Large Unilamellar Vesicle Systems Research Articles

Anti-proliferative effect on a colon adenocarcinoma cell line exerted by a membrane disrupting antimicrobial peptide KL15

The antimicrobial and anticancer activities of an antimicrobial peptide (AMP) KL15 obtained through in silico modification on the sequences of 2 previously identified bacteriocins m2163 and m2386 from Lactobacillus casei ATCC 334 by us have been studied. While significant bactericidal effect on the pathogenic bacteria Listeria, Escherichia, Bacillus, Staphylococcus, Enterococcus is exerted by KL15, the AMP can also kill 2 human adenocarcinoma cells SW480 and Caco-2 with measured IC50 as 50 μg/ml or 26.3 μM. However, the IC50 determined for KL15 on killing the normal human mammary epithelial cell H184B5F5/M10 is 150 μg/ml. The conformation of KL15 dissolved in 50% 2,2,2-trifluroroethanol or in 2 large unilamellar vesicle systems determined by circular dichroism spectroscopy appears to be helical. Further, the cell membrane permeability of treated SW480 cells by KL15 appears to be significantly enhanced as studied by both flow cytometry and confocal microscopy. As observed under a scanning electron microscope, the morphology of treated SW480 cells is also significantly changed as treating time by 80 μg/ml KL15 is increased. KL15 appears to be able to pierce the cell membrane of treated SW480 cells so that numerous porous structures are generated and observable. Therefore, KL15 is likely to kill the treated SW480 cells through the necrotic pathway similar to some recently identified AMPs by others.

Establishing the Synergy of Forces Governing TIM3 Binding to Lipid Membranes

T cell/transmembrane immunoglobulin mucin protein 3 (TIM3) is an integral membrane protein on the plasma membranes of phagocytes and other cells that participates in the recognition of surface-marked cells by the innate immune system. It recognizes its targets by specifically binding phosphatidylserine (PS) on their surfaces. Thus, a detailed characterization of the TIM3/lipid interaction is key to understanding its function. Our group has recently identified the binding orientation of TIM3 to lipid membranes, leading us to hypothesize a synergy of different forces in play in the TIM3/lipid interaction. Here we established hydrophobic insertion and electrostatic attraction as two other forces, in addition to the well-known calcium-mediated binding to PS at a pocket on the protein surface. We have further quantified these three forces in terms of dissociation constants in different large unilamellar vesicle systems, using fluorescence spectral shift as the tool. Our quantitative analysis has led to the surprising discovery of a three- to four- fold affinity enhancement effect of minute amounts of the negatively charged phosphatidic acid (PA) are present in the membrane. Study of this PA enhancement effect has been extended to TIM1 and TIM4, two other members of the TIM protein family, and the results will be discussed.

RBPI 21 Causes (HEMI)Fusion and Leakage of Membranes and Promotes Massive Lipopolysaccharide Aggregation

Bacteria are infectious agents that seem to have difficulties in acquiring resistance against natural occurring antimicrobial peptides (AMPs). These positively charged polypeptides were then developed as an alternative to conventional therapy against bacterial infections. rBPI21 is a 21 kDa peptide based on the N-terminal region of the neutrophil bactericidal/permeability-increasing protein (BPI). It has bactericidal effects on Gram-negative bacteria and higher affinity for lipopolysaccharide (LPS), neutralizing its effect. rBPI21 is in phase III clinical trials against meningitis. This work aimed at elucidating the mechanism of action of rBPI21 at the molecular level. We demonstrate that rBPI21 promotes the aggregation of negatively charged large unilamellar vesicles (LUV) and LPS aggregates, by dynamic light scattering, while for zwitterionic phosphatidylcholine (POPC) LUV the size remains unchanged. The aggregation increases with peptide concentration until promotion of massive aggregation followed by sample flocculation/precipitation [1,2]. Through a Förster resonance energy transfer (FRET) assay, the aggregation is followed by (hemi)fusion of the negatively charged vesicles, culminating at their leakage (also assessed by fluorescence spectroscopy). With the rBPI21-lipid interaction, there is a progressive change in the zeta-potential of the LUV systems and LPS aggregates [2]. LUV systems composed of phosphatidylglycerol (POPG) and POPC:POPG mixtures have higher zeta-potential variations than POPC LUV. For the LPS aggregates, rBPI21 neutralizes the surface charge and, at higher peptide concentrations, overcompensates it [2]. The results demonstrate that rBPI21 mechanism of action at the molecular level involves the interaction with the LPS of the outer membrane of Gram-negative bacteria, followed by internalization and leakage induction through the (hemi)fusion of the bacterial outer and inner membranes, both enriched in phosphatidylglycerol. [1] Domingues, MM et al., Biophys. J. 2009, 96, 987-996. [2] Domingues, MM et al., PLoS ONE 2009, 4, e8385.

