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BBA - Biomembranes (v.1758, #4)

Editorial Board (pp. ii).

Assembly of nucleic acid-lipid nanoparticles from aqueous-organic monophases by M.E. Hayes; D.C. Drummond; K. Hong; J.W. Park; J.D. Marks; D.B. Kirpotin (pp. 429-442).

Effective, reproducible, and scalable methods for DNA–lipid assembly are important for the success of non-viral vectors in in vivo gene therapy. We hypothesized DNA–lipid assembly would be optimal if started from a liquid monophase where both DNA and lipids separately form molecular or micellar solutions prior to mixing, without preexisting condensed lipid phases, thus allowing DNA–lipid assembly under conditions close to equilibrium. Previously, we found that mixing plasmid DNA, 1-palmitoyl-2-oleoyl-3- sn-phosphatidylcholine (POPC), cholesterol and a cationic lipid, 1, 2-dioleoyl-3-(trimethylammonio) propane (DOTAP) in 50% (v/v) aqueous ethanol spontaneously produced an optically transparent solution. Upon ethanol removal, DNA–lipid nanoparticles (Genospheres™) were formed. For comparison with well-known technologies, different DNA–lipid particles were prepared by interaction of plasmid DNA and stable or ethanol-destabilized lipid vesicles by combining the components in water or 30% (v/v) aqueous ethanol, respectively. Among the three studied DNA–lipid assembly methods, only Genospheres combined the properties of small size (less than or around 100 nm), high incorporation of both lipid and DNA, high degree of DNA protection (dye accessibility 5–12%), a narrow distribution of particle density and when immuno-targeted, the highest transfection efficiency in HER2-overexpressing cells in vitro. We conclude that the Genosphere assembly methodology offers advantages for the development of effective, scalable and targetable non-viral gene delivery vectors.

Keywords: Abbreviations; DMSO; dimethyl sulfoxide; FRET; fluorescence resonance energy transfer; HEPES; 2-(4-[2-hydroxyethyl]piperazino)ethanesulfonic acid]); HBS; 5 mM HEPES, pH 7.4, 144 mM NaCl; PEDL; particles formed from ethanol-destabilized liposomes; PEG-DSPE; N-[ω-methoxy-(poly(oxyethylene)-α-oxycarbonyl]-DSPE (PEG mol. weight 2,000); PEG-DSG; 1,2-distearoyl-sn-glycerol 1-(methoxypoly(ethylene glycol) ether. (PEG Mol. weight 2,000)Non-viral gene delivery; DNA–lipid complex; Nucleic acid encapsulation; HER2 targeting

The TatA_d component of the Bacillus subtilis twin-arginine protein transport system forms homo-multimeric complexes in its cytosolic and membrane embedded localisation by Martin Westermann; Ovidiu I. Pop; Roman Gerlach; Thomas R. Appel; Wiebke Schlörmann; Sandra Schreiber; Jörg P. Müller (pp. 443-451).

The twin arginine translocation (Tat) system has the capacity to transfer completely folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. The most abundant TatA protein of this system has been suggested to form the protein conducting channel. Here, the molecular organisation of soluble and membrane embedded Bacillus subtilis TatA_d was analysed using negative contrast and freeze-fractured electron microscopy. In both compartments, the protein showed homo-oligomerisation. In aqueous solution, TatA_d formed homo-multimeric micelle-like complexes. Freeze-fracture analysis of proteoliposomes revealed self association of membrane-integrated TatA_d independent from TatC_d, the second component of this transport system. Immunogold labelling demonstrated that the substrate prePhoD was co-localised with membrane-integrated TatA_d complexes.

Keywords: Protein export; Twin-arginine translocation; Bacillus subtilis; Complex formation; TatA

Nystatin-induced lipid vesicles permeabilization is strongly dependent on sterol structure by Liana Silva; Ana Coutinho; Alexander Fedorov; Manuel Prieto (pp. 452-459).

