Structural investigations of a human calcitonin-derived carrier peptide in a membrane environment by solid-state NMR

Previous studies have shown that human calcitonin (hCT) and its C-terminal fragment hCT(9-32) translocate in nasal epithelium. Moreover, hCT(9-32) was used as a carrier to internalize efficiently the green fluorescent protein, drugs, and plasmid DNA. To understand the mechanism of the membrane crossing process, we determined structural parameters of the carrier peptide hCT(9-32) in a membrane environment using solid-state NMR. For that purpose, we synthesized a multiply labeled hCT(9-32) peptide comprising four positions with fully (15)N- and (13)C-labeled amino acids. Multilamellar vesicle samples containing varying mixing ratios of hCT(9-32) and phospholipids found in the plasma membrane of nasal epithelium were prepared. The typical axially symmetric powder patterns of (31)P NMR spectra confirmed the presence of lamellar bilayers in our samples. The chemical shift anisotropy of the (31)P NMR spectra of the samples in the presence of hCT(9-32) is slightly reduced, revealing weak interaction of the peptide with the lipid headgroups. The peptide does not penetrate the lipid membrane as indicated by very similar (2)H NMR order parameters of the phospholipid fatty acid chains in the absence and presence of the carrier peptide. This membrane topology was confirmed by measurements of paramagnetic enhancement of relaxation rates. The conformation of hCT(9-32) was investigated by cross polarization magic angle spinning NMR methods. All peptide signals were resolved and fully assigned in two-dimensional proton-driven (13)C spin diffusion experiments. The isotropic chemical shifts of (13)CO, (13)Calpha, and (13)Cbeta provide information about the secondary structure of the carrier peptide. The conformation of hCT(9-32) was further corroborated by quantitative phi torsion angle measurements. Two monomeric structural models are consistent with the data: (i) a linear backbone conformation of hCT(9-32) and (ii) an antiparallel beta-sheet structure. These structures are maintained over a wide range of peptide:lipid mixing ratios. No direct indications for fibril formation of hCT(9-32) were found. Dipolar coupling measurements indicate rather high amplitudes of motion of the peptide.     

Interactions of the human calcitonin fragment 9-32 with phospholipids: a monolayer study

Human calcitonin and its C-terminal fragment 9-32 (hCT(9-32)) administered in a spray translocate into respiratory nasal epithelium with an effect similar to intravenous injection. hCT(9-32) is an efficient carrier to transfer the green fluorescent protein into excised bovine nasal mucosa. To understand the translocation of hCT(9-32) across plasma membranes, we investigated its interactions with phospholipids and its interfacial structure using model lipid monolayers. A combination of physicochemical methods was applied including surface tension measurements on adsorbed and spread monolayers at the air-water interface, Fourier transform infrared, circular dichroism, and atomic force microscopy on Langmuir-Blodgett monolayers. The results disclose that hCT(9-32) preferentially interacts with negatively charged phospholipids and does not insert spontaneously into lipid monolayers. This supports a nonreceptor-mediated endocytic internalization pathway as previously suggested. Structural studies revealed a random coil conformation of hCT(9-32) in solution, transforming to alpha-helices when the peptide is localized at lipid-free or lipid-containing air-water interfaces. Atomic force microscopy studies of monolayers of the peptide alone or mixed with dioleoylphosphatidylcholine revealed that hCT(9-32) forms filaments rolled into spirals. In contrast, when interacting with dioleoylphosphatidylglycerol, hCT(9-32) does not adopt filamentous structures. A molecular model and packing is proposed for the spiral-forming hCT(9-32).     

