Gilles Ponchel

Examinateurs Aurélie Malzert-Fréon

Anne Sapin-Minet

Franck Hansmannel

David Moulin

Hui Ming

Wen Wu

Philippe Maincent

Marianne Parent

Caroline Gaucher

Volume 9: Organic particles Polymer nanocomposites for drug oral delivery: development strategies and potentialities

Keywords: GSNO, Ussing Chamber, intestinal barrier integrity

Results:

The result showed that GSNO can modulate intestinal barrier integrity in a dose-dependent manner.

Conclusion:

In conclusion, low concentrations of GSNO may be a novel treatment to prevent barrier disruption and IBD relapses.

start my PhD study here. Pr. Hu provided the research platform for me during the two years of master study in the lab, which helped me improve the experimental skills and inspired my research enthusiasm. Pr. Maincent gave me some really useful suggestions of the formulation part. The conversation with him was always relaxed and lively. His smile comforted me a lot when I did the presentation during the formulation meeting.

He looks always optimistic. His friendly encouragement made me stronger when I lost my confidence.

I feel grateful to Pr. Leroy (the director of EA3452 CITHEFOR) for his critical opinions.

His strong sense of responsibility, serious and detailed attitude of work influenced me a lot.

My deepest gratitude goes to my supervisors, Anne and Franck, for their instructive and valuable advices on my thesis. Anne helped me a lot not only in the guidance of thesis but also in the manipulation of experiment part. She is always nice and patient in analyzing and solving experimental problems with me. Besides the life in the lab, Anne also helped me to make my casual life easier in France. I learned a lot from Franck's profound knowledge in physiology part and analysis of data. He was very patient in explaining basic knowledge to me, such as western blot, statistic study, intestine physiology and pathology. Without their patient instruction, insightful criticism and expert guidance, the completion of this thesis would not have been possible.

I would like to express my gratitude to Caroline G. and Isabelle F. for their useful suggestion and helpful manipulation in Caco-2 experiment part. Caroline's smart mind and efficient work capacity impressed me a lot.

Great thanks shall be transported to Philippe G. for his work on GSNO preparation, apparatus setting and validation. His easygoing character and warm introduction helped me get used to this lab more easily and quickly.

My grateful words should go to Marianne as well for her sharing of interesting and useful literatures. She is very efficient in literature searching, analyzing and summarizing, which inspired me a lot.

I also own a truthful gratitude to Romain whose thesis is crossed with mine under the same supervisor of Anne. We shared a lot of happy moments together. He helped me a lot also in computer technical part. He is very good at communication which I can learn a lot from him.

Heartful gratitude is sent to Pascale, Nathalie and Ghizlane for their friendly smile every time and help in administrative things. Even though the language barrier exists, we still make us understandable to each other. Special gratitude should go to my dear friends Haiyan and Yi, who accompanied me, comforted me and encouraged me a lot. I also own my sincere gratitude to all my colleges for their help at any time for any cases.

At last, my thanks would go to my beloved family for their loving consideration and The differences between the innate and adaptive immunity. 5 Table 2 GSNO related delivery system. 54 Table 3 Optimization of alginate nanocomposite. 117 Table 4 Experiment of interaction between TPP and Eudragit ® E 100.

150

Table 5 Characterization of GSNO-aeNCP. 152 Article Table 1 Simplified summary of drugs, nanoplateforms, polymer matrix and their efficiencies.

68

Table 2 Simple summary of proceeding methods to prepare polymer nanocomposites.

72

Table 3 Elimination of burst effect by polymer nanocomposites. 76 Article Table 1 Evolution of the apparent permeability coefficient (Papp) of sodium fluorescein (NaFlu, 1 mg/mL).

99

Article

Table 1 Characterization of IV In vivo metabolism of GSNO by enzymes: 1. GSNO reductase (GSNOR) and carbonyl reductase 1 (CR1); 2. thioredoxin system (Trx); 3. protein disulfide isomerase (PDI); 4, γglutamyltranspeptidase (GGT).

LIST OF FIGURES

46

Figure A model for cellular GSNO uptake 1: GSNO was metabolised by GGT forming S-nitrosocysteinylglycine, which further deliver NO spontaneously and then diffused into cell, or V transfer NO to L-cysteine. 2: PDI catalyzes transnitrosation and denitrosation of GSNO to release NO. 3: GSNO transfers NO to L-cysteine forming CysNO, which is transported into the cell through the L-amino acid transporter system (L-AT). Figure 22 Function of GSH as an antioxidant. Figure 23 Strategies of polymer matrix. Figure 24 Drug (GSNO and degradation product nitrite ions) release from GSNO-NCP (free GSNO, ionotropic gelation, polyelectrolyte complexation) in acidic buffer (pH 1.2, 37 °C).

Figure 25

Advantages and drawbacks of two different alginate-NCP. Figure 26 Possible mechanism of GSNO release in different alginate-NCP in in vitro environment.

Figure 27 Perspectives of in cellulo study to explore the mechanism of NO localization in intestine.

Figure 28 Mechanism of Interaction between TPP and chitosan. Figure 29 Possible mechanism of Interaction between TPP and Eudragit ® E 100.

Figure 30 SEM images of GSNO-aeNCPs: a) surface morphology and b) zoomed in nanoscale.

Figure 31

In vitro cytotoxicity of GSNO-aeNCP on Caco-2 cells. Caco-2 cells were treated with indicated polymer concentrations.

Figure 32 A crucial goal for the patients: Inflammation prevention. Figure 33 Perspectives of NO action with intestine. Figure 34 In vivo evaluation of GSNO loaded formulations in inflammation model.

Article 1

Figure 1 Representative scheme of polymer nanocomposites: drug, nanoplatforms and polymer matrix. They may present as A) core-shell structures, B) particles and C) bulk/film.

Article 2

Figure 1 Criteria of NaFlu permeability and TEER result during 2 h in Ussing chamber.

Figure 2 Correlation between NaFlu permeability and TEER result during 2 h in Ussing chamber.

Article 3

Figure 1 Scanning Electronic Microscopy images of GSNO-NCPs: A, GSNO-NCP prepared by ionotropic gelation method; B, GSNO-NCP prepared by polyelectrolyte complexation.

Figure 2 Drug (GSNO and degradation product nitrite ions) release from GSNO-NCPs.

Figure 3 GSNO-NCPs (ionotropic gelation, polyelectrolyte VI complexation) swell in PBS (0.148 M, pH 7.4, 37 °C). Figure 4 In vitro cytotoxicity of GSNO-NCPs (A: ionotropic gelation; B: polyelectrolyte complexation) on Caco-2 cells. Caco-2 cells were treated with indicated concentrations of GSNO-NCP for 24 h at 37 °C.

131

Figure 5 GSNO permeability evaluation of free GSNO and GSNO-NCP (ionotropic gelation, polyelectrolyte complexation) in Caco-2 cells monolayers.

VII

LIST OF ABBRIEVIATIONS

Résumé francophone du manuscrit

Les maladies inflammatoires chroniques de l'intestin (MICI) sont un groupe d'affections idiopathiques incluant la rectocolite hémorragique (RCH) et la maladie de Crohn (MC) qui affectent 200.000 personnes en France et 2,2 millions de personnes en Europe. La prévalence des MICI a augmenté principalement dans les pays occidentaux dans les 50 dernières années jusqu'à 200 / 100.000 pour les RCH et 200 / 100.000 pour la MC [START_REF] Cosnes | Epidemiology and natural history of inflammatory bowel diseases[END_REF]. Elles apparaissent chez des sujets jeunes (15-35 ans) et sont incurables actuellement.

Elles représentent un véritable problème de santé publique compte tenu de leurs fréquences, de leurs pronostics à court et à long termes, du coût de leurs prises en charge ainsi que de leurs répercussions sur la qualité de vie des malades. Elles se caractérisent par des épisodes inflammatoires (poussées) répétés et invalidants pour le patient au cours desquels il ressent toutes les manifestations cliniques de la maladie. Actuellement la prise en charge des patients MICI se limite à la prise en charge de ces poussées inflammatoires. A plus ou moins long terme, les MICI conduisent fréquemment à des chirurgies visant à éliminer la zone enflammée [START_REF] Cosnes | Epidemiology and natural history of inflammatory bowel diseases[END_REF]. De plus, l'inflammation chronique de la muqueuse du colon (colite) du patient MICI peut conduire à la mise en place d'un cancer colorectal (CCR) [START_REF] Jess | Risk of colorectal cancer in patients with ulcerative colitis: a meta-analysis of population-based cohort studies[END_REF].

Leurs étiologies sont inconnues, mais la pathogénie des MICI reposerait sur une activation inappropriée du système immunitaire intestinal, dirigé contre la flore intestinale de l'hôte chez des patients prédisposés (génétiquement ou épigénétiquement), dans un contexte de dysbiose. Cette dérégulation dans les interactions hôte-microbiote conduit à l'inflammation intestinale.

Des approches chirurgicales lourdes permettent de réaliser l'ablation de la zone lésée chez les malades lorsque le traitement médical n'est plus efficace, ou en cas de complications graves liées à la sévérité de la lésion. Ces interventions ne peuvent être envisagées qu'en seconde instance car elles sont d'une part très traumatisantes et d'autre part elles n'ont pas d'effet curatif réel puisque les récidives sont nombreuses après la chirurgie. Le développement de nouveaux axes de recherche et de nouveaux traitements chroniques permettant de prévenir les récidives inflammatoires améliorerait la qualité de vie des patients atteints de ces maladies et de diminuer le risque de ii développement de cancer. Les traitements conventionnels des MICI visent à limiter l'inflammation de la muqueuse des patients et incluent les aminosalicylates, les corticostéroïdes, les antibiotiques et les agents immunosuppresseurs, qui sont nécessairement administrés pendant de longues périodes et par conséquent, entraînent des effets secondaires [START_REF] Meissner | Alternative drug delivery approaches for the therapy of inflammatory bowel disease[END_REF]. De plus, les dernières innovations thérapeutiques proposées comme l'utilisation d'agent anti-Tumor Necrosis

Factor (anti-TNF) ont de puissants effets anti-inflammatoires mais une perte de réponse est observée lors d'un traitement au long cours et seulement un tiers des patients présente une rémission clinique totale après un an. L'identification de nouvelles cibles, de nouveaux agents, produits naturellement par l'organisme sain, pourrait ouvrir de nouvelles perspectives thérapeutiques pour ces pathologies.

Lors d'une inflammation, l'oxyde nitrique (NO) joue un rôle pivot et paradoxal, discuté de façon récurrente dans de nombreuses études. Ainsi, le NO intervient à la fois dans des processus physiologiques se déroulant dans des tissus sains et dans des processus physiopathologiques. C'est particulièrement le cas au niveau du tractus gastro-intestinal, où le NO intervient à la fois comme médiateur important dans la préservation et la réparation de la muqueuse (Figure 1) mais contribue également directement aux dommages constatés dans les pathologies digestives, par une action pro-inflammatoire.

Compte tenu de cette dualité, le développement de principes actifs pouvant moduler la synthèse ou l'action du NO constitue évidemment un véritable challenge et le rôle pivot du NO justifie les recherches de nouvelles stratégies thérapeutiques autour de ce composé. Le rôle bénéfique du NO dans ce contexte pathologique implique une action préventive en délivrant de faibles concentrations en NO (nM à µM) dans un environnement peu engagé dans l'inflammation (la balance redox doit être peu perturbée) afin qu'il exerce son action anti-inflammatoires.

L'apport de NO à l'état gazeux est particulièrement délicat pour remplir ces conditions, en raison d'une demi-vie très courte (de l'ordre de la seconde) et de sa très grande réactivité qui l'amène à interagir très rapidement avec les éléments de son environnement, de façon non contrôlable. Toutefois, différentes molécules libérant le NO ou des espèces apparentées, appelées « donneurs de NO », sont décrites voire même utilisées en thérapeutique (dans un contexte cardiovasculaire notamment) (Parent et al., 2013c). Certains donneurs de NO ont ainsi été étudiés dès les années 90, dans le contexte des MICI pour promouvoir les actions bénéfiques du NO au niveau intestinal iii (tableau 1). Préclinique [START_REF] Wallace | Enhanced anti-inflammatory effects of a nitric oxide-releasing derivative of mesalamine in rats[END_REF] Diminue les dommages de la muqueuse intestinale :

• Diminution infiltration des leucocytes • Augmentation de la sécrétion de mucus • Maintien du flux sanguin local Glyceryltrinitrate Clinique [START_REF] Lund | A randomised, prospective, double-blind, placebo-controlled trial of glyceryl trinitrate ointment in treatment of anal fissure[END_REF] Améliore la cicatrisation de fissures anales associées à la maladie de Crohn NO-NSAIDS Préclinique [START_REF] Muscará | Effect of a nitric oxide-releasing naproxen derivative on hypertension and gastric damage induced by chronic nitric oxide inhibition in the rat[END_REF][START_REF] Wallace | Enhanced anti-inflammatory effects of a nitric oxide-releasing derivative of mesalamine in rats[END_REF] Potentialise action du NSAIDS SNAP Préclinique (Lefer and Lefer, 1999) • Evite augmentation de la P-Sélectine sur la membrane des neutrophiles • Inhibe le dysfonctionnement de l'endothélium NO gaz et NaNO2 acidifié Préclinique [START_REF] Aoki | Beneficial effects of two forms of NO administration in feline splanchnic artery occlusion shock[END_REF] Diminue l'adhérence des neutrophiles au niveau de l'endothélium mésentérique iv Figure 1 Rôles protecteurs de l'oxyde nitrique dans les conditions physiologiques : le maintien de l'intégrité de la barrière intestinale (Wallace andMiller, 2000, Heanue and[START_REF] Heanue | Enteric nervous system development and Hirschsprung's disease: advances in genetic and stem cell studies[END_REF] v En effet, quand le NO est combiné avec un donneur (R) au niveau d'un groupement thiol pour former un RSNO, la demi-vie du NO se trouve augmentée (plusieurs heures) et le NO est alors délivré aux cellules et aux tissus de façon plus contrôlée que sous la forme d'anion peroxynitrite. Ceci est d'autant plus intéressant dans un contexte inflammatoire lorsque le donneur (R) est une molécule antioxydante naturellement produite par l'organisme, par exemple le glutathion formant ainsi le S-nitrosoglutathion (GSNO). Il a d'ailleurs été démontré que le GSNO sécrété par les cellules gliales entériques à la suite d'une stimulation du nerf vague permet de prévenir un épisode inflammatoire au niveau intestinal et de préserver l'intégrité de la barrière (Savidge et al., 2007, Cheadle et al., 2013). Ce projet de thèse répond à une réelle attente des patients pour améliorer leur confort mais répond aussi à un réel besoin économique, proposant une solution pour diminuer le taux de récidives des MICI après un épisode aigu et pour réduire la période et le coût des traitements chroniques actuels. Le traitement préventif des MICI tel qu'il a été envisagé ci-dessus requiert donc la vérification de plusieurs points autour desquels s'articule cette thèse.

La
La première étape de cette thèse consiste à vérifier que le GSNO apporté dans la lumière intestinale (donc administré par voie orale) maintient l'intégrité de la barrière intestinale au même titre que le GSNO produit de manière endogène par les cellules gliales vi entériques présentes dans la muqueuse intestinale (Savidge et al., 2007, Cheadle et al., 2013). Pour ce faire, une méthode expérimentale ex vivo a été utilisée pour suivre la capacité du GSNO à préserver l'intégrité de la barrière intestinale. Nous avons utilisé la chambre de Ussing qui est un dispositif permettant l'étude de la diffusion, du transport de molécules à travers une membrane. Dans ce dispositif, du tissu intestinal peut-être positionné de façon à séparer deux compartiments : un compartiment donneur (mimant la lumière intestinale) et un compartiment accepteur (mimant le tissu conjonctif sous-jacent voire le compartiment sanguin). Cette étude a été réalisée sur des tissus intestinaux prélevés chez des rats Wistar sains. Pour ce faire, différentes concentrations en GSNO ont été déposées (de 0,1 µM à 100 µM) dans le compartiment donneur de la chambre de Ussing et nous avons évalué plusieurs critères :

-la perméabilité d'une molécule fluorescente « de référence » (la fluorescéine sodique) au cours du temps, -la mesure de la résistance transépithéliale (TEER), -le niveau d'expression des protéines de jonctions serrées et de cytokines proinflammatoires (par Western Blot). directe du GSNO par différentes techniques de formulation (Parent et al., 2013c, Wu et al., 2015a, Diab et al., 2016) par exemple démontré une encapsulation efficace du GSNO (54% d'encapsulation de GSNO, soit 40 mmol de GSNO/L de suspension) dans des nanoparticules d'Eudragit ® RL (un polymère polycationique de type méthacrylique utilisé couramment dans de très nombreuses formes orales sous forme de film de pelliculage et actuellement sur le marché), libérant un GSNO actif de façon plus prolongée dans le temps et limitant sa dégradation en ions nitrite et nitrate (Wu et al., 2015a). Afin d'augmenter la stabilité et de prolonger la libération du GSNO au cours du temps, l'incorporation des nanoparticules dans une matrice polymérique pour former les systèmes particulaires composites (Wu et al., 2015b) a été proposée (Figure 2). ix Ce type de système est recommandé dans la littérature pour réduire la libération initiale massive de principe actif [START_REF] Hassan | Composite microparticles with in vivo reduction of the burst release effect[END_REF]. Des systèmes particulaires pouvant mesurer jusqu'à plusieurs dizaines de micromètre ont été préparés par un procédé de gélification ionique, menant à des particules de chitosane ou d'alginate de calcium [START_REF] Nasti | Chitosan/TPP and chitosan/TPP-hyaluronic acid nanoparticles: systematic optimisation of the preparative process and preliminary biological evaluation[END_REF][START_REF] Koukaras | Insight on the formation of chitosan nanoparticles through ionotropic gelation with tripolyphosphate[END_REF] Un autre type de formulation a été proposé en parallèle de cette étude. Il s'agissait de proposer une nouvelle association de polymères, non décrite dans la littérature et répondant au même cahier des charges, pour constituer la matrice des particules composites par un procédé de gélification ionotropique (Wu et al., 2016). Le chitosan a été remplacé par un autre polymère polycationique (l'Eudragit ® E) pour constituer la matrice en association avec l'alginate. Contrairement au chitosan qui est connu pour ouvrir les jonctions serrées, donc inadapté à notre problématique, [START_REF] Thanou | Oral drug absorption enhancement by chitosan and its derivatives[END_REF], Wu et al., 2016), l'Eudragit ® E pourrait limiter la libération du GSNO au niveau intestinal. L'encapsulation de GSNO dans ces systèmes particulaires est efficace Pour présenter ces travaux de thèse, le manuscrit s'articule de la manière suivante : A ce stade du travail, nous avons proposé, caractérisé et évalué plusieurs méthodologies de formulation dont les avantages et les limites sont discutées dans une dernière partie du manuscrit. L'étude de ces formulations peut maintenant être poursuivie in vivo par l'évaluation de leur capacité à prévenir les récidives inflammatoires au niveau intestinal.

Nos études, conduites pendant

Inflammation bowel disease

Inflammatory bowel diseases (IBD) are chronic and relapsing disorders affecting gastrointestinal (GI) tract and associated with intestinal mucosa lesions and inflammation. The most known subtypes are Crohn's disease (CD) and ulcerative colitis (UC). While both subtypes can lead to symptoms such as abdominal pain, cramping, diarrhea or weight loss, there are differences between them. It is important to note that CD can occur all over the digestion tract, from the mouth to the anus, but most often affects the large and small intestines, whereas UC primarily affects only the colon and rectum. As chronic disease, IBD are disabling, lead to significant health care costs and cause patients to suffer a lot in life quality and work capacity [START_REF] Bannaga | Inflammatory bowel disease and anxiety: links, risks, and challenges faced[END_REF].

IBD epidemiology

IBD has become a worldwide disease. Incidence and prevalence of IBD are higher in developed countries than in developing countries, and in urban areas than in rural areas [START_REF] Molodecky | Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review[END_REF]. Currently, the highest annual incidence of CD is in Canada (20.2 per 100 000), New Zealand (16.5 per 100 000), northern Europe (10.6 per 100 000), and Australia (29.3 per 100 000) [START_REF] Torres | Crohn's disease[END_REF]. Meanwhile the highest incidences of UC have been reported in northern Europe (24.3 per 100 000), Canada (19.2 per 100 000), and Australia (17.4 per 100 000) (Ungaro et al., 2016b). The prevalence of both UC and CD are highest in Europe (505 and 322, per 100,000 respectively), Canada (319 and 248 per 100 000 respectively), and the USA (214 per 100 000 for both) [START_REF] Molodecky | Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review[END_REF][START_REF] Ponder | A clinical review of recent findings in the epidemiology of inflammatory bowel disease[END_REF][START_REF] Torres | Crohn's disease[END_REF], Ungaro et al., 2016b). A significant increasing trend of IBD incidence was observed with developing countries, such as Asian, Middle East, South America and Africa while a plateaued of incidence was achieved for the western areas (Ng et al., 2013, Ng et al., 2016[START_REF] Torres | Crohn's disease[END_REF], Ungaro et al., 2016b). IBD has become more common in Asia over the past few decades. The rate of increase in prevalence of the disease varies greatly in Asia, with several countries in East Asia experiencing a more than doubled increase in IBD prevalence over the past decade (Ng et al., 2016).

IBD physiopathology

Till now, the exact physiopathology remains unknown. Genetic susceptibility, environment factors, intestinal microbial flora dysbiosis and immune dysfunction are all correlated with the pathogenesis.

Genetic susceptibility

About 12% of patients have a family history of CD while the ratio for UC is 8-14% of patients [START_REF] Moller | Familial risk of inflammatory bowel disease: a population-based cohort study 1977-2011[END_REF][START_REF] Torres | Crohn's disease[END_REF], Ungaro et al., 2016b). Ashkenazi Jews have higher rates of IBD than in non-Jewish populations, and African-American and Asian ancestries are associated with the lowest risk [START_REF] Ananthakrishnan | Epidemiology and risk factors for IBD[END_REF], Huang et al., 2015). Genome-wide association studies have identified more than 200 alleles associated with IBD, of which most genes contributing to both ulcerative colitis and Crohn's disease phenotypes [START_REF] Jostins | Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease[END_REF], Liu et al., 2015b). The discovery of genes associated with bacterial sensing, innate immunity, Th17-cell function (NOD2, ATG16L1, LRRK2, IRGM, Il23R, HLA, STAT3, JAK2, and Th17 pathways) and an altered mucus layer (MUC2), human leukocyte antigen, barrier function, such as HNF4A and CDH1 brought major insights into disease pathogenesis [START_REF] Barrett | Genome-wide association study of ulcerative colitis identifies three new susceptibility loci, including the HNF4A region[END_REF][START_REF] Jostins | Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease[END_REF][START_REF] Mcgovern | Genetics of inflammatory bowel diseases[END_REF]. However, only 13.1% of CD and 7.5% of UC heritability is explained by genetic variation [START_REF] Torres | Crohn's disease[END_REF], Ungaro et al., 2016b). The importance of epigenetic and other nongenetic environmental factors are highlighted [START_REF] Huang | Characterization of genetic loci that affect susceptibility to inflammatory bowel diseases in African Americans[END_REF]. Therefore, genetics alone has little predictive capacity for phenotype, and currently are of limited clinical use.

Environment factors

The increasing incidence of IBD worldwide suggests the importance of environmental factors in its development. Smoking is one of the well-studied factor and strongest environmental risk for CD and UC. The protective effect of current smoking on the development of UC was confirmed. The active smokers are less likely to develop UC compared with former and non-smokers and have a milder disease course [START_REF] Birrenbach | Inflammatory bowel disease and smoking. A review of epidemiology, pathophysiology, and therapeutic implications[END_REF]Böcker, 2004, Cosnes, 2004). Contrary to its effect on UC, smoking is associated an increasing risk of developing CD by two-fold (Mahid et al., 2006). However, the mechanism how smoking impacts IBD remains unclear as does the reason for its protective effect in UC but deleterious impact on CD [START_REF] Cosnes | Epidemiology and natural history of inflammatory bowel diseases[END_REF]. The medications such as aspirin, non-steroidal anti-inflammatory drugs, oral contraceptives and antibiotics are potentially associated with increased risk [START_REF] Alberts | Environmental Triggers for Inflammatory Bowel Disease[END_REF], whereas statins have been related with a decreased risk, especially in older people (Ungaro et al., 2016a). Stress is another environment factor that plays a role in the pathogenesis of CD [START_REF] Mawdsley | The role of psychological stress in inflammatory bowel disease[END_REF]. Mood components of perceived stress including depression and anxiety exacerbated the disease related to stress [START_REF] Cámara | Mood and nonmood components of perceived stress and exacerbation of Crohn's disease[END_REF] while individuals with lower levels of stress and better coping mechanisms had a reduced risk of disease flare [START_REF] Bitton | Predicting relapse in Crohn's disease: a biopsychosocial model[END_REF]. Micronutrients (zinc and iron) and vitamin D has also been proposed for a role of environmental role [START_REF] Ananthakrishnan | Epidemiology and risk factors for IBD[END_REF]. Diet has been implicated with an important factor of IBD pathology. An increasing risk of UC and CD was observed with reduction in dietary fiber and increase in saturated fat intake [START_REF] Ananthakrishnan | Long-term intake of dietary fat and risk of ulcerative colitis and Crohn's disease[END_REF]. Many environmental factors need to be proven in the further study.

Intestinal microbial flora dysbiosis

The involvement of the intestinal microbial flora alteration in the pathophysiology of IBD has recently been highlighted [START_REF] Matsuoka | The gut microbiota and inflammatory bowel disease[END_REF]. The altered balance of the gut microbiota is referred to dysbiosis. Dysbiosis of IBD patient includes reduced diversity of the gut microbiota [START_REF] Tong | A modular organization of the human intestinal mucosal microbiota and its association with inflammatory bowel disease[END_REF], a decrease in Bacteroides and Firmicutes bacteria and an increase in Gammaproteobacteria, Actinobacteria, Enterobacteriaceae and Proteobacteria [START_REF] Matsuoka | The gut microbiota and inflammatory bowel disease[END_REF][START_REF] Torres | Crohn's disease[END_REF], Ungaro et al., 2016b). The reduced diversity of the gut microbiota observed in IBD patients is largely owing to a reduction in the diversity of Firmicutes. An increase abundance of Escherichia coli which is mucosa associated adherent-invasive bacteria

were observed in about one third CD patient [START_REF] Darfeuille-Michaud | High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn's disease[END_REF][START_REF] Lapaquette | Crohn's disease-associated adherent-invasive E. coli are selectively favoured by impaired autophagy to replicate intracellularly[END_REF][START_REF] Torres | Crohn's disease[END_REF]. These strains trigger high amount of tumor necrosis factor-α (TNF-α) secretion. The mechanism is that Escherichia coli goes through the intestinal mucosal barrier, adhere to and invade intestinal epithelial cells, then survive and replicate within macrophages [START_REF] Torres | Crohn's disease[END_REF]. Although the alternation of intestinal microbial flora is a highly active research area, it has not yet applied to practices, because of the difficulties in microbiota manipulation [START_REF] Torres | Crohn's disease[END_REF].

Immune dysfunction

Immunity is one of physiological functions in humans, which can be divided into innate and adaptive immunity [START_REF] Huang | Inflammatory bowel disease related innate immunity and adaptive immunity[END_REF]. Two major processes of the innate immunity are usage of a large set of different pattern recognition receptors (PRRs) and a system for random and nonselective generation of antigen specific receptors. The adaptive immunity has been considered as a complement to the innate immunity and a definitive solution to pathogen recognition [START_REF] Huang | Inflammatory bowel disease related innate immunity and adaptive immunity[END_REF]). The differences between innate immunity and adaptive immunity are presented in Table 1.

Table 1 The differences between the innate and adaptive immunity [START_REF] Huang | Inflammatory bowel disease related innate immunity and adaptive immunity[END_REF])

Item

Innate immunity Adaptive immunity

Acquired form

Innate immunity defects in IBD

The innate intestinal immune system is constituted of intestinal mucosal barrier (see paragraph 1.2.1), innate immune cells, innate immune molecules… Innate immunity defects in IBD are associated with intestinal barrier by apoptosis of intestinal epithelial cells and increasing intestinal permeability.

Immune cells in inflammatory intestinal mucosa of IBD patients induce expression of epithelial cell apoptosis related proteins, such as Caspase-1 by producing inflammation factors, such as tumor necrosis factor-α (TNF-α) and interferons-γ (IFN-γ), which, and inhibit expression of anti-apoptotic proteins, such as Bcl-2, which lead to induced epithelial cell apoptosis, weakened function of epithelial cells resisting pathogens and increased permeability of intestinal mucosa [START_REF] Bouma | The immunological and genetic basis of inflammatory bowel disease[END_REF]. During an active stage of IBD, junction protein and its corresponding mRNA in intestinal tissue decrease significantly, resulting in the disruption of intestinal barrier integrity [START_REF] Gassler | Inflammatory bowel disease is associated with changes of enterocytic junctions[END_REF]. Innate immune cells include Macrophages, Dendritic cells (DCs) and Natural killer (NK) cells. Macrophages can decompose engulfed pathogens into specific antigenic determinants, such as peptides and lipopolysaccharide, using proteases and oxygen free radicals [START_REF] Huang | Inflammatory bowel disease related innate immunity and adaptive immunity[END_REF]. During the acute phase of IBD, quantity of macrophages in intestinal mucosa dramatically increases, leading to secretion of macrophage pro-inflammatory factors [START_REF] Baj-Krzyworzeka | Tumour-derived microvesicles carry several surface determinants and mRNA of tumour cells and transfer some of these determinants to monocytes[END_REF].

Mature DCs are antigens presenting cells. In colonic mucosa of IBD patients, interactions between DCs and T cells increase the secretion of inflammatory cytokines and cause inflammation [START_REF] Drakes | Colon lamina propria dendritic cells induce a proinflammatory cytokine response in lamina propria T cells in the SCID mouse model of colitis[END_REF]. NK cells can control viral and bacterial infections, produce cytokines, kill tumor cells, and build a relationship between innate immunity and acquired immunity [START_REF] Colucci | What does it take to make a natural killer?[END_REF]. NK cells that express interleukin 22 (IL-22) have a protective effect on initiation of IBD [START_REF] Zenewicz | Innate and adaptive interleukin-22 protects mice from inflammatory bowel disease[END_REF].

Innate immune molecules contain defensins and PRRs. Defensins, which contain two types: α and β, have lots of functions. They play an important role in cell immunity by serving as a bridge between innate immunity and adaptive immunity. Thus, a decline of defensins not only weakens innate immunity, but also influences initiation and regulation of adaptive immunity [START_REF] Huang | Inflammatory bowel disease related innate immunity and adaptive immunity[END_REF]. More bacteria invasion of intestinal mucosa was observed with a loss of mucosal defensins, which resulted in inflammation [START_REF] Wehkamp | Human defensins in Crohn's disease. Mechanisms of Epithelial Defense[END_REF]. PRRs contain Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain like receptors (NLRs) [START_REF] Huang | Inflammatory bowel disease related innate immunity and adaptive immunity[END_REF]. PRRs also act as a bridge between innate immunity and adaptive immunity. They can activate innate immunity by secreting cytokines to fight against pathogens [START_REF] Huang | Inflammatory bowel disease related innate immunity and adaptive immunity[END_REF].

Adapted immune response in IBD

Adapted immunity works as a complementary immune response of innate immunity against the invasion of pathogenic microorganisms. After antigens stimulating, the original T cells amplify and are differentiated into different subsets such as Th1, Th2, Th17 and Treg cells [START_REF] Huang | Inflammatory bowel disease related innate immunity and adaptive immunity[END_REF]). Th1 cells eliminate pathogens inside cells;

Th2 cells protect human body from harmful parasites and adjust allergic reactions; Th17 cells remove extracellular bacteria and fungi; Treg cells help tissue repairs [START_REF] Huang | Inflammatory bowel disease related innate immunity and adaptive immunity[END_REF]. However, disorders of T cell responses and imbalance of Th1/Th2 cells and Th17/Treg cells promote excessive secretion of cytokines and chemokines, resulting inflammation [START_REF] Huang | Inflammatory bowel disease related innate immunity and adaptive immunity[END_REF]. Thl and Th2 cells are in dynamic equilibrium under normal conditions, and are regulated and inhibited by cytokines produced by each other [START_REF] Huang | Inflammatory bowel disease related innate immunity and adaptive immunity[END_REF]. Imbalance of Th1/Th2 cells is associated with pathogenesis of IBD [START_REF] Kanai | TH1/TH2mediated colitis induced by adoptive transfer of CD4+ CD45RBhigh T lymphocytes into nude mice[END_REF]. Th1 cells release proinflammatory cytokines, such as IL-1, IL-2, IL-6 and IL-8 which involved in cellular immune response, while Th2 cells secret anti-inflammatory cytokines, such as IL-4, IL-10 and IL-13 which is correlated with humoral immune responses [START_REF] Huang | Inflammatory bowel disease related innate immunity and adaptive immunity[END_REF]. Thus, balance of Thl and Th2 cells decides balance of pro-inflammatory and anti-inflammatory cytokines, which then leads to whether and which kinds of immune reactions occur [START_REF] Huang | Inflammatory bowel disease related innate immunity and adaptive immunity[END_REF]. Under normal condition, balance of Th17 cells and Treg cells exist in dynamic way. If the balance was broken, intestinal mucosa damages occur. In IBD patients, the balance is disrupted with over-increases of Th17 cells, over-enhancements of immunogenicity and decreases or abnormal functions of Treg cells [START_REF] Huang | Inflammatory bowel disease related innate immunity and adaptive immunity[END_REF].

The disruption of intestinal barrier has been implicated in the pathology of IBD as well.

The intestinal barrier plays a crucial role in observed in the preventing microbiota invasion. This barrier defect was observed in IBD patients (Söderholm et al., 1999), which caused by decreased expression and redistribution of tight junction proteins, including occludin, claudin-3, -5, -8, and JAM-1, and increased expression of poreforming claudin-2, and cytoskeletal dysregulation caused by alternation of MLCK expression and MLC phosphorylation (Zeissig et al., 2007, Vetrano et al., 2008, Blair et al., 2006). Therefore, the improvement of intestinal mucosal barrier represents a potential therapeutic target of IBD.

Intestine physiology

The intestine is a remarkable organ. It is filled with 10 13 -10 14 bacteria and also with digestive enzymes, which has two important function for human health: digestion and protection from external environment. For these functions, intestine covers a surface of about 400 m 2 and requires approximately 40% of the body's energy expenditure [START_REF] Bischoff | Intestinal permeability-a new target for disease prevention and therapy[END_REF]. The body has two types of intestine: small intestine which begins at the pyloric sphincter of the stomach and extends to the cecum with a length of approximately 6.7 meters; large intestine which is also called colon with a length of 1.8 meters. The small intestine is divided in three major segments: the duodenum, the jejunum, and the ileum. The large intestine is composed of four parts: the ascending colon, the transverse colon, the descending colon and the sigmoid colon.

The intestine tissue is composed of four concentric layers: the serosa, muscularis, submucosa, and mucosa (Figure 2) [START_REF] Rubin | Small intestine: anatomy and structural anomalies[END_REF]. The serosa (the outmost layer) is a smooth membrane that is also a thin layer of connective tissue. helps the absorption of nutrients, electrolytes and water into vascular, followed by circulation in the whole body. Secondly, the intestinal mucosa plays a crucial role of barrier as it prevents the uncontrolled translocation of luminal contents such as microorganisms or toxins into the intestinal tissue circulatory system and in the end in the whole organism [START_REF] Groschwitz | Intestinal barrier function: molecular regulation and disease pathogenesis[END_REF]. This barrier function is particularly important in the defense against the invasion by microorganisms of the gut microbiota and to avoid septic shock. The intestinal barrier is a complex biological system including several components with two main functions: the prevention of the mucosa invasion and the elimination of the microorganism who succeeded in the invasion.

Intestinal barrier components

Intestinal barrier is a complex multilayer system, consisting in an external "physical" barrier and an inner "functional" immunological barrier. The tight interaction of these two barriers maintains an equilibrated permeability to nutrients, electrolytes and avoids the entry of pathogens, toxins, antigens [START_REF] Bischoff | Intestinal permeability-a new target for disease prevention and therapy[END_REF]. To understand this complex barrier, the functions of its components is very crucial to be described. Several components are assembled to form a functional intestinal barrier (Figure 3).

Figure 3 Elements comprising the intestinal mucosal barrier [START_REF] Salim | Importance of disrupted intestinal barrier in inflammatory bowel diseases[END_REF].

First, the endothelial cell layer is covered with two mucus layers, a thick, unstirred and relatively sterile mucus layer and an outer layer in direct contact with the cell which forms a protective area. The single layer of epithelial cells interspersed with mucus producing goblet cells, anti-microbial peptide producing Paneth cells, and specialized luminal sampling enterocytes M-cells forms the main barrier between the outside world and the internal body proper. In the lamina propria, innate and adaptive immune cells such as T cells, B cells, eosinophils, mast cells, dendritic cells, and macrophages, comprise the mucosal immune system that responds with "active" eradication or "toleragenic-reaction" towards foreign antigens. In addition, the degradative properties of gastric acids, pancreatic and biliary juices, and the intestinal propulsive motility have also been recognized to be important factors in intestinal barrier function. It is imperative to keep in mind that although these components might be presented individually, they are inextricably linked and can affect the functional responses of each other [START_REF] Salim | Importance of disrupted intestinal barrier in inflammatory bowel diseases[END_REF].

The role of the commensal microbiota

More than 500 different microbial species constituting up to 100 trillion (10 14 ) microorganisms per human colonize the intestinal tract (Figure 4) [START_REF] Konturek | Emerging role of fecal microbiota therapy in the treatment of gastrointestinal and extra-gastrointestinal diseases[END_REF]. The gut microbiota maintains a symbiotic relationship with the gut mucosa and imparts substantial metabolic, immunological and gut protective functions in the healthy individual [START_REF] Jandhyala | Role of the normal gut microbiota[END_REF]. The normal human gut microbiota comprises of two major phyla, namely Bacteroidetes and Firmicutes [START_REF] Jandhyala | Role of the normal gut microbiota[END_REF].

Figure 4 Spatial and longitudinal variations in microbial numbers and composition across the length of the gastrointestinal tract [START_REF] Konturek | Emerging role of fecal microbiota therapy in the treatment of gastrointestinal and extra-gastrointestinal diseases[END_REF]. The number of bacterial cells present in the mammalian gut shows a continuum that goes from 102 to 103 bacteria per gram of contents in the stomach and duodenum, progressing to 104 to 107 bacteria per gram in the jejunum and ileum and culminating in 109 to 1012 cells per gram in the colon.

The composition of the gut microbiota remains relatively unchanged from late childhood to old age, the changes occur when aging process starts (Petschow et al., 2013, Sun and[START_REF] Sun | Exploring gut microbes in human health and disease: pushing the envelope[END_REF]. Gut microbiota plays a crucial role in host health through different aspects.

