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Hijacking cell signalling pathways with magnetic nanoactuators for remote-controlled stem cell therapies of neurodegenerative disorders

Deliverables

MAGNEURON brochure

The MAGNEURON brochure will be a dissemination way with public information on the project activities, objectives and progress that will be distributed whenever possible. Quantity of printed brochures will be defined by the Dissemination Plan. The logo, the design and the graphical chart will be consistent with the Website and other communication supports (internal and external).

Dispersions of silica core shell MNPs

D2.2 is dedicated to the coating of maghemite nanoparticles synthesized in task 1 by a silica shell. If this synthesis pathway is well established for smaller particles, the protocol must be adapted to the big (12-14, up to 20 nm) ones that will be used in this project. Amines functional groups will be included on the surface of core-shell nanoparticles so as to be used by the other members of the consortium. Near infrared Fluorescent probes (Cyanine 5,5 for instance) will be included in the silica shell during synthesis for optical detection of nanoparticles in cells. The quantities deliverables will be in the magnitude of 20-50 mL at 0.1 M of iron content. Sample should be available in the first 6-8 months after the beginning of the project.

Activation of intracellular membrane-anchored small G-proteins by cytosolic MNPs

MNPs functionalized with activated Ras/Rac (or their GEF activators) will be delivered into the cytoplasm of cells transfected with biosensors (Raichu-Ras, Raichu-Rac). Using the spaceMode approach, we will test for asymmetrical activation of downstream cytoskeletal targets using confocal fluorescence microscopy. Nonfunctionalized MNP will serve as negative control.

Characterization of magnetic manipulation in cells

We will characterize the forces required to manipulate cells inside cells, the kinetics with which they can be displaced through the cells. These results will be analyzed as a function of the colloidal and biofunctional properties of the nanoparticles.

Dispersions of stabilized MNPs

This deliverable is synthesized big magnetic nanoparticles (from 12-14 and up to 20 nm in diameter) using an alternative Massart’s synthesis pathway. The necessary size sorting will be achieved in order to gain in monodispersity. For the full project, 200 mL at 1M of iron content is expected for each diameter selected. This will need several synthesis batches, as long as size sorting decreases strongly the quantity of expected nanoparticles. Concomitantly, different coatings will be applied to these nanoparticles. Citrate ligands, as well as poly(sodium acrylate) (PAA) and poly(sodium acrylate-co-sodium maleate) (PAAMA) polyelectrolytes will be used to stabilize these nanoparticles in biocompatible solvents. These coated nanoparticles will be used in task 2.2 and 2.3. Sample should be available in the first 4 months after the beginning of the project.

Identification and assessment of neuronal differentiation and axonal outgrowth

Cells will be identified using immunocytochemistry with the following specific antibodies: to assess neuronal differentiation - beta-III-tubulin, Map2ab; and midbrain dopaminergic neurons – tyrosine hydroxylase, vesicular monoamine transporter (VMAT), dopamine transporter, Aromatic L-amino acid decarboxylase (AADC), and Nurr1. Axonal outgrowth will be measured and quantified from digital images of beta-III-tubulin labeled cells.

Data Management Plan

Data Management Plan will be written on the base of the EC tool. All the non-sensitivity data will be stored in an open repository.

Dissemination Strategy plan

This document will report on the dissemination and communication strategies implemented by the MAGNEURON consortium, with regard to the described communication activities in DoA. A consistent graphical chart will be proposed for communication and dissemination supports. It shall be consistent with the IPR strategy plan.

Protocols for activation of neuronal differentiation through key receptor tags

Key receptors will be tested which can control neuronal cell differentiation e.g. TREK channels, Fz receptors. Magnetic particles will be coated with receptor tags and attached to receptors on the cell membrane. In response to oscillating fields, the cells will be induced to differentiate in vitro.

Recombinant ferritin-based MNPs

We will deliver a protocol describing (i) expression and purification of recombinant ferritin particles, (ii) loading with magnetic iron oxide core and (iii) biochemical functionalization of magneto ferritin particles.

Systematic analysis of the cell differentiation response to signalling perturbations

In vitro tests to define the differentiation responses of dopamine neurons in culture and in 3D systems including brain slices. Different regimes in 2D and 3D will be tested and optimized to promote neuronal regeneration.

Intermediate workshop

The MAGNEURON consortium will organise an intermediate workshop during the project to communicate the intermediate results and generate synergies with the scientific community.

Dissemination Strategy report 1

This document will report on the dissemination and communication activities carried out in the period from M1 to M12 by the MAGNEURON consortium, with regard to the described communication activities in DoA.

Final workshop

The final workshop will allow communicating the project results to a large scientific and industrial community.

Dissemination Strategy report 2

This document will report on the dissemination and communication activities carried out in the period from M13 to M30 by the MAGNEURON consortium, with regard to the described communication activities in DoA.

Final Dissemination Strategy report

This document will report on the dissemination and communication activities carried out in the period from M31 to M48 by the MAGNEURON consortium, with regard to the described communication activities in DoA.

Device for parallelized stimulation and high-throughput assay

We will develop a device for parallelized magnetic stimulation of multiple cells, based on microfabrication of micromagnets combined to micropatterning, enabling high-throughput assays. We will characterize the magnetic and optical performances of the devices.

