Optimización de una plataforma analítica para el aislamiento y carga de vesículas extracelulares pequeñas para el transporte de doxorrubicina.
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Date
2023
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Universidad de Concepción
Abstract
Las vesículas extracelulares (VEs) son pequeñas estructuras de origen celular. Participan en la comunicación intercelular y tienen gran un potencial como biomarcadores y para el transporte de moléculas. Sin embargo, los métodos de aislamiento y carga no están uniformemente establecidos. En esta tesis, se optimizaron las condiciones para purificar VEs pequeñas menores a 200 nm y cargarlas con Doxorrubicina. Se aplicó una metodología basada en ultrafiltración acoplada a cromatografía de exclusión por tamaño, y la carga por electroporación, mediante el Diseño de Experimentos (DoE). El método optimizado permitió aislar sobre un 70% de VEs entre 30-150 nm, siendo positivas para marcadores exosomales. Además, las fracciones de suero ricos en VEs exhibieron bajos niveles de Albúmina y Apo-B100. Durante la carga, el DoE reveló la importancia de la concentración inicial de la droga (DOX), demostrando la presencia de interacciones entre los factores que modifican su incorporación (DOXin). Además, se demostró que la agregación no sólo depende del campo eléctrico aplicado, sino también de la forma del pulso. En su conjunto, estos resultados sientan las bases de futuras investigaciones para lograr la carga efectiva y eficiente de moléculas en VEs.
Extracellular 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.
Extracellular 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.
Description
Tesis presentada para optar al grado de Doctor en Ciencias y Tecnología Analítica