Visualization and Identification of Bioorthogonally Labeled Exosome Proteins Following Systemic Administration in Mice

Abstract

Exosomes transport biologically active cargo (e.g., proteins and microRNA) between cells, including many of the paracrine factors that mediate the beneficial effects associated with stem-cell therapy. Stem cell derived exosomes, in particular mesenchymal stem cells (MSCs), have been shown previously to largely replicate the therapeutic activity associated with the cells themselves, which suggests that exosomes may be a useful cell-free alternative for the treatment of cardiovascular disorders. However, the mechanisms that govern how exosomes home to damaged cells and tissues or the uptake and distribution of exosomal cargo are poorly characterized, because techniques for distinguishing between exosomal proteins and proteins in the targeted tissues are lacking. Here, we report the development of an in vivo model that enabled the visualization, tracking, and quantification of proteins from systemically administered MSC exosomes. The model uses bioorthogonal chemistry and cell-selective metabolic labeling to incorporate the non-canonical amino acid azidonorleucine (ANL) into the MSC proteome. ANL incorporation is facilitated via expression of a mutant (L274G) methionyl-tRNA-synthetase (MetRS∗) and subsequent incubation with ANL-supplemented media; after which ANL can be covalently linked to alkyne-conjugated reagents (e.g., dyes and resins) via click chemistry. Our results demonstrate that when the exosomes produced by ANL-treated, MetRS∗-expressing MSCs were systemically administered to mice, the ANL-labeled exosomal proteins could be accurately and reliably identified, isolated, and quantified from a variety of mouse organs, and that myocardial infarction (MI) both increased the abundance of exosomal proteins and redistributed a number of them from the membrane fraction of intact hearts to the cytosol of cells in infarcted hearts. Additionally, we found that Desmoglein-1c is enriched in MSC exosomes and taken up by ischemic myocardium. Collectively, our results indicate that this newly developed bioorthogonal system can provide crucial insights into exosome homing, as well as the uptake and biodistribution of exosomal proteins.

Nanoengineering facilitating the target mission: targeted extracellular vesicles delivery systems design

“…The advantage of using EVs of specific parental cell origin is that the secreted EVs carry some of the properties of the parental cells, however, the modification of the secreted EVs can alter or even lose these endogenous properties [145]. The use of click chemistry allows a selective, rapid and effective method that is independent of the origin of the EV cells and is suitable for clinical applications but may inactivate EV surface proteins [146]. The overall negative charge of EVs is critical for uptake by target cells, and targeting approaches developed based on multiple charge interactions can reduce non-specific uptake by receptor cells.…”

Exosomes: Potential Disease Biomarkers and New Therapeutic Targets

“…Conjugation reactions can covalently and stably modify the amine groups of exosome surface proteins with alkyne groups. They can now be conjugated with azide-containing motifs through "click chemistry" reactions [355]. This allows conjugation of small molecules and macromolecules that would act as ligands on target cells.…”

Reporter Systems for Assessments of Extracellular Vesicle Transfer

“…Azide–alkyne cycloaddition (click chemistry) is a powerful tool that permits covalent conjugation, thus tagging, of exosomes. Azide or alkyne group can be incorporated into EVs by supplementing azide or alkyne bearing amino acids (e.g., AHA) ( 50 , 51 ) or glycans/proteoglycans ( 52 ) to EV-producing cells, in vitro or in vivo , or by directly adding azide or alkyne bearing chemicals to the isolated EVs ( 53 ). The click reaction is catalyzed by Cu, but also can occur in Cu-free physiological fluids permitting in vivo labeling ( 54 , 55 ).…”

“…The ANL-labeled (i.e., MSC exosome-derived) proteins were isolated with click-catalyzed alkyne-agarose capture from various organs at different time points and identified with mass spectrometry; the MSC exosomal proteins were also localized histologically via fluorescent non-canonical amino-acid tagging (FUNCAT) in situ . We found that MSC exosomal proteins distributed in different organs are highly diverse, that ischemic injury significantly augments the tissue intake of exosomes, and that in the injured tissue, the exosomal proteins are predominantly associated with cytosol vs. membrane ( 51 ).…”