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Cationic peptides are efficiently taken up by biological cells through different pathways, which can be exploited for delivery of intracellular drugs. For example, their endocytosis is known since 1967, and this typically produces entrapment of the peptides in endocytotic vesicles. The resulting peptide (and cargo) degradation in lysosomes is of little therapeutic interest. Beside endocytosis (and various subtypes thereof), cationic cell-penetrating peptides (CPPs) may also gain access to cytosol and nucleus of livings cells. This process is known since 1988, but it is poorly understood whether the cytosolic CPP appearance requires an active cellular machinery with membrane proteins and signaling molecules, or whether this translocation occurs by passive diffusion and thus can be mimicked with model membranes devoid of proteins or glycans. In the present chapter, protocols are presented that allow for testing the membrane binding and disturbance of CPPs on model membranes with special focus on particular CPP properties. Protocols include vesicle preparation, lipid quantification, and analysis of membrane leakage, lipid polymorphism ((31)P NMR), and membrane binding (isothermal titration calorimetry). Using these protocols, a major difference among CPPs is observed: At low micromolar concentration, nonamphipathic CPPs, such as nona-arginine (WR(9)) and penetratin, have only a poor affinity for model membranes with a lipid composition typical of eukaryotic membranes. No membrane leakage is induced by these compounds at low micromolar concentration. In contrast, their amphipathic derivatives, such as acylated WR(9) (C(14), C(16), C(18)) or amphipathic penetratin mutant p2AL (Drin et al., Biochemistry 40:1824-1834, 2001), bind and disturb lipid model membranes already at low micromolar peptide concentration. This suggests that the mechanism for cytosolic CPP delivery (and potential toxicity) differs among CPPs despite their common name.

Methods in Membrane Lipids: Table of Contents Introductory Chapters: 1 A glance at the structural and functional diversity of membrane lipids Alejandro M. Dopico and Gabor J. Tigyi 2 Membrane lipid polymorphism: relationship to bilayer properties and protein function Richard Epand Lipid distribution, membrane lipid structure, and lipid-lipid interactions: 3 Acrylodan-labeled intestinal fatty acid binding protein to measure concentrations of unbound fatty acids Jeffrey R. Simard, Frits Kamp, and James A. Hamilton 4 Measuring molecular order and orientation using Coherent-anti Stokes Raman scattering microscopy Hilde A. Rinia, George W.H. Wurpel, and Michiel Muller 5 Preparation of oriented, fully hydrated lipid samples for structure determination using X-ray scattering Stephanie Tristram-Nagle 6 Nuclear Magnetic Resonance investigation of oriented lipid membranes Olivier Soubias and Klaus Gawrisch 7 Molecular dynamics simulations as a complement to Nuclear Magnetic Resonance and X-ray diffraction measurements Scott Feller 8 Use of inverse theory algorithms in the analysis of biomembrane Nuclear Magnetic Resonance data Edward Sternin 9 Statistical thermodynamics via computer simulation to characterize phospholipid interactions in membranes Mihaly Mezei and Pal Jedlovszky 10 Fluorometric assay for detection of sterol oxidation in liposomal membranes Parkson L-G. Chong and Michelle Olsher 11 Fluorescence detection of signs of sterol superlattice formation in lipid membranes Parkson L-G. Chong, Berenice Venegas, and Michelle Olsher Characterization of lipid phases: 12 Differential scanning calorimetry in the study of lipid phase transitions in model and biological membranes: practical considerations Ruthven N. A. H. Lewis, David A. Mannock, and Ronald N. McElhaney 13 Pressure-perturbationcalorimetry Heiko Heerklotz 14 Fourier transform infrared spectroscopy in the study of lipid phase transitions in model and biological membranes: practical considerations Ruthven N. A. H.Lewis and Ronald McElhaney 15 Optical dynamometry to study phase transitions in lipid membranes Rumiana Dimova and Bernard Pouligny Lipid movements and diffusion: 16 Fluorescence assays for measuring fatty acid binding and transport through membranes Kellen Brunaldi, Jeffrey R. Simard, Frits Kamp, Charu Rewal, Tanong Asawakarn, Paul O'Shea, and James A. Hamilton 17 Measurement of lateral diffusion rates in membranes by Pulsed Magnetic Field Gradient, Magic Angle-Spinning Proton Nuclear Magnetic Resonance Klaus Gawrisch and Holly Gaede 18 Using Fluorescence Recovery After Photobleaching to measure diffusion in membranes Conrad W. Mullineaux and Helmut Kirchhoff 19 Single molecule fluorescence microscopy to determine phospholipid lateral diffusion Michael J. Murcia, Sumit Garg, and Christoph Naumann 20 Modeling two- and three-dimensional diffusion Michael Saxton 21 Measurement of water and solute permeability by stopped-flow fluorimetry John C. Mathai and Mark L. Zeidel Pressure between lipids, mono- and bilayer lipid curvature and stress: 22 Fluorescence microscopy to study pressure between lipids in giant unilamelar vesicles Anna Celli, Claudia Y.C. Lee, and Enrico Gratton 23 X-ray scattering and solid-state deuterium Nuclear Magnetic Resonance probes of structural fluctuations in lipid membranes Horia I. Petrache and Michael F. Brown 24 Determination of lipid spontaneous curvature from X-ray examinations of inverted hexagonal phases Michael Kozlov 25 Shape analysis of giant vesicles with fluid phase coexistence by laser scanning microscopy to determine curvature, bending elasticity and line tension Samuel T. Hess, M

Bilayers are the most familiar arrangement of phospholipids. However, even as bilayers, phospholipids can arrange themselves in a variety of morphologies from essentially flat structures found in large liposomes or when adhered to a flat solid support, to the curved structures found in small liposomes or as bicontinuous cubic phases. Phospholipids can also arrange themselves as curved monolayers, such as in the hexagonal phase, and they can even form spherical or ellipsoid-shaped micelles. A number of factors will determine the final morphology of a lipid aggregate including the structure of the lipid, the nature of the lipid headgroup and its degree of hydration, and the temperature. In addition to being interesting in its own right, the property of lipid polymorphism can be applied to understand how fundamental intrinsic curvature properties of a membrane alter the physical properties of a membrane bilayer. This, in turn, will affect the functional characteristics of membrane proteins, with several possible mechanisms explaining the coupling of membrane properties with protein function.