Betty Gourdon

Caroline Chemin

Xavier Declèves

Oral delivery of BCS III molecules: An overview of the existing approaches and strategies. Focus on the intestinal epithelium transporters and receptors targeting

Keywords:

Ce travail a été effectué à l'Université Paris Descartes, au sein de la Faculté de Pharmacie dans l'UMR-S1144, et en collaboration avec Technologie SERVIER à Orléans. Je tiens tout d'abord à remercier les membres du jury pour avoir accepté d'évaluer mon travail. Je remercie Véronique Préat et Hervé Hillaireau pour m'avoir fait l'honneur d'être les rapporteurs de ma thèse. Je remercie aussi Patrick Hindlet pour avoir examiné ce travail de recherche. J'adresse une profonde reconnaissance à Nathalie Mignet pour avoir présidé ce jury. Je remercie par ailleurs Xavier Declèves et Caroline Chemin, mes directeurs de thèse, pour la confiance témoignée au cours de ces trois ans de doctorat. Merci pour vos expertises chacun dans votre domaine, pour votre aide dans ce travail et pour toutes les connaissances que vous m'avez transmises et qui m'ont permis de faire avancer ce projet. Je tiens également à remercier le Pr Jean-Louis Laplanche directeur du laboratoire « Variabilité de réponses aux Psychotropes » pour m'avoir accueillie au sein de l'unité et

 Philippe Baumy et Jean-Marie Delbos pour leur expertise en pharmacocinétique et leur aide jusqu'au dernier jour.

 L'équipe de toxicologie et en particulier Hélène Aerts pour sa réactivité.

 Fabienne Munari, Florie Chenaye et Olivier Drillaud pour vos très nombreuses analyses RMN et votre réactivité.

 Antoine Raguet pour avoir assuré le SAV en HPLC même à 19h30 depuis son canapé, et Cyrielle Lemaire pour le SAV mais cette fois en LC/MS, parce que parfois un bout de carton peut résoudre beaucoup de choses.

Je tiens à remercier les équipes de biologie cellulaire, de pharmacocinétique et de radiopharmacie de Paris Descartes. Même si mes séjours parmi vous étaient parfois éclairs, je vous remercie pour votre aide, vos explications sur la vie du labo et surtout pour votre accueil.

Merci également à tous les doctorants ou post-doctorants parisiens qui ont pu m'aider, me conseiller, et surtout m'intégrer. Même si je ne suis pas souvent venue aux afterworks, je garderai de très bons souvenirs de nos repas du midi ou de réunions tous ensembles.

Un merci tout particulier à toute l'équipe de galénique d'Orléans. Ces trois années à vos côtés ont été enrichissantes tant du point de vue scientifique qu'humain. Merci de m'avoir si bien intégrée parmi vous, merci pour vos conseils, nos discussions… La pause café du matin va me manquer.

Je remercie également Claire et Lorine pour m'avoir apporté l'aide qui me manquait pendant votre stage ou apprentissage. Je vous souhaite le meilleur pour la suite.

Merci à tous les stagiaires, doctorants, post-doctorants que j'ai croisés sur mon chemin à Orléans, les années se suivent et ne se ressemblent pas. Impossible d'oublier de remercier la coloc. Merci de m'avoir soutenue, écoutée, aidée pendant ces trois années à vos côtés dans ce bureau. Merci de m'avoir gardé patiemment ma place, ça n'aurait pas été pareil sans vous.

Merci à tous mes amis d'Orléans, Paris, Lyon : Coralie, Fabienne, Camille, Céline, Marion, Laure… Ces trois années n'auraient pas été les mêmes sans nos lundi sportifs, nos mardi ciné, nos weekends parisiens, angevins, ski et j'en passe.

INTRODUCTION GÉNÉRALE CONTEXTE & OBJECTIFS

Introduction

L'administration d'une molécule thérapeutique par la voie orale est le graal de tout développeur depuis des années. Aussi, il est prévu que ce marché dépasse les 100 milliards de dollars en Amérique du nord en 2018, sachant qu'il représentait déjà 38% du marché pharmaceutique en 2012 (American Pharmaceutical Review, 2012). C'est sa simplicité et son confort, permettant une bonne observance du traitement par les patients, mais aussi son développement simplifié par comparaison aux formes parentérales stériles, qui en font tout son attrait.

Cependant, toutes les molécules ne peuvent être administrées par cette voie privilégiée. En effet, en raison de la barrière intestinale, leur biodisponibilité peut être limitée. Afin de rejoindre la circulation systémique, un principe actif doit non seulement être soluble dans le milieu gastrointestinal mais doit également être capable de traverser la barrière intestinale. Ainsi, les molécules appartenant à la classe III du Système de Classification Biopharmaceutique (BCS III) sont très hydrophiles et donc très solubles dans le milieu aqueux gastro-intestinal, mais elles ont peu d'affinité pour la membrane intestinale et sont donc faiblement absorbées (Amidon, Lennernas et al., 1995). Cette faible biodisponibilité par voie orale entraîne non seulement une efficacité limitée et des variabilités inter-et intra-individuelles importantes mais également la nécessité d'administrer des doses plus conséquentes pouvant engendrer une toxicité locale au niveau de l'intestin. C'est pourquoi ces molécules sont généralement administrées par voie parentérale ce qui leur permet de rejoindre directement la circulation sanguine, mais ce qui alourdi considérablement les traitements.

Les molécules appartenant à la classe BCS III représentent une part non négligeable des molécules thérapeutiques. Avec l'arrivée sur le marché de Nouvelles Entités Moléculaires (NEMs), tels que les peptides ou les protéines, cette classe connait un très grand essor. En 2015, on comptait environ 140 molécules en essais cliniques et plus de 500 en précliniques. Le marché global des peptides est en réelle explosion et devrait atteindre 25.4 milliards de dollars en 2018 (Fosgerau and Hoffmann, 2015). Leur utilisation est cependant limitée car leur faible biodisponibilité requiert le développement de formulations complexes afin de les rendre administrables par la voie orale. L'administration de peptides par la voie orale est un sujet d'étude majeur pour la communauté scientifique depuis des années. Elle connaît même actuellement un regain d'attractivité pour les entreprises pharmaceutiques qui s'explique en plusieurs points selon Brayden et Alonso [START_REF] Brayden | Oral delivery of peptides: opportunities and issues for translation[END_REF].

 Tout d'abord, la fabrication de peptides synthétiques et recombinants est devenue moins onéreuse. L'accès à des molécules plus puissantes, qui même avec une faible biodisponibilité offrent une réponse thérapeutique intéressante est désormais possible.

 Des peptides de faibles masses moléculaires sont rendus plus stables et des pro-drogues lipidiques de peptides voient le jour. De plus, il apparait une biodisponibilité non négligeable pour certaines molécules ne répondant pas exactement aux règles de Lipinski.

 L'argument de l'observance du traitement se révèle ne pas être le seul avantage d'une administration per os. En effet, certaines molécules offrent une meilleure réponse clinique par voie orale que par voie systémique.

 Des avancées majeures ont été réalisées dans les domaines des nouvelles technologies, des nouvelles techniques de caractérisation, en imagerie ou dans la connaissance des modèles in vitro et in vivo. Ceci permet une meilleure connaissance des mécanismes d'actions, du rôle de chaque composant, et offre une meilleure efficacité dans le design de nouvelles formulations.

Tout ceci conduit à un nombre croissant de formulations de peptides pour la voie orale en essais cliniques, ce qui est non seulement encourageant mais également prometteur.

De nombreuses stratégies permettant l'amélioration de la biodisponibilité des molécules par voie orale ont été développées ces dernières décennies. Elles agissent sur l'augmentation du transport passif ou actif à travers la membrane intestinale. Leur mode d'action peut consister à augmenter le transport des molécules ou bien à les protéger de l'environnement gastro-intestinal extérieur. Il est possible d'agir directement sur la structure de la molécule, par co-administration avec des agents propices à l'amélioration du transport, ou par la formulation de systèmes galéniques adaptés.

C'est dans ce contexte que s'inscrit le sujet de cette thèse consistant à « Concevoir un système d'administration pour la voie orale interagissant avec les cellules gastro-intestinales pour améliorer les performances biopharmaceutiques d'un principe actif ».

L'enjeu est alors de trouver une nouvelle approche innovante, adaptée aux molécules BCS III et plus particulièrement à l'administration d'un peptide.

Une des approches largement exploitées et des plus prometteuses est le ciblage de récepteurs ou de transporteurs de la membrane intestinale par des pro-drogues ou des nanoparticules vectorisées. Dans le cas des pro-drogues, la molécule d'intérêt est directement fonctionnalisée avec un ligand reconnu par la membrane intestinale, ce qui permet d'augmenter son transport en favorisant son internalisation. Dans le cas des nanoparticules, le principe actif faiblement absorbé est encapsulé, et c'est l'objet qui est fonctionnalisé avec un ligand. Une lecture approfondie de la littérature a permis de conclure sur le fait que le ciblage de récepteurs est réalisé à la fois avec des pro-drogues et des nano-objets tandis que les transporteurs sont uniquement ciblés avec des prodrogues. Ceci s'explique par le fait que les transporteurs intestinaux permettent le passage de petites entités et non de macro-molécules telles que des nanoparticules.

Désormais, toutes ces approches ont déjà largement été étudiées et l'intérêt de cibler un énième transporteur avec une nouvelle pro-drogue ou un énième récepteur avec un nouveau design de nanoparticules reste limité. Aussi, l'idée de ce travail de recherche est d'utiliser les acquis développés sur des stratégies bien connues jusqu'alors, et de coupler plusieurs approches afin d'aboutir à un nouveau système d'administration pour la voie orale pour des molécules faiblement absorbées.

Ainsi, l'objectif de ce travail de thèse est de mettre en place une stratégie de ciblage du transporteur intestinal PepT1 avec des nanoparticules polymères fonctionnalisées encapsulant une molécule BCS III. Le but initial n'étant pas de favoriser le passage du nano-objet mais de cibler le transporteur afin de créer une surconcentration de la nanoparticule et donc de la molécule encapsulée près de la membrane intestinale, et ainsi augmenter son absorption. Le transporteur PepT1 ayant été choisi car il est bien connu et déjà largement étudié dans des stratégies de ciblage avec des pro-drogues (cf. Valacyclovir, pro-drogue de l'agent antiviral Acyclovir).

Au cours de cette investigation, plusieurs questions ont pu être soulevées :  Est-il possible de cibler un transporteur intestinal avec des nanoparticules fonctionnalisées ?

 Cette stratégie permet-elle l'amélioration du transport d'une molécule hydrophile in vitro et in vivo ?

 Est-il possible d'encapsuler un peptide dans des nanoparticules polymères selon plusieurs techniques de fabrication ?  La technique de fabrication des nanoparticules influe-t-elle sur la structure des nanoobjets, sur leur capacité de ciblage et donc d'amélioration de la biodisponibilité de la molécule encapsulée ?

Le corps de ce manuscrit se compose d'une première partie bibliographique présentant le contexte scientifique nécessaire à la compréhension des objectifs et de la stratégie adoptée lors de cette thèse. Une seconde partie expérimentale présente les résultats obtenus répondant aux objectifs et interrogations soulevés par le sujet et précédemment exposés.

L'étude bibliographique de ce manuscrit est divisée en deux parties. Le premier chapitre présente une revue des différentes approches et stratégies développées pour l'administration orale de molécules de classe BCS III. Une attention particulière est portée sur la stratégie de ciblage des récepteurs et transporteurs intestinaux permettant l'amélioration du transport actif. Une présentation des technologies déjà présentes sur le marché sera finalement faite pour conclure ce chapitre. Le second chapitre de l'étude bibliographique est consacré à la formulation des nanoparticules polymères pour l'encapsulation de molécules de classes BCS III. Y sont détaillées les techniques de fabrication et de caractérisation des nanoparticules.

L'étude expérimentale est divisée en trois parties présentées sous forme d'articles scientifiques.

Le premier chapitre est la preuve de concept de la stratégie envisagée. Ainsi, il y sera tout d'abord étudié la capacité de nanoparticules fonctionnalisées à cibler le transporteur intestinal PepT1. Puis, la possibilité d'améliorer le transport in vitro et in vivo d'une molécule BCS III modèle, encapsulée dans ces nanoparticules, sera explorée. Le second chapitre consiste à formuler des nanoparticules polymères pour l'encapsulation d'un peptide par deux techniques différentes. Pour cela, l'influence de paramètres de formulation et de procédés sur la taille des nanoparticules, leur stabilité et le taux d'encapsulation sera étudiée grâce à des plans de criblage.

Le troisième chapitre permettra de s'interroger sur l'influence de la technique de fabrication des nanoparticules polymères sur leur capacité à cibler PepT1 et donc à améliorer le transport de la molécule encapsulée in vitro et in vivo.

Les principaux résultats obtenus seront repris et discutés dans la partie discussion générale, perspectives et conclusion.

Introduction

Cette première partie, présentée sous la forme d'une revue scientifique, introduit le contexte scientifique nécessaire à la compréhension des objectifs et de la stratégie adoptée lors de cette thèse.

Dans un premier chapitre présenté sous forme de revue scientifique seront décrites les différentes approches et stratégies existantes pour l'administration par voie orale de molécules BCS classe III. Des exemples précis seront donnés afin d'illustrer l'intérêt de chacune de ces stratégies. Une attention particulière sera portée sur le ciblage des récepteurs et transporteurs intestinaux afin d'augmenter la biodisponibilité des molécules classées BCS III. Des exemples de pro-drogues et de nanoparticules fonctionnalisées encapsulant des molécules d'intérêt seront donnés afin de montrer l'apport de cette stratégie sur leur absorption. Pour conclure, ce chapitre mettra en lumière les différentes techniques présentes sur le marché et repositionnera les enjeux du sujet de thèse au regard de l'existant.

Le second chapitre sera consacré plus particulièrement à l'encapsulation de molécules BCS III dans des nanoparticules polymères. Les polymères utilisés, les procédés de formulations et les méthodes de caractérisation seront détaillés et illustrés par des exemples appliqués aux molécules BCS III. L'objectif de ce chapitre est d'apporter les connaissances nécessaires à la compréhension de toute la partie formulation présente dans ce travail de thèse.

Introduction

The oral administration route is the privileged administration pathway for medication mainly because of its simplicity which allows a better patient compliance. In this way, orally administered drugs represent 38% of the North American drug delivery market in 2012 (American Pharmaceutical Review, 2012). It is attended to cross $100 billion by 2018. On the other hand, BCS III drugs represent a non-negligible part of current therapeutic molecules and two main molecule characteristics are controlling oral drug absorption according to Amidon et al. (Amidon, Lennernas et al., 1995): the aqueous solubility and the gastrointestinal permeability.

They designed a Biopharmaceutics Classification System (BCS) and drugs were thus categorized in four classes according to these two parameters (Figure 1). The main objective of this classification was to predict in vivo pharmacokinetic performance of molecules from measurements of permeability and solubility. A drug is considered as "highly soluble" if the highest dose strength is soluble in 250 mL or less of aqueous media over a pH range of 1 to 7.5 at 37°C. It is considered to be "highly permeable" when the absorption in humans is at least 90% of an administered dose based on a mass balance determination or in comparison to an intravenous reference dose [START_REF] Wu | Predicting drug disposition via application of BCS: transport/absorption/ elimination interplay and development of a biopharmaceutics drug disposition classification system[END_REF]. For Class III molecules permeation rate through the intestinal membrane is limited leading to low bioavailability which required permeability enhancements. Small entities could be classified as BCS III such as acyclovir, neomycin B, captopril, enalaprilate, alendronate or ranitidine. But in this regard, New Molecular Entities (NMEs) including peptides and proteins are part of this BCS III class [START_REF] Nikhil | Biopharmaceutical classification system: A strategic tool for oral drug delivery technology[END_REF]. With approximately 140 therapeutic peptides currently evaluated in clinical trials and more than 500 in preclinical development, the global peptide drug market has been predicted to increase from US$14.1 billion in 2011 to an estimated US$25.4 billion in 2018 (Fosgerau and Hoffmann, 2015). While the peptide and protein therapeutic market has developed significantly in the past decades, their administration to the body remains an issue, especially by the oral route. Indeed, most of NMEs does not reside within Lipinski's Rule of 5 (Ro5) space proposed in 1997 [START_REF] Lipinski | Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings[END_REF]. Molecules following the guidelines (Mw ≤ 500 Da, cLogP ≤ 5, Hydrogen Bond Donors ≤ 5 and Hydrogen Bond Acceptors ≥ 10) are more likely orally absorbed and 90% of molecules from oral drug design of the time respect three out of four of these rules. In this way, conventional formulation strategy cannot be applied due to very limited oral bioavailability for these NMEs. Thus, highly sophisticated formulations have to be developed. Companies are actively working on this topic such as ENTERIS (Boonton, USA) or EMISPHERE (Roseland, USA), with one product currently in phase 3. This review gives an insight of the different approaches explored in order to improve oral permeability of poorly permeable molecules. A rapid overview of strategies allowing the improvement of passive transport will be first presented. Then, the review will particularly focus on the intestinal receptors or transporters targeting approach meaning the enhancement of the active transport. Specific examples involving BCS III therapeutic molecules will be given to illustrate the current advance carried on until now.

Approaches and strategies

Oral administration of drugs has to face the gastrointestinal epithelium which behaves as a chimico-physical barrier for molecules (Catnach, Fairclough et al., 1994;[START_REF] Muheem | A review on the strategies for oral delivery of proteins and peptides and their clinical perspectives[END_REF][START_REF] Renukuntla | Approaches for enhancing oral bioavailability of peptides and proteins[END_REF]. Bioavailability of drugs depends on their ability to cross the intestinal barrier and reach the systemic circulation (Johnson, 1994;Kwan, 1997).

Intestine is divided in several segments consisting themselves of several parts (Fig. 2).

Gastrointestinal pH varies from 1.0 to 3.0 in stomach, 6.0 to 6.5 in duodenum and 5.5 to 7.0 in large intestine (Van de graaff K.M, 1986). As main site of absorption for drugs and more than 90% of nutrients, the small intestine supplies a large surface area allowing to reach the bloodstream or lymphatic circulation [START_REF] Tortora | Principles of Anatomy and Physiology[END_REF]. Its inner surface is made of mucosa, a secreting layer containing many projections (villi) responsible for the absorption of nutrients and drugs. Drug transport across the intestinal epithelium is mediated by passive and active transport processes according to the molecules properties (Fig. 3). Passive diffusion is described as a combination of paracellular and transcellular transport. Paracellular process consists in a transport through pores or tight junctions between cells. It is favored for low molecular weight hydrophilic compounds as paracellular spaces is between 10 and 30-50 Å. Transport of peptides and proteins via paracellular route is thus limited as most of them are more than 7000 Da (20 Å). The bioavailability of drugs decreases with the increase of the molecular weight [START_REF] Antosova | Therapeutic application of peptides and proteins: parenteral forever?[END_REF]. In this way, modification of drug molecules and modulation of tight junctions are new investigated strategies in order to improve the penetration of macromolecules through the paracellular pathway [START_REF] Lane | Paracellular and transcellular pathways facilitate insulin permeability in rat gut[END_REF].

Transcellular transport of a drug consists in the drug diffusion into or across the cell membrane according to the concentration gradient. Cell membrane consists of lipid bilayer and is thus the favored route for lipophilic drugs (Camenisch, Alsenz et al., 1998). Several models were set up to study transcellular transport of drugs according to their molecular size, charge, hydrogen bonding and lipophilicity [START_REF] Rautio | Prodrugs: design and clinical applications[END_REF]. Contrarily to small entities, permeability of peptides through transcellular route was found to depend more on the number of polar groups that require desolvatation than on lipophilicity (Burton, Conradi et al., 1996).

Active transport, also described as carrier mediated transport, allows the transport of molecules thanks to membrane proteins. Few numbers of proteins and peptides reach the bloodstream through active transport by cell surface receptors of the small intestine (Bastian, Walton et al., 1999).

Several in vitro models of the intestinal epithelium have been developed in order to study these mechanisms and the most currently used is the Caco-2 one. Caco-2 cells are heterogeneous human epithelial colorectal adenocarcinoma cells that differentiate into human enterocytes after 21 days of culture on Transwells ® inserts. Many studies pointed out the correlation between drug absorption in human and those obtained with the in vitro model [START_REF] Artursson | Caco-2 monolayers in experimental and theoretical predictions of drug transport[END_REF].

Finally, both passive and active transports can be investigated for the improvement of BCS III molecules permeability. It composes the two main parts of this review. Main strategies such as permeation enhancers, mucoadhesive systems, enzyme inhibitors, Cell Penetrating Peptides, chemical modifications or drug carriers are developed in this first section illustrated with specific examples involving BCS III therapeutic molecules.

Passive transport improvement

Permeation enhancers

Permeation enhancers are functional excipients allowing improvement of oral drug bioavailability by either the increase of paracellular permeability thanks to modification of tight junction properties (Aungst, 2012;[START_REF] Jitendra | Noninvasive routes of proteins and peptides drug delivery[END_REF]Williams and Barry, 2004), or the increase of transcellular permeation by disrupting the intestinal membrane or the formation of membrane permeable complex with carrier or ion pairing [START_REF] Liu | Dodecylphosphocholine-mediated enhancement of paracellular permeability and cytotoxicity in Caco-2 cell monolayers[END_REF].

Permeation enhancers have to be safe, non-toxic and inert [START_REF] Senel | Drug permeation enhancement via buccal route: possibilities and limitations[END_REF].

Initially, enhancement of paracellular permeability was considered as the privileged strategy [START_REF] Maher | Safety and efficacy of sodium caprate in promoting oral drug absorption: from in vitro to the clinic[END_REF]. This strategy was studied for oral insulin delivery with the addition of fatty acids and biliary salts, and more recently with Zonula occludens toxin (ZOT) [START_REF] Salama | Tight junction modulation and its relationship to drug delivery[END_REF]. ZOT is a single polypeptide chain which reversibly alters the tight junctions. It targets ZOT receptors, an intracellular modulator of the cytoskeleton and of the tight junction complex. It was shown to enhance insulin intestinal absorption by 72% in rabbit (Fasano and Uzzau, 1997).

The second strategy consists in the improvement of the transcellular transport. The disruption of the structure of the membrane layer to make it more permeable enhances drug adsorption. For instance, molecules such as insulin, human growth hormone (HGH) or calcitonin are administered simultaneously with lipophilic carriers which favor access to the membrane [START_REF] Sood | Peroral route: an opportunity for protein and peptide drug delivery[END_REF]. For instance, the absorption of insulin in combination with SNAC (sodium N-[8-(2-hydroxybenzoyl)amino] caprylate) showed a glucose-lowering effect [START_REF] Kidron | A novel per-oral insulin formulation: proof of concept study in non-diabetic subjects[END_REF]. Another mechanism developed to enhance drug absorption is the formation of membrane permeation complex. For instance, zanamivir heptyl ester and guanidino oseltamivir used ion pairing by inclusion of 1-hydroxy-2-naphthoic acid as a counter-ion, in order to improve membrane permeability. It was found that log P of drugs was increased up to 3.7 log units [START_REF] Miller | Enabling the intestinal absorption of highly polar antiviral agents: ion-pair facilitated membrane permeation of zanamivir heptyl ester and guanidino oseltamivir[END_REF]. Indeed, complexing the molecule of interest with oppositely charged ionic species neutralizes the charges on the drug which increases lipophilicity of the complex and improves its ability to cross biological membranes. Also, calcium chelators were investigated as they act by complex formation in order to improve permeation of proteins [START_REF] Hochman | Mechanisms of absorption enhancement and tight junction regulation[END_REF].

Finally, sodium caprate absorption enhancer was found to not only open tight junctions (paracellular permeation enhancement) but also increase cell membrane penetration (transcellular permeation enhancement) [START_REF] Hochman | Mechanisms of absorption enhancement and tight junction regulation[END_REF]. However, it can induce the release of membrane phospholipids and cause cytotoxicity in vitro (Aungst, 2012).

Mucoadhesive systems

The use of mucoadhesive agent allows improving bioavailability of drugs by prolonging their residence time. It was demonstrated by Longer et al. who described mucoadhesion as the interaction between the mucin surface and a polymer (Longer, Ch'ng et al., 1985). Mucoadhesive agents must present several characteristics such as hydrophilicity to form adhesive bonds with mucosal membrane, high molecular weight to multiply the bonding sites, optimum surface tension, presenting hydrogen bond-forming group, they must be non-toxic and inert (Shaikh, Raj Singh et al., 2011). They are synthetic or semi-natural polymers and some of them are presented in Table 1. The mucoadhesive polymers can be divided into several categories according to their mechanism of action. The mechanism can be the adherence through non-specific or non-covalent electrostatic interactions, the interaction via hydrophilic functional bounds or the binding to specific receptor sites on the cell or mucus surface. Mucoadhesive polymers can thus have specific behavior depending on ionic strength, electric field, light, temperature or pH of the dispersing media [START_REF] Park | Oral protein delivery: Current status and future prospect[END_REF]. For instance, administration of insulin encapsulated with pH sensitive mucoadhesive polymer such as poly(methacrylic acid-g-ethylene glycol) presents a 10% improvement of bioavailability compared to free insulin [START_REF] Peppas | Controlled release by using poly(methacrylic acid-g-ethylene glycol) hydrogels[END_REF]. Formulation of chitosan nanocapsules encapsulating insulin allowed the improvement of the transport across the intestinal mucosa and a significant decrease of glycemia levels in rats [START_REF] Prego | Transmucosal macromolecular drug delivery[END_REF]. It was also demonstrated that thiolated polymers have more largely improved (2-to 140folds) mucoadhesive properties than unmodified mucoadhesive polymers. For instance, mucoadhesive chitosan-thiobutylamidine conjugate is essential for calcitonin absorption. Indeed, only tablets formulated from thiolated chitosan allowed a decrease of plasma calcium level of more than 5% for several hours (Bernkop-Schnurch, 2005).

Enzyme inhibitors

This type of absorption enhancement concerns more particularly BCS III macromolecules such as proteins and carbohydrates. Their absorption is naturally low (<1%) because they are degraded into amino acids and sugars in the gut by various types of enzymes which are responsible for the cleavage of amino acid chains, such as trypsin, chymotrypsin, elastase, pepsin or carboxypeptidases. In this way, the improvement of the drug bioavailability consists in the reduction of its metabolism by adding a protease-inhibiting excipient in the formulation and potentially encapsulating it for protection (Mahato, Narang et al., 2003;Malik, Baboota et al., 2007). For instance, trypsin, bestatin, aprotinin, boroleucine, amastatin, camostat mesilate, chromostatin soybean, or puromycin inhibitors have been widely studied but their administration for chronic or long duration applications can be toxic as they induce the deficiency of these enzymes in humans and affect the absorption of other proteins that normally would have been degraded [START_REF] Watanabe | Mediation of trypsin inhibitor-induced pancreatic hypersecretion by secretin and cholecystokinin in rats[END_REF][START_REF] Yamamoto | Effects of various protease inhibitors on the intestinal absorption and degradation of insulin in rats[END_REF].

Cell-penetrating peptides (CPPs)

CPPs are used to improve the internalization of small molecules as well as macro-biomolecules or carriers into cells. They are short peptide sequences (<30 amino acids) like HIV-1 Tat peptide, oligoarginine or penetratin peptide [START_REF] Munyendo | Cell Penetrating Peptides in the Delivery of Biopharmaceuticals[END_REF][START_REF] Kristensen | Cell-Penetrating Peptides as Carriers for Oral Delivery of Biopharmaceuticals[END_REF].

CPP is covalently conjugated or simply co-administered with the drug or the carrier involving intermolecular interactions. It allows endocytosis or a perturbation of the lipid layer of the membrane in order to enhance the intestinal absorption according to the type of CPP-cargo interactions [START_REF] Zorko | Cell-penetrating peptides: mechanism and kinetics of cargo delivery[END_REF][START_REF] Khafagy | Oral biodrug delivery using cell-penetrating peptide[END_REF]. It has been demonstrated

that CPPs could safely enhance peptide and protein absorption by non-covalent cargo interaction approach. For instance, administration of insulin with a CPP allows an enhancement of oral bioavailability up to 30% [START_REF] Kamei | Noninvasive insulin delivery: the great potential of cell-penetrating peptides[END_REF].

Chemical modification

Chemical modification of poorly absorbed molecules can be divided in two categories:

substitution or derivatization [START_REF] Hamman | Oral delivery of peptide drugs: barriers and developments[END_REF].

The first strategy consists in the replacement of existing amino acids by an analog one. For instance, for desmopressin, a vasopressin analog, L-amino acid is interchanged with D-amino acid [START_REF] Vilhardt | In vitro intestinal transport of vasopressin and its analogues[END_REF]. Similarly, an atom can be replaced by another one such as nitrogen atom by sulfur for thytropar [START_REF] Frank | Comparison of thyrotropin-releasing hormone (TRH) to thyrotropin (TSH) stimulation for evaluating thyroid function in dogs[END_REF].

The second strategy consists in drug derivatization or prodrug design. A prodrug is composed by a drug substance and a transport moiety covalently linked. Inactive or less active than the initial drug, they are turned into their initial active form by enzymatic transformations after crossing the membrane [START_REF] Hsieh | Current prodrug design for drug discovery[END_REF][START_REF] Jana | Prodrug design to improve pharmacokinetic and drug delivery properties: challenges to the discovery scientists[END_REF]. Main objectives of a prodrug for passive transport enhancement are the improvement of either drug water solubility or drug absorption, and the reduction of drug metabolism [START_REF] Abet | Prodrug approach: An overview of recent cases[END_REF]. BCS III molecules are concerned by the two last objectives since they are highly soluble. Drug derivatization with PEG allows protecting the molecule from enzymatic degradation or improving the solubility. For instance, PEG-insulin was evaluated by Tuesca et al. [START_REF] Tuesca | Synthesis, characterization and in vivo efficacy of PEGylated insulin for oral delivery with complexation hydrogels[END_REF]. Drug can also be derivatized with fatty acid in order to increase the lipophilicity of the molecule and improve membrane permeability [START_REF] Zhang | Converting peptides into drug leads by lipidation[END_REF].

Drug carriers

Encapsulation of molecules in carriers was largely explored in order to protect them from the intestinal environment (pH and metabolism) and thus to enhance their bioavailability. Carriers are mainly liposomes, microparticles or nanoparticles (NPs). They are characterized through their size, surface charge, encapsulation efficiency and drug release.

Liposomes

Liposomes are 0.01 to 10 µm vesicles composed by a phospholipid bilayer separating an aqueous core from the external aqueous media (Fig. 4). According to the preparation method, liposomes can differ by their size, structure and encapsulation capacity. Liposome membrane is composed by natural or synthetic, possibly saturated phospholipids. Negatively charged phospholipids, antioxidant agents or cholesterol can be added in order to avoid aggregation and oxidation issues, or to increase the stability. According to the properties expected or the molecule encapsulated, liposomes can be cationic, pH-or temperature-sensitive, coupled with PEG or ligand for furtive or targeting liposomes. Moreover, liposomes allowed to obtain improved absorption properties following oral administration of BCS III drugs. For instance, positive surface charge showed enhanced biological properties thanks to better interaction with intestinal mucosa. This was observed by Hashimoto et al. reporting a significant reduction in blood glucose in rats after administration of insulin-loaded positively charged liposomes compared to neutral or negatively charged [START_REF] Hashimoto | Effects of Oral Administration of Positively Charged Insulin Liposomes on Alloxan Diabetic Rats: Preliminary Study[END_REF]. Inclusion of excipients into the liposome formulation can also allow the improvement of drug permeation properties. Generally, they are inserted into lipid bilayer or they can be used as liposomal coating. Recently, heparin (LMWH) was encapsulated in Eudragit S 100-coated positively charged liposomes. It was found a 3-fold increase of AUC in vivo in rats after oral administration [START_REF] Lavanya | Preparation and evaluation of a novel oral delivery system for low molecular weight heparin[END_REF].

Polymeric particles

Polymeric particles are currently used for passive transport improvement as encapsulation of molecules not only protects them from pH and enzymatic degradation, but also allows a control of their release. It includes micro-and nano-particles.

Microparticles are between 1 and 1000 µm and can be microspheres or microcapsules depending on the materials and the manufacturing process used (Fig. 5). Microspheres are composed by a tangle of polymers or lipids in which the active ingredient is dissolved or dispersed.

Microcapsules are composed by a crown and a liquid, solid or gaseous core. Transcellular transport of molecule can be thus improved.

For instance, incorporation of hydrophilic polymers such as poly(ethylene glycol) (PEG) or chitosan allows to enhance NP transport thanks to specific interactions with the intestinal epithelium. PEG is mainly used for its stabilizing properties. For example, poly(methyl vinyl esther-co-maleic anhydride) (PVM/MA) NPs were coated with PEG 1000 or PEG 2000 and it was found a deeper penetration through the mucus due to better permeation properties for pegylated NPs [START_REF] Yoncheva | Pegylated nanoparticles based on poly(methyl vinyl ether-co-maleic anhydride): preparation and evaluation of their bioadhesive properties[END_REF]. In another hand, chitosan was widely studied for its mucoadhesive properties. In this way, chitosan coated PLGA NPs were developed and it was found a better insulin response compared to classical PLGA NPs [START_REF] Vila | Design of biodegradable particles for protein delivery[END_REF].

Active transport improvement

In order to improve the oral delivery of BCS III molecules, it is possible to increase the active transport of drugs. In this way, receptors or transporters of the intestinal membrane can be targeted using prodrugs or functionalized NPs. As described above, prodrugs consist in ligands directly linked on the active molecules. Nevertheless, this chemical modification can inactivate the molecule. In these cases, functionalization of a carrier encapsulating the molecule is favored.

The present section will review the intestinal receptors and transporters currently targeted for active transport improvement and will give examples from the literature of prodrugs or functionalized NPs encapsulating BCS III molecules.

Receptors targeting

Receptors are membrane-associated proteins and they are expressed at the intestinal cell surface in the gastro intestinal tract. They are responsible for the internalization of nutrients through a receptor-mediated endocytosis process and that is a well-known process for molecule absorption.

The mechanism of internalization consists of first a binding of the substrate to the receptor. The specific part of the substrate recognized by the receptor is called the ligand. The ligand can be directly linked to the active molecule (prodrug) or it can be linked on the surface of a carrier.

Phagocytosis and pinocytosis endocytotic mechanisms are responsible for the transport of prodrugs or functionalized carriers. Phagocytosis involves phagocytotic immune cells and M cells, whereas pinocytosis can occur through macropinocytosis, caveolae-or clathrin-mediated or independent endocytosis [START_REF] Beloqui | Mechanisms of transport of polymeric and lipidic nanoparticles across the intestinal barrier[END_REF] (Table 2, Fig. 6).

Endocytosis pathway Description

Phagocytosis

Depend on the solute concentration surrounded the cell.

Formation of membrane protusions collapsing with the membranegenerating endocytotic vesicles.

Macropinocytosis

Caveolae-mediated or independent endocytosis

From specific or non-specific receptor-ligand interaction.

Membrane invagination from the fission of caveolae, a dimeric protein, forming caveolar vesicles.

Clathrin-mediated or independent endocytosis

From specific or non-specific receptor-ligand interaction.

Formation of clathrin-coated vesicles transported to the nucleus. The endocytosis pathways across the cells depend not only on molecule or particle size but also on the ligand and on the receptor targeted.

Main receptors of the intestinal membrane already targeted for oral absorption improvement are presented below with accurate examples of drug delivery systems designed for BCS III drugs.

Vitamin receptors

Vitamin B12, folate and biotin transcytosis-mediated receptors are widely expressed in the gastrointestinal tract and especially in the small intestine. Targeting these receptors by conjugation of specific ligand directly to the drug or on loaded-nanocarrier is an efficient strategy to enhance drug absorption. Vitamin B12 receptor was largely exploited in order to increase oral drug bioavailability. In contrast, folate receptors were studied mainly because of their overexpression in cancerous cells as they were only recently exploited for oral drug delivery or intestinal targeting. Finally, biotin receptor was also lately used in this purpose and few examples can be found in the literature (Russel-Jones, 2004).

Vitamin B12

The process of vitamin B12 (VB12) endocytosis is initiated by the complexation of VB12 with intrinsic factor (IF), a protein produced in the stomach. The complex binds to IF receptors localized in the luminal surface of the small intestine, which induced internalization via receptormediated endocytosis. VB12 is then released from IF into the circulation with the action of cathepsin L (Fig. 7) [START_REF] Hamman | Targeting receptors, transporters and site of absorption to improve oral drug delivery[END_REF]. VB12 was largely used as a ligand for both prodrug and functionalized carrier strategies.

Habberfield et al. covalently linked VB12 on erythropoietin (EPO) for the transport from the small intestine to the circulation in rats. It was found a 4-times increase in plasma level of the protein compared to free EPO [START_REF] Habberfield | Vitamin B12-mediated uptake of erythropoietin and granulocyte colony stimulating factor in vitro and in vivo[END_REF]. VB12 was recently linked to solid lipid NPs (SLN) loaded with insulin, and showed almost 2-fold increase of bioavailability compared to non-modified insulin SLN. A 60% reduction of blood glucose concentration was maintained from 6 h to 12 h [START_REF] He | VB12-coated Gel-Core-SLN containing insulin: Another way to improve oral absorption[END_REF]. This strategy was also explored by Francis et al. with a linkage of VB12 on the micelles encapsulating cyclosporin A (CsA). The apical to basolateral absorption of CsA through Caco-2 cells after 24 h was found 1.8 time higher when loaded in VB12-micelles compared to free CsA [START_REF] Francis | Exploiting the vitamin B12 pathway to enhance oral drug delivery via polymeric micelles[END_REF].

Conjugation of VB12 was also realized on dextran NPs. Influence of the insulin load and of the dextran molecular mass on the drug pharmacological availability were tested. It was found that 70 kDa VB12-conjugated dextran NPs containing 2, 3 and 4% w/w of insulin, improved the molecule pharmacological availability by 1.1, 1.9 and 2.6 fold, respectively, compared to NPs without VB12. Furthermore, decreasing the molecular mass from 70 kDa to 10 kDa decreased the pharmacological availability by 1.4 [START_REF] Chalasani | Effective oral delivery of insulin in animal models using vitamin B12-coated dextran nanoparticles[END_REF].

