Introduction .1 Mucin

The intestinal mucus is synthesized by specialized goblet cells and secreted by the epithelial GIT surface and is a water insoluble viscoelastic gel that adheres to the epithelia of the GIT (Atuma et al., 2001; Strugala et al., 2003; Andrews et al., 2009).

The thickness of the GIT mucus varies between 50-500 µm in the stomach and decrease distally to a range of 15-150 µm in the colon (Pullan et al., 1994; J et al., 1991; Bickel

& Kauffman, 1981; Bravo-Osuna et al., 2007). A balance between synthesis and secretion rates and abrasion through enzymatic digestion and/or mechanical shear maintains the thickness. Any imbalance may led to pathological conditions such as ulcerative colitis (Atuma et al., 2001). Despite the low pH of the luminal cavity of the stomach, the pH of the mucosal surfaces ranges from 5.23-8.1 throughout the entire GIT (Atuma et al., 2001; Bahari et al., 1982; Flemstrom & Kivilaakso, 1983).

About 95% of the mucus gel comprises of water along with sulphated glycoproteins (up to 5%), and to a lesser extent free proteins, mineral salts, and lipids (Allen & Snary, 1972; Allen & Garner, 1980). The gel-forming properties of the mucus are manifested due to the high molecular weight of mucin. The glycoproteins are rich in amino acid residues such as serine, proline, and threonine. Moreover, glycoproteins contain fructose, glucosamine, galactose, galactosamine and sialic acid. Glycoprotein units are joined by disulphide bridges covalently attached to protein cores. In addition to glycoproteins, 5-10% of mucin consists of non-covalently bonded proteins. The sialic acid units of the glycoproteins (pKa =2.6) and the sulphate groups are responsible for the negative surface charge of mucin at neutral pH (Andrews et al., 2009).

108 Functionally, the mucus layer protects epithelia from the degradation and erosive effect of gastric acid, digestive enzymes, free radicals, and bacterial and ingested toxins and abrasion. Furthermore, it acts as lubricant, facilitating the passage of food through the GIT and protects it from mechanical injury. In the colon, the mucosal layer serves as a favourable environment for the colonic microflora, whilst at the same time, prevents bacteria from adhering onto it. This way, bacterial infections are prevented (Atuma et al., 2001; Bickel & Kauffman, 1981; Carbajal et al., 2000;

Strugala et al., 2003).

3.1.2 Mucoadhesion process

The term “adhesion” refers to the molecular interaction at the interface between materials (Marshall et al., 2010). When, at least, one of these materials is a biological surface, it is called “bioadhesion”. When the biological material is particularly restricted to the mucus layer, the term “mucoadhesion” is used (Smart, 2005; Bravo-Osuna et al., 2007; Chickering & Mathiowitz, 1999). Recently, researchers have shown interest in taking advantage of mucoadhesion in localized and systemic drug delivery due to the extended contact time (Smart, 2005) of formulation with mucosa.

Researchers have proposed several theories explaining the mucoadhesion mechanism including adsorption, diffusion, electronic, fracture, mechanical, and wetting theories (Dodou et al., 2005; Smart, 2005; Peppas & Sahlin, 1996; Chickering

& Mathiowitz, 1999). Therefore, we may conclude that mucoadhesion is a complex process that cannot be explained based on a single theory. The phenomenon of mucoadhesion is best explained by a combination of theories. Firstly, the contact stage, where the mucoadhesive compound binds to the mucus (mechanical theory), gets wetted and swells (wetting theory). Through this wetting stage, the mucus-material

109 interfaces are physically bonded (electronic and adsorption theories) so that the material and mucin chains interpenetrate and entangle (diffusion theory) forming additional covalent and non-covalent bonds (diffusion, electronic, and adsorption theories) (Dodou et al., 2005; Smart, 2005; Bravo-Osuna et al., 2007; Chickering & Mathiowitz, 1999). Mucoadhesive materials bind to mucus through a variety of forces including van der Waal’s, hydrophobic, hydrogen, ionic, or covalent bonds (Dodou et al., 2005;

Bravo-Osuna et al., 2007; Peppas & Sahlin, 1996; Marshall et al., 2010; Chickering &

Mathiowitz, 1999).

Factors affecting the mucoadhesive propensity of a material include intrinsic (structural) factors such as optimum molecular weight, degree of cross-linking, high chain flexibility, optimum surface tension, or external (environmental) factors such as the environment pH and temperature, length of contact time, presence of metal ions, and the shear rate of the environment (Dodou et al., 2005; Bravo-Osuna et al., 2007;

Smart, 2005; Chickering & Mathiowitz, 1999).

3.1.3 High Performance Liquid Chromatography

The High Performance Liquid Chromatography (HPLC) technique was proposed in the late 1960s and has undergone several modifications since (Ornaf &

Dong, 2005). It is a physical separation and quantification technique operated by carrying the analyte in a liquid phase. The separation is achieved by the distribution of the constituents between the mobile phase (MP) (liquid phase) and immobilized stationary phase (column) (Ornaf & Dong, 2005). There are several other chromatographic techniques however HPLC is the most versatile (Sandie Lindsay, 1997; Ornaf & Dong, 2005; Zhang et al., 2008; White, 1981) because of its superior sensitivity, precision, resolution, reliability, reproducibility, shorter analysis time, and

110 lower cost (White, 1981; Zhang et al., 2008). HPLC technique is widely used in food, forensic, environmental, clinical, and pharmaceutical industries (Zhang et al., 2008).

Physical separation is achieved in the stationary phase (column), which consists of uniform silica particles with spherical or irregular shape with sizes that range between 3-50 µm. They may be coated with various chemical groups in order to impart the desired level of polarity. This in turn is the basis of separation between analytes and the bonded phase of the column (Engelhardt, 1979; Lindsay, 1997; White, 1981). Based on the stationary phase polarity, two separation modes are available, namely, normal phase and reversed phase (RP) chromatography. The former comprises of a polar (silica) stationary phase, whereas the latter consists of non-polar (C18) stationary phase (Lindsay, 1997). The MP is carefully chosen in order to match the right balance between retention of analyte of interest against a matrix background.

Separated constituents are detected by a wide variety of detectors such as IR, refractive index, fluorescence, and ultraviolet light (UV/visible) detectors. The latter is the most frequently used detector due to its reasonable prices and sensitivity (Zhang et al., 2008; Lacourse, 2002; Christie, 1992). The choice of the detector is aligned to the maximum sensitivity obtained for the analyte. In the present pursuit, a UV detector was used for CUR because of the sensitivity of this technique to maximum absorption at specified wavelength.

3.1.3.1 HPLC analysis of CUR

Several HPLC methods have been proposed for the quantification of CUR, mostly employing UV-vis for detection (Wichitnithad et al., 2009; Wang et al., 1997;

Syed et al., 2015; Pak et al., 2003; Hsu et al., 2001; Ireson et al., 2002; Ma et al., 2007;

111 Garcea et al., 2004). In this chapter, we used the method proposed by Wichitnithad et al., (2009) due to simplicity and sensitivity, albeit after minor modification.

3.1.4 Aims and Objectives

CUR-CS-PEC-NPs were successfully formulated and characterized as described in chapter 2. This chapter was dedicated to studying the mucoadhesion properties of the NPs, CUR release in various media as well as the stability of CUR-CS-PEC-NPs.

3.2 Materials and Methods