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.

Pharmacokinetic stability of macrocyclic peptide triazole HIV-1 inactivators alone and in liposomes.

Previously, we reported the discovery of macrocyclic peptide triazoles (cPTs) that bind to HIV-1 Env gp120, inhibit virus cell infection with nanomolar potencies, and cause irreversible virion inactivation. Given the appealing virus-killing activity of cPTs and resistance to protease cleavage observed in vitro, we here investigated in vivo pharmacokinetics of the cPT AAR029b. AAR029b was investigated both alone and encapsulated in a PEGylated liposome formulation that was designed to slowly release inhibitor. Pharmacokinetic analysis in rats showed that the half-life of FITC-AAR029b was substantial both alone and liposome-encapsulated, 2.92 and 8.87 hours, respectively. Importantly, liposome-encapsulated FITC-AAR029b exhibited a 15-fold reduced clearance rate from serum compared with the free FITC-cPT. This work thus demonstrated both the in vivo stability of cPT alone and the extent of pharmacokinetic enhancement via liposome encapsulation. The results obtained open the way to further develop cPTs as long-acting HIV-1 inactivators against HIV-1 infection.

Impact of HIV-1 Membrane Cholesterol on Cell-Independent Lytic Inactivation and Cellular Infectivity.

Peptide triazole thiols (PTTs) have been found previously to bind to HIV-1 Env spike gp120 and cause irreversible virus inactivation by shedding gp120 and lytically releasing luminal capsid protein p24. Since the virions remain visually intact, lysis appears to occur via limited membrane destabilization. To better understand the PTT-triggered membrane transformation involved, we investigated the role of envelope cholesterol on p24 release by measuring the effect of cholesterol depletion using methyl beta-cyclodextrin (MßCD). An unexpected bell-shaped response of PTT-induced lysis to [MßCD] was observed, involving lysis enhancement at low [MßCD] vs loss of function at high [MßCD]. The impact of cholesterol depletion on PTT-induced lysis was reversed by adding exogenous cholesterol and other sterols that support membrane rafts, while sterols that do not support rafts induced only limited reversal. Cholesterol depletion appears to cause a reduced energy barrier to lysis as judged by decreased temperature dependence with MßCD. Enhancement/replenishment responses to [MßCD] also were observed for HIV-1 infectivity, consistent with a similar energy barrier effect in the membrane transformation of virus cell fusion. Overall, the results argue that cholesterol in the HIV-1 envelope is important for balancing virus stability and membrane transformation, and that partial depletion, while increasing infectivity, also makes the virus more fragile. The results also reinforce the argument that the lytic inactivation and infectivity processes are mechanistically related and that membrane transformations occurring during lysis can provide an experimental window to investigate membrane and protein factors important for HIV-1 cell entry.

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.

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.

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.

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.

Selective Enhancement of Swine Myocardium with a Novel Ultrasound Enhancing Agent During Transthoracic Echocardiography.

Ultrasound enhancing agents are approved to delineate the endocardial border and opacify the left ventricle cavity (LVC). We present a nested phase change agent (NPCA) designed to enable selective myocardial enhancement without enhancing the LVC by employing a dual-activation mechanism dependent on sufficient ultrasound intensity and the microenvironment of the myocardium. Swine received bolus injections of NPCA while echocardiograms were collected and processed to determine background-subtracted acoustic intensities (AI) in the LVC and septal myocardium. At mechanical index (MI) ≥ 0.8, the NPCA enhanced the myocardium selectively (p < 0.001) while the LVC remained at baseline AI. A 5-mL bolus of NPCA enhanced swine myocardium and enhancement persisted for > 5 min at 1.4 MI, while hemodynamics and EKG remained normal. Our findings demonstrate that the NPCA enhances swine myocardium selectively without enhancing the LVC. The NPCA could have utility for functional and structural echocardiographic studies with clinical ultrasound using standard settings.

An Ultrasound Enhancing Agent with Nonlinear Acoustic Activity that Depends on the Presence of an Electric Field.

The nonlinear acoustic properties of microbubble ultrasound enhancing agents have allowed for the development of subharmonic, second harmonic, and contrast-pulse sequence ultrasound imaging modes, which enhance the quality, reduce the noise, and improve the diagnostic capabilities of clinical ultrasound. This study details acoustic scattering responses of perfluorobutane (PFB) microbubbles, an un-nested perfluoropentane (PFP) nanoemulsion, and two nested PFP nanoemulsions-one comprising a negatively charged phospholipid bilayer and another comprising a zwitterionic phospholipid bilayer-when excited at 1 or 2.25 MHz over a peak negative pressure range of 200 kPa to 4 MPa in the absence and presence of a 1-Hz, 1-V/cm electric field. The only sample that exhibited an increase in nonlinear activity in the presence of an electric field at both excitation frequencies was the negatively charged nested PFP nanoemulsion; the most pronounced effect was observed at an excitation of 2.25 MHz. Interestingly, the application of an electric field not only increased the nonlinear acoustic activity of the negatively charged nested PFP nanoemulsion but increased it beyond that seen when the nanoemulsion is un-nested and on the same scale as PFB microbubbles.

