Dr Philippe Barthélémy

Ling Peng

Le Karine Pr Elias Andrieux

Fattal

Harivardhan Le Dr

Reddy

Dr Fabienne Testard pour avoir accepté de porter un regard critique sur cette thèse en tant qu'examinateurs.

Je tiens également à remercier très chaleureusement Elias pour son accueil au sein du laboratoire. Tu ne m'as pas uniquement accueillie en tant que directeur de l'institut mais aussi en tant que simple collègue, et tu as discrètement veillé sur moi tout au long de ces trois ans (et peut-être encore plus au cours de ces derniers mois). Merci pour cette attention.

Un immense merci à Patrick, mon directeur de thèse, pour sa disponibilité au cours de ces trois ans. Votre emploi du temps est chargé mais vous avez une « disponibilité d'esprit » exceptionnelle : vous avez toujours été extrêmement réactif quand j'avais besoin de vous, et aviez toujours parfaitement à l'esprit l'état d'avancement de mes travaux. C'est une qualité exceptionnelle chez un responsable et je tenterai de la reproduire dans ma vie professionnelle future. Merci également pour toutes nos discussions, scientifiques ou non.

J'aime écouter des histoires et vous savez en raconter de fameuses ! Je souhaite également témoigner toute ma gratitude envers Sinda, ma co-directrice de thèse. Tu cherches avant tout à soutenir et faire progresser les gens qui t'entourent, et tu le fais avec beaucoup d'humilité. Les personnes d'une telle générosité pour l'humain sont rares et précieuses. J'essaierai de garder en moi cet apprentissage, car je suis convaincue qu'une de nos missions professionnelles les plus importantes (mais également des plus faciles à oublier) est de nous aider les uns les autres à grandir. Merci à Karine, Mélanie et toute leur équipe pour leur accueil particulièrement chaleureux à la plateforme animalerie de l'IGR. Malgré toutes les galères qu'on a traversées, ça a toujours été un bonheur de venir chez vous car je savais que j'y retrouverais votre bonne humeur inaltérable. Merci de m'avoir tant appris sur la souris et de m'avoir fait confiance dès le début pour la chirurgie. qui aurait été extrêmement triste pour l'ensemble de nos fans. Marion, qui a la capacité extraordinaire de râler toute la journée avec motivation, entrain et bonne humeur. Je n'ai toujours pas compris comment tu faisais ! Gianpiero, qui a illuminé de son amitié mes longs trajets en bus entre Paris et la lointaine Châtenay ; Jiao, for kindly sharing some of your culture (and some of your office !) with me ; Elise, notre maman qu'on aurait bien gardée un an de plus ; Daniele, qui me comprend maintenant même quand je n'articule pas ; Julie (eh oui c'est ton côté un peu schizo, tu es à la fois avec les statutaires et les doctorants), ta persévérance va payer ; Justin, j'ai une immense admiration pour toi, tous les efforts que tu fais pour apprendre le français, ta curiosité, ton attention à l'autre… tu es l'une des personnes qui m'a le plus impressionnée ici et je ne te l'ai sûrement pas suffisamment dit ! ; Flavio, cher compagnon d'adénosine-squalène, bon courage pour la fin ; Romain, garde ta bonne humeur et ta simplicité, elles sont des atouts extraordinaires ; Fanny et Annaëlle, avec qui ça a été un plaisir de partager le bureau ; Alex, toujours de bonne humeur, toujours motivé, toujours attentif à l'autre ; Tanguy, mon pharmacien préféré ; Raul, mon herboriste préféré ; Quentin, mon véto préféré ; et Julien, que j'aurais bien croisé plus souvent. Je ne peux pas citer tous les doctorants, post-doctorants et stagiaires de l'institut dont j'ai croisé la route, pourtant vous avez chacun contribué à créer cette ambiance si joyeuse et dynamique au sein du labo. Merci d'avoir fait de mon environnement de travail un environnement affectif. J'ai également une pensée amicale pour l'équipe iGEM Paris Saclay 2016, que j'ai suivie dans la grande aventure iGEM lors de ma première année de thèse. J'ai adoré participer à toutes ces réunions avec vous, à ce mois de manips en juillet (où j'ai probablement appris autant que vous sur la biologie synthétique !), vous coacher pour la présentation finale à Boston, bref vous apporter mon maigre soutien pour ce projet fabuleux ! Vous suivre a été l'une des expériences qui m'a le plus appris en termes d'encadrement au cours de cette thèse, et je vous en suis très reconnaissante.

Je remercie ma famille et mes amis, qui m'ont entourée tout au long de ces trois ans, qui m'apportent chaque jour un nouvel entrain, de nouveaux projets, et qui m'aident à grandir tout au long de ma vie.

Enfin, je ne remercierai jamais assez mon mari, Bertrand, pour m'apporter le bonheur extraordinaire de partager sa vie avec moi. Au cours de ces trois ans de thèse, tu m'as offert par deux fois la beauté d'un engagement à vie l'un avec l'autre, en m'épousant et en me donnant un enfant.

I would also like to thank Pr. Adriaan P. IJzerman for welcoming me in his lab in Leiden for two weeks and taking time to discuss with me. Xue, you have been a wonderful teacher and host, and this collaboration could not have been as successful without you. I also would like to thank Rongfang for making preliminary experiments before I came, Henk de Vries, the one who knows everything about the lab, and Anna for giving me some tips on adenosine agonists and antagonists.

Je n'aurais pu obtenir tous ces résultats sans l'aide de mes stagiaires, master 2 ou BTS. Ils m'ont beaucoup appris et m'ont permis de faire mes premiers pas en tant qu'encadrante sans être trop sévères avec moi. Merci Manon pour tout ce boulot en culture cell, et Ophélie qui est maintenant bien plus calée en HPLC que moi ! Merci d'avoir gardé votre bonne humeur et votre confiance en ma capacité à vous guider malgré les moments difficiles. Une petite pensée également pour Yannis, mon stagiaire de troisième, qui m'a rappelé à quel point je trouve la biologie extraordinaire.

J'ai été entourée de personnes remarquables au sein de cet institut : Simona, la mémoire vivante de tout ce qui a été fait sur le squalène depuis la nuit des temps (et surtout depuis que le labo a développé la squalénisation), toujours prête à prendre du temps pour écouter les difficultés et aider ; Juliette, la pro du Western (en tournant ça comme ça on pourrait t'imaginer avec deux colts à la ceinture et un chapeau de cowboy, mais ça ne serait pas une image très fidèle à ta gentillesse !) ; Stéphanie, avec qui on a vécu les coupures d'électricité à répétition (et on n'a même pas fait de burn out !), merci pour les petits dessins sur les flasques ; Julie, the HPLC expert comme dirait Ophélie, et aussi celle qui sait à chaque instant où est Sinda ; Claudie, à l'écoute si attentive ; Didier, qui déjeune tous les jours au CROUS ; Assia et Ghozlene, grâce à qui j'ai pu faire toutes mes manips en radioactivité (et ce jusqu'au bout !) ; Jean-Philippe, dont la patience n'a d'égale que sa dextérité à l'AFM.

Merci également aux responsables des plateformes de la faculté de pharmarcie, notamment Delphine, toujours prête à aider, même dans le bus, et Valérie Nicolas, aux explications limpides concernant la microscopie confocale.

Vient ensuite la foule des doctorants que j'ai côtoyés avec grand plaisir au cours de ces trois dernières années. Johanna, bien sûr, sans qui je ne me serais pas lancée dans ma grande carrière artistique à l'institut. Merci d'avoir proposé de m'aider pour le film des 30

Introduction générale

Le développement de nouvelles techniques de délivrance du médicament permet d'adapter la stratégie d'administration des substances actives en fonction de leurs propriétés physico-chimiques et de la cible thérapeutique envisagée. En particulier, l'introduction des nanomédecines (nanotechnologies appliquées au domaine de la médecine) dans les années 60 a été la source d'importants espoirs pour le traitement de maladies graves telles que le cancer.

Cependant, bien qu'une quinzaine de ces nanoformulations aient été mises sur le marché, le développement des nanomédecines en général a rencontré plusieurs verrous technologiques.

En effet, les nanomédicaments présentent souvent un très faible taux de charge en substance active, relarguent parfois de manière massive et précoce le principe actif (phénomène de « burst release ») et peuvent exhiber une certaine toxicité due au matériel de vectorisation utilisé.

De plus, la transposition d'échelle pour une production industrielle se révèle parfois trop compliquée et/ou coûteuse.

La stratégie de « squalénisation », mise au point depuis 2004 par l'équipe du Professeur

Couvreur, a permis de lever certains de ces verrous. En effet, cette technique consiste à coupler une substance active à un dérivé du squalène -un lipide endogène, biocompatible et biodégradable, précurseur du cholestérol -afin de former un bioconjugué ayant la capacité de s'auto-assembler en nanoparticules d'une centaine de nanomètres de diamètre en milieu aqueux. Ces nanomédicaments présentent notamment un fort taux de charge en principe actif, une très bonne biocompatibilité, une pharmacocinétique nettement améliorée par rapport à la substance active libre et une biodistribution beaucoup plus ciblée de celle-ci.

L'application de cette stratégie à l'adénosine, une molécule au potentiel thérapeutique important mais limité par un temps de demi-vie plasmatique extrêmement court, a fourni des résultats particulièrement prometteurs dans le cadre du traitement de l'ischémie cérébrale et du traumatisme de la moëlle épinière. L'objectif de cette thèse était donc de faire progresser le développement pré-clinique de ces nanoparticules d'adénosine-squalène selon deux axes majeurs : l'étude de leur mécanisme d'action et l'optimisation de leur formulation.

Dans une première partie bibliographique, nous présenterons les différentes associations créées jusqu'à aujourd'hui entre l'adénosine et les lipides. Nous aborderons notamment la délivrance de l'adénosine et de ses dérivés phosphorylés via des liposomes, puis nous décrirons les différents bioconjugués lipidiques formés à base d'adénosine ainsi que leurs capacités d'auto-organisation supramoléculaire.

Le premier chapitre expérimental décrira ensuite les résultats obtenus lors de l'étude in vitro du mécanisme d'action des nanoparticules d'adénosine-squalène. L'interaction directe entre l'adénosine-squalène (sous forme de nanoparticule ou de bioconjugué) et les récepteurs à l'adénosine a été étudiée grâce à une expérience de déplacement d'un radioligand. Cette interaction s'étant révélée négligeable, l'étude de l'adénosine-squalène en tant que prodrogue a été réalisée sur des hépatocytes. L'adénosine-squalène est en effet retrouvé en quantités importantes dans le foie après injection des nanoparticules par voie intraveineuse chez la souris. Ainsi, l'internalisation par les cellules HepG2 de nanoparticules d'adénosine-squalène radiomarquées ou fluorescentes, suivie du relargage d'adénosine, ont été étudiés. Cette étude a permis de mettre en lumière la formation d'un réservoir intracellulaire d'adénosine due à l'internalisation puis à l'accumulation des nanoparticules d'adénosine-squalène.

Le second chapitre expérimental présentera enfin l'optimisation des conditions de lyophilisation des nanoparticules d'adénosine-squalène. La bonne conservation de leurs propriétés physico-chimiques a été vérifiée par différentes techniques analytiques, dont la diffusion dynamique de la lumière, la diffraction des rayons X et des neutrons aux petits angles, ou encore la microscopie à force atomique. L'influence de la présence d'éthanol résiduel (utilisé lors de la formulation) sur le résultat de la lyophilisation est un point intéressant qui a été soulevé lors de cette étude. La lyophilisation a permis une bonne stabilité des nanoparticules dans le temps avec une reconstitution très aisée de celles-ci, les rendant ainsi très facilement manipulables en vue d'une administration en cas d'urgence.

L'ensemble de ces travaux constitue ainsi une avancée significative dans le développement pré-clinique des nanoparticules d'adénosine-squalène, par la mise en lumière de leur mécanisme d'action et l'établissement d'une nouvelle formulation, plus pratique et reproductible.

General Introduction

Ongoing advances in drug delivery help to adapt the administration strategy of the active ingredient taking into account its physico-chemical properties as well as the therapeutic target.

In particular, the introduction of nanomedicines (or nanotechnologies applied to medicine) in the 1960's has brought hope for enhancing the effectiveness of the treatments against severe diseases such as cancer, through a better targeted approach. However, even though around fifteen nanoformulations are already used in the clinic, the development of nanomedicines in general has suffered from serious drawbacks. Indeed, nanomedicines often present extremely low drug loadings, may prematurely and massively release the active compound before reaching the target (phenomenon of "burst release") and exhibit some toxicity due, among other things, to the vector per se. Moreover, scaling-up the production for clinical needs is sometimes challenging and/or too costly.

The so-called « squalenoylation » strategy, developed since 2004 by Pr. Couvreur's team, has overcome some of these limitations. This original technology consists in coupling an active ingredient with a derivative of squalene -an endogenous, biocompatible and biodegradable lipid which is a precursor of cholesterol -to form bioconjugates able to self-assemble as nanoparticles of around 100 nm of diameter in aqueous medium. The resulting nanomedicines generally present high drug loadings, very good biocompatibility, enhanced pharmacokinetics compared to the free drug as well as a more targeted biodistribution.

This strategy was applied to adenosine, a molecule which high therapeutic potential is severely impaired by its very short half-life in blood circulation. Squalene-adenosine nanoparticles have shown extremely promising results for the treatment of cerebral ischemia and spinal cord injury. Hence, the objective of this thesis was to further develop squaleneadenosine nanoparticles at the pre-clinical stage, pursuing two major goals: studying their mechanism of action and optimizing their formulation.

First, in a bibliographic part, we will present the different associations which have so far been designed between adenosine and lipids. More precisely, we will address liposomal delivery of adenosine and its phosphorylated derivatives, before describing adenosine-based lipid bioconjugates and their supramolecular self-assemblies.

The first experimental chapter will then describe the mechanism of action of squaleneadenosine nanoparticles through in vitro studies. Direct interaction between squaleneadenosine (whether as a nanoparticle or bioconjugate) with adenosine receptors was studied by radioligand displacement assays. As this interaction was found to be negligible, we therefore investigated whether squalene-adenosine could act as a prodrug. This mechanism was studied on hepatocytes, as high quantities of squalene-adenosine are found in the liver after intravenous injection of squalene-adenosine nanoparticles in mice. HepG2 cellular uptake of radio-or fluorescently labeled squalene-adenosine nanoparticles was studied, as well as the resulting adenosine release. This study highlighted the presence of an adenosine intracellular reservoir due to the internalization and intracellular accumulation of squalene-adenosine nanoparticles.

Finally, the second experimental chapter will present the optimization of squaleneadenosine nanoparticles formulation through the use of the best freeze-drying conditions. The preservation of the nanoparticles' physico-chemical properties was checked by different analytical techniques, such as dynamic light scattering, small-angle X-ray or neutrons scattering or atomic force microscopy. In addition, the presence of residual ethanol (used during the nanoparticles' formulation) was shown to influence the lyophilization success. Of note, this finding had rarely been studied before. In that respect, the freeze-drying of nanoparticles led to their long-term stability as well as to their easy and rapid reconstitution in ready-to-use nanoparticulate suspension which could thus prove quite convenient for administration in emergency cases.

Overall, this work represents a major step forward the pre-clinical development of squalene-adenosine nanoparticles by bringing to light their mechanism of action and establishing a new, easy-to-use and reproducible formulation.

Introduction bibliographique

Adenosine and lipids: a forced marriage or a love match?

Accepted in Advanced Drug Delivery Reviews

Adenosine and lipids: a forced marriage or a love match?

Abstract

Adenosine is a fascinating compound, crucial in many biochemical processes: this ubiquitous nucleoside serves as an essential brick for building RNA, is also a component of ATP and regulates numerous pathophysiological mechanisms via binding to four extracellular receptors. Due to its hydrophilic nature, it belongs to a different world than where lipids come from, and has no affinity for them. Since the 1970's, however, new discoveries have emerged and prompted the scientists to associate adenosine with the lipid family, especially via liposomal preparations and bioconjugation. This seems to be an arranged marriage, but could it turn into a true love match? This review clustered all types of unions which were established between adenosine and lipids. Even though exciting supramolecular structure were observed with adenosine-lipid conjugates, as well as, with liposomal preparations which resulted in promising pre-clinical results, the translation of these technologies to the clinic is still limited.

Adenosine is a nucleoside involved in multiple essential biochemical processes both intra-and extracellularly (Figure 1). In the cell, adenosine can be phosphorylated into adenosine monophosphate (AMP), which is used by the cell as an RNA building block. 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 [1], which govern major pathophysiological functions. Equilibrative nucleoside transporters (ENTs), which are widely expressed by the cells [2], 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 [3] and its prompt transformation into adenosine by ectonucleotidases CD39 and CD73.

Adenosine is a hydrophilic molecule that has no affinity with lipids. Like Romeo and Juliet, they come from two separate worlds that rarely mingle. But, if forced to join, would they repel each other, accommodate or start a fusional relationship? Scientific discoveries in the 70's and 80's encouraged research on that topic.

The development of liposomes for drug delivery in the early 1970's [4] was seen by some specialists as a very suitable match for adenosine and its derivatives. Indeed, adenosine and ATP have very short plasmatic half-life [5,6] due to their degradation by plasmatic enzymes and rapid uptake by erythrocytes and endothelial cells, which could be circumvented by liposomal encapsulation. First proposed applications were the delivery of liposomal ATP to treat rat ischemic brains [7] and liposomal adenosine to examine its effect on the contraction of rat aortic smooth muscle cells [8]. As will be presented in the first part of this review, the first matchmakers' intuition was accurate and liposomal forms of adenosine, ATP and even ADP have been developed and studied since this consensual marriage with expected successful outcomes.

Completely independently, a few naturally occurring nucleolipids were discovered in the 1970's, such as tunicamycins, which were isolated from Streptomyces lysosuperficus [9].

This drove chemists to create multiple artificial nucleolipids a few years later, including adenosine nucleolipids like adenosine-5'-alkylphosphates [10]. There were two main rationales for studying such adenosine nucleolipids: (i) authors saw them as possible intermediates in enzymatic reactions involving ATP and lipids and wanted to study their physiological significance [10,11], or (ii) they wanted to explore the existence of hydrophobic regions in the active sites of ATP-dependent enzymes [12].

With a totally different mindset, a third rationale for developing adenosine nucleolipids was established in the late 1980's. Adenosine congeners were functionalized by covalently attaching lipids at a permissive site (e.g. N6 or C2 positions of adenosine) with the idea to alter the drug distribution and metabolism in vivo while conserving good binding properties without any prior cleavage [13,14].

Even though these approaches have opened the field of adenosine nucleolipids and given some interesting results, no author really anticipated that this forced marriage would become a perfect love match between adenosine and lipids. Nucleolipids, and adenosine nucleolipids in particular, present remarkable properties that will be exposed in the second part of this review.

I. Adenosine and adenosine derivates loaded liposomes

Liposomes are lipid-bilayer vesicles made of combinations of natural or/and synthetic lipids.

These lipids are perfectly biocompatible and biodegradable and have already been used as excipients in numerous vaccines. As a result, liposomes can be used as safe delivery systems for both hydrophilic and hydrophobic molecules. Up until now, more than 15 liposomal formulations already reached the market, Doxil® being the most well-known among them [15].

When adenosine, ADP or ATP are loaded into the liposomes' hydrophilic core, they are protected from the blood stream and their pharmacokinetics and biodistributions are completely modified, offering new therapeutic possibilities. Table 1 describes the composition and size of the liposomes that will be described in this part, as well as their drug loading and/or encapsulation efficiency when available. Regrettably, not all reports provide a full and comprehensive characterization of their liposomal system, which leads to uncertainties in terms of dosage, especially because liposomal formulation often present very poor drug loadings.

