Characterization of changes in the viscosity of lipid membranes with the molecular rotor FCVJ

Membrane viscosity is a key parameter in cell physiology, cell function, and cell signaling. The most common methods to measure changes in membrane viscosity are fluorescence recovery after photobleaching (FRAP) and fluorescence anisotropy. Recent interest in a group of viscosity sensitive fluorophores, termed molecular rotors, led to the development of the highly membrane-compatible (2-carboxy-2-cyanovinyl)-julolidine farnesyl ester (FCVJ). The purpose of this study is to examine the fluorescent behavior of FCVJ in model membranes exposed to various agents of known influence on membrane viscosity, such as alcohols, dimethyl sulfoxide (DMSO), cyclohexane, cholesterol, and nimesulide. The influence of key agents (propanol and cholesterol) was also examined using FRAP, and backcalculated viscosity change from FCVJ and FRAP was correlated. A decrease of FCVJ emission was found with alcohol treatment (with a strong dependency on the chain length and concentration), DMSO, and cyclohexane, whereas cholesterol and nimesulide led to increased FCVJ emission. With the exception of nimesulide, FCVJ intensity changes were consistent with expected changes in membrane viscosity. A comparison of viscosity changes computed from FRAP and FCVJ led to a very good correlation between the two experimental methods. Since molecular rotors, including FCVJ, allow for extremely easy experimental methods, fast response time, and high spatial resolution, this study indicates that FCVJ may be used to quantitatively determine viscosity changes in phospholipid bilayers.     

Characterization of changes in the viscosity of lipid membranes with the molecular rotor FCVJ

Membrane viscosity is a key parameter in cell physiology, cell function, and cell signaling. The most common methods to measure changes in membrane viscosity are fluorescence recovery after photobleaching (FRAP) and fluorescence anisotropy. Recent interest in a group of viscosity sensitive fluorophores, termed molecular rotors, led to the development of the highly membrane-compatible (2-carboxy-2-cyanovinyl)-julolidine farnesyl ester (FCVJ). The purpose of this study is to examine the fluorescent behavior of FCVJ in model membranes exposed to various agents of known influence on membrane viscosity, such as alcohols, dimethyl sulfoxide (DMSO), cyclohexane, cholesterol, and nimesulide. The influence of key agents (propanol and cholesterol) was also examined using FRAP, and backcalculated viscosity change from FCVJ and FRAP was correlated. A decrease of FCVJ emission was found with alcohol treatment (with a strong dependency on the chain length and concentration), DMSO, and cyclohexane, whereas cholesterol and nimesulide led to increased FCVJ emission. With the exception of nimesulide, FCVJ intensity changes were consistent with expected changes in membrane viscosity. A comparison of viscosity changes computed from FRAP and FCVJ led to a very good correlation between the two experimental methods. Since molecular rotors, including FCVJ, allow for extremely easy experimental methods, fast response time, and high spatial resolution, this study indicates that FCVJ may be used to quantitatively determine viscosity changes in phospholipid bilayers.     

A novel approach to blood plasma viscosity measurement using fluorescent molecular rotors

Molecular rotors, a group of fluorescent molecules with viscosity-dependent quantum yield, were tested for their suitability to act as fluorescence-based plasma viscometers. The viscosity of samples of human plasma was modified by the addition of pentastarch (molecular mass 260 kDa, 10% solution in saline) and measured with a Brookfield viscometer. Plasma viscosity was 1.6 mPa x s, and the mixtures ranged up to 4.5 mPa x s (21 degrees C). The stimulated light emission of the molecular rotors mixed in the plasma samples yielded light intensity that was nonoverlapping and of significantly different intensity for viscosity steps down to 0.3 mPa x s (n = 5, P < 0.0001). The mathematical relationship between intensity (I) and viscosity (eta) was found to be eta = (kappaI)(nu). After calibration and scaling the fluorescence based measurement had an average deviation versus the conventional viscometric measurements that was <1.8%. These results show the suitability of molecular rotors for fast, low-volume biofluid viscosity measurements achieving accuracy and precision comparable to mechanical viscometers.     

