Sylviane Muller

Le Professeur

Maria-José Blanco-Prieto

Hervé Juliette Gilles

Nicolas, Stéphanie, François, Catherine, Kawthar. Laurence

Marie Jiao

Alexandre Jiao

Elena Noueirata

Brice

F Testard

Patrick Couvreur

M.-J Prieto-Blanco

Couvreur

CEA Bernard Rousseau

Y Capan

T Dalkara

Catherine Dubernet

Simona Mura

Didier Desmaele

Sinda Lepetre-Mouelhi

Lucien Bildstein

Joachim Caron

Elise Lepeltier

Dunja Sobot

L Harivardhan Reddy

Andrei Maksimenko

Fatima Zouhiri

Eric Sliwensky

Hélène Chacun

Julie Mougin

Stéphanie Denis

Luigi Cattel

Barbara Stella

Flavio Rocco

Jean-Christophe Cintrat

and liquid chromatography/tandem mass spectrometry » -(45 k€

Keywords: ♦ Porteur des projets scientifiques suivants:, LabEx « Nano-Saclay », Projet NanoLenkSqual Squalene-Adenosine nanoparticles for heart ischemia/reperfusion injuries treatment Collaboration : Nanoparticules d'adénosine-squalène et ischémie cérébrale : caractérisation du passage de la Barrière hémato-encéphalique, évaluation de

Professeur Elias Fattal pour me faire l'honneur d'évaluer mon travail en tant que rapporteurs ; le Professeur Janick Ardisson et le Dr. Ling Peng pour avoir accepté de porter un regard critique sur mon travail recherche en tant qu'examinateurs. Je tiens également à remercier tout particulièrement le Professeur Patrick Couvreur pour m'avoir d'abord fait confiance en me recrutant en tant que Maître de Conférences au sein de son équipe de recherche. Travailler à vos côtés tout au long de ces années, a été pour moi une chance inestimable. J'ai grâce à vous énormémment appris sur cette « magic bullet » qui au départ pour un chimiste comme moi, représentait un objet appartenant à l'univers de la science-fiction. Vous m'avez petit à petit amenée dans votre univers de l'infiniment petit en me montrant que la fiction pouvait devenir réalité avec de l'imagination, de l'audace, de la persévérance, de la passion, la curiosité et de la chance. Merci infiniment de m'avoir transmis votre culture pharmaceutique, votre passion pour la nanomédecine, et merci de m'avoir poussée au-delà de mes limites toujours avec beaucoup de bienveillance et de disponibilité. Vous avez aussi su créer une atmosphère très conviviale et de confiance au sein de notre équipe, grâce à votre esprit fédérateur, en valorisant chacun d'entre nous en fonction de nos compétences et points forts respectifs. Et merci également pour votre ouverture d'esprit et pour toutes nos discussions toujours très enrichissantes et passionnantes sur la science et la vie en générale. Un grand merci également au Professeur Elias Fattal pour tout son soutien, sa bienveillance et ses précieux conseils tout au long ces années. Tu m'as dès le début ouvert grand ta porte, prêt à aider et toujours à l'écoute. Tu as toujours été plus comme un collègue ami que chef, et c'est très appréciable. Merci pour ta confiance. Tu t'es toujours soucié du bien-être et de la carrière de chacun de nous, personnels de l'Unité ainsi qu'étudiants. Tu as su diriger l'Unité avec beaucoup d'intelligence et d'implication, en fédérant toutes les équipes dans un esprit de solidarité tout en créant une atmosphère très conviviale. Ça nous fait toujours plaisir lorsque tu viens nous faire un petit coucou à notre bureau. A Julie, ma co-bureau qui partage mon quotidien. Merci pour ta compagnie apaisante, ta gentillesse et pour tes petites astuces en informatique qui me dépannent toujours. Merci à Julien, d'avoir repris le flambeau après Patrick et de gérer au quotidien notre équipe et de veiller à ce que tout se passe bien. Bravo pour ton implication car ce n'est pas une mince affaire.

Je remercie également mes collègues de l'équipe, Mariana, que j'ai le plaisir de côtoyer au quotidien et avec qui j'ai partagé de supers moments durant nos voyages dans le cadre des colloques en compagnie de Patrick. Merci également à Simona, présente sur tous les fronts et toujours prête à aider et à Didier avec qui j'ai travaillé au début de mon recrutement.

I. SYNTHÈSE DE LA CARRIÈRE :

1. Diplômes :

Après avoir effectué mon premier et 2 ème cycle à l'Université de Montpellier II Sciences et Techniques du Languedoc (USTL) à l'issu desquels j'ai obtenu le Diplôme d'Etudes Universitaires Générales en Sciences avec spécialité Biochimie (DEUG B), la Licence et Maîtrise de Chimie, mention Chimie Moléculaire, j'ai souhaité m'orienter vers la recherche en entreprenant des études de 3 ème cycle. J'ai tout d'abord obtenu en 1997 le Diplôme d'Etudes Approfondies (DEA) en Hétérochimie, Polymères et Catalyse. Durant cette année de DEA, j'ai effectué un stage de recherche de 6 mois au sein du laboratoire du Pr. J L. Montéro, dont le sujet s'intitulait « Synthèse d'agents de réticulation à motifs osidiques en vue d'obtenir un film alimentaire biodégradable ». Passionnée par la recherche, j'ai entrepris de poursuivre ensuite mes études de 3ème cycle en débutant en Septembre de la même année une thèse de doctorat en chimie organique à l'Université de Cergy-Pontoise (UFR Sciences et Techniques) que j'ai soutenue en Mars 2001. Mon travail de recherche, dirigé par le Pr. M. Bellassoued au sein du Laboratoire de chimie organométallique, a consisté à mettre au point une nouvelle voie d'accès direct aux acides (E)-α,β-éthyléniques au moyen d'un synthon trisilylé. Ce travail a donné lieu à 4 publications internationales et un chapitre de livre.

Postes occupés :

Après obtention de ma thèse de doctorat, j'ai eu l'opportunité d'effectuer deux missions (Avril 2001 à Mars 2002) au sein des Nations Unies au Kosovo (UNMIK) qui m'ont appris à gérer et conduire plusieurs aspects d'un projet (politique, technique, humain). En 2003 j'ai marqué un arrêt d'un an dans ma carrière pour congé de maternité et recherche d'emploi.

De Mars 2004 à Février 2005 j'ai effectué un premier stage post-doctoral en chimie médicinale à la faculté de Pharmacie de Châtenay-Malabry au sein de l'Unité BIOCIS sous la direction du Pr. S. Sicsic et du Pr. S. Ongeri. Mon travail a consisté à synthétiser de nouveaux ligands spécifiques pour le récepteur NOP/ORL-1 de la Nociceptine.

De Mars 2005 à Mai 2005 j'ai été recrutée sur une mission d'intérim de 3 mois en tant que chercheur scientifique en chimie médicinale chez SANOFI-AVENTIS (Chilly-Mazarin) où j'ai remplacé le Team leader (Dr S. Mallart) au sein du département Cardiovasculaire. Le projet de recherche sur lequel j'ai été recrutée a consisté à mettre au point des stratégies de synthèse de molécules à visée thérapeutique.

De Juin 2005 à Août 2006, j'ai effectué un 2 ème stage post-doctoral en chimie thérapeutique et formulation à la faculté de Pharmacie de Châtenay-Malabry dans le cadre d'une collaboration entre l'UMR 8076-BioCIS et l'UMR 8612-Institut Galien Paris-Sud, sous la direction du Pr. P. Couvreur et du Dr. D. Desmaële. Mon travail a consisté à mettre au point la synthèse de nouveaux matériaux à base de squalène pour la vectorisation d'analogues nucléosidiques anticancéreux et antiviraux (anti-HIV) sous forme de nanoparticules et a donné lieu à 1 publication.

En Septembre 2006 j'ai été recrutée en tant que Maître de Conférences en Pharmacotechnie et biopharmacie toujours dans l'équipe du Pr. Couvreur au sein de l'Institut Galien à la faculté de Pharmacie de Châtenay-Malabry.

Principales responsabilités et activités :

A mon recrutement, j'ai effectué une mobilité thématique en prenant en charge de nombreux aspects de formulation en plus de mon apport à l'équipe en tant que chimiste. Durant mes premières années en tant que Maître de Conférences, j'ai continué à développer l'aspect chimie et formulation de ma 1ère thématique de recherche au sein de l'équipe « Nanomédicaments pour le traitement des maladies graves », et par la suite, mes compétences se sont même étendues à la pharmacologie pour traiter les troubles neurologiques tels que l'accident vasculaire cérébral, les traumas de la moelle épinière et plus récemment la douleur. Ainsi, ma 1 ère thématique a consisté à prendre en charge la conception et la synthèse des agents de vectorisation correspondant à de molécules de type isoprènes à longueurs de chaînes variables, puis à les coupler (directement ou via des bras espaceurs) à des molécules anticancéreuses et antivirales pour former des prodrogues que j'ai ensuite formulées en nanoparticules puis caractérisées. Depuis 2011, j'ai développé de nouvelles thématiques au sein de l'équipe. Ainsi, j'ai été chargée de la synthèse de nouveaux nanomédicaments à visée neuroprotectrice (système nerveux central et périphérique) à partir de bioconjugués d'adénosine-squalène pour traiter l'ischémie cérébrale et le trauma de la moelle épinière. Cette thématique m'a permis de co-diriger 2 doctorantes Alice Gaudin à 30% qui a soutenu sa thèse en Novembre 2014 avec 3 publications et Marie Rouquette à 50% qui a soutenu sa thèse en Février 2019 avec 3 publications. Je développe également depuis 2014 des nanomédicaments squalénisés qui traitent la douleur à partir de neuropeptides naturels sans avoir à traverser la barrière hémato-encéphalique. Sur cette thématique j'ai co-dirigé Jiao Feng à 50% et qui a soutenu sa thèse en Décembre 2018 avec 2 publications et un brevet. Actuellement, je poursuis ma recherche dans le domaine de la douleur en explorant une nouvelle voie d'administration plus confortable pour le patient, la voie transdermique, pour cibler les douleurs chroniques. Sur ce projet, j'encadre à 100% un étudiant en 1 ère année de Thèse, Alexandre Da Silva, en attendant de soutenir très prochainement mon Habilitation à Diriger les Recherches (HDR). Mes travaux de recherche depuis mon recrutement ont donné lieu à 40 publications dans des revues internationales à comité de lecture, (dont Science Advances, Nature Nanotechnology, etc…) ainsi qu'un brevet et un chapitre de livre. Mon h-index est de 20 avec en moyenne 31 citations par publication et 1300 citations globales. Je suis régulièrement invitée à donner des conférences (dont 7 depuis septembre 2018). En 2019, j'ai obtenu la prime d'encadrement doctorale et de recherche (PEDR) et en 2020 j'ai été promue à la Hors Classe. Cette mobilité thématique s'est également traduite au niveau de mes enseignements étant donné que j'ai choisi d'enseigner la Pharmacie Galénique qui n'était pas ma discipline de départ. Je dispense ainsi mes enseignements dans cette discipline depuis 2006 (CM, ED et TP) à la Faculté de Pharmacie de Châtenay-Malabry, aux étudiants de 2 ème , 3 ème , 4 ème année et 5 ème année (industrie et officine) et 6 ème année officine. Je dirige également depuis quelques années 4 Unités d'Enseignement.

II. ACTIVITÉ PÉDAGOGIQUE :

1. Présentation de l'activité d'enseignement :

Enseignements en cours :  Enseignement en Projet professionnel et personnel :

• UE 19 : « Projet Professionnel, Communication, Organisation-Gestion » pour les étudiants de 3 ème année de pharmacie. (Direction à 50%)

Enseignement dispensé depuis 2006 (Enseignements dirigés)

Cet enseignement a pour but de pousser l'étudiant à adopter une démarche active par rapport à son avenir professionnel. Il l'accompagne dans un travail réflexif pour construire son parcours (choix d'orientation adéquat) et donner sens à son projet. Il consiste à pousser l'étudiant à effectuer un bilan introspectif sur lui-même, à faire ensuite un vrai bilan de compétences et enfin à valoriser les expériences d'apprentissage et de formation pour pouvoir rédiger son CV et lettre de motivation. Il sera également amené à entreprendre un travail bibliographique sur un métier ou secteur d'activité lié à la pharmacie qui pourrait l'intéresser ou qu'il souhaiterait tout simplement découvrir. Pour compléter ce travail bibliographique, l'étudiant devra également aller à la rencontre de professionnels pour les interviewer. Ce travail se concrétisera par une soutenance orale devant ses camarades. Tous les étudiants auront ainsi l'occasion de découvrir par ce biais là les différents métiers du pharmacien.

 Enseignements en pharmacie galénique :

• UEL 294 « Formulation des préparations parentérales » pour les étudiants de 5 ème année industrie/recherche en pharmacie. (Création et direction à 100%) Enseignement dispensé depuis 2014 (Cours Magistraux, Enseignements dirigés et Travaux Pratiques)

Les objectifs de cette UE consistent à approfondir les connaissances des étudiants sur la formulation des préparations parentérales en tenant compte des exigences de la pharmacopée quant à la préparation de ces formes pharmaceutiques notamment en termes de stérilité, isotonie, limpidité pour les solutions, neutralité et apyrogénicité. Les étudiants devront décider eux-mêmes de la meilleure formulation à proposer au travers d'études de cas pour lesquelles solubilité et stabilité des molécules actives sont problématiques. Ils devront être en mesure de proposer un schéma de fabrication approprié qu'ils mettront en oeuvre pour chaque étude de cas lors de séances de TP. Lors de cette UEL, les étudiants auront l'occasion d'expérimenter diverses opérations pharmaceutiques telles que l'autoclavage, la filtration stérilisante sous hotte à flux laminaire, la lyophilisation, le conditionnement en ampoules et sous atmosphère inerte.

• UE 135 : « Préparations galéniques en officine » pour les étudiants de 6 ème année Officine. (Direction à 50%). Cet enseignement est issu de la fusion de l'UE 115 et de l'UE120.

Enseignement dispensé depuis 2019 (Cours Magistraux, Enseignements Dirigés et Travaux Pratiques) Dans cette UE, les étudiants voient plus en détail les bonnes pratiques de préparation (BPP) en officine au travers d'études de cas et auront à développer une réflexion logique pour exécuter des préparations magistrales et officinales à partir d'une ordonnance de médecin. Cette UE les met en situation de décider d'eux-mêmes du protocole à établir en tenant compte des caractéristiques physico-chimiques des principes actifs et excipients, de leurs compatibilités, etc… Les étudiants mettront ensuite en pratique leurs protocoles et exécuteront des préparations magistrales selon les bonnes pratiques de préparation (BPP). Durant cet enseignement en grande majorité pratique, les étudiants auront à préparer des gélules (avec et sans enrobage gastro-résistant), des sirops, des potions, des lotions, des pommades, des crèmes, des gels. Ils réaliseront ensuite les contrôles nécessaires pour garantir la qualité de ces préparations et enfin rempliront un document de préparation qui est un gage d'assurance qualité. Cet enseignement les amènera à prendre conscience de l'importance de la démarche qualité à chaque étape de la fabrication et du contrôle et notamment au travers des BPP.

Enseignements anciennement dispensés :

• UE 120 : « Préparations galéniques en officine » pour les étudiants de 6 ème année Officine. (Direction à 50% depuis 2013) Enseignement dispensé de 2006 à 2019 (Cours Magistraux et Enseignements Dirigés) Dans cette UE, les étudiants voient plus en détail les bonnes pratiques de préparation (BPP) en officine au travers d'études de cas et auront à développer une réflexion logique pour exécuter des préparations magistrales et officinales à partir d'une ordonnance de médecin. Cette UE les met en situation de décider d'eux-mêmes du protocole à établir en tenant compte des caractéristiques physico-chimiques des principes actifs et excipients, de leurs compatibilités, etc…

• UE 115 : « Préparations officinales et magistrales : pratique » pour les étudiants de 5 ème année Officine (Transformation en 2017 à partir de l'UE120 aboutissant à un enseignement pratique) (Direction à 50% depuis 2017) Type d'enseignements dispensés depuis 2017 à 2019 (Cours Magistraux et Travaux Pratiques) Les objectifs de cette UE visent à apprendre aux étudiants à exécuter une préparation magistrale à partir d'une ordonnance de médecin selon les bonnes pratiques de préparation (BPP). Durant cet enseignement en grande majorité pratique, les étudiants auront à préparer des gélules (avec et sans enrobage gastro-résistant), des sirops, des potions, des lotions, des pommades, des crèmes, des gels. Ils réaliseront ensuite les contrôles nécessaires pour garantir la qualité de ces préparations et enfin rempliront un document de préparation qui est un gage d'assurance qualité. Cet enseignement les amènera à prendre conscience de l'importance de la démarche qualité à chaque étape de la fabrication et du contrôle et notamment au travers des BPP.

• UE 26 : « Formulation, fabrication et aspects pharmaceutiques » pour les étudiants de 3 ème année de pharmacie.

Enseignement dispensé sur les périodes 2012-2013, 2016-2017, 2018-2019 (Travaux pratiques) Les étudiants auront à proposer plusieurs formules pour fabriquer un gel cutané à base de caféine (gels aqueux et hydroalcooliques) puis exécuteront les essais de préformulation avant de choisir la préparation la plus adéquate.

• UEM PG2 : « Formulation et génie des procédés pharmaceutiques» pour les étudiants de 5 ème année Industrie. Type d'enseignements dispensés de 2012 à 2015 (Cours Magistraux et Travaux Pratiques) Formulation d'une solution injectable stable de vitamine C en tenant compte des propriétés physico-chimiques de l'acide ascorbique, des critères de qualité des préparations injectables et des coûts des contraintes de production. Les étudiants auront à réaliser un plan d'expériences, des essais de préformulation, un dosage direct de la Vitamine C, le choix de la formulation et l'évaluation de l'impact des paramètres de stabilité. Ils procèderont ensuite à la fabrication d'un lot pilote.

• UE 13 : « Formulation et préparation des médicaments» pour les étudiants de 2 ème année de pharmacie.

Enseignement dispensé de 2012 à 2014 (Enseignements dirigés) Cet enseignement permet aux étudiants de définir les stratégies à utiliser suivant la substance active et le site d'action. J'étais en charge de la partie « Formulation des préparations parentérales ». L'enseignement se présentait sous la forme d'études de cas. Les étudiants devaient proposer des formulations et évaluer de l'impact des paramètres de stabilité.

• Travaux pratiques de pharmacie galénique pour les étudiants de 3 ème année de pharmacie.

Enseignement dispensé de 2006 à 2012 (Travaux pratiques)

Au cours de cet enseignement, les étudiants effectuent d'abord des essais de pré-formulation, avant de fabriquer les gélules, comprimés, suspensions et suppositoires puis procèdent aux contrôles exigés par la pharmacopée européenne pour valider leurs préparations. Les étudiants auront également à reconnaitre les produits chimiques couramment utilisés en officine par un examen organoleptique de ceux-ci.

• Travaux pratiques coordonnés « Du gène au médicament : Insuline » pour les étudiants de 4 ème année de pharmacie.

Enseignement dispensé de 2008 à 2013 (Travaux pratiques au niveau de la formulation) Approche pluridisciplinaire permettant de découvrir quelques étapes de la genèse d'un médicament : Préparation de différentes formes galéniques, contrôle (identité, pureté, microbiologie), caractérisation biologique in vitro et in vivo.

Présentation des formations suivies concernant mes activités pédagogiques :

Dans le cadre de l'UE19 « Projet Professionnel, Communication, Organisation-Gestion » : Participation aux ateliers sur la construction du bilan des compétences, l'accompagnement des étudiants dans leur projet professionnel (coaching), l'utilisation de l'outil pédagogique PEC dans le but de le présenter ensuite aux étudiants en DFGSP3 (L3), et enfin consignes sur la rédaction du CV et de la lettre de motivation.

•
Impact sur la pratique de l'enseignement: Mise en place dans l'UE 19 d'un mini atelier pour les étudiants, autour du bilan des compétences impliquant un travail d'introspection (questionnaire), une analyse des aptitudes, des motivations, des compétences, en vue de faciliter les choix d'orientation et les prises de décisions au cours de leur parcours de formation. Présentation de l'outil PEC aux étudiants.

• Formation « Professionnalisation des enseignants et des enseignants-chercheurs : l'approche par les compétences » 14-15 Novembre 2012 à l'ESEN (Ecole supérieure de l'éducation nationale) à Poitiers.

Objectifs: Percevoir les enjeux liés à l'approche par les compétences dans les universités. Savoir identifier une compétence et accompagner les étudiants dans l'identification de leurs compétences (acquises ou à acquérir). Etre en capacité de formaliser le lien entre enseignement-recherche et compétences (notamment au travers des fiches RNCP (Répertoire national de la certification professionnelle).

Impact sur la pratique de l'enseignement: Amener les étudiants vers une démarche d'identification des compétences avant de commencer à rédiger leur CV et lettre de motivation en vue d'une demande de stage.

Responsabilités pédagogiques :

Responsabilité des Unités d'enseignement suivantes : Particularité : Approche théorique puis mise en pratique de la préparation de gels, pommades, sirops et gélules en appliquant les bonnes pratiques de préparation (BPP).

III. ACTIVITÉ SCIENTIFIQUE :

1. Présentation synthétique des thématiques de recherche :

Notre équipe a développé de nouveaux nanomédicaments à base de squalène, un lipide triterpène endogène, biocompatible et biodégradable (Couvreur et al. Nano Lett 2006). Ces nanomédicaments sont obtenus à partir d'un couplage chimique entre le squalène (SQ) et un principe actif (PA), via une liaison biodégradable, donnant lieu à une prodrogue ayant la capacité de s'auto-assembler spontanément dans l'eau sous forme de nanoparticules (NPs) d'une 100 aine de nanomètres (nm) de diamètre compatibles avec une administration par voie intraveineuse (IV). Dans la plupart des cas, ces nanomédicaments dont le taux de charge en PA avoisine les 50%, ont significativement augmenté l'activité pharmacologique du PA en le protégeant de la métabolisation rapide et ont modifié favorablement sa biodistribution, diminuant ainsi sa toxicité. Il s'agit d'un projet important qui a fait l'objet d'un ERC Advanced Grant dans lequel j'ai été très impliquée en apportant mes compétences de chimiste mais aussi en encadrant des étudiants en thèse ou des chercheurs post-doctoraux.

Mon travail de recherche a donc consisté dans un premier temps à prendre la responsabilité de la synthèse d'une bibliothèque de dérivés de squalène pouvant être ensuite couplés à différents type de PA pour former les prodrogues squalénisées. Ces dérivés ont tout d'abord été obtenus par fonctionnalisation chimique d'une des 2 doubles liaisons terminales du squalène permettant ainsi d'introduire une fonction acide, amine, alcool, thiol, etc… sur celui-ci. Ces dérivés de squalène sont constitués de 5 unités isoprènes. J'ai par la suite étendu la synthèse à des analogues de SQ à longueurs de chaînes variables allant du mono jusqu'aux polyisoprènes.

Le couplage de ces dérivés de squalène aux PA a été réalisé soit directement via un lien chimique biodégradable (amide, ester, hydrazone, disulfure, etc…), soit par introduction d'un bras espaceur entre le PA et les dérivés de squalène dans le but de moduler par la suite la libération du PA à partir des nanoparticules (NPs). Une fois le couplage réalisé, j'ai pris en charge la formulation de ces prodrogues squalénisées en NPs puis de leur caractérisation. J'ai procédé ensuite à l'étude de leur efficacité pharmacologique in vitro et in vivo.

Les premiers nanomédicaments que j'ai synthétisés étaient destinés au traitement du cancer. Par la suite, j'ai orienté mon travail de recherche vers le traitement des désordres neurologiques, tels que l'ischémie cérébrale, les lésions de la moelle épinière et la douleur. Le traitement de la douleur est devenu mon domaine principal de recherche.

La description de mon travail de recherche se divise en 3 axes principaux : nanomédicaments pour le traitement du cancer, nanomédicaments neuroprotecteurs et nanomédicaments pour le traitement de la douleur.

Nanomédicaments à base de squalène pour le traitement du cancer (2005-2011):

La gemcitabine (dFdC), analogue nucléosodique anticancéreux, a été la première molécule à être couplée à l'acide squalénique (5 unités isoprènes). Ce couplage s'est fait via un lien amide et le bioconjugué issu de ce couplage (SQdFdC) s'est spontanément auto-assemblé dans l'eau en NPs (130 nm). La diffusion de rayons X aux petits angles (SAXS) ont mis en évidence une structure hexagonale inverse (Couvreur et al. SMALL 2008). Les NPs de SQdFdC ont montré une cytotoxicité très élevée sur différentes lignées cancéreuses humaines et murines (y compris résistantes) par rapport à la dFdC libre. In vivo, ces NPs ont montré une meilleure inhibition de la croissance tumorale et un meilleur taux de survie par rapport à la dFdC libre sur des modèles expérimentaux de cancer (Couvreur et al. Nano Lett 2006). L'étude du mécanisme d'action a révélé une forte interaction entre les NPs de SQdFdC et les lipoprotéines (LPs), permettant ainsi un ciblage indirect des cellules cancéreuses qui surexpriment les récepteurs aux LPs (Sobot et al. Nat. Commun. 2017). Dans le but d'étudier l'influence de la longueur de la chaîne polyisoprénique sur la formation des NPs et sur l'efficacité anti-cancéreuse, une série de prodrogues de gemcitabine avec des longueurs de chaines allant de 1 à 6 unités isoprènes ont été synthétisées (Maksimenko et al. Cancer Lett. 2013). Toutes ont pu s'auto-assembler en NPs, mais avec des stabilités variables nécessitant l'adjonction de squalène-PEG à 20% (m/m) (pour les chaînes de 1 à 3 unités isoprènes). Bien que les essais sur différentes lignées cellulaires cancéreuses aient montré que la prodrogue à 3 unités isoprènes était la plus active (confirmé également in vivo), la nanoformulation nécessite cependant d'être optimisée.

Nanomédicaments neuroprotecteurs à base de squalène (Depuis 2011-en cours):

L'adénosine qui est un neuromodulateur endogène du système nerveux central, agit en activant des récepteurs spécifiques A1, A2A, A2B et A3. Elle intervient dans les processus de transfert d'énergie, dans la transduction de signal, agit sur les vaisseaux sanguins, etc… Elle présente un fort potentiel neuroprotecteur dans les troubles neurologiques, notamment au niveau de l'ischémie cérébrale et le trauma de la moelle épinière. Cependant, son usage clinique est limité par son temps de demi-vie plasmatique extrêmement court qui limite de manière sérieuse son efficacité. Le couplage de l'adénosine (Ad) à l'acide de squalène acétique SQ (chaîne à 6 unités isoprènes) via une liaison amide a permis de résoudre ces problèmes. Ainsi, la prodrogue issue de ce couplage a donné des NPs de 120 nm et l'analyse de leur structure par SAXS a révélé une phase de type hexagonale inverse. Ces NPs Ad-SQ ont tout d'abord été appliquées à l'ischémie cérébrale. De ce fait, l'administration par voie IV de ces NPs en pré-ou post-ischémie a permis de diminuer significativement le volume de l'infarct dans un modèle d'ischémie cérébrale murin. Nous avons pu également démontrer que ces NPs avaient significativement prolongé le temps de circulation de l'Ad dans le sang, favorisant ainsi une microcirculation sanguine améliorée dans le cerveau, d'où une reperfusion plus efficace et un meilleur effet neuroprotecteur (Gaudin et al. Nat. Nanotechnology 2014). Le mécanisme d'action de ces NPs d'Ad-SQ a été étudié à partir de cellules endothéliales immortalisées de cerveau humain et a révélé que celles-ci n'interagissaient pas directement avec les récepteurs à l'adénosine, mais formaient un réservoir intracellulaire d'adénosine. En effet, après internalisation, le bioconjugué d'AdSQ est clivé pour libérer l'adénosine. Celle-ci finit par être effluée par les cellules vers le milieu extracellulaire, où elle peut ainsi activer les récepteurs spécifiques situés au niveau des membranes des cellules avoisinantes (Rouquette et al. J. Pharmacol. Exp. Ther. 2019). L'effet neuroprotecteur de ces NPs d'Ad-SQ a été également évalué sur un modèle de rat sujet à une lésion de la moelle épinière. L'étude a montré une récupération motrice spectaculaire chez les rats lésés contrairement à l'Ad libre. Ceci a été confirmé par l'analyse de la moelle épinière par microscopie électronique en transmission montrant une régénération des axones myélinisés (Gaudin et al. Nat. Nanotechnology 2014).

• Perspective actuelle : Nous souhaitons à présent tester le potentiel neuroprotecteur et neurorégénérateur de ces NPs Ad-SQ sur la régénération du nerf sciatique après écrasement (grade 3) sur un modèle de rat après leur administration par voie intraveineuse versus l'injection intraneurale.

Nanomédicaments à base de squalène ciblant la douleur (Depuis 2014):

Dans cette étude, nous avons pu montrer que pour la première fois les Leu-enképhalines (LENK) au temps de demi-vie plasmatique court, pouvaient devenir efficaces pharmacologiquement une fois couplées au squalène SQ (5 unités isoprènes), via une liaison amide (Am), ou via un bras espaceur, tel que le dioxycarbonyl (Diox), ou le diglycolate (Dig). Les prodrogues LENK-SQ résultant de ce couplage ont toutes montré des propriétés d'auto-assemblage en milieu aqueux. Cette nanoformulation à base de squalène a permis, d'une part de protéger la LENK de la métabolisation rapide dans le plasma et d'autre part, de lui conférer un effet antinociceptif significatif dans un modèle de douleur inflammatoire chez le rat (test de Hargreaves). Il est important de souligner que cet effet antinociceptif a duré 3 fois plus longtemps qu'avec la morphine. Un prétraitement avec un antagoniste des récepteurs opioїdes ne traversant pas la BHE, comme la méthylnaloxone a complètement antagonisé l'effet antinociceptif des nanoparticules de LENK-SQ, démontrant ainsi que celles-ci agissent via les récepteurs opioïdes périphériques (Feng et al. Sci. Adv. 2019). De plus, l'étude de biodistribution des NPs LENK-SQ fluorescentes a montré une forte accumulation des celles-ci au niveau de la patte oedémateuse, mais aussi dans le foie, la rate et les poumons alors qu'aucun signal n'a pu être détecté au niveau cérébral, ce qui confirme bien l'effet périphérique de ces nanoparticules. Enfin, une étude toxicologique a montré que malgré l'accumulation des NPs dans le foie, les taux de transaminases étaient normaux, tout comme les coupes histologiques, confirmant ainsi l'innocuité de ces NPs. Cette étude représente une approche innovante et prometteuse permettant une distribution ciblée du neuropeptide endogène LENK dans les tissus oedémateux pour soulager efficacement la douleur inflammatoire. Je viens de déposer un projet ANR sur ce sujet qui a passé la première phase de l'évaluation.

• Recherche actuelle : La douleur chronique est un véritable phénomène de société. Un Français sur deux souffre de douleurs aiguës ou chroniques. Cela représente, pour des millions de personnes, une dégradation considérable de la qualité de vie au quotidien. Le vieillissement de la population et l'accroissement des maladies chroniques sont autant de tendances qui appellent à l'amplification, dans les années à venir, des problématiques relatives à la prise en charge de la douleur. Dans ce contexte, un grand nombre de personnes âgées souffrent de problèmes d'insuffisance rénale, résultant d'une consommation chronique d'anti-inflammatoires non stéroїdiens. En tenant compte de cette problématique, j'ai entrepris d'orienter ma recherche afin de répondre aux besoins de cette catégorie de patients trop souvent négligée. Ainsi, en explorant une voie d'administration plus adaptée aux douleurs chroniques telle que la voie transdermique, j'envisage de développer un patch transdermique antidouleur à base de NPs de LENK-SQ. Celui-ci devrait permettre au patient de contrôler par lui-même l'administration de la LENK en toute sécurité. Mon travail actuel a pour objectif de promouvoir un effet systémique de ces LENK à partir de NPs de LENK-SQ en procédant à des essais de pré-formulation et formulation de ces NPs puis en évaluant le passage transcutané des prodrogues LENK-SQ sur des cellules de Franz avec des biopsies de peau d'oreilles de cochon congelées. Comparativement à l'utilisation des dérivés morphiniques, un tel patch est susceptible de générer un effet anti-douleur prolongé tout en étant dénué d'effets secondaires.

Encadrement doctoral et scientifique :

♦ Thèse en cours : (1 en cours)

Etudiants de niveau M2 8

Etudiants de niveau L3 ou M1 7

Etudiants de niveau L2 : BTS ou DUT 9

Etudiants post-doctorants 2

Communications

• Conférences en tant qu'orateur invité dans un congrès national et international :

• Congrès de la Société Française d'étude et de traitement de la douleur (SFETD, chapitre Français de l'IASP), du 18 au 20 novembre 2020 (congrès virtuel). « Un nouveau nanomédicament à base de Leuenképhaline pour traiter efficacement la douleur sans créer d'addiction » Lepetre-Mouelhi S.

(Conférence). • Communications orales : • Translational Nanomedicine International Meeting, Angers, France, August 2014 « Squalenoyl adenosine nanoparticles and neuroprotection : first steps from bench to bedside » Gaudin, A., Lepetre-Mouelhi, S., Yemisci, M., Tagit, O., Hildebrandt, N., Andrieux, K., Dalkara, T. and Couvreur, P. (Prix meilleur poster)

• 14 èmes Journées de l'Ecole Doctorale ED 425, Châtenay-Malabry, France, Juin 2014 « Squalene-based nanoparticles and plasma lipoproteins: characterization of molecular interactions and evaluation of their involvement in the therapeutic response. » Sobot, D., Mura, S., Dalbin, L., Lepetre-Mouelhi, S. and Couvreur, P. (Prix meilleur poster).

