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

Periodic Reporting for period 1 - MAGNEURON (Hijacking cell signalling pathways with magnetic nanoactuators for remote-controlled stemcell therapies of neurodegenerative disorders)

Reporting period: 2016-01-01 to 2016-12-31

The MAGNEURON project aims at a breakthrough in stem cell therapies of neurodegenerative diseases by developing a novel technology for magnetic actuation of cellular functions. The innovative concept of our technology is to remote-control cellular signalling pathways by means of biofunctionalised magnetic nanoparticles (bMNPs) engineered to function as intracellular signalling nanoplatforms. Once delivered into the cytoplasm of target cells, bMNPs can activate signalling pathways in response to external magnetic fields. The remote magnetic actuation will be achieved with either of two modalities, allowing spatial and/or temporal control of the signalling perturbation.

With this novel technology, our long-term goal is to fundamentally advance cell therapies and regenerative medicine in the brain by remote-controlling the differentiation and oriented growth of transplanted cells. In the context of Parkinson’s disease, we will focus on the control of signalling pathways (Wnt/Frizzled, as well as Ras, and Rho GTPases) known to promote the survival, differentiation and growth of transplanted DA precursor neurons. By the end of the project, we will have reached a conceptual and practical expertise for the magnetic control of signalling activity in cells, with a demonstrated proof-of-concept for the differentiation and growth of stem cell derived neuronal cells in cultures, tissues and animals.
Over the last year, our consortium of groups has launched a pluridisciplinary effort with the goal of developing and applying a new technology for the control of cellular functions. Our work has been focused on setting up novel tools and methods coming from chemistry, physics, nanosciences, biochemistry, cell biology and regenerative medicine to design and characterize magnetic nanoparticles that can be used for remote actuation of oriented cell growth and differentiation.

For the conversion of magnetic fields into biochemical signals, a critical element is to use magnetic nanoparticles (MNPs) with well-controlled chemical, colloidal and biofunctional properties. A major challenge is to achieve an optimal trade-off between their magnetic response (dictated by the size and composition of the magnetic core), their intracellular mobility (affected by their surface passivation and size) and their ability to activate signalling events.

To this end, we are following two complementary routes. On the one hand, we are using synthetic MNPs made laboratory by the co-precipitation of Fe2+ and Fe3+ under alkaline conditions by the Massart method. The maghemite (γFe2O3) nanoparticles currently have a size between 7 and 9 nm, with the goal of preparing 12-14 nm nanoparticles. These MNPs have excellent magnetic properties with a magnetization saturation (measured by SQUID) between 70 and 78 emu/g of particles, which is very close to the saturation magnetization of bulk maghemite, measured between 78 and 85 emu/g. The magnetic nanoparticles are subsequently encapsulated in a silica shell for biocompatibility and covered with PEG molecules used as anti-fouling agents. Furthermore, organic dyes (such as Rhodamine or Cyanine) are incorporated into the silica shell to make the MNPs fluorescent. On the other hand, we have developed semi-synthetic protein-based nanoparticles. For that purpose, we are taking advantage of ferritin cages that constitute well-defined, stable supramolecular assemblies naturally designed for iron storage. In our experiments, ferritin heavy-chain, fused to GFP, are first expressed in bacteria and purified. Next, after surface pegylation, a magnetite core was synthesized in the cage at 65°C in oxygen depleted aqueous solution. Overall this yields highly biocompatible, well-monodispersed MNPs with a hydrodynamic diameter of 27.7 ± 1.1nm and a magnetization saturation around 87 emu/g.

In our approach, MNPs, once internalized into cells, are used as functional signalling nanoplatforms that can be displaced or remotely activated. To this end, we have developed two highly efficient targeting strategies in order to bind functional molecules at their surface (such as RhoGTPases or guanine-exchange factors): (i) covalent coupling to Halo-tagged proteins using a specially designed Halo-ligand grafted to the MNP surface. (ii) strong affinity coupling of GFP (at the MNP surface) to antiGFP-nanobody fused to a protein of interest. We noted that the latter strategy enables extremely fast (~ 1 s) and highly specific attachment to intracellular targets.

To characterize the behavior of MNPs inside cells, a key element for their use as controllable signalling platforms, we have implemented a novel assay based on the analysis of single nanoparticle trajectories. With this assay, we could probe the role of size and surface properties on the cytosolic mobility of the MNPs. We identified several important elements for the design of nanoparticles: (i) we observed that there is a critical size (around 50-75 nm) above which MNPs are not diffusing but are trapped by an elastic intracellular meshwork. (ii) we found that non-specific interactions (rather than geometric crowding) are the main factor controlling the diffusivity and can dramatically reduce the mobility by several orders of magnitude. This assay will be key for the desing and in situ characterization of MNPs in the future.
In the last couple of months, we have already made progress that goes beyond the state-of-the art. In particular, we have developed semi-synthetic ferritin-based protein cages, a new class of magnetic nanoparticles. These ferritin nanoparticles exhibit remarkable properties in terms of biocompatibility, dispersity and magnetic response and will find use well beyond the framework of our project. In the context of the MAGNEURON project, ferritin provide a robust and versatile tool to target and manipulate functional molecules. In parallel, we have largely improved our understanding of the behavior of MNPs inside cells. In particular, we have established a powerful single-nanoparticle assay that enables a quantitative characterization of the dynamic properties of MNPs in the cytosol. With this assay, which can be used to address a variety of questions in nanobiosciences, we have probed key colloidal parameters that underlie the intracellular mobility. Thereby, we have identified an upper critical size for the MNPs and revealed how surface modification can dramatically affect the intracellular dynamics of nanoparticles.

In the future, all our current tools will be further optimized and combined to achieve reliable spatial and temporal control of signalling pathways, especially in the context of Parkinson’s disease. We expect that we will be able to recover functional activity either by injecting degenerating neurons with MNPs or by transplanting MNP-loaded cells and stimulating their differentiation and growth by magnetic actuation.

Overall, our project represents a novel approach that will improve the efficacy of cell-based therapies, especially in the context of neurodegenerative diseases, which represent a major medical and societal challenge in the coming years.