Self-quenching Fluorescent Dye Research Articles

Lipid-Modified Peptide Nucleic Acids: Synthesis and Application to Programmable Liposome Fusion.

Peptide nucleic acids (PNAs) can be modified with aliphatic lipid chains and designed to be water soluble and able to spontaneously insert into phospholipid bilayers. Liposomes with 1.5% negatively charged POPG can be driven to fuse and mix their inner content volumes via functionalization with such lipidated peptide nucleic acids (LiPNAs). During fusion, only low amounts of leakage occur (<5%). We describe here the synthesis and purification of such LiPNAs using an automated peptide synthesizer and the preparation of LiPNA functionalized liposomes. Further, we describe the measurement of LiPNA-induced fusion using a fluorescence-based assay for the content mixing between a liposome population with an encapsulated self-quenching fluorescent dye (SRB) and a buffer-filled liposome population.

DNA-Mediated Liposome Fusion Observed by Fluorescence Spectrometry.

DNA-programmed and controlled fusion of lipid membranes have recently been optimized to reliably mix the contents between two populations of liposomes, each functionalized with complementary lipidated DNA (LiNA) oligomer. In this chapter we describe a procedure for DNA-controlled fusion of liposomes mediated by LiNAs that are designed to force bilayers into close proximity. Using a self-quenching fluorescent dye (Sulforhodamine B) to monitor both the mixing of the internal volumes and leakage of the dye into the outer volume we measure the efficiency of content mixing in the bulk population, allowing for direct comparison between different LiNA designs. By generating samples for calibration corresponding to different amounts of content mixing, the average number of fusion events per labeled liposome can be estimated.

Choice of cuvette material can influence spectroscopic leakage and permeability experiments with liposomes

Liposome solute permeability experiments are widely performed to gain information about lipid membrane characteristics. Spectroscopic methods are often used for this purpose, usually monitoring the leakage of a self-quenching fluorescent dye (e.g., carboxyfluorescein, CF) from the liposomes. Hereby, we investigate the effect of liposome-cuvette interactions, a seldom considered detail, on the results obtained from liposomal permeability experiments. The spontaneous leakage of CF from liposomes with different surface properties and phase states is followed using quartz and polystyrene cuvettes, and the results are compared. It is shown that for most lipid compositions the leakage profiles vary notably between different cuvette materials. Reproducibility of the measurements also varies depending on the cuvettes used, with polystyrene providing with more robust results. To explain these observations, the interaction of liposomes with polystyrene and quartz-like surfaces was characterized with the help of the quartz crystal microbalance with dissipation monitoring (QCM-D). Our results show that, while liposomes seldom interact with polystyrene, quartz-liposome interactions are almost unavoidable and have a large impact on the leakage experiments mainly via two mechanisms: i) the rupturing of liposomes on the cuvette surface causing a fast release of encapsulated CF, and ii) the disruption of adsorbed liposomes caused by magnetic stirring. Depending on their composition, the liposomes interact in different ways with quartz, affecting thus the extent of each proposed mechanism. The experiments demonstrate the importance of considering the cuvette material when planning and conducting spectroscopic experiments with liposomes.

Membrane Leakage: Further Insight by Advanced Statistical Analysis of Vesicle Leakage

The action of membrane-active compounds involves permeabilizing the lipid portion of membranes. For example, this applies to antimicrobial, anticancer and cell penetrating peptides and their synthetic mimics, transfection agents, and drug delivery vehicles. Membrane permeabilization is modelled by the release of an entrapped self-quenching fluorescent dye from lipid vesicles. This allows to determine the total leakage but also the distribution of leakage over the vesicle population. By a statistical concept and kinetic studies, we characterize the strength of an individual leakage event and discuss the molecular mechanisms such as asymmetry stress, curvature stabilization (toroidal pores, defects), large (oligomeric) pores and combinations of these. We apply the concept to leakage induced by detergents, antimicrobial compounds and leakage upon phase transition of lipids. Surprisingly, the strength of the individual leakage event can vary for a given molecular mechanism. For example, the strength of leakage in asymmetric binding and cracking-in can depend on the ratio of electrostatic to hydrophobically driven interactions and varies with both, active agent and lipid composition. The superposition or succession of different mechanisms (e.g. asymmetry stress and formation of defined pores at higher concentrations of active agent) can be assessed and may contribute to selectivity and activity. We better relate results from model studies to biological cases and improve our understanding of processes impossible to study directly, such as membrane-mediated antimicrobial action.

