Fonctionnalisation de nanoparticules plasmoniques pour le ciblage de cellules du cancer du sein in vitro

Le cancer du sein représente un quart des cas de cancer diagnostiqués chez les femmes au Canada. De nos jours, le moyen le plus efficace de diagnostiquer le cancer du sein consiste à effectuer une biopsie de la zone suspecte chez le patient et à l’analyser en laboratoire. L’étalon d’or utilisé par les pathologistes pour déterminer si les tissus sont cancéreux ou non est la coloration par immunohistochimie (IHC). Cette méthode présente plusieurs inconvénients, tels que la dégradation possible des antigènes ciblés en raison de l’utilisation d’agents de fixation (Formaline, etc.) ou la difficulté à cibler plusieurs antigènes dans un même échantillon. Dans ce projet sera présenté un moyen de répondre à ces problèmes en utilisant des nanoparticules (NP) plasmoniques fonctionnalisées avec des anticorps (Ac) pour cibler spécifiquement des cellules du cancer du sein. Outre une grande stabilité qui permet d’éviter l’utilisation d’agents de fixation avant le marquage des cellules, les NPs biocompatibles ont des propriétés optiques particulières qui permettent de les visualiser et de les compter facilement. L’objectif final est de développer et appliquer un protocole robuste qui pourra être appliqué directement par les pathologistes au Canada et montrera des résultats capables de rivaliser avec l’IHC. La première partie du mémoire concerne la fonctionnalisation des NPs et leur caractérisation. Trois approches de fonctionnalisation ont été testées : EDC/(Sulfo-)NHS (N-3- (dimethylamino)propyl-N-ethylcarbodimide hydrochloride/ N - Hydroxy(sulfo)succinimide), NHS et Hydrazide. Ces noms font référence au groupe fonctionnel qui se lie à l’Ac. Dans le cas des approches utilisant des esters NHS, la liaison se fait aux amines primaires des Ac qui sont réparties dans toute leur structure. On parle donc de fonctionnalisation non orientée car la position du site de liaison sur les Ac est aléatoire et les domaines Fab peuvent ainsi être liées aux NPs. C’est pourquoi le protocole Hydrazide, spécifique au domaine Fc des Ac, présentait un intérêt particulier. Les trois procédures ont été adaptées pour des NPs d’or (AuNPs) sphérique de 100 nm de diamètre et celles-ci ont été caractérisées par des mesures des spectres d’absorbance, de la taille hydrodynamique, du potentiel zêta ainsi que des techniques d’imageries et de dosage à l’aide d’Ac conjugués à des fluorophores. Tous ces résultats ont mené à la conclusion que le protocole EDC/NHS effectué à pH 7 était le plus robuste et assurait la plus grande couverture en Ac sur les NPs. Une fois la fonctionnalisation maîtrisée, la seconde partie de ce mémoire présentera comment utiliser ces NPs pour évaluer quantitativement l’expression de certains antigènes (CD44, HER2 et CD24) sur trois lignées cellulaires de cancer du sein (MDA-MB-231, MDA-MB-453 et BT-474). La méthode développée dans ce projet de maîtrise présente la nouveauté d’incuber les cellules en suspension et non plus en pétri comme dans les modèles de marquage bidimensionnels classiques. Les résultats de l’immunomarquage avec les AuNPs (immunoplasmonique) sont directement comparés aux résultats d’immunofluorescence pour la quantification des antigènes. Une bonne corrélation a été trouvée entre les deux tests pour les MDA-MB-231 (tous antigènes confondus) et pour les MDA-MB-453 (excepté pour le CD24). Les BT-474 ont démontré des résultats incohérents avec l’immunofluorescence avec un faible nombre de nanoparticules par cellules pour tous les antigènes. Les expériences d’immunoplasmoniques ont également été réalisées en présence de sérum humain et les mêmes tendances ont été observées. Au vu de ces résultats, il est recommandé pour de futures études de se pencher sur l’optimisation des techniques de purification des AuNPs fonctionnalisées ainsi que sur le développement d’un contrôle qualité pour mesurer l’activité des anticorps. Aussi, en vue d’une standardisation des procédés, il faudrait augmenter le nombre de AuNPs par cellules pour réduire la variabilité de la mesure.----------Breast cancer accounts for 25% of all cancer cases diagnosed in women in Canada. Nowadays, the most effective way to diagnose breast cancer is to perform a biopsy of the suspect area in the patient and analyze it in laboratory. The gold standard used by pathologists to determine whether the tissue is cancerous or not is immunohistochemical staining (IHC). This method has several disadvantages, such as the possible degradation of targeted antigens due to the use of fixating agents (Formalin, etc.) or the difficulty of targeting several antigens in the same sample. In this project will be presented a way to address these problems by using functionalized nanoparticles (NP) with antibodies (Ab) to specifically target breast cancer cells. In addition to high stability that avoids the use of fixating agents before labelling the cells, biocompatible NPs have special optical properties that make it easy to visualize and to count. The ultimate goal of this work is to develop and test a robust protocol that can be applied directly by pathologists in Canada and show results capable of competing with IHC. The first part of the thesis will be about the functionalization of NPs and their characterization. Three functionalization approaches were tested: EDC/(Sulfo-)NHS (N-3-(dimethylamino) propyl-N-ethylcarbodimide hydrochloride/N-Hydroxy(sulfo)succinimide), NHS and Hydrazide. These names refer to the functional group that binds to the Ab. In the case of approaches using NHS esters, the binding is to the primary amines of the Ab which are distributed throughout their structure. We therefore speak of non-oriented functionalization because the position of the binding site on the Ab is random and the Fab domains can thus be bound to NPs. This is why the Hydrazide protocol, specific to the Fc domain of Ab, was of particular interest. The three procedures were adapted for 100 nm diameter spherical gold nanoparticles (AuNPs) and these were characterized by measurements of absorbance spectra, hydrodynamic size, zeta potential, and imaging and assay using Ac conjugated to fluorophores. All these results led to the conclusion that the EDC/NHS protocol performed at pH 7 was the most robust and provided the greatest coverage of Ab on NPs. Once the functionalization is under control, the second part of this thesis will present how to use these NPs to quantitatively evaluate the expression of certain antigens (CD44, HER2 and CD24) on three breast cancer cell lines (MDA-MB-231, MDA- MB-453 and BT-474). The method developed in this master’s project presents the novelty of incubating suspended cells and no longer kneading them as in conventional two-dimensional marking models. The results of immunolabeling with AuNPs (immunoplasmonic) are directly compared with immunofluorescence results for the quantification of antigens. A good correlation was found between the two tests for MDA-MB-231 (all antigens combined) and for MDA-MB-453 (except for CD24). BT-474 demonstrated inconsistent results with immunofluorescence with a very low number of nanoparticles per cell for all antigens. Immunoplasmonic experiments were also performed in the presence of human serum and the same trends were observed. In view of these results, it is recommended for future studies to focus on the optimization of purification techniques for functionalized AuNP as well as the development of quality control to measure antibody activity. Also, in order to standardize processes, the number of AuNPs per cell should be increased to reduce the variability of the measurement

