ABSTRACT
The stability of active compounds encapsulated in nanoparticles depends on the resistance of the particles to diffusion of environmental degradation agents. In this paper, off-lattice Monte Carlo simulations are used to investigate a suspension of nanostructured lipid carriers (NLC) composed of interspaced liquid and solid lipid domains, immersed in a solution containing molecules representing oxidative or other degradation agents. The simulations examine the diffusion of the degradation agents into the nanoparticles as a function of nanoparticle size, solid domain fraction, and domain size. Two types of suspensions are studied: one (representing an infinitely dilute nanoparticle suspension) where the concentration of oxidative agents is constant in the solution around the particle and the other, finite system where diffusion into the nanoparticle causes depletion in the concentration of degradation agents in the surrounding solution. The total number of degradation agent molecules in the NLCs is found to decrease with the solid domain fraction, as may be expected. However, their concentration in the liquid domains is found to increase with the solid domain fraction. Since the degradation reaction depends on the concentration of the degradation agents, this suggests that compounds encapsulated in nanoparticles with high liquid content (such as emulsions) will degrade less and be more stable than those encapsulated in NLCs with high solid domain fraction, in agreement with previous experimental results.
ABSTRACT
Kappa-casein (κCN) and beta-casein (ßCN) are disordered proteins present in mammalian milk. In vitro, ßCN self-assembles into core-shell micelles. κCN self assembles into similar micelles, as well as into amyloid-like fibrils. Recent studies indicate that fibrillization can be suppressed by mixing ßCN and κCN, but the mechanism of fibril inhibition has not been identified. Examining the interactions of native and reduced kappa-caseins (N-κCN and R-κCN) with ßCN, we expose a competition between two different self-assembly processes: micellization and fibrillization. Quite surprisingly, however, we find significant qualitative and quantitative differences in the self-assembly between the native and reduced κCN forms. Specifically, thermodynamic analysis reveals exothermic demicellization for ßCN and its mixtures with R-κCN, as opposed to endothermic demicellization of N-κCN and its mixtures with ßCN at the same temperature. Furthermore, with time, R-κCN/ßCN mixtures undergo phase separation into pure ßCN micelles and R-κCN fibrils, while in the N-κCN/ßCN mixtures fibril formation is considerably delayed and mixed micelles persist for longer periods of time. Fibrils formed in N-κCN/ßCN mixtures are shorter and more flexible than those formed in R-κCN/ßCN systems. Interestingly, in the N-κCN/ßCN mixtures, the sugar moieties of N-κCN oligomers seem to organize on the mixed micelles surface in a manner similar to the organization of κCN in milk casein micelles.
Subject(s)
Caseins/chemistry , Micelles , Milk/chemistry , Amyloid/metabolism , Animals , Caseins/metabolism , Temperature , ThermodynamicsABSTRACT
At the critical micelle concentration (CMC), amphiphiles self-assemble into spherical micelles, typically followed by a transition at the second CMC to cylindrical micelles that are uniform in width but are polydispersed in length and have swollen ends. In this Letter, we report on a new structural path of self-assembly that is based on discoidal (coin-like), rather than spherical, geometry; the nonionic sterol ChEO10 is shown to form monodisperse equilibrium disc assemblies at the first CMC, transitioning at the second CMC into flat ribbons that (like the cylindrical micelles) have uniform width, polydispersed length, and swollen ends. Increase in ChEO10 concentration or the temperature leads to ribbon elongation, branching, and network formation. This self-assembly path reveals that (1) surfactants can form equilibrium nonspherical assemblies at the CMC and (2) aggregate progression around the second CMC is similar for the disc and sphere geometries.