RBPI21 Promotes Lipopolysaccharide Aggregation and Exerts Its Antimicrobial Effects by (Hemi)fusion of PG-Containing Membranes

Antimicrobial peptides (AMPs) are important potential alternatives to conventional therapies against bacterial infections. rBPI21 is a 21 kDa peptide based on the N-terminal region of the neutrophil bactericidal/permeability-increasing protein (BPI). This AMP possesses highly selective bactericidal effects on Gram-negative bacteria and have affinity for lipopolysaccharide (LPS) which is believed to be at the origin of its neutralizing effect of the LPS segregated into the bloodstream. We aim at understanding the molecular bases of rBPI21 bactericidal and LPS neutralization actions, using biomembrane model systems. Using dynamic light scattering spectroscopy we demonstrate that rBPI21 promotes aggregation of negatively charged large unilamellar vesicles (LUV), even in the absence of LPS, and LPS aggregates, while for zwitterionic phosphatidylcholine (POPC) LUV the size remains unchanged. The peptide also promotes the fusion (or hemifusion) of membranes containing phosphatidylglycerol (POPG). The aggregation and fusion of negatively charged LUV are peptide concentration-dependent until massive aggregation is reached, followed by sample flocculation/precipitation. Concomitantly, there is a progressive change in the zeta-potential of the LUV systems and LPS aggregates. LUV systems composed of phosphatidylglycerol (POPG) and lipid mixtures with POPG have higher zeta-potential variations than in the absence of POPG. The interaction of rBPI21 with lipid vesicles is followed by leakage, with higher effect in POPG-containing membranes. LPS aggregation can be related with a decreased toxicity, possibly by facilitating its clearance by macrophage phagocytosis and/or blocking of LPS specific receptor recognition. Our data indicate that rBPI21 mechanism of action at the molecular level involves the interaction with the LPS of the outer membrane of Gram-negative bacteria, followed by internalization and leakage induction through the (hemi)fusion of the bacterial outer and inner membranes, both enriched in phosphatidylglycerol.

A new model system for lipid interactions in stratum corneum vesicles: effects of lipid composition, calcium, and pH.

We prepared large unilamellar vesicles (LUVs) with three different stratum corneum lipid compositions: constant amounts of ceramides (55 wt %) and fatty acids (15%) with varying amounts of cholesterol sulfate (0-15%) and cholesterol (15-30%). One of the compositions served as a model for normal stratum corneum, while the second one served as a model for recessive X-linked ichthyosis stratum corneum. The third composition consisted of no cholesterol sulfate. Intervesicle lipid interactions in these LUVs were monitored by fluorescence methods for content leakage, and contents mixing at pH 9, in the absence and presence of Ca2+, and at pH 6. Since the content leakage and contents mixing assays were originally developed for phospholipid vesicles, we characterized the probe binding and the probe quenching properties for stratum corneum LUV systems, and modified the assays slightly accordingly. The time-dependent fluorescence intensity changes in the probe-containing LUVs at pH 9 and 6 and in response to the addition of calcium were monitored. Our results demonstrated that all three types of LUVs were relatively stable at pH 9. Addition of Ca2+ or decreasing the pH to 6 activated intervesicle lipid mixing followed by vesicle fusion and lysis. We found that the LUVs with no cholesterol sulfate and 30% cholesterol exhibited a more extensive Ca2+- or low-pH-activated intervesicle lipid interaction than LUVs with either 5% cholesterol sulfate and 25% cholesterol or 15% cholesterol sulfate and 15% cholesterol. These results suggest that fusogenic agents such as Ca2+ and H+ act to neutralize the fatty acids in the lipid bilayer of stratum corneum vesicles. The inclusion of 5-15% cholesterol sulfate helps to prevent the collapse of fused vesicles into other structures.