• 40th Annual Meeting & Exposition of the Controlled Release Society, Honolulu, Hawaii, USA, July 21 -24, 2013 «Polyisoprenoyl gemcitabine conjugates self assemble as nanoparticles, useful for cancer therapy» Maksimenko A., Mura S., Nicolas J., Harrisson S., Sliwinski E., Zouhiri F., Mougin J., Bui D.T., Lepetre-Mouelhi S., Lepeltier E., Bourgaux C., Desmaële D., Couvreur P.

• NAD Project Annual Meeting, Milan (Italy), July 2013, « Passage of nucleoside analogues accross the Blood-Brain Barrier using squalenoyl nanovectors » Gaudin, A., Lepetre-Mouelhi S., Sobot D., Tajit O., Hildebrandt N., Andrieux K., Couvreur P.

• Journées de Chimie Organique 2013, Palaiseau (France) September 24-26, 2013 17-18 juin 2013 «Polyisoprenoyl Gemcitabine conjugates self assemble as nanoparticles useful for cancer therapy » Lepetre-Mouelhi S., Zouhiri F., Sliwinsky E., Maximenko A., Mougin J., Mura S., Lepeltier E., Bourgaux C., Couvreur P.

• 13 èmes Journées de l'Ecole Doctorale ED 425, Faculté de Pharmacie de Châtenay-Malabry (Prix du meilleur poster) 17-18 juin 2013 « Passage of nucleoside analogues across the Blood-Brain Barrier using squalenoyl nanovectors» Gaudin A., Lepetre-Mouelhi S., Sobot D., Tajit O., Hildebrandt N., Andrieux K., Couvreur P.

• 24ème colloque de l'IFSBM, Villejuif, 21 mai 2013 «Passage of nucleoside analogues accross the Blood-Brain Barrier using squalenoyl nanovectors» Sobot D., Gaudin A., Lepetre-Mouelhi S., Andrieux K., Couvreur P.

• Joint Franco-Belgian-British multinational meeting on blood-brain interfaces, Arras, 15-17 may 2013 « Passage of nucleoside analogues across the Blood-Brain Barrier using squalenoyl nanovectors» Gaudin A., Lepetre-Mouelhi S., Tajit O., Hildebrandt N., Andrieux K., Couvreur P.

• 12 èmes Journées de l'Ecole Doctorale ED 425, Faculté de Pharmacie de Châtenay-Malabry (Prix du meilleur poster) juin 2012 «Nanoparticules Adénosine-Squalène et ischémie cérébrale : caractérisation du passage de la BHE, évaluation de l'efficacité pharmacologique et théragnostique» Gaudin A., Lepetre-Mouelhi S., Boucher, M., Mériaux, S., Andrieux K., Couvreur P. Conventional chemotherapy is often hampered by poor diffusion through biological barriers such as epithelium, endothelium, cell membranes, and a rapid metabolization and clearance from the body. In addition, it frequently leads to a non-specific biodistribution of the drug in the body thus generating severe side effects due to drug losses in healthy organs and tissues.

One promising approach to circumvent these drawbacks is the use of nanoscaled drug delivery systems. In that respect, nanotechnology in drug delivery has gained great attention in recent years. Nanocarriers can be used to increase drug stability and solubility, deliver drug to specific site in the body, thus minimizing the side effects. The concept of nanocarrier was inspired by the idea of the "magic bullet", proposed by Paul Ehrlich at the beginning of 20th century. [START_REF] Strebhardt | Paul Ehrlich's Magic Bullet Concept: 100 Years of Progress[END_REF] However, it was only until the 1970s that the first nanoparticles were developed by the group of Peter Speiser. 2 Nanocarriers have been extensively studied since then.

Practically, the drug can be encapsulated into nanoparticles (NPs). This nanoparticular formulation allows protection to the drug from degradation, an efficient targeting towards diseased tissues, a better intracellular diffusion and a controlled release. 3,4 A nanoparticle is composed of a core surrounded by a corona (Figure 1). Typically, the core material encapsulates the drug. It is constituted of either organic or inorganic compounds from natural and synthetic sources, such as polymeric materials, lipids, metal oxides, etc… The corona, for its part, is a natural protein layer spontaneously formed around nanoparticles when exposed to biological media. The corona is constituted of plasma proteins, such as Albumin, immunoglobulin G (IgG), fibrinogen, apolipoproteins, coagulation factors, complement proteins... The formation of the protein corona around nanoparticles is dependent on physico-chemical properties of NPs including nanoparticle size, surface charge, hydrophobicity, surface chemistry and is characteristic of the environment in which are dispersed NPs. The corona can also be synthetic when designed to (i) confer stealth properties to the nanoparticle against immune system or/and (ii) enhance their targeting ability. In the first case, the corona is composed of Polyethylene Glycol (PEG) chains covalently linked or adsorbed onto the surface of the NP in order to prevent interactions between the particle surface and the plasma proteins (thus avoiding recognition by reticuloendothelial system). 5,6 In the second case, for a better targeting, the corona may be constituted of PEG-chains grafted to specific ligands or only specific ligands adsorbed on the surface of the nanoparticle. 7,8 The corona affects the fate of the nanoparticle in the body. It determines the rate of clearance from the bloodstream, the volume of distribution, the organ disposition, and the rate and route of clearance from the body.

These drug delivery nanosystems may be classified in 2 categories: organic NPs (e.g., polymeric, liposomes, micelles, etc.) and inorganic NPs (e.g., gold, silica, iron oxide, etc.).

Currently, there are a number of nanomedicines that have been approved for clinical use, either by the Food and Drug Administration (FDA) in the United States (51 FDA-approved nanomedicines), or the European Medicines Agency (EMA) in the European Union and more than 77 nanomedicines are in clinical trials. 9,10 Among FDA-approved nanomedicines, most of organic NPs are liposomal systems and are mainly used in cancer therapy.

The liposomes are spherical-shaped vesicles that are composed of one or more phospholipid bilayers, which closely resembles the structure of cell membranes. The ability of liposomes to encapsulate hydrophilic or lipophilic drugs have allowed these vesicles to become useful drug delivery systems. Liposomes became the first nanomedicines in FDA clinical trials. The first approved nanomedicine was Doxil® (FDA 1995), encapsulating the doxorubicin for cancer treatment. 11 This PEGylated liposomal nanomedicine was proven effective in the reduction of cardiotoxic side effects of doxorubicin treatment. Doxil® has been approved for Karposi's sarcoma, ovarian cancer, and multiple myeloma, as well as for metastatic breast cancer. 12 Then, other liposomal carriers were also developed for cancer therapy such as DaunoXome® (daunorubicin), Myocet® (doxorubicin), Marqibo® (vincristine) and more recently, Onivyde® (irinotecan). [13][14][15][16] Onivyde is a topoisomerase I inhibitor approved as a second line treatment for metastatic pancreatic cancer. There are still very few options for these patients and no consensus of care has been established. 17,18 Vyxeos® (FDA 2017) is the first clinically approved liposome to deliver a synergistic combination of 2 drugs, daunorubicin and cytarabine for the treatment of acute myeloid leukemia. 19,20 The majority of these formulations are not PEGylated, with the exception of Doxil® and Onivyde®. 21 All of these nanoformulations are passively targeted with the ability to preferentially accumulate at tumor sites via the enhanced permeation and retention (EPR) effect. 22 The liposomes are also used in some others clinical applications, such as general anesthesy with Diprivan®, 23,24 or for the treatment of some bacterial/fungal infections, with Ambisome® loading amphotericin B. 25,26 The FDA-approved Visudyne®, a light-activated liposome loading verteporfin, was used in photodynamic therapy for choroidal neovascularization due to age-related macular degeneration. 27 Two other liposomes Inflexal V and Epaxal were commercialized successfully as vaccines. These virosome-based vaccines incorporated inactivated influenza and hepatitis A virus respectively. 28 It should be noted that the virosomes Inflexal V and Epaxal have since been phased out of the clinic.

Hence, liposomal drug delivery has become a clinical staple and it is clear that with improvements in nanotechnology, they will continue to evolve into next generation nanomedicines into the future.

Other non liposomal organic nanoformulations were also approved such as protein-based nanoparticles. Abraxan®, is a protein nanoparticle which is used in cancer therapy with paclitaxel formulated as albumin bound. This formulation avoid the use of toxic solvent such as Cremophor, normally required for paclitaxel delivery. 29 Since its approval in 2005, there has been a shift from unmodified protein to the utilization of more highly engineered particle complexes in order to gain active targeting functionality. Ontak®, (Denileukin Diftitox) (approved in 2008), is an example of an approved engineered fusion protein combining cytotoxic molecule (diphteria toxin) with targeting proteins (IL-2 receptor antagonist). 30,31 Ontak® (approved in 2008), appeared to be an effective treatment to treat the aggressive form of non-Hodgkin's Peripheral T-cell Lymphomas.

Regarding to polymeric nanoparticles, they can be divided in two categories: (a) polymer-drug conjugates for increased drug half-life and bioavailability, and (b) degradable polymer architectures for controlled release applications. Polymeric nanomedicines can be used as direct drugs. The best example is Copaxone® (glatiramer acetate), which is a random copolymer composed of L-glutamic acid, L-alanine, L-lysine and L-tyrosine approved in 1996, and was a revolutionary treatment that acted as an immunomodulator in the treatment of multiple sclerosis. 32 The hydrophilic polymers have been most extensively utilized in nanomedicine. More frequently, drugs are attached to a hydrophilic polymer to increase circulation or improve biocompatibility/solubility and the most well established polymer is poly (ethylene glycol) (PEG). 33 . Such systems include the extremely popular and successful drug Neulasta® (approved in 2002), a PEGylated granulocyte colony stimulating factor which has been for chemotherapy induced neutropenia, or Cimzia® composed of PEGylated Certolizumab antibody for the treatment of Crohn disease ( 2008), Rheumatoid arthritis ( 2009), Psoriatic Arthritis and Ankylosing Spondylitis (2013). 10,34 In 2014, Plegridy® (PEGylated IFN beta-1a) 35 was approved for the treatment of relapsing multiple sclerosis and in 2015, Adynovate®, a PEGylated anti-hemophilic factor VIII, was approved for the treatment of hemophilia A, both in terms of preventing bleeding episodes, or treating acute bleeding. 36 More recently, Rebinyn®, the PEGylated glyco-protein drug, received its FDA approval in 2017 and is used for patients suffering from factor IX (FIX) deficiency also known as hemophilia. 37 It consists of PEG attached to recombinant DNA-derived coagulation FIX concentrate. Beyond just extending the circulation time of established drugs, biocompatible polymeric nanoparticles can be developed based on hydrophobic materials that facilitate controlled release of the therapeutic. In that respect, a long established polymer nanoparticle that has had significant success is based upon incorporation of leuprolide, a testosterone inhibiting drug, into a polylactide-co-glycolic acid (PLGA) nanoparticle. This drug is sold under the tradename Eligard® (FDA 2002) and is indicated as an effective treatment for the symptoms of prostate cancer. 38 Micellar nanoparticles are self-assembled polymeric amphiphiles nanoparticles in which internal core is hydrophobic and can be used to encapsulate poorly water soluble drugs, whereas the exterior surface is polar enough to allow dissolution in aqueous solution. To date, a traditional micellar formulation of estradiol (Estrasorb™) is the only FDA-approved micelle, indicated as a topical treatment for moderate to severe vasomotor symptoms of menopause. 10 In the past few years, lipid nanoparticles have emerged as one of the most advanced and efficient mRNA delivery platforms. The lipid nanoparticles Onpattro® (Patisiran®) delivering siRNA for the silencing of a specific gene responsible for expression of transthyretin (which can cause hereditary transthyretin amyloidosis), were recently approved (2018). 39 In 2020, an effective vaccine against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (which emerged in late 2019) received a fast-track approval. In that respect, BNT162b2 (Pfizer/BioNTech) and mRNA1273 (Moderna) received conditional marketing authorisation (CMA) approval in the EU after clinical trials to fulfil the unmet medical need. [START_REF] Jackson | An MRNA Vaccine against SARS-CoV-2 -Preliminary Report[END_REF] The candidate vaccine mRNA-1273 and BNT162b2 are constituted of lipid nanoparticles encapsulating a nucleoside-modified messenger RNA (mRNA) that encodes the SARS-CoV-2 spike (S) glycoprotein. Hence, the approval of the first vaccine effective against the devastating SARS-CoV-2 pandemic using lipid nanoparticles represents a major milestone for nanomedicines.

Crystalline nanoparticles, for their part, are a particular case of nanomedicines because the NPs themselves are composed of 100% drug compound. The increased surface area for dissolution, owing to the nanoscale dimensions, leads to increased dissolution velocity and also increased saturation. This last point is particularly important -at the nanoscale, saturation solubility increases with decreasing particle size, leading to enhanced driving forces for diffusion-based mass transfer through biological structures (e.g. walls of the gastro-intestinal tract). [START_REF] Shegokar | Nanocrystals: Industrially Feasible Multifunctional Formulation Technology for Poorly Soluble Actives[END_REF] This approach is expected to be broadly applicable to a range of drugs that suffer from solubility issues. Rapamune®, the first FDA-approved nanocrystal in 2000, is an organic nanocrystalline drug containing the active component sirolimus (a bacterial derived 914 Da macrocyclic immunosuppressant) used to prevent organ rejection following transplantation, specifically for kidneys. [START_REF]Sirolimus: AY 22989, NSC 226080, NSC 606698, Rapamycin, Rapamune??[END_REF] Following the approval of Rapamune several other approved nanocrystalline drug formulations have been produced in this manner : Megace® (2001, anti-anorexic), Ritalin LA® (psychostimulant, 2002), EquivaBone (bone substitut, 2009), Sustenna (2014, schizophrenia therapy). 10 Approved inorganic nanoparticles, for their part, are mainly iron-based colloids. Iron oxide nanoparticles have an emerging research profile as contrast enhancement reagents for magnetic resonance imaging, however the majority of FDA-approved materials are indicated as iron replacement therapies. There are several approved compounds such as Feraheme® (FDA 2009), Venofer® (FDA 2000), Ferrlecit® (FDA 1999), Dexferrum® (FDA 1996), which are employed to treat anemia related to chronic kidney disease (CKD). [START_REF] Coyne | Anemia Management in Chronic Kidney Disease: Intravenous Iron Steps Forward[END_REF] Each of these is composed of an iron oxide core, coated with hydrophilic polymers (e.g. dextran, sucrose), which provide slow dissolution of the iron following intravenous injection. [START_REF] El-Din | Magnetite Nanoparticles as a Single Dose Treatment for Iron Deficiency Anemia[END_REF] Feraheme (ferumoxytol) is currently being investigated as an imaging agent. [START_REF] Bashir | Emerging Applications for Ferumoxytol as a Contrast Agent in MRI: Emerging Applications of Ferumoxytol[END_REF] In contrast to iron replacement therapies, Nanotherm™, which consists of aminosilane-coated superparamagnetic iron oxide NPs (SPIONs) received an EU-wide regulatory approval in 2001. These particular iron oxyde NPs were designed for tumor therapy (glioblastoma) using local tissue hyperthermia induced by alternating magnetic field. [START_REF] Thiesen | Clinical Applications of Magnetic Nanoparticles for Hyperthermia[END_REF] The local heating of the tumor environment leads to programmed and non-programmed cell death.

As regards the diagnostic imaging, starting with iron oxide nanoparticles, Feridex® (Endorem®, FDA 1996) and Resovist® (EMA 2001) were primarily designed for liver imaging. Due to the lack of users, Feridex® has been withdrawn from the market in 2008, and Resovist® is current available in only limited countries, such as Japan. 9,10,[START_REF] Klibanov | Microbubble Contrast Agents: Targeted Ultrasound Imaging and Ultrasound-Assisted Drug-Delivery Applications[END_REF] Silica coated superparamagnetic iron oxide (SPIO) Ferumoxsil (Lumirem®, or GastroMARK®) was approved as a useful oral contrast agent for darkening of bowel loops, but was also taken off the market in 2009 due to lack of users. [START_REF] Wáng | A Comprehensive Literatures Update of Clinical Researches of Superparamagnetic Resonance Iron Oxide Nanoparticles for Magnetic Resonance Imaging[END_REF] Sienna plus® is an organically coated SPIO particles with a particle size of 59 nm which is used to detect lymph nodes, was CEapproved for marketing and sales in Europe, and ARTG (Australian Register of Therapeutic Goods) listed for Australasia. [START_REF] Klibanov | Microbubble Contrast Agents: Targeted Ultrasound Imaging and Ultrasound-Assisted Drug-Delivery Applications[END_REF] In addition to MRI contrast enhancers, particles can be used as intravenous ultrasound enhancing agents. In these cases, particles typically take the form of micron-sized microbubbles. [START_REF] Schutt | Injectable Microbubbles as Contrast Agents for Diagnostic Ultrasound Imaging: The Key Role of Perfluorochemicals[END_REF][START_REF] Klibanov | Microbubble Contrast Agents: Targeted Ultrasound Imaging and Ultrasound-Assisted Drug-Delivery Applications[END_REF] Few of these products are approved and currently used in the clinic, for example, Definity (FDA 2001) and SonoVue (EMA 2001) are fluorocarbons or sulfur hexafluoride encased in lipid shells, respectively. 9 Optison (FDA 1997 and EMA 1998) is another ultrasound contrast agent formulated as human serum albumin encased perflutren. 9 Recently, BTXR3/Hensify a crystalline hafnium oxide nanoparticle with negatively charged phosphate coating, received aproval in 2019. [START_REF] Bonvalot | NBTXR3, a First-in-Class Radioenhancer Hafnium Oxide Nanoparticle, plus Radiotherapy versus Radiotherapy Alone in Patients with Locally Advanced Soft-Tissue Sarcoma (Act.In.Sarc): A Multicentre, Phase 2-3, Randomised, Controlled Trial[END_REF] These inorganic nanoparticles enhances external radiotherapy via a physical mode of action that relies on hafnium's natural radioenhancing properties.

Through these bibliographic data, we could highlight the significant expansion in clinically-validated nanomedicine-based drugs and suggest that we are likely to see an increase in the number of FDAapproved therapeutic, especially with (i) lipid nanoparticles carrying oligonucleotide-based drugs (such as SiRNA and mRNA) for gene therapy, (ii) nanomedicines with active targeting, (iii) smart nanoparticles releasing the drug "on demand" and (iv) sophisticated imaging agents, over the foreseeable future. It is however important to note that nanoparticles were introduced to decrease some of the challenges of traditional drug delivery, but come with their own difficulties and limitations that will continue to be an important area of research. Therfore, development of new nanoformulations is still urgently needed to address current global health challenges, which requires collaborative efforts of scientists arising from different disciplines. In this context, Couvreur's team developed a very promising nanomedicine plateform for the treatment of severe diseases, using natural and biocompatible materials such as squalene lipid.

2.

The squalenoylation nanotechnology: an innovative and efficient nanoformulation : Squalene (SQ) 1, a natural endogenous lipid belonging to the terpenoid family, is an intermediate metabolite in the synthesis of cholesterol. It is widespread in nature and with reasonable amounts found in shark liver oil, olive oil, wheat germ oil and rice bran oil. It received its name because of its occurrence in shark liver oil (Squalus spp.). [START_REF] Kelly | Squalene and Its Potential Clinical Uses[END_REF] In human body, squalene is produced in the liver and the skin, transported by very low density lipoproteins (VLDL) and low density lipoprotein (LDL) in the blood, and then secreted in large quantities by the sebaceous glands. [START_REF] Simonen | The Distribution of Squalene and Non-Cholesterol Sterols in Lipoproteins in Type 2 Diabetes[END_REF][START_REF] Reddy | Squalene: A Natural Triterpene for Use in Disease Management and Therapy[END_REF] Furthermore, SQ is well tolerated whether injected intravenously or consumed orally and is distributed to various tissues. [START_REF] Miettinen | Serum Concentration and Metabolism of Cholesterol during Rapeseed Oil and Squalene Feeding[END_REF][START_REF] Gylling | Postabsorptive Metabolism of Dietary Squalene[END_REF] Because it is of biological origin and frequently used as excipient in several pharmaceutical formulations for oral and parenteral administration, as well as a dietary supplement, SQ is favourably predisposed from a toxicological standpoint. However, it has never been used as covalent partner for drug delivery and targeting purposes before the introduction in 2006 of the «Squalenoylation nanotechnology» by our team which was led by Patrick Couvreur. [START_REF] Couvreur | Squalenoyl Nanomedicines as Potential Therapeutics[END_REF] This new strategy consists in coupling the SQ to therapeutic agents using a chemical biocleavable linkage. Concretely, the SQ has to be first fonctionnalized, by introducing a functional group on one of its extremities. In that respect, the selective oxidation of one of the 2 terminal double bonds of SQ (C30) gives in 2 steps the corresponding monoepoxide 3 according to the Van Tamelen method with a global yield of 35% (scheme 1). [START_REF] Van Tamelen | The Selective Oxidation of the Terminal Double Bonds in Squalene[END_REF] Scheme 1 : Functionalization of squalene Then, starting from monoepoxysqualene 3, we can have access to a large library of SQ derivatives such as squalenic acid, squalenol, squalenamine, SQ maleimide, etc… allowing to link SQ moiety to a huge variety of drug molecules using a chemical biocleavable linkage, such as ester, amide, hydrazone, carbamate, disulfide, etc… In order to modulate the release of the drug, different chemical linkers i.e. succinate, PEG, diglycolate, maleimide, etc… were also introduced between SQ and drug moieties. Owing to the rigid structural property of SQ, these SQ-drug conjugates may self-assemble in aqueous media without the use of any surfactant into compacted nanoparticles (100 nm) with high drug loadings (Figure 2). The squalenoylation was first applied to small hydrophilic molecules such as nucleoside analogues (ie Gemcitabine), and was then extended to a wide variety of drugs such as highly charged macromolecules. [START_REF] Feng | Preparation and Characterization of Modular Squalene-Based Nanosystems for Controlled Drug Release[END_REF] This new concept offers numerous advantages in comparison with the conventional nanocarriers. Unlike polymer nanoparticles or liposomes nanoformulations which encapsulate by physical means the active principles into their matrices, the SQ-drug conjugates nanoparticles employ a chemical encapsulation approach, thus avoiding the "burst" release phenomenon corresponding to the premature release of the drug. In all cases, the bioreversible SQ based bioconjugates (prodrugs) which constitute the nanoparticles, undergo an enzymatic and/or a chemical transformation in vivo to release the active drug. The nanoparticle conformation allows protection to drug molecules from degradation/metabolization which results in greater plasmatic half-life, when a controlled and prolonged release of the drug may also be obtained. Moreover, these nanosystems facilitated drug's passage through biomembranes due to the lipophilic properties of SQ, resulting in an enhanced drug concentration within targeted tissues and/or cells, thus generating dramatic increase in drug pharmacological activity (in comparison with the parent compound) and a reduced toxicity. [START_REF] Feng | Preparation and Characterization of Modular Squalene-Based Nanosystems for Controlled Drug Release[END_REF] Concretely, since my recruitement by Institut Galien Paris-Sud in 2005 as postdoctoral fellow and then as an associate professor in 2006, my research work aimed first at developing the «squalenoylation technology». I took the responsability of the synthesis and formulation of a library of squalene-based prodrugs, and their formulation into nanoparticles. Then, my interests gradually turned toward the pharmacological evaluation in vitro and in vivo. This research work will be presented under 2 chapters. The first chapter corresponds to my previous work related to cancer field (2005-2010) with squalenoylation of anti-cancer gemcitabine (dFdC). The second chapter which represents my ongoing research (2011-ongoing), deals with neurological disorders with squalenoylation of neuroprotective drug and painkillers.

My research work will be presented in the form of questionnaire that we will try to answer.

• ANTICANCER SQUALENE-BASED NANOMEDICINES

Nucleoside analogues are a class of therapeutic agents with significant anticancer properties. However, these molecules possess some serious limitations that often restrict their use, such as short plasma half-life, rapid metabolism, the induction of resistance, and the advent of severe side effects. [START_REF] Galmarini | Nucleoside Analogues: Mechanisms of Drug Resistance and Reversal Strategies[END_REF] Gemcitabine is an anticancer nucleoside analogue active against various solid tumors, including colon, lung, pancreatic, breast, bladder, and ovarian cancers. [START_REF] Lund | Clinical and Preclinical Activity of 2′,2′-Difluorodeoxycytidine (Gemcitabine)[END_REF] However, it possesses important drawbacks like a poor biological half-life and the induction of resistance. With the objective of overcoming the above drawbacks, we designed a new squalene-based nanomedicine of gemcitabine and studied its anticancer efficacy in vitro in human and murine cancer cells and in vivo in experimental models of mice after intravenous administration. In a second step, we focused our efforts on understanding the mechanism of action of this nanomedicine (loading gemcitabine), and then, we decided to elaborate strategies to improve their anticancer efficacy. Finally, we designed a library of new bioconjugates for nanoformulation of gemcitabine using other terpenes and studied their anticancer activity.

State-of-art on Gemcitabine (dFdC) : mechanism of action and limitations

What is the mechanism of action of gemcitabine (dFdC) ?

Gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdC), is a deoxycytidine (dC) analog, active against a wide variety of solid tumors, such as pancreas, lung, and bladder cancers. [START_REF] Bildstein | Transmembrane Diffusion of Gemcitabine by a Nanoparticulate Squalenoyl Prodrug: An Original Drug Delivery Pathway[END_REF] Marketed as Gemzar, it acts as an antimetabolite, inhibiting ribonucleotide reductase and DNA synthesis leading to apoptosis. [START_REF] Cappella | Cell Cycle Effects of Gemcitabine[END_REF] Too hydrophilic to freely diffuse through plasma membranes, dFdC enters the cells mainly by facilitated diffusion through the human equilibrative nucleoside membrane transporters (hENTs). Once in the cytoplasm, dFdC is then phosphorylated into its active form, primarily into monophosphate mainly by deoxycytidine kinase and, to a lower extent, by other intracellular kinases. The monophosphate is further phosphorylated into di-and triphosphate forms, the latter being the only active metabolite which inhibits DNA synthesis (figure 3). However, the dFdC 4 has a narrow therapeutic index due to its short halh-life. Indeed, the dFdC undergoes rapid enzymatic deamination in the bloodstream by deoxycytidine deaminase (dCDA) into its corresponding inactive uracil derivative (2',2'-difluoro-2'-deoxyuridine ; dFdU), thus leading to a short half-life (figure 4). [START_REF] Abbruzzese | A Phase I Clinical, Plasma, and Cellular Pharmacology Study of Gemcitabine[END_REF][START_REF] S.; R | Change in Pharmacokinetic and Pharmacodynamic Behavior of Gemcitabine in Human Tumor Xenografts upon Entrapment in Vesicular Phospholipid Gels[END_REF] Therefore, the administration of high doses of drug is needed to achieve the desired therapeutic response but simultaneously, results in various adverse effects mainly myelosuppression. [START_REF] S.; R | Change in Pharmacokinetic and Pharmacodynamic Behavior of Gemcitabine in Human Tumor Xenografts upon Entrapment in Vesicular Phospholipid Gels[END_REF] Another limitation associated with the use of gemcitabine is the induction of anticancer drug resistance which is due to (i) the downregulation of deoxycytidine kinase, the enzyme responsible for the primary phosphorylation of gemcitabine, (ii) the downregulation of hENTs which limits the entry into the cells and (iii) the overexpression of deaminases that increases dFdC inactivation. [START_REF] Amrutkar | Pancreatic Cancer Chemoresistance to Gemcitabine[END_REF] For instance, in the case of leukemia, the cellular resistance to gemcitabine due to nucleoside transporter and dCK (deoxycytidine kinase) deficiency has been well documented. [START_REF] Galmarini | Resistance to Gemcitabine in a Human Follicular Lymphoma Cell Line Is Due to Partial Deletion of the Deoxycytidine Kinase Gene[END_REF] Thus, it appears clearly that the drug resistance is an important clinical concern in the gemcitabine-based chemotherapy.

What approaches had been developed to improve the therapeutic potential of gemcitabine?

When we started this project in 2005, many attempts have been made to improve dFdC stability to chemical and enzymatic hydrolysis. In that respect, long chain saturated and monounsaturated fatty acids were coupled to dFdC on its amino or hydroxyl group through amide or ester bond respectively. 67 In addition to the dFdC protection, this lipidation improved its lipophilicity, and thus, increased its interactions with the lipidic membranes and its cellular uptake, resulting in an increased cytotoxicity. The lipophilic derivatives of dFdC were found to have a higher cytotoxicity than gemcitabine itself. However, these chemical modifications led sometimes to more lipophilic compounds with poor solubility in water, which was an obstacle to the intravenous administration of these molecules. 68,[START_REF] Bergman | Antiproliferative Activity and Mechanism of Action of Fatty Acid Derivatives of Gemcitabine in Leukemia and Solid Tumor Cell Lines and in Human Xenografts[END_REF] PEG-dFdC water soluble conjugates were also developed and displayed a greater influence on the cellular cytotoxicity of pancreatic cancer cell lines in comparison to native gemcitabine. 68 In order to improve the half-life of dFdC and by the same time increase the concentration of the drug at the tumor site by EPR effect, nanoparticulate systems were used. One strategy consisted in incorporating the dFdC into biodegradable polymer chitosan, while another one consisted in encapsulating C18-dFdC prodrugs in liposomes. [START_REF] Arias | Superior Preclinical Efficacy of Gemcitabine Developed As Chitosan Nanoparticulate System[END_REF][START_REF] Brusa | Antitumor Activity and Pharmacokinetics of Liposomes Containing Lipophilic Gemcitabine Prodrugs[END_REF] The dFdC prodrugs (sometimes encapsulated into NPs) were also developed with the aim of increasing oral bioavailability of gemcitabine, resulting in either linear or branched unsaturated or saturated acid ester or amide derivative of gemcitabine (dFdC), some of which already reached clinical trials. [START_REF] Wang | Oral 4-( N )-Stearoyl Gemcitabine Nanoparticles Inhibit Tumor Growth in Mouse Models[END_REF] 2. Application of squalenoylation nanotechnology to dFdC : synthesis and characterization 2.1. How can we obtain nanoparticles of dFdC using squalene?

Using the « squalenoylation nanotechnology », the squalene derivative was coupled to dFdC and the resulting bioconjugates were able to self-organize in water as nanoassemblies which were compatible with intravenous administration.

2.1.1.

Synthesis of the squalene-based vector : the tris-nor-squalenic acid

For this purpose, 1,1',2-tris-norsqualenic acid (SQ Ac) 6 (C27) was synthesized in 2 steps from epoxysqualene 3 (C30) (scheme 2).

O 2,3-epoxysqualene H 5 IO 6, Et 2 O O H O OH CrO 3, H 2 SO 4 , 81% 79% 2 h, 20 °C Acetone, 0 °C1,1',2-trisnor-squalenic acid (SQAc) 3 5 6
Scheme 2 : Synthesis of 1,1',2-tris-norsqualenic acid (SQ Ac)

First, the epoxysqualene 3 underwent an oxidative cleavage in presence of periodic acid (H5IO6) leading to 1,1',2-tris-norsqualenaldehyde 5 (C27). Then, this latter was easily converted into the C27 tris-norsqualenic acid 6 (SQ Ac) using Jones reagent.

2.1.2.

Lipidization of dFdC by squalene : Synthesis of SQdFdC conjugate

The SQ Ac 6 was then activated into its mixed anhydride prior to be coupled via an amide bond to the 4-amino group of dFdC 4 (particularly subjected to deaminase). The mixed anhydride of SQ Ac was obtained in situ using ethyl chloroformate 7 in the presence of triethylamine. Then, condensation of dFdC with the mixed anhydride provided the tris-norsqualenoyl-gemcitabine (SQdFdC) conjugate 8 (55%) (scheme 3). [START_REF] Feng | Preparation and Characterization of Modular Squalene-Based Nanosystems for Controlled Drug Release[END_REF] Scheme 3 : Synthesis of SQdFdC

Formulation of SQdFdC conjugate into nanoparticles

For the formulation step, the squalenoyl-gemcitabine bioconjugate (SQdFdC) underwent nanoprecipitation technique. This original technique consists in a drop-wise addition of ethanolic solution of SQdFdC bioconjugate to a 5% aqueous dextrose solution under magnetic stirring leading to nanoassemblies without any added surfactant, which from a toxicological point of view is a major advantage. Then, the ethanol was completely evaporated to obtain an aqueous suspension of SQdFdC NPs, with a mean diameter of 130 nm (± 9 nm) with a unimodal size distribution (polydispersity index corresponding to 0.12) and a zeta potential of -20 mV. Because dFdC is not physically encapsulated but is covalently linked to the SQ lipid nanocarrier, a high drug loading of 41% wt was reached. [START_REF] Couvreur | Squalenoyl Nanomedicines as Potential Therapeutics[END_REF] 2.2. What is the shape and supramolecular structure of SQdFdC NPs?

The supramolecular structure of SQdFdC nanoassemblies was investigated by X-ray diffraction and electron microscopy. The cryogenic transmission electron microscopy (cryoTEM) revealed hexagonal or multifaceted shape with size ranging from 27 to 125 nm and an average value of approximately 104 nm (Figure 5A andB). Each nanoassembly was surrounded by an external shell (approximately 3.27 nm thick) and displayed an internal structure made of reticular planes. Depending their orientation, the nanoassemblies clearly exhibited a sixfold symmetry or reticular planes (Figure 5A-D). 73 The SQdFdC nanoassemblies were also examined by X-ray diffraction at both small and wide angles and displayed an inverse hexagonal structure formed by close-packed cylinders in which the aqueous cores containing the hydrophilic head-group of SQdFdC molecule were surrounded by hydrophobic squalene moieties (Figure 5E). [START_REF] Couvreur | Discovery of New Hexagonal Supramolecular Nanostructures Formed by Squalenoylation of an Anticancer Nucleoside Analogue[END_REF] 3. Biological activity of SQdFdC nanoparticles : in vitro and in vivo results

Does the squalenoylation nanotechnology improve the anticancer activity of dFdC in vitro?

The anticancer activity of SQdFdC NPs was first evaluated in vitro in comparison with dFdC free on several types of cancer cell lines : MCF-7 (human breast adenocarcinoma), KB3-1 (human nasopharyngeal carcinoma), L1210wt (murine leukemia), L1210 10K (a resistant variant of L1210), CEM/ARAC8C (a hENT1-deficient human leukemia).

