Multilamellar Phospholipid Vesicles Research Articles

NaCl increases the dielectric constant of nanoconfined water in phospholipid multilamellar vesicle by enhancing intermolecular orientation correlation rather than rotational freedom of individual molecules

We investigate how the NaCl affects the dielectric constant of nanoconfined water in phospholipid multilamellar vesicles using dielectric relaxation spectroscopy. An anomalous increase in the dielectric constant is observed near the physiological NaCl concentration (0<c<0.3M), which has been consistently proposed in molecular dynamic simulations but is first confirmed by experiment. NaCl accelerates the reorientation motion of surface-bound water, which implies an increase of the rotational degree of freedom. However, for water beyond the hydration layer, retarded collective reorientation motion and increased dielectric strength are observed, suggesting NaCl enhances intermolecular orientation correlation rather than rotational freedom.

Phospholipid multilamellar vesicles entrapping phenothiazine photosensitizers. Preparation, characterization and evaluation of their photodynamic properties

The aim of the present paper was to optimize the properties of phenothiazine photosensitizers (PSs), Azure B and its monobrominated derivative. They were entrapped in multilamellar liposomes of egg phosphatidylcholine (EPC) and dipalmitoylphosphatidylcholine (dpPC). The higher partition coefficient of both PSs in the EPC/water system compared with that of both PSs in the dpPC/water system allowed the selection of EPC to be used for the development of third generation photosensitizers. The optimal phospholipid/phenothiazine ratio and the location of these dyes in the lipid bilayer systems were established. These dyes were found to be located within the polar head group region of the lipid bilayer, which would enhance the effectiveness of the irradiation procedure. In addition, the encapsulation of both photosensitizers in liposomes led to a decrease in the aggregation of these compounds, and consequently to an increase in their singlet oxygen quantum yield (ϕΔ). The encapsulation of the PSs in lipid vesicles significantly increased their photochemical reactivity, doubling the ϕΔ value of AzBBr and increasing that of AzB by 60%. The results obtained demonstrate that the vehiculization of these PSs in liposomes allowed the development of third generation photosensitizers with better photochemical properties to be used in potential therapeutic applications.

Apolipoprotein A-I glycation by Glucose and Reactive Aldehydes Alters Phospholipid Affinity but Not Cholesterol Export from Lipid-Laden Macrophages

Increased protein glycation in people with diabetes may promote atherosclerosis. This study examined the effects of non-enzymatic glycation on the association of lipid-free apolipoproteinA-I (apoA-I) with phospholipid, and cholesterol efflux from lipid-loaded macrophages to lipid-free and lipid-associated apoA-I. Glycation of lipid-free apoA-I by methylglyoxal and glycolaldehyde resulted in Arg, Lys and Trp loss, advanced glycation end-product formation and protein cross-linking. The association of apoA-I glycated by glucose, methylglyoxal or glycolaldehyde with phospholipid multilamellar vesicles was impaired in a glycating agent dose-dependent manner, with exposure of apoA-I to both 30 mM glucose (42% decrease in kslow) and 3 mM glycolaldehyde (50% decrease in kfast, 60% decrease in kslow) resulting is significantly reduced affinity. Cholesterol efflux to control or glycated lipid-free apoA-I, or discoidal reconstituted HDL containing glycated apoA-I (drHDL), was examined using cholesterol-loaded murine (J774A.1) macrophages treated to increase expression of ATP binding cassette transporters A1 (ABCA1) or G1 (ABCG1). Cholesterol efflux from J774A.1 macrophages to glycated lipid-free apoA-I via ABCA1 or glycated drHDL via an ABCG1-dependent mechanism was unaltered, as was efflux to minimally modified apoA-I from people with Type 1 diabetes, or controls. Changes to protein structure and function were prevented by the reactive carbonyl scavenger aminoguanidine. Overall these studies demonstrate that glycation of lipid-free apoA-I, particularly late glycation, modifies its structure, its capacity to bind phospholipids and but not ABCA1- or ABCG1-dependent cholesterol efflux from macrophages.

