Figure 11.2 provides an overview of the main thiolation techniques on the backbone of chitosan. The main targets for the covalent attachment of thiol compounds are functional groups on the polymeric backbone, such as amino groups on chitosan and polyallylamine or carboxylic acid moieties on poly(acrylic acid) and carboxymethylcellulose.

In the case of chitosan, the most convenient synthesis technique is presented by usage of coupling reagents such as 2-iminothiolane (Traut’s reagent) and isopropyl-S-acetylthioacetimidate, yielding chitosan-4-thiobutylamidine and chitosan-thioethylamidine, respectively.

Both chemical compounds react readily with the primary amino groups of chitosan under formation of amidine bonds via a simple one step reaction. A further advantage of this thiolation technique is based on the protection of the thiol groups based on the chemical properties of the reagents. The sulfhydryl moiety of 2-iminothiolane is located within the tetrahydrothiophene ring and the thiol group of isopropyl-S-acetylthioacetimidate is acetylated, which ensures their protection against oxidation during synthesis [10,11].

The second possibility to graft mercaptane molecules on polymeric materials is the use of carbodiimides. Compounds containing the carbodiimide functionality are applied to catalyse the formation of amide bonds by activating the carboxylic acid moiety to form an O-acylurea derivative as intermediate product that reacts with primary amino groups [12]. Hence, this method allows the immobilization of cysteine or cysteamine to activated poly(acrylic acid) as well as the attachment of activated thioglycolic acid or cysteine to chitosan [13,14]. Performing the reaction under inert conditions excludes unintended oxidation of thiol groups during the synthesis process. Alternatively, the synthesis can be conducted at a pH below five, where the concentration of reactive thiolate anions is low and the formation of disulfide bonds can be almost prevented [15]. Disulfide bonds formed during the synthesis can be cleaved by addition of reducing agents such as sodium borohydride [16] or tris-(2-carboxyethyl)-phosphine hydrochloride [17].

To improve the mucoadhesive and permeation enhancing properties and to increase the degree of thiol groups, a further thiolation technology was developed. For this purpose polysaccharides such as chitosan or hydroxyethylcellulose were oxidized by means of periodate under cleavage of their fundamental structure. The intermediate polymer was coupled with cysteamine and reduced with sodium cyanoborohydride to obtain the secondary amine moiety [18,19]. The amount of immobilized free and oxidized sulfhydryl groups can be quantified with Ellman’s reagent after reducing the entire amount of oxidized thiol groups with sodium borohydride [20]. The ratio of reduced thiols and disulfide bonds can be determined by omitting the reduction process during the Ellman’s assay [21].

11.2.2 Cationic Thiomers

The most important polymeric material for the development of cationic thiomers is chitosan. Chitosan is a natural polysaccharide derived from chitin by partial deacetylation of its acetamido groups by alkaline treatment. It has been widely used as an excipient for drug delivery systems based on its excellent mucoadhesive properties [22]. The primary amino group at the C2 position of the glucosamine subunits is readily accessible for the covalent attachment of sulfhydryl bearing ligands. Depending on the immobilized thiol compound the cationic character can be increased or lowered. In the case of chitosan–thiobutylamidine, the positive charge was improved due to the formation of the amidine group [10]. On the contrary, the grafting of chitosan with thioglycolic acid leads to an uncharged amide bond and reduces the total quantity of amino groups that can be protonated. Thiol moieties with cationic neighbour groups react to a greater extent with disulfide bonds within the mucus layer having anionic substructures [23]. According to this, chitosan–thiobutylamidine exhibits stronger adhesive properties compared to chitosan–thioglycolic acid.

Besides chitosan, the cationic nonbiodegradable polymer poly(allylamine) was modified with N-acetylcysteine to develop a further positively charged thiomer [24]. Recently, a novel cationic thiolated polymer based on hydroxyethylcellulose was designed and characterized. The polysaccharide structure was altered by oxidative ring opening with periodate and reductive amination to immobilize cysteamine to hydroxyethylcellulose. This alternative thiolated cationic polymer showed similar properties to sulfhydryl tethered chitosan but was soluble in a broader pH range [18].

11.2.3 Anionic Thiomers

Anionic thiomers developed so far exhibit carboxylic acid moieties as ionic substructures, which offer the advantage that thiol compounds bearing a primary amino group can be easily immobilized to this polymer via the formation of amide bonds. Ligands as cysteamine, cysteine [13], thioaminophenol [25] and homocysteine [26] can be attached mediated by carbodiimide. The most commonly used anionic polymers are poly(acrylic acid) and polycarbophil, but pectin, alginate, carboxymethylcellulose and hyaluronic acid have also been thiolated and characterized. A disadvantage of anionic mucoadhesive polymers is their incompatibility with multivalent such as like Ca²⁺, Mg²⁺ and Fe³⁺, in which presence these polymers precipitate and coagulate [27], leading to a strong reduction in their bioadhesive properties.

