In this study, the effect of zinc on insulin stability during the primary emulsification step of poly(lactide-co
-glycolide) microspheres preparation by the water-in-oil-in-water (w/o/w) double emulsion solvent evaporation technique was evaluated. Insulin was emulsified at homogenization speeds of 5000 and 10,000 rpm. Insulin was extracted from the primary w/o emulsion by a method previously reported from our laboratory and analyzed by comprehensive analytical techniques. The differential scanning calorimetry thermograms of insulin with zinc showed a single peak around 83 °C with calorimetric enthalpy values similar to native insulin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of extracted insulin showed a single intense band around 6 kDa, demonstrating the preservation of primary structure. High performance liquid chromatography (HPLC) analysis revealed that no degradation products were formed during the homogenization process. Insulin aggregates residing at the w/o interfaces were found to be of non-covalent nature. In addition, observation of a single characteristic peak for insulin at m
5808 in the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrum confirmed the absence of insulin degradation products and covalent dimers. Presence of zinc preserved the secondary structure of insulin as indicated by circular dichroism. In conclusion, these results show that with the addition of zinc, insulin stability can be improved during the primary emulsification step.
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