A platform for stop-flow gradient generation to investigate chemotaxis

The ability of artificial microswimmers to respond to external stimuli and the mechanistical details of their origins belong to the most disputed challenges in interdisciplinary science. Therein, the creation of chemical gradients is technically challenging, because they quickly level out due to diffusion. Inspired by pivotal stopped flow experiments in chemical kinetics, we show that microfluidics gradient generation combined with a pressure feedback loop for precisely controlling the stop of the flows, can enable us to study mechanistical details of chemotaxis of artificial Janus micromotors, based on a catalytic reaction. We find that these copper Janus particles display a chemotactic motion along the concentration gradient in both, positive and negative direction and we demonstrate the mechanical reaction of the particles to unbalanced drag forces, explaining this behaviour

Colloidal active matter mimics the behavior of biological microorganisms-an overview

This article provides a review of the recent development of biomimicking behaviors in active colloids. While the behavior of biological microswimmers is undoubtedly influenced by physics, it is frequently guided and manipulated by active sensing processes. Understanding the respective influences of the surrounding environment can help to engineering the desired response also in artificial swimmers. More often than not, the achievement of biomimicking behavior requires the understanding of both biological and artificial microswimmers swimming mechanisms and the parameters inducing mechanosensory responses. The comparison of both classes of microswimmers provides with analogies in their dependence on fuels, interaction with boundaries and stimuli induced motion, or taxis

Fluorescent Multifunctional Organic Nanoparticles for Drug Delivery and Bioimaging: A Tutorial Review

