Triglycerides Stabilize Water/Organic Interfaces of Changing Area via Conformational Flexibility.

The role of triglycerides (TGs) in both natural and synthetic biological membranes has long been the subject of study, involving metabolism, disease, and colloidal synthesis. TGs have been found to be critical components for successful liposomal encapsulation via a water/oil/water double emulsion, which this work endeavors to explain. TGs can occupy multiple positions in biological membranes. The glycerol backbone can reside at the water/organic interface, adjacent to phospholipid headgroups ("m" conformation), typically with relatively low (<3%) solubility. The glycerol backbone can also occupy hydrophobic regions, where it is isolated from water ("h" or "oil" conformation). This can occur in either midmembrane positions or phospholipid-coated lipid droplets (LDs). These conformations can be distinguished using 13C-nuclear magnetic resonance spectroscopy (NMR), which determines the degree of hydration of the TG backbone. Using this method, it was revealed that TGs transition from "m" to "h" conformation as the organic solvent is removed via evaporation. A new transitional TG backbone position has been identified with a level of hydration between "m" and "h". These results suggest that TGs can temporarily coat and stabilize the large water/organic interfaces present after emulsification. As the organic solvent is removed and interfaces shrink, the TGs recede into midmembrane spaces or bud off into LDs, which are confirmed via transmission electron microscopy (TEM) and can be removed via centrifugation. Encapsulation efficiency is found to be inversely related to both the saturation and length of the TG acyl chains, indicating that membrane fluidization is a key property arising from the presence of TGs. Beyond clarification of a mechanism for high-efficiency liposomal encapsulation, these results implicate TGs as components that are able to stabilize biological membrane transitions involving a changing interfacial area and curvature. This role for TGs may be of use in the formulation of drug delivery systems as well as in the investigation of membrane transitions in life sciences.

Enhanced antimicrobial effect of ultrasound by the food colorant Erythrosin B.

The synergistic combination of the food colorant Erythrosin B (E-B, FD&C 3) (0, 25, and 50µM) and low-frequency ultrasound (20kHz, 0.86-0.90WmL-1) was evaluated against Listeria innocua. Although E-B was antibacterial by itself, the inactivation rate significantly increased in a concentration-dependent manner upon exposure to ultrasound and followed a sigmoidal behavior. The enhanced antimicrobial effect of E-B in the presence of ultrasound can be explained in part from a microbubble disappearance study in which it was confirmed that the presence of E-B enhances inertial cavitation, thereby enhancing the antimicrobial effect of ultrasound. The inactivation rate in a sequential treatment, where L. innocua was sonicated for 4min followed by exposure to 25µM Erythrosin B, was comparable to that obtained by the simultaneous treatment, indicating complementary mechanisms of inactivation. Fluorescence microscopy showed attachment of E-B to the cells, which may explain its intrinsic antimicrobial property. Other mechanism may include the confirmed decrease in the cavitation threshold of water by addition of E-B, resulting in more effective cavitation. The study offers a proof-of-concept of a novel approach to complement ultrasound treatment for enhanced microbial inactivation.

Investigating the spatial extent of acoustically activated echogenic liposomes.

The purpose of this work was to investigate the ability of bubbles entrapped within echogenic liposomes (ELIP) to serve as foci for cavitational events that would cause leakage in neighboring non-echogenic liposomes (NELIP). Previous studies have shown that entrapping bubbles into liposomes increases ultrasound-mediated leakage of hydrophilic components at ultrasound settings known to induce inertial cavitation, specifically 20kHz and 2.2W/cm2. Using tone-burst approach and pulse repetition frequency of 10Hz would bring this intensity level to the one accepted (220mW/cm2) in clinical imaging. Mixed populations of ELIP and NELIP were simultaneously exposed to ultrasound at varying ratios to examine the effect of ELIP concentration on release of a hydrophilic dye, calcein, from NELIP. Calcein release from NELIP was observed to be independent of ELIP concentration, suggesting that the release enhancement from echogenicity is strictly a localized event. Additionally, it was observed that the release mechanisms independent of echogenicity were active for the duration of experiment whereas those associated with echogenicity were active for only the initial 1-2min.