Folate

Folic acid also known as vitamin B9, is an essential nutrient which is absorbed via a saturable, pH-and sodium-dependent route. It is a well-known targeting agent in formulation since folate receptors were found to be over-expressed in many type of cancer cells [START_REF] Weitman | Distribution of the folate receptor GP38 in normal and malignant cell lines and tissues[END_REF]. Numerous examples targeting folate receptors for targeting tumor cells by IV route exist in comparison with folate uptake systems for oral drug delivery. Nevertheless, folate absorption from the intestinal tract has been described and can be used for promoting folate-linked molecule or carrier absorption [START_REF] Rosenberg | Herman Award lecture. Folate absorption: clinical questions and metabolic answers[END_REF]. Folic acid receptor is known to induce caveolaemediated endocytosis. Folic acid can be chosen as a ligand because of its ability to be transcytosed and its stability in simulated gastric and intestinal fluids for at least 6 h. Roger et al. investigated the potential of folic acid receptor-targeted PLGA-NPs to improve the oral bioavailability of paclitaxel using a Caco-2 cell monolayer model. It was found that simple encapsulation of paclitaxel in PLGA-NPs increased its transport across Caco-2 cells monolayers by 5-folds, whereas encapsulation in folic acid functionalized NPs further increased the transport by 8-folds (Roger, Kalscheuer et al., 2012). Folic acid decorated PEG-PLGA NPs were also explored for enhancing the oral absorption of insulin. Prepared by double emulsion, solvent evaporation and freeze dried, folic acid-PEG-PLGA NPs loaded with insulin exhibited a 2-fold increase in oral bioavailability compared to free drug. It was considered as a promising formulation as the freeze dried NPs can be filled in a capsule and a daily administration was sufficient to control diabetes for the entire day [START_REF] Jain | Folate-decorated PLGA nanoparticles as a rationally designed vehicle for the oral delivery of insulin[END_REF]. Not only polymeric NPs but also liposomes were explored as folic acid functionalized carriers. For instance, Ling et al.

described the oral improvement of the uptake and transport of folic acid-coupled liposomes loaded with the poorly absorbed hydrophilic cefotaxime drug model. The bioavailability of cefotaxime increased up to 2.0-fold as compared with folic acid-free liposomes [START_REF] Ling | Oral bioavailability enhancement of a hydrophilic drug delivered via folic acid-coupled liposomes in rats[END_REF]. Finally, improvement of peptide bioavailability thanks to oral folic acid-mediated liposomal delivery was also studied with vancomycin as a peptide model. It was found a 5.7-fold increase in Caco-2 cell uptake compared to free vancomycin. In vivo, delivery through folic acid functionalized liposomes increased the area under the curve of oral vancomycin by 12.5-fold compared with an oral drug free solution [START_REF] Anderson | Formulation and evaluation of a folic acid receptor-targeted oral vancomycin liposomal dosage form[END_REF].

Biotin

Also known as vitamin B7, Biotin is not synthetized by mammalian system and has to be brought as a nutrient from plant or bacterial systems. Its transport is accomplished through two pathways (i) a high-affinity biotin receptor and (ii) a sodium-dependent multivitamin transporter (SMVT).

Biotin receptors are non-specific and are distributed throughout the small intestine [START_REF] Ren | Recent development of biotin conjugation in biological imaging, sensing, and target delivery[END_REF]. Biotinylation of drug was firstly studied. For instance, this strategy was explored for the oral delivery of glucagon-like peptide-1 (GLP-1). Biotinylated GLP-1s were prepared and it was found that biotin-GLP-1 had 5.6-higher apparent permeability than GLP-1 on Caco-2 cells [START_REF] Youn | Improved peroral delivery of glucagon-like peptide-1 by site-specific biotin modification: Design, preparation, and biological evaluation[END_REF]. The potential of biotin as nanocarrier ligand for oral drug delivery was investigated recently with biotinylated liposomes for the oral delivery of insulin. It was pointed out a specific clathrin-mediated endocytosis. The insulin oral bioavailability was measured at 3.3% and 8.3% when encapsulated in classical or biotinylated liposomes, respectively. It was thus shown that the biotin modification enhanced the insulin transport across Caco-2 cells monolayer [START_REF] Zhang | Biotinylated liposomes as potential carriers for the oral delivery of insulin[END_REF].

Other receptors

Transferrin

The transferrin receptor 1 (TfR1) is a transmembrane homodimer receptor composed of two identical monomers with a molecular mass of approximately 90 kDa. It is expressed in all nucleated cells such as blood cells, hepatocytes, blood brain barrier or intestinal cells. It is not expressed at the same level everywhere as they are intensively found on tumor cells whereas they are nearly undetectable in nonproliferating cells [START_REF] Qian | Targeted drug delivery via the transferrin receptormediated endocytosis pathway[END_REF]. This receptor appears to be a good candidate for targeting strategy because it is expressed in appreciable amount in small intestinal epithelium [START_REF] Jeffrey | Molecular regulation of transferrin receptor and ferritin expression in the rat gastrointestinal tract[END_REF][START_REF] Banerjee | Transferrin receptors in the human gastrointestinal tract. Relationship to body iron stores[END_REF]. Shah and Shen demonstrated that human insulin conjugated to transferrin (In-Tf) via a disulfure bond was transported across cultured epithelial cells via TfR-mediated transcytosis and improved 5-to 15fold insulin transport compared to free drug [START_REF] Shah | Transcellular delivery of an insulin-transferrin conjugate in enterocyte-like Caco-2 cells[END_REF]. Later, it was observed a hypoglycemic effect in diabetic mice after oral administration of In-Tf, which was slow but prolonged in diabetic rats [START_REF] Wang | Oral delivery of insulin-transferrin conjugate in streptozotocin-treated CF/1 mice[END_REF][START_REF] Xia | Hypoglycemic effect of insulin-transferrin conjugate in streptozotocin-induced diabetic rats[END_REF]. Recently, exploiting the high affinity of 7peptide (7pep, Histidine-Alanine-Isoleucine-Tyrosine-Proline-Arginine-Histidine) obtained through phage display for TfR, Du et al. conjugated 7pep on the surface of polymeric micelles (M) loaded with coumarin 6 (C6). It was found that 7pep-M-C6 was internalized into Caco-2 cells faster and exhibited higher fluorescence intensity than M-C6 alone [START_REF] Lee | Receptor mediated uptake of peptides that bind the human transferrin receptor[END_REF]Du, Fan et al., 2013).

FcRn

The neonatal Fc receptor (FcRn) is present in neonatal intestine and allows the Immunoglobulin G (IgG) transport from mother breast milk to offspring (Brambell, 1969) (Fig. 8). It is also expressed in adult small intestine and plays an important role in the function of IgG in immune surveillance and host defense at mucosal surface [START_REF] Israel | Expression of the neonatal Fc receptor, FcRn, on human intestinal epithelial cells[END_REF]. The Fc-domain of IgG is sufficient to be recognized by FcRn receptor. Thus, Fc-fusion protein allows the delivery of proteins across intestinal barrier [START_REF] Sockolosky | The neonatal Fc receptor, FcRn, as a target for drug delivery and therapy[END_REF]. For instance, oral delivery of a follicle stimulating hormone fusion protein (FSH-Fc) in a neonatal rat model induced a significant increase of testicular weight compared to free FSH [START_REF] Low | Oral and pulmonary delivery of FSH-Fc fusion proteins via neonatal Fc receptor-mediated transcytosis[END_REF]. Fc fragment can also be conjugated to NPs to induce their transport across the intestinal membrane through a transcytosis pathway. This strategy was explored by Pridgen et al. with PLA-PEG-Mal-Fc NPs loaded with insulin (Pridgen, Alexis et al., 2013). They found that orally administered FcRn-targeted insulin-loaded NPs crossed the intestinal epithelium and reached systemic circulation which involved a prolonged hypoglycemic response (15 versus 1.5 hours) compared to free insulin solution in mice.

ICAM-1

The intercellular adhesion molecule 1 (ICAM-1) is a transmembrane glycoprotein expressed on the apical surface of intestinal epithelium [START_REF] Rothlein | A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1[END_REF]. ICAM-1 has affinities for specific antibodies anti-ICAM. Mane et al, explored the ligand specific targeting of rat IgG antibodies coupled to the surface of polymer nanocarriers. Anti-ICAM nanocarriers demonstrated significantly enhanced retention of nanocarriers confirming intestinal targeting. This was confirmed by microscopy and X-ray spectroscopy which revealed anti-ICAM nanocarriers in vesicular compartments [START_REF] Mane | Biodistribution and endocytosis of ICAM-1-targeting antibodies versus nanocarriers in the gastrointestinal tract in mice[END_REF]. According to these observations, Ghaffarian et al. coated PLGA NPs with IgG or anti-ICAM, and loaded them with the enzyme α-galactosidase as model. They firstly confirmed the rapid and efficient uptake of anti-ICAM nanocarriers (80% after 1 h on Caco-2 cells) and the vesicular transport. Indeed, they observed large vesicular compartment formation and transcytosis without any disruption of tight junctions. Then, they highlighted the significant delivery capacity of the ICAM receptor on the range of clinical dose (14 mg for a 70 kg person). Nevertheless, they insisted on the low level of release of αgalactosidase from the NPs which had to be improved [START_REF] Ghaffarian | Transport of nanocarriers across gastrointestinal epithelial cells by a new transcellular route induced by targeting ICAM-1[END_REF].

Lectin

Lectins are proteins or glycoproteins from nonimmunological origin, which can specifically recognize sugar molecules and are capable of binding glycosylated membrane components. They are characterized by a relatively low binding specificity and a high binding capacity. When a substrate is linked to the cell membrane it induced an active vesicular transport process of transcytosis. Many studies have been carried out in order to enhance intestinal uptake of orally administered lectin-functionalized polystyrene particles [START_REF] Irache | Bioadhesion of lectin-latex conjugates to rat intestinal mucosa[END_REF][START_REF] Irache | In vitro study of lectin-latex conjugates for specific bioadhesion[END_REF], but these particles had nondegradability issues. Recently biodegradable particles have been investigated for lectin conjugations. Nontoxic lectins from plants such as wheat germ agglutinin (WGA) were conjugated to alginate microparticles loaded with insulin. It was found that alginate-WGA microparticles improved the stability of the entrapped insulin by protecting it from degradation, and the hypoglycemic effect was doubled after 12 h compared to free insulin solution [START_REF] Kim | Bioadhesive interaction and hypoglycemic effect of insulin-loaded lectin-microparticle conjugates in oral insulin delivery system[END_REF]. Another study pointed out the efficacy of thymopentin encapsulation in PLGA-WGA NPs on its oral absorption. It suggested that the uptake improvement was correlated with the WGA amount on the NPs [START_REF] Yin | Preparation and evaluation of lectinconjugated PLGA nanoparticles for oral delivery of thymopentin[END_REF]. Zhang et al. used Solid Lipid NPs (SLNs) functionalized with WGA to improve the oral absorption of insulin. It was found that the relative bioavailability of insulin after oral administration of SLNs and WGA-SLNs were 4.99% and 7.11%, respectively. These results confirmed the enhancement of the oral absorption of insulin thanks to lectin receptors targeting [START_REF] Zhang | Lectin-modified solid lipid nanoparticles as carriers for oral administration of insulin[END_REF]. Finally, WGA can also be linked directly on the molecule. This was demonstrated by Gabor et al. studying BSA-WGA conjugates. Caco-2 monolayer binding was found to be significantly more important with BSA-WGA compared to BSA (19% versus 1%), which confirmed the importance of the lectin conjugation [START_REF] Gabor | Lectin-mediated drug delivery: binding and uptake of BSA-WGA conjugates using the Caco-2 model[END_REF].

Finally, a lot of receptors have been targeted by prodrugs or functionalized NPs. All of them are summed up in the table below associated to the drug substance (Table 3). [START_REF] Habberfield | Vitamin B12-mediated uptake of erythropoietin and granulocyte colony stimulating factor in vitro and in vivo[END_REF] Insulin VB12-SLN (Solid Lipid NPs) [START_REF] He | VB12-coated Gel-Core-SLN containing insulin: Another way to improve oral absorption[END_REF] Cyclosporin A (CsA) VB12-Micelles [START_REF] Francis | Exploiting the vitamin B12 pathway to enhance oral drug delivery via polymeric micelles[END_REF] Insulin VB12-dextran NPs [START_REF] Chalasani | Effective oral delivery of insulin in animal models using vitamin B12-coated dextran nanoparticles[END_REF] Folate Paclitaxel Folic acid-PLGA NPs (Roger, Kalscheuer et al., 2012) Insulin Folic acid-PEG-PLGA NPs [START_REF] Jain | Folate-decorated PLGA nanoparticles as a rationally designed vehicle for the oral delivery of insulin[END_REF] Cefotaxime Folic acid-liposomes [START_REF] Ling | Oral bioavailability enhancement of a hydrophilic drug delivered via folic acid-coupled liposomes in rats[END_REF] Vancomycin Folic acid-liposomes [START_REF] Anderson | Formulation and evaluation of a folic acid receptor-targeted oral vancomycin liposomal dosage form[END_REF])

Biotin

Glucagon-like peptide-1 (GLP-1) Biotin-GLP-1 prodrug [START_REF] Youn | Improved peroral delivery of glucagon-like peptide-1 by site-specific biotin modification: Design, preparation, and biological evaluation[END_REF] Insulin Biotin-liposomes [START_REF] Zhang | Biotinylated liposomes as potential carriers for the oral delivery of insulin[END_REF] Transferrin Insulin Tf-insulin prodrug [START_REF] Shah | Transcellular delivery of an insulin-transferrin conjugate in enterocyte-like Caco-2 cells[END_REF]) [START_REF] Wang | Oral delivery of insulin-transferrin conjugate in streptozotocin-treated CF/1 mice[END_REF] Coumarin 6 (C6) 7pep-C6 micelles [START_REF] Lee | Receptor mediated uptake of peptides that bind the human transferrin receptor[END_REF]) (Du, Fan et al., 2013)

FcRn

Follicle stimulatin hormone (FSH) Fc-FSH prodrug [START_REF] Low | Oral and pulmonary delivery of FSH-Fc fusion proteins via neonatal Fc receptor-mediated transcytosis[END_REF] Insulin Fc-Mal-PEG-PLA (Pridgen, Alexis et al., 2013) ICAM-1 α-galactosidase IgG-or anti-ICAM-PLGA NPs [START_REF] Ghaffarian | Transport of nanocarriers across gastrointestinal epithelial cells by a new transcellular route induced by targeting ICAM-1[END_REF] Lectin Insulin WGA-alginate microparticles [START_REF] Kim | Bioadhesive interaction and hypoglycemic effect of insulin-loaded lectin-microparticle conjugates in oral insulin delivery system[END_REF] Thymopentin WGA-PLGA NPs [START_REF] Yin | Preparation and evaluation of lectinconjugated PLGA nanoparticles for oral delivery of thymopentin[END_REF] Insulin WGA-SLN [START_REF] Zhang | Lectin-modified solid lipid nanoparticles as carriers for oral administration of insulin[END_REF] BSA WGA-BSA [START_REF] Gabor | Lectin-mediated drug delivery: binding and uptake of BSA-WGA conjugates using the Caco-2 model[END_REF]

Transporters targeting

Intestinal transporters are membrane proteins able to transfer substrates through cell membranes by a facilitated or active process. They can be divided into two gene superfamilies: the adenosine tri-phosphate binding cassette (ABC) transporters requiring energy from adenosine tri-phosphate (ATP) hydrolysis, and the solute carriers (SLC) dependent of the co-transport of an inorganic counter-ion (Na + , H + , Cl -) or organic molecules and using their concentration gradient as a driving force, or forming solute selective channels for diffusion across the membrane (facilitated diffusion). Efflux by ABC transporters in the apical side limits the absorption of molecules, whereas SLC facilitate the transport in both directions. Both superfamilies differ by the mechanism induced to translocate their substrates. More than 400 membrane transporters have been inventoried (Varma, Ambler et al., 2010;[START_REF] Giacomini | Membrane transporters in drug development[END_REF]Estudante, Morais et al., 2013). Among them, 49 human ABC proteins are classified into 7 subfamilies. Three of them are particularly relevant in the transport of drug-like molecules: the ABCB1 (hosting the Pglycoprotein transporter, P-gp), the ABCCs (hosting multidrug resistance-associated proteins)

and the ABCG2 (hosting the breast cancer resistance protein, BCRP) families. SLC family includes 395 members classified into 52 subfamilies. They are at the origin of the transport of nutrients, ions and drugs. Targeting intestinal transporters to improve oral bioavailability of poorly absorbed molecules is a largely explored strategy. The drug molecule is designed as a prodrug with conjugation with a ligand in order to be recognized as a substrate at the apical membrane surface. The drug-ligand complex is generally cleaved within the intracellular environment [START_REF] Hamman | Targeting receptors, transporters and site of absorption to improve oral drug delivery[END_REF]. As they are responsible of the transport of a wild range of substrates, SLC transporters considered for drug targeting strategies are described in the following part of this review.

Amino acid transporters

Amino acid transporters are largely expressed in the apical membrane of the small intestine.

Some of them are Na + -dependent and allow the transport of large neutral amino acids (Steffansen, Nielsen et al., 2004). Seven amino acids transport systems have been identified and are classified according to genetic family, driving force, substrate specificity and cellular localization. For instance, they differ in their Na + -dependence or in the involvement of other ions in the transport process such as Cl -and K + . System L is described as the main mechanism allowing transport across the small intestine of large, branched or aromatic amino acids from extracellular fluids [START_REF] Hidalgo | Carrier-mediated transport and efflux mechanisms in Caco-2 cells[END_REF][START_REF] Hamman | Targeting receptors, transporters and site of absorption to improve oral drug delivery[END_REF]. Because of its broad substrate specificity, system L is largely explored to transport amino-acid related compounds.

Val-quinidine was studied as quinidine prodrug to improve its intestinal permeability. As an inhibitor of P-gp, the intestinal absorption of quinidine from the distal small intestine is low because it is recognized as a substrate for P-gp. To overcome this phenomenon, valine ester prodrug of quinidine was investigated as a substrate of amino acid transporter and not a P-gp recognized complex [START_REF] Jain | Circumventing P-glycoproteinmediated cellular efflux of quinidine by prodrug derivatization[END_REF].

Glucose transporters

Two types of transporters are involved in the monosaccharide intestinal transport according to their Na + dependence: SGLT which is sodium-dependent Na + /glucose co transporter and GLUT transporters which are sodium independent. They are distributed in the small intestine. Several glycosides, glucoses conjugated aromatic rings, glycosylated tetrapeptides and glucose conjugated acetaminophen were found to be substrate of SGLT-1 (Steffansen, Nielsen et al., 2004;[START_REF] Murakami | A Minireview: Usefulness of Transporter-Targeted Prodrugs in Enhancing Membrane Permeability[END_REF]. For instance, Nomoto et al. glycosylated a tetrapeptide (Gly-Gly-Tyr-Arg, GGYR) not transported by di-or tri-peptide transporters, with p-(succinylamido)phenyl αor β-D-glucopyranoside (α,β-SAPG) in order to target SGLT-1. Free GGYR do not penetrate the intestinal membrane because of enzymatic degradation and very low permeability. Thus, SLGT-1 was found to take part in the uptake of both α-and β-SAPG-GGYR, which is confirmed by an inhibition of the transport in the presence of D-glucose and in the absence of a Na + -gradient [START_REF] Nomoto | Improvement of intestinal absorption of peptide drugs by glycosylation: transport of tetrapeptide by the sodium ion-dependent Dglucose transporter[END_REF]. The same team demonstrated few years before the improvement of intestinal insulin absorption targeting SLGT-1 with α-SAPG conjugation. When SAPG-INS was administered intra-intestinally, blood glucose level decreased to 56% of initial value [START_REF] Haga | Hypoglycemic Effect of Intestinally Administered Monosaccharide-Modified Insulin Derivatives in Rats[END_REF]. It was also demonstrated an improve of p-nitrophenol (p-NP) absorption rate when conjugated with β-disaccharide in rat small intestine [START_REF] Mizuma | Intestinal Na+/glucose cotransporter-mediated transport of glucose conjugate formed from disaccharide conjugate[END_REF].

Monocarboxylate transporters

Monocarboxylate transporter is responsible for the uptake of weak organic acids such as benzoic acid, nicotinic acid, pravastatin, valproate acid, baclofen and salicylic acid. Lactic acid generated during the metabolic reactions involving the production of ATP is also transported into and out the cells by monocarboxylate transporters through H + -dependent gradient. MCT1 is the most studied form of the transporter and was found to be expressed in Caco-2 cells and rat intestine [START_REF] Ritzhaupt | Identification and characterization of a monocarboxylate transporter (MCT1) in pig and human colon: its potential to transport L-lactate as well as butyrate[END_REF]Tamai, Takanaga et al., 1995). An example of prodrug exploiting monocarboxylate acid transport system is carindacillin (CIPC), obtained from the modification by ester formation of the chemical structure of carbenicillin (CBPC), a dicarboxylic acid. CBPC is currently administrated intravenously according to its poor stability and absorption in the GI tract. CIPC was orally administered and was studied in rat intestinal brush border membrane vesicles. It was found a 70-fold improvement of apparent uptake clearance of CIPC compared to CBPC thanks to the carrier mediated absorption [START_REF] Li | Role of the monocarboxylic acid transport system in the intestinal absorption of an orally active beta-lactam prodrug: carindacillin as a model[END_REF].

Bile acid transporters

Bile acids are produced in the liver and are secreted into the duodenum after the ingestion of meals in order to facilitate the digestion and to absorb fats. Approximately 90% of the secreted bile acids are recycled back to the liver. Re-absorption of bile acids occurs in the jejunum and ileum and is made through passive diffusion and active transport, respectively. Active transport bile carriers are proteins located in the apical membrane of ileum cells and involve Na +dependent transport. Caco-2 cells were validated as a model to study intestinal bile transport. In order to improve the bioavailability of poorly absorbed molecules, they are conjugated to bile acid known to be transported by the bile acid carrier. The size of the molecule appears to play an important role in the complex ability to be transported by the carriers [START_REF] Hidalgo | Carrier-mediated transport and efflux mechanisms in Caco-2 cells[END_REF]Dawson, 2011). From the 1990's, Kramer and Wess largely explored the strategy by coupling small drugs entities or peptides to bile acid [START_REF] Kramer | Bile acid transport systems as pharmaceutical targets[END_REF]. More recently and considered as the most relevant example of increased oral bioavailability using a prodrug targeting bile acid transporter is the Tolle-Sander et al. study. They designed bile acid acyclovir prodrug by conjugation with chenodeoxycholate (CDCA), deoxycholate, cholate, and ursodeoxycholate. The best affinity for the transporter was found with CDCA linked to acyclovir thanks to a valine side chain linker (acyclovir-valyl-CDCA). It was then found a 2-fold increase in oral bioavailability of acyclovir in rats with prodrugs, compared to acyclovir free (Tolle-Sander, Lentz et al., 2004). Also, bile acid-gabapentin prodrug was investigated in order to improve gabapentin oral bioavailability as high doses are required in the treatment of neuropathic pain. CDCA-glu-gabapentin methyl ester was found to be an interesting novel prodrug. Indeed, gabapentin apparent permeability appeared to be largely increased compared to gabapentin alone [START_REF] Rais | Synthesis and in vitro evaluation of gabapentin prodrugs that target the human apical sodium-dependent bile acid transporter (hASBT)[END_REF].

Organic anion transporting polypeptides

Organic anion transporting polypeptides (OATPs) are expressed all along human tissues. For instance, OATP1B1 and OATP1B3 are exclusively expressed in human liver, OATP4C1 in human kidney and OATP1C1 in brain and testis. Little information on intestinal OATPs is known, and it is only recently that OATP1A2 and OATP2B1 were suggested to be responsible of intestinal absorption of several drugs. Immunohistochemical studies in human small intestinal tissues confirmed the presence of OATP2B1 and OATP1A2 proteins at the apical membrane of intestinal epithelial cells, but the OATP1A2 expression level was disputed. In this way, OATP2B1 is described as responsible of the functional OATP in small intestine. Thanks to its broad substrate specificity, it could be targeted for oral delivery (Steffansen, Nielsen et al., 2004;Tamai, 2012). This strategy was explored by Kato et al. with the synthesis of tebipenem pivoxil (TBPM-PI), a prodrug of carbapenem antibiotic. In vitro studies confirmed the contribution of carrier-mediated transport mechanisms as the TBPM-PI uptake decreased with ATP suppression and at low temperature. The prodrug was shown to be OATP2B1 substrate and to improve carbapenem bioavailability up to 80% [START_REF] Kato | Intestinal absorption mechanism of tebipenem pivoxil, a novel oral carbapenem: involvement of human OATP family in apical membrane transport[END_REF].

Ascorbate and other vitamins transport systems

Vitamin C is obtained from vegetables or fruits and is essential in the growth and the repair of tissues. Its absorption is carried out by two transporters: SVCT1 and SVCT2 (Sodium-dependent Vitamin C Transporter). Whereas SVCT2 is largely expressed in brain, SVCT1 is found in intestine and is expressed by Caco-2 cells. Vitamin C transport is mediated by Na + -gradient.

Conjugation of drugs with ascorbic acid, the reduced form of the vitamin, allowed to obtain prodrug for transporter targeting strategy in order to improve the bioavailability of the molecule [START_REF] Anand | Current prodrug strategies via membrane transporters/receptors[END_REF][START_REF] Murakami | A Minireview: Usefulness of Transporter-Targeted Prodrugs in Enhancing Membrane Permeability[END_REF]. In this way, a prodrug of saquinavir (SQV) was synthetized by Luo et al. to improve the oral absorption of the molecule with the conjugation of ascorbyl-succinic and obtain the AA-Su-SQV chemical complex. Apparent permeability of SQV was 5-fold increased on Caco-2 cells. The involvement of SVCT in the transport was confirmed as the prodrug permeability decreased with the addition of free ascorbic acid in competition [START_REF] Luo | Targeting SVCT for enhanced drug absorption: synthesis and in vitro evaluation of a novel vitamin C conjugated prodrug of saquinavir[END_REF].

Others vitamins such as biotin or pantothenic acid are transported by sodium-dependent multivitamin transporter (SMVT). They are expressed in several human tissues such as brain, cornea, heart, kidney, liver, lung and intestine. Substrate absorption could be different according to the region (duodenum > jejunum > ileum) and the concentration. Thanks to its low affinity and high capacity along with wide substrate specificity, SMVT became an interesting transporter to target. For instance, biotin-derivative for the intestinal delivery was studied by Vadlapudi et al. with the conjugation of the vitamin on acyclovir (ACY) in order to improve its oral bioavailability. It was found a better recognition by SMVT transporter of biotin-targeted lipid prodrugs than biotin-targeted prodrugs, and an ACY absorption increase by biotin-ricinoleic acid-ACY or biotin-12hydroxystearic acid-ACY compared to free ACY [START_REF] Vadlapudi | Novel biotinylated lipid prodrugs of acyclovir for the treatment of herpetic keratitis (HK): transporter recognition, tissue stability and antiviral activity[END_REF].

Peptide transporters

The proton-coupled oligopeptide transporter family (POT) includes several peptides transporters such as PepT1 or PepT2, peptide/histidin transporter PHT1, PHT2 and L1-cadherin transporter PT1. Some of them were found predominantly in kidney (PepT2), brain and eye (PHT1), or were not so largely studied. PepT1 is one of the most extensively investigated transporters and the present section will focus on it. Firstly cloned and characterized by Fei et al. in rabbit (Fei, Kanai et al., 1994), it was later isolated from other species such as human [START_REF] Liang | Human intestinal H+/peptide cotransporter. Cloning, functional expression, and chromosomal localization[END_REF], rat [START_REF] Saito | Cloning and characterization of a rat H+/peptide cotransporter mediating absorption of beta-lactam antibiotics in the intestine and kidney[END_REF] and mouse (Fei, Sugawara et al., 2000). Encoded as a 708 amino acids protein in human (78 kDa), PepT1 is localized in the apical membrane of small intestine. It is also distributed on Caco-2 cells. Characterized by high-capacity, low-affinity and broad substrate specificity, it allows the transport of di-, tri-peptides and peptidomimetics, but not free amino acids or tetra-peptides, according to H + -gradient [START_REF] Brandsch | Drug transport via the intestinal peptide transporter PepT1[END_REF]) (Fig. 9). The action of Na + -K +

ATPases system located in the basolateral membrane and the Na + -H + exchanger (NHE) on the apical membrane allow maintaining the inward proton gradient. The uptake of PepT1 is thus dependent on the extracellular pH which must be comprised between 4.5 and 6.6 to optimize substrate transport. Once in the intestinal cells, substrates are hydrolyzed and free amino acids are generated, reaching the systemic circulation thanks to basal amino acid transporters. PepT1 substrates do not need to express typical peptide-like structure with a peptide bond, but preferential features are known such as a free N-terminal α-amino group, an electron-rich terminal carboxylic group and hydrophobic side chains. Preferential 3D features have even been described [START_REF] Rubio-Aliaga | Mammalian peptide transporters as targets for drug delivery[END_REF]. The minimal requirements for PepT1 substrates are two oppositely charged head groups separated by a spacer carbon unit with a distance > 500 pm and < 650 pm and it was reported that PepT1 is stereo selective preferring trans-rather than cisconformation [START_REF] Sai | Transporter-mediated drug delivery: recent progress and experimental approaches[END_REF]Majumdar, Duvvuri et al., 2004;[START_REF] Hamman | Targeting receptors, transporters and site of absorption to improve oral drug delivery[END_REF]Steffansen, Nielsen et al., 2004;Varma, Ambler et al., 2010;[START_REF] Smith | Proton-coupled oligopeptide transporter family SLC15: physiological, pharmacological and pathological implications[END_REF]. Good substrates of PepT1 are well known and are currently used for competition experiments, such as Glycylsarcosine (GlySar).

Different approaches have been developed to use the high capacity of PepT1 transporter. The most widely employed is to form amino acid or dipeptide conjugated prodrugs by linking the poorly absorbed molecule with amino acids or dipeptides ligands, sometimes with the addition of a linker. The entire prodrugs can be then recognized by PepT1 transporter as a peptidomimetic substrate and be transported, involving an improvement of the drug absorption [START_REF] Anand | Current prodrug strategies via membrane transporters/receptors[END_REF]. Numerous examples of prodrugs targeting PepT1 transporters exist and some of them are developed below. They are divided according to the moiety conjugated on the drug.

Amino acids ester prodrugs

Most of the examples found in the literature described the improved absorption results obtained from the conjugation of an amino acid esterified entity on a poorly absorbed BCS III drug. The most promising amino acid ester ligand is the valine, as the majority of prodrugs are obtained from its chemical linkage to the drug.

Indeed, a first relevant example is the valacyclovir prodrug of acyclovir. Acyclovir (ACY) is an antiviral drug prescribed for the treatment of infection from herpes virus. Its oral bioavailability is approximately 10-20%. It is transported in the intestine mainly by passive diffusion. The oral bioavailability of ACY in human is reported low and variable. In order to improve its membrane permeability, the L-Valyl ester prodrug of ACY targeting PepT1 was synthetized in order to obtain valacyclovir (ValACY). It was found in Caco-2 cells that ValACY showed 7-fold higher apical-to-basolateral transport than ACY, and inhibited the uptake of GlySar in competition experiment confirming its ability to target PepT1 in a saturable, concentration-and pH-dependent manner (de Vrueh, Smith et al., 1998). In rats, 3-to 5-fold improvement of oral bioavailibity was found after ValACY administration compared to free ACY. Similarly, in human, the oral bioavailability was measure closed to 55% with the prodrug. It was confirmed that PepT1 transporters were responsible of the permeation improvement by using PepT1 knockout (KO) mice. It was thus found a 4-to 6-fold and 2-to 3-fold decrease of the C max and AUC, respectively, after oral administration of ValACY compared with classical wild type mice (Yang and Smith, 2013;[START_REF] Yang | Impact of Peptide Transporter 1 on the Intestinal Absorption and Pharmacokinetics of Valacyclovir after Oral Dose Escalation in Wild-Type and PepT1 Knockout Mice[END_REF].

Cytarabine (Ara-C) is a chemotherapy agent for the treatment of acute myeloid leukemia and non-Hodgkin lymphoma. Conjugation with valine involves a 5-fold increase of the intracellular accumulation of Val-Ara-C compared to Ara-C in Caco-2 cells. Nevertheless, in vivo in rats, oral bioavailability of Ara-C after administration of the prodrug is only 4% because of low metabolic conversion of Val-Ara-C to the parent drug (Cheon, Hong et al., 2006).

Decitabine is an anticancer chemotherapy drug currently administered intravenously. In vivo in rats at a dose of 15 mg/mL, oral bioavailability was 27%. In order to improve it, amino aciddecitabine prodrugs were designed for PepT1 transporter targeting. Val-decitabine and Phedecitabine showed higher improvement of decitabine permeability in Caco-2 cells, and it was found oral bioavailability on rats of 47 and 51%, respectively. As both prodrugs were shown to inhibit GlySar uptake, PepT1 targeting was confirmed [START_REF] Zhang | A carrier-mediated prodrug approach to improve the oral absorption of antileukemic drug decitabine[END_REF].

Didanosine (DDI) is prescribed in the treatment of HIV to avoid replication. Whereas the oral bioavailability of DDI in rats was 7.9%, it increased up to 47.2% after administration of the Val-DDI prodrug. Moreover, it decreased to 33.9% when GlySar was administered with the prodrug, confirming the involvement of PepT1 transporter in the enhancement of the bioavailability [START_REF] Yan | Bifunctional peptidomimetic prodrugs of didanosine for improved intestinal permeability and enhanced acidic stability: synthesis, transepithelial transport, chemical stability and pharmacokinetics[END_REF].

Floxuridine, which is used to treat metastatic liver disease by infusion directly into the hepatic artery, can see an improvement of its Caco-2 cells membrane permeability by 2 to 11 folds when treated with amino acid ester prodrugs. Among them, D-Val-floxuridine is the most inhibited prodrug when incubated with GlySar, thus the best PepT1targeting conjugate [START_REF] Tsume | Enhanced absorption and growth inhibition with amino acid monoester prodrugs of floxuridine by targeting hPEPT1 transporters[END_REF].

Gancyclovir is prescribed for the treatment of cytomegalovirus. Valine ester prodrug of gancyclovir was synthetized in order to improve the oral bioavailability initially reported at 8%. Thus, it was found a 82.5% bioavailability for the prodrug and a 10-fold increase of the gancyclovir plasma concentration after valgancyclovir administration compared to the parent drug (Sugawara, Huang et al., 2000).

Levovirin is used to treat hepatitis C. Its oral bioavailability is less than 10%. Several amino acid prodrug of levovirin were developed and the best permeability improvement was observed for val-levovirin with a 48-fold increase. Furthermore, competition with GlySar on Caco-2 cells confirmed that the prodrug was a substrate of PepT1 [START_REF] Li | Transport of levovirin prodrugs in the human intestinal Caco-2 cell line[END_REF].

Zanamivir is prescribed to treat influenza infection. Usually administrated by inhalation, its oral bioavailability is very low (2%). To improve it, L-Val-, D-Val-or Ile-were tested as ligand in order to target PepT1 transporter. It was found a 9-fold higher influx permeability for L-Valzanamivir compared to free zanamivir in vitro, on Caco-2 cells (Gupta, Gupta et al., 2011).

Other amino acid ligands than valine such as Gly-or Phe-were also found to have promising results in the synthesis of amino acid ester prodrugs in order to target PepT1 transporter.

DMAE (1-(2',5'-Dimethoxyphenyl)-2-Aminoethanol) is used to treat orthostatic hypotension.

Tsuda et al. studied the possibility to improve its oral bioavailability to 100% with the conjugation of an amino acid to the drug by a peptide bond. Gly-ligand was found to form the most stable prodrug called Midodrine, with the best affinity for PepT1 transporter (Tsuda, Terada et al., 2006).

L-Dopa is used in the treatment of Parkinson's disease. Its oral bioavailability was reported to be 35% in patient which is not sufficient for appropriate medication. Thus, amino acid prodrug of L-Dopa was developed and it was found a 40-fold increase of in vitro uptake when targeting PepT1

with Phe-L-Dopa prodrug (Tamai, Nakanishi et al., 1998).

Dipeptides prodrugs

Most of the examples found in the literature describing prodrugs obtained from the conjugation of dipeptide ligands concerned BCS IV drugs. The dipeptide moieties not only allow to improve parent drug permeability but also to increase solubility. This is the case for Val-Val-prodrug of lopinavir, used to treat HIV infection. Among tested amino acids Ile, Trp and Met, or tested dipeptides Val-Val, Gly-Val and Val-Ile, Val-Val-lopinavir showed better solubility, stability and permeability [START_REF] Patel | Amino Acid Prodrugs: An Approach to Improve the Absorption of HIV-1 Protease Inhibitor[END_REF]. Similar results were found for saquinavir (SQV), as the intestinal absorption rates of Val-Val-SQV was 4.7-fold improved compared to SQV, and was significantly inhibited in presence of GlySar, which confirmed that the improvement was a consequence of PepT1 targeting [START_REF] Jain | Intestinal absorption of novel-dipeptide prodrugs of saquinavir in rats[END_REF]. An example of dipeptide-prodrug from BCS III drug is Gly-Val-glucosamine prodrug of Glucosamine developed by Gilzad-Kohan et al. It was found a 3-fold increase of C max and AUC when orally administered in rats compared to free Glucosamine (Gilzad-Kohan H., [START_REF] Gilzad-Kohan | Synthesis and characterization of a new peptide prodrug of glucosamine with enhanced gut permeability[END_REF].

Finally, a lot of transporters have been targeted by prodrugs. All of them are summed up in the table below associated to the drug substance (Table 4). [START_REF] Jain | Circumventing P-glycoproteinmediated cellular efflux of quinidine by prodrug derivatization[END_REF] Glucose, SGLT-1 Gly-Gly-Tyr-Arg (GGYR) α,β-SAPG-GGYR [START_REF] Nomoto | Improvement of intestinal absorption of peptide drugs by glycosylation: transport of tetrapeptide by the sodium ion-dependent Dglucose transporter[END_REF] Insulin α-SAPG-insulin [START_REF] Haga | Hypoglycemic Effect of Intestinally Administered Monosaccharide-Modified Insulin Derivatives in Rats[END_REF] p-nitrophenol (p-NP) β-disaccharide-p-NP [START_REF] Mizuma | Intestinal Na+/glucose cotransporter-mediated transport of glucose conjugate formed from disaccharide conjugate[END_REF] Monocarboxylate, MCT1 Carbenicillin Carindacillin [START_REF] Li | Role of the monocarboxylic acid transport system in the intestinal absorption of an orally active beta-lactam prodrug: carindacillin as a model[END_REF] Bile Levovirin Val-levovirin [START_REF] Li | Transport of levovirin prodrugs in the human intestinal Caco-2 cell line[END_REF] Zanamivir L-val-zanamivir (Gupta, Gupta et al., 2011) DMAE Gly-DMAE or midodrine (Tsuda, Terada et al., 2006) L-Dopa Phe-L-Dopa (Tamai, Nakanishi et al., 1998) Lopinavir Val-Val-lopinavir (Patel, Gohel et al., 2014) Saquinavir (SQV) Val-val-SQV [START_REF] Jain | Intestinal absorption of novel-dipeptide prodrugs of saquinavir in rats[END_REF] Glucosamine Gly-Val-glucosamine (Gilzad-Kohan H., [START_REF] Gilzad-Kohan | Synthesis and characterization of a new peptide prodrug of glucosamine with enhanced gut permeability[END_REF] Bolognese et al., 2012). Peptelligence ® was also used for parathyroid hormone oral delivery and it was demonstrated in Phase 2 clinical trial promising results in the improvement of bone density despite a lower efficacy than with injectable reference treatment [START_REF] Henriksen | Evaluation of the efficacy, safety and pharmacokinetic profile of oral recombinant human parathyroid hormone [rhPTH(1-31)NH(2)] in postmenopausal women with osteoporosis[END_REF].

