Research paper Sustained release of bioactive glycosylated glial cell-line derived neurotrophic factor from biodegradable polymeric microspheres E. Garbayo a , E. Ansorena a , J.L. Lanciego b , M.S. Aymerich b , M.J. Blanco-Prieto a, * a Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Navarra, Pamplona, Spain b Neuromorphology Tracers Laboratory, CIMA, University of Navarra, Pamplona, Spain Received 28 November 2007; accepted in revised form 18 February 2008 Available online 23 February 2008 Abstract Glial cell-line derived neurotrophic factor (GDNF), a potent neurotrophic factor for dopaminergic neurons, appeared as a promising candidate for treating Parkinson’s disease. GDNF microencapsulation could ensure protection against degradation due to the fragile nature of the protein. Poly(lactide-co-glycolide) (PLGA) microparticles loaded with recombinant glycosylated GDNF obtained in a mammalian cell line were prepared by TROMS, a semi-industrial technique capable of encapsulating fragile molecules maintaining their native properties. The effects of several parameters as PLGA copolymer type, PEG 400 quantity co-encapsulated with GDNF or drug loading, on the properties of the particles were investigated. Microparticles showed a mean diameter between 8 and 30 lm, compatible with their stereotaxic implantation. The drug entrapment efficiency ranged from 50.6% to 100% depending on the microsphere compo- sition. GDNF was better encapsulated using hydrophilic polymers with high molecular weight such as RG 503H. In vitro drug release was influenced by the polymer type as well as by the amount of PEG 400 co-encapsulated with GDNF. Microparticles prepared using PLGA RG 503H released 67% of the total protein content within 40 days. Moreover, very low concentrations of poly(vinyl alcohol) were detected after microparticles washing and freeze-drying. Finally, a PC-12 bioassay demonstrated that the in vitro GDNF released was bioactive. Ó 2008 Elsevier B.V. All rights reserved. Keywords: Rat recombinant GDNF; Biodegradable microparticles; PLGA; TROMS; PEG 400 1. Introduction Therapeutic proteins are a new type of drug that repre- sents a growing sector in the drug market [1]. Numerous therapeutic proteins have been approved or are in advanced clinical testing [2]. In particular, a total of 16 new therapeutic proteins were approved by the FDA dur- ing 2004 and 2005 [3]. In contrast to other therapeutic agents that are normally used, these macromolecules have high specificity and activity at relatively low level concen- trations. However, protein administration presents other serious limitations due to their short in vivo half-life, phys- ical and chemical instability and low oral bioavailability [4]. Several years ago, the use of microspheres as carriers of these compounds was proposed as an attractive way to overcome these problems. Microspheres would offer sev- eral advantages since they could provide protein protection from degradation and a prolonged delivery. The encapsula- tion of a wide range of proteins has been studied, although the development of delivery systems for protein as drugs is still a major challenge [5]. Among the methods described to prepare microparticles, multiple emulsion solvent evapora- tion technique (W/O/W) is widely accepted as the most suitable for encapsulation of labile hydrophilic compounds such as peptides and proteins [6]. However, there are two critical steps in this process that could affect the protein activity; shear stress and the water/organic interface. Microsphere preparation by Total Recirculation One- 0939-6411/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejpb.2008.02.015 * Corresponding author. Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Navarra, C/Irunlarrea 1, E-31080 Pamplona, Spain. Tel.: +34 948 425 600x6519; fax: +34 948 425 649. E-mail address: mjblanco@unav.es (M.J. Blanco-Prieto). www.elsevier.com/locate/ejpb Available online at www.sciencedirect.com European Journal of Pharmaceutics and Biopharmaceutics 69 (2008) 844–851