An assay detecting and quantifying cholesterol nucleation from low-density lipoproteins has been established. Förster resonance energy transfer between dehydroergosterol and dansylated lecithin becomes significantly alleviated as a consequence of conucleation of dehydroergosterol and cholesterol. The assay, in combination with dynamic light scattering, absorbance spectroscopy, and fluorescence microscopy, can be used to study aggregation and nucleation in model blood systems. Human plasma LDL was labeled with dehydroergosterol and dansylated lecithin by incubation with donor multilamellar liposomes and isolated by centrifugation. Exposure of labeled LDL (0.5 mg/mL of total lipids) to sphingomyelinase (0.0-0.2 unit/mL) led to modest particle aggregation but produced no changes in energy transfer and no crystallization. However, addition of sphingomyelinase produced significant particle aggregation, nucleation, and crystallization, in a dose-dependent fashion, in samples that were previously treated with the enzyme, cholesterol esterase (0.2 unit/mL). The combination of cholesterol esterase and sphingomyelinase led to a significant alleviation of energy transfer, which preceded by 24 h the appearance of fluorescent, microscopic sterol crystals. These results point to a synergistic effect between cholesterol esterase and sphingomyelinase, suggesting that mere aggregation of LDL is insufficient to promote nucleation, and crystal formation likely proceeds in the intracellular space after LDL uptake by macrophages.
"Ultrasound of 20 kHz is commonly used to prepare small, unilamellar vesicles from large, multi-lamellar vesicles . This process is highly destructive to the membrane, resulting in membrane fragments being displaced from the parent membrane and complete leakage of vesicle contents. "
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: This work examines three related, but previously unexplored, aspects of membrane biophysics and colloid science in the context of atherosclerosis. First, it is shown that Sphingomyelinase (SMase)-induced aggregation of low density lipoproteins (LDL), coupled with LDL exposure to cholesterol esterase (CEase), results in nucleation of cholesterol crystals; long considered the hallmark of atherosclerosis. This is measured utilizing a time dependent Förester Resonance Energy Transfer (FRET) assay using the membrane probes ergosta-5,7,9(11),22-tetraen-3b-ol (DHE) and 1-acyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-sn-Glycero-3-phosphocholine (DL). In particular, this study reveals that cholesterol nucleation from LDL can be quantified and the order of enzyme addition does not affect the propensity of LDL to nucleate cholesterol crystals. This raises the possibility that nucleation can proceed from either the intra- or extra-cellular space. Second, using a combination of dynamic light scattering and UV/Vis absorbance spectroscopy to measure aggregation kinetics and particle sizes, a mass action model was developed to describe the aggregation process of LDL upon their exposure to SMase. It is found that LDL aggregation is independent of the relative concentrations of LDL and SMase, but rather depends on the LDL-to-SMase molar ratio. An important finding of this work was that the aggregate size was found to be a critical factor in foam cell formation as determined by an increase in cellular cholesterol content upon incubation with J774A.1 cells. Finally, the interactions between cholesterol (Chol) and sphingomyelin (SM) were investigated. Specifically, it is demonstrated that ceramide-rich aggregates of LDL release cholesterol to neighboring vesicles far more rapidly, and to a greater extent, than does native LDL. A likely explanation for this observation is due to the loss of the SM-Chol interaction and the displacement of cholesterol from SM-Chol rafts by “raft-loving” ceramide. Moreover, a time-independent FRET assay is used to measure SM-Chol raft sizes in model membrane systems. Taken together, these findings point to the possibility of an extracellular nucleation mechanism and underscore the important role that biological colloids play in human disease.
[Show abstract][Hide abstract] ABSTRACT: The fields of ultrasound bioeffects and membrane properties have been studied extensively in the past. However, the effects of membrane properties on ultrasound susceptibility have not been systematically studied. This work investigated the changes in membrane permeation (as indicated by fluorophore leakage) in vitro during exposure to ultrasound applied at center frequencies of 20 kHz, 1 MHz, and 1.55 MHz. Model membranes were used for the studies described here. Vesicles of various sizes, ranging from 100 nm to 1 μm, and various compositions were examined. The effect of incorporation of polyethylene glycol (PEG) into the bilayer membrane was studied using PEG concentrations ranging from 0-8 mol%. This work also gave the leakage profiles of samples containing cholesterol concentrations up to 65 mol%. In addition to PEG and cholesterol, the bilayer membranes were also composed of a combination of egg phosphatidylcholine, 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC), and 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC). Leakage was quantified in terms of temporal fluorescence intensity changes observed during carefully controlled ultrasound ON/OFF time intervals. In some experiments, the ON intervals were kept constant throughout. There were also experiments where the ON interval was allowed to vary. The OFF intervals were the same for all experiments. Results shown here demonstrated that while the ultrasound parameters were important in the ultrasound/membrane interaction, the properties of the membrane, as determined by composition and size, were equally as important. The major purpose of this work was to enhance the understanding of the interaction between ultrasound and cell membranes by studying the ultrasound parameters and membrane properties that govern this interaction. This work will prove to be helpful in diagnostic and therapeutic ultrasound applications.
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