Influence of Apolipoprotein (Apo) A-I Structure on Nascent High Density Lipoprotein (HDL) Particle Size Distribution

The principal protein of high density lipoprotein (HDL), apolipoprotein (apo) A-I, in the lipid-free state contains two tertiary structure domains comprising an N-terminal helix bundle and a less organized C-terminal domain. It is not known how the properties of these domains modulate the formation and size distribution of apoA-I-containing nascent HDL particles created by ATP-binding cassette transporter A1 (ABCA1)-mediated efflux of cellular phospholipid and cholesterol. To address this issue, proteins corresponding to the two domains of human apoA-I (residues 1-189 and 190-243) and mouse apoA-I (residues 1-186 and 187-240) together with some human/mouse domain hybrids were examined for their abilities to form HDL particles when incubated with either ABCA1-expressing cells or phospholipid multilamellar vesicles. Incubation of human apoA-I with cells gave rise to two sizes of HDL particles (hydrodynamic diameter, 8 and 10 nm), and removal or disruption of the C-terminal domain eliminated the formation of the smaller particle. Variations in apoA-I domain structure and physical properties exerted similar effects on the rates of formation and sizes of HDL particles created by either spontaneous solubilization of phospholipid multilamellar vesicles or the ABCA1-mediated efflux of cellular lipids. It follows that the sizes of nascent HDL particles are determined at the point at which cellular phospholipid and cholesterol are solubilized by apoA-I; apparently, this is the rate-determining step in the overall ABCA1-mediated cellular lipid efflux process. The stability of the apoA-I N-terminal helix bundle domain and the hydrophobicity of the C-terminal domain are important determinants of both nascent HDL particle size and their rate of formation.

Solid-state 2H and 15N NMR studies of side-chain and backbone dynamics of phospholamban in lipid bilayers: Investigation of the N27A mutation

Phospholamban (PLB) is an integral membrane protein regulating Ca 2+ transport through inhibitory interaction with sarco(endo)plasmic reticulum calcium ATPase (SERCA). The Asn27 to Ala (N27A) mutation of PLB has been shown to function as a superinhibitor of the affinity of SERCA for Ca 2+ and of cardiac contractility in vivo. The effects of this N27A mutation on the side-chain and backbone dynamics of PLB were investigated with 2H and 15N solid-state NMR spectroscopy in phospholipid multilamellar vesicles (MLVs). 2H and 15N NMR spectra indicate that the N27A mutation does not significantly change the side-chain or backbone dynamics of the transmembrane and cytoplasmic domains when compared to wild-type PLB. However, dynamic changes are observed for the hinge region, in which greater mobility is observed for the CD 3-labeled Ala24 N27A-PLB. The increased dynamics in the hinge region of PLB upon N27A mutation may allow the cytoplasmic helix to more easily interact with the Ca 2+-ATPase; thus, showing increased inhibition of Ca 2+-ATPase.

Mechanism of Antibacterial Action of Dermaseptin B2: Interplay between Helix−Hinge−Helix Structure and Membrane Curvature Strain

Dermaseptin B2 (Drs B2) is a 33-residue-long cationic, alpha-helical antimicrobial peptide endowed with membrane-damaging activity against a broad spectrum of microorganisms, including bacteria, yeasts, fungi, and protozoa, but its precise mechanism of action remained ill-defined. A detailed characterization of peptide-membrane interactions of Drs B2 was undertaken in comparison with a C-terminal truncated analogue, [1-23]-Drs B2, that was virtually inactive on bacteria despite retaining the cationic charge of the full-length peptide. Both peptides were tested on living cells using membrane permeabilization assays and on large unilamellar and multilamellar phospholipid vesicles composed of binary lipid mixtures by dye leakage assay, fluorescence spectroscopy, circular dichroism, and differential scanning calorimetry and also on SDS micelles using NMR spectroscopy. The results indicate that Drs B2 induces a strong perturbation of anionic lipid bilayers, resides at the hydrocarbon core-water interface, parallel to the plane of the membrane, and interacts preferentially with the polar head groups and glycerol backbone region of the anionic phospholipids, as well as the region of the lipid acyl chain near the bilayer surface. The interfacial location of Drs B2 induces a positive curvature of the bilayer and clustering of anionic lipids, consistent with a carpet mechanism, that may lead to the formation of mixed peptide-phospholipid toroidal, transient pores and membrane permeation/disruption once a threshold peptide accumulation is reached. In constrast, the truncated [1-23]-Drs B2 analogue interacts at the head group level without penetrating and perturbing the hydrophobic core of the bilayer. NMR study in SDS micelles showed that [1-23]-Drs B2 adopts a well-defined helix encompassing residues 2-20, whereas Drs B2 was previously found to adopt helical structures interrupted around the Val(9)-Gly(10) segment. Thus the antibacterial activity of Drs B2 depends markedly on a threshold number of hydrophobic residues to be present on both extremities of the helix. In a membrane environment with a strong positive curvature strain, Drs B2 can adopt a flexible helix-hinge-helix structure that facilitates the concomitant insertion of the strongly hydrophobic N- and C-termini of the peptide into the acyl core of the membrane.

