphere-like shapes, an average particle size of 140 nm, a polydispersity index of 0.24, and a zeta potential of −11 mV. The PZONs demonstrated good stability in both PBS and serum. PZONs exhibited significant cellular uptake, which was mainly mediated by clathrin-dependent endocytosis. Compared to free OVA, PZONs significantly up-regulated the expression of co-stimulatory molecules (CD40, CD80, and MHC-II) on the surface of BMDCs and enhanced antigen cross-presentation. Furthermore, PZONs elicited significantly higher titers of OVA-specific IgG, IgG1, and IgG2a, with an increased IgG2a/IgG1 ratio indicating a Th1-based immune response.Conclusion The PZONs platform successfully induce potent, Th1 biased antigen-specific immune responses, highlighting its significant potential for anti-tumor immunotherapy.