Technologies currently under development

Another oral tablet formulation named GIPET ® was developed by Merrion pharmaceuticals (Dublin, Ireland). In this case, sodium caprate is used to open intestinal tight junction and an enteric coating is added to deliver the drug substance in small intestine. This technology was tested in a Phase 1 clinical study for insulin and GLP-1 analogs.

The last example of oral tablet for peptide administration was developed by Emisphere Technologies (Roseland, NJ, US) and is named Eligen ® . With vitamin B12 as active compound

(Eligen B12 ® ) SNAC (sodium N-[8-(2-hydroxybenzoyl)amino] caprylate
) is added as absorption enhancer. They have shown that SNAC does not cause histological damage to the intestinal epithelium. Eligen ® technology was recently applied to other peptides such as insulin or calcitonin and is currently in Phase 1 clinical studies.

Capsules were also explored as administration forms. They can contain the drug with additional agents or the drug pre-formulated as micro-or nano-particles.

For instance, POD ® technology developed by Ormaed (Jerusalem, Israel) was designed for insulin delivery. The drug was suspended in oil with the addition of an enzyme inhibitor (soya bean trypsin) and an absorption enhancer (EDTA), and filled into an enteric coated capsule.

Phase 2a and Phase 2b clinical trials reported a reduction of glycaemia to patient with uncontrolled type 1 diabetes.

Octreolin ® formulation uses the transient permeation enhancer technology developed by Chiasma, Inc (Newton, MA, US). Octreotide drug is suspended in oil and encapsulated in microparticles with sodium caprylate, well-known for its tight junction opening properties.

Microparticles are themselves suspended in castor oil or medium chain glyceride and administered in enteric-coated capsules. Phase 3 clinical trials demonstrated protection of bioactivity of the peptide and similar pharmacokinetic between oral administration of 20 mg Octreolin ® and 0.1 mg of subcutaneous administered octreotide [START_REF] Tuvia | Oral octreotide absorption in human subjects: comparable pharmacokinetics to parenteral octreotide and effective growth hormone suppression[END_REF].

NOD ® technology consists in calcium phosphate NPs with enteric coating and eventually containing carbomer as bioadhesive polymer which were mixed with exenatide and included into capsules.

Finally, a much more complex capsule design, named "Robotic Pill Technology", was recently developed by Reni therapeutics and Traverso et al. consisting in oral peptide injection [START_REF] Traverso | Microneedles for Drug Delivery via the Gastrointestinal Tract[END_REF]. The capsule is composed of a chemical compartment containing citric acid and sodium carbonate, an inflatable balloon-like structure with microneedles made of sugar and loaded with the drug. The structure remains intact in stomach but when reaching the intestinal pH 6.5-7, the capsule dissolves and a chemical reaction occures releasing CO 2 . Inflation of the balloon is thus induced allowing the apparition of the needles. The needles can then hook to the intestinal wall and the drug is thus delivered to the body (Fig. 10).

A.

B. Besides, recent studies present a combination between several approaches still with a will of improving drug permeability. For instance, two examples of hybrid complexes combining the ion-pairing approach and nanoencapsulation can be quoted from the literature. The ion-pairing is used to enhance the absorption, and the nanoparticles protect the drug from enzymatic degradation. Fattal et al. developed polyalkylcyanoacrylate NPs loaded with antisense oligonucleotides and coated with a cationic hydrophobic detergent which is an ion-pairing agent.

Lipophilic NPs-ion paired complex were then obtained. It was demonstrated an increase of the cellular transport of the complex and of its stability in plasma in vitro and in vivo, respectively.

Nevertheless, drug permeability was not found to be significantly improved (Fattal, Vauthier et al., 1998). Few years later, this hybrid strategy was also explored by Sun et al. with promising results. They formed an insulin-deoxycholate ion-pairing complex increasing the lipophilicity of insulin, and encapsulated it in PLGA NPs to protect it from degradation. It was then found in vivo a significant decrease of plasma glucose concentration after administration of the complex compared to the free drug (Sun, Liang et al., 2011).

Conclusion and future prospects

Crossing the intestinal membrane for hydrophilic and poorly absorbed BCS III compounds remains the first oral drug delivery challenge. The approaches developed to improve oral bioavailability are diverse. They consist in both protection of the molecule from the gastro intestinal tract in order to prevent from drug degradation by enzymes, and increase the drug permeability by using permeation enhancers, mucoadhesive systems, Cell Penetration Peptides, chemical modification or drug carriers. Although these strategies have shown some improvement in increasing absorption in the GI tract, they could result in undesirable effects. For instance, inflammation or irritation of the membrane may be caused by permeation enhancers as they are able to modify the phospholipid bilayer. Whereas, increasing the paracellular transport by opening the tight junctions may allow the entrance of toxic and pathogens molecules. In this way, future enhancement will be certainly found from targeting approaches. Achieving site specific and controlled delivery of the molecules preserving their physico-chemical, biological and therapeutic properties appear as very promising strategy.

Thus, developing an effective oral delivery system require to consider (i) the modification of the molecule itself in order to provide it a minimum of lipophilicity or enzyme resistance, for instance, (ii) the addition of novel functionality such as the ability to be recognize by an intestinal receptor, and (iii) the use of a delivery nanocarrier system. A combination of these approaches is now the most interesting alternative for drug absorption improvement. That is why many hybrid strategies appeared on the literature, but a lot of combinations still remain unexplored. For instance, coupling targeting option for the improvement of passive transport could be interesting to think about. The actual challenge is thus now to combine the right strategy in the same technology together with good clinical results, easy scale-up production and reasonable manufacturing cost in order to bring the formulation in the market.

Introduction

For many years, a lot of studies have been carried out to improve the oral bioavailability of therapeutics BCS III molecules. Various strategies have been developed and among them their encapsulation in polymer NPs. In 2012, the TRANS-INT consortium was funded by the European Union's FP7 programme. Composed of 17 academical and industrial partners they wondered about how NPs could be used for oral peptide delivery (http://www.trans-int.eu/, 2016). Particularly stable in the organism they offer a strong protection against gastrointestinal environment (pH, enzyme…). NPs have an important specific surface area due to their small size and as it is possible to control NPs physicochemical characteristics, drug release properties or biological behavior, they represent a serious option to consider for the improvement of poorly absorbed molecules. They have been first developed in 70s' as an alternative to liposomes that are less stable. NPs are described as colloidal systems between 10 and 1000 nm. Their surface can be modified and adapted by absorption or chemical grafting in order to improve their absorption through the intestinal membrane. Several manufacturing processes exist for NPs preparation depending on the active ingredient properties (solubility) to be encapsulated, on the nature of material used (polymer or monomer) and on the required NP properties (des Rieux A., Fievez et al., 2006). This chapter presented as a short review will first describe polymer materials usually used for NP formulation, then the different manufacturing processes developed with a special focus on the encapsulation of BCS III molecules, and the characterization methods.

Polymer materials

Polymers responding to bioavailability, biocompatibility and controlled degradation are authorized by FDA or European pharmacopeia to be part of the formulation composition of drug delivery devices. They influence not only the NP size but also the release profile of the molecule encapsulated. Generally, natural polymers induce a quick release whereas synthetic ones allow a sustained liberation of the drug over days (Soppimath, Aminabhavi et al., 2001) ., Fievez et al., 2006). The aim of NPs manufacturing methods is to add a polymer previously dissolved in a solvent into a non-solvent in order to obtain solid NPs. This short review will focus on the two main processes which have been largely developed, namely the nanoprecipitation and the double emulsion solvent evaporation processes. NP structure, mean particle size, advantages and drawbacks are detailed in Table 2 for these manufacturing processes. A rapid overview of the methods for both techniques will be then exposed.

Nanoparticle formulation

Nanoprecipitation

This method was initially developed by Fessi et al. and is particularly interesting as it is a one step process, requiring low amount of energy to obtain NPs between 100 and 500 nm. It consists in dissolving both polymer(s) and active ingredient in an organic solvent. Usually, the solvent is chosen as semi polar and must be miscible to water. This solution is added under moderate stirring to an aqueous phase potentially containing a hydrophilic surfactant and in which the polymer(s) is (are) not soluble (Fessi, Puisieux et al., 1989). NPs are thus instantaneously formed by self-assembly of the polymer(s) with the diffusion of the solvent to the aqueous phase. Indeed, the polymer(s) which is (are) not soluble in water precipitates inducing the formation of nanospheres encapsulating the active ingredient. Solvent is then removed by evaporation or filtration. NPs can be obtained by nanoprecipitation without any surfactant (Danafar, Rostamizadeh et al., 2016). Nevertheless, to avoid stability issue, small amount is added in aqueous phase such as poloxamer P188 or polysorbate 80 [START_REF] Fonseca | Paclitaxel-loaded PLGA nanoparticles: preparation, physicochemical characterization and in vitro anti-tumoral activity[END_REF]. Figure The nanoprecipitation method is preferentially used to encapsulate lipophilic drug as hydrophilic ones easily diffuse to the aqueous phase inducing low encapsulation rate. Barichello et al.

compared the encapsulation efficiencies of drugs presenting different degree of solubility [START_REF] Barichello | Encapsulation of hydrophilic and lipophilic drugs in PLGA nanoparticles by the nanoprecipitation method[END_REF]. As a result, they obtained low levels of encapsulation whatever the hydrophilic drug, compared to lipophilic ones. However, Bilati et al. adapted it for PLA and PLGA NPs encapsulating hydrophilic molecules (Bilati, Allemann et al., 2005). Their work focuses on parameters of the nanoprecipitation manufacturing process which can be modified to extend the technique to hydrophilic drugs. They explored different solvent or nonsolvent. It was found that using alcohol as non-solvent was interesting because of its low dielectric constant. It induces a lower dissolution of hydrophilic compounds avoiding drug leakage to the external phase. In this respect, ethanol or n-propanol was described as the most suitable. Concerning the solvent, the dielectric constant is also decisive as it impacts the NP size.

Indeed, the lower solvent dielectric constant is, the larger NPs are. To improve the encapsulation of hydrophilic drug, solvent has to be a polar aprotic solvent; ketones or esters are known as the most suitable. DMSO is also particularly appreciated for protein encapsulation as it is non-toxic for cells at controlled amount and provides a stable environment. Characteristics of protein-loaded NPs obtained by nanoprecipitation are described in Table 3. Significant drug load and encapsulation efficiency were obtained for hydrophilic molecules (Bilati, Allemann et al., 2005). Few years later, Patel et al. also get interest in the encapsulation of hydrophilic molecules.

Thanks to a statistical experimental design, they explored formulation and process parameters impacting the encapsulation of a BCS III drug (acyclovir) into PLA NPs. They found all parameters inducing a decrease of the solubility of the drug in the system can increase the encapsulation efficiency. Indeed, the affinity of the drug for the polymer increases if the drug is present in undissolved form in the system. In this way, suitable pH, low temperatures, osmotic gradient and ratio between phases are efficient parameters to explore. Responses of experiments carried on are proposed in Table 4 for acyclovir loaded PLA NPs obtained by nanoprecipitation.

Double emulsion

The double emulsion solvent evaporation method was developed to encapsulate both hydrophilic and hydrophobic molecules. For BCS III, drugs are dissolved in an internal aqueous phase with a surfactant. Polymer is dispersed under sonication into an organic solvent which is non-miscible to water in order to form a first W/O emulsion. This first emulsion is then added to an external aqueous phase in order to obtain W/O/W emulsion. Whereas surfactant is facultative for nanoprecipitation method, direction and stability of the emulsions can be favored according to the surfactant properties. Hydrophilic-Lipophilic Balance (HLB) allows the prediction of the emulsion direction as low HLB induces O/W emulsions whereas high HLB induces W/O emulsion. Finally, solvent is commonly removed by evaporation under reduced pressure and each droplet induces the formation of a NP after evaporation of the organic solvent [START_REF] Legrand | Sphéroïdes et formes vectorisées[END_REF]. Figure 2 gives a quick overview of the process.

Several parameters can be explored in order to improve the encapsulation of hydrophilic molecules by double emulsion process. For instance, Govender et al. increased the aqueous phase pH to 9.3 or incorporated pH-responsive excipients such as poly(methyl methacrylate-comethacrylic acid) PMMA-MAA), lauryc and caprylic acid to enhance hydrophilic drug encapsulation [START_REF] Govender | PLGA nanoparticles prepared by nanoprecipitation: drug loading and release studies of a water soluble drug[END_REF]. In another hand, Murakami et al. modified the solvent diffusion technique in order to improve the encapsulation. They used two water-miscible solvents, one with more affinity for PLGA and one with more affinity for the surfactant PVA, such as acetone and ethanol. The addition of alcohol in the PLGA solution dispersed the solution and induced the formation of nano-droplets. The alcohol diffused out of the droplets since the affinity of alcohol is better with acetone than with PLGA [START_REF] Murakami | Preparation of poly(DLlactide-co-glycolide) nanoparticles by modified spontaneous emulsification solvent diffusion method[END_REF].

Eventually, double emulsion manufacturing process was largely explored to encapsulate both hydrophilic small molecules and peptides. For instance, the co-delivery of doxorubicin and paclitaxel was studied and a 9.67 ± 0.22% drug load was obtained in 244 ± 12 nm PEG-PLGA NPs (Wang, Zhao et al., 2011). More recently, encapsulation of 5-fluorouracil in PLA NPs for oral delivery resulted in 283 ± 10 nm NPs 4.2%-loaded (Mattos, Altmeyer et al., 2016). 120 nm PLA-PEG-PLA micelles encapsulating lisinopril with a drug load of 8.2% were also obtained (Danafar, Rostamizadeh et al., 2016). Concerning the encapsulation of peptides or proteins, Tomar et al. succeeded in the encapsulation of insulin in PLA-PEG in 168.5 to 181.9 nm NPs with encapsulation efficiency from 38.4 to 58.5%, respectively, depending on the PEG length (Tomar, Tyagi et al., 2013). Similarly, Jain et al. obtained 4.5% insulin-loaded PLGA NPs of 209 ± 3.9 nm (Jain and Jain, 2015). Also, alpha-1 antitrypsin (α1AT) was loaded in PLGA NPs with a drug load close to 16% by the double emulsion technique (Pirooznia, Hasannia et al., 2012).

Finally, development of innovative but complex techniques gave even further enhancements. For instance, Gao et al. developed a strategy based on PLA NPs containing reverse micelles [START_REF] Gao | Preparation of aminated core-shell fluorescent nanoparticles and their application to the synchronous fluorescence determination of gammaglobulin[END_REF]. Composed of poly(glycerolmethacrylate) they act like reservoirs for hydrophilic molecules. In the same idea, Liau et al. developed double emulsion process with incorporation of pegylated liposomes for the encapsulation of ovalbumine [START_REF] Liau | A lipid based multi-compartmental system: Liposomes-in-double emulsion for oral vaccine delivery[END_REF].

Nanoparticle characterization

Characterization of NPs is essential as their physico-chemical properties explain not only their ability to encapsulate the active ingredient but also their behavior in contact of the biological membrane. There are currently characterized by their size, surface properties, encapsulation capacity, drug release rate and stability.

Size and structure

NP size is currently measured by Dynamic Light Scattering (DLS). A laser is crossing a suspension of NPs and when light hits particles, it scatters in all direction. The scattering intensity fluctuates over time as it is due to the Brownian motion of NPs. It is described as natural and perpetual and it follows a randomized movement. From these intensity fluctuations, information is obtained about the time scale of movement of the scatters. As it is well-known that small particles move faster than bigger ones, variation of laser intensity is then directly linked to NP size. NP imaging can be used to confirm DLS results but also to obtain details on particle conformation. Electronic microscopy is the only way to obtain a sufficient resolution for the observation. Scanning transmission electron microscopy is favored as it allows the observation of the NP structure meaning the nanosphere or nanocapsule conformation or the membrane thickness.

Surface properties

It is interesting to characterize NP surface properties as they have an important specific surface. It influences not only the stability of the suspension but also the behavior of the NPs in contact to the targeted site. Surface charge is described by zeta potential value, calculated from the movement of NPs in an electric field. It was demonstrated that a stable suspension is characterized by a zeta potential over |30| mV (Hans and Lowman, 2002).

Drug loading and entrapment efficiency

Measurements of drug load and entrapment efficiency are made after separation of NPs from external media by filtration or centrifugation. They must be carrying out under gentle conditions in order to not induced additional release of the drug from the NPs. The drug encapsulated can be evaluated directly by dosing the drug in the NPs, or indirectly by dosing the drug in the external media.

Drug loading is then calculated as follows:

Direct:

( ) Indirect:

( )
Then, the entrapment efficiency is described as follows:

( )
Drug loading and entrapment efficiency depend mainly of the drug solubility, of the initial amount of drug in the formulation and on the eventual interactions between the drug and the polymer. Indeed, Pridgen et al. found that due to its slight hydrophilic nature, the presence of PLGA in NPs formulation favored the interaction with hydrophilic molecules, improving their encapsulation (Pridgen, Alexis et al., 2013).

Drug release

Drug release from NPs is particularly difficult to measure mainly because of the technical separation of the drug released from the NPs. This step can be made by filtration or centrifugation with a risk to enhance the natural release. Dialysis is an alternative method with the condition of an absence of interaction between the drug and the dialysis membrane. Drug release depends on the molecule solubility in the external media, on the volume of this phase and on the NPs properties.

Stability

Stability of NPs suspension consists in the lack of sedimentation or agglomeration of the particles. It is controlled by size measurement overtime. Moreover, drug loading must stay constant without any degradation of the drug. This is controlled similarly as for drug release measurement. In order to stabilize NPs, suspension can be dried by a final freeze-drying step with addition of cryo protecting agent and a perfect control of pressure and temperature.

Conclusion

Finally, encapsulation of BCS III molecules in polymer NPs remains a challenging issue to face.

Promising results were obtained from the investigation and the optimization of classical manufacturing processes such as nanoprecipitation or double emulsion solvent evaporation.

Improvement of polymer NPs manufacturing process for the encapsulation of hydrophilic molecules will remain as long as new molecular entities will increasingly multiplicate.

Bastian,S.E., Walton,P.E., Ballard,F.J., Belford,D.A., 1999. Transport (Beauchamp et al., 1992;Bei Yang, 2012). De ce fait, l'acyclovir a été choisi comme molécule modèle pour la réalisation de cette preuve de concept dans l'idée de comparer les performances biopharmaceutiques de la molécule libre (acyclovir), de la pro-drogue (valacyclovir) et des nanoparticules fonctionnalisées encapsulant l'acyclovir.

Dans un premier temps, ce travail a exploré la possibilité de réaliser des NPs de PLA-PEG par la technique de nanoprécipitation, avec la contrainte d'obtenir des nanoparticules de taille inférieure à 200 nm afin qu'elles puissent traverser le mucus et rejoindre la paroi intestinale (Ensign et al., 2012). L'influence de paramètres de formulation et de fabrication sur cette contrainte a été étudiée par le biais d'un plan d'expérience.

Puis, différents ligands ont été sélectionnés en fonction de leur stabilité et de leur affinité pour le transporteur PepT1 et ont été couplés sur le polymère PLA-PEG (Anand et al., 2003). La première partie de ce travail de thèse est développée sous forme d'un article scientifique publié dans International Journal of Pharmaceutics (Gourdon et al., 2017).

Graphical abstract 1. Introduction

With the development of peptides as efficient therapeutics, potential strategies multiplied and diversified for increasing oral bioavailability of hydrophilic and poorly absorbed drugs. The bioavailability of these molecules depends on their ability to cross the intestinal epithelium and reach the systemic blood circulation (Johnson, 1994;Kwan, 1997). Formulation strategies have been developed in the past decades in order to increase their intestinal uptake, such as absorption enhancers or mucoadhesive systems (Aungst, 2012;Williams and Barry, 2004;Longer et al., 1985;Shaikh et al., 2011). Finally, targeting specialized intestinal cells allowed a more efficient oral delivery of drugs (Russell-Jones, 2004;Varma et al., 2010). This method was widely explored with the use of prodrugs targeting either intestinal receptors or transporters (Majumdar and Mitra, 2006;Sun, 2010).

Among them, PepT1, which is well known to promote the uptake of dipeptides, tripeptides and peptidomimetics due to an inward proton gradient, is the most widely studied. Thanks to its broad substrate specificity, it is still an attractive transporter to target by modifying a drug having a low oral bioavailability with a peptide-like ligand allowing its intestinal transport by PepT1 into the bloodstream. Midodrine, gabapentin enacarbil, zanamivir and oseltamivir are some of the molecules that have been chemically modified to target PepT1 [START_REF] Terada | Peptide transporters: structure, function, regulation and application for drug delivery[END_REF][START_REF] Vig | Amino acids as promoieties in prodrug design and development[END_REF]Tsuda et al., 2006;Gupta et al., 2011;[START_REF] Wang | Biological transporters as targets for new drug design[END_REF]). The most relevant example is valacyclovir (ValACY), an acyclovir (ACY) prodrug linked to valine. It was shown that 90% of ValACY was transported by PepT1, resulting in a 3-to 5fold increase in ACY human oral bioavailability (Beauchamp et al., 1992;Bei Yang, 2012;Soul-Lawton, 1995). Another strategy for intestinal targeting is the use of functionalized nanocarriers: the targeting ligand is then linked to the carrier (micelle, liposome, polymer nanoparticle) and not to the drug. Functionalized nanocarriers targeting transferrin, vitamin B12 or folate receptors have been widely studied for the oral delivery of drugs (Du et al., 2013;Gosselin and Lee, 2002;Roger et al., 2012;Stella et al., 2000).

Nevertheless, the potential of nanoparticles to target intestinal transporters has not been investigated yet. Even if nanoparticles have a particle size under 200 nm allowing them to go through the intestinal mucus layer and reach the targeted plasma membrane transporter, they are obviously not expected to cross the intestinal barrier by transport mechanism (Ensign et al., 2012). By targeting an intestinal transporter, the aim was rather artificially increase the residence time of loaded nanoparticles, and consequently the drug, close to the intestinal membrane surface, thus increasing its chance to cross the intestinal membrane by its own mechanism of membrane permeability. This strategy was recently proven to be effective using an external magnetic field to maintain magnetic carriers close to the intestinal membrane [START_REF] Seth | Performance of magnetic chitosan-alginate core-shell beads for increasing the bioavailability of a low permeable drug[END_REF]. In the present first proof-of-concept study, the ability to target an intestinal transporter with functionalized nanoparticles in order to improve the bioavailability of a poorly absorbed hydrophilic drug was explored. As PepT1 was largely studied in order to enhance oral drug absorption with a prodrug strategy, it was thus chosen for the current one. But the main challenge concerned poorly absorbed hydrophilic molecules which could not be designed as prodrugs targeting PepT1. For the latter, a new strategy consisting in encapsulating them into functionalized nanoparticles targeting PepT1 transporters was designed.

Polymer nanoparticles made from PLA, PLGA or PLA-PEG, safe biodegradable polymers, were frequently used in order to encapsulate hydrophilic molecules [START_REF] Peltonen | Improved entrapment efficiency of hydrophilic drug substance during nanoprecipitation of poly(l)lactide nanoparticles[END_REF][START_REF] Rodrigues | Primaquine-loaded poly(lactide) nanoparticles: physicochemical study and acute tolerance in mice[END_REF]. Moreover, by using a PLA-PEG polymer expressing a terminal amine function, various ligands can be linked on its polar end, as Pridgen et al. did with the antibody fragment Fc (Pridgen et al., 2013). Nanoparticles were obtained by a nanoprecipitation method (Fessi et al., 1989). Briefly, this method is based on a water miscible solvent, dissolving both polymers and active ingredient, and when added to the aqueous phase involves self-assembly of polymers and formation of nanoparticles.

First of all, formulation and process parameters influencing the particle size of PLA-PEG-NH 2 nanoparticles were studied. PLA-PEG-NH 2 polymer was chemically functionalized with Valine, Tyrosine-Valine, Valine-Glycine and Glycylsarcosine ligands, chosen according to their enzymatic stability, their ability to target PepT1 and their capacity of being coupled to the PLA-PEG-NH 2 polymer (Anand et al., 2003). The ability of functionalized nanoparticles to target PepT1 was then investigated. For that purpose competition experiments were carried out in vitro with a Caco-2 cell model. Finally, drug-loaded nanoparticles were prepared with PLA-PEG having the most relevant ligand. ACY was selected as a tool compound because of its capacity to be encapsulated in polymer nanoparticles and in light of the promising results obtained by the prodrug strategy with ValACY targeting PepT1 [START_REF] Gupta | Galactose decorated PLGA nanoparticles for hepatic delivery of acyclovir[END_REF]Patel et al., 2014). Based on the functionalized nanoparticles concept, we hypothesized that acyclovir when prepared as acyclovir-loaded nanoparticles with a dipeptide-targeting ligand would have better oral performance than when administered alone. By doing so, the dipeptide ligand would direct the acyclovir-loaded nanoparticles to PepT1 at the luminal membrane, increase its residence time at the membrane surface, and then allow acyclovir to be released from the nanoparticles with a greater driving force (i.e., concentration difference) for absorption than what might be achieved when administered alone. This approach might be useful for hydrophilic drugs such as ACY or those that cannot be chemically modified as prodrugs targeting directly PepT1, a strategy which is already quite well demonstrated and the most efficient way to improve oral bioavailability of hydrophilic drugs.

Transport experiments were conducted in vitro and in vivo in mice to conclude on the input of PepT1 targeting with functionalized nanoparticles to improve the transport of poorly absorbed drugs.

Materials and Methods

Materials

Poly were obtained from Sigma-Aldrich.

Nanoparticle preparation and characterization

Preparation of nanoparticles

PLA-PEG-NH 2 drug-free nanoparticles were prepared by nanoprecipitation. Polymers were dissolved in DMSO. The aqueous phase solution was prepared dissolving a surfactant in water. The organic phase was added to the aqueous phase under stirring. DMSO and water were removed by successive tangential filtrations during 1 h at 700 × g until a 100 µL residual volume of nanoparticle suspension was obtained. Nanoparticles were re-suspended in aqueous phase and stocked as a suspension.

Formulation and process parameters were studied using a factorial experimental design with PLA-PEG-NH 2 drug-free nanoparticles. The studied parameters are presented in Table 1 and the experimental plan is given in Supplementary Material (Table S1). The studied formulation parameters were the amount and ratio of polymers in the organic phase, the ratio of organic versus aqueous phases, the amount of surfactant in the aqueous phase and the surfactant type. The process parameters included needle diameter, stirring duration, stirring rate, and addition time. Each parameter was studied at 3 or 4 levels. Design of multivariate experiments and calculations were performed with the software NEMRODW ® . Data collected were the median particle size X 50 (i.e. 50% of the particles are smaller and 50% are larger) and the X 99 (particle dimension corresponding to 99% of the cumulative undersize distribution). Thirty-two experiments were carried out and one of them was repeated 3 times in order to assess the experimental error. Results are presented as a graphical analysis. The length and direction of each bar are related to the difference between the effect of the factor at levels 'i' and 'j'. A short bar means that the particle size is slightly altered by a change of level. At the opposite, a high bar indicates that one of the levels increased the response more than the other one. A positive bar (i.e. positive difference of the effect) means larger particle size at level 'i'. On the contrary, a negative bar (negative difference of the effect) means larger particle size at level 'j'. Significance levels, calculated from the experimental errors (repeated experiment), are indicated with dotted lines.

Then, the impact of PLGA addition on the actual drug loading was studied for set conditions, as well as increasing the theoretical loading from 25 to 50%. ACY-loaded nanoparticles were finally prepared by nanoprecipitation after dissolving 10 mg of PLA-PEG-ligand polymer, 5 mg of PLGA RG502 and 15 mg of ACY in 1 mL of DMSO. The aqueous phase solution was prepared dissolving 1% (w/v) of the poloxamer surfactant Kolliphor ® P188 in 4 mL of water.

The polymer solution was added rapidly to the aqueous poloxamer phase under magnetic stirring (600 rpm, 2 min), using a syringe and a needle (0.5 mm diameter) immersed in the solvent. The solvent and the non-entrapped ACY were removed by tangential filtration during 1 h at 700 g.

Size, shape, zeta potential and stability determination

Nanoparticle size was characterized by dynamic light scattering (DLS) using a Nanophox ® instrument (Sympatec, Germany). Zeta potential was measured with a NanoZ ® instrument (Malvern, UK). Scanning transmission electron microscopy (STEM, JEOL ® , Japan) was used to characterize the shape and the size of the nanoparticles. A ten-fold dilution was operated before dropping off 1 to 2 µL of the suspension on a copper grid covered with a Formvar/Carbon film. The sample was dried at room temperature and was negatively stained with uranic acetate for observation. Size measurements were regularly made over a 24-hour period in transport media in order to evaluate the stability of the nanoparticles in suspension overtime. As a precautionary principle, for both in vitro and in vivo experiments, nanoparticles were prepared just before the trials.

Drug loading and entrapment efficiency

Total amount of drug dissolved in the organic phase was determined by HPLC dosage after addition of 800 µL of DMSO to 200 µL of nanoparticle suspension in order to destruct the nanoparticles. Then, we measured the concentration of free ACY in the filtered medium collected from tangential filtration of the nanoparticles. The amount of encapsulated ACY was evaluated indirectly by calculating the difference between the total amount of ACY and the amount found in the filtrate. The drug loading was defined as the percentage of encapsulated drug compared to the total amount of nanoparticles. The entrapment efficiency was defined as the percentage of encapsulated drug compared to the total amount of drug dissolved in the organic phase. The ACY loading and entrapment efficiency were calculated using the following formulae:

Drug loading (%) = (Amount of encapsulated drug/Total amount of nanoparticles) × 100

Entrapment efficiency (%) = (Amount of encapsulated drug/Amount of drug dissolved in the organic phase) × 100

All samples were assayed using HPLC. The system comprised a Water 2695 Separation module associated with a Waters UV 2487 dual λ Absorbance UV detector at 300 nm. The column used was a C18 Inertsil ODS (4.6 x 250 mm, 5 µm). The mobile phase consisted in water/methanol (90:10 v/v) at pH 2.5 adjusted with orthophosphoric acid. The flow rate was 0.8 mL/min and the injected volume 20 µL.

Polymer synthesis

Typical procedure for polymer deprotection

PLA-PEG-NH 2 and PLA-PEG-Val (Fig. 1) were obtained from the deprotection of PLA-PEG-NH-Boc and PLA-PEG-Val-Boc by reacting in a mixture (50:50, v/v) of trifluoroacetic acid and dichloromethane (Walls et al., 2014). The reaction was continued for 2 hours under constant magnetic stirring at room temperature. Solvent and reagent were evaporated under reduced pressure. The residual oil was washed three times with dichloromethane followed by evaporation. Final product was triturated with water and pentane until obtaining a white powder. The powder was frozen and lyophilized for 24 h. Covalent linkage of the ligand on the polymer was carried out as described by Zhu H. et al. [START_REF] Zhu | Synthesis and characterization of well-defined lactic acid-PEG cooligomers and its tricarbonyl rhenium conjugates[END_REF]. Final functionalized polymer was deprotected from the terminal Boc group as described above. The residual oil was washed three times with dichloromethane followed by evaporation. Final product was triturated with water and pentane until obtaining a white powder. The powder was frozen and lyophilized during 24 h. The successive steps of polymer deprotection, protection of ligand and conjugation are described in Figure 2. Ligands, polymers, ligand-conjugated polymers and all products from conjugation, protection and deprotection steps were analyzed by NMR spectroscopy. Spectra were recorded on a Bruker Avance 600 (work frequency 600.13 MHz, spectra width 10200 Hz, probe temperature 27°C, scan length 3.21 s).

In vitro PepT1 targeting

Cell culture

Cultures were maintained in a humidified incubator at 37°C with 5% carbon dioxide in air atmosphere. Caco-2 cells were obtained from Servier Biology Research Department (Orléans, France). Cells were grown in plastic culture flasks (Corning ® ) in culture medium containing DMEM, 10% FBS (heat-inactivated), 1% NEAA, 1% L-Glutamine 200 mM and 1% P/S (10,000 UI/mL) and used between passages 77 and 90. When confluency reached 80%, these cells were detached by treating them with trypsin/EDTA, and then plated at a density of 30,000 cells/unit on 24-well plates (polycarbonate membrane -0.4 µm -0.33 cm 2 ). Cells were then grown in culture medium for 21 to 30 days. [ 14 C]Sucrose (1 nM) or Lucifer Yellow (100 µM) transports were used as markers of cell monolayer integrity. An apparent permeability of sucrose or Lucifer Yellow over 0.2 x 10 -6 cm.s -1 invalidated the results obtained for the well.

The integrity of the cell layer was also evaluated before permeability experiments by

Caco-2 permeability experiments

Apical transport medium is composed of HBSS supplemented with 0.1% BSA, 1 mM MES, and 1% Kolliphor ® P188, adjusted to the required pH. Basolateral transport medium is composed of HBSS supplemented with 0.1% BSA, 1 mM HEPES, adjusted to pH 7.4. After TEER measurements, culture medium was eliminated and replaced with respective transport medium for 30 min of pre-incubation in the same volume as for transport experiments (150 µL in the apical chamber, 500 µL in the basolateral chamber). At t 0 min , pre-incubation medium was eliminated and replaced in the apical side by transport medium containing the tested product and the marker of cell monolayer integrity, and in the basolateral side by fresh transport medium at 37°C. At t 5 min , 20 µL of apical medium was withdrawn for initial apical concentration measurement. At t 35 min and t 95 min , 90 µL of basolateral medium was withdrawn for transport measurement.

The functional expression of PepT1 on Caco-2 cells was evaluated by testing in competition a tracer amount of [ 3 H]Glycylsarcosine (10 nM) with increasing amounts (0 to 10 mM) of Glycylsarcosine at pH 6.0 and 7.4. Apparent permeability (Papp, cm.s -1 ) of [ 3 H]Glycylsarcosine was calculated as follows:

Papp (cm/s) = (dQ/dt)/(A.C 0 ), with dQ/dt the amount of drug transported from the apical chamber to the basolateral chamber per unit of time, A the membrane surface, and C 0 the initial drug amount in the apical chamber. The IC 50 (mM) of Glycylsarcosine was calculated using GraphPad Prism ® software and according to a simple mechanism of competition with Hill constant chosen as 1.

In order to evaluate the in vitro PepT1 targeting with nanoparticles, a tracer amount of [ 3 H]Glycylsarcosine (10 nM) was tested in competition with increasing amounts (0 to 10 mg/mL) of drug-free PLA-PEG nanoparticles, without any functionalization or functionalized with -NH 2 , -Val, -Val-Gly, and -Gly-Sar, at pH 6.0, after 30 min of preincubation of the cells with nanoparticles and under gentle stirring (60 rpm). Papp of [ 3 H]Glycylsarcosine was evaluated and its inhibition percentage was calculated using the following formula: 2E) and Caco-2 cells at late passages 87-112 (Caco-2L) (Lu et al., 1996).

Toxicity of NPs and ACY to Caco-2 cells

When confluency reached 80%, Caco-2 cells were detached by treating them with trypsin/EDTA and seeded at a density of 5,000 cells/unit on 96-well plates [START_REF] Kakinen | Solubility-driven toxicity of CuO nanoparticles to Caco2 cells and Escherichia coli: Effect of sonication energy and test environment[END_REF][START_REF] Patel | Cytotoxicity of folic acid conjugated hollow silica nanoparticles toward Caco2 and 3T3 cells, with and without encapsulated DOX[END_REF]. Cells were then grown in culture medium for 48 h (37°C, 5% CO 2 ).

100 µL of valine-functionalized nanoparticle suspensions concentrated at 0.5, 2.5 and 10 mg/mL, and ACY solutions concentrated at 10, 50 and 150 µM were incubated for 90 min with the cells. Experiments were performed in triplicate. The CellTiter Glo assay kit (Promega ® ) was used to determine the ATP content in the cells after exposure. After incubation with the compounds, the cells were washed twice with PBS and added with fresh PBS and CellTiter Glo solution in equal volumes. The cells were placed in a plate shaker for 2 min to induce lysis, and left to incubate for 10 min at room temperature. The luminescent signal was measured with a luminometer (Clariostar, BMG Labtek) and results were compared to control (cells without formulation and treated with ATP).

In vivo experiments

In vivo study plan was reviewed and approved by Servier ethical committee registered in the French Ministry of Research. It was in general accordance with the animal health and welfare guidelines, and standard operating procedures. C57Bl/6J female mice of approximately 11 weeks were used for the present study.

Study design

Thirty mice were marked and split into three groups, each composed of 10 mice. Free ACY, free ValACY and ACY encapsulated in nanoparticles functionalized with valine were administered by oral gavage to the animals of groups G1, G2 and G3, respectively. An equivalent dose of 25 mg/kg of ACY was applied for each group as performed in a previous study (de [START_REF] De Miranda | The disposition of acyclovir in different species[END_REF]. Blood samples were collected at room temperature from hindlimb marginal vein into sodium heparinized capillaries at 0.083, 0.25, 0.5, 1, 2, 4 and 8 h after dosing. Blood samples were centrifuged (10 min, 3500 rpm, + 4°C) and plasma samples were separated and transferred (exactly 10 µL) into tubes. Tubes were stored at -80°C until analysis.

Test items

The test items were prepared extemporaneously and protected from light. Free ACY, free

ValACY and ACY encapsulated in nanoparticles were dissolved (G1 and G2) or suspended (G3) in purified water. They were prepared at an ACY concentration of 2.5 mg/mL.

Administration volumes were determined according to the animal weight following the recommendation for oral administration on mice of 10 mL/kg, which correspond approximately to 200 µL for C57Bl/6J female mice.

Pharmacokinetic analysis

For each animal, plasma ACY concentrations versus time were submitted to a noncompartmental PK analysis using WinNonlin® (version 5.1, Pharsight Corporation, Cary, NC, USA). The area under plasma concentration-time curve (AUC 0-8h ) from time zero to 8 h after dosing was calculated using the linear-up and log-down trapezoidal rule. Values for the maximum plasma concentration (C max ) and time to C max (T max ) were obtained directly from the observed individual plasma concentration-time courses. For both in vitro and in vivo analyses, an analytical run consisted in a set of calibration standards analyzed in duplicate (one at the beginning and one at the end of the run), a blank sample without and with internal standard, quality control samples at 4 concentrations, all analyzed in duplicate and interspersed among the study samples. Peak area of analyte and internal standard were determined by the Analyst 1.6.2 (AB Sciex) integration system. ACY concentrations were evaluated using the internal standard method. The standard curves were generated from the peak area ratios of ACY/deuterated ACY (y) and the nominal ACY concentrations (x) using linear regression analysis with 1/x 2 weighting. For calculation of the accuracy, the precision and the relative error, the following formulae were respectively used:

Analyte quantification

Acc (%) = (

Statistical analysis

For the factorial experimental design performed to screen formulation parameters (factors), one of the experiments was repeated three times in order to assess the experimental error.

According to the result of each experiment, a mathematical model was set up. For each experimental result, a theoretical value was calculated. The model was considered as valid if the difference between the experimental values for the studied response and the values calculated by the model were not statistically different (ANOVA, p > 0.05). The effects of the factors were evaluated comparing the experimental error with the variation of the studied response calculated by the model.

