The influence of protein solubilisation, conformation and size on the burst release from poly(lactide-co-glycolide) microspheres Gayle Duncan a , Thomas J. Jess b , Farahidah Mohamed a , Nicholas C. Price b , Sharon M. Kelly b , Christopher F. van der Walle a, * a University of Strathclyde, Pharmaceutical Sciences, Glasgow, G4 0NR, Scotland b University of Glasgow, Institute of Biomedical and Life Sciences, Glasgow, G12 8QQ, Scotland Received 7 July 2005; accepted 15 September 2005 Available online 12 October 2005 Abstract Encapsulation of proteins in poly(lactide-co-glycolide) microspheres via emulsion is known to cause insoluble protein aggregates. Following protein emulsification and encapsulation in PLGA microspheres, we used circular dichroism to show that the recoverable soluble protein fraction also suffers subtle conformational changes. For a panel of proteins selected on the basis of molecular size and structural class, conformational stability measured by chemical denaturation was not indicative of stability during emulsion-encapsulation. Partial loss of structure was observed for a-helical proteins released from freeze-dried micro- spheres in aqueous buffer, with dramatic loss of structure for a h-sandwich protein. The addition of sucrose (a lyoprotectant) did not prevent the loss of protein conformation upon encapsulation. Therefore, the conformational changes seen for the released soluble protein fraction originates during emulsification rather than microsphere freeze-drying. Analysis of the burst release for all proteins in buffer containing denaturant or surfactant showed that the degree of protein solubilisation was the dominant factor in determining the initial rate and extent of release. Our data for protein release into increasing concentrations of denaturing buffer suggest that the emulsion-denatured protein fraction remains insoluble; this fraction may represent the protein loss encountered upon comparison of protein encapsulated versus protein released. D 2005 Elsevier B.V. All rights reserved. Keywords: Poly(lactide-co-glycolide) microspheres; Protein conformation; Burst release; Circular dichroism 1. Introduction Poly(lactide-co-glycolide) (PLGA) microspheres are often employed to encapsulate proteins with the intention to bring about controlled release [1]. Unfortunately, a dburst releaseT is commonly observed wherein the majority of protein (N 40%) is released from microspheres within the first 12 h [2–5]. Release of the remaining protein may be observed during microsphere degradation but often a fraction of the protein encapsulated remains unac- counted for. Understanding the mechanism behind 0168-3659/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2005.09.007 * Corresponding author. Tel.: +44 141 548 5755; fax: +44 141 552 6443. E-mail address: chris.walle@strath.ac.uk (C.F. van der Walle). Journal of Controlled Release 110 (2005) 34 – 48 www.elsevier.com/locate/jconrel