UC Davis

Spinal cord injury (SCI) results in a wide range of clinical manifestations varying in severity from sensory deficits to irreversible paralysis, with approximately 12,500 new cases occurring every year in the US1. Recently, stem cell-based therapy, specifically with placenta-derived mesenchymal stem/stromal cells (PMSCs), has been suggested as a promising approach for the treatment of SCI, as PMSCs can secrete multifunctional therapeutic factors for remyelination, neuroprotection, and immunomodulation2–4. However, many studies have also shown poor PMSC survival and integration within the host after transplantation, suggesting that PMSCs exert their therapeutic functions mainly via a paracrine mechanism5,6. PMSCs secrete numerous factors that orchestrate key biological functions and cell behaviors including a significant number of extracellular vesicles (EVs)7. EVs are small membranous vesicles, ranging from 50-150nm in diameter, derived from the cell’s plasma membrane or endosomes and recognized to play an important role in long range cell-cell communication transporting various functional molecules, including proteins, lipids, microRNAs, and mRNAs8. Compared to cell-based therapy, EVs display long term storage stability, reduced immune rejection, and natural targeting surface markers that allow them to cross the blood brain barrier (BBB), a major obstacle for most cell-based and drug therapies for central nervous system (CNS) disease and injury9. Although EVs pose a promising alternative to cell-based therapy, targeted delivery in vivo – especially to the CNS - is lacking10,11. To correct for this limitation, researchers have modified their surface to endow them with active targeting molecules to enable specific cell uptake and tailor EV biodistribution12–16. A dominant paradigm has been to evaluate the EV surface functionalization using bulk analysis assays, such as western blotting and bead-based flow cytometry17,18. However, to our knowledge, there are no standardized practices to confirm the successful surface modification of EVs or calculate the degree of conjugation on EV surfaces (conjugation efficiency). Considering the many single-nanovesicular analysis technologies developed, the potential to inform the optimization of conjugation and to correlate the degree of surface conjugation required to see therapeutic effects in vivo is endless. Thus, the objectives of this dissertation were to 1) find the proper channels through which to confirm and calculate EV conjugation efficiency after surface modification, 2) use the identified methods to optimize conjugation of EVs for the improved targeting ability, and 3) test the optimized surface-modified EVs in relevant in vitro and ex vivo assays within the context of developing a targeted SCI therapy. First, we conducted a literature review to explore the bulk and single nanoparticle analysis techniques used to confirm that EVs or nanoparticles were successfully conjugated with a desired targeting molecule or fluorescent tag19. Then, we identified the reported conjugation efficiency calculation methods to figure out either 1) the % EV population modified with said molecules or 2) the estimated number of molecules modified on each EV. Then, we engineered the EV surface at the single-vesicle level. We applied orthogonal platforms with single vesicle resolution to determine and optimize the efficiency of conjugating the myelin-targeting aptamer LJM-3064 to single EVs (Apt-EVs). The aptamers were conjugated using either lipid insertion or covalent protein modification, followed by an assessment of single-EV integrity and stability. We observed unique aptamer conjugation to single EVs that depend on EV size. Finally, we tested the collective myelin-targeting, oligodendrocyte (OL) protection, and regenerative properties of the optimized Apt-EVs using relevant in vitro and ex vivo models. We used optimized lipid insertion-aided conjugation strategies to modify PMSC-EVs and observed 1) improved mature OL targeting, 2) improved rescue of Ols under oxidative stress, and 3) minimally altered intrinsic PMSC-EV functions (neuroprotection and immunomodulation) post-modification through various well-established in vitro assays. We also observed improved uptake in myelin-expressing oligodendrocytes compared to neurons in an ex vivo rat brain tissue model. This work study underscores the importance of use of single vesicle analysis technology for surface engineering EVs and provides a novel single-EV-based framework for informing optimized EV surface modification. This work is also essential in the study to effectively deliver therapeutic EVs to CNS injury sites to decrease off-target effects and increase treatment efficacy.