The potential of thiolated biomaterials depends markedly on the chemical and physical properties of the immobilized mercaptane bearing ligand. The pK_a value of the thiol group influences the concentration of the reactive thiolate anion and determines the extent of disulfide bond formation [8]. Figure 11.3 provides an overview of chitosan and poly(acrylic acid) with aliphatic, aromatic and preactivated sulfhydryl bearing compounds. Up to now, a considerable number of thiolated polymers has been synthesized using alkyl thiol bearing compounds. Most of these aliphatic ligands, such as thioglycolic acid, N-acetylcysteine or thiobutylamidine, possess a sulfhydryl moiety with a pK_a value in the range 8–10 [28].

Consequently, alkyl thiolated polymers reveal the strongest mucoadhesive properties in a pH slightly above the physiological intestinal pH. Besides the pK_a value, charge, lipophilicity and hydrophilicity of the attached thiol bearing ligand affect success and degree of mucoadhesive bonding. Focusing on the structure of glutathione as the aliphatic ligand, the tripeptide structure and the high negative redox potential improved the mucoadhesive features of chitosan [29]. A comparative study of chitosan with different sulfhydryl ligands showed that hydrophilic molecules with additional cationic charge increase the residence time of thiomers at mucosal surfaces [28].

11.3.2 Aromatic Thiomers

Recently, the adhesive properties of thiolated polymers were improved by immobilizing aromatic thiol ligands. For this purpose, 4-mercaptobenzoic acid-functionalized chitosan was synthesized to accomplish enhanced affinity to mucin-containing surfaces. This introduced hydrophobic entity is supposed to show higher reactivity due to a low pK_a (5–7) value of sulfhydryl functional groups. At intestinal pH values from 5.5–7.5 aryl thiol groups are represented in the reactive form of thiolate anions, which enhance the formation of disulfide bridges [17]. A further improvement was achieved by attachment of 6-mercaptonicotinic acid to the chitosan’s backbone. In addition to the low pH of the aromatic mercaptane, mercaptonicotinamide allows the formation of tautomeric structures, leading to a pH-independent disulfide formation [30].

11.3.3 Preactivated Thiomers

Despite all the advantages of thiomers, they show comparatively low stability in solutions and gels, as they are subject of thiol oxidation at pH above six unless sealed under inert conditions. This fast oxidation of the sulfhydryl groups restricts their application in body compartments where the pH is raised [31]. Disulfide formation occurs before the polymer comes into contact with the mucus layer and diminishes the interactions between thiomer and mucus layer, thereby resulting in reduced efficacy of the dosage form.

Therefore, preactivated polymers were designed and developed in order to enhance the stability, mucoadhesion and cohesive properties of thiolated biomaterials. According to this, pyridyl substructures were coupled to chitosan–thioglycolic acid mediated by disulfide bond formation. Pyridyl disulfides react very rapidly with thiol moieties over a broad pH range to form disulfide bridges. The disulfide exchange occurs between the mercaptane group and the pyridylthiol group, that is the thiomer forms disulfide bonds with cysteine-rich subdomains of mucins, with the pyridyl thiol moiety as the leaving group [32].

Rheological experiments verified the theory of the in situ gelling process. The sol–gel transition of thiolated polymers was completed after two hours, when a highly cross-linked gel was formed (Figure 11.4). Also the ascertained decrease in the free thiol group amount indicated the formation of disulfide bonds. Investigations demonstrated also a correlation between the degree of thiol groups on the polymeric backbone and the change in the viscoelastic properties of the formed gel. A high amount of mercaptane ligands leads to a significant increase in the elastic modulus [10,35].

Recently, it was demonstrated by in vitro and in vivo studies that thiomers can inhibit efflux pumps. The transmucosal transport of the P-gp substrate rhodamine-123 was strongly improved in the presence of thiolated polymeric agents. In vivo studies in rats with chitosan–thiobutylamidine/glutathione tablets exhibited a threefold improved oral bioavailability of rhodamine-123 compared to the administration of this P-gp substrate given in solution. The results of this study are depicted in Figure 11.5 [40]. The mechanism of efflux pump inhibition is based on the interaction of the polymeric thiol moieties with the cysteine subunit located within the channel forming transmembrane region of P-gp. The theory is supported by the size-dependent activity of thiolated polymers and the observation that corresponding unthiolated polymers exhibit no or a negligible effect on the efflux pump activity [41].

11.6.2 Permeation-Enhancing Effect

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