Extracellular Vesicle Heterogeneity and its Translational Implications for Cardiac Repair

Abstract

Extracellular vesicles (EVs) are small vesicles naturally produced by cells to transfer bioactive content to other cells. This thesis addresses key challenges in translating EV therapeutics in the context of cardiac repair. This work encompasses multiple projects with a focus on enhancing their functional delivery, purification, preservation, and understanding of their functional heterogeneity. The study begins by demonstrating the potential of EV-liposome hybrid nanoparticles for delivering therapeutic cargo, showcasing their promise as future carriers for siRNA. The development of novel EV isolation methods is crucial for large-scale applications; a multimodal flow-through chromatography (MFC) method is introduced as a scalable alternative to conventional methods. Furthermore, preserving EV stability during concentration and storage is explored, resulting in optimized conditions that significantly boost EV recovery rates while maintaining functionality. The thesis also delves into the complexity of EV heterogeneity and its impact on their therapeutic potential. By purifying EV subpopulations using the combination of MFC and size-exclusion chromatography, differences in properties, proteomic composition, and functionality among subpopulations were unveiled, particularly in the context of cardiac regeneration. Asymmetric flow field flow fractionation is introduced as an alternative method for EV separation, offering higher yields and better preservation of EV function compared to MFC, with the added capability of subpopulation separation. Collectively, these findings provide valuable insights for advancing EV-based therapeutics. By addressing critical challenges in delivery, isolation, preservation, and understanding heterogeneity, this work contributes to the potential clinical translation of EVs. The study underscores the promise of EVs in regenerative medicine and drug delivery, offering tailored approaches to optimize their therapeutic potential. Advances in these areas will enhance the efficiency and reproducibility of EV treatments and ultimately accelerate the translation of EV-based therapeutics towards clinical application.