Membrane surface-associated helices promote lipid interactions and cellular uptake of human calcitonin-derived cell penetrating peptides

hCT(9-32) is a human calcitonin (hCT)-derived cell-penetrating peptide that has been shown to translocate the plasma membrane of mammalian cells. It has been suggested as a cellular carrier for drugs, green fluorescent protein, and plasmid DNA. Because of its temperature-dependent cellular translocation resulting in punctuated cytoplasmatic distribution, its uptake is likely to follow an endocytic pathway. To gain insight into the molecular orientation of hCT(9-32) when interacting with lipid models, and to learn more about its mode of action, various biophysical techniques from liposome partitioning to high-resolution NMR spectroscopy were utilized. Moreover, to establish the role of individual residues for the topology of its association with the lipid membrane, two mutants of hCT(9-32), i.e., W30-hCT(9-32) and A23-hCT(9-32), were also investigated. Although unstructured in aqueous solution, hCT(9-32) adopted two short helical stretches when bound to dodecylphosphocholine micelles, extending from Thr10 to Asn17 and from Gln24 to Val29. A23-hCT(9-32), in which the helix-breaking Pro23 was replaced by Ala, displayed a continuous alpha-helix extending from residue 12 to 26. Probing with the spin label 5-doxylstearate revealed that association with dodecylphosphocholine micelles was such that the helix engaged in parallel orientation to the micelle surface. Moreover, the Gly to Trp exchange in W30-hCT(9-32) resulted in a more stable anchoring of the C-terminal segment close to the interface, as reflected by a twofold increase in the partition coefficient in liposomes. Interestingly, tighter binding to model membranes was associated with an increase in the in vitro uptake in human cervix epithelial adenocarcinoma cell line cells. Liposome leakage studies excluded pore formation, and the punctuated fluorescence pattern of internalized peptide indicated vesicular localization and, in conclusion, strongly suggested an endocytic pathway of translocation.     

Novel daunorubicin-carrier peptide conjugates derived from human calcitonin segments

Severe and often therapy-limiting side effects are a major obstacle in cancer chemotherapy. New delivery concepts reducing systemic side effects are needed in order to optimize anticancer therapies. Several approaches have been followed, most of them concentrating on macromolecular carriers like liposomes, monoclonal antibodies, serum proteins or polyethylene glycol. We present here a novel type of anthracycline conjugate, using a small carrier peptide derived from the peptide hormone human calcitonin (hCT). The carrier peptide hCT(9-32) has so far been shown to be capable of transporting fluorophores or proteins across cellular membranes. Two different carrier peptide-daunorubicin conjugates were prepared, one with an acid-stable amide bond, the second with an acid-labile hydrazone bond. In vitro studies with daunorubicin linked to the carrier peptide via an acid-labile hydrazone bond demonstrated comparable cytotoxicity to daunorubicin in various daunorubicin sensitive cell lines (neuroblastoma cell lines SK-N-MC and SMS-KAN; HEK 293 T cells). In addition, fluorescence microscopy provided further insight into the mechanism of uptake of the carrier peptide hCT(9-32), indicating that endosomal compartments with reduced pH are involved in the intracellular release of daunorubicin.     

The polar nature of 7-ketocholesterol determines its location within membrane domains and the kinetics of membrane microsolubilization by apolipoprotein A-I