Phospholipid-bound molecular rotors: synthesis and characterization

Molecular rotors are fluorescent molecules with a viscosity-sensitive quantum yield that are often used to measure viscosity changes in cell membranes and liposomes. However, commercially available molecular rotors, such as DCVJ (1) do not localize in cell membranes but rapidly migrate into the cytoplasm leading to unreliable measurements of cell membrane viscosity. To overcome this problem, we synthesized molecular rotors covalently attached to a phospholipid scaffold. Attaching the rotor group to the hydrophobic end of phosphatidylcholine (PC) did not affect the rotor's viscosity sensitivity and allowed adequate integration into artificial bilayers as well as complete localization in the plasma membrane of an endothelial cell line. Moreover, these new rotors enabled the monitoring of phospholipid transition temperature. However, attachment of the rotor groups to the hydrophilic head of the phospholipid led to a partial loss of viscosity sensitivity. The improved sensitivity and exclusive localization in the cell plasma membrane exhibited by the phospholipid-bound molecular rotors suggest that these probes can be used for the study of membrane microviscosity.     

Environment-sensitive behavior of fluorescent molecular rotors

Molecular rotors are a group of fluorescent molecules that form twisted intramolecular charge transfer (TICT) states upon photoexcitation. When intramolecular twisting occurs, the molecular rotor returns to the ground state either by emission of a red-shifted emission band or by nonradiative relaxation. The emission properties are strongly solvent-dependent, and the solvent viscosity is the primary determinant of the fluorescent quantum yield from the planar (non-twisted) conformation. This viscosity-sensitive behavior gives rise to applications in, for example, fluid mechanics, polymer chemistry, cell physiology, and the food sciences. However, the relationship between bulk viscosity and the molecular-scale interaction of a molecular rotor with its environment are not fully understood. This review presents the pertinent theories of the rotor-solvent interaction on the molecular level and how this interaction leads to the viscosity-sensitive behavior. Furthermore, current applications of molecular rotors as microviscosity sensors are reviewed, and engineering aspects are presented on how measurement accuracy and precision can be improved.     

A molecular rotor as viscosity sensor in aqueous colloid solutions

BACKGROUND: Molecular rotors exhibit viscosity-dependent quantum yield, allowing non-mechanical determination of fluid viscosity. We analyzed fluorescence in the presence of viscosity-modulating macromolecules several orders of magnitude larger than the rotor molecule. METHOD OF APPROACH: Fluorescence of aqueous starch solutions with a molecular rotor in solution was related to viscosity obtained in a cone-and-plate viscometer. RESULTS: In dextran solutions, emission intensity was found to follow a power-law relationship with viscosity. Fluorescence in hydroxyethylstarch solutions showed biexponential behavior with different exponents at viscosities above and below 1.5 mPa s. Quantum yield was generally higher in hydroxyethylstarch than in dextran solutions. The power-law relationship was used to backcalculate viscosity from intensity with an average precision of 2.2% (range of -5.5% to 5.1%). CONCLUSIONS: This study indicates that hydrophilic molecular rotors are suitable as colloid solution viscosity probes after colloid-dependent calibration.     

Membrane electropermeabilization effects of frequency and membrane surface order on liposome leakage

Liposomes containing fluorescence marker were exposed to an alternating electric field of 80 V peak to peak square electric waves at different frequencies 0.01, 1, and 100 kHz to perturb the liposome permeation. The efflux of fluorescence dye after application of the electric field was measured by recording the fluorescence emission due to the complex formation reaction between the fluorescence dye and calcium ions in the bulk medium solution. Two independent sets of experiments were conducted: 1) calcium ions were present during electropulsation; and 2) they were added after electric field application. Two parameters, fluorescence emission intensity and increment of temperature of the solution in the chamber, were studied. The effect of membrane surface order on the fluorescence dye leakage from the liposomes was studied by addition of urea at threshold concentration before the liposomes sealed. The data demonstrate the existence of frequency dependency window at 1 kHz. Furthermore, the data were interpreted according to the theory of interactions of electromagnetic fields with highly polarized and deformed materials such as liposome particles. The urea caused an enhancement of the fluorescence dye leakage at frequency of 100 kHz. This effect could be explained as a decrease of the membrane binding rigidity due to the disordering effect of urea on the membrane lipid surface. Our conclusion is that the frequency and the membrane surface order are additional parameters that influence the processes of membrane electropermeabilization.     

