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Titre du projet:

Functionalization and evaluation of antioxidant cerium oxide nanoparticles for stroke treatment

Sujet

Ecole Doctorale: ED 563 - Médicament,Toxicologie, Chimie, Imageries

Titre du projet:

Functionalization and evaluation of antioxidant cerium oxide nanoparticles for stroke treatment

Structure d’accueil principale:

INSERM UMR_S1140 Innovations Thérapeutiques en Hémostase

Structure d’accueil secondaire:

LIST (Luxembourg Institute of Science and Technology) - Materials Research and Technology (MRT) department

Directeur·ice de thèse pressenti·e

Nom: Margaill

Prénom: Isabelle

Adresse électronique: isabelle.margaill@u-paris.fr

Co-directeur·ice de thèse pressenti·e (le cas échéant)

Nom: Roques (co-encadrante)

Prénom: Caroline

Adresse électronique: caroline.roques@u-paris.fr

Encadrant·e dans la structure d’accueil secondaire

Nom: Thomann

Prénom: Jean-Sébastien

Adresse électronique: jean-sebastien.thomann@list.lu

Description

Description du projet de thèse:

Stroke is the second cause of mortality and the first cause of acquired disability in France and Europe (around 150 000 new cases per year in France) [1]. Ischemic stroke refers to neurological dysfunction that results from the thrombotic obstruction of cerebral arteries and represents 85% of all stroke. The primary goal of ischemic stroke treatment is to restore blood flow in the occluded artery. Current guidelines prescribe pharmacological thrombolysis, obtained through intravenous injection of recombinant tissue plasminogen activator (r-tPA), and/or mechanical thrombectomy.

However, less than 15% of patients can receive this treatment and 20-30% of thrombi are resistant to current retrieval approaches [2]. The mechanism underlying resistance to r-tPA thrombolysis remains unknown. Emerging evidence suggests a potential role for polymorphonuclear neutrophils and more particularly neutrophil extracellular traps (NETs) that may partly explain the failure of r-tPA to lyse thrombi. We thus recently demonstrated that NETs are responsible for the thrombolytic failure of endogenous tPA in septic shock patients [3].

As NETs might compromise thrombus lysis, our project is to investigate new therapeutic opportunities in ischemic stroke by targeting NETs components of the occluding thrombus.

However, systemic administration of those NET-degrading molecules would require high doses to reach the clot. We therefore propose to use nanoparticulate carrier-based systems that may protect them from degradation/inhibition and induce their accumulation into the clot. For that purpose, CNP, cerium oxide nanocrystals (CeO2), seem particularly interesting due to their antioxidant properties. In stroke, an oxidative stress indeed occurs and is worsened by recanalization (either spontaneous or induced by r-tPA) leading to a burst in reactive oxygen species ROS [4]. This oxidative stress is a major contributor to neuronal loss and to vascular lesions causing further dramatic hemorrhages.

In the present PhD project, we aim to develop a new generation of CNPs functionalized with NETs-degrading molecules. In association with r-tPA, these innovative, functionalized antioxidant cerium nanovectors could increase its effectiveness and safety. Our objective is to improve recanalization for a better management of acute ischemic stroke patient’s treatment.

This programme is linked to a larger project recently funded by the ANR (STRIC-ON; ANR-20-CE18-0022), and involving both academic and industrial partners. The PhD student assigned to the project is expected to carry out the following tasks :

1: Cerium-oxide nanoparticles coating and functionalization

To reach their target, bare CNPs must be coated using organic coating. This organic layer will assume three functions: (i) decrease aggregation of CNPs, (ii) increase circulation time in blood, (iii) anchor functionalization of the nanoparticles by using specific bioconjugates chemistry.  In this aim, the PhD candidate will investigate coatings from synthetic innovative polymers produced by ANR partners (Specific Polymers® company and Jean-François Berret, Matières et Systèmes Complexes, UMR 7057, Université de Paris), lipidic surfactant or biobased polymers (from Luxembourg Institute of Science and Technology (LIST)). As the bio-functionalization of sub 10 nm cerium oxide nanoparticles still  remains a challenge, a collaboration between our laboratory and the Nano Engineering group from the LIST (Luxembourg Institute of Science and Technology) has indeed been established to investigate (i) biobased and lipidic coating on cerium oxide using LIST patented technologies, (ii) the grafting of NETs-degrading molecules at the surface of coated CNPs using thiol-maleimides chemistry. For that purpose, NETs-degrading molecules will be derivatized with Sulfo SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) and fluorescent probes (such as NHS-cyanine 5 or 7) and then grafted on activated Mercapto “lipids or (Bio) polymers”. The grafting quantification will use LC-MALDI-MS or fluorescence quantification. The most robust technology will be upscaled and then transferred to the INSERM UMR_S1140 for evaluation of these functionalized CNP.