Spin labeling EPR studies of the properties of oxidized phospholipid-containing lipid vesicles

This study aims at characterizing the structure and some properties of phospholipid multi-lamellar vesicles (MLVs) containing the oxidized species γ-palmitoyl-β-(9-hydroperoxy-10,12-octadecanedienoyl)-lecithin (HPPLPC), γ-palmitoyl-β-(9-hydroxy-10,12-octadecanedienoyl)-lecithin (HOPLPC), γ-palmitoyl-β-glutaroyl-lecithin (GlPPC) and γ-palmitoyl-β-azelaoyl-lecithin (AzPPC). Sepharose 4B gel-chromatography was used to ensure and check that only MLVs are used in EPR measurements. Gel-solid to gel-liquid transition temperature (T m), lateral phase separation, fluidity gradient and polarity profile were studied by use of EPR spectroscopy of enclosed n-doxylstearoyl lecithin spin labels. Contrarily to conjugate dienes and normal phospholipids, pure carboxyacyl species yielded aqueous suspensions showing gel-chromatography elution profile resembling that of lysolecithin micelles. Conjugate dienes/DPPC MLVs showed lateral phase separation at room temperature and T m value lower than pure DPPC MLVs. Pure conjugate dienes MLVs resembled more PLPC MLVs and displayed free miscibility with PLPC in mixed MLVs. Pure HPPLPC MLV bilayer appeared to be slightly more rigid, while that of HOPLPC and the polarity profile of MLVs made of the pure conjugate dienes species were similar to those of normal PLPC. It is concluded that carboxyacyl lecithins in MLVs tend to disrupt vesicle structure, while conjugated dienes lecithins are more able to affect some physical properties of the bilayer, and that DPPC in MLVs enhances these effects while PLPC shows a better compatibility with the lipoperoxides.

In vitro and In vivo Characterization of a Novel Liposomal Butorphanol Formulation for Treatment of Pruritus

As yet no transdermal topical formulations have been developed for the treatment of chronic itch. We developed a formulation containing 2 mg butorphanol tartrate in 100 microl purified water encapsulated into multilamellar phospholipid vesicles. Drug permeation experiments were studied with Franz diffusion chambers using human skin in vitro and on rat skin in vivo. Histological analysis of rat skins was performed to evaluate skin irritation of the formulation in vivo. Physical properties showed stable formulation with desirable viscosity. In vitro dermal penetration rate data suggest that there was significant permeation at time-points 2 h and 4 h, and a steady state was achieved afterwards to 24 h. Maximal plasma butorphanol concentration was noted at 2 h and steady state was achieved at 8 h. Visual skin assessment as well as histological analysis of excised rat skin did not demonstrate any evidence of inflammation and irritation. In vitro and in vivo analysis demonstrated release of a consistent amount of butorphanol in a sustained manner for 24 h. This liposomal transdermal delivery formulation could serve as a method to deliver butorphanol for patients with chronic pruritus.