Matrix Metalloproteinase-Assisted Triggered Release of Liposomal Contents

We offer a novel methodology for formulating liposomes by incorporating sequence-specific collagen-mimetic peptides such that they are specifically "uncorked" by a matrix metalloproteinase, MMP-9. By encapsulating carboxyfluorescein (as a self-quenching fluorescent dye), we demonstrate that the time-dependent release of the dye from liposomes is due to the specific enzymatic cleavage of the surface-exposed collagen-mimetic peptides. The specificity of such cleavage is attested by the fact that the liposomal "uncorking" and their content release occur only by MMP-9 and not by a general proteolytic enzyme, trypsin, despite the fact that the collagen mimetic peptides contain the trypsin cleavage site. The mechanistic details underlying the formulations of liposomes and their enzyme-selective "uncorking" and content release are discussed. Arguments are presented that such liposomes can be fine-tuned to serve as the drug delivery vehicles for the detection and treatment of various human diseases, which occur due to the overexpression of a variety of pathogenic matrix metalloproteinases.

Detection of polychlorinated biphenyls using an antibody column in tandem with a fluorescent liposome column: Effect of albumin on phospholipase A 2-catalyzed membrane leakage

Phospholipase A 2 (PLA 2)-catalyzed membrane leakage can be detected by immobilized liposomes containing a self-quenching fluorescent dye, 3,3-bis[ N, N-di(carboxymethyl)aminomethyl]fluorescein (calcein). This enzymatic reaction was applied as signal amplification for biosensor detection of low concentrations of polychlorinated biphenyls (PCBs). In order to increase the fluorescent signal for improvement of PCBs detection, the effect of BSA on optimal lipid composition for PLA 2-catalyzed membrane leakage from fluorescent liposomes has been investigated in this report. Various kinds of calcein-entrapped liposomes were immobilized in Sephacryl S1000 gel beads using avidin–biotin binding. In a contrast, free calcein was removed by size exclusion chromatography on Sephacryl S300 for free liposome suspensions. The PLA 2-catalyzed membrane leakage was detected both in these gel-bead-immobilized liposomes and in free liposome suspensions. In both systems, the fluorescent release from the liposomes by PLA 2 hydrolytic action significantly increased with increasing albumin concentration. The most rapid and greatest membrane leakage by PLA 2 hydrolysis was found in anionic liposomes in the presence of albumin, both in free liposome suspensions and gel-bead-immobilized liposomes. Finally, the stabilities of various free liposomes and gel-bead-immobilized liposomes were monitored. Immobilized 1-palmitoy-2-oleoylphosphatidylcholine (POPC)/1-palmitoy-2-oleoylphosphatidylglycerol (POPG) liposome gel was chosen due to its excellent stability and large dye leakage by PLA 2. A concentration of PCBs as low as 0.1 ng/mL was detectable using this tandem column system.

Effect of lipid composition on phospholipase A2‐catalyzed membrane leakage in immobilized liposomes: Sensitization for polychlorinated biphenyls detection with antibody affinity column tandem with fluorescent liposome column

