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

Abstract

The effects of solvent treatments and physical stresses encountered by two proteins, ribonuclease A and adenosine deaminase, during the process of microencapsulation into biodegradable polymeric microspheres were examined. The protein stability at each step of the microencapsulation procedure was monitored by using fluorescence, circular dichroism and sodium dodecyl sulfate-polyacrylamide gel electrophoresis methods to characterize the tertiary structure, secondary structure and aggregation characteristics, respectively. The enzymatic activity of the proteins was determined by spectrophotometric assays. The microencapsulated product was characterized for size distribution, surface morphology, encapsulation efficiency, protein release profiles and protein activity. For both the proteins, solvent treatment did not lead to aggregate formation. For ribonuclease A, heptane treatment caused a change in the environment of the tyrosine residues of the protein which was manifested as an additional peak in the fluorescence emission spectrum. However, the secondary structure remained intact. Adenosine deaminase showed a significant change in the secondary structure following solvent treatment. The fraction of {dollar}\alpha{dollar}-helix and {dollar}\beta{dollar}-sheet showed a decrease with a corresponding increase in random structure. No changes were observed in the fluorescence spectrum. These changes did not affect the enzymatic activity of either protein and it was concluded that the active site of the enzyme which is responsible for substrate binding was not affected by the stresses of microencapsulation. The microspheres had smooth surfaces and a mean diameter of 24 {dollar}\mu{dollar}m. The encapsulation efficiency was high in the case of ribonuclease A but less than 50% for adenosine deaminase. During release studies, a burst release of about 60% of the encapsulated protein was seen in both cases. Complete release of ribonuclease A from the microspheres occurred in a week. Sustained but incomplete release of adenosine deaminase from the microspheres occurred over 25 days. Both proteins showed loss of enzymatic activity in the release samples. Although proteins may be resistant to inactivation due to solvent treatment and physical stresses, interactions with the polymer matrix during release may result in loss of activity and inability to obtain sustained release of active protein.