Dynamic Molecular Structure and Phase Diagram of DPPC−Cholesterol Binary Mixtures: A 2D-ELDOR Study

This paper is an application of 2D electron-electron double resonance (2D-ELDOR) with the "full Sc- method" to study model membranes. We obtain and confirm the phase diagram of 1,2-dipalmitoyl-sn-glycerophosphatidylcholine (DPPC)-cholesterol binary mixtures versus temperature and provide quantitative descriptions for its dynamic molecular structure using 2D-ELDOR at the Ku band. The spectra from the end-chain 16-PC spin label in multilamellar phospholipid vesicles are obtained for cholesterol molar concentrations ranging from 0 to 50% and from 25 to 60 degrees C. This phase diagram consists of liquid-ordered, liquid-disordered, and gel phases and phase coexistence regions. The phase diagram is carefully examined according to the spectroscopic evidence, and the rigorous interpretation for the line shape changes. We show that the 2D-ELDOR spectra differ markedly with variation in the composition. The extensive line shape changes in the 2D-plus-mixing-time representation provide useful information to define and characterize the membrane phases with respect to their dynamic molecular structures and to determine the phase boundaries. The homogeneous T2's are extracted from the pure absorption spectra and are used to further distinguish the membrane phases. These results show 2D-ELDOR to be naturally suitable for probing and reporting the dynamic structures of microdomains in model membrane systems and, moreover, providing a very detailed picture of their molecular dynamic structure, especially with the aid of the "full Sc- method".

Solution structures and model membrane interactions of lactoferrampin, an antimicrobial peptide derived from bovine lactoferrin

Bovine lactoferrampin (LFampinB) has been identified as a novel antimicrobial peptide, which is derived from the N-terminal lobe of bovine lactoferrin. In this study, the solution structure of LFampinB bound to negatively charged sodium dodecyl sulphate micelles and zwitterionic dodecyl phosphocholine micelles was determined using 2-dimensional nuclear magnetic resonance (NMR) spectroscopy. The interaction between LFampinB and multilamellar phospholipid vesicles, containing choline and glycerol head groups, was examined using differential scanning calorimetry (DSC). In addition, the interaction between the N-terminal tryptophan residue and model membranes of varying composition was analyzed by fluorescence spectroscopy. LFampinB adopts an amphipathic alpha-helical conformation across the first 11 residues of the peptide but remains relatively unstructured at the C-terminus. The hydrophobic surface of the amphipathic helix is bordered by the side chains of Trp1 and Phe11, and is seen in both micelle-bound structures. The fluorescence results suggest that Trp1 inserts into the membrane at the lipid/water interface. The phenyl side chain of Phe11 is oriented in the same direction as the indole ring of Trp1, allowing these two residues to serve as anchors for the lipid bilayer. The DSC results also indicate that LFampinB interacts with glycerol head groups in multilamellar vesicles but has little effect on acyl chain packing. Our results support a two step model of antimicrobial activity where the initial attraction of LFampinB is mediated by the cluster of positive charges on the C-terminus followed by the formation of the N-terminal helix which binds to the surface of the bacterial lipid bilayer.

Encapsulation of Bowman-Birk soybean proteinase inhibitor within zwitterionic phospholipid multilamellar vesicles

In the present document we describe the possibility for encapsulating a water-soluble protein lacking α-helices into multilayer liposomes made of soybean zwitterionic phospholipid mixtures (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)) in order to prepare proteoliposomes. The influence of the PC/PE w/w ratio on the incorporation efficiency of the Bowman-Birk soybean proteinase inhibitor (BBI) into liposomes was studied. Increase in ionic strength did not inhibit the proteoliposome formation process. BBI encapsulation did not affect liposome sizes, surface charge and structural phospholipid organization. Confocal laser scanning microscopy showed that BBI was located in the central part of the spherical particle and between bilayers. The fluorescence intensity after anthracene-labelled phospholipids interaction with Rhodamin B-labelled BBI confirmed that the protein is deeply immersed into liposomes. The biological activity (antitrypsin and antichymotrypsin) estimation of the protein entrapped in liposomes showed that the protein active centers are spatially shielded. The effect of an ionic detergent on the activity of the encapsulated BBI confirms this hypothesis and suggests that this shielding is reversible. The proteoliposomes prepared seem to be promising formulations for BBI delivery.

