In situ study of insulin aggregation induced by water-organic solvent interface.

Abstract

The aim of this study was to assess insulin stability by monitoring in situ time-course of insulin aggregation induced by a water-organic solvent (o/w) interface that occurs during the microencapsulation process. Insulin aggregation at a simple o/w interface was monitored spectrophotometrically by detecting the percentage of turbidity changes (%T) at 350 nm. The effects of protein concentration and agitation and the presence of poly (lactic-co-glycolic acid) (PLGA) in methylene chloride (MC) on insulin aggregation were observed. For the 0.72 mg/ml insulin in phosphate-buffered saline (PBS), the effect of nonionic (dodecyl maltoside [DDM]) and anionic (sodium dodecyl sulfate [SDS]) surfactant in PBS were also evaluated at various protein/surfactant mol ratios. The conformation of insulin protected by a 10-fold molar excess of SDS recovered after 1 h of contact with MC was evaluated via circular dichroism (CD) spectroscopy. A typical turbidity-time profile was represented by a sigmoidal curve. Greater change in %T was observed with increasing insulin concentration, in the presence of PLGA in MC and in the presence of agitation. DDM failed to delay insulin aggregation at all ratios used, whereas a less than 10% change in %T was observed in 1 h when a 10- - approximately 20-fold excess of SDS was used. CD spectra indicated that the presence of insulin in SDS after 1 h of contact with MC qualitatively retained its secondary structure integrity. An experimental method was designed for an in situ assessment of protein stability at the o/w interface.