Hydrophobic drug concentration affects the acoustic susceptibility of liposomes.

The purpose of this study was to investigate the effect of encapsulated hydrophobic drug concentration on ultrasound-mediated leakage from liposomes. Studies have shown that membrane modifications affect the acoustic susceptibility of liposomes, likely because of changes in membrane packing. An advantage of liposome as drug carrier is its ability to encapsulate drugs of different chemistries. However, incorporation of hydrophobic molecules into the bilayer may cause changes in membrane packing, thereby affecting the release kinetics. Liposomes containing calcein and varying concentrations of papaverine, a hydrophobic drug, were exposed to 20 kHz, 2.2 Wcm(-2) ultrasound. Papaverine concentration was observed to affect calcein leakage although the effects varied widely based on liposome phase. For example, incorporation of 0.5mg/mL papaverine into Ld liposomes increased the leakage of hydrophilic encapsulants by 3× within the first minute (p=0.004) whereas the same amount of papaverine increased leakage by only 1.5× (p<0.0001). Papaverine was also encapsulated into echogenic liposomes and its concentration did not significantly affect calcein release rates, suggesting that burst release from echogenic liposomes is predictable regardless of encapsulants chemistry and concentration.

Acoustically active liposome-nanobubble complexes for enhanced ultrasonic imaging and ultrasound-triggered drug delivery.

Ultrasound is well known as a safe, reliable imaging modality. A historical limitation of ultrasound, however, was its inability to resolve structures at length scales less than nominally 20 µm, which meant that classical ultrasound could not be used in applications such as echocardiography and angiogenesis where one requires the ability to image small blood vessels. The advent of ultrasound contrast agents, or microbubbles, removed this limitation and ushered in a new wave of enhanced ultrasound applications. In recent years, the microbubbles have been designed to achieve yet another application, namely ultrasound-triggered drug delivery. Ultrasound contrast agents are thus tantamount to 'theranostic' vehicles, meaning they can do both therapy (drug delivery) and imaging (diagnostics). The use of ultrasound contrast agents as drug delivery vehicles, however, is perhaps less than ideal when compared to traditional drug delivery vehicles (e.g., polymeric microcapsules and liposomes) which have greater drug carrying capacities. The drawback of the traditional drug delivery vehicles is that they are not naturally acoustically active and cannot be used for imaging. The notion of a theranostic vehicle is sufficiently intriguing that many attempts have been made in recent years to achieve a vehicle that combines the echogenicity of microbubbles with the drug carrying capacity of liposomes. The attempts can be classified into three categories, namely entrapping, tethering, and nesting. Of these, nesting is the newest-and perhaps the most promising.

Nanoscale phase separation in DSPC-cholesterol systems.

The lipid arrangement of eukaryotic cell membranes has been shown to be heterogeneous, with domains enriched in cholesterol and saturated phospholipids, coexisting with a continuous phase that is enriched in unsaturated phospholipids. While the existence of these domains is well-established, there is still a lack of consensus regarding domain size and the factors influencing it. In this work, we investigate model membranes consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)-1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-cholesterol (Chol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, 18:1-16:0)-DSPC-Chol with a steady-state fluorescence assay and report the influence of phospholipid chain saturation and chain length on domain size. The spectral shifts of 1-myristoyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (DAN-PC) and a Förster resonance energy transfer (FRET) assay were used, along with an analytical model, to estimate domain sizes. A region of nanoscale domain existence was observed in both ternary systems; however, the domains formed in the system containing the asymmetric lipid (POPC, 18:1-16:0) were larger than those formed in the diunsaturated lipid (DOPC, 18:1-18:1). This is a new finding, as domains were not previously known to exist in similar POPC-based systems.

Size distribution of microbubbles as a function of shell composition.

The effect of modifying the shell composition of a population of microbubbles on their size demonstrated through experiment. Specifically, these variations include altering both the mole fraction and molecular weight of functionalized polymer, polyethylene glycol (PEG) in the microbubble phospholipid monolayer shell (1-15 mol% PEG, and 1000-5000 g/mole, respectively). The size distribution is measured with an unbiased image segmentation program written in MATLAB which identifies and sizes bubbles from micrographs. For a population of microbubbles with a shell composition of 5 mol% PEG2000, the mean diameter is 1.42 µm with a variance of 0.244 µm. For the remainder of the shell compositions studied herein, we find that the size distributions do not show a statistically significant correlation to either PEG molecular weight or mole fraction. All the measured distributions are nearly Gaussian in shape and have a monomodal peak.