-It can impact on host nutrient, xenobiotic and drug metabolism [START_REF] Jandhyala | Role of the normal gut microbiota[END_REF]. For example, the gut microbiota ferment non-digestible dietary carbohydrates into short chain fatty acids (SCFA) such as butyrate, propionate and acetate, which are rich sources of energy for the host epithelial cells [START_REF] Macfarlane | Regulation of short-chain fatty acid production[END_REF]Macfarlane, 2003, Sartor, 2008). Microbiome induced drug metabolism function is reflect by an interesting example: the microbial βglucoronidase induced deconjugation of the anticancer drug irinotecan that can contribute to its toxic adverse effects, such as diarrhea, inflammation and anorexia [START_REF] Wallace | Alleviating cancer drug toxicity by inhibiting a bacterial enzyme[END_REF].

-Gut microbiota is furthermore implied in maintenance of structural integrity of the gut mucosal barrier, immunomodulation, and protection against pathogens.

For example, the gut microbiota, via its structural components and metabolites, has been shown to promote the mucosal barrier function via a pattern recognition receptor (PRR) mediated mechanism, which results in activation of several signaling pathways that are essential for the production of antimicrobial proteins (AMP) such as cathelicidins, C-type lectins, and (pro)defensins by the host Paneth cells, mucin glycoproteins and IgA (Salzman et al., 2007[START_REF] Hooper | Do symbiotic bacteria subvert host immunity?[END_REF]. Bacteroides thetaiotaomicron is reported to induce expression of the small proline-rich protein 2A (sprr2A), which is required for maintenance of desmosomes at the epithelial villus [START_REF] Lutgendorff | The role of microbiota and probiotics in stress-induced gastrointestinal damage[END_REF]. Furthermore, the Lactobacillus rhamnosus GG strain produces two soluble proteins namely p40 and p75 that can prevent cytokine induced apoptosis of the intestinal epithelial cells in an epithelial growth factor receptor (EGFR) and protein kinase C (PKC) pathway dependent manner [START_REF] Yan | Colon-specific delivery of a probioticderived soluble protein ameliorates intestinal inflammation in mice through an EGFR-dependent mechanism[END_REF]. Finally, the gut microbiota contributes to the structural development of the gut mucosa by inducing the transcription factor angiogenin-3, which has been implicated in the development of intestinal microvasculature [START_REF] Stappenbeck | Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells[END_REF]. [START_REF] Carding | Dysbiosis of the gut microbiota in disease[END_REF].

Role of mucus layer

The production of a thick mucus layer that overlies the entire intestinal epithelium prevents large particles, including most bacteria, from direct contact with the epithelial cell layer. Goblet cells secrete large amounts of mucin glycoprotein, via the expression of the MUC2 gene. Mucin forms into a protective gel-like layer that can extend up to as much as 800 µm thick in the colon [START_REF] Atuma | The adherent gastrointestinal mucus gel layer: thickness and physical state in vivo[END_REF]. As mentioned previously, the majority of microorganisms in the lumen can be found in the outer mucus layer, there is an inner, protected, and unstirred layer that is directly adjacent to the epithelial surface and is relatively sterile (Figure 3). The sterility of this layer contributes to the retention of a high concentration of antimicrobial proteins (such as cathelicidiens, defensins) produced by various intestinal epithelial lineages, including enterocytes and Paneth cells (see paragraph 1.2.1.3). The existence of unstirred layer can slow down the diffusion of small molecule (ions and small solutes…) which can pass through the heavily outer layer with relative ease (Turner, 2009). The crucial role of the mucus layer in maintaining the intestinal barrier has been illustrated in MUC2 knockout mice, where bacteria were found to be in direct contact with the epithelial cells (Van der [START_REF] Van Der Sluis | Muc2-deficient mice spontaneously develop colitis, indicating that MUC2 is critical for colonic protection[END_REF][START_REF] Johansson | The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria[END_REF]. This subsequently led to the exacerbation of intestinal inflammation. Defective mucus layers resulting from the lack of MUC2 mucin, mutated MUC2 mucin Willebrand factor domains, or from deletion of core mucin glycosyltransferase enzymes in mice increased bacterial adhesion to the epithelium surface, intestinal permeability, and enhanced susceptibility to colitis caused by dextran sodium sulfate (Kim and Ho, 2010).

Role of the intestinal epithelial cells

The intestinal epithelial cells (IECs) are by far the most integral part of the mucosal barrier. Epithelial cells form a continuous, polarized monolayer that is linked together by a series of dynamic junctional complexes. The pluripotent intestinal epithelial stem cells that are located at the crypt region of the intestine include enterocytes, Paneth cells, the mucus-secreting Goblet cells and enteroendocrine cells [START_REF] Barker | The intestinal stem cell[END_REF].

Enterocytes are columnar cells, primarily functioning as absorptive cells, regulating nutrients and ions absorption [START_REF] Salim | Importance of disrupted intestinal barrier in inflammatory bowel diseases[END_REF]. At the apical surface, these epithelial cells possess an extensive network of microvillar extensions that form the brush border and contain many of the digestive enzymes and transporter receptors that are essential in metabolism and uptake of dietary antigens. The enterocytes have also developed intricate transport mechanisms (Passage Routes, described in paragraph 1.2.2) while maintaining an effective barrier to harmful macromolecules and microorganisms. The enterocytes take up and process antigen via two function pathways. In the major pathway, the antigens are degraded by enzymes in the lysosomes.

In the other pathway, antigens are released into the interstitial space without degradation.

Moreover, the enterocytes are often directly involved in immune processes by presenting processed antigens directly to T cells [START_REF] Snoeck | The role of enterocytes in the intestinal barrier function and antigen uptake[END_REF]. In addition to constitute a physical barrier, epithelial cells maintain a mucosal defense system through the expression of a wide range of pattern recognition receptors (PRRs), such as Tolllike receptors (TLRs) and intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). These PRRs form the backbone of the innate immune system through the rapid response and recognition of the unique and conserved microbial components, the microbial-associated molecular patterns (MAMPs) (Kumar et al., 2011, Chu and[START_REF] Chu | Innate immune recognition of the microbiota promotes host-microbial symbiosis[END_REF] In general, the detection of bacteria or its components results in the ligands binding of TLRs and/or NLRs. And triggers a cascade of events that activates signaling molecules such as nuclear factor-κB (NF-κB), resulting in the expression of proinflammatory cytokines, chemokines, and antimicrobial peptides. The result is the eradication of any invading microbes. The enormous bacterial populations and proximity of epithelial PRRs presents the paradox of how gut epithelia avoid continuous activation of inflammatory responses to resident symbiotic bacteria. ''Physiological inflammation'' where the intestine is at a constant state of low-grade inflammation has been identified to constitute part of the intestine mucosal barrier [START_REF] Fiocchi | What is "physiological" intestinal inflammation and how does it differ from "pathological" inflammation?[END_REF]. However, aberrant commensal-mediated signaling of PRRs can lead to the stimulation of diverse inflammatory responses that may result in "pathological inflammation" [START_REF] Fiocchi | What is "physiological" intestinal inflammation and how does it differ from "pathological" inflammation?[END_REF].

Paneth cells also participate to this response by sensing luminal bacteria release a host of AMPs, including defensins, cryptidines, and lysozymes, forming a biochemical barrier that limit bacterial access to and penetration into the gut epithelial surfaces [START_REF] Gersemann | Crohn's diseasedefect in innate defence[END_REF][START_REF] Duerkop | Immune responses to the microbiota at the intestinal mucosal surface[END_REF]. These AMPs disrupt highly conserved and essential features of bacterial biology, such as surface membranes, which are targeted by pore-forming defensins and cathelicidins, and Gram-positive cell wall peptidoglycans, which are targeted by C-type lectins [START_REF] Gallo | Epithelial antimicrobial defence of the skin and intestine[END_REF]Hooper, 2012, Mukherjee et al., 2014). Paneth cells deficient mice exhibit an enhanced translocation of bacteria into the host tissues [START_REF] Vaishnava | Paneth cells directly sense gut commensals and maintain homeostasis at the intestinal host-microbial interface[END_REF]. It also illustrates the ability of epithelial cells to monitor the total bacterial census, thereby activating antimicrobial responses that are density-dependent. This in turn allows epithelial cells to directly control ''surface-associated'' bacterial populations without disrupting the luminal microbiota that are crucial for host metabolic health.

Goblet cells can not only produce the mucin glycoproteins to form a physical barrier, but also producing factors like trefoil factor 3 (TFF3) and the resistin-like molecule-β (RELMβ) that can further stabilize and regulate this physical barrier of intestine. TFF3 stabilizes structural integrity to mucus through mucin crosslinking and act as a signal that promotes epithelial repair, migration of IECs and resistance to apoptosis [START_REF] Dignass | Trefoil peptides promote epithelial migration through a transforming growth factor beta-independent pathway[END_REF][START_REF] Taupin | Intestinal trefoil factor confers colonic epithelial resistance to apoptosis[END_REF]. RELMβ works by promoting MUC2 secretion, regulate macrophage and adaptive T cell responses during inflammation and, in the case of nematode infection, directly inhibit parasite chemotaxis [START_REF] Artis | RELMβ/FIZZ2 is a goblet cell-specific immune-effector molecule in the gastrointestinal tract[END_REF][START_REF] Nair | Goblet cell-derived resistin-like molecule β augments CD4+ T cell production of IFN-γ and infection-induced intestinal inflammation[END_REF]. Study showed that goblet cells dysfunction is an epithelial-autonomous defect in the cystic fibrosis intestine that could be responsible of intestinal manifestations of obstruction and inflammation (Liu et al., 2015a).

Enteroendocrine cells help the linkage between the central and enteric neuroendocrine systems by secreting numerous hormones and other signal molecules of digestive function [START_REF] Peterson | Intestinal epithelial cells: regulators of barrier function and immune homeostasis[END_REF]. The cells are involved in the efficient absorption of food and participate in sensing hunger and thirst.

The epithelial cells are tightly joined with junctional complexes. The perfect communication between cells to assume role of barrier is essential and possible by establishment of many junctional complexes.

Junctional complexes between epithelial cells

Junctional complexes are symmetrical structures between the epithelial cells. There are three different junctional complexes: from the luminal side to the basolateral side of the epithelial cell layer, there is firstly, tight junction (TJ) where claudin, occludin, zonula occluden (ZO), junctional adhesion molecule (JAM) and F-actin interact; secondly, adherens junction (AJ) where E-cadherin, α-catenin 1 and β-catenin interact; and thirdly, desmosomes where the interaction is between desmoglein, desmocollin, desmoplakin and keratin filaments (Figure 5) (Turner, 2009[START_REF] Pavelka | Junctional Complex[END_REF]. As the apical member of the junctional complexes, the TJ forms a continuous, circumferential, belt-like structure at the luminal end of the intercellular space [START_REF] Schneeberger | The tight junction: a multifunctional complex[END_REF]. Thanks to the development of electron microscopy in the 1960's, the TJ biology evolution emerged. In the morphology study, these so-called "kissing point" in TJ are different from AJ and desmosomes, where the adjacent cell membranes remain 15-20 nm apart [START_REF] Tsukita | Multifunctional strands in tight junctions[END_REF]. TJ is a multiple protein complex which consist of three integral transmembrane proteins (occludins, claudins, junctional adhesion molecules (JAM)) associated with the cytoplasmic proteins [START_REF] Groschwitz | Intestinal barrier function: molecular regulation and disease pathogenesis[END_REF]. The intracellular domains of these transmembrane proteins interact with cytosolic scaffold proteins, such as ZO proteins, which in turn anchor the transmembrane proteins to the actin (F-actin) cytoskeleton (Figure 6).

Figure 6 Molecular structures of TJ proteins and their interaction [START_REF] Robinson | Regulation of the Intestinal Barrier Function by Host Defense Peptides[END_REF].

Occludin

Occludin is a tetraspan membrane protein with a molecule weight around 60~82 kDa.

It forms two extracellular loops, one intracellular loop, a short cytoplasmic N-terminus and a long cytoplasmic C-terminus project into the intracellular compartment (Figure 6) [START_REF] Harhaj | Regulation of tight junctions and loss of barrier function in pathophysiology[END_REF]Antonetti, 2004, Hartsock and[START_REF] Hartsock | Adherens and tight junctions: structure, function and connections to the actin cytoskeleton[END_REF]. The long C-terminal domain of occludin interacts with several intracellular TJ proteins, such as ZO proteins, which are requested to link occludin to the actin cytoskelet (Figure 5) [START_REF] Furuse | Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions[END_REF], Li et al., 2005a). Investigations have also confirmed roles for the extracellular loops and at least one transmembrane domain in occludin localization and TJ stability by expressing mutant and chimeric occludin that exert a dominant negative effect on paracellular permeability. [START_REF] Balda | Multiple domains of occludin are involved in the regulation of paracellular permeability[END_REF]. Numerous studies showed that occludin has crucial roles in the maintenance and assembly of TJ structure [START_REF] Al-Sadi | Occludin regulates macromolecule flux across the intestinal epithelial tight junction barrier[END_REF].

Study showed that knockdown of occludin in in vitro (filter-grown Caco-2 monolayers)

and in vivo (recycling perfusion of mouse intestine) intestinal epithelial models induces an increase in paracellular permeability to macromolecules [START_REF] Al-Sadi | Occludin regulates macromolecule flux across the intestinal epithelial tight junction barrier[END_REF]. In the intact epithelium, occludin is highly phosphorylated on the serine (Ser) and threonine (Thr) residues [START_REF] Sakakibara | Possible involvement of phosphorylation of occludin in tight junction formation[END_REF]. Dynamic phosphorylation of occludin seems to play an important role in the assembly and disassembly of tight junctions. The phosphorylation rate of occludin depends of Protein kinase Cs (PKCs)

and Ser/Thr phosphatase. [START_REF] Seth | Protein phosphatases 2A and 1 interact with occludin and negatively regulate the assembly of tight junctions in the CACO-2 cell monolayer[END_REF], Jain et al., 2011). PKCη plays an important role in occludin phosphorylation and assembly of tight junctions [START_REF] Suzuki | PKCη regulates occludin phosphorylation and epithelial tight junction integrity[END_REF].

PKCζ directly interacts with the C-terminal domain of occludin and phosphorylates it on specific Thr residues, which help assembly into tight junctions [START_REF] Jain | Protein kinase Cζ phosphorylates occludin and promotes assembly of epithelial tight junctions[END_REF].

Disruption of tight junctions by calcium depletion [START_REF] Seth | Protein phosphatases 2A and 1 interact with occludin and negatively regulate the assembly of tight junctions in the CACO-2 cell monolayer[END_REF], phorbol ester [START_REF] Clarke | Protein kinase C activation leads to dephosphorylation of occludin and tight junction permeability increase in LLC-PK1 epithelial cell sheets[END_REF] and pathogens [START_REF] Simonovic | Enteropathogenic Escherichia coli activates ezrin, which participates in disruption of tight junction barrier function[END_REF] results in dephosphorylation of occludin on Ser/Thr residues. Protein Ser/Thr phosphatases, PP2A and PP1, are involved in occludin dephosphorylation and destabilization of tight junctions [START_REF] Seth | Protein phosphatases 2A and 1 interact with occludin and negatively regulate the assembly of tight junctions in the CACO-2 cell monolayer[END_REF].

Claudins

Claudins, similar with occcludin, are tetraspan integral membrane proteins with a molecular weight 20~27 kDa. They have four hydrophobic transmembrane domains, two extracellular loops, a short intracellular loop and N-and C-terminal cytoplasmic domains (Figure 6) [START_REF] Turksen | Barriers built on claudins[END_REF][START_REF] Van Itallie | Claudins and epithelial paracellular transport[END_REF][START_REF] Lee | Intestinal Permeability Regulation by Tight Junction: Implication on Inflammatory Bowel Diseases[END_REF]. The extracellular loops are critical for TJ protein-protein interactions and the formation of ion-selective channels [START_REF] Van Itallie | Claudins and epithelial paracellular transport[END_REF]. The intracellular C-terminal domain is involved in anchoring claudin to the cytoskeleton via interactions with cytosolic scaffold proteins, including ZO-1, and ZO-2 and -3 (less investigated) [START_REF] Itoh | Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins[END_REF], Morita et al., 1999). Twenty four members of claudin family have been identified [START_REF] Mitic | Occludin and claudins: transmembrane proteins of the tight junction[END_REF]Van Itallie, 2001, Tsukita et al., 2001). As occludin, some claudin isoforms are phosphorylated in the cells, which is associated with localization and paracellular permeability [START_REF] Agarwal | Phosphorylation of claudin-3 at threonine 192 by cAMP-dependent protein kinase regulates tight junction barrier function in ovarian cancer cells[END_REF][START_REF] Indig | Phosphorylation of claudin-4 by PKCε regulates tight junction barrier function in ovarian cancer cells[END_REF]. Claudin-1 knockout mice die within 24 hours of birth because of a dramatic loss of fluid and electrolytes through the impaired epidermal barrier. Therefore, Claudins play a very important role in the barrier formation and paracellular permeable selectivity in various tissues [START_REF] Tamura | Loss of claudin-15, but not claudin-2, causes Na+ deficiency and glucose malabsorption in mouse small intestine[END_REF].

Junctional Adhesion Molecules

The junctional adhesion molecules (JAMs) family is integral membrane protein which belongs to the immunoglobulin (Ig) superfamily and is characterized by 2 extracellular Ig-like domains, one transmembrane domain, and one intracellular C-terminal domain (Figure 6) [START_REF] Cunningham | A Novel Protein with Homology to the Junctional Adhesion Molecule CHARACTERIZATION OF LEUKOCYTE INTERACTIONS[END_REF]. They are subdivided in two classes based on the expression of Type I or II binding motifs in the intracellular C-terminus, which suggests that the two types interact with specific scaffolding and cytoplasmic proteins [START_REF] Ebnet | Junctional adhesion molecules (JAMs): more molecules with dual functions[END_REF]. JAM-1, -2 and -3 have Type II binding motifs, while the atypical JAMs, including JAM-4, coxsackie and adenovirus receptor (CAR) and endothelial selective adhesion molecule (ESAM) contain Type I binding motifs [START_REF] Ebnet | Junctional adhesion molecules (JAMs): more molecules with dual functions[END_REF][START_REF] Ebnet | Organization of multiprotein complexes at cell-cell junctions[END_REF]. Homophilic JAM-1 or -2 interactions regulate the formation of functional TJs and cell-cell border formation [START_REF] Bazzoni | Homophilic interaction of junctional adhesion molecule[END_REF][START_REF] Babinska | Two regions of the human platelet F11-receptor (F11R) are critical for platelet aggregation, potentiation and adhesion[END_REF], while heterophilic JAM interactions play a role in leukocyte-endothelial cell adhesion [START_REF] Bazzoni | The JAM family of junctional adhesion molecules[END_REF]. The extracellular N-terminal domains of the JAM family members bind to multiple ligands through homophilic and heterophilic interactions which are proposed to regulate the cellular functions and paracellular permeability of JAMs [START_REF] Bazzoni | The JAM family of junctional adhesion molecules[END_REF][START_REF] Ebnet | Junctional adhesion molecules (JAMs): more molecules with dual functions[END_REF].

In intestinal epithelial cells, JAM-1 and JAM-4 are expressed and associated with TJ regulation. In vitro and in vivo studies indicate that JAM-1 is important in the maintenance of the TJ barrier. Treatment of intestinal epithelial cells with monoclonal JAM-1 antibodies inhibited the resealing of the TJs, as indicated by delays in transepithelial electrical resistance (TEER) recovery and occludin assembly [START_REF] Liu | Human junction adhesion molecule regulates tight junction resealing in epithelia[END_REF]. JAM-1 knockout mice show a higher permeability to dextran and an increase of myeloperoxidase activity (an inflammation marker) in the colon compared to wild type mice. In addition, the colonic injury and inflammation induced by dextran sodium sulfate are more severe in the JAM-1 knockout mice than in wild type mice [START_REF] Laukoetter | JAM-A regulates permeability and inflammation in the intestine in vivo[END_REF]. Furthermore, the colonic mucosa of JAM-1 deficient mice shows increased paracellular permeability, leukocyte infiltration and lymphoid aggregates, and, consequently, these mice are more susceptible to experimentally induced colitis (Luissint et al., 2014) 1.2.1.4.1.4 Zonula Occludens ZO proteins contain multiple domains that bind a diverse set of junction proteins. To date, three ZO proteins, ZO-1, -2, and -3, have been identified [START_REF] Haskins | ZO-3, a novel member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and occludin[END_REF].

These multi-domain proteins carry three post-synaptic density 95/Drosophila disc large/zona-occludens (PDZ) domains, a Src homology-3 (SH-3) domain and a region of homology to guanylate kinase (GUK) from the side of the N-terminus (Figure 7) [START_REF] Lee | Intestinal Permeability Regulation by Tight Junction: Implication on Inflammatory Bowel Diseases[END_REF]. Unlike occludins and claudins which are integral membrane proteins that form inter cellular homophilic and heterophilic adhesions, ZO proteins are cytoplasmic proteins that form a scaffold between the transmembrane proteins and the actin cytoskeleton [START_REF] Mcneil | Zonula occludens-1 function in the assembly of tight junctions in Madin-Darby canine kidney epithelial cells[END_REF]. Interestingly, many TJ proteins bind to the N-terminal half region of ZO proteins, while the C-terminal region interacts with the actin cytoskeleton and cytoskeleton-associated proteins [START_REF] Fanning | Isolation and functional characterization of the actin binding region in the tight junction protein ZO-1[END_REF]. Among the ZO protein, the functions of ZO-1 was better investigated compared with ZO-2 and -3. Investigators demonstrate that gene silencing of ZO-1 causes a delay of ∼3 h in tight junction formation in Madin-Darby canine kidney (MDCK) epithelial cells, but mature junctions seem functionally normal even in the continuing absence of ZO-1 (McNeil et al., 2006). Therefore, ZO-1 plays a vital role in the regulation of TJ assembly.

ZO-2 was demonstrated to serve as a simple structural component of the "cytoplasmic plaque" which is involved in TJ assembly and function [START_REF] Traweger | Beyond cell-cell adhesion: Emerging roles of the tight junction scaffold ZO-2[END_REF]. Protein kinases, signaling molecules and other regulatory proteins including cytokines which directly or indirectly target ZO-2 complete the dynamic network of the ZO-2 scaffold.

These lead to phosphorylation of ZO-1 and ZO-2 resulting in disruption of TJs and an increase in paracellular permeability [START_REF] Sabath | Gα12 regulates protein interactions within the MDCK cell tight junction and inhibits tight-junction assembly[END_REF]. ZO-1 and ZO-2 play pivotal roles in the final establishment of the TJs with paracellular barrier function, thereby providing the general basis for selective paracellular permeability in epithelial and endothelial cells [START_REF] Tsukita | Roles of ZO-1 and ZO-2 in Establishment of the Belt-like Adherens and Tight Junctions with Paracellular Permselective Barrier Function[END_REF].

The importance of ZO-1 and ZO-2 was proved by respective knockout mice embryonic lethality [START_REF] Katsuno | Deficiency of zonula occludens-1 causes embryonic lethal phenotype associated with defected yolk sac angiogenesis and apoptosis of embryonic cells[END_REF][START_REF] Xu | Early embryonic lethality of mice lacking ZO-2, but Not ZO-3, reveals critical and nonredundant roles for individual zonula occludens proteins in mammalian development[END_REF]. In contrast, ZO-3 seems to be not as important as them. Although ZO-3 exhibited a prominent localization and was specifically expressed in epithelial-type cells ZO-3 knockout mice showed no phenotypes, suggesting that ZO-3 was not essential in vivo [START_REF] Adachi | Normal establishment of epithelial tight junctions in mice and cultured cells lacking expression of ZO-3, a tight-junction MAGUK protein[END_REF][START_REF] Xu | Early embryonic lethality of mice lacking ZO-2, but Not ZO-3, reveals critical and nonredundant roles for individual zonula occludens proteins in mammalian development[END_REF]. In addition, ZO-3 could not target junctional areas by itself and required the preexistence of ZO-1 and/or ZO-2 [START_REF] Tsukita | Roles of ZO-1 and ZO-2 in Establishment of the Belt-like Adherens and Tight Junctions with Paracellular Permselective Barrier Function[END_REF].

Adherens junction and desmosomes

AJ is immediately subjacent to the TJ and consists of the transmembrane protein Ecadherin, and intracellular components, include β-catenin and α-catenin (Figure 5 and 8). It occurs at points of cell-cell contact in a belt-like way and formed by cadherincatenin interactions which is called cadherin-catenin complex (Figure 8) [START_REF] Hoffman | Towards a Dynamic Understanding of Cadherin-Based Mechanobiology[END_REF]. Localisation of cadherins to the plasma membrane is regulated by p120catenin. Mechanical linkages between cadherin and actin are mediated by α-and βcatenin [START_REF] Hoffman | Towards a Dynamic Understanding of Cadherin-Based Mechanobiology[END_REF]. AJ provides a strong mechanical attachment between adjacent cells. Classical cadherins, such as E-cadherin, are the major transmembrane protein of the AJ and initiate intercellular contacts through trans-pairing between cadherins on opposing cells [START_REF] Mehta | Defects in the adherens junction complex (E-cadherin/ beta-catenin) in inflammatory bowel disease[END_REF]. One of the main function of AJ is to provide the dynamic adhesive connection between epithelial cells. Classical cadherins bind directly and indirectly to many cytoplasmic proteins. particularly members of the catenin family (α-catenin and β-catenin). This binding locally regulates the organization of the actin cytoskeleton, cadherin stability. Another function is involved in intracellular signaling pathways that control gene transcription [START_REF] Perez-Moreno | Catenins: keeping cells from getting their signals crossed[END_REF]. AJ is necessary for the TJ assembly. Cadherin-catenin complexes are important not only for linking adjacent cells, but also for maintaining cell polarity, regulating epithelial migration and proliferation and the formation of other adhesive complexes such as desmosomes [START_REF] Perez-Moreno | Sticky business: orchestrating cellular signals at adherens junctions[END_REF][START_REF] Ebnet | Organization of multiprotein complexes at cell-cell junctions[END_REF]. As a consequence, loss of AJ results in disruption of cell-cell and cell-matrix contacts, ineffective epithelial cell polarization and differentiation, and premature apoptosis [START_REF] Hermiston | In vivo analysis of cadherin function in the mouse intestinal epithelium: essential roles in adhesion, maintenance of differentiation, and regulation of programmed cell death[END_REF]. Desmosome is an adhesive junction that holds two adjacent epithelial cells together.

Desmosome has similar structure with AJ but is like a button instead of a belt (Figure 9) [START_REF] Alberts | Environmental Triggers for Inflammatory Bowel Disease[END_REF]. It consists of transmembrane linker glycoproteins (e.g. superfamily that mediate calcium-dependent cell-cell adhesion [START_REF] Thomason | Desmosomes: adhesive strength and signalling in health and disease[END_REF][START_REF] Saito | Classical and desmosomal cadherins at a glance[END_REF]. Emerging evidence suggests important roles of desmosomal cadherins for these expression patterns in driving epithelial patterning and differentiation [START_REF] Thomason | Desmosomes: adhesive strength and signalling in health and disease[END_REF][START_REF] Saito | Classical and desmosomal cadherins at a glance[END_REF]. The second major gene family is armadillo family proteins which represent cadherin binding partners. They play important roles in tissue integrity and cell signaling. Two types of armadillo proteins are present in desmosomal plaque represent cadherin binding partners [START_REF] Hatzfeld | Plakophilins: Multifunctional proteins or just regulators of desmosomal adhesion?[END_REF][START_REF] Carnahan | The molecular evolution of the p120-catenin subfamily and its functional associations[END_REF]. They are plakoglobin and plakophilins which play important roles in tissue integrity and cell signaling [START_REF] Kowalczyk | Structure, Function and Regulation of Desmosomes[END_REF].

The third major gene family encoding desmosomal proteins is the plakin family [START_REF] Sonnenberg | Plakins in development and disease[END_REF]. Among its members, desmoplakin is an obligate desmosomal protein that couples intermediate filaments to the desmosomal plaque [START_REF] Bornslaeger | Breaking the connection: displacement of the desmosomal plaque protein desmoplakin from cell-cell interfaces disrupts anchorage of intermediate filament bundles and alters intercellular junction assembly[END_REF][START_REF] Gallicano | Desmoplakin is required early in development for assembly of desmosomes and cytoskeletal linkage[END_REF]. AJ, along with desmosome, provide the strong adhesive bonds that maintain cellular proximity and are also a site of intercellular communication.

Subepithelial immune cells

Intrinsic immune system which exists in intestinal tract is to ensure the selective absorption of nutrients and fluids, the prevention of commensals translocation and pathogenic microorganisms across this barrier. Figure 10 showed the different role of immune cells in intestine. Most scattered intestinal immune cells and tissue-specific organized lymphoid structures, such as Peyer's patches (PPs), cryptopatches and isolated lymphoid follicles (ILFs), are located just below the epithelial layer in the lamina propria (LP). The intestinal immune system also consist of intraepithelial effector lymphocytes interspersed in the epithelial lining and extra-intestinal outposts, such as the mesenteric lymph nodes (mLNs) [START_REF] Tomasello | Intestinal innate immune cells in gut homeostasis and immunosurveillance[END_REF]. Although effector T and B lymphocytes provide the host with potent, adaptive immune responses against the vast numbers of antigens that are continuously encountered [START_REF] Maynard | Reciprocal interactions of the intestinal microbiota and immune system[END_REF], the intestinal immune system relies to mainly on innate immune cells and their rapid effector functions to maintain intestinal homeostasis.

Figure 10 Key feature of intestinal immune system [START_REF] Macpherson | Interactions between commensal intestinal bacteria and the immune system[END_REF] Bacteria that cross the enterocyte epithelial layer and invade the intestinal mucosa are rapidly killed by the macrophages in the LP [START_REF] Macpherson | Interactions between commensal intestinal bacteria and the immune system[END_REF]. The others which penetrate through the PPs are not only rapidly killed by macrophages, but also transported to underlying immune cells. The PPs are the major immune inductive sites for the intestinal mucosal immune responses [START_REF] Salim | Importance of disrupted intestinal barrier in inflammatory bowel diseases[END_REF]. PPs, found mostly at the distal ileum, consist of aggregated lymphoid follicles. PPs can be considered as the immune sensors of the intestine because of their ability to detect process and present luminal antigens to other immune cells.

Microfolds cells (M Cells)

in the FAE firstly transport macromolecules and microorganisms through endocytosis, and delivers them to underlying immune cells. Antigen-presenting cells and lymphocyte continuously pick up them. Among the subsets of antigen-presenting cells, dendritic cells (DCs) serve as the key sentinels that can mount appropriate responses, determining whether to ignore or react to the transported foreign antigens [START_REF] Salim | Importance of disrupted intestinal barrier in inflammatory bowel diseases[END_REF]. The presence of either pathogenic bacteria or disruption of the epithelial-cell barrier results in activation and migration of DCs to the mesenteric lymph nodes, where they activate naive T cells. Finally, the T cells undergo differentiation under the influence of factors released by DCs and other stromal elements [START_REF] Cho | The genetics and immunopathogenesis of inflammatory bowel disease[END_REF].

The only aim of the subepithelial immune system is to maintain intestinal homeostasis.

The net effect is to eradicate the unwanted foreign antigens by releasing corresponding cytokines, chemokines and the recruitment of innate immune cells.

Epithelial passage routes

Molecules pass through the intestinal epithelial monolayer either through the cells via the transcellular pathway, or between the cells through the paracellular pathway (Figure 11). The transcellular pathway is active or passive movement across cell membranes, usually involves in specific transport channels. The paracellular pathway is a passive movement through the space between adjacent cells. Low molecular weight molecules (≤ 600 Da in vivo; ≤ 10 kDa in vitro in the cell lines), such as small ions and water pass TJ in the paracellular route, while larger macromolecules such as protein pass through the epithelial cells via the transcellular route [START_REF] Watson | Functional modeling of tight junctions in intestinal cell monolayers using polyethylene glycol oligomers[END_REF][START_REF] Krug | Tricellulin forms a barrier to macromolecules in tricellular tight junctions without affecting ion permeability[END_REF].

Transcellular pathway

Transcellular pathway allows macromolecules transporting from intestinal lumen into subepithelial compartment. Epithelial cells take up luminal contents non-selectively through phagocytosis or pinocytosis, or selectively through receptor-mediated endocytosis (Figure 12). Non-selective phagocytosis, mostly utilized by M cells, transport luminal contents such as bacterial antigens to the underlying basement membrane. (Owen et al., 1986, Conner and[START_REF] Conner | Regulated portals of entry into the cell[END_REF]. The initial process of both pinocytosis and receptor-mediated endocytosis involves the formation of clathrincoated pits. Most of the contents in the vesicles of nonselective pinocytosis fuse with lysosomes and undergo degradation, although low amounts of intact proteins escape degradation and are transcytosed to the basolateral compartment [START_REF] Hewlett | The coated pit and macropinocytic pathways serve distinct endosome populations[END_REF].

Fluid-phase macromolecular transport normally mediates the uptake of nutrients and soluble protein antigens. Receptor-mediated endocytosis is a highly specific process that involves the binding of ligands to their specific receptor. Internalized antigens bound to receptors either undergo the degradation or are directed to the underlying basement membrane and into the lamina propria [START_REF] Schwartz | Receptor cell biology: receptor-mediated endocytosis[END_REF][START_REF] Mukherjee | Endocytosis[END_REF].

Paracellular pathway

Paracellular route across the epithelial barrier is highly regulated and allow the access not only to small molecules such as Ca 2+ and Mg 2+ through the "pore pathway" but also to larger molecules, including small peptides and bacterial lipopolysaccharides through the "leak pathway" in an intact intestinal barrier.

Pore pathway is a high-capacity, size-and charge-selective paracellular route that permeable to molecules with radii of ~4 Å or less [START_REF] Shen | Tight junction pore and leak pathways: a dynamic duo[END_REF]. Claudins expression change is responsible for the ion selectivity and modulation of pore pathway permeability. This pathway can be regulated by interactions among claudin proteins to form pores, interactions between specific claudins and other tight junction proteins, which finally regulate pore opening and closing events [START_REF] Shen | Tight junction pore and leak pathways: a dynamic duo[END_REF]. IL-4, IL-6, IL-9 and IL-13 have been reported to activate pore pathway by inducing increases in claudin-2 expression, which finally enhance the intestinal permeability (Shen et al., 2011, Odenwald andTurner, 2017). Some bacterial pathogens, such as Clostridium perfringens enterotoxin, binding to specific claudins (claudins-3 and -4, here) can also lead to a disruption of intestinal barrier [START_REF] Saitoh | Structural insight into tight junction disassembly by Clostridium perfringens enterotoxin[END_REF].

Leak pathway is a low-capacity, paracellular route that does not discriminate between solutes and allows limited flux of large molecules. Leak pathway is regulated by ZO-1, occludin and myosin light chain kinase (MLCK) (Shen et al., 2011, Odenwald andTurner, 2017). The width of the lateral space below the TJ is around 75 Å, being wider in comparison to 4-9 Å for the TJ pores in villi or 50-60 Å for the TJ pores in crypts (Ćirković [START_REF] Veličković | Intestinal Permeability and Transport of Food Antigens[END_REF]. The main rate-limiting factor for paracellular transport pathway is the junctional complex between epithelial cells as mentioned above. Leak pathway flux of Madin-Darby canine kidney (MDCK) cells monolayer was increased after ZO-1 knockdown [START_REF] Van Itallie | ZO-1 stabilizes the tight junction solute barrier through coupling to the perijunctional cytoskeleton[END_REF]. MDCK cells lacking ZO-1 also showed increased sensitivity to barrier disruption induced by actin depolymerization, extracellular Ca 2+ chelation, or myosin II motor inhibition [START_REF] Van Itallie | ZO-1 stabilizes the tight junction solute barrier through coupling to the perijunctional cytoskeleton[END_REF]. Occluding internalization induced by TNF diminished intestinal barrier function trough activate leak pathway [START_REF] Marchiando | Caveolin-1dependent occludin endocytosis is required for TNF-induced tight junction regulation in vivo[END_REF]. ZO-1 and occludin, but not claudins, are redistributed following MLCK activation in cultured monolayers as well as after immune activation in vivo [START_REF] Van Itallie | ZO-1 stabilizes the tight junction solute barrier through coupling to the perijunctional cytoskeleton[END_REF].

Although the leak and pore pathways are separable across the paracellular barrier, the two pathways are often affected in parallel. For example, MLCK-dependent increases in intestinal epithelial leak pathway permeability are also able to induce mucosal IL-13 production, which, in turn, increases claudin-2 expression and pore pathway permeability [START_REF] Weber | Epithelial myosin light chain kinase activation induces mucosal interleukin-13 expression to alter tight junction ion selectivity[END_REF]. During homeostasis and less active inflammatory disease, the intestinal barrier is generally intact and barrier function primarily reflects flux across the paracellular pore and leak pathways (Odenwald and Turner, 2017).

At sites of intestinal mucosa damage in active diseases, such as erosions and ulcers, luminal contents cross the intestinal barrier through a third pathway, named as "unrestricted pathway" (Odenwald and Turner, 2017). In these cases, tight junctions are lost and therefore cannot contribute to local barrier function. Therefore, the unrestricted pathway is high-capacity and nonselective with respect to solute size and charge (Odenwald and Turner, 2017). Large proteins and even whole bacteria can cross the unrestricted pathway, which partially explains the severe disease initiated by intestinal mucosa damage. Nonsteroidal anti-inflammatory drug (NSAID) treatment lead to epithelial damage by increasing flux across the unrestricted pathway, which probably provokes colitis in IL10 -/-mice [START_REF] Berg | Rapid development of colitis in NSAID-treated IL-10-deficient mice[END_REF].

Intestinal permeability measurement

The concept of intestinal permeability is defined as a functional feature of the intestinal barrier at given sites, measurable by analyzing flux rates across the intestinal barrier as a whole or across barrier components of defined molecules that are largely inert during the process [START_REF] Bischoff | Intestinal permeability-a new target for disease prevention and therapy[END_REF]. And it can be adequately measured in different settings (in vitro, ex vivo or in vivo). Intestinal permeability tests give researchers valuable information about the status of epithelial barrier integrity, the effectiveness of barrier-targeting therapeutics, and the pathophysiology of diseases [START_REF] Galipeau | The complex task of measuring intestinal permeability in basic and clinical science[END_REF].

In vivo functional test

The gold standard for functional measurements of intestinal permeability in humans is the test of urinary excretion of the two orally administered non-metabolized sugars (lactulose and mannitol) over a 6 h period. The intestinal permeability is expressed by lactulose/mannitol ratio. Both lactulose and mannitol are paracellular marker, whereas mannitol permeability is independent of intestinal barrier damage or dysfunction. By expressing lactulose excretion as a percentage of mannitol excretion, it is possible to correct for variables unrelated to intestinal permeability [START_REF] Ziegler | Attachment 1: Article-Polymer nanocomposites enhance Snitrosoglutathione intestinal absorption and promote the formation of releasable nitric oxide stores in rat aorta[END_REF]. Apart from oral gavage of sugars, fluorescently labeled dextrans, PEG, or 51 Cr-EDTA followed by detection in the urine or blood are commonly used methods in animal studies.

