Extracellular vesicles (EVs) are under evaluation as therapeutics or as vehicles for drug delivery. Preclinical studies of EVs often use mice or other animal models to assess efficacy and disposition. However, as most EVs under evaluation are derived from human cells, they may elicit immune responses which may contribute to toxicities or enhanced EV clearance. Furthermore, EVs from different cell sources or EVs comprising various cargo may differ with respect to immunogenicity or toxicity. To assess EV-induced immune response and toxicity, we dosed C57BL/6 mice with EVs intravenously and intraperitoneally for 3 weeks. EVs were harvested from wild type or engineered HEK293T cells which were modified to produce EVs loaded with miR-199a-3p and chimeric proteins. Blood was collected to assess hematology, blood chemistry, and immune markers. Spleen cells were immunophenotyped, and tissues were harvested for gross necropsy and histopathological examination. No signs of toxicity were observed, and minimal evidence of changes in immune markers were noted in mice dosed with engineered, but not with wild type EVs. This study provides a framework for assessment of immunogenicity and toxicity that will be required as EVs from varying cell sources are tested within numerous animal models and eventually in humans.

细胞外囊泡（EVs）正在作为治疗剂或药物输送的载体进行评估。电动汽车的临床前研究通常使用小鼠或其他动物模型来评估功效和处置。但是，由于大多数被评估的EV都源自人类细胞，因此它们可能会引发免疫反应，这可能会导致毒性或增强EV清除率。此外，来自不同细胞来源的电动汽车或包含各种货物的电动汽车的免疫原性或毒性可能有所不同。为了评估EV诱导的免疫反应和毒性，我们给C57BL / 6小鼠静脉内和腹膜内注射EV，持续3周。从野生型或工程改造的HEK293T细胞中收获EV，将其进行修饰以产生载有miR-199a-3p和嵌合蛋白的EV。收集血液以评估血液学，血液化学和免疫标记。对脾细胞进行免疫表型分析，并收集组织用于大体尸检和组织病理学检查。没有观察到毒性迹象，并且在用工程改造的小鼠但未用野生型EV的小鼠中观察到免疫标志物变化的最小证据。这项研究提供了评估免疫原性和毒性的框架，因为在许多动物模型中以及最终在人类中对来自不同细胞来源的电动汽车进行测试。