Subject(s)
Micelles , Sterols/chemistry , Ethylene Oxide/chemistry , Models, Molecular , Molecular Conformation , Solutions , Surface-Active Agents/chemistryABSTRACT
ß-Casein is a 24 kDa amphiphilic and unstructured protein that self-assembles into small core-shell micelles at a wide range of concentrations, pH values and temperatures. We recently developed the micelles as nanocarriers for oral delivery of hydrophobic drugs. In this paper we examined the effect of the hydrophobic non-steroidal anti-inflammatory drug (NSAID) ibuprofen on the micellar structure, as a function of temperature and loading. Using cryo-transmission electron microscopy (cryo-TEM) we find two routes of organization mixed micellization and co-assembly (aggregation). The time-dependent events that characterize the second routes has been examined in detail. At 25 °C we find coexistence of small assemblies and larger aggregates of irregular (but defined) structures that contain the drug. Increasing the drug loading increases the relative number of the larger aggregates and their dimensions, leading eventually to the formation of long then branched structures, like in amphiphilic block copolymer solutions. Similar trends were identified for changes in the temperature. Combined, our results suggest that ibuprofen acts as a co-surfactant that possibly is localizes to the interface rather than being encapsulated in the micellar core as other NSAID hydrophobic drugs.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Caseins/chemistry , Drug Carriers/chemistry , Ibuprofen/administration & dosage , Micelles , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Cattle , Ibuprofen/chemistry , Models, Molecular , Protein AggregatesABSTRACT
Electrical, ultrasound and other types of external fields are known to induce the formation of pores in cellular and model membranes. This paper examines drug release through field induced liposome pores using Monte Carlo simulations. We find that drug release rates vary as a function of pore size and spacing, as well as the overall fraction of surface area covered by pores: The rate of release from liposomes is found to increase rapidly with pore surface coverage, approaching that of the fully ruptured liposome at fractional pore areas. For a given pore surface coverage, the pore size affects the release rate in the limit of low coverage, but not when the pores cover a relatively high fraction of the liposome surface area. On the other hand, for a given pore size and surface coverage, the distribution of pores significantly affects the release in the limit of high surface coverage: The rate of release from a liposome covered with a regularly spaced array of pores is, in this limit, higher than the release rate from (most) systems where the pores are distributed randomly on the liposome surface. In contrast, there is little effect of the pore distribution on release when the pore surface coverage is low. The simulation results are in good agreement with the predictions of detailed diffusion models.
Subject(s)
Liposomes/chemistry , Pharmaceutical Preparations/chemistry , Molecular Dynamics Simulation , Monte Carlo Method , Surface PropertiesABSTRACT
Emulsions, solid lipid nanoparticles (SLN), and nanostructured lipid carriers (NLC) containing a mix of liquid and solid domains are of interest as encapsulation vehicles for hydrophobic compounds. Studies of the release rate from these particles yield contradictory results: Some find that increasing the fraction of solid phase increases the rate of release and others the opposite. In this paper we study the release of encapsulated materials from lipid-based nanoparticles using Monte Carlo simulations. We find that, quite surprisingly, the release rate is largely insensitive to the size of solid domains or the fraction of solid phase. However, the distribution of the domains significantly affects the rate of release: Solid domains located at the interface with the surrounding solution inhibit transport, while nanoparticles where the solid domains are concentrated in the center enhance it. The latter can lead to release rates in NLCs that are faster than in the equivalent emulsions. We conclude that controlling the release rate from NLCs requires the ability to determine the location and distribution of the solid phase, which may be achieved through choice of the surfactants stabilizing the particles, incorporation of nucleation sites, and/or the cooling rates and temperatures.