The selectivity of the antibiotic nystatin towards ergosterol compared to cholesterol is believed to be a crucial factor in its specificity for fungi. In order to define the structural features of sterols that control this effect, nystatin interaction with ergosterol-, cholesterol-, brassicasterol- and 7-dehydrocholesterol-containing palmitoyloleoylphosphocholine vesicles was studied by fluorescence spectroscopy. Variations in sterol structure were correlated with their effect on nystatin photophysical and activity properties. Substitution of cholesterol by either 7-dehydrocholesterol or brassicasterol enhance nystatin ability to dissipate a transmembrane K⁺ gradient, showing that the presence of additional double bonds in these sterols–carbon C7 and C22, plus an additional methyl group on C-24, respectively–as compared to cholesterol, is fundamental for nystatin–sterol interaction. However, both modifications of the cholesterol molecule, like in the fungal sterol ergosterol, are critical for the formation of very compact nystatin oligomers in the lipid bilayer that present a long mean fluorescence lifetime and induce a very fast transmembrane dissipation. These observations are relevant to the molecular mechanism underlying the high selectivity presented by nystatin towards fungal cells (with ergosterol) as compared to mammalian cells (with cholesterol).

Keywords: Abbreviations; 7-DHC; 7-Dehydrocholesterol; AmB; Amphotericin B; bras; brassicasterol; BrPC; 1-Palmitoyl-2-stearoyl (11–12)dibromo-; sn; -glycero-3-phosphocholine; chol; cholesterol; erg; ergosterol; FCCP; (carbonyl cyanide; p; -(trifluormethoxy)phenylhydrazone); LUV; large unilamellar vesicle; nys; nystatin; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; Pyranine; (8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt); TMA-DPH; (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene; p; -toluenesulfonate); val; valinomycinNystatin; Sterol structure; Membrane permeabilization; Nystatin–sterol complexes; Antifungal agent; Fluorescence

Raft-like domain formation in large unilamellar vesicles probed by the fluorescent phospholipid analogue, C12NBD-PC by Virginie Coste; Nicolas Puff; Daniel Lockau; Peter J. Quinn; Miglena I. Angelova (pp. 460-467).

The liquid-ordered/disordered-phase domain co-existence in large unilamellar vesicle membranes consisting of phosphatidylcholine:sphingomyelin (2:1) with different amounts of cholesterol has been examined using a concentration-dependent self-quenching of a single reporter molecule, C12NBD-PC. A temperature-dependent decrease of fluorescence intensity was associated with the expected formation and increase of l_o-phase membrane fraction in the vesicles. The result is consistent with exclusion of the fluorescent probe from the liquid-ordered phase which partitions preferentially into the liquid-disordered phase membrane domains. This leads to an increase of the local concentration of fluorophore in the liquid-disordered phase and a decrease of the quantum yield. This effect was used to obtain a quantitative estimation of the fraction of the vesicle membrane occupied by the liquid-ordered phase, Φ_o, as a function of temperature and cholesterol content between 0 and 45 mol%. The value of Φ_o was related to the assumed partition coefficient k_p of probe between liquid-ordered/disordered phases. For large unilamellar vesicles containing 20 and 4 mol% cholesterol and probe, respectively, with k_p=0 (probe completely excluded from liquid-ordered phase), Φ_o=0.16 and with k_p=0.2, Φ_o=0.2. The results are relevant to the action of detergent in the fractionation of detergent-resistant membrane from living cells.

Keywords: Abbreviations; LUV; large unilamellar vesicle; GUV; giant unilamellar vesicle; SUV; small unilamellar vesicle; MLV; multilamellar vesicle; PC; egg yolk; l; -α-phosphatidylcholine; SM; egg yolk sphingomyelin; Chol; cholesterol; PC*; fluorescent lipid analogue C12NBD-PC (1-acyl-2-[12-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]dodecanoyl]-; sn; -glycero-3-phosphocholine); C; _M; mean molar concentration of PC*; C; _L; local molar concentration of PC*; T; _M; main phase transition temperature; l; _d; and l; _o; liquid disordered and liquid ordered (cholesterol containing) L; _α; phases; Φ; _d; and; Φ; _o; membrane fraction occupied by l; _d; and l; _o; phases; k; _p; l; _o; /l; _d; phase partition coefficient; DRMs; detergent resistant membranesMembrane phase co-existence; Liquid-ordered domains; C12NBD-PC; Cholesterol; Unilamellar vesicles

Influence of the chain length of ubiquinones on their interaction with DPPC in mixed monolayers by Yann Roche; Pierre Peretti; Sophie Bernard (pp. 468-478).

