Inactivation of lysozyme by sonication under conditions relevant to microencapsulation

Controlled release dosage forms of proteins and other biomolecules can be p repared by microencapsulating them in polymeric microspheres. Proteins are subjected to potentially damaging effects of sonication and exposure to org anic solvents during the microencapsulation process. The relatively stable enzyme lysozyme was dissolved in aqueous buffer and sonicated in the presen ce of methylene chloride to mimic the initial step of the microencapsulatio n process. The stability of lysozyme was evaluated by determining the enzym e activity before and after sonication? size-exclusion chromatography, nati ve polyacrylamide gel electrophoresis, and by measuring the amount of preci pitates formed. Following sonication, the total protein introduced was dist ributed between a soluble and an insoluble fraction. Sonication of lysozyme solutions in the presence of methylene chloride led to an increase in prec ipitates. The precipitates were enzymatically inactive, did not dissolve ea sily, and were held by non-covalent interactions. No fragments or aggregate s of lysozyme were detectable in the soluble fraction. Sonicating aqueous l ysozyme solutions with and without methylene chloride decreased the specifi c activity of the enzyme in the soluble fraction. Excipients such as dimeth yl sulfoxide (DMSO). mannitol, sucrose, and tween 80 were included in the s onication mixtures containing lysozyme. With the exception of tween 80, the addition of the excipients to aqueous solutions of lysozyme led to a great er decrease in the specific activity of lysozyme when sonicated in the pres ence of methylene chloride. DMSO caused the greatest loss of enzyme activit y following sonication. Sonication of lysozyme with water, methylene chlori de, and DMSO yielded methyl] radicals, which were trapped with a-phenyl N-t ert-butylnitrone and detected by ESR spectroscopy. (C) 2000 Elsevier Scienc e B.V. All rights reserved.