Structural stability and in vitro biological activity of ribonuclease a during microencapsulation into biodegradable polymers

The effects of solvent treatment and physical stresses encountered by the model protein ribonuclease A (RNase A) during microencapsulation into biodegradable polymeric microspheres were examined. Stability of the protein at each step of the microencapsulation procedure was monitored by using fluorescence, circular dichroism, and sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE) methods to characterize the tertiary structure, secondary structure, and aggregation characteristics, respectively. Enzymatic activity was determined by a spectrophotometric assay. The microspheres were characterized for size distribution, surface morphology, encapsulation efficiency, protein release profiles, and protein activity. Heptane treatment caused a change in the fluorescence spectrum of RNase A but the secondary structure remained intact. No gross aggregation or change in the activity of the solvent-treated samples was detected. Uniformly spherical microspheres in the 20–60 µm size range with smooth surfaces and high encapsulation efficiencies were prepared by the solvent extraction method. The microspheres showed an initial burst release followed by complete release of RNase A in 8 days. However, the released RNase A showed loss of enzymatic activity. Interaction of the protein with the polymer matrix may have resulted in its inactivation.