Influence of shell composition on the resonance frequency of microbubble contrast agents.

The effect of variations in microbubble shell composition on microbubble resonance frequency is revealed through experiment. These variations are achieved by altering the mole fraction and molecular weight of functionalized polyethylene glycol (PEG) in the microbubble phospholipid monolayer shell and measuring the microbubble resonance frequency. The resonance frequency is measured via a chirp pulse and identified as the frequency at which the pressure amplitude loss of the ultrasound wave is the greatest as a result of passing through a population of microbubbles. For the shell compositions used herein, we find that PEG molecular weight has little to no influence on resonance frequency at an overall PEG mole fraction (0.01) corresponding to a mushroom regime and influences the resonance frequency markedly at overall PEG mole fractions (0.050-0.100) corresponding to a brush regime. Specifically, the measured resonance frequency was found to be 8.4, 4.9, 3.3 and 1.4 MHz at PEG molecular weights of 1000, 2000, 3000 and 5000 g/mol, respectively, at an overall PEG mole fraction of 0.075. At an overall PEG mole fraction of just 0.01, on the other hand, resonance frequency exhibited no systematic variation, with values ranging from 5.7 to 4.9 MHz. Experimental results were analyzed using the Sarkar bubble dynamics model. With the dilatational viscosity held constant (10(-8) N·s/m) and the elastic modulus used as a fitting parameter, model fits to the pressure amplitude loss data resulted in elastic modulus values of 2.2, 2.4, 1.6 and 1.8 N/m for PEG molecular weights of 1000, 2000, 3000 and 5000 g/mol, respectively, at an overall PEG mole fraction of 0.010 and 4.2, 1.4, 0.5 and 0.0 N/m, respectively, at an overall PEG mole fraction of 0.075. These results are consistent with theory, which predicts that the elastic modulus is constant in the mushroom regime and decreases with PEG molecular weight to the inverse 3/5 power in the brush regime. Additionally, these results are consistent with inertial cavitation studies, which revealed that increasing PEG molecular weight has little to no effect on inethe rtial cavitation threshold in the mushroom regime, but that increasing PEG molecular weight decreases inertial cavitation markedly in the brush regime. We conclude that the design and synthesis of microbubbles with a prescribed resonance frequency is attainable by tuning PEG composition and molecular weight.

Bubble nucleation in lipid bilayers: a mechanism for low frequency ultrasound disruption.

Recent experiments have shown that low frequency ultrasound (LFUS) induces leakage from lipid vesicles. However, the mechanism by which LFUS disrupts the lipid bilayer structure is not clear. In this paper we develop a theoretical model to test the possibility that gas molecule partitioning from the aqueous media into the lipid bilayer core can lead to the nucleation of microscale gas bubbles. If those can, indeed, form, then their presence in the lipid bilayer and interactions with an ultrasound field can cause bilayer disruption and leakage. The model derived here for the nucleation of stable bubbles accounts for the 'surface tension' that the lipid bilayer exerts on the bubble, a result of the associated disruption of the lipid packing. The model predicts that the probability of bubble nucleation is highly sensitive to the bilayer thickness, and largely insensitive to the bilayer phase. The probability of stable bubble formation is shown to correlate with experimentally measured sensitivity of lipid bilayers to LFUS, suggesting that membrane disruption may be due to embedded bubbles that nucleated in the bilayer.

Low-frequency ultrasound-induced transport across non-raft-forming ternary lipid bilayers.