7-Ketocholesterol is an oxidized derivative of cholesterol with numerous physiological effects. In model membranes, 7-ketocholesterol and cholesterol were compared by physical measures of bilayer order and polarity, formation of detergent resistant domains (DRM), phase separation, and membrane microsolubilization by apolipoprotein A-I. In binary mixtures of a saturated phosphatidylcholine (PC), dipalmitoyl-PC (DPPC), and cholesterol or 7-ketocholesterol, the sterols modulate bilayer order and polarity and induce DRM formation to a similar extent. Cholesterol induces formation of ordered lipid domains (rafts) in tertiary mixtures with dioleoyl-PC (DOPC) and DPPC, or DOPC and sphingomyelin (SM). In tertiary mixtures, cholesterol increased lipid order and reduces bilayer polarity more than 7-ketocholesterol. This effect was more pronounced when the mixtures were in a miscible liquid-disordered (L(d)) phase. Substitution of 7-ketocholesterol for cholesterol dramatically reduced the extent of DRM formation in DOPC/DPPC and DOPC/SM bilayers and ordered lipid phase separation in mixtures of a spin-labeled PC with DPPC and with SM. Compared to cholesterol, 7-ketocholesterol decreased the rate for the microsolubilization of dimyristoyl-PC multilamellar vesicles by apolipoprotein A-I. The membrane effects of 7-ketocholesterol were dependent on the phospholipid matrix. In L(d) phase phospholipids, a model for 7-ketocholesterol indicates that the proximity of the 7-keto and 3beta-OH groups puts both polar moieties at the lipid-water interface to tilt the sterol nucleus to the plane of the bilayer. 7-Ketocholesterol was less effective in forming ordered lipid domains, in decreasing the level of bilayer hydration, and in forming phase boundary bilayer defects. Compared to cholesterol, 7-ketocholesterol can differentially modulate membrane properties involved in protein-membrane association and function.     

Epifluorescence microscopic studies of monolayers containing mixtures of dioleoyl- and dipalmitoylphosphatidylcholines.

Monolayers of dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and some mixtures of these lipids were investigated using an epifluorescence microscopic surface balance. Monolayers were visualized at 23 +/- 1 degree C through the fluorescence of 1 mol% of two different fluorescent probes, 1-palmitoyl-2-(12-[(7-nitro-2-1,3-benzoxadizole-4- yl)amino]dodecanoyl)phosphatidylcholine (NBD-PC), which partitions into the liquid expanded (LE) or disordered lipid phase and 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO-C18), which preferentially associates with the liquid condensed (LC) phase or lipid with ordered chains. LC domains were observed in pure DPPC monolayers at relatively low surface pressures (pi), and these domains grew with increasing surface pressure. Only liquid expanded phase was observed in pure DOPC monolayers up to the point of monolayer collapse. In monolayers containing 29:70:1, 49:50:1, and 69:30:1 (mol/mol/mol) of DPPC:DOPC:probe the domains of LC phase were smaller than those seen in DPPC monolayers at equivalent surface pressures. Quantitative analysis of the visual fields shown by the mixed monolayers showed a distribution of sizes of condensed domains at any given pi. At pi = 30 mN m-1, liquid-expanded, or fluid, regions occupied more than 70% of the total monolayer area in all three mixtures studied, whereas DPPC monolayers were more than 75% condensed or solid at that pressure. For monolayers of DPPC:DOPC:NBD-PC 49:50:1 and 69:30:1 the average domain size and the percentage of the total area covered with LC, or rigid, areas increased to a maximum at pi around 35 mN m-1 followed by a decrease at higher pi.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coexisting stripe- and patch-shaped domains in giant unilamellar vesicles

We report a new type of gel-liquid phase segregation in giant unilamellar vesicles (GUVs) of mixed lipids. Coexisting patch- and stripe-shaped gel domains in GUV bilayers composed of DOPC/DPPC or DLPC/DPPC are observed by confocal fluorescence microscopy. The lipids in stripe domains are shown to be tilted according to the DiIC18 fluorescence intensity dependence on the excitation polarization. The patch domains are found to be mainly composed of DPPC-d62 according to the coherent anti-Stokes Raman scattering (CARS) images of DOPC/DPPC-d62 bilayers. When cooling GUVs from above the miscibility temperature, the patch domains start to appear between the chain melting and the pretransition temperature of DPPC. In GUVs containing a high molar percentage of DPPC, the stripe domains form below the pretransition temperature. Our observations suggest that the patch and stripe domains are in the Pbeta' and Lbeta' gel phases, respectively. According to the thermoelastic properties of GUVs described by Needham and Evans [(1988) Biochemistry 27, 8261-8269], the Pbeta' and Lbeta' phases are formed at relatively low and high membrane tensions, respectively. GUVs with high DPPC percentage have high membrane surface tension and thus mainly exhibit Lbeta' domains, while GUVs with low DPPC percentage have low membrane surface tension and form Pbeta' domains accordingly. Adding negatively charged lipid to the lipid mixtures or applying an osmotic pressure to GUVs using sucrose solutions releases the surface tension and leads to the disappearance of the Lbeta' gel phase. The relationship between the observed domains in free-standing GUV bilayers and those in supported bilayers is discussed.     

