rXXXX American Chemical Society A dx.doi.org/10.1021/ct100528u | J. Chem. Theory Comput. XXXX, XXX, 000–000 ARTICLE pubs.acs.org/JCTC The Influence of Cholesterol on the Properties and Permeability of Hypericin Derivatives in Lipid Membranes Emma S. E. Eriksson and Leif A. Eriksson* School of Chemistry, National University of Ireland;Galway, Galway, Ireland b S Supporting Information ABSTRACT: The promising photosensitizing properties of hypericin, a natural quinine substituted with hydroxyl and alkyl groups, have led to the proposal that it can be utilized in photodynamic therapy. Neither the detailed mechanism behind the powerful action of hypericin, arising as a result of light excitation, nor the intracellular localization and transportation of the molecule is yet fully understood. The behavior of hypericin derivatives in a pure dipalmitoylphosphatidylcholine (DPPC) lipid membrane has recently been studied theoretically by means of molecular dynamics simulations. Natural membranes however contain many important constituents;cholesterol being one of the most essential;that influence the function and structure of the membrane, and thereby also the behavior of drug molecules therein. In the present study, we investigated hypericin and its brominated derivatives in membranes containing 9 and 25 mol % cholesterol. The results show that the presence of cholesterol in the membrane affects the permeability of the hypericin molecules and does so differently for the various molecules in the two membranes. Hypericin containing one bromine was found to exhibit the lowest free energy profile for the transport process into the lipids, and also the highest permeability coefficients, indicating that this molecule displays the fastest and easiest diffusion in the membranes. All three molecules were found to accumulate most preferably close to the polar headgroup region in both membranes. 1. INTRODUCTION 1.1. Cholesterol in Lipid Membranes. Cholesterol (Figure 1) is an important compound in nature, possessing many essential properties, not only as precursor to several vitamins and hormones but also as an important constituent in biological membranes, besides phospholipids and glycolipids, in which it for example increases mechanical strength, regulates phase behavior, and reduces the passive permeability of water and other small molecules. The cholesterol molecule is made up of three groups that have all proven essential for their effect on membranes: the fused rigid steroid rings, the hydroxyl group attached to one of the rings, and the short flexible hydrocarbon chain. 1 Although intracellular synthesis of cholesterol takes place in the endoplasmic reticulum (ER) and external choles- terol is transported to the lysosomes where it is hydrolyzed, the majority of the cellular cholesterol is found in the plasma membrane, 2 and an equilibrium process in which cholesterol is being transported between the cell membrane and the cytosol has been proposed. 3 Cholesterol is found in a wide range of concentrations in various animal membranes, normally around 20-30 mol %, but plasma membranes of some cells contain up to 50 mol %. 4 In membranes in which the cholesterol concentration is high (>25 mol %), an additional phase exists besides the solid-ordered and liquid-disordered ones: the liquid-ordered phase. 5,6 In this phase, the lipids are translationally disordered and conformationally ordered, i.e., a combination of the two other phases in which the lipids are either completely ordered or disordered. In the liquid- ordered state, both ordering 7,8 and condensing 9,10 effects caused by cholesterol are observed. Cholesterol has also been exten- sively studied in the controversial field of lipid rafts and its presence in those. Lipid rafts are dynamic liquid-ordered domains made up by cholesterol, sphingolipids, and proteins important in, for example, signaling. 11 1.2. Computational Studies of Cholesterol-Containing Membranes. Basic initial computational studies of cholesterol/ lipid membranes were performed more than two decades ago 12-14 and have been followed by numerous more extended and detailed studies carried out by Monte Carlo (MC) and molec- ular dynamics (MD) simulation techniques in recent years. The improvement of computers and algorithms has enabled progres- sion to study larger systems, including more than 1000 lipids, and the use of longer simulation time scales. 15,16 Many proper- ties of cholesterol-containing membranes as seen experimentally have been reproduced theoretically, thereby also enabling the evaluation of detailed properties that are difficult to observe experimentally. It has been clearly shown, both experimentally and theoreti- cally, that cholesterol has a crucial effect on the properties of the membrane and that the cholesterol concentration plays an important role. A wide range of concentrations of cholesterol has been included in the computational studies to cover for the occurrence in natural membranes. Cholesterol has an ordering and condensing effect, two features that are closely related and that have been observed in numerous computational studies at varying cholesterol concentrations. 15-22 The ordering and con- densing effect results in a decreased membrane surface area and thereby a reduced area per lipid. Chui et al. performed simula- tions of DPPC bilayers with cholesterol concentrations ranging from 4 to 50 mol % and found a linear relationship between Received: September 15, 2010