In vitro and in vivo results were analyzed by ANOVA followed by Tukey's multiple comparison tests to determine their statistical significance compared to control; p < 0.05 was considered to be statistically significant. All in vitro experiments were conducted independently at least three times and performed in triplicate; the results are expressed as mean ± S.D.

Results

Nanoparticle formulation

Effect of formulation factors on particle size

PLA-PEG-NH 2 nanoparticles were prepared using nanoprecipitation method. Formulation and process parameters were studied using a factorial experimental design with X 50 and X 99 as studied responses. Results are presented as a graphical analysis (Fig. 3). Since experimental error was found negligible, dotted lines and x axis are almost merged together. The model was considered valid for the X 50 response since the differences between experimental values and the values calculated by the model were not statistically significant (ANOVA, p = 0.17).

In other words, these differences could be attributed to experimental error. For X 99 response, significant differences were found between experimental values and the values calculated by the model (ANOVA, p = 0.01). This result was due to particularly small experimental error (S.D. = 23.4 nm). It was thus considered that the model adequately depicted the effect of parameters on the response.

Then, the effect of the different factors on the studied responses was compared to the effect of experimental error. All studied factors appeared to be influent on the responses (ANOVA, p < 0.01 for both responses). The polymer concentration in DMSO had an influence on the particle size: the use of 5.71 mg/mL of polymer (level 1) led to lower X 50 and X 99 compared to 15 mg/mL (level 2) and 30 mg/mL (level 3). Regarding DMSO/water ratio, the graphical analysis showed a lower X 50 and X 99 for ratio 1:4 (level 2) than 1:2 (level 1) and 1:10 (level 3). It also appeared that 0.01% (w/v) of surfactant in water (level 2) induced lower X 50 than 0% (level 1) or 0.1% (level 3). Concerning X 99 , the lowest value was obtained with 0.1% of surfactant (level 3). As expected, increasing the needle diameter from 0.5 mm (level 1) to 0.8 mm (level 2) and to 1.2 mm (level 3) increased the X 50 and the X 99 . The graphical analysis also indicated that adding PLGA RG502 into the formulation increased the particle size.

Similarly, increasing the stirring duration increased the particles X 50 and X 99 . Concerning the addition time, the graphical analysis showed lower X 50 and X 99 for an instantaneous addition of the organic phase (level 1) into the aqueous phase. The surfactant type also had an influence on the particle size: the use of poloxamer P188 (level 3) led to lower X 50 and X 99 compared to the other surfactants. Finally, the slower the stirring rate was, the lower X 50 and X 99 were. According to these results, formulation and process parameters were set up for the preparation of drug-free nanoparticles. A 10 mg/mL of PLA-PEG-NH 2 solution in DMSO was prepared as well as a 0.05% (w/v) solution of the poloxamer surfactant Kolliphor ® P188 in water. The ratio between the organic and the aqueous phase solution was 1:4. The organic phase was added instantaneously to the aqueous phase with a 0.5 mm diameter needle immersed directly into the solvent. The suspension was kept under 600 rpm magnetic stirring for 2 min.

Nanoparticles X 50 of 32.8 ± 7.1 nm were obtained and Zeta potential was 22.6 ± 1.6 mV. In order to avoid flocculation in conventional transport medium during in vitro assays, the amount of surfactant in aqueous phase was increased to 1%. Zeta potential for the PLA-PEG-NH 2 nanoparticle suspension in conventional transport medium was then -0.039 ± 0.35 mV.

The X 50 of nanoparticles was 29.3 ± 7.2 nm and regular particle shapes were visible in TEM images (Fig. 4).

Encapsulation efficiency

With PLA-PEG-NH 2 without PLGA as core material of nanoparticles, disappointing results in terms of ACY loading and entrapment efficiency were observed. When PLGA was added at a 1:2 ratio (PLGA RG502/PLA-PEG-NH 2 ), higher drug loading was obtained. Indeed, for a theoretical loading of 25%, it was measured that an addition of PLGA in the formulation improved the actual drug load by two fold. For a targeted loading of 50%, an improvement of the actual loading by a factor 3.6 was observed. Characteristics of the loaded nanoparticles are summarized in Table 2. Nanoparticles size remained stable for 24 h and the X 50 mean was found 57.1 ± 2.0 nm.

Polymer derivation with ligand

Characterization of the polymers was performed by 1 H NMR analysis at each step of the synthesis. In order to validate each step (deprotection, coupling and final deprotection), the attention was focused on the terminal -NH 2 region and the integration of peaks was compared.

NMR spectra of PLA-PEG-NH 2 presented the characteristic peak of terminal -NH 2 at 7.72 ppm. When coupled to a ligand, the peak integration decreased to one proton and appeared at 6.6 ppm (Fig. 5). Similar results and spectra were obtained for PLA-PEG-GlySar and PLA-

Terminal -NH 2 Conjugated -NH-

PepT1 activity in Caco-2 cells and interaction with nanoparticles

Before testing the ability of functionalized nanoparticles to target PepT1, the functional activity of PepT1 in Caco-2 cells, as well as its proton dependency, had to be confirmed. At pH 7.4 in the apical chamber, the apparent permeability of [ 3 H]Glycylsarcosine was 2.2 times lower than that at pH 6.0. Increasing amounts of unlabeled Glycylsarcosine from 0 to 10 mM were also tested in competition with a tracer amount of [ 3 H]Glycylsarcosine. Unlabeled

Glycylsarcosine 10 mM was found to significantly decrease the apparent permeability of [ 3 H]Glycylsarcosine from 1.07 ± 0.08 x 10 -5 cm.s -1 to 2.73 ± 0.59 x 10 -6 cm.s -1 when pH 6.0 was applied in the apical chamber. Increasing amounts of unlabeled Glycylsarcosine reduced the [ 3 H]Glycylsarcosine Papp in a dose-dependent manner with an IC 50 at 4.1 mM, in accordance with the literature (Anand et al., 2003) (Fig. 6). 3). **) ligands. In vitro ACY Papp was 2.9 times higher when encapsulated in NPs-Val as compared to free ACY for an initial apical concentration of 50 µM. Nevertheless, free

ValACY prodrug strategy remained more efficient, as it increased 8.9-fold the Papp of free ACY when incubated at 50 µM in the apical compartment (Fig. 7).

Pharmacokinetics of ACY loaded in NPs-Val

After single oral administration of free ACY, encapsulated ACY in NPs-Val and free

ValACY at 25 mg/kg to female C57Bl/6J mice, the PK of ACY was characterized by a rapid absorption with a maximal plasma concentration reached between 0.25 h and 1 h. C max of ACY was measured to be 9.1 and 7.4 times increased when administered as ValACY prodrug compared to ACY free or encapsulated in NPs-Val, respectively. C max of ACY in NPs-Val was itself 1.2-times higher than ACY free, but not statistically different. Whereas a 4-time increase in AUC 0-8h was observed when the prodrug ValACY was administered compared to both ACY free and ACY encapsulated, no difference of AUC was found between free and encapsulated ACY. ACY was eliminated more slowly in its encapsulated form as its t 1/2 was 1.7 and 1.3 times higher than the elimination times observed after ValACY and free ACY forms, respectively. Regarding the MRT, it was found 2.6 and 1.3 times longer for ACY NPs-Val than for ValACY and ACY free, respectively (Table 4, Fig. 8).

Discussion

The intestinal epithelium is the most critical barrier to cross for an effective systemic effect for orally absorbed molecules. There have been many attempts to develop oral drug delivery systems that overcome this barrier. Currently, one of them consists in targeting intestinal transporters with prodrugs. The current proof of concept study undertook a novel approach to address this issue by developing functionalized drug-encapsulated nanoparticles as "cargo" capable of targeting PepT1 and to over-concentrate the drug on the intestinal epithelium apical surface. The two main advantages of targeting PepT1 are its well-known high capacity of transport as well as its high small intestine expression. Furthermore, the nanoparticles are not expected to be internalized by the cell through transport mechanism because of their size and the lack of known receptor-mediated transcytosis.

Nanoparticle size is a significant determinant of the formulation efficacy since only particles smaller than 200 nm are capable of penetrating mucus layer of the intestinal membrane and thus reach PepT1 transporter (Ensign et al., 2012). A factorial experimental design is a common way to study the impact of several parameters on specific characteristics. In our case, it was pointed out that not only formulation parameters but also process parameters could affect the size of the nanoparticles. Indeed, particle size was influenced both by the amount of polymer in the solvent phase and by the ratio between the aqueous and the solvent phases, since larger particles were obtained with high polymer content or a small proportion of the solvent phase in regards to the aqueous phase. These effects are in accordance with results previously observed by Bilati et al. (Bilati et al., 2005). They can be due to the high viscosity of the polymer solution, slowing down the diffusion of the solvent toward the nonsolvent phase and inducing the formation of aggregates. Similarly, process parameters such as the stirring duration and rate, and the addition duration of the solvent in the aqueous phase affected the particle size because of their influence on the solvent diffusion, inducing modification of the structure of the nanoparticles. Even if, as expected, the addition of PLGA in the formulation had an effect on the particle size, it could double the drug loading when added at a (1:2) ratio to PLA-PEG-NH 2 . With its more hydrophilic nature, PLGA can interact with both the drug and the PLA moiety of the PLA-PEG polymer, allowing a better encapsulation in the nanoparticles. These results have already been observed by Pridgen et al.

for insulin encapsulation in PLA-PEG nanoparticles (Pridgen et al., 2013). Because of the -NH 2 terminal function of the polymer, nanoparticles form a cationic suspension unstable in physiological medium. Thus, 1% of surfactant (w/v) was added to the formulation in order to neutralize the nanoparticle surface charge and stabilize the suspension. Influence of the nature of the surfactant used on the nanoparticle size could be partially explained by the surfactant molecular weights (poloxamer P188 Mw=768-9,510, PVA Mowiol 4-88 = 31,000 Da). The surfactant dispersed in the aqueous phase and inserted between the PEG fragments of the amphiphilic polymer neutralized the cationic charge but also increased the size of the nanoparticles.

In order to prove the intestinal transporter targeting strategy, PepT1 ligands were covalently linked to polymer nanoparticles. Valine, Tyrosine-Valine and Valine-Glycine were chosen according to their enzymatic stability and their known affinities toward PepT1 on the human intestinal Caco-2 cell line (Anand et al., 2003). Glycylsarcosine, a well-known substrate of PepT1, was also tested as a ligand. All of these ligands could be easily conjugated with PLA-PEG-NH 2 in an organic solvent as they could be dissolved in DCM. Amino acids were conjugated with PLA-PEG-NH 2 by peptide coupling with EDC. The conjugation between PLA-PEG-NH 2 and the ligand was confirmed by NMR spectroscopy and arbitrarily, only polymers effectively derivatized with the ligands (50% and over conjugative efficiency) were selected for PepT1 targeting study. The low efficiency obtained for PLA-PEG-Tyr-Val was explained by its degradation during the conjugation step. Nanoparticles were obtained from PLA-PEG-ligand by the nanoprecipitation method and we observed that conjugation of peptide ligands on PLA-PEG-NH 2 did not change the physico-chemical properties of the nanoparticles.

For a proof of concept of intestinal PepT1 transporters targeted with functionalized nanoparticles, human Caco-2 cell line was used as an in vitro human intestinal model (Anand et al., 2003;[START_REF] Liu | Uptake, transport and regulation of JBP485 by PEPT1 in vitro and in vivo[END_REF]. The expression of PepT1 and its H + /peptide symporter character was first confirmed for the cell-line used in the present study thanks to a competition experiment between a tracer nanomolar amount of [ 3 H]Glycylsarcosine and increasing amounts of unlabeled Glycylsarcosine (Bei Yang, 2012;[START_REF] Brandsch | Drug transport via the intestinal peptide transporter PepT1[END_REF][START_REF] Brodin | Transport of peptidomimetic drugs by the intestinal Di/tri-peptide transporter, PepT1[END_REF][START_REF] Newstead | Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2[END_REF][START_REF] Terada | Peptide transporters: structure, function, regulation and application for drug delivery[END_REF][START_REF] Ziegler | Distribution of the H+/peptide transporter PepT1 in human intestine: up-regulated expression in the colonic mucosa of patients with short-bowel syndrome[END_REF]. When functionalized nanoparticles instead of Glycylsarcosine were studied in competition with

[ 3 H]Glycylsarcosine, a significant decrease in the radioactive molecule transport was observed independently of the ligand, evidencing the ability of functionalized nanoparticles to interact and compete with PepT1 transport activity. The best inhibition implying the highest affinity for the transporter was observed for the valine ligand. These results could be compared with those obtained by Anand et al. regarding the apparent permeability of ACY prodrugs in Caco-2 cells (Anand et al., 2003). Papp of Gly-Val-ACY and Val-ACY were very similar at 3.01 ± 0.21 x 10 -6 and 2.99 ± 0.59 x 10 -6 cm/s, respectively. On the contrary, the results found in the present study for functionalized nanoparticles showed a significantly better inhibition of PepT1 activity for NPs-Val than for NPs-Gly-Val. Surprisingly, the present results also showed that the terminal amine function expressed on the surface of the nanoparticles was even enough to slightly inhibit the transport of the substrate and to be recognized by PepT1, suggesting the ability of PepT1 to consider a PEG monomer as potential substrate. Furthermore, functionalization with GlySar, a high capacity substrate of PepT1, did not allow a better affinity. Finally, high concentrations of PLA-PEG NPs, even without any ligand, could disturb the transport of the substrate, probably because of nonspecific steric hindrance.

According to these results, transport experiments were carried out with the poorly absorbed drug ACY. Improvement of its Papp was observed in vitro when encapsulated in PLA-PEG-Val nanoparticles. These results can be correlated with the inhibition results, suggesting that this new strategy targeting PepT1 and overconcentrating ACY-loaded nanoparticles close to the membrane might increase ACY intestinal permeability. Nevertheless, the improvement still remained 8.9-higher with the prodrug ValACY. In contrast to in vitro results, in vivo improvement of ACY AUC and C max when the drug was encapsulated in functionalized nanoparticles was not significant compared to free ACY. A part of the drug released quickly from the nanoparticles only after a few minutes because of the diffusion of the molecule through the polymer core, which induced similar C max and AUC to free ACY (Fig. S2). This burst release rapidly led to a free drug solution with no input of the nanoparticls strategy in enhancing intestinal drug absorption. This fast escape of hydrophilic drugs from PLA-PEG nanoparticles was already observed by Pridgen et al. when encapsulating insulin (Pridgen et al., 2013). Furthermore, PepT1 is expressed in the small intestine with a variable distribution along its entire length. An increase is observed from the duodenum to the ileum. Therefore, the drug could be released from the nanoparticles because of the burst even before reaching PepT1 transporters mostly expressed in the ileum (Drozdzik et al., 2014). However, ACY exposure still remained more important after ValACY prodrug administration. Furthermore, it was found here a more important increase of the C max and the AUC for ValACY compared to ACY free (9.1 and 4.0-times, respectively). Nevertheless, t 1/2 and MRT of ACY were significantly increased when encapsulated in NPs-Val as compared to free ACY. These results suggest that NPs-Val might extend the ACY absorption. Even if a large part of ACY was likely released quickly from the nanoparticle, another part of ACY might stay stuck in the nanoparticles and diffuse slowly, which would increase the t 1/2 and the MRT.

Conclusion

In conclusion, the present work resulted in a successful production of valine-functionalized nanoparticles able to target the intestinal PepT1 transporters. Nanoparticles were recognized by PepT1, which might lead to an increase of their residence time, of their local concentration and thus of the drug close to the membrane after release from the NPs, consequently improving the drug permeability. Valine-nanoparticles showed improved in vitro ACY permeability and longer in vivo mean residence time, as compared to free ACY. Nevertheless, the current findings demonstrated much higher AUC and C max of ACY for ValACY prodrug.

Therefore, no doubt, this strategy using functionalized-NPs cannot compete with prodrug delivery strategies targeting PepT1 that have been successful for many hydrophilic drugs.

ACY was chosen here as a tool compound in order to demonstrate the proof of concept of the strategy and its potential to increase oral drug delivery might be evaluated as an alternative for hydrophilic drugs that are not easily targetable as prodrugs.

Résumé des Résultats Principaux et Perspectives

L'objectif de cette première partie était de mettre en place une stratégie d'amélioration de transport d'une molécule BCS III. La stratégie choisie était le ciblage du transporteur intestinal PepT1 avec des NPs fonctionnalisées encapsulant une molécule modèle, l'acyclovir.

Cette preuve de concept a permis de mettre en lumière les résultats suivants :

 La possibilité de réaliser des NPs placebo de PLA-PEG par la technique de nanoprécipitation, avec une contrainte de taille inférieure à 200 nm afin qu'elles puissent traverser le mucus et rejoindre la paroi intestinale (Ensign et al., 2012). Ces résultats ne sont pas forcément corrélés à leur stabilité et à leur affinité pour le transporteur PepT1 en tant que dipeptides seuls (Anand et al., 2003)

Introduction

Advances in biotechnologies induced the discovery of several bioactive molecules based on peptides and proteins. Suitable carriers had to be developed because of challenges entailed by these molecules such as limited bioavailability and gastrointestinal degradation by digestive enzyme. In this way, nanoparticles (NPs) became a very attractive strategy for their application in biology and medicine. They are generally measured from 10 to 1000 nm and are defined as solid, submicron-sized drug carriers which may be biodegradable [START_REF] Reis | Nanoencapsulation I. Methods for preparation of drug-loaded polymeric nanoparticles[END_REF]. Oral delivery of drugs by NPs induced significant improvements in terms of bioavailability, biodistribution and residence time (Mahapatro and Singh, 2011). For instance, insulin-loaded NPs were found to not only preserve insulin activity, but also reduced blood glucose concentration in diabetic rats for up to 14 days after oral administration [START_REF] Damgé | Nanocapsules as carriers for oral peptide delivery[END_REF]. Depending on the needed NPs criteria, method of preparation differs. The present study firstly paid attention to the double emulsion solvent evaporation technique for the encapsulation of peptide. In that method, the hydrophilic molecule and polymers are dissolved in an internal aqueous phase and in an organic solvent, respectively.

The organic phase should not be miscible to water. The solubilization step is followed by an emulsion of the mixture to form water in oil emulsion. The resulting emulsion is then emulsified with an external aqueous phase in order to obtain water in oil in water emulsion.

Solvents are then usually removed under vacuum (Iqbal, Zafar et al., 2015;Vauthier and Bouchemal, 2009). The most extensively used biodegradable polymers for preparation of NPs are poly-D-L-lactide-co-glicolide (PLGA) and polylactic acid (PLA). They are broken down to monomeric units of lactic acid and glycolic acid in the organism. Furthermore, polyethylene glycol (PEG) grafting on the polymer chain is possible, allowing the self-assembly of NPs according to its amphiphilic properties and the possible expression of PEG-grafting ligand at the surface. These polymers were largely explored through double emulsion process to encapsulate peptide or hydrophilic molecules. For instance, the co-delivery of doxorubicin and paclitaxel was studied and a 9.7 ± 0.2% drug load was obtained in 244 ± 12 nm PEG-PLGA NPs (Wang, Zhao et al., 2011). More recently, encapsulation of 5-fluorouracil in PLA NPs for oral delivery resulted in 4.2%-loaded 283 ± 10 nm NPs (Mattos, Altmeyer et al., 2016). One hundred and twenty nm PLA-PEG-PLA micelles encapsulating lisinopril with a drug load of 8.2% were also obtained (Danafar, Rostamizadeh et al., 2016). Encapsulation of peptides or proteins by double emulsion process was also explored. Indeed, Tomar et al.

succeeded in the encapsulation of insulin in PLA-PEG in 168.5 to 181.9 nm NPs with encapsulation efficiency from 38.4 to 58.5%, respectively, depending on the PEG length (Tomar, Tyagi et al., 2013). Similarly, Jain et al. obtained 4.5% insulin-loaded PLGA NPs of 209 ± 3.9 nm (Jain and Jain, 2015). Also, alpha-1 antitrypsin (α1AT) was loaded in PLGA NPs with a drug load close to 16% by the double emulsion technique (Pirooznia, Hasannia et al., 2012). Nevertheless, the double emulsion method is energy-and time-consuming because of the two emulsion steps and requires a perfect formulation control of each phase.

Thus, the present study challenged the double emulsion solvent evaporation technique for peptide encapsulation by a simple emulsion process. Although it is a well-known NPs manufacturing process, simple emulsion process is not so largely exploited for the nanoencapsulation of hydrophilic molecules as very low drug load was obtained, and to our knowledge, no example of peptide encapsulation was found in the literature. Some examples of small entities encapsulation can be given such as, Rhodamine B which was encapsulated in PEG-PLA NPs with a drug load from 0.024 to 0.17% (Essa, Rabanel et al., 2011), or nisin in PLA NPs with a drug load from 0.5 to 6.0% (Ji, Lu et al., 2014). This one step process consists in the formation of nanocapsules by simple water in oil emulsion. Polymers, active ingredient and surfactant were dissolved in an organic phase which was added under sonication to an aqueous phase containing another surfactant. The organic phase was then removed by evaporation under vacuum.

According to the final objective of using these NPs for oral administration forms, the particle size is an important formulation constraint as they have to remain under 200 nm to be able to penetrate the mucus layer of the intestinal membrane (Ensign, Cone et al., 2012). Other formulation constraints were the drug load which was wanted as high as possible, and the suspension stability characterized by a zeta potential over |30| mV (Hans and Lowman, 2002).

Thus, the present study firstly developed a formulation of placebo NPs by the double emulsion process, and then investigated their ability to encapsulate a small peptide, oxytocin (OXY). Finally, the possibility of obtaining OXY-loaded polymer NPs from simple emulsion manufacturing process was explored. It was largely described that several parameters were found to be important to study for affecting the characteristics of the final suspension (particle size, zeta potential, drug load). Not only formulation parameters such as the type and amount of surfactant in the different phases, the polymers amount, the type and amount of additional excipient, the ratio between the phases, the type and the amount of viscosifying agent were evaluated, but also process parameters as sonication duration [START_REF] Budhian | Haloperidol-loaded PLGA nanoparticles: Systematic study of particle size and drug content[END_REF]

Materials and Methods

Material

Poly(lactic acid)-poly(ethylene glycol)-NH-Boc (PLA-PEG-NH-Boc) (PLA (Mw: 10 kDa)-PEG (Mw: 1 kDa)) polymer was synthetized by Specific Polymers. Dichloromethane (DCM), pentane, orthophosphoric acid and trifluoroacetic acid (ATFA) were purchased from Merck.

Oxytocin

Polymer preparation

PLA-PEG-NH 2 was obtained from the deprotection of PLA-PEG-NH-Boc by reacting in a mixture (50:50, v/v) of ATFA and DCM (Walls, Gupta et al., 2014) (Fig. 1). The reaction was continued for 2 h under constant magnetic stirring at room temperature. Solvent and reagent were evaporated under reduced pressure. The residual oil was washed three times with dichloromethane followed by evaporation. Final product was triturated with water and pentane until obtaining a white powder. The powder was frozen and lyophilized for 24 h. ATFA/DCM

Experimental plan strategy

Experimental plan strategy was used in order to identify influent formulation and process parameters for the obtention of PLA-PEG-NH 2 NPs from double or simple emulsion process.

Each parameter was studied from 2 to 4 levels. Design of multivariate experiments and calculations were performed with the software NEMRODW ® . Data collected were the median particle size X 50 , (i.e. 50% of the particles are smaller and 50% are larger), the drug load (DL) and zeta potential. Defined experiments were carried out and one of them was repeated 2 times in order to assess the experimental error. Results are presented as a graphical analysis.

The length and the direction of each bar are related to the difference between the effect of the factor at the level 'i' and at the level 'j'. A short bar means that the response was slightly altered by a change of level. On the opposite, a high bar indicates that one of the level increased more the response than the other one. A positive bar (i.e. positive difference of the effect) means larger particle size, higher drug load and zeta potential at level 'i'. On the contrary, a negative bar (negative difference of the effect) means larger particle size, higher drug load and zeta potential at the level 'j'. Significance levels, calculated from the experimental errors (repeated experiment), are indicated with dotted lines.

Nanoparticles preparation and characterization

Double emulsion process

A first factorial experimental design was carried out with PLA-PEG-NH 2 drug-free NPs in order to evaluate the feasibility of stable NPs suspensions with median particle size under 200 nm from double emulsion manufacturing process.

A primary W/O emulsion was prepared by adding an internal aqueous phase (WI) containing a surfactant into an organic phase (O) containing polymers and surfactant dissolved in ethyl acetate, under sonication (40 W, continuous mode, in an ice bath) using a 3.1 mm probe (Vibracell ® , Biobloch Scientific, France). This primary emulsion was added to a second aqueous phase (WE) containing a surfactant, under sonication (40 W, continuous mode, in an ice bath). The organic solvent was then evaporated from the double emulsion under vacuum (37°C, 100 rpm, 200 mbar) for 15 min. NPs were stored as a suspension.

Formulation parameters evaluated were the amount and the nature of surfactant in the three phases, the amount and ratio of polymers in the organic phase, the ratio of internal aqueous phase versus organic phase and the ratio of the first emulsion (WI/O) versus external aqueous phase. Sonication duration was tested as process parameters. Each parameter was studied at 2 or 3 levels. Twenty-eight experiments were carried out and one of them was repeated 2 times in order to assess the experimental error. The observed responses were the X 50 and the zeta potential reflecting suspension stability. The studied parameters and the experimental plan are presented in Table 1 andTable 2, respectively. A second factorial experimental design was carried out in order to evaluate the impact of the type and amount of viscosifying agent in the aqueous internal phase, and the type of PLGA, the surfactant amount and the total polymers amount in the organic phase, on the OXY load and on the median particle size of PLA-PEG-NH 2 NPs obtained from double emulsion manufacturing process. Drug load was targeted as maximal and median particle size under 200 nm.

A primary W/O emulsion was prepared by adding 0.5 mL of an aqueous internal phase containing 1% (w/v) of poloxamer P188 and OXY (theoretical loading set at 30%) and a viscosifying agent into 2 mL of organic phase containing Montane 80 and PLA-PEG-NH 2 /PLGA (2:1) dissolved in ethyl acetate, under sonication (40 W, continuous mode, 60 s, in an ice bath) using a 3.1 mm probe (Vibracell ® , Biobloch Scientific, France). This primary emulsion was added to a second aqueous phase (6.25 mL) containing 1% (w/v) of Montanox 80, under sonication (40 W, continuous mode, 60 s, in an ice bath). The organic solvent was then evaporated from the double emulsion under vacuum (37°C, 100 rpm, 200 mbar) for 15 min. Unentrapped OXY was removed by tangential filtration during 1 h at 800 × g until a 100

µL residual volume of NPs suspension was obtained.

Studied parameters were the nature and the amount of viscosifying agent in the internal aqueous phase, the amount of total polymers, the nature of PLGA and the amount of surfactant in the organic phase. Each parameter was studied at 2 or 3 levels. For PEG viscosifying agent, ratio 1 and ratio 2 were (3:2) and (2:3) of water/PEG, respectively. For HEC viscosifying agent, ratio 1 and ratio 2 were 1% and 2.5% (w/v) in water, respectively.

Thirteen experiments were carried out and one of them was repeated 2 times in order to assess the experimental error. The observed responses were the X 50 and the OXY load. The studied parameters and the experimental plan are presented in Table 3 andTable 4, respectively.

Simple emulsion process

Factorial experimental design was carried out in order to evaluate the feasibility of the manufacturing of PLA-PEG-NH 2 OXY-loaded NPs with median particle size under 200 nm and a maximal drug load from simple emulsion manufacturing process. Formulation parameters evaluated thanks to the experimental design were the phase ratios, the amount of surfactant in the organic and aqueous phases, the OXY/polymers ratio, the PLA-PEG-NH 2 /additional polymer ratio and the nature of additional polymer. Each parameter was studied at 3 or 4 levels. Seventeen experiments were carried out and one of them was repeated 2 times in order to assess the experimental error. The observed responses were the X 50 as NPs and the drug load. The studied parameters and the experimental plan are presented in Table 5 and Table 6, respectively.

Drug release

Residual suspension of OXY-loaded NPs (100 µL) was diluted in 3 mL of Caco-2 cells apical conventional transport medium (HBSS, 1 mM MES, 0.1% BSA, adjusted to pH 6.0) and incubated at 37°C under stirring (60 rpm) for 24 h. Initial amount of drug in the suspension after dilution at t = 0 min was determined by HPLC assay after addition of 800 µL of DMSO to 200 µL of suspension in order to destruct the NPs. Successive 500 µL samplings of NPs suspension were made at t = 10, 30, 60, 90 min and 24 h. Then, they were centrifugated in tangential filtration tubes for 5 min at 800 g. The concentration of free OXY in the filtered medium collected from tangential filtration of NPs was measured. The amount of OXY released was evaluated indirectly by calculating the difference between the total amount of OXY and the amount found in the filtrate.

Statistical analysis

For the factorial experimental design performed to screen formulation parameters (factors), one of the experiments was repeated 2 times in order to assess the experimental error.

According to the result of each experiment, a mathematical model was set up. For each experimental result, a theoretical value was calculated. The model was considered as valid if the difference between experimental values for the studied response and the values calculated by the model were not statistically different (ANOVA, p > 0.05). The effects of the factors were evaluated comparing the experimental error with the variation of the studied response calculated by the model.

Results

Double emulsion manufacturing method

Nanoparticle formulation development

Placebo PLA-PEG-NH 2 NPs were prepared using a double emulsion manufacturing method.

A factorial experimental design was carried out in order to identify the formulation and process parameters impacting the studied responses X 50 and zeta potential, and in which way they influence the response.

For X 50 response Fig. Response results for all the experiments are given in Table 2.

Internal aqueous phase composition

It was studied in this part the influence of the nature (parameter b7) and amount (parameter b8) of surfactant in the internal aqueous phase composition on NP size (Fig. 2.A and 2.B.) and zeta potential (Fig. 2.C. and 2.D.). PVA, poloxamer P188 and Montanox 80 at 0, 1 and 2.8%

(w/v) were evaluated.

Regarding the impact on NP size (Fig. 2.B.), it was found that poloxamer P188 (b7, level 2) led to smaller NPs than PVA (b7, level 1). Montanox 80 (b7, level 3) increased the X 50 compared to the two other surfactants. Then, smaller NPs were obtained with 1% (w/v) of surfactant (b8, level 2) compared to without any surfactant (b8, level 1) and 2.8% (b8, level 3) increased the NP size compared to the two other levels.

Concerning the impact on zeta potential (Fig. 2.D.), it was found that poloxamer P188 (b7, level 2) led to lower zeta values than PVA (b7, level 1). Montanox 80 (b7, level 3) decreased the zeta potential compared to PVA and poloxamer P188. Then, 1% (w/v) (b8, level 2) of surfactant increased zeta value compared with no surfactant. Similarly, 2.8% (w/v) (b8, level

3) increased zeta value compared to 0 (b8, level 1) and 1% (w/v).

Thus, parameters levels conducting to a compromise between the smallest NP size and the highest zeta potential values were poloxamer P188 at 1% (w/v).

Organic phase composition

The first emulsion was obtained after addition of the internal aqueous phase into the organic phase. It was studied in this part the influence of the nature (parameter b1) and amount (parameter b2) of surfactant, the addition of PLGA as second polymer in the NP core (parameter b3), the total polymer amount (parameter b4) and the ratio between the internal aqueous phase (WI) and the organic phase (O) (parameter b5), on NP size (Fig. 2.A and 2.B.)

or zeta potential (Fig. 2.C. and 2.D.). Poloxamer P188 and Montane 80 at 0.5, 1 and 2% (w/v) were evaluated. Ratios PLA-PEG-NH 2 /PLGA of (1:0), (2:1) and (4:1), for total polymer amounts of 0.5, 1 and 2% in the organic phase (w/v) were tested. Finally, (1:2), (1:3) and

(1:4) WI/O ratios were considered.

Concerning the impact on NP size (Fig. 2.B.), it was observed that using Montane 80 (b1, level 2) highly influence the particle size compared to poloxamer P188 (b1, level 1).

Decreasing their amount from 1% (b2, level 2) to 0.5% (w/v) (b2, level 2) slightly decreased the particle size, whereas using 2% (w/v) of surfactant largely increased NP size compared to 0.5 and 1% (w/v). It was also observed an important decrease of NP size for PLA-PEG-NH 2 /PLGA (2:1) ratio (b3, level 2) compared with a formulation without PLGA (b3, level 1).

Similarly, it was noticed an important decrease of NP size for PLA-PEG-NH 2 /PLGA (4:1) ratio (b3, level 3) compared with a formulation without PLGA. Significant but small increase was found for PLA-PEG-NH 2 /PLGA (4:1) compared to (2:1) ratio. Larger NPs were obtained with a total amount of polymers of 1% (b4, level 2) compared to 0.5% (w/v) (b4, level 1). It was observed a slight decrease of NP size with 2% (w/v) (b4, level 3) compared to 0.5% (w/v), which was more important compared to 1% (w/v). It was found that decreasing the amount of organic phase in the ratio WI/O from (1:3) (b5, level 2) to (1:2) (b5, level 1), induced a decrease of the NP size. This increase was also observed when studying the ratio WI/O (1:4) (b5, level 3) compared to the two other ratios.

Regarding the impact on zeta potential (Fig. 2.D.), it was observed that using Montane 80 (b1, level 2) rather than poloxamer P188 (b1, level 1) as surfactant led to higher zeta values.

Decreasing the amount from 1% (b2, level 2) to 0.5% (b2, level 2) slightly decreased the zeta potential, whereas using 2% (w/v) (b2, level 3) of surfactant induced a more significant decrease of zeta potential compared to 0.5 and 1% (w/v). It was observed a decrease of zeta potential for PLA-PEG-NH 2 /PLGA (2:1) (b3, level 2) ratio compared with a formulation without PLGA (b3, level 1). Similarly, it was also observed a decrease of zeta value for PLA-PEG-NH 2 /PLGA (4:1) ratio (b3, level 3) compared with a formulation without PLGA.

Significant but small decrease was found for PLA-PEG-NH 2 /PLGA (4:1) compared to (2:1) ratio. Higher zeta potentials were obtained with a total amount of polymers of 1% (level 2) compared to 0.5% (w/v). It was also observed an increase of zeta value with 2% (w/v) compared to 0.5% (w/v), whereas no difference was observed with 1% (w/v). It was found that increasing the amount of organic phase in the ratio WI/O from (1:2) (b4, level 1) to (1:3) (b4, level 2), induced an increase of zeta potential. This increase was also observed when studying the ratio WI/O (1:4) (b4, level 3) compared to (1:2), whereas a decrease was found compared to (1:3).

Thus, parameters levels conducting to a compromise between the smallest NP size and the highest zeta potential values were Montane 80 surfactant at 1% (w/v), PLA-PEG-NH 2 /PLGA

(2:1) ratio, total polymer amount of 2% (w/v) and WI/O ratio of (1:4).

External aqueous phase composition

It was studied in this part the influence of the nature (parameter b9) and amount (parameter b10) of surfactant, and the ratio between the first emulsion and the external aqueous phase (parameter b11), on NP size (Fig. 2.A. and 2.B.) and zeta potential (Fig. 2.C and 2.D.). PVA, poloxamer P188 and Montanox 80 at 1, 2 and 3% (w/v) were evaluated.

Concerning the impact on NP size (Fig. 2.B.), it was found that smaller NPs were obtained with poloxamer P188 (b9, level 2) compared to PVA (b9, level 1). Similarly, Montane 80 (b9, level 3) compared to both poloxamer P188 and PVA induced a decrease of NP size. 2% (w/v) (b10, level 2) of surfactant allowed obtaining larger NP size than 1% (w/v) (b10, level 1).

Finally, 3% (w/v) of surfactant (b10, level 3) induced larger NP size compared to 1% (w/v) and smaller NP size compared to 2% (w/v). It was found that (WI/O)/WE (1:2) ratio (b11, level 2) induced an increase of NP size compared to (2:5) (b11, level 1). Similarly, (3:10) ratio (b11, level 3) increased the size compared to (2:5) but decreased it compared to (1:2).

Regarding the impact on zeta potential (Fig. 2.D.), higher zeta values were obtained with poloxamer P188 (b9, level 2) compared to PVA (b9, level 1). Similarly, Montane 80 (b9, level 3) compared to PVA induced an important increase of zeta potential, and a very slight decrease compared to poloxamer P188. 2% (w/v) (b10, level 2) of surfactant allowed obtaining higher zeta values than with 1% (w/v) (b10, level 1). Finally, 3% (w/v) (b10, level

3) amount decreased zeta potential compared to 1% (w/v) and even more compared to 2% (w/v). It was found that (WI/O)/WE (1:2) ratio (b11, level 2) induced a decrease of zeta potential compared to (2:5) (b11, level 1). Similarly, (3:10) ratio (b11, level 3) decreased zeta value compared to (2:5). No significant difference was observed between (1:2) and (1:3) ratios.

Thus, parameters levels conducting to a compromise between the smallest NP size and the highest zeta potential values were Montanox 80 at 1% (w/v) and a first emulsion/WE (2:5) ratio.

Influence of the process parameters

It was studied in this part the influence of the process parameters and more specifically of the sonication durations for the manufacturing of the two emulsions (parameters b6 and b12), on NP size (Fig. 2.A. and 2.B.) and zeta potential (Fig. 2.C. and 2.D.). 30, 60 and 90 seconds were tested for both processes.

Concerning the impact on NP size (Fig. 2.B.), it was observed for the first emulsion that 60 s (b6, level 2) of sonication led to larger NPs than 30 s (b6, level 1). Increasing the duration until 90 s (b6, level 3) increased the NP size compared to 30 s but decreased it compared to 60 s. For the second emulsion, 60 s (b12, level 2) of sonication led to smaller NPs than 30 s (b12, level 1). Increasing the duration until 90 s (b12, level 3) increased the NP size compared to 30 s and even more compared to 60 s.

Regarding the impact on zeta potential (Fig. 2.D.), it was noticed that for the first emulsion, 60 s (b6, level 2) of sonication led to higher zeta potential than 30 s (b6, level 1). Increasing the duration until 90 s (b6, level 3) decreased zeta potential compared to 30 s and 60 s. For the second emulsion, 60 s (b12, level 2) of sonication led to higher zeta potential than 30 s (b12, level 1). Increasing the duration until 90 s (b12, level 3) increased the NP size compared to 30 s and 60 s.

Thus, 60 s of sonication for the two emulsion processes conducted to a compromise between the smallest NP size and the highest zeta potential values.

NPs formulation selected

All studied parameters appeared to be influent on NP size and zeta potential. Thus, levels conducting to the smallest NP size and to the highest zeta potential values were selected for the preparation of drug-free NPs by double emulsion process.