Ethanol effects on binary and ternary supported lipid bilayers with gel/fluid domains and lipid rafts

Ethanol-lipid bilayer interactions have been a recurrent theme in membrane biophysics, due to their contribution to the understanding of membrane structure and dynamics. The main purpose of this study was to assess the interplay between membrane lateral heterogeneity and ethanol effects. This was achieved by in situ atomic force microscopy, following the changes induced by sequential ethanol additions on supported lipid bilayers formed in the absence of alcohol. Binary phospholipid mixtures with a single gel phase, dipalmitoylphosphatidylcholine (DPPC)/cholesterol, gel/fluid phase coexistence DPPC/dioleoylphosphatidylcholine (DOPC), and ternary lipid mixtures containing cholesterol, mimicking lipid rafts (DOPC/DPPC/cholesterol and DOPC/sphingomyelin/cholesterol), i.e., with liquid ordered/liquid disordered (ld/lo) phase separation, were investigated. For all compositions studied, and in two different solid supports, mica and silicon, domain formation or rearrangement accompanied by lipid bilayer thinning and expansion was observed. In the case of gel/fluid coexistence, low ethanol concentrations lead to a marked thinning of the fluid but not of the gel domains. In the case of ld/lo all the bilayer thins simultaneously by a similar extent. In both cases, only the more disordered phase expanded significantly, indicating that ethanol increases the proportion of disordered domains. Water/bilayer interfacial tension variation and freezing point depression, inducing acyl chain disordering (including opening and looping), tilting, and interdigitation, are probably the main cause for the observed changes. The results presented herein demonstrate that ethanol influences the bilayer properties according to membrane lateral organization.     

Preferential accumulation of Abeta(1-42) on gel phase domains of lipid bilayers: an AFM and fluorescence study

Peptide-membrane interactions have been implicated in both the toxicity and aggregation of beta-amyloid (Abeta) peptides. Recent studies have provided evidence for the involvement of liquid-ordered membrane domains known as lipid rafts in the formation and aggregation of Abeta. As a model, we have examined the interaction of Abeta(1-42) with phase separated DOPC/DPPC lipid bilayers using a combination of atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRF). AFM images show that addition of Abeta to preformed supported bilayers leads to accumulation of small peptide aggregates exclusively on the gel phase DPPC domains. Initial aggregates are observed approximately 90 min after peptide addition and increase in diameter to 45-150 nm within 24 h. TIRF studies with a mixture of Abeta and Abeta-Fl demonstrate that accumulation of the peptide on the gel phase domains occurs as early as 15 min after Abeta addition and is maintained for over 24 h. By contrast, Abeta is randomly distributed throughout both fluid and gel phases when the peptide is reconstituted into DOPC/DPPC vesicles prior to formation of a supported bilayer. The preferential accumulation of Abeta on DPPC domains suggests that rigid domains may act as platforms to concentrate peptide and enhance its aggregation and may be relevant to the postulated involvement of lipid rafts in modulating Abeta activity in vivo.     