Influence of transbilayer area asymmetry on the morphology of large unilamellar vesicles

The morphological consequences of differences in the monolayer surface areas of large unilamellar vesicles (LUVs) have been examined employing cryoelectron microscopy techniques. Surface area was varied by inducing net transbilayer transport of dioleoylphosphatidylglycerol (DOPG) in dioleoylphosphatidylcholine (DOPC):DOPG (9:1, mol:mol) LUVs in response to transmembrane pH gradients. It is shown that when DOPG is transported from the inner to the outer monolayer, initially invaginated LUVs are transformed to long narrow tubular structures, or spherical structures with one or more protrusions. Tubular structures are also seen in response to outward DOPG transport in DOPC:DOPG:Chol (6:1:3, mol:mol:mol) LUV systems, and when lyso-PC is allowed to partition into the exterior monolayer of DOPC:DOPG (9:1, mol:mol) LUVs in the absence of DOPG transport. Conversely, when the inner monolayer area is expanded by the transport of DOPG from the outer monolayer to the inner monolayer of non-invaginated LUVs, a reversion to invaginated structures is observed. The morphological changes are well described by an elastic bending theory of the bilayer. Identification of the difference in relaxed monolayer areas and of the volume-to-area ratio of the LUVs as the shape-determining factors allows a quantitative classification of the observed morphologies. The morphology seen in LUVs supports the possibility that factors leading to differences in monolayer surface areas could play important roles in intracellular membrane transport processes.

Generation and characterization of iron- and barium-loaded liposomes

Previous work (Veiro and Cullis (1990) Biochim. Biophys. Acta 1025, 109–115) has shown that Ca 2+ can be accumulated into large unilamellar vesicles (LUVs) in the presence of a transmembrane pH gradient (inside acidic) and the Ca 2+-ionophore A23187. Here, the ability of A23187 to mediate the uptake of iron and barium into LUVs has been investigated. It is shown that under appropriate conditions of temperature and A23187 concentration, iron (in the form of Fe 2+) can be accumulated into EPC and DSPC/cholesterol (55:45; mol/mol) LUVs with an acidic interior. This uptake is dependent on the internal buffer concentration, with maximum levels of uptake in the range of 300 nmol of cation per μmol lipid. The DSPC-cholesterol LUV systems exhibit superior retention properties compared to the EPC systems. It is demonstrated that Ba 2+ can also be loaded by similar methods. It is also shown that the maximally loaded Fe 2+- and Ba 2+-containing LUVs exhibit increased densities. This is expressed by enhanced gravimetric properties, as an increased proportion of the loaded LUVs can be pelleted by low speed centrifugation, and by enhanced electron densities, in that the Ba 2+-loaded systems can be directly visualized employing cryo-electron microscopy.

