WP2: Preparation of synthetic and recombinant magnetic nanoparticles
In this work package, we are synthesizing different types of magnetic nanoparticles. Our aim is to provide particles that have the correct size for the studied mode of magnetic actuation (“temp mode” or “space mode”) but with strong magnetic properties, that appear due to the size of the particles.
These particles prepared for this project are all based on magnetic iron oxide, with adapted size, shape and surface coating and functionality. Indeed, these nanoparticles must move freely in their environment but have to recognize specific targets intracellularly or on the cell membrane.
For the “temp mode”, large nanoparticles of a few hundreds of nanometers can be used. They are synthesized by encapsulating clusters of iron oxide nanoparticles in a silica shell. For the “space mode”, it is necessary to have nanoparticles that can diffuse in the complex intracellular medium.
For such reason we developed magnetic nano-objects with different passivation strategies and different shapes, with a size always smaller than 70 nm, so they can diffuse freely inside the cell.
Finally, all the particles used in the project are fluorescent to be localized in cells, and colocalized with their targeted proteins. At this time, the particles developed for the project are:
WP4: Tools for manipulation of functionalized magnetic nanoparticles in single-cell assays
In this work package, we are working on single isolated cells as a model system to develop and optimize the “space” mode of magnetic actuation. We are using high magnification microscopes to follow in live the behaviour of nanoparticles in the cytoplasm of living cells. For the success of our project, we need first to understand how nanoparticles move inside cells. Indeed, the cell interior is very crowded by many molecules, and if our nanoparticles are not “inert” enough they will be stuck and not able to move under the application of a magnetic force. We recently developed an assay to measure the biocompatibility of nanoparticles based on the tracking of single nanoparticles. Our assay provides a way to benchmark the nanoparticles, and thus find the best candidate for further applications within the MAGNEURON project. We found that the nanoparticles need to be small enough to move within the maze of the intracellular cytoskeleton and need also to be well passivated to move freely without being stuck to other intracellular molecules.
We also working on the biofunctionalization of magnetic nanoparticles, to be able to control signalling activity inside cells. The behaviour of cells is indeed controlled by many molecules that forms cascades of chemical reactions, called signalling pathways. These signalling pathways can be viewed as the mean by which cells transduce information about the environment and about themselves. Our idea is to hijack these pathways using active molecules attached to the surface of magnetic nanoparticles that are able to activate signals within cells.
In order to assess the success of our tool, we are using fluorescent biomolecules as reporters of the signalling pathway activities. In the movie bellow, the first channel shows a zoom of a part of a cell that have been microinjected with magnetic nanoparticles. The green channel shows the attraction of magnetic nanoparticles toward the tip of the cell, thanks to a magnetic tip which is approached very close to the cell. The red channel reports for the recruitment on the nanoparticle surface of a biomolecule that have been expressed by the cell itself. Eventually, the cyan channel shows the local activation of the targeted signalling pathway, observed by the accumulation of a signalling protein.
HERE MOVIE SPACE MODE
In addition to working with single cells, we are also developing new magnetic devices to manipulate many cells at the same time. To attract magnetic nanoparticles, it is necessary to use high magnetic forces which cannot be obtained with a big magnet. Instead, we use many small magnets, almost of the same size of a cell. Each of these small magnets is then able to attract the magnetic nanoparticles inside cells that are close by. Such a device allows our observations and measurements to be repeated many times in a single shot, thus increasing greatly the throughput of our studies.