The results showed that SQdFdC NPs were about 6-to 8-fold more cytotoxic than the gemcitabine itself on both MCF-7 and KB3-1 cell lines using the MTT assay. [START_REF] Couvreur | Squalenoyl Nanomedicines as Potential Therapeutics[END_REF] Interestingly, in L1210 wt cell line, cytotoxicity of SQdFdC NPs, in comparison to dFdC, was lower (Table 1).

The efficacy of dFdC NPs was also tested on resistant cancer cell lines. Indeed, control over the spread of cancer cells and metastasis is obviously a very important challenge in chemotherapy, since it accounts for 90% of lethality in cancer patients. In many clinical situations, gemcitabine like other anticancer agents is not efficient in inhibiting metastasis, because it doesn't reach the metastasized tissues in sufficient concentrations. A typical example is leukemia, with abnormal cells which generally spread to the tissues such as bone marrow, spleen and liver. [START_REF] Behrmann | Acute Myeloid Leukemia and the Bone Marrow Niche-Take a Closer Look[END_REF] In that respect, the L1210 10K cell line, which is a resistant variant of L1210 cell line, was first tested. This resistant cell line is ∼5000-fold less sensitive to gemcitabine (dFdC) than the parent cell line because of the deficiency in dCK responsible for the phosphorylation of gemcitabine. Indeed, when tested in L1210 10K cells, a dramatic lowering in the cytotoxicity of dFdC compared to that of the parent cell line L1210 wt was observed, thus confirming a high resistance of these cells to dFdC (Table 1; Figure 6A). [START_REF] Reddy | A New Nanomedicine of Gemcitabine Displays Enhanced Anticancer Activity in Sensitive and Resistant Leukemia Types[END_REF] However, the cytotoxicity of SQdFdC NPs, in comparison to dFdC, was impressively higher on L1210 10K cells (Table 1). Although SQdFdC NPs showed a lower cytotoxicity (3.26-folds) on L1210 10K cells in comparison to that on the L1210 wt cells, the decrease in cytotoxicity in resistant cells was appreciably lower than with dFdC. [START_REF] Reddy | A New Nanomedicine of Gemcitabine Displays Enhanced Anticancer Activity in Sensitive and Resistant Leukemia Types[END_REF] The anticancer activity of SQdFdC NPs was also tested in CEM/ARAC8C, a hENT1 transporterdeficient human leukemia cell line. Here again, the SQdFdC NPs exhibited 3.22-folds greater cytotoxicity than dFdC free (Figure 6B). Therefore, the formulation of dFdC with squalene, significantly improved its cytotoxicity in resistant leukemia cell lines.

Does the squalenoylation nanotechnology improve the anticancer activity of dFdC in vivo?

The anticancer activity of SQdFdC NPs was assessed in L1210 wt leukemia bearing mice. As apparent from the figure (Figure 7A), both the groups of SQdFdC NPs-treated mice (5 mg/kg and especially 15 mg/kg equivalent dFdC) showed greater control of the body weight loss, compared to the equivalent doses of dFdC, or control groups. These results correlated with survival curves corresponding to untreated and treated groups (Figure 7B). Indeed, both the doses of SQdFdC NPs caused significantly greater increase in the survival of mice than the dFdC free. Especially 15 mg/kg SQdFdC NPs exhibited impressive long-term survival of almost half of the mice (~43%)(Figure 7B). [START_REF] Reddy | A New Nanomedicine of Gemcitabine Displays Enhanced Anticancer Activity in Sensitive and Resistant Leukemia Types[END_REF] Hence, while in vitro, SQdFdC NPs appeared less cytotoxic on L1210 wt than dFdC free, in vivo, these NPs showed a greater efficacy than the free drug. Based on the fact that in culture cell media, the gemcitabine is not subjected to rapid metabolization as it was the case in vivo, for this reasons, it appeared more efficient in vitro than dFdC NPs which probably exhibited a delayed effect, due to progressive release the drug.

The anticancer activity of SQdFdC nanoassemblies after intravenous administration was also evaluated in P388 subcutaneous-tumour-bearing mice and compared with dFdC free (Figure 8). The SQdFdC nanoassembly treatment caused significant control over the tumour growth in mice in comparison with all the other treatments such as dFdC and saline (Figure 8A and8C). The same results were reflected from the survival curves constructed for untreated and treated groups. The survival curve of the dFdC-treated group was similar to that of the untreated and saline-treated groups, whereas the SQdFdC nanoassembly-treated group exhibited significantly longer survival of the tumour-bearing mice (Figure 8B). Couvreur et al. 2008). [START_REF] Couvreur | Discovery of New Hexagonal Supramolecular Nanostructures Formed by Squalenoylation of an Anticancer Nucleoside Analogue[END_REF] All these data clearly highlighted the high potential of SQdFdC NPs in vivo, in comparison with dFdC free.

Why SQdFdC NPs are more effective on resistant cancer cell lines than gemcitabine free ?

Gemcitabine (dFdC) is a hydrophilic molecule and its cellular uptake occurs by means of active transport through nucleoside transporters, among which hENT type is one of the most important. [START_REF] Aoyama | Predictive Role of Human Equilibrative Nucleoside Transporter 1 in Patients with Pancreatic Cancer Treated by Curative Resection and Gemcitabine-Only Adjuvant Chemotherapy[END_REF] Thus, the cells with deficiency in hENT1 transporters represent an interesting model to study resistance to gemcitabine. As mentioned above, in such CEM/ARAC8C cells, the SQdFdC NPs still displayed a significant anticancer activity. This suggested that these nanoassemblies of SQdFdC should penetrate into the CEM/ARAC8C cells even in the absence of hENT1 transporter, which was not the case with gemcitabine (Figure 6B). [START_REF] Reddy | A New Nanomedicine of Gemcitabine Displays Enhanced Anticancer Activity in Sensitive and Resistant Leukemia Types[END_REF] With regard to L1210 10K cells, a lower expression of cytoplasmic dCK represents another major resistance factor to dFdC. In this leukemia cell line, the SQdFdC NPs were found to lower the resistance by a factor 32, compared to dFdC (Figure 6A). The probable mechanism for this remarkable resistance reversal could be explained by the fact that SQdFdC NPs were slowly releasing the dFdC intracellularly, never overflowing the low intracellular dCK enzymatic activity. On the other hand, as long as the dFdC remained covalently bound to squalene through its amino group, it is expected to resist the deamination by intracellular deaminases. In the case of dFdC free, the picture is quite different. Indeed, dFdC is mainly degraded intracellularly by deamination, since the dCK activity is too low to convert a sufficient amount of dFdC into its active phosphorylated counterpart. Thus, based on those results, it clearly appears that the SQdFdC NPs were able to bypass various types of resistance mechanisms in leukemia cells, resulting in greater cytotoxicity compared to the dFdC free.

Mechanism of action of SQdFdC nanoparticles :

4.1. Do the SQdFdC NPs act under nanoparticular form or bioconjugate form?

During the evaluation of anticancer activity of SQdFdC NPs toward KB3-1 and MCF-7 cell lines, it was shown that even when separated from the cells by inserts with a pore size smaller than the nanoparticles diameter, the SQdFdC NPs maintained their cytotoxicity up to 72 h incubation. Thus, even when physically separated from the cells, the SQdFdC NPs were able to deliver the active drug. The molecular form would be expected to diffuse freely through the insert membrane, while the nanoassemblies are excluded. [START_REF] Couvreur | Squalenoyl Nanomedicines as Potential Therapeutics[END_REF] These observations clearly show that SQdFdC NPs act by releasing SQdFdC bioconjugate.

Does the SQdFdC bioconjugate act as a prodrug by releasing dFdC ?

The biodegradability of the amide linkage of SQdFdC was tested in the presence of two lysosomal enzymes (extracted from spleen cells), the cathepsin B (a cysteine protease) and the cathepsin D (an aspartic protease). [START_REF] Cavallo-Medved | Encyclopedia of Signaling Molecules[END_REF][START_REF] Barrett | PROTEINASES AND THEIR SPECIFICITY[END_REF][START_REF] Conner | Handbook of Proteolytic Enzymes[END_REF] Although they do not represent the entire complement of lysosomal enzymes, they are those which play a key role in degradation of amide-bearing drugs, some polypeptides and proteins within the cells. Therefore, the selective cleavage of an amide-conjugate by these enzymes is a good indicator of their probable degradation inside the target cells. The percentage of unmodified drug as a function of time was determined by HPLC analysis and free dFdC was used as a control in this experiment. As expected, dFdC was not modified by the action of cathepsins, whereas the SQdFdC underwent a rapid cleavage by the two spleen extracts leading to the release of dFdC. [START_REF] Couvreur | Squalenoyl Nanomedicines as Potential Therapeutics[END_REF] This clearly suggests that the SQdFdC NPs could act as a prodrug and release gemcitabine intracellularly after cleavage by proteases.

Does the squalenoylation modify the pharmacokinetics of dFdC?

The pharmacokinetics of the SQdFdC NPs were assessed after i.v. administration in healthy mice, compared with dFdC, by simultaneous quantification of the drug concentrations in plasma using liquid chromatography (LC)-tandem mass spectrometry. It was observed that the dFdC released from SQdFdC NPs displayed a ~3.9-fold higher t1/2 and ~7.5-fold higher main resident time (MRT), showing that SQdFdC NPs considerably modified the pharmacokinetics of dFdC with prolonged blood retention and increased volume of distribution (Figure 9A). On the other hand, the plasmatic concentrations of dFdU (the inactive uracil metabolite of dFdC) were also quantified and indicated that deamination by deoxycytidine deaminase was significantly delayed (Figure 9B). In vitro and in vivo interaction of SQdFdC NPs with plasma components :

To determine the fate of SQdFdC NPs, the dFdC bioconjugate was radiolabeled by tritium. The tritiated SQdFdC NPs, were obtained by the coupling of commercially available 3 H-dFdC with tris-norsqualenic acid (SQ Ac) following the same procedure as was used for the synthesis of cold SQdFdC.

The resulting radiolabeled 3 H-SQdFdC were mixed with cold SQdFdC before being formulated into nanoparticles using nanoprecipitation technique.

 In vitro interaction :

In the blood, NPs encounter a complex biological environment composed of a plethora of endogenous molecules. Depending on their physico-chemical properties such as material, size, surface charge and functionalization, NPs immediately interact with a specific set of biomolecules, thereby acquiring certain biological identity. [START_REF] Tenzer | Nanoparticle Size Is a Critical Physicochemical Determinant of the Human Blood Plasma Corona: A Comprehensive Quantitative Proteomic Analysis[END_REF][START_REF] Lundqvist | Nanoparticle Size and Surface Properties Determine the Protein Corona with Possible Implications for Biological Impacts[END_REF] This identity will govern the in vivo fate of NPs in terms of biodistribution, pharmacokinetics, therapeutic efficacy and potential toxicity. [START_REF] Monopoli | Biomolecular Coronas Provide the Biological Identity of Nanosized Materials[END_REF][START_REF] Tenzer | Rapid Formation of Plasma Protein Corona Critically Affects Nanoparticle Pathophysiology[END_REF] Among proteins capable of interacting with NPs, apolipoproteins have received special attention. [START_REF] Tenzer | Rapid Formation of Plasma Protein Corona Critically Affects Nanoparticle Pathophysiology[END_REF][START_REF] Göppert | Plasma Protein Adsorption of Tween 80-and Poloxamer 188-Stabilized Solid Lipid Nanoparticles[END_REF] Apolipoproteins are amphipathic molecules, which associate with different plasma lipids to form complex structures called lipoproteins (LPs), acting as macromolecular vehicles of waterinsoluble lipids in the circulation (ie. cholesterol, triglycerides and so on). [START_REF] Schumaker | Circulating Lipoproteins[END_REF] LPs can be classified into chylomicrons, very low density LPs (VLDL), low density LPs (LDL) and high density LPs (HDL). Apolipoproteins play a major role in determining LPs structural stability, metabolism, as well as interaction with cells since they act as ligands for LP receptors. [START_REF] Mahley | Lipoprotein Receptors and Their Ligands: Structure and Function of Apoproteins E and B[END_REF] The capacity of LPs to transport hydrophobic molecules on one hand, and the lipid nature of squalene as well as its bio-relation to cholesterol on the other hand, led our team to explore the interaction between the SQdFdC NPs and LPs. Thus, the interaction of SQdFdC NPs with the different plasma fractions was first investigated in vitro after incubation of radiolabelled 3 H-SQdFdC NPs with human blood samples at 37°C (in comparison with 3 H-dFdC). It was found that 3 H-SQdFdC was mainly distributed between LP fractions (51%) and albumin (20%). Among the LP fractions, the LDL was the predominant 3 H-SQdFdC-interacting LP (37%) followed by VLDL (11%) and HDL (3%) while 3 H-dFdC was almost entirely recovered in the water phase (70%), even if some radioactivity was also measured in HDL (24%) (Figure 10 a and 10b). The interaction of SQdFdC and free dFdC with plasma components was also investigated in vivo after intravenous injection of 3 H-SQdFdC NPs and 3 H-dFdC in rats. In this set of experiments, a strong association of 3 H-SQdFdC with LPs was observed once again (49%) but, unlike in human blood, the main SQdFdC-interacting LPs in rats was HDL (32%), rather than LDL (10%) (Figure 10c). Of note, there are important qualitative and quantitative differences in the LP system of rodents and humans, in particular with HDL being more abundant and LDL being almost depleted in rats comparatively to humans (Figure 10 c). Regarding to free 3 H-dFdC, no interaction of this drug was observed with LPs, thus confirming once again, that in the case of SQdFdC, the interaction with LPs was mediated through the SQ moiety. [START_REF] Sobot | Conjugation of Squalene to Gemcitabine as Unique Approach Exploiting Endogenous Lipoproteins for Drug Delivery[END_REF]  Colloidal stability of SQdFdC NPs in blood :

In order to determine the stability of dFdC NPs in the blood circulation, another experiment was performed using Förster resonance energy transfer (FRET) technique. FRET is a non-radiative process, in which energy is transferred from an excited molecule (donor, D) to an acceptor molecule (acceptor, A) in close proximity (between ca. 1 and 20 nm), after excitation of the donor at a specific wavelength. In that respect, these 2 fluorescent probes (Donnor and Acceptor) were together encapsulated inside SQdFdC NPs. Considering that the energy transfer between these 2 fluorescent probes could occur only if the NPs remained intact, hence, an acceptor emission signal under donor excitation could be considered as a validated marker of NPs colloidal integrity. [START_REF] Gravier | FRET Imaging Approaches for in Vitro and in Vivo Characterization of Synthetic Lipid Nanoparticles[END_REF] For this experiment, SQGem NPs were fluorescently labeled with two squalene derivatives of the cyanine fluorophores : SQCy5.5 and SQCy7.5 which behaved as FRET donor and acceptor respectively, after excitation of the donor at 640 nm. Taking advantage of the dependence of FRET signal on the distance between the FRET pair (SQCy5.5/SQCy7.5) this approach enabled to monitor the integrity of SQGem NPs over time in rat blood (Figure 11). In this experiment, it was observed after incubation in blood, a fast disassembly of the NPs, resulting in the release of individual SQdFdC molecules able to insert into LPs, probably as a result of the bio-relation between the squalene moiety and the cholesterol. Thus, the interaction of SQdFdC NPs with LPs might be imagined as a dynamic process which first involves the adsorption of the small LDLs at the NPs surface as soon as they enter into contact. Then, the fast disassembly of the NPs as shown by FRET experiments leads to the release of the SQdFdC bioconjugates, which will further insert into the LPs.

4.4.2.

Mechanism of cellular uptake of 3 H-SQdFdC NPs :

Based on the fact that the large majority of cancer cells require large amounts of cholesterol for the synthesis of new membranes, thus many of them overexpress LDL receptors (LDLR) on their surface. In this context, the ability of LDL particles to mediate the SQdFdC cellular uptake via the LDLR was investigated in vitro on two breast cancer cell lines MDA-MB-231 and MCF-7 (Figure 12A). It was observed that the cellular uptake of 3 H-SQdFdC was highly dependent on the LDLR activity. Indeed, incubation of SQdFdC NPs with MDA-MB-231 cells, which were pre-incubated with an excess of LDL, led to a significant decrease in NPs uptake due to down-regulation of LDL receptors (LDLR) by the cells. In contrary, incubation of these NPs in lipoprotein deficient serum (LPDS) led to an increase of NPs uptake, as a result of overexpression of LDLR by LDL-deprived cells (Figure 12A). Moreover, the higher uptake of 3 H-SQdFdC in the MDA-MB-231 cells (high LDLR expression) compared to the MCF-7 (low LDLR expression) further confirmed the key role of this receptor in the cellular internalization of the SQdFdC bioconjugates (Figure 12B). Similar observation was also done in vivo, when increased uptake of 3 H-SQdFdC occurred in tumour xenografts originating from the breast cancer cell line with the highest expression of LDLR (Figure 12C). In view of these elements, it seemed obvious that endogenous LDL particles may function as carriers for SQdFdC molecules, thus allowing the indirect targeting of cancer cells displaying high expression and activity of LDLR, without the need to functionalize NPs surface with specific ligands. [START_REF] Zheng | Rerouting Lipoprotein Nanoparticles to Selected Alternate Receptors for the Targeted Delivery of Cancer Diagnostic and Therapeutic Agents[END_REF]

4.4.3.

Biodistribution study of 3 H-SQdFdC NPs in mice :

A biodistribution study was performed in mice after intravenous administration of 3 H-SQdFdC NPs versus 3 H-dFdC. A lower radioactivity was observed in kidney and urine with 3 H-SQdFdC NPs suggesting a slow elimination and, hence, longer body retention compared with the 3 H-dFdC radioactivity, which is in accordance with the plasma pharmacokinetic data (Figure 9). The significantly higher accumulation of 3 H-SQdFdC NPs radioactivity (in comparison with 3 H-dFdC) in the reticuloendothelial organs such as the spleen, liver, and lung may possibly be due to the uptake of the dFdC nanomedicine by resident macrophages, which results from opsonization phenomenon (Figure 13). [START_REF] Reddy | Squalenoylation Favorably Modifies the in Vivo Pharmacokinetics and Biodistribution of Gemcitabine in Mice[END_REF] This biodistribution pattern suggests that a significant amount of SQdFdC was directly captured by these tissues as nanoassemblies, before cleavage. Otherwise, a quite similar organ/tissue distribution as for 3 H-dFdC should have been observed. This SQdFdC NPs biodistribution may explain the increased activity against cancer metastasis because the lymphoid organs, such as the spleen and liver, are the major organs of metastasis. Based on all these results, we can deduce that once administered intravenously, SQdFdC NPs interact with plasma proteins, majorly with LDL, and are then uptaken by resident macrophages of the reticuloendothelial system (RES) which allow their accumulation mainly in spleen, liver and lung tissues. Then, within these tissues, the SQdFdC NPs decorated with LDL may penetrate the cancer cells (which generally overexpress LDLR) by 2 simultaneous ways. Either the NPs enter intact by LDL receptor-mediated endocytosis, or they progressively disassemble and release the SQdFdC bioconjugates which are taken in charge by LDL receptors before being uptaken by the cells (via LDL receptors-mediated endocytosis). Once in the endosome-lysosome compartment of the cancer cell, the SQdFdC is metabolized by cathepsins into the active dFdC which is effluxed in the cytoplasm.

Could the anticancer activity of SQdFdC NPs be improved by dFdC monophophorylation ?

As mentioned before, dFdC acts by mimicking physiological nucleosides in terms of uptake and metabolism and is then incorporated into newly synthesized DNA resulting in synthesis inhibition causing premature chain termination. However, to be active, the dFdC has to be first bioconverted into its monophosphate derivative by the action of cellular kinases prior to undergo further phosphorylations into the diphosphate and finally into the active triphosphate form. Thus, the monophosphorylation by kinases becomes a rate-limiting step for the activation of this drug. Indeed, the dFdC is a poor substrate for the cellular kinases needed for its activation and down-regulation of kinases represents an important mechanism of cellular resistance. [START_REF] Galmarini | Nucleoside Analogues: Mechanisms of Drug Resistance and Reversal Strategies[END_REF] For all the above reasons, a direct delivery of the dFdC monophosphate (dFdC-MP) into the cells has been considered as a potential strategy for overcoming the rate-limiting primary phosphorylation step. Unfortunately, dFdC-MP was found to be inefficient because it is not transported by nucleoside transporters and thus poorly penetrate freely through the cellular membranes owing to its high polarity. [START_REF] Lichtenstein | The Metabolism of Exogenously Supplied Nucleotides by Escherichia Coli[END_REF] In this context, we investigated the possibility of delivering dFdC-MP into the cells by taking advantage of the squalenoylation methodology.

4.5.1.

Monophosphorylation of SQdFdC (SQdFdC-MP):

Many very efficient procedures are available for the phosphorylation of nucleosides, [START_REF] William | Phosphorus Methods in Nucleotide Chemistry[END_REF] however we discovered at the outset of the work that the presence of the isoprenylated side chain bound to the cytosine nucleus of the nucleoside was much more troublesome than we originally thought. For example, attempts at phosphorylating the 5'-hydroxyl group of squalenoyl deoxycytidine (SQdC) or squalenoyl dideoxycytidine (SQddC) 10 used as a simplified surrogate of SQdFdC 8 with various phosphore (V) reagents were unsuccessful. For example, condensation of ddC with POCl3/triethylphosphate 93 induced the competitive cleavage of the squalene chain. On the other hand diethylphosphorochloridate [START_REF] Corriu | Silicon-Phosphorus Analogies. Nucleophilic Catalysis in the Alcoholysis of Chlorophosphorus Derivatives[END_REF] and dibenzylphosphorochloridate [START_REF] Atherton | Studies on Phosphorylation. Part I. Dibenzyl Chlorophosphonate as a Phosphorylating Agent[END_REF][START_REF] Ozaki | Encyclopedia of Reagents for Organic Synthesis[END_REF] smoothly reacted with squalenoylated ddC to provide the expected triester phosphates in 90% (12a) and 48% (12b) yield respectively. Unfortunately attempts to remove ethyl or benzyl groups using TMSBr (Trimethylsilyl bromide) [START_REF] Brossette | Synthesis of Haptens for the Development of Immunoassays for the Monitoring of Intracellular Anti-HIV Nucleosides and Nucleotides[END_REF] met with failure once again, because of the participation of the double bonds of the squalene chain.

The use of phosphoramidite reagents for the introduction of the desired phosphate group was therefore investigated. [START_REF] Beaucage | Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach[END_REF] Because of the potentially easy deprotection of the resulting phosphate through retro-Michael process, bis(2-cyanoethyl) N,N-diisopropylchlorophosphoramidite [START_REF] Sinha | β-Cyanoethyl N,N-Dialkylamino/N-Morpholinomonochloro Phosphoamidites, New Phosphitylating Agents Facilitating Ease of Deprotection and Work-up of Synthesized Oligonucleotides[END_REF] was chosen. In the event, sequential treatment of SQddC 10 with the phosphoramidite in the presence of 1H-tetrazole, followed by oxidative work up using hydrogen peroxide (30%), gave the expected phosphotriester 12c in 80% yield. Then, the final deprotection of phosphotriester through βelimination was attempted with DBU. Unfortunately, even in the presence of TMSCl (Trimethylsilyl chloride) as catalyst, only one cyanoethyl group could be removed. After some unsuccessful attempts, we turned to the use of bis(9H-fluoren-9-ylmethyl) diisopropylamidophosphite 11. Indeed, the 9fluorenylmethyl group was demonstrated by Watanabe et al. to be an efficient base-labile protecting group allowing for complete deprotection of phosphotriesters by means of an excess of a tertiary amine under anhydrous conditions. 100 The phosphoramidite 11 was prepared in a one-pot process from phosphorus trichloride (PCl3) via N-diisopropylphosphoramidous dichloride according to a slight modification of the reported procedure. 101 Thus condensation of SQddC 10 with the phosphoramidite 11 in the presence of 1H-tetrazole followed by oxidative work up gave the expected phosphotriester 12d in 52% yield. 102 Compound 12d upon treatment with a large excess of triethylamine in acetonitrile gave rise to the desired triethylamonium phosphate salt 13 in 98% yield (Scheme 4). 102 With a viable synthetic route in hand, we turned our attention to the more challenging case of gemcitabine. Although we were aware that both hydroxyl groups of dFdC could react, we first check the direct condensation of phosphoramidite 11 with SQdFdC 8 assuming that we could expect such a bulky reagent to phosphorylate selectively the less hindered primary alcohol (Scheme 5). Unfortunately, treatment of SQdFdC 8 with phosphoramidite 11 followed by oxidative work up gave the desired 5'-phosphorylated SQdFdC with only 10% yield together with the 3'-isomer and 3',5'diphosphorylated compound.

4.5.2.

Preparation and characterization of SQdFdC-MP NPs:

The nanoassemblies of trialkylammonium SQdFdC-MP conjugates 19a and 19b were obtained by nanoprecipitation technique. These particles had a polydispersity index lower than 0.30, mean diameters around 100 nm with negative zeta potentials as measured by quasi-elastic light scattering.

Transmission electron microscopy (TEM) experiments were performed on SQdFdC-MP NPs which showed rather spherical particles with mean diameters of 100 nm and 150 nm, respectively (Figure 14). Triethylammonium SQdFdC-MP (19a) nanoassemblies were imaged by cryo-TEM, revealing a population of unilamellar liposomes with diameters ranging from 100 to 300 nm (Figure 14). 102,106 Both cryo-TEM and SAXS provided evidence that triethylammonium SQdFdC-MP bioconjugates selforganized as liposomal vesicles. In contrast, it has previously been shown that SQdFdC NPs displayed an inverse hexagonal structure formed by close-packed cylinders in which the aqueous cores were surrounded by hydrophilic gemcitabine molecules linked to the hydrophobic squalene moieties. [START_REF] Couvreur | Discovery of New Hexagonal Supramolecular Nanostructures Formed by Squalenoylation of an Anticancer Nucleoside Analogue[END_REF] The difference in structure between SQdFdC and SQdFdC-MP NPs could be explained by simple geometrical packing considerations. The molecular shape of the SQdFdC molecule can be approximated to a truncated cone, with a small polar headgroup interfacial area and a bulky hydrophobic SQ chain. The addition of the 5'-phosphate group should result in a bulkier and probably more hydrated headgroup. Furthermore, electrostatic interactions between polar groups are believed to increase their interfacial areas. In a nutshell, this should correspond to a more cylindrical shape of the SQdFdC-MP molecules, stabilizing bilayers upon self-assembly in water. The electrostatic repulsion between bilayers should favour the existence of unilamellar liposomes.

4.5.3.

In vitro evaluation of anticancer activity of SQdFdC-MP NPs :

The anticancer activity of SQdFdC-MP of 19a and 19b liposomes was tested against L1210 wt (a dFdC-sensitive murine lymphoid leukemia cell line). On this cell line, dFdC exhibited an efficient anticancer activity, displaying a 50% inhibitory concentration (IC50) value of 3.2 nM (Table 2). Owing to the prodrug nature, SQdFdC nanoassemblies showed a higher IC50 (38 nm) and thus a lower anticancer activity than free dFdC, probably because of the time lag associated with cellular penetration, dFdC leakage from SQdFdC and further primary phosphorylation, before resulting in the formation of the active triphosphate. In contrast, the phosphorylated derivatives of SQdFdC NPs (i.e., the triethylammonium salt 19a and the dimethylethanolammonium salt 19b liposomes) displayed a considerably lower IC50 value (6.1 nm and 6.7 nm, respectively) and thus a more efficient anticancer activity than SQdFdC nanoassemblies, whereas interestingly the activities of these phosphorylated derivatives were quite similar to that of dFdC (Table 2). 102 To rule out the possibility of the interference of the triethylammonium cation of 19a with its anticancer activity, the cytotoxicity of the aqueous triethylammonium dihydrogen phosphate solution (as a placebo) on this cell line was studied. This placebo did not elicit any toxicity toward the cancer cell line, thus pointing to the absence of any interference of triethylammonium cations in the observed activity of 19a liposomes. As drug resistance is a serious clinical concern in the treatment of cancer, the anticancer activity of the SQdFdC-MP derivative 19a was tested also against L1210 10K murine lymphoid leukemia cell line, the resistant variant of L1210 wt cell line (Table 3). 107 As previously mentioned, the L1210 10K cell line thus exhibits ca. 5000-fold resistance to dFdC measured in terms of the IC50 values, due to the downregulation of deoxycytidine kinase, the enzyme mediating the primary phosphorylation step necessary for the activation of dFdC.

As expected, on the L1210 10K cell line, dFdC exhibited a considerably lower activity, displaying an IC50 value of 19.4 µM (relative to the 3.2 nM on the L1210 wt cell line) (Table 3). 102 In contrast, and unlike in the case of L1210 wt cell line, the SQdFdC NPs displayed an improved activity (IC50 value of 9.8 µM) relative to free dFdC (19.4 µM) (Table 3). As explained before, this lower IC50 value was attributed to the fact that SQdFdC NPs were slowly releasing the gemcitabine intracellularly, not overwhelming the low intracellular dCK enzymatic activity, which was not the case with free dFdC. [START_REF] Reddy | A New Nanomedicine of Gemcitabine Displays Enhanced Anticancer Activity in Sensitive and Resistant Leukemia Types[END_REF] Interestingly, liposomal formulations of 19a were still more efficient (IC50 value of 3.7 µM) than those of the parent compound SQdFdC NPs.

The superiority of triethylammonium SQdFdC-MP liposomes has thus been clearly demonstrated, because they significantly reduced the resistance displayed by L1210 10K cell line in relation to dFdC and SQdFdC. It is hypothesized that the observed improved anticancer activity of 19a liposomes is the result of by-passing the primary phosphorylation step, thereby presenting dFdC for the formation of the active triphosphate form of dFdC.

This platform thus opens exciting prospects for the discovery of new anticancer nanomedicines with improved pharmacological activities on resistant cancer cells.

4.6.

Is the squalene structure essential for the formation of nanoparticles ? May other polyisoprene-dFdC NPs also improve the pharmacological activity of dFdC?

With the aim to investigate whether the "squalenoylation" approach could also work using other terpenes for drug conjugation and further nanoassemblage, we decided to design a series of polyisoprenoyl prodrugs of dFdC. In other words, the aim was to explore the influence of the isoprene length on the ability (or disability) of the resulting bioconjugates to form nanoassemblies and to emphasize the impact of terpene's nature on the in vitro and in vivo pharmacological activity, using the gemcitabine as a drug model.

4.6.1.

Synthesis of polyisoprenoyl dFdC bioconjugates :

For this purpose, in order to modulate the length of the terpenoyl side chain of dFdC prodrugs, we started with the synthesis of various polyisoprenic acids with 1 to 6 isoprene units (6, 6b-g). These acids were reacted with ethyl chloroformate in THF to give the intermediate mixed anhydrides which then reacted with 3',5'-bis tert-butyl silyl dFdC (20) to afford the tresulting protected bioconjugates. Then, conventional TBAF (tert-butyl ammonium fluoride) deprotection of these compounds led to the corresponding conjugates 8 and 8b-g (50-80% yields) (scheme 6). 108 The synthesis of monoisoprenyl acid 6b started with the alkylation of diethyl malonate 21 with prenyl bromide, to give 22, followed by saponification and then decarboxylation. According to the method developed by Kuawajima and Doi 109 and later utilized by Coates, 110 alkylation of the copperlithium enolate of ethyl acetate 23 with geranyl bromide gave geranyl acetic acid ethyl ester 24 which was next saponified to provide geranyl acetic acid 6c. The same process using farnesyl bromide gave farnesyl acetic acid 6d (scheme 7). 108 To access the four isoprenyl-units acid 6e, the lithium derivative of geranyl tolyl sulfone 25 was alkylated with the bromide 26 available in three steps from geranyl acetate. The acetate group of the sulfone 27 was hydrolysed in basic conditions, and the resulting alcohol 28 was then treated with a catalytic amount of [PdCl2(dppp)] in THF, to give a π-allyl complex that was reduced in situ with an excess of LiBHEt3 applying the conditions described by Mohri and co-workers. 111 The obtained tetraisoprenol 29 was then reacted with PBr3 in THF. The reaction of the resulting bromide with an excess of the copper-lithium enolate of ethyl acetate in THF using the Kuwajima procedure provided ester 30 that was hydrolysed with lithium hydroxide to give the corresponding acid 6e (Scheme 7). 108,109 Similarly, farnesyl tolyl sulfone 31 was reacted with 26, providing the penta-isoprenyl secondary sulfone 27 in 83% yield. Reduction of the sulfonyl group and two-carbon elongation as described above provided the five-isoprenyl units acid 6f (Scheme 8). 108 Scheme 8 : Synthetic approach to polyisoprenic acid 6 f. This technique was also successfully applied to the synthesis of longer polyisoprenyl chains, bearing 6 isoprene units. Thus, alcohol 28 was quantitatively converted to the bis-sulfone 32 via the bromide 31 in 73% yield. Bis-sulfone 32 was in turn reacted with 26, after treatment with n-butyllithium in the aforementioned conditions to give the hexa-isoprenyl bis-sulfone 33 in 78% yield. The reduction as well as the functionalisation of sulfone 33 to the corresponding polyisoprenic acid 6g was achieved as described for the four-and five-isoprenyl unit acids 6e-f (scheme 9). 108 Scheme 9 : Synthetic approach to polyisoprenic acid 6 g. 4.6.2. Preparation and characterization of polyisoprenoyl-dFdC NPs (8, 8b-g):

HO O F OH F N N NH 2 O HO O F OH F N N HN O R O 4 8, 8b-g R = b, R = c, R = d, R = e, R = f, R = g, R = 1) RCO 2 H 2) n-Bu
Each conjugate (8, 8b-g) was submitted to nanoprecipitation conditions in order to assess the minimal requisite size of the polyisoprenoyl side chain to allow the formation of nanoassemblies. Only bioconjugates 8 and 8e-8g, containing from 4 to 6 isoprene units, were able to form stable NPs in pure distilled water. On the contrary, the bioconjugates with a polyisoprenic moiety less than 4 monomeric units (ie. 8b, 8c and 8d) didn't produce stable nanoassemblies in pure water: 8b and 8c prodrugs, containing respectively 1 and 2 isoprene units, allowed the formation of NPs only in a 10% trehalose aqueous solution whereas the bioconjugate 8d (bearing 3 units of isoprene) was able to form NPs only in the presence of the surface active SQ-PEG (Table 4). 108 Thus, bioconjugates with small isoprenoyl chains (less than 4 isoprene units) were less prone to form stable nanoassemblies than those bearing longer chains, which could be due to the fact that the amphiphilic character of these prodrugs was less pronounced. Indeed, theoretical calculation of log P gave values between 5.37 and 8.43 for bioconjugates made with isoprene units 4-6, whereas they were comprised between 3.85 and 0.79 for bioconjugates made with less than four isoprene units. On the other hand, conjugate 8f gave nanoassemblies quite similar to SQdFdC 8 revealing that the symmetrical distribution of the double bonds found in the squalene chain is not required for the NPs formation and that a regular polyisoprenyl chain is equally effective.

