(275f) Cellular Microenvironment and Material Properties Regulate Extracellular Vesicle Endocytosis in Bone Marrow MSC

One of the major challenges in regenerative medicine remains, to date, the repair of critical size bone defects, particularly through underlying conditions, such as post-menopausal osteoporosis. Bone marrow derived mesenchymal stromal cells (MSC) have been used in a variety of clinical trials to promote bone regeneration, due to their elevated secretion of regenerative factors (growth factors, chemokines, extracellular vesicles (EVs)). EVs are nanosized membranous particles that mediate the complex transmission of molecular signals by delivering biomolecular cargo between cells. Their therapeutic potential lies in the ability to propagate signal transduction between cells and tissues independently of the presence of parent cells. Although several biomaterial scaffolds (hydrogels) have been proposed in order to prevent rapid EV clearance and promote retention of EVs at the lesion site, the regulation of EV uptake by the cellular microenvironment and the properties of biomaterials is not understood. In this study, we investigate the mechanisms of EV uptake (clathrin dependent pathways, membrane tension, raft mediated internalization) as a function of changes in the cellular microenvironment and material properties.

Specifically, we utilize Strain-Promoted Azide-Alkyne Click Chemistry reaction (SPAAC) to fabricate 2D hydrogels with excess DBCO groups. Subsequently, we metabolically glycoengineer MSCs and their secreted EVs through a ManNAz analog of ManNAc that replaces sialic acid membrane sugars with Azide groups, thus allowing to click EVs on DBCO-excess 2D hydrogels. Furthermore, the EVs are labeled with a DBCO-488 dye to allow fluorescent visualization during EV uptake. To overcome the challenge of the visualization of EVs under an epifluorescence microscope, due to their small size (~100nm), we use photo-expansion microcopy (photo-ExM), a method that physically enlarges our samples up to ~8x, thus allowing for high resolution images using conventional confocal microcopy (Fig. 1). By varying the %wt of our hydrogels we tested the effect of hydrogel stiffness on EV uptake and discovered that stiff substrates significantly facilitate the endocytosis of our labeled EVs (Fig. 2). Similar results were observed when our 2D hydrogels were functionalized with HAVDI, a N-Cadherin mimetic peptide, signifying that Cadherin interactions enhance endocytosis mechanisms. Next, we measured the cellular membrane tension utilizing Flipper-TR fluorescence tension probe and positively correlated cell membrane tension with EV uptake, utilizing cholesterol as a positive control and membrane stiffening agent. Finally, we investigated the signal transduction of endocytosis pathways and negatively correlated membrane stiffening with clathrin mediated EV uptake, through clathrin chemical inhibition (Pitstop).

Overall, we have utilized a 2D hydrogel scaffold that presents clickable EVs to investigate cell endocytosis mechanisms and uncovered that cellular microenvironment and biomaterial properties greatly influences EV uptake by MSCs. This information is pivotal to the efficacy of EV-based therapeutics, as EV intake can regulate MSC fate towards an osteogenic phenotype for in vivo bone regeneration.