Mapping the locations of estradiol and potent neuroprotective analogues in phospholipid bilayers by REDOR

AbstractEstrogens are potent antioxidants and neuroprotectants. They have become the focus of drug development efforts aimed at treating and preventing neuronal damage in stroke‐related brain damage, Alzheimer's disease, and Parkinson's disease. 13C{31P} and 13C{19F} rotational‐echo double‐resonance NMR have been used to map the locations of 17β‐estradiol (estradiol), 17β‐estradiol 17‐benzoate (estradiol benzoate), and 17β‐2‐(1‐adamantyl)estradiol (adamantyl estradiol) in 19F‐labeled DPPC phospholipid multilamellar vesicles. Placement of the estrogen molecules in lipid bilayers is correlated with the differences in their neuroprotective potency. Drug Dev. Res. 66:99–102, 2006. © 2006 Wiley‐Liss, Inc.

Oxidized phospholipids induce phase separation in lipid vesicles

The thermal behaviour of phospholipid multilamellar vesicles (MLV) made of various molar percentages of DPPC and LPPC, containing also oxidized LPPC (LPPCox), was studied by use of EPR spectroscopy and n-DSPC spin label in order to determine variations in the membrane fluidity brought about by lipid oxidation. Experimental variables were temperature, ranging from 4 to 44 °C, and molar percentage composition of DPPC/LPPC/LPPCox ternary mixture. We found that the presence of LPPCox in a percentage higher than both normal phospholipids’ heavily hindered membrane formation, while lower percentage of the oxidized lipid with higher DPPC percentages yielded two-components EPR spectra, showing the presence of two different fluidity domains, indicative of membrane phase separation. When LPPC was the dominant lipid in the ternary mixture, simple EPR spectra were observed, indicating homogeneity of MLV membranes. Phase separation observed in the presence of LPPCox was better visible at lower temperature (12 °C or less), and almost disappeared with increasing temperature (36 °C or more). Furthermore, the correlation time of 16-DSPC in ternary mixture MLVs with higher LPPC percentage (homogeneous membranes) was not affected by the presence of LPPCox, while it normally increased upon DPPC percentage increase, as readily calculated from the EPR spectra featuring simple bands at 24 °C. It is concluded that oxidized lipid induces phase separation in more rigid DPPC-rich membranes, while leaving fluidity unaffected in more fluid LPPC-rich membranes, and at higher temperature.

Structural modification of plasma HDL by phospholipids promotes efficient ABCA1-mediated cholesterol release

It has been suggested that ABCA1 interacts preferentially with lipid-poor apolipoprotein A-I (apoA-I). Here, we show that treatment of plasma with dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles generates prebeta(1)-apoA-I-containing lipoproteins (LpA-I)-like particles similar to those of native plasma. Isolated prebeta(1)-LpA-I-like particles inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than HDL(3) (IC(50) = 2.20 +/- 0.35 vs. 37.60 +/- 4.78 microg/ml). We next investigated the ability of DMPC-treated plasma to promote phospholipid and unesterified (free) cholesterol efflux from J774 macrophages stimulated or not with cAMP. At 2 mg DMPC/ml plasma, both phospholipid and free cholesterol efflux were increased ( approximately 50% and 40%, respectively) in cAMP-stimulated cells compared with unstimulated cells. Similarly, both phospholipid and free cholesterol efflux to either isolated native prebeta(1)-LpA-I and prebeta(1)-LpA-I-like particles were increased significantly in stimulated cells. Furthermore, glyburide significantly inhibited phospholipid and free cholesterol efflux to DMPC-treated plasma. Removal of apoA-I-containing lipoproteins from normolipidemic plasma drastically reduced free cholesterol efflux mediated by DMPC-treated plasma. Finally, treatment of Tangier disease plasma with DMPC affected the amount of neither prebeta(1)-LpA-I nor free cholesterol efflux. These results indicate that DMPC enrichment of normal plasma resulted in the redistribution of apoA-I from alpha-HDL to prebeta-HDL, allowing for more efficient ABCA1-mediated cellular lipid release. Increasing the plasma prebeta(1)-LpA-I level by either pharmacological agents or direct infusions might prevent foam cell formation and reduce atherosclerotic vascular disease.

