Blank PLGA microspheres, were prepared for the study using the water-in-oil-in-water(w/o/w) double emulsion technique: First, 500 mL of a 0.5% aqueous solution of polyvinyl alcohol (PVA) were emulsified in 12.5 mL of a 5% (w/v) PLGA dissolved in dichloromethane (DCM) by stirring at a rotation speed of 22, 000 pm for 15 seconds using an ultra-turrax (IKA T-25, Cole-Parmer, UK). This primary emulsion was emulsified into 0.5% aqueous PVA under stirring at 200 rpm, on a stirring plate, for four hours. For the preparation of the peptide-loaded microspheres, exendin-4 was dissolved into a buffer consisting of citrate, citric acid, (pH = 4.5) and 0.5% PVA. This peptide solution was emulsified into the 5% (w/v) dispersion of PLGA in DCM, which was stirred at rotation speed 22, 000 rpm for 15 seconds using the ultra-turrax. Different peptide loadings in the microspheres were achieved using exendin-4 concentrations of 1% and 10% (w/v) in the primary emulsion. Conceptually, the same method was followed to produce the peptide loaded microspheres, as that for the blank microspheres. The solid microspheres were collected by centrifugation at 4000 g for 5 minutes, then washed with distilled water and centrifuged once more at 4000 g for 5 minutes. Finally, after removal of the aqueous supernatant and dispersion, the microspheres were freeze-dried using a lyophiliser, by the following steps: freezing was performed by cooling the shelf to 233 K, maintaining this temperature for 30 minutes, then raising the temperature to 253 K; primary drying was performed at 253 K for 650 minutes at a pressure of 133 mbar; secondary drying was subsequently performed at 313 K for 360 minutes, also at 133 mbar. The vials were closed under a pressure of 850 mbar at 298 K using a rubber stopper, removed from the lyophiliser and crimped with aluminum seals. Vials were stored at 278 K until all further measurements and analysis. Exendin-4 concentration was determined using the bicinchoninic acid protein assay kit (Sigma Aldrich, MO, USA) following the manufacturer's instructions, with bovine serum albumin (BSA) used as the standard. The exendin-4 concentration was then analysed using a UV-VIS spectrophotometer (Agilent Cary 60 UV-Vis, CA, USA) at 562 nm. The water content for each lyophilised microsphere was determined using Karl Fischer (Mettler Toledo, Leicester, UK) coulometric titration.