A Voltage-Sensitive Ultrasound Enhancing Agent for Myocardial Perfusion Imaging in a Rat Model.

Echocardiographers with specialized expertise sometimes perform myocardial perfusion imaging using U.S. Food and Drug Administration-approved microbubbles in an off-label capacity, correlating microbubble replenishment in the near field with blood flow through the myocardium. This study reports the in vivo clinical feasibility of a voltage-sensitive ultrasound enhancing agent (UEA) for myocardial perfusion imaging. Four UEAs were injected into Sprague-Dawley rats while ultrasound images were collected to quantify brightness in the left ventricular (LV) cavity, septal wall, and posterior wall in systole and diastole. Formulation IV, a phase change agent nested within a negatively charged phospholipid bilayer, increased the tissue-to-cavity ratio in both systole and diastole in the septal wall, 6 dB, and in the posterior wall, 5 dB, while leaving the LV cavity at baseline. This outcome improves the signal of the myocardium relative to the LV cavity and shows promise as a myocardial perfusion UEA.

Inactivation of foodborne pathogens based on synergistic effects of ultrasound and natural compounds during fresh produce washing.

Ultrasound has potential to be used for disinfection, and its antimicrobial effectiveness can be enhanced in presence of natural compounds. In this study, we compared the antimicrobial effects of ultrasound at 20 kHz (US 20 kHz) or 1 MHz (US 1 MHz) in combination with carvacrol, citral, cinnamic acid, geraniol, gallic acid, lactic acid, or limonene against E. coli K12 and Listeria innocua at a constant power density in water. Compared to the cumulative effect of the individual treatments, the combined treatment of US 1 MHz and 10 mM citral generated >1.5 log CFU/mL additional inactivation of E. coli K12. Similarly, combined treatments of US 1 MHz and 2 mM carvacrol (30 min), US 20 kHz and 2 mM carvacrol, 10 mM citral, or 5 mM geraniol (15 min) generated >0.5-2.0 log CFU/mL additional inactivation in L. innocua. The synergistic effect of citral, as a presentative compound, and US 20 kHz treatment was determined to be a result of enhanced dispersion of insoluble citral droplets in combination with physical impact on bacterial membrane structures, whereas the inactivation by US 1 MHz was likely due to generation of oxidative stress within the bacteria. Combined ultrasound and citral treatments improved the bacterial inactivation in simulated wash water in presence of organic matter or during washing of inoculated blueberries but only additive antimicrobial effects were observed. Findings in this study will be useful to enhance fresh produce safety and shelf-life and design other alternative ultrasound based sanitation processes.

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.

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.

Atherosclerotic plaque progression and vulnerability to rupture: angiogenesis as a source of intraplaque hemorrhage.

Observational studies of necrotic core progression identify intraplaque hemorrhage as a critical factor in atherosclerotic plaque growth and destabilization. The rapid accumulation of erythrocyte membranes causes an abrupt change in plaque substrate characterized by increased free cholesterol within the lipid core and excessive macrophage infiltration. Neoangiogenesis is associated closely with plaque progression, and microvascular incompetence is a likely source of intraplaque hemorrhage. Intimal neovascularization is predominantly thought to arise from the adventitia, where there are a plethora of pre-existing vasa vasorum. In lesions that have early necrotic cores, the majority of vessels invading from the adventitia occur at specific sites of medial wall disruption. A breech in the medial wall likely facilitates the rapid in-growth of microvessels from the adventitia, and exposure to an atherosclerotic environment stimulates abnormal vascular development characterized by disorganized branching and immature endothelial tubes with "leaky" imperfect linings. This network of immature blood vessels is a viable source of intraplaque hemorrhage providing erythrocyte-derived phospholipids and free cholesterol. The rapid change in plaque substrate caused by the excessive accumulation of erythrocytes may promote the transition from a stable to an unstable lesion. This review discusses the potential role of intraplaque vasa vasorum in lesion instability as it relates to plaque rupture.

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.

Interactions between sphingomyelin and cholesterol in low density lipoproteins and model membranes.