In conclusion, both advantages and disadvantages exist in the in vivo functional test.

The advantage of these tests is non-invasive real-life setting where the host-microbe interactions can be taken into account and it can be conducted in clinic permeability study. However, the drawbacks are time-consuming, existences of several confounding factors which can hamper interpretation of results, such as big individual differences in motility, intestinal cell surface, epithelial cell integrity, renal function, bacterial degradation, gastric dilution, and diet, and no standardized protocols [START_REF] Quigley | Leaky gut-concept or clinical entity?[END_REF].

Even though, the in vivo functional test is still very useful in the applications because it can reflect the reality and complexity of process which cannot be replaced by ex vivo and in vitro testes.

Ex vivo functional test

Ussing Chamber is usually used in ex vivo functional test of intestinal permeability. The system mimics the compartmentalization between the lumen of the intestine and the mucosal part of the intestine in vivo. It can be separated by intestine segment into two compartments (equal volume): a donor compartment (also referred to as the apical, mucosal or luminal-side membrane) and an acceptor compartment (also referred to as the basolateral, serosal, nutrient, or mucosal-side membrane) (Figure 14). The permeability is assayed by the ability of markers to cross the intestine tissue and to accumulate in the acceptor compartment. Different permeability markers, such as 51 Cr-EDTA, mannitol, sodium fluorescein (paracellular), horseradish peroxidase (HRP) and propranolol (transcellular), can be deposited in donor compartment and harvested in acceptor compartment to measure the intestinal permeability. At the same time, electrodes are used to measure the potential difference across the membrane, and determine the short circuit current, which reflects active ion transport taking place across the membrane. From this, transepithelial electrical resistance (TEER) can be calculated or it can be immediately measured, which reflects the integrity of the tissue in relation to paracellular permeability (Clarke, 2009).

One of the advantage is the ability to measure the specific region of intestine. The main shortcoming is that the study is limited in time because the viability of ex vivo tissue limited in usually 2 h. Furthermore, it may not reflect the complexity of process that occurs in vivo.

In vitro functional test

For in vitro permeability study, cultured cells monolayer and parallel artificial membrane permeability assay (PAMPA) are wildly used.

Cultured cells monolayer offers a reductionist approach, where differentiated intestinal epithelial cells such as Caco-2, T-84, and HT-29 cells, are grown on semipermeable filter supports forming apical and basolateral compartments (Figure 15). This has been especially advantageous for assaying drug delivery systems (Wu et al., 2016) and screening potential drug compounds. The in vitro model has been used to rapidly predict a drug candidate's physiochemical characteristics toward cellular permeability, the pathways utilized during transport, and identify potential toxic effect that could disrupt the epithelial barrier [START_REF] Meunier | The human intestinal epithelial cell line Caco-2; pharmacological and pharmacokinetic applications[END_REF][START_REF] Grès | Correlation between oral drug absorption in humans, and apparent drug permeability in TC-7 cells, a human epithelial intestinal cell line: comparison with the parental Caco-2 cell line[END_REF][START_REF] Press | Permeability for intestinal absorption: Caco-2 assay and related issues[END_REF].

TEER and permeability of specific marker can also be measured in this system. It has longer experiment durations (up to 24 h) compared with Ussing Chamber. So it can be applied in the drug absorption in the particles (Wu et al., 2016), which extended the application. But it has the same limitation with Ussing Chamber, which lacks the complexity of process that occurs in vivo. It takes longer time, because cells need to be grown on filters up to several weeks before they can be used. Furthermore differences in cell culture conditions can also cause significant differences of permeability results. phosphatidylinositol, cholesterol, 1,7-octadiene membrane-PAMPA [START_REF] Yu | A new PAMPA model proposed on the basis of a synthetic phospholipid membrane[END_REF].

Moreover, similar with cell models cultivated on the Transwell® system, a filter separates one side containing the test molecule (apical compartment) from a receiver side (initially free of the molecule) (basolateral compartment).

IBD therapy

As incurable disease, the major aim of the IBD therapy is to induce and maintenance of remission. Current clinical drugs for the treatment of IBD include aminosalicylates, corticosteroids, classic immunosuppressive drugs and anti-TNF agent (Neurath, 2017).

Aminosalicylates

Aminosalicylates (ASA) are a group of drugs containing the active ingredient "5aminosalicylic acid (5-ASA)" [START_REF] Sales-Campos | Classical and recent advances in the treatment of inflammatory bowel diseases[END_REF]. They usually are the first medicines used to treat IBD within several decades. They are widely used to relieve symptoms and reduce inflammation in treatment of mild to moderate UC in active and remission period or mild active CD by oral or topical administration.

The beneficial effect of ASA is known as a scavenger of reactive oxygen metabolites [START_REF] Ahnfelt-Rønne | Clinical evidence supporting the radical scavenger mechanism of 5aminosalicylic acid[END_REF], inhibition of microphage chemotaxis (Nielsen et al., 1988) and decreasing of different inflammation molecules production such as tumor necrosis factor (TNF)-α effects, interleukin(IL)-1β and nuclear factor (NF)-κB [START_REF] Mahida | 5-Aminosalicylic acid is a potent inhibitor of interleukin 1 beta production in organ culture of colonic biopsy specimens from patients with inflammatory bowel disease[END_REF][START_REF] Kadian | Mesalamine blocks tumor necrosis factor growth inhibition and nuclear factor κB activation in mouse colonocytes[END_REF]. They can also activate peroxisome proliferator activated receptor-γ which is typically expressed in epithelial and immune cells in the colonic mucosa and is important in the regulation of IBD [START_REF] Rousseaux | Intestinal antiinflammatory effect of 5-aminosalicylic acid is dependent on peroxisome proliferator-activated receptor-γ[END_REF][START_REF] Dubuquoy | PPARγ as a new therapeutic target in inflammatory bowel diseases[END_REF].

The frequently used class of aminosalicylates as therapeutic agents for IBD are basolazide, mesalamine, olsalazine and sulfasalazine. Even though ASA are well tolerated and safe in most cases, some side effects were presented in different symptoms such as headache, nausea, abdominal pain and cramping, loss of appetite, vomiting, rash, fever, diarrhea and rare kidney injury. Another drawback of this treatment is that lack of responsive is also observed for some patient.

Therefore, ASA represent a good treatment of IBD but the present of significant side effects and poorly responsive patients limit this therapy application.

Corticosteroids

Corticosteroids are known as the most effective drugs at inducing remission in both ulcerative colitis and Crohn's disease but bot suitable in maintaining remission in IBD.

They have been used for more than half a century. They are commonly prescribed for treatment of acute exacerbation of IBD. Corticosteroids profoundly work on both immunologic and inflammatory responses. As mentioned in the review [START_REF] Sands | Therapy of inflammatory bowel disease[END_REF], for the immunologic part, they can inhibit lots of leukocyte functions, including adherence, chemotaxis and phagocytosis, and interfere with metabolism of arachidonic acid and production of eicosanoids. For the inflammation response part, it can downregulate the production of proinflammation cytokines (e.g., IL-1and IL-6, the chemokine IL-8, the Th1 cytokines IL-2 and IFN-γ, and the Th2 cytokines IL-4 and IL-5).

However, this therapeutic class presents a well-known serious side effects when used daily and long-term or even low-dose. The adverse effects are mostly reported to be related with systemic administration, include sleep disturbance, mood disturbance, acne, striae, hirsutism, adrenal suppression, proximal myopathy, glucose intolerance, hypertension, narrow angle glaucoma, cataracts, pseudotumor cerebri, infection, edema, impaired wound healing, growth retardation, osteoporosis, and aseptic necrosis [START_REF] Sands | Therapy of inflammatory bowel disease[END_REF]. Therefore, topical administration is an alternative treatment for corticosteroids.

Combined treatment with other agents such as ASA and immunosuppressive drugs are also effective strategy for UC or CD therapy [START_REF] Mulder | Beclomethasone dipropionate (3mg) versus 5-aminosalicylic acid (2g) versus the combination of both (3mg/2g) as retention enemas in active ulcerative proctitis[END_REF][START_REF] Markowitz | A multicenter trial of 6-mercaptopurine and prednisone in children with newly diagnosed Crohn's disease[END_REF][START_REF] Hartmann | Clinical trial: controlled, open, randomized multicentre study comparing the effects of treatment on quality of life, safety and efficacy of budesonide or mesalazine enemas in active left-sided ulcerative colitis[END_REF].

In summary, corticosteroid therapy is one of the best choice for acute exacerbation treatment. But the significant side effects by systemic administration limit the continuous use. To reduce the occurrence of side effects, topical administration and combined treatment present a promising method.

Immunosuppressive drugs

Immunosuppressive drugs are the mainstay in the long-tern treatment of steroiddependent or steroid-refractory IBD. Several immunosuppressive agents have been successfully used for induction and maintenance treatment in IBD. These include The thiopurine immunosuppressants azathioprine (AZA) and 6-mercaptopurine (6MP), methotrexate (MTX), anti-alpha 4 integrins, and anti-IL-12/23 agent.

AZA and 6MP have been effectively used for induction and maintenance of IBD. The mechanisms of thiopurine are not completely clear. The action has been involved with nucleotide or protein synthesis and lymphocytes proliferation. Patients refractory to AZA treatment is occurred by the fact that half of the patients are intolerant to AZA [START_REF] Hindorf | Mercaptopurine treatment should be considered in azathioprine intolerant patients with inflammatory bowel disease[END_REF].

MTX may be prescribed for patients' refractory or intolerant to thiopurine therapy, and is generally preferred for induction and maintenance of remission in CD. The antiinflammatory properties of MTX is associated with increasing of the extracellular level of adenosine which is caused by the inhibition of some enzymes related to the folate pathway and involved in purine and pyrimidine synthesis [START_REF] Cronstein | The antiinflammatory mechanism of methotrexate. Increased adenosine release at inflamed sites diminishes leukocyte accumulation in an in vivo model of inflammation[END_REF]. The limitation of MTX is low efficacy during long-term use [START_REF] Suares | Efficacy and tolerability of methotrexate therapy for refractory Crohn's disease: a large single-centre experience[END_REF].

Anti-TNF agents

Anti-TNF therapy is prescribed as a new therapeutic class with high efficacy, rapid onset of action prolonged effect, and improved tolerance [START_REF] Sandborn | Antitumor necrosis factor therapy for inflammatory bowel disease: a review of agents, pharmacology, clinical results, and safety[END_REF].

A number of different anti-TNF antibodies are available, such as infliximab, Anti-TNF therapy have shown great efficacy, represent improvement and advance in management of IBD. However, loss of response and side-effects were appeared as well.

Over the past 10 years, ~30-50% of patients with IBD do not respond to anti-TNF therapy (Neurath, 2017). These side effects include infection, other autoimmune phenomena, heart failure, malignancies such as non-melanoma skin cancers, melanoma, leukemia, and lymphoma, which have been previously reported to be associated with biologics (Mariette et al., 2011, McLean and[START_REF] Mclean | Adverse events in IBD: to stop or continue immune suppressant and biologic treatment[END_REF].

Innovative therapy

As mentioned above, lots of limitations, such as significant side effects, low efficiency and loss of response, exist in current treatment, which indicates the need of new therapies. Modulations of intestinal barrier, gut microbiota, fibrosis and tissue remodeling, macrophage and lymphocyte activation, homing and retention, as well as angiogenesis, are emerging new targets for therapy in clinic trials (Neurath, 2017).

Among all the molecules work on these different targets, nitric oxide (NO) is good candidate. Because it can work on several targets involving in new therapies of IBD [START_REF] Magierowski | Gaseous mediators nitric oxide and hydrogen sulfide in the mechanism of gastrointestinal integrity, protection and ulcer healing[END_REF]. Firstly, it can regulate the intestinal barrier integrity by increasing mucus secretion, tight-junction proteins localization and expression.

Secondly, it targets on different immune cells to reduce cytokines and mediators releasing. At last, NO can also target on angiogenesis to increase new vessels formation, which can finally help inflamed intestinal mucosa healing. In a word, NO is a promising candidate for the innovative treatment of IBD.

Nitric oxide

Nitric oxide (NO) is a free radical that has been involved in a wide range of biochemical reaction to evoke plenty of biological responses. It has been recognized as a powerful relaxing factor derived from the endothelial lining (endothelial derived relaxing factor: EDRF) of blood vessels in response to agonists such as acetylcholine and bradykinin at first place [START_REF] Furchgott | The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine[END_REF]Zawadzki, 1980, Palmer et al., 1987). However this endogenous messenger molecules plays important roles not only in vascular system but also in neural and immune systems [START_REF] Ignarro | Nitric oxide: biology and pathobiology: Academic press[END_REF]. NO generated by neurons acts as a neurotransmitter and is implicated with memory formation [START_REF] Snyder | Nitric oxide and neurons[END_REF]. NO produced by immune system works as a immunomodulator and intracellular signaling molecule [START_REF] Thomas | The chemical biology of nitric oxide: implications in cellular signaling[END_REF]. Besides, NO generated by macrophages in response to invading microbes acts as an antimicrobial agent [START_REF] Fang | Perspectives series: host/pathogen interactions. Mechanisms of nitric oxide-related antimicrobial activity[END_REF]. Endogenous NO is synthesized by nitric oxide synthases (NOS) using L-arginine, oxygen and cofactors as substrates (Figure 16). Three isoforms of the enzymes are identified: endothelial (eNOS) and neuronal (nNOS) isozymes are constitutively expressed and regulated to generate relatively small amounts of NO for signaling, while the inducible isoform (iNOS) is expressed in response to inflammatory mediators leading to high and cytotoxic local concentrations of NO [START_REF] Knowles | Nitric oxide synthases in mammals[END_REF]. The primary target for low levels of endothelium derived NO is the soluble guanylate cyclase (sGC). Stimulation of sGC by NO coordination to the ferrous prosthetic group of the haem moiety induces the conversion of guanosine-5'-triphosphate (GTP) to cyclic guanosine monophosphate (cGMP), that activates the cGMP-dependent kinase: the protein kinase G [START_REF] Francis | cGMP-dependent protein kinases and cGMP phosphodiesterases in nitric oxide and cGMP action[END_REF]. Over this traditional mechanism, NO is also involved in other cellular signaling pathways including the phospholipase C/calcium signaling systems [START_REF] Sterin -Borda | Endogenous nitric oxide signalling system and the cardiac muscarinic acetylcholine receptor-inotropic response[END_REF], tyrosine kinase signaling pathways [START_REF] Monteiro | Tyrosine Phosphorylation in Nitric Oxide-Mediated Signaling Events[END_REF], G-protein linked receptors [START_REF] Garthwaite | Concepts of neural nitric oxidemediated transmission[END_REF], ion channels [START_REF] Gonzalez | S-Nitrosylation of cardiac ion channels[END_REF], and the apoptotic pathway [START_REF] Singh | Apoptotic neuronal death in Parkinson's disease: involvement of nitric oxide[END_REF], often at multiple sites in the same pathway. NO presents both beneficial and detrimental biological effects, which tightly dependents on concentration and location (Figure 17).

Role of nitric oxide in intestinal mucosal defense

NO plays an important role in mucosal defense (Wallace and Miller, 2000) which is disordered in IBD patients. Firstly, NO which was produced by epithelial cells can increase mucus (Price et al., 1994) and fluid (Dow et al., 1994) functions. The first one is that NO causes relaxation of the vascular smooth muscle [START_REF] Ignarro | Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites, nitroprusside and nitric oxide: evidence for the involvement of S-nitrosothiols as active intermediates[END_REF], and as a result of the dilation of submucosal arterioles, the increase of mucosal blood flow and the removal of any toxins that had across the epithelium. The other function is that NO inhibits expression of the β-2 adhesion molecules on neutrophils (Banick et al., 1997) and P-selectin on the vascular endothelium [START_REF] Davenpeck | Inhibition of endothelial-derived nitric oxide promotes P-selectin expression and actions in the rat microcirculation[END_REF]. It also down-regulates neutrophil aggregation and secretion [START_REF] May | The role of nitric oxide as an endogenous regulator of platelet and neutrophil activation within the pulmonary circulation of the rabbit[END_REF]. In this way, NO acts as a homeostatic regulatory molecule designed to attenuate leukocyte-endothelium interaction and thus attenuate local inflammation. In addition, NO is an important modulator of mucosal repair, because of vascular smooth muscle relaxation causing blood flow increasing, enhancement of collagen deposition by fibroblasts and angiogenesis stimulation [START_REF] Schäffer | Nitric oxide regulates wound healing[END_REF][START_REF] Schäffer | Nitric oxide, an autocrine regulator of wound fibroblast synthetic function[END_REF], Jadeski and Lala, 1999). NO also has potent effects on immunocytes within the gastrointestinal lamina propria. NO secreted by mast cell appears to reduce the release of inflammatory mediators, including histamine, platelet-activating factor PAF, TNF [START_REF] Salvemini | Synthesis of a nitric oxide-like factor from L-arginine by rat serosal mast cells: stimulation of guanylate cyclase and inhibition of platelet aggregation[END_REF][START_REF] Bissonnette | Potentiation of tumor necrosis factor-alpha-mediated cytotoxicity of mast cells by their production of nitric oxide[END_REF][START_REF] Masini | Rat mast cells synthesize a nitric oxide like-factor which modulates the release of histamine[END_REF][START_REF] Hogaboam | Modulation of rat mast cell reactivity by IL-1 beta. Divergent effects on nitric oxide and platelet-activating factor release[END_REF], Van Overveld et al., 1993). Macrophage function is modulated by NO, with important implications for mucosal defense. For example, the production of various immunomodulatory cytokines (e.g., IL-12 and IL-1) can be inhibited by NO (Huang et al., 1998[START_REF] Obermeier | Interleukin-1 production by mouse macrophages is regulated in a feedback fashion by nitric oxide[END_REF]. Thus, the overall action of NO could be characterized as anti-inflammatory.

NO donors

NO, as a free radical, is very sensitive to the environment. It can react with a variety of atoms and radicals, such as oxygen superoxide anion, which leads to its very short halflife (within seconds). Due to its instability, treatment with drugs, which can deliver NO, has been used for more than a century (e.g. nitroglycerin (GTN)). In cardiovascular disease, for prevention and treatment of angina pectoris and hypertension, organic nitrates and sodium nitroprusside respectively have been used (Al-Sa'doni and Ferro, 2000;Ignarro, 2002). The most commonly used drugs in therapy of cardiovascular disease are organic nitrate and nitrate esters, including GTN, amyl nitrate, isosorbide dinitrate (ISDN), isosorbide mononitrate (ISMN) and pentaerythrityl nitrate (PETN) (Ignarro, 2002;Schade et al., 2010). Organic nitrates are indirect NO donors; for example GTN is used in acute angina pectoris is a prodrug, which needs enzymatic activation to generate bioactive NO (Ignarro, 2002). For the treatment of patients suffering from chronic stable angina, slower NO-releaser nitrate such as ISMN, ISDN

or PETN are used, as well as retarded GTN in the form of transdermal patches.

Isosorbide mononitrate and ISDN are useful for the treatment of chronic heart failure, especially for patient with concomitant coronary insufficiency. Disadvantages of conventional organic nitrates are their short therapeutic half-life, drug tolerance, hemodynamic effects, lack of selectivity, systemic absorption and limited bioavailability (Ignarro, 2002).

Among direct NO donors, only sodium nitroprusside (SNP) and NO gas have clinical relevance. Sodium nitroprusside is used in intensive care medicine to provide rapid lowering of high blood pressure crises (Al-Sa'doni and Ferro, 2000;Miller and Megson, 2007;Schade et al., 2010). In SN, NO is coordinated, as a nitrosyl group, to iron in a square bipyramidal complex (Ignarro, 2002). Side effects associated with toxic metabolites, cyanide and thiocyanide appear with prolonged administration. Nitric oxide in gaseous phase can be used therapeutically only by inhalation in pulmonary hypertension (Creagh-Brown et al., 2009), and in neonates inhalation (Miller and Megson, 2007).

Novel NO donors including diazeniumdiolates and S-nitrosothiols have been widely investigated for biomedical applications. Diazeniumdiolates are synthesized by exposing nucleophiles to NO. These compounds are generally stable in solid form but decompose spontaneously in solution to generate up to two molar equivalents of NO (Megson and Webb, 2002). Diazeniumdiolates present the major bioactivities through activation of sGC and depend on the rate of NO generation (Megson and Webb, 2002).

S-nitrosothiols (RSNO) such as S-nitrosoglutathion (GSNO), may be more effective than currently available NO donor drugs [START_REF] Hogg | Biological chemistry and clinical potential of S-nitrosothiols[END_REF][START_REF] Richardson | Potential therapeutic uses for S-nitrosothiols[END_REF], Parent et al., 2013a[START_REF] Dahboul | S, S′-dinitrosobucillamine, a new nitric oxide donor, induces a better vasorelaxation than other S-nitrosothiols[END_REF]. They have a number of potential advantages over other classes of NO donors. They do not appear to engender vascular tolerance (Miller and Megson, 2007) and are characterized by an in vitro half-life ranging from 10 to 38 h (Mancuso et al., 2003). S-nitrosothiols do not induce oxidative stress and, in arteries with endothelial cells dysfunction, play a role in the storage and transport of NO (Sarr et al., 2007). S-nitrosothiols have made a promise for the future as interesting tools as complementary therapy.

S-Nitrosoglutathione: a potent S-nitrosothiol

S-Nitrosoglutathione (GSNO, Figure 19) is the S-nitrosated derivative of the most abundant cellular thiol, glutathione (GSH), which is a tripeptide of gamma-glutamine, L-cysteine, and glycine. It is one of the physiological RSNO and an attractive therapeutic candidate:

a) It has been reported to be involved in the chemical biology and physiological functions of NO in intestine (mentioned above);

b) It has been discovered as endogeneously present in extracellular fluids and tissues [START_REF] Gaston | Endogenous nitrogen oxides and bronchodilator S-nitrosothiols in human airways[END_REF][START_REF] Snyder | Acute effects of aerosolized S-nitrosoglutathione in cystic fibrosis[END_REF]; c) Compared to other NO donor such as organic nitrates, GSNO presents non reported tolerance [START_REF] Sarr | Formation of releasable NO stores by S-nitrosoglutathione in arteries exhibiting tolerance to glyceryl-trinitrate[END_REF].

Figure 19 Chemical structure of S-nitrosoglutathione (GSNO)

Synthesis

Three strategies to synthesize GSNO were described in the literature: ⅰ) the most popular method to produce GSNO is reacting glutathione (GSH) with acidified sodium nitrite [START_REF] Hogg | Biological chemistry and clinical potential of S-nitrosothiols[END_REF][START_REF] Heikal | Characterisation of the decomposition behaviour of S-nitrosoglutathione and a new class of analogues: S-Nitrosophytochelatins[END_REF], ⅱ) GSNO can also be synthesized by reacting GSH with nitrous acid (HONO) [START_REF] Kettenhofen | Proteomic methods for analysis of S-nitrosation[END_REF], ⅲ) simply bubbling NO through GSH solution produces GSNO as well (Seabra et al., 2004b).

Unlike most of other low-molecular-weight S-nitrosothiols, GSNO can be precipitated with acetone and purified as a solid [START_REF] Hart | Some observations concerning the S-nitroso and S-phenylsulphonyl derivatives of L-cysteine and glutathione[END_REF], and this has promoted its use as an experimental tool. The molar extinction coefficient of GSNO has been variously reported, with a wide range of values. [START_REF] Hart | Some observations concerning the S-nitroso and S-phenylsulphonyl derivatives of L-cysteine and glutathione[END_REF] gave an extinction coefficient of 922 M -1 cm -1 at 335 nm, with agreement to this result, we measured the value as 922 M -1 cm -1 at 334 nm (Parent et al., 2013a).

In vitro stability

The limitation of GSNO application is its fast and often unpredictable rate of decomposition in aqueous solutions. In aqueous solutions, the stability of GSNO may be explained by the S-NO bond mesomerization (Eq. 1). The distribution of those mesomers depends on the electrophile and nucleophile groups in the S-NO bond environment.

Published half-lives of GSNO are not unified and are clearly condition-dependent.

Basically, GSNO is subject to several mechanisms of decomposition, including S-NO bond homolysis (Eq. 2) metal ion-catalyzed (mainly Cu 2+ ) decomposition (Eq. 3), heterolysis (Eq. 4), and hydrolysis (Eq. 4).

Those reactions may explain why, despite the proven benefits of GSNO, this tripeptide

is not yet present in any pharmaceutical composition. Hence it is important to develop dosage forms to protect GSNO from environmental decomposition for further applications, especially for chronic medical application.

In vivo metabolism

Figure 20 In vivo metabolism of GSNO by enzymes: 1. GSNO reductase (GSNOR) and carbonyl reductase 1 (CR1); 2. thioredoxin system (Trx); 3. protein disulfide isomerase (PDI); 4, γ-glutamyltranspeptidase (GGT).

As described above, GSNO is unstable in vitro, and it is susceptible to many enzymes as well. In the literature, GSNO reductase (GSNOR), carbonyl reductase 1 (CR1), the thioredoxin (Trx) system, the protein disulfide isomerase (PDI) and γglutamyltranspeptidase (GGT) are the main enzymes reported to be involved in GSNO decomposition (Figure 20).

S-Nitrosoglutathione reductase and carbonyl reductase 1

The GSNOR reduces GSNO to an unstable intermediate, S-hydroxylaminoglutathione, which then rearranges to form glutathione sulfonamide, or in the presence of GSH, forms oxidized glutathione (GSSG) and hydroxylamine (NH2OH) (Figure 20) [START_REF] Koivusalo | Evidence for the identity of glutathione-dependent formaldehyde dehydrogenase and class III alcohol dehydrogenase[END_REF][START_REF] Jensen | S-Nitrosoglutathione is a substrate for rat alcohol dehydrogenase class III isoenzyme[END_REF]. Through this catabolic process, GSNOR regulates the cellular concentrations of GSNO and plays a central role in regulating the levels of endogenous S-nitrosothiols and controlling protein S-nitrosation-based signaling [START_REF] Höög | Mammalian alcohol dehydrogenases-a comparative investigation at gene and protein levels[END_REF]. In fact, GSNOR-dependent oxidation of Shydroxylaminoglutathione is increased 8-fold in the presence of GSNO in vitro and more than 20-fold in crude lung and liver lysates. These results highlight the potential impact of alternative GSNO reductase substrates on the biological activity and degradation of GSNO [START_REF] Staab | Reduction of S-nitrosoglutathione by alcohol dehydrogenase 3 is facilitated by substrate alcohols via direct cofactor recycling and leads to GSH-controlled formation of glutathione transferase inhibitors[END_REF].

Similar to GSNOR, CR1 metabolizes GSNO to an intermediate product which can then react with GSH to produce NH2OH and GSSG, thus there is no NO liberation in its catalytic reaction as well [START_REF] Staab | Studies on reduction of S-nitrosoglutathione by human carbonyl reductases 1 and 3[END_REF].

The thioredoxin system

The Thioredoxins (Trx) system comprises three main players, Trx, thioredoxin reductase (TrxR), and NADPH, and it plays a critical role in the control and maintenance of redox homeostasis (Nishiyama et al., 2001). Thioredoxins, with a dithiol/disulfide active site are the major cellular protein disulfide reductases [START_REF] Arnér | Physiological functions of thioredoxin and thioredoxin reductase[END_REF]. Two isoforms of Trx localize to subcellular compartments including the cytosol (Trx1), nucleus (Trx1), and mitochondria (Trx2) and they are responsible for reduction of protein disulfides via the active site vicinal dithiols.

Reduced Trx (rTrx) reacts directly with a protein disulfide through a thiol-switching mechanism to yield a reduced protein dithiol and oxidized Trx (oTrx) (Figure 20).

Although Trx has long been recognized as a target of S-nitrosation [START_REF] Haendeler | Redox regulatory and anti-apoptotic functions of thioredoxin depend on S-nitrosylation at cysteine 69[END_REF], it has also been identified as a major player in the reduction of low-molecular weight and protein S-nitrosothiols where it participates in both denitrosation and transnitrosation reactions [START_REF] Mitchell | Thioredoxin catalyzes the S-nitrosation of the caspase-3 active site cysteine[END_REF][START_REF] Sengupta | Thioredoxin catalyzes the denitrosation of low-molecular mass and protein S-nitrosothiols[END_REF][START_REF] Barglow | Site-specific and redox-controlled S-nitrosation of thioredoxin[END_REF]. Through denitrosation, rTrx reacts directly with either a low-molecularweight or protein S-nitrosothiol (including GSNO) to liberate NO and thiols (Nikitovic and Holmgren, 1996). Through transnitrosation, the active-site thiol for Trx becomes S-nitrosated, leaving behind a low-molecular weight or protein thiol. The nitroso group now residing on Trx will then be turned over through unclear mechanism with release of nitroxyl (HNO) and oxidation of Trx (Figure 20). Oxidized Trx is then reduced by TrxR and nicotine adenine disphosphonucleotide (NADPH). Compared to GSNOR and CR1, the Trx system has ubiquitous expression, hence it has been proposed as the primary regulator of the S-nitroso proteome in most tissues [START_REF] Sengupta | Thioredoxin and thioredoxin reductase in relation to reversible S-nitrosylation[END_REF].

Protein disulfide isomerase

Another enzyme involved in NO transfer is PDI, which catalyzes thio-disulfide exchange reactions, mechanistically similar to RSH-RSNO exchange reaction [START_REF] Zai | Cell-surface protein disulfide isomerase catalyzes transnitrosation and regulates intracellular transfer of nitric oxide[END_REF]. The protein disulfide isomerase catalyzes transnitrosation and denitrosation regulating intracellular transfer of NO (Figure 20). The protein disulfide isomerase has been reported to denitrosate GSNO and it is postulated that NO released via this reaction combines with dioxygen in the hydrophobic environment of either the cell membrane or the PDI protein structure, to form N2O3 [START_REF] Sliskovic | Characterization of the S-denitrosation activity of protein disulfide isomerase[END_REF] (Figure 20). This "NO-charged PDI" can perform intra-and intermolecular S-nitrosation reactions similar to that proposed for serum albumin. Reduced PDI was able to denitrosate S-nitrosated PDI (PDI-SNO) resulting in the release of NO.

γ-Glutamyl transpeptidase

γ-Glutamyltranspeptidase is an extracellular cell surface enzyme involved in the catabolism of GSH adducts and the metabolism of thiol-based leukotrienes. The enzyme transfers the γ-glutamyl group of GSH through either hydrolysis or in the presence of co-substrates (an amino acid, a peptide) leaving behind cysteinyl-glycine (Figure 20) [START_REF] Neil | S-Nitrosoglutathione as a substrate for γ-glutamyl transpeptidase[END_REF]. It was shown that GGT used GSNO as a substrate, generating S-nitrosocysteinylglycine to accelerate the decomposition of GSNO. GGT is membrane bound and appears to act during the translocation of GSH across cell membranes to deliver extracellular cysteine. Many substituted derivatives of GSH, and GSSG, are also substrates for GGT, and this enzyme acts to degrade and recycle both GSSG and the products of glutathione S-transferase activity [START_REF] Abbott | Gamma-glutamyl-glutathione. Natural occurrence and enzymology[END_REF].

Cell transport of S-nitrosoglutathione

The S-nitrosoglutathione itself is not directly taken up into cells because of molecular weight and its good hydrophily; however, GSNO treatment increases cell S-nitrosothiol levels in many conditions. Initially, it was hypothesized that GSNO decomposes in the extracellular space to release NO which is then able to diffuse across the cell membrane to S-nitrosate protein targets [START_REF] Ramachandran | N-Dansyl-S-nitrosohomocysteine a fluorescent probe for intracellular thiols and S-nitrosothiols[END_REF][START_REF] Ramachandran | Mechanism of transfer of NO from extracellular S-nitrosothiols into the cytosol by cellsurface protein disulfide isomerase[END_REF].

Enzymes like GGT and PDI are involved in the transmembrane GSNO transportation.

As previously described, GGT can use GSNO as a substrate to generate Snitrosocysteinylglycine (CysGlyNO), which is not stable, further deliver NO spontaneously or transfer NO to L-cysteine, prior to uptake. In a similar way, PDI catalyzes transnitrosation and denitrosation of GSNO to release NO. Besides the NO dependent mechanism, the NO-independent mechanism was also involved in GSNO uptake into cells. This mechanism requires the transfer of the nitroso group from GSNO to another thiol containing amino acid, L-cysteine. This transnitrosation produces GSH and a new low-molecular weight S-nitrosothiol, S-nitroso-L-cysteine (L-CysNO) which is a good substrate for uptake through the L-amino acid transporter system (L-AT) (Rassaf et al., 2003, Zhang and[START_REF] Zhang | The mechanism of transmembrane S-nitrosothiol transport[END_REF]. L-CysNO is readily transported into cells and can either S-nitrosate cellular GSH to reform GSNO inside the cell or directly S-nitrosate protein thiols to elicit cellular responses. It was observed that the presence of cystine in cell culture media was required for the cellular metabolism of GSNO [START_REF] Zeng | Metabolism of S-nitrosoglutathione by endothelial cells[END_REF]. This is summarized in Figure 21.

Figure 21 A model for cellular GSNO uptake [START_REF] Broniowska | S-nitrosoglutathione[END_REF][START_REF] Gaucher | Snitrosation/denitrosation in cardiovascular pathologies: facts and concepts for the rational design of S-nitrosothiols[END_REF]. 1: GSNO was metabolised by GGT forming S-nitrosocysteinylglycine, which further deliver NO spontaneously and then diffused into cell, or transfer NO to L-cysteine. 2: PDI catalyzes transnitrosation and denitrosation of GSNO to release NO. 3: GSNO transfers NO to L-cysteine forming CysNO, which is transported into the cell through the L-amino acid transporter system (L-AT).

S-nitrosoglutathione and intestinal barrier integrity maintenance

GSNO which is secreted from enteric glia cells (EGCs) has been shown a protection and maintenance function in intestinal barrier (Savidge et al., 2007, Cheadle et al., 2013[START_REF] Yu | Enteric glial cells and their role in the intestinal epithelial 202 barrier[END_REF]. Savidge and co-workers showed that intraperitoneally administrated GSNO obviously attenuated the disruption of intestinal barrier induced by enteric glial cell ablation in transgenic mice. In their work, The Ussing Chamber result further confirmed the idea by GSNO significantly restored mucosal barrier function in colonic biopsy specimens from patients with Crohn's disease (Savidge et al., 2007). The mechanism might be implicated with improving expression of peri-junctional F-actin and TJ proteins such as zonula occludens-1 (ZO-1) and occludin after GSNO treatment (Savidge et al., 2007). The same function of GSNO preventing intestinal barrier breakdown was observed by Cheadle. The in vitro model demonstrated that the maintenance of the intestinal barrier function by increasing the localization of the intestinal tight junction proteins, such as ZO-1, occudin and phosphorylated MLC (Cheadle et al., 2013). Furthemore, GSNO can attenuate the intestine inflammatory response by redox-sensitive S-nitrosylation of nuclear factor κB (NF-κB) inflammatory signaling, inhibiting the transcription of proinflammatory mediators such as TNF-α [START_REF] Reynaert | Nitric oxide represses inhibitory κB kinase through S-nitrosylation[END_REF]. Changing NF-κB inflammatory signaling also plays vital roles in the inhibition of endothelial cell adhesion molecules that accelerate leukocyte infiltration [START_REF] Awad | Cold induces reactive oxygen species production and activation of the NF-kappa B response in endothelial cells and inflammation in vivo[END_REF]. However, it is noteworthy that GSNO did not regulate the intestinal barrier integrity in a dose-dependent manner. It was reported that disruptive function of GSNO on the epithelial integrity was obtained at relatively higher concentrations (Savidge et al., 2007). The in vitro study showed that GSNO promotes a significant increase in Caco-2 transepithelial electrical resistance (TEER) at 5-100 µmol/L. This TEER is reversed at concentrations ≥150 µmol/L. In addition, Tetramethylrhodamine isothiocyanate phalloidin-labeling of MDCK cells in the presence of different GSNO concentration (10 µmol/L, and 250 µmol/L) for 24 hours demonstrated that at low micromolar concentrations GSNO promotes tight-junctionassociated proteins to associate with cytoskeletal components, whereas at higher and potentially pathogenic doses it directly disrupts this cytoskeletal F-actin network.

However, the molecular mechanism remains unclear. It could be attributed to altered NO production. GSNO is a potent nitric oxide donor, which can function to Snitrosylate proteins and play an important role in proper epithelial ion transport [START_REF] Jaffrey | Protein S-nitrosylation: a physiological signal for neuronal nitric oxide[END_REF][START_REF] Marshall | S-nitrosylation: Physiological regulation of NF-κB[END_REF]. GSNO works not only by releasing NO but also by the residual, reduced glutathione (GSH) which is known as an antioxidant cytoprotective molecule [START_REF] Dringen | Glutathione metabolism in brain -Metabolic interaction between astrocytes and neurons in the defense against reactive oxygen species[END_REF][START_REF] Yap | Determination of GSH, GSSG, and GSNO Using HPLC with Electrochemical Detection[END_REF]. GSH reacts nonenzymatically with radicals (R•) and is the electron donor for the reduction of peroxides (ROOH) in the reaction catalyzed by GPx. GSH is regenerated from GSSG by GR which uses NADPH as cofactor (Figure 22).

Figure 22 Function of GSH as an antioxidant [START_REF] Dringen | Glutathione metabolism in brain -Metabolic interaction between astrocytes and neurons in the defense against reactive oxygen species[END_REF].

GSNO is not available as a prescription drug in any format till now. The apparent lack of drug testing and clinic application investigation may be due to its instability and thus a sufficiently stable drug formulation is difficult to be achieved. Compared with NO it has better stability. However it's still too unstable as they are susceptible to physical factor such as light, temperature, divalent cations and biochemical factors such as enzymes [START_REF] Gaucher | Snitrosation/denitrosation in cardiovascular pathologies: facts and concepts for the rational design of S-nitrosothiols[END_REF]. Therefore, if GSNO want to be used for oral administration, a delivery system is necessary to protect GSNO from the harsh environment of gastrointestinal tract (oxygen, enzyme...), which can also release effective amount of GSNO in a controlled way.

S-nitrosoglutathione related delivery system

The delivery of GSNO poses substantial challenges owing to its susceptibility to degradation and small size (easy to diffusion, which may cause low encapsulation efficiency and burst release). The aim of GSNO encapsulation is not only to protect GSNO from degradation but also to control GSNO release in effective concentration.

The bioactivity of GSNO are also location dependent, so the encapsulation strategy can reveal targeting in functional site as well with another goal of minimum the systemic side effect. Because of the real challenges, there are not a lot of publications mentioned about GSNO formulations (Table 2). After a careful literature searching, all the approaches can be clarified into three strategies:

1) Development of GSNO conjugated formulation 2) Encapsulation of glutathione (precursor of GSNO), thereby constructing a thiolloaded formulation, followed by S-nitrosation

3) Direct encapsulation of GSNO in delivery system such as liposomes, polymeric nanoparticles/microparticles, polymeric film/matrix/hydrogel and polymer nanocomposite particles…

Each strategy has merits and drawbacks. For illustration, the first one can extend the half-life of GSNO and easily overcome the disadvantage of small molecule, leading to GSNO diffusion limitation, resulting in higher GSNO loading capacity (depending on the S-nitrosation yield) and sustained release. However, the main issues are the potent toxicity induced by reagents or organic solvent used in synthesis, complicated synthesis and purification process. The second strategy provide the large opportunities to avoid S-NO bound cleavage during the preparation process with a S-nitrosation at the step.