Phospholipase A(2) (PLA(2))-catalyzed membrane leakage can be detected by immobilized liposomes containing a self-quenching fluorescent dye, calcein, on an open column using off-line analysis with a fluorescent spectrophotometer. The calcein release was found to be affected by the pH value, incubation time, and liposome compositions. The fluorescent signal from the negatively charged liposomes hydrolyzed by PLA(2) was 5 times higher than that from neutral liposomes. We utilized this enzymatic reaction to amplify signal to detect polychlorinated biphenyls (PCBs). To achieve this goal, we conjugated an analogue of PCB, 3,4-dichloroaniline, to PLA(2). The competitive immunoreaction between the 3,4-dichloroaniline-PLA(2) conjugate and PCB samples on the anti-PCB antibody column caused the release of the bound PLA(2) conjugates in proportion to the PCB concentration. The released PLA(2) conjugates was then passed through the tandem fluorescent liposome column causing release of fluorescent dye from the liposomes. Therefore, the signal of immunocompetitive assay was amplified on the fluorescent liposome column. The tandem column system achieves a high sensitivity by detecting the PCB concentration as low as 0.5 ng/mL in less than 20 min. It has great potential in detecting other pollutants, and has been used for sensitive immunoassays.

Peg-Containing Surfactants Enhance the Ultrasonic Permeabilizability of Liposomes

AbstractThe susceptibility of phosphatidylcholine liposomes to rupture by ultrasound was investigated. The liposomes were treated with (or had incorporated) a surface active dopant. The dopants studied here all contain polymers or oligomers of ethylene glycol as their hydrophilic “headgroup” component. All dopants strongly increased the ultrasonic permeabilizability of liposomes, as measured by the rate of release of a self-quenching fluorescent dye, at concentrations that caused no increase in permeability in the absence of ultrasound. The surface active dopants reached maximal effectiveness at about 1% of their critical micelle concentrations (CMCs). Using the roughly inverse relationship between CMC and membrane partition coefficient, we estimate that these maximally effective concentrations correspond approximately to the onset of headgroup contact among the surfactants in the membrane. Two surfactants, a PEG-lipid and a Pluronic triblock polymer, can be irreversibly incorporated into liposomes to give formulations that could in principle be used as drug delivery vehicles. The Pluronic polymer offers the possibility of additional temperature responsivity, owing to its highly temperaturedependent CMC.

Phospholipase A2-Catalyzed Membrane Leakage Studied by Immobilized Liposome Chromatography with Online Fluorescent Detection

Unilamellar liposomes composed of phosphatidylcholine with an entrapped self-quenching fluorescent dye, calcein, were immobilized in chromatographic gel beads by avidin–biotin binding. Bee venom phospholipase A2 (PLA2) was applied in a small amount onto the immobilized liposome column. The release of calcein from the immobilized liposomes resulting from the catalyzed hydrolysis of the phospholipids was detected online by immobilized liposome chromatography (ILC) using a flow fluorescent detector. The PLA2-catalyzed membrane leakage of the immobilized liposomes as studied with ILC was found to be affected by the gel pore size used for immobilization, by liposome size, and as expected by the concentration of calcium, but was unaffected by the flow rate of ILC. The largest PLA2-induced calcein release from the liposome column was detected on large unilamellar liposomes immobilized on TSK G6000PW or Sephacryl S-1000 gel in the presence of 1 mM Ca2+ in the aqueous mobile phase. Comparison with the PLA2-catalyzed membrane leakage in free liposome suspensions, we conclude that the fluorescent leakage from liposomes hydrolyzed by PLA2 can be rapidly and sensitively detected by ILC runs using large amount of immobilized liposomes with entrapped fluorescent dye.

Quantitative studies on the melittin-induced leakage mechanism of lipid vesicles.

We have investigated, both experimentally and theoretically, the efflux of carboxyfluorescein (a self-quenching fluorescent dye) from vesicles of different sizes and lipid species (POPC, DOPC) after having added the bee venom peptide melittin. This comprises quantitative analyses regarding the extent of lipid-associated peptide, the mode as well as the temporal progress of dye release and the possible leakage mechanism. Our results indicate a graded efflux characterized by a single-pore retention factor reflecting the formation of pores whose lifetimes are rather small (millisecond range). The observed fluorescence signal arising from the dequenching of effluent dye has been converted to the number of pore openings over the course of time. All the resulting curves exhibit a pronounced slowing down of the pore formation rate revealing two distinct relaxation steps at about 20 and 200 s, respectively, being largely independent of vesicle type and peptide to lipid ratio. The pore formation rate itself increases in proportion to the amount of membrane bound peptide. We give a quantitative account of our experimental findings based on a novel reaction scheme applicable to any of our various liposome systems. It implies that the pore formation rate is controlled by a passage through two intermediate monomeric peptide states. These states are thought to become well populated in the initial stage of lipid bilayer perturbation, but would practically die out after some time owing to a restabilization of the membrane system.