Subject(s)
Drug Carriers/chemistry , Lipids/chemistry , Models, Chemical , Nanoparticles/chemistry , Surface-Active Agents/chemistry , EmulsionsABSTRACT
The structure and function of lipid-based complexes (lipoplexes) have been widely investigated as cellular delivery vehicles for nucleic acids-DNA and siRNA. Transfection efficiency in applications such as gene therapy and gene silencing has been clearly linked to the local, nano-scale organization of the nucleic acid in the vehicle, as well as to the global properties (e.g. size) of the carriers. This review focuses on both the structure of DNA and siRNA complexes with cationic lipids, and the kinetics of structure evolution during complex formation. The local organization of the lipoplexes is largely set by thermodynamic, equilibrium forces, dominated by the lipid preferred phase. As a result, complexation of linear lambda-phage DNA, circular plasmid DNA, or siRNA with lamellae-favoring lipids (or lipid mixtures) forms multi-lamellar L(α)(C) liquid crystalline arrays. Complexes created with lipids that have bulky tail groups may form inverted hexagonal HII(C) phases, or bicontinuous cubic Q(II)(C) phases. The kinetics of complex formation dominates the large-scale, global structure and the properties of lipoplexes. Furthermore, the time-scales required for the evolution of the equilibrium structure may be much longer than expected. In general, the process may be divided into three distinct stages: An initial binding, or adsorption step, where the nucleic acid binds onto the surface of the cationic vesicles. This step is relatively rapid, occurring on time scales of order of milliseconds, and largely insensitive to system parameters. In the second step, vesicles carrying adsorbed nucleic acid aggregate to form larger complexes. This step is sensitive to the lipid characteristics, in particular the bilayer rigidity and propensity to rupture, and to the lipid to nucleic acid (L/D) charge ratio, and is characterized by time scales of order seconds. The last and final step is that of internal rearrangement, where the overall global structure remains constant while local adjustment of the nucleic acid/lipid organization takes place. This step may occur on unusually long time scales of order hours or longer. This rate, as well, is highly sensitive to lipid characteristics, including membrane fluidity and rigidity. While the three step process is consistent with many experimental observations to date, improving the performance of these non-viral vectors requires better understanding of the correlations between the parameters that influence lipoplexes' formation and stability and the specific rate constants i.e., the timescales required to obtain the equilibrium structures. Moreover, new types of cellular delivery agents are now emerging, such as antimicrobial peptide complexes with anionic lipids, and other proteins and small-molecule lipid carriers, suggesting that better understanding of lipoplex kinetics would apply to a variety of new systems in biotechnology and nanomedicine.
Subject(s)
Lipids/chemistry , Nucleic Acids/chemistry , Kinetics , Molecular StructureABSTRACT
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.
Subject(s)
Lipid Bilayers/radiation effects , Ultrasonics , Lipid Bilayers/chemistryABSTRACT
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.
Subject(s)
Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Ultrasonics , 1,2-Dipalmitoylphosphatidylcholine/chemistry , 1,2-Dipalmitoylphosphatidylcholine/metabolism , Diffusion , Kinetics , Liposomes/chemistry , Liposomes/metabolism , Phase Transition , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolismABSTRACT
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.
Subject(s)
Lipid Bilayers/chemistry , Microbubbles , Animals , Humans , Polyethylene Glycols/chemistry , Ultrasonics , Unilamellar Liposomes/chemistryABSTRACT
Coating hydrogel films or microspheres by an adsorbed colloidal shell is one synthesis method for forming colloidosomes. The colloidal shell allows control of the release rate of encapsulated materials, as well as selective transport. Previous studies found that the packing density of self-assembled, adsorbed colloidal monolayers is independent of the colloidal particle size. In this paper we develop an equilibrium model that correlates the packing density of charged colloidal particles in an adsorbed shell to the particle dimensions in monodisperse and bidisperse systems. In systems where the molar concentration in solution is fixed, the increase in adsorption energy with increasing particle size leads to a monotonic increase in the monolayer packing density with particle radius. However, in systems where the mass fraction of the particles in the adsorbing solutions is fixed, increasing particle size also reduces the molar concentration of particles in solution, thereby reducing the probability of adsorption. The result is a nonmonotonic dependence of the packing density in the adsorbed layer on the particle radius. In bidisperse monolayers composed of two particle sizes, the packing density in the layer increases significantly with size asymmetry. These results may be utilized to design the properties of colloidal shells and coatings to achieve specific properties such as transport rate and selectivity.
