Antibiotic interaction with phospholipid monolayers F. Gambinossi a , B. Mecheri a , G. Caminati a, * , M. Nocentini b , M. Puggelli a , G. Gabrielli a a Dipartimento di Chimica, Universita ` di Firenze, Polo Scientifico, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze, Italy b Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana Dipartimento di Firenze, Via di Castelpulci 41, 50010 San Martino alla Palma, Firenze, Italy Abstract We studied the interactions of tetracycline (TC) antibiotic molecules with phospholipid monolayers with the two-fold aim of elucidating the mechanism of action and providing a first step for the realization of bio-mimetic sensors for such drugs by means of the Langmuir – Blodgett technique. We examined spreading monolayers of three phospholipids in the presence of tetracycline in the subphase by means of surface pressure– area and surface potential – area isotherms as a function of bulk pH. We selected phospholipids with hydrophobic chains of the same length but polar head groups differing either in dimensions and protonation equilibria, i.e. dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE) and dipalmitoylpho- sphatidic acid (DPPA). The interaction of tetracycline with the three phospholipids was found to be highly dependent on the electric charge of the antibiotic and on the ionization state of the lipid. Significant interactions are established between the negatively charged form of dipalmitoylphosphatidic acid and the zwitterionic form of tetracycline. The drug was found to migrate at the interface where it is adsorbed underneath or/and among the head groups, depending on the surface pressure of the film, whereas penetration through the hydrophobic layer was excluded for all the three phospholipids. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Tetracyclines; Monolayers; Phospholipids; Bio-mimetic sensor 1. Introduction Tetracyclines are a subclass of the polyketide antibiotics strongly used in veterinary area whose presence in animal- derived foods induces allergic syndrome and strain resist- ance to drugs in the population [1]. The activity of these compounds is related to their penetration through biological membranes [2] but little is known on the details of such processes. The paper is a part of a systematic investigation on mimetic membrane/antibiotic interactions; we examined spreading monolayer of three phospholipids in the presence of tetracycline (TC) in the subphase by means of surface pressure – area and surface potential – area isotherms focusing on the expanded phase domains of the phospholipid mono- layer where higher TC effects were observed [3,4]. We selected dipalmitoylphosphatidylcholine (DPPC), dipal- mitoylphosphatidylethanolamine (DPPE), and dipalmitoyl- phosphatidic acid (DPPA) in order to isolate the contribution of the polar head group. Since the species distribution of both tetracycline and DPPA is pH dependent, we investigated the spreading isotherms of DPPA monolayers on two buffer solutions, at pH = 7.2 and pH = 4. We analysed the experimental results at low surface pressure on the basis of two different binding models: continuous equilibrium partition of TC between the mono- layer and the bulk phase as well as Langmuir adsorption isotherm. The results showed that tetracycline induces significant changes on phospholipid monolayers and that the strongest interactions are observed for the system DPPA on pH = 7.2 buffer due to specific dipole–dipole or ion– dipole interactions. 2. Experimental 2.1. Materials Tetracycline hydrochloride (TC) was supplied by Sigma (purity>99%). Dipalmitoylphosphatidic acid (DPPA), dipal- mitoylphosphatidylcholine (DPPC) and dipalmitoylphos- 0928-4931/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII:S0928-4931(02)00187-X * Corresponding author. E-mail address: caminati@unifi.it (G. Caminati). www.elsevier.com/locate/msec Materials Science and Engineering C 22 (2002) 283 – 288