Undetectable apolipoproteinA-Igene expression suggests an unusual mechanism of dietary lipid mobilisation in the intestine ofCyprinus carpio

High density lipoprotein (HDL) has been shown to play an important role in the dietary lipid mobilisation in the carp. In spite of this, previous studies have failed to demonstrate the synthesis of the major protein component of HDL, apolipoprotein A-I (apoA-I), in the proximal intestine of the carp. Therefore, the aim of the present study was to evaluate the expression of apoA-I throughout the entire intestine. Curiously, no transcription of the apoA-I gene could be detected either by northern blot or RT-PCR assays in the intestinal mucosa, in clear contrast with the abundant cytosolic immunoreactive apoA-I detected in almost all intestinal segments, which suggests a different origin for this protein. In addition, the detection of specific, but low affinity, binding sites for apoA-I in the carp intestinal brush-border membranes (BBM), and the strong interaction with BBM, which is highly dependent on temperature, points to an important contribution of membrane lipids in apoA-I binding to the intestinal mucosa. This idea was reinforced by the ability of carp apoA-I to associate with multilamellar phospholipid vesicles.

Incorporation of isoniazid into liposomes with different lipid composition.

Liposomes containing isoniazid were obtained by extrusion of multilamellar phospholipid vesicles containing isoniazid, by ultrasound treatment, and from proliposomes. Optimal conditions for isolation of stable liposomes (200 nm in diameter) and the amount of incorporated isoniazid were determined by varying concentrations of phospholipids and isoniazid and by changing phospholipid composition.

Complexing of basic pancreatic proteinase inhibitor with soybean phospholipid multilamellar vesicles.

The formation of complexes of basic pancreatic proteinase inhibitor (BPTI) with multilamellar vesicles (MLV) from six preparations of soybean phospholipids of various composition was studied. When BPTI, a non-membrane protein, interacts with MLV, the vesicles aggregate, forming a precipitate of protein-lipid complexes. The BPTI content in the protein-lipid complexes increases with decreasing pH of the medium and on addition of negatively charged components into the lipid mixture. The protein-induced aggregation of the phospholipid vesicles is suggested to be mainly determined by electrostatic forces. The antiproteinase activity of BPTI in the complexes was rather low but increased up to 70% of the initial activity on addition of an ionic detergent (sodium deoxycholate).

Oxidation of Methionine Residues to Methionine Sulfoxides Does Not Decrease Potential Antiatherogenic Properties of Apolipoprotein A-I

The initial stage of oxidation of high density lipoproteins (HDL) is accompanied by the lipid hydroperoxide-dependent, selective oxidation of two of the three Met residues of apolipoprotein A-I (apoA-I) to Met sulfoxides (Met(O)). Formation of such selectively oxidized apoA-I (i.e. apoA-I(+32)) may affect the antiatherogenic properties of HDL, because it has been suggested that Met(86) and Met(112) are important for cholesterol efflux and Met(148) is involved in the activation of lecithin:cholesterol acyl transferase (LCAT). We therefore determined which Met residues were oxidized in apoA-I(+32) and how such oxidation of apoA-I affects its secondary structure, the affinity for lipids, and its ability to remove lipids from human macrophages. We also assessed the capacity of discoidal reconstituted HDL containing apoA-I(+32) to act as substrate for LCAT, and the dissociation of apoA-I and apoA-I(+32) from reconstituted HDL. Met(86) and Met(112) were present as Met(O), as determined by amino acid sequencing and mass spectrometry of isolated peptides derived from apoA-I(+32). Selective oxidation did not alter the alpha-helicity of lipid-free and lipid-associated apoA-I as assessed by circular dichroism, and the affinity for LCAT was comparable for reconstituted HDL containing apoA-I or apoA-I(+32). Cholesteryl ester transfer protein mediated the dissociation of apoA-I more readily from reconstituted HDL containing apoA-I(+32) than unoxidized apoA-I. Also, compared with native apoA-I, apoA-I(+32) had a 2- to 3-fold greater affinity for lipid (as determined by the rate of clearance of multilamellar phospholipid vesicles) and its ability to cause efflux of [(3)H]cholesterol, [(3)H]phospholipid, and [(14)C]alpha-tocopherol from lipid-laden human monocyte-derived macrophages was significantly enhanced. By contrast, no difference was observed for cholesterol and alpha-tocopherol efflux to lipid-associated apolipoproteins. Together, these results suggest that selective oxidation of Met residues enhances rather than diminishes known antiatherogenic activities of apoA-I, consistent with the overall hypothesis that detoxification of lipid hydroperoxides by HDL is potentially antiatherogenic.