4.6.3.

In vitro evaluation of anticancer activity of polyisoprenoyl-dFdC NPs (8, 8b-g):

In vitro biological assessment showed that all polyisoprenoyl-dFdC nanoassemblies displayed notable cytotoxicity (IC50 below the micromolar range) on the tested cancer cell lines, including murine melanoma cell line B16F10, human pancreatic carcinoma cell line MiaPaCa-2, human lung carcinoma cell line A549 and human breast adenocarcinoma cell line MCF-7 (Table 5). 108 Interestingly, it was observed that the anticancer efficacy of these NPs depended on the length of the polyisoprenoyl moiety. The polyisoprenoyl prodrug of gemcitabine containing three isoprene units (8d) was more active, displaying a cytotoxicity comparable to dFdC free (and even better in case of the B16F10 melanoma cell line). In particular, 8d NPs were found to display a dramatically improved cytotoxicity comparatively to SQdFdC NPs (depending on the cell line, IC50 of 8d NPs was five-times to 36-times lower than that of 8 NPs) (Table 5). This may be explained in the light of the drug release experiments, showing that the release of dFdC was faster for geranylacetyl-(8c) and farnesylacetyl-(8d) dFdC nanoassemblies comparatively to SQdFdC (8) NPs (Figure 15). 108 gemcitabine faster than 8d although being less cytotoxic. 108 However, other parameters like the size could also influence cytotoxicity. Indeed, it has been observed that the volume of encapsulated drug is correlated to the third power of nanoparticles radius. 112 Therefore, although the extent of uptake of nanoassemblies 8d is probably lower than that of smaller nanoassemblies, their higher entrapment efficiency led to higher intracellular drug content, responsible of the enhanced cytotoxicity compared to the other nanoassemblies. 113,114 4.6.4.

In vivo evaluation of anticancer activity of polyisoprenoyl-dFdC NPs (8, 8d):

The antitumor efficacy of the most active 8d NPs has then been investigated on human pancreatic (MiaPaca2) carcinoma xenograft model in mice, comparatively to the treatment using the SQdFdC NPs ( 8), dFdC as a free drug, empty carrier (6, 6d), or saline. Noteworthy, all treatments were performed at the equimolar doses (7 mg of dFdC equivalent/kg). The nanoassemblies constructed using gemcitabine bioconjugates bearing three isoprene units (ie. 8d) showed important antitumor efficacy whereas the parent drug (ie. dFdC free) didn't display any antitumor activity on human MiaPaca2 pancreatic cancer (Figure 16). 8d NPs were also significantly more active than the SQdFdC ( 8) NPs (the tumor nodule was decreased, on day 34, by 76% with 8d versus only 41% with 8). Noteworthy, 8d NPs revealed lower toxicity than SQdFdC as seeing in body weight loss, which was expected since SQdFdC NPs were administered at maximum tolerated dose (MTD) (Figure 16). 108 Thus, this study demonstrated that more efficient terpene based anticancer nanomedicines may be designed by adjusting the length of the polyisoprenyl chain, allowing moving from the "squalenoylation" concept to the "terpenoylation" concept.

Conclusion :

Through this study, we could show that the single chemical linkage of squalene to gemcitabine (dFdC) and formulation into nanoparticles favorably modified the pharmacokinetics and biodistribution of this anticancer drug.

The SQdFdC NPs were found to be more cytotoxic than dFdC free on human and murine gemcitabine-resistant lines. In vivo, SQdFdC NPs also exhibited superior anticancer activity on several experimental cancer models, showing higher inhibition of tumor growth as well as greater survival rates in comparison with those treated with free dFdC. The study of the mechanism of action revealed that after intravenous administration, the SQdFdC NPs interact with plasma proteins, among which LDL, resulting in their opsonization. Then, a high amount of opsonized NPs is uptaken by resident macrophages of the reticuloendothelial system (RES) which allowed their accumulation mainly in lymphoid organs such as spleen, liver and lungs which are the major organs of metastasis and also represent the reservoir of abnormal white blood cells. This explains why SQdFdC NPs appeared very effective in drug-resistant leukemia. In case of solid tumor, the remained fraction of NPs (which was not uptaken by the RES), is able to reach the tumor site by EPR effect (passive targeting of NPs). Once in tumor tissues, the NPs are then uptaken by cancer cells via LDLR-mediated endocytosis either as intact nanoassemblies, or under monomer form inserted inside LDLs. Once in the endosomelysosome compartment of the cancer cell, the SQdFdC prodrug is metabolized by cathepsins into dFdC which is then progressively released in the cytoplasm. The dFdC is then triphosphorylated into its active metabolite prior to penetrate the nucleus and integrate the growing DNA as a "false" nucleoside. It should be noted that depending on the type of cancer, the high efficacy of the SQdFdC NPs observed in the in vivo studies can be correlated with (i) the uptake by RES, (ii) the probable overexpression of cathepsin B and (iii) overexpression of LDL receptors by cancer cells (Figure 17). [START_REF] Sobot | Conjugation of Squalene to Gemcitabine as Unique Approach Exploiting Endogenous Lipoproteins for Drug Delivery[END_REF]115 Figure 17 : Mechanism of action of SQdFdC NPs Furthermore, in order to improve the SQdFdC NPs activity and overcome the monophosphorylation limiting step before integration into DNA, monophosphorylated SQdFdC conjugates were synthesized and formulated as nanoparticles. The nanoassemblies of these derivatives displayed interesting anticancer activities demonstrating that the squalene conjugation promoted efficient cellular delivery of this negatively charged phosphate esters. The exact mechanism by which these prodrugs enter the cells either by passive diffusion by LDL through cell membranes or endocytosis remains to be addressed.

Lastly, we also demonstrated that by adjusting the length of the polyisoprenyl chain, the « squalenoylation » concept can be extended to the « terpenoylation » concept.

The squalenoylation and terpenoylation platform thus opens exciting prospects for the discovery of new anticancer nanomedicines with improved pharmacological activities on resistant cancer cells. In that respect, the squalenoylation was extended to other anticancer drugs going from nucleoside analogues (araC) to more lipophilic drugs (paclitaxel) or highly hydrophilic macromolecules such as SiRNA. All the resulting bioconjugates were able to self assemble into NPs with high drug loadings. In all cases, the squalenoylation provided a remarkable improvement in the drug's therapeutic index. 116 Adriaan P. IJzerman, Xue Yang

NEUROPROTECTION AND NEUROREGENERATION

• NEUROPROTECTIVE AND NEUROREGENERATIVE SQUALENE-BASED NANOMEDICINES :

Central nervous system (CNS) disorders represent the largest area of unmet medical need, with more than 1.5 billion people affected worldwide. Apart from the undeniable suffering that they generate, cerebral lesions have immediate consequences on motor and mental functions. These handicaps create a loss of autonomy and induce dependency, with considerable social and economic consequences. 117 In particular, with an event occurring every forty seconds, stroke represents the fourth leading cause of death in developed countries, and the first cause of long-term handicap. The high unmet medical need in this area has driven academic research groups as well as pharmaceutical and biotechnology companies to pool their competences for the discovery of news treatments. Despite great progress in understanding the structure and functions of the CNS, the discovery of new drugs and their clinical development for many CNS disorders has been problematic. As a result, CNS drug discovery and development programs have been subjected to significant cutbacks and eliminations over the last decade. CNS drugs in general have higher failure rates than non-CNS drugs, both preclinically and clinically, and in some areas, such as the major neurodegenerative diseases, the clinical failure rate for disease-modifying treatments has been 100%. The development times for CNS drugs are significantly longer for those drugs that are approved, and post-development regulatory review is longer. 118 The major contributing factor for the poor translation of neuroscience research into medicines is the high degree of complexity of the human CNS, and the presence of the bloodbrain barrier (BBB), which hinder many biologically active molecules to reach the brain.

State-of-art on Adenosine :

1.1. What is the mechanism of action of adenosine ?

Adenosine (6-amino-9-β-D-ribofuranosyl-9-H-purine) represents a class of potential therapeutic agent with significant beneficial activity in several severe neurological disorders, such as stroke, spinal cord injury or multiple sclerosis. [119][120][121] Adenosine is a nucleoside involved in multiple essential biochemical processes both intra-and extracellularly (Figure 18).

In the cell, adenosine can be phosphorylated into adenosine monophosphate (AMP), which is used by the cell as an RNA brick. This phosphorylation process can be repeated in order to generate adenosine triphosphate (ATP), the "molecular currency" furnishing energy to the cell. ATP can also be converted by adenylyl cyclase (AC) into cyclic AMP (cAMP), a very common second messenger which triggers intracellular signal transduction cascade.

Outside the cell, adenosine regulates essential processes by binding to four G-protein-coupled receptors: A1, A2A, A2B and A3 78 , which govern major pathophysiological functions. Equilibrative nucleoside transporters (ENTs), which are widely expressed by the cells, maintain the balance between intra-and extracellular concentrations of adenosine. Under pathological conditions, extracellular adenosine concentrations dramatically rise due to the release of abundant intracellular ATP by damaged cells 123 and its prompt transformation into adenosine by ectonucleotidases CD39 and CD73. Given that pharmacological effects of adenosine are not just limited to neurological level, but are also well recognized in diverse other physiological functions including cardiovascular and immunological, as well as in pathological situations like inflammation, pain or cancer, 125,126 adenosine is therefore sometimes described as a "multi-signalling guardian angel". 127

What are the limitations of adenosine ?

Despite its potentially significant beneficial activity in several severe neurological disorders, 119,128 adenosine has never been used for the treatment of cerebral diseases because of its short plasma halflife (t1/2 ~ 10 s) and its inability to cross the BBB and the BSCB (blood-spinal cord barrier). 129,130 Indeed, adenosine undergoes rapid enzymatique deamination in the bloodstream by adenosine deaminase into its corresponding inosine, which in turn is metabolized into hypoxanthine. This latter is then oxidized into xanthine and then uric acid by xanthine oxidase (Scheme 10). 131 Scheme 10 : Metabolization of adenosine Therefore, to be pharmacologically active, adenosine must be administered at high doses, resulting in severe side effects which limit its the clinical use. Indeed, adenosine receptor subtypes in the brain and periphery play complicated and sometimes opposing roles in cerebral ischaemia, and even if central administration or continuous intravenous infusion of adenosine have been shown to be protective against stroke, 132 the side effects offset any potential improvement in penumbral residual blood flow. 133 Moreover, the development of stable agonists and antagonists of the Adenosine receptors (ARs) to therapeutically target adenosine signaling led to side effects and/or the lack of real pharmacological efficiency in clinical trials. [134][135][136] Therefore, innovative pharmaceutical strategies are still needed to fully exploit adenosine's tremendous therapeutic potential. In this context, the so-called « squalenoylation » approach may represent a promising strategy for protecting adenosine from rapid metabolization and sustaining its release.

What about nanomedicine approach ?

With the aim of prolonging plasma half-life of adenosine and enhance the quantity of active drug at the site of action, different liposomal formulations were proposed. 124 In that respect, Gutman et al. developed adenosine loaded liposomes to target inflammation. This group demonstrated that these liposomes were internalized by human monocytes and reduced inflammation in vitro. However their influence on inflammation in vivo was not monitored. 137 Takahama et al. developed PEGylated adenosine liposome for the treatment of myocardial ischemia and reperfusion. Hence, this nanoformulation prolonged circulation time of adenosine and allowed its high accumulation in the necrotic area leading to a reduction of the infarct size which was not associated with usual adenosine side effects (hypotension and bradycardia). 138 Based on the fact that extracellular adenosine, acting at A2A receptor, has a critical function in the maintenance of cartilage homeostasis, Corciulo et al. recently developed a liposomal formulation of adenosine to treat or prevent osteoarthritis. 139 Hence, the intraarticular injection of liposomal adenosine prevented and reduced joint swelling, as well as allowed protection to cartilage and joint. It seems however unfortunate that liposomal formulations were so poorly defined with no mention of physicochemical parameters.

Application of squalenoylation nanotechnology to adenosine : synthesis and characterization

Can we obtain nanoparticles of adenosine using squalenoylation approach?

In order to better promote the entry of adenosine in the brain, we decided to use a new squalene derivative with 6 isoprene units : the squalenylacetic acid 6L. In that respect, it was intended to couple the squalene moiety on the 6-amino group of adenosine (subjected to deamination) through an amide bond and formulate this bioconjugate into nanoparticles.

2.1.1.

Synthesis of elongated squalene-based vector : the squalenylacetic acid For this purpose, the squalenylacetic acid was prepared in three steps from 2,3-epoxysqualene 3. First, aluminium isopropoxide-catalyzed ring opening 26 of the 2,3-epoxysqualene 3 to give the allylic alcohol 34. The alcohol 34 was then subjected to standard Johnson-Claisen rearrangement 27 to provide the predominantly (E) unsaturated ester 35 which was subsequently saponified into squalenylacetic acid 6L quantitatively (scheme 11).

Scheme 11 : Synthesis of the squalenylacetic acid 6L.

Conjugation of squalenyl acid to adenosine : Synthesis of SQAd bioconjugate

The squalenylacetic acid (SQ ac acid) 6L was then coupled to the 6-amino group of 2',3',5'-tris TBDMS-protected adenosine 36 through an amide bond using HATU uranium coupling agent (2-(1H-7azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) and HOBt (1hydroxybenzotriazole hydrate) in presence of diisopropylethylamine (DIPEA). The resulting bioconjugate 37 underwent conventional TBAF deprotection to provide N 6 -squalenylacetic acidadenosine (SQAd) 38 (scheme 12). 140

Squalenylacetic acid adenosine

Scheme 12 : Synthesis of N 6 -squalenylacetic acid adenosine (SQAd) 38.

Formulation of SQAd conjugate into nanoparticles

The nanoassemblies were prepared by nanoprecipitation of an ethanolic solution of squalenoyl adenosine (SQAd) 38 in a 5% (w/v) aqueous dextrose solution. After evaporation of ethanol, the aqueous suspension of SQAd NPs was obtained at a concentration of 2mg/mL or 8mg/mL (Figure 19). Because adenosine is covalently linked to the lipid nanocarrier (squalenylacetic acid 6L), a drug loading of 37% was reached. Transmission electron microscopy (TEM) images of SQAd NPs (Figure 19b) showed monodisperse nanoassemblies (the PdI < 0.15) with a mean diameter of ∼120 nm, as also confirmed by dynamic light scattering (DLS) (Figure 19a). The average surface charge of the nanoassemblies was found to be approximately -25 mV, ensuring colloidal stability. 140

What is the supramolecular structure of SQAd NPs?

The supramolecular organization of SQAd nanoassemblies was revealed by small-angle X-ray scattering (SAXS) and displayed an inverse hexagonal phase, as was the case with SQdFdC NPs. 141,142 3. Evaluation of neuroprotective potential of SQAd nanoparticles in CNS injuries (in vivo experiments) : ISCHEMIC STROKE 3.1. May SQAd NPs be effective for the treatment of stroke?

3.1.1.
State-of-art on stroke :

With an event occurring every forty seconds, stroke represents the fourth leading cause of death in developed countries, and the first cause of long-term handicap.

An ischemic event begins with the local impairment of cerebral blood flow that restricts the delivery of substrates to the brain tissue, and particularly oxygen and glucose. Immediately after stroke onset, the loss of energy stores results in ionic imbalance and membrane depolarization. Excitatory-aminoacid release and inhibition of their reuptake, drive the accumulation of glutamate in the extracellular space, and the subsequent intra-cellular calcium (Ca 2+ ) overload thus leading to mitochondrial dysfunction 143 and dramatic reduction in cellular ATP abundance in neuronal and glial cells (Figure 20). 144 Then, under glutamate-mediated activation, monovalent ions enter the neurons and water follows passively leading to a cytotoxic-brain-edema. This phenomenon is considered as a marker of irreversible injury (necrosis) and is followed by production of reactive oxygen species (ROS). 145 On the other hand, Intra-cellular Ca 2+ overload also leads to the activation of enzymes able to produce reactive oxygen species (ROS), such as superoxide anion radical (O2 .-), hydroxyl radical (HO . ) and hydrogen peroxide (H2O2). 146 To «save» the organ, the reperfusion (restoration of normal blood flow) remains the most effective way, but paradoxically, it creates still more severe damages. Indeed, reperfusion in turn, generates the production of ROS, but also nitric oxide (NO). On the other hand it generates the production of mediators of inflammation (platelet-activating factor, tumor necrosis factor α (TNFα) and interleukin-1β (ILB1β)(Figure 20). Consequently, neutrophils cross the vascular wall (endothelial cells) and enter the parenchyma, followed by macrophages and monocytes. The ensuing inflammatory response causes a high degree of cytotoxicity that amplifies neuronal death in the penumbra. Furthermore, the oxidative stress causes sustained contraction of pericytes which leads to narrowing of the microvasculature lumen. The lumenal narrowing is further worsened by compression from swollen end-feet of activated astrocytes. 146 Evaluation of neuroprotective effect of SQAd NPs :

Based on the fact that squalenoylation of adenosine should allow protection from rapid metabolization, increased concentrations of adenosine in the blood circulation and consequently a higher exposure to BBB, we decided to directly test in vivo the neuroprotective potential of SQAd NPs in an ischaemia-reperfusion model in mice.

In that respect, 2 models were developed: transient and permanent cerebral ischaemia. In transient ischaemia, mice were subjected to 2 h of middle cerebral artery occlusion (MCAo) (Figure 21A) and 22 h of reperfusion, while with permanent ischaemia mice were subjected to 24 h of permanent MCAo. With transient MCAo, the SQAd NPs were administered intravenously either just before ischaemia or 2 h post-ischaemia.

It was observed that intravenous bolus administration of SQAd nanoassemblies dose-dependently decreased the infarct volume in mice (Figure 21B). Pre-ischemic injection of 7.5 mg. kg -1 or 15 mg. kg -1 of SQAd NPs (equiv. respectively to 2.75 and 5.5 mg. kg -1 of adenosine) significantly reduced the stroke size by 38% and 65%, respectively, compared to the control groups treated with dextrose solution, free adenosine (5.5 mg kg -1 ) or with unconjugated squalenoyl (SQ) nanoassemblies (9.45 mg kg -1 ) (Figure 21B). 140 Then, SQAd nanoassemblies (15 mg kg -1 ) were also administered at reperfusion, 2 h after stroke. Again, the infarct volume significantly decreased by 47% compared to the controls (Figure 21B). To evaluate if this neuroprotective effect was preserved in the absence of reperfusion, a common clinical setting, 147 mice were subjected to 24 h of permanent MCAo. SQAd nanoassemblies (15 mg kg -1 ) administered 2 h after the induction of ischaemia significantly decreased the infarct volume by 56% compared to the vehicle-treated control group (Figure 21B). 140 The cell death processes were also analyzed in SQAd nanoassemblies treated group (15 mg kg -1 , pre-ischaemia) and showed significant reductions at 24 h in the number of cells exhibiting apoptotic nuclei with clumped chromatin or caspase-3 activation in the ischaemic areas destined to infarct. 140 Given the vasodilatory properties of adenosine, we investigated if the protective action of SQAd nanoassemblies could result from a primary vascular mechanism leading to a secondary parenchymal neuroprotection. We observed that most of the ischaemic microvessels were patent 6 h after reopening of the MCA in SQAd nanoassemblies administered mice, in contrast to untreated animals in whom ischaemic capillaries were still clogged with trapped erythrocytes (Fig. 23 A,B), possibly due to ischaemia-induced pericyte contractions. 148 In addition, astrocyte endfeet around microvessels and endothelial nuclei were not swollen in treated animals, unlike untreated mice (Fig. 23 C,D). The infarct area was also measured in SQAd nanoassemblies treated mice compared to the untreated controls after 6 h of reperfusion (instead of 22h), that is before significant activation of inflammatory and astrogliotic mechanisms. The significant decrease of infarct area in SQAd NPs treated mice may suggest that adenosine dissociated from nanoassemblies (which penetrated into the vascular endothelium) maybe reached the brain compartment and interacted with adenosine receptors located on pericytes of the cerebral microvessels and astrocytes endfeet, thereby improving microcirculation. 149 Moreover, adenosine has been shown to be cytoprotective in low intracellular adenosine triphosphate (ATP) states by promoting ATP generation independently of adenosine receptors, which may have prevented the observed swelling in the endothelium capillaries and astrocyte endfeet. 150 It is noteworthy that the concentration of adenosine achieved within the capillary wall around the neurovascular unit appears to be sufficient to improve microcirculation, without inducing systemic vasodilation and resulting hypotension. In that respect, the arterial blood pressure was measured in mice subjected to ischaemia and treated with SQAd NPs versus untreated or treated with only adenosine. In all cases the blood pressure was constant (88 ± 3 mmHg). 140 In the case of permanent MCAo, adenosine may promote microcirculatory perfusion in the periphery of the MCA area, supported by collateral flow with a similar mechanism.

Biodistribution study of SQAd nanoparticles :

In order to monitor the SQAd NPs distribution and check if adenosine or SQAd NPs were able to cross the BBB and reach the brain compartment, a biodistribution study was performed. In that respect, dual-labeling strategy was employed, using two types of radiolabeled nanoassemblies: one with 14 C labelling located on the squalene moiety and the other with 3 H labeling on the adenosine moiety. Tissue profiles of both [ 3 H]-SQAd and [ 14 C]-SQAd were examined with comparison to the distribution profile of [ 3 H]-Adenosine injected as a free drug.

What is the best strategy to radiolabel SQAd bioconjugate by tritium and 14 C ?

For this purpose, the adenosine moiety of SQAd was radiolabeld by tritiation of the position 8 of the nucleobase, and the squalene moiety by 14 C on the carbonyl group (Figure 24). (a: oxalyl chloride, toluene, 3h)

Preparation and characterization of radiolabeled SQAd nanoassemblies :

The radiolabeled SQAd NPs were prepared by co-nanoprecipitation of cold SQAd and radiolabeled SQAd ([ 3 H]-SQAd 38*A or [ 14 C]-SQAd 38*B) in 5% dextrose solution. In that respect, cold SQAd and radiolabeled SQAd (38*A or 38*B) were together solubilized in ethanol prior to undergo nanoprecipitation process (the same as previously described for non radiolabelled SQAd NPs).

The radiolabeled [ 3 H]-or [ 14 C]-SQAd NPs were monodisperse but little bit larger than pure cold SQAd NPs (120 nm Ø), with diameters going from 144 to 180 (same range of magnitude) and with negative zeta potential. These data clearly demonstrated that the NPs did not translocate from the blood circulation to the brain parenchyma. It remains to be established if the [ 3 H]-radioactivity related to [ 3 H]-SQAd NPs corresponded to Adenosine or its metabolites (as it was probably the case with [ 3 H]-Ad NPs with its short half-life.

Mechanism of action of SQAd nanoparticles :

The mechanism behind the observed pharmacological activity of SQAd NPs was then investigated in more detail by performing different experiments. As a first step, the pharmacokinetics of SQAd NPs was performed using tritiated SQAd NPs and the plasma profile of [ 3 H]-SQAd was examined in comparison with the distribution profile of [ 3 H]-Adenosine injected as a free drug. Then, by using both liquid scintillation counting and radio-HPLC as analytical methods, we were able to assess the fate of the SQAd prodrug. The integrity of SQAd NPs, for its part, was studied using DLS and FRET. Therefore, on one hand the radiolabeling strategy combined with high performance liquid chromatography (HPLC), and on the other hand, imaging strategy using flow cytometry and confocal microscopy, represent an essential source of information on the nanoformulation's integrity and stability, which may provide a better understanding of the therapeutic efficacy.

5.1. Did adenosine reach the brain to exhibit pharmacological activity on ischemic stroke?

5.1.1.
Drug metabolism in the brain :

In order to identify the radioactive content observed in the brain, the experiments using highperformance liquid chromatography with radioactive detection (radio-HPLC) were performed. (Figure 24). As expected, adenosine was not detected in the brain 1 h following administration of free [ 3 H]adenosine (Figure 26C). Interestingly, no intact adenosine could be detected in the brain following [ 3 H]-SQAd NPs administration too. These observations clearly show that the detected radioactivity in the brain corresponded only to the metabolites derived either from SQAd or from released adenosine, suggesting that both molecules were metabolized before reaching the brain parenchyma (Figure 26B and 26C).

All these data clearly indicate that the SQAd nanoassemblies efficacy could not be explained by a central pharmacological activity, but rather by a primary vascular mechanism leading to a secondary parenchymal neuroprotection, as already indicated by the improved microcirculation in the stroke study (Figure 23A and 23B). This also suggested that the effectiveness of SQAd nanoassemblies in brain ischaemia could result from a multitargeted pleiotropic action on the cells of the neurovascular unit or other peripheral cell types, 152,153 thanks to a sustained systemic circulation. Thus, our next goal was to understand how exactly these SQAd NPs operated on ischemic stroke? In vitro colloidal stability of SQAd NPs :

The integrity of the NPs in mouse plasma was first investigated in vitro using DLS and FRET. Colloidal stability is of crucial importance as it may affect biodistribution and metabolization profiles and, in turn, therapeutic efficacy and toxicity.

 Evaluation of SQAd NPs integrity using DLS :

Initially, the mean diameter of SQAd NPs in water as measured by DLS was 110±3. When incubated in water at 37 °C, the mean diameter of the NPs did not significantly change for at least 24 h of incubation (Fig. 27A). But when the NPs were dispersed in freshly collected mouse plasma pre-warmed at 37 °C, the mean diameter significantly increased to reach 175 ± 31 nm, likely due to the formation of a protein corona (Fig. 27A) and then decreased progressively till complete disaggregation at 24h. 151  Evaluation of SQAd NPs integrity using FRET :

In order to confirm this observation, NPs structure integrity was studied using FRET. For this experiment, two fluorophores (a green cholesteryl ester BODIPY-FL donor probe and a red cholesteryl ester BODIPY-542/563 acceptor probe) were co-nanoprecipitated together with SQAd in order to fluorescently label the SQAd NPs. The cholesteryl ester chains made these non polar BODIPY fluorophores intrinsically lipophilic, which allowed an excellent insertion into the nanoassemblies without significantly modifying their physicochemical properties. Indeed, size and polydispersity index (PDI) of the fluorescently labeled NPs were measured by dynamic light scattering (DLS), showing diameters below 150 nm with negative zeta potential and PDI below 0.2, an indicator of a monodisperse size distribution. 151 When these NPs were excited at 490 nm (donor [i.e. BODIPY-FL] excitation wavelength), an emission signal at 574 nm (acceptor [i.e. BODIPY-542/563] emission wavelength) was measured, highlighting an energy transfer from the donor to the acceptor. Therefore, when NPs were dispersed in mouse plasma, a strong acceptor signal was measured the first hours confirming NP integrity in plasma (Fig. 27B) and the acceptor signal then decreased over time. 151 Altogether, these data suggest that in vitro, the SQAd NPs were stable during the first 2 h of incubation in mouse plasma at 37 °C, thus allowing to preserve a significant amount of adenosine over this period, before to progressively disassemble (figure 27B). This disassembly is probably due to the interaction with plasma proteins such as lipoproteins (LPs), as previously described for SQdFdC NPs. [START_REF] Sobot | Conjugation of Squalene to Gemcitabine as Unique Approach Exploiting Endogenous Lipoproteins for Drug Delivery[END_REF] Indeed, we previously demonstrated that once interacted with SQ-based NPs, LPs were able to detach the SQ-based conjugates from the nanoparticle in order to incorporate it, leading to the progressive dissociation of the NP.

5.2.2.

In vitro chemical stability of SQAd bioconjugate:

 Evaluation of chemical stability SQAd NPs integrity using HPLC :

The chemical stability of the SQAd prodrug was then investigated up to 24 h incubation in mouse plasma (at 37 °C) using HPLC. As shown in Figure 28, more than 80% of the prodrug remained intact until 2 h, and 54% after 24 h of incubation. To be noted, after 4 h of incubation in plasma, 25% of the SQAd prodrug was metabolized, which remained in the same order of magnitude than the SQAd NPs diameter decrease (i.e. 33% as described above). Hence, these in vitro observations suggest that in the plasma, when the SQAd NPs started to disassemble, they progressively released the SQAd prodrug which was then metabolized likely by the amidases present in plasma. 154 This metabolization may also occur in vivo in the bood circulation after interaction of the nanoassemblies with circulating macrophages. 151

5.2.3.

In vivo stability of SQAd NPs : Pharmacokinetics of SQAd NPs

The in vivo fate of SQAd NPs was studied in the blood circulation using dual-radiolabeled SQAd NPs. The resulting plasma radioactivity concentration profiles for each radiolabel (i.e. [ 3 H] on the adenosine moiety or [ 14 C] on the squalene moiety) are reported in Figure 29A and expressed as the percentage of injected dose per milliliter of plasma. Plasma profiles for [ 3 H] and [ 14 C] appeared quite divergent. Indeed, [ 14 C]-radioactivity found in plasma rapidly decreased and 1 h post-injection only 0.6 ± 0.03% of the injected dose/mL was still detected, when the amount of tritium was 2.8-fold greater. These results clearly suggested that the SQAd prodrug underwent metabolization in systemic circulation. It is interesting to note that in vivo, the nanoassembly disaggregation was significantly faster than observed in vitro, likely because the body is an open system, with other rapidly occurring events such as tissue distribution. Moreover, the presence of shear stress and other mechanical constraints in the bloodstream may increase the disaggregation rate of SQAd NPs. The in vivo metabolization of the SQAd prodrug has been further investigated by radio-HPLC analysis of the plasma radioactivity at different time points (Figure 29B-D) post-injection of [ 3 H]-SQAd NPs. The SQAd peak intensity (retention time (RT)=22.5 min) significantly decreased over time, confirming a progressive metabolization of the prodrug in the circulation. To be noted that when injected as a free molecule, no adenosine (RT = 12.7 min) could be detected at those time points, which was in accordance with the extremely fast metabolization of this molecule in systemic circulation. 155 Thus, SQAd NPs represent a unique approach to significantly improve blood retention of adenosine under the form of SQAd prodrug. Inosine (RT = 7.1 min), the first circulatory metabolite of adenosine, was not detected either, but a peak corresponding to unidentified highly polar metabolites (RT = 2.1 min) strongly increased over time while the SQAd peak decreased (Figure 29B-D).

Altogether, these results suggest that the linkage of adenosine to squalene and the assembly of the bioconjugate as nanoparticles provided a dramatic protection of adenosine from rapid metabolization both in vitro after incubation with mouse plasma, and in vivo after intravenous injection. SQAd NPs provided a reservoir of adenosine in vivo, by prolonging the stay of SQAd bioconjugate and subsequently the adenosine in the bloodstream, thus maximizing the chances for adenosine or SQAd bioconjugate to reach the endothelial cells of the BBB to further provide the cerebrovascular protection.

Is SQAd NP or SQAd bioconjugate able to directly bind adenosine receptor as do adenosine and activate them?

We are aiming now to check if SQAd bioconjugates or SQAd NPs may act directly on adenosine receptors (ARs) as a specific ligand AR agonists (or antagonists) by binding them via their adenosine moiety. Indeed, as a remainder, the surface of SQAd NP is constituted of adenosine moiety which is thus exposed to the extra and intracellular environment. Thus, SQAd interaction with AR was checked by radioligand displacement assays on membrane fractions of human cells overexpressing ARs. In that respect, specific radioligands were used for each adenosine receptor, and their displacement was measured in the presence of increasing concentrations of SQAd bioconjugate or NPs. Up to 1 mM, SQAd showed no interaction with any of the ARs in the assay buffer, whether as a bioconjugate or assembled as NPs (Figure 30A and 30B respectively). At and above 10 mM, some displacement became detectable, with both SQAd bioconjugates and NPs showing a slight preference for the adenosine A3 receptor (Figure 30A and 30B). To be as close as possible to the in vitro and in vivo conditions, SQAd bioconjugates and SQAd NPs were also preincubated for 4 hours at 37°C with FBS (fetal bovine serum) prior to the radioligand displacement assay. It should be noted that the interaction with the adenosine A2B receptor could not be evaluated under these conditions due to unspecific interactions between the membranes and the FBS. Regarding the three other receptors, an interaction with SQAd bioconjugates or NPs could no longer be detected, even at the highest concentration (Figure 30C and30D). 156 This was attributed to the interaction of SQAd with plasma proteins, especially with cholesterol-rich lipoproteins, i.e., low-density lipoproteins (LDLs) in humans and high-density lipoproteins in rodents. 88 Hence, it was concluded that the preincubation with serum prevented interaction of SQAd with adenosine receptors. Thus, LDLs may hinder the possible interaction of SQAd with ARs. As a result, the hypothesis that SQAd may act directly on ARs as a ligand could be rejected, thus confirming an exclusive indirect activation of ARs by SQAd prodrug releasing adenosine. 156 5.4. Did SQAd NPs interact with human brain endothelial cells (BBB)?