1) Liposomal adenosine

The main objective of adenosine encapsulation into liposomes is to prolong its half-life in the blood stream. Liposomal formulations can also enhance the quantity of active drug at the site of action by passive or active targeting.

Based on this background knowledge, Gutman et al. developed adenosine loaded liposomes to target inflammation [16]. They showed that negatively charged liposomes composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), distearoylphosphatidylglycerol (DSPG) and cholesterol could be prepared by reverse phase evaporation method and loaded with adenosine (reaching a drug loading charge of 12.9 %), and that these monodisperse liposomes of around 170 nm of diameter were internalized by human monocytes and reduced inflammation in vitro. Regrettably, the only parameter assessed in vivo was the absence of adenosine loaded liposomes toxicity on monocytes, whereas their influence on inflammation was not monitored.

Another approach for passive targeting via liposomal formulation was developed by Takahama et al. for myocardial ischemia and reperfusion [17]. It is known that cellular permeability is enhanced in the zone of infarct due to the disruption of the vascular endothelial integrity. As a result, the authors decided to prepare PEGylated adenosine liposomes with prolonged circulation time for reaching higher concentration of adenosine in the target zone.

For this purpose, neutral monodisperse liposomes were prepared by the hydration method, with hydrogenated soy phosphatidylcholine, cholesterol and 1,2-distearoyl-sn-glycero-3phosphoethanolamine-n-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000). The use of fluorescent liposomes showed that they indeed accumulated in the necrotic area, which correlated with a prolonged residence time of liposomal radiolabeled adenosine in the same zone. This led to a reduction of the infarct size which was not associated with usual adenosine side effects (hypotension and bradycardia), confirming the advantage of using liposomal delivery.

Very recently, Corciulo et al. developed the idea of using liposomal adenosine for treating osteoarthritis [18], based on the fact that extracellular adenosine downregulation was known to be implicated in the development of the disease, more specifically via its interaction with A2A receptor. Unexpectedly, they observed that injection of A2AR agonists did not protect rats against a model of post-traumatic osteoarthritis. Hence, they decided to load adenosine in liposomes made of soybean oil, phosphatidylcholine, cholesterol and glycerin and injected them locally in the joint. Using this formulation of adenosine, they witnessed important prevention and protection effects against the disease, with reduced joint swelling, cartilage and joint protection, as well as lowered OARSI scores, and demonstrated that these effects depended on A=R activation. Even though these results constitute a promising pre-clinical proof-of-concept, it seems unfortunate that liposomal formulations were so poorly defined (basic information on their composition can only be found in the corresponding patent [START_REF] Cronstein | Methods and compositions for treating osteoarthritis and promoting cartilage formation, US20180036238A1[END_REF]) and scarcely characterized in this work, with no mention of physico-chemical parameters.

2) Liposomal ADP

An original liposomal ADP system has been developed for 10 years in Takeoka's lab for platelet substitution. Pegylated liposomes were loaded with ADP in the intraliposomal aqueous core and functionalized with a fibrinogen γ-chain peptide. This peptide corresponded to the carboxyl terminal 12 amino acids of the fibrinogen γ-chain and is a ligand for glycoprotein IIb/IIIa receptors which are expressed on activated platelets at the site of injury. As a result, these "H12-(ADP)-liposomes" specifically accumulated within the sites of injuries after intravenous injection [22] and reduced the bleeding time in thrombocytopenic rats [21] and rabbits [20,[START_REF] Hagisawa | Fibrinogen γ-Chain Peptide-Coated Adenosine 5' Diphosphate-Encapsulated Liposomes Rescue Mice From Lethal Blast Lung Injury via Adenosine Signaling[END_REF] by playing two different roles: (i) they enhanced platelet aggregation via crosslinking between activated platelets thanks to their functionalization with the fibrinogen γ-chain peptide and (ii) they enhanced platelet activation by releasing ADP, which acts through platelet surface receptors P2Y1, P2Y12 or P2X1. Interestingly, authors could modulate this ADP release by tuning the vesicles lamellarity and flexibility [21].

Recently, this team made a brilliant conceptual and experimental move: instead of continuing to consider ADP only as a drug for platelet activation, they started looking at it as an adenosine prodrug. This led them to evaluate the pharmacological efficacy of H12-(ADP)liposomes on blast lung injury, where ADP would be useful in treating the hemorrhage, while adenosine would exert anti-inflammatory protective effects [START_REF] Hagisawa | Fibrinogen γ-Chain Peptide-Coated Adenosine 5' Diphosphate-Encapsulated Liposomes Rescue Mice From Lethal Blast Lung Injury via Adenosine Signaling[END_REF] (Figure 2). They obtained an important increase in mice survival after lung injury in the group treated with H12-(ADP)liposomes, going with reduced tissue damage/hemorrhage and inflammation. Moreover, they proved the implication of adenosine in this phenomenon, as treatment with A2A and A2B antagonists abrogated the beneficial effects of the H12-(ADP)-liposomes.

3) Liposomal ATP

Like adenosine, ATP cannot be efficiently delivered intracellularly by itself. It is very rapidly metabolized in the bloodstream and the hydrophilic nature of this nucleoside prevents from crossing cell membranes. As a result, ATP-loaded liposomes have been used for several decades to improve ATP delivery (Figure 3A).

As ATP is the main energy currency in the cell, intracellular ATP delivery has mainly been used for pathologies involving a phenomenon of energy failure. The main application has been ischemia, i.e. the lack of blood supply in a tissue, leading to a shortage in oxygen, decreased cell metabolism and ATP depletion. Ischemic insults can virtually affect any organ or tissue, the most common and severe cases being heart, brain and liver ischemia. Early proof of concept studies on ATP-liposomes pharmacological efficacy against cerebral ischemia were conducted on rats in the 1990's. Liposomes efficiently protected ATP from plasma degradation, could cross the blood-brain-barrier under hypoxic conditions [24] and protected the rats against cerebral ischemic insults [7,23]. A closely related model of retinal ischemia [START_REF] D'onofrio | What can we learn about stroke from retinal ischemia models?[END_REF] was later used to better understand the mechanism of action of these ATPliposomes. ATP delivery led to increased cell survival and decreased cell necrosis in primary retinal ganglion submitted to glucose-oxygen deprivation in vitro, and to decreased neurons death in the ganglion cell layer in vivo after ischemia-reperfusion. Pro-inflammatory genes were also down-regulated in ischemic retinas following ATP-liposomes treatment [25,26]. These simple ATP-liposomes were recently redesigned into pH-sensitive "energy formula"-loaded liposomes [START_REF] Liu | Acute bioenergetic intervention or pharmacological preconditioning protects neuron against ischemic injury[END_REF] (Figure 3B). The lipid composition was chosen in such a way that liposomes responded to pH and released their content at the penumbral intracellular pH. In addition, ATP was co-loaded with pentobarbital, which inhibits energy consumption, and with suramin, for blocking ATP toxic extracellular actions. Even though these liposomes protected a neuronal cell line against ATP depletion in vitro, they failed to protect astrocytes or endothelial cells under the same conditions and no in vivo data were reported. Hence, the superiority of these "energyformula"-liposomes, compared to simple ATP-liposomes, remains to be proven.

ATP-liposomes were also shown to accumulate in myocardial infarct tissues in dogs [27].

Torchilin's team confirmed this result in isolated rat hearts perfused with a Langendorff instrument and showed that ATP-liposomes could protect the mechanical functions of the myocardium during ischemia/reperfusion [29]. Similarly, ATP-liposome treatment reduced the fraction of the irreversibly damaged heart area within the total area at risk in a model of myocardium infarction in rabbits [28]. Further improvement of the liposomes was realized by functionalizing them with the monoclonal 2D4 antibody which specifically binds cardiac myosin [30] (Figure 3C). Intracellular cardiac myosin become, indeed, exposed to the bloodstream after sarcolemmal disruption happening during ischemia. Hence, targeting these proteins should lead to higher accumulation of antibody-coated ATP-liposomes compared to bare ATP-liposomes. Even though this mechanism was not demonstrated in vivo, it was shown that the pharmacological efficacy of antibody-coated ATP-liposomes was enhanced compared to bare ATP-liposomes on isolated rat hearts perfused with a Langendorff instrument [31].

Regarding liver ischemia, ATP liposomes have shown protective activity on rat models of both warm ischemia in vivo after a hypovolemic shock [32] and cold ischemia ex vivo during liver preservation for transplantation [33,34]. In both cases, authors witnessed an enhanced preservation of the ATP cellular content. Functionalizing ATP-liposomes with an asialoglycoprotein receptor ligand did not show any improved hepatocytes targeting or delivery efficacy so far [35], and their freeze-drying led to ATP leakage [START_REF] Vincourt | Freeze-drying of ATP entrapped in cationic, low lipid liposomes[END_REF].

Regarding organ preservation, protective effects of ATP-liposomes were also tested on a model of ischemic pancreatic β cells for possible application on islet transplantation [START_REF] Atchison | Maintenance of ischemic β cell viability through delivery of lipids and ATP by targeted liposomes[END_REF].

ATP-liposomes were functionalized with a fibronectin-mimetic peptide, which favored their cell internalization. Surprisingly, metabolic protection against the ischemic insult was shown to arise from both ATP and the lipids constituting the liposome (l,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC), cholesterol, PEG2000). However, ATP-liposomes could not restore insulin production from ischemic islet cells.

Finally, in a model of mesenteric ischemia/reperfusion, authors showed that liposomal ATP could restore crypt proliferation but did not prevent cell death in the ischemic gut, arguing towards important complementary cytoprotective roles of other glucose metabolites like pyruvate [START_REF] Huang | Distinct cytoprotective roles of pyruvate and ATP by glucose metabolism on epithelial necroptosis and crypt proliferation in ischaemic gut[END_REF]. In terms of commercial applications, highly fusogenic ATP-liposomes have been developed by the company Energy Delivery Solutions, a spin-off from Chien's lab at the University of Louisville, under the product name of ATPv (formerly VitaSol). These liposomes are composed of a stable vesicle former, for example 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or soy phosphatidylcholine (PC), and an unstable vesicle former, like 1,2-dioleoly-snglycero-3-ethylphosphocholine (DOPC-e) or 1,2-dioleoyl-3-trimethyl-ammonium-propane (DOTAP) [START_REF] Ehringer | Direct cellular energy delivery system[END_REF]. Dressings were saturated with these ATPv and applied to wounds to study the liposomes effect on skin healing. Very promising results were obtained on wounds performed on mice heads [START_REF] Chiang | Enhancing skin wound healing by direct intracellular ATP delivery[END_REF] and on ischemic ears of normal [START_REF] Howard | Rapid Granulation Tissue Regeneration by Intracellular ATP Delivery-A Comparison with Regranex[END_REF][START_REF] Wang | Intracellular Adenosine Triphosphate Delivery Enhanced Skin Wound Healing in Rabbits[END_REF] or diabetic [START_REF] Wang | Intracellular delivery of adenosine triphosphate enhanced healing process in full-thickness skin wounds in diabetic rabbits[END_REF] rabbits. A very special finding was the discovery of a completely new healing process, with extremely fast granulation attributed to massive macrophage migration, their in situ proliferation and direct collagen production [START_REF] Howard | Rapid Granulation Tissue Regeneration by Intracellular ATP Delivery-A Comparison with Regranex[END_REF][START_REF] Sarojini | Rapid tissue regeneration induced by intracellular ATP delivery-A preliminary mechanistic study[END_REF][START_REF] Wang | Intracellular delivery of adenosine triphosphate enhanced healing process in full-thickness skin wounds in diabetic rabbits[END_REF][START_REF] Wang | Intracellular Adenosine Triphosphate Delivery Enhanced Skin Wound Healing in Rabbits[END_REF]. On a different therapeutic area, using a model of hemorrhagic shock on rats, these ATP-liposomes also improved survival when injected during resuscitation [START_REF] Zakaria | Direct energy delivery improves tissue perfusion after resuscitated shock[END_REF][START_REF] Zakaria | Improved Post-Resuscitation Survival Time with Adjuvant Cytosolic Energy Replenishment in a Murine Model of Hemorrhagic Refractory Shock[END_REF]. ATPv liposomes are currently sold as holding solutions for hair transplants. Clinical trials on other applications are eagerly awaited.

In a completely different mindset, ATP-liposomes have also been used as a helper system for ATP-responsive nanocarriers. In this strategy, the delivery of an anticancer drug by fusogenic liposomes containing ATP-responsive DNA scaffold was enhanced by the coadministration of ATP-liposomes [START_REF] Mo | Enhanced Anticancer Efficacy by ATP-Mediated Liposomal Drug Delivery[END_REF].

All these studies show that there are still interesting perspectives in ATP-liposomes and ATP-vesicles development, particularly by tuning the cargo composition with other energetic molecules, by decorating the carrier surface with specific peptides or antibodies for enhanced targeting, and by finding new areas of applications. A better understanding of the ATPliposomes mechanism of action may help move this technology to the clinic. In particular, it would be interesting to confirm whether ATP-liposomes act only by delivering intracellular ATP or if this ATP delivery leads to adenosine extracellular delivery [START_REF] Erga | Role of A2A-adenosine receptor activation for ATP-mediated coronary vasodilation in guinea-pig isolated heart[END_REF][START_REF] Maldonado | Administration of exogenous adenosine triphosphate to ischemic skeletal muscle induces an energy-sparing effect: Role of adenosine receptors[END_REF] as in the case of ADP-liposomes [START_REF] Hagisawa | Fibrinogen γ-Chain Peptide-Coated Adenosine 5' Diphosphate-Encapsulated Liposomes Rescue Mice From Lethal Blast Lung Injury via Adenosine Signaling[END_REF].

II. Adenosine-lipid conjugates or nucleolipids

Nucleolipids are composite molecules formed by the assembly of a lipidic component and a nucleobase, nucleoside or nucleotide.

1) General synthetic strategies for adenosine nucleolipids

Different chemical approaches can be employed to obtain artificial adenosine nucleolipids. A commonly employed strategy was first proposed by Shuto and colleagues in 1987 [START_REF] Shuto | A facile one-step synthesis of 5′phosphatidylnucleosides by an enzymatic two-phase reaction[END_REF] for obtaining 5'-phosphatidyl-nucleosides (Figure 4A). This method's popularity arises from its simplicity: a single-step enzymatic reaction in a two-phase system (chloroform and buffer) is necessary, in which phospholipase D catalyzes the transfer reaction of the phosphatidyl residue to the primary hydroxyl group of nucleosides. One of the first artificial adenosine nucleolipid, l,2-dipalmitoyl-sn-glycero-3-phosphatidyl-adenosine (DPPA), was synthetized by this method with a yield of 52 %. A few years later, Itojima et al. made use of this technique for creating a library of four 5'-phosphatidyl-adenosines with different alkyl chain lengths [START_REF] Itojima | Spontaneous formation of helical structures from phospholipid-nucleoside conjugates[END_REF].

Other organic strategies consist of activating either the adenosine monophosphate or the lipid moiety before coupling them. For example, 5'-phosphatidyl-adenosines can also be obtained by condensation of fatty alcohols with AMP activated by N,N'dicyclohexylcarbodiimide [10,[START_REF] Shiraishi | Selective inhibition of lignoceroyl-CoA synthetase by adenosine 5′alkylphosphates[END_REF] or by reaction of AMP tetrabutylammonium salt with alkyl bromides [START_REF] Machida | Inhibitory effects of nucleoside 5′-alkylphosphates on sexual agglutination in Saccharomyces cerevisiae[END_REF]. A completely different strategy was recently introduced by Ceglie's team using micellar catalysis. Practically, the 1,2-epoxydodecane was coupled to 5'-phosphoribonucleotide in an aqueous cationic microemulsion. The cationic interface favored the coupling between the water insoluble epoxide and the anionic nucleotides [START_REF] Angelico | Catanionic Systems from Conversion of Nucleotides into Nucleo-Lipids[END_REF].

2) Adenosine nucleolipids self-assembly

The attraction for nucleolipids in general (and adenosine nucleolipids in particular) has increased with the discovery of their capacity to form macro-or nanostructures. This unique property arises from both members of the couple: lipids have self-association ability and are known to organize in membrane layers and other structures such as liposomes, while nucleoside have base-pairing properties via hydrogen bonding or π-stacking.

Ahlers et al. were the first to explore this intriguing aspect. They compared single-chain or double-chain adenosine lipid monolayers and found that, to form stable structures, a balance had to be found between the lipophilicity procured by the alkyl chain and the size of the headgroup. Indeed, single-chain adenosine lipid did not form stable monolayers, while single-

A B C D

chain adenine and double-chain adenosine did [START_REF] Ahlers | Orientation, recognition, and photoreaction of nucleolipids in model membranes[END_REF]. Some of these adenosine nucleolipids were even found to form various helical structures, with an impact of the alkyl chain length on the efficacy of the formation of these supramolecular assemblies [START_REF] Itojima | Spontaneous formation of helical structures from phospholipid-nucleoside conjugates[END_REF]. It was also reported that the pH of the solution greatly affected the structure: as an example, 1,2-dimyristoyl-sn-glycero-3phosphatidyl-adenosine (DMPA) formed multihelical strands in alkaline conditions, while it formed tubular cigar-like scrolls in acidic conditions (Figure ).

Since these early explorations, adenosine nucleolipids have been described to form many different structures including micelles and liposomes (Table 2), depending on the conjugated lipid and the buffer conditions. Compared to other nucleolipids, adenosine lipids possess some special properties. In addition to forming hydrogen bonds, adenosine, like other purines, has a high stacking tendency (Table 3), which leads to a negative Gibbs free energy for adenosine-adenosine association in aqueous solution [START_REF] Milani | Structural Investigation of Bilayers Formed by 1-Palmitoyl-2-Oleoylphosphatidylnucleosides[END_REF]. These specificities affect several structural parameters of adenosine lipid self-assemblies, thus contributing to the final supramolecular structure.

Table 3 Computed RNA nucleoside pairs stacking free energies [START_REF] Brown | Stacking free energies of all DNA and RNA nucleoside pairs and dinucleoside-monophosphates computed using recently revised AMBER parameters and compared with experiment[END_REF] Indeed, when comparing l,2-dilauroyl-sn-glycero-3-phosphatidyl-adenosine (DLPA) and l,2-dilauroyl-sn-glycero-3-phosphatidyl-uridine (DLPU) wormlike aggregates, Bombelli et al.

reported very different behaviors upon aging: while DLPU structures remained identical, DLPA aggregates self-assembled into giant twisted helicoidal aggregates [START_REF] Bombelli | Light Scattering and Cryo-Transmission Electron Microscopy Investigation of the Self-Assembling Behavior of Di-C12P-Nucleosides in Solution[END_REF]. The authors attributed this dissimilarity to higher association stacking constants of adenosine compared to uridine.

This property also induced tighter nucleolipid arrangement in micelles in the case of 1,2dioctanoyl-sn-glycero-3-phosphatidyl-adenosine (diC8PA) compared to 1,2-dioctanoyl-snglycero-3-phosphatidyl-uridine (diC8PU) [67]. Yet, this is not the case with all adenosine nucleolipids, as 2′,3′-O-16-hentriacontanyliden-adenosine-5′-phosphocholine (PAPC), for example, displayed a larger surface area per lipid compared to its equivalent 2′,3′-O-16hentriacontanyliden-uridine-5′-phosphocholine (PUPC) [START_REF] Taib | Chemical Details on Nucleolipid Supramolecular Architecture: Molecular Modeling and Physicochemical Studies[END_REF].