The thermodynamic behavior of representative short (UQ2), middle (UQ4 and UQ6) and long-chain (UQ10) ubiquinones (UQ) mixed with dipalmitoyl-phosphatidylcholine (DPPC) was studied in monolayers at the air–water interface. The influence of isoprenoid chain-length of UQ on miscibility of both lipids was investigated by analysis of surface pressure-area isotherms and using fluorescence microscopy. Analysis of excess areas ( A_ex) and free energies of mixing (Δ G_m), calculated from compression isotherms in the full range of ubiquinones concentrations, has given evidences for UQ-rich constant-size (UQ6, UQ10) or less growth limited (UQ2, UQ4) microdomains formation within mixed films. Fluorescence microscopy observation revealed that ubiquinones are preferentially soluble in the expanded phase. When lateral pressure increased, concomitant evolutions of A_ex and Δ G_m parameters, and composition dependence of collapse surface pressures, argue for an evolution towards a total segregation, never reached due to expulsion of ubiquinones from the film. The possible significance of these observations is discussed in relation to ubiquinones organization and similar chain length effects in membranes.

Keywords: Mixed monolayer; Ubiquinone; Fluorescence microscopy; Molecular interaction; Miscibility

Direct measurement of VDAC–actin interaction by surface plasmon resonance by Inge Roman; Jurgen Figys; Griet Steurs; Martin Zizi (pp. 479-486).

VDAC – a mitochondrial channel involved in the control of aerobic metabolism and apoptosis – interacts in vitro and in vivo with a wide repertoire of proteins including cytoskeletal elements. A functional interaction between actin and Neurospora crassa VDAC was reported, excluding other VDAC isoforms. From a recent genome-wide screen of the VDAC interactome, we found that human actin is a putative ligand of yeast VDAC. Since such interaction may have broader implications for various mitochondrial processes, we probed it with Surface Plasmon Resonance (SPR) technology using purified yeast VDAC (YVDAC) and rabbit muscle G-actin (RGA). We show that RGA binds to immobilized YVDAC in a reversible and dose-dependent manner with saturating kinetics and an apparent K_D of 50 μg/ml (1.2 μM actin). BSA does not bind VDAC regardless of the concentrations. Alternatively, VDAC binds similarly to immobilized RGA but without saturating kinetics. VDAC being known to interact with itself, this latter interaction was directly measured to interpret the RGA signals. VDAC could bind to VDAC without saturating kinetics as expected if higher order binding occurred, and could account for maximally 66% of the non-saturating behavior of VDAC binding onto RGA. Hence, actin–VDAC interactions are not a species-specific oddity and may be a more general phenomenon, the role of which ought to be further investigated.

Keywords: VDAC; Mitochondria; SPR; Actin; Cytoskeleton; Protein–protein interaction

AFM study of interaction forces in supported planar DPPC bilayers in the presence of general anesthetic halothane by Z. Leonenko; E. Finot; D. Cramb (pp. 487-492).

In spite of numerous investigations, the molecular mechanism of general anesthetics action is still not well understood. It has been shown that the anesthetic potency is related to the ability of an anesthetic to partition into the membrane. We have investigated changes in structure, dynamics and forces of interaction in supported dipalmitoylphosphatidylcholine (DPPC) bilayers in the presence of the general anesthetic halothane. In the present study, we measured the forces of interaction between the probe and the bilayer using an atomic force microscope. The changes in force curves as a function of anesthetic incorporation were analyzed. Force measurements were in good agreement with AFM imaging data, and provided valuable information on bilayer thickness, structural transitions, and halothane-induced changes in electrostatic and adhesive properties.