Nevertheless, GSNO encapsulation efficiency was limited by the poor presence of active and available thiol group. Unlike the two above strategies, the last one is independent from chemical reaction, which facilitates the manufacture process, avoids limited S-nitrosation yield. As a consequence, GSNO encapsulation efficiency is relatively higher. But it is challenging to preserve the S-NO bond integrity throughout the formulation process.

S-nitrosoglutathione conjugated delivery system

As mentioned previously, GSNO directly applicated as a therapy is limited because of the lack of stability mainly due to light, temperature, metal ions and enzymes… Thus, some researchers focused on the development of conjugation GSNO with macromolecular to aim at improving GSNO stability and achieving sustained release or increase biological activity. For example, binding chitosan to GSNO has been reported as a method to increase the storage and delivery of GSNO [START_REF] Shah | Synthesis and characterization of S-nitrosoglutathione-oligosaccharide-chitosan as a nitric oxide donor[END_REF].

With this new macromolecular, up to 525.08 ± 151.35 μmol of GSNO per gram polymer was obtained. When contact with intestine tissue, 20% S-NO bond integrity was preserved for this new macromolecular while 100% GSNO was degraded during 6 h.

In another study, GSNO was chemically linked to alginate, which leaded to a loading capacity of GSNO up to 468 ± 23 μmol/g polymer [START_REF] Shah | Synthesis of S-nitrosoglutathionealginate for prolonged delivery of nitric oxide in intestines[END_REF]. Snitrosoglutathione-alginate finally showed better stability and sustained release of NO.

S-nitrosophytochelatins(SNOPCs) are oligopeptide analogues of GSNO [START_REF] Heikal | Characterisation of the decomposition behaviour of S-nitrosoglutathione and a new class of analogues: S-Nitrosophytochelatins[END_REF], Heikal et al., 2011). with by increasing the NO "load" per mol compound, the SNOPCs exhibited more potent than GSNO in inhibiting platelet aggregation, stimulating vasorelaxation and targeting intracellular. However, the rate of NO release in buffer was greater for SNOPCs in comparison to GSNO. For further stability and sustained released improvement, the GSNO conjugated macromolecular can be assembled as nanostructures to present also a better site-specific delivery. Selfassembled polymeric nanoparticles by GSNO conjugated diblock copolymers which includes oligoethylene glycol-methacrylate and 2-vinyl-4,4-dimethyl-5-oxazolone monomer, resulted in an 5 times more enhanced stability compared with GSNO, which were also non-toxic and could efficiently release NO intracellularly [START_REF] Duong | Intracellular nitric oxide delivery from stable NOpolymeric nanoparticle carriers[END_REF].

S-nitrosation of glutathione related delivery system

Encapsulation of GSH (precursor of GSNO) and further S-nitrosation to form GSNO presented an option to delivery GSNO. As reported, GSH was encapsulated into mucoadhesive alginate/chitosan nanoparticles by polyelectrolyte complexation firstly, followed by thiol group S-nitrosation, leading to GSNO encapsulation (Marcato et al., 2011, D. Marcato et al., 2013, Pereira et al., 2015). The rates of NO release from GSNO are greatly reduced due to GSNO encapsulation into biodegradable and non-toxic nanoparticles. Furthermore, the GSNO encapsulation decreased the cytotoxic effect of GSNO in fibroblast V79 cells. Thus, the better stability of encapsulated GSNO compared to free GSNO and the absence of cytotoxicity of alginate/chitosan/GSNO nanoparticles enables the use this new NO-nanocarrier in pharmaceutical applications, or other uses where the NO effects are required, without severe side effects. In another study, the surfaces of the superparamagnetic iron oxide nanoparticles (SPIONs) was coated with GSH firstly, followed by free thiol group S-nitrosation (Santos et al., 2016).

The amounts of NO released from GSNO-SPIONs were 124 ± 1.0 μmol of NO per gram of nanoparticles (NP), which is considered to have therapeutic effects in different biomedical applications, such as the inhibition of platelet adhesion, promotion of wound healing and increased blood flow [START_REF] Ignarro | Nitric oxide: biology and pathobiology: Academic press[END_REF][START_REF] Georgii | Topical S-nitrosoglutathione-releasing hydrogel improves healing of rat ischaemic wounds[END_REF][START_REF] Molina | Nitric oxide donor superparamagnetic iron oxide nanoparticles[END_REF]. Alternatively, in in vitro/in vivo, GSH-coated nanoparticles are reported to facilitate nanoparticle internalization, since cells intake of GSH is rapidly increasing during pathological conditions including inflammation and cancer. Therefore, the remaining un-nitrosated GSH residual may help the NP serve as targeted nano-vehicle and/or diagnostic tool in biomedical applications.

Direct S-nitrosoglutathione encapsulation

Through Table 2, it demonstrated that most studies concentrated on direct encapsulation of GSNO. Different formulation forms were developed to encapsulate GSNO, such as polymeric nanoparticles, in situ microparticles, liposomes, films, stents/scaffolds, hydrogels, and polymer nanocomposites… For a topical wound healing treatment. GSNO incorporated into different polymeric films made of poly(vinyl alcohol), poly(vinyl pyrrolidone) (Seabra and De Oliveira, 2004[START_REF] Seabra | Solid films of blended poly (vinyl alcohol)/poly (vinyl pyrrolidone) for topical Snitrosoglutathione and nitric oxide release[END_REF], Simoes and de Oliveira, 2010) or chitosan (Kim et al., 2015).

These polymeric films imposed a great protection of GSNO from decomposition, stabilizing the storage and controlling local NO release. Pluronic ® F-127 hydrogel incorporate GSNO with consistent, sustained and biologically effective release of NO was developed for different topical treatment application such as thermal and photochemical delivery of GSNO to target areas [START_REF] Shishido | Thermal and photochemical nitric oxide release from S-nitrosothiols incorporated in Pluronic F127 gel: potential uses for local and controlled nitric oxide release[END_REF], inflammation pain and impaired dermal blood flow (Seabra et al., 2004a[START_REF] Vercelino | Skin vasodilation and analgesic effect of a topical nitric oxidereleasing hydrogel[END_REF] and ischaemic wounds [START_REF] Georgii | Topical S-nitrosoglutathione-releasing hydrogel improves healing of rat ischaemic wounds[END_REF]. For a female sexual dysfunction application of GSNO, it was encapsulated in a gel Carbopol 934P/HPMC/Polyethylene glycol films to achieve improved GSNO stability (at least 30 days) and increased duration of GSNO action (at least 6 h) (Yoo et al., 2009). Respectable researched were focused on more complex GSNO delivery systems such as on poly(lactide-co-glycolide)/poly(εcaprolactone) implants (Parent et al., 2015a) (Acharya et al., 2012) . All these scaffolds were developed for treatment of cardiovascular diseases and demonstrate a stable release trend of NO over al least 24 h, some even up to 28 days. Furthermore, innovative lipid and polymer particles using more complex formulation processes were also proposed. Phospholipids and cholesterol liposomes incorporating GSNO demonstrated sustained release (less than 30% of encapsulated GSNO was released within 8 h) and macrophage targeting behavior (Diab et al., 2016).

With GSNO loaded polymer Eudragit ® RL nanoparticles, improved stability, delayed protein S-nitrosation and enhanced cellular uptake were observed compared with nonencapsulated GSNO (Wu et al., 2015a). With a further incorporation of GSNO-Eudragit ® RL nanoparticles within alginate or chitosan or the blend polymer matrix, a stronger protection and better sustained release were obtained, leading in vivo to enhanced GSNO intestinal absorption and promotion of a releasable NO store into the rat aorta (Wu et al., 2016). Nanocomposite formulations presents advantages, such as control of NO loading, spherical shape favor exchanges with environment et al. and disadvantages, such as difficulties to peptide encapsulation in lipophilic tools (limitation in loading), risk of degradation during process…it will be further investigated in the following part, focusing efficient GSNO oral delivery.

Polymer nanocomposites for drug oral delivery: development strategies and potentialities

Polymer nanocomposites are complex drug delivery systems, which combine nanoscale tool embedded into a polymer matrix. The following part (reviews proposed as a chapter book) clarifies the development and potentialities of polymer nanocomposites adapted to oral delivery. Advantages and inconvenient of these drug delivery systems for drug controlled release will be discussed.

LIST OF ABBREVIATIONS

Introduction

In the past two decades, the improvement and application of nanotechnology in the biomedical field has resulted in enormous work leading to hundreds of articles with new ideas, trends and methods for the development of a suitable drug delivery system.

Polymer nanocomposites (NC) have been designed to combine the merits of both polymer matrix and nanoplatforms in one system, improving mechanical properties (such as tensile strength, elastic modulus or varying stiffness), permeability, crystallinity, thermal stability, biodegradability, and biocompatibility (Bhattacharya et al., 2008). Not a mixture of two materials, the NC may be presented as a core-shell, a particle or a bulk (Figure 1). These carriers are composed of three main components: i) the drug to be released, ii) the core material or nanoplatform, iii) the shell material or polymer matrix. The properties of drug such as solubility, lipophilicity, surface charge and therapeutic index determine the core materials nature to form nanoplatforms. The drug can be encapsulated in the nanoplatforms, adsorbed on the surface of nanoplatforms or conjugated with polymer matrix. The unique location allows controlling the manner and rate of the release process. Organic nanoplatforms consist of polymeric nanoparticles, solid lipid nanoparticles, polymeric micelles, dendrimers or liposomes (Figure 1). The polymer matrix can be based on synthetic (controlled degradation and well-defined physicochemical properties) and natural polymers (low toxicity, biocompatibility and biodegradability).

This chapter will not attempt to cover all areas, including mixtures of two or more materials forming a homogeneous mixture (so not composites), neither the entrapment of nanocrystals composed of pure drug in polymer matrix. In this case, no nanocarriers were present and the nanocomposites were limited to only two components.

The main objective of this chapter is to describe the formulation strategies and potentialities of polymer NC made by the incorporation of nanoplatforms into a polymer matrix for improved delivery efficiency.

Influence on drug properties

As previously described, polymer NC generally consist of three main components. The selection of the different materials and the fabrication methodology depend on the properties of drug that will be encapsulated, such as solubility, lipophilicity, surface charge and therapeutic index… Polymer NC have turned into a versatile platform especially for applications in the field of biomedicine thanks to their capacity to load different substances such as hydrophilic and hydrophobic drugs, peptides and proteins, and nucleic acids (Table 1).

With a careful selection of the nanoplatforms, the polymers and the methodology of production, polymer NC can be exploited to incorporate both hydrophilic and hydrophobic drugs. The hydrophobic drugs were often limited by their solubility, the application of hydrophilic polymeric encapsulation contributed to improve their solubility (Wang et al., 2014, Kang and Ko, 2015, Delmar and Bianco-Peled, 2016, Mansuri et al., 2016). Whereas hydrophilic drug delivery was often challenged by poor cellular permeability, the encapsulation in hydrophobic polymeric or lipidic nanoparticles was presented as promising strategies to improve the bioavailability (Garcıa-Fuentes et al., 2003[START_REF] Vrignaud | Strategies for the nanoencapsulation of hydrophilic molecules in polymer-based nanoparticles[END_REF].

This strategy was also developed for peptides and proteins. The short half-life and fragility of peptides and proteins reveal the need to develop sustained delivery systems with very controlled burst effects. However, the challenge in protein drug delivery is to conserve its conformation, crucial for the bioactivity. Exposure of proteins to unfavorable conditions during the manufacturing process tended to attenuate their bioactivity (Yeo et al., 2001). Two general approaches have been used to avoid protein denaturation during the production of polymer nanocomposites: ⅰ) incorporation of solid protein nanoparticles into hydrophobic polymeric microparticles (Leach et al., 2005, Han et al., 2009, Klingler et al., 2009, Kakizawa et al., 2010); ⅱ) combination with hydrophilic polymers, which are biocompatible with peptide and protein drugs, and avoid direct contact with hydrophobic polymers, which might induce unfolding of peptide or protein drugs [START_REF] Sluzky | Kinetics of insulin aggregation in aqueous solutions upon agitation in the presence of hydrophobic surfaces[END_REF]. Thus, peptides or proteins could be primarily encapsulated in nanoparticles made of hydrophilic polymers such as dextran or gelatin (Li et al., 1997) or alginate [START_REF] Schoubben | Novel composite microparticles for protein stabilization and delivery[END_REF], for better encapsulation efficiency and preservation of bioactivities, and then microencapsulated in a hydrophobic polymer like poly(lactic-co-glycolic acid) (PLGA) to reach sustained release (Li et al., 1997[START_REF] Schoubben | Novel composite microparticles for protein stabilization and delivery[END_REF].

Besides peptides and proteins, nucleic acid-based macromolecules such as siRNA or DNA-plasmids were also adapted to combine with NC for the protection of these substances from degradation in the GI tract. One of the most important advantages of using polymer NC is the ability to increase nucleic acid loading. Moreover, polymer NC might permit the specific delivery of the nucleic acid in the GI tract such as the intestine. Researchers have encapsulated plasmid DNA containing gelatin nanoparticles into poly(epsilon-caprolactone) (PCL) matrix to form polymer NC with a size less than 5 µm (Bhavsar et al., 2006). In the presence of degrading enzymes, the PCL matrix provided protection for gelatin nanoparticles and supported the DNA specific delivery in intestines. In this way, the plasmid DNA survived from the harsh environment in the stomach and preserved its bioactivity (Bhavsar and Amiji, 2007). When radioactively labeled polymeric NC were administered orally to rats, there was preferential longlasting accumulation in the small and large intestine. In contrast, simple gelatin nanoparticles travelled very quickly along the GI tract. The longer residence of polymer NC in intestine as compared to the unencapsulated gelatin nanoparticles may translate into enhanced transfection efficiency of the plasmid DNA (Bhavsar and Amiji, 2008).

Physicochemical characteristics of polymer matrix

As in traditional composite devices, the role of the matrix in nanocomposites is to support and protect their nanoplatforms. Matrix molecules can be anchored to the nanoplatforms surface by chemical reactions or adsorption, which determines the strength of interfacial adhesion. The polymer NC can be formulated with both synthetic and natural polymers. Synthetic polymers can be tailor-made for specific application, having controlled degradation and well-defined physicochemical properties, such as poly(L-lactic acid) (PLA), PLGA, and PLA/ PLGA-b-poly(ethylene glycol) (PEG).

Natural polymers including chitosan, gelatin, alginate, hyaluronic acid (HA), starch, and dextran present a low toxicity, biocompatibility and biodegradability. The choice of a matrix depends on several factors like application, compatibility with drug and nanoplatform, technique of processing, and costs. In the present section, we will overview the most popular polymers used for the production of polymer NC adapted for oral delivery (Table 1).

Processing technologies

Several methodologies have been developed to prepare polymer nanocomposites.

Generally, this process involves two main steps: first, the production of the nanoplatforms by any of the available methods, it depends on the type of nanoplateform.

In this review, five kinds of nanoplateform were introduced. Polymeric meric/solid lipid nanoparticles, liposome, micelles, dendrimer (Figure 1).

For polymeric nanoparticles, water in oil in water (W/O/W) double emulsion was widely used (Sheikh [START_REF] Hassan | Composite microparticles with in vivo reduction of the burst release effect[END_REF], Sun et al., 2015, Delmar and Bianco-Peled, 2016). The emulsions were formed by dispersing primary emulsion which was obtained by dispersing an aqueous phase into immiscible liquid into aqueous phase. The emulsification is rarely a spontaneous process. Therefore, the input of energy is necessary. Normally, the energy is provided by mechanical shear which comes from different kinds of mixers. The final size of the emulsion is caused not only the chemistry but also power of energy used. To stabilize the emulsion, the use of emulsifiers or emulsion stabilizers are also important. The emulsifiers and emulsion stabilizers are mostly amphiphilic molecules with a hydrophilic "head" attached to a hydrophobic "tail". Nanoprecipitation method was also common applied in prepare the polymeric nanoparticle [START_REF] Reis | Nanoencapsulation I. Methods for preparation of drug-loaded polymeric nanoparticles[END_REF], Joshi et al., 2010, Garrait et al., 2014).

Nanoprecipitation is a facile, mild, and low energy input process for the preparation of polymeric nanoparticles. Generally, the method describes the precipitation of a dissolved material as nanoscale particle after exposure to a non-solvent that is miscible with the solvent [START_REF] Schubert | Nanoprecipitation and nanoformulation of polymers: from history to powerful possibilities beyond poly(lactic acid)[END_REF]. The polymeric nanoparticles whave the drawbacks of the lack of safe polymers with regulatory approval and high cost. Solid lipid nanoparticles (SLNs) were developed to overcome these limitation with advantage of improved drug absorption in the oral delivery system (Mukherjee et al., 2009). They can enhance the mucosal adhesion because of small particles size and long gastrointestinal tract (GIT) resident time. Hydrophilic drugs were usually encapsulated in the solid lipid nanoparticles to acquire better controlled release profile and good encapsulation efficiency. SLNs are consist of solid lipid, emulsifier and water/solvent (Mukherjee et al., 2009). Different techniques were introduced in a review wrote by S. Mukherjee, Such as high shear homogenization, Hot homogenization, cold homogenization, ultrasonication or high speed homogenization, solvent emulsification/evaporation, micro emulsion, supercritical fluid, spray drying method and double emulsion method (Mukherjee et al., 2009). Therefore, no more details will be explained in this chapter.

A liposome is a spherical vesicle consisting of a lipid bilayer surrounding an aqueous core (Liu et al., 2016). The liposome was widely used as a delivery system of nutrients and pharmaceutical drugs. The preparation methods of liposome were described in a review in detail by A. Akbarzadeh. All the preparation methods involve four basic stages: 1). Drying down lipids from organic solvent; 2). Dispersing the lipid in aqueous media; 3). Purifying the resultant liposome; 4). Analyzing the final product (Akbarzadeh et al., 2013). In the review, the preparation techniques include 3 different methods: mechanical dispersion method, solvenr dispersion method and detergent removal method (removal of non-encapsulated material). More details could be found in the review.

Polymeric micelles are formed by self-assembling of amphiphilic block copolymers containing hydrophilic "head" and hydrophobic "tail" in aqueous phase at or above the critical micelle concentration (CMC). Micelles are widely used in encapsulating scarcely water-soluble drugs owing to the core-shell structure. This effective drug delivery system has a lot of merits such as small particle size (<100 nm), targeting ability, prolonging blood circulation duration and easy preparation [START_REF] Sezgin | Preparation and characterization of polymeric micelles for solubilization of poorly soluble anticancer drugs[END_REF].

Due to the properties and interrelations of drug and polymer, drugs can be incorporated into micellar core by chemical conjugation or physical encapsulation trough dialysis or emulsification (Ahmad et al., 2014). In chemical conjugation technique, the formation of covalent bond between the specific group of the drug and the hydrophobic core of the micelles cause incorporation of the hydrophobic drug inside the polymeric micelle core. Such micelles can avoid renal clearance and Reticuloendothelial system (RES) phagocytosis. In comparison to the chemical method, physical method is more favorable for drug incorporation. Polyionic compounds can be incorporated through the formation of polyion complex micelles. Physical entrapment of drugs is generally done by dialysis or oil-in-water emulsion procedure. In dialysis, the drug and polymer are brought from the selective solvent to a solvent that is selective only for the hydrophilic part of the polymer. By the replacement of good solvent with selective one, the hydrophobic portion of the polymer associates to form the micellar core, thus, incorporating insoluble drug. Extending the dialysis over several days can ensure the complete removal of the organic solvent. The oil-in-water emulsion method consists of preparing an aqueous solution of the copolymer to which a solution of the drug in a water-insoluble volatile solvent is added in order to form an oil-in water emulsion. The micelle-drug conjugate is formed as the solvent evaporates. The main advantage of the dialysis procedure over the latter method is that the use of potentially toxic solvents can be avoided.

Dendrimers are nano-sized, radially symmetric molecules with well-defined, homogeneous, and monodisperse structure consisting of tree-like arms or branches [START_REF] Abbasi | Dendrimers: synthesis, applications, and properties[END_REF]. Dendrimers are generally prepared using either a divergentapproach or a convergent one. In the divergent approach, used in early periods, the synthesis starts from the core of the dendrimer to which the arms are attached by adding building blocks in an exhaustive and step-wise manner [START_REF] Abbasi | Dendrimers: synthesis, applications, and properties[END_REF], Mansuri et al., 2016). In the convergent approach, synthesis starts from the exterior, beginning with the molecular structure that ultimately becomes the outermost arm of the final dendrimer. In this strategy, the final generation number is pre-determined, necessitating the synthesis of branches of a variety of requisite sizes beforehand for each generation [START_REF] Grayson | Convergent dendrons and dendrimers: from synthesis to applications[END_REF].

There second, the encapsulation of such nanoplatforms into polymer matrix is usually based on emulsification, gelation and polyelectrolyte complexation. The

Potentialities of polymer nanocomposites

Major goals of polymer NC application in drug delivery are the maximization of drug bioavailability and efficacy, the control of pharmacokinetics, pharmacodynamics, nonspecific toxicity, immunogenicity as well as the overcoming of obstacles arising from low drug solubility, degradation, fast clearance rates, relatively short-lasting biological activity and inability to cross biological barriers.

Solubility improvement

Almost 40% of commercial drugs are poorly water-soluble. In particular, in the biopharmaceutical classification system, class II drugs (poorly soluble and highly permeable drugs) have limited oral absorption because of their inappropriate dissolution rates (Löbenberg and Amidon, 2000). Various methods to improve the dissolution behavior and bioavailability of drugs have been developed (Gulsun et al., 2009[START_REF] Singh | Oral formulation strategies to improve solubility of poorly water-soluble drugs[END_REF], Krishnaiah, 2012, Kesarwani et al., 2014). One of the effective strategies improving drug solubility and drug dissolution rates is reducing the particles size to the nanoscale, either drug nanocrystals or the accociation with nanocarrier (Ozeki and Tagami, 2013; S.R. Krishnaiah, 2010). The surface area of drug particles increases with decreasing particle size and consequently, the large surface area increases the drug dissolution rate. In addition, decreasing the size of drug particles enhances the saturation solubility (Hu et al., 2004, Keck andMüller, 2006).

Cyclodextrins (CD) have been described as "an important tool in the formulator's armamentarium to improve drug solubility and dissolution rate for poorly water-soluble drug candidates" and efforts have been made to predict whether cyclodextrins will be of benefit in creating a dosage form for a particular drug candidate (Loftsson and Brewster, 2010).

The work of Sangwai and coworkers [START_REF] Sangwai | Amorphous ternary cyclodextrin nanocomposites of telmisartan for oral drug delivery: Improved solubility and reduced pharmacokinetic variability[END_REF], explored an innovative ternary beta-CD NC to deliver telmisartan (TEL) having exceedingly pH dependent and poor solubility profile. In comparison with plain drug and marketed formulation, TEL NC exhibited remarkable improvement in in vitro dissolution characteristics in multimedia and biorelevant media. Beside CD, natural carriers like gelatin, acacia, cassia and ghatti gum with less swelling and low viscosity were also used to improve drug solubility thus improving the oral bioavailability (Kushare and Gattani, 2013).

Drug protection

The bioavailability of labile drugs such as peptides and proteins was limited by their poor stability, which was highlighted while encapsulating them in various devices (De Rosa et al., 2000, Woo et al., 2001, Yu et al., 2002, Hinds et al., 2005). The application of polymer NC is beneficial to protect the drug during fabrication procedure and also against enzymatic and hydrolytic degradation in the GI tract by preventing the direct contact between the drugs and the harsh, digestive environments. G. Garrait developed a polymer nanocomposite carrier to protect drug from gastric acid and pepsin, based on two natural polymers, namely chitosan and alginate (Garrait et al., 2014). Chitosan nanoparticles were developed to protect a model molecule (amaranth red) from enzymatic attack in the small intestine. However, the glycosidic bonds of chitosan are rapidly hydrolyzed under gastric conditions (George and Abraham, 2006). Sodium alginate is more resistant, and since it is an anionic polysaccharide with charges opposed to those of chitosan, it was chosen as a natural polymer to protect chitosan nanoparticles from gastric hydrolysis. The intention was to ensure protection of the nanoparticles until they reach the lumen of the small intestine, where they may be released in order to deliver the entrapped active compound. As presented in their study, polymer dissolution and drug release into the gastric environment are slowed down. This polymer NC system allowed a pH-sensitive or polypeptidic drug to pass intact through the stomach (Garrait et al., 2014). For more resistance to the acidic conditions in the stomach, PLGA was used as the polymer matrix. Schoubben developed polymer NC as the delivery vectors for bovine insulin [START_REF] Schoubben | Novel composite microparticles for protein stabilization and delivery[END_REF]. They loaded bovine insulin into alginate nanoparticles, a hydrophilic polymer to produce a friendly environment preventing denaturation by stress conditions. Then these nanoparticles were embedded into PLGA polymer matrix to avoid the degradation of alginate nanoplatform in acidic condition. Compared with the conventional PLGA microparticles, these polymer NC presented higher encapsulation efficiency and protection of the drug with improved stability during the in vitro release under acidic pH [START_REF] Schoubben | Novel composite microparticles for protein stabilization and delivery[END_REF].

Modification of drug release

In drug therapy, it is important to provide and maintain therapeutic levels of pharmaceutically active agents to the site of action throughout the treatment.

Furthermore, it is necessary to minimize temporal variations in drug concentration that can lead to periods of over-dosing. As described previously, the main target of drug absorption for oral delivery is the intestine. However, in physiological conditions, the delivery system has to survive in extreme acidic condition of stomach before reaching the desired target. So when, where and how to release the drug from the delivery system is an important consideration in the design of formulations. To these considerations, modified release technologies are employed to deliver drugs in a controlled manner, providing some actual sustained therapeutic doses within the desired time frame and/or spatial control of drug release. In this respect, polymer NC could be potential carrier for controlled drug delivery due to their enhanced functionality and more complex structures, compared with single-component counterparts. Mainly, the release of drug from polymer NC depended on the diffusion alone or was accompanied with the erosion of polymer matrix. Different combination of nanoplatform and polymer matrix could achieve the reduction of burst release, sustained release, stimulate release and target release.

Reduction of burst release

One of the prominent merits of polymer NC as delivery system is the reduction of the burst effect, especially when the drug was previously encapsulated in lipidic or polymeric nanoparticles then incorporated into the polymer matrix. The association of polymer matrix in NC provides prolonged diffusion distance that slows down the release speed and achieves sustained delivery, because first the drug has to overcome the nanoparticle barrier, and then cross the polymer matrix. It was demonstrated by different researchers that the polymer matrix made with hydrophobic polymers such as PLA, PLGA, PCL, Eudragit ® RS 100 or ethylcellulose (EC) diminished the burst effect more effectively than hydrophilic polymers such as gelatin, alginate, and chitosan.

These hydrophilic polymers used for nanoparticles might favor water uptake by the microparticles and consequently induce a faster release and/or a faster hydrolysis of the non-soluble polyester polymer, as it was shown in the case of the PCL microparticles.

For instance, PCL encapsulated ibuprofen nanoparticles presented obvious burst effect in vitro release. The further incorporation of PCL nanoparticles into polymer matrix (PLGA, RS, EC or the blend of RS and EC) reduced the initial burst release efficiently.

These systems were also adapted to triptorelin acetate, ibuprofen and insulin (Hasan et al., 2007, Sun et al., 2015) (Table 3).

Target delivery

Drug targeting is a useful tool for achieving selective and efficient delivery of drug at the anticipated site of action with minimized unwanted side effects. Different strategies based on physical environment leading to time or pH dependent delivery systems or based on biological environment such as microflora or enzymes [START_REF] Prasad | Colon targeted drug delivery systems: A review[END_REF] have been proposed.

The most investigated targeting system is pH-dependent polymer NC devoted to protect the drug in acidic environment (as described in protection of drug) and release in the intestine, the promising absorption site, or localized targeting to colonic region (Chourasia and Jain, 2003). The systems are suitable for oral administration leading systemic activities, and the treatment of colonic region diseases like colorectal cancer or inflammation bowel disease. As described by Huang, indomethacin polymer NC, constructed by incorporation of drug loaded micelles into alginate beads, presented modified release behavior when compared to indomethacin loaded simple micelles and indomethacin loaded simple alginate beads (Huang et al., 2012). Specifically, indomethacin was released at neutral pH instead of acidic pH, thus, its side effect in stomach was diminished.

Conclusion:

This chapter summarized the formulation strategies and potentialities of polymer nanocomposites in oral delivery. According to the drug properties, different materials and processing technologies will be selected. Compared with oral drug administration alone, the nanoplateform can provide good protection from the harsh gastrointestinal environment. With the further encapsulation of polymer matrix, oral administrated drug can have further protection, higher loading efficiency, better controlled release, reduced burst release and specific target delivery. Thus, the combination of nanoplateform and polymer matrix strengthened the drug therapeutic efficiency. In conclusion, polymer nanocomposites are promising candidates for oral delivery.

Introduction

In direct contact with external environment, intestinal mucosa plays the crucial role of barrier that can avoid the invasion of the mucosa by microbiota. As described in chapter 1 (paragraph 1.2.1). The functional intestine mucosal barrier consists of a mucus layer on the surface of epithelial cells, strong junctions between adjacent epithelial cells, and immune cells involved in the innate immune response. In healthy physiological conditions, this intestine barrier is efficient and keep intestine homeostasis. In contrast, this barrier is disrupted in IBD patients. The disruption of the intestinal barrier is an important cause of IBD because it allows pathogen and toxin penetration and triggers an excessive and uncontrolled inflammation. The disruption of tight junction structure is always followed with intestinal barrier defects, proinflammatory cytokines production (such as TNFa, IL-1β), activation of NF-κB signaling pathway, expression of MLCK expression, which subsequently increases the intestinal epithelial permeability by disrupting epithelial TJs in intestinal inflammation (Li et al., 2016).

Therefore, a potent therapeutic approach of preventing intestinal inflammation is to improve the intestinal mucosal barrier function by reducing intestinal permeability.

S-Nitrosoglutathione (GSNO) is the endogenous S-nitrosylated derivative of the major antioxidant glutathione (GSH). GSNO, which is secreted by Enteric Glia Cells (EGCs) from Enteric Nervous System (ENS), has been shown to maintain and protect intestinal mucosal barrier (Savidge et al., 2007[START_REF] Flamant | Enteric glia protect against Shigella flexneri invasion in intestinal epithelial cells: a role for S-nitrosoglutathione[END_REF], Cheadle et al., 2013).

Savidge and co-workers showed that glial-derived GSNO was identified as a potent GSNO effects on intestine exhibited a dose-dependent way in both in vitro and in vivo studies (Savidge et al., 2007[START_REF] Goncalves | Treatment of bowel in experimental gastroschisis with a nitric oxide donor[END_REF]. Different concentrations of GSNO (5 μM -250 μM) exposure to Caco-2 cells monolayer. Dose-response curves showed that GSNO promoted a significant increase in Caco-2 TEER at 5-100 μM. This TEER was reversed at concentrations >150 μM. At low micromolar concentrations GSNO promotes tight-junction-associated proteins to associate with cytoskeletal components, whereas at higher and potentially pathogenic doses it directly disrupts this cytoskeletal F-actin network. An in vivo study showed also dose effect (0.05 μM-5 μM) on intestine. Fetal treatment with lower concentration of GSNO (0.05 μM) resulted in significant better improvement of bowel morphology in gastroschisis [START_REF] Goncalves | Treatment of bowel in experimental gastroschisis with a nitric oxide donor[END_REF].

To summarize all these literatures, the source of GSNO is from the mucosal side of intestine (secreted by EGCs and femoral venous injection…) However, no publication investigates GSNO which comes from the luminal side of intestine (after oral administration) to date. Therefore, the first aim of present study is to prove the concept that luminal GSNO can also has a protective effect on intestine as mucosal GSNO.

Study of the impact of the GSNO on the intestinal barrier permeability

To confirm our first hypothesis, GSNO impacts on the intestinal barrier permeability was investigated in Ussing chamber by following a paracellular marker permeability after different concentration GSNO treatment in luminal side of the chamber (donor compartement). And the mechanism was studied by measuring the junction protein expression with western blot. That constitute the article presented below (to be submitted in Journal of Gastroenterology).

Introduction

Several actors ensure integrity and permeability of the intestinal barrier which both play a critical role in the defense of the body against external environmental aggressions.

The intestine mucosa allows renewing of the epithelium through stem cells every 3 to 5 days (Potten et al., 2002). Cell junctions -which are divided, from intraluminal to deeper parts of the intestinal wall, into tight junctions, adherent junctions and desmosomes -are the main elements involved in the maintenance of integrity (desmosomes) and function of the intestinal barrier: tight junctions and adherent junctions especially regulate intestinal permeability and absorption, as entry of luminal elements through conjunctive tissues to intestinal vessels (Turner, 2000). Under physiological conditions, intercellular crossing is limited to small molecules. A selective crossing through enterocytes is also possible using transporters. Mucuscomposed of glycoproteins (mainly mucins) and forming a gel recovering the epithelium (Kim and Ho, 2010) -also contributes to intestine integrity. Immune cells, in Peyer's patches and in conjunctive tissue, reinforce the role and the maintenance of the barrier integrity by an innate immune response and a control of inflammation (Hooper and Macpherson, 2010). Finally, the intestine microbiota takes over food components that cannot be absorbed such as fibers and digestible polysaccharidic compounds and contribute to the renewing and contributes to maturation and renewing of the epithelium, and to cellular differentiation (Payros et al., 2014).

Amongst the different models used to evaluate intestine permeability, the Ussing diffusion Chamber model developed by Hans Henriksen Ussing in 1946, allows ex vivo study of the transport of ions, nutrients or drugs through a donor to an acceptor compartments separated by an insert on which is fixed the intestinal tissue (Clarke, 2009). Both sides of the intestinal tissue are immersed in an osmotic solution maintained at 37°C. This technique permits to study absorption differences, by dosage of components in both compartments and inside the tissue, according to the different intestinal sections. This requires low amounts of molecules. In this model, morphological and functional modifications can be observed during the experiment.

Intestinal permeability is witnessed by measuring the crossing (flux) of a substance through the intestinal barrier, while intestinal integrity is reflected by the trans-epithelial electrical resistance (TEER). The later traduces the viability of the tissue and witnesses the open state of cell junctions (Srinivasan et al., 2015). The effect of an active substance on the barrier itself can be evaluated using markers of intestinal permeability (sodium fluorescein, NaFlu, medium permeability, paracellular transport).

At the intestinal level, nitric oxide (NO) plays an important but paradoxical role: in one hand, NO has also beneficial effects: at smaller concentrations than above, it is the most important neurotransmitter at the gastro-intestinal level, allowing to control gastric motility. It also allows to control intestinal secretion of mucus and to improve absorption in physiological conditions (Kochar et al., 2011). In the other hand, excessive production of NO has been correlated with sustained inflammation in the colon, particularly observed for epithelial cells near to the inflammation site (Kolios et al., 2004). This is due to an increased production of peroxynitrites ions (ONOO -).

Therefore, new therapeutics of IBD, with the challenges to maintain low concentrations of NO during remission period of IBD (Kochar et al., 2011) remain to be developed.

Under its gaseous form, NO is unstable (half-life within seconds) and is easily and uncontrollably reacted in its environment (Dattilo and Makhoul, 1997). Thus, NO donors are necessary to avoid this drawback. NO donors show longer half-life and some of them are already used in therapeutic (i.e. cardiovascular context (Parent et al., 2013b)). One family of NO donors is the S-nitrosothiols (RSNO) with the Snitrosoglutathione (GSNO) as the leader molecule. GSNO is a very interesting molecule as it is naturally produced in the organism. This GSNO is derived from the most important intracellular thiol, glutathione (GSH, a tripeptide gamma-glutamylcysteinyl-glycine that intervenes in RedOx regulation by the glutathione/disulfide glutathione ratio). GSNO secreted by enteric glial cells after stimulation of the vagus nerve prevents inflammatory events and preserves barrier integrity (Savidge et al., 2007, concentration is of 10 -2 M with a tolerance of ± 5 %.

Animals and ethical statement

Intestinal barrier integrity was evaluated on ileum isolated from 12-week-old, male, normotensive, Wistar rats (Janvier Laboratories, Le Genest-St-Isle, France; 400-500 g).

All experiments were performed in accordance with the European Community guidelines (2010/63/EU) for the use of experimental animals in the respect of the 3 Rs' requirements for Animal Welfare. The project untitled "Nitro-Vivo" was positively evaluated by the regional ethical committee for animal experiments and approved by the French Ministry of Research (n°APAFIS#1614-2015090216575422v2). Animals were kept under standard conditions (temperature: 21 ± 1 °C, hygrometry 60 ± 10%, light on from 6 am to 6 pm) and had diet ad libitum (A04, Safe, Villemoisson-sur-Orge, France) and water (reverse osmosis system, Culligan, Brussells, Belgium).

Wistar rats were sacrificed by exsanguination after anesthesia (sodium pentobarbitone 60 mg/kg, intraperitoneal, Sanofi Santé Nutrition Animale®, Libourne, France). An ileum segment (around 12 cm) of the rat intestine was removed and immediately placed in cold Krebs-Henselheit buffer (NaCl 119, NaHCO3 24, KCl 4.7, MgSO4 1.2, KH2PO4

1.2, CaCl2:2H2O 1.6 and glucose 10 mM; pH adjusted to 7.4 and equilibrated with 95 % O2 / 5 % CO2 for 30 minutes before use). Intestine pieces were cleaned from fat tissue and cut into 2-cm long segments (6 segments per rat). The segments were opened along the mesenteric vein, cleaned with cold Krebs-Henselheit buffer, pinned on insert and immediately placed in Ussing Chamber system. A 6-Ussing Chamber bench (P2404 Physiologic Instrument®, Easymount LVSYS, Harvard Apparatus) was used. In each chamber, compartments were filled with Krebs-Henselheit buffer (2.5 mL), temperature was maintained at 37°C in continuous circulation (using a water bath) and pH was maintained at 7.4 with gas infusion (12 mL/min using a peristaltic pump).

J in μg•cm -2 •s -1 , Papp in cm•s -1 ; Q in mg; t in s; A in cm², C0 in mg•cm -3 ).

Evaluation of the effects of GSNO on intestine permeability and integrity

First, stability of GSNO was evaluated by measuring its concentration all over the 2 hours under experimental conditions, without intestine (100 µM = 500 nmol added to the donor compartment at 37 °C, Krebs-Henselheit buffer with 95% O2 / 5% CO2 at 12 mL/min).

