(H-304) Stem Cell-Derived Theranostic Nanovesicles for Preventing Atherosclerosis

Introduction:: Atherosclerosis is a critical cause of mortality, but current diagnostic methods are not capable of detecting the early pathogenic signals associated with irreversible pathogenic cascades. One of the earliest atherogenic events is the generation of disturbed blood flow, which occurs at the branch points, curved regions, or distal to the stenosis, resulting in dysfunction of vascular endothelial cells (ECs). Human mesenchymal stem cells (MSC)-derived exosomes have emerged as therapeutic nanocarriers with a prolonged circulation time that promotes cellular interactions while retaining the cell-originated anti-inflammatory and pro-regenerative properties from MSCs, but poor lesion-targeting efficiency and low productivity have become major issues for clinical translation. To address these challenges, we developed exosome-mimetic nanovesicles derived from hMSC that are functionalized with GSPREYTSYMPH (PREY), a disturbed flow-targeting peptide that was previously identified from the in vivo phage display screening of a hundred million candidates. We demonstrate the effective anti-atherogenic effects of these disturbed flow-targeting therapeutic nanovesicles in mouse and porcine atherogenic models, as well as human microfluidic disturbed flow models.


Materials and Methods:: The plasmid used to express and localize PREY outward on the cell membrane consists of an outward N-terminal-promoter-signal peptide-PREY-V5-CD271-GFP-inward C-terminal structure. In the plasmid sequence, the signal peptide guided the localization of the PREY peptide outside the cell membrane, and the V5 tag and GFP were added to monitor the location and expression level of PREY. On day 3 post-transfection, the transfected cells were extruded six times with 10-µm, 5-µm, and 400-nm pore-sized polycarbonate membrane filters in sequence using an extruder kit, then the NVs were collected by centrifuging at 15 000 g for 30 min. The in vivo models used in this study include mouse and porcine partial carotid ligation (PCL) models, inducing acute disturbed flow and atherosclerosis under atherogenic diet. In addition, human artery endothelial cells were tested using microfluidic devices subjecting 10 dyne/cm2 of shear stress for normal flow, and pulsatile back-and-forth flow to mimic disturbed flow.


Results, Conclusions, and Discussions:: The nanovesicles were effectively produced from PREY-expressing human mesenchymal stem cells, designed to functionalize the cell membrane by displaying PREY on the outer surface of the nanovesicles. We confirmed the potent anti-inflammatory and pro-endothelial recovery effects in vitro using artery endothelial cells and monocytes, similar to those of the parent MSCs. In addition, employing mouse and porcine partial carotid artery ligation models as well as a microfluidic disturbed flow model with human carotid artery-derived endothelial cells confirmed the targeting and therapeutic effect on early-stage atherosclerosis in multiple species including humans, suggesting the potential for clinical translation. This nanovesicle platform is expected to contribute to the development of new theragnostic strategies for preventing the progression of atherosclerosis.


Acknowledgements (Optional): : This research was supported by the International Research & Development Program of the National Research Foundation of Korea(NRF) funded by the Ministry of Science and ICT(NRF-2021K1A3A1A74099704) and Korea government(MSIT)(RS-2023-00213691).

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