Dielectric Imaging of Fixed HeLa Cells by In-Liquid Scanning Dielectric Force Volume Microscopy

Mapping the dielectric properties of cells with nanoscale spatial resolution can be an important tool in nanomedicine and nanotoxicity analysis, which can complement structural and mechanical nanoscale measurements. Recently we have shown that dielectric constant maps can be obtained on dried fixed cells in air environment by means of scanning dielectric force volume microscopy. Here, we demonstrate that such measurements can also be performed in the much morechallenging case of fixed cells in liquid environment. Performing the measurements in liquid media contributes to preserve better the structure of the fixed cells, while also enabling accessing the local dielectric properties under fully hydrated conditions. The results shown in this work pave the way to address the nanoscale dielectric imaging of living cells, for which still further developments are required, as discussed here

Towards Cellular Ultrastructural Characterization in Organ-on-a-Chip by Transmission Electron Microscopy

Organ-on-a-chip technology is a 3D cell culture breakthrough of the last decade. This rapidly developing field of bioengineering intertwined with microfluidics provides new insights into disease development and preclinical drug screening. So far, optical and fluorescence microscopy are the most widely used methods to monitor and extract information from these models. Meanwhile transmission electron microscopy (TEM), despite its wide use for the characterization of nanomaterials and biological samples, remains unexplored in this area. In our work we propose a TEM sample preparation method, that allows to process a microfluidic chip without its prior deconstruction, into TEM-compatible specimens. We demonstrated preparation of tumor blood vessel-on-a-chip model and consecutive steps to preserve the endothelial cells lining microfluidic channel, for the chip’s further transformation into ultrathin sections. This approach allowed us to obtain cross-sections of the microchannel with cells cultured inside, and to observe cell adaptation to the channel geometry, as well as the characteristic for endothelial cells tight junctions. The proposed sample preparation method facilitates the electron microscopy ultrastructural characterization of biological samples cultured in organ-on-a-chip device

Fast Label-Free Nanoscale Composition Mapping of Eukaryotic Cells Via Scanning Dielectric Force Volume Microscopy and Machine Learning

Mapping the biochemical composition of eukaryotic cells without the use of exogenous labels is a long-sought objective in cell biology. Recently, it has been shown that composition maps on dry single bacterial cells with nanoscale spatial resolution can be inferred from quantitative nanoscale dielectric constant maps obtained with the scanning dielectric microscope. Here, it is shown that this approach can also be applied to the much more challenging case of fixed and dry eukaryotic cells, which are highly heterogeneous and show micrometric topographic variations. More importantly, it is demonstrated that the main bottleneck of the technique (the long computation times required to extract the nanoscale dielectric constant maps) can be shortcut by using supervised neural networks, decreasing them from weeks to seconds in a wokstation computer. This easy-to-use data-driven approach opens the door for in situ and on-the-fly label free nanoscale composition mapping of eukaryotic cells with scanning dielectric microscopy

Real-time ratiometric imaging of micelles assembly state in a microfluidic cancer-on-a-chip

The performance of supramolecular nanocarriers as drug delivery systems depends on their stability in the complex and dynamic biological media. After administration, nanocarriers are challenged by physiological barriers such as shear stress and proteins present in blood, endothelial wall, extracellular matrix, and eventually cancer cell membrane. While early disassembly will result in a premature drug release, extreme stability of the nanocarriers can lead to poor drug release and low efficiency. Therefore, comprehensive understanding of the stability and assembly state of supramolecular carriers in each stage of delivery is the key factor for the rational design of these systems. One of the main challenges is that current 2D in vitro models do not provide exhaustive information, as they fail to recapitulate the 3D tumor microenvironment. This deficiency in the 2D model complexity is the main reason for the differences observed in vivo when testing the performance of supramolecular nanocarriers. Herein, we present a real-time monitoring study of self-assembled micelles stability and extravasation, combining spectral confocal microscopy and a microfluidic cancer-on-a-chip. The combination of advanced imaging and a reliable 3D model allows tracking of micelle disassembly by following the spectral properties of the amphiphiles in space and time during the crucial steps of drug delivery. The spectrally active micelles were introduced under flow and their position and conformation continuously followed by spectral imaging during the crossing of barriers, revealing the interplay between carrier structure, micellar stability, and extravasation. Integrating the ability of the micelles to change their fluorescent properties when disassembled, spectral confocal imaging and 3D microfluidic tumor blood vessel-on-a-chip resulted in the establishment of a robust testing platform suitable for real-time imaging and evaluation of supramolecular drug delivery carrier's stability