We have previously shown that the neuroprotective effect of SQAd NPs on brain ischaemia resulted from an interaction of these NPs or their molecular components with the microcirculation and the neurovascular unit (NVU), mainly composed by endothelial cells, pericytes, astrocytes endfeet, and neurons, thereby allowing an indirect central effect. 140 We intended now to clarify the interaction of these SQAd NPs with the brain endothelial cells of the BBB, in order to understand if the SQAd NPs were able to be uptaken by these cells, if they were handled intracellularly and if the transcytosis process occured. To this aim, the hCMEC/D3 cell line, a well-characterized human brain capillary endothelial cell line, were used. This model has been demonstrated to mimic most of the basic characteristics of the BBB, even in the absence of co-culture with glial cells. 157 Moreover, it has been shown that hCMEC/D3 cell monolayers display highly restricted permeability to standard drugs over a wide range of hydrophobicity, in good correlation with in vivo permeability values from brain perfusion studies. Furthermore, many of the drug transporters and efflux pumps that are found in the human BBB in vivo could be detected in this cell line, [158][159][160] making it a powerful tool to study both passive and active transport of molecules or nanoparticles across the BBB. To monitor and visualize the interaction of SQAd NPs with hCMEC/D3 cell line (cell internalization and transcytosis), we fluorescently labeled these NPs by incorporating either one fluorescent dye (green BODIPY-FL) or the two fluorescent BODIPY-cholesteryl ester dyes (green BODIPY-FL and red BODIPY-542/563 fluorophores as described previously) depending on the experiments.

Thus, uptake kinetics of the BODIPY-FL-labeled NPs by hCMEC/D3 cells was investigated by flow cytometry (Figure 31A) and confocal microscopy (Figure 31B-D). We thus could demonstrate by these experiments that brain endothelial cells (hCMEC/D3) were able to internalize the SQAd nanoassemblies. The confocal microscopy revealed the presence of fluorescence spots inside the cells suggesting an increasing internalization of SQAd NPs with time (Figure 31). These observations were confirmed by the presence of intracellular radioactivity after incubation of these cells with [ 3 H]-SQAd NPs. 140 In a set of experiments using flow cytometry we could show that internalization of fluorescently labeled SQAd NPs by hCMEC/D3 cells dramatically decreased at 4°C compared to 37 °C (Figure 32A) suggesting that SQAd NPs were endocytosed via energy-dependent pathway. 161 On the other hand, because surface coating of nanoparticles with blood proteins such as apolipoproteins was previously shown to promote organ targeting and drug delivery across the BBB, 162 we investigated the impact of varying serum concentrations on the cell capture of NPs (Figure 32B). In the absence of serum, only a few NPs were internalized by hCMEC/D3 cells even after 24 h of incubation while serum supplementation significantly increased the cell uptake (Figure 32B). The interaction of SQAd NPs with Lipoproteins (LPs) was also confirmed by the biodistribution study of SQAd NPs distribution among plasma fractions. Indeed, after intravenous injection of [ 3 H]-SQAd NPs, the tritium was found in the LDL, HDL and the VLDL fractions while as expected, [ 3 H]-Ad did not interact significantly with the LPs. [START_REF] Sobot | Conjugation of Squalene to Gemcitabine as Unique Approach Exploiting Endogenous Lipoproteins for Drug Delivery[END_REF] As was the case with SQdFdC NPs, here again, LPs probably acted as a natural endogenous carrier that transported SQAd NPs towards the endothelial cells, characterized by high LDL receptors activity, thus enabling their uptake. Moreover, the presence of low-density lipoprotein (LDL) receptors at the BBB and their important role in the delivery of essential lipids and also of some nanocarrier systems has been reported by several groups. 163,164 showing that cellular association is significantly decreased at 4 °C (***p < 0.001, N = 3). B) Cellular internalization of fluorescently labeled SQAd NPs in the presence of different concentrations of serum. Cellular uptake of fluorescent SQAd NPs was quantified by flow cytometry in the presence of different percentage of serum (0%, 1%, 2.5% and 5%) after 1h, 2h, 4h, 6h and 24h of incubation. The serum % clearly influenced the cell capture of NPs: in absence of serum, no significant cell internalization could be observed, whereas the serum supplementation induced NPs uptake. Noteworthy, the internalization increased when adding 2.5% compared to 1%, but the uptake profile was similar with 2.5% and 5% of serum, suggesting a saturation of the protein adsorption at the nanoparticles surface. Results are presented as mean ± SEM (N = 3 independent experiments run in duplicate) (fig. adapt. from Gaudin et al. 2015). 161 Furthermore, to confirm this hypothesis, hCMEC/D3 cells were pre-treated with excess of LDL (which led to overexpression of LDLR), prior to being incubated with SQAd NPs. The experiment revealed that SQAd NPs cell uptake (at 1 h 30) was reduced by 32% (Figure 33A). 161 On the other hand, the co-incubation of red BODIPY-542/563 labeled SQAd NPs and green fluorescently-labeled LDL with hCMEC/D3 cells led to a perfect colocalization of both dyes (yellow spots) when the cells were observed by confocal microscopy after 1, 2, and 4 h incubation (Figure 33B). These results highlighted the implication of the LDL receptors in the uptake of SQAd NPs by the hCMEC/D3 cells. 161 All together, these results clearly indicated that NPs internalization occurs via energy-dependent pathway involving LDL receptors and is mediated by the formation of a protein corona (lipoproteins), which has been shown to be determinant for nanoparticle-receptor interac on. 165 5.6. Once in the brain endothelial cells, in which subcellular compartment accumulated SQAd NPs ?

It was previously reported that following endocytosis, an energy-dependent intracellular trafficking of nanoparticles among endosomes, lysosomes, Golgi complex, and endoplasmic reticulum (ER) was observed. 166 Thus, fluorescently labeled SQAd NPs were incubated during 24h with hCMEC/D3 cells and their localization was examined after the labeling of the different subcellular compartments using specific markers for late endosomes and lysosomes, the ER, and the Golgi apparatus. 161 Thus, it was observed that SQAd NPs fluorescence colocalized only with the late endosomal/lysosomal marker (figure 34) at all incubation times and were found in close vicinity of the Golgi apparatus. 161 Lysosomes are critical structures regarding the degradation of materials arriving via endocytosis, as their acidic environment and enzymatic content can be involved in intracellular drug liberation and/or degradation. We thus hypothesize that once in the late endosomes and then lysosomes, SQAd NPs may disassemble and release SQAd bioconjugates which are metabolized into adenosine.

Do SQAd NPs disaggregate inside brain endothelial cells after being endocytosed?

Using FRET SQAd NPs (encapsulating green cholesteryl ester BODIPY-FL probe and a red cholesteryl ester BODIPY-542/563 probe), we then investigated by flow cytometry the integrity of the NPs during internalization by hCMEC/D3 cells during 72 h. During the first hours of incubation, a linear increase of FRET signal was observed, clearly indicating that both BODIPY dyes could be found inside the cells (Figure 35). Then, degradation occurred inside the cells, allowing the liberation of the BODIPY dyes as revealed by the progressive disappearance of the FRET signal. 140,161 These observations mean that SQAd NPs undergo degradation several hours after internalization by brain endothelial cells.

Is it possible that a fraction of SQAd NPs was able to translocate intact into the brain during transcytosis phenomenon?

The passage of SQAd NPs across the hCMEC/D3 cells monolayer has been also analyzed using a transwell system 167 (Figure 36A). Within such an experimental setup, the cells were grown on a collagen-coated membrane to form the barrier and the FRET SQAd NPs (used as a marker of NP integrity) were added in the apical compartment (modeling the blood compartment), whereas the translocation toward the basolateral compartment (modeling the brain compartment) was measured by spectrofluorimetry upon donor excitation. 161 The experiment showed that the FRET signal in the apical compartment was present during the whole course of the experiment (72 h), even if the relative FRET intensities only slightly decreased (black curves in Figure 36B-D) over me, as his was a ributed to a partial degradation of the NPs within the apical compartment. But more importantly, no FRET signal could be observed in the basolateral compartment at all incubation times, which clearly confirmed previous observations, and led to the conclusion that the NPs were internalized as intact nanostructures but were not able to reach the brain intact. 161 5.9. After intracellular disaggregation of SQAd NPs, does SQAd prodrug undergo bioconversion thus contributing to increase the adenosine intracellular content?

Assuming that once in the cell, SQAd would undergo hydrolysis in acidic endo-lysosomal compartments, SQAd degradation was then evaluated over 24 h in acidic conditions (pH 4.8) in the presence or in the absence of cathepsin B, a typical lysosomal enzyme able to cleave amide bonds (Figure 37A). Although almost no bioconversion of the SQAd bioconjugate could be observed until 6 h, up to 20 % of SQAd was degraded after 24 h of incubation irrespective of wheteher or not the cathepsin B were present. Interestingly, the degradation of SQAd led to the release of free adenosine (retention time 3.9 min), which was characterized by HPLC analysis (Figure 37B). In a nutshell, all these results strongly suggest the following series of events: SQAd NPs were taken up by the cells and remained intact the first hours, before undergoing degradation, thus allowing the release of SQAd prodrug from the NPs inside the cells. Then, SQAd may be further metabolized into adenosine, under acidic conditions in lysosomes (low pH conditions) by various mechanisms. 168 Either SQAd prodrug is released from the nanoassemblies prior to undergo intracellular hydrolysis, or the prodrug is hydrolyzed directly from the surface of the nanoassemblies.

Hence, SQAd intracellular bioconversion should increase the adenosine intracellular content. It remains now to be checked if some adenosine could be further released in the extracellular compartment as a result of the equilibrium between the intracellular and the extracellular adenosine levels.

5.10.

Once released intracellularly, may the exogenous adenosine be effluxed in the extracellular medium ?

The efflux of adenosine from hCMEC/D3 cells was then evaluated after incubation with SQAd NPs from 5-minute to 4-hours (in comparison with free adenosine or 5% dextrose solution). The cell culture medium was then removed and replaced by new simplified medium (containing glucose and inorganic salts but without vitamins and amino-acids) in order to remove adenosine (eventually resulting from the release from SQAd NPs before their capture by the cells). The extracellular medium was then analysed 1 h following cell culture medium renewal, by HPLC in the presence of EHNA (erythro-9-(2hydroxy-3-nonyl)adenine), an inhibitor of adenosine deaminase, to determine the quantity of adenosine released from the cells (Figure 38). After 5-minute incubation, cells treated with free adenosine released higher concentrations of adenosine (around 300 nM) than cells treated with SQAd NPs (around 100 nM) or dextrose. This clearly highlighted the almost instantaneous ability of the cells to uptake free adenosine, whereas SQAd NP uptake and cleavage (which results in adenosine metabolite release) required more time (Figure 38). Interestingly, after a longer incubation time with free adenosine, a decrease in adenosine release was observed, which likely resulted from a consumption of the overall pool of adenosine by the cells. On the contrary, after a longer incubation time with SQAd NPs, higher levels of released adenosine were observed, which correlated well with an increased intracellular content of SQAd over time. 156 Hence, these results clearly highlight the reservoir effect resulting from the intracellular accumulation of SQAd NPs followed by the progressive cleavage of the SQAd bioconjugate, allowing a progressive release of free adenosine in the extracellular medium. Then, the effluxed adenosine may instantaneously interact with adenosine receptors located at the surface of brain endothelial cells, pericytes or astrocytes end-feet, leading to the therapeutical observed effect.

6. Biodistribution study of SQAd NPs in other organs : 6.1. How SQAd NPs are biodistributed in other vital organs?

6.1.1. Biodistribution study : heart, liver, spleen, kidneys Tissue distribution of [ 3 H]-SQAd and [ 14 C]-SQAd was also investigated in other vital organs of mice after intravenous injection of radiolabeled [ 3 H]-SQAd NPs and [ 14 C]-SQAd NPs respectively and compared to the distribution of free [ 3 H]-Adenosine. Concerning the lungs, the results suggested that SQAd NPs or SQAd bioconjugate may penetrate entire which was not the case for the heart. Notably, in the heart the radioactivity raising from [ 3 H]-Adenosine administration was relatively constant up to 5 day post-injection, when the one resulting from NPs decreased constantly to reach a very low level, suggesting the absence of accumulation of SQAd NPs or free SQAd prodrug in this organ. These distinct profiles confirm that the squalenoylation of adenosine allows to modify the drug biodistribution pattern. 151 As expected, the highest radioactivity content was observed in the liver and in the spleen (Figure 39) 169 after intravenous administration of radiolabeled-SQAd NPs (while the radioactivity found in the kidneys was more important after injection of Adenosine free). 151 This biodistribution pattern showing equal [ 3 H] and [ 14 C] contents suggested the uptake of intact SQAd by these tissues which was in accordance with previous studies showing that colloids are preferentially captured by the liver, due to opsonization, 170 while free adenosine was mainly eliminated by the kidneys as commonly observed for small hydrophilic molecules. Maximum accumulation of NPs in the liver and spleen occurred at 15 min post-injection, and then constantly decreased with time. These results were confirmed by radio-HPLC analysis of the same organs. Indeed, intact SQAd was found in the liver at 1 h post-injection (Figure 39B) but was no more detectable after 24 h (Figure 39C). In the spleen, SQAd accounted for 96 ± 1% of the total radioactivity found in this organ at 1 h post-injection (Figure 39E) and still for 66 ± 4% after 24 h (Figure 39F). Interestingly, adenosine released from SQAd NPs was detected in the liver 1 h (accounting for 27 ± 7% of radioactivity detected in this organ) and 24 h (accounting for 47 ± 8% of radioactivity detected in this organ) post-injection, but not in the spleen. SQAd metabolization in the liver was probably due to the activity of amidases, and in particular fatty acid amide hydrolases (FAAHs), 171,172 as the incubation of the SQAd prodrug in the presence of FAAH enzymes in vitro was shown to induce a sustained release of adenosine. 151 Moreover, FAAHs were shown to be 100-times more active in the liver compared to the spleen, probably explaining the absence of metabolization in this organ 24 h post-administration. The emergence of side effects is an important limitation for the use of most CNS medicines. In this context, given that SQAd NPs accumulated in high quantity in the liver, the levels of aspartate transaminase (AST) and alanine transaminase (ALT) were analyzed. The normal levels in the SQAd nanoassemblies group, compared to control animals (dextrose 5%) indicated no damage to the liver. This was confirmed by histological analysis of this tissue at 24 h and 28 days post-injection. 140 7.2. Do SQAd NPs may be considered as safe upon systemic intravenous administration?

A complete blood count was also performed and didn't reveal any increase or decrease in the white or red blood cell counts after SQAd administration. The levels of creatinine and urea were also checked and didn't show any increase in the SQAd NPs group, indicating no damage to the kidneys too. The observations by histological analysis of kidneys, spleen and lungs showed no morphological damages after 24 h and 28 days post-injection of SQAd NPs. Therefore, all these results show that SQAd NPs may be considered as safe upon systemic intravenous administration at the therapeutic dose of 15 mg/kg. 140 On, the other hand, given that adenosine is known to be a neuromodulator involved in numerous neurological processes, 128 the potential effect of SQAd NPs on food intake, body weight and sleep cycle was evaluated in comparison with control group and didn't reveal any side effect. Together, these results confirm that the dramatic pharmacological efficiency of SQAd nanoassemblies was obtained without triggering any side effect commonly associated with central administration of adenosine. 140

Conclusion :

We could demonstrate through this study the mechanism of action of SQAd NPs. Once in the blood circulation, the SQAd NPs interact with plasma proteins (among them LDL lipoproteins). A significant amount of SQAd NPs is captured by the reticulo-endothelium system, such as resident macrophages (ie. kupffer cells) and accumulates mainly in the liver and spleen. Nevertheless, a fraction of NPs could reach the brain endothelial cells, either as intact SQAd NPs (surrounded by LDL corona) or as SQAd bioconjugates which are inserted in LDLs, and both forms are then internalized through LDLRmediated endocytosis by the brain endothelial cells. Once in the endo-lysosomal compartment, SQAd NPs (or SQAd bioconjugate) behave as an intracellular reservoir, progressively releasing adenosine after enzymatic cleavage of SQAd bioconjugate. Then, some amount of adenosine is finally effluxed outside the cell and interacts instantaneously with ARs located either on endothelial cells or on the pericytes and the end feet of astrocytes (which are located in the close viscinity of endothelial cells) as an autocrine or paracrine signal (Figure 40). Biodistribution study in mice subjected to ischemic mice It is important to note that the biodistribution study was performed in healthy animals with intact BBB which was not the case with mice subjected to MCAo. This may explain the absence of intact adenosine detected in the brain. It would have been more relevant to study the biodistribution of SQAd NPs on mice subjected to ischemic stroke. Indeed, in the absence of any insult, the BBB is a highly organized structure, composed of different cellular types that cooperate to protect neurons from external agents circulating in the blood. In the ischemic situation, within infarct area, the alteration of BBB leads to a significant increase in paracellular permeability at the level of the cerebral microvasculature (a remarkable pathological feature of stroke). 173 Thus, it would be interesting to explore under those conditions if a fraction of SQAd NPs and/or SQAd bioconjugate and/or exogenous adenosine was able to reach the brain, by using dual-radiolabeled SQAd NPs as was done before.

Thus, the absence of radiolabeled adenosine or SQAd in the brain revealed by the biodistribution study could not reflect the reality in the pathological situation. The promising results obtained in the cerebral ischaemia model triggered our curiosity regarding further extending the pharmacological application of SQAd nanoassemblies to the treatment of traumatic injury of the spinal cord. It has been shown that adenosine could provide neuroprotection after spinal trauma, especially through peripheral and central effects on its A2A receptors. 152 To determine whether the SQAd nanoassemblies could improve functional outcome in a spinal cord injury (SCI) model, we performed a behavioural and ultrastructural study in Sprague-Dawley rats undergoing a T9 contusion injury, which is the most relevant model of human SCI (Figure 41 a). 174 Within 5 min post-injury, we injected intravenously either SQAd NPs (32 mg kg -1 ; equiv. dose of 11.5 mg kg -1 adenosine and 20.2 mg kg -1 squalene), SQ NPs (20.5 mg kg -1 ), free adenosine (11.5 mg kg -1 ) or a solution of dextrose 5% as control. Functional evaluations were performed 175 at 24, 48 and 72 h post-injury, and then regularly up to 28 days post-injury. The scores of animals in all groups are presented in Figure 41b. At 72 h after trauma, animals injected post-injury with either dextrose 5% solution or free adenosine solution were unable to voluntarily move their hindlimbs, in accordance with the presence of the important traumatic area observed on the cords (Figures 42a and42b). In contrast, animals injected with the SQAd NPs did not present any visible traumatic area on their cords (Fig. 42c) and were fully able to walk with consistent coordination between the hindlimbs and forelimbs. As soon as 9 days after trauma, SQAd nanoassemblies treated animals achieved a score of 14.4 (Figure 41b), which is widely accepted as promising and clinically meaningful 174 as it is a sign of axonal transduction through the lesion site. 176 All other treatment groups did not exceed a score of 5 until 28 days post-trauma (Figure 41b).

Because it has been shown that the preservation of only 5-10% of axons in each individual tract is sufficient to achieve meaningful locomotor recovery, 117 we further investigated the ultrastructure of the myelinated axons 2 and 28 days post-injury, using TEM (Figure 42 d-g). Examination of the white matter of control animals (SQ NPs or free adenosine solution) revealed that the small, medium and large myelinated axons had interruptions in myelin configuration (Figures 42e 42f). In the case of animals treated with the SQAd NPs, axons with separations in myelin configuration were detected, but most of these neurons were medium or large in size, with a few of the medium and most of the small myelinated axons being normal (Figure 42g). Quantification of these observations showed that the SQAd NPs treatment significantly decreased damage to the small (Figure 42h) and medium myelinated axons, as soon as 2 days post-trauma. d-g) After 48 h, the ultrastructure of the myelin configuration of the axons was studied by TEM. For animals treated with the solution of free adenosine (e) or only squalene NPs (f), the small, medium and large myelinated axons presented separations and/or interruptions in their myelin configuration (red arrows) compared with healthy animals (d). For SQAd NPs-injected animals (g), examination of the white matter showed medium and large myelinated axons with separations in the myelin configuration, with a few of the medium and most of the small myelinated axons found to be ultrastructurally normal (blue arrows). m, myelin; s, myelinated axons with separation in myelin configuration. h, Quantification of damage on the small myelinated axons showed that the SQAd NPs dramatically reduced the damage score (data are presented as mean ± s.d., ****P < 0.0001) compared to all groups (fig. adapt. from Gaudin et al. 2014). 140

Conclusion :

Through this study, we could confirm once again the highly potent neuroprotective and neuroregenerative effect of SQ-Ad NPs in spinal cord injury rat model. SQAd NPs were able to significantly reduce the damage in the injured spinal cord of the rats by regenerating the myelinated axons in the affected area and this allowed an impressive recovery of the clinical score of the injured rats, especially concerning the complete recuperation of their walking capabilities. an impressive recovery of their hindlimbs. Overall, these results allowed us to further enlarge the therapeutic prospects of squalene nanoparticles to the treatment of peripheral nervous system pathologies.

12. Perspectives :

12.1.
Could the neuroprotective and neuroregenerattive effects of SQAd NPs be also extended to peripheral nerve injury? 12.1.1. Evaluation of neuroprotective and neuroregenerative effects of SQAd NPs in peripheral nerve injury model in rats.

Peripheral nerve injuries (300 000 cases each year in Europe) caused by accidental trauma can have devastating consequences by compromising the patient's quality of life due to lifelong morbidity such as chronic neuropathic pain and permanent disability. 177,178 These injuries also have important consequences for the social welfare system 179,180 because nerve injuries of the upper extremities predominantly affect the young and economically active population. 181 Outcome after traumatic nerve injuries and reconstructive surgery is primarily based on motor and sensory recovery of individual nerves. It is estimated that 66% patients with traumatic nerve injuries recover to normal or average function, the other 34% exhibit incomplete motor and sensory nerve recovery and poor functional outcomes. 181 Both proximal and distal nerve injuries led to long-term disability, subsequent sick-leave and in 30% to permanent disability pension.

Nerve injury may cause nerve displacement, stretching, crushing, partial or complete section. Based on Seddon and Sunderland classifications, nerve injuries can be classified into different categories based on the presence of demyelination and the extent of damage to the axons and the connective tissues of the nerve (Figure 43). The mildest form of injury is called neurapraxia, defined by focal demyelination without damage to the axons or the connective tissues. Neurapraxia (Grade I) typically occurs from mild compression or traction of the nerve and results in a decrease in conduction velocity. The next level is called axonotmesis (Grade II-IV), which involves direct damage to the axons in addition to focal demyelination while maintaining continuity of the nerve's connective tissues. The most severe form of injury is called neurotmesis (Grade V), which is a full transection of the axons and connective tissue layers wherein complete discontinuity of the nerve is observed. The presence of an intact endoneurial tube more often leads to a better outcome in nerve regeneration. Thus Grade II lesions, which confer damage to the axons alone without any damage to the surrounding connective tissue, have optimal conditions for axonal regrowth. Grade III and IV lesions however, not only have a disrupted endoneurial tube making it difficult to form appropriate bands of Bungner, they also have increased scar tissue formation that can be a considerable deterrent to the growth cone, leading to disorganized outgrowth (Figure 44). Given the very impressive results obtained with SQAd NPs in spinal cord injury model, we aim now at evaluate the neuroprotective and neuroregenerative properties of SQAd NPs in peripheral nerve injury rat model.

In that respect, the neuroprotective and neuroregenerative properties of squalene-adenosine nanoparticles (SQAd NPs) will be evaluated using the most common injury model which is the sciatic nerve injury. Although sciatic nerve injuries in humans are rare due to the deep anatomical location within the lower extremity, the animal model provides a plethora of information regarding recoveries of particular nerve types and their potential for recovery as they attempt to reach endoneurial tubules. In that respect, peripheral nerve injury rat model will be used, and the injury will be induced by two nerve crushes (spaced of 0.7 cm apart) on the sciatic nerve (Figure 45). 182 The degree of nerve injury will correspond to grade III, according to Seddon and Sunderland classification, corresponding to axon and endonerieum damage, leaving perineurium and epineurium intact. The neuroregenerative and neuroprotective effects of SQ-Ad NPs will be evaluated through histological and behavioral studies following acute or chronic treatment. Acute treatment of SQ-Ad NPs :

• Histology:

The study will be performed on male Sprague-Dawley rats with nerve injury according to experimental design (Figure 46). SQAd NPs will be injected (single administration) 5 minutes after injury, whether intravenously (32 mg/kg; equiv. adenosine 11.5 mg/kg) or intraneurally (0.32 mg/kg; equiv. adenosine 0.11 mg/kg), between the two crushes. Two weeks post-injury, the nerve will be sectioned (between the two crushes), and a Fluoro-Gold (FG) solution will be applied during 30 minutes to the proximal nerve stump. Five days later, the rats will be sacrified, and then reperfused. The lumbar spinal cord (L4 to L6) (Figure 45) and their corresponding dorsal root ganglia (DRG) will be collected and compared with those of rats treated with daily subcutaneous injection of progesterone (positive control) by quantification of the retrogradely labelled neurons using confocal fluorescence microscopy. The regeneration of sensory neurons in DRG and motor neurons in spinal cord will be evaluated.

• SQ-BASED NANOMEDICINES FOR PAIN MANAGEMENT :

LEU-ENKEPHALIN-SQUALENE NPS

Pain represents an important global health challenge for many reasons, including high prevalence, serious associated sequelae and the relative lack of efficient treatment, especially for neuropathic pain alleviation. Pain relevant disorders such as arthritis, cancer and pathological changes in nervous system are highly prevalent and bring great inconvenience and distress to the patients. 184 Chronic pain has a significant impact not only on the patients themselves, but also on the broader community and economy. By activating μ-opioid receptors, the most powerful and widely used painkillers in current clinical practice are morphine and the related synthetic opioids. But morphinic treatments are associated with severe side effects, such as respiratory depression and addiction linked to the development of opioid tolerance and dependence. 185 According to CDC/NCHS (Center for Disease Control and Prevention/National Center for Health Statistics) , National Vital Statistics System, 186 every day, more than 115 people in the United States die after overdosing on opioids. The misuse of and addiction to opioids, especially morphine, represents a serious national crisis in the US (and probably also in other countries) that affects public health, as well as, social and economic welfare. This highlights the need to urgently find new painkillers as effective as morphine but devoided of the severe side effects. In this context, endogenous neuropeptides, such as enkephalins, represent a very promising alternative to morphin derivatives and synthtic opioids.

1. State-of-art on Enkephalins : mechanism of action and limitations 1.1. What is the mechanism of action of enkephalins ?

Leucine-enkephalins (Leu-enkephalins) and Methionine-enkephalins (Met-enkephalins) are endogenous pentapeptides that are distinguished by their carboxy-terminal amino acids, leucine, or methionine : Tyr-Gly-Gly-Phe-Leu/Met (Figure 47). Enkephalins activate both μ-and δ-opioid receptors, but with a ten-fold higher affinity towards δ-opioid receptors. 187 Compared with μ-opioid receptor agonists, δ-opioid receptor ligands are believed to have reduced side effects such as a much lower abuse potential. [188][189][190][191][192][193] At the pre-synaptic level, the binding of enkephalins to the opioid receptors (seven transmembrane domain G-protein coupled receptors) results in conformational changes of these receptors allowing their coupling to heterotrimeric G proteins (consisting of Gα, Gβ and Gγ subunits). The Gαβγ heterotrimer which was initially bounded to GDP (Guanosine diphosphate) through its Gα subunit, dissociates from GDP in favour of GTP-bound. This results in activation of the trimeric G-protein complex, which dissociates into Gα (binded to GTP) and Gβγ subunits (Figure 48). Then, Gα•GTP inhibits adenylyl cyclases (ACs) which results in reduced production of intracellular cAMP (cyclic adenosine monophosphate), and leads to a decrease of protein kinase A (pkA) activity. Concequently, the opening of potassium channels (efflux of potassium) as well as inhibition of the opening of calcium channels (inhibition of calcium entry) cause hyperpolarization which inhibits the release of excitatory neurotransmitters (Figure 49). 195,196 At the postsynaptic level, opioid receptors mediate hyperpolarization by opening K + channels, thereby preventing excitation and/or propagation of action potentials. 197 These co-ordinated changes lead to analgesia. Although very effective, these endogenous opioid peptides are often not directly suitable for use as convenient therapeutics because of their intrinsic weaknesses, including poor chemical and physical stability, and a short circulating plasma half-life (from a few second to 2-3 minutes). 198 In this respect, due to brain enzymatic activity, it was reported that extremely high quantities (120 to 200 μg) of enkephalins needs to be injected into rat cerebral ventricles and brain to induce analgesia. 199 These limiting aspects must be seriously taken in account for their use as medicines.

Which approaches have been used to exploit the analgesic activity of endogenous neuropeptides ?

To date, the two main approaches to enhance the analgesic activity of opioid peptides relied on (i) the increase of the stability of endogenous peptides using enkephalinase inhibitors, or (ii) the chemical synthesis of exogenous peptides with enhanced lipophilicity and degradation resistance. However, enkephalinase inhibitors present some limitations such as insufficient enzymatic specificity and they also depend primarily on the amount of endogenous enkephalins released in response to a noxious stimuli. 200,201 On the other hand, the derivatization of peptides often ends-up with biologically inactive compounds, and their lipidation often led to poor water solubility. 202 This explains why none of the research efforts performed since decades ago has resulted in marketed medicines.

What about nanomedicine approach ?

Although nanoparticulate drug delivery systems represent an efficient approach for protecting drug molecules from rapid metabolization, only few were applied to enkephalins and enkephalins derivatives. In a primary study, Kreuter et al. showed that polybutylcyanoacrylate (PBCA) nanoparticles coated with polysorbate 80, a non-ionic surfactant, were able to transport various drugs to the CNS across the blood brain barrier (BBB), including dalargin, a LENK analogue peptide. [203][204][205] Even though the in vivo studies demonstrated that dalargin-loaded PBCA particles exhibited an anti-nociceptive effect, the mechanism of action did not appear completely clear. 206 In addition, the potential toxicity associated with polysorbate 80-coated PBCA nanoparticles is one of the important concerns remaining with this approach since polysorbate 80 was shown to induce BBB disturbance following intravenous injection. 206,207 The biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA) polymer was also used for the vectorization of drugs, proteins, peptides and various other macromolecules such as DNA and RNA. In that respect, among neuropeptides, only Dalargin was incorporated into PLGA or PEGylated PLGA NPs. 208 However, no in vivo studies have been reported so far. Chitosan-based NPs encapsulating the dalargin were also developed and formulated with anionic derivatives of either βcyclodextrin or dextran sulfate to improve stability of this neuropeptide and to modulate its release kinetics. However, these stability studies were only performed in PBS media and there was no followup concerning this approach since then. 209 Quaternary ammonium palmitoyl glycol chitosan (GCPQ) nanoparticles were also proposed for the delivery of analgesic peptides to the brain. Thus, TPLENK ester prodrug (tyrosinyl-palmitate-leu-enkephalin) was formulated with GCPQ into nanofibers. This nanoformulation developed by Uchegbu avoid plasma protein binding and liver uptake but the amount of TPLENK in the brain was less than 0.1% of the injected dose. 210 Another approach using oral and nasal route for the brain delivery of Leu-enkephalins was described by the same group. In that respect, oral and intra-nasal administration of the neuropeptide loading quaternary ammonium palmitoyl glycol chitosan (GCPQ) nanoparticles was performed in mice and rats respectively. Via oral route, these nanoparticles encapsulating leu-enkephalin or its Palmitoyl-derivative, increased brain drug levels by 50% and 67% respectively, and significantly increased antinociceptive activity in mice using tail-flick test. 211 The intra-nasal route, for its part, reversed hypersensitivity in the Complete Freund's adjuvant (CFA) model of inflammation-induced tactile hypersensitivity and produced relief of on going pain in a nerve injury model of neuropathic pain. 212 However, it's important to note that this model is not necessarily transposable in humans, as the nasal anatomy in animals differs widely in relation to humans. Indeed, the ratio of surface area to luminal volume in the nasal cavity of a rat is very different from that in humans (i.e., rat is 3350 mm 2 /cm 3 and human is 820 mm 2 /cm 3 ). 213 Grinberg et al (2005) developed novel bolavesicles employed to deliver analgesic peptides (kyotorphin or leu-enkephalin) to the brain mice after iv administration. These bolavesicles are monolayer membrane vesicles that are assembled from cationic amphiphilic compounds synthesised from vernonia oil, with 2 acetylcholine head groups that are connected to both sides of a lipophilic chain. These nano-sized vesicles, formulated with and without chitosan, were able to cross the BBB and release the analgesic peptides in the brain in quantities sufficient to elicit efficient and prolonged analgesic activity. 214 The use of liposomes as drug carriers for transdermal delivery of Leu-Enkephalin (LENK) using iontophoretic transport was also investigated ex vivo by Banga. 215,216 This preliminary study indicated that the liposomal formulation provided a significant protection of the LENK from proteolysis degradation into the skin. However, when absorbed into the blood stream the neuropeptide was rapidly degraded.

As toxicity and elimination of nanoparticles from the brain parenchyma remain a major issue and this is especially true for the above mentioned cationic nanodrugs, thus, the design of safe analgesic nanoformulations able to restrict their activity peripherally should overcome these issues. Indeed, avoiding brain penetration while optimizing drug concentration at the site of injury may represent a a highly promising strategy. Another advantage of targeting peripheral opioid receptors is to prevent and reverse the effects of multiple excitatory agents expressed within damaged tissue. 217 2. Application of squalenoylation nanotechnology to Leu-Enkephalin : synthesis and characterization 2.1. Can we obtain nanoparticles of Leu-enkephalin peptide using squalenoylation approach?

The aim of this project was to develop novel nanomedicine approach allowing the specific delivery of Enkephalin neuropeptide into inflamed tissues for pain control, using "squalenoylation" concept. It is important to note that the conjugation of a peptide to squalene has been an innovative, rather tricky achievement for the following reasons: (i) peptides are hydrophilic molecules, whereas squalene is a lipid, insoluble in water. This makes the chemical reaction rather uncertain and (ii) the chemical modification of a peptide often results in a loss of its pharmacological activity. The chemical approach used in the present study has overcome all these complications, allowing the design of a small library of innovative enkephalin-squalene bioconjugates with a preserved pharmacological activity.