Then, a second series of intestinal segments was exposed to GSNO (0.1 µM -100 µM, donor compartment), with 10 min equilibrium before starting the experiment on 2 hours, in order to measure permeability marker and TEER. In parallel, for each series of 6

Ussing Chambers, one or two were dedicated to control conditions (therefore, the control group includes the highest number of experiments, see Figure 3), in order to evaluate quality and viability of the segments and allow comparison of results between each series of Ussing experiments. Quality criteria were fixed according to NaFlu flux at t = 2 h, and TEER values, on control condition, to have more reliable results. At the end of the experiment, intestinal tissues were taken out of the Ussing chambers and placed in plastic tubes, frozen in liquid nitrogen and conserved at -80°C until western blot analysis.

Cell junction proteins expression by Western blot

Intestinal piece was taken out from -80°C and mashed into powder, using mortar and pestle, in liquid nitrogen. Proteins were extracted from the tissue in RIPA buffer (NaCl 140; NaH2PO4 12; KH2PO4 2 mM; sodium deoxycholate 0.5%; nonidet P-40 1%, SDS 1%) with additional polymethylsulfonyl fluoride, sodium orthovanadate and protease inhibitor cocktail. Firstly, tissue powder was incubated with RIPA buffer (ice, 10 mins), then homogenized. Mixture was frozen and thawed, for 3 cycles, in liquid nitrogen and 37°C water bath. After centrifugation (12 000 × g, 4 •C, 30 mins), supernatant was used for proteins quantification by bicinchoninic acid (BCA) assay.

Protein samples were mixed with an equal volume of Laemmli 2X sample buffer and heated to 95°C for 5 mins. Total proteins (30 µg) were separated with sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes.

The membranes were blocked with 5% bovine serum albumin or non-fat milk/TBST

for 1 hour at room temperature and then incubated with primary antibodies (occludin,1:600; claudin-1, 1:600; E-cadherin, 1:800; β-catenin, 1:700) overnight at 4°C. After washing in TBST, the appropriate HRP-conjugated secondary antibody was added and the membrane was incubated for 1h at room temperature. After further washing in TBST, the proteins were detected using an ECL or ECL PLUS kit (Amersham, Velizy-Villacoublay, France), and with the "fusion" software (Vilber®).

Densitometric analysis was performed with "Image J" software.

β-actin was used as a reference protein, and so the relative amount was expressed with the ratio of protein /beta-actin expressions. Results were normalized from "wild type" group and expressed in percentage: "wild type" is a tissue harvested from animal and directly frozen; control+Ussing is a tissue that has been set in Ussing system and frozen after 2-hours experiment without treatment.

Statistical analysis

Results are expressed as mean ± standard error of the mean (sem). Statistical comparisons were performed using a one-way (groups: controls and GSNO at different concentrations) or two-way (groups and time) ANOVA, or a linear regression analysis for correlation test (p < 0.05). Statistical analyses were performed using the GraphPad Prism software (GraphPad Software version 5.0, San Diego, USA).

Results

Assessment of quality criteria

Before the real experiment starts, quality criteria of Ussing Chamber should be validated to obtain reliable result: in the present study, the ex vivo intestine viability is crucial. A preliminary study was performed for 3 h, following NaFlu flux and TEER values that significantly changed at t=3 h, indicating a loss of intestinal barrier integrity.

Therefore, the Ussing Chamber duration was limited to 2 h, to have more reliable result:

this drastic intestine tissue viability decreasing after 2 hours was also observed in the literature (Forsgård et al., 2014).

The results of NaFlu permeability and TEER values of intestine tissue without any treatment in Ussing Chamber (control condition) were given in Figure 1. After calculation of NaFlu flux and statistical analysis, the cut-off was fixed for NaFlu and TEER, this leads to a cut-off values of 15 µg•cm -²•s -1 for NaFlu flux. Moreover, a lower TEER value than 30 Ω•cm² at the beginning of the experiment (t = 0 h) and a loss of TEER more than 50% after 0.5 h during the experiment, witnessed too high extension or rupture of the tissue during setting. Those results were also in the range of other values from the literature (Srinivasan et al., 2015). If NaFlu flux and TEER values were out of criteria that has been mentioned above, it means that the tissue has taken too much time to be set in the system, or that the tissue was too much extended and was broken during the setting. The correlation between markers of permeability and TEER values in this first series of tissues was confirmed by using a linear regression analysis (p < 0.05; Figure 2). Results of further study were analyzed according to these criteria.

Evaluation of the effects of GSNO on intestine permeability and integrity

GSNO is a very fragile and sensitive molecule which can be degraded easily by light, temperature, oxygenation by bubbling during Ussing Chamber experiment. Thus, to start the experiment with the presence of GSNO in Ussing Chamber system. The stability in Ussing Chamber was tested. 100 μM GSNO was adding in the system without presence of intestine tissue. All the other conditions were exactly the same with permeability study. After 2 hours, GSNO concentration is not significantly affected.

The result showed a stable state during our experiment with a condition of 37 • C and oxygenation with 95% O2 / 5% CO2 at 12 mL•min -1 .

After the confirmation of GSNO stability in this experimental system. The GSNO effects on intestinal barrier integrity was evaluated by adding GSNO (0.1, 10, 100 μM) and permeability markers in the donor (luminal) compartment and the marker permeation to acceptor (mucosal) compartment was followed during 2 h. The flux of markers and TEER result was given in Figure 3 and Papp values were shown in Table 1. Through the NaFlu permeability result (Figure 3 and Table 1), dose-dependent effect was observed. On healthy tissue, high concentrations of GSNO (100 µM) showed a trend to increase the NaFlu flux (or Papp) compared to the Control group; while low concentration of GSNO (0.1 µM) showed a trend to decrease the NaFlu flux (or Papp, Table 1) compared to Control. A significant difference was observed between high concentration of GSNO (100 µM) and Control at t=2 h for both NaFlu flux and Papp value. There was observed a significant increase of this paracellular marker after a highconcentration treatment of GSNO (100 µM), in comparison to low-concentration GSNO (0.1 µM). NaFlu flux and apparent permeability coefficient showed similar results. The TEER values showed in Figure 3 showed no significant difference in different groups. At the beginning of the experiment, the intestine tissue was discarded if the TEER values were less than 30 Ω×cm² which indicated the intact intestine tissues.

But with experiments going, the TEER decreased, which showed a decreased tissue integrity during experiments.

Cell junction proteins expression after GSNO treatment in Ussing Chamber

The result was shown in the Figure 4. The relative amount of Claudin-1 and Occludin did not significantly change in tissue during the experiment (Control) when compared with fresh tissue (WT). The similar result was observed with E-cadherin proteins.

Unexpectedly, the apparent molecular weight of E-cadherin appears to be smaller in the control tissue compared with fresh tissue, which might be linked with protein degradation in this ex vivo model.

Discussion and conclusion

The Ussing chamber system is a simple, but powerful technique to investigate the intestinal permeability. In the present study, Ussing Chamber system was chosen to investigate the GSNO effects on intestinal barrier integrity by following the intestinal permeability to a paracellular marker (NaFlu) because of its convenience, effectiveness (6 tissues in parallel) and better mimicking in vivo environment in compared with Caco-2 cells monolayer. Upon removal from the animal, an ex vivo intestinal preparation and incubation in Krebs-Henselheit buffer has limited viability (Clarke, 2009). Thus, the limitation of this system is the difficulties to ensure the tissue viability. There are not uniform quality criteria to control the tissue viability in the literatures. TEER and paracellular marker permeability is mostly used as the main indicators. For example, TEER remaining within 15% of the starting value was considered as indicator of viable tissue, and tissues were excluded where TEER fell by more than this in some literatures (Stephens et al., 2002, Fortuna et al., 2012). Marker permeated less than 0.01% was indicated in the literature as tissue viability indicator (Woitiski et al., 2011). There are not standard criteria. Thus, to ensure that we can obtain the reliable result with Ussing chamber system, the validation of quality criteria was performed by both TEER value and paracellular marker (NaFlu) permeability. The result indicated that the tissue with a TEER value lower than 30 Ω•cm² at the beginning of experiment or a decrease more than 50% of the initial TEER value after 0.5 h should be discarded because of destroyed barrier integrity during the preparation. To control the tissue viability during experiment, the NaFlu permeability in the control group at every experiment without any treatment was used as a reference. If the flux of NaFlu for control group at 2 h was less than 15 µg•cm -²•s -1 , the data from the whole experiment was exclude. Therefore, all the experiments and data were performed under these quality criteria to get reliable and analyzable result.

The dynamic equilibrium between all the actors of the barrier and the interactions between mucus, epithelial cells, microbiota and immune defense, are essential for the maintenance of the barrier integrity and the intestinal homeostasis. The disruption of this homeostasis induced an increase of the permeability, and leads to an inflammation and strong damages of the mucosa which is not able anymore to ensure the vital function of protection and absorption. It is what happened in the context of Inflammatory Bowel Diseases (IBD).

Our study showed that a low concentration of GSNO in the luminal compartment of the using chamber tend to delay and limit the permeability of the intestinal mucosa while high concentration aggravates the permeability. GSNO protective action on intestinal barrier was published by different authors (Savidge et al., 2007, Cheadle et al., 2013, Li et al., 2016). Enteric glia cells derived GSNO was identified by Savidge in 2007 as a potent inducer of intestinal barrier function ex vivo and in vivo and of attenuated tissue inflammation by an increased expression of perijunctional F-actin and tight-junctionassociated proteins zonula occludens-1 and occludin (Savidge et al., 2007). Same idea was published by Cheadle and co-workers in 2013 with in vitro cells model. GSNO secreted by enteric glia cells prevented intestinal barrier failure after injury and improved expression and localization of tight junction proteins including ZO-1 and occludin (Cheadle et al., 2013). Li and co-workers showed that exogeneous femoral venous injected GSNO protected intestinal barrier under endotoxin-induced inflammation by preserving tight junction protein ZO-1 expression and reducing inflammation marker releasing (IL-1β and TNF-α) through inhibiting NF-κB pathway (Li et al., 2016). But all these GSNO can be identified as a source from mucosal side of intestine. Thus, the present work is the first one that mentioned about the GSNO derived from oral administration route (luminal side) effects on intestinal integrity. The permeability study and western blot results suggested that GSNO can affect intestinal barrier in a dose-dependent manner, which proved the same idea as mentioned by Savidge (Savidge et al., 2007). This is in accordance with our hypothesis that the delivery of a low concentration of NO donor on the luminal side of the intestinal mucosa might be a good approach to prevent a disruption of the mucosal barrier integrity. The exact mechanism hasn't been investigated yet, but it could be associated with hypothetic action like post-translational modifications of proteins caused by addition of NO (Figure 5). NF-κB ability to induce the production of proinflammatory cytokines is reduced after a S-nitrosation step and thus this might be associated with the potential protective action in intestinal barrier integrity of NO (Li et al., 2016). Another target of S-nitrosation caused by NO could be adherens junction proteins. As reported in literature, Snitrosation of β-catenin leads to changes in microvascular permeability after administration of vascular endothelial growth factor (VEGF) for 15 min (Thibeault et al., 2010): S-nitrosation amino acid of β-catenin was Cysteine 619 which is located at the interaction site of β-catenin with VE-cadherin. Its S-nitrosation causes dissociation of the complex and an increase in microvascular permeability (Thibeault et al., 2010). p120-catenin was considered as another target of S-nitrosation (Marin et al., 2012). Cys 579 as the main S-nitrosated-residue was the responsible amino acid in p120-catenin.

Cys 579 is also located in the interaction site with VE-cadherin. Thus, S-nitrosation regulated protein-protein interactions and promoted the dissociation of the adherens junction complex. S-nitrosation of β-catenin and p120-catenin destabilizes the adherens junction complex, which leads to changes in the localization and interaction between these proteins at the adherens junction, which finally induce the internalization of VEcadherin. All these actions were well investigated within microvascular. Thus, we hypothesize that similar mechanism could occurs in intestinal mucosa. S-nitrosation of β-catenin caused by NO incubation leads to E-cadherin internalization, which finally increased the intestinal permeability.

Occludin was reported that could be nitrated by NO through the tyrosine residue, which may finally lead to the internalization and destroy the tight-junction between epithelial cells [START_REF] Rocha | Dietary nitrite induces occludin nitration in the stomach[END_REF].

For further study of mechanism exploration, more work need to be done, such as identification of post-translational modifications (S-nitrosation or nitration) targets and quantification of the modification level to understand the modulation process of the intestinal barrier integrity in the presence of NO.

To conclude about the present study, different GSNO concentrations (0.1, 10 and 100 µM) were added in the luminal side of intestine to investigate its effect on intestinal barrier integrity in Ussing chamber system by following the intestinal permeability to a paracellular marker (NaFlu). The preliminary mechanism was studied by western blot to follow the expression of tight junction proteins (ZO-1 and occludin) and adherens junction proteins (E-cadherin and β-catenin). Finally, a dose-dependent effect was observed for GSNO effects on intestinal barrier integrity. High concentration of GSNO (100 µM) showed deleterious impact in intestinal barrier while low concentration of GSNO (0.1 µM) preserving intestinal permeability with healthy intestine tissue.

Through the current study, we can conclude that the secure concentration of GSNO for an application of IBD prevention should be less than 0.1 μM. In order to confirm the feasibility of luminal GSNO preventing intestinal barrier disruption, further study is necessary to continue with an ex vivo inflammation model that can induce the disruption of intestinal barrier (need to be explored, because no existing model in the literatures)

or with a proposal of formulation to oral deliver GSNO in an in vivo inflammation model which has been well investigated. Meanwhile, the mechanism of GSNO regulating intestinal barrier integrity will also be studied by following the GSNO metabolism (involved enzymes) in contact with intestine tissue and the functionalization of GSNO by post-translation (nitrosation/nitrosylation/nitration) of proteins. Therefore, an innovative chronic treatment of IBD -in order to prevent relapses of inflammation -may rely on supplementation of small amounts of nitric oxide to preserve the intestinal barrier integrity.

Introduction

Concentration of GSNO is crucial for its application in pharmacology, as previously described in chapter 2. Thus, formulations are necessary to control GSNO concentration at target site. As showed in Table 2 from chapter 1, there is not a lot of research about GSNO formulation. Among all the development, they are mostly concentrated on topical treatment for wound healing [START_REF] Shishido | Thermal and photochemical nitric oxide release from S-nitrosothiols incorporated in Pluronic F127 gel: potential uses for local and controlled nitric oxide release[END_REF], Seabra et al., 2004a, Seabra and De Oliveira, 2004, Seabra et al., 2004b[START_REF] Seabra | Solid films of blended poly (vinyl alcohol)/poly (vinyl pyrrolidone) for topical Snitrosoglutathione and nitric oxide release[END_REF], Simoes and de Oliveira, 2010[START_REF] Vercelino | Skin vasodilation and analgesic effect of a topical nitric oxidereleasing hydrogel[END_REF] or implants for vascular disease treatment [START_REF] Heikal | Characterisation of the decomposition behaviour of S-nitrosoglutathione and a new class of analogues: S-Nitrosophytochelatins[END_REF], Heikal et al., 2011, Acharya et al., 2012, de Mel et al., 2014, Parent et al., 2015a, Parent et al., 2015b). Few publications introduced about GSNO formulations for oral treatment (Wu et al., 2016). In fact, oral administration is the best choice for chronic and incurable disease such as cardiovascular disease and IBD, because of its apparent advantages, such as convenience, cost-effectiveness, good patient compliance, non-injections, and decreased risk of infection [START_REF] Sarmento | Alginate/chitosan nanoparticles are effective for oral insulin delivery[END_REF]. However, the unique article mentioned about GSNO oral delivery was published by our laboratory (Wu et al., 2016). The nanocomposite particles were developed based on natural polymers alginate and chitosan with an enhanced GSNO bioavailability adapted to oral administration for the application of cardiovascular disease (Wu et al., 2016). The encapsulation strategy is based on a double incorporation: GSNO is firstly entrapped in nanoparticles, followed by a second encapsulation in polymer matrix. This double encapsulation provides not only a strong protection of GSNO but also sustained release over 24 h. Therefore, the strategy of polymer nanocomposites is good candidate for GSNO oral delivery.

As described in chapter 1, polymer nanocomposites showed lots of advantages, such as improved drug bioavailability and efficiency, controlled of pharmacokinetics, targeted delivery as well as the overcoming of obstacles arising from low drug solubility, degradation, fast clearance rated, relatively short biological activity and inability to cross biological barriers. All these advantages of polymer nanocomposites make it an adapted delivery system for fragile molecules, such as GSNO. As delivery vehicles of GSNO, nanoplatforms and polymer matrix are not conflict but complementary.

Nanoplatforms were capable of efficient entrapment of drug but quick release within 3 h (Wu et al., 2015a). Whereas, polymer matrix presents prominent sustained release as well as stabilization effect, but are limited by burst release. Application of polymer nanocomposites for GSNO delivery represent a promising strategy to merge the advantages of nanoplatforms and polymer matrix in one system to improve the therapeutic effect.

To develop GSNO formulation adapted to oral administration for IBD application, several requirements need to be followed:

1) Polymer adapted to oral route 2) Size less than 100 µm (adapted to gavage experiment of rat)

3) Efficient GSNO encapsulation 4) Sustained release profile

5) Local delivery in intestine (mucoadhesion)

To answer to the first requirement, different polymers classically used by industry (pharmaceutic or food) and authorized by authorities were preferred.

Eudragit ® RL is synthetic cationic copolymers of ethyl acrylate, methyl methacrylate and a low content of methacrylic acid ester with quaternary ammonium groups [START_REF] Patra | Pharmaceutical significance of Eudragit: A review[END_REF]. It's a cationic and non-degradable polymer which is not soluble in water [START_REF] Kadian | Mesalamine blocks tumor necrosis factor growth inhibition and nuclear factor κB activation in mouse colonocytes[END_REF]. Eudragit ® RL characterized with mucoadhesion property is mainly used for sustaining drug release in diverse drug delivery systems like nanoparticles [START_REF] Gandhi | In-vitro release of acyclovir loaded Eudragit RLPO® nanoparticles for sustained drug delivery[END_REF], Wu et al., 2015a), mucoadhesive tablets [START_REF] Singh | Iron oxide induced enhancement of mucoadhesive potential of Eudragit RLPO: formulation, evaluation and optimization of mucoadhesive drug delivery system[END_REF] and patches (Pandey et al., 2013), solid dispersions [START_REF] Varshosaz | Preparation and characterization of metoprolol controlled-release solid dispersions[END_REF][START_REF] Dahiya | Development of extended release coevaporates and coprecipitates of promethazine HCl with acrylic polymers: formulation considerations[END_REF][START_REF] Sahoo | Formulation of sustained-release dosage form of verapamil hydrochloride by solid dispersion technique using eudragit RLPO or Kollidon® SR[END_REF]. Singh et al investigated the effect of iron oxide in the development of mucoadhesive tablets of cinnarizine utilizing Eudragit ® RL [START_REF] Singh | Iron oxide induced enhancement of mucoadhesive potential of Eudragit RLPO: formulation, evaluation and optimization of mucoadhesive drug delivery system[END_REF]. Eudragit ® RL and iron oxide showed potential for gastroretentive and mucoadhesive drug delivery systems. Pandey et al tried site-specific drug delivery by formulating bi-layered gastro-retentable and mucoadhesive patch (stomach) (Pandey et al., 2013). Both Eudragit ® RS and RL were used for formulation of patch. Patches could achieve sustained drug release up to 12 h, with mucoadhesion. Sahoo et al formulated solid dispersion of verapamil utilizing Eudragit ® RL or Kollidon SR to control drug release [START_REF] Sahoo | Formulation of sustained-release dosage form of verapamil hydrochloride by solid dispersion technique using eudragit RLPO or Kollidon® SR[END_REF] and prolonged the drug release up to 12 h was obtained based on Eudragit ® RL.

Eudragit ® E is synthetic cationic copolymer based on dimethylaminoethyl methacrylate, butylmethacrylate and methyl methacrylate [START_REF] Patra | Pharmaceutical significance of Eudragit: A review[END_REF]. It is soluble in gastric pH up to 5.0. Eudragit ® E has characteristic properties such as low viscosity, high pigment binding capacity, good adhesion and low polymer weight. It has been widely used in oral delivery systems such as nanoparticles [START_REF] Eerikainen | Preparation of polymeric nanoparticles containing corticosteroid by a novel aerosol flow reactor method[END_REF], Khan and Schneider, 2014[START_REF] Chaurasia | Curcuminpolymeric nanoparticles against colon-26 tumor-bearing mice: cytotoxicity, pharmacokinetic and anticancer efficacy studies[END_REF], microparticles [START_REF] Shishu | Development of Taste Masked Oral Formulation of Ornidazole[END_REF], solid dispersions [START_REF] Chella | Formulation and Pharmacokinetic Evaluation of Polymeric Dispersions Containing Valsartan[END_REF][START_REF] Dangre | Improved Dissolution and Bioavailability of Eprosartan Mesylate Formulated as Solid Dispersions using Conventional Methods[END_REF], tablets and films [START_REF] Shishu | Taste Masking and Formulation of Ofloxacin Rapid Disintegrating Tablets and Oral Suspension[END_REF][START_REF] Shams | Pharmacokinetics of a losartan potassium released from a transdermal therapeutic system for the treatment of hypertension[END_REF][START_REF] Brniak | Orodispersible films and tablets with prednisolone microparticles[END_REF], solid complex [START_REF] Moustafine | Characteristics of interpolyelectrolyte complexes of Eudragit E 100 with sodium alginate[END_REF] and pellets [START_REF] Avachat | Feasibility studies of concomitant administration of optimized formulation of probiotic-loaded Vancomycin hydrochloride pellets for colon delivery[END_REF].

Chaurasia et al prepared Eudragit ® E nanoparticle by emulsification-diffusionevaporation method to enhance the bioavailability of curcumin [START_REF] Chaurasia | Curcuminpolymeric nanoparticles against colon-26 tumor-bearing mice: cytotoxicity, pharmacokinetic and anticancer efficacy studies[END_REF].

Brniak et al developed Eudragit ® E coated microparticle to obtain an efficient taste masking of a bitter drug substance [START_REF] Brniak | Orodispersible films and tablets with prednisolone microparticles[END_REF]. Avachat et al fomulated pellets coated with Eudragit ® E 100 and Eudragit ® L 100 multi-particulates systems for colon delivery [START_REF] Avachat | Feasibility studies of concomitant administration of optimized formulation of probiotic-loaded Vancomycin hydrochloride pellets for colon delivery[END_REF].

Alginate is an anionic polysaccharide linear unbranched polymer which contains varying amounts of 1,4'-linked β-D-mannuronic acid and α-L-guluronic acid residues, isolated from brown seaweed (Phaeophyceae) (Tonnesen and Karlsen, 2002). Alginate has several unique properties that have enabled it to be used as a matrix for the entrapment and/or delivery of a variety of bioactive compounds. These properties include: (i) mucoadhesive, biodegradable and low toxicity properties [START_REF] Sonavane | Preparation of Alginate Nanoparticles Using Eudragit E100 as a New Complexing Agent: Development, <I>In-Vitro</I>, and <I>In-Vivo</I> Evaluation[END_REF]Devarajan, 2007, Simonoska Crcarevska et al., 2008); (ii) pH-sensitive property (not soluble in gastric pH less than 3) [START_REF] Aguero | Alginate microparticles as oral colon drug delivery device: A review[END_REF]; (iii) a mild room temperature encapsulation process free of organic solvents [START_REF] Gombotz | Protein release from alginate matrices[END_REF]. pH-sensitivity and mucoadhesiveness presented the key properties for oral intestine application.

Figure 23 Strategies of polymer matrix

In our project, we purchase the GSNO nanocomposite particles formulation We focused on the polymer matrix composition and behavior of this nanocomposite particles, by two different strategies (Figure 23).

Strategy1: Polyelectrolyte complexation is the first step of this matrix establishment.

It consists in electrolyte interactions between positively charged nanoparticles and negatively charged alginate chains. A cross-linker can be added in the following step to reinforce the polymer matrix structure. In our case, it is calcium ions. These divalent ions can act as a bridge between alginate chains, which can result in tighter structure.

The ionotropic gelation process is not complicated but required many steps which can lead to GSNO degradation, GSNO leakage and finally decrease the encapsulation efficiency. The proposition of a simpler process based on polyelectrolyte complexation can optimized alginate nanocomposite. A comparison of both process will be presented in the first part of this chapter (article submitted in Journal of Microencapsulation in September 2017).

Strategy2:

In parallel, another strategy was conducted to optimize the polymer matrix of the nanocomposites. In a previous study from our team, a complementary "coating" of Ca-alginate nanocomposite with chitosan was proposed (Wu et al., 2016). This natural well-known polymer, form delivery system by ionotropic gelation process and cross-linking with sodium Tripolyphosphate (TPP) [START_REF] Yang | Microfluidic emulsification and sorting assisted preparation of monodisperse chitosan microparticles[END_REF]. Chitosan with positive charge can also interact directly with alginate chain [START_REF] Simonoska Crcarevska | Chitosan coated Ca-alginate microparticles loaded with budesonide for delivery to the inflamed colonic mucosa[END_REF]. Chitosan was reported to reduce the transepithelial electrical resistance and to open tight junction between epithelial cells transiently, which resulted in intestinal drug absorption enhancement [START_REF] Dodane | Effect of chitosan on epithelial permeability and structure[END_REF][START_REF] Thanou | Oral drug absorption enhancement by chitosan and its derivatives[END_REF], Wu et al., 2016). This tight-junction opening is not desirable in our case because totally opposite to the aim of barrier reinforcement. Fragile intestine in presence of chitosan could be further overrun by commensal bacteria which could finally induce inflammation.

Furthermore, the aim of present GSNO delivery is to localize the drug in intestine instead of permeation through intestine. Taking into consideration of above reasons,

Eudragit ® E 100 which present similar properties and widely used for colon delivery was used to replace chitosan to conserve the gel reinforcement property and achieve GSNO local effect in intestine. Alginate/Eudragit ® E 100 nanocomposite formulation will be presented as complementary data in this chapter:

Alginate nanocomposite particles optimization

The major aims of the first strategy based on alginate nanocomposite particles are: 1) simplify the preparation process; 2) increasing encapsulation efficiency; 3) achieve GSNO local effect in intestine.

Pre-optimization

GSNO-loaded alginate nanocomposite particles (GSNO-aNCP) was formulated as described previously (Wu et al., 2015b). Briefly, GSNO was firstly encapsulate in to Eudragit ® RL nanoparticle (NP) by double-emulsion and solvent-evaporation method.

Briefly, an aqueous solution of GSNO (10 mg) in 500 mL of 0.1% (w/w) Pluronic1 F-68 solution was emulsified by sonication for 60s (100% amplitude, Vibra-cell TM , France) in ice bath in 5 mL of dichloromethane (DCM) containing 500 mg of Eudragit ® RL PO.

This primary emulsion was further emulsified in 20 mL of 0.1% (w/w) Pluronic1 F-68 solution by 30 s sonication (30 W, Vibra-cell TM , France), in ice bath to form a wateroil-water emulsion. Finally, the GSNO-NP were hardened by solvent evaporation. The second encapsulation was based on ionotropic gelation of alginate with Ca 2+ . Sodium alginate (20 mg) was completely dissolved in 20 mL GSNO-NP suspension with magnetic agitation at a speed 100. On ice, 1.5 mL of calcium chloride solution (CaCl2, 2 M) was added dropwise into this mixture to cross-link the alginate. During this step, this mixture was homogenized by sonication (40 W, ultrasonic processor, France).

Finally, two consecutive encapsulation steps are performed in this process: GSNO is firstly encapsulated in NP and GSNO-NP are encapsulated in polymer matrix. During the second step of encapsulation (NP into polymer matrix), a part of non-encapsulating GSNO can be entrapped in the matrix, increasing the final GSNO loading. However, in reality, a half percentage of encapsulated GSNO (around 20% in total GSNO) was leaked after the second encapsulation step. We speculated that GSNO was diffused during strong energy intervention to form the polymer matrix such as strong mechanical agitation to dissolve sodium alginate and ultrasonication step during Ca² + cross-linking.

GSNO could also diffused in the aqueous environment or could be degraded during the formulation process. Therefore, a simple experiment was conducted to confirm this hypothesis. GSNO encapsulation efficiency (EE) was measured at three stages during the process: 1) after nanoparticles preparation (at the end of the first encapsulation step);

2) after alginate dissolution in NP suspension; 3) after Ca 2+ cross-linking (at the end of the process). The result showed that around 10% GSNO was diffused at stage "2)alginate dissolution" and 7% GSNO at stage "3) cross-linking", respectively. No increase of nitrite ions level was remarked. These results confirm the hypothesis of a GSNO diffusion in the aqueous environment, a leakage outside the delivery system.

Thus, different attempts were performed to optimize EE and avoid the GSNO leakage during the process (Table 3). The EE in the table was calculated by indirect method as below:

EE = 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝐺𝑆𝑁𝑂 𝑎𝑚𝑜𝑢𝑛𝑡 -𝑢𝑛𝑒𝑛𝑐𝑎𝑝𝑠𝑢𝑙𝑎𝑡𝑒𝑑 𝐺𝑆𝑁𝑂 𝑎𝑚𝑜𝑢𝑛𝑡 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝐺𝑆𝑁𝑂 𝑎𝑚𝑜𝑢𝑛𝑡 × 100%
The first attempt was to reinforce the gel by increasing alginate concentration. 40 mg alginate was used instead of 20 mg. Finally, no increase GSNO encapsulation efficiency was obtained (around 20% GSNO diffused compared with the EE of NP-43%).

The second attempt was to avoid the leakage during alginate dissolution step. Two strategies were developed. Firstly, sodium alginate was dissolve in only 5 mL nanosuspension (1/4 of final volume). For a homogeneous dissolution of alginate, a strong agitation was performed (magnetic agitation with a speed of 1300 rpm for 10 min). The remaining nanosuspension was added after alginate was totally dissolved with a reduced agitation speed (200 rpm for 5 min). The second one is dissolving sodium alginate in 10 mL ultrapure water instead of nanosuspension, at the same time different concentration of Ca 2+ was tested (2 M and 0.05 M). Finally, for the first method we got EE of 37.7%, for the second one, for 2 M and 0.05 M Ca 2+ concentration respectively, which decrease the GSNO diffusion with losing less than 15% of GSNO encapsulation. However, there are apparent drawbacks for these two methods (1.

dissolving alginate in only one part of nanosuspension 2. dissolving alginate in water):

for the first one, it's not so easy to dissolve alginate in only 5 mL suspension; for the second one, the final particle sizes were more than 100 µm, which is not adapted for gavage experiment.

The third attempt is to avoid leakage during cross-linking step. We continued to optimize the formulation based on the less complicated process (method 2 in first idea)

with improved GSNO leakage. A gentle energy intervention, magnetic agitation was used. In order to homogenize the mixture in a better way to obtain adapted particle size (less than 100 μm) after reducing the intervention energy level, lower concentration but higher volume of Ca 2+ was introduced, at the same time, the order of mixing was reversed. EE of 35.9% and 37.1% were achieved for 0.1 and 0.05 M Ca 2+ respectively.

Finally, the method not only obtained smaller particle size (86.1 μm and 84.4 μm) but also improved the process by decreasing GSNO leaking around 5%.

The fourth attempt is to remove Ca 2+ cross-linking step directly. Because as mentioned in chapter 1, divalent ions may induce the breakdown of S-NO bond. In addition, alginate chain with negative charge can interact direct with the positively charged NP surface which was formed by Eudragit ® RL PO by polyelectrolyte complexation. Thus, with a direct mixing of alginate solution with NP suspension can be good method to conserve the GSNO encapsulation efficiency in a maximum way. Different energy intervention methods were involved for the development of this attempt: ultrasonication and mild agitation. The result showed that 48.9% of GSNO EE and acceptable size (66 μm) were observed with a mild agitation while only 30.5% of GSNO EE was obtained with ultrasonication.

To conclude, the last trial of alginate nanocomposite particles based on polyelectrolyte complexation with a simple process improves the GSNO encapsulation efficiency in a best way. More details about physical-chemical characterization and in vitro evaluation of this formulation is introduced in the following context with a submitted paper. In this paper, alginate nanocomposite particles base on ionotropic gelation was also fully characterize to compare with the optimize method of polyelectrolyte complexation. Simpler process mild agitation 66 ± 2 0.980 ± 0.043 48.9 ± 7.1

S-nitrosoglutathione loaded alginate nanocomposite particles development

Two different preparation methods were introduced to form the outer polymer matrix: ionotropic gelation and polyelectrolyte complexation. The comparison of physicalchemical characterization, in vitro release and swelling study, in cellulo study of permeability through Caco-2 cells monolayer has been done and presented in the following article (submitted to Journal of Microencapsulation).

Introduction

Inflammatory bowel diseases (IBD), in which the most known subtypes are Crohn's disease and ulcerative colitis, are disabling pathologies affecting young patients caused by the conjunction of genetic susceptibility, environment factors, intestinal microbial flora alteration and immune dysfunction. IBD current treatment present the particularity that most of the current agents act by down regulating chronic inflammation in the intestine mucosa but cannot cure the disease (Kirchgesner et al., 2017, Schumacher et al., 2017). Conventional drugs for the treatment of IBD include aminosalicylates, corticosteroids, antibiotics and immunosuppressive agents, which are necessarily long term administrated and consequently lead to adverse effects (Pithadia andJain, 2011, Neurath, 2017). Novel therapeutic agents like anti-Tumor Necrosis Factor (anti-TNF)

have potent anti-inflammatory effects but a loss of response is frequent over time and only one third of patients achieve clinical remission after one year (Pithadia andJain, 2011, Neurath, 2017). It is therefore a real challenge to develop novel strategies for decreasing relapses frequency and improving the quality of patient's life.

Nitric oxide (NO) is a free-radical gas and one of the smallest endogenous molecules with the ability to function as a chemical messenger. NO plays a critical role in regulating a diverse range of physiological processes. Among those functions, NO was found to be implied into regulation of immunity and inflammation (Kobayashi, 2010). Depending on NO concentrations, this mediator can favour or prevent inflammation. Large amounts of NO generated primarily by inducible NO synthase increase the formation of peroxynitrites ions. In contrast, low concentration produced by constitutive and neuronal NO synthases inhibit adhesion molecule expression, cytokine and chemokine synthesis and leukocyte adhesion and transmigration (Guzik et al., 2003).

Nitric oxide plays an important role in mucosal defense (Wallace and Miller, 2000) which is defective in IBD. For example, NO produced by epithelial cells can increase mucus (Price et al., 1994) and fluid (Dow et al., 1994) secretions in the gastrointestinal tract, which reduces bacterial adherence to the epithelium. Furthermore, NO secreted by enteric glial cells can also locally enhance intestinal barrier integrity by improving i) the expression and localization of tight-junction proteins (occludin, zonula occludens-1 (ZO-1) protein), ii) the phosphorylation of myosin light chain which are implied in tight-junction structure (Savidge et al., 2007, Cheadle et al., 2013). In addition, NO acts as a homeostatic regulatory molecule designed to attenuate leukocyte-endothelium interaction (Banick et al., 1997, Lefer andLefer, 1999), immune response (Van Overveld et al., 1993, Huang et al., 1998[START_REF] Obermeier | Interleukin-1 production by mouse macrophages is regulated in a feedback fashion by nitric oxide[END_REF] and thus limit the local inflammation.

Reduction of NO with a reactive thiol group in cysteine residues in peptides and proteins, leads to the formation of S-nitrosothiols (RSNO), such as S-nitrosoglutathione (GSNO) and represents an endogenous form of storage and transport of NO in the body [START_REF] Noble | Nitric oxide release from S-nitrosoglutathione (GSNO)[END_REF]. To propose GSNO (and specifically NO) in a therapeutic of IBD requires a strict controlled supplement with low concentrations in a sustained way.

Different innovative formulations of GSNO such as polymeric nanoparticles (D. Marcato et al., 2013, Pereira et al., 2015), inorganic nanoparticles (Santos et al., 2016), polymer matrix (Seabra et al., 2004b, Cariello et al., 2013) polymeric stent (Acharya et al., 2012), polymer films (Yoo et al., 2009, Simoes and de Oliveira, 2010, Kim et al., 2015), liposomes (Diab et al., 2016) and polymer nanocomposites (de Mel et al., 2014) were developed to improve the stability of GSNO and to propose sustained release.

Among all the GSNO formulations, few are focused on oral delivery. We have already developed alginate/chitosan polymer nanocomposites adapted to oral route and presenting high GSNO encapsulation efficiency, effective drug protection and a sustained drug release, for a systemic action (Wu et al., 2016).

In the present study, we proposed a novel strategy to limit chronic inflammation and prevent of barrier failure in patients by using alginate nanocomposites (alginate matrix embedding Eudragit ® RL nanoparticles) as GSNO oral delivery system. Eudragit ® RL, is a synthetic cationic copolymer based on ethyl acrylate, methyl methacrylate widely 123 used as retardant of drug release in sustained released formulation (Apu et al., 2009).

A low content of methacrylic acid ester with quaternary ammonium groups leads to positively charged nanoparticles by double emulsion process. Alginate is a natural anionic polysaccharide of (1-4)-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G). It was chosen as the external matrix material, because of its well-documented biocompatibility, mucopenetration and mucoadhesion properties (Tonnesen andKarlsen, 2002, Arora et al., 2011). Alginates are natural polysaccharide polymers isolated from brown seaweed (Tonnesen and Karlsen, 2002). The alginate molecule will undergo an almost immediate hydration to create a hydrocolloidal layer of high viscosity, which builds up a diffusion barrier decreasing and delaying the migration of small molecules (Tonnesen and Karlsen, 2002). Ionotropic gelation and polyelectrolyte complexation, two formulation processes classically described for alginate delivery systems (Lopes et al., 2017), are compared in the present study, in terms of GSNO protection, GSNO controlled delivery and GSNO local effect in intestine. Physicochemical characterization and kinetic release study were performed. GSNO permeability through Caco-2 cells monolayer was tested to preliminarily prove the local and sustained effects of GSNO loaded nanocomposite particles (GSNO-NCP).

Methods

Acrylates / ammonium methacrylate copolymer (Eudragit ® RL PO) was a generous gift from Evonik industries (Germany). Alginate sodium (medium viscosity, mannuronic acid/guluronic acid ratio of 1.56), polyoxyethylene-polyoxypropylene block copolymer (Pluronic ® F-68), calcium chloride and all other reagents were obtained from Sigma-Aldrich (France). S-nitrosoglutathione (GSNO) was synthesized by reaction of reduced glutathione (GSH) and an equimolar ratio of sodium nitrite in acidic medium as a previously described method (Parent et al., 2013a).

Formulation of GSNO loaded alginate nanocomposite particle

Preparation of GSNO-NCP GSNO-NCP were constituted by inner cores and external polymer matrix (Wu et al., 2015b). GSNO-nanoparticles were prepared by a double emulsion and solvent evaporation method (Wu et al., 2015a). Briefly, 10 mg GSNO were dissolved in 500 µL of 0.1% Pluronic ® F-68 solution as the inner aqueous phase. In a separate step, 500 mg of Eudragit ® RL PO were dissolved in 5 mL of dichloromethane (DCM) constituting organic phase, and then poured into inner aqueous phase and homogenized by sonication (60 seconds, over an ice bath, 100 % amplitude, probe 3 mm, Vibra-cell TM , France). The obtained water-in-oil emulsion was poured into 20 mL of 0.1% Pluronic ® F-68 solution and homogenized by sonication (30 seconds, over an ice bath, 30 W, probe 6 mm, Vibra-cell TM , France) to obtain the water-in-oil-in-water emulsion. The nanoparticles were hardened by solvent evaporation (100 mbar, 10 min, under vacuum, Evaporator Heidolph, Laborota 4000, Pump CVC 2000, Minichiller Huber). The size (289 ± 14 nm) and zeta-potential (40 ± 6 mV) were controlled by Zetasizer ® Nano ZS (Malvern ® Instrument, France) (Wu et al., 2015a).