This work examines three related, but previously unexplored, aspects of membrane biophysics and colloid science in the context of atherosclerosis. First, we show that sphingomyelinase (SMase)-induced aggregation of low density lipoproteins (LDLs), coupled with LDL exposure to cholesterol esterase (CEase), results in nucleation of cholesterol crystals, long considered the hallmark of atherosclerosis. In particular, this study reveals that the order of enzyme addition does not effect the propensity of LDL to nucleate cholesterol crystals, raising the possibility that nucleation can proceed from either the intra- or extracellular space. Second, we demonstrate 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 displacement of cholesterol from SM-Chol rafts by "raft-loving" ceramide. Third, we demonstrate that a time-independent Förster resonance energy transfer (FRET) assay, based on dehydroergosterol and dansylated lecithin and used previously to study cholesterol nanodomains, can be used to measure raft sizes (on the order of 10 nm) in model membrane systems. Taken together, these observations point to the possibility of an extracellular nucleation mechanism and underscore the important role that biological colloids play in human disease.

Interaction of apolipoprotein A-I with lecithin-cholesterol vesicles in the presence of phospholipase C.

Here we study the anti-nucleating mechanism of apolipoprotein A-I (apo A-I) on model biliary vesicles in the presence of phospholipase C (PLC) utilizing dynamic light scattering (DLS), steady-state fluorescence spectroscopy, cryogenic transmission electron microscopy (cryo-TEM), and UV/Vis spectroscopy. PLC induces aggregation of cholesterol-free lecithin vesicles from an initial, average size of 100 nm to a maximal size of 600 nm. The presence of apo A-I likely inhibits vesicle aggregation by shielding the PLC-generated hydrophobic moieties, which results in vesicles of an average size of 200 nm. A similar phenomenon is observed in cholesterol-enriched lecithin vesicles. Whereas PLC alone produces aggregates of 300 nm, no aggregation is observed when apo A-I is present along with PLC. However, the ability of apo A-I to inhibit aggregation is temporary, and after 8 h, a broad particle size distribution with sizes as high as 800 nm is observed. Apo A-I possibly induces the formation of small apo A-I/lecithin/cholesterol complexes of about 5-20 nm similar to the discoidal pre-HDL complexes found in blood when it can no longer effectively shield all the DAG molecules. Concomitant with formation of complexes, DAG molecules coalesce into large oil droplets, which account for the large particles observed by light scattering. Thus, apo A-I acts as an anti-nucleating agent by two mechanisms, anti-aggregation and microstructural transition. The mode of protection is dependent on the cholesterol content and the relative amounts of DAG and apo A-I present. This study supports the possibility of apo A-I solubilizing lipids in bile in a similar fashion as it does in blood and also delineates the mechanism of formation of the complexes.

Temperature and cholesterol composition-dependent behavior of 1-myristoyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-sn-glycero-3-phosphocholine in 1,2-dimyristoyl-sn-glycero-3-phosphocholine membranes.

We present a steady-state and time-resolved fluorescence emission spectra analysis of the membrane probe 1-myristoyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (DANSYL) in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol multi-lamellar vesicles (MLV) prepared by modified rapid solvent exchange. We report that the dose-dependent cholesterol-induced blue shifts in the steady-state fluorescence emission spectra observed in DMPC MLV are due to complex solvent effects that include time-dependent dipolar relaxation and the formation of internal charge transfer (ICT) states. A key finding of this investigation is identification of two distinguishable DANSYL populations existing at both shallow and deep locations in the membrane; these two DANSYL populations are evidence of laterally phase-separated domains at cholesterol compositions between X(chol) = 0.30 and 0.60 at 30 degrees C in DMPC MLV.

Aggregation kinetics of low density lipoproteins upon exposure to sphingomyelinase.

The response-to-retention hypothesis in atherosclerosis states that subendothelial retention of cholesterol-rich, atherogenic lipoproteins is the central pathogenic event that is both necessary and sufficient to provoke lesion initiation in an otherwise normal artery. Sphingomyelinase-induced aggregation of low density lipoproteins (LDL) is known to facilitate LDL retention, and the only available measurements of LDL aggregates suggest LDL aggregate size is approximately 100 nm. This study investigates the hypothesis that LDL aggregate size is determined by the relative rates of sphingomyelinase hydrolysis and LDL collisions. 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. It is found that LDL aggregation is sensitive to the relative amounts of sphingomyelinase and LDL and to pH. Model rate parameters were fit to experimental data in vitro and used to predict LDL aggregate sizes in vivo. The value of 100 nm in vivo does not appear to be fixed; rather, it is the value expected for the prevailing enzyme-to-LDL molar ratio.