Mohamed Dribek 1 Isabelle Le Potier 1 Arnaud Rodrigues 1 Antoine Pallandre 1 Elias Fattal 2, 3 Myriam Taverna 1 1 University of Paris-Sud, Group of Proteins and Nanotechnologies in Separation Sciences, Châtenay-Malabry, France 2 University of Paris-Sud, Group of Drug Targeting and Delivery of Poorly Stable Drugs, Châtenay-Malabry, France 3 CNRS, Châtenay-Malabry, France Received November 26, 2006 Revised March 14, 2007 Accepted March 15, 2007 Research Article Determination of binding constants of vasoactive intestinal peptide to poly(amidoamine) dendrimers designed for drug delivery using ACE The purpose of the present paper was to study at physiological pH the affinity between vasoactive intestinal peptide (VIP) and four poly(amidoamine) dendrimers (PAMAMs) designed for drug delivery. Therefore, a fast and reproducible CE method was first devel- oped to analyze the strongly basic peptide. To allow an accurate determination of binding constant (K) values, the ability to suppress peptide adsorption onto the silica capillary of nonpermanent coatings (poly(ethylene oxide) (PEO), low and medium relative molecular masses poly(diallyldimethylammonium chloride) (PDDA)) or poly(acrylamide) permanent coating (PAA) was evaluated. Very good intraday repeatability of VIP migration times and peak areas (0.1–0.6 and 2.9–4.9% RSD, respectively) was obtained using two of the investi- gated coatings (PEO and PDDA with medium molecular mass). ACE combined with these dynamic coatings was then employed to evaluate K between VIP and two amine-terminated PAMAM dendrimers of generation 2 and 5 (G2.NH2, G5.NH2) and two carboxyl-termi- nated PAMAM derivatives of generation 2 and 5 (G2.COOH, G5.COOH). Binding constant of (6.7 6 1.1)610 4 /M could be determined for the couple VIP/G5.NH2, while no affinity was evidenced between VIP and all other dendrimers investigated. These results suggest that G5.NH2 might be an interesting carrier for the delivery of VIP. Keywords: ACE / Drug delivery / Peptide adsorption / Poly(amidoamine) dendrimers / Vasoactive intestinal peptide DOI 10.1002/elps.200600768 Electrophoresis 2007, 28, 2191–2200 2191 1 Introduction Vasoactive intestinal peptide (VIP), a 28-aa neuropeptide widely distributed in the central and peripheral nervous sys- tem, exhibits many interesting anti-inflammatory and immunomodulatory in vitro and in vivo properties [1–5]. Pre- vious studies have shown that treatment with VIP reduces the severity of inflammation in experimental models of auto- immune encephalomyelitis [6], arthritis [7] and Crohn’s dis- ease [8]. However, the half-life of exogenous VIP in biological fluids is very short, most likely due to degradation and inacti- vation of the peptide [9–11]. The rapid degradation of VIP is a major factor limiting the potential clinical applications of this peptide. To overcome this problem, strategies have been pro- posed to improve VIP efficacy and/or stability in biological fluids by the mean of new delivery systems, using for instance, the potential of liposomes. VIP encapsulation within sterically stabilized liposomes was reported to prevent its degradation in vitro and in vivo and also to preserve its ac- tivity by maintaining its a-helix conformation [9, 12]. Never- theless, due to their poor stability, liposomes are not suitable to retain efficiently the VIP peptide for a long time. During the last decade, dendrimers have emerged as macromolecular vectors capable of drug delivery and allow- ing biomedical applications [13, 14]. In particular, commer- cially available poly(amidoamine) dendrimers (PAMAMs) are one of the most widely used dendrimer scaffolds in biol- ogy [15–17]. In order to avoid the toxicity and liver accumu- lation associated with their polycationic surface or to offer Correspondence: Dr. Isabelle Le Potier, University of Paris-Sud, JE 2495, IFR 141, F-92296, Châtenay-Malabry, France E-mail: isabelle.le-potier@u-psud.fr Fax: 133-1-46 83-54 58 Abbreviations: Gx.COOH, carboxyl-terminated PAMAM dendri- mer of generation X; Gx.NH2, amine-terminated PAMAM dendri- mer of generation X; PAA, poly(acrylamide); PAMAM, poly(ami- doamine) dendrimer; PDDA, poly(diallylmethylammonium chlo- ride); PEO, poly(ethylene oxide); VIP, vasoactive intestinal peptide;  2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com