Stabilization of insulin against agitation-induced aggregation by the GMO cubic phase gel

The main objective of the study was to evaluate if the liquid crystalline cubic phase gel of glyceryl monooleate (GMO) protects insulin from agitation induced aggregation. The aggregation of Humulin(R), Regular Iletin I(R) and Regular Iletin II(R), in cubic phase GMO gels at 30 U/g of gel was compared with that in PBS at 100 oscillations/min at 37 degrees C using optical density at 600 nm. The effect of agitation on the secondary structure of insulin in solution and in the gels was determined with circular dichroism (CD) spectroscopy, and the time course of aggregation was also followed by HPLC. A sigmoidal increase in optical density of solution with time indicated formation of increasing amounts of insoluble insulin aggregates. However, in the gels, optical density values stayed at, or around, the initial optical density value, comparable with that of a blank gel suggesting that insulin had not aggregated in the gel. CD spectroscopy of the soluble insulin showed a total loss of native conformation upon aggregation of insulin in solution. In contrast, CD spectra of insulin in the gel were unaltered suggesting protection from aggregation during agitation. Furthermore, agitation of insulin in gels for a duration as long as 2 months at 37 degrees C, had very little adverse effect on the native conformation of insulin, as indicated by the lack of a significant change in its CD spectrum. Therefore, the cubic phase gel was indeed able to protect insulin from agitation-induced aggregation and subsequent precipitation. Although the majority of insulin in solution appeared to have aggregated and precipitated after 8 days by UV and CD spectroscopy, RP-HPLC results indicated the presence of some soluble aggregates of insulin. In summary, the liquid crystalline cubic phase gel of GMO protects peptides, like insulin, from agitation-induced aggregation.