Two strategies were used to formulate the external polymer matrix. For ionotropic gelation process, sodium alginate (20 mg) was dissolved in 20 mL GSNO nanosuspension previously prepared. Over an ice bath, 2 mL calcium chloride solution (CaCl2, 2 M) was added drop wisely (needle 26 G, 0.45 x 10 mm; syringe 2.5 mL) into the mixture to cross-link alginate with homogenization by sonication in ice bath (40% amplitude, 40 W, generator 130 W, Bioblock Scientific 7501D, probe CV18-7 mm, France). For polyelectrolyte complexation process, sodium alginate (20 mg) was dissolved in 10 mL ultrapure water. GSNO nanosuspension was poured in the sodium alginate solution. The mixture was homogenized by gently shaking for 1 min and then 10 mins standing before characterization.

Characterization of GSNO-NCP

Determination of particle size

The volume particle size distribution of GSNO-loaded polymer nanocomposite particles (GSNO-NCP) was determined by laser diffraction method (Mastersizer 2000, Malvern Instruments, France). GSNO-NCP were suspended in ultrapure water. The particle size of the GSNO-NCP was described by the volume mean diameter performed in triplicate. The uniformity of the GSNO-NCP was expressed by the absolute deviation from the median.

Morphology study

The surface morphology and shape of GSNO-NCP were observed by using the scanning electron microscopy (SEM). Briefly, the diluted suspension of particles was dropped on a SEM stub and dried at room temperature. Then, the images were captured by a Hitachi S-4800 SEM (Hitachi, Japan) at 2 kV (accelerating voltage) and 2 µA (emission current). The working distance was 10 mm.

Determination of GSNO loading

GSNO loading describes the quantity of the GSNO loaded within GSNO-NCP compared with the initial drug quantity plus polymer quantity. It was determined according to the following equation ( 1), where meGSNO represents encapsulated GSNO amount, miGSNO means initial GSNO weight and mipolymer is initial polymer weight, respectively:

GSNO loading (mg GSNO g ⁄ polymer) = 𝑚𝑒𝐺𝑆𝑁𝑂 (𝑚𝑔) 𝑚𝑖𝐺𝑆𝑁𝑂(𝑔) + 𝑚𝑖𝑝𝑜𝑙𝑦𝑚𝑒𝑟 (𝑔)

(1)

The encapsulated GSNO within GSNO-NCP was evaluated by a two-step-liquid-liquid extraction. Briefly, the external matrix of GSNO-NCP composed of sodium alginate was disrupted by mechanical stirring for 6 min in sodium citrate solution (0.1 M). The resulting suspension was centrifuged at 15,000 g for 20 min at 4 °C, and the GSNO remaining in the supernatant was quantified by the Griess-Saville assay (Sun et al., 2003). Briefly, the sample was incubated in a 0.6% (w/v) sulfanilamide solution supplemented with 0.2% (w/v) of HgCl2 (to cleave the S-NO bond) in 0.4 M HCl. The formed diazonium salt was reacted with a 0.6% (w/v) N-(1-napthyl) ethylenediamine solution to form a chromophoric azo product that absorbs at 540 nm. Free nitrite ions quantified by Griess assay were subtracted from the resulting concentration calculated above.

The resulting pellet, which corresponds to GSNO Eudragit ® RL nanosuspension, was dissolved in 2 mL DCM and GSNO was extracted in 9 mL phosphate buffered saline (PBS, 135 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8 mM K2HPO4 , pH 7.4). The GSNO amount in the nanoparticles was also determined by the Griess-Saville assay as described above. All the samples were prepared in triplicate.

In vitro study

Kinetic release study

One milliliter GSNO-NCP suspension was placed in cellulose dialysis tubing (average flat width 10 mm (0.4 in), cut-off 14 kDa). The kinetic release study was performed in 200 mL of PBS pH 7.4 at 37°C with protection from light. All studies were conducted in triplicate. The GSNO and nitrite ions released were monitored at t=0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 24 h and immediately quantified with fluorometric method (DAN/DAN-Hg 2+ Assay) (Misko et al., 1993).

Swelling study

The same condition as used in kinetic release study was selected for swelling study. In cellulo study

Cytotoxicity study of GSNO-NCP

The cytotoxicity of GSNO-NCPs was evaluated on intestinal Caco-2 cells (ATCC ® HTB-37™). The cytotoxicity study was conducted as previously described (Wu et al., 2016). Caco-2 cells were seeded in 96-wells plates at 20,000 cells/well 24 h before experiment. They were then exposed to 0.5, 1.0, 5.0, 10.0 or 50.0 g/L of GSNO-NCP

for 24 h at 37°C, complete medium being used as control. Cytotoxicity was checked with the MTT assay and expressed by metabolic activity. Briefly, 0.5 mg/mL of MTT was incubated with the cells for 3 h. DMSO (100 µL) was then added under stirring for 10 min to extract the formazan crystals. The absorbance was measured at 570 nm with a reference at 630 nm (in order to subtract the background) using EL 800-microplate reader (Bio-TEK Instrument, Inc®, France). Metabolic activity at the lowest concentration (0.5 g/L) was considered as 100%.

GSNO permeability in NCP across the Caco-2 cells monolayer

The permeability of GSNO across the Caco-2 monolayer was evaluated in the apical to basolateral direction in Hanks Balanced Salt Solution (HBSS, pH 7.4). Caco-2 cells were seeded at 1.0×10 6 cells/well on polycarbonate membranes (pore size: 0.4 µm).

after reaching confluency, differentiated cell monolayers were obtained within 10-15 days after seeding. The transepithelial electrical resistance (TEER) gradually increased and reached a plateau at (1976 ± 681 Ω•cm 2 ) indicating the formation of the cell monolayer. Free GSNO, or GSNO-NCPs (50 µM of GSNO) prepared in HBSS without phenol red were placed in the apical compartment. GSNO, nitrite and nitrate ions were quantified in harvested medium from basolateral compartment by DAN/DAN-Hg 2+ assay after 1, 4 and 24 h. Then each basolateral compartment was filled with the same volume of fresh HBSS. Finally, after 4 and 24 h of incubation, the remaining quantities of GSNO, nitrite ions in the apical compartment were also quantified by DAN/DAN-Hg 2+ assay. At the end of the experiment, monolayer integrity was assessed by measuring TEER across the cell monolayer using a Millicell ® -Electrical Resistance system (Millipore, USA). Monolayer integrity was also evaluated by the ability of sodium fluorescein (5 µM), a low permeable marker, to cross the mucosa (Khan and Schneider, 2014). Fluorescein was detected in the basolateral compartment after 2 h by excitation at 490 nm and fluorescence emission at 514 nm.

Statistical analysis

Results are shown as either mean ± standard deviation (SD) or mean ± standard error of mean (SEM). All the statistical comparisons were performed with two-way ANOVA and post-test of Bonferroni's multiple comparisons test. p < 0.05 was considered significantly different. Statistical analyses were performed using the GraphPad Prism software (GraphPad Software, San Diego, USA).

Results

Physico-chemical characterization of GSNO-NCP after formulation

Size, uniformity and GSNO loading, are shown in Table 1. GSNO loaded nanocomposites (GSNO-NCP) formulated according to ionotropic gelation method present smaller size and better uniformity than GSNO-NCP prepared by polyelectrolyte complexation method (size: 24 ± 5 μm vs 66 ± 2 μm and uniformity: 0.48 ± 0.06 vs 0.98 ± 0.04, respectively). Nevertheless, with polyelectrolyte complexation method the GSNO loading is twice higher than ionotropic gelation, leading to 4.4 ± 0.4 mg and 2.7 ± 0.2 mg GSNO/g polymer, respectively. The GSNO-NCP were morphologically analysed by SEM as shown in Figure 1.

Distinct spherical shape with rough surface was observe for both formulations (Figure A1 and B1). In nanoscale, picture enlarging show that nanoparticles are dispersed homogeneously in the matrix (Figure A2 and B2). No difference with blank particle is observed.

Figure 1 Scanning Electronic Microscopy images of GSNO-NCPs: A, GSNO-NCP prepared by ionotropic gelation method; B, GSNO-NCP prepared by polyelectrolyte complexation.

In vitro study

Kinetic release study

Drug (GSNO and degradation product nitrite ions) release profile from the two formulations prepared by ionotropic gelation and polyelectrolyte complexation is given in Figure 2. A burst release of GSNO reaching around 100% within the first 3 h is observed for GSNO-NCP prepared by polyelectrolyte complexation method (Figure 2).

At the opposite, the GSNO-NCP formulated by ionotropic gelation method release only 28% ± 5% GSNO within 3 h and sustaine the release over 24 h (44% ± 9% GSNO and 17% ± 4% nitrite ions). After 24 h, 20% ± 4% GSNO and 20% ± 7% nitrite ions remain in the dialysis bag for this formulation method.

Swelling study

Drug release rate from the alginate particles is highly dependent on particle swelling.

As shown in Figure 3, the particle size decrease during 4 h and stabilized until 24 h for GSNO-NCP prepared by polyelectrolyte complexation method. However, the size of the particles prepared by ionotropic gelation slightly increases from the beginning to 24 h (the end of experiment).

In cellulo study

Cytotoxicity study

In the present study, Caco-2 cells were incubated with GSNO-NCP for 24 h. The results showed in Figure 4 do not fit with a dose-response curve model and a low mortality rate is observed under a polymer concentration less than 1 g/L for both formulations (ionotropic gelation, Figure 4A; polyelectrolyte complexation, Figure 4B). Even for the highest polymer concentration (50 g/L) tested (corresponding to log[polymer concentration] value of 1.5), more than 50% cells were still viable .

Permeability of Caco-2 cells monolayer to GSNO delivered by GSNO-NCP

Caco-2 cell monolayers were incubated with 50 µM (25 nmol) free GSNO or GSNO-NCP in apical compartment to investigate the GSNO permeability. An obvious decrease of RSNO permeation in basolateral compartment is observed for both GSNO-NCPs regardless the formulation methods used, after 4 h of incubation (0.50 ± 0.04 nmol for free GSNO and 0.04 ± 0.02 nmol for ionotropic gelation, 0.04 ± 0.02 nmol for polyelectrolyte complexation, respectively) (Figure 5A). With both types of formulation, GSNO degradation forms (nitrite ions and nitrate ions) are much less than with free GSNO within 4 h (Figure 5B and5C). In the apical compartment, significant less RSNO is observed by formulation groups at 4 h than control group, whereas no difference is seen at 24 h (Figure 5D). Interestingly, GSNO-NCP prepared by ionotropic gelation remain less RSNO in the apical compartment than GSNO-NCP prepared by polyelectrolyte complexation at t=4 h (2.0 ± 0.2 nmol for ionotropic gelation, 7.5 ± 1.6 nmol for polyelectrolyte complexation, respectively, Figure 5D). Meanwhile, the nitrite amount in the apical compartment remain unchanged after 4h and only a slight decrease is observed with polyelectrolyte complexation GSNO-NCP (Figure 5E).

Discussion

The role of NO, and related NO donors, in intestinal homeostasis has been clearly demonstrated: maintaining redox balance (Kubes and McCafferty, 2000), helping mucosal healing (Jadeski and Lala, 1999), working on immune cells by inhibiting various immunomodulatory cytokines [START_REF] Obermeier | Interleukin-1 production by mouse macrophages is regulated in a feedback fashion by nitric oxide[END_REF], protecting intestinal barrier integrity by increasing junction protein expression (Savidge et al., 2007, Cheadle et al., 2013, Li et al., 2016). S-nitrosoglutathione, a naturally produced NO donor, was recently considered as a candidate drug able to prevent intestinal barrier disruption after femoral venous injection (Li et al., 2016) or in ex vivo study (Savidge et al., 2007). Oral delivery of GSNO with tune-up concentrations and timing controls necessarily require novel pharmaceutical technologies. With that in mind, different strategies of GSNO formulations were already elaborated, mainly for vascular applications (Heikal et al., 2011, de Mel et al., 2014, Parent et al., 2015b).

Nanocomposite particles as proposed in the present study, relay on a double entrapment, which improves GSNO encapsulation and sustains the release of GSNO. Firstly, GSNO was efficiently encapsulated in Eudragit ® RL nanoparticles (5.7 mg GSNO/g polymer),

which protected GSNO from degradation (Wu et al., 2015a). GSNO loaded nanoparticles were further entrapped by an external matrix to form polymer nanocomposites, which improved the protection of GSNO in gastrointestinal tract, the control of GSNO delivery and the adhesion with intestine tissue. Our previous study demonstrated a systemic NO delivery (establishment of a stock of NO in vascular wall)

after oral administration of GSNO loaded nanocomposites (15 mg/kg, rat) composed from Eudragit ® RL nanoparticles embedded in an alginate/chitosan polymer matrix formulated by ionotropic gelation process (Wu et al., 2016).

In the present study, alginate nanocomposite particles are proposed for oral GSNO delivery in intestine and adapted to a luminal protective effect. Two formulation processes were compared (ionotropic gelation with CaCl2 as cross-linker and polyelectrolyte complexation) in order to fulfil project requirements and with the objective to limit step number in favour of GSNO encapsulation. To our knowledge, a direct complexation between formulated cationic Eudragit ® RL nanoparticles and sodium alginate chains leading to microparticles has never been described before. The literature was focused essentially on tablet coating based on polyelectrolyte complexation film between sodium alginate (anionic) and the cationic Eudragit ® RL dispersion. The resulting quaternary polymethacrylate-sodium alginate films have a strong potential for use in sustained-release tablets (Khuathan andPongjanyakul, 2014, Pongjanyakul andKhuathan, 2016).

In our case, higher GSNO loading is obtained with polyelectrolyte complexation method than with ionotropic gelation method. Because GSNO is a very small hydrophilic molecule, it can easily diffuse into the aqueous phase. This GSNO leakage might be explained by the intervention of strong mechanical strength during the matrix formation step: indeed, 7% of GSNO leaked from NP during additional step of strong agitation to dissolve sodium alginate in nanosuspension and 10% of GSNO leaked during the last ultrasonication step of adding calcium ions to cross-link alginate chain (data not shown). In contrast, polyelectrolyte complexation method was performed in a single step of gently mixing. Consequently, GSNO encapsulation in Eudragit ® RL nanoparticles (5.7 mg GSNO/g polymer was preserved (Wu et al., 2015a)). The difference of the matrix network between both processes was not visible in SEM (figure 1), where the same distribution of nanoparticles was observed.

Nevertheless, as shown in Figure 2, a burst release of GSNO until almost 100% within the first 3 hours was observed for GSNO-NCP formulated with polyelectrolyte complexation method, which is comparable to previous observation with GSNO loaded Eudragit ® RL nanoparticles (Wu et al., 2015a). On the contrary, ionotropic gelation led to a sustained release (still > 24 h). This big difference of release kinetics profile between these two methods may be attributed to tight complex structure of the particle prepared by ionotropic gelation method with much smaller particle size (24 ± 15 µm).

In this tight complex structure, calcium ions induce alginate chain-chain association leading to the well-known "egg-box model" (Li et al., 2007). With polyelectrolyte complexation method, alginate was only coated on the nanoparticle surface by electrostatic interaction between alginate and Eudragit ® RL which led to larger particle size (66 ±2 µm). Eudragit ® RL is positively charged because of quaternary ammonium groups of methacrylic acid ester while alginate is negatively charged by carboxyl group existing in the chain (Li et al., 2005b, Vysloužil et al., 2013). However, Eudragit ® RL is insoluble in aqueous phase while alginate is a hydrophilic polymer. Thus, the interaction between alginate and Eudragit ® RL is limited because alginate cannot really get into contact very efficiently with Eudragit ® RL. The reaction is much more like a coating on the surface of nanoparticle which results in a burst release due to the lack of effective interactions.

The swelling profile was matched with the release study. The swelling characteristics are principally related to the hydration level of hydrophilic groups [START_REF] Nikoo | Controlling the morphology and material characteristics of electrospray generated calcium alginate microhydrogels[END_REF]. The size decrease observed in particles prepared by polyelectrolyte complexation method may be caused by the detachment of alginate chain from nanoparticle. Meanwhile the continuous swelling behaviour of GSNO-NCP prepared from ionotropic gelation method might be explained by the water uptake of calcium alginate in PBS (Bajpai and Sharma, 2004).

The cytotoxicity result of GSNO loaded Eudragit ® RL nanoparticles has already been published that a good cytocompatibility was observed a working concentration 3 g/L (containing 50 µM GSNO) (Wu et al., 2015a).. With the nanocomposite particles introduced in the present study, an addition of polymer alginate participated in the formation of particle. Alginate is regarded as generally non-toxic and biocompatible polymer (Tonnesen and Karlsen, 2002). It is widely used in the pharmaceutical, cosmetic, and food industry (Onsoyen, 1996, Skjåk-Braek andEspevik, 1996). In different cytotoxicity test with alginate microparticles, non-cytotoxic effects were observed on different cell lines (Rossi et al., 2009, D. Marcato et al., 2013, Martins et al., 2015). In the present study, cell viability was studied as metabolic activity by the MTT assay. Therefore, the working concentration 6.2 g/L (ionotropic gelation), 3.8 g/L (polyelectrolyte complexation) delivering 50 µM GSNO, preserved more than 80% cell viability and was used for further investigations on GSNO permeability study through Caco-2 cells monolayer.

Transport experiments were performed from the apical to the basolateral compartment of an insert system. A delay in RSNO and degradation products (nitrite ions and nitrate ions) permeabilities (basolateral compartment) was observed at least up to 4 h in the case of formulations when compared to free GSNO (figure 5), whatever the formulation process. This sustained release was already mentioned in a previous study, describing a GSNO-NCP formulated by ionotropic gelation process (Wu et al., 2015b). In apical compartment, significant decrease of RSNO amount was observed with both GSNO-NCP formulations. Remaining amount of GSNO in NCP could be progressively released from 4 to 24 h. With alginate matrix /cells interactions, Caco-2 cells monolayer could accumulate RSNO and be considered as RSNO store, consequently participated in the observed delay.

In the case of GSNO-NCP formulated by ionotropic gelation, sustained release was particularly observed: less RSNO were quantified in the apical compartment at 4 h when compared to polyelectrolyte complexation. The tight alginate matrix obtained by ionotropic gelation may retain GSNO and sustain more efficiently the release than complexation process, as observed in in vitro release study.

Surprisingly, the clear difference between release profile from ionotropic gelation and polyelectrolyte complexation observed in vitro (PBS) disappeared totally in permeability study in cellulo, leading to the similar amount of RSNO and degradation ions crossing the barrier (basolateral compartment). We hypothesize that, opposite to in vitro experiment in PBS, presence of proteins secreted by Caco-2 and divalent cations in the culture medium may reinforce alginate hydrocolloidal layer. The resulting high viscosity of the matrix may finally delay GSNO release from polyelectrolyte complexation GSNO-NCP. Further experiment should be investigated for the exact mechanism of this alginate matrix modification.

Unlike to other natural polymer such as chitosan, alginate is not known to present a direct action on enterocytes junction (Yeh et al., 2011), but more on mucus organization (Mackie et al., 2016). It could be interesting to further investigate the mucopenetration ability of the GSNO-NCP formulation by the single-step process proposed in this study.

Supplementary study: GSNO alginate nanocomposite particles in vitro release in acidic pH

For better understanding of GSNO loaded alginate NCP behavior all along the gastrointestinal tract, the kinetic release study was also performed under an acidic pH which is corresponding to the simulated gastric fluid pH.

Method

A similar release study than the previous study performed in PBS at pH 7.4 was proposed. free GSNO solution or GSNO-loaded alginate NCP suspension (1 mL) was placed in cellulose dialysis tubing (average flat width 10 mm (0.4 in), cut-off 14 kDa).

The kinetic release study was performed in 200 mL of acidic buffer (1g/L NaCl, 0.7% v/v HCl) at pH 1.2 (simulated gastric fluid pH) at 37°C with protection from light. All studies were conducted in triplicate. The GSNO and nitrite ions released were monitored at t=0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 24 h and immediately quantified with fluorometric method (DAN/DAN-Hg 2+ Assay) (Misko et al., 1993).

Result and discussion

Drug (GSNO and degradation product nitrite ions) release profile from the two formulations prepared by ionotropic gelation and polyelectrolyte complexation is given in Figure 24. As shown in Figure 24, a burst release of GSNO till almost 100% within the first 3 hours was observed for all the formulations. No difference with free GSNO and no sustained release were observed for both formulation. The nitrite ions were not detectable with both nanocomposite particles at pH 1.2 (less than limit of quantification = 0.25 μM). Finally, GSNO loaded alginate NCP prepared by polyelectrolyte complexation method still got the same release profile for both acidic pH (1.2) and neutral pH (7.4). Electrostatic interactions between carboxyl group from alginate chain and quaternary ammonium group from Eudragit ® RL on the surface of NP were expected. The structure could be not tight enough to prevent small hydrophilic molecules (GSNO and nitrite ions) diffusion, which leads to a quick release in both conditions. What is interesting is the totally different release behavior for GSNO loaded alginate NCP prepared by ionotropic gelation method in different pH (sustained release (28% ± 5%) at pH 7.4 and burst release (almost 100%) at pH 1.2 within 3h). It might be explained by that alginate is more stable near neutral pH, since with extreme pH, its degradation increases [START_REF] Takka | Evaluation of chitosan/alginate beads using experimental design: formulation and in vitro characterization[END_REF]. Below pH 5, a proton-catalyzed hydrolysis and alginate gel shrinks lead to in alginate instability (Lopes et al., 2017).

As a result, the alginate monomers linkages can be cleaved in this acidic pH environment (pH 1.2), which finally caused as rapid GSNO diffusion as Eudragit ® RL NP release profile (Wu et al., 2015a).

Figure 24 Drug (GSNO and degradation product nitrite ions) release from GSNO-NCP ( free GSNO, ionotropic gelation, polyelectrolyte complexation) in acidic buffer (pH 1.2, 37 °C). Data are shown as mean ±SD (n=6).

Discussion

For alginate-NCP, we finally obtained 2 different particles through ionotropic gelation method and polyelectrolyte complexation method. The comparison was done between these two formulations through the different experiments introduced in paragraph 3.2.2.

With ionotropic gelation method, the final particles have sustained GSNO release in vitro but lower GSNO loading and longer preparation process with high energy intervention (potential to destroy S-NO bond of GSNO). With polyelectrolyte complexation method, the final particles have higher GSNO loading and simpler process but fast GSNO release in vitro. The possible mechanism of different release behaviors observed in vitro might be explained with different structure characteristics (figure 26). With ionotropic gelation method, the structure of the final particles is very tight. Water uptake of the particles [START_REF] Nikoo | Controlling the morphology and material characteristics of electrospray generated calcium alginate microhydrogels[END_REF] leads to slow and continuous swelling during release experiment, which can explain the slowly continuous size increasing during 24 h experimental duration. As a result, GSNO can be diffused from the particles sustainably. But with polyelectrolyte complexation method, bigger size is obtained for these particles, which might be explained with the loose network between the nanoparticles and alginate chain.

It's not easy to have very tight interaction between the positively charged hydrophobic polymer (Eudragit ® RL) on the surface of nanoparticles and negatively charged hydrophilic polymer (alginate) chain. Thus, the risk of detachment of alginate chain from the surface of nanoparticles exists, and this might explain the quick size decreasing within 4 h in the beginning of experiment. GSNO and even GSNO-NP can be diffused easily into the outer aqueous phase from the loose network of these particles.

As published in laboratory previously (Wu et al., 2015a), fast release of GSNO-NP was observed. All these reasons might finally explained the fast release of alginate-NCP prepared by polyelectrolyte complexation in in vitro condition. A following in cellulo study was performed to understand the behavior of different alginate-NCPs in contact with cells. The permeability study trough Caco-2 cells monolayer in paragraph 3.2.2 showed that both formulations obtain delayed release in contact with cells for minimum 4 h, but better efficiency for ionotropic gelation. This might be explained by a more complex condition in contact with cells may help particles prepared by polyelectrolyte complexation to retain GSNO. But the exact mechanism need to be further explored.

The following step is to study NO storage into the cells. For this study, two different aspects could be interesting to investigate (figure 27). The first one is the quantification of NO species into the cells. This study will be performed by HPLC-MS (Highperformance liquid chromatography-mass spectrometry) with 15 N labeled GSNO after derivatization by DAN (2,3-diaminonaphthalene) which just developed in the laboratory. With this method, we can distinguish the endogenous NO produced naturally by cells from the exogenous NO added during the experiment. Thus, the accuracy of the detection is increased. Besides, this method also significantly increased the sensitivity by 20 times through decrease the LOQ (limit of the quantification) to 5 nM compared with the traditional detection method (fluorescence) with the LOQ at 100 nM. The second aspect that has to be studied is to know the duration of NO can localized into the cells. This study will be further investigated by kinetic study between 4 h and 24.

Figure 27 Perspectives of in cellulo study to explore the mechanism of NO localization in intestine.

Alginate/Eudragit ® E 100 nanocomposite particles development

As described in the introduction part of this chapter, there are two purposes of this second strategy: 1) reinforce of the polymer matrix to limit GSNO diffusion; 2) achieve GSNO local effects in intestine.

Pre-experiment

Before the formulation step, a pre-experiment is necessary to explore the interaction between Eudragit ® E 100 and sodium Tripolyphosphate (TPP) (cross-linker of chitosan).

The mechanism of TPP cross-linking with chitosan is the interaction between a positively charged amino group (NH 3+ ) of chitosan and P3O10 5-anions (Figure 28) [START_REF] Yang | Microfluidic emulsification and sorting assisted preparation of monodisperse chitosan microparticles[END_REF].

Figure 28 Mechanism of Interaction between TPP and chitosan [START_REF] Yang | Microfluidic emulsification and sorting assisted preparation of monodisperse chitosan microparticles[END_REF] As described already, Eudragit ® E 100 is also a positively charge polymer which consists of dimethylaminoethyl methacrylate, butylmethacrylate and methyl methacrylate (Figure 29). But there is no publication mentioned a 3D matrix based on Eudragit ® E 100 and cross linker association. We speculated that the positively charged dimethylamino groups of Eudragit ® E 100 can still interact with P3O10 5-anion by electrostatic interaction. The possible mechanism is shown in Figure 26 Eudragit ® E 100-TPP cross-linking and ionotropic gelation process were explored.

Briefly, 220 mg TPP was dissolved in 20 mL 0.1% pluronic ® F-68, while 20 mg Eudragit ® E 100 was dissolved in 20 mL 1% acetic acid. TPP solution was added drop wisely in Eudragit ® E 100 under agitation of 1000 rpm. Twenty mL 0.1% pluronic ® F-68 without TPP or 1% acetic acid without Eudragit®E 100 was used as controls. During the experiment, pH of each solution before reaction and mixture after reaction was measured, at the same time, the final mixture state was observed visually. The result is given in Table 4. From Table 4, an interaction between TPP and Eudragit ® E 100 existed obviously, proved by white precipitates formation, obtained only if the both presence of TPP and

Eudragit ® E 100. Without TPP or Eudragit ® E 100, the final mixture was still transparent without any opalescence or precipitation. Thus, the hypothesis at the beginning was confirmed. The preparation of GSNO loaded alginate/Eudragit ® E 100 nanocomposite particles can be prepared in the same way as described for GSNO loaded alginate/chitosan nanocomposite particles (Wu et al., 2016).

Methods

Preparation of GSNO loaded alginate/Eudragit ® E 100 nanocomposite particles

GSNO-aeNCP were constituted by inner cores and external polymer matrix (Wu et al., 2015b). GSNO-nanoparticles (GSNO-NP) were prepared by a double emulsion and solvent evaporation method as previously described (Wu et al., 2015a). Then, alginate sodium (20 mg) and sodium tripolyphosphate (TPP, 220 mg) was fully dissolved in 20 mL GSNO-NP suspension. Over an ice bath, 2 mL calcium chloride solution (CaCl2, 2 M) was added dropwisely into this mixture to cross-link sodium alginate and homogenized by sonication (40 W, ultrasonic processor, France). Meanwhile, 20 mg

Eudragit ® E 100 was dissolved in 20 mL 1% (v/v) acetic acid. GSNO-loaded alginate

Eudragit ® E 100 nanocomposites (GSNO-aeNCP) were formed by dropwise addition of GSNO-nanoparticle-alginate mixture into chitosan solution under mechanical stirring (1000 rpm) over ice bath.

Characterization of GSNO-aeNCP

Determination of particle size

The volume particle size distribution of GSNO-aeNCP was determined by laser diffraction method (Mastersizer 2000, Malvern Instruments, France). The GSNO-acNCP were suspended in ultrapure water. The size of GSNO-acNCP was described by the volume mean diameter performed in triplicate.

Morphology study

The surface morphology and shape of GSNO-aeNCP were carried out by using the scanning electron microscopy (SEM) as previously mentioned in article 3 of chapter 3.

Determination of GSNO loading

GSNO loading is calculated in the same way as described in article 2.

The encapsulated GSNO within GSNO-NCP was evaluated by a two-step-liquid-liquid extraction. Briefly, the external matrix of GSNO-NCP composed of sodium alginate and Eudragit ® E 100 were disrupted by vortex for 5 min in sodium citrate/HCl solution.

The resulting suspension was centrifuged at 15,000 g for 20 min at 4 °C, and the GSNO remaining in the supernatant was quantified by the Griess-Saville assay as previously described (Sun et al., 2003).

The resulting pellet, which corresponds to GSNO-NP, was dissolved in 1 mL DCM and GSNO was extracted in 9 mL of pH 7.4 phosphate buffered saline (PBS, 135 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8 mM K2HPO4). The amount of GSNO in the nanoparticles was also determined by the Griess-Saville assay as previously described.

All the samples were prepared in triplicate.

Cytotoxicity study

The cytotoxicity of GSNO-aeNCP was evaluated on intestinal Caco-2 cells ATCC® HTB-37™ as previously described in article 3 of chapter 3.

Result and discussion:

Physical-chemical characterization of GSNO-aeNCP

The size measured by Mastersizer and GSNO loading was shown in table 5. Smaller size (12 ± 1 µm) but similar loading (2.7 ± 0.2 mg GSNO/g polymer) was obtained after one more step of gelation with Eudragit ® E 100 compared with only alginate gelation (23.6 ± 4.7 µm for size and 2.5 ± 0.6 mg GSNO/g polymer for loading). The smaller size can be involved in tighter-network of polymer matrix. Calcium ions cross-linked alginate and TPP cross-linked Eudragit ® E 100, beyond that Eudragit ® E 100 chain can be bonded directly with alginate chain by polyelectrostatic interaction. These multiple interactions between different polymers resulted in tighter structure of polymer matrix, which might limit GSNO diffusion. The GSNO-aeNCPs are morphologically analyzed by SEM as shown in Figure 30. The images showed distinct spherical shape with smooth surface, which further confirmed of the tighter network. In nano-scale, it was observed by zooming the picture that nanoparticles were dispersed homogeneously in the matrix. Through figure 30 b), the polymer matrix formation was observed within the mark of red circle. The particles sizes are found to be smaller than those obtained from Mastersizer analysis. This may be explained by hydrodynamic diameter of freshly prepared particles are generally measured by Mastersizer, whereas SEM images can nullify the swelling effects with dry samples [START_REF] Mukhopadhyay | pH-sensitive chitosan/alginate core-shell nanoparticles for efficient and safe oral insulin delivery[END_REF].

Cytotoxicity result

The concentration-response curve built from the MTT metabolic activity assay (Figure 31) gave an IC50 at 20.39 g/L for GSNO-aeNCP. Therefore, a concentration of 6.7 g/L (containing 50 μM GSNO) that maintained more than 80% of cell viability can be envisaged for Caco-2 permeability studies.

Conclusion

The GSNO potential in oral chronic treatment in IBD relapses prevention is limited by its poor stability and high hydrophilicity. In the second strategy, the polymer nanocomposite particles based on alginate/ Eudragit ® E 100 blend polymers approved by the pharmaceutical authorities as suitable for oral delivery has been developed. This GSNO-aeNCP showed efficient encapsulation efficiency related to application (effective concentration for intestinal barrier protection).

Conclusion and perspectives:

To develop GSNO formulation adapted to oral administration for IBD application, we fixed several requirements in the beginning of this chapter:

1) Polymer adapted to oral route 2) Size less than 100 µm (adapted to gavage experiment of rat)

3) Efficient GSNO encapsulation 4) Sustained release profile

5) Local delivery in intestine (mucoadhesion)

During the study, all the attempts are made to answer these requirements. In conclusion, three formulations based on two strategies adapted to oral administration for IBD application have been developed: alginate nanocomposite particles based on ionotropic gelation method and alginate nanocomposite particles based on polyelectrolyte complexation method; alginate/Eudragit ® E 100 blend nanocomposite particles.

1) All the formulations are prepared with polymers approved by the pharmaceutical authorities as suitable for oral delivery, which has been defined in the beginning of this chapter.

2) The size of all the formulations (24±5 μm, 66±2 μm and 12±1 μm respectively) is less than 100 µm which is adapted to further investigation.

3) From the chapter 2, we know that the effective concentration of GSNO for intestinal barrier protection is 0.1 µM. According to this result, a calculation related to effective concentration per cm 2 surface area of intestine to transfer the ex vivo dose to in vivo dose. The final theoretical calculated dose of oral administration for preclinical experiment is 0.91 mg GSNO/kg rat weight. One formulation containing 1.4 mg GSNO to 2.3 mg GSNO depending on formulations, which cover finally what could be needed in preclinical study.

4) The sustained release till 24 h has been achieved with alginate nanocomposite particles based on ionotropic gelation method and polyelectrolyte complexation method when contacted to Caco-2 cells. With alginate nanocomposite particles, polyelectrolyte complexation method has revealed process simplification adapted to industry application, which will be further standardized. Release profile of alginate/Eudragit ® E 100 blend nanocomposite particles need to be performed.

5) Finally, the mucoadhesion ability of these GSNO delivery system should be further investigated to complete the requirement we fixed at the beginning of this project. The mucoadhesion study can be carried out by measurement of the mucosal residence time of particles with excised intestine tissue according the published methods [START_REF] Mukhopadhyay | pH-sensitive chitosan/alginate core-shell nanoparticles for efficient and safe oral insulin delivery[END_REF][START_REF] Menzel | Preactivated thiolated nanoparticles: A novel mucoadhesive dosage form[END_REF].

Discussion

S-nitrosoglutathion (GSNO), the main endogenous low-molecular-weight Snitrosothiol, plays a dual role in the inflammation modulation depending on the its concentration (Savidge et al., 2007[START_REF] Flamant | Enteric glia protect against Shigella flexneri invasion in intestinal epithelial cells: a role for S-nitrosoglutathione[END_REF], Cheadle et al., 2013[START_REF] Goncalves | Treatment of bowel in experimental gastroschisis with a nitric oxide donor[END_REF], Li et al., 2016). The physiological levels of GSNO may promote beneficial action, whereas higher concentration exacerbate pathological condition. Low "physiological" GSNO concentrations (< 10 μM) were found to promote NF-κB activation and IL-6 expression in human peripheral blood mononuclear cells.

Conversely, higher GSNO concentrations (> 100 μM) exhibited opposite effect [START_REF] Siednienko | Nitric oxide affects IL-6 expression in human peripheral blood mononuclear cells involving cGMPdependent modulation of NF-κB activity[END_REF]. A biphasic effect was also observed in epithelial cell, at low micromolar concentrations GSNO (10 μM) promotes tight-junction-associated proteins (ZO-1) to associate with cytoskeletal components, whereas at higher and potentially pathogenic doses (250 μM) it directly disrupts this cytoskeletal F-actin network (Savidge et al., 2007). Similarly, low concentration of GSNO (0.05 μM) inhibited the iNOS expression, improved morphological bowel changes in gastroschisis while higher concentration (5 μM) was not as effective in the treatment of bowel morphology changes [START_REF] Goncalves | Treatment of bowel in experimental gastroschisis with a nitric oxide donor[END_REF]. Furthermore, 100 μM GSNO was reported to be able to decrease CoCl2-induced hypoxia-inducible factor-1α stabilization while 1 mM GSNO was ineffective [START_REF] Sandau | Accumulation of HIF-1α under the influence of nitric oxide[END_REF]. Low concentrations of GSNO (<10 μM) promoted Sp3 and inhibited Sp1 binding to 'housekeeping' genes. On the other hand, nitrosative stress-associated high concentration of GSNO (10 μM-100 μM) resulted in a complete reversal of the observed effects [START_REF] Zaman | Concentration-dependent effects of endogenous S-nitrosoglutathione on gene regulation by specificity proteins Sp3 and Sp1[END_REF]. Altogether, if GSNO is produced in small amounts, it generally exerts physiological functions, whereas production of high amounts of GSNO can result in a number of pathophysiological processes. In this context, the delivery of controlled low amount of GSNO is crucial to promote beneficial action of GSNO in intestine.

Impact of luminal GSNO on the integrity of the intestinal mucosa

Our study is in accordance with these results as we showed a dual and dose dependent effect on the maintenance of the integrity of the mucosal barrier: As expected, high concentration of GSNO accelerated the loss of integrity of the mucosal barrier when we studied the permeability of the intestinal mucosa to a paracellular marker in the Ussing chamber system. This result indicates that the cell junctions might be disrupt in the presence of a such a high concentration of NO and it was confirmed by the decrease of the amount of occludin. Although low concentration of GSNO (0.1 µM) do not change the amount of cell junction proteins in the intestinal mucosa, it delayed the loss of integrity of the intestinal barrier during the permeability study.

Therefore, this low concentration of NO on the luminal side of the mucosa preserved the integrity of the intestinal barrier. That is the reason why we proposed NO donor administrated by oral route as a good methodology to prevent relapses in IBD patients.

The mechanism of this protective action of NO was not investigated yet. Nevertheless, it might be involved in post-translational or proteolytic processing or regulation of protein kinase A (PKA) pathway, which can modulate occludin redistribution or degradation [START_REF] Feldman | Occludin: structure, function and regulation[END_REF][START_REF] Tai | Pen ch 13 allergen induces secretion of mediators and degradation of occludin protein of human lung epithelial cells[END_REF][START_REF] Runswick | Pollen proteolytic enzymes degrade tight junctions[END_REF][START_REF] Kawedia | The protein kinase A pathway contributes to Hg2+-induced alterations in phosphorylation and subcellular distribution of occludin associated with increased tight junction permeability of salivary epithelial cell monolayers[END_REF] or post-translation of adherens junction proteins as mentioned in the discussion part of article 2 in chapter 2.