Synthesis of Leu-enkephalin-SQ bioconjugates

In this context, the conjugation of SQ to the currently unusable Leu-enkephalin (LENK) was considered in order to exploit its analgesic potential. Conjugation of SQ to LENK was then performed by exploiting the 2 terminal sites of the neuropeptide : C-terminal acid and N-terminal amine (Figure 50). Although it is well known that the N-terminal of LENK is required for binding to opioid receptors, 218 the conjugation on N-terminal was envisaged based on the fact that the amide bond on this position is susceptible to be cleaved by overexpressed peptidases within inflammation site. 219 In that respect, various squalenoyl-Leu-enkephalin (LENK-SQ) conjugates were designed by also using chemical linkers (Figure 51). The linkers with different sensitivity to hydrolysis were used in addition to a simple amide bond in order to modulate the release kinetics of LENK from NPs. It was expected that LENK-SQ-Diox 40 (with dioxycarbonyl linker) released faster the LENK neuropeptide than LENK-SQ-Dig 41 (with diglycolic spacer), and LENK-SQ-Am 42 was supposed to trigger the slower release as reported in the literature. 220,221 Scheme 17 : Synthesis of Leu-enkephalin-squalene with amide bond (LENK-SQ-Am) 42.

Preparation and characterization of Leu-enkephalin-SQ nanomedicines

The LENK-SQ bioconjugates were then formulated as nanoparticles using the nanoprecipitation technique without the aid of any surfactant. In that respect, LENK-SQ ethanolic solutions were nanoprecipitated in 5% dextrose solution (2 mg/mL) and the LENK-SQ NPs were obtained with high drug payloads (i.e. 53 to 59%) (Figure 52). The size of the NPs varied from 61 nm to 112 nm, based on one hand on the linkage between SQ and enkephalin, and on the other hand on the conjugation site. The NPs displayed spherical and monodisperse structures with net positive or negative surface charge (Figure 52), which could be attributed to the free terminal function of the peptide depending on the bioconjugation mode. Indeed, in case of the LENK-SQ-Diox bioconjugate, the SQ conjugation on the C-terminus LENK peptide let its N-terminus site free (primary amino group), leading to a net positive charge. On the contrary, the zeta potential became negative when the conjugation with SQ was performed on the N-terminus LENK peptide (LENK-SQ-Dig and LENK-SQ-Am). 223 3. Release kinetics of LENK-SQ NPs :

3.1. Are LENK-SQ NPs able to release LENK in serum ?

In order to ascertain that free LENK peptide could be released from the LENK-SQ nanoparticles, the chemical stability of the different linkers (i.e., direct amide or dioxycarbonyl or diglycolate spacers) was studied after incubation of the LENK-SQ NPs with mouse serum (Figure 53).

The corresponding experiments clearly showed that the concentration of the bioconjugate decreased gradually, while LENK-SQ-Diox NPs progressively released the free LENK peptide. The peptide was then slowly degraded by the peptidases of the serum but still lasted beyond 10 h postincubation (Figure 53A). The incubation of LENK-SQ-Dig in serum resulted in a decrease of the bioconjugate until completely disappearance at 2 h, but no presence of free peptide was detected. The RP-HPLC analyses, however, highlighted a slow release of the peptide still attached to its linker. This release reached a maximum at 45 min followed by progressive degradation of the peptide-linker fragment which could still be detected over 10h (Figure 53B). On the contrary, LENK-SQ-Am remained stable in serum, without significant decrease during 48 h, and no peptide was released in the course of the experiment (Figure 53C). Of note, in the case of the LENK-SQ-Diox bioconjugate, both the LENK and the SQ moieties were each linked through an ester bond to the dioxycarbonyl linker. In the case of the LENK-SQ-Dig bioconjugate, the diglycolate linker was attached on one side to SQ by an ester bond and on the other side to the LENK through an amide bond when in the LENK-SQ-Am bioconjugate a direct amide bond connected the LENK to the SQ moiety. The absence of release of LENK from LENK-SQ-Am was not surprising given that, generally, amide bond is chemically and enzymatically more stable than ester bond. 223 Furthermore, it is well known that N-terminal modification of linear peptides increases peptidase resistance, which is the case for LENK-SQ-Am and LENK-SQ-Dig. 224 Finally, as mouse serum is particularly rich in esterases, it was expected that the release of LENK from LENK-SQ NPs mainly resulted from enzymatic hydrolysis of ester bond. 220 Despite these in vitro data showing that depending on the type of linker, whether or not the LENK peptide was released, all the 3 conjugates were recruited for anti-hyperalgesia experiments. It was expected that the more aggressive in vivo enzymatic content, particularly rich in proteolytic enzymes at the inflammation site, will contribute to the release of LENK from all the bioconjugates. Indeed, the presence of high concentrations of proteolytic enzymes like chymotrypsin, cathepsin D and other proteases in inflammatory exudates has been reported which also indicates their important role in the inflammatory process. The antinociceptive properties of LENK-SQ NPs were evaluated using a carrageenan-induced paw edema model in rats. The inflammation was generated by intraplantar injection of λ-carrageenan into the right hind paw thus inducing a local inflammatory response, characterized by marked edema, hyperthermia, and hyperalgesia restricted to the injected right hind paw. Then, the pain sensation was evaluated by the Hargreaves test, which consists in the measurements of paw withdrawal latencies (PWL) to a noxious thermal stimulus according to experimental design illustrated in Figure 54. The increase in PWL is characteristic of an analgesic effect. Indeed, rats take more time to remove their inflamed paw after noxious stimulus (which means that they are less sensitive to this stimulus). Baseline measurements of paw withdrawal latencies (PWL) were first made prior to carrageenan injection using Hargreaves test [START_REF] El-Din | Magnetite Nanoparticles as a Single Dose Treatment for Iron Deficiency Anemia[END_REF] and presented a mean value ± SEM (n = 8) of 6.65 ± 0.37 s (Figure 55). The baseline measurements correspond to the « normal » response to the induced noxious stimulus (with absence of inflammation). Then, thermal sensitivities were evaluated 3 h after carrageenan injection, which corresponded to the peak inflammatory response, leading to a mean decrease of 52.48% of PWL compared to the basal PWLs in naïve rats (Figure 55). This decrease in PWL indicates that rats are hyperalgesiated (more sensitive to pain due to inflammation).

Anti-hyperalgesic effects were assessed using the same test at various times during 4 hours, after acute intravenous administration of the different drug treatments at this 3 h inflammation peak (Figure 54). The acute treatment with morphine which was used as positive control (1 mg/kg), reduced the thermal hyperalgesia as shown by the resulting significant increase in PWLs. Indeed, 10 min postmorphine injection, the PWL reached 12.87 ± 1.38 s, while it remained at 3.05 ± 0.20 s after treatment with a control dextrose solution (Figure 55A). However, morphine's anti-hyperalgesic pharmacological activity disappeared rapidly and no longer significant effect was observed as soon as 100 min postmorphine administration (Figure 55A).

Then, the anti-hyperalgesic effect of LENK-SQ NPs with the 3 different linkers was evaluated (Figure 55C-H). All LENK-SQ NPs displayed a significant anti-hyperalgesic effect on inflamed hind paw leading to a significant reduction of thermal hyperalgesia, as expressed by a dramatic increase of respective AUC values in comparison with λ-carrageenan-treated rats injected with either the free LENK peptide or the blank SQ NPs (Figure 55D,55F and 55H). The anti-hyperalgesic effect was less intense than after morphine treatment, but nanoparticles evidenced a much longer lasting effect. In particular, the antihyperalgesic activity was significant at all time points from 10 min to 130 min in rats injected with LENK-SQ Diox NPs or LENK-SQ Am NPs (Figure 55C and55G). Surprisingly, LENK-SQ-Am NPs, which was expected to release the peptide slower in comparison with the other two NPs, exhibited a stronger effect with a shorter duration, probably because the enzymatic serum capability is not predictive of the enzymatic ecosystem in the inflamed paw. For their part, LENK-SQ-dig NPs also displayed a significant anti-hyperalgesic effect (Figure 55E) which lasted twice as long as morphine, with a maximum increase in PWL maintained from 10 min to 130 min post-injection, and a progressive decline down to baseline at 220 min.

Interestingly, maximal PWL values reached after administration of LENK-SQ NPs in λ-carrageenantreated rats corresponded to basal PWL values measured in control naïve rats, before λ-carrageenan treatment (Figure 55C, 55E and 55G), indicating a pure anti-hyperalgesic action of these nanoparticles. In addition, blank SQ NPs (without the LENK, corresponding to the SQ carrier) did not demonstrate any anti-hyperalgesic activity (Figure 55), which indicated that the analgesic response to LENK-SQ NPs administration resulted from the release of LENK peptide. 223 In contrast, morphine injection in λcarrageenan-treated rats resulted in PWL values twice as high as those found in control naïve rats (Figure 55A), as expected of not only an anti-hyperalgesic effect but also the well established analgesic effect of the opiate agonist. This would suggest that LENK nanoparticles are devoid of analgesic properties but are especially potent to counteract hyperalgesia in long lasting pain suffering subjects. However, further studies are required to assess this hypothesis. Thus, through this experiment, we could demonstrate that when formulated into squalene-based NPs, LENK neuropeptide was able to exibit an anti-hyperalgesic effect in vivo which lased up to twice longer than with morphine.

Do LENK-SQ NPs act on the peripheral or central nervous system ?

In order to ascertain the involvement of central or peripheral opioid receptors during the antihyperalgesic effect of LENK-SQ NPs, naloxone (Nal, brain-permeant opioid receptor antagonist) or naloxone methiodide (Nal-M, brain-impermeant opioid receptor antagonist) 225 were subcutaneously injected 15 min prior to the injection of morphine or NPs (Figure 54). Pre-administration of the nonselective opioid receptor antagonist Nal (0.5 mg/kg s.c.) abolished the amplitude and the duration of the anti-hyperalgesic effect of morphine (PWL 3.20 ± 0.59 s vs 12.87 ± 1.38 s at 10 min) and decreased the corresponding AUC value by 81% in comparison with the morphine group (Figure 55B). The peripheral opioid receptor antagonist, Nal-M, was markedly less effective since it reduced the morphine's effect by only 13%. (Figure 55A and55B). Interestingly, pre-administration of either Nal or its quaternary derivative Nal-M, abrogated the anti-hyperalgesic effect of the three LENK-SQ NPs (Figure 55C-H).

Indeed, Nal pre-treatment caused a reduction of 66%, 105% or 73% of the AUC values compared to these found in rats injected with LENK-SQ-Diox, LENK-SQ-Dig and LENK-SQ-Am NPs alone, respectively. The corresponding reductions in AUC values with Nal-M reached 81%, 99% and 96%, respectively, indicating that the selective blockade of peripheral opioid receptors only was enough to abrogate the anti-hyperalgesic effects of LENK-SQ NPs. This clearly demonstrated that all three LENK-SQ NPs acted exclusively through peripherally located opioid receptors. 223 5. Biodistribution study LENK-SQ NPs : 5.1. Are LENK-SQ NPs able to reach the inflamed tissues?

In order to investigate the ability of LENK-SQ NPs to address the peptide towards the inflamed tissue, biodistribution studies were performed using in vivo fluorescence imaging. In that respect, LENK-SQ NPs were fluorescently tagged by insertion of DID-fluorescent probe (1,1′-dioctadecyl-3,3,3′,3′ tetramethylindodicarbocyanine,4-chlorobenzenesulfonate salt) inside the NPs.

The in vivo biodistribution of LENK-SQ-Am NPs was assessed after intravenous injection of the DiDfluorescently labeled LENK-SQ-Am NPs in a murine λ-carrageenan-induced paw edema model (right hind paw). The fluorescence in tissues was monitored up to 24h, non-invasively, from the abdomen side using an IVIS Lumina (Figure 56). Mice injected with saline into the paw were used as non-inflamed control.

The real-time in vivo imaging showed, in comparison with the healthy paw, a dramatic increase of fluorescence by 2-3 times of the average radiant efficiency within the inflamed paw after iv injection of fluorescent LENK-SQ-Am NPs (Figure 56A, 56D, 56F). In a control experiment, when the λcarrageenan administered mice were intravenously injected with a single DiD solution, no significant accumulation of fluorescence was observed in the inflamed paw (Figure 56C and 56F). In another control experiment, mice were injected locally with saline in the hind paw and intravenously treated with fluorescent LENK-SQ NPs. No significant accumulation of fluorescence at hind paw level was also observed under this condition (Figure 56B and 56E) showing that the accumulation of fluorescence in the λ-carrageenan-inflamed paw was not due to the local hind paw injection per se. 5.2. Are LENK-SQ NPs able to reach intact the inflamed tissues to exert their pharmacological activity?

In order to confirm that observed fluorescence in the inflamed paw was not due to DID accumulation (which could be released from NPs), we perfomed an additional experiment to check that LENK-SQ NPs were able to reach intact the inflamed tissues. In that respect, LENK-SQ NPs containing 2 fluorescent probes, a DID fluorescent dye and a DIR fluorescent quencher (1,1′dioctadecyltetramethyl indotricarbocyanine iodide), were incubated in serum. In the same way as for FRET, while NPs are intact, the DIR probe (which is close enough to the DID probe) quench the DID emission (Figure 57). Thus, the donor emission evidence the disassembly of the NPs. The experiment showed that the incubation in serum of LENK-SQ NPs containing both DiD and DiR, resulted in the progressive appearance of fluorescence, the dynamics of which indicating a relatively slow dissociation of LENK-SQ nanoparticles in serum: 20% after 5 min and 50% after 30 min. These observations suggeste that under our in vivo conditions, a significant proportion of intact NPs could reach the inflamed tissue.

How are LENK-SQ NPs distributed in other organs?

In a separate experiment, 4 h after the intravenous injection of either fluorescent NPs (containing DID alone) or DiD solution, animals were euthanized and transcardially perfused with 40 mL of saline to remove the fluorescence which was in the blood circulation. After collection of tissues, a strong ex vivo fluorescence signal was again observed in the inflamed paw, but also in the liver, the spleen and the lungs, whereas no detectable accumulation of fluorescence occurred in the brain of the animals (Figure 58). These results confirmed that the anti-hyperalgesic displayed by LENK-SQ NPs resulted from the targeting of these NPs toward peripheral opioid receptors in inflamed tissue, rather than central opioid receptors. 223 The overall toxicity of LENK-SQ NPs was investigated 24 h and 48 h after their intravenous administration (20 mg/kg) in rats and compared to control animals injected with 5% dextrose solution. As nanoparticles accumulated in high amounts in liver (as expected), the levels of aspartate transaminase (AST) (Figure 59A) and alanine transaminase (ALT) (Figure 59B) were measured and indicated normal levels in the LENK-SQ NPs group, thus indicating no toxicity towards the liver. The safety of LENK-SQ NPs was also confirmed by histological analysis of this tissue at 24h and 48 h (Figure 59C-F intravenously administered in rats. The AST (A) and ALT (B) levels in plasma showed no differences compared with dextrose solution (data presented as mean UI/L ± SED, N = 3 animals per group). Histological analysis of organs after intravenous administration of LENK-SQ-Am NPs (20mg/kg) did not show any signs of cell or tissue damage at 24 h and 48h, comparatively to a control 5% dextrose solution. Liver (C-F), spleen (G-J), kidneys (K-N), lungs (O-R) and heart (S-V). All tissue images were analyzed by microscopy at 10× magnification except for kidneys which were at 5× magnification (Zeiss). (fig. adapt. from Feng et al. 2019). 223 Together, these results show that the LENK-SQ NPs may be considered as safe upon systemic intravenous administration at the therapeutic dose (20 mg/kg). 223

Conclusion :

Based on the bioconjugation of LENK to SQ, we discovered a new nano-formulation capable of precise and efficient delivery of LENK for pain control associated with inflammatory events without the need to cross or "break" the BBB. The resulting LENK-SQ NPs exhibited an anti-hyperalgesic effect which lasted longer than with morphine. We could demonstrate that LENK-SQ NPs were able to reach intact the inflammatory site probably through the EPR-like effect. Within the inflamed tissues, the LENK-SQ NPs progressively disassemble and release the LENK-SQ prodrug. Due to the high concentrations of proteolytic enzymes as well as macrophages at the inflammation site, the prodrug undergoes an enzymatic hydrolysis and release the active LENK. Locally, the LENK was able to bind the peripheral opioid receptors to exert its pharmacological activity. By circumventing the CNS, we believe that the very promising LENK-SQ NPs will be devoided of the severe side effects associated with the morphine derivatives such as tolerance, addiction and respiratory depression. Moreover, the nanoprecipitation approach used for the preparation of these nanoparticles is simple and easy and doesn't require any complicated nanoparticle surface functionalization, which should facilitate further pharmaceutical development and clinical translation. However, further studies are needed to explore (i) the supramolecular structure of these nanoassemblies, (ii) their pharmacokinetics and phamacodynamics, (iii) other administration routes more adapted for chronic pain and (iv) the efficacy of the LENK-SQ NPs in other types of pain (not only inflammatory pain). Noteworthy, due to the versatility of the approach, the application to this delivery system to other therapeutic peptide molecules may be reasonably envisioned.

Perspectives :

• In vivo fate of LENK-SQ NPs (LabEX project NANOLENKSQUAL, Granted):

8.1. Based on the physico-chemical properties of LENK-SQ NPs, are we able to predict their analgesic profile?

The basic understanding of the physicochemical properties of NPs is decisive for the development of well-characterized nanomaterials. Indeed, it is well known that the composition of protein corona dramatically influences the colloidal stability of the NPs in vivo, and consequently the biological responses, including NPs pharmacokinetics, biodistributions, and therapeutic results.

Our preliminary study on LENK-SQ NPs already explored the influence of the chemical conjugation between squalene and LENK (i.e., direct amide or dioxycarbonyl or diglycolate spacers) on the chemical stability of the 3 LENK-SQ NPs after incubation in mouse serum and the release profile of LENK peptide from these NPs could be highlighted (Figure 52 and 53). Through this study, we clearly showed that the release of LENK from LENK-SQ NPs depended on the type of linkage between LENK and the SQ moiety (Figure 53). 223 The absence of release of LENK from LENK-SQ-Am was expected given that, in general, an amide bond is chemically and enzymatically more stable than an ester bond. 220,226 Interestingly, these in vitro experiments (concerning the bioconversion of LENK-SQ NPs into LENK) did not really correlate with in vivo antinociceptive experiments (Figure 60). Indeed, LENK-SQ-Am NPs, which was expected to release the peptide slower in comparison with the other two NPs, exhibited a stronger effect with a shorter duration, showing that the chemical stability of the linker was not the only parameter that govers the in vivo fate of these NPs. Indeed, other parameters have to be also taken in consideration such as the supramolecular organization of the NPs and the interaction of these NPs with plasma proteins which depends on their surface charge, shape, size, lipophilicity. All these parameters highly influence the pharmacokinetics and biodistribution of these NPs.

In that respect, we intend to investigate the fate of these NPs in vivo to better understand the differences in the analgesic profiles exhibited by LENK-SQ NPs after their intravenous administration. Furthermore, this approach would be also very helpful to predict the analgesic profile of new types of squalene-based nanoparticles carrying other analgesic peptides.

In this context, by combining complementary techniques such as SAXS (Small angle X-ray scattering), SANS (Small-angle neutron scattering ) and LC/MS-MS (liquid chromatography/tandem mass spectrometry) 227 , we will study on one hand the supramolecular organization as well as the composition of protein corona of LENK-SQ NPs (i.e., direct amide or dioxycarbonyl or diglycolate spacers), and on the other hand the pharmacokinetics of these NPs, to determine their prodrug metabolism. Thus, a better mastering of the mechanism of action of these promising LENK-SQ NPs will allow to bridge the gap between in vitro and in vivo outcomes while ensuring their safety. This step is essential if we whish to accelerate the application of these nanomedicines in clinics while ensuring their safety.

This project (for which I'm the coordinator) recently received funding from Labex NanoSaclay (45 k€). Partnership: This multidisciplinary project will gather 3 complementary partners, Institut Galien Paris-Sud UMR 8612 (Partner 1), LIONS/NIMBE-UMR 3685 (Partner 2) and ICP-UMR 8000 (Partner 3), whose matched competences will allow to establish the in vivo fate of the very promising Enkephalinbased nanoparticles targeting pain, thus enabling us to understand how they operate and thus optimize their efficiency. The project acts at the interface between chemical synthesis, nanoformulation, physico-chemistry, analytical chemistry and in vivo experiments. This strategic partnership will involve researchers with different expertises (organic and analytical chemists, physico-chemists, pharmacists and biologists), therefore ensuring the successful outcome of the project.

• Optimization of LENK-SQ NPs in order to facilitate further pharmaceutical development and clinical translation (ANR Proposal NANOPAINKILL): 8.2. Is it possible to still improve the anti-nociceptive efficacy by also developing new nanomedicines such as Opiorphin-SQ NPs, or multidrug NPs combining LENK-SQ with OPN-SQ?

Indeed, until now, the approach focused exclusively on LENK. It is also intended to go a step ahead by exploiting a multidrug approach, combining the therapeutic potential of the endogenous pentapeptide Opiorphin (an enkephalinases inhibitor) with Enkephalin. Opiorphin has already been tested as a potential painkiller, however, it presents a limitation such as insufficient enzymatic specificity. It is expected that when conjugated with squalene and under the form of nanoparticles, this peptide could be released in higher amounts at the peripheral injured tissues, thus decreasing more specifically the enkephalin breakdown. In that respect, in order to manage very severe pain, a combination of LENK-SQ NPs and OPN-SQ NPs will be developed to provide a proof of concept of a possible synergistic effect.

In that respect, the squalene moiety (tris-nor-squalenic acid 6) can be coupled on the primary alcohol group of serine via an ester bond using a linker to decrease the steric hindrance with squalene, after protecting N-terminal glutamine and side chain of arginines by Alloc group. Following the same Alloc protection strategy, the squalene moiety (chloromethyl ester of squalenic acid 44) can also be conjugated to C-terminal of opiorphine.

8.3. Do administration routes other than the intravenous one result in similar antinociceptive effects after treatment with LENK-SQ NPs?

Especially, transdermal and subcutaneous administrations will be considered because they allow the patient to manage his (her) pain by his own and this may greatly improve patient comfort. In case of success, it would also represent significant progress because, on the contrary to intravenous administration, these routes do not require expensive patient hospital stay.

Assuming that only a few molecules are able to cross the skin due to the stratum corneum layer which serves as a physical and enzymatic barrier, a specific formulation based on LENK-SQ NPs is aimed at (i) creating a reservoir of LENK within the skin, (ii) allowing the peptide protection due to conjugation to squalene , (iii) generating a controlled drug delivery and (iv) increasing drug lipophilicity for efficient transdermal diffusion.

The transdermal delivery of LENK-SQ bioconjugates will be determined using standard permeation experiments using freshly excised porcine ear skin or synthetic membranes mounted in a Franz diffusion cell. The topical application will be first considered using first hydro-alcoholic lotion (or gel) of LENK-SQ NPs, and then other permeation enhancers will be tested, such as azones, oleic acid, pyrrolidones, urea and various terpenes. 228 In addition, combination with iontophoresis will be considered, in order to increase the percutaneous penetration of LENK-SQ NPs.

If such topical administration of LENK-SQ does not allow a significant transdermal delivery of LENK, we will use microneedles systems to physically promote the delivery of LENK-SQ NPs. This technology, suitable for self-administration, should trigger the skin translocation of LENK-SQ NPs by disruption of the skin layer, thus creating micron size pathways and allowing LENK-SQ NPs and/or the LENK-SQ prodrug to reach the epidermis or the upper dermis region before absorption in the systemic circulation. Many studies have already documented the efficacy to use microneedles for promoting the transdermal delivery of several fragile macromolecules such as insulin, oligonucleotides, albumin and other proteins, antigens, DNA and even latex nanoparticles as large as 100 nm diameter. 229,230 We are therefore confident that the application of microneedles to the skin should trigger the absorption of LENK-SQ.

As last resort, the subcutaneous administration of LENK-SQ NPs will be envisioned. Although this administration route is more invasive, it remains nevertheless more appropriate for repeated administration than the intravenous route. Of note, several other peptidic drugs, such as insulin, are administered by subcutaneous injection since it allows the self-administration by the patient, does not require hospital admission, improves compliance, quality of life and is associated with a lower risk of systemic infection than intravenous injection. Besides, new medical devices are currently available on the market, allowing simple, accurate, and convenient delivery of medicines by the patient itself. For instance, insulin prefilled and disposable pens make life easier, particularly for older adults and children. It allows to deliver highly accurate doses with small and thin needle sizes that reduce fear and pain.

8.4. Is the anti-nociceptive effect of LENK-SQ, OPN-SQ or the multidrug LENK-SQ/OPN-SQ NPs restricted to pure inflammatory pain or capable to alleviate other pain episodes, like neuropathic, post-incisional, cancerous bone, visceral etc.?

Hence, different rat models, including neuropathic pain model based on nerve ligation, metastatic bone pain induced by breast cancer cell injection in rat tibia and inflammatory pain model generated by carrageenan-or complete Freund's adjuvant injection will be tested. The anti-nociceptive effect of LENK-SQ NPs will be evaluated using Randall-Selitto and Von Frey methods for mechanical sensitivity, Hargreaves method for thermal sensitivity.

8.5. Do LENK-SQ, OPN-SQ and multidrug LENK-SQ/OPN-SQ NPs avoid opioid-induced side effects and central sensitization which leads to delayed secondary hyperalgesia, as observed with morphine and its derivatives?

Even if LENK-SQ NPs were found to act peripherally, additional investigations are needed to confirm that the nanoformulation of LENK associated or not with OPN, does not cause any opioid-induced side effects.

In that respect, hemodynamic and respiratory side effects will be explored throughout the antinociceptive experiments on the plantar incision pain model using OPN-and LENK-squalene based NPs, over a 3-day postoperative period using a non-invasive carotid artery plethysmographic Collarclip and a tail cuff sphygmomanometer coupled to a Student oscillograph.

For gastro-intestinal disorders, rat faeces will be accounted every day during the seven days experiment to evaluate possible constipation.

As opioid administration generally induces delayed hyperalgesia due to central sensitization, leading to the need to increase the dose of analgesics administered to the patients, our purpose will be to investigate whether the administration of LENK-SQ NPs or LENK-SQ/OPN-SQ NPs leads or not to induced hyperalgesia. The experiment will involve repeated administrations of LENK-SQ NPs or LENK-SQ/OPN-SQ NPs in comparison with fentanyl, a synthetic opioid known to induce hyperalgesia, used as a positive control. The nociceptive thresholds will be monitored in non-injured rats and in rats with carrageenan-induced inflammatory pain.

The drug addiction in rats will be evaluated using the conditioned place preference paradigm test. 231 In this test, during a preconditioning phase, the rats which stay in two different colored plexiglas boxes, will receive LENK-SQ NPs or LENK-SQ/OPN-SQ NPs every day. Then, after preconditioning phase, the time spent to access the preferred compartment will be evaluated.

This project (for which I'm the coordinator) aims at addressing a major medical challenge related with the treatment of pain without the side effects associated with morphine and other opioids derivatives. Partnership: The multidisciplinary project which was recently submitted to ANR, and the letter of intend selected for evaluation of the full project. NanoPainKill will gather two partners with an evident complementarity, Institut Galien Paris-Sud UMR 8612 (Partner 1) and INSERM 1195, (Partner 2), for the management of a pluridisciplinary proposal at the interface between chemical synthesis, nanoformulation, pharmaceutical technology, ex vivo experiments (pig skin to explore transdermal route of administration) and in vivo pharmacological experiments (various experimental models of pain). It represents a new strategic partnership (the two teams have never collaborated before) in an effort to improve the management of pain care.

10. 1 .

 1 May SQAd NPs be effective in other neurological injuries such as spinal cord injury? ....................................................................................................................................... 11. CONCLUSION ...................................................................................................................... 12. PERSPECTIVES .................................................................................................................. 12.1. Could the neuroprotective and neuroregenerative effects of SQAd NPs be also extended to peripheral nerve injury ? .................................................................................. 12.1.1. Evaluation of neuroprotective and neuroregenerative effects of SQAd NPs in peripheral nerve injury model in rats ......................................................................................................... 12.1.2. Acute treatment of SQAd NPs ...................................................................................... 12.1.3. Chronic treatment of SQAd NPs ................................................................................... • SQUALENE-BASED NANOMEDICINES FOR PAIN MANAGEMENT : ......................... 1. STATE-OF-ART ON ENKEPHALINS ..................................................................................... 1.1. What is the mechanism of action of enkephalins ? ..................................................... 1.2. What are the limitations of enkephalins ? ................................................................... 1.3. Which approaches have been used to exploit the analgesic activity of endogenous neuropeptides ? ..................................................................................................................... 1.4. What about nanomedicine approach ? ........................................................................ 2. APPLICATION OF SQUALENOYLATION NANOTECHNOLOGY TO LEU-ENKEPHALIN : SYNTHESIS AND CHARACTERIZATION .......................................................................................................... 2.1 Can we obtain nanoparticles of Leu-enkephalin peptide using squalenoylation approach? ............................................................................................................................... 2.1.1 Synthesis of Leu-Enkephalin-SQ bioconjugates ................................................................. 2.1.2 Preparation and characterization of Leu-Enkephalin-SQ nanomedicines ........................... 3. REALEASE KINETICS OF LENK-SQ NPS ............................................................................ 3.1 Are LENK-SQ NPs able to release LENK in serum ? ...................................................... 4. EVALUATION OF ANTINOCICEPTIVE EFFECT OF LENK-SQ NPS IN INFLAMMATORY PAIN MODEL ..................................................................................................................................... 4.1 Are LENK-SQ NPs able to display an anti-hyperalgesic effect in inflammatory pain model? .................................................................................................................................... 4.2 Do LENK-SQ NPs act on the peripheral or central nervous system ? ......................... 5. BIODISTRIBUTION STUDY OF LENK-SQ NPS ................................................................... 5.1 Are LENK-SQ NPs act locally within the inflamation site? ........................................... 5.2 Are LENK-SQ NPs able to reach intact the inflamed tissues to exert their pharmacological activity? ...................................................................................................... 5.3 How are LENK-SQ NPs distributed in other organs?.................................................... 6. TOXICITY STUDY ............................................................................................................... 6.1 Could accumulation of LENK-SQ NPs in vital organs generate toxicity? .................... 7. CONCLUSION .................................................................................................................... 8. PERSPECTIVES .................................................................................................................. 8.1 Based on the physico-chemical properties of LENK-SQ NPs, are we able to predict their analgesic profil? ............................................................................................................ 8.2 Is it possible to still improve the antinociceptive efficacy by also developing new nanomedicines such as opiorphin-SQ NPs, or multidrug NPs combining LENK-SQ with OPN-SQs? ................................................................................................................................ 8.3 Do administration routes other than the intravenous one result in similar antinociceptive effect after treatment with LENK-SQ NPs? ............................................... 8.4 Is the antinociceptive effect of LENK-SQ, OPN-SQ or the multidrug LENK-SQ/OPN-SQ NPs restricted to puree inflamatory pain or capable to alleviate other pain episodes, like neuropathique, post-incisional, cancerous bone, visceral, etc?......................................... 8.5 Do LENK-SQ, OPN-SQ and multidrug LENK-SQ/OPN-SQ NPs avoid opioid-induced side effects and central sensitisation which leads to delayeh secondary hyperalgesia as observed with morphine and its derivatives? ...................................................................... IV. REFERENCES ........................................................................................................... de la barrière hématoencéphalique -Formulation -Chimie des bioconjugués -Pharmaco-cinétique -Biodistribution du médicament Domaines de recherche : Anti-ischémiques -Analgésiques -Anti-cancéreux -Antiviraux 30 Publications dans des revues internationales • INFORMATIQUE : Mac, Windows, Microsoft Office (Word, Excel, PowerPoint), ChemDraw, ISIS Draw, Beilstein, SciFinder • COMPÉTENCES PÉDAGOGIQUES : Enseignement : Cours de galénique à la Faculté de pharmacie (cours magistraux, enseignements dirigés et travaux pratiques : étudiants de 3 ème , 5 ème et 6 ème année de pharmacie • COMPÉTENCES MANAGÉRIALES : Gestion de projets : Demandes de financement : projets de recherche Encadrement doctoral et scientifique : Doctorants, Master 2, Post-doctorants • LANGUES : Anglais (courant), Russe (maternel), Arabe (maternel), Italien (courant). Maître de Conférences à la Faculté de Pharmacie de Châtenay-Malabry (Université Paris-Sud) -UMR-CNRS 8612 « Institut Galien » -Equipe P. Couvreur Post-doctorat en chimie thérapeutique et galénique à la Faculté de Pharmacie de Châtenay-Malabry (Université Paris-Sud) -UMR-CNRS 8612 « Institut Galien »-Mission d'intérim en chimie médicinale chez SANOFI-AVENTIS (Chilly-Mazarin). Remplacement du responsable d'unité (Dr S. Mallart) au sein du département cardiovasculaire 2004-2005 (1 an) 2003 Post-doctorat en chimie médicinale à la Faculté de Pharmacie de Châtenay-Malabry (Université Paris-Sud) -UMR-CNRS 8076 « BioCIS » -(S. Ongéri, S. Sicsic) Congé de maternité -Recherche d'emploi 2001-2002 (1 an) International Trainer à l'ONU au Kosovo (UNMIK) ► Participation au projet d'enregistrement des minorités Serbes au Kosovo en vue des élections de Septembre 2001 : Recrutement, formation et supervision de collaborateurs 1997-2001 (3 ans et demi) Doctorat en chimie organique à l'Université de Cergy-Pontoise Formation et diplômes 2001 Doctorat en chimie organique à l'Université de Cergy-Pontoise. 1997 DEA Hétérochimie, Catalyse et Polymères à l'Université de Montpellier II. 1996 Licence et Maîtrise en chimie moléculaire à l'Université de Montpellier II.