Fluorescent organic nanoparticles (FONs) are a large family of nanostructures constituted by organic components that emit light in different spectral regions upon excitation, due to the presence of organic fluorophores. FONs are of great interest for numerous biological and medical applications, due to their high tunability in terms of composition, morphology, surface functionalization, and optical properties. Multifunctional FONs combine several functionalities in a single nanostructure (emission of light, carriers for drug-delivery, functionalization with targeting ligands, etc.), opening the possibility of using the same nanoparticle for diagnosis and therapy. The preparation, characterization, and application of these multifunctional FONs require a multidisciplinary approach. In this review, we present FONs following a tutorial approach, with the aim of providing a general overview of the different aspects of the design, preparation, and characterization of FONs. The review encompasses the most common FONs developed to date, the description of the most important features of fluorophores that determine the optical properties of FONs, an overview of the preparation methods and of the optical characterization techniques, and the description of the theoretical approaches that are currently adopted for modeling FONs. The last part of the review is devoted to a non-exhaustive selection of some recent biomedical applications of FONs.This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowka-Curie grant agreement No. 101007804 (Micro4Nano). M.K., N.V., and I.R. acknowledge financial support from the Spanish Ministry of Science and Innovation through the Severo Ochoa Program for Centers of Excellence in R&D (SEV-2015-0496 and CEX2019-000917-S) and through the grant “MOL4BIO” (PID2019-105622RB-I00). K.D.B acknowledges support from the US National Science Foundation (EFMA-2203704) and the National Institutes of Health (R21AA028340).With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).Peer reviewe

A platform for stop flow gradient generation to investigate chemotaxis

The ability of artificial microswimmers to respond to external stimuli and the mechanistical details of their origins belong to the most disputed challenges in interdisciplinary science. Therein, the creation of chemical gradients is technically challenging, because they quickly level out due to diffusion. Inspired by pivotal stopped flow experiments in chemical kinetics, we show that microfluidics gradient generation combined with a pressure feedback loop for precisely controlling the stop of the flows, can enable us to study mechanistical details of chemotaxis of artificial Janus micromotors, based on a catalytic reaction. We find that these copper Janus particles display a chemotactic motion along the concentration gradient in both, positive and negative direction and we demonstrate the mechanical reaction of the particles to small forces deviations, explaining this behaviour

A Platform for Stop-Flow Gradient Generation to Investigate Chemotaxis

The ability of artificial microswimmers to respond to external stimuli and the mechanistical details of their origins belong to the most disputed challenges in interdisciplinary science. Therein, the creation of chemical gradients is technically challenging, because they quickly level out due to diffusion. Inspired by pivotal stopped flow experiments in chemical kinetics, we show that microfluidics gradient generation combined with a pressure feedback loop for precisely controlling the stop of the flows, can enable us to study mechanistical details of chemotaxis of artificial Janus micromotors, based on a catalytic reaction. We find that these copper Janus particles display a chemotactic motion along the concentration gradient in both, positive and negative direction and we demonstrate the mechanical reaction of the particles to unbalanced drag forces, explaining this behaviour

Entwicklung einer Plattform zur Generierung von Stop-Flow- Gradienten zur Untersuchung von Chemotaxis

Die Fähigkeit künstlicher Mikroschwimmer, auf äußere Reize zu reagieren und deren mechanistische Ursprünge, gehören zu den umstrittensten Fragen der interdisziplinären Wissenschaft. Die Erzeugung chemischer Gradienten ist dabei eine technische Herausforderung, da sie aufgrund von Diffusion schnell abflachen. Inspiriert von ‘Stop-flow’ Experimenten aus der chemischen Kinetik zeigen wir, dass die Erzeugung eines mikrofluidischen Gradienten durch Kombination mit einer Druckrückkopplungsschleife zur präzisen Kontrolle des Stoppens erfolgen kann. Das ermöglicht es uns, die mechanistischen Details der Chemotaxis von künstlichen katalytischen Janus-Mikromotoren zu untersuchen. Wir stellen fest, dass diese Kupfer-Janus-Partikel eine chemotaktische Bewegung entlang des Konzentrationsgradienten sowohl in positiver als auch in negativer Richtung zeigen, und wir demonstrieren die mechanische Reaktion der Partikel auf unausgewogene Widerstandskräfte, die dieses Verhalten erklären

Effect of Viscosity on Microswimmers: A Comparative Study

Although many biological fluids like blood and mucus exhibit high viscosities, there are still many open questions concerning the swimming behavior of microswimmers in highly viscous media, limiting research to idealized laboratory conditions instead of application-oriented scenarios. Here, we analyze the effect of viscosity on the swimming speed and motion pattern of four kinds of microswimmers of different sizes which move by contrasting propulsion mechanisms: two biological swimmers (bovine sperm cells and Bacillus subtilis bacteria) which move by different bending patterns of their flagella and two artificial swimmers with catalytic propulsion mechanisms (alginate microtubes and Janus Pt@SiO2 spherical microparticles). Experiments consider two different media (glycerol and methylcellulose) with increasing viscosity, but also the impact of surface tension, catalyst activity and diffusion coefficients are discussed and evaluated