Mechanical properties of vesicles. I. Coordinated analysis of osmotic swelling and lysis

To determine how transmembrane osmotic gradients perturb the structure and dynamics of biological membranes, we examined the effects of medium dilution on the structures of osmolyte-loaded lipid vesicles. Our preparations were characterized by dynamic light scattering (DLS) and nuclear magnetic resonance (NMR) spectroscopies. Populations of Escherichia coli phosphatidylethanolamine (PE) or dioleoylphosphatidylglycerol (DOPG) vesicles prepared by the pH jump technique were variable and polymodal in size distribution. Complex and variable structural changes occurred when PE vesicles were diluted with hypotonic buffer. Such vesicles could not be used as model systems for the analysis of membrane mechanical properties. NaCl-loaded, DOPG vesicles prepared by extrusion through 100 nm (diameter) pores were reproducible and monomodal in size distribution and unilamellar, whereas those prepared by extrusion through 200-, 400-, or 600-nm pores were variable and polymodal in size distribution and/or multilamellar. Time and pressure regimes associated with osmotic lysis of extruded vesicles were defined by monitoring release of carboxyfluorescein, a self-quenching fluorescent dye. Corresponding effects of medium dilution on vesicle structure were assessed by DLS spectroscopy. These experiments and the accompanying analysis (Hallett, F.R., J. Marsh, B.G. Nickel, and J.M. Wood. 1993. Biophys. J. 64:000-000) revealed conditions under which vesicles are expected to reside in a consistently strained state.

Kinetics of melittin induced pore formation in the membrane of lipid vesicles

We have investigated the permeabilization of POPC unilamellar vesicle bilayers upon the addition of melittin. This process was measured in an early time range of a few minutes by means of monitoring the release of an entrapped marker, the self-quenching fluorescent dye car☐yfluorescein. Pore formation is indicated by an apparent ‘all-or-none’ efflux out of individual vesicles and a higher than linear dependence on melittin concentration. Applying a recently developed evaluation procedure, the data are readily converted into the gross number of pores per vesicle formed within the elapsed measuring time t. The results can be generally described in terms of a fast initial rate of pore formation that slows down to a much lower value after a period of about 1 to 2 minutes, following a single exponential time course. The three rate parameters involved are shown to be power functions of the concentration of melittin that is actually associated with the vesicle membrane. These findings are in excellent quantitiative agreement with a proposed scheme of reaction steps where the formation of lipid associated peptide dimers becomes rate determining once an initial fast deposit is exhausted.

Interactions of target-sensitive immunoliposomes with herpes simplex virus. The foundation of a sensitive immunoliposome assay for the virus.

