(F-224) Engineering Adiponectin-Loaded Microparticles to Stimulate Stem Cells to Secrete Exosomes & Subsequent Characterization for Treatment of Metabolic Disease

Introduction:: There is significant interest in the use of exosomes for cell-free treatment of disease because of the advantages over cell therapy. Exosomes have notable treatment potential for cell-free therapies due to their natural origin. Given different stimuli, exosome production may be facilitated. Metabolic diseases present with a wide diversity of overlapping symptoms. The majority of metabolic diseases present with inflammation making it extremely difficult to diagnose and control. Adiponectin is a well-known protein that is secreted by adipose tissue, which can regulate inflammatory responses in patients. Alginate is a highly studied biomaterial that provides a superb immobilization matrix for encapsulating cells and drugs within chemically crosslinked hydrogel microparticles. Small extracellular vehicles (ECVs) are a group of secretory vesicles that contain multiple intracellular signaling molecules, playing a role in reducing inflammation and restoring tissue homeostasis. It is hypothesized that due to adiponectin’s interaction with cells, encapsulating the protein in alginate beads will stimulate exosome production that can contain anti-inflammatory signals to be utilized for improving inflammatory conditions outcome.

Materials and Methods:: The goal of the present study is to determine the optimal procedure utilizing air extrusion methodology to encapsulate adiponectin in alginate microbeads crosslinking in 100 mM CaCl₂ solution and use it to stimulate and isolate exosomes from cells. Additionally, this study characterized extracellular vesicles secreted by adipose derived mesenchymal stem cells (Ad-MSCs) after prolonged exposure (3 days and 7 days) to adiponectin. This characterization comprised of three assays including quantifying protein content by bicinchoninic acid assay (BCA); identification of exosome biomarkers CD63, ALIX and Syntenin-1 by Western Blot; and nanoparticle tracking analysis (NTA) to analyze particle concentrations and size distribution.



Results, Conclusions, and Discussions:: This study was one of the first to encapsulate adiponectin utilizing air extrusion to engineer exosomes for potential therapeutic use. The experiments in this study illustrated that at a minimum, exosome secretion was equivalent for both Ad-MSCs and PSCs after exposure to adiponectin when compared to controls. Despite no differences in NTA analysis between control, adiponectin bolus, and adiponectin beads, detailed analysis of exosome contents may clarify differences of microRNAs, proteins and lipid packaging secondary to adiponectin exposure. Furthermore, as illustrated by NTA data, small extracellular vesicles were isolated in high proportions in both cell types, with Ad-MSCs providing a higher homogeneity in vesicle size. These results demonstrate that Ad-MSCs have a more robust exosome secretion compared to PSCs. Additionally, the low proportion of large vesicles support utilizing Ad-MSCs for future research. Furthermore, this cell type is readily available for isolation from adipose tissue biopsies with low-cost culture media. In contrast, PSCs need to be isolated from placental tissue which is limited by donation frequency and high-cost media required to grow these cells. Due to these results and the ease of harvesting Ad-MSCs with low-cost media, this provides an opportunity to select cell types to study engineering exosomes utilizing different signaling molecules to produce novel therapies utilizing exosomes. Encapsulated adiponectin can be used to stimulate exosomes to incorporate specific cargos (differential expression of proteins and microRNAs) for treatments that could improve defects in lipid metabolism, dysfunctional energy regulation, regulate overactive immune responses and provide protection of neurons and neural stem cells to prevent further damage within the CNS. By stimulating relevant cell types to encapsulated adiponectin, the isolation and characterization protocol from this study can provide the foundation for multiple potential studies for clinical application of the engineered exosomes. These findings support utilizing the engineered Ad-MSCs small ECV for the next step towards clinical work utilizing anti-inflammatory analyses prior to studying their effect upon metabolic diseases. This investigation has laid the foundation for stem cell selection and the optimization of sustained release adiponectin from 1.5% alginate beads to characterization of engineered exosomes for future applications.



Acknowledgements (Optional): : I could not have undertaken this journey without Dr. Opara. Many thanks to my committee members for their essential input, including Dr. Deep, Dr. Levi, and Dr. Kerr. I would like to recognize Dr. Alwan for his training and oversight into my research and Mitu Sharma for her essential work in providing the experiments to strengthen my thesis.


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