Interactions of Novel, Nonhemolytic Surfactants with Phospholipid Vesicles Per E. G. Thore ´n,* ,† Olle So ¨derman, § Sven Engstro ¨m, † and Christian von Corswant ‡ Department of Chemical and Biological Engineering, Chalmers UniVersity of Technology, SE-412 96 Gothenburg, Sweden, AstraZeneca R&D Mo ¨lndal, SE 431 83 Mo ¨lndal, Sweden, and Department of Physical Chemistry 1, Lund UniVersity, Box 124, SE 221 00 Lund, Sweden ReceiVed December 21, 2006. In Final Form: March 28, 2007 PEG-12-acyloxystearates constitute a novel class of pharmaceutical solubilizers and are synthesized from polyethylene glycol and 12-hydroxystearic acid, which has been esterified with a second acyl chain. The hemolytic activity of these surfactants decreases drastically with increasing pendant acyloxy chain length, and surfactants with an acyloxy chain of 14 carbon atoms or more are essentially nonhemolytic. In this paper, the interactions of PEG-12-acyloxystearates (acyloxy chain lengths ranging from 8 to 16 carbon atoms) with phosphatidylcholine vesicles, used as a model system for erythrocyte membranes, were studied in search of an explanation for the large variations in hemolytic activity. Surfactant-induced alterations of membrane permeability were investigated by studying the leakage of vesicle-entrapped calcein. It was found that all of the surfactants within the series interact with the vesicle membranes and cause slow leakage at elevated surfactant concentrations, but with large variations in leakage kinetics. The initial leakage rate decreases rapidly with increasing pendant acyloxy chain length. After prolonged incubation, on the other hand, the leakage is not a simple function of acyloxy chain length. The effect of the surfactants on membrane integrity was also investigated by turbidity measurements and cryo-transmission electron microscopy. At a surfactant/lipid molar ratio of 0.4, the vesicle membranes are saturated with surfactant. When the surfactant/lipid molar ratio is further increased, the vesicle membranes are progressively solubilized into mixed micelles. The rate of this process decreases strongly with increasing acyloxy chain length. When comparing the results of the different experiments, it can be concluded that there is no membrane permeabilization below saturation of the vesicle membranes. The large variations in the kinetics suggest that several steps are involved in the mechanism of leakage induced by PEG-12-acyloxystearates and that their relative rates vary with acyloxy chain length. The slow kinetics may in part be explained by the low critical micelle concentrations (CMCs) exhibited by the surfactants. The CMCs were found to be in the range of 0.003-0.025 µM. Introduction Surfactants are widely used in pharmaceutical applications as solubilizers for sparingly soluble active compounds. When the concentration of a surfactant in aqueous solution is above the critical micelle concentration (CMC), micelles, which have the ability to increase the solubility of sparingly soluble active compounds, are formed. The use of micelles as drug delivery vehicles also helps to minimize drug degradation and adverse drug responses. 1 Certain types of micelles may have long circulation times in the bloodstream, allowing gradual accumula- tion of the drug within the required area, and their size permits them to accumulate in areas with affected and leaky vasculature, such as tumors and inflammations. 2,3 Micellar systems are most commonly used for parenteral or oral administration, but they can also be useful for ophthalmic, topical, rectal, and nasal delivery. 1 For intravenous administration, the generally low viscosity exhibited by solutions containing spherical micelles makes them particularly suitable, since injection of solutions with high viscosity may cause pain. 1 Surfactants may, however, cause a variety of adverse side effects when used as pharmaceutical solubilizers. 4,5 In particular, surfactants used for parenteral formulation may induce hemolysis, that is, rupture of red blood cells resulting in the release of hemoglobin, as a result of surfactant incorporation into the erythrocyte membrane. The hemolytic activity of a number of surfactants has been investigated. 6-10 It is generally accepted that two types of surfactant-induced hemolysis exist. 11-15 Osmotic hemolysis occurs at low surfactant concentrations, where membrane absorption of surfactant monomers may lead to increased selective permeability to small solutes, followed by water penetration and cell swelling. At higher surfactant concentrations, the surfactant causes complete or partial solu- bilization of membrane lipids and proteins by the formation of mixed micelles. In general, nonionic surfactants have exhibited less negative side effects than ionic surfactants, and surfactants based on poly(ethylene glycol) (PEG) are frequently employed. * To whom correspondence should be addressed. E-mail: thoren@ chalmers.se. † Chalmers University of Technology. ‡ AstraZeneca. § Lund University. (1) Lawrence, M. J. Chem. Soc. ReV. 1994, 23, 417-424. (2) Torchilin, V. P. J. Controlled Release 2001, 73, 137-172. 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Colloids Surf., B 2002, 27, 215- 222. (15) Shalel, S.; Streichman, S.; Marmur, A. J. Colloid Interface Sci. 2002, 255, 265-269. 6956 Langmuir 2007, 23, 6956-6965 10.1021/la063700b CCC: $37.00 © 2007 American Chemical Society Published on Web 05/22/2007