Keywords: Planar bilayer; Halothane; Microdomains; Force spectroscopy

Redox-regulated ion channel activity of a cysteine-containing gramicidin A analogue by Yuri N. Antonenko; Tatyana B. Stoilova; Sergey I. Kovalchuk; Natalya S. Egorova; Alina A. Pashkovskaya; Alexander A. Sobko; Elena A. Kotova; Andrey Y. Surovoy (pp. 493-498).

According to recent data, gramicidin A analogues having positively charged amino acid sequences at the C-termini exhibit two types of channel activity in lipid membranes: classical cation-selective channels and large unselective pores. The induction of unselective pores was shown here to strongly depend on the redox state of the membrane-bathing solution, if the gramicidin analogue contained a cysteine residue in the sequence GSGPKKKRKVC attached to the C-terminus. In particular, the addition of H₂O₂ led to an increase in the transmembrane current and the loss of cationic selectivity on planar bilayer lipid membranes and an increase in the carboxyfluorescein leakage of liposomes. The effect was observed at high concentration of the peptide while was absent at the single-channel level. It was concluded that oxidation led to possible formation of dimers of the peptide, which promoted the formation of large unselective pores.

Keywords: Abbreviations; BLM; bilayer lipid membrane; gA; gramicidin A; P4C; peptide gramicidin-βA-GSGPKKKRKVC; P5C; peptide acetyl-gramicidin-βA-GSGPKKKRKVG; P10C; peptide acetyl-gramicidin-βA-GSGPKKKRKVC; CF; carboxyfluorescein; DPhPC; diphytanoylphosphatidylcholine; DPhPG; diphytanoylphosphatidylglycerolIon channel; Transmembrane peptide; Phospholipid membrane

Preferable stimulation of PON1 arylesterase activity by phosphatidylcholines with unsaturated acyl chains or oxidized acyl chains at sn-2 position by Su Duy Nguyen; Dai-Eun Sok (pp. 499-508).

To examine the effect of phospholipids on PON1 activities, purified PON1 was exposed to phospholipids prior to the determination of arylesterase and paraoxonase activities. Phosphatidylcholines with saturated acyl chains (C10–C16) showed a stimulation of both activities, chain length-dependent, with a greater stimulation of arylesterase activity, suggesting the implication of lipid bilayer in the stimulatory action. Such a preferable stimulation of arylesterase activity was more remarkable with phosphatidylcholines with polyunsaturated acyl chains or oxidized chains at sn-2 position, implying that the packing degree of acyl chain may be also important for the preferable stimulation of arylesterase activity. Separately, 1-palmitoyl-lysoPC also stimulated arylesterase activity preferably, indicating that the micellar formation of lipids around PON1 also contributes to the stimulatory action. Additionally, phosphatidylglycerols slightly enhanced arylesterase activity, but not paraoxonase activity. In contrast, phosphatidylserine and phosphatidic acid (≥0.1 mM) inhibited both activities Further, such a preferable stimulation of arylesterase activity by phosphatidylcholines was also reproduced with VLDL-bound PON1, although to a less extent. These data indicate that phosphatidylcholines with polyunsaturated acyl chains or oxidized chain, or lysophosphatidylcholine cause a preferable stimulation of arylesterase activity, thereby contributing to the decrease in the ratio of paraoxonase activity to arylesterase activity.

