S-nitrosoglutathione loaded alginate nanocomposite particles development

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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) (Cunningham et al., 2000). 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 (Ebnet et al., 2004). 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 (Ebnet et al., 2004, Ebnet, 2008). Homophilic JAM-1 or -2 interactions regulate the formation of functional TJs and cell-cell border formation (Bazzoni et al., 2000, Babinska et al., 2002), while heterophilic JAM interactions play a role in leukocyte-endothelial cell adhesion (Bazzoni, 2003). 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 (Bazzoni, 2003, Ebnet et al., 2004).
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 (Liu et al., 2000). 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 (Laukoetter et al., 2007). 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)

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 (Haskins et al., 1998). 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) (Lee, 2015). 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 (McNeil et al., 2006).
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.
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 (Fanning et al., 2002). 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.

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) 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, Gerald A. Cheadle, 2012) which play important roles in tight-junction. If the mucosal barrier failed to prevent microbial invasion, NO still works on it from two systemic functions. The first one is that NO causes relaxation of the vascular smooth muscle (Ignarro et al., 1981), 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 (Davenpeck et al., 1994). It also down-regulates neutrophil aggregation and secretion (May et al., 1991). 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 (Schäffer et al., 1996, Schäffer et al., 1997, 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 (Salvemini et al., 1990, Bissonnette et al., 1991, Masini et al., 1991, Hogaboam et al., 1993, 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, Obermeier et al., 1999). Thus, the overall action of NO could be characterized as anti-inflammatory.

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) (Koivusalo et al., 1989, JENSEN et al., 1998). 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 (Höög and Östberg, 2011). In fact, GSNOR-dependent oxidation of S-hydroxylaminoglutathione 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 (Staab et al., 2008). 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 (Staab et al., 2011).

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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 (Ramachandran et al., 1999, Ramachandran et al., 2001).
Enzymes like GGT and PDI are involved in the transmembrane GSNO transportation. As previously described, GGT can use GSNO as a substrate to generate S-nitrosocysteinylglycine (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 Hogg, 2004). 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 (Zeng et al., 2001). This is summarized in Figure 21.

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, Yu and Li, 2014). 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 (Reynaert et al., 2004). Changing NF-κB inflammatory signaling also plays vital roles in the inhibition of endothelial cell adhesion molecules that accelerate leukocyte infiltration (Awad et al., 2013). 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-junction–associated 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 S-nitrosylate proteins and play an important role in proper epithelial ion transport (Jaffrey et al., 2001, Marshall et al., 2004). GSNO works not only by releasing NO but also by the residual, reduced glutathione (GSH) which is known as an antioxidant cytoprotective molecule (Dringen et al., 2000, Yap et al., 2010). 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).

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 physical-chemical 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).

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.

Table of contents :

Chapter 1. Introduction
1.1 Inflammation bowel disease
1.1.1 IBD epidemiology
1.1.2 IBD physiopathology
1.2 Intestine physiology
1.2.1 Intestinal barrier components
1.2.2 Epithelial passage routes
1.2.3 Intestinal permeability measurement
1.3 IBD therapy
1.3.1 Aminosalicylates
1.3.2 Corticosteroids
1.3.3 Immunosuppressive drugs
1.3.4 Anti-TNF agents
1.3.5 Innovative therapy
1.4 Nitric oxide
1.4.1 Role of nitric oxide in intestinal mucosal defense
1.4.2 NO donors
1.4.3 S-Nitrosoglutathione: a potent S-nitrosothiol
1.5 S-nitrosoglutathione related delivery system
1.5.1 S-nitrosoglutathione conjugated delivery system
1.5.2 S-nitrosation of glutathione related delivery system
1.5.3 Direct S-nitrosoglutathione encapsulation
1.6 Polymer nanocomposites for drug oral delivery: development strategies and potentialities
Chapter 2. Luminal GSNO effects on the intestinal barrier integrity
2.1 Introduction
2.2 Study of the impact of the GSNO on the intestinal barrier permeability
Chapter 3. Formulations of GSNO nanocomposites adapted to oral delivery
3.1 Introduction
3.2 Alginate nanocomposite particles optimization
3.2.1 Pre-optimization
3.2.2 S-nitrosoglutathione loaded alginate nanocomposite particles development
3.2.3 Supplementary study: GSNO alginate nanocomposite particles in vitro release in acidic pH
3.3 Alginate/Eudragit®E 100 nanocomposite particles development
3.3.1 Pre-experiment
3.3.2 Methods
3.3.3 Result and discussion:
3.3.4 Conclusion
3.4 Conclusion and perspectives:
Chapter 4. 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


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