Real-time imaging of drug-membrane interactions by atomic force microscopy

Understanding drug-biomembrane interactions at high resolution is a key issue in current biophysical and pharmaceutical research. Here we used real-time atomic force microscopy (AFM) imaging to visualize the interaction of the antibiotic azithromycin with lipid domains in model biomembranes. Various supported lipid bilayers were prepared by fusion of unilamellar vesicles on mica and imaged in buffer solution. Phase-separation was observed in the form of domains made of dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (SM), or SM/cholesterol (SM/Chl) surrounded by a fluid matrix of dioleoylphosphatidylcholine (DOPC). Time-lapse images collected following addition of 1 mM azithromycin revealed progressive erosion and disappearance of DPPC gel domains within 60 min. We attribute this effect to the disruption of the tight molecular packing of the DPPC molecules by the drug, in agreement with earlier biophysical experiments. By contrast, SM and SM-Chl domains were not modified by azithromycin. We suggest that the higher membrane stability of SM-containing domains results from stronger intermolecular interactions between SM molecules. This work provides direct evidence that the perturbation of lipid domains by azithromycin strongly depends on the lipid nature and opens the door for developing new applications in membrane biophysics and pharmacology.     

Temperature-pressure phase diagram of a heterogeneous anionic model biomembrane system: results from a combined calorimetry, spectroscopy and microscopy study

By using Fourier transform infrared (FT-IR) spectroscopy in combination with differential scanning calorimetry (DSC) coupled with pressure perturbation calorimetry (PPC), ultrasound velocimetry, Laurdan fluorescence spectroscopy, fluorescence microscopy and atomic force microscopy (AFM), the temperature and pressure dependent phase behavior of the five-component anionic model raft lipid mixture DOPC/DOPG/DPPC/DPPG/cholesterol (20:5:45:5:25 mol%) was investigated. A temperature range from 5 to 65 °C and a pressure range up to 16 kbar were covered to establish the temperature-pressure phase diagram of this heterogeneous model biomembrane system. Incorporation of 10-20 mol% PG still leads to liquid-ordered (l(o))-liquid-disordered (l(d)) phase coexistence regions over a wide range of temperatures and pressures. Compared to the corresponding neutral model raft mixture (DOPC/DPPC/Chol 25:50:25 mol%), the p,T-phase diagram is - as expected and in accordance with the Gibbs phase rule - more complex, the phase sequence as a function of temperature and pressure is largely similar, however. This anionic heterogeneous model membrane system will serve as a more realistic model biomembrane system to study protein interactions with anionic lipid bilayers displaying liquid-disordered/liquid-ordered domain coexistence over a wide range of the temperature-pressure plane, thus allowing also studies of biologically relevant systems encountered under extreme environmental conditions.     

Real-time imaging of drug-membrane interactions by atomic force microscopy

Understanding drug-biomembrane interactions at high resolution is a key issue in current biophysical and pharmaceutical research. Here we used real-time atomic force microscopy (AFM) imaging to visualize the interaction of the antibiotic azithromycin with lipid domains in model biomembranes. Various supported lipid bilayers were prepared by fusion of unilamellar vesicles on mica and imaged in buffer solution. Phase-separation was observed in the form of domains made of dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (SM), or SM/cholesterol (SM/Chl) surrounded by a fluid matrix of dioleoylphosphatidylcholine (DOPC). Time-lapse images collected following addition of 1 mM azithromycin revealed progressive erosion and disappearance of DPPC gel domains within 60 min. We attribute this effect to the disruption of the tight molecular packing of the DPPC molecules by the drug, in agreement with earlier biophysical experiments. By contrast, SM and SM-Chl domains were not modified by azithromycin. We suggest that the higher membrane stability of SM-containing domains results from stronger intermolecular interactions between SM molecules. This work provides direct evidence that the perturbation of lipid domains by azithromycin strongly depends on the lipid nature and opens the door for developing new applications in membrane biophysics and pharmacology.     

