Abstract:
Essentially all bacteria secrete nano-sized (~20-200 nm) bacterial extracellular vesicles (bEVs) loaded with proteins, lipids, glycans, and nucleic acids. bEVs facilitate interactions among cells of the same species, different microbial species, and even with cells of multicellular organisms in the context of colonization or infection. Their interactions with host organism immune cell receptors vary depending on the producing bacterial species and are now being harnessed for the development of bEVs as a potential immunotherapeutic platform. Both basic/mechanistic and preclinical therapeutic development studies are thus increasing in number and require implementation of methods for multiparametric analytical characterization as well as in vivo administration in preclinical animal models of disease. We summarize a variety of analytical methods that can be used to calculate bEV dose for preparations made from diverse bacterial sources (including sterility testing, total protein concentration, particle concentration, and lipopolysaccharide concentration). We also describe basic methodology for intravenous administration of bEV preparations via tail vein injection in laboratory mice. Throughout the description of methodology, we highlight potential pitfalls and alternatives to further equip the reader for troubleshooting should challenges arise. Robust and reproducible characterization is a prerequisite of bEV preparation quality control and consistent dosing during preclinical development. This will allow for more streamlined testing of candidate therapeutic bEVs within a given research laboratory, and furthermore facilitate reproducibility of findings across laboratories.
摘要:
基本上所有细菌都分泌纳米大小(〜20-200nm)的细菌胞外囊泡(bEV),脂质,聚糖,和核酸。BEV促进同一物种细胞之间的相互作用,不同的微生物种类,甚至在定殖或感染的情况下,多细胞生物的细胞也是如此。它们与宿主生物体免疫细胞受体的相互作用取决于产生的细菌种类,现在正被用于开发bEV作为潜在的免疫治疗平台。因此,基础/机理和临床前治疗开发研究的数量都在增加,并且需要在疾病的临床前动物模型中实施多参数分析表征以及体内给药的方法。我们总结了各种分析方法,可用于计算从不同细菌来源制成的制剂的bEV剂量(包括无菌测试,总蛋白质浓度,颗粒浓度,和脂多糖浓度)。我们还描述了在实验室小鼠中通过尾静脉注射静脉内施用bEV制剂的基本方法。在整个方法论的描述中,我们强调潜在的陷阱和替代方案,以便在出现挑战时进一步装备读者进行故障排除。稳健和可重复的表征是临床前开发期间bEV制剂质量控制和一致给药的先决条件。这将允许在给定的研究实验室内对候选治疗bEV进行更简化的测试。并进一步促进跨实验室发现的可重复性。