MAGNEURON website

The MAGNEURON web site will provide public information about the project, its objectives, the partners, the advancements, and the organised workshop and provide an entry contact point with any industry or organism interested in the project results.

Consolidated Data Management Plan

Updated Data Management Plan will be consolidated at the end of the project.

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Publications

Rheb in neuronal degeneration, regeneration, and connectivity


Published in: ISSN 1431-6730
DOI: 10.1515/hsz-2016-0312

Non-specific interactions govern cytosolic diffusion of nanosized objects in mammalian cells

Author(s): Fred Etoc, Elie Balloul, Chiara Vicario, Davide Normanno, Domenik Liße, Assa Sittner, Jacob Piehler, Maxime Dahan, Mathieu Coppey
Published in: Nature Materials, Issue 17/8, 2018, Page(s) 740-746, ISSN 1476-1122
DOI: 10.1038/s41563-018-0120-7

Magnetic control of cellular processes using biofunctional nanoparticles

Author(s): Cornelia Monzel, Chiara Vicario, Jacob Piehler, Mathieu Coppey, Maxime Dahan
Published in: Chemical Science, Issue 8/11, 2017, Page(s) 7330-7338, ISSN 2041-6520
DOI: 10.1039/c7sc01462g

Engineered Ferritin for Magnetogenetic Manipulation of Proteins and Organelles Inside Living Cells

Author(s): Domenik Liße, Cornelia Monzel, Chiara Vicario, John Manzi, Isabelle Maurin, Mathieu Coppey, Jacob Piehler, Maxime Dahan
Published in: Advanced Materials, Issue 29/42, 2017, Page(s) 1700189, ISSN 0935-9648
DOI: 10.1002/adma.201700189

Lethal Factor Domain-Mediated Delivery of Nurr1 Transcription Factor Enhances Tyrosine Hydroxylase Activity and Protects from Neurotoxin-Induced Degeneration of Dopaminergic Cells

Author(s): Dennis Paliga, Fabian Raudzus, Stephen H. Leppla, Rolf Heumann, Sebastian Neumann
Published in: Molecular Neurobiology, Issue no. 12035, 2018, ISSN 0893-7648
DOI: 10.1007/s12035-018-1311-6

Perspectives of RAS and RHEB GTPase Signaling Pathways in Regenerating Brain Neurons

Author(s): Hendrik Schöneborn, Fabian Raudzus, Mathieu Coppey, Sebastian Neumann, Rolf Heumann
Published in: International Journal of Molecular Sciences, Issue 19/12, 2018, Page(s) 4052, ISSN 1422-0067
DOI: 10.3390/ijms19124052

Protection of Oligodendrocytes Through Neuronal Overexpression of the Small GTPase Ras in Hyperoxia-Induced Neonatal Brain Injury

Author(s): Meray Serdar, Josephine Herz, Karina Kempe, Elke Winterhager, Holger Jastrow, Rolf Heumann, Ursula Felderhoff-Müser, Ivo Bendix
Published in: Frontiers in Neurology, Issue 9, 2018, ISSN 1664-2295
DOI: 10.3389/fneur.2018.00175

Novel Tools towards Magnetic Guidance of Neurite Growth: (I) Guidance of Magnetic Nanoparticles into Neurite Extensions of Induced Human Neurons and In Vitro Functionalization with RAS Regulating Proteins

Author(s): Hendrik Schöneborn, Fabian Raudzus, Emilie Secret, Nils Otten, Aude Michel, Jérome Fresnais, Christine Ménager, Jean-Michel Siaugue, Holm Zaehres, Irmgard D. Dietzel, Rolf Heumann
Published in: Journal of Functional Biomaterials, Issue 10/3, 2019, Page(s) 32, ISSN 2079-4983
DOI: 10.3390/jfb10030032

Optical Magnetometry of Single Biocompatible Micromagnets for Quantitative Magnetogenetic and Magnetomechanical Assays

Author(s): Loïc Toraille, Koceila Aïzel, Élie Balloul, Chiara Vicario, Cornelia Monzel, Mathieu Coppey, Emilie Secret, Jean-Michel Siaugue, João Sampaio, Stanislas Rohart, Nicolas Vernier, Louise Bonnemay, Thierry Debuisschert, Loïc Rondin, Jean-François Roch, Maxime Dahan
Published in: Nano Letters, Issue 18/12, 2018, Page(s) 7635-7641, ISSN 1530-6984
DOI: 10.1021/acs.nanolett.8b03222

Zwitterionic polymer ligands: an ideal surface coating to totally suppress protein-nanoparticle corona formation?

Author(s): Manon Debayle, Elie Balloul, Fatimata Dembele, Xiangzhen Xu, Mohamed Hanafi, Francois Ribot, Cornelia Monzel, Mathieu Coppey, Alexandra Fragola, Maxime Dahan, Thomas Pons, Nicolas Lequeux
Published in: Biomaterials, Issue 219, 2019, Page(s) 119357, ISSN 0142-9612
DOI: 10.1016/j.biomaterials.2019.119357

Nanowerkzeuge für regeneratives Nervenwachstum.

Author(s): Marie Kappen, Domenik Liße, Jacob Piehler
Published in: Gesundheit für Mensch, Tier und Pflanze, Issue 2019, 2019, Page(s) 16