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WP7: Magnetic manipulation of cellular signalling in organotypic brain slices and in vivo models of Parkinson’s disease

Parkinson’s Disease is characterised by the loss of dopaminergic neurons in the brain. Current strategies of treating Parkinson’s Disease include transplantation of dopaminergic neurons into the brain. However, one of the major challenges in the transplantation field is the ability to control the outgrowth and integration of dopaminergic cells in their target area of the brain.


In this work package, we have developed a model of Parkinson’s Disease using rat brain slices grown in a dish. The brain is cut into slices using a fast vibrating blade and these brain slices are then placed on special membranes that allow access to nutrients from specially formulated media below the slice and oxygen from above. These slices remain healthy for several weeks but show degeneration of dopaminergic neurons, the principal hallmark of Parkinson’s Disease.


The aims of WP7 are to be able to control the differentiation and outgrowth of transplanted cells in slices using MNPs and magnetic fields. The slice model enables us to apply the magnetic nanoparticle (MNP) “temp” mode and “space” mode strategies developed in single cells to a model system that retains the structures and connections of the brain, thus mimicking the whole brain more closely. This will provide for a more informed translation to in vivo models of Parkinson’s Disease that seek to improve the efficacy of cell transplantation therapy in Parkinson’s Disease.

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:

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WP4: Tools for manipulation of functionalized magnetic nanoparticles in single-cell assays

 

wp4-img1In 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.

wp4-img2We 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  icone-video

wp4-img4In 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.

 

WP3: Design and Engineering of Semi-Synthetic Nanoparticles for Application Inside Neuronal Cells

wp3-imgMagnetic nanoparticles (MNP) suitable for biomedical applications require specifically designed physicochemical properties. Most importantly, biological compatibility represents the key prerequisite for production of safe nanobiomaterials and, thus for unbiased applications inside neuronal cells. Biochemical recognition elements at the nanoparticle surfaces enables site-specific functionalization with biologically active molecules. Furthermore, engineered membrane permeability facilitates nanoparticles delivery into the cytoplasm. Finally, MNPs are required to sensitively respond to magnetic field gradient to allow remote control of cellular functions inside patients. Fluorescent reporter for localizing MNPs inside cells are ideal for systematically optimizing intracellular properties and magnetic responses.

The combination of all these features in one object that is ~5000 times smaller than the diameter of a hair is the challenge of WP3. To this end, we are developing the production of semi-synthetic MNP based on combining engineered biomaterials. We utilize naturally occuring protein nanocages as a scaffold for synthesis of magnetic nanoparticles (MNPs) by mimicking a natural biochemical process in vitro. This approach yields MNPs with intrinsically biological identity. The green fluorescent protein (GFP), which is proven to be non-toxic, is fused to the surface of MNPs by genetic engineering. GFP is exploited as fluorescent reporter and for site-specific capturing of proteins to the MNP surface.
For this purpose, proteins to manipulate neuronal signalling are fused to a nanobody against GFP, which ensures fast and highly specific capturing to the GFP-coated MNPs. By engineering a viral protein transduction domain into the GFP, we furthermore aim to achieve efficient transfer across the cell outer membrane. Similar strategies are adapted to synthetic MNP with the aim to produce and to establish safe and potent magnetic nanobiomaterials for remote-controlled actuation of signalling pathways inside living cells.

WP5: Biomagnetic control of stem cell differentiation

Magnetic particles have been employed to study the way mechanical forces effect neuronal cells as they allow the application of defined levels of force, locating the particles to specific regions of the cell membrane using particle coatings. Using these techniques, it is possible to apply forces of physiological stresses without either detachment of the particles or cell membrane damage. We have screened mechanoreceptors and demonstrated activation of receptors which control stem cells to form mature neuronal tissues.  These strategies for combining magnetic fields and nanoparticles can be used for stimulating the differentiation of neurons and promoting neuronal fibre outgrowth to different regions in the brain.

Our aims within WP5 are to use novel cross-disciplinary methods by which specific mechano-receptors in the cell membrane can be tagged with magnetic particles for the purpose of controlling neuronal cell differentiation. Development of these strategies are very challenging but if successful will ultimately allow us to direct cell differentiation and proliferation using tissue engineering approaches and cell therapy treatments for Parkinson’s disease

WP5: Biomagnetic control of stem cell differentiation:

 

Magnetic particles have been employed to study the way mechanical forces effect neuronal cells as they allow the application of defined levels of force, locating the particles to specific regions of the cell membrane using particle coatings. Using these techniques, it is possible to apply forces of physiological stresses without either detachment of the particles or cell membrane damage. We have screened mechanoreceptors and demonstrated activation of receptors which control stem cells to form mature neuronal tissues.  These strategies for combining magnetic fields and nanoparticles can be used for stimulating the differentiation of neurons and promoting neuronal fibre outgrowth to different regions in the brain.

 

Our aims within WP5 are to use novel cross-disciplinary methods by which specific mechano-receptors in the cell membrane can be tagged with magnetic particles for the purpose of controlling neuronal cell differentiation. Development of these strategies are very challenging but if successful will ultimately allow us to direct cell differentiation and proliferation using tissue engineering approaches and cell therapy treatments for Parkinson’s disease