Controlled microfluidic encapsulation of cells, proteins, and microbeads in lipid vesicles

Cells have been encapsulated inside lipid vesicles by using a new microfluidic lipid vesicle formulation technique. Lipid vesicles are formulated within minutes without using toxic lipid solvents. The encapsulation efficiency inside the vesicles is controlled by the microfluidic flows. Green fluorescent proteins (GFP), carcinoma cells, and bead encapsulated vesicles have mean diameters of 27.2 mum, 62.4 mum, and 55.9 mum, respectively. The variations of vesicle sizes are approximately 20% for the GFP and cell encapsulated vesicles and approximately 10% for the bead encapsulated vesicles.     

Vesicle and bilayer formation of diphytanoylphosphatidylcholine (DPhPC) and diphytanoylphosphatidylethanolamine (DPhPE) mixtures and their bilayers' electrical stability

Lipid bilayers are of interest in applications where a cell membrane mimicking environment is desired. The performance of the lipid bilayer is largely dependent on the physical and chemical properties of the component lipids. Lipid bilayers consisting of phytanoyl lipids have proven to be appropriate choices since they exhibit high mechanical and chemical stability. In addition, such bilayers have high electrical resistances. Two different phytanoyl lipids, 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE), and various combinations of the two have been investigated with respect to their behavior in aqueous solutions, their interactions with solid surfaces, and their electrical stability. Dynamic light scattering, nuclear magnetic resonance diffusion, and cryogenic transmission electron microscopy measurements showed that pure DPhPC as well as mixtures of DPhPC and DPhPE consisting of greater than 50% (mol%) DPhPC formed unilamellar vesicles. If the total lipid concentration was greater than 0.15g/l, then the vesicles formed solid-supported bilayers on plasma-treated gold and silica surfaces by the process of spontaneous vesicle adsorption and rupture, as determined by quartz crystal microbalance with dissipation monitoring and atomic force microscopy. The solid-supported bilayers exhibited a high degree of viscoelasticity, probably an effect of relatively high amounts of imbibed water or incomplete vesicle fusion. Lipid compositions consisting of greater than 50% DPhPE formed small flower-like vesicular structures along with discrete liquid crystalline structures, as evidenced by cryogenic transmission electron microscopy. Furthermore, electrophysiology measurements were performed on bilayers using the tip-dip methodology and the bilayers' capacity to retain its electrical resistance towards an applied potential across the bilayer was evaluated as a function of lipid composition. It was shown that the lipid ratio significantly affected the bilayer's electrical stability, with pure DPhPE having the highest stability followed by 3DPhPC:7DPhPE and 7DPhPC:3DPhPE in decreasing order. The bilayer consisting of 5DPhPC:5DPhPE had the lowest stability towards the applied electrical potential.     

The contrasting kinetics of peroxidation of vitamin E-containing phospholipid unilamellar vesicles and human low-density lipoprotein

It is well established that alpha-tocopherol, TocH, is an outstanding lipid-soluble, peroxyl radical trapping antioxidant in homogeneous systems. It is also well established that TocH functions as a prooxidant in human low-density lipoprotein, LDL, subjected to attack by peroxyl radicals generated in the aqueous phase by, for example, thermal decomposition of the azo compound, ABAP. This tocopherol-mediated peroxidation, TMP, of LDL involves a three-step chain reaction, one step being hydrogen atom abstraction from the LDL lipids by the tocopheroxyl radical, Toc*. The occurrence of TMP has been attributed to three factors, (i) translocation by TocH of radical character from the aqueous phase into LDL lipid, (ii) isolation of the water-insoluble Toc* in the LDL particle in which it is formed for times sufficient to permit it to react with the lipid, and (iii) the small lipid volume of LDL which ensures that no particle can contain more than a single radical for a significant length of time. This consensus view of TMP implies that it should occur in any TocH-containing dispersion of small lipid particles. However, the present examination of the kinetics of the ABAP-initiated peroxidation of small unilamellar vesicles, SUVs, made from palmitoyllinoleoylphosphatidylcholine and cholesterol with a composition designed to mimic the surface coat of LDL, has shown that TocH functions as an antioxidant in such systems and that TMP does not occur under conditions where it would have occurred if the particles had been LDL. Several possible reasons for the kinetic differences between SUVs and LDL have been considered and ruled out by experiment. It is concluded that TMP can occur in LDL because these particles contain a lipid core in which the Toc* radical "hides" for much of its lifetime well away from the peroxyl radicals in the aqueous phase. In contrast, because SUVs have no lipid core, the Toc(*) radical is always "exposed" and available to aqueous peroxyl radicals with which it reacts rapidly and is destroyed before it can abstract a hydrogen atom from the lipid.     