Kinetics for the subgel phase formation in DPPC/DOPC mixed bilayers

We analyzed the kinetics for the subgel (SGI) phase formation in DPPC/DOPC binary bilayers paying attention to DOPC-induced modification of the bilayer physical properties. Differential scanning calorimetry and X-ray diffraction revealed that addition of DOPC reduced the apparent initial lag time to start the SGI phase formation, and that the SGI phase in the binary bilayers had basically the same structure as that in pure DPPC bilayers though addition of DOPC markedly increased the peak temperature and enthalpy of the subtransition in heating. Moreover, addition of DOPC abolished the prolongation of the initial lag time in pure DPPC bilayers induced by lowering the incubation temperature from 0 to ?5 °C. Our results suggested that DOPC molecules work as a diffusion enhancer to promote the nucleation of the SGI phase, and relatively destabilize the gel phase so that the formed SGI phase transforms into the ripple phase in heating.     

Developing novel hCT derived cell-penetrating peptides with improved metabolic stability

Many promising therapeutics are currently awaiting their clinical application. Due to their low capability of cell membrane crossing, these compounds do not reach their site of action. One way to overcome this problem might be the fusion of these agents to cell-penetrating peptides (CPP), which are able to shuttle various cargoes across cellular membranes. One disadvantage in using CPP in drug delivery is their low metabolic stability. The aim of our work was to increase the proteolytic resistance of the CPP hCT(9-32), a truncated C-terminal fragment of human calcitonin. Thus, we synthesised six modified N-terminally carboxyfluorescein labelled hCT(9-32) derivatives by replacing positions 12 and/or 16 of hCT(9-32) with either N-methylphenylalanine or d-phenylalanine, respectively. By using confocal laser scanning microscopy we showed that the modifications did neither affect the peptide internalisation efficiency in HeLa nor HEK 293T cells. The metabolic stability of the peptides was investigated in human blood plasma and HEK 293T cell culture supernatant. To analyse the degradation patterns, we used RP-HPLC and MALDI-TOF mass spectrometry. However, we found for all of the new derivatives high metabolic stabilities. In blood plasma, the half-lives for five of the six peptides increased compared to unmodified hCT(9-32). The degradation patterns showed a distinct stabilisation in the N-terminal part of the modified peptides, in the C-terminal part, we found some cleavage to a minor extent. Furthermore, we studied the conformation of the peptides by CD spectroscopy and demonstrated that they possess no cell toxicity. Since our metabolically more stable compounds are still able to pass the cell membrane they provide powerful tools as drug delivery vectors.     

Developing novel hCT derived cell-penetrating peptides with improved metabolic stability

Many promising therapeutics are currently awaiting their clinical application. Due to their low capability of cell membrane crossing, these compounds do not reach their site of action. One way to overcome this problem might be the fusion of these agents to cell-penetrating peptides (CPP), which are able to shuttle various cargoes across cellular membranes. One disadvantage in using CPP in drug delivery is their low metabolic stability. The aim of our work was to increase the proteolytic resistance of the CPP hCT(9-32), a truncated C-terminal fragment of human calcitonin. Thus, we synthesised six modified N-terminally carboxyfluorescein labelled hCT(9-32) derivatives by replacing positions 12 and/or 16 of hCT(9-32) with either N-methylphenylalanine or d-phenylalanine, respectively. By using confocal laser scanning microscopy we showed that the modifications did neither affect the peptide internalisation efficiency in HeLa nor HEK 293T cells. The metabolic stability of the peptides was investigated in human blood plasma and HEK 293T cell culture supernatant. To analyse the degradation patterns, we used RP-HPLC and MALDI-TOF mass spectrometry. However, we found for all of the new derivatives high metabolic stabilities. In blood plasma, the half-lives for five of the six peptides increased compared to unmodified hCT(9-32). The degradation patterns showed a distinct stabilisation in the N-terminal part of the modified peptides, in the C-terminal part, we found some cleavage to a minor extent. Furthermore, we studied the conformation of the peptides by CD spectroscopy and demonstrated that they possess no cell toxicity. Since our metabolically more stable compounds are still able to pass the cell membrane they provide powerful tools as drug delivery vectors.     