  Formation de formateur en « Techniques de recherche d'emploi » (TRE) les 17 et 18 Mars 2008 à l'UFR Pharmacie de Châtenay-Malabry. Formation organisée avec le Service Insertion Professionnelle (SIP) et l'APEC au service des enseignants de l'UE 19. Objectif : savoir enseigner aux étudiants les techniques de recherche de stage ou d'emploi Impact sur la pratique de l'enseignement: Mise en place dans l'UE19 d'une séance spécifique sur les techniques de recherche de stage ou d'emploi pour les étudiants. Particularité de cette approche pédagogique en TRE : Interactivité, dimension humaine. • Participation à la journée séminaire PPPE « Panorama d'ici et d'ailleurs » (Projet professionnel et personnel étudiant) le 20 Mai 2010 au Campus d'Orsay Objectif : Favoriser la réussite étudiante en licence par le projet personnel et professionnel de l'étudiant. Mon intervention : Présentation de l'UE Projet Professionnel et personnel tel qu'il est enseigné à la Faculté de pharmacie de Châtenay-Malabry. Echanges avec les intervenants d'autres universités également responsables de l'UE Projet Professionnel et personnel afin de réfléchir aux évolutions possibles et à l'harmonisation du PPPE sur l'Université Paris XI.

•

  UE19 A et B « Projet Professionnel, Communication, Organisation-Gestion » pour les étudiants de L3 (3 ème année pharmacie), (2ECTS). Direction à 100% de l'UE19 de Septembre 2007 à Juillet 2017 puis co-direction à 50% à partir de Septembre 2017 (avec Juliette Vergnaud) Nombre d'enseignants impliqués dans l'UE 19 : 12 enseignants-chercheurs (dont moi-même) Coordination d'intervenants extérieurs participant dans cette Unité d'Enseignement Types de cours : Cours magistraux (responsables de filières + intervenants extérieurs) et Enseignements dirigés Particularité : A partir des formations suivies (voir pages 5 et 7), mise en place de : techniques de recherche de stage ou d'emploi ; mise à disposition de l'outil PEC (Portefeuille d'Expériences et de Compétences) ; Mise en place dans l'UE 19 d'un mini atelier pour les étudiants, autour du bilan des compétences impliquant un travail d'introspection (questionnaire), une analyse des aptitudes, des motivations, des compétences, en vue de faciliter les choix d'orientation et les prises de décisions au cours de leur parcours de formation ; conseils à la rédaction d'un CV et d'une lettre de motivation pour les étudiants. • UEL294 « Formulation des préparations parentérales » pour les étudiants de 5 ème année industrie/recherche en pharmacie (3 ECTS). Montage de l'UE294 Direction à 100% de l'UE294 depuis Septembre 2014 Nombre d'enseignants impliqués dans l'UE 294 : 2 enseignants-chercheurs (dont moi-même) et 1 moniteur Types de cours : Cours magistraux, Enseignements dirigés et travaux pratiques Particularité : Mise en place d'un mini atelier pour les étudiants en salle informatique pour leur apprendre à exploiter les monographies de la Pharmacopée Européenne (version électronique) en vue de proposer un schéma de fabrication pour des préparations parentérales qu'ils auront à réaliser dans le respect des bonnes pratiques de fabrication (BPF). • UE 135 « Préparations galéniques en officine» pour les étudiants de 6 ème année Officine (2 ECTS). Co-direction à 50% depuis Septembre 2013 (Jean-Philippe Michel puis Mariana Varna) Nombre d'enseignants impliqués dans l'UE120 : 2 enseignants-chercheurs Types de cours : Cours magistraux, Enseignements dirigés et Travaux Pratiques.

Figure 1 :

 1 Figure 1 : Protein corona surrounding the nanoparticle

Figure 2 :

 2 Figure 2 : Nanoprecipitation process

Figure 3 :

 3 Figure 3 : Mechanism of entry of dFdC in the cells (fig. adapt. from Bildstein et al. 2010).62

62

 62 Figure 3 : Mechanism of entry of dFdC in the cells (fig. adapt. from Bildstein et al. 2010).62

1. 2 .

 2 What are the limitations of dFdC in cancer therapy ?

Figure 4 :

 4 Figure 4 : Deamination process of dFdC

Figure 5 :

 5 Figure 5 : CryoTEM images of nanoassemblies of SQdFdC nanoassemblies. The supramolecular organization is clearly visible. Nanoassemblies of hexagonal shape (A and B) are surrounded by an external shell and display an internal structure. The TEM images after freeze-fracture of SQdFdC (C and D). ) Schematic drawing of the hexagonal (HII) packing of amphiphilic molecules (E) (fig. adapt. from Couvreur et al. 2008).73

Figure 6A :

 6A Figure 6A : Cytotoxicity of SQdFdC NPs ( ) and dFdC ( ) in L1210 wt and L1210 10K (dCK-deficient) murine leukemia cell lines (fig. adapt. fromReddy et al. 2007).[START_REF] Reddy | A New Nanomedicine of Gemcitabine Displays Enhanced Anticancer Activity in Sensitive and Resistant Leukemia Types[END_REF]

Figure 6B :

 6B Figure 6B : Cytotoxicity of dFdC (-o-) and SQdFdC NPs (-■-) CEM/ARAC8C transporter-deficient human leukemia cell line. (fig. adapt. fromReddy et al. 2007).[START_REF] Reddy | A New Nanomedicine of Gemcitabine Displays Enhanced Anticancer Activity in Sensitive and Resistant Leukemia Types[END_REF]

Figure 7A :

 7A Figure 7A: Weight loss. Differences in weights of the leukemia bearing mice either untreated (-•-), squalene-treated (-o-), or treated with dFdC 5 mg/kg (-■-), squalene + dFdC 5 mg/kg (--), SQdFdC NPs equ. to 5 mg/kg dFdC (-▲-), dFdC 15 mg/kg (-□-) or SQdFdC NPs equ. to 15 mg/kg dFdC (-Δ-). n=7-8 (fig. adapt. fromReddy et al. 2007).[START_REF] Reddy | A New Nanomedicine of Gemcitabine Displays Enhanced Anticancer Activity in Sensitive and Resistant Leukemia Types[END_REF]

Figure 7B :

 7B Figure 7B: Survival rates. Survival of the mice after treatment. Untreated (-), squalene nanoparticles (---), squalene + dFdC 5 mg/kg ( ▬ ), dFdC 5 mg/kg (---), SQdFdC NPs equ. to 5 mg/kg dFdC ( ▬ ), dFdC 15 mg/kg (⋯⋯), SQdFdC NPs equ. to 15 mg/kg dFdC (-• -•), n=7-8. *Kaplan-Meier test was significant for the L1210 wt leukemia bearing mice at both 5 mg/kg and 15 mg/kg (Pb0.05) doses. (fig. adapt. from Reddy et al.2007).[START_REF] Reddy | A New Nanomedicine of Gemcitabine Displays Enhanced Anticancer Activity in Sensitive and Resistant Leukemia Types[END_REF]

73

 73

Figure 8 :

 8 Figure 8 : Anticancer activity of SQdFdC in vivo. Anticancer activity of SQdFdC in nanoassembly form and dFdC (5 mg.kg -1 equiv. doses) following i.v. treatment (on days 0, 4, 8, and 13) of mice bearing P388 subcutaneous tumours. A) Tumour progression: Control (black spheres), saline (white spheres), dFdC (black diamonds), SQdFdC nanoassemblies (white diamonds). B) Survival curve of mice: Control (solid line), saline (dotted line), dFdC (dashed line), SQgem nanoassemblies (heavy solid line). * indicates P<0.05, as assessed by Kaplan-Meier test. C) Photograph showing the difference in tumour growth in mice following the completion of indicated treatment. (fig. adapt. fromCouvreur et al. 2008).[START_REF] Couvreur | Discovery of New Hexagonal Supramolecular Nanostructures Formed by Squalenoylation of an Anticancer Nucleoside Analogue[END_REF]

  [START_REF] Reddy | Squalenoylation Favorably Modifies the in Vivo Pharmacokinetics and Biodistribution of Gemcitabine in Mice[END_REF]

Figure 9A :

 9A Figure 9A : Comparative plasma concentration of dFdC and SQdFdC versus time curves. dFdC ( -Δ-; 15 mg/kg ), SQdFdC ( --; 15 mg/kg mol Eq of dFdC), and released dFdC (-■-) (i.e., the dFdC released from SQdFdC nanoassemblies) in mice. The values are the mean S.D. of four mice (fig. adapt. from Reddy et al. 2008).80

Figure 9B :

 9B Figure 9B : Appearance of the deaminated metabolite dFdU as indicated by the plasma concentration-time curves after treatment with dFdC (-Δ-;15 mg/kg) or SQdFdC nanoassemblies (--; 15 mg/kg mol Eq of dFdC). The values are the mean S.D. of four mice (fig. adapt. from Reddy et al. 2008).80

4. 4 .

 4 What is the fate of SQdFdC NPs after intravenous administration? 4.4.1.

88

 88

Figure 10 88 

 1088 Figure 10. 3 H-SQGem and 3 H-dFdC plasmatic distribution in vitro (a and b) and in vivo (c) . The distribution of 3 H among the different plasma fractions was analysed after incubation (5 min) of (a) 3 H-SQdFdC NPs or (b) 3 H-dFdC with human blood. Results are expressed as a percentage (mean values) of total plasma radioactivity. (c) Radioactivity (magenta lines) and cholesterol (blue line) distribution among the collected fractions of plasma obtained from rats treated with 3 H-SQdFdC (solid magenta line) or free 3 H-dFdC (dashed magenta line), 5 min post administration. Results are expressed as relative radioactivity (mean values) compared to total plasma (fig. adapt. from Sobot et al. 2017).88

88

 88

Figure 11 .

 11 Figure 11. In vitro stability of FRET NPs. In vitro stability study of FRET NPs after dilution with MilliQ® water, rat blood or ethanol. Results are expressed as mean ± standard error of the mean (s.e.m.) (fig. adapt. from Sobot et al. 2017).88

  [START_REF] Sobot | Conjugation of Squalene to Gemcitabine as Unique Approach Exploiting Endogenous Lipoproteins for Drug Delivery[END_REF]

Figure 12 :

 12 Figure 12 : 3 H-SQdFdC NPs cellular (A and B) and tumor (C) uptake. (A) 3 H-SQGem NPs uptake in MDA-MB-231 cells cultured in medium supplemented with FBS, LPDS or preincubated with an excess of LDL in LPDS-supplemented medium at 37° C. Results are expressed as nanomoles of Gem per one million of cells (bars represents mean values±s.e.m.***Po0.001 by analysis of variance, Tukey's multiple comparison test).(B) 3 H-SQGem uptake in MDA-MB-231 and MCF-7 cells. Comparison of 3 H-SQGem NPs uptake in MDA-MB-231 and MCF-7 breast cancer cells after 6 h incubation at 37 °C. Results are expressed as nanomoles of Gem per million of cells. Bars represent mean ± standard error of the mean (s.e.m.) (* indicates p < 0.05 by unpaired, twotailed test with Welch's correction) (C) Tumor uptake of 3 H-SQGem NPs. Radioactivity found in MDA-MB-231 or MCF-7 tumor xenografts excised from athymic nude mice 6 h after treatment with 3 H-SQGem NPs (5 mg kg-1 eq. Gem). Bars represent means ± s.e.m. (fig. adapt. from Sobot et al. 2017).88

Figure 13 :

 13 Figure 13 : Biodistribution of 3 H-SQdFdC NPs. Organ distribution of radioactivity after the completion of four i.v. doses of 3 H-dFdC ( ) or 3 H-SQdFdC NPs ( ) (10 mg/kg on days 0, 4, 8, and 13). Sampling of organs was performed on day 15. The values are the mean ± S.D. of three mice. *, p < 0.05 compared with that of 3 H-dFdC (fig adapt. from Reddy et al. 2008).80

Scheme 5 :

 5 Scheme 5 : Synthetic approach to the tertiary ammonium phosphate salts 19a,b. Reagents and conditions: a) DMTrCl, DMAP cat., Py, RT, 3d, 83%; b) TBDMSCl, DBU, CH2Cl2, RT, 36 h, 90%; c) HCO2H/MeOH/CH2Cl2 (2:1:1), 0 °C, 45 mn, 77%; d) i. 11, 1H-tetrazole, CH3CN, CH2Cl2 (1:1), RT, 3d; ii. 30% H2O2, 0 °C, 4 h, 80%; e) HF.py, py, RT, 4h, 69%; f) R 1 R 2 R 3 N, CH3CN, 24 h, RT, (19a: 98%; 19b, 85%).

Figure 14 :

 14 Figure 14 : TEM (A,B) and Cryo-TEM (C) of SQdFdC-MP. (A) 19a (3 mg.mL-1); (B) 19b (4 mg.mL-1); (C) 19a (2 mg.mL-1). dFdC (fig. adapt. from Caron et al. 2011). 102

Scheme 6 :

 6 Scheme 6 : Synthetic approach to polyisoprenoyl dFdC bioconjugates 8 b-g.

Figure 15 :

 15 Figure 15 : Release of gemcitabine from 8, 8b, 8c, 8d, 8f and 8g nanoassemblies at 37 °C in solution containing 90% FBS. Released gemcitabine was separated from polyisoprenoyl gemcitabine nanoassemblies 8, 8b-8d, 8f and 8g by centrifugation and quantified using HPLC (fig. adapt. from Maksimenko et al. 2013).108

Figure 16 :

 16 Figure 16 : Tumor volume and body weight. Tumor growth inhibition after farnesylacetyl-(8d) and squalenoyl-(8) gemcitabine nanoassemblies treatment of mice bearing human pancreatic (MiaPaCa-2) tumor. Tumor volume (a) and body weight (b) were regularly measured during the experimental period. The mice were treated with prodrug 8 NPs (4 x 7 mg/kg gemcitabine equivalent, MTD) (o) and prodrug 8d NPs (4 x 7 mg/kg gemcitabine equivalent) (•), gemcitabine (4 x 7 mg/kg) ( ), farnesylacetic acid NPs (6d NPs, 4 x 50 mg/kg) (∆), 1,10 ,2-tris-nor-squalenic acid NPs (6 NPs, 4 x 50 mg/kg) (■) or, saline (NaCl 0.9%) (□), (P < 0.1; n = 8).

Figure 18 :

 18 Figure 18 : Intracellular and extracellular adenosine. In the cell, adenosine is degraded by adenosine deaminase (ADA) into inosine or phosphorylated by adenosine kinase (AdK) into adenosine monophosphate (AMP), diphosphate (ADP) or triphosphate (ATP), which is used as substrate by adenylyl cyclase (AC) for cyclic AMP (cAMP) formation. Outside the cell, adenosine binds four receptors involved in various pathophysiological processes (fig. adapt. from Rouquette et al. 2019). 124

Figure 19 :

 19 Figure 19 : Characterization of SQAd NPs. a) Hydrodynamic diameter, polydispersity index (PdI), zeta potential and b) morphological appearance (cryoTEM) of SQAd NPs.

Figure 20 :

 20 Figure 20 : Schematic model of neurovascular mechanism of postischemic reperfusion injury. During reperfusionafter ischemia, overproduction of reactive oxygen species (ROS) causes oxidative stress. The oxidative stress damages the endothelial cells, resulting in an exposure of the subendothelial extracellular matrix to blood flow. The exposure triggers adhesion and activation of platelets in microvasculature causing thrombosis. The injured endothelial cells release metalloproteinase that attacks basal lamina causing leakage of the blood-brain barrier (BBB). The damaged endothelial cells interact with regulatory T cells and activated leukocytes to exacerbate intravascular thrombosis. In addition, a large amount of inflammatory factors including cytokines, chemokines, NO, TNF-α, IL-1b, IL-6, ROS, prostanoids are released from activated astrocytes and microglia. The ensuing inflammatory response causes a high degree of cytotoxicity that amplifies neuronal death in the penumbra. Furthermore, the oxidative stress causes sustained contraction of pericytes which leads to narrowing of the microvasculature lumen. The lumenal narrowing is further worsened by compression from swollen end-feet of activated astrocytes. RBC, red blood cell; NO, nitric oxide; IL, interleukin (fig. adapt. fromBai et al. 2015).146

Figure 21 :

 21 Figure 21: Systemic administration of SQAd nanoassemblies (NPs) provides significant neuroprotection in a mouse model of cerebral ischaemia. A) MCAo was performed in mice under anaesthesia by the intraluminal filament method. A filament was advanced in the internal carotid artery through a small incision proximal to the bifurcation up to the origin of the MCA to interrupt blood flow to the MCA territory. B) Ischaemic volumes in control and treated mice subjected to transient (2 h MCAo and 22 h reperfusion) and permanent (24 h MCAo) focal cerebral ischaemia were identified by reduced Nissl staining under a light microscope (magnification ×10, insets) (data are presented as mean (mm3) ± s.d., N=6 animals per group; † and * indicate P < 0.05 compared to respective controls). Intravenous administration of 7.5 mg kg-1 or 15 mg kg-1 SQAd nanoassemblies just before ischaemia or 2 h post-ischaemia significantly decreased the infarct volume compared with control groups that received vehicle (dextrose 5%), adenosine-unconjugated SQ nanoassemblies (9.45 mg kg-1) or free adenosine (5.5 mg kg-1). A significant therapeutic effect was also observed when SQAd nanoassemblies were administered 2 h post-ischaemia in the permanent MCAo model (fig. adapt. from Gaudin et al. 2014).140

Figure 22 :

 22 Figure 22 : Neurologic deficit scores after systemic administration of SQAd NPs The significant neuroprotective effect of SQAd nanoassemblies was also evident in neurologic evaluation (data presented as mean ± s.d., N = 6 animals per group; † and * indicate P < 0.05 compared to respective controls). Neurologic deficit scores, assessed 24 h after the induction of stroke, paralleled the changes in the infarct volume, although the decreases in deficit scores were less striking, possibly because some pyramidal fibres descending from the recovered cortex could not escape damage in the ischaemic subcortical area. d,e, In untreated mice, capillaries in the ischaemic brain were filled with trapped erythrocytes (fig. adapt. from Gaudin et al. 2014). 140

Figure 23 :

 23 Figure 23 : Microvessels in the ischaemic area of SQAd NPs treated mice (B, D) or untreated mice (A, C). A, red fluorescence, arrowheads) 6 h after re-opening of the MCA following 2 h of occlusion, whereas the majority of capillaries were not clogged in SQAd nanoassemblies-treated mice (B). The treatment also reduced ischaemic tissue changes such as swelling of axon bundles (E, asterisk). C) and D) Similarly, capillary structure was well preserved in treated animals (D), unlike controls (C), in which astrocyte endfeet ensheathing capillaries as well as endothelial nuclei were swollen (arrowheads) in addition to oedematous neurophil and several dark-stained degenerating cells surrounded by swollen endfeet (C, asterisks). In treated mice, parenchymal ischaemic changes were mild, limited to light swelling and scattered degenerating neurons (D, asterisks). (fig. adapt. from Gaudin et al. 2014). 140

Figure 24 :

 24 Figure 24 : Chemical structures of radiolabelled SQAd : [ 3 H]-SQAd 38*A (✽ site of 3 H-radiolabeled proton) and [ 14 C]-SQAd 38*B (✽ site of 14 C-radiolabeled carbon).

4. 1 . 1 .

 11 Synthesis of [ 3 H]-SQAd 38*A and [ 14 C]-SQAd 38*B : The [ 3 H]-SQAd 38*A was obtained by hydrogen-tritium (H-T) exchange in position 8 at 105°C in presence of tritiated water (HTO). For the synthesis of [ 14 C]-SQAd 38*B, [ 14 C]-squalenylacetic acid 6L* was coupled to protected adenosine 39 to give sililated [ 14 C]-SQAd 40 which was then deprotected to give [ 14 C]-SQAd 38B* (Scheme 13).

Scheme 13 :

 13 Scheme 13 : Radiolabeling of SQAd 38*BThe [ 14 C]-squalenylacetic acid 6L* was obtained in 3 steps from the squalenylacetic acid 6L. In that respect, the acid 6L first underwent a bromodecarboxylation followed by Hermann Kolbe reaction with 14 C-radiolabeled potassium cyanide ([ 14 C]-KCN) resulting in [ 14 C]-nitrile derivative. This latter was then hydrolyzed under basic conditions to give the [ 14 C]-squalenylacetic acid 6L* (Scheme 14).151 For the bromodecarboxylation step, the reaction mechanism involved homolytic cleavage of the nitrogen-oxygen bond of thiohydroxamate ester 41 (Barton ester) initiated by heat, leading to the acyloxy radical (SQCOO • ) 42 and the 2-pyridylthiyl radical 43. Then, the radical decarboxylation of 42 generated the squalenyl radical (SQ • ) 44, which reacted with a radical-trapping species CBrCl3, resulting in the formation of bromomethylsqualene 45 (Scheme 14).151

  151

4. 2 .

 2 Did SQAd NPs reach the brain to exhibit pharmacological activity on ischemic stroke? 4.2.2. Biodistribution study : Brain Tissue distribution of [ 3 H]-SQAd and [ 14 C]-SQAd was investigated in mice after intravenous injection of radiolabeled [ 3 H]-SQAd NPs and [ 14 C]-SQAd NPs respectively and compared to the distribution of free [ 3 H]-Adenosine (Figure 25). Total radioactivity of [ 3 H] and [ 14 C] rapidly diverged in the brain following [ 3 H]-SQAd NPs or [ 14 C]-SQAd NPs administration (Figure 25). Indeed, following intravenous injection of [ 3 H]-SQAd NPs, [ 3 H]radioactivity content progressively increased with time to reach a plateau 8 h post-administration corresponding to 1.4 ± 0.1% of injected dose/g of organ. Meanwhile, following intravenous injection of [ 14 C]-SQAd NPs, [ 14 C]-radioactivity content stayed constant and equal to ca. 0.1% of injected dose/g of tissue up to 5 day post-injection. 151

Figure 25 :

 25 Figure 25 : Distribution of the radioactivity found in the brain after intravenous injection of free [ 3 H]-Adenosine, [ 3 H]-SQAd NPs or [ 14 C]-SQAd NPs. Radioactivity found in the brain was expressed as the percentage of injected dose per gram of tissue following intravenous administration of [ 3 H]-Adenosine (5.55 mg/kg, 60 μCi/kg), [ 3 H]-SQAd NPs (15 mg/kg, equiv. 5.55 mg/kg adenosine, 42.4 μCi/kg) or [ 14 C]-SQAd NPs (15 mg/kg, equiv. 5.55 mg/kg adenosine, 9.2 μCi/kg). Results are mean ± SEM from N = 6 animals/time-point/group (fig. adapt. from Gaudin et al. 2015). 151

Figure 26 :

 26 Figure 26 : Radio-HPLC chromatograms of brain homogenates. The analytical detection of [ 3 H]-Adenosine and [ 3 H]-SQAd has been performed using a C18 Nucleosil 100-5 of 150 mm x 4.6 mm x 5 μm column and a C18 pre-column, gradient KH2PO4 10 mM pH 4/MeOH, flow 1 mL/min, injection 50 μL, tritium detection. (A) Reference chromatogram after a brain homogenate has been overloaded with [ 3 H]-Adenosine and [ 3 H]-SQAd. Retention times were 7.3 min for [ 3 H]-Adenosine and 26.9 min for the [ 3 H]-SQAd. Representative chromatograms of the brain homogenates obtained 1 h after intravenous injection of [ 3 H]-SQAd (B) and 1 h after intravenous injection of [ 3 H]-Adenosine (C) in mice (fig. adapt. from Gaudin et al. 2014). 140

Figure 27 :

 27 Figure 27 : In vitro colloidal and chemical stability of SQAd NPs in mouse plasma. SQAd NPs were dispersed in freshly isolated mouse plasma or water, and NA integrity was assessed over incubation time by measuring the NPs mean diameter using DLS (A). Diluting FRET SQAd NPs in mouse plasma, the acceptor emission fluorescence under donor excitation was measured to assess colloidal status (B) (fig. adapt. from Gaudin et al. 2015). 151

Figure 28 :

 28 Figure 28: Chemical stability of SQAd NPs in plasma. Incubation of SQAd NPs at different time points in plasma and stability of SQAd was assessed by HPLC. Results are presented as mean ± SEM from N = 3 independent samples (fig. adapt. from Gaudin et al. 2015). 151

Figure 29 :

 29 Figure 29 : Plasma concentrations of SQAd NPs. (A) [ 3 H]-and [ 14 C]-radioactivity plasma time profiles expressed as the percentage of the injected dose per mL of plasma after intravenous injection of [ 3 H]-SQAd NPs (15mg/kg, 42.4 μCi/kg) or [ 14 C]-SQAd NPs (15mg/kg, 9.2 μCi/kg). Results are presented as mean±SEM from N=6 animals/time-point/group. Representative radio-chromatograms obtained after radio-HPLC analysis of plasma (B) 1 min, (C) 15 min and (D) 1 h postinjection of [ 3 H]-SQAd NPs (15 mg/kg, 600 μCi/kg) (fig. adapt. from Gaudin et al. 2015). 151

Figure 30 :

 30 Figure 30 : Displacement of specific radioligand binding from the human adenosine receptors A1 (red circle), A2A (orange square), A2B (yellow triangle), and A3 (green diamond) by increasing concentrations of SQAd bioconjugate (A and C) or SQAd NPs (B and D) diluted in assay buffer (A and B) or preincubated for 4 hours at 37°C in FBS (C and D). Displacement experiments were performed at 25°C. NSB, non specific binding; TB, total binding. Data represent mean values 6 S.D. of three independent experiments (fig. adapt. from Rouquette et al. 2019). 156

Figure 31 :

 31 Figure 31: Internalization of the SQAd nanoassemblies (NPs) by human cerebral endothelial cells (hCMEC/D3). A) The cell capture of SQAd nanoassemblies by hCMEC/D3 cells was quantified by flow cytometry and compared to cell internalization of the fluorescent probe alone (N=3 independent experiments run in duplicate, data presented as mean (mean fluorescence intensity, MFI) ± s.e.m., **P < 0.01, ***P<0.001). B) and C) The effective internalization of the nanoassemblies was confirmed by confocal microscopy after 2 h (B) and 6 h (C) of incubation. D), It was observed that the nanoassemblies mainly localized in the cell cytoplasm using markers for the nucleus (blue, SYTO61) and for the cellular membrane (red, WGA AlexaFluor 555) (fig. adapt. from Gaudin et al. 2014).140

Figure 32 :

 32 Figure 32: SQAd NPs internalization by the hCMEC/D3 cells. A) The internalization was quantified by flow cytometry,

Figure 33 :

 33 Figure 33: Involvement of LDL receptors in the SQAd NA cell uptake. (A) Pretreatment of hCMEC/D3 cells with an excess of LDL (100 μg/mL, 30 min) followed by incubation (1 h 30) with fluorescent SQAd NPs significantly decreased the SQAd NA internalization, as quantified by flow cytometry (**p < 0.001). BSA (bovine serum albumin) as a protein control did not show any significant inhibitory effect on SQAd NP cell uptake. (B) The coincubation of green fluorescently labeled LDL (LDL-f) and red fluorescent SQAd NPs led to a strong colocalization as observed by confocal microscopy after 1 h (Manders coefficients: M1 (fraction of green pixels overlapping with red pixels) = 0.646 and M2 (fraction of red pixels overlapping with green pixels) = 0.654) and 4 h (Manders coefficients: M1 = 0.881 and M2 = 0.868) incubation, confirming the involvement of the LDL receptors in the energy-dependent contribution to SQAd NP cell uptake. Differential interference contrast (DIC) images showed the healthy morphology of the cells during NP internalization. Scale bars = 10 μm (fig. adapt. from Gaudin et al. 2015). 161

Figure 34 :

 34 Figure 34 : Intracellular trafficking of SQAd NPs. The colocalization of subcellular organelles (green) and SQAd NPs (red) was examined after (A) 1h, (B) 4h, (C) 12h and (D) 24 h of incubation using specific LAMP-2 antibody for lysosome compartment. (A-D) Colocalization with lysosomes. Scale bars = 10 μm (fig. adapt. from Gaudin et al. 2015). 161

Figure 35 :

 35 Figure 35: Cell internalization of SQ-3H-Ad NPs by the hCMEC/D3 cells. Internalization of FRET SQAd nanoassemblies showed that the nanoassemblies disassembled inside the cells after 6 h of incubation (data presented as mean (MFI) ± s.e.m.) (fig. adapt. from Gaudin et al. 2014). 140

Figure 36 :

 36 Figure 36 : Transcytosis of SQAd NPs across the hCMEC/D3 monolayer. (A) Transcytosis of SQAd NPs was studied using a transwell system. hCMEC/D3 cells were grown on a collagen-coated membrane to form a monolayer, and fluorescent SQAd NPs were added in the apical compartment (corresponding to the blood side). The incubation with FRET SQAd NPs did not show any detectable acceptor signal in the baso-lateral compartment after (B) 24, (C) 48, and (D) 72 h, confirming that the NPs were internalized as intact nanostructures but disassembled inside the cells and were exocytosed in a molecular form (fig. adapt. from Gaudin et al. 2015). 161

  161

  156

Figure 37 :

 37 Figure 37: (A) Percentage of intact SQAd after incubation until 24 h, with or without cathepsin B (pH 4.8, 37 °C). Data represent mean values ± standard deviation of two independent experiments. (B) Representative HPLC chromatogram of partial activation of SQAd into adenosine (Ad) (24 h incubation, pH 4.8, 37 °C, in the presence of cathepsin B). Of note, the peak corresponding to SQAd is not visible in the elution conditions used for adenosine detection (fig. adapt. from Rouquette et al. 2019). 156

Figure 38 :

 38 Figure 38 : Concentrations of adenosine in the extracellular medium from hCMEC/D3 cells cultures. Cells were incubated for indicated times with 100 µM of SQAd NPs (red bars), adenosine (Ad) (orange bars) or 5 wt. % dextrose (yellow bars) and released adenosine concentrations were measured 1 h following cell culture medium renewal (fig. adapt. from Rouquette et al. 2019). 156

Figure 39 :

 39 Figure 39 : Distribution of the radioactivity found in liver and spleen after intravenous injection of free [ 3 H]-Adenosine, [ 3 H]-SQAd NPs or [ 14 C]-SQAd NPs and metabolic profiles obtained by radio-HPLC. (A) Radioactivity found in the liver expressed as the percentage of injected dose per gram of tissue following intravenous administration of [ 3 H]-Adenosine (5.55 mg/kg, 60 μCi/kg), [ 3 H]-SQAd NPs (15 mg/kg, equiv. 5.55 mg/kg adenosine, 42.4 μCi/kg) or [ 14 C]-SQAd NPs (15 mg/kg, equiv. 5.55 mg/kg adenosine, 9.2 μCi/kg). Results are presented as mean ± SEM from N = 6 animals/time-point/group. Representative radio-chromatograms obtained after radio-HPLC analysis of liver (B) 1 h and (C) 24 h post-injection of [ 3 H]-SQAd NPs (15 mg/kg, 600 μCi/kg). Retention times in liver were RT([ 3 H]-SQAd) = 22.5 min and RT([ 3 H]-Adenosine) = 12.9 min. (D) Radioactivity found in the spleen expressed as the percentage of injected dose per gram of tissue following intravenous administration of [ 3 H]-Adenosine (5.55 mg/kg, 60 μCi/kg), [ 3 H]-SQAd NPs (15 mg/kg, equiv. 5.55 mg/kg adenosine, 42.4 μCi/kg) or [ 14 C]-SQAd NPs (15 mg/kg, equiv. 5.55 mg/kg adenosine, 9.2 μCi/kg). Results are mean ± SEM from N = 6 animals/time-point/group. Representative radio-chromatograms obtained after radio-HPLC analysis of spleen (E) 1 h and (F) 24 h post-injection of [ 3 H]-SQAd NPs (15 mg/kg, 600 μCi/kg). Retention times in spleen were RT([ 3 H]-SQAd) = 22.5 min and RT([ 3 H]-Adenosine) = 13.4 min (fig. adapt. from Gaudin et al. 2015). 151

Figure 40 :

 40 Figure 40 : Mechanism of action of adenosine

10 .

 10 Evaluation of neuroprotective potential of SQAd nanoparticles in CNS injuries (in vivo experiments) : SPINAL CORD INJURY 10.1. May SQAd NPs be effective in other neurological injuries such as spinal cord injury?

Figure 41 :

 41 Figure 41 : Pharmacological efficiency of the SQAd nanoassemblies (NPs) in a model of spinal cord injury in rats. a, The pharmacological efficiency of the SQAd NPs was assessed in a T9 contusion SCI model. Within 5 min, the animals were intravenously injected with either dextrose 5%, SQAd NPs (32 mg kg -1 , equivalent adenosine 11.5 mg kg -1 , N = 10 animals), SQ NPs (20.2 mg kg -1 , N = 10 animals) or free adenosine (11.5 mg kg -1 , N = 10 animals). b, After 24 h, 48 h and 72 h and up to 28 days post-trauma, the animals were functionally graded using the Basso, Beattie and Bresnahan grading scale (data presented as mean ± s.e.m., #, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) (fig. adapt. from Gaudin et al. 2014).140

Figure 42 :

 42 Figure 42 : Evaluation of neuroprotective effect of SQAd NPs on spinal cord after injury. a-c) Absence of visible traumatic area on the cord of healthy mice (c) compared with the trauma group (a) and the adenosine treated group (b) after 72h.d-g) After 48 h, the ultrastructure of the myelin configuration of the axons was studied by TEM. For animals treated with the solution of free adenosine (e) or only squalene NPs (f), the small, medium and large myelinated axons presented separations and/or interruptions in their myelin configuration (red arrows) compared with healthy animals (d). For SQAd NPs-injected animals (g), examination of the white matter showed medium and large myelinated axons with separations in the myelin configuration, with a few of the medium and most of the small myelinated axons found to be ultrastructurally normal (blue arrows). m, myelin; s, myelinated axons with separation in myelin configuration. h, Quantification of damage on the small myelinated axons showed that the SQAd NPs dramatically reduced the damage score (data are presented as mean ± s.d., ****P < 0.0001) compared to all groups (fig. adapt. fromGaudin et al. 2014). 140

Figure 43 :

 43 Figure 43: Seddon and Sunderland classifications of nerve injury (fig. adapt. from Menorca et al. 2013).182

Figure 44 :

 44 Figure 44 : Peripheral nerve organization (fig. adapt. from 2012 Pearson Education, Inc. -Ch14)

Figure 45 :

 45 Figure 45 : Sciatic nerve injury (fig. adapt. from Martin et al. 2019) 183

Figure 46

 46 Figure 46 : Experimental design

Figure 47 :

 47 Figure 47 : Structures of Leu-Enkephalin and Met-Enkephalin

Figure 48 :

 48 Figure 48 : General principle of the GPCR signaling system (fig. adapt. from Wiseman et al. 2020). 194

Figure 49 :

 49 Figure 49 : Mechanism of morphine on blocking pain process (fig. adapt. from education site for healthcare professionals from Grünenthal)

Figure 50 :

 50 Figure 50 : Conjugation sites on Leu-Enkephalin

Figure 51 :

 51 Figure 51 : Chemical structures of the bioconjugates. Leu-enkephalin-squalene with dioxycarbonyl linker (LENK-SQ-Diox) 40, Leu-enkephalin-squalene with diglycolic linker (LENK-SQ-Dig) 41, and Leu-enkephalin-squalene with direct amide bond (LENK-SQ-Am) 42.

