Development and Brain Delivery of Chitosan-PEG Nanoparticles Functionalized with the Monoclonal Antibody OX26 Yes ¸ im Aktas ¸ , †,⊥ Muge Yemisci, X Karine Andrieux, ⊥ R. Neslihan Gu ¨ rsoy, † Maria Jose Alonso, § Eduardo Fernandez-Megia, # Ramo ´n Novoa-Carballal, # Emilio Quin ˜ oa ´, # Ricardo Riguera, # Mustafa F. Sargon, | H. Hamdi C ¸ elik, | Ayhan S. Demir, ‡ A. Atilla Hıncal, † Turgay Dalkara, X Yılmaz C ¸ apan, † and Patrick Couvreur ⊥, * Department of Pharmaceutical Technology, Faculty of Pharmacy, Hacettepe University, 06100 Ankara, Turkey, Physico-Chimie, Pharmacotechnie, Biopharmacie, Faculte ´ de Pharmacie, Universite ´ Paris Sud, UMR CNRS 8612, 92296 Chatenay Malabry, France, Department of Neurology, Faculty of Medicine, and Institute of Neurological Sciences and Psychiatry, Hacettepe University, 06100 Ankara, Turkey, Departament of Pharmaceutical Technology, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain, Departamento de Quı ´mica Orga ´ nica, Facultad de Quı ´mica, and Unidad de RMN de Biomoleculas asociada al CSIC, Universidad de Santiago de Compostela, Avda. de las Ciencias S.N. 15782 Santiago de Compostela, Spain, Department of Anatomy, Faculty of Medicine, Hacettepe University, 06100 Ankara, Turkey, and Department of Chemistry, Faculty of Arts and Sciences, Middle East Technical University, 06531 Ankara, Turkey. Received July 21, 2005; Revised Manuscript Received September 16, 2005 The inhibition of the caspase-3 enzyme is reported to increase neuronal cell survival following cerebral ischemia. The peptide Z-DEVD-FMK is a specific caspase inhibitor, which significantly reduces vulnerability to the neuronal cell death. However, this molecule is unable to cross the blood-brain barrier (BBB) and to diffuse into the brain tissue. Thus, the development of an effective delivery system is needed to provide sufficient drug concentration into the brain to prevent cell death. Using the avidin (SA)-biotin (BIO) technology, we describe here the design of chitosan (CS) nanospheres conjugated with poly(ethylene glycol) (PEG) bearing the OX26 monoclonal antibody whose affinity for the transferrin receptor (TfR) may trigger receptor-mediated transport across the BBB. These functionalized CS-PEG-BIO-SA/OX26 nanoparticles (NPs) were characterized for their particle size, zeta potential, drug loading capacity, and release properties. Fluorescently labeled CS-PEG-BIO- SA/OX26 nanoparticles were administered systemically to mice in order to evaluate their efficacy for brain translocation. The results showed that an important amount of nanoparticles were located in the brain, outside of the intravascular compartment. These findings, which were also confirmed by electron microscopic examination of the brain tissue indicate that this novel targeted nanoparticulate drug delivery system was able to translocate into the brain tissue after iv administration. Consequently, these novel nanoparticles are promising carriers for the transport of the anticaspase peptide Z-DEVD- FMK into the brain. INTRODUCTION The blood-brain barrier (BBB) is a diffusion barrier essential for the normal function of the central nervous system. The BBB endothelial cells differ from endothelial cells in the rest of the body by the absence of fenestra- tions, more extensive tight junctions (TJs), and sparse pinocytic vesicular transport. Endothelial cell tight junc- tions limit the paracellular flux of hydrophilic molecules across the BBB (1). In fact, only nonionic, lipophilic, and low molecular weight molecules can diffuse freely through the endothelial membrane and may passively cross the BBB. On the other hand, a large number of drugs that possess a favorable lipophilicity, which normally should enable an easy transport across these cells, are rapidly pumped back into the bloodstream by effective efflux pump systems (2, 3). These efflux pump systems include multiple organic anion transporter (MOAT) and espe- cially P-glycoprotein (P-gp) sometimes referred to as multidrug resistance protein (mdr). Other essential compounds such as amino acids, hexoses, neuropeptides, and proteins need specific carriers or transporter systems to permeate the brain (2). Consequently, very few mol- ecules may be transported efficiently into the brain which may compromise the efficient treatment of neurological and psychiatric disorders. Vector-mediated drug delivery to the brain employs the chimeric peptide technology, wherein the drug is conju- gated to a molecular carrier of protein nature (i.e. cationized albumin or OX26 monoclonal antibody). Al- though this approach has been found useful for the brain delivery of a vasoconstrictive intestinal peptide analogue (VIP), the amount of drug transported with the aid of this technology remains generally limited since the ratio * Corresponding author. Tel:+33 1 46 83 53 96. Fax:+33 1 46 61 93 34. E-mail: patrick.couvreur@cep.u-psud.fr. † Department of Pharmaceutical Technology, Faculty of Phar- macy, Hacettepe University. ⊥ Universite ´ Paris Sud. X Department of Neurology, Faculty of Medicine, and Institute of Neurological Sciences and Psychiatry, Hacettepe University. § Departament of Pharmaceutical Technology, University of Santiago de Compostela. # Departamento de Quı ´mica Orga ´ nica, University of Santiago de Compostela. | Department of Anatomy, Hacettepe University. ‡ Middle East Technical University. 1503 Bioconjugate Chem. 2005, 16, 1503-1511 10.1021/bc050217o CCC: $30.25 © 2005 American Chemical Society Published on Web 10/20/2005