2: In vitro evaluation of functionalized CNP

First experiments will aim to evaluate the degradation of NETs by functionalized CNP. NETs will be prepared from neutrophils isolated from anti-coagulated blood and stimulated using purified lipopolysaccharide or PMA (phorbol 12-myristate 13-acetate). Different concentrations of NETs-degrading molecules, free or grafted onto CNPs, will be used to study their effect on isolated NETs. Selected enzyme protein/activity i.e. neutrophil elastase released from NETs, will be quantitated using a chromogenic substrate (MeO-Suc-AAPV-pNA) selective for human neutrophil elastase. A second set of experiments will investigate whether functionalization of CNPs do not alter their antioxidant activity This antioxidant activity is important to protect brain from ROS burst due to reperfusion and in particular to protect from vascular lesion and avoid dramatic brain hemorrhages. For this purpose, cerebral endothelial cells (bEnd.3) will be stressed by glutamate, a neurotransmitter responsible for excitotoxicity after stroke notably through triggering oxidative stress. Antioxidant efficacy of functionalized and non-functionalized CNP will be then addressed using available commercialized kits dosing the formation of peroxides (Peroxi-Detect kit, Sigma) or and flow cytometry using the dihydro-rhodamine 123 compound.

3: Biodistribution and in vivo toxicity

Functionalization of CNP with a fluorophore (NHS-cyanine 7) will allow studying the in vivo biodistribution of the CNPs in mice, in particular their stealthiness, their half-life time in plasma and their tissue distribution. Blood samples and sections of kidney, liver, spleen and brain tissues will be collected at day 1 and 4 weeks after injection of the CNPs. Plasma extracted from the blood will be used for routine dosage of hepatic enzymes ASAT, ALAT and γGT. Sections of kidney, liver and spleen tissues collected will be stained with haematoxylin, eosin and saffron, and subsequently analyzed by a pathologist. Cyanine 7 will allow for ex vivo detection of CNPs in the harvested organs. CNPs present in the organs will also be quantified by inductively coupled plasma (ICP) elementary analysis.

Overall, task 3 will allow selecting the functionalized particles that will finally be tested in stroke.

4: In vivo cerebral ischemia

Cerebral ischemia will be performed by injection of thrombin into the left middle cerebral artery (surgery will be performed by an engineer). CNP will be injected intravenously (tail vein) to mice at 0.1, 0.5, 1.5 mg/kg 4 hours after ischemia onset with or without r-tPA (Actilyse®, Boehringer-Ingelheim, tail vein, 10 mk/kg, 10% bolus-90% 30 minutes perfusion). Cerebral blood flow in MCA territory will bre followed by laser Doppler to evaluate the impact of CNPs administration on reperfusion. Moreover, hemorrhagic transformations are due to the disruption of blood brain barrier following degradation of junction proteins on endothelial cells. We will thus examine the effect of our CNP by immunochemistry and western blotting on brain from ischemic and non-ischemic mice treated with CNP ± r-tPA to assess the vascular protection provided by CNPs.

The PhD candidate will spend the first 6 months at the Faculty of Pharmacy (UMR-S1140 and UTCBS) getting familiar with the scientific context of the project, as well as starting handling the CNPs and the polymer coatings. The next 6 months will be carried out at the LIST (Luxembourg) to test alternative coatings and achieve the successful functionalization of the CNPs. The following period will be dedicated to the in vitro and in vivo assessment of the functionalized CNPs (performed at the Faculty of Pharmacy), as well as the dissemination of the project (articles, poster and/or oral presentations,…).

 

[1] W. Johnson, et al., Bull World Health Organ, 94 (2016) 634-634A [2] A.J. Yoo, et al., J Stroke, 19 (2017) 121-130 [3] D. Barbosa da Cruz, et al., FASEB J, 33 (2019) 14270-14280 [4] M. Kanazawa, et al., J. Atheroscler. Thromb., 24 (2017) 240-253

Fichier de description du projet de thèse: Description_du_sujet_de_th__se.pdf

Description de l’équipe d’encadrement principale:

Isabelle Margaill (54 years old, PharmD, PhD, H-factor 27, 82 publications) is a professor of physiology at at the Faculté de Pharmacie de Paris since 2006. She has strong expertise in in vivo models of stroke in mice and focuses on the vascular lesions subsequent to ischemic stroke in the context of thrombolysis. She has directed the thesis of 15 PhD students, and is currently co-director of one PhD student. She was the coordinator the ANR project ICONS “Innovative Cerium Oxide Nanoparticles for Stroke” (ANR-15-CE18-0024 –482 114 euros) and is the coordinator of the recently funded project STRIC-ON (STROKE THROMBOLYSIS WITH r-tPA IMPROVED BY CERIUM OXIDE NANOPARTICLES - ANR-20-CE18-0022 - 563 228, 14 euros). She is also a member of the scientific committee of the French network Strokavenir and president of the French society “Cerveau et Maladies cérébrovasculaires”.

In 2019, I. Margaill joined the IThEM unit (Innovations Thérapeutiques en Hémostase (ItHem), INSERM UMR_S1140, Université de Paris) for its strong expertise in haemostasis. Among the members of the unit, E. Anglés-Cano is an MD, ScD expert in the fields of vascular biology, haemostasis and thrombosis with a particular focus on fibrinolytic activity and plasminogen activation in blood and vascular cells. The resistance to lysis of fibrin clots containing NETs as a result of immuno-thrombosis is his current field of interest.