We examined the effect of bilayer composition on membrane sensitivity to low-frequency ultrasound (LFUS) in bilayers composed of ternary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), dipalmitoyl-phosphocholine (DPPC), and cholesterol. The phase diagram of this system does not display macroscopic phase coexistence between liquid phases (although there are suggestions that there is coexistence between a liquid and a solid phase). Samples from across the composition space were exposed to 20 kHz, continuous wave ultrasound, and the response of the bilayer was quantified using steady-state fluorescence spectroscopy to measure the release of a self-quenching dye, calcein, from large unilamellar vesicles. Dynamic light scattering measurements indicate that, in this system, release proceeds primarily by transport through the vesicle bilayer. While vesicle destruction might account, at least in part, for the light scattering trends observed, evidence of destruction was not as obvious as in other lipid systems. Values for bilayer permeability are obtained by fitting release kinetics to a two-film theory mathematical model. The permeability due to LFUS is found to increase with increasing DPPC content, as the bilayer tends toward the solid-ordered phase. Permeability, and thus sensitivity to LFUS, decreases with either POPC or cholesterol mole fractions. In the liquid regime of this system, there is no recorded phase transition; thus cholesterol is the determining factor in release rates. However, the presence of domain boundaries between distinctly differing phases of liquid and solid is found to cause release rates to more than double. The correlation of permeability with phase behavior might prove useful in designing and developing therapies based on ultrasound and membrane interactions.

Bursting bubbles and bilayers.

This paper discusses various interactions between ultrasound, phospholipid monolayer-coated gas bubbles, phospholipid bilayer vesicles, and cells. The paper begins with a review of microbubble physics models, developed to describe microbubble dynamic behavior in the presence of ultrasound, and follows this with a discussion of how such models can be used to predict inertial cavitation profiles. Predicted sensitivities of inertial cavitation to changes in the values of membrane properties, including surface tension, surface dilatational viscosity, and area expansion modulus, indicate that area expansion modulus exerts the greatest relative influence on inertial cavitation. Accordingly, the theoretical dependence of area expansion modulus on chemical composition-- in particular, poly (ethylene glyclol) (PEG)--is reviewed, and predictions of inertial cavitation for different PEG molecular weights and compositions are compared with experiment. Noteworthy is the predicted dependence, or lack thereof, of inertial cavitation on PEG molecular weight and mole fraction. Specifically, inertial cavitation is predicted to be independent of PEG molecular weight and mole fraction in the so-called mushroom regime. In the "brush" regime, however, inertial cavitation is predicted to increase with PEG mole fraction but to decrease (to the inverse 3/5 power) with PEG molecular weight. While excellent agreement between experiment and theory can be achieved, it is shown that the calculated inertial cavitation profiles depend strongly on the criterion used to predict inertial cavitation. This is followed by a discussion of nesting microbubbles inside the aqueous core of microcapsules and how this significantly increases the inertial cavitation threshold. Nesting thus offers a means for avoiding unwanted inertial cavitation and cell death during imaging and other applications such as sonoporation. A review of putative sonoporation mechanisms is then presented, including those involving microbubbles to deliver cargo into a cell, and those--not necessarily involving microubbles--to release cargo from a phospholipid vesicle (or reverse sonoporation). It is shown that the rate of (reverse) sonoporation from liposomes correlates with phospholipid bilayer phase behavior, liquid-disordered phases giving appreciably faster release than liquid-ordered phases. Moreover, liquid-disordered phases exhibit evidence of two release mechanisms, which are described well mathematically by enhanced diffusion (possibly via dilation of membrane phospholipids) and irreversible membrane disruption, whereas liquid-ordered phases are described by a single mechanism, which has yet to be positively identified. The ability to tune release kinetics with bilayer composition makes reverse sonoporation of phospholipid vesicles a promising methodology for controlled drug delivery. Moreover, nesting of microbubbles inside vesicles constitutes a truly "theranostic" vehicle, one that can be used for both long-lasting, safe imaging and for controlled drug delivery.

Mechanistic roles of lipoprotein lipase and sphingomyelinase in low density lipoprotein aggregation.

