General Framework. As an alternative to polymers or hydrogels that are commonly used when soft substrates are needed for migration or uptake studies, we developed a new class of materials, oil-in-water emulsion droplets, which are deformable particles that can be functionalized with biological ligands (e.g. igGs or Fab’2) freely-diffusing at the interface. Having a stiffness comparable to those of cells, emulsions can be used as synthetic cell mimics to probe biological phenomena such as phagocytosis and to measure mechanical stresses produced by cells while migrating under confinement.
High-throughput Induction of immune cells Polarisation
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Droplets bumpers as mechanical sensors for cell migration under confinement. Mechanical cell forces play determinant roles in cell growth and tissue development. Cell migration is a relevant context to explore single cell forces, since mechanotransduction and cell morphology are dynamically and biochemically co-regulated in order to modulate cell shape changes minute to minute. In this work we present the design of a hybrid microdevice made from a set of parallel PDMS microchannels in which oil emulsion droplets are introduced to be used as biocompatible and deformable cell forces transducers upon HL-60 human leukemia cell line migration. We show that the mechanical stress exerted by the cells can be measured from the analysis of the droplets deformations at the single cell level. Finally we show that acto-myosin contraction plays a role in the cell ability to cross obstacles in confinement conditions.
Reference : D. Molino, S. Quignard, C. Gruget, F. Pincet, Y. Chen, M. Piel, J. Fattaccioli*
On-chip quantitative measurement of mechanical stresses during cell migration with emulsion droplets. Scientific Reports, Scientific Reports 6, 29113 (2016)[pdf][doi]
Nanometric emulsions encapsulating solid particles as alternative carriers for intracellular delivery. We developed a multi-functional platform based on oil-in-water emulsions. These nano-vehicles (360 nm) are composed of an edible oil core stabilised by a biocompatible surfactant and encapsulate hydrophobic-functionalised silica nanoparticles (60 nm). The concept is depicted below. The silica nanoparticles were rendered fluorescent by covalent grafting to use fluorescence microscopy to track the particles during cell studies. After characterisation, the particles were incubated with model epithelial cells HeLa to determine the effect of solid particles encapsulation within an oil droplet on their interaction with the cells, their internalisation pathway and subsequent intracellular fate as compared to free solid nanoparticles.
Reference : S. Quignard*, G. Frébourg, Y. Chen, J. Fattaccioli*
Nanometric emulsions encapsulating solid particles as alternative carriers for intracellular delivery
Nanomedicine, 11 (16), 2059-2072 (2016) [pdf][doi].
Phagocytosis of Emulsion Droplets. Phagocytosis by macrophages represents a fundamental process essential for both immunity and tissue homeostasis. The size of targets to be eliminated ranges from small particles as bacteria to large objects as cancerous or senescent cells. Most of our current quantitative knowledge on phagocytosis is based on the use of solid polymer microparticles as model targets that are well adapted to the study of phagocytosis mechanisms that do not involve any lateral mobility of the ligands, despite the relevance of this parameter in the immunological context. Herein we designed monodisperse, IgG-coated emulsion droplets that are efficiently and specifically internalized by macrophages through in-vitro FcγR-mediated phagocytosis. We show that, contrary to solid polymeric beads, droplet uptake is efficient even for low IgG densities, and is accompagnied by the clustering of the opsonins in the zone of contact with the macrophage during the adhesion step. Beyond the sole interest in the design of the material, our results suggest that lateral mobility of proteins at the interface of a target greatly enhances the phagocytic uptake.
Functionalization and Characterization of Emulsion Droplets. Biotechnological applications of emulsions, such as micro-reactors or drug carriers, demand accurate characterization techniques, able to measure the size and the biochemical content of the droplets at the individual level. Since no available characterization technique fulfilled completely these needs, we extended the use of flow cytometer, which was originally developed for cell studies, to the straightforward and quantitative characterization of micron-sized emulsions . We developped a self-consistent numerical method able to determine the size of microscopic oil droplets from flow cytometric measurements of forward scattering and side scattering intensities. This technique, associated to the fluorescence of each droplet measured at the same time by the instrument, allows characterizing the surface-density of proteins on the droplets as a function of their size at the droplet level with no influence of the emulsion polydispersity.
Emulsion Adhesion on Biomimetic Substrates. Following all the studies that have been made with liposomes on the biophysics of cell adhesion, we studied the adhesion phenomenon of streptavidin functionnalized emulsion droplets on glass substates covered by a mixture of poly-ethylene-glycol and biotin molecules. We first showed that non-specific interactions significantly enhance the proteins density within the contact zone between the droplets and the surface and then that the binding of the streptavidins is partially inhibited by the high protein density. Moreover, we confirmed that the density of specific bonds sets the adhesive energy and therefore the final contact angle of the droplets. Finally, we showed that specific binding in our system is always associated to the existence of a positive line tension, which linearly increases with the density of receptors. Although emulsion droplets are different from lipidic bilayers and naturally from cells, their high level of control allows using them as a new model surface for the study of the physics of cell adhesion.
Liquid Nanoparticles for Imaging. Compared to other colloidal particles, emulsion droplets have unique properties regarding the encapsulation and the transportation of nanoparticles or molecules. We have shown several years ago, in collaboration with Swapan Mandal, that we were able to design magnetic and fluorescent emulsion droplets, loaded by quantum dots and ferromagnetic nanoparticles, resulting in the creation of multimodal liquid nanoparticles.
Convinced that emulsions could constitute a good choice for an efficient drug carrier, and after the development of the functionnalization protocols leading to a control of the droplets adhesion on biomimetic substrates, we started the development of liquid nanoparticles for bioimaging, in collaboration with Isabelle Texier and Mathieu Goutayer (CEA-LETI, Grenoble, France). These nanoemulsions, functionnalized by biomolecules directed toward specific tumoral cells, were designed and tested in-vivo on mice to evaluate their biocompatibility, their dynamics of biodistribution and their lifetime.
Reference: M. Goutayer, I. Texier-Nogues, J. Fattaccioli and J. Bibette.
Émulsions fluorescentes pour l’imagerie optique. Patent Number : WO2008125747 (A2) [pdf]