The UTCBS (Unité des Technologies Chimiques et Biologiques pour la Santé) research laboratory is dedicated to nanomedecine, advanced drug delivery and nanotechnologies as imaging contrast agents. The laboratory is composed of multidisciplinary teams gathering chemists, physico-chemists, analytical chemists, pharmacists, and biologists. Within UTCBS, the team nanovectors for molecular imaging and targeted therapy (VICT) leaded by Dr. Nathalie Mignet has a long expertise in the design and in vitro/in vivo evaluation of nanocarriers for both therapeutic and imaging applications. Moreover, the VICT team has created a platform dedicated to optical imaging and MRI on small animals (http://piv.parisdescartes.fr/modalites-imagerie/optique/).

Caroline Roques, PharmD, PhD (Age 41) is Associate Professor of Pharmaceutical Technology in the VICT team. She has signed 14 articles, 2 book chapters and 1 NIN (new invention notification). She has dedicated her research to the design, characterization, and biological evaluation of nanocarriers, especially nanovectors based on amphiphilic molecules or lipids. Her approach is transdisciplinary, associating formulation, physico-chemistry, cell biology and in vivo evaluations. Recently, she was part of both ICONS and STRIC-ON ANR-funded projects, dedicated to innovative antioxidant cerium oxide nanoparticles as therapeutic agents in stroke. She has been mentoring more than 15 Master students and participated in the supervision of 3 PhD students.

Overall, the PhD student will benefit from a large and diverse scientific environment through frequent meetings between Ithem and UTCBS teams.

Description de la structure d’accueil secondaire:

The Luxembourg Institute of Science and Technology (LIST) is a mission-driven Research and Technology Organization (RTO) that develops competitive and market-oriented product/service prototypes for public and private stakeholders.

With its 600 employees, 75% of whom are researchers or innovation experts from all around the world, LIST is active in the fields of informatics, materials and environment and works across the entire innovation chain: fundamental/applied research, incubation and transfer of technologies.

Through its research into advanced materials and processes, the “Materials Research and Technology” (MRT) department, with its 200 researchers and engineers, contributes to the emergence of enabling technologies that underpin the innovation processes of local and international industry. MRT’s activities hinge on four thematic pillars: nanomaterials and nanotechnology, scientific instrumentation and process technology, structural composites, and functional polymers.  The department also includes four high-tech platforms, focusing on composites, prototyping, molecular characterization and testing. These platforms serve both LIST research staff, and other stakeholders in Luxembourg.

 

The Nano Engineering unit from MRT has developed a unique portfolio of Nanocarriers and Nanocoatings over the past years. In the context of this PhD project, the Nano Engineering research unit is proposing to develop an innovative nanocoating for antioxidant nanoceria. These coatings will be based on the LIST’s patented coatings (lipids or polymers) or on the poly(2-oxazolines) designed by Specific Polymers. In addition, the PhD candidate will take advantage of the expertise of the LIST’s Nano Engineering team to investigate different synthetic processes to efficiently conjugate functional moieties at the surface of the coated nanoceria. The challenge posed by the grafting of NETs-degrading molecules onto sub 10 nm cerium oxide nanoparticles is requiring the participation to the project of experts in the domain. LIST owns a significant IP portfolio of Nanoparticles and colloids for multiple kinds of application. Most of these capsule / particle technologies are covered by granted or filed patents (ZnO and CuO Nanorods, silica hybrid structure, Kraft Lignin nanoparticle, Lipid calcium carbonate NPs…). LIST infrastructure for colloidal synthesis is at the cutting edge in this domain. LIST chemistry laboratory is equipped with high end chemical screening reactors (Radleys Mya4, Carousel tornado set up), an advanced flow chemistry set up (ACR Co flore), high capability batch synthetic reactors (50 L, syris orb pilot) and a large capacity evaporator (Buchi, 25 L). For the characterization of nanoparticles, LIST is equipped with DLS, NTA and TRPS characterization tools, as well as SEM, a helium beam microscope, and TEM for dry characterization. The molecular analytical platform is equipped with high end LC-MS technology. The PhD will be supervised at the LIST by Dr. Jean-Sébastien Thomann (senior researcher, https://orcid.org/0000-0003-1381-8349) who is an expert in the field of nano chemistry and has already developed multiple organic and inorganic nanosystems for healthcare and composite applications. He has a mixed profile between research and technology (20 publications; 13 patents), and will be complementary to the academic supervision at Paris University.  He currently supervises one PhD student (FNR PhD fellowship, end 2022) and co-supervises two others (PPP PhDs, end 2022). He is currently leading 2 bilateral Industrial projects (Grants confidential) and recently got a public grant from FNR (Fond National de la Recherche - 400000 euros) under the bridge program for developing new bio based nano carrier for skin care application.

From this PhD joint programme, LIST expects to get valorization by developing new IP and/or new publications in partnership with the UMR_S1140. The international collaboration will prepare future project between LIST and Faculty of Pharmacy of Paris, especially in the context of Horizon Europe programs.