Other phenomena attributed to adenosine high base-stacking potential were observed on several occasions by Baglioni's team with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyladenosine (POPA) structures. First, anhydrous POPA bilayers displayed a powder-like nature, which could be explained by a specific orientation of the stacked adenosine moieties, compared to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-uridine (POPU) [START_REF] Milani | Structural Investigation of Bilayers Formed by 1-Palmitoyl-2-Oleoylphosphatidylnucleosides[END_REF] (Figure 6). Secondly, POPA/POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) bilayers exhibited the presence of POPA-enriched microdomains, due to partial demixing of POPA and POPC [START_REF] Milani | Intercalation of single-strand oligonucleotides between nucleolipid anionic membranes: a neutron diffraction study[END_REF]. The adenosine stacking interactions responsible for this behavior could be detected by hypochromism [START_REF] Montis | Complexes of nucleolipid liposomes with single-stranded and double-stranded nucleic acids[END_REF]. Finally, POPA/POPC vesicles were found to be more stable than the equivalent POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-glycerol)/POPC, where the RNA nucleoside pair AA GG CC UU ΔGstack (kcal/mol) -2.41 -2.77 -1.76 -1.51 adenosine polar head was replaced by a glycerol head group bearing the same charge.

According to the authors, only the base stacking interactions could explain this behavior [START_REF] Montis | Nucleolipid bilayers: A quartz crystal microbalance and neutron reflectometry study[END_REF].

Mixing adenosine nucleolipids with uridine nucleolipids can also lead to original supramolecular architectures. Indeed, besides π-stacking, the presence of a nucleoside headgroup on a nucleolipid can also drive molecular recognition through hydrogen bonding with the complementary nucleoside on another nucleolipid. A very striking example of this phenomenon was presented by Moreau et al. by mixing PAPC and PUPC. While individual nucleolipids formed large lamellar structures, mixed nucleolipids self-assembled into small vesicles which could be visualized in real-time at the micrometer scale [START_REF] Moreau | Real Time Imaging of Supramolecular Assembly Formation via Programmed Nucleolipid Recognition[END_REF]. A similar phenomenon could be observed at the macroscopic scale between 2',3'-O-18pentatriacontanyliden-adenosine-5'-phosphocholine (SAPC) and 2',3'-O-18pentatriacontanyliden-uridine-5'-phosphocholine (SUPC): when separate, these two nucleolipids form hydrogels and fluid lamellar phases above a certain temperature. When mixed, however, they form a new stable combined supramolecular system, which forms a precipitate upon heating [START_REF] Taib | Formation of supramolecular systems via directed Nucleoside-Lipid recognition[END_REF].

3) Adenosine nucleolipids as delivery systems a. Adenosine nucleolipids lipoplexes for gene delivery

As presented above, adenosine headgroups on nucleolipids can lead to molecular recognition with complimentary nucleolipids bearing uridine headgroups. Interestingly, this phenomenon of molecular recognition was also described between adenosine headgroups on nucleolipids and nucleic acids such as RNA or DNA. As a result, self-assembled adenosine nucleolipids may be considered as promising platforms for gene delivery [START_REF] Allain | Self-assembled nucleolipids: from supramolecular structure to soft nucleic acid and drug delivery devices[END_REF]. A great advantage of this system is that it relies on specific Watson-Crick pairings (hydrogen bonding and π-stacking) between nucleolipids and polynucleotides, offering an interesting alternative to other non-viral technologies for gene deliveries which are based on aspecific electrostatic interactions [START_REF] Al-Dosari | Nonviral Gene Delivery: Principle, Limitations, and Recent Progress[END_REF].

It was discovered by Baglioni's team that when adenosine nucleolipid and polyuridilic acid (polyU) were brought together, the formation of nucleolipoplexes occurred without the presence of divalent cations (Figure 7). The proof-of-concept was obtained with diC8PA globular micelles decorated with single-stranded complimentary RNA: this association resulted in a polynucleotide-micelle adduct that could be characterized by SAXS as a hexagonal phase (Figure 7A) [START_REF] Banchelli | Molecular Recognition Drives Oligonucleotide Binding to Nucleolipid Self-Assemblies[END_REF]. Other supramolecular structures were obtained with adenosine derivatives bearing a longer lipidic chain. Indeed, POPA bilayers swollen with a polyU-containing buffer exhibited an increase in their smectic period due to the appearance of a 1D lattice of polynucleotides between the POPA lamellar stacks (Figure 7B) [START_REF] Milani | Nucleolipoplexes: A New Paradigm for Phospholipid Bilayer-Nucleic Acid Interactions[END_REF]. The formation of these nucleolipoplexes was specific to the presence of adenosine as a functional headgroup leading to selective molecular recognition of nucleic acids. For example, POPG [START_REF] Montis | Complexes of nucleolipid liposomes with single-stranded and double-stranded nucleic acids[END_REF][START_REF] Montis | Nucleolipid bilayers: A quartz crystal microbalance and neutron reflectometry study[END_REF] and POPU were shown to be less able than POPA to associate with polynucleotides [START_REF] Milani | Association of polynucleotides with nucleolipid bilayers driven by molecular recognition[END_REF].

Despite these interesting observations, the in vitro and in vivo proofs of concept for using these nucleolipoplexes as gene delivery systems remain extremely limited. In particular, enhanced transfection efficacies at high lipid concentrations were obtained for two uridine nucleolipids compared to conventional lipid agents (DOTAP, TransFast TM and Lipofectamine® 2000) [START_REF] Chabaud | Cationic nucleoside lipids for gene delivery[END_REF][START_REF] Moreau | Nucleoside phosphocholine amphiphile for in vitro DNA transfection[END_REF], but so far, no study has been published regarding adenosine nucleolipids for gene delivery on pre-clinical models and no data are available in the literature regarding their transfection efficacy.

The most advanced study regarding the capacity of adenosine nucleolipid to deliver DNA acids for gene delivery. However, this association is only driven by electrostatic interaction, which is not very specific and could happen with any other negatively charged surface or molecule. In addition, due to their positive charge, PAMAM dendrimers exhibited some toxicity.

In that respect, as alternative, non-covalent association of anionic DLPA is a simple way of giving PAMAM dendrimers molecular recognition ability for specific association with nucleic acids [START_REF] Arteta | Molecular recognition of nucleic acids by nucleolipid/dendrimer surface complexes[END_REF].

As seen so far, this field of research tends to exclusively see adenosine nucleolipids as a new material for gene delivery. However, it seems to omit that adenosine itself is more than a polar head with base recognition ability and could be worth delivering for itself.

b. Adenosine nucleolipids nanoparticles for adenosine delivery

Other nucleolipids than adenosine ones are sometimes considered prodrugs, where the lipid moiety positively affects the drug biodistribution and leads to lowering toxicity and increasing pharmacological efficacy (e.g., anticancer or antiviral agents). Surprisingly, although adenosine is often acclaimed for its potential as therapeutic molecule whereas the main limitation of this molecule lies in its hydrophilicity and short half-life in vivo, adenosine nucleolipids have rarely been considered with the aim of delivering adenosine itself [13,14].

Squalene-adenosine (SQAd) is, to our knowledge, the only example of currently developed adenosine nucleolipid prodrug. The so-called "squalenoylation" technology consists in linking a derivative of squalene -a natural, biocompatible and biodegradable triterpene -to a hydrophilic active compound. The resulting bioconjugate showed the unique ability to selfassemble into stable nanoparticles (NPs) [START_REF] Couvreur | Squalenoyl Nanomedicines as Potential Therapeutics[END_REF].

In the case of SQAd, two slightly different compounds were studied by Couvreur's team:

a SQAd bioconjugate with a short squalene-based chain constituted of 5 isoprene units [START_REF] Lepeltier | Self-Assembly of Squalene-Based Nucleolipids: Relating the Chemical Structure of the Bioconjugates to the Architecture of the Nanoparticles[END_REF] and a SQAd bioconjugate with a longer squalene-based chain constituted of 6 isoprene units [START_REF] Gaudin | Squalenoyl adenosine nanoparticles provide neuroprotection after stroke and spinal cord injury[END_REF].

Both compounds were found to self-assemble as NPs into an aqueous phase after nanoprecipitation (consisting in the addition of an ethanolic solution of SQAd into a water phase). The SQAd bioconjugate with 5 isoprene units was shown to form sponge-like macrostructures [START_REF] Lepeltier | Self-Assembly of Squalene-Based Nucleolipids: Relating the Chemical Structure of the Bioconjugates to the Architecture of the Nanoparticles[END_REF], while the SQAd with 6 isoprene units self-assembled into lamellar or hexagonal phase NPs, depending on the water-to-ethanol ratio during the nanoprecipitation process [START_REF] Lepeltier | Influence of the nanoprecipitation conditions on the supramolecular structure of squalenoyled nanoparticles[END_REF].

The SQAd NPs with 6 isoprene units were used for in vivo pharmacological evaluation.

Interestingly, the so-called "squalenoylation" of adenosine and its self-assembly into NPs dramatically affected adenosine pharmacokinetics and biodistribution in mice [START_REF] Gaudin | Pharmacokinetics, biodistribution and metabolism of squalenoyl adenosine nanoparticles in mice using dual radio-labeling and radio-HPLC analysis[END_REF].

Importantly, adenosine plasma half-life was significantly increased after intravenous administration of SQAd NP, compared to adenosine injected free. Even though SQAd NPs did not show the ability to cross the blood-brain barrier, they displayed high neuroprotective pharmacological effect on cerebral ischemia in mice, as well as on spinal cord injury in rats [START_REF] Gaudin | Squalenoyl adenosine nanoparticles provide neuroprotection after stroke and spinal cord injury[END_REF].

Of note, free adenosine did not display any therapeutic activity in these models. Importantly, a complete toxicological study demonstrated the perfect innocuity of SQAd NPs in mice [START_REF] Gaudin | Squalenoyl adenosine nanoparticles provide neuroprotection after stroke and spinal cord injury[END_REF].

Nevertheless, disposing of an easy-to-handle formulation would be a step further in the preclinical development of this system.

On the other hand, the exact mechanism of action of SQAd NPs urgently deserves a deeper investigation, both at cellular and molecular levels. It was already shown that, after intravenous administration, the SQAd NPs disassembled before the bioconjugate inserted into low-density lipoproteins (LDLs), which explained the prolonged circulation time of SQAd in the blood [START_REF] Sobot | Conjugation of squalene to gemcitabine as unique approach exploiting endogenous lipoproteins for drug delivery[END_REF]. The LDLs loaded with SQAd were further observed to be cell internalized through LDL receptor-dependent pathway [START_REF] Gaudin | Transport Mechanisms of Squalenoyl-Adenosine Nanoparticles Across the Blood-Brain Barrier[END_REF]. But several questions remain: does the nanoparticle or SQAd bioconjugate itself act directly on adenosine receptors before internalization or is there a release in the extracellular space of free adenosine which bind to these receptors? If the second hypothesis is confirmed, how and where does this release happens? A better understanding would help to develop this promising technology further and extend its use to other therapeutic areas.

III. Conclusion

Adenosine and lipids form a fertile couple with many different offspring. ATP-liposomes seem to be the most advanced technology in terms of clinical applications, even though they are far from being FDA-approved yet and they are only sold as cosmetics. Covalent conjugation between adenosine and lipids generated completely unexpected materials with self-assembly abilities, but they probably still need several years to mature as gene delivery systems. Use of these adenosine nucleolipids as adenosine delivery systems may be an elegant way of developing this technology faster towards clinical applications, as the example of squaleneadenosine nanoparticles shows.

Bringing nanomedicines into the clinic is generally challenging [START_REF] Ragelle | Nanoparticle-based drug delivery systems: a commercial and regulatory outlook as the field matures[END_REF], especially because they are generally developed at the pre-clinical stage in academic laboratories and in an environment far from the conditions for the pharmaceutical development rules. Most works cited in this review lack a comprehensive approach, with a rigorous reflection on chemistry, manufacturing and controls as well as thorough in vitro and in vivo evaluation. Extremely basic characterization such as size, size distribution, surface characterization, drug loading and drug encapsulation are missing in most studies, while animal testing, when mentioned, generally focuses only on efficacy without describing detailed pharmacology and toxicology issues.

Another difficulty comes from the fact that both adenosine and nanomedicines may be considered as risky bets from an industrial point of view. Indeed, the development of adenosine agonists and antagonists has often been synonymous with important clinical failures so far [START_REF] Massie | Rolofylline, an Adenosine A1-Receptor Antagonist, in Acute Heart Failure[END_REF],

creating some misgivings from investors, while the development of nanomedicines was still at an early stage some decades ago. However, as the first generation of nanomedicines has been successfully commercialized for more than 20 years now, industrials may become less reluctant to invest on these better-known technologies, under the condition that they can rely on solid preclinical studies. Hence, it is now time for them to fully exploit adenosine's therapeutic potential.

Chapitre 1

Squalene-adenosine nanoparticles: ligands of adenosine receptors or adenosine prodrug?

Marie Rouquette a , Sinda Lepetre-Mouelhi a , Ophélie Dufrançais a , Xue Yang b , Julie In the present study, two hypotheses were discussed: (i) either SQAd bioconjugates, which constitute the NPs building blocks, act directly as ARs ligands, or (ii) adenosine, once released from intracellularly processed SQAd NPs, interacts with these receptors. The first hypothesis was rejected, using radioligand displacement assays, as no binding to human ARs was detected up to 100 µM of SQAd, in the presence of plasma. Hence, the second hypothesis was examined. SQAd NPs uptake by HepG2 cells, which was followed using radioactive and fluorescence tagging, was found to be independent of equilibrative nucleoside transporters but rather mediated by low-density lipoproteins receptors.

Interestingly, it was observed that after cell internalization, SQAd NPs operated as an intracellular reservoir of adenosine, followed by a sustained release of the nucleoside in the extracellular medium. This resulted in a final paracrine-like activation of ARs pathway, evidenced by fluctuations of the second messenger cAMP. This deeper understanding of SQAd NPs mechanism of action provides now strong rational for extending the pharmaceutical use of this nanoformulation.

I. Introduction

Adenosine is an endogenous purine acting on four different G protein-coupled receptors (GPCRs) identified as A1, A2A, A2B and A3. The wide distribution of these adenosine receptors (ARs) is associated with a great diversity of pathophysiological effects, including regulation of cardiovascular, nervous and immune systems (Fredholm et al., 2011). Although considerable Currently, the so-called "squalenoylation" approach may represent a promising technological platform for delivering hydrophilic compounds with stability issues or off-target toxicity [START_REF] Couvreur | Squalenoyl nanomedicines as potential therapeutics[END_REF]. This methodology consists of the conjugation of a fragile and quickly metabolized drug molecule to the squalene, a natural and biocompatible lipid.

Interestingly, the resulting drug-squalene bioconjugates have the unique ability to selfassemble in water as nanoparticles (NPs). It was found that these squalene-based NPs favorably modified the in vivo pharmacokinetics and biodistribution of the conjugated drug molecules [START_REF] Reddy | A new nanomedicine of gemcitabine displays enhanced anticancer activity in sensitive and resistant leukemia types[END_REF]. In particular, the delivery of adenosine via squalene-adenosine (SQAd) NPs showed an impressive neuroprotective efficacy in brain ischemia in mice and in spinal cord injury in rats (Gaudin et al., 2014). This effect was explained by a prolonged circulation of SQAd NPs into the bloodstream compared to free adenosine (Gaudin et al., 2015), and appeared to originate from the interaction of SQAd NPs with the brain endothelial cells. However, the nature of the interaction between SQAd NPs and cells still remains totally unknown.

Thus, the current study aims at elucidating the mechanism of action of SQAd NPs in vitro.

Two hypotheses were tested: (i) either the SQAd NPs or SQAd bioconjugates would act as direct ARs agonists (or antagonists) by binding them via their adenosine moiety; or (ii) they would function as prodrugs, by slowly releasing adenosine intracellularly and into the extracellular space.

The first hypothesis was checked by radioligand displacement assays on membrane fractions of cells overexpressing human ARs. The second hypothesis was tested on the HepG2 cell line, a well-characterized in vitro human hepatocyte model. Liver cells were chosen for this study as it has previously been shown that SQAd NPs accumulate in high quantities in the liver after their intravenous injection in mice (Gaudin et al., 2015). The cell capture of SQAd NPs was followed by radiolabeling and fluorescent labeling, while adenosine release was determined by high-performance liquid chromatography (HPLC). Finally, the activation of the adenosine receptors was also evaluated. All other chemicals were of analytical grade and obtained from standard commercial sources.

II. Materials and methods

1) Materials

2) SQAd NPs preparation

SQAd NPs were prepared as previously described (Gaudin et al., 2014). Briefly, 333 µL of an ethanolic solution of 6 mg/mL SQAd was added dropwise into 1 mL of a solution of 5 wt. % dextrose under stirring. Ethanol was then removed by evaporation using a Rotavapor® (Buchi, Rungis, France) to obtain a final concentration of 2 mg/mL SQAd NPs. Radiolabeled NPs were obtained following the same procedure, with the addition of SQ- The mean NPs size and polydispersity index (PdI) were checked from 1 mg/mL suspensions by dynamic light scattering (DLS) using a Zetasizer Nano ZS (173° scattering angle, 25 °C) (Malvern, UK).

3) Radioligand displacement assay

CHO or HEK293 cells stably expressing the human A1, A2A, A2B or A3 ARs were cultured and membranes were prepared as previously described (Heitman et 1). Incubations were terminated by dilution with ice-cold assay buffer, immediately followed by separation of bound from free radioligand by rapid filtration through 96-well GF/B filter plates using a Perkin Elmer Filtermate-harvester (PerkinElmer, Groningen, Netherlands). Filters were subsequently washed three times with 2 mL of ice-cold washing buffer (Supplementary Table 1). The filter-bound radioactivity was determined by scintillation spectrometry using a P-E 1450 Microbeta Wallac Trilux scintillation counter (PerkinElmer). IC50 values were obtained by fitting the curves with a one-site binding model using GraphPad Prism 7.00 software. Ki values were deduced using the Cheng-Prusoff equation [START_REF] Cheng | Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction[END_REF].

4) Cell culture

HepG2 cells were cultured in DMEM high glucose medium supplemented with 10 % FBS, penicillin (50 U/mL) and streptomycin (50 µg/mL). If not stated otherwise, cells were plated Chapitre 1 53 on 24-well plates at a density of 200 000 cells per well and left to adhere overnight before any further procedure.

hCMEC/D3 cells were cultured in EBM-2 medium supplemented with 5% FBS, penicillin (50 U/mL) and streptomycin (50 µg/mL), 1.4 μM hydrocortisone, 5 μg/mL ascorbic acid, 1%

Chemically Defined Lipid Concentrate, 10 mM HEPES and 1 ng/mL bFGF. Cells were used between passages 4 and 8. Cells were plated on 24-well plates at a density of 100 000 cells per well and left to adhere overnight before any further procedure.

All cells were maintained at 37 °C, 5 % CO2, 100 % humidity. Cells were passaged twice a week.

5) Cell uptake of radiolabeled SQAd NPs and free adenosine

HepG2 cells were treated with radiolabeled SQAd NPs or radiolabeled free adenosine at a concentration of 10 µM and 0.1 µCi/mL. At different time points, cells were washed twice with PBS and detached with trypsin before been pelleted and resuspended in PBS. Samples radioactivity was measured using a β-scintillation counter (Beckman Coulter LS6500) after overnight incubation with 1 mL Soluene-350 at 50 °C and addition of 10 mL Hionic Fluor.