The primary W/O emulsion was prepared by adding 1 mL of an aqueous internal phase containing 1% (w/v) of poloxamer P188 into 4 mL of organic phase containing 1% (w/v) of Montane 80, 53.3 mg of PLA-PEG-NH 2 and 26.7 mg of PLGA RG502 dissolved in ethyl acetate, under sonication for 60 s. This primary emulsion was added to the second aqueous phase (12.5 mL) containing 1% (w/v) of Montanox 80, under sonication for 60 s. The organic solvent was then evaporated from the double emulsion under vacuum for 15 min. NPs were stored as a suspension. NPs obtained had X 50 = 185 ± 5 nm, X 99 = 249 ± 6 nm and ZP = 34.1 ± 0.99 mV (Table 7), and thus totally fit with the targeted formulation properties. Thanks to the experimental design, formulation and process parameters were set up in order to manufacture polymer NPs having size less than 200 nm and zeta potential over |30| mV.

OXY-loaded nanoparticles

From this polymer NPs formulation development by double emulsion process, the next objective was to encapsulate OXY as active ingredient. A second factorial experimental design was carried out in order to identify formulation parameters affecting the NP size and the drug load, and in which way they influence the response. X 50 and DL responses were studied.

For X 50 response Fig. 3.A., dotted lines and x axis were almost merged together as experimental error was found negligible. The effect of the different factors on the studied responses was compared to the experimental error. All studied factors appeared to be influent on the responses (ANOVA, p < 0.01): responses variations were consequences of factors effects and not of the experimental error. Moreover, the model was considered valid since the differences between experimental values and values calculated by the model were not statistically significant (ANOVA, p = 1.04). It was considered that the model depicted adequately the effect of parameters on the response and the results could be interpreted.

Concerning drug load response Fig. 3.C., some factors levels do not appeared to be influent on the responses (ANOVA, p < 0.5). Nevertheless, the model was considered valid since the differences between experimental values and the values calculated by the model were not statistically significant (ANOVA, p = 9.4).

Response results for all the experiments are given in Table 4.

Internal aqueous phase composition

It was studied in this part the influence of the addition of a viscosifying agent in the internal aqueous phase composition, on NP size (Fig. 3.A. and 3.B.) and OXY drug loading (DL) (Fig.

3.C. and 3.D.

). The nature of the agent (parameter b1) and its amount (parameter b3) were more specifically studied. For PEG, none, (3:2) and (2:3) ratios of water/PEG (ratio 1 and ratio 2, respectively) were tested. For HEC, none, 1% and 2.5% (w/v) (ratio 1 and ratio 2, respectively) were evaluated.

Concerning the impact on NP size (Fig. 3.B.), it was not found any significant influence of the addition of a viscosifying agent. NPs were observed significantly smaller whatever the ratio of viscosifying agent added (b3, level 2 and 3) than without any agent (b3, level 1).

Nevertheless, the same influence was found for the two ratios (b3, level 2 and 3).

Regarding the impact on the DL (Fig. 3.D.), it was found no significant impact of the nature of the agent. Surprisingly, addition of viscosifying agent at ratio 1 (b3, level 2) largely decreased the DL compared to without any agent addition (b3, level 1). No impact on the DL was observed between the addition of a viscosifying agent at the ratio 2 (b3, level 3) or without any agent. However, it increased the DL compared to the first ratio.

Finally, the impact of the nature of the viscosifying agent on the size or on the DL was not significant even if a slight increase of the DL could be observed with HEC. The lack of a viscosifying agent or its addition at a ratio 2 did not impact the DL whereas it significantly decreased the NP size at a ratio 2. Thus, 2.5% of HEC (w/v) seemed to conduct to the smallest NPs with the highest DL.

Organic phase composition

It was studied in this part the influence of the nature of PLGA added (parameter b2), the amount of surfactant (parameter b4) and the total amount of polymers (PLA-PEG-NH 2 + PLGA) (parameter b5), on NP size (Fig. 3.A. and 3.B.) and OXY drug loading (DL) (Fig.

3.C. and 3.D.). PLGA RG502 and PLGA RG502H were tested, as well as 1, 1.5 and 2% (w/v) of surfactant in the phase and 2, 3 and 4% (w/v) of polymers.

Concerning the impact on NP size (Fig. 3.B.), it was previously evidenced an influence of the addition of PLGA in the formulation, and it appeared that the nature of PLGA (b2) was also influent. Indeed, larger NPs were obtained with PLGA RG502H (b2, level 2) in comparison with PLGA RG502 (b2, level 1). No significant difference between 1 and 1.5% (w/v) (b4, level 1 and 2, respectively) of Montane 80 surfactant was observed. Nevertheless, larger NPs were obtained with 2% (w/v) (b4, level 3) of Montane 80 in the organic phase compared to the two other amounts. 3% (w/v) (b5, level 2) of total polymers concentration in the organic phase largely increased NP size compared to 2 and 4% (w/v) (b5, level 1 and 3) of polymers.

No difference was observed between 2 and 4% (w/v).

Regarding the impact on the DL (Fig. 3.B.), higher DL were obtained with PLGA RG502H (b2, level 2) than PLGA RG502 (b2, level 1). Increasing the amount of surfactant from 1%

(w/v) (b4, level 1) to 1.5% (w/v) (b4, level 2) largely increased the DL. Similar result was found from 2 (b4, level 3) to 1% (w/v) of surfactant. Nevertheless, no significant difference was observed between 1.5 and 2% (w/v). Increasing the total amount of polymers in the organic phase from 1 (b5, level 1) to 3% (w/v) (b5, level 2), or from 1 to 4% (w/v) (b5, level

3) largely increased the DL. The improvement was not significant from 3 to 4% (w/v).

Thus, regarding its significant impact on the NP size, PLGA RG502 appeared to be most relevant. As no difference was observed between 1 and 1.5% (w/v) of surfactant on the size and as 1.5% (w/v) led to higher DL, 1.5% was found to be the best compromise. Finally, no difference was observed between 2 and 4% (w/v) of total polymer amount on the size and 4%

(w/v) led to higher DL so it was considered as the best level to fit with the targeted results.

NPs formulation selected

Levels conducting to the smallest NP size and to the highest DL values were selected for the preparation of OXY-loaded NPs by double emulsion process.

The primary W/O emulsion was prepared by adding 1 mL of an aqueous internal phase containing 1% (w/v) of poloxamer P188, 68.6 mg of OXY (theoretical DL = 30%) and 2%

(w/v) of HEC, into 4 mL of organic phase containing 1.5% (w/v) of Montane 80, 106.7 mg of PLA-PEG-NH 2 and 53.3 mg of PLGA RG502 dissolved in ethyl acetate, under sonication for 60 s. This primary emulsion was added to the second aqueous phase (12.5 mL) containing 1%

(w/v) of Montanox 80, under sonication for 60 s. The organic solvent was then evaporated from the double emulsion under vacuum for 15 min. NPs were stored as a suspension. NPs obtained had X 50 = 195 ± 5 nm, X 99 = 260 ± 6 nm, ZP = 12.9 ± 1.0 mV and DL = 4.0 ± 0.7% (Table 7) and thus totally fit with the targeted formulation.

Thanks to this second experimental design, formulation parameters were improved and set up in order to manufacture polymer NPs having a size under 200 nm and an optimized DL. The drug release from the NPs was found biphasic and was characterized by a burst release up to 8% in less than 10 minutes followed by a slow release (Fig. 5).

Simple emulsion manufacturing method

OXY-loaded PLA-PEG-NH 2 NPs were prepared using a simple emulsion manufacturing method. A factorial experimental design was carried out in order to identify the formulation and process parameters impacting the studied responses X 50 and DL, and in which way they influence the response.

For X 50 response Fig. Response results for all the experiments are given in Table 6.

Organic phase composition

It was studied in this part the influence of the amount of surfactant in the organic phase (parameter b2), the ratio between total amount of polymer and drug (parameter b3), the addition of a second polymer in the organic phase (parameter b4) and its nature (parameter b6), on NP size (Fig. 4A. and 4.B.) and on the DL (Fig. 4.C and 4.D.). 1, 1.5 and 2% (w/v) of surfactant were tested, as well as (1:4), (1:3) and (1:2) ratios of OXY/polymers, and the addition of PLGA RG502 or PDLG 7502 at (2:1), (3:2) and (1:1) ratios compared to PLA-PEG-NH 2 alone. Concerning the impact on NP size (Fig. 4.B.), it was observed that increasing the amount of surfactant from 1 to 1.5% (w/v) (b2, level 1 and 2, respectively), slightly decreased the NP size, and 2% (w/v) (b2, level 3) largely increased NP size compared to the two other amounts. Decreasing the amount of polymer compared to the amount of drug from (1:4) (b3, level 1) to (1:3) (b3, level 2) and (1:2) (b3, level 1) increased the NP size.

Concerning the impact on NP size (Fig. 4.B.), it was found a very slight decrease of NP size when increasing the amount of water from (1:3) (b1, level 1) to (1:5) (b1, level 2) ratio. The increase of the NP size was more important with (1:10) (b1, level 3) ratio compared to the two other ratios. The amount of surfactant in the aqueous phase (b5) also had an influence since smaller NPs were obtained with 1.5% (w/v) (b5, level 2) of Montanox 80 surfactant compared to 1% (w/v) (b5, level 1). Addition of 2% (w/v) (b5, level 3) of Montanox 80 significantly increased the NP size compared to the two other amounts.

Regarding the impact on the DL (Fig. 4.D.), it was showed no difference between (1:3) (b1, level 1) or (1:5) (b1, level 2) internal aqueous phase/organic phase ratios. An increase of the DL was observed with (1:10) (b1, level 3) ratio compared to the two others. Finally, the amount of Montanox 80 in the aqueous phase (b5) also had an influence since lower DLs were obtained with the increase of the amount of surfactant.

Finally, a Montanox 80 surfactant amount of 1.5% (w/v) conducted to a compromise between the smallest NP size and to the highest DL. Regarding the W/O ratio, (1:5) led to smallest NPs whereas (1:10) to highest DL. An intermediary (1:7) ratio could be a good compromise.

NPs formulation selected

All studied parameters appeared to be influent on NP size and on the DL. Thus, levels conducting to the smallest NP size and to the highest DL values were selected for the preparation of OXY-loaded NPs by simple emulsion process.

The emulsion was prepared by adding 570 µL of an organic phase containing 5.7 mg of PLA-PEG-NH 2 , 5.7 mg of PDLG 7502, 5.7 mg of OXY and 2.3 mg of Montane 80 as a surfactant dissolved in a solvent blend EtAc/EtOH (3:2), into 4 mL of aqueous phase (W) containing 1.5% (w/v) of Montanox 80 surfactant, under sonication for 45 s. The organic solvent was then evaporated from the simple emulsion under vacuum. Unentrapped OXY was removed by tangential filtrations during 1 h at 800 × g until a 100 µL residual volume of NPs suspension was obtained. NPs were stored as a suspension. NPs obtained had X 50 = 226 ± 5 nm, X 99 = 301 ± 12 nm, ZP = -0.33 ± 4.75 mV and DL = 3.26 ± 0.87% (Table 7).

Thanks to the experimental design, formulation parameters were improved and set up in order to manufacture polymer NPs having size close to 200 nm and optimized DL.

The drug release from the NPs was found biphasic and was characterized by a burst release up to 17% in less than 10 minutes followed by a slow release (Fig. 5).

A. B. C. D.

Discussion

Various manufacturing processes have been previously developed in order to obtain polymeric NPs for the encapsulation of hydrophilic molecules. Although they have been applied for peptide encapsulation, it was not specifically developed for oxytocin entrapment.

The objective of this study was to obtain a stable suspension (|ZP| > 30 mV) with NP size under 200 nm and a drug load as high as possible. Thus, double emulsion process was firstly studied and adapted from the literature. Then, this well-known double emulsion manufacturing process was challenged by a simple emulsion process. As factorial experimental design is a common way to study the influence of formulation and process parameters on specific characteristics such as NP size, zeta potential and DL, it was not only used in order to set up the double emulsion process but also to design the simple emulsion process. Two factorial experimental designs were realized for the double emulsion process in order to obtain stable suspension with NPs under 200 nm and then loaded with oxytocin. A last experimental design allowed to obtain a challenging simple emulsion process for polymer NPs encapsulating oxytocin. Main characteristics of loaded NPs from the two processes were compared and effects of parameters levels on the size and the drug load were discussed.

Effect of surfactant type and concentration

It is well-known that depending on its physico-chemical properties, surfactant could promote the direction of the emulsion. Indeed, water in oil (W/O) emulsions are obtained with surfactant having a low HLB, whereas a high HLB surfactant is in favor of oil in water (O/W) emulsions. In this way, three surfactant with different HLB were tested for emulsion investigations, such as Montane 80 (HLB = 4.3), Montanox 80 (HLB = 15) or poloxamer P188 (HLB = 29). For double emulsion process, the first emulsion was WI/O. Theoretically, it was promoted by using a low HLB surfactant in the organic phase. This was confirmed experimentally as smaller and more stable NPs were obtained with Montane 80 surfactant instead of poloxamer P188. The second emulsion was O/WE emulsion which was induced by a high HLB surfactant in the external aqueous phase. It was also confirmed experimentally as more stable NPs were obtained with poloxamer P188 and Montanox 80. Furthermore, NPs size can be influenced by the molecular weight of the surfactant. Larger NPs were obtained with poloxamer P188 (Mw = 7,680-9,510 g/mol) than with Montanox 80 (Mw = 1,310 g/mol). Montanox 80 was thus chosen for the tested formulation after experimental design.

From these results, Montane 80 and Montanox 80 were chosen as organic and aqueous phase surfactants for the simple emulsion process, respectively.

Finally, the amount of surfactant in the different phases had an influence on both the NP size and zeta potential. When more surfactant was added, larger NPs was obtained. Indeed, the surfactant is both dispersed in the aqueous phase and inserted between the PEG fragments, so it neutralized the cationic charge which reduced zeta potential but also increased NP size. In this way, a compromise between the size and the stability of the suspension had to be made.

That is why 1% of poloxamer P188, 1.5% of Montane 80 and 1% of Montanox 80 were selected for WI, O and WE phases, respectively in the double emulsion process. Also, 0.4% of Montane 80 and 1.5% of Montanox 80 were selected for O and WE phases, respectively in the simple emulsion.

Effect of aqueous/organic phase ratio

Bilati et al. reported that larger particles are obtained with a smaller proportion of organic phase in regards to aqueous phase (Bilati, Allemann et al., 2005). Indeed, with small proportion of organic phase the diffusion of the solvent toward a large amount of aqueous phase is slowed down which induces the formation of bigger NPs or even aggregates. In this way, increasing amounts of organic and aqueous phases have been tested for double and simple emulsion processes, respectively. For the first WI/O emulsion of the double emulsion process, results were in accordance with the theory as larger particles were obtained with small proportion of organic phase in regards to aqueous phase.

Finally, it was demonstrated that a high amount of internal aqueous phase in which the molecule is dissolved, induces an increase of the amount of drug dispersing in the aqueous phase and, therefore, the increase of the encapsulation [START_REF] Budhian | Haloperidol-loaded PLGA nanoparticles: Systematic study of particle size and drug content[END_REF].

Furthermore, as NP size increased with the amount of external aqueous phase which allows higher encapsulation, higher DL was observed with higher amount of aqueous phase.

Effect of phase composition

In order to limit the OXY release from NP core, the composition of the different phases was explored.

It was firstly tested the addition of a viscosifying agent in the internal aqueous phase of the double emulsion process in order to slow down the diffusion of the drug from the NPs. HEC and PEG 400 have been tested as they are soluble or miscible to water. HEC was found more efficient on increasing the drug load than PEG. This result was explained by rheological behavior of these two viscosifying agents. For different amounts in the external aqueous phase (Fig. S1), it was found similar viscosity with 1% of HEC or with a ratio WI/PEG (3:2).

The ratio WI/PEG (2:3) induced a slight increase of the viscosity, but less important that the apparent viscosity of 2.5% of HEC. This explained why HEC was found more efficient on increasing the drug load than PEG whatever the ratio tested. Furthermore, the higher the amount of viscosifying agent was, the higher the obtained drug load was, without any impact on the NP size. significantly increase the DL (Pridgen, Alexis et al., 2013;Gourdon, Chemin et al., 2017).

Indeed, PLGA can interact with both the drug and the PLA moiety of the PLA-PEG polymer allowing a better encapsulation in the NPs. Also, its termination can influence the NP size and the drug load [START_REF] Cheng | Formulation of functionalized PLGA-PEG nanoparticles for in vivo targeted drug delivery[END_REF]. Indeed, ester or COOH terminations are more hydrophobic or hydrophilic, respectively. Acidic one is then expected to interact with the hydrophilic encapsulated molecule and induce a better encapsulation by retaining OXY in the NP core. Thus, experimental design for double emulsion process investigated on the influence of the use of PLGA RG502 (ester termination) or PLGA RG502H (acid termination) and it pointed out a better DL with COOH terminal function. Nevertheless, it induced a large increase of NP size.

Influence of PLGA lactide/glycolide ratio was investigated for simple emulsion process.

PDLG 7502 ((75:25) DL-lactide/glycolide copolymer, 0.2 dl/g, 17 kDa) and PLGA RG502 ((50:50) DL-lactide/glycolide copolymer, 0.2 dl/g, 7-17 kDa) was chosen as they have same molecular weight and viscosity but different lactide/glycolide ratios. PDLG is more hydrophobic than PLGA, so better peptide encapsulation is expected with PLGA. This hypothesis was explored with simple emulsion process and it was observed a significant improvement of the DL with both PLGA RG502 and PDLG 7502, but much more important with PLGA RG502. Moreover, it also largely increased the NP size whereas no significant change was observed with PDLG 7502. A compromise had to be done and PDLG 7502 was thus chosen for this simple emulsion process as NP size fit with the objective < 200 nm.

Finally, Zweers et al. demonstrated that increasing the amount of total polymer amount in the organic phase increased the drug load (Zweers, Grijpma et al., 2003). The more polymers there are, the more the organic phase viscosity is increased. Droplets are more resistant to breakdown which induces larger NPs. Concerning the influence of the amount of polymer on the DL, Budhian et al. observed than the more polymers there were, the faster they selforganized which induced a larger amount of drug encapsulated in NPs [START_REF] Budhian | Production of haloperidol-loaded PLGA nanoparticles for extended controlled drug release of haloperidol[END_REF]. Furthermore, thanks to a higher viscosity of the organic phase, the drug diffused more slowly to the aqueous phase (Görner, Gref et al., 1999). All these conclusions were confirmed for the simple emulsion process.

Effect of sonication duration

Sharma and coll. have shown that increasing the sonication time brought more energy in the process leading to a rapid dispersion of the organic phase and smaller NPs (Sharma, Madan et al., 2016). This was evaluated on double emulsion process. Sonication duration impact was measured for the two emulsifying steps, and its effect on the size and the zeta potential were not found linear. It appeared that the longest sonication duration resulted in the lowest zeta potential probably because of a difficult control of the formation of foam and air bubbles. A compromise had to be done between small NPs and stable suspension. The effect of process parameters on NPs obtained by simple emulsion were deduced from the conclusions obtained for double emulsion process.

Double or simple emulsion process

Finally, it was found that whatever the emulsion process, the DL was directly correlated with the particle size. It was thus possible to obtain OXY-loaded NPs from double and simple emulsion processes respecting the < 200 nm size condition and with a DL close to 5%. Then, biphasic drug release typical of polymeric NPs was observed for both processes: an important burst followed by a slow release over days. This fast escape of hydrophilic drug from PLA-PEG nanoparticles is known and was already observed by Pridgen et al. when encapsulating insulin (Pridgen et al., 2013).

Consequently, similar characteristics were found for OXY-loaded NPs obtained from double or simple emulsion processes. The median particle sizes (X 50 ) were under 200 nm or really close to and DL were 3 or 4% with a burst release within the first minutes. The way formulation and process parameters influenced the response appeared to be similar whatever the process used. Indeed, for both processes, NP size increased with the molecular weight of the surfactant and its amount in the different phases, with the amount of organic phase compared to the aqueous one, and with the sonication duration. The stability of the emulsions depended on the HLB of the surfactant which determined the direction of the emulsion, but also on the amount of surfactant. Finally, for both processes, the drug load differed according to the composition of the organic phase. Additional PLGA or an increase of the global amount of polymer can enhance the encapsulation of the active ingredient.

Conclusion

In conclusion, the present work allowed obtaining peptide-loaded NPs by double and simple emulsion processes. Influence of formulation and process parameters on NPs characteristics obtained from both processes were evaluated. It was highlight that similar results were obtained from both processes. Thus, with a simplifying perspective, a simple step process can be now favored, which is time and cost saving.

Résumé des Résultats Principaux et Perspectives

Introduction

Multiple strategies have already been explored in order to improve the oral bioavailability of hydrophilic molecules. The low bioavailability of these compounds is directly linked to their poor ability to reach the systemic blood circulation because of limitations to cross the intestinal epithelium and/or hydrolysis by peptidases (Johnson, 1994;Kwan, 1997). Among the different strategies already described, one of them consists in targeting specific transporters or receptors of the intestinal membrane (Russell-Jones, 2004;Varma, Ambler et al., 2010). This method was largely explored for prodrugs (Majumdar and Mitra, 2006;Sun, 2010). In the last few years, it was also investigated for nanocarriers (e.g. micelles, liposomes and polymeric nanoparticles (NPs)) that were functionalized with specific ligands and loaded with poorly absorbed drugs. Several intestinal receptors such as transferrin (Du, Fan et al., 2013), vitamin B12 [START_REF] Chalasani | Effective oral delivery of insulin in animal models using vitamin B12-coated dextran nanoparticles[END_REF] or folate (Roger, Kalscheuer et al., 2012) were targeted. The ability of functionalized nanocarriers to target intestinal drug transporters was evidenced only recently with valine-functionalized PLA-PEG NPs. In this study, the PepT1 transporter was targeted and when acyclovir, a hydrophilic poorly absorbed tool compound, was loaded into such functionalized NPs, a 3-times increase of its in vitro apparent permeability through Caco-2 cells was observed (Gourdon, Chemin et al., 2017a).

PLA-PEG-Valine NPs were obtained by nanoprecipitation and NPs had a diameter under 200 nm which allow them to cross the intestinal mucus barrier (Ensign, Cone et al., 2012). In this method, a water miscible solvent, dissolving both polymers and drug, was added to an aqueous phase in order to form instantaneously NPs (Fessi, Puisieux et al., 1989). NPs manufacturing processes have been described in the literature. The most widely studied for polymer NPs encapsulating hydrophilic molecules is the double emulsion solvent evaporation process. It consists in solubilizing the drug in an aqueous phase which is added under sonication into an organic phase containing the polymers and surfactants. This first water in oil emulsion is then added to another aqueous phase containing a surfactant leading to a water in oil in water double emulsion. Solvents are usually removed under vacuum (Iqbal, Zafar et al., 2015;Vauthier and Bouchemal, 2009).This manufacturing process was, for example, explored with PEG-PLGA copolymer NPs for the co-delivery of doxorubicin and paclitaxel (Wang, Zhao et al., 2011), with PLA NPs for oral delivery of 5-fluorouracil (Mattos, Altmeyer et al., 2016), with PLA-PEG-PLA micelles encapsulating lisinopril (Danafar, Rostamizadeh et al., 2016) and with PEG-PLGA NPs for the encapsulation of gemcitabine [START_REF] Zhao | Co-delivery of HIF1alpha siRNA and gemcitabine via biocompatible lipid-polymer hybrid nanoparticles for effective treatment of pancreatic cancer[END_REF]. Polymeric NP encapsulating a hydrophilic compound can also be obtained by a simple emulsion process. This process is not so largely exploited but some examples are quoted from the literature like rhodamine B encapsulation in PEG-PLA NPs (Essa, Rabanel et al., 2011) and nisin entrapment in PLA particles (Ji, Lu et al., 2014). This simple emulsion process consists in the formation of nanocapsules by dissolving polymers, the active ingredient and a surfactant in an organic phase not miscible to water, which is added under sonication to an aqueous phase containing a surfactant. The organic phase is then removed by evaporation under vacuum. Simple emulsion process was recently developed for the encapsulation of the small peptide oxytocin (OXY) (Gourdon, Declèves et al., 2017b).

Based

Material and Methods

Materials

Poly

Nanoparticles preparation and characterization

Polymer preparation

PLA-PEG-Val (Fig. 1) and PLA-PEG-NH 2 were obtained from the deprotection of PLA-PEG-Val-Boc and PLA-PEG-NH-Boc by reacting in a mixture (50:50, v/v) of trifluoroacetic acid and dichloromethane (Walls, Gupta et al., 2014). The reaction was continued for 2 h under constant magnetic stirring at room temperature. Solvent and reagent were evaporated under reduced pressure. The final oil was washed three times with dichloromethane followed by evaporation. Final product was triturated with water and pentane until obtaining a white powder. The powder was frozen and lyophilized for 24 h.

Formulation of NPs by nanoprecipitation

Drug-free NPs were prepared by nanoprecipitation after dissolving 10 mg of PLA-PEG-Val or PLA-PEG, and 5 mg of PLGA RG502 in 1 mL of DMSO. The aqueous phase solution was prepared dissolving 1% (w/v) of the poloxamer surfactant Kolliphor ® P188 in 4 mL of water.

The organic phase was added instantaneously to the aqueous phase under magnetic stirring (600 rpm, 2 min), with a 0.5 mm diameter needle immersed directly in the solvent. DMSO and water were removed by successive tangential filtrations at 700 × g until a 100 µL residual volume of NP suspension was obtained. NPs were re-suspended in aqueous phase and stocked as a suspension.

Formulation of NPs by double emulsion solvent evaporation

A primary W/O emulsion was prepared by adding 500 µL of an aqueous internal phase containing 1% (w/v) of poloxamer P188 and 2% (w/v) of HEC into 2 mL of organic phase containing 1.5% (w/v) of Montane 80, 53 mg of PLA-PEG-Val or PLA-PEG and 27 mg of PLGA RG502 dissolved in ethyl acetate, under sonication (40 W, continuous mode, 60 s, in an ice bath) using a 3.1 mm probe (Vibracell ® , Biobloch Scientific, France). This primary emulsion was added to a second aqueous phase (6.25 mL) containing 1% (w/v) of Montanox 80, under sonication (40 W, continuous mode, 60 s, in an ice bath). The organic solvent was then evaporated from the double emulsion under vacuum (37°C, 100 rpm, 200 mbar) for 15 min. NPs were stocked as a suspension.

Formulation of NPs by simple emulsion solvent evaporation

The simple emulsion was prepared by adding 570 µL of an organic phase containing 0.4% (w/v) of Montane 80, 5.7 mg of PLA-PEG-Val or PLA-PEG and 5.7 mg of PDLG 7502 dissolved in a mixture of ethyl acetate and ethanol (6:4), into 4 mL of an aqueous phase containing 1.5% of Montanox 80, under sonication (40 W, continuous mode, 45 s, in an ice bath). The organic solvents were then evaporated from the emulsion under vacuum (37°C, 100 rpm, 200 mbar) for 8 min. NPs were stocked as a suspension.

Size, shape, and zeta potential determination

NPs size was characterized by dynamic light scattering (DLS) using a Nanophox ® instrument (Sympatec, Germany). Zeta potential was measured with a NanoZ ® instrument (Malvern, UK). Scanning transmission electron microscopy (STEM, JEOL, Japan) was used to characterize the shape and the size of NPs. A ten-fold dilution was operated before dropping off 1 to 2 µL of the suspension on a cooper grid covered with a Formvar/Carbon film. The sample was dried at room temperature and was negatively stained with uranic acetate for observation. In order to avoid stability matter, for in vitro or in vivo experiments, NPs were prepared just before the experiments.

In vitro PepT1 targeting

Cell culture

Cultures were maintained in humidified incubator at 37°C with a 5% carbon dioxide in air atmosphere. Caco-2 cells were obtained from Servier Biology Research Department (Orléans, France). Cells were grown in plastic culture flasks (Corning) in culture medium containing DMEM, 10% FBS (heat-inactivated), 1% NEAA, 1% L-Glutamine 200 mM and 1% P/S (10,000 UI/mL) and used between passages 77 and 90. When confluency reached 80%, these cells were removed by treating them with trypsin/EDTA.

In vitro PepT1 targeting with nanoparticles

Caco-2 permeability experiments

Cells were plated at a density of 30,000 cells/unit on Transwell ® 24-well plates (polycarbonate membrane -0.4 µm -0.33 cm 2 ). They were then grown in conventional culture medium for 21 to 30 days. [ 14 C]Sucrose (1 nM) or Lucifer Yellow (100 µM) were used as markers of cell monolayer integrity. An apparent permeability of [ 14 C]sucrose (for radiolabelled experiments) or Lucifer Yellow (for OXY permeability experiments) over 0.2 x 10 -6 cm.s -1 invalidated the results obtained for the well. The integrity of the cell monolayer was also evaluated by measuring transepithelial electrical resistance (TEER). Monolayers with TEER over 1000 Ω.cm -2 were used for the experiments. Apical conventional transport medium is composed of HBSS supplemented with 0.1% BSA, 1 mM MES, and 1% poloxamer P188, adjusted to pH 6.0. Basolateral conventional transport medium is composed of HBSS supplemented with 0.1% BSA, 1 mM HEPES, adjusted to pH 7.4.

After TEER measurements, culture medium was discarded and replaced with conventional transport medium for 30 min of pre-incubation in the same volume than for transport experiments (150 µL and 500 µL in the apical and basolateral chambers, respectively). At t 0 min , pre-incubation medium was eliminated and replaced in apical side by transport medium containing the tested product and the marker of cellular integrity, and in basolateral side by fresh transport medium at 37°C. At t 5 min , 20 µL of apical medium was withdrawn for initial apical concentration measurement. At t 35 min and t 95 min , 90 µL of basolateral medium was withdrawn for transport measurement.

In order to evaluate the in vitro PepT1 targeting with NPs, a tracer amount of All in vitro experiments were carried out with Caco-2 cells at the same passage range (77-90).

The influence of the passage on PepT1 expression was previously studied by Lu et al.; it was not found any difference for [ 3 H]Glysar apparent permeability across Caco-2 cells at early passages 35-47 (Caco-2E) and Caco-2 cells at late passages 87-112 (Caco-2L) (Lu, Gough et al., 1996).

Caco-2 fluorescence intensity experiments

One equivalent of PLA-PEG-NH 2 and 0.8 equivalent of FITC were dissolved in anhydrous DMF [START_REF] Zheng | A water-soluble, low-cytotoxic and sensitive fluorescent probe based on poly(ethylene glycol) for detecting sulfide anion in aqueous media and imaging inside live cells[END_REF] (Fig. 2). The reaction was continued overnight under constant magnetic stirring at room temperature, protected from light. DMF was evaporated under reduce pressure. The final oil was triturated with a large amount of water until obtaining a white powder. The powder was frozen and lyophilized for 24 h. Final product was analyzed by NMR spectroscopy. NP formulations were prepared according to the 2.2 section with an addition of 1% (w/w) of PLA-PEG-FITC in the organic phase in order to obtain FITC-labeled PLA-PEG-Val and PLA-PEG NPs from the three manufacturing processes.

In order to assess the ability of functionalized NPs to target PepT1, in vitro fluorescent experiments were carried on. The Caco-2 cells were seeded into Corning ® 24-well multi-wells plates at a density of 5,000 cells/unit and cultured in conventional culture medium for 14 days. Before experiment, the cells were washed three times with PBS and pre-incubated for 30 min with apical conventional transport medium at pH 6.0 (500 µL). A second preincubation with a 10 mM GlySar solution in apical conventional transport medium at pH 6.0 (500 µL) could be applied for 30 min in order to saturate PepT1 transporters. Pre-incubation medium was eliminated and replaced by tested formulations concentrated at 2.5 mg/mL in NPs. Formulations were incubated with cells for 90 min, and then eliminated. Cells were finally washed 3 times with PBS. Fluorescence was measured for each well with absorption and emission filters set at 485 and 535 nm, respectively, with a multilabel plate reader (VICTOR ® , Perkin Elmer, USA). The remaining fluorescence linked to the cell membrane or within the cells was compared to the initial fluorescence of the tested NP formulations.

These results were confirmed by fluorescence confocal imaging. Caco-2 cells were seeded at a density of 30,000 cells/unit on Transwell ® 24-well plates and cultured similarly than for competition experiments for 21 to 30 days. After TEER measurements, culture medium was eliminated and replaced with conventional transport medium for 30 min of pre-incubation in the same volume than for competition experiments (150 µL and 500 µL in the apical and basolateral chambers, respectively). Cells were then incubated with fluorescent NPs (150 µL)

for 90 min at 37°C. After incubation, they were washed with PBS for 10 min, and 4% of paraformaldehyde was applied to fix the cells for 15 min at room temperature. Each polycarbonate membrane loading the cells was cut out from the filter and disposed into a well of a Corning ® 24-well multi-dishes. Then a solution of PBS, 1% BSA, 0.1% TX100 was applied followed by a dipping in PBS. The cell nuclei and membranes were dyed with TO-PRO-3 and phalloidin 555 for 20 min, respectively. After two 5 min-PBS washing, the membranes loading the cells were mounted on microscope slides with glycerol. They were kept at 4°C until confocal microscopy imaging.

dosing, detector was set at 220 nm. The column used was a Merck's LiChrospher 60 RPselect B C8 (4 x 1,250 mm, 5 µm) and was heated at 40°C. The mobile phase consisted in water/acetonitrile (85:15 v/v) at pH 2.1 adjusted with orthophosphoric acid. The flow rate was 1.25 mL/min and the injected volume 20 µL. Samples were analyzed against a set of calibration standards prepared in the mobile phase. The determination of drug concentrations was carried out over a calibration range obtained from a control sample T 1 (1 mg/mL), and a 10%, 1% and 0.1% dilution of T 1 in mobile phase. An analytical run consisted of T 1 and blank samples interspersed among the study samples and a set of calibration standards (one at the beginning and one at the end of the run). Analyte concentrations were evaluated using the internal standard method. The standard curves were generated from the peak area ratios of analyte/internal standard (y) and the nominal analyte concentrations (x) using linear regression analysis with 1/x 2 weighting.

Samples from in vitro experiments were defrosted at room temperature and mixed. They were transferred into UPLC vials without any dilution and drug concentration was determined using UPLC/MS/MS detection (Xevo, Acquity Waters). The column was an Acquity BEH Phenyl (Waters) (2.1 x 100 mm, 1.7 µm) and was set à 25°C for both compounds. The injection volume was 3 µL, and the flow rate was 0.55 mL/min. The mobile phase consisted in 90% of water, ammonium formate 10 mM, formic acid 0.1% and 10% of acetonitrile.

Parameters of the detector (TQD, Waters) were as follows: capillary tension 1 kV, source temperature 120°C, desolvatation temperature 500°C, gas flow for desolvatation 1,100 L/h, gas flow for cone 20 L/h, nebulizer pressure 7 bar, collision energy 5 eV and cone voltage 30

V. Samples were analyzed against a set of calibration standards prepared in conventional transport medium. The determination of drug concentration was carried out over a calibration range of 0.5 ng/mL (LLOQ) to 1,000 ng/mL (ULOQ) in conventional transport medium. The standard curve consisted of 12 calibration standards. An analytical run consisted of a set of calibration standards analyzed in duplicate (one at the beginning and one at the end of the run), a blank sample without and with internal standard, quality control samples at 4 concentrations, all analyzed in duplicate and interspersed among the study samples. Peak area of analyte and internal standard were determined by the Analyst 1.6.2 (AB Sciex) integration system. Analyte concentrations were evaluated using the internal standard method. The standard curves were generated from the peak area ratios of analyte/internal standard (y) and the nominal analyte concentrations (x) using linear regression analysis with 1/x 2 weighting.

For calculation of the accuracy, the precision and the relative error, the following formulae were respectively used:

Acc (%) = (

Statistical analysis

In vitro results were analyzed by ANOVA followed by Tukey's multiple comparison tests to determine their statistical significance compared to control and p < 0.05 was considered to be statistically significant. All in vitro experiments were conducted independently at least three times and performed in triplicate and the results are expressed as mean ± S.D. NPs surface was also characterized measuring zeta potential of PLA-PEG-Val NP suspensions for the three manufacturing processes (Table 2). Zeta potentials of NP suspensions obtained by emulsion processes were 2-folds higher than those obtained from nanoprecipitation process.

In vitro PepT1 interaction with nanoparticles

Drug-free PLA-PEG-Val and PLA-PEG NPs were prepared by nanoprecipitation, simple or double emulsion processes. All these NP formulations were incubated at 2.5 mg/mL together with [ 3 H]GlySar with Caco-2 cells for a competition study in conventional transport medium at pH 6 in the apical side. The [ 3 H]GlySar apparent permeability is presented in Figure 4. A significant decrease in [ 3 H]GlySar apparent permeability was observed when PLA-PEG-Val NPs were added to the media whatever the NP manufacturing process used. Inhibitions of 31.5 ± 4.0%, 31.6 ± 6.8%, and 42.7 ± 7.7% for nanoprecipitation, simple emulsion and double emulsion, respectively were found. With PLA-PEG NPs, the decrease in the [ 3 H]GlySar apparent permeability was found significant for the two emulsion processes, but not for the nanoprecipitation. Valine-functionalized NPs allowed a significant higher inhibition of [ 3 H]GlySar apparent permeability compared with not functionalized PLA-PEG NPs. Indeed, PLA-PEG-Val NPs decreased [ 3 H]GlySar apparent permeability 1.5, 1.6 and 1.3 times more than without any ligand when the NPs were prepared by nanoprecipitation, double emulsion and simple emulsion, respectively. However, the [ 3 H]GlySar inhibition difference observed between the three manufacturing processes was not found significant whatever the NPs polymer PLA-PEG-Val or PLA-PEG.

The ability of NPs to target PepT1 depending on their manufacturing process was also explored measuring fluorescence intensity of Caco-2 cells after incubation with fluorescent FITC-NPs. NPs were obtained by nanoprecipitation, simple or double emulsion, with 1% (w/v) of PLA-PEG-FITC added to the formulations as described in materials and methods.

PLA-PEG-FITC linkage was confirmed by 1 H NMR spectroscopy as well as the lack of residual free FITC. Firstly, the effect of the addition of PLA-PEG-FITC on the surface charge of the NPs was evaluated by comparing zeta potentials of PLA-PEG-Val NP suspensions (Table 2). A 1.2 mV-averaged decrease was observed for the three manufacturing processes which was significant for both nanoprecipitation and simple emulsion processes (p = 0.0031 and p = 0.006, respectively), but not for the double emulsion process (p = 0.0562). Caco-2 cells at 2.5 mg/mL concentration, with or without a 10 mM GlySar preincubation for 30 min. The ability of NPs to bind and/or penetrate the cells thanks to their interaction with PepT1 was evaluated comparing the amount of residual fluorescence intensity on Caco-2 cells after incubation with either functionalized NPs or with non-functionalized NPs (Fig. 5). Residual fluorescence was significantly higher for PLA-PEG-Val NPs whatever the manufacturing process used compared to PLA-PEG NPs (3.3 times for double emulsion process, 6.7 times for simple emulsion process and 6.0 times for nanoprecipitation). By comparing manufacturing processes between each other's for PLA-PEG-Val NPs, residual fluorescence differences were all significant. The highest residual fluorescence was observed for double emulsion (39.1 ± 3.3%), followed by simple emulsion (19.6 ± 3.1%) and then by nanoprecipitation (10.0 ± 0.9%), 2 and 4-fold weaker, respectively.

