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.