Oral administration of GSNO

According to the result of chapter 2, to control the GSNO concentration is crucial in the application in IBD treatment. In addition, GSNO is a very fragile molecule which is easily degraded by many factors, such as light, pH, temperature, enzymes… Therefore, the development of GSNO delivery system for the chronic treatment in IBD is necessary. Oral administration is a better choice for chronic treatment because of good patient compliance and convenience. However, the drastic environment, such as various pH, temperature and enzymes, exists in gastrointestinal tract is the first obstacle to be overcome for GSNO oral administration. The second challenge is to control and sustain GSNO release in small amount and achieve GSNO local effect in intestine. As a small hydrophilic molecule, GSNO can be easily diffused, which cause low encapsulation efficiency and rapid penetration to external aqueous phase in encapsulation process. Besides, the sensitivity of GSNO to light, pH, temperature, metal ions and mechanical agitation leads to greater challenge in GSNO encapsulation.

Double emulsion technique is commonly used for encapsulation of hydrophilic molecules. In our study, GSNO is firstly encapsulated in Eudragit ® RL nanoparticle by double emulsion-solvent evaporation method, which presented favorable encapsulation efficiency, increased stability and preserved bioavailability of GSNO (intracellular protein S-nitrosation) (Wu et al., 2015a). However, no sustained release was observed in vitro. It might be caused by rapid diffusion in aqueous phase. Therefore, a second barrier to capture GSNO from penetration was developed by an extra entrapment in polymer matrix. This double encapsulation structure was called polymer nanocomposites. As described in chapter 1, with one more encapsulation layer, which may also promote the encapsulation efficiency by re-entrapment of GSNO with the polymer matrix. In the previous study in our laboratory, nanocomposite particles (NCP) based on alginate/chitosan blend polymers was developed to increase GSNO bioavailability for the application of cardiovascular disease. As chitosan was identified to open tight-junction between intestinal epithelial cells, it was finally characterized as a pro-inflammatory polymer [START_REF] Thanou | Oral drug absorption enhancement by chitosan and its derivatives[END_REF], Wu et al., 2016). The main idea of our study is to optimize this developed formulation for the application of IBD. Two strategies were carried out in parallel:

Firstly, chitosan was avoided in the construction of the polymer matrix. Two methods were utilized to form this alginate NCP: polyelectrolyte complexation and ionotropic gelation. With polyelectrolyte complexation method, the alginate NCP exhibited bigger size and simpler preparation process with higher GSNO loading capacity, but burst release in an in vitro artificial environment (pH 7.4 PBS at 37 • C) was observed.

Meanwhile, with ionotropic gelation method, the alginate NCP showed smaller size with better uniformity, less GSNO loading capacity but sustained release for at least 24 hours in in vitro. The smaller size and better uniformity were caused by stronger energy intervention (ultrasonication instead of mechanical agitation) and stronger network after calcium ions cross-linking with ionotropic gelation method. Burst release of polyelectrolyte complexation might be caused by the loose structure of polymer matrix layer which was formed by a simple electrostatic interaction between negatively charged alginate chain and positively charged Eudragit ® RL. Furthermore, Eudragit ® RL is a hydrophobic polymer, which leads to only parts of the polymer existed on the surface of NP can contact with alginate. Thus, the interaction is not tight enough to capture GSNO diffusion in a good way. But with ionotropic gelation, alginate not only react with Eudragit ® RL on the surface of NP but also interact with calcium ions to form an "egg-box" structure, which result in stronger structure. Calcium ion is small and divalent ion. It can be diffused to anywhere of the alginate chain and replace sodium ions, act as a bridge between two chains. There exists lots of binding sites for calcium ions in alginate chain. Thus, the interaction in the alginate NCP based on ionotropic gelation is much stronger and tighter, which leads to better capacity of GSNO capture.

However, the GSNO permeability study in Caco-2 cells monolayer showed that similar apparent delayed release and same behavior were obtained for both NCPs. One possible reason could be the calcium ions existed in the medium and cells may enhance the structure of alginate NCP based on polyelectrolyte complexation method. Because calcium ions can penetrate into the polymer matrix, then replace sodium ions and connect those alginate chains which finally preventing GSNO penetration. Therefore, we hypothesis that the only shortcoming of alginate nanocomposites particles based on polyelectrolyte complexation can be overcome when it will be applied in in vivo study.

The second strategy is to replace chitosan with another positively charged polymer

Eudragit ® E which is often used in formulation for targeting inflamed colon sites [START_REF] Avachat | Feasibility studies of concomitant administration of optimized formulation of probiotic-loaded Vancomycin hydrochloride pellets for colon delivery[END_REF]. It is widely used in oral delivery system for film coating, odour and taste masking, moisture and light protection [START_REF] Patra | Pharmaceutical significance of Eudragit: A review[END_REF]. The resulting alginate/Eudragit ® E NCP showed smaller size than alginate/chitosan NCP. The polymer molecular weight and viscosity are the key factors influencing the particle size, higher molecular weight and higher viscosity resulting in bigger particle size. Thus, the smaller size might be explained by lower polymer weight (47 kDa for Eudragit ® E and 50-190 kDa for chitosan) and lower viscosity.

With the optimization of NCP polymer matrix, not really increased GSNO encapsulation efficiency was achieved compared with NP. As mentioned before, GSNO, as a small hydrophilic molecule, can be easily diffused into aqueous phase. During the second entrapment, one more preparation step in aqueous medium with strong mixing method (ultrasonication) leads to rapid diffusion of GSNO, which finally leaded to less GSNO encapsulation efficiency than NP. Nevertheless, the burst release of the particle was improved. The application of NCP for GSNO delivery emerged the merits of both nanoparticles (considerable GSNO encapsulation efficiency) and polymer matrix (controlled and sustained release) and overcome the shortcomings of each other (burst release for NP and low GSNO capture ability) in one system.

Conclusion and Perspectives

Formulations

In conclusion, Four formulations have been developed for oral delivery of GSNO:

1. Eudragit [START_REF] Sun | The Caco-2 cell monolayer: usefulness and limitations[END_REF]. Following TEER, marker permeability and by visualization of junction protein by immunostaining during and after the permeability study through Caco-2 cells monolayer, the definitive utility of this formulation will be demonstrated. The aim of our study was to find an innovative treatment to prevent intestinal inflammation relpases in IBD patients (figure 32). We developed formulations with no described toxic effect of the particle and good patient compliance achieved by daily administration with easy treatment (oral route).

We have also the possibility to associated our treatment with other treatments.

Combination therapy has been published as a novel strategy of IBD treatment [START_REF] Hanauer | Combination Therapy for Inflammatory Bowel Disease[END_REF]. It was shown that combination therapy with infliximab (anti-TNF agent) and azathioprine (immunosuppressive drug) is superior to induce corticosteroid-free clinical remission than monotherapy with either agent in both Crohn's disease and ulcerative colitis [START_REF] Colombel | Infliximab, azathioprine, or combination therapy for Crohn's disease[END_REF], Panaccione et al., 2014). Therefore, drugs synergistic effect is promising strategy in IBD treatment. Our formulation strategy can be a good candidate to improve this combination therapy by co-delivery GSNO with drug that can induce remission, such as corticosteroids. Corticosteroids was reported to inhibit endogenous growth factors, thus preventing angiogenesis at the local tissue [START_REF] Patil | Concurrent delivery of dexamethasone and VEGF for localized inflammation control and angiogenesis[END_REF] while GSNO can promote angiogenesis through mediating induction of vascular endothelial growth factor [START_REF] Khan | Promoting endothelial function by S-nitrosoglutathione through the HIF-1alpha/VEGF pathway stimulates neurorepair and functional recovery following experimental stroke in rats[END_REF]. Corticosteroids and GSNO can act as supplementary treatment for each other. Corticosteroids can reinforce the anti-inflammation function while GSNO can accelerate the therapeutic effect by helping mucosa healing. Furthermore, local effect can be achieved to reduce the side effect and enhance the therapeutic function with the developed nanocomposite delivery strategy. A co-delivery of dexamethasone (a type of corticosteroid medication) and VEGF using PLGA microsphere/PVA hydrogel composites has already been developed

for localized inflammation control and angiogenesis [START_REF] Patil | Concurrent delivery of dexamethasone and VEGF for localized inflammation control and angiogenesis[END_REF]. In the present study, the strategy could be GSNO and corticosteroids firstly encapsulated in NPs separately, followed by a further encapsulation of polymer matrix. Thus, GSNO codelivered with anti-inflammation drugs in polymer NCP can be a promising strategy in IBD treatment. With our NCP strategy, it gives us potential to co-delivery the two different drugs. As previously mentioned in the conclusion part of chapter 3, main requirements of the formulations have already been achieved. But the main advantages of the NCP have not been explored yet: co-encapsulation of two drugs in the same delivery system. Different strategies could be proposed: a). different drug-loaded-NP, prepared separately, entrapped in the same matrix system; b). one drug loaded in NP and a second one included into the matrix system. The choice of strategies a) or b) may induce modification of physical-chemical characterization and drug release profile. The strategy will be selected according to the drug.

However, for a further application in clinical trial for human, more investigations of the formulation part need to be done. Additional "difficulties" will rise when theses formulations will be tested in clinical trial for human. Stable conservation form like powder need to be developed for longer and convenient storage. For the further economic consideration, we still have to optimize the formulation to increase encapsulation efficiency. The strategy could be increasing the interaction between GSNO and polymer by electrostatic interaction or covalently bonding. For example, Snitrosoglutathione-alginate (SNA) which has been developed in the laboratory can be used to replace sodium alginate in the present study. SNA is a novel NO-donor by crosslinking alginate with S-nitroglutathione, which can deliver NO in a sustained manner [START_REF] Shah | Synthesis of S-nitrosoglutathionealginate for prolonged delivery of nitric oxide in intestines[END_REF]. With the chemical bonding, GSNO loading capacity can be absolutely increased without worrying about GSNO diffusion. In addition, we expect that the release behavior will be further controlled and sustained by replace alginate with SNA. Because, the remaining GSH in the SNA can react with NO released by GSNO and there might be transnitrosation reaction between GSNO and SNA to retain GSNO in a better way.

The final goals of our study are to decrease the treatment cost and to increase quality of patients' life. With our proposed simple preparation process, it could be easy to scaleup in industry, which can finally improve the low cost-benefits ratio of current treatments. Meanwhile, to decrease patient daily handicap and decrease the risk of colorectal cancer associated with relapses could finally increase quality of patients' life.

Effects of released GSNO in inflammation

For the application part, we have confirmed our concept that luminal GSNO (oral administration) also showed a dose-dependent effects on intestinal barrier integrity as published mucosal source GSNO. However, the limitation of our study is the absence of ex vivo inflammation model. The application of our study is to prevent the inflammation in IBD patient. To assess the luminal GSNO ability of preventing inflammation, we used the healthy intestine tissue in Ussing Chamber as a stress model with loss of TJ expression (occludin and beta-catenin) compared with wild-type rat intestine tissue. But the mechanism was not deeply investigated yet. This investigation should be performed with a model closer to the pathophysiological context of inflammation in vivo. There are lots of in vivo inflammation models described in the literature that trigger inflammation process similar to what is observed in IBD patients.

No similar model has been developed in Ussing chamber as an ex vivo model for better mimicking the therapy process as in vivo process. We tried to induce inflammation in the Ussing chamber with addition of colitogenic agent (such as Dextran Sodium Salt)

and LPS [START_REF] Emmanuel | Acidosis and lipopolysaccharide from Escherichia coli B:055 cause hyperpermeability of rumen and colon tissues[END_REF]) in the luminal compartment in order to study the intestinal permeability to the paracellular marker, the expression of cell junction proteins and of inflammation markers (data not shown). Unfortunately, no differences were observed between colitogenic and control conditions. The time limitation of this ex vivo study probably explain this negative result. Because, it is probably not suitable for a study of inflammation process involving transcription, translation, posttranslational modification, protein transportation and mediator releasing and so on… When the developed formulations reach the target-intestine, the interaction between formulation and mucus is essential and it would be important to further investigate mucoadhesion and mucopenetration.

Another question that has to be considered is the bioavalability of GSNO in the intestine in vivo. After GSNO release, it can be used by cells and bacteria as well. The proportion of bacterial and cells contribute in the GSNO metabolism is interesting to be known the effective GSNO bioavailability. GSNO cannot be transported directly into cells. But different enzyme presenting in the cells can help the transportation of NO. Firstly, GSNO can be catalyzed to CysGlyNO by GGT. But CysGlyNO is not stable, which The goal of our study is to check if oral administrated GSNO can prevent IBD relapses or not. The ex vivo experiments in order to mimic inflamed condition in the intestinal mucosa seems to be not adapted, which finally impose to use in vivo models. Now that we have developed three formulations that could be adapted to oral administration, we are able to plan in vivo studies in order to check that the protection of the integrity of the mucosa is conserved in physiological conditions, and to check if in inflamed condition, it can prevent, delay or protect from the inflammation. DSS inflammation model will be used. Alginate nanocomposite particles based on polyelectrolyte complexation method will be firstly tested in this DSS model rat because of higher GSNO encapsulation efficiency but simpler preparation process. The experiment schedule is shown in Figure 34. The in vivo model is used to determine the formulation efficiency and GSNO therapeutic potential as a chronic oral treatment of IBD prevention. GSNO loaded NCP, free GSNO, and corresponding blank-NCP or PBS as controls will be administrated by gavage to rats before inflammation induction. Dextran sodium sulfate (5%) in drinking water will be used to induce colitis [START_REF] Melhem | Methyl-deficient diet promotes colitis and SIRT1-mediated endoplasmic reticulum stress[END_REF]. The treatment of GSNO, such as duration and repetition times, should be further confirmed to maximize the efficiency. Meanwhile, the exact duration of inflammation induction should be defined according to the severity of inflammation. Seven days of DSS induction was known to finally result in severe inflammation in rat intestine [START_REF] Melhem | Methyl-deficient diet promotes colitis and SIRT1-mediated endoplasmic reticulum stress[END_REF]. In order to make sure we can see difference between GSNO treatment group and control group.

We need to adapt the inflammation severity to our study in advance, maybe 2 days, or 4 days... The inflammation severity will be visually determined by clinical scoring changes in weight, hemoccult positivity or gross bleeding, and stool consistency during the induction [START_REF] Murthy | Treatment of dextran sulfate sodium-induced murine colitis by intracolonic cyclosporin[END_REF] and endoscopic, histological scores at the end of the induction [START_REF] Melhem | Methyl-deficient diet promotes colitis and SIRT1-mediated endoplasmic reticulum stress[END_REF][START_REF] Kökten | TREM-1 inhibition restores impaired autophagy activity and reduces colitis in mice[END_REF]. The integrity of intestinal barrier will be followed by permeability study to paracellular marker in Ussing Chamber system after DSS induction. The biochemical studies containing quantification of junction proteins and inflammation markers will be fulfilled by western blot and ELISA as well.

169 GSNO loaded NCPs adapted to oral administration has been developed, presenting a promising methodology for the care of patients, that could be proposed in parallel or as an alternative of classical treatment in order to ameliorates the quality of life of patients.

Nanoparticle hardening:

Immediately evaporate the secondary emulsion (100 mbar, 10 min; water-bath 25°C, chiller 10 °C, 60 rpm). When the pump reaches 100 mbar, press immediately the "Mode" button to set this as target value (NB: if the pump has been previously used the same day and not switched off, there is no need to press "Mode" button).

In the case of composite particles preparation, prepare the chitosan solution during evaporation: in a 50 mL brown bottle, weight 20 mg of low molecular weight chitosan and dissolve in 20,0 mL (18,0 mL of ultrapure water and 2,0 mL of acetic acid, glass pipettes) under magnetic stirring at 1000 rpm. Keep on ice before using.

After evaporation, measure the volume of suspension in a cylinder (usually ̴ 18 mL). For preparation of nanocomposites particles, adjust the volume at 20 mL with ultrapure water (this volume is necessary to ensure a correct dissolution of alginate).

The protocol for preparation of GSNO-NP is schematized in the following Figure.

Western Blot protocol

Principle:

Western blotting uses specific antibodies to identify proteins that have been separated based on size by gel electrophoresis. The immunoassay uses a membrane made of nitrocellulose or PVDF (polyvinylidene fluoride). The gel is placed next to the membrane and application of an electrical current induces the proteins to migrate from the gel to the membrane. The membrane can then be further processed with antibodies specific for the target of interest, and visualized using secondary antibodies and detection reagents.

Solutions preparation:

RPN1675K

All Solutions are prepared with ultrapure water (18,2 MΩ•cm). β-catenin, MA1-2001, PIERCE, 1:800 5% non-fat milk/TBST;E-cadherin, SC-7870, Santa Cruz, 1:700 5% BSA/TBST;Occludin, 71-1500, Invitrogen, 1:600 5% non-fat milk/TBST;Claudin-1, 37-4900, Invitrogen, 1:600 5% non-fat milk/TBST;IL-1β, ab9722, abcam, 1:1000 5% non-fat milk/TBST;TNF-α, ab6671, abcam, 1:1000 5% BSA/TBST Reference proteins: If the reference protein is conjugated with Horseradish Peroxidase (HRP), it is not necessary to incubate with another second antibody. Then, use the dilution which is recommended by the company and follow the same protocol above.

β-actin, ab197277, abcam, 1:10,000 5% BSA/TBST; room temperature, 1h

• Place the membrane in the plastic bag • Pour the first antibody solution carefully inside the bag, remove all the bubbles inside the bag and seal it. • Incubate at 4 °C overnight in a tray, use adhesive tape to fix all the bags, switch on the agitator at a speed 15 rpm. • Conserve the antibody solution at -20 °C for repeated use (normally 3 times maximum).

Washing:

• Take out the membrane from the plastic bag. Then put it in a small tray.

• Conserve the remaining antibody solution in the plastic bag at -20 °C. It can be reused for 2-3 times. • Rinse it rapidly with TBST firstly to remove most of the residual liquid. Then rinse the membrane 10 min, 4 times, on a shaker with a speed of 70 rpm at room temperature.

Reference protein:

For reference proteins which is conjugated with HRP, the membranes can go directly revelation ECL step.

Secondary antibody incubation:

• Prepare a plastic bag which is bigger than the membrane and write antibody information, dilution times, dilution medium date and experimentor's name on the bag. • Dilute the second antibody solution in TBST/5 % non-fat milk (or BSA) (recommend 1:10,000) • Incubate it on a shaker with a speed of 15 rpm at room temperature for 1 hour

Washing:

• Same process with the first antibody washing.

Gel preparation (SDS-PAGE Tris/Glycine) Développement de formulations polymériques de S-nitrosoglutathion comme traitement per os pour prévenir les maladies inflammatoires chroniques de l'intestin Les maladies inflammatoires chroniques de l'intestin (MICI) représentent un problème de santé publique majeur touchant de jeunes patients. Aux récidives inflammatoires, à la mauvaise qualité de vie et à l'espérance de vie réduite des patients viennent s'ajouter la durée, l'efficacité parfois limitée et le coût des traitements actuellement proposés. La recherche de nouvelles stratégies permettant de prévenir les récidives inflammatoires est primordiale. Ainsi, le rôle du S-nitrosoglutathion (GSNO, donneur et forme de stockage naturelle de NO) dans le maintien de l'intégrité de la barrière intestinale est étudié dans cette thèse, en décrivant : i) l'effet concentration-dépendante du GSNO sur la perméabilité intestinale (modèle de chambre de Ussing, expression des protéines de jonctions cellulaires…), ii) des formulations innovantes de GSNO (nanoparticules composites) à base de nanoparticules d'Eudragit ® RL, elles-mêmes encapsulées dans une matrice polymérique à base d'alginate. Différents procédés de formulation ont été testés. Une caractérisation physicochimique des objets développés et des études de perméabilité sur des modèles cellulaires (Caco-2) ont conduit à l'obtention de trois formulations, adaptées à la voie orale et permettant de délivrer de façon prolongée le GSNO. Cependant, la prise en charge du GSNO libre ou formulé, l'identification des cibles du NO au niveau intestinal, mais aussi les doses et durées du traitement in vivo restent encore à définir avant de proposer ce donneur de NO comme candidat pour un traitement préventif des MICI. Mot clefs : S-nitrosoglutathion, nanoparticules composites, prévention, barrière intestinale.

Development of S-nitrosoglutathione loaded particles adapted to oral administration for

preventing Inflammatory Bowel Disease relapses Inflammatory bowel diseases (IBD) (Crohn's disease, ulcerative colitis…), are disabling pathologies affecting young patients and presenting the particularity that most of the current agents act by down regulating chronic inflammation in the intestine mucosa and cannot cure the disease. As the pivotal role of S-nitrosoglutathione (GSNO) in preventing intestinal inflammation and gut barrier failure has been clearly pointed out, therapies based on pharmaceutical technology to limit chronic inflammation and prevent relapses of barrier failure by an optimized dosage form of GSNO appears as an interesting challenge. The objective of this project relies on i) studying GSNO concentration/response functions in intestine by using Ussing chamber ex vivo model and following protein cell junction expression, ii) developing polymer nanocomposite particles encapsulating GSNO and adapted to the oral route, with GSNO protection, controlled delivery and local effect in intestine as major requirements. GSNO loaded polymeric (Eudragit ® RL) nanoparticles were included in polymer matrix based on alginate; different processes were tested and particles were characterized: high GSNO loading, GSNO sustained release and local retention for 4 h in cellulo study (Caco-2) were obtained. Thus, therapies based on GSNO administration should represent a novel strategy to limit chronic inflammation and prevent relapses of barrier failure in IBD patients. Key words: S-nitrosoglutathione, intestinal barrier, oral administration, nanocomposites

  General discussion and perspectives .......................................................................... 4.1 Discussion ........................................................................................................ 4.1.1 Impact of luminal GSNO on the integrity of the intestinal mucosa ......... 4.1.2 Oral administration of GSNO ................................................................... 4.2 Conclusion and Perspectives............................................................................ Reference ................................................................................................................... Attachments ............................................................................................................... Attachment 1: Article-Polymer nanocomposites enhance S-nitrosoglutathione intestinal absorption and promote the formation of releasable nitric oxide stores in rat aorta .. Attachment 2: Protocols ............................................................................................. III LIST OF TABLES

  FigureDescription d'un système particulaire composite délivrant un RSNO.viii

Figure 3

 3 Figure 3 NaFlu flux: Evolution of sodium fluorescein (NaFlu, 1 mg/mL) flux; TEER: Evolution of trans-epithelial electrical resistance Figure 4 Expression of cell junction proteins by western blotting after different concentration of GSNO exposure in Ussing Chamber for 2 h.

Figure 5

 5 Figure 5Possible mechanism of intestinal barrier integrity modulation by NO.

  mise en place d'un traitement chronique préventif, basé sur l'administration orale de S-nitrosoglutathion (GSNO) représente une nouvelle stratégie pour limiter les récidives inflammatoires et les dommages au niveau de la barrière intestinale chez des patients atteints de MICI. Cependant, le développement d'un traitement chronique préventif des MICI, par voie orale, à base de GSNO représente un véritable challenge. Il s'agit de proposer une forme pharmaceutique stable adaptée à la voie orale, administrée à distance d'un épisode inflammatoire aigu, contrôlant la concentration de NO délivré au niveau intestinal pour assurer une action locale bénéfique.

  2 h, ont montré pour la première fois, que la perméabilité de la fluorescéine sodique (marqueur du passage entre les cellules intestinales, au travers des jonctions cellulaires) est significativement diminuée, en présence d'une faible concentration en GSNO (0,1 µM), par rapport aux plus fortes concentrations testées (10 et 100 µM). En revanche, aucune modification significative de la résistance transépithéliale (TEER) et de la quantité des protéines de jonction étudiées n'a été observée. Seule une diminution de 50 % de la quantité d'occludine (protéine constitutive des jonctions serrées) a été constatée en présence de 100 µM de GSNO. Ainsi, nos résultats indiquent qu'une faible concentration en GSNO (0,1 µM) retarderait la perte d'intégrité de la muqueuse intestinale dans ce système ex vivo, alors que les fortes concentrations en GSNO (100 µM) accélèreraient cette perte d'intégrité et altèreraient les jonctions cellulaires responsables de cette intégrité. vii Une étude plus précise de l'expression des protéines de jonction (par la technique de Western Blot sur extraits membranaires uniquement, et non sur lysats tissulaires totaux ou par ELISA) pourrait affiner ces résultats. Pour compléter ces travaux, l'internalisation des protéines des jonctions cellulaires pourrait également être étudiée par immunofluorescence en microscopie confocale. Le TEER et l'expression des protéines de jonction à la membrane sont finalement des marqueurs tardifs des effets précoces du GSNO. Dans un tissu sain présentant une balance RedOx contrôlée, la capacité de la cellule à lutter contre le stress pourrait masquer ces effets précoces du NO. A l'inverse, dans un tissu inflammatoire, cette capacité de la cellule est altérée et l'effet du NO pourrait être observé plus aisément car ils pourraient résulter d'une nitrosation modérée de protéines impliquées dans l'intégrité de la barrière. Ce résultat constitue la première « preuve de concept », la première étude de faisabilité quant à l'utilisation de GSNO comme traitement préventif des MICI. L'administration orale requiert une mise en forme adaptée du GSNO et constitue une seconde partie majeure de ce manuscrit. Si le NO voit sa demi-vie augmentée sous la forme GSNO (de quelques minutes à plusieurs heures), ces donneurs de NO n'en demeurent pas moins fragiles et très réactifs vis-à-vis de leur environnement. Sensible à différents facteurs environnementaux comme par exemple la lumière, les variations de température ou la présence d'ions métalliques divalents, la liaison S-NO est maintenue dans le temps très difficilement (Gaucher et al., 2013). L'établissement d'un traitement chronique avec ce type de molécules nécessite une mise en forme galénique adaptée afin de protéger le NO jusqu'à son lieu d'action. De plus, cette forme galénique doit permettre un contrôle des doses de NO libéré. Les systèmes particulaires à libération contrôlée, tel que des nano-ou microparticules polymériques, peuvent tout à fait répondre à ces contraintes puisqu'elles sont basées sur l'utilisation de polymères biocompatibles, approuvés par les autorités de santé, et que les particules développées offrent une surface d'échange avec leur environnement particulièrement adaptée à la muqueuse intestinale.viii L'encapsulation directe de GSNO est un véritable challenge, liée notamment à la difficulté de préserver la liaison S-NO durant le procédé de formulation des systèmes particulaires. Cette stratégie offre la possibilité d'obtenir des charges en NO très intéressantes pour minimiser les quantités de particules à administrer, pour protéger le GSNO véhiculé et pour proposer des libérations et des actions plus prolongées dans le temps. Pour une application orale et un effet au contact de l'intestin, une libération sur 24 h serait plus adaptée. Ce travail de thèse fait suite à différents travaux entrepris au sein de l'équipe CITHEFOR ayant démontré la faisabilité de cette encapsulation

Figure 2

 2 Figure 2 Description d'un système particulaire composite délivrant un RSNO.

( 2 ,

 2 5±0,6 mg de GSNO/g de polymère) et équivalente à celles des particules composites à base d'alginate seul ou associé avec le chitosan. La caractérisation physico-chimique de ces particules composites doit être complétée par des études in vitro et in cellulo de xi libération du GSNO. En conclusion, ce travail s'attache donc au développement et à la caractérisation, limitée à la détermination des paramètres physico-chimiques des particules, à l'évaluation de leur cytotoxicité et à l'étude de la perméabilité du GSNO délivré sur un modèle de barrière intestinale en culture. A l'issue de ces travaux de thèse, trois formulations adaptées à la délivrance orale de GSNO sont proposées. La démarche entreprise devra être poursuivie par : -des études de stabilité des formulations proposées -des études in vivo (modèles animaux de MICI) évaluant la capacité des systèmes particulaires composites de GSNO les plus efficaces à prévenir le développement et à limiter l'apparition d'épisodes inflammatoires aigus. Il s'agira d'administrer oralement (par gavage) les formulations (sous la forme de suspensions) de manière préalable à l'induction de l'inflammation induite par des composés chimiques (sulfate de dextran sodique par exemple) (Tran CD et al., 2012) (Figure 3).

Figure 3 :

 3 Figure 3 : Schéma expérimental des expériences qui pourront être menées in vivo.

  substances, inflammatory cytokines, phagocytic cells, NK cells, NK T cells T cells (cell immunity) B cells (humoral immunity) Inflammation happens when the immune system dysfunction occurs. The Figure 1 showed the link of IBD and immune dysfunction. The innate immune abnormalities of inflammatory bowel disease result in adaptive immune disorders (Th1/Th2 regulation imbalance and Th17/Treg transformation imbalance). The inflammatory cytokines in turn increase the innate immune damages (apoptosis of intestinal epithelial cells, reduction of connection protein expression, decrease of antibacterial peptides secreted by Paneth cells, etc.), abate the intestinal barrier function, and aggravate inflammation.

Figure 1

 1 Figure1IBD and immune dysfunction[START_REF] Huang | Inflammatory bowel disease related innate immunity and adaptive immunity[END_REF]).

  It is constituted of a thin layer of cells (epithelium) that produce serous fluid. Serous fluid is a lubricating fluid which decrease friction from the muscularis movement. The muscularis is in charge of the movement or peristalsis of the intestine. It normally has two obvious layers of smooth muscle: circular and longitudinal. The submucosa is the dense layer of irregular connective tissue that supports the mucosa, as well as joins the mucosa to the bulk of underlying smooth muscle. The mucosa is a mucous membrane, which is the innermost tissue layer of the small intestine It includes digestive enzymes and hormones. The intestinal villi are part of the mucosa, which has bigger surface that can enhance the nutrients absorption. The mucosal layer of the gut consists of epithelium overlying the lamina propria, and resting on a narrow layer of smooth muscle, the muscularis mucosae. It represents the obvious physical boundary between the external and internal environment to constitute an important defense barrier of human body.

Figure 2

 2 Figure 2 Anatomy of intestine

Figure 5

 5 Figure5The junctional complexes (a)The junctional complexes, comprised of tight junctions, adherens junctions, gap junctions, and desmosomes.(Turner, 2009) (b) Electron micrograph of apical junctional complex between two intestinal epithelial cells of human ileal mucosa.[START_REF] Salim | Importance of disrupted intestinal barrier in inflammatory bowel diseases[END_REF]

Figure 7 .

 7 Figure 7. The multiple domains of ZO proteins. PDZ: large/zona-occludens domains, SH-3:a Src homology-3 domain; GUK: a region of homology to guanylate kinase.

Figure 8

 8 Figure8AJ belt-like connection structure formed by cadherin-catenin complex(Hoffman and Ya p, 2015)

  demosgleins and desmocollins -which are cadherin proteins) attached to desmosomal plaque linked by desmoplakin with the intermediate filaments (called keratin in epithelial cells) cytoskeleton (Figure5 and 9). There are three major gene families encoding desmosomal proteins. The first one is desmosomal cadherins, comprising two subtypes called desmogleins and desmocollins. They are a subfamily of the cadherin

Figure 9

 9 Figure 9 Molecular components and model of desmosomal protein organization (Kowalczyk and Green, 2013); DP: desmoplakin, PG: plakoglobin, PKP: plakophilins.

Figure 11

 11 Figure 11 Epithelial pathway route: transcellular pathway and paracellular pathway.

Figure 12

 12 Figure 12 Transcellular pathway: non-selective phagocytosis, non-selective pinocytosis and receptor-mediated endocytosis[START_REF] Salim | Importance of disrupted intestinal barrier in inflammatory bowel diseases[END_REF]

Figure 13 .

 13 Figure 13. Three distinct paracellular epithelial permeability pathways in homeostasis and active disease state (Odenwald and Turner, 2017). EGF: epidermal growth factor; IL-13R:

Figure 14

 14 Figure 14 Ussing Chamber system

Figure 15

 15 Figure 15 Transwell ® system: cell culture monolayer

  adalimumab, certolizumab pegol, and golimumab[START_REF] Sales-Campos | Classical and recent advances in the treatment of inflammatory bowel diseases[END_REF]. Anti-TNF antibodies can suppress intestinal inflammation in IBD through several mechanisms(Neurath, 2017): inhibit macrophages to produce proinflammatory cytokines; prevent Paneth cell death from necroptosis; reduce intestinal epithelial cells apoptosis42; regulates T-cell apoptosis; and increase production of tissue inhibitor of matrix metalloproteinases (MMPs) by fibroblasts to mediate tissue injury via preventing activation of MMPs45.

Figure 16

 16 Figure 16 Endogenous synthesis of nitric oxide using L-arginine, oxygen and cofactors as the substrates.

Figure 17

 17 Figure17Concentration dependency of NO functions.[START_REF] Thomas | Superoxide fluxes limit nitric oxide-induced signaling[END_REF][START_REF] Thomas | The chemical biology of nitric oxide: implications in cellular signaling[END_REF]

Figure 18

 18 Figure18Major beneficial actions of NO in intestine[START_REF] Magierowski | Gaseous mediators nitric oxide and hydrogen sulfide in the mechanism of gastrointestinal integrity, protection and ulcer healing[END_REF]

  secretion in the gastrointestinal tract. The secretion of mucus and fluid can not only reduce bacterial adherence to the epithelial but also dilute and flush away any potentially noxious substance in the lumen. If microbes or antigens are able to penetrate through the mucus and fluid secreted, the next barrier they will encounter is the tight-junction between adjacent epithelial cells. NO secreted by enteric glia cells can also locally enhance this barrier by improving the expression and localization of occludin, zonula occludens-1 (ZO-1), and phosphorylation of myosin light chain (P-MLC)(Savidge et al., 2007[START_REF] Cheadle | Enteric glia cells attenuate cytomix-induced intestinal epithelial barrier breakdown[END_REF] which play important roles in tight-junction. If the mucosal barrier failed to prevent microbial invasion, NO still works on it from two systemic

Figure 1

 1 Figure 1 Representative scheme of polymer nanocomposites: drug, nanoplatforms and polymer matrix. They may present as A) core-shell structures, B) particles and C) bulk/film

  inducer of mucosal barrier function in vitro and in vivo and of attenuated tissue inflammation after ablation of enteric glia gene in mice. GSNO regulation of mucosal barrier function was associated directly with an increased expression of perijunctional F-actin and TJ associated proteins ZO-1 and occludin. GSNO also significantly restored mucosal barrier function in colonic biopsy specimens from patients with Crohn's disease in the ex vivo Ussing Chamber experiment. Flamant et al investigated that both EGCs and GSNO but not GSH significantly reduced barrier disruption and inflammatory response induced by shigella flexneri infectious in Caco-2 monolayers. Cheadle et al further investigated the mechanism of GSNO secreted by EGCs prevent intestinal mucosal barrier disruption through an in vitro model. The result showed both GSNO and co-culture with EGCs prevented Cytomix-induced increases in permeability by improving expression and localization of occludin, ZO-1, and P-MLC (phosphorylated myosin light chain). Recently, Li et al tested the potential therapeutic effect and mechanism of femoral venous injected GSNO on lipopolysaccharide (LPS)induced inflammatory response and intestinal mucosal barrier injury in a rat model of endotoxemia(Li et al., 2016). GSNO (1mg/kg) was administered by femoral venous injection 15 mins after LPS injection. The result demonstrated that GSNO addition reduced the intestinal injury observed in histologic sections, decreased permeability to fluorescein isothiocyanate dextran, attenuated damage of the junction between epithelia, and protected against the LPS-induced expression decrease of ZO-1. Furthermore, addition of GSNO reduced plasma and intestinal TNF-α and IL-1β levels as well as inhibited the LPS-induced up-regulation of myosin light-chain kinase expression and NF-κB p65 level in the intestine.

  A current electrode and a voltage electrode(multimeter, Millicell-ERS2, Millipore®; electrodes P2020, Physiologic Instrument®) were placed in each compartment for TEER measurement. TEER (Ω×cm²) was determined by the U = R × I law after inducing a current from one compartment to the other, with U the potential gradient, I the induced current by the apparatus and R the resistance of the tissue (Ω) multiplied by the tissue area separating both compartments (cm²).Permeability experimentsNaFlu (2.7 mM) permeability (from donor/luminal compartment to acceptor/mucosal compartment) were assessed by measuring changes in concentration of the compounds with time. Samples (250 µL of medium) were harvested in the acceptor compartment at different times (every 30 min, until 2 hours; each removed volume was replaced by an equal volume of 37°C pre-heated Krebs-Henselheit buffer) for the quantification of permeability markers flux. TEER measurement was performed at these same time intervals to follow intestine tissue integrity. [NaFlu] was evaluated with a spectrofluorimeter (BioTek®, ex = 460 nm; em = 515 nm)The flux (J) of compounds or their apparent permeability (Papp) through the intestine were calculated according to the following formulae: slope of the steady state portion of the amount of the drug permeated during time t, A the tissue area separating both compartments and C0 the initial concentration in active molecule; units:

Figure 1 Figure 2

 12 Figure1Criteria of NaFlu permeability and TEER result during 2 h in Ussing chamber (--criteria: NaFlu flux at 15 µg×cm -²×s -1 at t=2 h, and TEER at 30 Ω×cm² at t=0). *p < 0.05, statistic difference s 0 h and 0.5 h; one-way ANOVA

Figure 3

 3 Figure 3 NaFlu flux: Evolution of sodium fluorescein (NaFlu, 1 mg/mL) flux, ---NaFlu flux criteria at 15 µg×cm -²×s -1 at t=2 h; TEER: Evolution of trans-epithelial electrical resistance, ---TEER criteria at 30 Ω×cm² at t=0, in Ussing chamber during 2 h. Control (n = 19; N = 17), 0.1 µM (n = 7; N = 5), 10 µM (n = 7; N = 5), 100 µM (n = 6; N = 5). *p < 0.05 νs control; # p < 0.05 νs 0.1 µM GSNO; two-way ANOVA (groups and time).

  in the amount of this cell junction proteins were observed with both low and high concentration of NO. Despite this lack of statistical significance, we noted that the amount of occludin is reduced by 50 % in the presence of 100 µM of GSNO.

Figure 4

 4 Figure 4 Expression of cell junction proteins by western blotting after different concentration of GSNO exposure in Ussing chamber for 2 h. The ratio value was normalized results from WT; two-way ANOVA (groups and proteins), n=5. WT: wild type, ctrl: control in Ussing chamber system.

  We speculate that different NO concentrations may cause different post-translational modifications of proteins according to the literatures. In the presence of low concentration of NO (0.1 µM), it might be a S-nitrosation caused by an interaction between NO and cystein residue of protein. The final product RSNO (S-nitrothione) could also act as a storage form of NO. In contrast, in the presence of high concentration of NO (100 µM) with oxidative stress condition, there might exist nitration of proteins on the tyrosine residue, which may provoke deleterious effect such as an increase of the intestinal permeability or a modulation of the activity of the immune response effectors and finally trigger the pathological inflammation process. Through different literatures, different proteins could be the interesting targets of different post-translational modification way caused by NO, which may finally modulate the intestinal barrier integrity.