Activation of cytokine production, tumoricidal properties, and tyrosine phosphorylation of MAPKs in human monocytes by a new synthetic lipopeptide, JBT3002.

We investigated the expression of cytokine genes and tumoricidal properties in human blood monocytes in response to a new synthetic immunomodulating lipopeptide, JBT3002. Incubation of peripheral blood monocytes with free-form JBT3002 or JBT3002 encapsulated in multilamellar phospholipid vesicles (liposomes, MLV-JBT3002) induced tumoricidal properties in a dose-dependent manner. Both MLV-JBT3002 and free-form JBT3002 induced production of tumor necrosis factor alpha, interleukin-1beta, and interleukin-6 in a dose-dependent manner with similar kinetics. Treatment of monocytes with interferon-gamma did not significantly alter the expression of cytokine genes but increased the expression of cytokines induced by MLV-JBT3002 and free-form JBT3002. In contrast to monocyte activation by lipopolysaccharide (LPS), activation by JBT3002 was independent of serum and was not inhibited by CD14-neutralizing antibody. Incubation of monocytes with JBT3002 induced a rapid increase in tyrosine phosphorylation of proteins with apparent molecular masses of 42 and 38 kDa, a migration band shift of c-Jun NH2-terminal kinase 1 (JNK1), and activation of extracellular signaling regulated kinases. Consistent with its effect on cytokine expression, stimulation of these intracellular signaling pathways by JBT3002 was not inhibited in serum-free conditions. Collectively, the data indicate that the synthetic lipopeptide JBT3002 is a potent monocyte activator that modulates monocyte function by mechanisms similar to LPS but by a distinct receptor.

Phase behavior and permeability properties of phospholipid bilayers containing a short-chain phospholipid permeability enhancer

The thermodynamic phase behavior and trans-bilayer permeability properties of multilamellar phospholipid vesicles containing a short-chain DC 10PC phospholipid permeability enhancer have been studied by means of differential scanning calorimetry and fluorescence spectroscopy. The calorimetric scans of DC 14PC lipid bilayer vesicles incorporated with high concentrations of DC 10PC demonstrate a distinct influence on the lipid bilayer thermodynamics manifested as a pronounced freezing-point depression and a narrow phase coexistence region. Increasing amounts of DC 10PC lead to a progressive lowering of the melting enthalpy, implying a mixing behavior of the DC 10PC in the bilayer matrix similar to that of a substitutional impurity. The phase behavior of the DC 10PC–DC 14PC mixture is supported by fluorescence polarization measurements which, furthermore, in the low-temperature gel phase reveal a non-monotonic concentration-dependent influence on the structural bilayer properties; small concentrations of DC 10PC induce a disordering of the acyl chains, whereas higher concentrations lead to an ordering. Irreversible fluorescence quench measurements demonstrate a substantial increase in the trans-bilayer permeability over broad temperature and composition ranges. At temperatures corresponding to the peak positions of the heat capacity, a maximum in the trans-bilayer permeability is observed. The influence of DC 10PC on the lipid bilayer thermodynamics and the associated permeability properties is discussed in terms of microscopic effects on the lateral lipid organization and heterogeneity of the bilayer.