可诱导双重免疫应答的硬乳液剂型构建及机制研究

The dual mucosal and systemic immune responses is of great significance to the effective prevention of mucosal infection disease. However, the existing vaccine formulations and administration methods can only induce one type of immune response and result in ineffective preventive effect. In order to solve above problems and induce the dual immune responses, the present study aims to develop potential injectable formulation based on solid emulsion. The specific strategy is to fabricate solid emulsion formulation containing all-trans retinoic acid (ATRA), which is prepared by polylactic acid and squalene. It is reported that ATRA can induce the homing of lymphocyte to intestinal mucosa. However, due to its strong hydrophobicity, unstable and narrow range for safe use, it is hard to inject ATRA in clinical application by using the traditional delivery system. This study proposes the solid formulation with the polymer shell and the internal oil phase to load ATRA. On the one hand, the solid emulsion formulation solves the delivery difficulties of ATRA with high loading efficiency and slow release profiles. On the other hand, solid emulsion combines the advantages of oil emulsion and particles as vaccine adjuvant in the humoral and cellular immune regulation. With this formulation as injection vaccine adjuvant, enhancement of mucosal and systematical immune response can be achieved with more effective, safe and comprehensive preventive effect against the infectious diseases of gastrointestinal mucous membranes.

诱导黏膜及系统双重免疫应答对有效预防经黏膜感染类疾病具有重要意义。但现有疫苗制剂及免疫方式只能诱导单方面应答，导致免疫效果不佳。针对上述问题，本研究拟采用生物工程的剂型化设计，构建可通过注射诱导双重免疫应答的硬乳液剂型加以解决。具体策略为构建载全反式维甲酸（ATRA）的硬乳液剂型（采用具有良好生物相容性的聚乳酸类聚合物材料及角鲨烯制备）。ATRA可诱导淋巴细胞肠黏膜归巢，但由于其强疏水性、不稳定及安全有效范围窄等给药难点，传统的给药剂型难以实现它的临床注射。本研究提出开发聚合物壳层、内部为油相的硬乳液剂型对其负载。一方面，硬乳液剂型可以解决全反式维甲酸的用药难点，实现其高效负载、缓控释放。另一方面，硬乳液剂型结合了油乳佐剂及颗粒佐剂分别在体液及细胞免疫调节方面的优势，可望具有黏膜及系统双重免疫增强特性，进而实现对经胃肠道感染疾病更为安全、高效、全面的免疫防护。

针对现有疫苗制剂及免疫方式难以同时诱导黏膜及系统双重免疫应答，导致对经黏膜感染类疾病的预防效果不佳这一难题，本研究构建了可通过注射诱导双重免疫应答的硬乳液剂型加以解决。硬乳液为核壳结构，内部为包埋全反式维甲酸（ATRA）的角鲨烯乳滴，外部为正电荷的聚合物壳层。将所制备的硬乳液制剂用于OVA、EV71手足口疫苗，通过细胞实验与动物实验，发现硬乳液组可以有效激活体液及细胞免疫应答，并在肠黏膜淋巴组织中有效激活树突状细胞、抗原特异性杀伤T细胞以及抗原特异性黏膜抗体IgA响应，可以同时激活系统性和肠黏膜免疫应答。此外，还从抗原储库、细胞摄取、细胞活化等多个方面对硬乳液的免疫作用机制进行了表征分析。本项目共发表SCI论文10篇，申请4项发明专利，获4项发明专利授权（含1项日本发明专利），培养博士生3名，硕士生2名。

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