Formulation and screening of drug nanocarriers using microfluidic technology

Abstract

[eng] Two decades ago, microfluidic technology begun to make its appearance in the fields of drug delivery and biomedical engineering to irrevocably revolutionize them. It was quickly realized how microchannels can aid formulation of microdroplets, microparticles and nanoparticles (NPs). They offer very small and controlled environment for reaction, that is unreproduced in bulk methods. As a result, the formulation is not limited only to the modification of compounds, but the flowing microvolumes open gates to the unexplored world of controllable mixing time and diffusion region impacting the formation of nanoparticles. Beyond the drug delivery systems formulation, the microfluidic technology is emerging as a gap-bridging element of the in vitro and in vivo tests in preclinical trials. Biocompatible and microscopy- friendly microfluidic chips are used to reconstruct physiological elements of human tissues (organ-on-a- chip). They recapitulate 3D, dynamic in vivo environment, that is lacking in 2D cell culture, revealing their relevance in understanding the development of a disease and screening of drug delivery candidates. This work presents the use of microfluidic technology in the formulation of tunable size amphiphilic block co-polymer nanoparticles for drug delivery. The particle diameter is modified in the response to studied phase flow rates. The impact of fluidic parameters on drug/dyes encapsulation efficiency and NP size are analyzed using traditional bulk methods, as well as techniques with single particle resolution, such as Transmission Electron Microscopy (TEM) and Total Internal Reflection Fluorescence (TIRF). Furthermore, the NPs are bioevaluated with in vitro tests performed on MCF-7 cell line. Following the NPs formulation, a chip for combinatorial mixing of NP precursors is presented. A passive micromixer is designed, prototyped and evaluated with fluorescent dyes, to visualize the mixing efficiency. Finally, the model is microfabricated in glass and re-assessed in terms of mixing and cleaning efficiency, which previously was poor due to the absorption of small molecules by PDMS. The micromixer is built into a platform for NPs formulation and first proof-of-concept experiments are performed, yielding monodisperse nanoparticles with encapsulated fluorescent dyes. The encapsulation of dyes is visualized in single particles with TIRF microscopy. The last part of the thesis takes the microfluidic technology into organ-on-a-chip, where a reconstruction of tumor blood vessel model is presented. It recapitulates elements of tumor 3D microenvironment such as blood vessel, endothelial barrier, extracellular matrix and cancer cell spheroid. Observed in vivo leakiness of endothelial barrier is reproduced here in the presence of cancer cells. In this work the microscopy- friendly chip is used as a platform for time- and space-resolved monitoring of micelles stability followed during their interaction with the reconstructed barriers mentioned above. The special optical properties of

perfused micelles allow to distinguish assembled from disassembled form. The results are consulted with previously reported observations in 2D cell culture, revealing significant difference in cellular uptake between the two studies. Overall, this work demonstrates how multidisciplinary approach of incorporation of microfluidic technology into formulation and screening of potential drug nanocarriers can accelerate development of nanomedicine. The proposed solutions deliver tunability of nanoparticle properties, combinatorial formulation to create library of NPs and a complementary method in in vitro screening.

[spa] Hace dos décadas, la tecnología microfluídica hizo su aparición en los campos de la industria farmacéutica y la ingeniería biomédica de manera revolucionaria. Rápidamente se descubrió cómo los microcanales pueden ayudar a la formulación de microgotas, micropartículas y nanopartículas. Ofrecen entornos de reacción muy pequeños y controlados comparados con la formulación de los métodos tradicionales. En consecuencia, la formulación no sólo se limita a la modificación de compuestos, sino que los flujos de microvolúmenes posibles con la tecnología abren puertas a un mundo inexplorado para la formulación de nanopartículas a través del control del tiempo de mezcla y el área de difusión. Más allá de la formulación de los sistemas de fármacos, la tecnología microfluídica está emergiendo como un elemento puente de las pruebas in vitro e in vivo en los ensayos preclínicos. Los chips de microfluidica biocompatibles y aptos para microscopía se utilizan para reconstruir elementos fisiológicos de tejidos humanos (órgano en un chip). Recapitulan el entorno dinámico in vivo en 3D, carente en el cultivo celular en 2D, desvelando su relevancia para comprender el desarrollo de una enfermedad y la detección de fármacos candidatos para la administración.

Este trabajo presenta el uso de la tecnología microfluídica en la formulación de nanopartículas de copolímeros de bloques anfifílicos de tamaño ajustable en respuesta a los caudales de las fases estudiadas. Se estudia el impacto de los parámetros de flujo sobre la eficiencia de encapsulación de fármacos/colorantes y el tamaño de NP. Además, se presenta un chip para la formulación combinatoria de nanopartículas fluorescentes, con potenciales aplicaciones en medicina personalizada. La última parte de la tesis traslada la tecnología de microfluidos a órgano en un chip, donde se presenta la reconstrucción del modelo de vaso sanguíneo tumoral. Recapitula las fugas observadas in vivo de la barrera endotelial en presencia de células tumorales. En este trabajo, se utiliza como una plataforma para el monitorización en el tiempo y en el espacio de la estabilidad de las micelas, mientras interactúan con las barreras reconstruidas que se encuentran en el cuerpo humano: vasos sanguíneos, barrera endotelial, matriz extracelular y esferoide multicelular de células cancerosas.