6) Cell uptake of fluorescently labeled SQAd NPs

For confocal microscopy imaging, HepG2 cells were plated onto sterile glass coverslips placed in 24-well plates, at a density of 50 000 cells per well, and left to adhere for 48 h. Cells were incubated with fluorescently labeled SQAd NPs (10 µg/mL) for 2 h at 37 °C. After several washing steps with PBS, membranes were stained for 20 min with 10 µg/mL of WGA Alexa Fluor 555. Cells were then washed again and fixed with 4 % paraformaldehyde for 15 min. After final washing, coverslips were mounted on slides using Vectashield® mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Labs) and sealed with nail polish. Images were acquired using a Leica TCS SP8 system, with a Leica HC Plan Apochromat 63×/1.4NO oil CS2 objective lens.

For studying the role of adenosine transporters, HepG2 cells were treated with fluorescently labeled SQAd NPs (10 µg/mL) and dipyridamole (20 µM). For studying the role of low-density lipoproteins receptors (LDLRs) in SQAd NPs cell uptake, 150 000 cells were plated by well. Cells were washed with PBS and medium was replaced with normal medium or

9) HPLC conditions

A Water™ 717 plus autosampler was employed equipped with a Waters™ 1525 binary HPLC pump, a Waters™ 2487 dual λ absorbance detector, and a Waters™ Breeze software. The analysis was performed using a Halo C18 column (4.6 x 250 mm, 5 µm; Interchim, France). The injection volume was 40 µL to 50 µL, the elution was carried out at 0.8 mL/min isocratically for 10 min with the column temperature maintained at 30 °C. Detection wavelengths were respectively 273 nm for SQAd and 260 nm for adenosine.

10) Intracellular cAMP levels

The day before the experiment, two plates were seeded with HepG2 cells: a 24-well plate with 200 000 cells per well and a 96-well plate with 5 000 cells per well.

On the day of the experiment, cells in the 24-well plate were treated with SQAd NPs, free adenosine or dextrose as previously described. After 2 h, extracellular medium was replaced with simplified medium containing 50 µM EHNA and broad-range phosphatase inhibitors (500 µM IBMX and 100 µM Ro 20-1724). After 1 h, 10 µL of this extracellular medium was used to stimulate cells plated in the 96-well plate for 15 min. cAMP intracellular levels were then determined using the cAMP-Glo TM assay from Promega. Briefly, cells were lysed with 10 µL cAMP-Glo™ Lysis Buffer for 15 min with shaking at room temperature. cAMP-Glo™ Detection Solution and Kinase-Glo Reagent were then added to all wells according to the manufacturer's protocol. Luminescence was detected using an EnSpire Alpha 2390 plate reader (Perkin-Elmer, USA).

III. Results

1) SQAd NPs

Prepared by nanoprecipitation, SQAd NPs suspensions were monodisperse, displaying a hydrodynamic diameter of 71 ± 15 nm, a PdI of 0.08 ± 0.02 and a drug loading of 37%.

2) SQAd interaction with human adenosine receptors

The direct interaction of SQAd with human ARs was checked by radioligand displacement assays. Specific radioligands were used for each adenosine receptor and their displacement was measured in the presence of increasing concentrations of SQAd bioconjugate or NPs. Results are shown in Figure 1 .

Up to 1 µM, SQAd showed no interaction with any of the ARs in the assay buffer, whether as a bioconjugate or assembled as NPs (Figure 1 Prusoff equation, the magnitude of SQAd (bioconjugate or NPs) affinity for each of these receptors was determined. Results are given in Table 1.

Table 1 SQAd bioconjugate and SQAd NPs pKi magnitude for each adenosine receptor.

The pKi values were determined from the radioligand displacement curves using the Cheng-Prusoff equation.

To be as close as possible to the in vitro and in vivo conditions, SQAd bioconjugates and SQAd NPs were also preincubated for 4 h at 37 °C with FBS, prior to performing the radioligand displacement assay. It should be noted that the interaction with 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, no interaction with SQAd bioconjugate or NPs could be detected anymore, even at the highest concentration (Figure 1 C and Figure 1 D). Hence, it was concluded that the preincubation with serum prevented interaction of SQAd with adenosine receptors.

3) SQAd NPs internalization

To follow the cell uptake of SQAd NPs, radiolabeled NPs were prepared by co-nanoprecipitation of SQAd and SQ-[ Both adenosine and SQAd NPs were internalized by HepG2 cells. However, the cell uptake of SQAd NPs occurred slower and to a lower extend than free adenosine. Indeed, after 30 min incubation, the intracellular content in adenosine (and its tritiated metabolites) was 13times higher than with SQAd (and its metabolites). After 24 h, however, the adenosine over SQAd ratio was reduced to a value of only 3.

For further experiments, fluorescently labeled SQAd NPs (diameter of 60.8 ± 0.4 nm, PdI of 0.13 ± 0.01) were prepared by co-nanoprecipitation of SQAd with a fluorescent BODIPYcholesteryl ester dye. Fluorescent nanoparticles maximum excitation and emission wavelengths were respectively of 484 nm and 520 nm (Supplementary Figure 1). NPs uptake by HepG2 cells was visualized by confocal microscopy and measured by flow cytometry under various conditions (Figure 3 ). The fluorescently labeled SQAd NPs were found to be taken up by HepG2 cells (2h incubation) (Figure 3 A), while no fluorescence was visible when the cells were incubated with the fluorescent probe alone (Figure 3 B). Hence, the intracellular fluorescence was considered to correlate well with SQAd NPs uptake.

Then, considering that adenosine enters the cells via equilibrative nucleoside transporters (ENTs) [START_REF] Baldwin | The equilibrative nucleoside transporter family, SLC29[END_REF], the cellular mechanism of uptake of SQAd NPs was also investigated using HepG2 cells treated with dipyridamole, an ENT inhibitor. After 30 min or 2 h of incubation with fluorescently labeled NPs, no difference in intracellular fluorescence was observed between cells pre-incubated with or without dipyridamole (Figure 3 C), which clearly indicated that SQAd NPs did not enter the cells via ENTs. The role of LDLRs was then evaluated by tuning their level of expression on HepG2 cells.

Cells were preincubated for 24 h with FBS-supplemented medium (containing normal level of lipoproteins), and LPDS-supplemented medium (containing low levels of lipoproteins). Cell starvation using LPDS induced an over-expression of LDLRs, as evident from Western Blot (Supplementary Figure 2). Noteworthy, in LPDS-supplemented medium, cells were found to internalize higher levels of fluorescently labeled SQAd NPs (Figure 3

4) SQAd degradation in acidic conditions

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. The percentage of intact prodrug under these conditions is shown in Figure 4 A.

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 in acidic conditions. The presence of cathepsin B did not influence the degradation profile over 24 h. Interestingly, the degradation of SQAd led to the release of free adenosine (retention time 3.9 min), which was characterized by HPLC analysis (Figure 4 B).

Hence, SQAd intracellular bioconversion into adenosine should increase the adenosine intracellular content. And, as a result of the equilibrium between the intracellular and the extracellular adenosine levels, it was hypothesized that some adenosine could be further released in the extracellular compartment.

5) Adenosine extracellular release

Thus, the concentration of adenosine released in the cell culture medium was then determined by HPLC in the presence of EHNA, an inhibitor of adenosine deaminase. After incubation of HepG2 (or hCMEC/D3) cells with SQAd NPs, free adenosine or 5 % dextrose solution, the extracellular medium was replaced by 1 mL of a new simplified medium (composition is given in Supplementary Table 2) to remove adenosine (eventually resulting from the release from SQAd NPs before their capture by the cells). After further 5 min to 4 h incubation, the extracellular medium was finally collected to determine the quantity of adenosine released from the cells after exposure to SQAd NPs or adenosine (Figure 5A).

After 5 min incubation, cells treated with free adenosine released higher concentrations of adenosine (around 600 nM) than cells treated with dextrose or SQAd NPs (100 nM to 200 nM). This clearly highlighted the almost instantaneous ability of the cells to uptake free adenosine, while SQAd NPs uptake and cleavage (which results in adenosine metabolite release) required more time (Figure 5A). Interestingly, after 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 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, hence producing a reservoir effect. Of note, very close results were obtained with hCMEC/D3 cells, a brain endothelial cell line derived from human temporal lobe microvessels (Supplementary Figure 3). To further study the intracellular reservoir effect of adenosine produced after incubation with SQAd NPs, cells were incubated for 2 h with free adenosine, SQAd NPs or dextrose and the kinetics of adenosine release from the cells into the extracellular medium was determined. Results are shown in Figure 5B. Interestingly, concentrations of released adenosine after incubation of the cells with dextrose or free adenosine did not increase over time, thus revealing that equilibrium was reached during the first minutes post incubation. On the contrary, when the cells were incubated with SQAd NPs, quantities of adenosine released by the cells increased over time. This clearly confirmed 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.

A remaining question was to know whether the released adenosine in the extracellular medium was capable of interaction with adenosine receptors on neighboring cells.

6) Adenosine receptors activation

Thus, adenosine receptors activation was evaluated by measuring the levels of intracellular cAMP. Indeed, adenosine receptors are G protein-coupled receptors (GPCR) known to activate or inhibit adenylate cyclase, hence tuning intracellular levels of cAMP. As in the previous set of experiments, HepG2 cells were incubated with dextrose, free adenosine or SQAd NPs for 2 h and then allowed to release adenosine during 1 h in their extracellular medium. This medium was then used as adenosine pool to investigate the interaction, through 15 min incubation, with other naïve cells by measuring their intracellular cAMP levels. As can be seen in Figure 6, the extracellular medium originating from cells treated with control vehicle (5 wt. % dextrose) or free adenosine did not induce major changes in intracellular cAMP levels. However, extracellular medium arising from cells treated with SQAd NPs induced a significant increase in cAMP, comparatively to vehicle. Interestingly, adenosine levels in extracellular medium originating from cells treated with SQAd NPs or free adenosine were very close under the conditions used (2 h of treatment and 1 h of release, cf. Figure 5A). Hence, adenosine might not be the only factor in the extracellular medium interacting with GPCRs.

IV. Discussion

This study aimed at clarifying the mechanism of action of SQAd NPs in vitro. The main question was to determine whether SQAd acted directly on ARs as a specific ligand, or indirectly as a prodrug releasing active adenosine.

By using radioligand displacement experiments, it was measured that SQAd as bioconjugate or as NPs displayed a rather weak affinity for human ARs, showing Ki superior to 10 -6 M for A3 and to 10 -5 M for all other ARs. As a matter of comparison, the native ligand adenosine has an affinity of around 10 -7 M for A1, A2A and A3 and 10 -6 M for A2B [START_REF] Yan | Adenosine receptor agonists: from basic medicinal chemistry to clinical development[END_REF].

More importantly, when SQAd bioconjugate or NPs were preincubated in serum to mimic the in vivo conditions, no more binding onto ARs could be detected. 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 (Sobot et al., 2017b). In silico simulations have confirmed that the interaction of the squalene-based bioconjugates with the LDL was driven by the squalene moiety, allowing the hydrophilic adenosine to insert deeply into the LDL hydrophobic core [START_REF] Yesylevskyy | Low-Density Lipoproteins and Human Serum Albumin as Carriers of Squalenoylated Drugs: Insights from Molecular Simulations[END_REF]. 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.

In order to assess the second hypothesis related to the progressive release of adenosine from SQAd NPs after cell capture, cultured hepatocytes were used. HepG2 cell line was chosen because SQAd NPs concentrated into the liver tissue after intravenous administration (Gaudin et al., 2015). It was observed that the cell capture of SQAd NPs was independent from the ENTs adenosine transporters. And as ENTs selectively mediate the bidirectional transport of nucleosides, nucleobases and nucleoside analogs (Boswell-Casteel and Hays, 2017), the presence of the squalene bulky moiety in SQAd was supposed to prevent the interaction of the adenosine fragment with ENTs. By contrast, the cell internalization of SQAd NPs was found to occur through the LDLRs.

As LDLRs are known to trigger the cell internalization via clathrin-dependent endocytosis [START_REF] Goldstein | Receptor-mediated endocytosis: concepts emerging from the LDL receptor system[END_REF], it was quite likely that SQAd followed the same pathway, thus ending up in endosomes and then lysosomes. This hypothesis is in perfect agreement with the confocal microscopy images obtained in this study (Figure 3 A). Once in the lysosomes, SQAd underwent a slow degradation under acidic conditions. Even though cathepsin B alone did not accelerate this proteolytic degradation under tested conditions, its combination with other enzymes like cathepsin D [START_REF] Couvreur | Squalenoyl nanomedicines as potential therapeutics[END_REF] and proteases may accelerate this phenomenon in vitro and in vivo. In addition, it was observed that the intracellular cleavage of SQAd into free adenosine resulted also in an extracellular release of adenosine, which gradually increased over time. In other words, SQAd behave as an intracellular reservoir, slowly releasing adenosine in the extracellular medium, probably via ENTs, since these transporters could act bidirectionally. Interestingly, the human brain endothelial hCMEC/D3 cell line showed similar adenosine extracellular release after SQAd NPs internalization which suggests that the interaction of SQAd NPs with HepG2 cells represents a more general trend occurring also in other cell types.

Finally, higher cAMP second messenger levels were observed in cells incubated with the extracellular medium originating from cells treated with SQAd NPs, which clearly indicated an increased activation of ARs. But since adenosine levels in the extracellular medium originating from cells treated with SQAd NPs or free adenosine were very close under the conditions used, adenosine might not be the only factor in the extracellular medium interacting with GPCRs.

In a nutshell, this study led to a better understanding of SQAd NPs mechanism of action as illustrated in

VI. Supplementary materials

I. Introduction

Adenosine is a ubiquitous purine nucleoside which is essential for cell metabolism. It plays many important biological roles intra and extracellularly. On one hand, intracellular adenosine is involved in nucleic acid and energy metabolism, as a major component of ATP. On the other hand, extracellular adenosine acts as a key intercellular signal by binding four widely expressed G-protein-coupled receptors: A1, A2A, A2B and A3. The pharmacological effects of adenosine are well recognized in diverse physiological functions including neurological, cardiovascular and immunological, as well as in pathological situations like inflammation, pain or cancer (1,2). In that respect, adenosine is sometimes described as a "multi-signalling guardian angel" (3).

However, the clinical use of adenosine has been hindered by its very-short plasmatic half-life (< 10 s). Considerable efforts have been devoted to therapeutically target adenosine signalling with stable agonists or antagonists but serious challenges remain, mostly due to the severe side effects observed after the systemic administration of these compounds (4,5).

Innovative drug delivery techniques offer a promising strategy for protecting adenosine from rapid metabolization and sustaining its release (6). The most promising strategy in terms of in vivo efficacy consists in conjugating adenosine to squalene (7), a human endogenous lipid.

This process results in the formation of an amphiphilic prodrug which can self-assemble into NPs in aqueous solutions after nanoprecipitation. These squalene-based NPs favourably modified the pharmacokinetic profile and biodistribution of adenosine (8), leading to dramatic neuroprotective effects on cerebral ischemia and spinal cord injury after intravenous administration, without producing any side effect (7).

If these impressive preclinical results suggest attractive prospects in clinical development, major obstacles remain. Squalene-adenosine (SQAd) NPs are formulated as liquid suspensions, which do not match industrial requirements in terms of transport and conservation. Indeed, NPs displayed a limited colloidal long-term stability, with NPs aggregation or fusion. In addition, nanoprecipitation requires appropriate equipment and know-how, restraining their direct preparation before administration.

Lyophilization is a common way to overcome these limitations prior to pharmaceutical distribution. The main goal of this method is to obtain a solid-state product as a "soft cake'' which can be easily redispersed when required. Such preparation has numerous advantages including easier manipulation, usually increased solubility in water and increased shelf-life of the product (9). This industrial process, also called lyophilization, consists in removing all water (or solvent) from a frozen suspension by sublimation under low pressure. But NPs will face several types of stress during freezing and dehydration, among which an increase in solutes local concentrations as pure water forms ice blocks and sublimes, and a mechanical stress due to ice crystallization and reduced pressure. The addition of cryo-and lyoprotectants (mainly sugars) usually protect NPs from these stresses by vitrifying the solution (10). However, surprisingly, the influence of freeze-drying on the NPs supramolecular organization, which may (11) or may not (12) be affected by removal of structural water, has rarely been discussed.

Thus, the present study aimed at investigating the physical, analytical and structural characterization of SQAd NPs prior and after lyophilization. The cryoprotective activity of four carbohydrates (dextrose, sucrose, trehalose and mannitol) at different concentrations was evaluated using dynamic light scattering techniques allowing to determine the mean particle size and size distribution before and after freeze-thawing. Best conditions were then used for freeze-drying the NPs. The influence of residual ethanol on the freeze-drying successful outcome was also studied. In order to evaluate NPs chemical and structural integrity, lyophilized samples were characterized respectively by nuclear magnetic resonance (NMR), atomic force microscopy (AFM) and small angle scattering. Furthermore, the long-term stability of the dried samples was evaluated after storage for up to three months. Finally, absence of toxicity after freeze-drying was checked in vitro on hepatocytes, as well as in vivo on mice.

II. Materials and methods

1) Materials

SQAd bioconjugate was provided by Holochem (Val-de-Reuil, France). D-(+)-glucose (≥ 99.5%), D-mannitol (≥ 98%), D-(+)-trehalose dihydrate (from Saccharomyces cerevisiae, ≥ 99%), sucrose (≥ 99.5%) and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Absolute ethanol (≥ 99.8%) originates from VWR Chemicals (Fontenay-sous-Bois, France). MilliQ ® water (resistivity 18.2 MΩ•cm) was produced by a water purification system (Millipore, Molsheim, France). Deuterium oxide (D2O) and

4) Freeze-thawing and freeze-drying experiments

NPs suspensions were mixed with 0.2 µm-filtered 10 wt. % carbohydrates solutions and water to obtain a final NPs concentration of 1 mg/mL and concentrations of sugars ranging from 0.5 wt. % to 5 wt. %. Thus, 500 μL were placed in 1.5 mL Eppendorf tubes and then frozen in a freezer (-30 °C, 24 h) or liquid nitrogen (-196 °C, 10 min). For freeze-thawing experiments, NPs were directly thawed after freezing step. For freeze-drying experiments, NPs were then dried overnight in an Alpha 2-4 LD plus freeze-dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Germany) (condenser < 70 °C, vacuum < 0.1 mbar). After lyophilization, samples were reconstituted by the addition of 500 µL MilliQ water and gently homogenized by pipetting.

Osmolality was measured by the freezing-point depression method on a micro-osmometer Autocal Type 13 (Roebling). For NMR analysis, SQAd was extracted from the lyophilisate using chloroform-d, and a drop of D2O was then added in the sample prior to analysis. 1 H spectrum was recorded on a Bruker Avance-300 spectrometer at 300 MHz.

5) Quantitative determination of residual ethanol

NPs suspensions were centrifuged (16,873 x g, 1 h, 4 °C). Ethanol concentrations (vol. %) in the supernatant were measured with the QuantiChrom TM Ethanol Assay Kit (BioAssay Systems, CA, USA), according to the manufacturer instructions. Absorbance at 570 nm was corrected with background absorbance at 650 nm.

6) Quantitative determination of residual moisture

Residual moisture was determined by the loss-on-drying technique. Briefly, clean annotated empty Eppendorf tubes were dried for over 30 min at 60 °C under vacuum (< 1 mbar), cooled down in a dry desiccator and weighted (mempty). Freeze-dried samples were then introduced in these empty Eppendorf tubes which were weighted again (mfull) and dried under the same conditions as before for over 3 h. Final weight (mdried) was determined.