Subject(s)
Colloids/chemistry , Particle Size , Adsorption , Hydrogels/chemistry , Polystyrenes/chemistry , Static Electricity , Surface PropertiesABSTRACT
Colloidosomes are aqueous cores surrounded by a shell composed of packed colloidal particles. Recent studies suggest that these colloidal shells reduce, or even inhibit, the transport of molecular species (diffusants). However, the effect of the colloidal shell on transport is unclear: In some cases, the reduction in transport of diffusants through the shell was found to be independent of the size of the colloidal particles composing the shell. Other studies find, however, that shells composed of small colloidal particles of order 100nm or less hindered transport of diffusants more than those composed of micro-scale colloidal particles. In this paper we present a simple diffusion model that accounts for three processes that reduce diffusant transport through the shell: (i) a reduction in the penetrable volume available for transport, which also increases the tortuousity of the diffusional path, (ii) narrow pore size which may hinder transport for larger diffusants through size exclusion, and (iii) a reduction in interfacial area due to 'blocking' of the surface by the adsorbed particles. We find that the colloidal particle size does not affect the reduction in transport through the colloidal shell when the shell is a monolayer. However, in closely packed, thick layers where the thickness of the multi-layer shell is fixed, the rate of transport decreases significantly with colloidal particle dimensions. These results are in excellent agreement with previously published experimental results.
Subject(s)
Colloids/chemistry , Diffusion , Models, Chemical , Particle SizeABSTRACT
Recent studies suggest that coating microcapsules by a shell composed of impenetrable colloidal particles (thereby forming 'colloidosomes') can be used to control surface porosity, and therefore, permeability. The voids between the particles in the coating define the size of the surface pores available for transport. However, to date, data demonstrating this selectivity has been largely qualitative. In this paper we examine, quantitatively, the effect of a surface coating (shell), composed of colloidal particles, on release from hydrogels. We find that the presence of a colloidal shell does indeed reduce the rate of transport of three model molecules: Aspirin, caffeine, and FITC-dextran with MW of approximately 3000-5000. Contrary to expectation, however, we find that for all three molecules the reduction in transport rate is largely independent of the dimensions of the particles composing the shell, despite differences that range over three orders of magnitude. In the case of the small molecules, caffeine and aspirin, the colloidal shell reduces the effective diffusion coefficient by a factor of 3. In the case of dextran, the suppression in the release rate due to the colloidal shell was much larger. These results are explained using a simple diffusion model that accounts for the volume fraction and diameter of the colloidal particles in the shell, and the size of the diffusing molecules.
ABSTRACT
Membrane budding has been extensively studied as an equilibrium process attributed to the formation of coexisting domains or changes in the vesicle area-to-volume ratio (reduced volume). In contrast, non-equilibrium budding remains experimentally widely unexplored, especially when timescales fall well below the characteristic diffusion time of lipids, tau. We show that localized mechanical perturbations, initiated by driving giant unilamellar vesicles (GUVs) through their lipid main phase transition from the gel to the fluid phase, lead to the immediate formation of rapidly growing, localized, non-equilibrium buds when the transition takes place at short timescales (Subject(s)
Unilamellar Liposomes/chemistry
, Molecular Dynamics Simulation
, Phase Transition
, Temperature
, Time Factors
, Viscosity
ABSTRACT
The compositional differences between domains in phase-separated membranes are associated with differences in bilayer thickness and moduli. The resulting packing deformation at the phase boundary gives rise to a line tension, the one dimensional equivalent of surface tension. In this paper we calculate the line tension between a large membrane domain and a continuous phase as a function of the thickness mismatch and the contact angle between the phases. We find that the packing-induced line tension is sensitive to the contact angle, reaching a minimum at a specific value. The difference in the line tension between a flat domain (that is within the plane of the continuous phase) and a domain at the optimal contact angle may be of order 40%. This could explain why previous calculations of the thickness mismatch based line tension tend to yield values that are higher than those measured experimentally.