三氯乙烯对脂质体膜通透性的影响及其破膜效应

本文通过测定脂质体膜对水的通透性及脂质体对被包裹血红蛋白的释放率,研究了三氯乙烯对脂质体的作用.观察到在低浓度时,三氯乙烯能增加蛋卵磷脂脂质体的通透性,随着浓度的增加有破膜释放的效应.     

Effects of solvent polarity and solvent viscosity on the fluorescent properties of molecular rotors and related probes

Fluorescent molecular rotors belong to a group of twisted intramolecular charge transfer complexes (TICT) whose photophysical characteristics depend on their environment. In this study, the influence of solvent polarity and viscosity on several representative TICT compounds (three Coumarin derivatives, 4,4-dimethylaminobenzonitrile DMABN, 9-(dicyanovinyl)-julolidine DCVJ), was examined. While solvent polarity caused a bathochromic shift of peak emission in all compounds, this shift was lowest in the case of molecular rotors. Peak intensity was influenced strongly by solvent viscosity in DMABN and the molecular rotors, but polarity and viscosity influences cannot be separated with DMABN. Coumarins, on the other hand, did not show viscosity sensitivity. This study shows the unique suitability of molecular rotors as fluorescent viscosity sensors.     

Effect of lipid composition changes on carbocyanine dye fluorescent response

Egg yolk phosphatidyl choline liposomes containing variable amounts of phosphatidyl ethanolamine, phosphatidyl inositol or phosphatidyl serine demonstrated important variations in the fluorescence of 3.3' dipropylthiodicarbocyanine. When the membrane contained no cholesterol, fluorescence was not correlated with membrane fluidity as measured by diphenyl hexatriene polarization. Increasing cholesterol concentration in valinomycin containing liposome membranes decreased the potassium induced apparent membrane potential and prevented sorption of dye to the membrane. Discontinuity in the apparent potential occurred at 30 mol% cholesterol but could not be correlated with changes in microviscosity. These results indicate that great care should be taken when correlating rapid variations of fluorescence to changes in membrane potential. We propose that changes in phospholipid metabolism could well explain fluorescent changes when monitoring the fluorescence of cyanine dye molecules sorbed to biological membranes.     

Interaction of fluorescent molecular rotors with blood plasma proteins

Many disease states have associated blood viscosity changes. Molecular rotors, fluorescent molecules with viscosity sensitive quantum yields, have recently been investigated as a new method for biofluid viscosity measurement. Current viscometer measurements are complicated by proteins adhering to surfaces and forming air-surface layers. It is unknown at this time what effects proteins may have on biofluid viscosity measurements using molecular rotors. To answer this question, binding affinities to blood plasma proteins were investigated by equilibrium dialysis for four hydrophilic molecular rotors. Aqueous solutions of 9-[(2-cyano-2-hydroxy-carbonyl)vinyl]julolidine (CCVJ) and three derivatives were prepared and dialyzed against solutions of bovine source albumin, fibrinogen and immunoglobulin G approximating normal physiologic concentrations and fresh-frozen human plasma. After equilibration, dye concentration on each side of the dialysis membrane was assessed by spectrophotometry. The relative binding affinity of the four dyes to the proteins and to the plasma was compared. Affinity of all dyes was highest for albumin. The bound dye fraction showed little change in relation to protein concentration in the physiological concentration range. Diol, the most hydrophilic molecular rotor tested showed the lowest affinity for albumin. This study indicates that hydrophilic molecular rotors are well-suited for biofluid viscosity measurement.     

Generic liposome reagent for immunoassays

We have derivatized liposomes with antibodies by using avidin to crosslink biotinylated phospholipid molecules in the liposome membranes with biotinylated antibody molecules. A comparison of the biotin binding activity of avidin in solution and avidin associated with liposomes shows that avidin bound to biotinylated phospholipid in liposome membranes retains full binding activity for additional biotin molecules. Changes in the fluorescence spectrum of avidin have been used to characterize the binding capacity of avidin for biotin in solution, and change in intensity of light scattered due to aggregation of liposomes was used to measure the biotin binding activity of avidin associated with liposomes. Relative amounts of the biotinylated phospholipid, avidin, and biotinylated antibody have been optimized to produce stable liposomes which are derivatized with up to 1.7 nmol of antibody/mumol of lipid. These derivatized liposomes are highly reactive to immunospecific aggregation in the presence of multivalent antigen. A linear increase in light scattering was recorded between 1 and 10 pmol of antigen. This work shows that liposomes containing biotinylated phospholipid can be a successful generic reagent for immunoassays.     