Keywords: Abbreviations; PON1; paraoxonase1; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PI; phosphatidylinositiol; PG; phosphatidylglycerol; PS; phosphatidylserine; PA; phosphatidic acid; PAF; 1-hexadecyl-2-acetyl-phosphatidylcholine; LPC; palmitoyl-lysophosphatidylcholine; HDL; high density lipoprotein; VLDL; very low density lipoprotein; Trp; tryptophanPON1; Arylesterase; Paraoxonase; Preferable stimulation; Phospholipid; Acyl chain; Unsaturation; Oxidized

Production and characterisation of recombinant forms of human pulmonary surfactant protein C (SP-C): Structure and surface activity by Dunja Lukovic; Inés Plasencia; Francisco J. Taberner; Jesús Salgado; Juan J. Calvete; Jesús Pérez-Gil; Ismael Mingarro (pp. 509-518).

Surfactant protein C (SP-C) is an essential component for the surface tension-lowering activity of the pulmonary surfactant system. It contains a valine-rich α helix that spans the lipid bilayer, and is one of the most hydrophobic proteins known so far. SP-C is also an essential component of various surfactant preparations of animal origin currently used to treat neonatal respiratory distress syndrome (NRDS) in preterm infants. The limited supply of this material and the risk of transmission of infectious agents and immunological reactions have prompted the development of synthetic SP-C-derived peptides or recombinant humanized SP-C for inclusion in new preparations for therapeutic use.We describe herein the recombinant production in bacterial cultures of SP-C variants containing phenylalanines instead of the palmitoylated cysteines of the native protein, as fusions to the hydrophilic nuclease A (SN) from Staphylococcus aureus. The resulting chimerae were partially purified by affinity chromatography and subsequently subjected to protease digestion. The SP-C forms were recovered from the digestion mixtures by organic extraction and further purified by size exclusion chromatography. The two recombinant SP-C variants so obtained retained more than 50% α-helical content and showed surface activity comparable to the native protein, as measured by surface spreading of lipid/protein suspensions and from compression π–A isotherms of lipid/protein films. Compared to the protein purified from porcine lungs, the recombinant SP-C forms improved movement of phospholipid molecules into the interface (during adsorption), or out from the interfacial film (during compression), suggesting new possibilities to develop improved therapeutic preparations.

Keywords: Abbreviations; DPPC; 1,2-dipalmitoyl phosphatidylcholine; LPC; lysophosphatidylcholine; LS; lung surfactant; NRDS; neonatal respiratory distress syndrome; POPG; palmitoyl oleyl phosphatidylglycerol; SDS-PAGE; sodium dodecylsulfate polyacryamide-gel electrophoresis; SP-C; surfactant protein C; rSP-C; recombinant SP-C; SN; nuclease A from; Staphylococcus aureus; TM; transmembranePulmonary surfactant; Recombinant membrain protein; SP-C; Lipid-protein interaction

Regulation of Na⁺/H⁺ exchanger-NHE3 by angiotensin-II in OKP cells by Liping Xu; Mehul P. Dixit; Kevin D. Nullmeyer; Hua Xu; Pawel R. Kiela; Ronald M. Lynch; Fayez K. Ghishan (pp. 519-526).

Previous studies have shown that circulating Angiotensin II (A-II) increases renal Na⁺ reabsorption via elevated Na⁺/H⁺ exchanger isoform 3 (NHE3) activity. We hypothesized that prolonged exposure to A-II leads to an increased expression of renal NHE3 by a transcriptionally mediated mechanism. To test this hypothesis, we utilized the proximal tubule-like OKP cell line to evaluate the effects of 16-h treatment with A-II on NHE3 activity and gene expression. A-II significantly stimulated NHE3-mediated, S-3226-sensitive Na⁺/H⁺ exchange. Inhibition of transcription with actinomycin D abolished the stimulatory effect of A-II on NHE3-mediated pH recovery in acid-loaded OKP cells. This prolonged exposure to A-II was also found to elevate endogenous NHE3 mRNA (by 40%)—an effect also abolished by inhibition of gene transcription. To evaluate the molecular mechanism by which A-II regulates NHE3 expression, the activity of NHE3 promoter driven reporter gene was analyzed in transient transfection assays. In transfected OKP cells, rat NHE3 promoter activity was significantly stimulated by A-II treatment, and preliminary mapping indicated that the A-II responsive element(s) is present between 149 and 548 bp upstream of the transcription initiation site in the NHE3 gene promoter. We conclude that a transcriptional mechanism is at least partially responsible for the chronic effects of A-II treatment on renal NHE3 activity.