The initiation of atherosclerosis involves retention of colloidal atherogenic lipoproteins, primarily low density lipoprotein (LDL), in the arterial intima. This retention occurs when LDL binds to smooth muscle cell extracellular matrix (SMC ECM), and is enhanced by lipoprotein lipase (LpL) and sphingomyelinase (Smase). Here we use a fluorescence assay and dynamic light scattering to study the individual and combined effects of these two enzymes on LDL aggregation. Our results show: (1) LpL is self-sufficient to induce LDL aggregation with aggregate sizes up to ~400 nm; (2) Smase induces LDL aggregation due to generation of ceramide and subsequent hydrophobic interactions; (3) Smase hydrolysis of LpL-induced LDL aggregates does not cause further aggregation and results in a ~3-fold diminished production of ceramide, while LpL treatment of Smase-induced aggregates does enhance aggregation; (4) The simultaneous addition of LpL and Smase causes increased variability in aggregation with final sizes ranging from 50 to 110 nm. Our data suggest a new proatherogenic function for LpL, namely, bridging between LDL particles causing their aggregation and consequently enhanced retention by SMC ECM. The mechanism of LpL-and-Smase-mediated LDL aggregation and binding to SMC ECM provides specific points of intervention to design novel effective antiatherogenic therapeutics.

Size-selective uptake of colloidal low density lipoprotein aggregates by cultured white blood cells.

This paper illustrates how principles of colloid science are useful in studying atherosclerosis. Accumulation of foam cells in the arterial intima is a key step in atherogenesis. The extent of foam cell formation is enhanced by low density lipoprotein (LDL) aggregates, and we have previously shown that the size of sphingomyelinase (Smase)-hydrolysis-induced aggregates depends directly on the concentration of ceramide generated in the LDL phospholipid monolayer, mediated by the hydrophobic effect. Here, we focus on the effect of LDL aggregate particle sizes on their subsequent uptake by macrophages. Our data show the first direct measurement of uptake as a function of aggregate size and the first direct comparison of uptake after Smase-catalyzed and vortex-mixing-mediated aggregation. Vortex-mixed aggregates with radii 20-77 nm showed maximal uptake approximately 118 microg sterol/mg protein at a 53 nm intermediate size, consistent with a mathematical model describing competition between aggregate surface area and volume. Smase-treated aggregates with radii 25-211 nm also showed maximal uptake at an intermediate size, approximately 58 microg sterol/mg protein for 132 nm particles, and fit a modified model that incorporated ceramide concentration expressed as aggregate size. This study shows that particle size is significant and composition may also be a factor in LDL uptake.

Gender differences in cholesterol nucleation in native bile: estrogen is a potential contributory factor.

The incidence of gallstone disease is two to three times higher in women than in men, and female sex hormones, particularly estrogens, have been implicated as contributory factors. Cholesterol nucleation is the initial step in gallstone pathogenesis and proceeds from cholesterol-rich phospholipid vesicles. The aim of this study was to investigate if there is a difference in cholesterol nucleation rates in male and female bile and whether estrogen influences nucleation rates by interacting with cholesterol-rich regions known as "lipid rafts" that exist within the cholesterol-phospholipid vesicles of the bile. Cholesterol nucleation from native prairie dog bile and the interaction of estrogens with lipid rafts in model bile solutions were investigated using Förster resonance energy transfer (FRET). Female native bile samples showed a greater reduction in energy transfer than did male native bile, indicating that cholesterol nucleation occurred more readily in female bile than in male bile. Model bile experiments demonstrated that the addition of estrogen has a significant effect, either cholesterol nucleation or raft disruption, but only in samples containing cholesterol-rich rafts. These results suggest that estrogen interacts with cholesterol-rich rafts in vesicles within bile to promote cholesterol nucleation and predispose females to gallstone formation.

Effect of sphingomyelinase-mediated generation of ceramide on aggregation of low-density lipoprotein.