Optical response of the indicator chlortetracycline to membrane potential

Chlortetracycline is a fluorescent, Ca2+ indicator commonly used to monitor the internal Ca2+ concentration of membrane vesicles and organelles. We have found that the intensity of chlortetracycline fluorescence in the presence of Ca2(+)-loaded liposomes is dependent on the membrane potential of the vesicles as well as the intravesicular Ca2+ concentration. The fluorescence of chlortetracycline was lower when an inside-negative membrane potential was placed across the liposome membrane. Since chlortetracycline diffuses across the membrane in the zwitterionic form, the distribution of chlortetracycline across the membrane should not be strongly dependent on the membrane potential. However, because the proton permeability of phospholipid vesicles is relatively high, the intravesicular proton concentration is dependent on the membrane potential. The binding of Ca2+ to chlortetracycline is dependent on pH in the range of pH 6 to pH 8. Therefore, changes in the intravesicular pH as a result of a change in the membrane potential causes relatively large changes in the chlortetracycline fluorescence signal even when there isn't a change in the Ca2+ concentration.     

Dual-color fluorescence-burst analysis to study pore formation and protein-protein interactions

Dual-color fluorescence-burst analysis (DCBFA) enables to study leakage of fluorescently labeled (macro) molecules from liposomes that are labeled with a second, spectrally non-overlapping fluorophore. The fluorescent bursts that reside from the liposomes diffusing through the focal volume of a confocal microscope will coincide with those from the encapsulated size-marker molecules. The internal concentration of size-marker molecules can be quantitatively calculated from the fluorescence bursts at a single liposome level. DCFBA has been successfully used to study the effective pore-size of the mechanosensitive channel of large-conductance MscL and the pore-forming mechanism of the antimicrobial peptide melittin from bee venom. In addition, DCFBA can be used to quantitatively measure the binding of proteins to liposomes and to membrane proteins. In this paper, we provide an overview of the method and discuss the experimental details of DCFBA.     

An aryleneethynylene fluorophore for cell membrane staining

The use of an amphiphilic aryleneethynylene fluorophore as a plasma membrane marker in fixed and living mammalian cells and liposome model systems is demonstrated. We show here that the optical properties of the novel dye are almost independent on pH, in the range 5.0-8.0. Spectroscopic characterization performed on unilamellar liposomes ascertained that the fluorescence intensity of the aryleneethynylene fluorophore greatly increases after incorporation in lipidic membranes. Experiments performed on different mammalian cells demonstrated that the novel membrane marker exhibits fast staining and a good photostability that make it a suitable tool for live cell imaging. Importantly, the aryleneethynylene fluorophore was also shown to be a fast and reliable blue membrane marker in classical multicolor immunofluorescence experiments. This study adds new important findings to the recent exploitation of the wide class of aryleneethynylene molecules as luminescent markers for biological investigations.     

Time-resolved fluorescence microscopy.

In fluorescence microscopy, the fluorescence emission can be characterised not only by intensity and position, but also by lifetime, polarization and wavelength. Fluorescence lifetime imaging (FLIM) can report on photophysical events that are difficult or impossible to observe by fluorescence intensity imaging, and time-resolved fluorescence anisotropy imaging (TR-FAIM) can measure the rotational mobility of a fluorophore in its environment. We compare different FLIM methods: a chief advantage of wide-field time-gating and phase modulation methods is the speed of acquisition whereas for time-correlated single photon counting (TCSPC) based confocal scanning it is accuracy in the fluorescence decay. FLIM has been used to image interactions between proteins such as receptor oligomerisation and to reveal protein phosphorylation by detecting fluorescence resonance energy transfer (FRET). In addition, FLIM can also probe the local environment of fluorophores, reporting, for example, on the local pH, refractive index, ion or oxygen concentration without the need for ratiometric measurements.     

