CE19 - Technologies pour la santé

Dévelopment of a Polydopamine Hybridized Iron Oxide Mussel Inspired Cluster for molecular MRI – pHySIOMIC

Development of a Polydopamine Hybridized Iron Oxide Mussel Inspired Cluster for molecular MRI

Ischemic stroke is a sudden neurological attack, representing the third leading cause of death and the leading cause of acquired disability in adults in industrialized countries. To guide clinical practitioners in the choice of these treatments, magnetic resonance imaging (MRI) is essential. But MRI has its limits and does not make it possible to diagnose microthrombi, which are nevertheless partly responsible for post-stroke sequelae.

This project consists in the development of a contrast agent to enable ultra-sensitive detection of microthrombi in ischemic stroke patients.

Despite significant progress in prevention and acute care over the past decades, ischemic stroke prevalence is on the rise with population aging and is expected to affect 1.3 million people per year in Europe by 2025.1 While the rapid management of strokes saves the lives of half of the patients, the resulting brain damages often remains dramatic for survivors and ischemic stroke is the leading cause of acquired disability in adults. <br />The current treatment for the acute phase of ischemic stroke consists of eliminating the thrombus obstructing the cerebral circulation by injecting a drug promoting its enzymatic degradation (thrombolysis) or, since 2015, by removing it mechanically by catheterization (thrombectomy). <br />However, even when successful recanalization of the main occluded vessel is achieved, dowstream microcirculation often remain occluded.2 The mechanisms explaining this incomplete microvascular reperfusion are not fully understood but we know that it is due to an obstruction by microthrombi and that it is exacerbated by the inflammatory consequences of ischemia, which induces a narrowing of the lumen of the microvessels.3 Several preclinical and clinical studies correlate the presence of such microthrombi with cognitive declines and dementia.4 Recent retrospective analysis of the implementation of thrombectomy in ischemic stroke care also underlines the importance of incomplete microvascular reperfusion. More than a third of the patients who benefit from successful thrombectomy do not recover to functional independence although successful recanalization is rapidly achieved.5 <br />Microthrombi in ischemic stroke are therefore of particular concern for patients surviving from ischemic stroke suffering permanent sequelae and thus represent a significant human, social and economic cost. <br />Despite this concern, the impact of microvascular thrombosis in ischemic stroke is currently not properly considered in clinical practice. The main obstacle being the absence of reliable methods for microthrombi diagnosis within the brain of stroke victims. <br />Here, we propose to develop a biodegradable iron oxide microparticles that could reveal microthrombi with the technique of molecular magnetic resonance imaging (MRI). This invention would have a strong commercial potential as a novel MRI contrast agent.

Synthesis of a biocompatible iron oxide contrast agent: pHySIOMIC
For the synthesis of the SPIO clusters, we will use carboxymethyl dextran coated iron oxide nanoparticles commercialised by the German company Bayer Schering (Resovist ®) which have been administered to patients for now 20 years. These nanoparticles were self assembled into sub-micrometer sized clusters via polymerization of dopamine into polydopamine. Although the project coordinator has previous experience with this type of emulsion process
The chemistry background behind this novel metal-phenol coordination technique is directly inspired from the study of mussel biology (1). We propose to include the bioinspiration origin of the particles in its name, calling this novel imaging tool pHySIOMIC, which stands for polydopamine hybridized iron oxide mussel inspired clusters.

Molecular imaging of microthrombi in ischemic stroke
Once the synthesis protocol optimized and the biodegradability confirmed, the pHySIOMIC particles will be used as a molecular imaging tool of microvascular thrombosis in ischemic stroke. We tested the imaging capacities in a mouse model of ischemic stroke developed in the PhIND laboratory. The molecular MRI experiments was led on a small animal 7T MRI (Brucker, Biospin) available within the facilities of Cyceron biomedical imaging platform where the PhIND laboratory is hosted.

Ref:
1. Lee, H., Dellatore, S. M., Miller, W. M. & Messersmith, P. B. Mussel-inspired surface chemistry for multifunctional coatings. Science 318, 426–430 (2007).

We have established a study of the presence of microthrombi in 2 mouse models of ischemic stroke. We have worked on a thromboembolic model induced by injection of thrombin into the middle cerebral artery (MCA) and a model where thrombosis is formed in situ at the same level of the MCA by deposition of a filter paper soaked in aluminum chloride. (AlCl3). We then examined the brains of these mice by brain MRI. We scanned them at the acute stage (30 minutes after stroke induction) with a preclinical 7T MRI (phramascan, Brucker). We studied the presence of anatomical modification in T1 and T2 weighted MRI and we see at this early stage no lesion or other cerebral damage. We do not see any bleeding on T2* MRI but we detect a lesion that is beginning to form on diffusion MRI (DWI). We also observe a perfusion deficit in perfusion MRI (PWI) as well as an extinguished signal in time-of-flight (TOF) MRI which corresponds to the ischemic zone. It is important to note that these ischemia-related deficits are identical in the 2 models. On the other hand, we have also studied the presence of thrombosis in the brain of these mice killed after these acquisitions (therefore always an acute time; 1 hour after the induction of the stroke) and we note a significant difference; in the thrombin model we observe a large number of microthrombi in the thrombin model throughout the ischemic zone whereas for the AlCl3 model we only see a thrombus at the level of the deposit of the filter paper.
We then worked on the development of pHySIOMIC microparticles and on its use as a molecular imaging tool for microthrombi in ischemic stroke.


