| Protein and peptide drugs which exhibited potent pharmacological activity, lasting therapeutic effect, and low toxicity have shown great potential in the treatment of diseases such as diabetes, cancers, and infectious diseases. However, the clinical applications are mostly restricted to injection formulations, which would lead to undesired patient compliance after frequent injection. When orally administrated, these hydrophilic biomacromolecules are readily degraded by various enzymes in gastrointestinal tract and barely penetrated through intestinal epithelium, resulting in poor bioavailability. Carriers bearing appreciable mucoadhesive property, unidirectional drug release fashion, and permeation enhancement were expected to improve in vitro and in vivo stability, absorption, and therapeutic efficiency of the loaded drugs. Consequently, a novel multilayered hydrogel film system containing a backing layer was developed, based on thiolated polymer which was proved to possess excellent mucoadhesion and permeation enhancement, with insulin as a model protein drug.Two thiolated polymers were developed, including hyaluronic acid-cysteamine (HA-Cym) and hyaluronic acid-cysteine (HA-Cys), through thiolation of HA. HA-Cym immobilezed 176.8±13.3μmol/g free thiol groups and 85.8±7.2μmol/g disulfide bonds, while HA-Cys immobilized 198.5±18.9μmol/g free thiol groups and 20.0±2.8μmol/g disulfide bonds. The optimized preparation procedure of thiolated HA was obtained after the investigation of different thiol substitute degree caused by varied synthesis conditions such as the kind and concentration of catalyst, molecular weight of HA, concentration of HA solution, weight ratio, pH value during the reaction. Gel-permeation chromatograph (GPC) showed that the molecular weight of HA was not significantly changed after Cym modification, while it was evidently decreased after Cys modification. Nuclear magnetic resonance (1H NMR) confirmed the covalent attachment between HA and thiol-bearing ligands. Differential scanning calorimeter (DSC) and thermogravimetric analysis (TGA) showed decreased thermal stability and reduced crystallinity of the polymer due to the introduction of thiol-bearing ligands.Compared with unmodified HA, the mucoadhesive property and resistance towards enzymatic degradation of thiolated HA was significantly improved. The thiol groups of thiolated HA could be oxidized at a neutral pH value, and the oxidative rate was depended on pKa value of thiol groups and environmental pH value. HA-Cym possessed a low thiol pKa value, resulting in a high thiol oxidative rate and a quick sol-gel transition. The hemolysis assay, MTT assay, and lactate dehydrogenase (LDH) assay in rat ileum showed desired biocompatibility of thiolated HA.A novel multilayered hydrogel film carrier was fabricated with layer-by-layer assembly (LbL) method based on HA-Cym and polyvinyl alcohol (PVA), containing a drug-impermeable backing layer, a supporting layer, a drug-loading layer, and a mucoadhesive layer. A high insulin loading capacity of 6.7%(w/w) was achieved.The influence on physical structure and in vitro and in vivo properties caused by the composition and preparation procedure was evaluated. Scanning electron microscopy (SEM) demonstrated the presence of distinct layers with no split and varied porous structure among different layers. DSC curves suggested the variation in thermal properties following glutaraldehyde (GA) crosslinking and vacuum drying. The mucoadhesion, in vitro drug release, swelling, erosion, and insulin stability were all influenced by film composition and drying method. Lyophilized hydrogel films with no or low amount of GA-crosslinked PVA showed appreciable mucoadhesion, high swelling ratio, high drug release rate, high insulin conformational stability and bioactivity. The ex vivo insulin permeation across rat ileum assay suggested that the apparent permeability coefficient (Papp) values of insulin was improved by 1.6 folds after application of hydrogel films. Heal administration of insulin-loaded multilayered hydrogel films showed hypoglycemic effect in normal rats compared with insulin solution, with a minimal blood glucose level of about 60% of the initial values. |
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