Liquid domains in vesicles investigated by NMR and fluorescence microscopy

We use (2)H-NMR, (1)H-MAS NMR, and fluorescence microscopy to detect immiscibility in three particular phospholipid ratios mixed with 30% cholesterol: 2:1 DOPC/DPPC, 1:1 DOPC/DPPC, and 1:2 DOPC/DPPC. Large-scale (>160 nm) phase separation into liquid-ordered (L(o)) and liquid-crystalline (L(alpha)) phases is observed by both NMR and fluorescence microscopy. By fitting superimposed (2)H-NMR spectra, we quantitatively determine that the L(o) phase is strongly enriched in DPPC and moderately enriched in cholesterol. Tie-lines estimated at different temperatures and membrane compositions are based on both (2)H-NMR observations and a previously published ternary phase diagram. (2)H- and (1)H-MAS NMR techniques probe significantly smaller length scales than microscopy experiments (submicron versus micron-scalp), and complex behavior is observed near the miscibility transition. Fluorescence microscopy of giant unilamellar vesicles shows micrometer-scale domains below the miscibility transition. In contrast, NMR of multilamellar vesicles gives evidence for smaller ( approximately 80 nm) domains just below the miscibility transition, whereas large-scale demixing occurs at a lower temperature, T(low). A transition at T(low) is also evident in fluorescence microscopy measurements of the surface area fraction of ordered phase in giant unilamellar vesicles. Our results reemphasize the complex phase behavior of cholesterol-containing membranes and provide a framework for interpreting (2)H-NMR experiments in similar membranes.     

Phospholipid-cationic lipid interactions: influences on membrane and vesicle properties

Liposomes composed of synthetic dialkyl cationic lipids and zwitterionic phospholipids such as dioleoylphosphatidylethanolamine have been studied extensively as vehicles for gene delivery, but the broader potentials of these cationic liposomes for drug delivery have not. An understanding of phospholipid-cationic lipid interactions is essential for rational development of this potential. We evaluated the effect of the cationic lipid DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium) on liposome physical properties such as size and membrane domain structure. DSC (differential scanning calorimetry) showed progressive decrease and broadening of the phase transition temperature of dipalmitoylphosphatidylcholine (DPPC) with increasing fraction of DOTAP, in the range of 0.4-20 mol%. Laurdan (6-dodecanolyldimethylamino-naphthalene), a fluorescent probe of membrane domain structure, showed that DOTAP and DPPC remained miscible at all ratios tested. DOTAP reduced the size of spontaneously-forming PC-containing liposomes, regardless of the acyl chain length and degree of saturation. The anionic lipid DOPG (dioleoylphosphatidylglycerol) had similar effects on DPPC membrane fluidity and size. However, DOTAP/DOPC (50/50) vesicles were taken up avidly by OVCAR-3 human ovarian tumor cells, in contrast to DOPG/DOPC (50/50) liposomes. Overall, DOTAP exerts potent effects on bilayer physical properties, and may provide advantages for drug delivery.     

Effect of the antibiotic azithromycin on thermotropic behavior of DOPC or DPPC bilayers

Azithromycin is a macrolide antibiotic known to bind to lipids and to affect endocytosis probably by interacting with lipid membranes [Tyteca, D., Schanck, A., Dufrene, Y.F., Deleu, M., Courtoy, P.J., Tulkens, P.M., Mingeot-Leclercq, M.P., 2003. The macrolide antibiotic azithromycin interacts with lipids and affects membrane organization and fluidity: studies on Langmuir-Blodgett monolayers, liposomes and J774 macrophages. J. Membr. Biol. 192, 203-215]. In this work, we investigate the effect of azithromycin on lipid model membranes made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Thermal transitions of both lipids in contact with azithromycin are studied by (31)P NMR and DSC on multilamellar vesicles. Concerning the DPPC, azithromycin induces a suppression of the pretransition whereas a phase separation between the DOPC and the antibiotic is observed. For both lipids, the enthalpy associated with the phase transition is strongly decreased with azithromycin. Such effects may be due to an increase of the available space between hydrophobic chains after insertion of azithromycin in lipids. The findings provide a molecular insight of the phase merging of DPPC gel in DOPC fluid matrix induced by azithromycin [Berquand, A., Mingeot-Leclercq, M.P., Dufrene, Y.F., 2004. Real-time imaging of drug-membrane interactions by atomic force microscopy. Biochim. Biophys. Acta 1664, 198-205] and could help to a better understanding of azithromycin-cell interaction.     