Figure 5

 5 Figure 5 Possible mechanism of intestinal barrier integrity modulation by NO.

  GSNO-NCP suspension (1 mL) was placed in cellulose dialysis tubing (average flat width 10 mm (0.4 in), cut-off 14 kDa). Then, the dialysis bag was immersed in 200 mL of phosphate buffered saline at pH 7.4 at 37°C with protection from light. The sample in the dialysis bag was harvested from the bag for the size measurement with Mastersizer 2000 (Malvern Instruments, France) at t=0, 2, 4, 6, 24 h. The swelling rate was presented by size change which was calculated to evaluate the swelling of the nanocomposite particles. The fractional size change was converted into percentage using equation (2), where St and Si were the particles size at different time intervals and initial time, respectively:

Figure 2

 2 Figure 2 Drug (GSNO and degradation product nitrite ions) release from GSNO-NCP ( free GSNO, ionotropic gelation, polyelectrolyte complexation) in PBS (0.148 M, pH 7.4, 37 °C). Free GSNO was used as control. Data are shown as mean ± SD (n=3). *p < 0.05, ionotropic gelation vs free GSNO & polyelectrolyte complexation, two-way ANOVA with post-test of Bonferroni's multiple comparisons test

Figure 3

 3 Figure 3 GSNO-NCPs ( ionotropic gelation, polyelectrolyte complexation) swell in PBS (0.148 M, pH 7.4, 37 °C). Data are shown as mean ± SD (n=3).

Figure 4

 4 Figure 4 In vitro cytotoxicity of GSNO-NCPs (A: ionotropic gelation; B: polyelectrolyte complexation) on Caco-2 cells. Caco-2 cells were treated with indicated concentrations of

Figure 5 :

 5 Figure 5: GSNO permeability evaluation of free GSNO ( ) and GSNO-NCP ( ionotropic gelation, polyelectrolyte complexation) in Caco-2 cells monolayers. A, B, C: GSNO, nitrite ions-and nitrate ions permeated from apical to basolateral compartment within 24 h. D, E: GSNO and nitrite ions remained in apical compartment after 4 h and 24 h of incubation. Values presented have been subtracted from control (cells without treatment for free GSNO, NCP without GSNO for GSNO-NCP. Data are shown as mean ± SEM (n ≥ 3). *p < 0.05 vs free GSNO, #p < 0.05 vs polyelectrolyte complexation, two-way ANOVA with post-test of Bonferroni's multiple comparisons test.

Figure 25

 25 Figure 25 Advantages and drawbacks of two different alginate-NCP.

Figure 26

 26 Figure 26 Possible mechanism of GSNO release in different alginate-NCP in in vitro environment.

  under acidic pH.

Figure 29

 29 Figure29Possible mechanism of Interaction between TPP and Eudragit ® E 100[START_REF] Moustafine | Characteristics of interpolyelectrolyte complexes of Eudragit E 100 with sodium alginate[END_REF]

Figure 30

 30 Figure 30 SEM images of GSNO-aeNCPs: a) surface morphology and b) zoomed in nano-scale

Figure 31

 31 Figure31In vitro cytotoxicity of GSNO-aeNCP on Caco-2 cells. Caco-2 cells were treated with indicated polymer concentrations of GSNO-aeNCP for 24 h at 37•C. viability was estimated by MTT assay. Data are expressed as mean ± SEM (n=4).

Figure 32 A

 32 Figure 32 A crucial goal for the patients: Inflammation prevention.

Figure 33

 33 Figure 33 Perspectives of NO action with intestine. (GGT: γ-Glutamyltransferase; PDI: Protein disulfide isomerase; L-AT: the L-amino acid transporter system)

  will finally be transnitrosated to CysNO. CysNO can be transported into cells by the Lamino acid transporter system (L-AT) to participate the transnitrosation of target proteins. A second transportation way is conducted by PDI, GSNO can be catalyzed to NO radical with PDI. Then this radical can be easily diffused into the cells, which may finally contribute in the S-nitrosation of target proteins. Thus, the proportion of GGT and PDI participate in the GSNO metabolism can be further studied for GSNO metabolism mechanism exploration in intestine in vivo. The identification of target proteins and NO storage into intestine are also necessary to completed the mechanism study. The identification of target proteins can be performed by Biotin Switch method. The investigation will start with the possible target proteins, such as NF-κB, β-catenin and occludin, which are mentioned in chapter 2. NO storage will be studied by DAF-2 DA (5,6-Diaminofluorescein diacetate) assay to check the localization of NO in intestine and NO amount quantification by HPLC-MS with 15 N labeled GSNO after DAN derivatization which mentioned in paragraph 3.3.4 of chapter 3.

Figure 34

 34 Figure 34 In vivo evaluation of GSNO loaded formulations in inflammation model

  preparation CaCl2 solution: dissolve 2.9 g CaCl2 in 10 mL water to obtain 2 M. 2.1.2 Sodium alginate dissolving 20 mg sodium alginate were dispersed on the surface of nanosuspension for 5 min (formation of a transparent gel, on the surface), then keep agitation (magnetic stirrer, 1000 rpm, Heidolph MR 3000D),) for 10 min (total dissolution of alginate) at room temperature (to avoid the solubility decrease of alginate at low temperature) 2.1.3 Cross-linking with calcium ions 2 mL CaCl2 (2M) are added drop-wisely (23 G) into the mixture of nanoparticles and sodium alginate under ultrasonication (ultrasonic system 1; 30 % amplitude, 40 W, over ice bath, with horizontal movement for homogenization, about 2 min 30s. The protocol for preparation of GSNO-alginate NCP based on IG method is schematized in the following Figure.

Table 1

 1

	N •	Captions

Le procédé de complexation de polyelectrolytes, plus simple et plus facilement industrialisable que le procédé de gélification ionotropique, est adapté à la libération du GSNO. Ces deux procédés répondent donc au cahier des charges mentionné plus haut, en termes d'encapsulation, de libération et de délivrance locale au niveau intestinal

  Cette différence de libération du GSNO observée in vitro, est minimisée au contact des cellules (cellules Caco-2 cultivées en système de Transwell ® ) : la perméabilité du GSNO et de ses produits de dégradation (ions nitrite et nitrate) est retardée de la même façon quelle que soit la formulation choisie.

	Procédés de formulation	Charge en GSNO (mg/g de polymère) Libération du GSNO à 3h (%)
	Complexation de polyélectrolytes	4,4 ± 0,4	27,9 ± 4,7
	Gélification ionotropique	. Ces systèmes particulaires composites ont 2,7 ± 0,2 97,8 ± 7,9
	été caractérisés en termes de taille, de taux d'encapsulation du GSNO, de cinétique de
	libération à pH 7,4 dans un milieu tamponné mais aussi de toxicité cellulaire par
	établissement de tests MTT sur une lignée d'adénocarcinome colique humaine (Caco-
	2).

Au cours de cette thèse, de nouveaux systèmes composites ont été formulés et caractérisés pour renforcer la protection du GSNO tout au long du tractus gastrointestinal et contrôler davantage la libération de ce dernier : les nanoparticules d'Eudragit ® RL préalablement développées au sein de l'équipe ont été elles-mêmes encapsulées dans une matrice polymérique à base d'alginate, polymère adapté à la voie orale et décrivant des propriétés favorisant la mucopénétration au niveau intestinal

(Laffleur and Bernkop-Schnürch, 2013, Kumar et al., 2014)

.

Pour ce faire, deux procédés de formulation de la matrice d'alginate ont été développés et comparés (article soumis dans Journal of Microencapsulation). Un procédé simple de complexation de polyélectrolytes (entre les nanoparticules d'Eudragit ® RL chargées positivement et l'alginate de sodium chargé négativement) a été comparé à un procédé plus classique de gélification ionotropique, utilisant le chlorure de calcium comme agent structurant des chaines d'alginate (« cross-linker »). Le procédé simple de complexation de polyélectrolytes a conduit à l'obtention de particules composites avec une charge en GSNO plus importante mais une libération in vitro plus rapide que celles formulées par gélification ionotropique (tableau 2).

x Tableau 2 : Charge et libération du GSNO dans les particules composites formulées par complexation de polyélectrolytes et par gélification ionotropique. . Il sera néanmoins nécessaire de confirmer ces caractéristiques par des essais chez l'animal, sur un tissu intestinal.

  Parmi ces derniers, le choix du GSNO et de l'oxyde nitrique est argumenté en décrivant les actions bénéfiques de ce dernier au niveau intestinal. La mise en forme du GSNO pour proposer cette molécule comme

	candidat à un traitement oral chronique est ensuite évoquée. L'intérêt des formulations
	appelées « nanoparticules composites » pour la délivrance de principe actif par voie
	orale est alors documentée au travers d'une revue bibliographique (Polymer
	nanocomposites for drug oral delivery: development strategies and potentialities. Hui
	Ming, Wen Wu, Philippe Maincent, Marianne Parent, Caroline Gaucher, Anne Sapin-
	Minet. Volume 9: Organic particles, Composites in Biomedical Engineering: Particles

Dans une première partie, les pathologies MICI, les traitements de références actuels xii et les traitements innovants sont présentés.

(Series C) -ELSEVIER, Edited by Alexandru Mihai GRUMEZESCU (in progress)).

La seconde partie rapporte l'étude du maintien, du renforcement de la barrière intestinale par le GSNO (non formulé) apporté dans la lumière intestinale : cette étude originale constitue la preuve de concept, l'étude de faisabilité de ce projet. Elle s'articule autour d'un article devant être prochainement soumis dans Journal of gastroenterology. La formulation de GSNO constitue la troisième partie du manuscrit. Un article soumis dans Journal of Microencapsulation décrit et compare deux systèmes particulaires composites. Un troisième système est ensuite décrit en données complémentaires.

Table 2

 2

	GSNO related delivery system

Table 1

 1 Simplified summary of drugs, nanoplateforms, polymer matrix and their efficiencies.

		Nanoplatforms	Polymer matrices
	Drugs					Efficiency	References
		Material	Form	Material	Form
	Vitamin C	Phospholipid and cholesterol	Liposomes	Chitosan/ alginate	Microencapsules	Increased stability	(Liu et al., 2016)
	Amaranth red	Chitosan	Nanoparticles	Alginate	Microparticles	Protection in GI Tract	(Garrait et al., 2014)
	5(6)-carboxyfluores cein	Phospholipid	Liposomes	Chitosan	Biopolymer-liposome composite	Increased stability and preserved permeability membrane	(Tan et al., 2015)
	triptorelin acetate/ Ibuprofen	PCL	Nanoparticles	Ethylcellulose /Eudragit ® RS 100	Microparticles	Sustained release and reduction of burst release	(Hasan et al., 2007)
						Sustained release	(Sheikh
	Ibuprofen	PCL	Nanoparticles	PLGA	Microparticles	and reduction of	Hassan et
						burst release	al., 2009)
	Nile red/Curcumin	IPM	Microemulsion	Chitosan	Hydrogel	Sustained release	(Delmar and Bianco-Peled, 2016)
						Protected in
	Docetaxel	PCEC	Micelles	PECA/MPEG MA /IA	Hydrogel	stomach and specific delivery in	(Wang et al., 2014)
						intestine
	Albendazole	PPI	Dendrimers	Chitosan	Matrix tablet	Prolonged the retention in the GI tract	(Mansuri et al., 2016)
	Probucol	PEGylated poly(amidoa mine)	Dendrimers	phospholipids	Solid lipids	Slower burst release rate and higher total release amount	(Qi et al., 2015)
	Bovine insulin	Alginate	Hydrogel particles	PLGA	Microparticles	Protected payload and sustained release	(Schoubben et al., 2009)
	Insulin	PLGA	Nanoparticles	Eudragit ® FS 30D	Microcapsules	Protected in stomach and released in intestine	(Sun et al., 2015)
	Plasmid DNA	Gelatin	Nanoparticles	PCL	Microspheres	Protected in stomach and specific delivery in intestine	(Bhavsar and Amiji, 2007, Bhavsar and Amiji, 2008)

Table 2

 2 simple summary of proceeding methods to prepare polymer nanocomposites

	nanoplatform

Table 3

 3 Elimination of burst effect by polymer nanocomposites. Drug release (%): the mean cumulative release of drug from simple nanoplatforms and polymer nanocomposites at first 15 min. Burst effect reduction was calculated by drug release from nanoplatforms (%)/drug release from nanocomposites (%).

							Burst
	Drug	Nanoplatfor m	Polymer matrix	Drug release (%)	effect reductio	References
							n ratio
		PCL	-		86 ± 7		1	Sheikh Hassan et al.,
	Ibuprofen	PCL PCL PCL PCL	PLGA RS EC RS/EC		9 ± 4 33 ± 7 28 ± 10 24 ± 4		10 2 3 3	2009 Hasan et al., 2007
		PCL	-		71 ± 11		1
	Triptorelin	PCL	RS		27 ± 2		3	Hasan et al.,
	acetate	PCL	EC		5 ± 8		20	2007
		PCL	RS/EC		27 ± 6		3
	Diclofenac	PCL/EC	-		88±1		1	Jelvehgari et
	sodium	PCL	EC		23.46±0.77	4	al., 2010
				pH1.2	pH6.8	pH7.4
	Insulin	PLGA PLGA	-Eudragit ® FS 30D	(0-2h) 50±2 59±4 90±4 (0-6h) (0-24 h) 22±1 38±2 90±4	1 2	Sun et al., 2015

  sciences, 71, 349. Prasad, K., Badarinath, A., Anilkumar, P., Reddy, B.R., Naveen, N., Nirosha, M. & Hyndavi, M., 2011. Colon targeted drug delivery systems: A review. Journal of Global Trends in Pharmaceutical Sciences, 2, 459-475.

	Reis, C.P., Neufeld, R.J., Ribeiro, A.J. & Veiga, F., 2006. Nanoencapsulation I. Methods
	for preparation of drug-loaded polymeric nanoparticles. Nanomedicine:
	Nanotechnology, Biology and Medicine, 2, 8-21.
	Sangwai, M. & Vavia, P., 2013. Amorphous ternary cyclodextrin nanocomposites of
	telmisartan for oral drug delivery: Improved solubility and reduced
	pharmacokinetic variability. International journal of pharmaceutics, 453, 423-
	432.
	Schoubben, A., Blasi, P., Giovagnoli, S., Perioli, L., Rossi, C. & Ricci, M., 2009. Novel
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	aerosol delivery to the lung. International Journal of Pharmaceutics, 269, 457-
	467.
	Sheikh Hassan, A., Sapin, A., Lamprecht, A., Emond, E., El Ghazouani, F. & Maincent,
	P., 2009. Composite microparticles with in vivo reduction of the burst release
	effect. Eur J Pharm Biopharm, 73, 337-44.
	Singh, A., Worku, Z.A. & Van Den Mooter, G., 2011. Oral formulation strategies to
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	delivery, 8, 1361-1378.
	Sluzky, V., Tamada, J.A., Klibanov, A.M. & Langer, R., 1991. Kinetics of insulin
	aggregation in aqueous solutions upon agitation in the presence of hydrophobic
	surfaces. Proceedings of the National Academy of Sciences, 88, 9377-9381.
	Sun, S., Liang, N., Yamamoto, H., Kawashima, Y., Cui, F. & Yan, P., 2015. pH-sensitive
	poly(lactide-co-glycolide) nanoparticle composite microcapsules for oral
	delivery of insulin. Int J Nanomedicine, 10, 3489-98.
	Vrignaud, S., Benoit, J.-P. & Saulnier, P., 2011. Strategies for the nanoencapsulation of
	hydrophilic molecules in polymer-based nanoparticles. Biomaterials, 32, 8593-
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	Wang, C.

-F., Mäkilä, E.M., Kaasalainen, M.H., Liu, D., Sarparanta, M.P., Airaksinen, A.J., Salonen, J.J., Hirvonen, J.T. & Santos, H.A., 2014a. Copper-free azidealkyne cycloaddition of targeting peptides to porous silicon nanoparticles for

Table 1

 1

	one-way ANOVA
	GSNO(µM)
		0	0.1	10	100
	Time
	0.5 h -1 h	1.1±0.2	0.7±0.1# 1.7±0.2 1.9±0.5
	1 h -2 h	2.0±0.3	1.4±0.2# 3.0±0.6 3.6±0.6
	0.5 h -2 h	1.7±0.2	1.2±0.1# 2.6±0.4 3.1±0.5*

Evolution of the apparent permeability coefficient (Papp) of sodium fluorescein (NaFlu, 1 mg/mL). *statistic difference vs Control; # statistic difference vs 100 µM; p<0.05;

Table 3

 3

	Optimization of alginate nanocomposite
	nanosuspension	Alginate dissolution weight dissolving (mg) medium	CaCl2 cross-linking concentration volume (M) (mL)	mixing order	intervention method	size (µm)	uniformity EE (%) n	aim
	20 mL nanosuspension	20 40	20 mL nanosuspension	2	2	CaCl2 / alginate	ultrasonication	23.6 ± 4.7 16.8	0.479 ± 0.058 0.575	21.6 ± 3.3 23.2	obtain stronger gel
	15mL + 5mL (alginate dissolution) 20 mL nanosuspension	20 20	5 mL nanosuspension 10 mL water	2 2 0.05	2 2	CaCl2 / alginate CaCl2 / alginate	ultrasonication 47.5 magnetic 124 agitation; 1000 rpm, 3 min 236 ± 4 0.661 ± 0.051 1.8 2.37	37.7 30 32.9 ± 1.2	Avoid GSNO leakage during alginate dissolution step
	20 mL nanosuspension	20	10 mL water	0.1 0.05	20	alginate/ CaCl2	magnetic agitation; 1000 rpm, 3 min	86.1 84.4	0.895 1.06	35.9 37.1	Avoid GSNO leakage during cross-linking step
	nanosuspension 20 mL	20	10 mL water	-	-	-	ultrasonication 4.2	0.398	30.5

Table 1

 1 Characterization of GSNO-NCP (mean ± SD, n=3)

	Formulation method	Size (µm) Uniformity	GSNO loading (mg
				GSNO/g polymer)
	Ionotropic gelation	24 ± 5	0.48 ± 0.06	2.7 ± 0.2
	Polyelectrolyte complexation	66 ± 2	0.98 ± 0.04	4.4 ± 0.4

Table 4

 4 Experiment of interaction between TPP and Eudragit ® E 100

	TPP/Eudragit ® E 100 reaction	TPP in 20 mL 0.1% pluronic ® F-68	Eudragit ® E 100 in 20 mL 1% acetic acid final pH	observation
		m (mg)	pH	m (mg)	pH
	Ex1	220	9.5	-	2.9	4.2	transparent
	Ex2	-	5.8	20	2.8	3.0	transparent
	Ex3	220	9.5	20	3.0	4.3	white precipitation
	Ex1: control group without Eudragit

® E 100; Ex2: control group without TPP; Ex3: experiment group.

Table 5

 5

	Characterization of GSNO-aeNCP (mean ± SD, n=3)
	Formulation	Size (µm) Uniformity	GSNO loading (mg
				GSNO/g polymer)
	GSNO-aeNCP	12 ± 1	0.63 ± 0.06	2.5 ± 0.6

  ® RL nanoparticle 2. Alginate NCPs based on ionotropic gelation; 3. Alginate NCPs based on polyelectrolyte complexation; 4. Alginate/ Eudragit ® E blend NCP. For the alginate/ Eudragit ® E blend NCP, in vitro release study and permeability study through Caco-2 cells monolayer need to be completed. Cytotoxicity evaluation is not enough. As the ability of Eudragit ® E to open tight junction is not yet reported, it is important to compare this new formulation we proposed with the alginate/ chitosan blend NCP. Caco-2 cells monolayer is really adapted to this exploration with a high expression of cell junctions in differentiated status

ACKNOWLEDGEMENTS

I would like to express my gratitude to all those who helped me to survive from these 3 years of PhD study here. I am sincerely grateful to Pr. Ponchel (Université Paris-Sud), Pr. Malzert-Fréon (Université de Caen Normandie) and Dr. Moulin (Université de lorraine): Pr. Ponchel and Pr. Malzert-Fréon for accepting to be my reviewers of this work; Pr. Ponchel and Dr. Moulin for their participation in my thesis committee and helpful suggestions. I gratefully acknowledge Pr. Hu (my supervisor during master study in Wuhan University) and Pr. Maincent (Université de lorraine) for giving me the opportunity to

Acknowledgements:

The authors acknowledge the program of Chinese Scholarships Council and University of Lorraine for their financial support. The work was co-financed by the European Union with the "Fonds Européen de Développement Régional (FEDER)".

Acknowledgement: The authors acknowledge the program of Chinese Scholarships

Council and University of Lorraine for their financial support. The work was cofinanced by the European Union with the "Fonds Européen de Développement Régional (FEDER)". The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Acknowledgements:

The authors are grateful to Andreï Lecomte (UMR 7359 GeoRessources -SCMEM) for helpful advice and manipulation in SEM study. The authors acknowledge the program of Chinese Scholarships Council and University of Lorraine for their financial support.

The work was co-financed by the European Union with the "Fonds Européen de Développement Régional (FEDER)".

Gastroenterol Hepatol, 14,[269][270][271][272][273][274][275][276][277][278] S.C., Tang, W., Ching, J.Y., Wong, M., Chow, C.M., Hui, A., Wong, T., Leung, V.K., Tsang, S.W. & Yu, H.H., 2013. Incidence and phenotype of inflammatory bowel disease based on results from the Asia-pacific Crohn's and colitis epidemiology study. Gastroenterology, 145, 158-165. e2. Ng, W.K., Wong, S.H. & Ng, S.C., 2016. Changing epidemiological trends of inflammatory bowel disease in Asia. Intestinal Research, 14, 111-119. Nielsen, O., Verspaget, H. & Elmgreen, J., 1988. Inhibition of intestinal macrophage chemotaxis to leukotriene B4 by sulphasalazine, olsalazine, and 5aminosalicylic acid. Alimentary pharmacology & therapeutics, 2, 203-211. Nikitovic, D. & Holmgren, A., 1996. S-nitrosoglutathione is cleaved by the thioredoxin system with liberation of glutathione and redox regulating nitric oxide. Journal of Biological Chemistry, 271, 19180-19185. Nikoo, A.M., Kadkhodaee, R., Ghorani, B., Razzaq, H. & Tucker, N., 2016. Controlling the morphology and material characteristics of electrospray generated calcium alginate microhydrogels. Journal of Microencapsulation, 33, 605-612. Nishiyama, A., Masutani, H., Nakamura, H., Nishinaka, Y. & Yodoi, J., 2001. Redox Regulation by Thioredoxin and Thioredoxin-Binding Proteins. IUBMB life, 52, 29-33. Noble, D. & Swift, H., 1999. Nitric oxide release from S-nitrosoglutathione (GSNO). Chemical Communications, 2317-2318. Obermeier, F., Gross, V., Schölmerich, J. & Falk, W., 1999. Interleukin-1 production by mouse macrophages is regulated in a feedback fashion by nitric oxide. Journal of leukocyte biology, 66, 829-836. Odenwald, M.A. & Turner, J.R., 2017. The intestinal epithelial barrier: a therapeutic target? Nat Rev Gastroenterol Hepatol, 14, 9-21. Onsoyen, E., 1996. Commercial applications of alginates. Carbohydr Eur, 14, 26-31. Owen, R.L., Pierce, N.F., Apple, R. & Cray, W.C., 1986. M Cell Transport of Vibrio cholerae from the Intestinal. Lumen into Peyer's Patches: A Mechanism for Antigen Sampling and for Microbial Transepithelial Migration. Journal of Infectious Diseases, 153, 1108-1118. Palmer, R.M., Ferrige, A. & Moncada, S., 1987. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature, 327, 524-526. Panaccione, R., Ghosh, S., Middleton, S., Márquez, J.R., Scott, B.B., Flint, L., Van Hoogstraten, H.J., Chen, A.C., Zheng, H. & Danese, S., 2014. Combination therapy with infliximab and azathioprine is superior to monotherapy with either agent in ulcerative colitis. Gastroenterology, 146, 392-400. e3. Pandey, S., Jirwankar, P., Mehta, S., Pandit, S., Tripathi, P. & Patil, A., 2013. Formulation and evaluation of bilayered gastroretentable mucoadhesive patch for stomach-specific drug delivery. Current drug delivery, 10, 374-383. Parent, M., Boudier, A., Fries, I., Gostynska, A., Rychter, M., Lulek, J., Leroy, P. & Gaucher, C., 2015a. Nitric oxide-eluting scaffolds and their interaction with

Abstract:

The association of polymers and nanocarriers in one system (i.e. nanocomposites) has been designed to address the challenges in oral drug delivery, combining drug delivery nano-system advantages (improvement of bioavailability, targeting…) and polymer matrix benefits in improving safety and drug release efficiency. This chapter describes the development and potentialities of polymer nanocomposites in oral delivery. In particular, the improvement of therapeutic efficiency is highlighted, including drug protection, drug solubility improvement, release profile modification, target delivery to specific area in gastrointestinal tract. The application of nanocomposite made by incorporation of organic nanoplatforms (such as polymeric or solid lipid nanoparticles, liposomes, micelles, cyclodextrins, dendrimers …) into a polymer matrix (based on polyacrylate, polymethacrylate, poly(lactic-co-glycolic acid), chitosan, alginate…) for improved drug delivery will be described. examined by western blot after the permeability study in Ussing chamber. Cheadle et al., 2013). In this context, it becomes an evidence to consider supplementation with low concentrations of GSNO as a potential therapeutic candidate in IBD pathologies.

Keywords

Before setting a prevention treatment of IBD using oral administration of GSNO, its ability to maintain or reinforce intestinal barrier integrity remains to be evaluated. This is the goal of the present study. Intestinal barrier permeability and integrity were evaluated by measuring, in the presence or not of GSNO, apparent permeability (Papp)

of NaFlu, and TEER. The expression of cell junction proteins was also evaluated by Western Blot. Occludin and claudin-1 were chosen as markers of tight junction, betacatenin and e-cadherin as markers of adherens junctions (Turner, 2009).

Material and Methods

Substances used and synthesis of GSNO

All reagents were of analytical grade and all solutions prepared with ultrapure deionized water (>18.2 mΩ.cm at 25°C). Sodium nitrite was purchased from Merck (Darmstadt, Germany), and all other reagents (including glutathione, sodium fluorescein) from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Antibodies for occludin (71-1500)

and claudin-1 (37-4900) were purchase from Invitrogen; antibody for E-cadherin (SC-7870) from Santa-Cruz; antibody for β-catenin (MA1-2001) from Thermo Fisher Scientific (Pierce Biotechnology, USA); antibody for β-actin loading control (HRP) (ab197277) from Abcam; antibody for HRP conjugated secondary goat-rabbit/mouse from Jackson ImmunoResearch Laboratories, Inc. S-nitrosoglutathione synthesis was performed in the laboratory according to a previously described method (Parent et al., 2013a) and all experiments involving GSNO were conducted under conditions of subdued light and at 4 °C in order to minimize light-and temperature-induced GSNO degradation. The concentration of GSNO was assessed by UV spectrophotometry (UV-1800, Shimadzu, France) using the specific molar absorbance of the S-NO bond at 334 nm using the Beer-Lambert law (molar absorbance 922 M -1 cm -1 ); the expected

Conclusion

In this study, GSNO, a small, fragile molecule, was successfully encapsulated in alginate-nanocomposite particles based on polyelectrolyte complexation. To our knowledge, it is the first description of a direct complexation between Eudragit ® RL nanoparticles (formulated by double emulsion process) and sodium alginate chain

Declaration of interest:

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare no conflict of interest.

Chapter 4. General discussion and perspectives

Attachments Preparation of polymer nanocomposite particles loaded with

S-nitrosoglutathione Principle

Nanoparticles are prepared with the double emulsion / solvent evaporation process. Formulation of nanocomposites is based on ionotropic gelation (cross-linker of alginate and Eudragit ® E100 are CaCl2, and tripolyphosphate respectively) and polyelectrolyte complexation (negatively charged alginate chain attached to the positively charged Eudragit ® RL from the nanoparticle by electrostatic interaction). During all the formulation process, it is necessary to protect GSNO from degradation (using fresh ultrapure water from the day, aluminum-foil paper, under ice bath). When using glass pipettes, be sure that they are of category A, A + or AS. 1.1 Prepare the organic phase:

Preparation of GSNO loaded nanoparticles based on Eudragit ® RL PO

Reagents

In a 30 mL brown bottle, weight 500 mg Eudragit ® RL PO and dissolve in 5,0 mL (glass pipette) of DCM (magnetic stirring 500 rpm, ̴ 10 min with parafilm over the cap to avoid DCM evaporation). NB: Add the magnetic stirrer after the DCM to avoid stickiness of the polymer.

1.2 Prepare the outer aqueous phase:

In a 30 mL brown bottle, weight 20 mg of Eudragit ® RL PO and dissolve in 20,0 mL (glass pipette 20 mL) of ultrapure water (magnetic stirring 500 rpm, ̴ 5 min).

Remove the stirrer and keep the bottle on ice before using.

Prepare the inner aqueous phase:

In a 5 mL brown bottle, weight 10 mg of GSNO and dissolve in 500 µL (automatic pipette P1000) of the Eudragit ® RL PO solution (taken from the outer aqueous phase) at room temperature.

If in winter it's not so easy to dissolve, it can be held in hand to warm a bit to accelerate the dissolution. If it's totally dissolved, usually in ̴ 5-10 min, the solution should be transparent.

Do not pause between the following steps 1.4 to 1.6, so check that everything is available and ready before starting: a chronometer, the two ultrasonic probes, the three phases, the balloon for evaporation, and the evaporator (water-bath 25 °C, chiller 10 °C, end of the exhaust gas tube outside).

For emulsification steps, the tip of the ultrasonic probe should be introduced halfway of the liquid height. Experimenter(s) must wear the ear protections provided.

Primary emulsion:

Retaining the magnetic stirrer with a bigger one, pour the organic phase into the inner aqueous phase and continue immediately by ultrasonication (ultrasonic system 1; 100 % amplitude, 60 s, over ice bath, with horizontal movement for homogenization).

Secondary emulsion:

Pour immediately the primary emulsion into the outer aqueous phase (if necessary use glass pipette to transfer the totality), followed by ultrasonication (ultrasonic system 2, amplitude maximum, corresponding to 30 W displayed on the screen, 30 s, over ice bath, with horizontal movement for homogenization). Transfer immediately in a 1 L brown balloon. The suspension should be milky, without aggregates.

Polyelectrolyte complexation (PEC) 2.2.1 Reagent preparation

Sodium alginate solution: dissolve 20 mg sodium alginate in 10 mL ultrapure water. 2.2.2 Reaction with NP GSNO nanosuspension was poured in the sodium alginate solution. The mixture was homogenized by gently shaking with hand through one direction for 1 minute and then 10 minutes standing before characterization. The protocol for preparation of GSNO-alginate NCP based on PEC method is schematized in the following Figure.

Preparation of alginate/Eudragit ® E100 blend nanocomposite particles loaded with S-nitrosoglutathione)

3.1 Reagent preparation CaCl2 solution: dissolve 2.9 g CaCl2 in 10 mL water to obtain 2 M. Eudragit ® E100: dissolve 20 mg Eudragit ® E100 in 20 mL water. 3.2 Alginate dissolution 20 mg sodium alginate were dispersed on the surface of nanosuspension for 5 min (formation of a transparent gel, on the surface), then keep agitation (magnetic stirrer, 1000 rpm, Heidolph MR 3000D),) for 10 min (total dissolution of alginate) at room temperature (to avoid the solubility decrease of alginate at low temperature). 3.3 Cross-linker of Eudragit ® E100 dissolution 220 of Sodium tripolyphosphate pentabasic (TPP) were added, agitation for another 5 min.

Interaction between alginate and calcium ions

2 mL CaCl2 (2M) are added drop-wisely (23 G) into the mixture of nanoparticles and sodium alginate under ultrasonication (ultrasonic system 1; 30 % amplitude, 40 W, over ice bath, with horizontal movement for homogenization, about 2 min 30s.

Interaction between Eudragit ® E100 and TPP

With agitation, the obtained emulsion was added into chitosan solution using syringe (18 G) drop-wisely. In ice bath, keep agitation at 1000 rpm for 30 min.

222

The protocol for preparation of GSNO-alginate/Eudragit ® E100 NCP is schematized in the following Figure.

Protein extraction from intestine Principal

Maximal extraction and solubilization of protein from tissue is important to make the whole protein complement available for proteomic analysis. It also helps to maximize reproducibility and to minimize waste. Minimal degradation of the protein amino acid backbone or dephosphorylation is essential to preserve the analytical utility of the extract.

RIPA buffer is the lysis buffer. PMSF (phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluoride) is a serine protease inhibitor. Na3VO4 (sodium orthovanadate) is a phosphatase inhibitor. As to protease inhibitor cocktail, just as the name suggests, it's used to inhibite protease. So all the components we use here is to obtain maximal extraction and solubilization of protein and minimal degradation of he protein amino acid backbone or dephosphorylation. • Mush the tissue with mortar-pestle in liquid nitrogen.

Material

• Weight the mass of the tissue.

• Calculate the volume of lysis buffer needed to extract the protein from tissue(1mg tissue/ 4μL buffer) • Defreeze all the components of lysis buffer: RIPA, PMSF, Na3VO4 and cocktail anti-protease in a flowing water bath • Prepare the lysis buffer: PMSF 10μL/mL RIPA Na3VO4 10μL/mL RIPA Protease inhibitor cocktail 10μL/mL RIPA • Add the corresponding volume of lysis buffer into the tissue.

• Incubate 10 min on the ice.

• Homogenize the mixture by a 1 mL syringe and blue needle. Pull and push the syringe 10 times. • Freeze and defreeze the mixture 3 times by liquid nitrogen and 37 °C water bath.

• Balance the tube and then centrifuge at 12000 rpm for 30 min at 4 °C.

• Transfer the supernatant and divide into different aliquots.

• Quantify the protein amount with BCA assay according to protocol

Solutions for gel preparation:

Tris-HCl 1.5 M pH 8.8 18.165 g of Tris/100 mL ultrapure water, adjust pH to 8.8 with HCl (37%), filter through 0.22 μm and conserve at 4 °C Tris-HCl 0.5 M pH 6.8 6.055 g of Tris/100 mL ultrapure water, adjust pH to 6.8 with HCl (37%), filter through 0.22 μm and conserve at 4 °C SDS 10% 10 g of SDS/100 mL ultrapure water, agitate gently to dissolve the SDS to avoid bubble, conserve at room temperature. APS (ammonium persulfate) 10% 1 g APS/100 mL ultrapure water, then aliquot in 400-µL tubes, conserve at -20°C. • Check the leakage of the glass plate with distilled water • Prepare the gel solution according to the table of gel preparation which is in the last page. • 10% APS and TEMED are used for solidifying the solution, so both gels solution can be prepared at the same time. Just pay attention that TEMED is added at the end just before pouring in the plates. • Pour separating gel solution into the plate till 2-3 cm away the top of thr shorter glass. Add 500 μL isopropanol to separate the media from air. Wait for 15-30 min till the gel is solidified. • Remove the isopropanol and rinse with distilled water.

• Overlay the separating gel with stacking gel till the plate is full • Insert the comb. Ensure that there is not bubble • Wait till the gel is solidified.

Sample preparation:

• Set the temperature of heating plate to 100 °C and switch on it.

• Add the same volume of Laemmli 2X sample buffer into each sample • Heat the sample with heating plate for 5 min at 100 °C • Make aliquots for samples and conserve them at -80 • C.

Electrophoresis

• Place gel inside the electroporator. (attention: bigger glass plate is face to outside) • Pour the migration buffer into the electroporator, firstly inside, then outside.

Ensure that the buffer covers the gel completely. Remove the comb carefully • Add sample and marker carefully to each well (according to the volume calculated before, to get 20-30 µg proteins in each well). Connect to a power supply (attention: red to red, black to black) • Run the stacking gel with low voltage (80V) for 15 min to 20 min till the loading buffer was concentrated in one line. Then turn up to higher voltage (110 V) for separating gel for around 1 h 30 min till the dye front runs off the bottom of the gel.

Transfer:

• Cut 6 filter paper sheets to fit the measurement of gel ( 6 cm * 8.5 cm minimum) and one membrane of 0.2 µm (nitrocellulose or PVDF) (or 0.45 μm for bigger protein) with the same dimension. If PVDF membrane is chosen, it should be immersed in methanol at least 30 sec to activate the positively charged groups on the membrane, in order to enhance reacting with negatively charged protein.

• Wet two sponges, the 6 filter papers and the membrane (5 min maximum) in pre-cold transfer buffer in a plate with transfer buffer, conserve at 4°C • Take out the glass plate from the electroporator, separate the plate and cut the stacking gel. Cut a corner of the gel to remember the sides. • Put the gel in transfer buffer (5 min maximum) • Prepare a transfer sandwich as follow: place the white side down and black side up  1 x sponge  x filter papers  1 x membrane  1 x gel  3 x filter papers  1 x sponge Ensure there is no bubbles between the gel and membrane, exclude the bubbles by finger slowly, squeeze out the extra liquid by a rotor

• Relocate the sandwich to transfer apparatus, which should be placed in ice to avoid del deformation because of heating produce by high electric current. Put a magnetic stirrer inside the apparatus. Add transfer buffer into the apparatus and ensure that the sandwich is covered by the buffer. Place the electrodes on the top of the apparatus. • Begin the transfer at 350 mA for 45 min.

• After the transfer, take out the sandwich from the apparatus, open it carefully and take out the membrane. Use different color pens (red, blue, green) to mark clearly all the bands: this is to avoid missing during the next steps. • Verification of transfer (in option): color of the membrane with a solution (rouge Ponceau S) for 5 min on a shaker with speed of 15 rpm, then remove the color with 5-6 distilled water till the membrane back to white color.

Blocking:

• Block the membrane with 25 mL TBST/5 % non-fat milk (or BSA) under agitation at room temperature for 1 hour.

Primary antibody incubation:

• Cut the redundant part of the membrane (to minimize the dosage of the antibody)

• Prepare a plastic bag which is bigger than the membrane and write antibody information, dilution times, dilution medium date and experimentor's name on the bag. • Dilute the first antibody solution in TBST/5 % non-fat milk (or BSA) (normally is 1:1000, but check the recommended dilution from the company and check in the literature which has the same sample and same antibody; the final volume for one membrane is around 5 mL in total)

• Take out the membrane from the plastic bag. Then put it in a small tray.

• Rinse it rapidly with TBST firstly to remove most of the residual liquid. Then rinse the membrane 10 min, 4 times, on a shaker with a speed of 70 rpm at room temperature.

Revelation ECL:

Principal: Enhanced chemiluminescence (ECL) is a method which provides highly precise detection of proteins from Western blots. In the chemiluminescence reaction horseradish peroxidase (HRP) catalyzes the oxidation of luminol into a reagent which emits light when it decays. Since the oxidation of luminol is catalyzed by horseradish peroxidase, and the HRP is complexed with the protein of interest on the membrane, the amount and location of light that HRP catalyzes the emission of, is directly correlated with the location and amount of protein on the membrane.

• Mix the ECL western blot substrats 500 μL + 500 Μl • Dry the membrane with tissue. Place it on a flat plastic membrane • Cover the membrane with 1 mL ECL solution • Incubate in dark for 5 min.

• Dry the membrane with tissue, put it on a plastic membrane.

• Expose the membranes by following the instructions of "FUSION" software.

• Put the membrane back into the tray • Conserve the membranes at -20 °C after exposure in a plastic bag without medium (not necessary to dry it)