Emam Sherif E, Elsadek Nehal E, Takata Haruka, Ando Hidenori, Ishida Tatsuhiro
Department of Pharmacokinetics and Biopharmaceutics, Institute of Biomedical Sciences, Tokushima University, 1-78-1 Sho-machi, Tokushima 770-8505, Japan; Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Zagazig University, Zagazig 44519 Egypt.
Department of Pharmacokinetics and Biopharmaceutics, Institute of Biomedical Sciences, Tokushima University, 1-78-1 Sho-machi, Tokushima 770-8505, Japan; Department of Pharmaceutics, Faculty of Pharmacy, Sinai University, Kantara, Egypt.
J Pharm Sci. 2025 Aug;114(8):103882. doi: 10.1016/j.xphs.2025.103882. Epub 2025 Jun 17.
Extracellular vesicles are attracting attention as delivery vehicles for vaccines due to the inherent capacity of these cellular nanoparticles to preserve the structure and antigenicity of membrane antigens, which allows them to elicit immune responses. This intrinsic characteristic addresses the challenges associated with traditional antigen-containing formulations in maintaining antigen integrity while achieving high levels of loading efficiency. In this study, extracellular vesicles expressing antigens on their surface were utilized as potential antigen-delivery vehicles in a two-step intravenous (i.v.) immunization method, which amounts to the sequential injections of empty PEGylated liposomes (PEG-Lip) followed by PEGylated extracellular vesicles within a prescribed interval. To obtain SARS-CoV-2 spike protein-expressing extracellular vesicles (Exo-S1), a plasmid encoding SARS-CoV-2 Spike S1, primarily comprised of N-terminal domain (NTD) and receptor-binding domain (RBD), was transfected into murine melanoma cancer (B16BL6) cells. The Exo-S1 vesicles were PEGylated via the post-insertion method. Mice were immunized three times, with two-week intervals, with sequential injections of empty PEG-Lip followed by PEGylated Exo-S1 in 3-day intervals. Under our two-step immunization method, we observed the spleen accumulation of injected PEGylated extracellular vesicles, most likely in the marginal zone. After immunization, the mice showed high serum levels of IgG to whole spike S1 and spike RBD. The induced anti-SARS-CoV-2 spike IgG inhibited the interaction of spike RBD to angiotensin-converting enzyme 2 (ACE2), which controls viral infection. PEGylated antigen-expressing extracellular vesicles achieved efficient antigen delivery and effective immune responses under our two-step i.v. immunization method, which could be adopted to prevent the spread of infectious diseases such as COVID-19.
细胞外囊泡作为疫苗的递送载体正受到关注，因为这些细胞纳米颗粒具有保持膜抗原结构和抗原性的内在能力，这使其能够引发免疫反应。这一固有特性解决了传统含抗原制剂在维持抗原完整性同时实现高负载效率方面所面临的挑战。在本研究中，表面表达抗原的细胞外囊泡被用作潜在的抗原递送载体，采用两步静脉注射免疫方法，即在规定间隔内依次注射空的聚乙二醇化脂质体（PEG-Lip），随后注射聚乙二醇化细胞外囊泡。为了获得表达严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白的细胞外囊泡（Exo-S1），将编码主要由N端结构域（NTD）和受体结合结构域（RBD）组成的SARS-CoV-2刺突S1的质粒转染到小鼠黑色素瘤癌（B16BL6）细胞中。Exo-S1囊泡通过后插入法进行聚乙二醇化修饰。小鼠每隔两周免疫三次，依次注射空的PEG-Lip，随后每隔3天注射聚乙二醇化的Exo-S1。在我们的两步免疫方法下，我们观察到注射的聚乙二醇化细胞外囊泡在脾脏中积累，最有可能在边缘区。免疫后，小鼠血清中针对全长刺突S1和刺突RBD的IgG水平较高。诱导产生的抗SARS-CoV-2刺突IgG抑制了刺突RBD与控制病毒感染的血管紧张素转换酶2（ACE2）的相互作用。在我们的两步静脉注射免疫方法下，聚乙二醇化表达抗原的细胞外囊泡实现了高效的抗原递送和有效的免疫反应，这一方法可用于预防如2019冠状病毒病等传染病的传播。
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