Exosomes for Wound Treatment: Purification Optimization, Bioactive Components Identification and Drug Loading
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Hettich, Britta Franziska. "Exosomes for Wound Treatment: Purification Optimization, Bioactive Components Identification and Drug Loading." PhD diss., ETH Zurich, 2021.
The application of exosomes as therapeutic agents and drug delivery systems has gained increasing popularity over the last decades owing to their natural functions in intercellular communication processes. Exosomes are nanosized membrane vesicles of endosomal origin, which are constitutively released by cells into the extracellular space. They consist of functional proteins, nucleic acids and lipids, which enable them to imitate the biological functions of their producing parent cells. While proteins and nucleic acids have been identified as key players in the biological activity of exosomes, potential contributions of constitutional lipids to these effects remain largely unknown. Moreover, the purification process of exosomes continues to be a critical issue in exosome research since the definition of standardized exosome purification conditions is still pending. Several isolation methods are currently available, yet their potential impact on the exosome functionality has been rarely assessed in sufficient detail. Finally, when investigated as drug delivery platforms, mostly hydrophobic drugs have been loaded into exosomes, therefore leaving the loading capacity of current processes for hydrophilic classical drugs largely unaddressed. Furthermore, the impact of the loading methods on the exosome integrity and intrinsic bioactivity remains incompletely understood as the vesicle characterization is often restricted to analyzing their basic physicochemical properties and cellular uptake as well as monitoring the drug response. This thesis is aimed at addressing central questions related to the characterization of stem cell-derived exosomes as therapeutic entities and drug carriers, mainly in the context of wound healing. The major objectives specifically encompass 1) the optimization of exosome production and purification parameters, and an investigation of their effect on the exosome properties, 2) the identification of the role of selected exosomal components in processes required for wound healing, and 3) a comprehensive appraisal of the impact of several drug loading methods on the exosome integrity and functionality. Chapter 1 introduces the research field of exosomes and presents currently available purification methods. Moreover, the therapeutic applications of stem cell-derived exosomes are portrayed, focusing on their potential use in wound healing. Chapter 2 presents a synopsis of currently available synthetic carriers and exosomes as drug delivery platforms. In addition, drug encapsulation techniques for exosomes are presented and discussed. In Chapter 3, a standardized exosome preparation protocol is described. Special attention was paid to the interplay between production/purification conditions and exosome 2 characteristics to ultimately establish the optimal conditions delivering a high yield of bioactive exosomes. Subsequently, the activity of stem cell-derived exosomes in skin wound healing was assessed both in vitro and in vivo. The potential involvement of the transmembrane enzyme CD73 and exosomal lipids in the wound healing-promoting effects of stem cell exosomes was reported. It was found that the extent of the different exosome components’ activities was dependent on the target cell type. Specifically, CD73 contributed significantly to the in vitro migratory/mitogenic activity of stem cell exosomes on keratinocytes, but had no effect on endothelial cells. Exosomal lipids, on the other hand, were involved in the in vitro and in vivo activity of stem cell exosomes in blood vessel formation and maturation, but did not promote proliferation/migration of keratinocytes or fibroblasts in vitro. Chapter 4 explores processes for the encapsulation of non-membrane permeable hydrophilic low molecular weight compounds (i.e. pyranine and pentoxifylline) into exosomes. The loading efficiency of several methods was compared, and the osmotic shock procedure was identified as the most efficient one. Subsequently, the potential impact of the loading processes on the functionality of stem cell-derived exosomes was assessed using physicochemical characterization and biological activity methods. Only two out of five tested encapsulation processes (i.e. freeze-thawing and osmotic shock) preserved the structural and biological integrity of stem cell exosomes. In Chapter 5, the main findings of the current work are recapitulated and discussed. In addition, an outlook on yet unsolved challenges in the exosome research area is provided.View full article