Residual moisture was calculated as

𝑚 𝑓𝑢𝑙𝑙 -𝑚 𝑑𝑟𝑖𝑒𝑑 𝑚 𝑓𝑢𝑙𝑙 -𝑚 𝑒𝑚𝑝𝑡𝑦 .

7) Small-angle neutron scattering (SANS)

NPs were nanoprecipitated in D2O at 2 mg/mL or resuspended in D2O after freeze-drying (1 mg/mL, 5 wt. % trehalose). D2O was used to enhance the scattering contrast between hydrogenated nanoparticles and deuterated solvent and to minimize the incoherent background from hydrogen.

SANS experiments were performed at Institut Laue Langevin on D33 beamline (proposal number DOI: 10.5291/ILL-DATA.9-13-781). Samples were measured in Quartz optical Hellma cell of 1 mm path length thermalized at 25 °C with the thermostated sample holder available on the beamline. Four configurations were used to cover a q range from 1.5 x 10 -3 Å -1 to 0.473 Å -1 . Detector distances of 2 m, 5.3 m and 12 m with the collimation 2.8, 5.3, 7.8 and 12.8 were used for respectively small angle (SA) (λ = 13 Å), medium angle (MA) (λ = 6 Å), wide angle (WA) (λ = 6 Å) and large angle (LA) (λ = 6 Å). Absolute intensities were obtained after radial average of the raw data and classical normalization using the LAMP software.

8) Small-angle X-ray scattering (SAXS)

NPs with low residual ethanol content were prepared as previously described but concentrated to 5 mg/mL after evaporation. These SQAd NPs were freeze-dried at 2.5 mg/mL in 5 wt. % trehalose as previously stated and concentrated twice during resuspension, reaching a final concentration of 5 mg/mL SQAd NPs in 10 wt. % trehalose. NPs with high residual ethanol content were prepared from nanoprecipitation of 333 µL SQAd ethanolic solution at 12 mg/mL into 1 mL of water and evaporated to reach a final concentration of 5 mg/mL.

SAXS experiments were performed on the SWING beamline at the SOLEIL synchrotron (Saint-Aubin, France). We have checked that identical patterns were recorded if samples were loaded into quartz capillaries (diameter 1.5 mm, Glas Müller, Berlin, Germany) or flowed through a quartz capillary during beam exposure to avoid possible degradation under X-ray irradiation. Data were recorded at 12 keV in the scattering vector q-range 0.004 < q < 0.35 Å -1 , using a bi-dimensional Eiger detector. The typical exposure time was 10 s. The beamline software Foxtrot was used for data collection and processing. For all samples, the scattering intensity was normalized with respect to the incident beam intensity, acquisition time and sample transmission. The scattering intensity I(q) was reported as a function of the scattering vector q = 4  sin  / where 2 is the scattering angle and  the X-ray wavelength.

9) Atomic force microscopy (AFM)

Resuspended freeze-dried SQAd NPs were diluted to 100 µg/mL into water. 1 mL of this suspension was deposited on a piranha treated SiO2 wafer and left to adsorb for 1 h before being imaged by AFM with a Nanowizard 3 Ultra Speed from JPK Instruments (Berlin, Germany).

The instrument was installed on an air-buffered table coupled to a dynamic antivibration device and enclosed in an acoustic box. Imaging was performed in water in AC mode with silicon nitride cantilevers of 0.8 ± 0.05 N/m spring constant and 230 ± 5 kHz resonance frequency (Bruker), with a radius of curvature less than 20 nm. Free amplitude oscillations of 10 to 15 nm were chosen, allowing a very high resolution of the imaged surface. Images were taken at scan rates of 1 Hz. Image processing (flatten, plane fit, edge and hole detection) was performed with the JPK Data Processing software (JPK Instruments).

10) Cytotoxicity study

HepG2 cells were grown in DMEM high glucose supplemented with 10 % FBS, penicillin (50 U/mL) and streptomycin (50 µg/mL) at 37 °C, 5 % CO2, and divided to the sixth every 3-4 days.

For cytotoxicity measurements, cells were seeded in 96-well plates at a density of 10,000 cells (in 100 µL) per well. After 24 h, 100 µL of medium containing increasing concentrations of NPs were added to each well. Cellular activity was assessed after 72 h of treatment by adding 20 µL of MTT solution (5 mg/mL in DPBS) and incubating 1 h before removing medium and dissolving formazan crystals with 200 µL DMSO. Absorbance was recorded at 570 nm.

11) Hepatotoxicity study

All procedures were conducted in compliance with the European legislation on animal experimentation (approved protocols 2013_0713_10166734 and 2016_085_6085). Male Hsd:ICR(CD-1) mice were obtained from Envigo (Gannat, France). Upon arrival, animals were housed in groups of five and allowed seven days to acclimatize under conventional housing conditions (water and food ad libitum, 12-hour light-dark cycle, controlled temperature). A mouse model of hepatic diseased mice was used for NPs hepatotoxicity assessment. First, blood samples were collected from the submandibular vein and mice were allowed seven days to recover before further procedure. Then, animals were anesthetized with 2 % isoflurane and injected intravenously (lateral tail vein) with SQAd NPs (2 mg/mL, 10 wt. % trehalose, 10 mL/kg). A mouse model of liver ischemia and reperfusion injury was performed according to the following procedure. After a midline laparotomy, the portal triad was gently isolated with cotton buds and the blood was clamped off using a 6 x 1 mm microvessel clip (Micro Serrefines, Fine Science Tools, Germany), leading to a global liver ischemia confirmed by the organ blanching. After 40 min, the clip was removed, and the abdominal wall and skin were closed. Before awaking, mice were given 3 μg of buprenorphine subcutaneously as post-operative analgesia. Body temperature was maintained during the whole procedure with a heating pad, and the mouse was covered with a survival blanket during ischemia.

24 h after reperfusion, blood was collected by cardiac puncture. Plasma samples were sent to Cerbavet (Wissous, France) for transaminases dosage.

III. Results

Nanoparticles (NPs) were prepared using the so-called nanoprecipitation process, which consisted in dropwise addition of an ethanolic solution of SQAd into water. SQAd NPs presented a hydrodynamic diameter of 62 ± 10 nm and a monodisperse distribution (polydispersity index or PdI < 0.1).

1) Optimization of nanoparticles freeze-thawing conditions using cryoprotectants

Freeze-thawing tests were conducted as a preliminary experiment with SQAd NPs at a concentration of 1 mg/mL. Two freezing methods were tested: slow freezing for 24 h using a freezer (-30 °C) and fast freezing for 10 min in liquid nitrogen (-196 °C). As expected, SQAd NPs showed irreversible aggregation upon freezing in water, with the appearance of macroscopic particles in suspension after thawing and important increases in diameter and PdI (Figure 1A).

To prevent NPs aggregation during freeze-drying process, cryoprotectants are generally added (13). Hence, four easily available carbohydrates were tested in this study for this purpose: a monosaccharide (dextrose), two disaccharides (sucrose and trehalose) and a polyol (mannitol) (14). Among them, trehalose was reported to be the most efficient cryoprotectant (9,15,16).

After thawing, samples frozen in sucrose, trehalose and dextrose appeared as limpid solutions displaying Tyndall effect, while samples frozen in mannitol presented macroscopic aggregates.

This was in good agreement with the values of the apparent hydrodynamic diameters measured after freeze-thawing: as shown in Figure 1A, addition of 5 wt. % sucrose or trehalose in the suspension perfectly preserved NPs diameter (diameter increase < 26%, PdI < 0.1), while dextrose has been less effective (238 % and 113 % diameter increase respectively after slow and fast freezing). With regards to mannitol, it was proved to be ineffective for our purpose, inducing instantaneous increase in diameter (+633 %) and PdI (0.2) even before freezing.

Because sucrose and trehalose demonstrated acceptable cryoprotective activity at a concentration of 5 wt. %, we decided to investigate their cryoprotective ability at lower concentrations. Both disaccharides presented similar trends, with poor protection up to 1 wt. % (macroscopic aggregates and final hydrodynamic diameter over 600 nm) and satisfying protection over 2.5 wt. % (limpid solution with slight Tyndall effect), particularly under slow freezing conditions (size increase < 14 %) (Figure 1B and Figure 1C).

2) Characterization of freeze-dried NPs after redispersion

Based on these preliminary tests, freeze-drying experiments were conducted in optimal conditions. It was observed that dry cakes from trehalose samples displayed well aerated appearance, while cakes from sucrose samples showed more compact forms (Supplementary Figure 1). However, resuspension occurred immediately after addition of water in all samples.

The final suspensions were perfectly limpid, with a slight Tyndall effect.

3) NPs size characterization by DLS

As shown in Figure 2A, both sucrose and trehalose exhibited excellent lyoprotective activities, with highly reproducible size conservation at 5 wt. %. However, trehalose displayed a slightly better colloidal conservation at 2.5 wt.%. Taking this into account and considering the differences between the two cryoprotectants in term of cake aspects, trehalose was chosen for further physico-chemical and biological investigations, even though NPs zeta potential was slightly increased by its addition (Figure 2B).

4) NPs size characterization by small-angle neutron scattering (SANS)

SANS patterns of freshly prepared SQAd NPs and freeze-dried SQAd NPs (after resuspension) are presented in Figure 3. Signals could be perfectly superimposed over a broad range of angles.

The minor signal difference at small angles might be explained by the presence of some larger particles in the freeze-dried sample or by an excluded volume effect, which could appear for higher concentrations (2 mg/mL for freshly prepared NPs vs. 1 mg/mL for freeze-dried NPs), thus inducing a decrease in the scattered intensity. Nevertheless, the supramolecular organization of NPs could be considered as mostly identical before and after freeze-drying.

Noteworthy, a small Bragg reflection around 0.075 Å -1 was visible on the diagram for both samples, indicating at the resolution of SANS experiments, a similar internal NPs structure for both preparations.

A diameter of 42 nm and a PdI of 0.2 were found when fitting the curves considering a log-norm population of spheres.

5) NPs morphology after freeze-drying

Then, freeze-dried NPs were imaged by AFM in liquid after resuspension (Figure 4). Two kinds of objects were visible: numerous small regular round-shape NPs with size lower than 100 nm, adsorbed onto the silica surface as a compact layer, but also some bigger stick-shape assemblies as depicted by the white objects on the height picture. It appeared that the NPs were highly aggregated: based on previous size characterization, this was likely due to artefactual effects, resulting from sample dilution and adsorption on the SiO2 surface. Freeze-dried NP behaved as a viscoelastic material as shown by the high phase shifts observed on the phase image. NPs were flattened by their interaction with the surface, inducing lower heights and increased diameters compared to that extracted from DLS measurements.

6) Influence of residual ethanol on NPs size

During the optimization of the experimental conditions described above, it was noted that immediately following nanoprecipitation and during the evaporation step, the time spent for removing ethanol strongly influenced the freeze-drying outcome, even though it did not affect the freeze-thawing after-effect. To explore this finding, the evaporation process was pushed to different limits and samples were characterized for residual ethanol content by a colorimetric assay relying on ethanol reduction by dichromate. As expected from the azeotropic nature of the water/ethanol mixture, a partial co-evaporation of water and ethanol occurred, and ethanol could not be fully evaporated. Depending on the evaporation time, residual ethanol content ranged from 0.1 % to 3.1 % (Figure 5A). The ethanol content after evaporation (before the freeze-drying step) could be fitted with a one-phase exponential decay model (r² = 0.96, 𝑐 𝑒𝑡ℎ𝑎𝑛𝑜𝑙 = 27 𝑒 -0.066 % 𝐸𝑣𝑎𝑝 ). Of note, NPs diameter was not affected by ethanol content after evaporation and before freeze-drying (68 ± 16 nm) (Figure 5B).

NPs samples from this library were then freeze-dried as previously optimized. As expected, the lyophilization drying step reduced the ethanol content in each sample (Figure 5A). However, ethanol remained detectable in most samples with concentration up to 1 vol. %, indicating that ethanol molecules may remain adsorbed onto NPs surfaces. Interestingly, two distinct behaviours could be identified from Figure 5B, which represents the NPs apparent diameter before and after lyophilization : if the ethanol content was under 0.8 vol. %, the increase in diameter was low and the diameter after freeze-drying (76 ± 11 nm) remained close to the initial diameter (62 ± 8 nm); on the contrary, when the ethanol content exceeded 0.8 vol. %, higher increases in diameter were witnessed, from + 44 % up to + 598 %. Hence, it was concluded that the ethanol content in NPs suspension dramatically impacted the freeze-drying outcome, as high ethanol content led to NPs aggregation during the freeze-drying process.

7) Influence of residual ethanol and freeze-drying on NPs supramolecular structure

To investigate the influence of residual ethanol and freeze-drying on NPs supramolecular structure, two types of samples were prepared: samples containing high levels of residual ethanol (evaporation under 40 %, leading to over 2.0 % of ethanol in the suspension according to the exponential decay model) and samples containing almost no residual ethanol (evaporation over 67 %, leading to less than 0.3 % of ethanol in the suspension). It was observed that high ethanol content hindered the resuspension of the NPs after lyophilization.

Thus, only ethanol-free samples were freeze-dried and analysed by small-angle X-ray scattering (SAXS).

The SAXS diffraction pattern of SQAd NPs with low residual ethanol content showed three Bragg peaks at 0.0825 Å -1 (q0), 0.138 Å -1 (~q0√3) and 0.164 Å -1 (~2q0) (Figure 6A), characteristic of an inverse hexagonal phase (HII) with a lattice parameter of 88 Å, in agreement with previous studies (17). The Bragg reflections in the SAXS pattern of SQAd NPs without residual ethanol are broader, revealing a lack of long-range order in the HII phase.

9) NPs innocuity a. Samples osmolality

Then, the osmolality of NPs solutions was measured before and after freeze-drying. Before freeze-drying, the low osmolality of the solution (14 ± 6 mOsm/kg) was attributed to the presence of NPs and traces of ethanol. The addition of 5 wt. % trehalose increased the tonicity to 151 mOsm/kg (theoretical calculation gives a contribution of 154 mOsm/kg). After freezedrying, a small decrease in tonicity was observed in 5 wt. % trehalose samples with or without NPs. Final NPs suspension was hypotonic (140 ± 4 mOsm/kg compared to 308 mOsm/kg for 0.9 % saline) (Table 1). Interestingly, resuspension in 250 µL instead of 500 µL did not seem to affect NPs, which leads to solutions with higher tonicity and concentrated NPs.

b. In vitro innocuity

Knowing that SQAd NPs mostly accumulate in the liver after parenteral injection (8), we

decided to carefully evaluate their potential hepatotoxicity. This was first done in vitro by checking NPs innocuity on HepG2, a human liver carcinoma cell line. Figure 8 shows that NPs induced no reduction on cellular activity after 72 h of treatment, even at very high concentration (400 µM). Importantly, no difference could be seen between freshly prepared

NPs and freeze-dried NPs.

c. In vivo innocuity

A mouse model of liver ischemia and reperfusion injury was used to investigate the safety of SQAd NPs in animals with a decreased hepatic function. In that respect, a solution of SQAd NPs (freshly prepared or resuspended after lyophilization, 2 mg/mL, 10 wt. % trehalose) or vehicle (10 wt. % trehalose) was injected into mice prior to ischemia. While all control mice survived for 24 h after the surgery, only 67 % of the ones who received the freshly prepared SQAd NPs where still alive at that time point, showing some toxicity of these NPs on injured mice. On the opposite, all mice injected with freeze-dried NPs survived and no significant difference was observed in terms of aspartate transaminase (AST) and alanine transaminase (ALT) levels when comparing with those treated with vehicle (Figure 9). Hence, freeze-dried NPs appeared to be perfectly safe for intravenous injection in mice at the tested dose (20 mg/kg).

IV. Discussion

Physical and chemical instabilities often occur during long-term storage of NPs in aqueous medium, which restricts their use (18). Freeze-drying NPs suspensions represents a useful solution to prevent aggregation and fusion of the particles, to maintain their size stability and to avoid their degradation during long-term storage.

The aim of the present study was to determine appropriate conditions for long-term storage of SQAd NPs in order to ensure their stability, which is one of the most important aspects for translation of these NPs into a clinical setting. Another important aspect consisted in the design of SQAd NPs easy to manipulate before their administration.

SQAd bioconjugates were first nanoprecipitated in water into NPs which were characterized by DLS technique. Of note, the size parameters reported in this study (diameter of 62 ± 10 nm, PdI < 0.1) were smaller than what was previously reported (diameter around 120 nm, PdI = 0.13 (7)) due to differences in preparation conditions and dilution before measurements.

As freezing is the first type of stress that NPs will face during the freeze-drying process, freeze-thawing experiments are often conducted as a preliminary simple and predictable approach for determining suitable cryoprotectants and their optimal concentrations (19). Most popular cryoprotectants used for freeze-drying nanosuspensions include sugars (glucose, sucrose, trehalose and mannitol), which are known to vitrify under a certain temperature (10).

As a result, they are thought to form a glassy matrix in which NPs are immobilized and cannot aggregate (glass dynamics hypothesis, formerly named vitrification theory (20)). With SQAd NPs, mannitol induced direct NPs aggregation and dextrose was unable to protect them properly from freezing stress, while sucrose and trehalose were found to display good cryoprotectant activities at this concentration. As expected, high concentrations of carbohydrates were beneficial (ie. 5 wt.%). Even though fast freezing is sometimes claimed as the more appropriate for freezing nanosuspensions (13), there is no real consensus on this question (21). In the present study, slow freezing at -30 °C revealed slightly better than fast freezing in liquid nitrogen for conserving NPs size. An explanation would be that, during freezing, two phases appear due to the moving of the freezing front: the first one constituted of ice crystals and the second one containing a cryo-concentrated solution. During this phenomenon, NPs accumulate in the cryo-concentrated liquid phase mainly through the cryoprotectants which physically adsorb on their surface. When the freezing rate is slow enough, cryoprotectants could move totally into the cryo-concentrated liquid phase. This led to increased concentration of cryoprotectant within the liquid phase, thus preventing aggregation.

During fast freezing rate, cryoprotectants have no time to fully migrate into the cryoconcentrated liquid phase. Therefore, their concentration is too low, and they are not efficient in preventing NPs aggregation. It must be noted that this explanation is valid only for low cryoprotectant concentration, which might explain why such a difference is observed between slow and fast freezing only at 2.5 wt. % of cryoprotectant, but not at 5 wt. % (13).

With regard to the choice of cryoprotectant, the best outcome was obtained with trehalose, which was already described as the best cryoprotectant for other lipid-based nanoparticles (14). This was attributed to its increased interaction with water molecules and its highest glass transition temperature (22).

After freeze-drying, the lyophilised NPs appeared as a white solid "soft cake" which could be easily redispersed in water without any aggregation. Freshly prepared SQAd NPs versus lyophilized and reconstituted ones were compared using different techniques. Size evaluation by DLS indicated conservation of their diameter and characterisation using SANS technique led to superimposed diagrams, confirming identical sizes. The scattering intensity was typical of a suspension of a log norm distribution of spherical particles (23). The discrepancy in size observed between the two techniques (~ 60 nm vs. ~ 40 nm for DLS and SANS respectively) may arise from their specificities: DLS gives access to the NPs hydrodynamic diameter, while SANS measures the contrast formed by hydrogen atoms in a deuterium background. The small increase in SANS signal intensity at small angles could be linked to the presence of some stick-shape assemblies, also evidenced by AFM. The Bragg peak visible around 0.075 Å -1 with SANS seemed to correspond to the one obtained for the first order of the hexagonal phase by SAXS (0.08 Å -1 ). However, the lamellar phase detected by SAXS after lyophilization was not visible by SANS. All these converging data confirmed the successful preservation of the NPs after freeze-drying.