Keywords: Slc9A3; Proximal tubule; Kidney; Antiport; Activity; Promoter

Use of a dialyzable short-chain phospholipid for efficient solubilization and reconstitution of influenza virus envelopes by Jørgen de Jonge; Pieter Schoen; Wouter terVeer; Toon Stegmann; Jan Wilschut; Anke Huckriede (pp. 527-536).

Virosomes are reconstituted viral envelopes that can serve as vaccines and as vehicles for cellular delivery of various macromolecules. To further advance the use of virosomes, we developed a novel dialysis procedure for the reconstitution of influenza virus membranes that is easily applicable to industrial production and compatible with encapsulation of a variety of compounds. This procedure relies on the use of 1,2-dicaproyl- sn-glycero-3-phosphocholine (DCPC) as a solubilizing agent. DCPC is a short-chain lecithin with detergent-like properties and with a critical micelle concentration of 14 mM. DCPC effectively dissolved the influenza virus membranes after which the nucleocapsids could be removed by ultracentrifugation. The solubilized membrane components were reconstituted either by removal of DCPC by dialysis or by a procedure involving initial dilution of the solubilized membrane components followed by dialysis. Both protocols resulted in removal of 99.9% of DCPC and simultaneous formation of virosomes. Analysis of the virosome preparations by equilibrium sucrose density gradient centrifugation revealed co-migration of phospholipid and protein for virosomes produced by either method. Moreover, both virosome preparations showed morphological and fusogenic characteristics similar to native influenza virus. Size, homogeneity and spike density of the virosomes varied with the two different reconstitution procedures employed. The recovery of viral membrane proteins and phospholipids in the virosomes was found to be higher for the dilution/dialysis procedure than for the simple dialysis protocol. This novel procedure for the production of virosomes is straightforward and robust and allows further exploitation of virosomes as vaccines or as drug delivery vehicles not only in academia, but also in industrial settings.

Keywords: Short-chain lecithin; Dialysis; Reconstitution; Influenza virus; Virosome

Pulling single bacteriorhodopsin out of a membrane: Comparison of simulation and experiment by Marek Cieplak; Sławomir Filipek; Harald Janovjak; Krystiana A. Krzyśko (pp. 537-544).

Mechanical unfolding of single bacteriorhodopsins from a membrane bilayer is studied using molecular dynamics simulations. The initial conformation of the lipid membrane is determined through all-atom simulations and then its coarse-grained representation is used in the studies of stretching. A Go-like model with a realistic contact map and with Lennard–Jones contact interactions is applied to model the protein–membrane system. The model qualitatively reproduces the experimentally observed differences between force-extension patterns obtained on bacteriorhodopsin at different temperatures and predicts a lack of symmetry in the choice of the terminus to pull by. It also illustrates the decisive role of the interactions of the protein with the membrane in determining the force pattern and thus the stability of transmembrane proteins.

Keywords: Mechanical stretching of protein; Go model; Molecular dynamic; Bacteriorhodopsin; Membrane; AFM

High-resolution electrophysiology on a chip: Transient dynamics of alamethicin channel formation by Markus Sondermann; Michael George; Niels Fertig; Jan C. Behrends (pp. 545-551).

Microstructured planar substrates have been shown to be suitable for patch clamp recording from both whole cells and isolated patches of membrane, as well as for measurements from planar lipid bilayers. Here, we further explore this technology with respect to high-resolution, low noise single-channel recording. Using solvent-free lipid bilayers from giant unilamellar vesicles obtained by electro-swelling, we recorded channels formed by the peptaibol alamethicin, a well-studied model system for voltage-dependent channels, focusing on the transient dynamics of single-channel formation upon application of a voltage step. With our setup, we were able to distinctly resolve dwell times well below 100 μs and to perform a thorough statistical analysis of alamethicin gating. Our results show good agreement with models that do not rely on the existence of non-conducting preaggregate states. Microstructured apertures in glass substrates appear promising with respect to future experiments on cellular ion channels reconstituted in suspended lipid membranes.

