Preservation of lysozyme structure and function upon encapsulation and release from poly(lactic-co-glycolic) acid microspheres prepared by the water-in-oil-in-water method

When proteins are encapsulated in bioerodible polymers by water-in-oil-in-water (w/o/w) encapsulation techniques, inactivation and aggregation are serious drawbacks hampering their sustained delivery. Hen egg-white lysozyme was employed to investigate whether stabilizing it towards the major stress factors in the w/o/w encapsulation procedure would allow for the encapsulation and release of structurally unperturbed, non-aggregated, and active protein. When it was encapsulated in poly(lactic-co-glycolic) acid (PLGA) microspheres without stabilizing additives, lysozyme showed substantial loss in activity and aggregation. It has been shown that by co-dissolving various sugars and polyhydric alcohols with lysozyme in the first aqueous buffer, interface-induced lysozyme aggregation and inactivation can be minimized in the first emulsification step [J. Pharm. Pharmacol. 53 (2001) 1217]. Herein, it was found that those excipients, which were efficient in preventing interface-induced structural perturbations, were also efficient in minimizing lyophilization-induced structural perturbations (e.g. lactulose). The efficient excipients identified also reduced structural perturbations upon lysozyme encapsulation in PLGA microspheres and this led to reduced lysozyme inactivation and aggregation. However, the data obtained also show that later steps in the encapsulation procedure are detrimental to lysozyme activity. Lysozyme inactivation was completely prevented only by employing the efficient excipients in the second aqueous phase also. In summary, protein aggregation and inactivation were minimized by rationally selecting excipients efficient in stabilizing lysozyme against the major stress factors of w/o/w encapsulation. (C) 2002 Elsevier Science B.V. All rights reserved.