Evaluation of enzyme stability during preparation of polylactide-co-glycolide microspheres

This work was aimed at studying enzyme prolidase stability and its interactions
with the reagents and the process conditions involved in preparation, by an
emulsi®cation process, of prolidase loaded poly(lactide-co-glycolide) (PLGA)
microparticulate systems. Enzyme stability was tested with respect to contact
with methylene chloride, ethyl acetate, PLGA polymers, and several agents
used as emulsi®ers such as polyvinyl alcohol (PVA), polyvinyl pyrolidone
(PVP), carboxymethyl cellulose (CMC) and sodium oleate (NaOl). Enzyme
stability to temperature and mechanical stirring was also evaluated. Prolidaseloaded
PLGA microspheres were prepared and evaluated in terms of protein
activity. The results obtained showed that the prolidase-loaded PLGA
microspheres can be prepared only upon eyzyme stabilization by addition of
both BSA and MnCl2 into its TRIS solution. Methylene chloride was the
suitable organic solvent to be used in the double emulsion process, together
with PVA as dispersing agent in the outer aqueous phase. Low temperatures
during the emulsi®cation step and very short process times are recommended,
in order to maintain enzyme activity at its maximum. In these conditions
spherical microspheres were obtained, releasing active prolidase for up to 15
days.