Tesis Doctorado
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Browsing Tesis Doctorado by Author "Contreras Moraga, Héctor Antonio"
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Item Optimización de una plataforma analítica para el aislamiento y carga de vesículas extracelulares pequeñas para el transporte de doxorrubicina.(Universidad de Concepción, 2023) Contreras Moraga, Héctor Antonio; Vergara Rosales, Carola Andrea; Vergara Rosales, Carola AndreaExtracellular vesicles (EVs) are small cellular structures that play a pivotal role in intercellular communication and exhibit significant potential as biomarkers and drug delivery systems. However, methods for their isolation and loading are not uniformly established. In this thesis, conditions were optimized to purify small EVs smaller than 200 nm, and load them with Doxorubicin. We employed a methodology based on ultrafiltration coupled with size-exclusion chromatography, and loading by electroporation, using a Design of Experiments (DoE) approach. The optimized method enabled the isolation of over 70% of EVs in the 30-150 nm size range, which were positive for exosomal markers. Furthermore, serum pools rich in small EVs exhibited low levels of Albumin and Apo-B100 contamination. During the loading process, the DoE revealed the significance of the initial drug concentration (DOX), demonstrating the presence of interactions among factors that modify its incorporation (DOXin). Additionally, it was demonstrated that EV aggregation is not solely dependent on the applied electric field but also on the pulse shape. Together, these results lay the foundation for future research endeavours aimed at achieving effective and efficient loading of molecules into EVs.