Protein encapsulation within poly(ethylene glycol)-coated nanospheres. II. Controlled release properties P. Quellec, 1 R. Gref, 1 * E. Dellacherie, 1 F. Sommer, 2 M. D. Tran, 3 M. J. Alonso 4 1 Laboratoire de Chimie Physique Macromole ´culaire UMR CNRS-INPL 7568, Groupe ENSIC, BP 451, 54001 Nancy Cedex, France 2 Biophy Research, Village des Entreprises de St Henri, 6 Rue Anne Gacon, 13016 Marseille, France 3 Universite ´ Claude Bernard, 69630 Villeurbanne Cedex, France 4 Universidad de Santiago, Laboratorio de Farmacia Gale ´nica, 15706 Santiago de Compostella, Spain Received 3 April 1998; accepted 21 April 1999 Abstract: The development of injectable nanoparticulate “stealth” carriers for protein delivery is a major challenge. The aim of this work was to investigate the possibility of achieving the controlled release of a model protein, human serum albumin (HSA), from poly(ethylene glycol) (PEG)- coated biodegradable nanospheres (mean diameter of about 200 nm) prepared from amphiphilic diblock PEG-poly(lactic acid) (PLA) copolymers. HSA was efficiently incorporated into the nanospheres, reaching loadings as high as 9% (w/ w). Results of the in vitro release studies showed that it is possible to control the HSA release by choosing the appro- priate nanosphere size, loading, and composition. These re- sults also revealed that, following their release, HSA mol- ecules readsorbed onto the nanospheres surfaces when they were not protected by a PEG coating. We were surprised to observe that in spite of the water uptake of the PLA-PEG nanospheres [11–29% (w/w)], the copolymer did not signifi- cantly degrade after a 15-day incubation period. Therefore, we concluded that during this time HSA release from PLA- PEG nanospheres followed a diffusion mechanism where bulk erosion and surface desorption were negligible. © 1999 John Wiley & Sons, Inc. J Biomed Mater Res, 47, 388–395, 1999. Key words: nanospheres; protein; poly(lactic acid); poly(eth- ylene glycol); albumin; controlled release; stealth drug de- livery systems INTRODUCTION Although the encapsulation and controlled release of bioactive macromolecules from microspheres has been the subject of numerous investigations, 1 only a few studies have considered the entrapment of pro- teins 2–7 and oligonucleotides 8 within nanospheres. On the other hand, the development of “stealth” nano- spheres for the delivery of macromolecules following intravenous administration seems to presently be a major challenge. In a previous article 6 we showed that a model pro- tein, human serum albumin (HSA), could be en- trapped with loadings up to 9% (w/w) into stealth poly(ethylene glycol) (PEG)-coated nanospheres pre- pared by a water/oil/water (w/o/w) method using poly(lactic acid) (PLA) homopolymers, PEG-PLA co- polymers, and their blends. In the present work our main goal was to study the principal parameters that govern the release of HSA from nanospheres (about 200 nm) either coated or not coated with PEG. Several benefits are expected from the use of co- polymers containing PEG blocks in the development of protein-loaded nanospheres. The presence of a PEG coating layer at the nanosphere’s surface hinders its opsonization and uptake by the mononuclear phago- cyte system (MPS), thus increasing its blood half-life. 9 The amphiphilic nature of the PEG-PLA copolymers allows a better dispersion of the protein-containing aqueous phase into the organic phase involved in the double emulsion process, thus improving the protein encapsulation properties. 10 The presence of PEG chains in the matrix could possibly reduce the inter- actions between the hydrophobic matrix and the en- trapped proteins and, thereby, their aggregation and inactivation; in this sense, some encouraging results were found in terms of preserving the activity of teta- nus toxoid by its encapsulation in PEG-PLA nano- spheres 3 and poly(lactic-co-glycolic acid) (PLGA)- *Present address: Faculte ´ de Pharmacie, UMR 8612, 5 Rue J. B. Cle ´ment, 92296 Cha ˆtenay–Malabry, France Correspondence to: R. Gref; e-mail: Ruxandra.Gref@cep. u-psud.fr © 1999 John Wiley & Sons, Inc. CCC 0021-9304/99/030388-08