Keywords: Patch clamp chip; Glass microstructure; Lipid bilayer; Electrophysiology; Alamethicin

Dipalmitoylation of a cellular uptake-mediating apolipoprotein E-derived peptide as a promising modification for stable anchorage in liposomal drug carriers by Ines Sauer; Heike Nikolenko; Sandro Keller; Khalid Abu Ajaj; Michael Bienert; Margitta Dathe (pp. 552-561).

Liposomes equipped with cellular uptake-mediating peptidic vector compounds have attracted much attention as target-specific drug delivery systems. Aside from the development of the target recognition motif itself, vector coupling to liposomes while conserving the active conformation constitutes an important element in carrier development. To elucidate the most efficient way for adsorptive peptide binding to liposomes, we synthesized and characterized two-domain peptides comprising a cationic sequence derived from the binding domain of apolipoprotein E (apoE) for the low-density lipoprotein receptor and different lipid-binding motifs, that is, an amphipathic helix, a transmembrane helix, single fatty acids or two palmitoyl chains. Peptide properties considered relevant for peptide–liposome complexes to initiate an endocytotic cellular uptake such as lipid binding, helicity, stability of anchorage, bilayer-disturbing activity, and toxicity showed that the dipalmitoyl derivative was the most suitable to associate the apoE peptide to the surface of liposomes. The peptide showed pronounced lipid affinity and was stably anchored within the lipid bilayer on a time scale of at least 30 min. The helicity of about 40% in the lipid-bound state and the location of the amphipathic helix on the liposomal surface provided the prerequisites for interaction of the complex with the cell surface-located receptor. The concentration of the dipalmitoylated peptide to permeabilize neutral lipid bilayers (lipid concentration 25 μM) was 0.06 μM and a 2 μM concentration reduced cell viability to about 80%. Efficient internalization of liposomes bearing about 180 peptide derivatives on the surface into brain capillary endothelial cells was monitored by confocal laser scanning microscopy. The concept of complexation using dipalmitoylated peptides may offer an efficient substitute to covalent vector coupling and a prospective way to optimize the capacity of liposomes as drug delivery systems also for different targets.

Keywords: Abbreviations; ApoE; apolipoprotein E; BBB; blood–brain barrier; CD; circular dichroism; CLSM; confocal laser scanning microscopy; DiBr-PSPC; 1-palmitoyl-2-stearoyl(6-7)dibromo-; sn; -glycero-3-phosphocholine; Dde; 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl; DMF; dimethyl formamide; DMSO; dimethyl sulfoxide; 5-DOX; 1-palmitoyl-2-stearoyl-(5-DOXYL)-; sn; -glycero-3-phosphocholine; EDTA; ethylenediaminetetraacetic acid; FITC; fluorescein isothiocyanate; Fmoc; 9-fluorenylmethoxycarbonyl; ITC; isothermal titration calorimetry; LUV; large unilamellar vesicle; LDL; low-density lipoprotein; MALDI-TOF; matrix-assisted laser desorption/ionization time-of-flight; MTT; (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl)tetrazolium bromide; PBS; phosphate-buffered saline; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; Rh-DPPE; 1,2-dipalmitoyl-; sn; -glycero-3-phosphoethanolamine-; N; -[lissamine rhodamine B sulfonyl]; RP-HPLC; reversed-phase high-performance liquid chromatography; SUV; small unilamellar vesicle; TEMPO; 1,2-dioleoyl-; sn; -glycero-3-phospho(TEMPO)choline; Tris; tris(hydroxymethyl)aminomethane; TFA; trifluoroacetic acid; TFE; trifluoroethanolLipid-binding domains; Apolipoprotein E; Peptide–liposome complex; Cellular uptake

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BBA - Biomembranes (v.1758, #4)