In the present study, we also investigated how the presence of residual ethanol (used during the nanoprecipitation process) in NPs suspensions could affect the lyophilization.

Several factors were previously reported to perturb nanomedicines structure during freezedrying such as mechanical stress during ice growth (24,25), change in osmolarity (26), interaction with sugar particles (27) or loss of structural water (11). However, the effect of removing residual solvent has never been discussed yet in the literature. Indeed, the presence of residual solvent after nanoprecipitation is most of the time overlooked, with no titration of residual solvent after evaporation or dialysis (28), and only few efficient methods for complete solvent elimination were reported (29). During this study, it was clearly observed that the presence of residual ethanol led to poor resuspension after freeze-drying. Since the presence of residual ethanol could affect the supramolecular structure of squalene based NPs ( 23) and since we anticipated that freeze-drying process would partly remove residual solvent, we decided to explore the NPs structure before and after this process. Therefore, it was observed that after the evaporation step, NPs in suspensions containing residual ethanol displayed an inverse hexagonal phase. This phase consisted of the assembly of SQAd bioconjugates within the NPs, forming thus cylinders whose aqueous core was surrounded by the hydrophilic adenosine moieties linked to the hydrophobic chains of squalene, which arranged on a two-dimensional hexagonal lattice (17). In that respect, increasing the evaporation step decreased the long-range order of the structure, which may be attributed to the small size of the NPs entailing short coherence lengths and structural distortions at the interface. Of note, a decrease in size of squalene based NPs upon withdrawal of ethanol has been previously reported (23). After freeze-drying and resuspension, the hexagonal phase appeared to coexist with a lamellar phase.

The occurrence of this lamellar phase has been previously linked to the increase in the mean Chapitre 2 92 area of the polar adenosine moieties at the water/SQAd interface (17). It might be hypothesized that the insertion of a few trehalose molecules between the adenosine head groups, promoted by hydration of the freeze-dried powder, could expand their mean lateral area as shown, for example, by Andersen et al. for sugar-phospholipid membrane interactions at low sugar concentrations (5-10 %) (30).

Regarding long-term stability, storage at 4 °C showed no modifications of NPs physicochemical characteristics after resuspension, which meets industrial requirements in terms of transport and conservation.

A final study was conducted to guarantee the innocuity of NPs both in vitro and in vivo.

Indeed, as the major aim in freeze-drying nanomedicines is to make NPs suitable for clinical applications (31), it seemed crucial to investigate this issue. As a first step, osmotic pressure measurements were performed on SQAd NPs solutions after resuspension and revealed hypotonic solutions. This is an interesting point because it allows for tonicity adjustment without dilution before parenteral injection.

Moreover, liver safety had to be considered as SQAd NPs accumulate in the liver after intravenous injection (8). Preliminary in vitro experiments were therefore performed on cultured hepatocytes. HepG2 cells were selected because they represent a well-accepted model of human hepatocytes and are easy to handle under in vitro conditions. In addition, the HepG2 cells have been used as a liver-specific cell line to predict hepatotoxicity of drugs in general (32) and nanoparticles in particular (33)(34)(35). Lyophilised SQAd NPs did not exhibit any toxicity in vitro after 72 h of treatment on HepG2 cell line. While intravenous injection of freshly prepared SQAd NPs induced some mortality on liver injured mice, freeze-dried SQAd NPs were shown to be perfectly safe on the same animal model. Higher residual ethanol contents in freshly prepared NPs may explain this difference. This result further strengthens the progress towards SQAd NPs clinical application.

V. Conclusion

Preserving the colloidal stability of nanoparticles before clinical translation is mandatory. This

I. SQAd NPs for the treatment of liver ischemia 1) SQAd NPs and liver ischemia

A model of global liver ischemia was set up as shown on Figure 1A. Briefly, mice portal triad was clamped for 25 min before reperfusion. SQAd NPs were injected at different time points and blood samples were collected 6 h after reperfusion for ALAT quantification (Figure 1B).

All mice survived to 6 h reperfusion, except when the treatment was injected 10 min before ischemia. In that case, an increase in the number of dead mice was observed during ischemia or reperfusion as a result of the injection of SQAd NPs, whatever the injected dose (Figure 1F). In terms of ALAT levels, no effect could be seen when the treatment was injected 30 min after reperfusion (Figure 1C) or 24 h before ischemia (Figure 1D) (same levels as control), while a significative increase was observed when the treatment was injected 10 min before ischemia at the highest dose (Figure 1E). Even though the number of mice was limited because these experiments were of preliminary nature, it likely appeared that the injection of SQAd NPs suspensions 10 min before ischemia resulted in some liver toxicity. Noteworthy, other preliminary experiments have shown that SQ NPs (NPs prepared with squalenylacetic acid under the same nanoprecipitation conditions as SQAd NPs) presented similar toxicity (data not shown). This toxicity on ischemic livers could arise from the different components of the NPs:

1. SQAd bioconjugates;

2. Ad released from NPs;

3. SQ released from NPs;

4. Uncompletely evaporated ethanol used during the nanoprecipitation process;

5. NPs assemblies due to complement activation.

But, previous detailed toxicity studies have demonstrated the overall safety of SQAd NPs on healthy mice and, interestingly, no significant complement activation was observed either (10). This is the reason why we have explored the possible involvement of residual ethanol in SQAd NPs arising from the nanoprecipitation process, which could eventually increase the toxicity in animals with ischemic liver.

2) Ethanol toxicity

Ethanol is listed by the FDA as a class 3 solvent, which means that it is considered as less toxic in acute or short-term studies in humans and negative in genotoxicity studies. Ischemic livers, however, might be more sensitive to ethanol, especially if it is delivered intracellularly in NPs.

In order to explore this hypothesis further, we measured the quantity of residual ethanol in NPs suspension. Results were presented in Chapter 2, Figure 5A. If evaporation was not pushed above 50 % (meaning evaporation of more than 0.5 mL when preparing 1 mL of final NPs suspension), residual ethanol was found to be over 1 vol. %. Of note, before this finding (and in particular for experiments on liver ischemia presented above), ethanol evaporation was not closely monitored and was considered to be complete over roughly 20 % evaporation.

A rapid calculation can give us an idea of the quantity of ethanol this represents once systemically injected in mice. Let us consider a suspension with 1 vol. % ethanol.

𝑐 𝑠𝑢𝑠𝑝𝑒𝑛𝑠𝑖𝑜𝑛 𝐸𝑡𝑂𝐻 = 1 𝑣𝑜𝑙. % = 0.8 𝑤𝑡. % = 8 𝑔/𝐿

The injected volume corresponds to 10 mL/kg (or 0.01 L/kg), and mice have a blood volume of around 60 mL/kg (0.06 L/kg).

As a result, the concentration of ethanol in the blood right after injection is

𝑐 𝑏𝑙𝑜𝑜𝑑 𝐸𝑡𝑂𝐻 = 𝑐 𝑠𝑢𝑠𝑝𝑒𝑛𝑠𝑖𝑜𝑛 𝐸𝑡𝑂𝐻 × 𝑣 𝑠𝑢𝑠𝑝𝑒𝑛𝑠𝑖𝑜𝑛 ÷ 𝑣 𝑏𝑙𝑜𝑜𝑑 = 8 × 0.01 ÷ 0.06 = 1.3𝑔/𝐿
Hence, the concentration of ethanol in mice blood right after NPs suspension injection is around 1.3 g/L, which is far to be negligible regarding toxicity in liver ischemic animals. Indeed, since SQAd NPs accumulate in the liver after intravenous injection, this may lead to the delivery of high doses of ethanol in the already ischemic tissue.

3) Freeze-drying as a way of removing ethanol

As described in Chapter 2, freeze-drying SQAd NPs represents an efficient way of minimizing residual ethanol (Chapter 2, Figure 5A).

Consequently, the effect of NPs containing no more residual ethanol (or at very low percentages) could be assessed again on mice with liver ischemia. As shown on Figure 2 (and Chapter 2, Figure 9), the previously described toxicity seemed to be abolished after ethanol removal by freeze-drying.

It is worth noting that the presence of residual ethanol (or other solvent like THF or acetone) in suspensions after nanoprecipitation was rarely looked at. Most of the time, residual solvent is removed by evaporation under reduced pressure based on the fact that its boiling point is lower than the one of water, and then considered to be absent even though no titration has been realized (13). Our study shows that, even if this solvent is considered as of low toxicity (ethanol, acetone and DMSO are class 3 solvents according to the FDA), overlooking this issue can lead to dramatic results in some circumstances (depending on the studied pathology, on NPs accumulation in an organ, etc). If dialysis is sometimes used to overcome this problem, we proposed here freeze-drying as a way to both remove ethanol and prepare NPs under a stable form and an easy-to-use formulation (Chapter 2). To our knowledge, such an approach had never been presented as a solution to rise above the problem of residual solvent in nanoparticles. Moreover, we could show during Chapter 2 that ethanol itself needed to be closely monitored for a complete success of the freeze-drying process (the presence of high residual quantities of ethanol induced NPs aggregation during freeze-drying). These findings on the reciprocal influence between ethanol and freeze-drying might be very useful for the nanomedicine community.

Getting back to our original aim, these SQAd NPs still had no efficacy in treating liver ischemia, as could be seen by ALAT titration (Chapter 2, Figure 9). This meant that our starting assumption was probably false, notwithstanding the strong rational presented above: SQAd NPs could not treat liver ischemia under tested conditions. Hence, a deeper understanding of SQAd NPs mechanism of action was urgently needed before moving into new applications.

II. Understanding SQAd NPs mechanism of action

As presented in the Chapter 1, SQAd NPs form an intracellular reservoir of adenosine (Figure 3A). The above in vitro study has led us to the important conclusion that SQAd NPs act as a prodrug and that the active substance is free adenosine released from this prodrug after intracellular processing.

Hence, we now have these two facts:

i. Free endogenous adenosine has protective effects on ischemic livers;

ii. SQAd NPs administration leads to free adenosine release.

A simplified reasoning would conclude again from these two facts that SQAd NPs should treat liver ischemia and reperfusion. However, this is not the case. How to explain this syllogism?

1) SQAd NPs vs. free adenosine vs. adenosine receptor agonists and antagonists

Even though endogenous adenosine was shown to be released during ischemia and have protecting effects, protective effects of exogenous adenosine on liver ischemia in vivo was only shown once 20 years ago (infusion of adenosine for 20 min prior to 90 min right lobe ischemia in rats led to reduced ALAT, ASAT and LDH levels) (14). The main reason why exogenous adenosine has shown so little results so far was due to its rapid metabolization in the blood circulation in vivo which do not allow it enough time to produce any effect on liver ischemia (Figure 3B).

The other studies that claim adenosine efficacy against liver ischemia and reperfusion injury actually target a single adenosine receptor with specific agonists or antagonists (Figure 3C). This case is very far from the use of free adenosine as this natural ligand binds the four receptors simultaneously, with a much more complex effect on the cells, eventually related to the dynamic overexpression or, on the contrary, downregulation of these receptors after adenosine binding. Moreover, using a specific agonist or antagonist can change the binding equilibria of endogenous adenosine with its four receptors and may lead to contradictory effects, too (15). As a result, it might be abusive to try to extrapolate studies using adenosine receptors agonists or antagonists to adenosine itself.

In the case of SQAd NPs, however, it seems that we should be rather close from the endogenous release observed during ischemia. Indeed, SQAd NPs were shown to act as an intracellular reservoir of adenosine which is released in the extracellular space, in the same way as endogenous adenosine is released from ischemic cells. The main difference between the two situations could however arise from the quantities that are released extracellularly and the equilibrium between intracellular and extracellular concentrations.

2) Adenosine release from SQAd NPs under ischemia

Another possible explanation for the lack of efficacy of SQAd NPs for the treatment of liver ischemia and reperfusion injury might be that, under ischemic or post-ischemic conditions, cells are not able to internalize SQAd NPs or release adenosine from SQAd bioconjugate. Indeed, ischemia induces hypoxia and cell energy depletion. SQAd NPs internalization was proven to require energy (16) and, even though SQAd cleavage was shown to happen simply under acidic conditions (Chapter 1 Figure 4), it may be more efficiently catalyzed by enzymes in cells and thus be slowed down during ischemia. It would be interesting to check these hypotheses in vitro by studying adenosine release from cells treated with SQAd NPs under hypoxic conditions.

3) Liver is not the brain

The last argument in favor of a possible protective effect of SQAd NPs on liver ischemia reperfusion injury was that NPs had already proven effective on cerebral ischemia and that both pathologies are very close at a cellular and molecular level, whatever the organ.

However, the lack of response from our therapeutic strategy on the liver means that this postulate was probably wrong.

A first difference between the brain and the liver may arise from SQAd biodistribution in vivo. Indeed, after SQAd NPs intravenous injection, very important quantities of the bioconjugate were found in the liver, while no bioconjugate could be found in the brain (12).

Liver accumulation is rather usual for NPs of this size but, in the case of SQAd NPs, internalization was also shown to occurs through LDL receptors (Chapter 1, Figure 3D) which are highly expressed in the liver (according to the Human Protein Atlas). This organ was shown to be responsible for a very large part of LDL clearance (17), so it is not surprising that it is also responsible for an important part of SQAd NPs clearance. This accumulation of SQAd into the liver was first seen as an advantage for delivering important quantities of adenosine to this organ and treating liver ischemia and reperfusion. Instead, it seems unfavorable (or ineffective) under these conditions.

Finally, another difference between the two organs is that different cell types may play crucial role in response to ischemia and reperfusion. In the brain, the protective action of SQAd NPs was shown to be mainly due to interactions with endothelial cells (10). Indeed, adenosine would induce vasodilation and thus a better oxygenation during reperfusion, due to a dramatic decrease in the number of clogged brain capillaries. In the liver, adenosine is claimed to protect from ischemia via its action on inflammatory cells (mainly NKT cells (7,8)) and on hepatocytes (9,18). And, still more importantly, adenosine is known to be vasodilator for most vascular tissues except hepatic portal vein (19).

III. Conclusion and perspectives

As a conclusion, we have to admit that predicting the therapeutic efficacy of SQAd NPs can be extremely hard, for different reasons:

• Adenosine pharmacology is extremely complex, with four receptors having different but complimentary actions and being expressed on different levels depending on space (the type of cell) and time (physiology vs. pathology);

• The delivery of adenosine as NPs changes the pharmacokinetics and biodistribution of this nucleoside to such a point that it is hard to extrapolate any results obtained with adenosine to SQAd NPs.

Even if some questions remain, our study has brought extremely valuable information for future applications:

1. A better understanding of SQAd NPs mechanism of action in vitro:

• We showed that SQAd NPs or SQAd bioconjugates had no direct interactions with adenosine receptors;

• We highlighted an original mechanism of entry dependent on LDL receptors;

• We unveiled the formation of an intracellular reservoir of adenosine via SQAd accumulation, SQAd cleavage under acidic conditions and adenosine release in the extracellular space;

• We checked that adenosine proceeding from this reservoir could activate adenosine receptors on neighboring cells;

• We established these results on a hepatocyte cell line and extended them to brain endothelial cells.

A method for enhancing SQAd NPs formulation by freeze-drying:

• We tuned freeze-drying conditions for obtaining perfect conservation of the NPs properties by varying the nature and concentrations of cryoprotectants, as well as the freezing process;

• We deeply characterized the physico-chemical properties of freeze-dried NPs via different techniques including dynamic light scattering, small-angle X-ray or neutrons scattering and atomic force microscopy;

• We investigated the influence of ethanol on the freeze-drying process and showed that, under some conditions, freeze-drying could be used as a way of removing ethanol from NPs preparations;

• We checked the innocuity of NPs after freeze-drying and resuspension both in vitro and in vivo;

• We followed freeze-dried NPs stability over several months.

These findings can be combined with the lack of efficacy observed on liver ischemia and reperfusion injury to better orientate future research on SQAd NPs therapeutic applications.

We believe that SQAd NPs should deserve to be investigated for beneficial effects on heart ischemia for the following reasons.

A first argument comes from SQAd NPs mechanism of action. As SQAd NPs act as a prodrug which main effect in vitro seems to be a prolonged release of extracellular adenosine, SQAd NPs efficacy could be evaluated on pathologies currently treated with medicines which also induce an increase of extracellular adenosine. Dipyridamole, which blocks adenosine entry into the cells, is one of these drugs. It inhibits platelet aggregation and induces vasodilation, and thus is used for treating thrombosis and heart ischemia after myocardial infarction.

Secondly, the discussion held above led us to conclude that the biodistribution of SQAd NPs in the targeted organ should not be too different from the one in the brain, and that endothelial cells should play an important role in the pathology. These two facts are verified for heart ischemia.

Last but not least, the freeze-dried formulation allows the use of SQAd NPs in emergency cases, which is a prerequisite in treating myocardial infarction.

Ô cher Zetasizer, que ferions-nous sans toi ? Abstract : Adenosine has a high therapeutic potential but its extremely short half-life in blood seriously impairs its efficacy.

As presented in the literature review, this difficulty can be overcome by using lipid-based drug delivery systems. Indeed, adenosine can be encapsulated into liposomes or conjugated to a lipid. In particular, among adenosine « lipidizations », the socalled « squalenoylation » has been shown to enhance adenosine biodistribution. This technique consists in coupling adenosine to a lipophilic squalene derivative, hence generating bioconjugates which are able to spontaneously self-assemble as nanoparticles of 100 nm of diameter in aqueous solution. Intravenous injection of these squalene-adenosine (SQAd) nanoparticles produced promising results for treating cerebral ischemia and spinal cord injury. Thus, the aim of this thesis was to push forward the preclinical development of these nanomedicines following two main directions: unveiling their mechanism of action and enhancing their formulation.

Thereby, the first chapter of this thesis presents results obtained when studying in vitro SQAd nanoparticles mechanism of action. This work has shown that SQAd nanoparticles did not interact directly with adenosine receptors, but formed an intracellular reservoir of adenosine. Indeed, after internalisation, SQAd bioconjugates are clived into free adenosine. This molecule is then released by the cells in the extracellular medium, where it can activate specific membrane receptors on neighbouring cells. After studying the mechanism of action, we explored how to enhance the formulation. Results are described in the second chapter. We focus our efforts on freeze-drying the nanoparticles suspension, in order to offer a stable and easy-to-use formulation. Selected conditions led to a good conservation of nanoparticles physico-chemical properties and obtention of solutions which could be safely injected in the animal. Overall, this work has widen the field of possible applications for SQAd nanoparticles thanks to a better understanding of their mechanism of action and the development of a more clinically-adapted formulation.

