Adsorption of a cationic amphiphilic drug on human serum albumin : characterization of the complex David Attwood,b Juan M. Ruso,a Pablo Taboada,a Luis M. Varelaa Mart•  n Pe  rez-Rodr•  guez,a and Mosquera*a V•  ctor a Grupo de de Coloides y Departamento de de la Materia Condensada y F•  sica Pol•  meros, F•  sica Departamento de Aplicada, Facultad de Universidad de Santiago de Compostela, F•  sica F•  sica, E-15706 Santiago de Compostela, Spain. E-mail : Fax : ]34 981 520676 ; fmvictor=usc.es; T el : ]34 981 563100 b School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, UK M13 9PL Received 26th January 2001, Accepted 7th March 2001 First published as an Advance Article on the web 4th April 2001 The complex formed by the interaction of the amphiphilic drug verapamil hydrochloride and human serum albumin (HSA) in water at 25 ¡C was investigated using a range of physico-chemical techniques. The colloidal dispersion was considered as a binary system in which water and verapamil molecules are regarded as the solvent for the HSAÈverapamil complex. Measurements of the solution conductivity and the electrophoretic mobility of the complexes showed an ionic adsorption of the drug on the protein surface leading to surface saturation at a verapamil concentration between 10 and 15 mmol kg~1. Measurements of the size of the complex and the thickness of the adsorbed layer by dynamic light scattering showed a gradual change in hydrodynamic radius of the complex with increasing drug concentration typical of a saturation rather than a denaturation process, the magnitude of the change being insufficient to account for any appreciable extension or unfolding of the HSA molecule. The interaction potential between the HSAÈverapamil complexes and their stability were determined from the dependence of the di†usion coefficients on protein concentration by application of the DLVO colloidal stability theory. The results indicate decreasing stability of the colloidal dispersion of the drugÈprotein complexes with increase in the concentration of added drug. 1. Introduction The interactions and structures of the complexes formed by proteins with surfactants in aqueous solutions have been the subject of extensive research in the past few decades.1,2 In general, the amphiphilic molecules chosen for these studies are ionic surfactants in view of their application in the area of membrane studies.3,4 In this study, we examined the inter- action between the globular anionic protein serum albumin, which is widely used as a protein model for studying such interactions, and the amphiphilic cationic drug verapamil hydrochloride, the formula of which is illustrated. Verapamil is a racemic mixture of dextro (R) and laevo (S) isomers that elicits a number of biological responses in susceptible tissues, including reduction of myocardial contractility, enhancement of atrioventricular conduction, relaxation of vascular smooth muscle and haemolysis.5 Most of these e†ects have been attributed to a selective inhibition of slow inward transport of calcium across cell membranes.6,7 Since the drug acts at the level of the cell membrane,8,9 its surface and self-association properties may have a strong inÑuence on its biological activity. Verapamil has been shown to be surface active10,11 and there is evidence for self- association from its ability to solubilise aromatic compounds in aqueous solution.12,13 Studies analysing the surface behav- iour and solubilising capacity of dexverapamil [the dextro (R) isomer] in aqueous solution have shown evidence of self- association.14,15 The results of our previous study13 support the conclusions from the solubility study of Surakitbanharn et al.12 that this drug dimerizes in water at a well-deÐned concentration. The main aim of this work was to study the nature and stability of the complex formed by verapamil and human serum albumin in water. Measurements of the electrophoretic mobility of the serum albuminÈverapamil complex in a wide range of verapamil concentrations have provided information on the adsorbed layer, the zeta potential of the complex and the energies of adsorption. Zeta potential and conductivity measurements were used to monitor changes in the onset of verapamil self-association caused by its adsorption on the albumin molecules. Static and dynamic light scattering tech- niques were used in the determination of the molar mass and charge interactions of the complexes. Finally, the interaction potential between complexes was quantiÐed by application of the DLVO theory of colloidal stability to di†usion data using the CortiÈDegiorgio model,16 assuming that the interpreta- tion of the proteinÈsurfactant interactions can be understood by a comparison with the self-assembly of free surfactant. In this respect, there are many similarities between the binding of surfactant to polyelectrolytes and micelle formation, both involving a cooperative process over a narrow concentration range.17 DOI : 10.1039/b100929j Phys. Chem. Chem. Phys., 2001, 3, 1655È1660 1655 This journal is The Owner Societies 2001 (