This study addresses the response-to-retention hypothesis, which states that the subendothelial retention of atherogenic lipoproteins is the necessary and sufficient condition for the initiation of atherosclerosis. Here we focus on the relationship between the generation of ceramide in the low-density lipoprotein (LDL) phospholipid monolayer and the resulting aggregation of LDL particles. This study provides the first measurement of neutral, Mg (2+)-dependent Sphingomyelinase (Smase)-mediated ceramide formation from LDL-sphingomyelin and does so for a range of enzyme concentrations (0-0.22 units Smase/mL). The kinetics of ceramide generation was measured using a fluorescence assay for the above enzyme concentrations with a fixed substrate concentration (0.33 mg LDL/mL). The kinetics of LDL aggregate formation was measured by dynamic light scattering (DLS, method of cumulants) for identical enzyme concentrations. Ceramide concentration profiles were fit with a modification of the Michaelis-Menten model ( k a = 1.11 x 10 (-1) microM (-1) min (-1), k -a = 6.54 x 10 (2) microM (-1) min (-1), k 1 = 3.33 x 10 (1) microM (-1) min (-1), k -1 = 1.41 x 10 (-2) min (-1), k cat = 8.05 x 10 (1) min (-1), K M = 2.418 microM, k deact = 4.66 x 10 (-2) microM (-1) min (-1)) that accounts for the effects of enzyme attachment to the LDL monolayer and for deactivation of Smase due to product inhibition. LDL aggregation is described by a mass action model as explained in previous studies. A key result of this work is the finding that LDL aggregate size depends directly on ceramide concentration and is independent of enzyme concentration. This study demonstrates how principles of colloid science are relevant to important biomedical problems.

Measuring raft size as a function of membrane composition in PC-based systems: Part 1--binary systems.

This work applied two steady-state fluorescence techniques to detect nanoscopic membrane domains in a binary dimyristoylphosphocholine (DMPC)-cholesterol system and a ternary dioleoylphosphocholine (DOPC)-dipalmitoylphosphocholine (DPPC)-cholesterol system. A polarity-induced spectral shift in the emission spectra of 1-myristoyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (DAN-PC) in combination with a Förster resonance energy transfer (FRET) assay agreed with the phase diagrams that have been published for these systems and were observed to be useful tools in the detection of membrane heterogeneities. The DAN-PC/dehydroergosterol (DHE) FRET pair was found to be best suited for use with these steady-state techniques because of their differential partitioning between phases, although a high acceptor concentration was needed to obtain accurate measurements. In the binary system, this high probe concentration was found to be perturbing, but in more representative ternary systems, the high probe concentration no longer disrupted the phase behavior of the system. This FRET pair allowed for the calculation of nanometer-scale domain sizes in model ternary systems, using the two steady-state fluorescence techniques along with a clear and straightforward model.

Measuring raft size as a function of membrane composition in PC-based systems: Part II--ternary systems.

The heterogeneity of cell membranes, specifically the presence of lipid rafts, has been hypothesized to play a role in a large number of cellular processes. Although extensive work has been carried out to show the function of lipid rafts in these processes, the characterization of lipid rafts has proven to be extremely difficult. It is known that raft size is relevant to the function of cellular processes and that raft coalescence may be a driving factor for these processes; however, it remains unclear what factors influence raft size and coalescence in natural cell membranes. In this work, we study two ternary model phospholipid and cholesterol systems using two steady-state fluorescent techniques to detect and characterize membrane domains. Domain size is determined through the use of a model to relate experimental Förster resonance energy transfer (FRET) measurements to domain size. Domains in the range of 3-15 nm were detected in a dioleoylphosphatidylcholine-dipalmitoylphosphatidylcholine-cholesterol (DOPC-DPPC-Chol) system, while only a very small region containing domains was detected in a 1-palmitoyl-2-oleoyl-phosphatidylcholine-dipamitoylphosphatidylcholine-cholesterol (POPC-DPPC-Chol) system. In addition, the polarity-dependent emission maximum shift of the acceptor 1-myristoyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (DAN-PC) was used to detect the type of liquid phase(s) present in the membrane. It was found that, even in the case in which no two-phase coexistence was observed (POPC-DPPC-Chol), two liquid phases are present, although not necessarily in coexistence. These steady-state fluorescent techniques provide a method for detecting the presence of very small domains in model membranes and provide previously inaccessible detail about the phase behavior of these two systems.

Effect of membrane microheterogeneity and domain size on fluorescence resonance energy transfer.