Formulation and screening of drug nanocarriers using microfluidic technology

[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

Formulation of tunable size PLGA-PEG nanoparticles for drug delivery using microfluidic technology

Amphiphilic block co-polymer nanoparticles are interesting candidates for drug delivery as a result of their unique properties such as the size, modularity, biocompatibility and drug loading capacity. They can be rapidly formulated in a nanoprecipitation process based on self-assembly, resulting in kinetically locked nanostructures. The control over this step allows us to obtain nanoparticles with tailor-made properties without modification of the co-polymer building blocks. Furthermore, a reproducible and controlled formulation supports better predictability of a batch effectiveness in preclinical tests. Herein, we compared the formulation of PLGA-PEG nanoparticles using the typical manual bulk mixing and a microfluidic chip-assisted nanoprecipitation. The particle size tunability and controllability in a hydrodynamic flow focusing device was demonstrated to be greater than in the manual dropwise addition method. We also analyzed particle size and encapsulation of fluorescent compounds, using the common bulk analysis and advanced microscopy techniques: Transmission Electron Microscopy and Total Internal Reflection Microscopy, to reveal the heterogeneities occurred in the formulated nanoparticles. Finally, we performed in vitro evaluation of obtained NPs using MCF-7 cell line. Our results show how the microfluidic formulation improves the fine control over the resulting nanoparticles, without compromising any appealing property of PLGA nanoparticle. The combination of microfluidic formulation with advanced analysis methods, looking at the single particle level, can improve the understanding of the NP properties, heterogeneities and performance

Towards Cellular Ultrastructural Characterization in Organ-on-a-Chip by Transmission Electron Microscopy

Organ-on-a-chip technology is a 3D cell culture breakthrough of the last decade. This rapidly developing field of bioengineering intertwined with microfluidics provides new insights into disease development and preclinical drug screening. So far, optical and fluorescence microscopy are the most widely used methods to monitor and extract information from these models. Meanwhile transmission electron microscopy (TEM), despite its wide use for the characterization of nanomaterials and biological samples, remains unexplored in this area. In our work we propose a TEM sample preparation method, that allows to process a microfluidic chip without its prior deconstruction, into TEM-compatible specimens. We demonstrated preparation of tumor blood vessel-on-a-chip model and consecutive steps to preserve the endothelial cells lining microfluidic channel, for the chip’s further transformation into ultrathin sections. This approach allowed us to obtain cross-sections of the microchannel with cells cultured inside, and to observe cell adaptation to the channel geometry, as well as the characteristic for endothelial cells tight junctions. The proposed sample preparation method facilitates the electron microscopy ultrastructural characterization of biological samples cultured in organ-on-a-chip device

Towards Cellular Ultrastructural Characterization in Organ-on-a-Chip by Transmission Electron Microscopy

Organ-on-a-chip technology is a 3D cell culture breakthrough of the last decade. This rapidly developing field of bioengineering intertwined with microfluidics provides new insights into disease development and preclinical drug screening. So far, optical and fluorescence microscopy are the most widely used methods to monitor and extract information from these models. Meanwhile transmission electron microscopy (TEM), despite its wide use for the characterization of nanomaterials and biological samples, remains unexplored in this area. In our work we propose a TEM sample preparation method, that allows to process a microfluidic chip without its prior deconstruction, into TEM-compatible specimens. We demonstrated preparation of tumor blood vessel-on-a-chip model and consecutive steps to preserve the endothelial cells lining microfluidic channel, for the chip’s further transformation into ultrathin sections. This approach allowed us to obtain cross-sections of the microchannel with cells cultured inside, and to observe cell adaptation to the channel geometry, as well as the characteristic for endothelial cells tight junctions. The proposed sample preparation method facilitates the electron microscopy ultrastructural characterization of biological samples cultured in organ-on-a-chip device

Data underlying the publication: Formulation of tunable size PLGA-PEG nanoparticles for drug delivery using microfluidic technology

Raw data associated to the article Formulation of tunable size PLGA-PEG nanoparticles for drug delivery using microfluidic technology. The work aims at the use of microfluidic to formulate PLGA particles in a more controlled and automated manner. The data includes the characterisation of nanoparticles formulated in bulk and with the microfluidic chip using DLA, TEM and TIRF microscopy