For experiments done with a 30 min GlySar preincubation, PLA-PEG-Val NP residual fluorescence was significantly decreased from 39.1% to 3.4% for double emulsion process.

The decrease was not significant for nanoprecipitation process while for simple emulsion, it induced a significant 1.6-decrease. No influence of GlySar preincubation was observed for non functionalized PLA-PEG NPs except for the double emulsion process with a 3.9decrease. For this double emulsion process with 30 min GlySar preincubation, the residual fluorescence after PLA-PEG-Val NPs incubation reached the residual fluorescence after PLA-PEG NPs incubation. These results clearly showed that targeting PepT1 with NPs significantly increase NP binding and/or penetration within the cells. This observation was well reversed with an excess of free Glysar in NPs produced by emulsion, suggesting that the mechanism of NPs interaction with cells was likely PepT1-mediated.

Finally, confocal microscopy was performed in order to observe NPs with Caco-2 cells. It was then possible to compare PLA-PEG-Val and PLA-PEG NPs according to their manufacturing process (Fig. 6). It was observed for PLA-PEG-Val NPs obtained by nanoprecipitation process a small amount of fluorescence inside the cell. However, nuclei and membrane remained blue and red, respectively (Fig. 6,A). For simple emulsion, fluorescent NPs were found mainly on the cell membrane. Nuclei remained perfectly blue (Fig. 6,B). For double emulsion process (Fig. 6, C), they were observed on the membrane and even in the cell reaching the nucleus. Indeed, nuclei were observed less blue than for simple emulsion which was explained by the presence of green NPs inside. For a same double emulsion process, the behavior of non-functionalized When OXY was dosed as NPs manufactured by the double emulsion process, a significant 2.1-fold increase of OXY apparent permeability was evidenced in comparison with free drug.

On the other hand, OXY encapsulation in NPs obtained by nanoprecipitation or simple emulsion processes had no effect on its apparent permeability.

According to in vitro results, OXY-NPs obtained by the double emulsion process were selected for in vivo experiments. After single intravenous (IV) administration of free OXY (G1 group, dosed at 0.01 mg/kg) and oral administration of free OXY (G2 group, dosed at 10 mg/kg) or encapsulated in NPs-Val obtained by double emulsion (G3 group, dosed at 10 mg/kg) to male C57Bl/6J mice, OXY pharmacokinetic was characterized. A rapid absorption of OXY was observed with a maximal plasma concentration reached between 0.08 h and 0.25 h after IV and oral administration, respectively. Per os, C max of OXY was measured to be 2.1 times higher when administered encapsulated in NPs-Val compared to free OXY (4167 ± 3436 vs 8872 ± 6049 pg/mL). Furthermore, a 6-time increase in AUC t was observed when OXY NPs-Val were orally administered compared to free OXY (1334 vs 8019 pg.h/mL).

Discussion

Among all the available routes of administration, oral route is the most convenient. However, new entities face many hurdles such as poor absorption, poor permeability, or rapid enzymatic or pH-induced degradation in the gastro intestinal tract. Various approaches have been designed to overcome these barriers including absorption enhancers, conjugation or chemical modifications, enzyme inhibitors and muco-adhesive polymers, often in combination. A targeted delivery approach was chosen in the present study, focusing on the valine functionalized polymeric NPs ability to target intestinal PepT1 transporter (Gourdon, Chemin et al., 2017a). In this previous work, NPs were obtained by nanoprecipitation. However, other manufacturing processes exist. Firstly, this study focused on the influence of the manufacturing process on ability of the polymeric NPs to target PepT1 transporter and on their behavior in contact with the intestinal cell. Then, the impact on the transport of the encapsulated OXY small peptide was evaluated in vitro and in vivo.

NPs were obtained by nanoprecipitation, double or simple emulsion, from PLA-PEG polymer chains functionalized or not with a valine ligand. It was observed on TEM imaging a contrasted shell and an empty core for the NPs obtained by emulsion processes. Furthermore for these NPs, the measure of zeta potential was higher than for NPs obtained by nanoprecipitation. These observations have been described in the literature by Vauthier and Bouchemal (Vauthier and Bouchemal, 2009). They pointed out that the NP configuration is different according to the manufacturing process and that nanospheres were obtained by nanoprecipitation whereas simple or double emulsions produced nanocapsules. It could be then hypothesized in the present study that a better expression of the PEG monomer of the copolymer chain could be found at the NP surface with emulsion processes than with nanoprecipitation, thanks to the core/shell structure. This was validated by a higher zeta potential for the emulsion NP suspensions. In this way, a better PepT1 targeting can be expected with NPs obtained by emulsion according to the better expression of the terminal valine ligand.

In order to verify the hypothesis, PLA-PEG-Val and PLA-PEG drug free NPs obtained by all the manufacturing processes were tested in competition with nanomolar amounts of Bioadhesion observed with PLA-PEG NPs was confirmed to be a non-specific interaction.

These observations confirmed the action of both specific and non-specific interactions.

These results are correlated with confocal observations. Whereas FITC labeled PLA-PEG-Val

NPs reached the membrane and the nuclei of the cells when obtained from double emulsion process, the effect was weaker for simple emulsion and non-observable for nanoprecipitation process. For PLA-PEG NPs obtained by double emulsion, they did not seem to stay linked to the membrane or to penetrate the cell as well. These pictures confirmed not only the better ability to target PepT1 transporters with PLA-PEG-Val NPs obtained by double emulsion, but also their capacity to penetrate the cell and reach the nuclei. This internalization was specifically induced by PepT1 targeting as it was observed in fluorescent experiments that the preincubation with free GlySar induced a decrease in the NP internalization. Furthermore, NP size played a non-negligible part in the internalization. Indeed, a similar organization was observed for NPs prepared by the two emulsion processes, and a similar capacity to inhibit the transport of a PepT1 substrate for both double and simple emulsions was obtained.

Nevertheless, only Val-NPs obtained by double emulsion fully penetrated the cell. Size of Val-NPs obtained by simple emulsion were measured larger than 200 nm, the upper limit for NPs to cross the cell mucus (Ensign, Cone et al., 2012).

From these results, the influence of the manufacturing process on the in vitro apparent permeability of hydrophilic and poorly absorbed OXY molecule was evaluated. OXY have been already tested on Caco-2 cells as free drugs and apparent permeability of 1.6 ± 0.3 x 10 6 cm/s was found for a drug incubation concentration of 0.5 mM [START_REF] Foger | Correlation of in vitro and in vivo models for the oral absorption of peptide drugs[END_REF][START_REF] Shah | In vitro assessment of acyclovir permeation across cell monolayers in the presence of absorption enhancers[END_REF]. Encapsulated in PLA-PEG-Val NPs by the 3 manufacturing processes, a significant 2-fold increase of its apparent permeability was observed in vitro on Caco-2 cells compared to free drug when they were encapsulated in nanoparticles obtained by double emulsion process. These results can be correlated with the conclusion made from competition, fluorescence and confocal experiments, suggesting the influence of the manufacturing process on Pept1 targeting. In a previous work, it was demonstrated the ability to improve the apparent permeability of the poorly absorbed drug acyclovir by encapsulation in PLA-PEG-Val NPs obtained by nanoprecipitation (Gourdon, Chemin et al., 2017a). It was hypothesized that this improvement was due to an overconcentration of the NPs bound to PepT1 and thus to an overconcentration of the drug at the cell membrane surface. In the present study, OXY overconcentration close to the membrane by encapsulation in Val-NPs obtained by nanoprecipitation were not efficient to improve its permeability as the increase was not significant compared to free drug. Only OXY encapsulation in NPs obtained by a double emulsion process allowed a significant increase of the OXY transport. That result could be probably directly related to the ability of these NPs to be internalized inside the cells.

Ability of valine-functionalized NPs to improve oral bioavailability of OXY was evaluated in vivo for the most promising manufacturing process, the double emulsion. A PK study was done in mice and important variability of the plasma concentrations was observed even by IV route. Nevertheless, a general trend was observed. OXY exposure was higher after OXY administration encapsulated in NPs than after free OXY administration. Improvement of OXY AUC t and C max when the drug was encapsulated in functionalized NPs and orally administered seemed to be higher in comparison with free OXY orally administered.

Nevertheless, these PK values remained less important than with free OXY intravenously administered. Oral administration of peptides is naturally low because they are degraded into amino acids and sugars in the digestive system by various types of enzymes which are responsible for the cleavage of amino acid chains. This phenomenon can be reduced by adding a protease-inhibiting excipient in the formulation or by encapsulation for protection (Mahato, Narang et al., 2003;Malik, Baboota et al., 2007). Results obtained for OXY, pointed out that not only the encapsulation in functionalized NPs allowing to target PepT1 transporter but also the protection of the peptide from enzyme degradation helped to significantly improved OXY bioavailability. However, it was demonstrated in a previous studied a fast release of OXY from NPs rapidly resolving to a free drug solution (8% in 10 min), followed by a very slow release over days (Gourdon, Decleves et al., 2017b). The burst release resulted in no input of the NPs strategy in enhancing intestinal OXY absorption and the fraction is probably directly degraded by enzymes. This suggested that a better control of OXY release from the NPs could induce an even better improvement of oral OXY bioavailability.

Cibler un transporteur avec des NPs polymères

Alors que le ciblage de récepteurs intestinaux avec des nano-objets fonctionnalisés est une stratégie bien connue, très peu de travaux mentionnent le ciblage de transporteurs avec des nanoparticules (Du et al., 2013;Gosselin and Lee, 2002;Roger et al., 2012;Stella et al., 2000). En effet, un transporteur n'est pas supposé favoriser le passage de macro-molécules mais plutôt de petites entités chimiques. C'est pourquoi l'objectif recherché dans la présente (Ensign et al., 2012).

Le premier objectif de ce travail de thèse a donc été de vérifier qu'il était possible de cibler un transporteur avec des nanoparticules fonctionnalisées.

Le choix du transporteur s'est arrêté sur PepT1 car il est bien connu et a largement été étudié.

De nombreuses pro-drogues ciblant ce transporteur et dont l'efficacité est démontrée ont été développées (valacyclovir, valgancyclovir, valdecitabine…). Ces études ont ainsi permis de sélectionner différents ligands susceptibles d'être reconnus par ce transporteur tels que la valine, la glycylsarcosine, la tyrosine-valine ou la glycyl-valine (Anand et al., 2003).

Concernant la formulation des NPs, la méthode la plus simple de fabrication, notamment pour une étude de preuve de concept est la technique de nanoprécipitation (Fessi et al., 1989). Le Aussi, ce devenir inattendu des NPs laisse supposer des résultats différents tant au niveau du transport de la molécule encapsulée, mais également vis-à-vis de la toxicité engendrée.

Contrôler la cytotoxité

La stratégie de départ visant à la surconcentration des NPs au contact de la membrane intestinale permet de s'affranchir en partie de la question de la toxicité des NPs dans La question de la toxicité des NPs demeure très présente au sein de la communauté scientifique, bien que les polymères de type PLA, PEG, PLGA soient utilisés depuis les années soixante-dix et soient approuvés comme biomatériaux compatibles chez l'homme. Les produits de dégradation sont en effet métabolisés et éliminés ce qui explique que la cytotoxicité mesurée est faible [START_REF] Sharma | Toxicological considerations when creating nanoparticle based drugs and drug delivery systems?[END_REF]. entière [START_REF] Shwarz | Freeze-drying of drug-free and drug-loaded solid lipid nanoparticles (SLN)[END_REF][START_REF] Holzer | Physicochemical characterisation of PLGA nanoparticles after freeze-drying and storage[END_REF].

A l'issue de ce travail de thèse, une première approche de lyophilisation a été réalisée sur des Différents groupements sensibles au pH sont couramment utilisés pour la formulation de systèmes d'administration répondants aux variations de pH tels que des groupements hydrazone, acétal, ortho ester et vinyl ester [START_REF] Yoshida | pH-and ion-sensitive polymers for drug delivery[END_REF]. L'utilisation de tels polymères pour la formulation de NPs permettrait de contrôler la libération de la molécule encapsulée qui aurait ainsi lieu uniquement une fois dans la cellule. Dans ce cas également, une protection des NPs dans l'estomac par un dispositif gastro-résistant doit être envisagée.

La stratégie mise en place est prometteuse mais un travail de formulation visant à un parfait contrôle de la libération du principe actif de la nanoparticule est à prévoir.

Améliorer la biodisponibilité in vitro et

Faire un choix de modèles in vitro et in vivo adaptés

De nombreuses études ont permis d'établir une corrélation entre les résultats obtenus chez l'homme pour une administration par voie orale et ceux mesurés sur modèle Caco-2 in vitro.

Cependant, un certain recul doit être pris vis-à-vis de ce modèle. En effet, certaines propriétés diffèrent entres les cellules Caco-2 et les cellules intestinales humaines. Tout d'abord, la résistance électrique trans-épithéliale (TEER) mesurée sur des cellules Caco-2 est beaucoup plus importante que celle mesurée au niveau de l'intestin chez l'homme. Ensuite, les cellules Caco-2 ne sont pas recouvertes de mucus comme peuvent l'être les cellules épithéliales humaines. Par conséquent, un modèle de co-culture entre des cellules Caco-2 et des cellules humaines HT29-MTX a été développé afin de se rapprocher des caractéristiques retrouvée chez l'homme [START_REF] Beloqui | Mechanisms of transport of polymeric and lipidic nanoparticles across the intestinal barrier[END_REF]. Récemment, des modèles 3D ont même été mis en place à partir de gels de collagène sur lesquels ont été ensemencées des cellules Caco-2 et HT29-MTX avec incorporation de fibroblastes et d'immunocytes [START_REF] Li | Development of an improved three-dimensional in vitro intestinal mucosa model for drug absorption evaluation[END_REF]. L'utilisation de ce type de modèle dans cette étude pourrait présenter un intérêt étant donné l'importance accordée au fait que les NPs doivent pouvoir traverser le mucus afin de rejoindre les transporteurs de la membrane intestinale.

Concernant le choix du modèle in vivo, tous les essais présentés ici ont été réalisés sur des souris C57Bl/6J. Cependant, au vue des importantes variabilités observées lors des essais avec l'ocytocine, la question de la pertinence de ce choix peut être soulevée. Il faut noter qu'il existe non seulement un taux circulant naturel d'ocytocine chez la souris femelle, mais également chez la souris mâle et ce taux diffère en fonction des animaux. De plus, l'ocytocine est produite de façon pulsative et ne dispose pas de dispositif régulateur, c'est pourquoi il est possible d'observer des pics importants qui peuvent être à l'origine de grandes variabilités inter-animaux. En outre, il est possible de s'interroger sur les limites de quantification de la méthode analytique sur des petits animaux tels que la souris. Ainsi, il faudrait pouvoir envisager des études in vivo sur des animaux déficients en ocytocine, et/ou sur de plus gros animaux tels que le rat, le mini-porc ou le chien.

Aussi, une attention toute particulière doit être portée au choix du modèle animal, d'autant plus lorsque l'étude porte sur une hormone naturellement présente chez l'individu.

Résumé

Si la voie orale reste la voie d'administration privilégiée, elle peut se révéler être un véritable défi lorsqu'il s'agit de molécules hydrophiles, faiblement absorbées au niveau de la barrière intestinale. L'objectif de cette thèse est ainsi de concevoir un nouveau système d'administration pour la voie orale interagissant avec les cellules gastro-intestinales pour améliorer les performances biopharmaceutiques d'un principe actif. Afin de répondre à cette problématique, il a été choisi de suivre une voie encore non explorée, consistant à cibler un transporteur intestinal avec des nanoparticules polymères encapsulant la molécule d'intérêt. Si l'internalisation de nanoparticules n'est pas attendue via un transporteur, il est plutôt recherché une surconcentration en nano-objets et donc en principe actif près de la membrane. La première partie de ce travail a permis de réaliser la preuve de concept et ainsi confirmer qu'il est possible de cibler le transporteur intestinal PepT1 avec des nanoparticules de PLA-PEG fonctionnalisées avec un ligand valine et réalisées par nanoprécipitation. Cette stratégie appliquée à l'acyclovir, molécule BCS III modèle encapsulée dans les nanoparticules, permet de tripler sa perméabilité in vitro et de multiplier par 1.3 son temps de rétention in vivo.

Cependant, une libération trop rapide du principe actif laisse supposer que de meilleurs résultats peuvent être atteints. Ainsi, la seconde partie de cette étude est consacrée à l'optimisation de la formulation des nanoparticules de PLA-PEG pour l'encapsulation d'une molécule hydrophile d'intérêt, l'ocytocine. Les techniques de double et simple émulsions évaporation de solvant ont été étudiées via des plans de criblage. Il a ainsi été mis en évidence que les paramètres de formulation et de procédé influaient significativement sur la taille des particules et le taux d'encapsulation. Par ailleurs, les deux techniques mènent à des résultats similaires en termes de taille de particules environ égales à 200 nm et de taux d'encapsulation proches de 4%. Finalement, la troisième et dernière partie de cette thèse s'est intéressée à l'influence que pouvait avoir la technique de fabrication des nanoparticules sur le ciblage du transporteur PepT1 et sur l'amélioration du transport de l'ocytocine in vitro et in vivo. La double émulsion s'est ainsi révélée être le procédé le plus efficace pour le ciblage du transporteur et permet de doubler le transport de la molécule encapsulée in vitro. Des résultats très intéressants sont obtenus in vivo mais avec une important variabilité inter-animaux. L'imagerie confocale a en outre permis de mettre en évidence une internalisation des nanoparticules fonctionnalisées avec le ligand valine et obtenues par double émulsion. Cependant, une libération très rapide d'une fraction d'ocytocine est observée suivie d'une libération très lente, profil qui n'est pas adapté à la stratégie de ciblage désirée. L'approche développée est ainsi prometteuse quant à l'administration de peptide par la voie orale et une optimisation du profil de libération de la molécule pourrait amener à des résultats encore plus convaincants.

Mots clés

Voie orale -Nanoparticules -Ciblage de transporteur -Peptide -Amélioration de la biodisponibilité

  INTRODUCTION AU CHAPITRE II .............................................................................................................. CHAPITRE II -PUBLICATION N°2 ............................................................................................................. INTRODUCTION AU CHAPITRE III ............................................................................................................. CHAPITRE III -PUBLICATION N°3 ...........................................................................................................

Figure 1 .

 1 Figure 1. Biopharmaceutics Classification System (BCS) designed by Amidon et al. (Amidon, Lennernas et al., 1995).

Figure 2 .

 2 Figure 2. Different segments of gastro-intestinal tract from the stomach to the rectum (Canonne-Hergaux, Gruenheid et al., 1999).

Figure 3 .

 3 Figure 3. Transport mechanism of drugs through the intestinal epithelium barrier.

Figure 4 .

 4 Figure 4. Liposome structure in aqueous media.

Figure 5 .

 5 Figure 5. Microparticles structure. A. Microsphere. B. Microcapsules.

Figure 6 .

 6 Figure 6. Scheme of endocytosis pathways across cells (Conner and Schmid, 2003).

Figure 7 .

 7 Figure 7. Transport mechanism of VB12-drug conjugates in the intestine (Russell-Jones, 2004).

Figure 8 .

 8 Figure 8. Transport mechanism of Fc-drug conjugates or Fc-NPs in the intestine (Sockolosky and Szoka, 2015). Access to the systemic circulation can be direct by crossing the intestinal epithelium or indirect by passing through the lymphatic system thanks to macrophages expressing Fc receptors.

Figure 9 .

 9 Figure 9. PepT1 transport pathway through intestinal epithelium membrane.

Figure 10 .

 10 Figure 10. Schematic diagram of intestinal injection strategy developed by Reni technology (Hassani L.N., Lewis A. et al., 2015). A. Capsule composition. B. Trafficking of the capsule into the intestinal tract.

  Numerous manufacturing processes exist and are dependent on the NPs materials, the encapsulated drug properties and the nature of NPs wanted. They consist in polymerization of monomers or in the use of preformed polymers avoiding the in situ polymerization issue. Most developed manufacturing processes use preformed polymers and NPs are obtained by nanoprecipitation, double or simple emulsion solvent evaporation, salting out or emulsiondiffusion processes. Two types of polymer NPs are described depending on the manufacturing process. The nanospheres with the active ingredient dispersed or dissolved in the polymer matrice or the nanocapsules with the active ingredient in the polymer core surrounded by a thin polymer shell (Fig.1).

Figure 1 .

 1 Figure 1. Nanoparticles structure depending on the nanosphere or nanocapsule configuration.

Figure 2 .

 2 Figure 2. Manufacturing processes for polymer nanoparticles formulation: nanoprecipitation and double emulsion solvent evaporation processes.

  Après confirmation de l'expression du transporteur PepT1 sur un modèle in vitro Caco-2, la capacité des NPs fonctionnalisées à cibler le transporteur intestinal a été vérifiée. Le ligand démontrant la meilleure affinité des NPs fonctionnalisées pour le transporteur PepT1 a alors été choisi. Enfin, l'acyclovir a été encapsulé dans des NPs fonctionnalisées, après optimisation de la formule pour l'encapsulation d'une molécule hydrophile. Il a alors été possible de comparer le transport de l'acyclovir, de la pro-drogue valacyclovir et de l'acyclovir encapsulé dans des NPs sur un modèle cellulaire in vitro Caco-2. Pour conclure, les mêmes formulations ont été testées in vivo chez la souris par voie orale.

  (lactic acid)-poly(ethylene glycol) (PLA-PEG), Poly(lactic acid)-poly(ethylene glycol)-NH-Boc (PLA-PEG-NH-Boc) and PLA-PEG-Valine-Boc (PLA (Mw: 10 kDa)-PEG (Mw: 1 kDa)) polymers were synthetized by Specific Polymers. H-Valine-Tyrosine-OH (H-Val-Tyr-OH) was purchased from Bachem. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was procured from Iris Biotech GmbH. Dichloromethane (DCM), pentane, orthophosphoric acid, dimethylsulfoxide (DMSO) and trifluoroacetic acid (ATFA) were purchased from Merck. Acyclovir (ACY) and Valacyclovir (ValACY) were obtained from Sigma Aldrich Chemical Sourcing and Apichem Technology, respectively. PLGA Resomer ® RG502 and RG502H (7-17 kDa) were graciously provided by EVONIK, Kolliphor ® P188 and P407 poloxamers by BASF. Tween 80 was purchased from Seppic. Methanol (MeOH) was obtained from VWR. Acetonitrile (ACN) was procured from Biosolve. Tangential filtration tubes Vivaspin ® were purchased from Sartorius Stedim. 24-well plates Transwells and T75 flasks were obtained from Corning. L-Glutamine and penicillin/streptomycin (P/S) were purchased from Invitrogen. Fetal bovine serum (FBS) was obtained from Lonza. Trypsine was procured from Life Technologie. [ 3 H]Glycylsarcosine and [ 14 C]Sucrose were purchased from Moravek Biochemicals and Perkin Elmer, respectively. Boc-Glycine-Valine-OH (Boc-Gly-Val-OH), Glycylsarcosine (GlySar), Di-tert-butyl dicarbonate (BOC 2 O), N,Ndiisopropylethylamine (DIEA), hydroxybenzotriazole (HOBt), polyvinyl alcohol (PVA) Mowiol ® 4-88, formic acid, ammonium formate, non-essential amino acids (NEAA), amphotericin B, bovine serum albumin (BSA), Dulbecco modified eagle medium (DMEM), Hanks buffer saline solution (HBSS), Lucifer Yellow (LY), 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) and 2-(N-morpholino)ethanesulfonic acid (MES)

Figure 1 .

 1 Figure 1. Chemical structures of co-polymers and dipeptide ligands. A: PLA-PEG-NH 2 . B: PLA-PEG-Val-NH 2 . C: NH 2 -Gly-Sar. D: NH 2 -Val-Tyr. E: NH 2 -Gly-Val.

Figure 2 .

 2 Figure 2. PLA-PEG-Val-Gly, PLA-PEG-Tyr-Val and PLA-PEG-Sar-Gly polymers synthesis by successive steps: deprotection of PLA-PEG-NH-Boc, protection of ligand, conjugation of ligands and polymer, and deprotection of the final product.

[ 3 H

 3 ]Glycylsarcosine inhibition (%) = [([ 3 H]Glycylsarcosine Papp -[ 3 H]GlycylsarcosinePapp with addition of NPs )/ [ 3 H]Glycylsarcosine Papp] x 100.Finally, 50 µM doses of free ACY, free ValACY and ACY loaded in nanoparticles were incubated in apical chambers under 60 rpm agitation during 90 min. ACY concentrations were determined by LC/MS/MS method, as described in paragraph 2.6., and apparent permeability was calculated for each group, as described above for Glycylsarcosine experiments. All in vitro experiments (expression of PepT1 and ACY transport) were carried out with Caco-2 cells at the same passage range (77-90). The influence of the passage on PepT1 expression was previously studied by Lu et al. No difference was found for [ 3 H]Glycylsarcosine apparent permeability across Caco-2 cells at early passages 35-47 (Caco-

  Radioactive samples from Caco-2 permeability experiments were analyzed by liquid scintillation counting (Tri-Carb 2810TR; Perkin Elmer) for [ 3 H]Glycylsarcosine and [ 14 C]Sucrose concentrations.Samples from in vitro experiments were defrosted at room temperature and mixed. They were transferred into UPLC vials without any dilution and ACY concentrations were determined using UPLC/MS/MS detection (Xevo, Acquity Waters). The column was an Acquity BEH Phenyl (Waters) (2.1 x 100 mm, 1.7 µm) and was set at 25°C. The injection volume was 2 µL and the flow rate was 0.2 mL/min. The mobile phase consisted in 90% of water, ammonium formate 10 mM, formic acid 0.1% and 10% of acetonitrile. Parameters of the detector (TQD, Waters) were as follows: capillary tension 1 kV, source temperature 150°C, desolvatation temperature 400°C, gas flow for desolvatation 800 L/h, gas flow for cone 150 L/h, nebulizer pressure 7 bar, collision energy 5 eV, cone voltage 30 V. Samples were analyzed against a set of calibration standards prepared in conventional transport medium. The determination of ACY concentration was carried out over a calibration range of 0.5 ng/mL (LLOQ) to 1000 ng/mL (ULOQ) in conventional transport medium. The standard curve consisted of 12 calibration standards.Samples from in vivo experiments were analyzed after solid-phase extraction followed by UPLC/MS/MS detection using deuterated ACY as internal standard (IS). Following a protein precipitation and phospholipid removal step (Ostro ™ ), ACY was measured in plasma by liquid chromatography coupled with tandem mass spectrometry detection. Plasma samples were defrosted at room temperature, mixed (Multi-vortex, 5 min, speed "1500") and centrifuged (5 min, 3500 rpm, +4°C). 10 µL of plasma were introduced into an Ostro ™ plate (Waters) with 100 µL of IS solution at 20 ng/mL in acetonitrile for extraction. The plate was mixed thoroughly with the Mixmate shaker for 1 min at 1200 rpm. The cap was replaced by an adhesive film placed on the empty wells. Samples were filtered with a negative pressure Manifold (Hamilton) of 300 mbar (5 cycles of 20 sec). The sample extracts were collected in a 96-well plate and then centrifuged for 5 min (1000 rpm) at 10°C before complete evaporation under nitrogen at 37°C. After evaporation, the residues were reconstituted in 50 µL with phase (water/acetonitrile/formic acid (90:10:0.1, V/V/V)) and mixed for 2 min with the Mixmate shaker (1800 rpm) with a polypropylene cap before analysis by UPLC-MS/MS (Acquity system WATERS with sample manager, sample organizer and binary solvent manager). Analyses were conducted by reverse phase liquid chromatography with an Acquity column Kinetex F5, 100 x 2.1 mm, 2.6 µm (Phenomenex). The column and the auto sampler temperatures were respectively set at 24°C and 10°C, the injection volume was 10 µL and the flow rate was 0.5 mL/min. The mobile phase A was composed of 0.1% of formic acid in water and 10 mM of ammonium formate and the mobile phase B was pure acetonitrile. A gradient was applied from 0% to 70% of phase B for the first 3.5 min, a plateau was maintained at 95% of phase B to 5 min, and then it came back to 100% of phase A until the end of the run (6 min). The MS/MS parameters (API 4000 system, AB Sciex, electrospray source and MRM detection) were characterized by a positive ion electrospray. The curtain gas (air) and the ion source gas 1 and 2 were respectively 20, 40 and 50 bars. The ion spray voltage was set at 5500 V and desolvatation temperature at 600°C. Nitrogen used as collision gas was set at 19 eV. Plasma samples were analyzed against a set of calibration standards prepared in blank C57Bl/6J mouse plasma. Stock solutions for calibration and internal standards were prepared by weighing appropriate amounts of ACY and deuterated ACY (IS) at a concentration of 1 mg/mL in DMSO. These stock solutions were stored at + 4°C. The determination of ACY concentration in mouse plasma samples (10 µL) was carried out over a calibration range from 1 ng/mL (LLOQ) to 1000 ng/mL (ULOQ) in plasma. The standard curve consisted of 7 calibration standards.

  mean measured concentration / nominal concentration) × 100 CV (%) = (standard deviation / mean measured concentration) × 100 RE (%) = [(measured concentrationnominal concentration) / nominal concentration] × 100.

Figure 3 .

 3 Figure 3. Graphical analysis of the fractional factorial design for the studied responses X 50 and X 99 . A. Significance levels are indicated with dotted lines. A short bar means that the response is slightly altered by a change of level. A positive bar indicates an increase of the response with a change of level. B. Effect of each level on studied responses. The response value increases with the height of the bar.

Figure 4 .

 4 Figure 4. (A) Size distribution of PLA-PEG-NH 2 nanoparticles by dynamic light scattering for the optimal formulation. Data was represented as differential distributions in volume (n=3). (B) Images of PLA-PEG-NH 2 nanoparticles for the optimal formulation by Transmission Electron Microscopy.

  PEG-Tyr-Val with peak characteristic of the ligand (data not shown). Deprotection yelds for PLA-PEG-NH-Boc and PLA-PEG-Val-Boc were evaluated close to 90%. Similarly, conjugation yield of -Val-Gly ligand to PLA-PEG-NH 2 was evaluated to 90 ± 5% and around 25% and 50% for -Tyr-Val and -Sar-Gly conjugation to PLA-PEG-NH 2 , respectively. Only -Val, -Val-Gly and -Sar-Gly were chosen for further investigation. Formulation of nanoparticles with functionalized polymer did not change their physical properties or their entrapment abilities.

Figure 5 .

 5 Figure 5. Proton NMR spectra of PLA-PEG-NH 2 (A) and PLA-PEG-Val-Gly (B) polymers. When coupled the chemical shift (ppm) of the peak associated to the terminal -NH 2 (A) decreased from 7.72 ppm to 6.60 ppm and the integration from 2 to 1 proton.

Figure

  Figure 6. Papp of [ 3 H]Glycylsarcosine (10 nM) in competition with increasing amounts of unlabeled Glycylsarcosine at pH 6.0 and 7.4. Each bar represents the mean ± S.D. of two independent experiments performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001 compared to [ 3 H]Glycylsarcosine Papp alone at pH 6.0.

[

  05, ** p < 0.01, *** p < 0.001 compared to control, NS = Non Significant In contrast, all functionalized nanoparticles significantly inhibited [ 3 H]Glycylsarcosine Papp through Caco-2 cells from 21.7 ± 9.5% to 46.4 ± 7.4%. The highest inhibition was observed for nanoparticles functionalized with Valine at a concentration of 10 mg/mL. These results indicate that the functionalized nanoparticles interfere with the transport of a substrate of PepT1. Valine functionalized nanoparticles (NPs-Val) were chosen to study the ability of PepT1-targeting nanoparticles to improve the Papp of ACY. Also, when comparing [ 3 H]Glycylsarcosine Papp inhibition by functionalized NPs each other's for 10 mg/mL concentration, only NPs-Val had a significant effect compared to the inhibition induced by -NH 2 (46.4 ± 7.4 vs 27.8 ± 7.1% *), -Val-Gly (vs 25.4 ± 4.2% **) or -GlySar (vs 27.0 ± 9.1%

Figure 7 .

 7 Figure 7. Papp (cm/s) of free ACY, ACY loaded in valine-nanoparticles NPs-Val-ACY and free ValACY at an apical dosing of 50 µM. Each data point represents the mean ± S.D. of three independent experiments performed in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to ACY free control.

Figure 8 .

 8 Figure 8. Mean plasma concentrations (ng/mL) of ACY in function of time (h), after single oral administration of ACY, ValACY and ACY encapsulated in NPs-Val (25 mg/kg, expressed as free compound) to female C57Bl/6J mice. Results are expressed as mean ± S.D. of n=10 mice per group.

Figure

  Figure S2. ACY release from NPs-Val (2.5 mg/mL of NPs in conventional transport medium at pH 6.0, 37°C, under 60 rpm stirring). At regular times, 500 µL samples were collected. The released ACY was removed by tangential filtration and evaluated by HPLC. Each bar represents the mean ± S.D. of two independent experiments performed in triplicate.

  demonstrated the highest Papp or C max in both Caco-2 cell model and in vivo in mice. Although Papp of ACY encapsulated in NPs-Val was significantly twice increased from ACY free in Caco-2 model, C max of ACY in vivo did not significantly differ from each other in mice. The hypothesis of a lower expression in PepT1 in our mice model as compared to the Caco-2 model is not supported by data with ValACY from our study and from the literature. Katragadda et al. found a 3-fold improvement of the in vitro ACY Papp from 1.5 to 4.43 x 10 -6 cm/s with the ValACY prodrug (Katragadda et al., 2008). Then, Yang et al. demonstrated that the C max and AUC 0-180min of ACY were 4-to 6-fold and 2-to 3-fold lower, respectively, in PepT1 knockout mice compared to wild type mice, for oral doses of 10to 100 nmol/g of ValACY(Yang et al., 2013). These results could be compared with those obtained in the present study for ValACY and ACY free. A 9-fold improvement of both ACY Papp and C max was observed after oral administration of the prodrug compared to the free drug, in vitro and in vivo, demonstrating the activity of PepT1 in both models. The reason why our strategy was not as effective in the in vivo study as in the Caco-2 study requires additional experiments.

L

  'influence de paramètres de formulation et de fabrication sur cette contrainte a été étudiée par le biais d'un plan d'expérience. Ainsi, il a été démontré que la concentration en polymère dans la phase organique ainsi que sa nature, le ratio entre les phases, la quantité et la nature du tensio-actif influaient sur la taille des NPs. Tout comme le temps et la vitesse d'agitation, le temps d'ajout et le diamètre de l'aiguille. L'ajout de PLGA dans une formulation de NPs de PLA-PEG permet une meilleure encapsulation de l'acyclovir. Son caractère légèrement plus hydrophile que le PEG permet l'interaction avec le PA hydrophile(Pridgen et al., 2013).  La capacité des NPs à cibler le transporteur PepT1dépend du ligand exprimé à leur surface. Ainsi, Tyr-Val, Val-Gly, GlySar et Valine ne permettent pas la même inhibition du passage de la Glycylsarcosine radiomarquée in vitro sur Caco-2, directement relié à la capacité des NPs fonctionnalisées à cibler le transporteur PepT1.

Figure 1 .

 1 Figure 1. Synthesis route for PLA-PEG-NH 2 polymer from PLA-PEG-NH-Boc.

  The emulsion was prepared by addition of an organic phase (O) containing polymers (PLA-PEG-NH 2 and an eventual additional polymer), OXY (theoretical loading set at 30%) and Montane 80 as a surfactant dissolved in a solvent mixture of EtAc/EtOH (3:2), into an aqueous phase (W) containing Montanox 80 surfactant, under sonication (45 s, 40 W, continuous mode, in an ice bath) using a 3.1 mm probe (Vibracell ® , Biobloch Scientific, France). The organic solvent was then evaporated from the double emulsion under vacuum (37°C, 100 rpm, 200 mbar). Unentrapped OXY was removed by tangential filtration during 1 h at 800 × g until a 100 µL residual volume of NPs suspension was obtained. NPs were stored as a suspension.

  Total amount of drug dissolved in the NP suspension was determined by HPLC assay after addition of 800 µL of DMSO to 200 µL of NP suspension in order to destruct the NPs. The amount of encapsulated OXY was evaluated indirectly by calculating the difference between the total amount of OXY and the concentration of free OXY in the filtered medium collected from tangential filtration of 500 µL of NPs suspension. The drug load was defined as the percentage of encapsulated drug compared to the total amount of NPs. The entrapment efficiency was defined as the percentage of encapsulated drug compared to the total amount of drug dissolved in the organic phase. They were calculated using the following formulae: Drug loading (%) = (Amount of encapsulated drug/Total amount of NPs) × 100 Entrapment efficiency (%) = (Amount of encapsulated drug/Amount of drug dissolved in the organic phase) × 100 All samples were assayed using HPLC. The system comprised a Water 2695 Separation module associated with a Waters UV 2487 dual λ Absorbance UV detector at 220 nm. The column used was a Merck's LiChrospher 60 RP-select B C8 (4 x 1250 mm, 5 µm) and was heated at 40°C. The mobile phase consisted in water/acetonitrile (85:15, v/v) at pH 2.1 adjusted with orthophosphoric acid. The flow rate was 1.25 mL/min and the injected volume 20 µL.

  2.A., dotted lines and x axis were almost merged together as experimental error was found negligible. The effect of the different factors on the studied responses was compared to the experimental error. All studied factors appeared to be influent on the responses (ANOVA, p < 0.01): responses variations were consequences of factors effects and not of the experimental error. Regarding the mathematical model, significant differences were found between experimental values and values calculated by the model (ANOVA, p = 0.04). But as particularly small experimental error was observed (S.D. = 16 nm), it was considered that the model depicted adequately the effect of parameters on the response and the results could be interpreted.Regarding zeta potential response Fig.2.C., dotted lines and x axis were also almost merged together as experimental error was found negligible. All studied factors appeared to be influent on the responses (ANOVA, p < 0.01). Moreover, the model was considered valid since the differences between experimental values and the values calculated by the model were not statistically significant (ANOVA, p = 2.01).

Figure 2 .

 2 Figure 2. NPs formulation by double emulsion manufacturing process: graphical analysis of the fractional factorial design for the studied responses X 50 (A, B) and zeta potential (C, D). W.I., W.E. and O. are the internal aqueous phase, external aqueous phase and organic phase, respectively. Fig.A. and C.: Are represented the direction and the significance of the change of level on the response for formulation and process parameters. Significant levels are indicated with dotted lines. Fig.B. and D.: Are represented the effect of each level on studied responses.

Figure 3 .

 3 Figure 3. OXY-loaded NPs formulation from double emulsion manufacturing process: graphical analysis of the fractional factorial design for the studied responses X 50 (A, B) and DL (C, D). W.I. and O. are the internal aqueous phase and organic phase, respectively. Fig. A. and C.: Are represented the direction and the significance of the change of level on the response for formulation and process parameters. Significant levels are indicated with dotted lines. Fig. B. and D.: Are represented the effect of each level on studied responses.