2 ) 3 )CholEsteryl 4 , 4 -

 2344 ans, je n'y serais pas arrivée toute seule et nos autres oeuvres n'auraient probablement pas vu le jour, ce Discussion Générale....................................................................... I. SQAd NPs for the treatment of liver ischemia ................................................................... 1) SQAd NPs and liver ischemia ................................................................................................................. 2) Ethanol toxicity .......................................................................................................................................... 3) Freeze-drying as a way of removing ethanol ............................................................................. II. Understanding SQAd NPs mechanism of action ....................................................... 1) SQAd NPs vs. free adenosine vs. adenosine receptor agonists and antagonists ...... Adenosine release from SQAd NPs under ischemia ................................................................ Liver is not the brain ............................................................................................................................... III. Conclusion and perspectives ............................................................................................. IV. References ................................................................................................................................... Annexes .........................................................................................difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3dodecanoate CPA N 6 -cyclopentyladenosine DAPI 4′,6-diamidino-2-phenylindole DDPA 1,2-didecanoyl-sn-glycero-3-phosphatidyl-adenosine DDPC 1,2-didecanoyl-sn-glycero-3-phosphocholine DHSG 1,5-dihexadecyl-N-succinyl-l-glutamate diC8PA 1,2-dioctanoyl-sn-glycero-3-phosphatidyl-adenosine diC8PC 1,2-dioctanoyl-sn-glycero-3-phosphocholine diC8PU 1,2-dioctanoyl-sn-glycero-3-phosphatidyluridine DLS Dynamic light scattering DLPA l,2-dilauroyl-sn-glycero-3-phosphatidyl-adenosine DLPC l,2-dilauroyl-sn-glycero-3-phosphocholine DLPU l,2-dilauroyl-sn-glycero-3-phosphatidyl-uridine DMPA 1,2-dimyristoyl-sn-glycero-3-phosphatidyl-adenosine DMPC l,2-dimyristoyl-sn-glycerodioleoyl-sn-glycero-3-phosphatidyl-adenosine DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine DOPC-E 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine DOTAP 1,2-dioleoyl-3-trimethyl-ammonium-propane DPPA l,2-dipalmitoyl-sn-glycero-3-phosphatidyl-adenosine DPPC l,2-dipalmitoyl-sn-glycero-3-phosphocholine DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine DSPE-PEG 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy(polyethylene glycol)] DSPG 1,2-distearoyl-sn-glycero-3-phosphorylglycerol -2-oleoyl-sn-glycero-3-phosphatidyl-adenosine POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine POPG 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-glycerol POPU 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-uridine PS Phosphatidylserine PUPC 2′,3′-O-16-hentriacontanyliden-uridine-5′-phosphocholine RLU -2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a}{1,3,5}triazin-5-ylamino]ethyl)phenol

Figure 1

 1 Figure 1 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), di-phosphate (ADP) or tri-phosphate (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.

Figure 2

 2 Figure 2 Hypothetic mechanism of action of H12-(ADP)-liposomes. (1) H12-(ADP)-liposomes accumulate at the site of injury by binding glycoprotein (GP) IIb/IIIa on activated platelets. (2) This also enhances platelet cross-linking, as the same liposome presents several fibrinogen γ-chain peptides moieties on its surface and can therefore bind several activated platelets. (3) ADP released from the liposomes (4) further activates platelets via P2Y1, P2Y12 or P2X1 receptors, and (5) is also metabolized into adenosine, which has anti-inflammatory protective effects.

Figure 3

 3 Figure 3 Different types of ATP-liposomes. (A) Basic ATP-loaded liposomes can be complexified by (B) co-loading with complementary energy molecules like pentobarbital and suramin or (C) by functionalization with targeting moieties like antibodies or receptor ligands. The lipid bilayer composition can also be tuned to provide pH-sensitivity, fusogenicity or stealthiness.

  Ahlers et al. created a new type of adenosine nucleolipids (Figure 4B, R'=H) by coupling N-hydroxysuccinimide actived esters of 2',3'-O-(3-carboxy-lmethylpropyliden)adenosine [58] with mono or dialkylamines. More recently, Moreau et al. synthesized other types of adenosine nucleolipids (Figure 4B, R'=H) by reacting adenosine with dimethoxy ketal of palmitone [59]. These adenosine nucleolipids were also reacted with 2-chloro-1,3,2-dioxaphospholane-2-oxide and trimethylamine in order to obtain lipid adenosine phosphocholines (Figure 4B, R'=phosphocholine) [59]. The same team also used imidazole activated fatty acids with 2',3'-(isopropylidene)-5'-(phosphocholine)-adenosine to form lipid adenosine phosphocholines (Figure 4C, R'=phosphocholine) [60]. Over the same period of time, Couvreur's team synthesized squalene-based adenosine conjugates [61,62](Figure4D).

Figure 4

 4 Figure 4 Adenosine nucleolipids. R = lipids, R' = H or phosphocholine. Lipids can be conjugated to adenosine by a phosphate diester link on the primary hydroxyl group of ribose to form 5'-phosphatidyladenosines (A), attached to 2',3'-hydroxyl groups of ribose through a ketal linkage (B) or ether bond (C), or coupled via an amide bond on the nucleobase (D).

Figure 5

 5 Figure 5 Electron micrograph of the formation process of cigar-like scrolls from DMPA in acidic solution (pH 5.0): (a) linear double-helical strand; (b) ribbon structure; (c) incomplete cigar-like scrolls from ribbon structure; (d) complete cigar-like scrolls. All scale bars represent 0.1 µm. Reprinted with permission from [55]. Copyright 1992 American Chemical Society.

  represents the total amount of Carbon atoms versus the number of Double unsaturated bonds in the conjugated lipid chain. AMPC16: adenosine 5'-hexadecylphosphate; DDPA: 1,2-didecanoyl-sn-glycero-3-phosphatidyl-adenosine; DDPC: 1,2-didecanoyl-sn-glycero-3phosphocholine; diC8PA: 1,2-dioctanoyl-sn-glycero-3-phosphatidyl-adenosine; diC8PC: 1,2-dioctanoyl-sn-glycero-3-phosphocholine; DLPA: l,2dilauroyl-sn-glycero-3-phosphatidyl-adenosine; DLPC: l,2-dilauroyl-sn-glycero-3-phosphocholine; DMPA: 1,2-dimyristoyl-sn-glycero-3phosphatidyl-adenosine; DMPC: l,2-dimyristoyl-sn-glycero-3-phosphocholine; DOAPC: di-oleyl-adenosine-phosphocholine; DOP-Ade: 1,2dioleoyl-sn-glycero-3-phosphatidyl-adenosine; DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine; PAPC: 2′,3′-O-16-hentriacontanylidenadenosine-5′-phosphocholine; POPA: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-adenosine; POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine; SAPC: 2',3'-O-18-pentatriacontanyliden-adenosine-5'-phosphocholine; SQAd: squalene-adenosine.

Figure 6

 6 Figure 6 Geometrical minimization for a group of POPU (A) and POPA (B) molecules performed with Hyperchem 5.1 using AMBER force field. The orientation of purinic rings is strongly altered by the interaction between bases, whereas the pyrimidinic bases keep, more or less, their original conformation. Reprinted with permission from [73]. Copyright 2006 Elsevier.

  inside cells was performed by Montis et al.. These authors investigated the adhesion and fusion processes of DNA-loaded POPA liposomes (formulated with POPC or 1,2-dioleoyl-sn-glycero-3phosphoethanolamine -DOPE -helper lipids) with giant unilamellar vesicles (GUVscomposed of POPC or 1:1=POPC:POPG) used as a model of biological cell membranes. Both nucleolipoplexes and GUV membranes could be labeled with fluorescent probes and the interaction followed by laser scanning confocal microscopy and fluorescence correlation spectroscopy. It was observed that DNA-loaded POPA liposomes entered in contact with GUVs and fused, simultaneously delivering their DNA payload. Authors claimed that they were able to reproduce in vitro a recently highlighted possible non-endocytic internalization pathway happening in vivo for nucleic acid delivery, based on the fusion of lipoplex with the cell plasma membrane [89]. For gene delivery purpose, a different approach was imagined by Arteta et al.: instead of making use of the nucleolipid self-assembling properties, these authors non-covalently decorated poly(amidoamine) (PAMAM) dendrimers with l,2-dilauroyl-sn-glycero-3phosphatidyl-adenosine (DLPA) which has the ability to associate with nucleic acids. It is known that PAMAM dendrimers have the excellent ability to condense and translocate nucleic

Figure 7

 7 Figure 7 Nucleolipoplexes formed by interactions between adenosine nucleolipid derivatives and polyU. (A) Hypothetical arrangement of a hexagonal phase formed by polyU (red helixes) and cylindrical diC8PA micelles (green cylinders). Adapted with permission from [84]. Copyright 2007 John Wiley & Sons. (B) Hypothetical polyU arrangements between POPA membranes. Adapted with permission from [85]. Copyright 2007 American Chemical Society.

  Mougin a , Grégory Pieters c , Sébastien Garcia-Argote c , Adriaan P. IJzerman b and Patrick Couvreur a a Institut Galien Paris-Sud, Université Paris-Sud, CNRS, Université Paris Saclay, Châtenay-Malabry, France; b Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research Leiden University, Leiden, the Netherlands; c Service de Chimie Bioorganique et de Marquage (SCBM), CEA, Université Paris Saclay, Gif-sur-Yvette, France.Accepted in the Journal of Pharmacology and Experimental TherapeuticsSqualene-adenosine nanoparticles: ligands of adenosine receptors or adenosine prodrug? AbstractAdenosine receptors (ARs) represent key drug targets in many human pathologies, including cardiovascular, neurological and inflammatory diseases. To overcome the very rapid metabolization of adenosine, metabolically stable ARs agonists and antagonists were developed. However, few of these molecules have reached the market due to efficacy and safety issues. Conjugation of adenosine to squalene to form squalene-adenosine nanoparticles (SQAd NPs) dramatically improved the pharmacological efficacy of adenosine, especially for neuroprotection in stroke and spinal cord injury. However, the mechanism by which SQAd NPs displayed therapeutic activity remained totally unknown.

  efforts have been devoted to identifying new compounds interacting with ARs(Borea et al., 2018), very few molecules have reached the clinic due to activity and/or toxicity issues. On the other hand, because of a very short half-life in the blood circulation (t1/2 ~ 10 s), adenosine must be administered at high doses which results in severe side effects, thus limiting the clinical use of this molecule. Therefore, innovative pharmaceutical strategies are still needed to fully exploit adenosine's tremendous therapeutic potential.

  SQAd bioconjugate originated from Holochem (Val-de-Reuil, France). D-(+)-glucose was obtained from Sigma (St Quentin Fallavier, France). Absolute ethanol and methanol came from VWR Chemicals (Strasbourg, France) while dimethyl sulfoxide was from Carlo Erba Reagents (Val-de-Reuil, France). MilliQ® water (resistivity 18.2 MΩ•cm) was purified on a system from Millipore (Molsheim, France). CholEsteryl 4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoate (CholEsteryl BODIPY™ FL C12) and wheat germ agglutinin (WGA) Alexa Fluor TM 555 conjugate were purchased from ThermoFisher Scientific (Karlsruhe, Germany). [2,8-3 H]-Adenosine (specific activity 20.3 Ci/mmol) was obtained from Moravek Biochemicals (Brea, CA, USA) while SQ-[ 3 H]-adenosine (SQ-[ 3 H]-Ad) (specific activity 2.8 Ci/mmol) was synthetized as previously reported(Gaudin et al., 2014). Soluene-350 and Hionic Fluor were provided by Perkin Elmer (Courtaboeuf, France). Radioligands 1,3-[ 3 H]-dipropyl-8cyclopentylxanthine ([ 3 H]DPCPX, specific activity of 120 Ci/mmol) and [2-3 H]-4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a}{1,3,5,}triazin-5-yl amino]ethyl)phenol ([ 3 H]ZM241385, specific activity of 50 Ci/mmol) were purchased from ARC Inc. (St.Louis, MO). [ 3 H]8-(4-(4-(4-Chlorophenyl)piperazide-1-sulfonyl)phenyl)-1-propylxanthine ([ 3 H]PSB603, specific activity 79 Ci/mmol) and [ 3 H]8-Ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one ([ 3 H]PSB11, specific activity 56 Ci/mmol) were kindly provided by Prof. C.E. Müller (University of Bonn, Germany). 5'-N-ethylcarboxamidoadenosine (NECA) was purchased from Sigma-Aldrich (Steinheim, Germany). N 6 -cyclopentyladenosine (CPA) was purchased from Abcam (Cambridge, UK). Unlabelled ZM241385 was a gift from Dr. S. M. Poucher (Astra Zeneca, Macclesfield, UK). Adenosine deaminase (ADA) was purchased from Boehringer Mannheim (Mannheim, Germany). Bicinchoninic acid (BCA) and BCA protein assay reagent were obtained from Pierce Chemical Company (Rockford, IL, USA). Chinese hamster ovary cells stably expressing the human A1, A2B or A3 ARs were provided by Prof. S. J. Hill (University of Nottingham, UK), Dr. S. Rees (AstraZeneca, Macclesfield, UK) and Dr. K-N. Klotz (University of Würzburg, Germany), respectively. HEK293 cells stably expressing the human A2A AR (HEK293 hA2AAR) were kindly provided by Dr. J. Wang (Biogen/IDEC, Cambridge, MA, USA). hCMEC/D3 cell line was a gift from Dr. Couraud (Hôpital Cochin, Paris, France). HepG2 cell line, Dulbecco's Modified Eagle's Medium (DMEM) high glucose, Dulbecco's phosphate buffered saline (PBS), lipoprotein deficient serum (LPDS) from fetal calf, trypsin and penicillin-streptomycin, HEPES, hydrocortisone, basic fibroblast growth factor (bFGF), ascorbic acid, adenosine, cathepsin B from human liver, theophylline, dipyridamole, erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) hydrochloride, paraformaldehyde, isobutyl-1-methylxanthine (IBMX) and 4-(3-butoxy-4methoxybenzyl) imidazolidone (Ro 20-1724) were obtained from Sigma (St Quentin Fallavier, France). Endothelial Basal Medium (EBM-2) medium was purchased from Lonza (Basel, Switzerland). Gibco TM fetal bovine serum (FBS) was obtained from Life Technologies (Saint-Aubin, France).

Figure 1

 1 Figure 1).

A and Figure 1 BFigure 1

 11 Figure 1 Displacement of specific radioligands binding from the human adenosine receptors A1 (red circle), A2A (orange square), A2B (yellow triangle) and A3 (green diamond) by increasing concentrations of squalene-adenosine (SQAd) bioconjugate (A and C) or SQAd nanoparticles (NPs) (B and D) diluted in assay buffer (A and B) or preincubated for 4 h at 37 °C in fetal bovine serum (FBS) (C and D). Displacement experiments were performed at 25 °C. TB: total binding; NSB: non-specific binding. Data represent mean values ± standard deviation of three independent experiments.

Figure 2

 2 Figure 2 Uptake of radiolabeled SQ-[ 3 H]adenosine (SQ-[ 3 H]-Ad) NPs (red circle) or [ 3 H]adenosine (orange square) by HepG2 cells (37 °C over 24 h). Data represent mean values ± standard deviation of two independent experiments.

  D), thus demonstrating an influence of LDLRs expression level on SQAd NPs uptake.

Figure 3

 3 Figure 3 Cell uptake of fluorescently labeled SQAd NPs by HepG2 cells. (A, B) Confocal microscopy images of HepG2 cells incubated for 2 h with (A) fluorescently labeled SQAd NPs or (B) the fluorescent tag BODIPY-cholesteryl ester dye as a control, prepared under the same conditions as NPs. Cell membranes were stained with wheat germ agglutinin Alexa Fluor 555 (red) while DAPI was used to visualize nuclei (blue). Scale bar = 50 µm. (C, D) Relative fluorescence measured by flow cytometry from HepG2 cells incubated for 30 min or 2 h with fluorescently labeled SQAd NPs. (C) Cells treated with NPs were incubated in the absence (bright red bars) or in the presence (dark red bars) of dipyridamole (dipy). (D) Cells were preincubated for 24 h with FBS-supplemented (bright red bars) or LPDS-supplemented DMEM (pink bars). Cell auto-fluorescence was subtracted from each data set. Data represent mean values ± standard deviation of three independent experiments. ** indicates p < 0.01, two-way ANOVA with Tukey's post hoc test.

Figure 4 (

 4 Figure 4 (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. AU: arbitrary unit.

Figure 5

 5 Figure 5 Concentrations of adenosine released in the extracellular medium from HepG2 cells exposed to various treatments. (A) 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. (B) Cells were incubated for 2 h with 100 µM of SQAd NPs (red circles), adenosine (Ad) (orange squares) or 5 wt. % dextrose (yellow triangles) and released adenosine concentrations were measured at different times. Data represent mean values ± standard deviation of three (A) or two (B) independent experiments.

Figure 6

 6 Figure 6 Changes in intracellular cAMP levels of naïve cells incubated during 15 min with extracellular medium collected from other cells exposed to SQAd NPs, free adenosine (Ad) or 5 wt. % dextrose (Dext). Changes in relative luminescence units (ΔRLU) were calculated as the difference in light units between treated and untreated cells. Data represent mean values ± standard deviation of three independent experiments. ** indicates p < 0.01, oneway ANOVA with Tukey's post hoc test.

Figure 7 .

 7 Unlike currently proposed agonists and antagonists of ARs, SQAd NPs do not interact directly with the ARs but function in a very original way, as a prodrug delivering high quantities of adenosine into the cells. Hence, cells dispose of an important intracellular reservoir of adenosine, progressively releasing the nucleoside which, in turn, can act as an autocrine or paracrine factor. In conclusion, SQAd NPs provide an efficient system for Chapitre 1 65 prolonged adenosine delivery resulting in AR activation. This new delivery pathway may open interesting prospects in terms of clinical applications.

Figure 7

 7 Figure 7 Schema of the interaction of SQAd NPs with cells. (1) The SQAd NPs are taken-up by the cells through the LDL receptors. (2) When located in the intracellular endo-lysosomal compartments, SQAd behaves as an intracellular reservoir, progressively releasing adenosine. (3) Adenosine is finally discharged outside of the cell and (4) interacts with ARs as an autocrine or paracrine signal. Ad: adenosine; AR: adenosine receptor; ENT: equilibrative nucleoside transporter; LDL: low-density lipoproteins; LDLR: LDL receptor; NPs: nanoparticles; SQAd: squalene-adenosine.

Figure 1

 1 Figure 1 Apparent SQAd NPs diameter and polydispersity index (PdI) after freeze-thawing, as measured by dynamic light scattering (DLS), (A) before and after freezing in water and 5 wt. % carbohydrates solutions or (B) with increasing concentrations of sucrose or (C) trehalose. Data represent mean ± standard deviation (n=3).

Figure 2

 2 Figure 2 Effect of freeze-drying on SQAd NPs (A) apparent diameter, polydispersity index (PdI) and (B) zeta potential (ZP). Characterization by dynamic light scattering (DLS) before (white bars) or after slow freezing (red bars). Data represent mean ± standard deviation (n=3), ** indicates p<0.01, one-way ANOVA with Tukey's post hoc test.

Figure 3

 3 Figure 3 Small-angle neutron scattering (SANS) patterns of freshly prepared nanoparticles (NPs) at 2 mg/mL in D2O (black) and of freeze-dried NPs (after resuspension) at 1 mg/mL, 5 wt. % trehalose in D2O (red). The intensity of the freshly prepared NPs pattern was divided by two to normalize signals for 1 mg/mL.

Figure 4

 4 Figure 4 AFM height (A) and phase (B) images of freeze-dried nanoparticles (1 mg/mL, 5 wt. % trehalose) after resuspension and dilution in water (100 µg/mL).

Figure 5

 5 Figure 5 Influence of ethanol evaporation on apparent NPs diameter before and after freeze-drying. (A) Residual ethanol concentration as a function of the evaporation percentage (defined as the percent change in liquid mass during the evaporation process) and (B) NPs apparent hydrodynamic diameter as a function of the residual ethanol concentration after evaporation (white dots) and after freeze-drying (red dots). Each dot represents a single sample. Curves correspond to least-squares regressions to one-phase exponential decay models.

Figure 7

 7 Figure 7 Colloidal stability of squalene-adenosine nanoparticles (SQAd NPs) after up to three months storage. (A) NPs apparent diameter, (B) PdI and (C) zeta potential, before freeze-drying (bef. FD), or after freeze-drying and storage at room temperature (RT) (circle, solid) or 4 °C (triangle, dotted) and resuspension. Data represent mean ± standard deviation (n=3), **** indicates p<0.0001, one-way ANOVA with Tukey's post hoc test for comparison of stored samples with freeze-dried samples.