Figure 52 :

 52 Figure 52 : NPs characterization. Representative cryo-TEM images showing the formation of NPs from different bioconjugates: (A) LENK-SQ-Diox NPs, (B) LENK-SQ-Dig NPs, and (C) LENK-SQ-Am NPs. Scale bars, 100 nm. Physicochemical characteristics of NPs [i.e., size, polydispersity index (PDI), zeta potential, and % drug loading] are shown in the table.

  223

Figure 53 :

 53 Figure 53 : In vitro bioconversion of LENK-SQ bioconjugates into LENK in the presence of serum. (A) LENK-SQ with dioxycarbonyl linker, (B) LENK-SQ with diglycolic linker, and (C) LENK-SQ with amide bond. Solid lines and dashed lines represent the bioconjugates and the released peptides, respectively (fig. adapt. from Feng et al. 2019).223

  219

4 .

 4 Evaluation of anti-nociceptive effect of SQ-LENK NPs in an inflammatory pain model : 4.1. Are LENK-SQ NPs able to display an anti-hyperalgesic effect in inflammatory pain model?

Figure 54 :

 54 Figure 54: Experimental design for algesimetry. The antinociceptive effect of NPs was tested in a pathophysiologicalcontext induced by an intraplantar carrageenan injection (2% saline, 100 μl). Involvement of central or peripheral opioid receptors was performed using a brain-permeant opioid antagonist, naloxone (Nal), and a brain-impermeant opioid receptor antagonist, naloxone methiodide (Nal-M). NP suspensions or control solutions were injected intravenously with a dose volume of 10 ml/kg during 30 s. The Hargreaves test was performed 10 min after the NP administration and then every 30 min up to a period of 250 min. The dose of LENK-SQ NPs (20 mg/kg) was equivalent to LENK (11.48 mg/kg) and to SQ NPs (8.28 mg/kg) and corresponded to 20.66 mmol/kg for bothLENK-SQ and LENK. s.c., subcutaneous; i.pl., intraplantar.

Figure 55 :

 55 Figure 55: Antihyperalgesic effect of LENK-SQ NPs and morphine. Antihyperalgesic effects of acute treatment with morphine (A and B), LENK-SQ-Diox NPs (C and D), LENK-SQ-Dig NPs (E and F), and LENK-SQ-Am NPs (G and H) in λcarrageenan-induced inflammatory pain injected rats. Administration of morphine, LENK-SQ NPs, Nal, Nal-M, LENK, blank SQ NPs, or dextrose solution (vehicle) was performed (arrows, 0 on abscissa) 3 hours after λ-carrageenan injection into the right hind paw. Morphine (A), LENK-SQ-Diox NPs (C), LENK-SQ-Dig NPs (E), and LENK-SQ-Am NPs (G) induced an increase in PWL (in seconds, means ± SEM of independent determinations in five to nine animals per group) in the Hargreaves test. *P < 0.05, **P < 0.01, ***P < 0.001, compared to dextrose solution or LENK solution; ### P < 0.001, compared to morphine; $ P < 0.05, $$ P < 0.01, $$$ P < 0.001, compared to LENK-SQ NPs. Two-way analysis of variance (ANOVA) with repeated measures, Bonferroni post test. Nal or Nal-M was administered 15 min before morphine or LENK-SQ NP injection. Basal on abscissa: Control (naïve) rats (before λ-carrageenan injection). (B, D, F, and H) Bars are the means ± SEM of AUCs (seconds × minutes) of the cumulative durations derived from the time course changes (A, C, E, and G) in PWL after the various treatments. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA, Tukey post test, compared to dextrose (vehicle) or LENK solution; $ P < 0.05, $$ P < 0.01, $$$ P < 0.001, compared to LENK-SQ NPs (fig. adapt. from Feng et al. 2019).223

Figure 56 :

 56 Figure 56 : IVIS Lumina scan of mice and of their organs after intravenous administration of fluorescent LENK-SQ-Am NPs or control fluorescent dye solution (ventral view). (A) Biodistribution of fluorescent LENK-SQ-Am NPs in mice with inflamed right hind paw. (B) Biodistribution of fluorescent LENK-SQ-Am NPs in mice with non-inflamed hind paw (saline injected only into the right hind paw). (C) Biodistribution of free dye in mice with inflamed right paw. (D) Zoom of group A at 2 hours. (E) Zoom of group B at 2 hours. (F) Quantitative analysis of the paws with the same region of interest (ROI). R, right hind paw; L, left hind paw (fig. adapt. from Feng et al. 2019).223

  223

Figure 57 :

 57 Figure 57 : In vitro colloidal stability of LENK-SQ-Am NPs in mouse serum. (A) Controls: When diluted in 5% dextrose LENK-SQ-Am NPs remained assembled (DiD: reporter dye; DiR: quencher). They disassembled in ethanol; (B) LENK-SQ-Am NPs (DiD: reporter dye; DiR: quencher) incubated in mouse serum (1:4). The fluorescence emission was measured to assess the progressive disassembly of the nanoparticles (fig. adapt. from Feng et al. 2019). 223

Figure 58 :

 58 Figure 58 : Biodistribution of fluorescent LENK-SQ-Am NPs or control fluorescent dye solution in mice with or without inflamed paw. 4 h after λ-carrageenan or saline injection into the right paw, fluorescent LENK-SQ NPs or free dye were intravenously introduced into the mice. At different time points, mice were deeply anesthetized with a mixture of ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) before euthanasia by transcardiac perfusion of 40 ml saline (8 mL/min), until the fluid exiting the right atrium was entirely clear. Then, liver, spleen, kidneys, heart, lungs, brain, and inflamed right hind paw were excised and immediately imaged with the imager. The fluorescence emitted was quantified with Living Image software over the region of interest (ROI) with the threshold of 20%. (A) Ex vivo fluorescence imaging of the harvested brain, heart, kidneys, lungs, liver and paw from fluorescent NPs or free dye-injected SWISS mice. (B) Average radiant efficiency of these organs after 4 or 24 h injection of NPs or free dye (fig. adapt. from Feng et al. 2019). 223

  ). The observations of the spleen (Figure59G-J), the kidneys (Figure59K-N), the lungs (Figure59O-R) and the heart (Figure59S-V) didn't show any morphological damage after LENK-SQ NPs administration, too.

Figure 59 :

 59 Figure 59: Toxicity study of LENK-SQ-Am NPs upon systemic administration. LENK-SQ-Am NPs (20mg/kg) were

  223

Figure 60 :

 60 Figure 60 : Effect of LENK-SQ on the paw withdrawal latencies using Hargreaves Test.

  • Alexandre Da Silva (Doctorant) : Depuis Septembre 2019 -Financement Allocation recherche (3 ans). Ecole doctorale 569. Avancées dans le développement pré-clinique des nanoparticules d'adénosine-squalène : mécanisme d'action et formulation. 3 publications : Adv. Drug Deliv. Rev. 2019 ; J. Drug Target., 2019 ; J. Pharmacol. Exp. Ther. 2019. • Jiao Feng : Octobre 2014-Décembre 2018 (49 mois) -Financement China Scholarship Council (4 ans). Ecole doctorale 569. Sci. Adv., 2019 ; Curr. Top. Med. Chem., 2017 ; Brevet Europe n° 18306002.9, 2018. • Alice Gaudin : Septembre 2011-Novembre 2014 (38 mois) -Financement Allocation recherche (3 ans). Ecole doctorale 569. Co-encadrement avec le Pr. P. Couvreur (40%) et le Pr. K. Andrieux (30%): 30% Thèse soutenue le 17 Novembre 2014 3 publications : J. Control. Release, 2015 ; Chem. Mater., 2015 ; Nat. Nanotechnol. 2014.

	Sujet : Co-encadrement avec le Pr. P. Couvreur: 50%
	Thèse soutenue le 14 Décembre 2018
	Sujet : Développement de nanomédicaments innovants pour vaincre la douleur : une alternative à la
	morphine.
	2 publications et 1 brevet : Encadrements	Total
	Doctorants	4
	Encadrement : 100%
	Sujet : Développement d'un patch transdermique à libération contrôlée de nanoparticules
	d'enképhaline-squalène ciblant les douleurs chroniques.
	♦ Thèses soutenues :
	• Marie Rouquette : Janvier 2016-Février 2019 (36 mois) -Financement AMX (3 ans). Ecole
	doctorale 569.
	Co-encadrement avec le Pr. P. Couvreur: 50%
	Thèse soutenue le 08 Février 2019
		20

•

  XII th Symposium JeCCo « New technologies Empowering Biomedical Research », du 9 au 10 décembre 2019 à Paris. « Nanotechnology application for pain therapy » Lepetre-Mouelhi S. (Conférence Plénière). • Congrès de la Société Française d'étude et de traitement de la douleur (SFETD, chapitre Français de l'IASP), du 27 au 29 novembre 2019 à Strasbourg. « A new painkiller nanomedicine to by-pass the blood-brain barrier and the use of morphine » Lepetre-Mouelhi S. (Conférence).

	Squalène pour le traitement des Accidents Vasculaires Cérébraux et des traumatismes de la moelle
	épinière » Lepetre-Mouelhi S. (Conférence Plénière)
	• Journée de la chimie et de la biologie-EA4566, le 05 Juin 2012, Ecole Nationale Supérieure de Chimie
	de Mulhouse «La squalénisation : Une méthode générale de formulation des principes actifs sous
	forme de nanoparticules » Lepetre-Mouelhi S. (Conférence Plénière)

• 4th Suprachem Lab Project Training "Biomaterials for therapeutic purposes" (Workshop), du 25 au 27 Septembre 2019 à l'Institute of Macromolecular Chemistry (ICM) Petru Poni, à Iasi, Roumanie, « Nanomedicines and biomedical applications » Lepetre-Mouelhi S. (2 Cours magistraux (4H)) • XV th Symposium of the French Pain Research Network, du 22 and 23, 2019 à l'Institut Cerveau Moelle (ICM), Hôpital de la Pitié Salpêtrière à Paris. « A new painkiller nanomedicine to by-pass the blood-brain barrier and the use of morphine ». Lepetre-Mouelhi S. (Conférence) • Congrès Ophtalmologie Perpignan (CORONA), L'odyssée de l'ophtalmologie : que nous réserve l'avenir ? Les 24 et 25 Mai 2019 à Argèles-sur-Mer. « Nanomédicaments et leurs applications » Lepetre-Mouelhi S. (Conférence Plénière) • Mini-Symposium on Nanomedicine for treating cancer and brain diseases (Euronanomed Horizon 2020), CINaM (Centre Interdisciplinaire de Nanoscience de Marseille), Université d'Aix-Marseille, le 08 Mars 2019 à Marseille. Intitulé de la conférence : « Squalene-based nanomedicine for cerebral ischemia, spinal cord injury and pain» Lepetre-Mouelhi S. (Conférence Plénière) • Séminaire UMR7209, Université Pierre et Marie Curie, le 06 Septembre 2018 à Paris. Intitulé de la conférence : « La squalénisation : une méthode générale de formulation des principes actifs sous forme de nanoparticules» Lepetre-Mouelhi S. (Conférence Plénière) • Séminaire à l'observatoire des Micro et Nanotechnologies (OMNT), Groupe Micro & Nanomédecines, Université Paris-Diderot, le 18 Mars 2015, Paris. « Nanoparticules d'Adénosine-

  Orléans (France), Décembre 2013 « Squalene nanoparticules and cerebral ischemia: toward a theranostic tool » Gaudin A., Lepetre-Mouelhi S., Andrieux K., Couvreur P. • 3 rd conference on «Innovation in Drug Delivery : Advances in Local Drug Delivery», Pise (Italie), Septembre 2013, « Passage of nucleoside analogues accross the Blood-Brain Barrier using squalenoyl nanovectors » Gaudin A., Lepetre-Mouelhi S., Andrieux K., Couvreur P.

	• The SFNano C'Nano joint meeting, Dijon, 10-12 Decembre 2019, Dijon « A new painkiller nanomedicine to by-pass the blood-brain-barrier and the use of morphine. » Lepetre-Mouelhi S., Feng j., Mura S. and Couvreur P. • 47 th IUPAC World Chemistry Congress, 5-12 Juillet 2019, Paris « A new painkiller nanomedicine to by-nanovectors» Gaudin A., Lepetre-Mouelhi S., Andrieux K., Couvreur P. Couvreur P. février 2013 «Passage of nucleoside analogues accross the Blood-Brain Barrier using squalenoyl pass the blood-brain-barrier and the use of morphine. » Lepetre-Mouelhi S., Feng j., Mura S. and • Doc&PostDoc Day, Orsay, France, 20 juin 2016. « Squalenoyl adenosine nanoparticles effect on hepatic ischemia-reperfusion injury. » Rouquette, M., Lepetre-Mouelhi, S. et Couvreur, P. • Doc&PostDoc Day, Orsay, France, juin 2015. «Development of novel nano-biotechnology tools targeting opioid receptors: Squalenoylation of enkephalin peptides.» Feng, J., Lepetre-Mouelhi, S. et • XXVIII GTRV Scientific Meeting, • Colloque de la Société du Cerveau et des Maladies Cérébrovasculaires (SCMC), Paris (France), 12 Couvreur, P.
	• 2 nd Annual Meeting "NanoTheRad", ISMO, Orsay, 9 th of April 2019, « Near Infrared irradiation of
	enkephalin nanomedicines for pain alleviation » Lepetre-Mouelhi S. and Couvreur P. • 27th GTRV Scientific meeting, SANOFI Research and developement Chilly-Mazarin (France) 03-05
	Décembre 2012, «Adenosine-squalene nanoparticles : passage of the blood brain barrier and
	• 5th Annual meeting of SFNano, Montpellier (France), December 2018, « A new painkiller neuroprotection in cerebral ischemia» Gaudin A., Lepetre-Mouelhi S., Yemiski O., Andrieux K., Dalkara
	nanomedicine to by-pass the blood-brain-barrier and the use of morphine. » Feng, J., Lepetre-T., Couvreur P.
	Mouelhi, S. and Couvreur, P.
	• 7 èmes Journées de Biologie Cellulaire du Grand Campus, Orsay (France), Mai 2012, « Nanoparticules • International Symposium "Drug Discovery and New Therapeutics", Orsay (France), April 2018, d'Adénosine-Squalène et ischémie cérébrale » Gaudin A., Lepetre-Mouelhi S., Yemiski O., Andrieux K., «Development of novel nano-biotechnology tools targeting opioid receptors: Squalenoylation of enkephalin peptides. » Feng, J., Lepetre-Mouelhi, S. and Couvreur, P. Dalkara T., Couvreur P.
	• Journées Communes LPS (laboratoire de Physique des Solides)-UMR 8612, CNRS Gif sur Yvette
	• 44 th Annual Meeting & Exposition of the Controlled Release Society, Boston, USA, July 2017 (France) 27 Septembre 2011 « Les terpènes pour la construction de nanomédicaments anticancéreux » «
	Development of novel nano-biotechnology tools targeting opioid receptors. » Feng, J., Lepetre-Lepetre-Mouelhi S.
	Mouelhi, S. and Couvreur, P.
	• Doc&PostDoc Day, Orsay, France, 09 juin 2017. « Squalene adenosine nanoparticles freeze-drying. » • Communications par affiche :
	Rouquette, M., Lepetre-Mouelhi, S. et Couvreur, P.
	• 24 th Young Research Fellow Meeting in Medicinal Chemsitry, Paris, France, 8-10 February 2017. • ULLA Summer School, Leuven, Belgium, 8-15 July 2017. « Development of novel nano-biotechnology «
	Squalenoylated adenosine nanoparticles freeze-drying.» Rouquette, M., Lepetre-Mouelhi, S. et tools targeting opioid receptors. » Feng, J., Lepetre-Mouelhi, S. and Couvreur, P.
	Couvreur, P. • ULLA Summer School, Leuven, Belgium, 8-15 July 2017. « Squalene-adenosine nanoparticles: a
	multifunctional agent for treating liver ischemia-reperfusion injury.» Rouquette, M., Lepetre-Mouelhi, • Doc&PostDoc Day, Orsay, France, 20 juin 2016. «Development of novel nano-biotechnology tools targeting opioid receptors: Squalenoylation of enkephalin peptides.» Feng, J., Lepetre-Mouelhi, S. et S., Beaujean, M., Ser-Le Roux, K., Polrot, M. and P. Couvreur
	Couvreur, P. • 24 th Young Research Fellow Meeting in Medicinal Chemsitry, Paris, France, February 2017.
	«Development of novel nano-biotechnology tools targeting opioid receptors: Squalenoylation of
	• 15 ème JED Innovation Thérapeutique, Paris, France, 16-17 Juin 2015. « Gemcitabine-squalene enkephalin peptides.» Feng, J., Lepetre-Mouelhi, S. et Couvreur, P.
	nanoparticles and lipoproteins: role in anticancer activity » Sobot, D., Mura, S.; Dalbin, L.; Lepetre-Mouelhi, S.; Couvreur, P. • 10 th PBP WORLD Meeting Glasgow, Scotland, 4-8 April 2016. « Development of novel nano-
	biotechnology tools targeting opioid receptors» Feng, J., Lepetre-Mouelhi, S. et Couvreur, P. • 36 th PAMM-EORTC Winter meeting, Marseille, France. 21-24 January 2015 « Gemcitabine-squalene nanoparticles: involvement of plasma lipoproteins in the therapeutic response » Sobot, D., Mura, S., • 16 èmes Journées de l'Ecole Doctorale ED 425, Châtenay-Malabry, France, 17 juin 2016. « Squalenoyl
	Dalbin, L., Lepetre-Mouelhi, S., Couvreur, P. (Award young researchers best oral communication) adenosine nanoparticles effect on hepatic ischemia-reperfusion injury. » Rouquette, M., Lepetre-
	• 41 st CRS Annual Meeting, Chicago, USA, 13-16 July 2014. « Squalenoyl Adenosine Nanoparticles and Mouelhi, S. et Couvreur, P.
	Cerebral Ischemia » Gaudin, A.; Lepetre-Mouelhi, S., Yemisci, M., Tagit, O., Hildebrandt, N., Andrieux, • 16 èmes Journées de l'Ecole Doctorale ED 425, Châtenay-Malabry, France, 17 juin 2016. «Development
	K., Dalkara, T. and Couvreur, P. of novel nano-biotechnology tools targeting opioid receptors: Squalenoylation of enkephalin
	• 14 èmes Journées de l'Ecole Doctorale ED 425, Châtenay-Malabry, France, Juin 2014. « Nanoparticules peptides.» Feng, J., Lepetre-Mouelhi, S. et Couvreur, P.
	d'adénosine squalène et ischémie cérébrale » Gaudin, A., Lepetre-Mouelhi, S., Yemisci, M., Tagit, O., • 24 th Young Research Fellow Meeting in Medicinal Chemsitry, Paris, France, 8-10 February 2017. « Hildebrandt, N., Andrieux, K., Dalkara, T. and Couvreur, P. Development of novel nano-biotechnology tools targeting opioid receptors.» Feng, J., Lepetre-
	Mouelhi, S. et Couvreur, P.

•

  International Symposium on Advances in Synthetic and Medicinal Chemistry, August 21-25, 2011, St Petersburg «Squalenoyl Gemcitabine Monophosphate Nanoassemblies : New Prodrug Strategy for the Delivery of Nucleotide Analogues » Caron J., Lepetre-Mouelhi S., Reddy L.H., Couvreur P., Desmaële D. • Colloque Chimie et Santé, de la molécule au Médicament, le 5 Mai 2011, Cité Internationale de Paris. « Reversion of resistance to gemcitabine by squalenoyl monophosphate nucleoside nanoassemblies » Caron J., Lepetre-Mouelhi S., Reddy L.H., Couvreur P., Desmaële D.

	Cy7.5 GCPQ LENK PEG TEA Abbreviations Cyanine7.5 (near-infrared fluorescence-emitting dye, acceptor) Quaternary ammonium palmitoyl glycol chitosan Leucine-enkephalin Poly(ethylene glycol) Triethylamine Cy5.5 Cyanine5.5 (near-infrared fluorescence-emitting dye, FRET donor) FRET Förster resonance energy transfer LDLR LDL receptors PdI Polydispersity Index TBDMSCl tert-butyldimethylsilyl chloride I. INTRODUCTION
	1.	dC GDP LENK-SQ-Am pkA TEM State-of-art on nanomedicines : deoxycytidine Guanosine diphosphate Squalenoyl-Leu-enkephalin with amide bond Protein kinase A Transmission Electron Microscopy
		dCDA GPCR LENK-SQ-Dig PLGA THF	deoxycytidine deaminase G protein-coupled receptors Squalenoyl-Leu-enkephalin with diglycolic linker Poly(lactic-co-glycolic acid) Tetrahydrofuran
		A549	Human breast adenocarcinoma cell line
		dCK GTP LENK-SQ-Diox PWL TMSBr	deoxycytidine kinase Guanosine triphosphate Squalenoyl-Leu-enkephalin with dioxycarbonyl linker Paw Withdrawal Latencies Trimethylsilyl bromide
		Ac	Acid
		dFdC HATU LPDS Py TMSCl	2',2'-difluoro-2'-deoxycytidine (Gemcitabine) (2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium Lipoprotein Deficient Serum Pyridine Trimethylsilyl chloride
		AC	Adenylyl Cyclase hexafluorophosphate)
		dFdC-MP LPs RES TNF	dFdC monophosphate Lipoproteins Reticuloendothelial System Tumor Necrosis Factor
		Ad hCMEC/D3	Adenosine Human brain microvascular endothelial cell line
		dFdU MCAo ROI TPLENK	2',2'-difluoro-2'-deoxyuridine Middle Cerebral Artery occlusion Region of interest Tyrosinyl-palmitate-leu-enkephalin
		Alloc HDL	Allyloxycarbonyl High Density Lipoproteins
		Dic MCF-7 ROS VLDL	Differential interference contrast Human breast cancer cell line Reactive Oxygen Species Very Low Density Lipoproteins
		ALT hENTs	Alanine transaminase human equilibrative nucleoside membrane transporters
		DID MDA-MB-231 RT XO	C18-based indodicarbocyanines fluorescent probe (donor for DIR) aggressive and invasive breast cancer cell line Retention Time Xanthine Oxydase
		araC HMPA	Cytarabine or Cytosine arabinoside Hexamethylphosphoramide
		DIPEA MiaPaca2 s.c.	diisopropylethylamine Human pancreatic cancer cell line subcutaneous
		ARs HOBt	Adenosine receptors (1-hydroxybenzotriazole hydrate)
		DIR MRT s.d.	C18-based indotricarbocyanine fluorescent probe (quencher of DID) Main Resident Time standard deviation
		AST HPLC	Aspartate transaminase High Performance Liquid Chromatography
		DLS MS SANS	Dynamic Light Scattering Mass Spectrometry Small-angle neutron scattering
		ATP AUC HTO i.n. DMF MTD SAXS	Adenosine Triphosphate Area Under the Curve Tritiated water intraneural Travaux de recherche Dimethyl formamide Maximum Tolerated Dose Small angle X-ray scattering
		DMT MTT SCI	Dimethoxytrityl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide Spinal Cord Injury
		B16F10 i.p.	Murine melanoma cell line intraperitoneal
		DRG mV SEM	Dorsal Root Ganglia Millivolts Standard Error of Measurement
		BBB i.pl.	Blood-Brain Barrier intraplantar
		EHNA Nal SQ ac acid	Erythro-9-(2-hydroxy-3-nonyl)adenine Naloxone squalenylacetic acid
		Bn IC50	Benzyl Half-maximal inhibitory concentration
		EMA Nal-M SQ Ac	European Medicines Agency Naloxone methiodide 1,1',2-tris-norsqualenic acid
		BODIPY-542/563 IgG	Red fluorescent probe Immunoglobulines
		ENTs NAs SQ	Equilibrative nucleoside transporters Nanoassemblies Squalene
		BODIPY-FL IL	Green fluorescent probe Interleukin
		EPR NCHS SQAd	Enhanced Permeation and Retention National Center for Health Statistics N6 -squalenylacetic acid-adenosine
		BSA KB3-1	Bovine Serum Albumine Human nasopharyngeal cancer cell line
		ER nm SQdC	Endoplasmic Reticulum nanometer Squalenoyl deoxycytidine
		cAMP L1210 10k	Cyclic Adenosine Monophosphate dFdC-resistant variant of L1210 cell line
		Et NO SQddC	Ethyl Nitric Oxide Squalenoyl dideoxycytidine
		CDC L1210wt	Center for Disease Control and Prevention Murine leukemic cell line
		FAAH NPs SQdFdC	Fatty Acid Amide Hydrolase Nanoparticles tris-norsqualenoyl-gemcitabine
		CEM/ARAC8C LC	hENT1-deficient human leukemic cell line Liquid Chromatography
		FBS NSB SQdFdC-MP	Fetal Bovine Serum Non Specific Binding tris-norsqualenoyl-gemcitabine-monophosphate
		CFA LC/MS-MS	Complete Freund's Adjuvant Liquid chromatography/tandem mass spectrometry
		FDA NVU SQ-PEG	Food and Drug Administration Neurovascular Unit 1,1',2-tris-norsqualene-polyethylene glycol
		Ci LC-MS	Curie (unit of radioactivity) Liquid Chromatography tandem Mass Spectrometry
		FG OPN TB	Fluoro-Gold Opiorphin Total Binding
		CMA LDA	Conditional Marketing Authorisation Lithium diisopropylamide
		FIX P388 TBAF	Factor IX Murine leukemia cell line Tetrabuthyl ammonium fluoride
		CNS LDL	Central nervous system Low Density Lipoproteins
		Fm PBCA TBDMS	9-Fluorenylmethyl Polybutylcyanoacrylate tert-butyldimethylsilyl
		Cryo TEM LDL-f	Cryogenic Transmission Electron Microscopy green fluorescently labeled LDL

Table 1 :

 1 In vitro anticancer activity of dFdC and nanoassemblies of SQdFdC against L1210 wt and L1210 10K murine leukemia cell line.

	Compound	IC50 on L1210 wt leukemia cell line	IC50 on L1210 10K leukemia cell line (µM)
		(µM)
	dFdC	0.0042 ± 0.0001	18.8 ± 1.13
	SQdFdC NPs	0.036 ± 0.001	5.0 ± 0.21

Table 2 :

 2 In vitro anticancer activity of dFdC and nanoassemblies of SQdFdC, 19a and 19b against L1210 wt (dFdC -sensitive) murine leukemia cell line.

	Compound	IC50 on L1210 wt leukemia	IC90 (nM))
		cell line (nM)
	dFdC	3.2	10
	SQdFdC NPs	38	73
	19a-NPs	6.1	31
	19b-NPs	6.7	28

Table 3 :

 3 In vitro anticancer activity of dFdC and nanoassemblies of SQdFdC and 19a against L1210 10K (dFdCresitant) murine leukemia cell line.

	Compound	IC50 on L1210 10K leukemia cell line (µM)
	dFdC	19,4
	SQdFdC NPs	9.8
	19a-NPs	3.7

Table 4 :

 4 Colloidal characteristics of the of polyisoprenoyl dFdC prodrugs.

	Compound	Number of	Overall	Calculated	Average size of	Polydispersity	Drug
		isoprenyl	chemical yield	log P c	nanoassemblies	index (PdI)	loading
		units	(%)		(nm)		(%)
	SQdFdC (8)	5	-	6.90	125	0.10	40.7
	8b	1	74	0.79	250 a	0.14	70.4
	8c	2	65	2.32	145 a	0.09	59.6
	8d	3	53	3.85	270 b	0.09	43.0 d
	8e	4	32	5.37	112	0.33	45.5
	8f	5	64	6.90	116	0.14	40.7
	8g	6	54	8.43	156	0.34	36.8

a Nanoassemblies were precipitated into 10% aqueous trehalose solution. b Co-nanoprecipitation of polyisoprenyol gemcitabine prodrugs with SQ-PEG into water. c ChemDrawBio Ultra 12.0 (CambridgeSoft). d Calculated taking account of the SQ-PEG added.

Table 5 :

 5 In vitro cytotoxicity (IC50) of nanoassemblies constructed with different polyisoprenoyl prodrugs of gemcitabine against various human and murine cancer cell lines.

  Maison du doctorat de l'Université Paris-Saclay

ème étage aile ouest, Ecole normale supérieure Paris-Saclay avenue des Sciences, Gif sur Yvette, France

Remerciements

Institut Galien staff involved in this project :

Main investigators : Patrick Couvreur, Karine Andrieux, Sinda Lepetre-Mouelhi Project participants : Simona Mura, Mariana Varna PhD students : Alice Gaudin, Dunja Sobot, Marie Rouquette, Grégory Pieters Flavio Dormont, Romain Brusini Post-doctorant students : Anne Gauthier, Hakan Eroglu Masters students Ophélie Dufrançais, Manon Beaujean Research engineers : Julie Mougin, Hélène Chacun, Catherine Cailleau

Main collaborations :

Institute of Neurological Sciences and Psychiatry, Hacettepe University, Ankara, Turkey.

Turgay Dalkara

Department of Pharmaceutical Technology, Hacettepe University (Pharmacy), Ankara Turkey.

Yilmaz Capan

CEA Saclay, iBiTecS-S/SCBM, Labex LERMIT, Gif-sur-Yvette, France Bernard Rousseau, Olivier Loreau, Sébastien Garcia Argote Leiden Academic Centre for Drug Research Leiden University, Leiden, the Netherlands

We therefore decided to protect the 3'-hydroxy moiety with a silyl group, assuming that it should be resistant to exposure to tetrazole. Transient protection of the 5'-hydroxy group was achieved through formation of the highly acid-labile dimethoxytrityl (DMT) ether by treatment of 8 with dimethoxytrityl chloride in pyridine (Scheme 5). 103 This C-5' protection was then followed by C-3' protection with a tert-butyldimethylsilyl (TBDMS) group, which provided the fully protected derivative 15. 102 Removal of the 5'-DMT group upon brief treatment with formic acid 104 led to the expected 5'free alcohol 16 (77% yield).

Condensation of 16 with the phosphoramidite 11 in the presence of 1H-tetrazole, followed by hydrogen peroxide work up, gave the phosphotriester 17 in 80% yield. At this stage the 3'-hydroxyl group was unmasked by treatment with HF.pyridine 105 to afford 18 in 69% yield. The final deprotection of the phosphate group was effected by treatment with a large excess of triethylamine in acetonitrile to give the triethylammonium phosphate salt 19a in 98% yield. 102 Since, a potential toxicity is associated with triethylamine, we looked for a pharmacological more suitable salt. Unfortunately, treatment with currently used amines such diethylethanolamine, 4-(2-hydroxyethyl)-morpholine or procaine turned out to be inefficient. We finally found that treatment of 18 with dimethylethanolamine smoothly gave the corresponding dimethylethanolammonium phosphate salt 19b in 85% yield (Scheme 5). 100 • Behavioral study (motor recovery and sensitivity):

The functional recovery of the rats after intravenous injection of SQAd NPs (32 mg/kg), is correlated with the reconnection of the injured DRG neurons to the spinal cord. This functional recovery will be assessed using motor tests such as behavioral score and locotronic test, which will be performed repeatedly over a 38-day period. The mechanical sensitivity will also be evaluated using Von Frey Filament test (mechanical allodynia) at day 27. Likewise, the thermal sensitivity will be assessed using Hargreaves test (thermal hyperalgesia) at day 29.

12.1.3.

Chronic treatment of SQ-Ad NPs :

• Behavioral study (motor recovery):

The functional recovery of the rats will be assessed repeatedly after 4 repeated injections of SQAd NPs (32 mg/kg), at days -4, 0, 3 and 8 after injury, using locotronic motor test, which will be performed over a 37-day period.

Through this study, we expect that SQAd NPs will prove to be neuroprotective and will allow regeneration of damaged sciatic nerve. In case of success, the squalene-based platform will open exciting perspectives in the treatment of peripheral nervous system pathologies.

Michel Hamon

Concerning the synthesis of LENK-SQ-Dig prodrug with 2′-diglycolate spacer (compound 41), the tris-nor-squalenol 47 reacted with diglycolic anhydride 48 leading to the squalene diglycolic acid 49. This latter was then activated by ethyl chloroformate into the corresponding mixed anhydride, prior to react with LENK, leading to the LENK-SQ bioconjugate 50 obtained in 69% yield (Scheme 16). 223 The oxa moiety of the linker was intended to enhance the susceptibility to hydrolysis by increasing the distance between LENK and SQ and so the accessibility to the linkage. The LENK-SQ-Am 42 conjugate was obtained by direct conjugation between LENK and tris-norsqualenic acid 6 via direct amide bond (73% yield). Prior to the conjugation step, the SQ Ac 6 was activated using ethyl chloroformate (Scheme 17). 223