Subject(s)
Membranes, Artificial , Models, ChemicalABSTRACT
Membrane inhomogeneity gives rise to a perturbation energy that may be manifested as line tension between regions of different compositions. Here we calculate the perturbation energy of a phase-separated fluid membrane composed of domains embedded in a continuous phase and relate it to the line tension in the system. We find that the effective line tension due to the thickness mismatch between the phases varies nonmonotonically as a function of the domain size and spacing when those decrease below about 20 nm. Although we find that the characteristic time scale for domain coalescence increases with domain size, the coalescence time for domains on the order of 25 to 50 nm may be as long as several days. The time scale for domain coalescence is found, quite surprisingly, to be maximal when the bending and area moduli of the domain and continuous phase are similar and to decrease when one phase is stiffer than the other. Domain stability is also found to vary as a function of spacing; as a result, small domains on the order of 5 nm become unstable and tend to coalesce when the domain-phase area fraction increases above 0.5, whereas larger domains on the order of 50 nm become unstable only when the area fraction increases above 0.8.
Subject(s)
Membranes, Artificial , Lipids/chemistry , Models, TheoreticalABSTRACT
We present a robust and straightforward approach for fabricating a novel colloidosome system where colloidal particles are assembled to form colloidal shells on the surface of stimuli-responsive microgel scaffolds. We demonstrate that the structural properties of the colloidal shells can be controlled through the colloidal particle size and modulus, and the state of supporting microgel particles. This technique offers a new way to engineer colloidosomes, enabling fine control over their permeability over a wide range of length scales.
Subject(s)
Colloids/chemistry , Crystallization/methods , Liposomes/chemistry , Nanostructures/chemistry , Nanostructures/ultrastructure , Nanotechnology/methods , Macromolecular Substances/chemistry , Materials Testing , Molecular Conformation , Particle Size , Permeability , Porosity , Surface PropertiesABSTRACT
Many lipids are composed of asymmetric tail chains that differ by their molecular weight (MW) and/or degree of saturation. Previous studies found that membrane moduli vary with the degree of lipid tail asymmetry. However, to date little is known regarding the effect (if any) of tail asymmetry on the membrane-induced interactions between embedded proteins. In this paper we use a self-consistent field model to examine the effect of lipid tail asymmetry on membrane proteins. We first examine the case where the overall tail length (sum of both chains) is held constant, which implies that the membrane thickness remains constant as well, independent of tail asymmetry. We find that, in these systems, the membrane area stretch and bending moduli decrease with increasing chain asymmetry, thereby reducing the magnitude of the membrane-induced barrier to protein aggregation. Since in symmetric lipid bilayers the energy barrier is typically of order approximately 1-2 times the thermal energy kT, the asymmetry-induced reduction in barrier height may increase the probability of protein aggregation significantly. In systems where one tail chain is held constant, increasing asymmetry involves changes in the bilayer thickness which are found to dominate any effect arising from the asymmetry.
Subject(s)
Membrane Lipids/chemistry , Membrane Proteins/chemistry , Membranes/chemistryABSTRACT
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.
Subject(s)
Membranes/chemistry , Biophysical Phenomena , Biophysics , Fluorescence Resonance Energy Transfer , Lipid Bilayers/chemistry , Membrane Microdomains/chemistry , Membranes, Artificial , Models, Molecular , Monte Carlo Method , NanotechnologyABSTRACT
Fluorescence resonance energy transfer (FRET) is sensitive to lateral heterogeneity in multicomponent membranes. Recently, we developed a model that enables the extraction of domain size from time-resolved FRET data, and here we examine the effects of domain polydispersity and spatial ordering on the accuracy of domain size determination. The model is applied to Monte Carlo calculations of membranes containing polydisperse domains, either randomly or hexagonally packed for three probe-partitioning schemes and three domain surface coverages. Statistical analysis of the model fitting results supports the conclusion that time-resolved FRET is a robust and precise technique for determining the size of small membrane domains (=4 times the Förster distance, approximately 20 nm, of the FRET pair), regardless of domain polydispersity and packing. The confidence estimates, presented here in dimensionless domain diameters (independent of the specific FRET pairs), provide a universal tool for domain size estimation in systems containing membrane-bound fluorophores.