Dopamine accumulation in large unilamellar vesicle systems induced by transmembrane ion gradients

Transmembrane movement of dopamine in response to K + or H + ion gradients has been investigated. It is shown that dopamine can accumulate rapidly into large unilamellar vesicles (LUVs) composed of egg phosphatidylcholine exhibiting either a K + diffusion potential (Δψ; negative inside) or a pH gradient (inside acidic). This can result in entrapped dopamine concentrations of 30–40 mM and inside-outside concentration gradients of nearly 300-fold. The transmembrane dopamine gradients formed in LUV systems exhibiting ΔpH (inside acidic) indicate that the transport process can be dictated by movement of the neutral form of dopamine which redistributes according to a simple Henderson-Hasselbach equilibrium. The mechanism of dopamine transport in response to a valinomycin-induced K + potential is more complex. Although generation of a K + diffusion potential results in acidification of the vesicle interior, the magnitude of the induced ΔpH (approx. 1 pH unit) is insufficient to account for the dopamine concentration gradient achieved (>200-fold). Further, data presented here suggest that higher uptake levels of dopamine can be achieved when certain anions (ATP and citrate) are entrapped within the LUV system. These anions may complex with the protonated form of dopamine creating a non-equilibrium trapping phenomena resulting in interior concentrations of dopamine in excess of that predicted by a simple Henderson-Hasselbach equilibrium.

Uptake of safranine and other lipophilic cations into model membrane systems in response to a membrane potential

Lipophilic cations such as safranine and methyltriphenylphosphonium (MTPP +) are commonly employed to obtain measures of the membrane potential (Δψ) exhibited by energized biological membrane systems. These probes reflect the presence of Δψ (inside negative) by accumulating in the interior of the membrane bound system to achieve transmembrane distributions dictated by the Nernst equation. In this work, we characterize the ability of model membrane large unilamellar vesicle systems to accumulate safranine and other biologically active lipophilic cations in response to a K + diffusion potential (interior negative) across the large unilamellar vesicle membrane. We show that safranine, MTPP +, chlorpromazine and vinblastine can be rapidly accumulated to achieve interior lipophilic cation concentrations which may be more than two orders of magnitude higher than exterior concentrations. In the case of safranine, for example, incubation of 2 mM safranine with large unilamellar vesicle systems exhibiting a Δψ of −100 mV or more can lead to interior safranine concentrations in excess of 120 mM. This accumulation appears to proceed as an antiport K +-safranine exchange process, and the optical ‘safranine response’ observed can be attributed to precipitation of the dye inside the vesicle as the interior concentrations of safranine exceeds its solubility (96 mM). These observations are discussed in terms of the utility of probes such as safranine and MTPP + for determinations of Δψ as well as their implications for the equilibrium transbilayer distributions of biologically active lipophilic cations in vivo.

Uptake of dibucaine into large unilamellar vesicles in response to a membrane potential.

Local amine anesthetics appear to exert their effects in the charged (protonated) form on the cytoplasmic side of excitable membranes. Two features of interest are the mechanism whereby these drugs move across the membrane to the inner monolayer and the actual membrane concentrations achieved. In this work, we have investigated the influence of a K+ diffusion potential, delta psi, on the transmembrane distribution and concentration of the local anesthetic dibucaine employing large unilamellar vesicle systems. It is demonstrated that egg phosphatidylcholine large unilamellar vesicles exhibiting a delta psi (interior negative) actively accumulate dibucaine to achieve high interior concentrations. 31P and 13C nuclear magnetic resonance studies show that the internalized drug is localized to the vesicle inner monolayer, and suggest that the protonated form of the anesthetic is the species that is actively transported. The inner monolayer anesthetic concentrations thus achieved can be an order of magnitude or more larger than predicted on the basis of anesthetic lipid-water partition coefficients. It is suggested that these effects may be related to the mechanisms whereby local anesthetics are localized and concentrated at their sites of action in nerve membranes.

Phosphatidic acid as a calcium ionophore in large unilamellar vesicle systems

The ionophoretic capabilities of dioleoylphosphatidic acid (DOPA) for transporting calcium across phospholipid bilayers have been investigated. Calcium uptake by large unilamellar vesicles is shown to depend on the presence of DOPA. This uptake is sensitive to the nature and concentration of calcium chelators in the vesicle interior, indicating that accumulation results from DOPA-mediated translocation of calcium across the membrane. Further, it is shown that characteristics of DOPA-mediated Ca 2+ uptake are similar to those observed for the fungal calcium ionophore, A23187.