  4.A., dotted lines and x axis were almost merged together as experimental error was found negligible. The effect of the different factors on the studied responses was compared to the experimental error. All studied factors appeared to be influent on the responses (ANOVA, p < 0.01): responses variations were consequences of factors effects and not of the experimental error. Regarding the mathematical model, significant differences were found between experimental values and values calculated by the model (ANOVA, p < 0.01). But as particularly small experimental error was observed (S.D. = 11 nm), it was considered that the model depicted adequately the effect of parameters on the response and the results could be interpreted. Concerning the drug load response Fig. 4.C., almost all studied factors appeared to be influent on the responses (ANOVA, p < 0.01). Moreover, the model was considered valid since the differences between experimental values and the values calculated by the model were not statistically significant (ANOVA, p = 3.66).

Figure 4 .

 4 Figure 4. OXY-loaded NPs from simple emulsion manufacturing process: graphical analysis of the fractional factorial design for the studied responses X 50 (A, B) and DL (C, D). W. and O. are the aqueous phase and organic phase, respectively. Fig. A. and C.: Are represented the direction and the significance of the change of level on the response for formulation and process parameters. Significant levels are indicated with dotted lines. Fig. B. and D.: Are represented the effect of each level on studied responses.

Figure

  Figure 5. OXY release from PLA-PEG-NH 2 NPs obtained from double or simple emulsion process (in conventional transport medium at pH 6.0, 37°C, under 60 rpm stirring). At regular times, 500 µL samples were collected. The released OXY was removed by tangential filtration and evaluated by HPLC. Each bar represents the mean ± S.D. of 3 independent experiments.

Figure S1 .

 S1 Figure S1. Apparent viscosity (Pas) of the viscofying agents HEC at 1% and 2.5% (w/v) and PEG 400 at (3:2) and (2:3) ratios with water, represented as function of shearing speed (1/s), in [water + poloxamer P188 1% (w/v)] aqueous phase.

L

  'objectif de cette seconde partie était d'améliorer le taux d'encapsulation d'un principe actif BCS III dans des NPs de PLA-PEG, ainsi que de contrôler sa libération. Pour cela, il a été choisi d'explorer les techniques de double et simple émulsions pour l'encapsulation de l'ocytocine. Ce chapitre a permis de mettre en lumière les résultats suivants :  La possibilité de réaliser des NPs placebo de PLA-PEG par la technique de double émulsion mesurant 185 nm (soit < 200 nm afin qu'elles puissent traverser le mucus et rejoindre la paroi intestinale (Ensign et al., 2012)) et de potentiel zeta égal à 34 mV (soit supérieur à |30| mV, condition pour une émulsion stable (Hans and Lowman, 2002)).  La possibilité de réaliser des NPs de PLA-PEG encapsulant de l'ocytocine par les techniques de double et simple émulsions, avec un diamètre moyen et un taux d'encapsulation respectivement de 195 nm et 226 nm et de 4% et 3.3%.  L'influence des paramètres de formulation et de fabrication sur la taille des NPs, leur stabilité et les taux d'encapsulation, a été étudiée par le biais de plans d'expérience. Ainsi, il a été pu être constaté l'importance de la quantité et de la nature des surfactants dans chacune des phases, du ratio entre les différentes phases aqueuses internes ou externes et phases organiques, de la composition des phases en polymères, principe actif et agent viscosifiant, et de la durée des ultrasons appliqués. Ainsi, ce chapitre a démontré qu'il était possible d'obtenir des NPs de PLA-PEG encapsulant de l'ocytocine par les techniques de double et simple émulsions avec des taux d'encapsulation non négligeables pour des molécules hydrophiles, tout en respectant des contraintes de taille et de stabilité. En outre, l'étude de la libération de l'ocytocine des NPs a montré une libération rapide sur les premières minutes (8% et 17% pour la double et la simple émulsion, respectivement) suivie d'une libération lente sur plusieurs heures. Finalement, les NPs obtenues ayant des caractéristiques très proches par les deux techniques de fabrication, la simple émulsion offre une alternative en une étape unique à la double émulsion. Cependant, il est largement décrit dans la littérature que la structure même des NPs différait selon la technique de fabrication. Il est alors possible d'obtenir de façon contrôlée des nanosphères ou des nanocapsules (Vauthier and Bouchemal, 2009). Se pose alors la question de l'organisation des NPs lorsque celles-ci sont réalisées à partir de polymères fonctionnalisés. Peut-on imaginer une meilleure organisation des NPs, une meilleure expression des ligands à leur surface et donc une meilleure stratégie de ciblage ? Introduction au Chapitre III Le premier chapitre de ce travail de thèse a permis de réaliser la preuve de concept d'une nouvelle stratégie consistant à cibler le transporteur intestinal PepT1 avec des NPs fonctionnalisées. Il a été démontré que malgré une libération très rapide de la molécule hydrophile encapsulée, une amélioration de son passage in vitro pouvait être observée. Ainsi, la seconde partie de cette thèse consistait à explorer les différentes techniques de formulation de NPs avec comme objectif l'amélioration du taux d'encapsulation et de la libération du principe actif. Appliqué à une molécule d'intérêt, il a été possible de formuler des NPs de PLA-PEG encapsulant de l'ocytocine par les techniques de double et simple émulsions. Il a été montré dans la littérature que la structure des NPs différait en fonction de la technique de fabrication. Ainsi, des nanosphères sont obtenues par nanoprécipitation, tandis que les techniques d'émulsions mènent à la formation de nanocapsules (Vauthier and Bouchemal, 2009). Ainsi, l'objectif de cette dernière partie est d'étudier l'influence de la technique de fabrication de NPs de PLA-PEG sur leur capacité à cibler le transporteur PepT1 et sur l'amélioration des performances biopharmaceutiques de l'ocytocine. Pour cela, des NPs de PLA-PEG-Val ont été fabriquées selon les trois techniques de fabrication mises en place : la nanoprécipitation, la double et la simple émulsion. Les différences de structure sont vérifiées par imagerie TEM. Dans un premier temps sans principe actif, il est alors mesuré la capacité des trois types de NPs à cibler le transporteur PepT1. Tous les essais étant menés en parallèle d'essais contrôles similaires avec des NPs de PLA-PEG sans ligand, l'intérêt de la fonctionnalisation avec le ligand valine est également vérifié pour les trois procédés. Ainsi, des études de compétition avec la Glycylsarcosine radiomarquée, ainsi que des études avec des NPs fluorescentes sont réalisées. La microscopie confocale permet aussi de mieux décrire le comportement des différentes NPs au contact de la membrane intestinale. Dans un second temps, les différentes NPs encapsulant de l'ocytocine sont testées dans des études de transport in vitro sur Caco-2 afin de vérifier encore une fois l'influence de la technique de fabrication sur le transport de la molécule encapsulée. Pour conclure, les mêmes formulations de NPs sont testées in vivo chez la souris par voie orale. Cette troisième et dernière partie de ce travail de thèse est présentée sous forme d'un article scientifique. Référence Vauthier,C., Bouchemal,K., 2009. Methods for the preparation and manufacture of polymeric nanoparticles. Pharm Res, 26, 1025-1058.

  on the ability of polymeric NPs functionalized with valine to target PepT1 transporter, the objective of the present work was to study the capacity of the NPs to target PepT1 for the oral delivery of oxytocin (OXY) according to the manufacturing process (nanoprecipitation, double emulsion and simple emulsion). Their ability to target PepT1 transporter was assessed for each process in vitro through competition experiments with [ 3 H]Glycylsarcosine, a wellknown substrate of PepT1, and by fluorescence assays measuring the residual fluorescence intensity of Caco-2 cells after incubation with functionalized NPs. Confocal microscopy was also performed to understand NPs interactions within the Caco-2 cells. Finally, OXY transport experiments were conducted in vitro with Caco-2 cells and in vivo in mice to evaluate the strategy of targeting PepT1 with functionalized NPs encapsulating OXY to enhance its oral absorption.

  (lactic acid)-poly(ethylene glycol) (PLA-PEG), Poly(lactic acid)-poly(ethylene glycol)-NH-Boc (PLA-PEG-NH-Boc) and PLA-PEG-Valine-Boc (PLA (Mw: 10 kDa)-PEG (Mw: 1 kDa)) polymers were synthetized by Specific Polymers. Dichloromethane (DCM), pentane, orthophosphoric acid, dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF) and trifluoroacetic acid (ATFA) were purchased from Merck. Oxytocin (OXY) was obtained from PHDS Sourcing. PLGA Resomer ® RG502 (7-17 kDa) was a gift from EVONIK and PDLG 7502 was procured from Purac Biomaterials. Poloxamer Kolliphor ® P188 was a gift from BASF. Montane 80 and Montanox 80 were purchased from SEPPIC. Methanol (MeOH) was obtained from VWR. Acetonitrile (ACN) was procured from Biosolve. Tangential filtration tubes Vivaspin ® were purchased from SartoriusStedim. 24-wells plates Transwells ® and T75 flasks were obtained from Corning. L-Glutamine and penicillin/streptomycin (P/S) were purchased from Invitrogen. Fetal bovine serum (FBS) was obtained from Lonza. Trypsine was procured from Life Technologie. [ 3 H]Glycylsarcosine ([ 3 H]GlySar) and [ 14 C]Sucrose were purchased respectively from Moravek Biochemicals and Perkin Elmer. Glycylsarcosine (GlySar), formic acid, ammonium formate, non-essential amino acids (NEAA), amphotericin B, bovine serum albumin (BSA), Dulbecco modified eagle medium (DMEM), Hanks buffer saline solution (HBSS), Dulbecco's Phosphate Buffer Saline (DPBS), Lucifer Yellow (LY), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-(N-morpholino)ethane sulfonic acid (MES) and fluorescein isothiocyanate (FITC) were obtained from Sigma-Aldrich. Hydroxy ethyl cellulose (HEC) was purchased from Dow Chemical. Ethyl acetate (AcEt) and ethanol (EtOH) were obtained from Carlo Erba. Triton X100 (TX100), TO-PRO ® -3 and phalloidin 555 were purchased from Thermo Fisher Scientific.

Final

  polymers were analyzed by MNR spectroscopy. Spectra were recorded on a Bruker Avance 600 (work frequency 600.13 MHz, spectra width 10,200 Hz, probe temperature 27°C, scan length 3.21 s).

Figure 1 .

 1 Figure 1. Chemical structures of PLA-PEG-NH 2 (A) and PLA-PEG-Valine (B) copolymers.

  [ 3 H]GlySar (10 nM) was tested alone and in competition with 2.5 mg/mL of drug-free PLA-PEG-Val or nonfunctionalized PLA-PEG NPs obtained from the three manufacturing processes. Experiments included 30 min of preincubation of the cells with the NPs and were conducted under gentle stirring (60 rpm). Apparent permeability (Papp, cm/s) of [ 3 H]GlySar was calculated as follow: Papp (cm/s) = (dQ/dt)/(A.C 0 ) with dQ/dt the amount of molecule transported from the apical chamber to the basolateral chamber per unit of time; A the membrane surface; and C 0 the initial molecule amount in the apical chamber. Radioactive samples from Caco-2 permeability experiments were analyzed by liquid scintillation counting (Tri-Carb 2810TR; Perkin Elmer) for [ 3 H]GlySar and [ 14 C]Sucrose concentrations.

FormulaFigure 2 .

 2 Figure 2. Synthesis route for PLA-PEG-FITC polymer from PLA-PEG and free FITC.

Figure 3 .

 3 Figure 3. TEM images of PLA-PEG-Val nanoparticles obtained by nanoprecipitation (A), simple emulsion (B) or double emulsion solvent evaporation (C) processes.

Figure 4 .

 4 Figure 4. Apparent permeability (Papp, cm/s) of [ 3 H]GlySar across Caco-2 cells without (control) or with 2.5 mg/mL of PLA-PEG and PLA-PEG-Val NPs obtained from nanoprecipitation, double or simple emulsion processes in competition (pH 6, 60 rpm agitation, 30 min of preincubation, mean ± S.D., n=4). * p < 0.05, ** p < 0.01, *** p < 0.001: [ 3 H]GlySar permeability in competition with NPs compared to control (without NPs). # p < 0.05, # # p < 0.01, # # # p < 0.001: PLA-PEG NPs compared to PLA-PEG-Val NPs.

Figure 5 .

 5 Figure 5. Residual fluorescence on Caco-2 cells compared to initial fluorescence of nanoparticle formulations (%) for the three manufacturing processes at 2.5 mg/mL, after 95 min of incubation. 10 mM GlySar 30 min preincubation could be applied. (mean ± S.D., n=3). * p < 0.05, ** p < 0.01, *** p < 0.001: PLA-PEG-Val NPs residual fluorescence compared to PLA-PEG NPs, without GlySar. # p < 0.05, # # p < 0.01, # # # p < 0.001: PLA-PEG-Val NPs residual fluorescence compared to PLA-PEG-Val NPs + 10 mM [ 3 H]GlySar.

Figure 6 .

 6 Figure 6. Confocal images of fluorescently labeled nanoparticles (green) after 90 min of incubation with Caco-2 cells. Cell nuclei and membranes were stained with TO-PRO-3 (blue) and phalloidin 555 (red), respectively. PLA-PEG-Val NPs obtained by nanoprecipitation (A), simple emulsion (B) and double emulsion (C), PLA-PEG NPs obtained by double emulsion (D) and control (E) were observed.

Figure 7 .

 7 Figure 7. Papp (cm/s) of free OXY or encapsulated in nanoparticles obtained by nanoprecipitation, simple or double emulsion manufacturing processes, at an apical dosing of 25 µM. Each data point represents the mean ± S.D. of three independent experiments performed in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to free OXY as control.

  the three groups is presented Figure8. No statistical analysis was done as important differences were observed between the animals. However, it gave a trend and the suggestion of an improvement of OXY transport when encapsulated in Val-NPs.

Figure 8 .

 8 Figure 8. Mean plasma concentrations (pg/mL) of OXY as a function of time (h), after single IV administration of free OXY (0.01 mg/kg, expressed as free compound), and oral administration of free OXY and encapsulated in NPs-Val obtained by double emulsion (10 mg/kg, expressed as free compound) to male C57Bl/6J mice. Results are expressed as mean ± S.D. of n=21 mice per group and n=3 per time point.

  [ 3 H]GlySar in order to assess and compare their ability to target PepT1 transporter in vitro in Caco-2 cells. Concerning PLA-PEG-Val NPs, no significant difference was found between the three manufacturing processes even if the highest inhibition of [ 3 H]GlySar apparent permeability was observed for NPs obtained by double emulsion. After incubation with PLA-PEG NPs, an inhibition of [ 3 H]GlySar transport was also observed when they were prepared by the emulsion processes, even if it remained significantly lower than with valine functionalized NPs. No effect was seen for PLA-PEG NPs obtained by nanoprecipitation. This result is in accordance with the fact that when prepared by the emulsion processes the PEG part of the copolymer is more distributed at the NP surface in comparison with NPs obtained by nanoprecipitation. Indeed, the bioadhesive properties of the pegylated NPs may induce a steric hindrance leading to non-specific competition with [ 3 H]GlySar. This bioadhesive property of pegylated NPs was demonstrated by Yoncheva et al.[START_REF] Yoncheva | Evaluation of bioadhesive potential and intestinal transport of pegylated poly(anhydride) nanoparticles[END_REF]. They compared the area under the curve of the adhered NPs (AUC adh , mg/h) and the mean residence time of the adhered fraction of the NPs (MRT adh , h) for pegylated NPs and non-modified NPs. They found higher AUC adh and longer MRT adh for PEG-NPs compared to non-modified NPs, 11.83 ± 2.0 vs 16.19 ± 2.29 mg/h and 2.77 vs 3.11 h, respectively.These competition results pointed out the intensive interactions of polymeric NPs with the intestinal membrane. It also confirmed the importance of a terminal valine ligand for active PepT1 targeting. However, it did not allow concluding about the impact of the manufacturing process.In this way, fluorescence assays were carried out with FITC labeled PLA-PEG-Val and PLA-PEG NPs obtained from the three manufacturing processes, and incubated with Caco-2 cells.The presence of PLA-PEG-FITC among classic PLA-PEG-Val NPs suspension was confirmed by a decrease of zeta potential of the emulsion with the addition of PLA-PEG-FITC polymer in the formulation. This decrease can be explained by the positive charge of the PLA-PEG-Val NPs suspensions and by the PLA-PEG-FITC electronic charge which depends on the pH of the solution and, as the FITC pKa is 6.4, it could be mainly in its neutral form below or in its negative form above. Best residual fluorescence on cells after incubation with valine-NPs was observed from the double emulsion process, followed by valine-NPs obtained by the simple emulsion process and then by nanoprecipitation. Functionalization of NPs with valine ligand allowed a specific adhesion of the NP to the cell. Targeting PepT1 more than doubled the residual fluorescence in comparison with PLA-PEG NPs. Pre incubation of the cells with 10 mM GlySar decreased significantly the residual fluorescence of PLA-PEG-Val for the three processes, whereas it did not change the residual fluorescence of PLA-PEG, which validated the specific adhesion to PepT1 transporter for valine-NPs. The Figure4confirmed an altered [ 3 H]GlySar transport by PLA-PEG NPs that were less linked to the cell membrane than PLA-PEG-Val NPs as after washing, the residual fluorescence decreased.

  étude était d'obtenir une surconcentration des particules près de la paroi intestinale grâce à leur reconnaissance par le transporteur ciblé, et non leur internalisation. Ainsi, la molécule encapsulée est libérée dans l'environnement proche de la membrane, permettant une augmentation de son passage. Pour pouvoir atteindre les transporteurs membranaires et ainsi s'y fixer, les NPs doivent pouvoir traverser le mucus. Pour cela, Ensign et al. ont évalué que leur diamètre moyen devait être inférieur à 200 nm

  copolymère PLA-PEG a été choisi comme polymère majoritaire car la conjugaison de ligand est possible sur la fonction amine terminale du PEG. De plus, il a déjà été utilisé par Pridgen et al. pour le ciblage du récepteur FcRn avec des NPs de PLA-PEG-Fc encapsulant de l'insuline (Pridgen et al., 2013). Des essais de compétition entre des NPs fonctionnalisées et de la Glycyclsarcosine, un très bon substrat de PepT1, ont pu mettre en évidence que la conjugaison du ligand valine sur les NPs obtenues par nanoprécipitation permettait le ciblage de PepT1 le plus efficace. En effet, les NPs-valine inhibent jusqu'à 40% de son passage. Mais d'autres techniques de fabrication de NPs existent telles que la double ou la simple émulsion. De la microscopie TEM a permis d'observer que la configuration des NPs était différente en fonction de la technique de fabrication utilisée. En effet, pour les NPs obtenues par nanoprécipitation, il est observé des nanosphères avec un coeur plein et uniforme. En revanche, les techniques d'émulsions mènent à des structures coeur/couronne caractéristiques des nanocapsules. Dans ce cas, il est observé une meilleure expression du PEG et donc du ligand à la surface des NPs. Il est alors supposé que ces différences de configuration peuvent influer le ciblage du transporteur PepT1 et que la structure coeur/couronne peut permettre un meilleur ciblage. Ceci a pu être confirmé par de nouveaux essais de compétition avec de la Glycylsarcosine qui ont montré une inhibition de son passage la plus significative avec les NPs-valine obtenues par double émulsion. De plus, lorsqu'elles sont marquées en fluorescence, après incubation avec des cellules intestinales, il est mesuré davantage de fluorescence résiduelle avec ces NPs. Enfin, des images obtenues par microscopie confocale montrent l'internalisation des NPs fonctionnalisées avec de la valine et obtenues par double émulsion, qui se retrouvent alors présentes dans le noyau de la cellule. Au contraire, les NPs obtenues par nanoprécipitation ou simple émulsion restent sur la membrane des cellules.

  l'organisme induite par leur internalisation et leur capacité à rejoindre la circulation systémique. En effet, une fois le transporteur ciblé et la molécule libérée, il est supposé que les NPs sont alors éliminées via le tractus gastro-intestinal.Cependant, il a été mis en évidence que les NPs fonctionnalisées avec de la valine et réalisées par double émulsion étaient capables de traverser la membrane cellulaire et de rejoindre le noyau. De ce fait, des études de toxicité utilisant des tests ATP ont dû être mises en place. Ces tests consistent au contrôle de la viabilité cellulaire par la mesure de l'ATP présent dans la cellule. En effet, la perte de l'intégrité des membranes des cellules induit une perte de leur faculté à synthétiser de l'ATP. Les résultats obtenus ont montré l'absence de toxicité induite par les NPs sur les cellules Caco-2.

  Mais l'internalisation des NPs est un phénomène bien connu lorsque celle-ci passent la barrière intestinale par un mécanisme de transcytose. C'est pourquoi Semete et al. ont étudié la toxicité de NPs de PLGA chez la souris Balb/C après administration orale. Il n'a été montré aucune altération des tissus, mais une forte accumulation des particules dans le foie a pu être observée, preuve d'une internalisation non spécifique des NPs (Semete et al., 2010). Récemment, deux équipes de chercheurs ont soulevés de nouvelles interrogations directement reliées à la présente étude. Tout d'abord, Ciappellano et al. ont étudié la pertinence d'appliquer des tests de toxicité communément utilisés pour des molécules classiques pour des NPs. Ainsi, ils ont approfondi les connaissances des modèles afin de relier la concentration et la durée d'exposition aux NPs pour différents types de cellules à ce qui peut être observé in vivo. Ils ont également identifié les principales causes de nano-toxicité (Ciappellano et al., 2016) (Fig. 1).

Figure 1 .

 1 Figure 1. Principales causes de nano-toxicité (Ciappellano et al., 2016).

  NPs de PLA-PEG obtenues par double émulsion. Le procédé est réalisé en trois étapes : une phase de congélation jusqu'à -40°C pendant 5 h à 800 µbar, suivie d'une phase de dessiccation primaire de -25 à -10°C pendant 14 h à 250 µbar, et enfin d'une phase de dessiccation secondaire à 25°C pendant 6 h à 41 µbar. Plusieurs agents cryoprotecteurs tels que le saccharose, le tréhalose ou le mannitol ont été testés à différents ratios. Il a alors été montré que l'ajout de 5% (m/v) de saccharose à une suspension de NPs concentrée à 10 mg/mL permettait d'obtenir des NPs de diamètre moyen 180 nm avant lyophilisation et de 220 nm après. Ces résultats sont prometteurs quant à la possibilité de lyophiliser ces NPs mais le procédé reste à développer pour des NPs chargées en principe actif.Il est également possible d'imaginer des designs de NPs beaucoup plus complexes. En effet, maintenant qu'il a été observé que les NPs étaient capables de pénétrer à l'intérieur de la cellule, l'utilisation de polymères sensibles au pH peut être intéressante. En effet, le pH intra cellulaire est mesuré à pH 5, ce qui est plus faible que la valeur observée au niveau intestinal.

  in vivo d'une molécule faiblement absorbée par le ciblage d'un transporteur La stratégie mise en place dans la preuve de concept consistant à cibler un transporteur intestinal avec des NPs fonctionnalisées permet de répondre à un objectif d'amélioration de la biodisponibilité d'une molécule faiblement absorbée. Cette amélioration peut se vérifier grâce à des études de transport sur des modèles in vitro ou par des études pharmacocinétiques sur des modèles in vivo. In vitro, les essais sont réalisés sur le modèle cellulaire Caco-2 largement utilisé pour les études d'absorption intestinale. Développées sur des puits Transwells ® pendant 21 jours, les cellules se différencient spontanément en cellules intestinales humaines. Les résultats obtenus dans la présente étude montrent qu'il est possible de multiplier respectivement par 3 et par 2 la perméabilité apparente in vitro de l'acyclovir et de l'ocytocine encapsulée dans des NPs de PLA-PEG-valine. In vivo, les essais ont été réalisés sur des souris C57Bl/6J. Ce modèle de souris est courant et est donc fréquemment utilisé pour une première approche pharmacocinétique sur une molécule. Les souris femelles sont privilégiées car elles cohabitent plus facilement. Cependant, il faut noter qu'il existe un taux circulant naturel de certaines molécules chez des individus. C'est le cas pour l'ocytocine chez la femelle, c'est pourquoi l'étude sur cette molécule a été réalisée avec des souris mâles. Les résultats des études in vivo montrent pour l'acyclovir que si la biodisponibilité n'est pas augmentée, le temps d'élimination l'est significativement (x 1.3). En effet, la molécule piégée dans la NP diffuse alors lentement, ce qui induit une augmentation du temps de rétention. Pour l'ocytocine, d'importantes variabilités sont observées entre les animaux, il est donc uniquement possible de conclure sur une tendance à l'amélioration de la biodisponibilité de la molécule. Dans tous les cas, cette stratégie offre des résultats intéressants pour l'augmentation de la perméabilité intestinale d'une molécule faiblement absorbée. De plus, elle ajoute une protection vis à vis du milieu extérieur pour la molécule encapsulée, ce qui est très intéressant pour des molécules de type peptide immédiatement dégradées par les enzymes du milieu gastro-intestinal.

Table 1 . Synthetic or semi-natural bioadhesive polymers used for oral drug bioavailability improvement.

 1

		Synthetic bioadhesive polymers	Semi-natural bioadhesive polymers
	Polyacrylic	Carbopol	Guar
	acid-based	Polycarbophil	Xanthan
		Polyacrylic acid	Crylamide-acrylate polymer (PHPA)
		Polyacrylate	Poly(vinylpyrrolidone)
		Poly(methylvinylether-co-methacrylic acid)	Poly (vinyl alcohol)
		Poly(2-hydroxyethyl methacrylate)
		Poly(methacrylate)
		Poly(alkylcyanoacrylate)
		Poly(isohexylcyanoacrylate)
		Poly(isobutylcyanoacrylate)
	Cellulose	Carboxymethyl cellulose
	derivatives	Hydroxyethyl cellulose
		Hydroxypropyl cellulose
		Sodium carboxymethyl cellulose
		Methyl cellulose
		Methylhydroxyethyl cellulose

Table 2 . Descriptions of the endocytosis pathways across cells.

 2

Table 3 . Intestinal receptor-targeted prodrugs or functionalized nanocarriers: Drug substances and strategies.

 3

	Receptor	Drug substance	Strategy	References
	Vitamin B12	Erythropoietin (EPO)	VB12-EPO prodrug

Table 4 . Intestinal transporter-targeted prodrugs: Drug substances and prodrug designs. Transporter Drug substance Prodrug designs References Amino acid, System L

 4

	Quinidine	Val-quinidine

Table 1 . Preparation methods of nanoparticles, in function of the polymeric matrice (des Rieux A., Fievez et al., 2006).

 1

Table 2 . Nanoparticles structure, mean particle size, advantages and drawbacks according to the manufacturing process (Iqbal, Zafar et al., 2015).

 2

	Manufacturing process	Structure	Size	Advantages	Drawbacks
					 Fast release of
	Double emulsion solvent evaporation	Nanocapsule 120 to 350 nm	 Encapsulation of both hydrophilic and lipophilic molecules	hydrophilic molecules to the external aqueous phase
					 Polydispersity
				 One step process
				 Monodispersity	 Fast release of
	Nanoprecipitation	Nanosphere	100 to 500 nm	 Encapsulation of both	hydrophilic molecules
				hydrophilic and	to the aqueous phase
				lipophilic molecules

Table 3 . Protein-loaded nanoparticles obtained by nanoprecipitation (Bilati, Allemann et al., 2005).

 3

Table 4 . Effect of formulation variables on physicochemical properties of PLA NPs (Patel, Gohel et al., 2014).

 4

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Cheon,E.P., Hong,J.H., Han,H.K., 2006. Enhanced cellular uptake of Ara-C via a peptidomimetic prodrug, L-valyl-ara-C in Caco-2 cells. J Pharm Pharmacol, 58, 927-932. Conner,S.D., Schmid,S.L., 2003. Regulated portals of entry into the cell. Nature, 422, 37-44. Couvreur,P., Kante,B., Roland,M., Guiot,P., Bauduin,P., Speiser,P., 1979. Polycyanoacrylate nanocapsules as potential lysosomotropic carriers: preparation, morphological and sorptive properties. J Pharm Pharmacol, 31, 331-332. Danafar,H., Rostamizadeh,K., Davaran,S., Hamidi,M., 2016. Drug-conjugated PLA-PEG-PLA copolymers: a novel approach for controlled delivery of hydrophilic drugs by micelle formation. Pharm Dev. Technol, 1-11. Fasano,A., Uzzau,S., 1997. Modulation of intestinal tight junctions by Zonula occludens toxin permits enteral administration of insulin and other macromolecules in an animal model. J Clin Invest, 99, 1158-1164. Fattal,E., Vauthier,C., Aynie,I., Nakada,Y., Lambert,G., Malvy,C., Couvreur,P., 1998. Biodegradable polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides. J Control Release, 53, 137-143.

Fei,Y.J., Kanai,Y., Nussberger,S., Ganapathy,V., Leibach,F.H., Romero,M.F., Singh,S.K., Boron,W.F., Hediger,M.A., 1994. Expression cloning of a mammalian proton-coupled oligopeptide transporter. Nature, 368, 563-566. Fei,Y.J., Sugawara,M., Liu,J.C., Li,H.W., Ganapathy,V., Ganapathy,M.E., Leibach,F.H., Sun,S., Liang,N., Kawashima,Y., Xia,D., Cui,F., 2011. Hydrophobic ion pairing of an insulinsodium deoxycholate complex for oral delivery of insulin.

Int J Nanomedicine., 6, 3049-3056. Swaminathan,J., Ehrhardt,C., 2012. Liposomal delivery of proteins and peptides. Expert Opin Drug Deliv, 9, 1489-1503. Tamai,I., 2012. Oral drug delivery utilizing intestinal OATP transporters. Adv Drug Deliv Rev, 64, 508-514. Tamai,I., Nakanishi,T., Nakahara,H., Sai,Y., Ganapathy,V., Leibach,F.H., Tsuji,A., 1998.

Improvement of L-dopa absorption by dipeptidyl derivation, utilizing peptide transporter PepT1. J Pharm

Sci, 87, 1542-1546. Tamai,I., Takanaga,H., Maeda,H., Sai,Y., Ogihara,T., Higashida,H., Tsuji,A., 1995.

récepteurs ou des transporteurs intestinaux

(Majumdar et al., 2004)

, ou bien même de nanoobjets fonctionnalisés reconnus par des récepteurs

(Du et al., 2013; Gosselin and Lee, 2002; Roger et al., 2012; Stella et al., 2000)

, il est difficile de trouver des exemples de ces mêmes nano-objets ciblant des transporteurs. En effet, les transporteurs intestinaux ne sont pas supposés favoriser le passage de grosses entités mais plutôt de petites molécules. Cibler un transporteur avec des nanoparticules fonctionnalisées permettrait davantage leur surconcentration près de la membrane intestinale plutôt que leur internalisation. Ainsi, le temps de résidence des nanoparticules et donc de la molécule d'intérêt encapsulée se trouverait augmenté, ce qui permettrait l'amélioration de ses performances biopharmaceutiques.

L'objectif de cette preuve de concept est de démontrer l'amélioration de la biodisponibilité d'une molécule modèle BCS III encapsulée dans des nanoparticules polymères administrées par voie orale, et ciblant un transporteur intestinal. Cette stratégie trouvant ainsi tout son intérêt pour des molécules ne pouvant être formulées comme des pro-drogues pour lesquelles le ligand est directement conjugué à la molécule.

Table 1 . Fractional factorial design to screen formulation and process parameters in terms of nanoparticle size: studied parameters.

 1

	Parameters (Xi)	Levels
	Polymers (PLGA RG502/PLA-PEG-NH 2 ) in DMSO (w/v %)	5.71 15 30
		1 : 2
	Ratio DMSO/water	1 : 4
		1 : 10
		0
	Surfactant in water (w/v %)	0.01
		0.1
		0.5
	Needle diameter (mm)	0.8
		1.2
		1 : 2
	Ratio PLGA RG502/PLA-PEG-NH 2	1 : 6.67
		0 : 1
		2
	Stirring duration (min)	5
		10
		Instantaneous
	Addition time (min)	1
		2
		Poloxamer 407
	Surfactant type	Poloxamer 188 Tween 80
		PVA Mowiol 4-88
		600
	Stirring rate (rpm)	1200 3200
		8000

Table S1 . Fractional factorial design to screen formulation and process parameters in terms of nanoparticles size: experimental plan.

 S1

	N°Exp	Polymers in DMSO (w/v %)	Ratio DMSO/water	Surfactant in water (w/v %)	Needle diameter (mm)	Ratio PLGA RG502/ PLA-PEG-NH2	Stirring duration (min)	Addition time (min)	Surfactant type	Stirring rate (rpm)
	1	5.71	1:2	0.1	0.8	0	2	instantaneous	PVA Mowiol 4-88
	2	5.71	1:2	0.1	0.8	0	2	instantaneous	PVA Mowiol 4-88
	3	5.71	1:2	0.1	0.8	0	2	instantaneous	PVA Mowiol 4-88
	4	15	1:2	0	1.2	1:6.67	10	1	PVA Mowiol 4-88
	5	30	1:2	0.01	0.5	0	5	instantaneous	Tween 80
	6	5.71	1:2	0	0.5	1:6.67	2	2	Tween 80
	7	5.71	1:2	0.01	1.2	1:2	2	2	Poloxamer 188	600
	8	15	1:2	0	0.8	1:2	10	instantaneous	Poloxamer 188
	9	30	1:2	0.1	0.5	1:2	5	1	Poloxamer 407
	10	5.71	1:2	0	0.5	1:2	2	instantaneous	Poloxamer 407	600
	11	5.71	1:4	0	0.5	1:2	10	1	Tween 80	600
	12	15	1:4	0.01	0.5	1:2	2	instantaneous	Tween 80
	13	30	1:4	0	0.8	1:2	2	2	PVA Mowiol 4-88
	14	5.71	1:4	0.1	1.2	1:2	5	instantaneous	PVA Mowiol 4-88	600
	15	5.71	1:4	0	0.5	0	10	instantaneous	Poloxamer 407
	16	15	1:4	0.1	0.5	1:6.67	2	2	Poloxamer 407
	17	30	1:4	0	1.2	0	2	instantaneous	Poloxamer 188
	18	5.71	1:4	0.01	0.8	1:6.67	5	1	Poloxamer 188
	19	5.71	1:10	0	0.5	1:2	5	2	Poloxamer 188
	20	15	1:10	0.1	0.5	1:2	2	instantaneous	Poloxamer 188
	21	30	1:10	0	1.2	1:2	2	1	Poloxamer 407
	22	5.71	1:10	0.01	0.8	1:2	10	instantaneous	Poloxamer 407
	23	5.71	1:10	0	0.5	1:6.67	5	instantaneous	PVA Mowiol 4-88
	24	15	1:10	0.01	0.5	0	2	1	PVA Mowiol 4-88	600
	25	30	1:10	0	0.8	1:6.67	2	instantaneous	Tween 80	600
	26	5.71	1:10	0.1	1.2	0	10	2	Tween 80
	27	5.71	1:2	0.01	1.2	1:6.67	2	instantaneous	Poloxamer 407
	28	15	1:2	0	0.8	0	5	2	Poloxamer 407	600
	29	30	1:2	0.1	0.5	1:6.67	10	instantaneous	Poloxamer 188	600
	30	5.71	1:2	0	0.5	0	2	1	Poloxamer 188
	31	5.71	1:2	0.1	0.8	1:2	2	1	Tween 80
	32	15	1:2	0	1.2	1:2	5	instantaneous	Tween 80
	33	30	1:2	0.01	0.5	1:2	10	2	PVA Mowiol 4-88
	34	5.71	1:2	0	0.5	1:2	2	instantaneous	PVA Mowiol 4-88

Table 2 . Effect of composition and theoretical loading on actual drug loading and particle size (mean ± S.D., n=3).

 2

	PLGA/PLA-PEG-NH 2	Theoretical drug loading (%)	Actual drug loading (%)	Entrapment efficiency (%)	Particle Size (nm) X 50 X 90
	0:1	25	1.3 ± 0.2	4.5 ± 0.8	33.6 ± 7.2	69.8 ± 3.6
	1:2	25	2.9 ± 0.1	8.8 ± 0.3	38.5 ± 3.9	56.0 ± 3.9
	1:2	50	10.2 ± 0.4	11.4 ± 0.5	46.3 ± 6.7	74.7 ± 15.5

Table 3 . Effect of increasing amounts of ligand-functionalized nanoparticles on the inhibition of

 3

Table 4 . Pharmacokinetic parameters of ACY after single oral administration of ACY (G1), ValACY (G2) and ACY encapsulated in NPs-Val (G3) to female C57Bl/6J mice (25 mg/kg, 2.5 mg/mL, expressed as free compound, 10 mice/group). C max , AUC 0-8h , t 1/2 and MRT are expressed as mean ± S.D. T max is expressed as median (min-max).

 4

	Group Id. Administered compound	C max (ng/mL)	T max (h)	AUC 0-8h (ng.h/mL)	t 1/2 (h)	MRT (h)
	G1 ACY	3279 ± 715	1 (0.5-1)	5761 ± 581	1.34 ± 0.08	1.85 ± 0.23
	G2 ValACY	29894 ± 13756 ***	0.25 (0.25-0.5)	22887 ± 4593 ***	1.06 ± 0.25	0.92 ± 0.16 ***
	G3
	NPs-Val-	4035 ±	0.25	5884 ±	1.80 ±	2.35 ±
	ACY	1561	(0.25-0.5)	1611	0.38 *	0.26 **
	* p < 0.05, ** p < 0.01, *** p < 0.001 compared to ACY (G1)

  Ainsi, cette preuve de concept a permis de démontrer qu'il était non seulement possible de cibler le transporteur PepT1 avec des nanoparticules fonctionnalisées avec le ligand valine, mais également d'augmenter la perméabilité apparente de l'acyclovir d'un facteur 3 en l'encapsulant dans ces NPs in vitro. In vivo, il est possible d'augmenter la demi-vie et le nombre réduit d'essais.[START_REF] Box | Statistics for experimenters: an introduction to design, data analysis, and model building[END_REF] Julienne M.C., Alonso et al., 1992 ; Lewis G.A., Mathieu D. et al., 1999). Un second plan de criblage est ensuite construit afin d'étudier l'influence de nouveaux paramètres de formulation sur la capacité des NPs à encapsuler l'ocytocine et les profils de libération en résultant.