Studies of multicomponent membranes suggest lateral inhomogeneity in the form of membrane domains, but the size of small (nanoscale) domains in situ cannot be determined with current techniques. In this article, we present a model that enables extraction of membrane domain size from time-resolved fluorescence resonance energy transfer (FRET) data. We expand upon a classic approach to the infinite phase separation limit and formulate a model that accounts for the presence of disklike domains of finite dimensions within a two-dimensional infinite planar bilayer. The model was tested against off-lattice Monte Carlo calculations of a model membrane in the liquid-disordered (l(d)) and liquid-ordered (l(o)) coexistence regime. Simulated domain size was varied from 5 to 50 nm, and two fluorophores, preferentially partitioning into opposite phases, were randomly mixed to obtain the simulated time-resolved FRET data. The Monte Carlo data show clear differences in the efficiency of energy transfer as a function of domain size. The model fit of the data yielded good agreement for the domain size, especially in cases where the domain diameter is <20 nm. Thus, data analysis using the proposed model enables measurement of nanoscale membrane domains using time-resolved FRET.

In vitro ultrasound-mediated leakage from phospholipid vesicles.

Interest in using ultrasound energy in wound management and intracellular drug delivery has been growing rapidly. Development and treatment optimization of such non-diagnostic applications requires a fundamental understanding of interactions between the acoustic wave and phospholipid membranes, be they cell membranes or liposome bilayers. This work investigates the changes in membrane permeation (leakage mimicking drug release) in vitro during exposure to ultrasound applied in two frequency ranges: "conventional" (1 MHz and 1.6 MHz) therapeutic ultrasound range and low (20 kHz) frequency range. Phospholipids vesicles were used as controllable biological membrane models. The membrane properties were modified by changes in vesicle dimensions and incorporation of poly(ethylene glycol) i.e. PEGylated lipids. Egg phosphatidylcholine vesicles with 5 mol% PEG were prepared with sizes ranging from 100 nm to 1 microm. Leakage was quantified in terms of temporal fluorescence intensity changes observed during carefully controlled ultrasound ON/OFF time intervals. Custom-built transducers operating at frequencies of 1.6 MHz (focused) and 1.0 MHz (unfocused) were used, the I(spta) of which were 46.9 W/cm2 and 3.0 W/cm2, respectively. A commercial 20 kHz, point-source, continuous wave transducer with an I(spta) of 0.13 W/cm2 was also used for comparative purposes. Whereas complete leakage was obtained for all vesicle sizes at 20 kHz, no leakage was observed for vesicles smaller than 100 nm in diameter at 1.6 or 1.0 MHz. However, introducing leakage at the higher frequencies became feasible when larger (greater than 300 nm) vesicles were used, and the extent of leakage correlated well with vesicle sizes between 100 nm and 1 microm. This observation suggests that physico-chemical membrane properties play a crucial role in ultrasound mediated membrane permeation and that low frequency (tens of kilohertz) ultrasound exposure is more effective in introducing permeability change than the "conventional" (1 MHz) therapeutic one. The experimental data also indicate that the leakage level is controlled by the exposure time. The results of this work might be helpful to optimize acoustic field and membrane parameters for gene or drug delivery. The outcome of this work might also be useful in wound management.

Sphingomyelinase-to-LDL molar ratio determines low density lipoprotein aggregation size: biological significance.

The subendothelial retention of low density lipoproteins (LDL) is believed to be the central pathogenic event in atherosclerosis, as stated by the response-to-retention hypothesis. Sphingomyelinase, an enzyme present in the arteries, has been proven to promote LDL aggregation. This study investigates the hypothesis that the extent of LDL aggregation is determined by the molar ratio of sphingomyelinase (SMase)-to-LDL, rather than the absolute concentrations. A mass action model is used to describe the aggregation process, and binding and dissociation rate constants are determined by fitting of dynamic light scattering data. The model predicts aggregate sizes that agree well with experimental observations. This study also tests the hypothesis that monocyte uptake of LDL correlates with aggregate size. LDL aggregates of three specific sizes (75, 100, and 150 nm) were incubated with J774A.1 cells and the net accumulation of LDL was monitored by measuring changes in the cellular cholesterol and protein content. Relative to a control sample, cholesterol accumulation was enhanced for aggregate sizes of 75 and 150 nm. The intermediate size aggregates, 100 nm, led to a very striking result demonstrating that cholesterol accumulation was markedly greater than the other samples, and was sufficient to cause cell death. These results underscore an important role of colloidal aggregation, and the influence of LDL aggregate size, in atherosclerosis.