We have therefore optimized the protocol for the synthesis of PHySIOMICs obtained by the process of self-assembly of iron oxide nanoparticles (average diameter of 70 nm) aggregated via the polymerization of dopamine into polydopamine in the form of sub-micrometric particles (diameter average of about 700 nm). We observed the PHySIOMICs by transmission electron microscopy, which allowed us to confirm their structure of aggregates of iron oxide nanoparticles trapped in polydopamine matrices. The relaxivity of PhySIOMICs is 413.2 mM-1.s-1, more than 2 times higher than the relaxivity of SPIOs (151.9 mM-1.s-1) which is very encouraging as regards the potential for detection of PhySIOMICs by magnetic susceptibility weighted MRI sequences.

The developed PHySIOMIC contrast agent combines strong magnetic and thrombosis targeting properties. We demonstrated its ability to reveal in an ultra-sensitive and high-resolution manner the presence of cortical microthrombi in an ischemic stroke mouse model in MRI. The PHySIOMIC contrast agent also exhibit a good biocompatibility, similar to FDA-approved SPIO nanoparticles. Although the mechanism of targeting on microthrombosis needs further investigation, we demonstrated that the protein corona formed at the surface is involve in the process of fixation. In vivo injection of PHySIOMIC in a thrombin-induced stroke model reveals microthrombosis in T2*-weighted images shortly after the stroke onset. Induced targeted hyposignal is disappearing after 24 hours. Furthermore, PHySIOMIC used in the context of a thrombolytic study shows a good ability to monitor microthrombosis degradation. Overall, this contrast agent could help to reveal and monitor microthrombosis in stroke patients, and guide treatment strategies.

A scientific publication gathering all the data acquired within the ANR project will be submitted to a peer review journal.

Ischemic stroke is a sudden neurological disorder which constitutes the third leading cause of death and the leading cause of acquired disability in adults in industrialized countries. The current treatment for the acute phase of ischemic stroke is to remove the blood clot obstructing the cerebral bloodflow by enzymatic degradation (thrombolysis) or by removing it mechanically through catheterization (thrombectomy). To guide clinical practitioners in their choice of treatment, magnetic resonance imaging (MRI) is essential. But MRI has its limits and does not allow the diagnosis of microthrombi, which are however partly responsible for post stroke sequelae.

This project aims to (i) study the physiopathology of microthrombi in ischemic stroke, (ii) develop a contrast agent to reveal microthrombi on MRI and (iii) establish a treatment for micrthrombi.

1. Study of microvascular thrombosis in ischemic stroke
The presence and impact of distal microthrombi will be studied in 2 models of ischemic stroke in mice. The first model we will use is thromboembolic, it is obtained by injection of thrombin into the middle cerebral artery (MCA). In the second model, cerebral ischemia is induced by occlusion of the MCA with a filament; removing the filament will allow us to reproduce the abrupt recanalization encountered in patients who benefited from thrombectomy. We will study precisely on histological sections the quantity, the stability over time and the composition of microthrombi in these 2 models.

2. Synthesis of a novel MRI contrast agent to reveal microthrombi
Previous work in the PhIND laboratory has demonstrated the great potential of the molecular magnetic resonance imaging (MRI) strategy with microparticles of iron oxide (MPIO). Despite the promises of this strategy, the MPIOs used in these studies are composed of inert and toxic materials. The development of biodegradable and non-toxic MPIOs is therefore necessary to make this technology applicable to humans.
In this project we propose to synthesize MPIOs from iron oxide nanoparticles assembled in a biodegradable matrix. We will use an emulsion coupled to a crosslinking process to cluster the iron oxide nanoparticles in the biodegradable matrix. We refer to these particles by the acronym PHysIOMIC. To optimize this synthesis and characterize the particles obtained, we will work with the organic chemistry department of the pharmaceutical research laboratory of Caen (CERMN).
We will then use this novel MRI contrast agent to reveal the microthrombi present in ithe schemic stroke models using the molecular MRI technique. Preliminary results confirm that this method is effective in revealing the occlusive microthrombi present in the ischemic thromboembolic stroke model. In order to increase the specificity of our system, we will work on functionalizing the PHysIOMIC with antibodies specific to the platelets contained in the microthrombi.

3. Preclinical study of a thrombolytic therapy for the treatment of microthrombi
We will test 3 different thrombolytic treatments that are known to be effective in degrading platelet and von Willebrand factor rich clots. We will test N-acetylcystein, the powerful thrombolytic effect of which has recently been demonstrated in the PhIND laboratory, and 2 treatments whose fibrinolytic activity is triggered by the presence of thrombin, which is generated in very large quantities by the activated platelets present in microthrombi.

Project coordination

Thomas Bonnard (Physiopathologie et imagerie des maladies neurologiques)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

PHIND Physiopathologie et imagerie des maladies neurologiques
CERMN CENTRE D'ETUDES ET DE RECHERCHE SUR LE MEDICAMENT DE NORMANDIE

Help of the ANR 220,417 euros
Beginning and duration of the scientific project: December 2020 - 48 Months

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