Characterization of a New Series of Fluorescent Probes for Imaging Membrane Order

Visualization and quantification of lipid order is an important tool in membrane biophysics and cell biology, but the availability of environmentally sensitive fluorescent membrane probes is limited. Here, we present the characterization of the novel fluorescent dyes PY3304, PY3174 and PY3184, whose fluorescence properties are sensitive to membrane lipid order. In artificial bilayers, the fluorescence emission spectra are red-shifted between the liquid-ordered and liquid-disordered phases. Using ratiometric imaging we demonstrate that the degree of membrane order can be quantitatively determined in artificial liposomes as well as live cells and intact, live zebrafish embryos. Finally, we show that the fluorescence lifetime of the dyes is also dependent on bilayer order. These probes expand the current palate of lipid order-sensing fluorophores affording greater flexibility in the excitation/emission wavelengths and possibly new opportunities in membrane biology.     

Ligand self-association at the surface of liposomes: A complication during equilibrium-binding studies

Daunomycin and carminomycin, two anthracycline antibiotics known to bind phospholipid bilayers, appear to self-associate at the surface of liposomes at high bound drug/lipid ratios (r). Fluorescence intensity, lifetime, and anisotropy measurements have been used to monitor the equilibrium binding of these drugs to small unilamellar solid-phase dipalmitoylphosphatidylcholine vesicles. Association of an anthracycline with excess liposome (low r) resulted in an increase in both the observed intensity and the fluorescence lifetime. At low vesicle concentrations (high r), a decrease in the total emission intensity was observed which was not paralleled by the excited-state lifetime. The data from these experiments are consistent with the formation of nonfluorescent anthracycline complexes at the surface of liposomes. Such ligand self-association is a potential complication in any studies on the interaction of amphipathic molecules with liposomes conducted at high r values. Because ligand self-association limits the collection of binding data over certain concentration ranges, this consequently results in greater uncertainty in the determination of the maximum value of r (n) in equilibrium binding studies.     

Cooperative partition model of nystatin interaction with phospholipid vesicles

Nystatin is a membrane-active polyene antibiotic that is thought to kill fungal cells by forming ion-permeable channels. In this report we have investigated nystatin interaction with phosphatidylcholine liposomes of different sizes (large and small unilamellar vesicles) by time-resolved fluorescence measurements. Our data show that the fluorescence emission decay kinetics of the antibiotic interacting with gel-phase 1,2-dipalmitoyl-sn-glycero-3-phosphocholine vesicles is controlled by the mean number of membrane-bound antibiotic molecules per liposome, . The transition from a monomeric to an oligomeric state of the antibiotic, which is associated with a sharp increase in nystatin mean fluorescence lifetime from approximately 7-10 to 35 ns, begins to occur at a critical concentration of 10 nystatin molecules per lipid vesicle. To gain further information about the transverse location (degree of penetration) of the membrane-bound antibiotic molecules, the spin-labeled fatty acids (5- and 16-doxyl stearic acids) were used in depth-dependent fluorescence quenching experiments. The results obtained show that monomeric nystatin is anchored at the phospholipid/water interface and suggest that nystatin oligomerization is accompanied by its insertion into the membrane. Globally, the experimental data was quantitatively described by a cooperative partition model which assumes that monomeric nystatin molecules partition into the lipid bilayer surface and reversibly assemble into aggregates of 6 +/- 2 antibiotic molecules.     

Preparation and characterization of immunoliposomes for targeting of antiviral agents

Antibodies specific to avian myeloblastosis virus envelope glycoprotein gp80 were raised. Immunoliposomes were prepared using anti-avian myeloblastosis virus envelope glycoprotein gp80 antibody. The antibody was palmitoylated to facilitate its incorporation into lipid bilayers of liposomes. The fluorescence emission spectra of palmitoylated IgG have exhibited a shift in emission maximum from 330 to 370 nm when it was incorporated into the liposomes. At least 50% of the incorporated antibody molecules were found to be oriented towards the outside in the liposomes. The average size of the liposome was found to be 300 A, and on an average, 15 antibody molecules were shown to be present in a liposome. When adriamycin encapsulated in immunoliposomes was incubated in a medium containing serum for 72 h, about 75% of the drug was retained in liposomes. In vivo localization studies, revealed an enhanced delivery of drug encapsulated in immunoliposomes to the target tissue, as compared to free drug or drug encapsulated in free liposomes. These data suggest a possible use of the drugs encapsulated in immunoliposomes to deliver the drugs in target areas, thereby reducing side effects caused by antiviral agents.