Interactions between target-sensitive (TS) immunoliposomes and herpes simplex virus (HSV) were investigated. Target sensitivity of phosphatidylethanolamine (PE) immunoliposomes is a result of the ability of acylated monoclonal anti-HSV glycoprotein D (gD) to stabilize the bilayer phase of PE, whereas by itself, PE does not form stable liposomes (Ho, R. J. Y., Rouse, B. T., and Huang, L. (1986) Biochemistry 25, 5500-5506). Upon binding of these immunoliposomes to HSV antigen-containing gD, destabilization of PE immunoliposomes was observed. By encapsulating either a self-quenching fluorescent dye, calcein, or alkaline phosphatase inside the liposomal compartment, the HSV-induced destabilization of TS immunoliposomes was shown to be target-specific. Neither Sendai, Semliki Forest, nor Sindbis virus could significantly destabilize the TS immunoliposomes. Moreover, HSV-induced liposome destabilization could be inhibited by free anti-gD (the same antibody used in TS immunoliposomes) but not by monoclonal anti-HSV glycoprotein B, indicating that the interaction was antigen-specific. Destabilization could also be induced by binding to truncated gD (tgD), but only when in a multivalent form immobilized on latex beads. Truncated gD is a cloned, 312-amino acid fragment of HSV-gD that lacks the transmembrane segment. Preincubation of soluble tgD with the TS immunoliposomes failed to induce destabilization and, in addition, abolished the tgD-bead-induced destabilization. This finding strongly indicated that multivalent binding is essential for TS immunoliposome destabilization. Using alkaline phosphatase encapsulated in the liposomes, TS immunoliposomes could be used to detect HSV in fluid phase with 50% signal recorded at 5 microliters of 3.2 x 10(3) pfu/ml; at least 10-fold more sensitive than the standard double-antibody sandwich enzyme-linked immunosorbent assay. The interactions described here may be useful in designing a homogeneous and sensitive immunoliposome assay.

Thermodynamic and phase characterizations of phosphatidylethanolamine and ganglioside G D1a mixtures

By employing diphenylhexatriene steady-state fluorescence anisotropy, pyrenedecanoic acid excimer formation, and high sensitivity scanning calorimetry we have demonstrated that the liposomes containing phosphatidylethanolamine (PE) and various mole fractions of ganglioside G D1a had a gel-liquid crystalline phase transition between 15 and 25° C. Calorimetric measurements indicated that these phase transitions were broad and centered between 17 and 21° C. The enthalpy change of the transition was linearly dependent on the ganglioside concentration up to 10.0 mol% and plateaued between 11.4–16.2 mol%. The high enthalpy change (37 kcal/mol of G D1a added into the PE bilayer) indicates the existence of PE-G D1a complex structure in the liposomal membrane. It is proposed that semi-fluid domains containing six PE and one ganglioside molecule are present in the PE-G D1a membranes at temperatures above gel-liquid crystalline phase transition. The Sendai virus induced leakage of PE-G D1a liposomes has been investigated by using an entrapped, self-quenching fluorescent dye, calcein. The leakage rate was dependent on the mole fraction of ganglioside G D1a and was maximal at 6.3 mol%. Arrhenius plots of the leakage rates showed breaks in the 20–25° C temperature range, which correspond to the gel-liquid crystalline phase transition of the target liposomes. These data suggest that the rate of Sendai virus-induced leakage can be regulated via fluidity modulation by changing the PE to G D1a ratio at constant temperatures.

Sendai virus induced leakage of liposomes containing gangliosides.

Sendai virus induced liposome leakage has been studied by using liposomes containing a self-quenching fluorescent dye, calcein. The liposomes used in this study were prepared by a freeze and thaw method and were composed of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine (1:2.60:1.48 molar ratio) as well as various amounts of gangliosides and cholesterol. The leakage rate was calculated from the fluorescence increment as the entrapped calcein leaked out of the liposomal compartment and was diluted into the media. It was shown that the target liposome leakage was virus dose dependent. Trypsin-treated Sendai virus in which the F protein had been quantitatively removed did not induce liposome leakage, indicating that the leakage was a direct result of F-protein interaction with the target bilayer membrane. The activation energy of this process was approximately 12 kcal/mol below 17 degrees C and approximately 25 kcal/mol above 17 degrees C. Gangliosides GM1, GD1a, and GT1b could serve as viral receptor under appropriate conditions. Liposome leakage showed a bell-shaped curve dependence on the concentration of ganglioside in the liposomes. No leakage was observed if the ganglioside content was too low or too high. Inclusion of cholesterol in the liposome bilayer suppressed the leakage rate of liposomes containing GD1a. It is speculated that the liposome leakage is a consequence of fusion between Sendai virus and liposomes.