Decoding the entry of two novel cell-penetrating peptides in HeLa cells: lipid raft-mediated endocytosis and endosomal escape

Cellular entry of peptide, protein, and nucleic acid biopharmaceuticals is severely impeded by the cell membrane. Linkage or assembly of such agents and cell-penetrating peptides (CPP) with the ability to cross cellular membranes has opened a new horizon in biomedical research. Nevertheless, the uptake mechanisms of most CPP have been controversially discussed and are poorly understood. We present data on two recently developed oligocationic CPP, the sweet arrow peptide SAP, a gamma-zein-related sequence, and a branched human calcitonin derived peptide, hCT(9-32)-br, carrying a simian virus derived nuclear localization sequence in the side chain. Uptake in HeLa cells and intracellular trafficking of N-terminally carboxyfluorescein labeled peptides was studied by confocal laser scanning microscopy and flow cytometry using biochemical markers in combination with quenching and colocalization approaches. Both peptides were readily internalized by HeLa cells through interaction with the extracellular matrix followed by lipid raft-mediated endocytosis as confirmed by reduced uptake at lower temperature, in the presence of endocytosis inhibitors and through cholesterol depletion by methyl-beta-cyclodextrin, supported by colocalization with markers for clathrin-independent pathways. In contrast to the oligocationic SAP and hCT(9-32)-br, interaction with the extracellular matrix, however, was no prerequisite for the observed lipid raft-mediated uptake of the weakly cationic, unbranched hCT(9-32). Transient involvement of endosomes in intracellular trafficking of SAP and hCT(9-32)-br prior to endosomal escape of both peptides was revealed by colocalization and pulse-chase studies of the peptides with the early endosome antigen 1. The results bear potential for CPP as tools for intracellular drug delivery.     

Fluorescent probe partitioning in GUVs of binary phospholipid mixtures: implications for interpreting phase behavior

The phase behavior of membrane lipids is known to influence the organization and function of many integral proteins. Giant unilamellar vesicles (GUVs) provide a very useful model system in which to examine the details of lipid phase separation using fluorescence imaging. The visualization of domains in GUVs of binary and ternary lipid mixtures requires fluorescent probes with partitioning preference for one of the phases present. To avoid possible pitfalls when interpreting the phase behavior of these lipid mixtures, sufficiently thorough characterization of the fluorescent probes used in these studies is needed. It is now evident that fluorescent probes display different partitioning preferences between lipid phases, depending on the specific lipid host system. Here, we demonstrate the benefit of using a panel of fluorescent probes and confocal fluorescence microscopy to examine phase separation in GUVs of binary mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Patch and fibril gel phase domains were found to co-exist with liquid disordered (l(d)) domains on the surface of GUVs composed of 40:60 mol% DOPC/DPPC, over a wide range of temperatures (14-25°C). The fluorescent lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl (NBD-DPPE), proved to be the most effective probe for visualization of fibril domains. In the presence of Lissamine(TM) rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Rh-DPPE) we were unable to detect fibril domains. This fluorophore also affected the partitioning behavior of other fluorescent probes. Overall, we show that the selection of different fluorescent probes as lipid phase reporters can result in very different interpretation of the phase behavior of DOPC/DPPC mixtures.