Figure 8

 8 Figure 8 HepG2 cells activity after 72 h incubation with freshly prepared or freezedried squalene-adenosine nanoparticles (SQAd NPs) at different concentrations. Data represent mean ± standard deviation (n=3).

Figure 9

 9 Figure 9 Plasma aspartate (AST) and alanine (ALT) transaminases levels in mice subjected (+) or not (-) to liver ischemia. NPs (20 mg/kg, 10 % wt. trehalose) or vehicle (10 % wt. trehalose) were injected 15 min before proceeding to a 40-min global hepatic ischemia. Plasma samples were collected 24 h after reperfusion on surviving mice. Bars represent mean ± standard deviation.

  has been successfully performed by lyophilization of squalene-adenosine nanoparticles and the Chapitre 2 93 demonstration has been done that the diameter, the zeta potential and the supramolecular structure of the particles were preserved. Additional in vitro and in vivo experiments have established the safety of this nanoformulation towards the liver tissue, the major organ of nanoparticles accumulation after systemic intravenous administration.

Figure 1

 1 Figure 1 Effect of SQAd NPs at different doses and times of injection on a mouse model of global liver ischemia. (A) Mice were anesthetized with isoflurane, opened by laparotomy and subjected to global ischemia by clamping the portal triad for 25 min. After 6 h of reperfusion, blood was collected and ALAT levels were measured. (B) NPs were injected at different time points. (C-E) Plasma ALAT levels (UI/L) of mice before (black dots) and after (colored dots) global ischemia and treatment by intravenous injection of SQAd NPs suspensions at different doses 24 h (C) or 10 min (D) before ischemia, or 30 min after ischemia (E). Each dot represents a mouse. Bars represent mean ± standard deviation. (F) All mice survived to 6 h reperfusion, except when the treatment was injected 10 min before ischemia. Each bar represents four mice.

Figure 2

 2 Figure 2 Mice survival after 40 min ischemia. Freshly prepared and freeze-dried SQAd NPs were injected 15 min before ischemia at a dose of 20 mg/mL.

Figure 3

 3 Figure 3 Proposed mechanisms of action of SQAd NPs (A), free adenosine (B) and adenosine receptor agonist (C). (A) After injection into the blood stream, SQAd NPs are internalized by cells and leading to an intracellular reservoir of SQAd bioconjugates, slowly releasing adenosine into the cell. Adenosine is then effluxed outside the cell, where it can bind adenosine receptors on neighbouring cells, inducing the activation of various signaling pathways which may interfer. (B) When free adenosine is injected in the blood stream, it is rapidly degraded and eventually only has an effect on cells located near the site of injection and during a very short period of time. (C) Specific adenosine receptor agonists or antagonists (e.g. A2A receptor agonist in the figure) usually present enhanced pharmacokinetics compared to free adenosine and reach their target all over the body. They selectively bind one of the adenosine receptors and induce the specific signaling pathway of this receptor.

  Tu déchaines la fureur d'Elias notre roi Car nous tous, pauvres gueux, doctorants et stagiaires Nous battons sans répit pour ton petit laser. Ô, que ne donnerais-je point pour ta compagnie, Quelques heures avec toi, ou bien toute une nuit, Regarder ton écran, mesurer mon zêta, Sont pour moi un bonheur qui ne se compare pas. Car oui, je vous l'avoue, j'aime tant ces mesures, Cette petite salle odorante et obscure Où l'on ne peut rien faire mis à part s'ennuyer, Que sur le GRR je réserve des heures Et tant pis pour tous ceux dont je fais le malheur, Le zêta est à moi, jamais vous ne l'aurez ! Université Paris-Saclay Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint-Aubin, France Titre : Avancées dans le développement pré-clinique des nanoparticules d'adénosine-squalène : mécanisme d'action et formulation Mots clés : nanomédicine ; adénosine ; squalène ; bioconjugaison ; prodrogue ; délivrance de médicament Résumé : L'adénosine est une molécule dotée d'un fort potentiel thérapeutique, mais présentant néanmoins un temps de demi-vie plasmatique extrêmement court qui limite de manière sérieuse son efficacité. Comme présenté dans l'introduction bibliographique de cette thèse, cette difficulté peut être surmontée grâce à l'utilisation de systèmes de délivrance de médicaments à base de lipides. L'adénosine peut en effet être soit encapsulée dans des liposomes, soit simplement couplée à un lipide. Parmi les « lipidizations » de l'adénosine, la « squalénisation », notamment, a favorablement modifié la biodistribution de cette substance active. Cette technique consiste à coupler l'adénosine à une molécule lipophile dérivée du squalène, générant ainsi des bioconjugués ayant la capacité de s'auto-assembler spontanément en milieu aqueux sous forme de nanoparticules d'une centaine de nanomètres de diamètre. L'injection de ces nanoparticules d'adénosine-squalène (AdSQ) par voie intraveineuse a donné des résultats très prometteurs pour le traitement de l'ischémie cérébrale et du traumatisme de la moëlle épinière. Ainsi, l'objectif de cette thèse a consisté à faire progresser le développement préclinique de ces nanomédicaments suivant deux axes principaux : l'étude du mécanisme d'action et l'amélioration de la formulation. De ce fait, le premier chapitre de cette thèse présente les résultats obtenus lors de l'étude in vitro du mécanisme d'action des nanoparticules d'AdSQ. Les travaux ont montré que ces nanoparticules d'AdSQ 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. Après étude du mécanisme, l'amélioration de la formulation de ces nanoparticules a été explorée et décrite dans le deuxième chapitre. Les efforts ont été principalement concentrés sur la lyophilisation de la suspension nanoparticulaire, afin de proposer une formulation stable dans le temps et facile d'utilisation dans le cadre médical. Les conditions utilisées ont aboutit au bon maintien des propriétés physicochimiques des nanoparticules et l'obtention de solutions injectables sans risque chez l'animal. Dans son ensemble, ce travail de thèse a permis d'élargir les perspectives d'application des nanoparticules d'AdSQ grâce à une meilleure compréhension de leur mécanisme d'action ainsi que la mise au point d'une formulation plus adaptée aux besoins cliniques. Title : Moving forward in the pre-clinical development of squalene-adenosine nanoparticles: mechanism of action and formulation Keywords : nanomedicine; adenosine; squalene; bioconjugation; prodrug; drug delivery

Table 1

 1 Adenosine, ADP and ATP-loaded liposomes.

	Drug loaded	Lipid composition	Liposome size (nm)	Drug loading (µmol drug / µmol lipid)	Encapsulation efficiency (%)	Therapeutic target	References
		DSPC:DSPG:chol	168 ± 18	n.d.	12.9
		DSPC:chol	206 ± 40	n.d.	8.6	Inflammation	[16]
	Adenosine	DSPC:DODAB:Chol HSPC:chol:DSPE-PEG	202 ± 27 134 ± 21	n.d. n.d.	10.5 n.d.	Myocardial ischemia	[17]
		Soybean oil:PC:chol:glycerin	n.d.	n.d.	n.d.	Osteoarthritis	[18]
		DMPC:chol:DHSG:DSPE-PEG:H12-PEG-Glu2C18	240 ± 68	n.d.	n.d.
	ADP	DPPC:chol:DHSG:DSPE-PEG:H12-PEG-Glu2C18	285 ± 78	n.d.	n.d.	Hemostasis	[19-22]
		DSPC:chol:DHSG:DSPE-PEG:H12-PEG-Glu2C18	298 ± 107	n.d.	n.d.
		PC:chol:SM4	~ 140	≤ 0.39	n.d.	Cerebral	[7,23]
		PC:chol	n.d.	n.d.	n.d.	ischemia	[24]
		PS		n.d.	n.d.
		PC	~ 100	n.d.	n.d.	Retinal ischemia [25,26]
		PS:PC		n.d.	n.d.
		PC:chol:stearylamine	> 2000	0.32 ± 0.02	n.d.		[27]
	ATP	PC:chol:DSPE-PEG:DOTAP PC:chol:DSPE-PEG:2G4-PEG-PE ~ 200 ~ 200 PC:chol:DSPE-PEG:DOTAP:2G4-PEG-PE 190 ± 45	c.a. 0.40 n.d. c.a. 0.40	n.d. n.d. n.d.	Myocardial ischemia	[28,29] [30] [31]
		PC:chol	n.d.	n.d.	8		[32]
		PC:chol:PG	~ 200	0.35 ± 0.06	n.d.		[33,34]
		PC:chol PC:chol:PG PC:chol:ODA	~ 100	n.d. n.d. ≤ 0.07 ± 0.01	n.d. n.d. n.d.	Liver ischemia	[35,36]
		PC:chol:DOTAP		n.d.	n.d.

CHEMS: cholesteryl hemisuccinate; Chol: cholesterol; DHSG: 1,5-dihexadecyl-N-succinyl-l-glutamate; DODAB: dimethyldioctadecyl ammonium bromide; DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOPC:

Table 2

 2 Adenosine nucleolipids and observed macrostructures.

	Chemical modification site	Conjugate name	Lipidic chains	C:D	Conjugated lipid	Type of link	Observed structures	Reference
		AMPC16	1	16:0	n-hexadecyl-phosphorylcholine		Micelles	[64,65]
							Vesicles aging into double
		diC8PA	2	8:0	diC8PC		helical strands	[55,66-68]
							Quasi-spherical micelles
	5'-hydroxyl group	DDPA	2	10:0 DDPC	Phosphate	Vesicles aging into double helical strands	[55]
	of ribose	DLPA	2	12:0 DLPC	diester	Long helicoidal micelles and wormlike structures	[55,67,69,70]
		DMPA	2	14:0 DMPC		Multihelical strands	[55]
		POPA	2	16:0 18:1	POPC		Liposomes	[71-74]
		DOP-Ade	2	18:1 DOPC		Liposomes	[71,75,76]
			1		Octadecylamine			[58]
			2		Dioctadecylamine		Stable monolayers	[58]
	2',3'-hydroxyl groups of ribose	PAPC	2 2	N,N-dioctadecyl-l,3-propanediamine 16:0 Palmitone	Ketal	Stable monolayers Large lamellar structures Hydrogels	[58] [59,77]
		SAPC	2	18:0 Stearone		Hydrogels Lamellar structures	[77]

Table 1 .

 1 al., 2009; Yang et al., 2017).Buffers compositions, membranes concentrations, radioligands and non-specific binding compounds used for each receptor type, as well as their concentrations, are listed inSupplementaryFor radioligand displacement assays, membrane aliquots at given concentrations

(Supplementary Table

1

) were incubated in a total volume of 100 µL assay buffer (Supplementary Table

1

) at 25 °C for 60 min (A1) to 120 min (other receptors). The dimethyl sulfoxide (DMSO) stock solution of SQAd as single bioconjugate molecules or the SQAd NPs preparation were diluted in assay buffer or FBS (DMSO final concentration < 1 %). Samples with serum were incubated for 4 h at 37 °C on an Eppendord Thermomixer before the experiment. Displacement experiments were performed in the presence of specific radioligands at given concentrations (Supplementary Table

1

), while nonspecific binding was determined in the presence of non-specific binding compounds at given concentrations (Supplementary Table

Table 1

 1 Samples osmolality (mOsm/kg) before and after freeze-drying (FD).

		Osmolality (mOsm/kg)
	Solution	Before FD	After FD
	5% trehalose	151 ± 2	142 ± 1
	NPs	14 ± 6	/
	NPs + 5% trehalose 165 ± 7	140 ± 4

Remerciements

Remerciements

Remerciements

Et merci finalement à cet enfant pour la confiance que tu m'accordes en grandissant jour Acknowledgements .............................................................................................................................................. Authors contributions......................................................................................................................................... V. Acknowledgements .............................................................................................................................................. VI. References ...................................................................................................................................... VII. Supplementary materials ....................................................................................................... Acknowledgements Authors would like to thank Ghozlene Mekhloufi and Assia Hessani, Stéphanie Denis, Juliette Vergnaud and Romain Brusini (Institut Galien, France) respectively for experiments involving radioactivity, cell culture experiments, Western blots and flow cytometry; Valérie Nicolas (IPSIT, France) for confocal microscopy; Delphine Courilleau (CIBLOT, IPSIT, France) for experiments on cAMP; Rongfang Liu (Division of Medicinal Chemistry, the Netherlands) for preliminary experiments on radiobinding and cell membrane preparation. Acknowledgements Special thanks to Professor Patrick Couvreur who has been greatly involved in this work. The authors also acknowledge the support provided by Patrick Guenoun (IRAMIS, CEA) and Isabelle Grillo (Institut Laue Langevin) in SANS experiments, and Javier Perez (Synchrotron SOLEIL) in SAXS experiments. Finally, authors are deeply grateful to Anne Beilvert (Occlugel) for her assistance with the freeze-dryer.

medium supplemented with LPDS instead of FBS. Cells were then incubated for 24 h to enhance LDLRs expression, as proven by Western Blot (protocol from (Sobot et al., 2017a), Supplementary Figure 2) and subsequently treated with fluorescently labelled SQAd NPs (10 µg/mL). In both cases, after 30 min or 2 h, cells were finally washed with PBS, detached with trypsin and resuspended in PBS. Cell fluorescence was evaluated using a BD Accuri TM C6 flow cytometer (BD Biosciences) with an excitation wavelength of 488 nm. Emission was recorded with the FL1 filter (533/30 nm).

7) SQAd degradation in acidic conditions

A solution of 1 µL SQAd (2.5 mg/mL dissolved in DMSO) was incubated at 37 °C under shaking in 100 µL of 0.3 M sodium acetate buffer (pH 4.8) containing cathepsin B (10 U/mL).

Degradation was stopped at different time points by the addition of 233 µL methanol. 5 µL gemcitabine-squalene (0.2 mg/mL dissolved in DMSO) were added as internal standard.

Samples were centrifuged (14 000 rpm, 10 min) and resulting supernatants were evaporated to dryness at 37 °C under air flow. The dried samples were then dissolved in 100 µL of mobile phase (methanol for SQAd detection; methanol/KH2PO4 (10 mM, pH 4) 20/80 v/v, for adenosine detection) prior to HPLC analysis.

8) Adenosine extracellular release

HepG2 or hCMEC/D3 cells were treated with SQAd NPs (100 µM), free adenosine dissolved in 5 wt. % dextrose (100 µM) or 5 wt. % dextrose. After 5 min to 4 h, extracellular medium was replaced by 1 mL of simplified medium (composition is given in Supplementary Table 2) supplemented with 50 µM EHNA (to avoid adenosine deamination) and 2 µM internal standard (theophylline). After 5 min to 4 h, the extracellular medium was collected and extracted with 2.3 mL of methanol. Samples were centrifuged at 17 000 x g for 10 min to remove insoluble materials and supernatants were filtered and evaporated to dryness at 37 °C under air flow.

The dried samples were dissolved in 100 µL of mobile phase (methanol/KH2PO4 (10 mM pH 4) 20/80 v/v) and filtered before HPLC analysis. The detection limit of the HPLC technique was 17 nM for the adenosine, while the quantification limit was 55 nM. This method exhibited linearity (R 2 = 0.99) over the assayed concentration range (100 -1000 nM) and demonstrated good precision with relative standard deviation (RSD) being all less than 5%. was obtained from Life Technologies (Saint-Aubin, France).

Authors contributions

2) SQAd NPs preparation

SQAd NPs were prepared by the nanoprecipitation method. Briefly, the SQAd bioconjugate was dissolved into pure ethanol (6 mg/mL) and 333 µL of this solution was added dropwise into 1 mL of water solution under stirring (500 rpm). The solution became spontaneously turbid with a slight Tyndall effect, indicating the formation of the nanoparticles. The total mass mi was evaluated. Ethanol was evaporated with a Rotavapor ® R-215 (Buchi, Switzerland): the pressure was slowly decreased to 43 mbar and the flask was immersed into the water-bath at 40 °C. The mass mf remaining in the flask after evaporation was evaluated and water was added to reach a final volume of 1 mL (considering a density of 1) and a concentration of 2 mg/mL.

The percentage of evaporation % 𝐸𝑣𝑎𝑝 was defined as:

If not stated, evaporation was over 50 %.

3) Dynamic light scattering (DLS)

The mean NPs size, polydispersity index (PdI) and zeta potential were evaluated from 1 mg/mL suspensions by DLS using a Zetasizer Nano ZS (173° scattering angle, 25 °C) (Malvern, UK). The measurements were performed in triplicate: size measurements consisted in 1 run of 60 s each, while zeta potential measurements were recorded in automatic mode. Due to limited information in the literature regarding carbohydrates solutions viscosities and refractive indexes at 25 °C, water values were used for all measurements. Hence, apparent size and zeta potential are reported.

The addition of trehalose did not impact the HII structure, as shown by the pattern of NPs with residual ethanol (Figure 6A). In contrast, the SAXS pattern of NPs rehydrated after freeze-drying displayed a broad band, around q~0.085 Å -1 , very close from the previously identified q0, and a new peak at 0.13 Å -1 (Figure 6B), corresponding to a different phase. Based on previous data, this suggests the coexistence of the initial HII phase and a lamellar phase with a d-spacing of 48 Å. Indeed, a lamellar phase with the same d-spacing has already been found in SQAd NPs (17).

8) Long-term conservation

Under the best conditions (in terms of cryoprotection and ethanol content), the freeze-drying process did not affect the chemical structure of SQAd, which was checked by NMR (see Supplementary Figure 2). The residual moisture after lyophilization was also measured by the weight-on-drying method and was found to be low (1,07 ± 0,57 wt. %).

As a result, freeze-dried samples could be characterized for stability upon long-term storage at room temperature (RT) or 4 °C. As can be seen on Figure 7, no change in apparent diameter, PdI or zeta potential could be recorded for up to two months of storage. However, a slight increase in size was noticed after three months storage at RT, which was not observed at 4 °C. Hence, SQAd NPs displayed a very good long-term stability when stored at 4 °C. This study is still ongoing, with the aim of characterizing NPs stability for up to two years.

VII. Supplementary materials

Supplementary Figure 1 SQAd NPs samples after freeze-drying, using 5 % wt. sucrose (left) or 5 % wt.

trehalose (right).

Discussion Générale

The original aim of this thesis was to evaluate the pharmacological efficacy of SQAd NPs on liver ischemia and reperfusion. The main hypothesis was that SQAd NPs would protect the liver from this pathology, which was supported by three main arguments.

First, an increase of endogenous extracellular adenosine was shown to happen during liver ischemia, as a result of transcriptional repression of ENTs (1) as well as CD39 (2) and CD73

(3) overexpression, and this increase in adenosine protected the liver against ischemiareperfusion injury (1,4). More specifically, adenosine acted via its four receptors (A1, A2A, A2B, A3) and particularly by A2A receptor which down-regulates inflammation (5)(6)(7)(8) and by A2B receptor which protects hepatocytes (1,9).

Secondly, SQAd NPs had previously been proven to be effective against cerebral ischemia on mice (10), which admittedly was another organ but had, however, similar pathological features including ATP depletion and cellular acidosis during ischemia, as well as, ROS generation and inflammation during reperfusion (11).

Thirdly, SQAd NPs had been found to accumulate in important quantities in the liver after intravenous injection (12) without triggering any hepatotoxic effect in healthy mice (10).

Moreover, this accumulation of SQAd NPs resulted in the release of free adenosine in the liver tissue (12).

Based on these evidences, it was decided to test the pharmacological efficacy of SQAd NPs on a mouse model of global liver ischemia.

Annexes