	Introduction au Chapitre II
	La preuve de concept présentée au Chapitre I a démontré qu'il était possible de cibler le
	transporteur intestinal PepT1 avec des NPs fonctionnalisées avec un ligand Valine. Cette
	temps de rétention moyen de la molécule. Ces résultats coïncident avec l'hypothèse de départ stratégie permet ainsi l'augmentation du transport in vitro de l'acyclovir, une molécule BCS Dans un second temps, la possibilité de simplifier la technique de double émulsion afin suggérant que le ciblage de transporteur permettrait l'augmentation du temps de résidence des III modèle. Cependant, une libération très rapide du principe actif des NPs ne rend pas cette d'aboutir à une émulsion unique est explorée. Pour cela, un dernier plan de criblage est mis en NPs au contact de la membrane et donc une augmentation du transport de la molécule encapsulée. Néanmoins, il a pu être constaté une libération très rapide du principe actif des amélioration perceptible in vivo chez la souris. place pour étudier l'influence des paramètres de formulation sur la taille et le taux
	NPs en quelques minutes (88% en 90 min). Ceci pourrait alors expliquer l'absence Ainsi, deux nouveaux axes de recherches découlent de cette preuve de concept : d'encapsulation de NPs de PLA-PEG encapsulant de l'ocytocine et obtenues par simple
	émulsion. Bien que ce type de procédé soit déjà bien connu, il a très peu été utilisé pour d'amélioration du transport chez la souris.  Le meilleur contrôle de la libération du principe actif des NPs. l'encapsulation de molécule hydrophiles et encore moins de peptides, à cause des faibles taux
	. Il a été alors Avec l'arrivée croissante des Nouvelles Entités Moléculaires sur le marché, il est désormais
	possible de définir le ligand permettant la meilleure affinité des NPs fonctionnalisées intéressant de remplacer la molécule modèle ayant servie à la preuve de concept par un
	pour le transporteur, soit la valine. peptide. L'ocytocine, petit peptide de neuf acides aminés, a été choisie comme molécule
	 L'augmentation de la perméabilité apparente de l'acyclovir par 3 in vitro quand il est d'intérêt. Cette molécule trouve ses applications thérapeutiques dans les domaines cognitifs et
	encapsulé dans des NPs fonctionnalisées. Cette amélioration reste cependant inférieure comportementaux.
	à celle observée avec la pro-drogue valacyclovir.
	Dans un premier temps, la technique de double émulsion est étudiée pour l'encapsulation de
	l'ocytocine. Il est tout d'abord évalué la faisabilité de NPs placebo de PLA-PEG par cette

 In vivo, les mêmes formulations ont été testées chez la souris par voie orale. Une étude pharmacocinétique sur huit heures n'a montré aucune amélioration du C max ou de l'AUC lorsque l'acyclovir est encapsulé dans des NPs-Val. Cependant, le t 1/2 et le TRM ont été multipliés par 1,3. De ce fait, cette preuve de concept a permis d'arriver à la conclusion que la stratégie mise en place était perfectible en agissant essentiellement sur la formulation des NPs afin d'arriver à un meilleur contrôle de la libération du principe actif.  Le remplacement de la molécule BCS III modèle par une molécule d'intérêt. Il a largement été démontré l'intérêt de l'utilisation des NPs pour des molécules faiblement absorbées ou dégradées dans l'intestin. De plus, de significatives améliorations de la biodisponibilité et des temps de résidence des molécules encapsulées ont pu être observées (Mahapatro and Singh, 2011). Plusieurs techniques de fabrication des NPs polymères existent telle que la nanoprécipitation déjà explorée dans la preuve de concept du Chapitre I, mais également les techniques d'émulsion. L'objectif de ce Chapitre II est donc d'explorer de nouvelles techniques de fabrication de NPs de PLA-PEG afin d'améliorer non seulement le taux d'encapsulation du principe actif mais également sa libération, tout en conservant une taille de NPs < 200 nm afin de pouvoir traverser le mucus et rejoindre la barrière intestinale après administration orale

(Ensign et al., 2012)

, et des émulsions stables avec un potentiel zéta idéalement supérieur à |30| mV

(Hans and Lowman, 2002)

. méthode, ainsi que l'influence des paramètres de formulation et de procédé sur la taille des NPs et sur la stabilité des émulsions, grâce à un plan de criblage. Ce type de plan d'expérience permet d'étudier l'influence d'un grand nombre de facteurs sur plusieurs niveaux avec un d'encapsulation obtenus. L'objectif de ce second chapitre en est d'autant plus intéressant et permettra de comparer les caractéristiques des NPs d'ocytocine obtenues par double ou simple émulsion, et de conclure sur l'intérêt d'une simplification de la première méthode. La seconde partie de ce travail de thèse est développée sous forme d'un article scientifique soumis dans Pharmaceutical Research (under review).

Table 1 . Fractional factorial design to screen formulation and process parameters in terms of NP size: studied parameters for double emulsion manufacturing process feasibility.

 1

		Parameters	Levels
	b1	(O) Surfactant	Poloxamer P188 Montane 80
			0.5
	b2	(O) Surfactant amount (%, w/v)	1
			2
			PLA-PEG-NH 2 /PLGA (1:0)
	b3	(O) Polymer	PLA-PEG-NH 2 /PLGA (2:1)
			PLA-PEG-NH 2 /PLGA (4:1)
			0.5
	b4	(O) Polymer amount (%, w/v)	1
			2
			1:2
	b5	(O) Ratio WI/O	1:3
			1:4
			30
	b6	(O) Sonication time (s)	60
			90
			PVA (Mowiol 4-88)
	b7	(WI) Surfactant	Poloxamer P188
			Montanox 80
			0
	b8	(WI) Surfactant amount (%, w/v)	1
			2.8
			PVA (Mowiol 4-88)
	b9	(WE) Surfactant	Poloxamer P188
			Montanox 80
			1
	b10	(WE) Surfactant amount (%, w/v)	2
			3
			2:5
	b11	(WE) Ratio (WI/O)/WE	1:2
			3:10
			30
	b12	(WE) Sonication time (s)	60
			90

Table 2 . Fractional factorial design to screen formulation and process parameters in terms of NP size: experimental plan for double emulsion manufacturing process feasibility.

 2

	N°Exp	(O) Surfactant	(O) Surfactant amount	(O) Polymer	(O) Polymer amount	(O) Ratio WI/O	(O) Sonication time	(WI) Surfactant	(WI) Surfactant amount	(WE) Surfactant	(WE) Surfactant amount	(WE) Ratio (WI/O)/WE	(WE) Sonication time	X50	Zeta potential
			(%, w/v)		(%,w/v)		(s)		(%, w/v)		(% w/v)		(s)	(nm)	(mV)
	1	Poloxamer P188	0.5	PLA-PEG-NH2/PLGA (1:0)	0.5	1:2	30	PVA (Mowiol 4-88)	0	PVA (Mowiol 4-88)	1	2:5		24	3.8
	2	Poloxamer P188	0.5	PLA-PEG-NH2/PLGA (1:0)	0.5	1:2	30	PVA (Mowiol 4-88)	0	PVA (Mowiol 4-88)	1	2:5		47	4.0
	3	Poloxamer P188	0.5	PLA-PEG-NH2/PLGA (1:0)	0.5	1:3	60	Poloxamer P188	1	Poloxamer P188	2	1:2		863	40.8
	4	Poloxamer P188	0.5	PLA-PEG-NH2/PLGA (1:0)	0.5	1:4	90	Montanox 80	2.8	Montanox 80	3	3:10		31	19.0
	5	Poloxamer P188	1.0	PLA-PEG-NH2/PLGA (2:1)	1.0	1:2	30	PVA (Mowiol 4-88)	1	Poloxamer P188	2	3:10		521	34.0
	6	Poloxamer P188	1.0	PLA-PEG-NH2/PLGA (2:1)	1.0	1:3	60	Poloxamer P188	2.8	Montanox 80	3	2:5		41	24.4
	7	Poloxamer P188	1.0	PLA-PEG-NH2/PLGA (2:1)	1.0	1:4	90	Montanox 80	0	PVA (Mowiol 4-88)	1	1:2		417	3.2
	8	Poloxamer P188	2.0	PLA-PEG-NH2/PLGA (4:1)	2.0	1:2	30	PVA (Mowiol 4-88)	2.8	Montanox 80	3	1:2		40	13.9
	9	Poloxamer P188	2.0	PLA-PEG-NH2/PLGA (4:1)	2.0	1:3	60	Poloxamer P188	0	PVA (Mowiol 4-88)	1	3:10		65	5.0
	10	Poloxamer P188	2.0	PLA-PEG-NH2/PLGA (4:1)	2.0	1:4	90	Montanox 80	1	Poloxamer P188	2	2:5		47	18.1
	11	Montane 80	0.5	PLA-PEG-NH2/PLGA (2:1)	2.0	1:2	60	Montanox 80	0	Poloxamer P188	3	2:5		37	23.5
	12	Montane 80	0.5	PLA-PEG-NH2/PLGA (2:1)	2.0	1:3	90	PVA (Mowiol 4-88)	1	Montanox 80	1	1:2		58	25.3
	13	Montane 80	0.5	PLA-PEG-NH2/PLGA (2:1)	2.0	1:4	30	Poloxamer P188	2.8	PVA (Mowiol 4-88)	2	3:10		566	31.0
	14	Montane 80	1.0	PLA-PEG-NH2/PLGA (4:1)	0.5	1:2	60	Montanox 80	1	Montanox 80	1	3:10		506	10.6
	15	Montane 80	1.0	PLA-PEG-NH2/PLGA (4:1)	0.5	1:3	90	PVA (Mowiol 4-88)	2.8	PVA (Mowiol 4-88)	2	2:5		32	30.1
	16	Montane 80	1.0	PLA-PEG-NH2/PLGA (4:1)	0.5	1:4	30	Poloxamer P188	0	Poloxamer P188	3	1:2		23174	10.5
	17	Montane 80	2.0	PLA-PEG-NH2/PLGA (1:0)	1.0	1:2	60	Montanox 80	2.8	PVA (Mowiol 4-88)	2	1:2		196528	25.1
	18	Montane 80	2.0	PLA-PEG-NH2/PLGA (1:0)	1.0	1:3	90	PVA (Mowiol 4-88)	0	Poloxamer P188	3	3:10		93341	12.6
	19	Montane 80	2.0	PLA-PEG-NH2/PLGA (1:0)	1.0	1:4	30	Poloxamer P188	1	Montanox 80	1	2:5		518	24.6
	20	Poloxamer P188	0.5	PLA-PEG-NH2/PLGA (4:1)	1.0	1:2	90	Poloxamer P188	0	Montanox 80	2	2:5		12	30.0
	21	Poloxamer P188	0.5	PLA-PEG-NH2/PLGA (4:1)	1.0	1:3	30	Montanox 80	1	PVA (Mowiol 4-88)	3	1:2		16	-0.01
	22	Poloxamer P188	0.5	PLA-PEG-NH2/PLGA (4:1)	1.0	1:4	60	PVA (Mowiol 4-88)	2.8	Poloxamer P188	1	3:10		65	25.5
	23	Poloxamer P188	1.0	PLA-PEG-NH2/PLGA (1:0)	2.0	1:2	90	Poloxamer P188	1	PVA (Mowiol 4-88)	3	3:10		503	0.01
	24	Poloxamer P188	1.0	PLA-PEG-NH2/PLGA (1:0)	2.0	1:3	30	Montanox 80	2.8	Poloxamer P188	1	2:5		10	36.9
	25	Poloxamer P188	1.0	PLA-PEG-NH2/PLGA (1:0)	2.0	1:4	60	PVA (Mowiol 4-88)	0	Montanox 80	2	1:2		80	28.6
	26	Poloxamer P188	2.0	PLA-PEG-NH2/PLGA (2:1)	0.5	1:2	90	Poloxamer P188	2.8	Poloxamer P188	1	1:2		393	0.2
	27	Poloxamer P188	2.0	PLA-PEG-NH2/PLGA (2:1)	0.5	1:3	30	Montanox 80	0	Montanox 80	2	3:10		25	15.5
	28	Poloxamer P188	2.0	PLA-PEG-NH2/PLGA (2:1)	0.5	1:4	60	PVA (Mowiol 4-88)	1	PVA (Mowiol 4-88)	3	2:5		30	-0.2

Table 3 . Fractional factorial design to screen formulation and process parameters in terms of NP size: studied parameters for OXY encapsulation in NPs obtained from double emulsion process. For PEG viscosifying agent, ratio 1 and ratio 2 were (3:2) and (2:3) of (water/PEG), respectively. For HEC viscosifying agent, ratio 1 and ratio 2 were 1% and 2.5% (w/v), respectively.

 3

		Parameters	Levels
	b1	(WI) Viscosifying agent	HEC PEG
			0
	b2	(WI) Viscosifying amount	Ratio 1
			Ratio 2
	b3	(O) PLGA	RG 502 RG 502H
			1
	b4	(O) Surfactant amount (%, w/v)	1.5
			2
			2
	b5	(O) Polymers amount (%, w/v)	3
			4

Table 4 . Fractional factorial design to screen formulation and process parameters in terms of NP size: experimental plan for OXY encapsulation in NPs obtained from double emulsion process. For PEG viscosifying agent, ratio 1 and ratio 2 were (3:2) and (2:3) of (water/PEG), respectively. For HEC viscosifying agent, ratio 1 and ratio 2 were 1% and 2.5% (w/v), respectively.

 4

	N°Exp	(WI) Viscosifying agent	(WI) Viscosifying amount	(O) PLGA type	(O) amount Surfactant	(O) amount Polymer	X 50	Drug Load
					(%, w/v)	(%, w/v)	(nm)	(%)
		HEC	0	RG502	1	2	39	4.5
		HEC	0	RG502	1	2	39	4.3
		HEC	Ratio 1	RG502	1.5	3	55	8.7
		HEC	Ratio 2	RG502	2	4	32	12.1
		PEG	0	RG50 2	1.5	4	39	13.6
		PEG	Ratio 1	RG502	2	2	28	3.4
		PEG	Ratio 2	RG502	1	3	34	6.5
		HEC	0	RG502H	2	3	4394	13.3
		HEC	Ratio 1	RG502H	1	4	31	8.0
	10	HEC	Ratio 2	RG502H	1.5	2	46	10.2
	11	PEG	0	RG502H	2	3	4394	13.3
	12	PEG	Ratio 1	RG502H	1	4	55	6.3
	13	PEG	Ratio 2	RG502H	1.5	2	53	6.9

Table 5 . Fractional factorial design to screen formulation and process parameters in terms of NP size: studied parameters for OXY encapsulation in NPs obtained from simple emulsion process.

 5

		Parameters	Levels
			1:3
	b1	O/W phase ratio	1:5
			1:10
			1
	b2	(O) Surfactant amount (%, w/v)	1.5
			2
			1:4
	b3	(O) OXY/Polymers ratio	1:3
			1:2
			2:1
	b4	(O) PLA-PEG-NH 2 /Additional polymer ratio	3:2
			1:1
			/
	b5	(O) Additional excipient	PLGA RG502
			PDLG 7502
			1
	b6	(W) Surfactant amount (%, w/v)	1.5
			2

Table 6 . Fractional factorial design to screen formulation and process parameters in terms of NP size: experimental plan for OXY encapsulation in NPs obtained from simple emulsion process.

 6

	N°Exp	O/W phase ratio	(O) Surfactant amount	(O) OXY/ Polymers ratio	(O) PLA-PEG/ Additional polymer ratio	(O) Additional excipient	(W) amount Surfactant	X 50	Drug Load
			(%, w/v)				(%, w/v)	(nm)	(%)
		1:3	1	1:4	2:1	/	1	21	0.65
		1:3	1	1:4	2:1	/	1	43	0.59
		1:3	1	1:4	2:1	/	1	34	0.53
		1:5	2	1:4	3:2	/	1	44	0.80
		1:5	1.5	1:3	3:2	/	1.5	23	2.05
		1:10	1	1:3	1:1	/	1.5	1003	1.56
		1:3	1.5	1:2	2:1	/	2	59	0.39
		1:10	2	1:2	1:1	/	2	6633	0.92
		1:10	1	1:2	3:2	PLGA RG502	1	4764	4.76
	10	1:3	1.5	1:4	1:1	PLGA RG502	1.5	65	1.61
	11	1:5	2	1:3	2:1	PLGA RG502	2	7700	0.48
	12	1:10	1.5	1:3	2:1	PDLG 7502	1	5969	1.95
	13	1:3	2	1:3	1:1	PDLG 7502	1	136	1.06
	14	1:5	2	1:2	2:1	PDLG 7502	1.5	106	0.45
	15	1:3	2	1:2	3:2	PDLG 7502	1.5	4499	2.00
	16	1:10	2	1:4	3:2	PDLG 7502	2	869	0.37
	17	1:5	1	1:4	1:1	PDLG 7502	2	71	0.32

Table 7 . Effect of manufacturing process on actual drug loading, entrapment efficiency, particle size and zeta potential for OXY-loaded PLA-PEG-NH 2 NPs (mean ± S.D., n=3).

 7

	Manufacturing process	Theoretical drug loading (%)	Actual drug loading (%)	Entrapment efficiency (%)	X 50	Particle size (nm)	X 99	Zeta potential (mV)
	Double emulsion	-	-	-	184.7 ± 5.0	248.9 ± 6.4	34.1 ± 0.99
	Double emulsion	30%	4.02 ± 0.69	13.4 ± 2.3	194.5 ± 4.8	260.4 ± 6.4	12.9 ± 1.00
	Simple emulsion	33.3%	3.26 ± 0.87	9.78 ± 2.61	226.4 ± 5.1	301.1 ± 12.4 -0.33 ± 4.75

  Samples from in vivo experiments were assayed using ELISA test. It is a widely used method to determine protein concentration in solution. Mice plasma samples were analyzed against a set of calibration standards provided in a kit. Standards and samples were pipetted into a clear microtiter plate coated with an antibody to capture rabbit antibodies. An OXY-peroxidase conjugate was added in the wells. The binding reaction was initiated by the addition of a polyclonal antibody to OXY to each well. After an overnight incubation at 4°C the plate was washed and supplied substrate (TMB 3,3',5,5'-Tetramethylbenzidine) was added. The substrate reacted with the bound OXY-peroxidase conjugate. After 30 min of incubation, the reaction was stopped and the intensity of the generated color was detected in a microtiter plate reader at 450 nm. The plate was shaken for 15 min at room temperature at 500 rpm to ensure adequate mixing of the reagents, then covered and stored at 4°C for 16-18 hours. The assay plate was washed four times, with 300 µL of wash buffer per well per time. 100 µL of TMB substrate was added to each well and the assay plate was covered and incubated for 30 min in the dark at room temperature without agitation. The reaction was stopped by adding 50 µL of 1 N hydrochloric acid to each well. The assay plate was read within 10 min at 450 nm (Bio-Tek EPOCH2). The concentration of OXY in test samples was interpolated by using the standard curve.

	mean measured concentration /nominal concentration) × 100
	CV (%) = (standard deviation/mean measured concentration) × 100
	RE (%) = [(measured concentration -nominal concentration)/nominal concentration] × 100.
	All solutions were equilibrated at room temperature before starting the assay. Assay Buffer
	5X-concentrate was diluted with deionized water by adding one part of the concentrate to four
	parts of deionized water. Calibration standards were obtained from a stock-solution of OXY
	at the concentration of 100,000 pg/mL diluted in assay buffer and ranged from 10,000 pg/mL
	to the lower limit of quantification (LLOQ) 16.4 pg/mL. No dilution was required for test
	samples. An extraction by protein precipitation was mandatory before use test samples
	(extraction solution ready-to-use X123). Prior to extraction, plasmas were centrifuged at
	4,500 rpm for 5 min at 4°C. One part of test sample was added to 1.5 part of extraction
	solution (320 µL of sample + 480 µL of extraction solution for triplicate analysis). Each
	sample was mixed with a vortex and kept at room temperature for 90 min. Then, they were
	centrifuged for 20 min at 4°C at 1,660 g. Samples were dried at 37°C, and reconstituted with
	320 µL of assay buffer. 100 µL of standards, controls and tests samples were placed into the
	plate. 25 µL of DetectX OXY Conjugate and 25 µL of DetectX OXY Antibodies were added
	to each well.

Table 2 . Influence of the addition of 1% (w/w) PLA-PEG-FITC in nanoparticles formulations, for the three manufacturing processes

 2

. (n=3 ± S.D.) * p < 0.05, ** p < 0.01, *** p < 0.

001: PLA- PEG-Val NPs compared to PLA-PEG-Val + 1% PLA-PAG-FITC NPs.

		Zeta potential (mV)
				Statistical
	Manufacturing process	PLA-PEG-Val NPs	PLA-PEG-Val + 1% (w/w) PLA-PEG-FITC NPs	analysis
	Nanoprecipitation	13.2 ± 0.4	9.8 ± 0.8	**
	Simple emulsion	29.0 ± 0.5	26.6 ± 0.6	**
	Double emulsion	26.4 ± 1.0	23.9 ± 1.3	NS
	PLA-PEG and PLA-PEG-Val NPs labeled with PLA-PEG-FITC were then incubated with

Inserm, U1144, Paris, F-75006, France

Université Paris Descartes, UMR-S 1144, Faculté de Pharmacie de Paris, Paris, F-75006, France

Remerciements 7

Un immense merci à tous les copains angevins. Merci d'avoir fait semblant de comprendre mon sujet et merci d'avoir tous répondu présents quand j'en avais le plus besoin, je ne l'oublierai jamais.

Enfin, je remercie sincèrement ma famille, mon frère de m'avoir accueillie à Paris ou Bruxelles pour le boulot ou juste pour me changer les idées, et je remercie mes parents car je sais que je pourrai toujours compter sur vous et que vous serez toujours là pour moi.

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Chapitre I -Publication n°1

Functionalized PLA-PEG Nanoparticles Targeting Intestinal Transporter PepT1 for

Oral Delivery of Acyclovir

Betty Gourdon 1,2,3 , Caroline Chemin 1 , Amélie Moreau 1 , Thomas Arnauld 1 , Philippe Baumy 1 , Salvatore Cisternino 2,3 , Jean-Manuel Péan 1 , Xavier Declèves 2,3 1 Technologie Servier, 27 rue Eugène Vignat, Orléans, France

Abstract

Targeting intestinal di-and tri-peptide transporter PepT1 with prodrugs is a successful strategy to improve oral drug bioavailability, as demonstrated with valacyclovir, a prodrug of acyclovir. The aim of this new drug delivery strategy is to over-concentrate a poorly absorbed drug on the intestinal membrane surface by targeting PepT1 with functionalized polymer nanoparticles. In the present study, poly(lactic acid)-poly(ethylene glycol)-ligand (PLA-PEGligand) nanoparticles were obtained by nanoprecipitation. A factorial experimental design allowed us to identify size-influent parameters and to obtain optimized ≈ 30 nm nanoparticles.

Valine, Glycylsarcosine, Valine-Glycine, and Tyrosine-Valine were chemically linked to PLA-PEG. In Caco-2 cell monolayer model, competition between functionalized nanoparticles and [ 3 H]Glycylsarcosine, a strong substrate of PepT1, reduced [ 3 H]Glycylsarcosine transport from 22 to 46%. Acyclovir was encapsulated with a drug load of ≈ 10% in valine-functionalized nanoparticles, resulting in a 2.7-fold increase in permeability as compared to the free drug. An in vivo pharmacokinetic study in mice compared oral absorption of acyclovir after administration of 25 mg/kg of valacyclovir, free or encapsulated acyclovir in functionalized nanoparticles. Acyclovir encapsulation did not statistically modify AUC or C max , but increased t 1/2 and MRT 1.3-fold as compared to free acyclovir. This new strategy is promising for poorly absorbed drugs by oral administration.

Conflict of interest

The authors declare no conflict of interest.

Chapitre II -Publication n°2

Double or Simple Emulsion Process to Encapsulate Hydrophilic Peptide in PLA-PEG

Nanoparticles.

Betty Gourdon 1,2,3 , Xavier Declèves 2,3 , Jean-Manuel Péan 1 , Caroline Chemin

Abstract

Oral delivery of drugs using nanoparticles (NPs) is a current strategy for poorly absorbed molecules. It offers significant improvement in terms of bioavailability, biodistribution and residence time. With the impressive development of New Molecules Entities (NMEs), the encapsulation of proteins and peptides in polymeric NPs is a challenge. Firstly, the present study focused on the double emulsion process in order to encapsulate the oxytocin (OXY) peptide. Then the technique was challenged by a one-step simplified process, the simple emulsion. In order to study the influence of formulation and process parameters at several levels, factorial experimental designs were carried on. The responses observed were the NP size (as it was for oral formulation, it had to remain < 200 nm in order to penetrate the intestinal mucus layer), the suspension stability (zeta potential < |30| mV) and the OXY loading. It was thus found that the amount and the nature of surfactant in the different phases, the ratio between the phases, the amount of PLA-PEG polymer and of OXY, the presence of a viscosifying agent, and the duration of the sonication could significantly influence the responses. Finally, it was possible to obtain OXY-loaded NPs from both processes with NP size of 195 and 226 nm and OXY loading of 4 and 3.3% for double and simple emulsions, respectively. The two processes appeared to be suitable and comparable in term of NP size, peptide drug load and release.

Keywords

Polymer nanoparticles -Double emulsion -Simple emulsion -Factorial experimental design Decreasing the ratio from (1:3) to (1:2) induced a slight decrease of the NP size. The addition of another polymer (PLGA RG502 or PDLG 7502) in the organic phase impacted NP size as increasing the ratio PLA-PEG-NH 2 /additional polymer (b4) from (2:1) to (3:2) and to (1:1) (b4, level 1, 2 and 3) decreased the NP size. Also, the nature of the additional polymer (parameter b6) was important as larger NPs were obtained with the addition of PLGA RG502 (b6, level 2) and not with PDLG 7502 (b6, level 3).

Regarding the impact on the DL (Fig. 4.D.), it was observed that increasing the amount of surfactant from 1 or 1.5% to 2% (w/v) (b2, level 1, 2 and 3, respectively) decreased the DL.

Increasing the total amount of polymer in comparison to the amount of drug from (1:4) (b3, level 1) to (1:3) (b3, level 2) and then to (1:2) (b2, level 2) increased the DL. Increasing the PLA-PEG-NH 2 /additional excipient ratio from (2:1) (b4, level 1) to (3:2) (b4, level 2) led to higher DL. Then, a ratio (1:1) (b4, level 3) also increased the DL compared to (2:1) ratio, whereas it induced smaller DL compared to (3:2) ratio. Finally, the nature of additional polymer (b6) was found important as higher DL were obtained with the addition of PLGA RG502 (b6, level 2) than without (b6, level 1). Slight increase of DL was observed with PDLG 7502 (b6, level 3) compared with PLA-PEG-NH 2 alone (b6, level 1), whereas DL were found largely lower with PDLG 7502 than with PLGA RG502. Thus, parameter levels conducting to the smallest NP size and to the highest DL were deducted from the results observed. 1% (w/v) of Montane 80 surfactant led to the smallest NPs and the highest DL. It was thus supposed that 0.4% of surfactant could allow to obtain NPs which fit even closer to wanted responses. OXY/polymer ratio of (1:4) led to smaller NP sizes but also smaller DL. As no difference was found between (1:3) and (1:2) ratios for the NP size, (1:2) was found to be more relevant as it induced higher DL. PDLG 7502 increased DL without increasing too much the NP size. Finally, even if increasing the amount of additional excipient decreased the NP size, (3:2) PLA-PEG-NH 2 /additional excipient ratio induced the highest DL.

Aqueous phase composition

The emulsion was obtained after addition of the organic phase into the aqueous phase. It was studied in this part the influence of the ratio between the internal aqueous phase (W) and the organic phase (O) (parameter b1) and the amount of Montanox 80 surfactant in the aqueous phase composition (parameter b5), on NP size (Fig. O/W ratio (1:3), (1:5) and (1:10) were tested and 1, 1.5 and 2% (w/v) of Montanox 80.

Conflict of interest

The authors declare no conflict of interest.

Chapitre III -Publication n°3

Influence of PLA-PEG Nanoparticles Manufacturing Process on Intestinal Transporter PepT1 Targeting and Oxytocin Transport

Betty Gourdon 1,2,3 , Caroline Chemin 1 , Amélie Moreau 1 , Thomas Arnauld 1 , Bruno Saubaméa 2,3 , Jean-Marie Delbos 1 , Jean-Manuel Péan 1 , Xavier Declèves 2,3 1 Technologie Servier, 27 rue Eugène Vignat, Orléans, France

Conclusion:

All these results demonstrated that NPs prepared by double emulsion allowed a better PepT1 targeting and is a promising approach for oral peptide delivery.

Keywords

Polymeric nanoparticles -PepT1 transporter -Manufacturing process -Intestinal targeting -

Oxytocin

Graphical abstract

Oxytocin transport

OXY-loaded nanoparticles formulation

OXY-loaded NPs were prepared according to the method described in 2.2 section for drug free PLA-PEG-Val NPs. OXY was added to the formulation as follow: 15 mg in the organic phase for the nanoprecipitation process, 34.3 mg in the internal aqueous phase for the double emulsion solvent evaporation process, and 10 mg in the organic phase for the simple emulsion process. Each formulation was followed by 1 hour of tangential filtration at 700 x g in order to remove non-entrapped drug.

Drug loading and entrapment efficiency

Total amount of drug dissolved in the organic phase was determined by HPLC dosage after addition of 800 µL of DMSO to 200 µL of NP suspension in order to destruct the NPs. Then, the concentration of free OXY in the filtered medium collected from tangential filtration of NPs was measured. The amount of encapsulated OXY was evaluated indirectly by calculating the difference between the total amount of OXY and the amount found in the filtrate. The drug loading was described as the percentage of encapsulated drug compared to the total amount of NPs. The entrapment efficiency was defined as the percentage of encapsulated drug compared to the total amount of drug dissolved in the organic phase. The OXY loading and entrapment efficiency were calculated using the following formulae:

Drug loading (%) = (Amount of encapsulated drug/Total amount of nanoparticles) × 100

Entrapment efficiency (%) = (Amount of encapsulated drug/Amount of drug dissolved in the organic phase) × 100

Caco-2 permeability experiments

Twenty five µM of free OXY, and OXY loaded in PLA-PEG-Val NPs made by the three manufacturing processes were incubated in apical chambers under 60 rpm agitation during 90 min. Samples were collected as described in paragraph 2.3.2.1. and conserved at -20°C until analysis. Drug concentrations were determined by UPLC/MS/MS and OXY apparent permeability was calculated for each group as described above for [ 3 H]GlySar experiments.

In vivo experiments

In vivo study plan was reviewed and approved by Servier ethical committee registered in the French Ministry of Research. It was in general accordance with the animal health and welfare guidelines, and standard operating procedures. C57Bl/6J male mice at approximately 11 weeks old were used for OXY study. Male were preferred because of the presence of a natural OXY rate in female species [START_REF] Kramer | Sex and species differences in plasma oxytocin using an enzyme immunoassay[END_REF].

Study design

Sixty-three mice were marked and split into three groups of 21 mice. The test items were prepared extemporaneously and protected from light. Free OXY was dissolved in 0.1 N solution of NaCl and was administrated intravenously to animals of G1 group at a dose of 0.01 mg/kg and at volume of 5 mL/kg (concentration of 2 µg/mL). Free OXY and OXY encapsulated in valine-NPs obtained by the double emulsion process were dissolved and suspended, respectively, in purified water and were administered by oral gavage to animals of G2 and G3 groups, respectively. Doses of 10 mg/kg of OXY and volume of 10 mL/kg (concentration of 1 mg/mL) were applied for these last two groups. Blood samples were collected at room temperature from jugular vein under isoflurane anesthesia at 0, 5, 10, 15, 30, 45 and 60 min after dosing for G1, and at 0, 15, 30, 45, 60, 120 and 180 min after dosing for G2 and G3. Blood was sampled on K 2 EDTA tubes, and then 0.5 mL was immediately transferred in polypropylene tubes containing 10 µL of 25,000 KIU/mL of aprotinin solution.

Blood samples were centrifuged (10 min, 3,500 rpm, + 4°C) and plasma samples were separated and transferred in tubes and stored at -80°C until analysis.

Pharmacokinetic analysis

For each animal, plasma drug concentrations versus time were submitted to a noncompartmental PK analysis using WinNonlin ® software (version 5.1, Pharsight Corporation, Cary, NC, USA). The area under plasma concentration-time curve (AUC t ) from time zero to the last time point after dosing was calculated using the linear-up and log-down trapezoidal rule. Values for the maximum plasma concentration (C max ) and for time of the C max (T max )

were obtained directly from the observed individual plasma concentration-time courses.

OXY samples quantification

OXY encapsulation in NPs was assayed using HPLC. The system comprised a Water 2695

Separation module associated with a Waters UV 2487 dual λ Absorbance UV. For OXY

Results

Nanoparticle formulation

Deprotection efficiency of PLA-PEG-Val-Boc was confirmed by 1 H NMR spectroscopy. The attention was focused on the -NH before the protection group which turned into -NH 2 after deprotection. NPs were prepared using nanoprecipitation, simple or double emulsion manufacturing processes. PLA-PEG-Val and PLA-PEG NP size for each method was summed up in Table 1. It was observed a slight increase of the NP size with the presence of a valine ligand. But the increase was only significant for the nanoprecipitation process. PLA-PEG-Val NP size increased from nanoprecipitation process to double and simple emulsions and this manufacturing process influence was significant compared to each other's.

TEM imaging was realized for PLA-PEG-Val NPs obtained with the three manufacturing processes (Fig. 3). Spherical particle shapes were observed for all of them. NPs obtained by nanoprecipitation (Fig. 3,A.) appeared as plain NPs, corresponding to nanospheres dense structure. NPs made by simple emulsion processes (Fig. 3,B.) were composed of an empty core and a darker shell. Double emulsion led to similar core/shell NPs (Fig. 3,C.) with an even darker shell representing PEG and ligand at the NP surface.

PLA-PEG NPs was different as they did not penetrate the cell core and seemed to not even stay close to the membrane (Fig. 6,D).

Drug transport

In order to carry out transport experiments in vitro and in vivo, NPs containing OXY were prepared either by nanoprecipiation, or by simple emulsion or by double emulsion. Depending on the manufacturing process used, the drug load, entrapment efficiency and particle size appeared to be different (Table 3). A slight difference of the particle size was observed.

Indeed when OXY-NPs were prepared by nanoprecipitation 1.3 and 1.5-fold smaller particles were obtained in comparison with double and simple emulsion, respectively. Regarding drug loading, a 1.5-and 1.8-fold higher drug load was obtained with nanoprecipitation in comparison with double and simple emulsion, respectively. Then, the best entrapment efficiency was observed when OXY was encapsulated with a double emulsion process (1.1and 1.3-fold higher than nanoprecipitation or simple emulsion processes respectively). In vitro experiments were performed in order to evaluate the influence of the manufacturing process on the drug transport. OXY transport was assessed by comparing its Papp as free compound or encapsulated in NPs obtained by nanoprecipitation, double or simple emulsion processes (Fig. 7).

Conclusion

In conclusion, the present work demonstrated the influence of the functionalized NPs manufacturing process on the PepT1 intestinal transporter targeting. It was found that double emulsion process allowed to obtain valine-NPs able to target the transporter but also to penetrate the cell and reach the nuclei. This strategy leads to an improvement of encapsulated drug permeability as it increased drug absorption in vitro and in vivo, but also offered a protection against enzymatic reaction. Therefore, IV administration still resulted in better absorption results but the developed strategy revealed potential to increase oral delivery of peptide.

Conflict of interest

The authors declare no conflict of interest.

Résumé des Résultats Principaux et Perspectives

L'objectif de cette dernière partie était d'étudier l'influence de la technique de fabrication de NPs de PLA-PEG sur leur capacité à cibler le transporteur PepT1 et sur l'amélioration des performances biopharmaceutiques de l'ocytocine.

Ce chapitre a permis de mettre en lumière les résultats suivants : (Majumdar and Mitra, 2006;Sun, 2010). Le valacyclovir, prodrogue de l'acyclovir en est un exemple probant (Beauchamp et al., 1992;Yang, 2012). Mais toutes les molécules ne peuvent pas être formulées sous forme de pro-drogues au risque de voir leur effet thérapeutique réduit. Ainsi la stratégie développée offre une alternative pour ces molécules.

Ce travail de thèse offre ainsi une nouvelle approche pour l'administration de peptides par la voie orale. Cette discussion met en lumière l'apport de ce travail dans ce domaine de recherche ainsi que les problématiques rencontrées.

Conclusion

Ce travail de thèse a permis de développer une nouvelle approche de vectorisation avec des nanoparticules polymères fonctionnalisées ciblant le transporteur PepT1 pour améliorer le passage intestinal de molécules hydrophiles de type peptide ayant une faible biodisponibilité par voie orale.

Désormais, il a été démontré qu'il était possible de cibler le transporteur intestinal PepT1 avec des nanoparticules de PLA-PEG fonctionnalisées avec de la valine et qu'en fonction de la technique de fabrication de ces nanoparticules elles étaient capables de se fixer à la membrane cellulaire ou bien même de pénétrer à l'intérieur de la cellule.

Ainsi, l'encapsulation dans ces vecteurs de molécules hydrophiles telles que l'acyclovir ou l'ocytocine, à des taux similaires à ceux trouvés dans la littérature, permet l'utilisation de cette nouvelle approche pour améliorer leur perméabilité intestinale.

Les résultats obtenus sont encourageants, tant sur le point de la formulation de nanoparticules, que sur les résultats obtenus in vitro et in vivo sur l'amélioration de leur perméabilité.

Néanmoins, le point faible de cette stratégie reste le contrôle de la libération du principe actif des nanoparticules. Un travail de formulation important reste à accomplir afin de maîtriser parfaitement cet aspect et ainsi obtenir des résultats aussi significatifs que cette approche est prometteuse.

ANNEXES

Annexes Abstract

If the oral route remains the favored administration pathway, it could be a real challenge when the molecule is hydrophilic and poorly absorbed by the intestinal barrier. The objective of the present work is thus to design a new oral administration system interacting with the gastrointestinal cells in order to improve the bioavailability of a therapeutic molecule. In order to answer this problem, it was chosen to follow a still unexplored route, consisting of targeting an intestinal transporter with polymer nanoparticles encapsulating the molecule of interest. If internalization of nanoparticles is not expected via a transporter, it is rather sought an overconcentration in nano-carriers and therefore in molecule near the membrane.

The first part of this work consists in the proof of concept and confirms that it is possible to target the intestinal transporter PepT1 with PLA-PEG nanoparticles functionalized with a valine ligand and obtained by nanoprecipitation. This strategy, applied to acyclovir a BCS III molecule encapsulated in nanoparticles, allows to triple its permeability in vitro and to multiply its retention time by 1.3 in vivo. However, a burst release of the molecule suggests that better results can be achieved. Then, the second part of this study is devoted to the optimization of the formulation of PLA-PEG nanoparticles for the encapsulation of a hydrophilic molecule of interest, oxytocin. The techniques of double and simple emulsions solvent evaporation were studied with factorial experimental designs. It was thus demonstrated that formulation and process parameters significantly influence the particle size and the drug load. Finally, the two manufacturing processes lead to similar results in terms of particle size of approximately 200 nm and drug load close to 4%. Finally, the third part of this work investigated the influence of the nanoparticle manufacturing process on PepT1 targeting and on the improvement of oxytocin transport in vitro and in vivo. The double emulsion appeared to be the most efficient for PepT1 transporter targeting and allows to double the encapsulated molecule permeability in vitro. Very promising results were obtained in vivo but with important variability inter-animals. Confocal imaging pointed out the internalization of the nanoparticles valine-functionalized obtained by double emulsion. However, a very fast release of a fraction of oxytocin was observed followed by a very slow release, a profile which is not adapted to the designed targeting strategy. The approach developed is thus promising regarding the oral administration of peptide and an optimization of the release profile of the molecule could lead to even more convincing results.