Enhancement of gemcitabine affinity for biomembranes by conjugation with squalene: differential scanning calorimetry and Langmuir-Blodgett studies using biomembrane models

Molecular interactions between gemcitabine, alone or conjugated with squalene to form the gem-squalene prodrug, with dimyristoylphosphatidylcholine have been investigated by differential scanning calorimetry and Langmuir film balance techniques to gain information about the interaction of gemcitabine and its prodrug with mammalian cell membranes and to evaluate the potential of liposomes as a delivery system for gemcitabine prodrugs. Phospholipids assembled as multilamellar vesicles or monolayers (at the air water interface) have been used as biomembrane models. Different interactions of gemcitabine, its prodrug, and squalene with the lipid were detected by dispersing the compounds in the MLV and were compared with kinetic experiments carried out to consider the ability of the examined compounds to dissolve in an aqueous medium, to migrate through it, and to be captured by multilamellar vesicles. Their ability to be released from drug-loaded liposomes and be taken up by empty vesicles mimicking biomembranes was also considered. Analysis of the differential scanning calorimetry curves reveals that gemcitabine has very little interaction with multilamellar vesicles whereas the gem-squalene prodrug strongly interacts with multilamellar vesicles. The kinetic experiments suggest that an aqueous medium does not permit the prodrug uptake by the biomembrane models, whereas it is allowed when gem-squalene is gradually released by the liposomes. The molecular area/surface pressure isotherms of the gemcitabine/lipid, gem-squalene/lipid, and pure compound monolayers, in agreement with the calorimetric results, indicate that gem-squalene interacts with the phospholipid monolayer with the squalene moiety in contact with the phospholipid chains and gemcitabine protruding in the aqueous medium.     

Complete budding and asymmetric division of primitive model cells to produce daughter vesicles with different interior and membrane compositions

Asymmetric cell division is common in biology and plays critical roles in differentiation and development. Unicellular organisms are often used as model systems for understanding the origins and consequences of asymmetry during cell division. Although basic as compared to mammalian cells, these are already quite complex. We report complete budding and asymmetric fission of very simple nonliving model cells to produce daughter vesicles that are chemically distinct in both interior and membrane compositions. Our model cells are based on giant lipid vesicles (GVs, 10-30 μm) encapsulating a polyethylene glycol (PEG)/dextran aqueous two-phase system (ATPS) as a crowded and compartmentalized cytoplasm mimic. Ternary lipid compositions were used to provide coexisting micrometer-scale liquid disordered (L(d)) and liquid ordered (L(o)) domains in the membranes. ATPS-containing vesicles formed buds when sucrose was added externally to provide increased osmotic pressure, such that they became not only morphologically asymmetric but also asymmetric in both their interior and their membrane compositions. Further increases in osmolality drove formation of two chemically distinct daughter vesicles, which were in some cases connected by a lipid nanotube (complete budding), and in others were not (fission). In all cases, separation occurred at the aqueous-aqueous phase boundary, such that one daughter vesicle contained the PEG-rich aqueous phase and the other contained the dextran-rich aqueous phase. PEGylated lipids localized in the L(o) domain resulted in this membrane domain preferentially coating the PEG-rich bud prior to division, and subsequently the PEG-rich daughter vesicle. Varying the mole ratio of lipids resulted in excess surface area of L(o) or L(d) membrane domains such that, upon division, this excess portion was inherited by one of the daughter vesicles. In some cases, a second "generation" of aqueous phase separation and budding could be induced in these daughter vesicles. Asymmetric fission of a simple self-assembled model cell, with production of daughter vesicles that harbored different protein concentrations and lipid compositions, is an example of the seemingly complex behavior possible for simple molecular assemblies. These compartmentalized and asymmetrically dividing ATPS-containing GVs could serve as a test bed for investigating possible roles for spatial and organizational cues in asymmetric cell division and inheritance.     

Molecular organization of the lipid-bound EDTA—chromium(III) complex

Ethylenediaminetetraacetic acid bound to a lipid (dioctadecyldimethylammonium bromide) has been used as an immobilized ligand system for extracting chromium(III) salts from an aqueous solution. The morphology and nature of the aggregation of the metal-ion-bound lipid—ligand complex has been studied by transmission electron microscopy and differential scanning calorimetry. A strong dependence of the vesicle size on the gel—crystalline phase transition has been observed and the results are rationalized in terms of higher-ordered packing of the large vesicles below the phase transition temperatures.     

Interaction of Natural and Modified β-Cyclodextrins with a Biological Membrane Model of Dipalmitoylphosphatidylcholine

Lipid vesicles made up of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were used as a biological membrane model to investigate the interaction between natural and modified β-cyclodextrins and these membrane bilayers. Differential scanning calorimetry was used to study the thermotropic behavior of the DPPC vesicles and any change caused by the presence of cyclodextrins. The presence of dimethyl-β-cyclodextrin (DM-β-CyD) triggered a reduction in the enthalpy values related to the main transition peak from gel state to liquid crystal phase of DPPC aqueous dispersions, as a function of the DM-β-CyD molar fraction: the larger the amount of DM-β-CyD, the greater the reduction in ΔHvalues. This effect was probably due to the ability of DM-β-CyD to extract and to complex the DPPC molecules forming the phospholipid vesicles. The presence of β-cyclodextrin (β-CyD) or hydroxypropyl-β-cyclodextrin (HP-β-CyD) caused no particular alteration in the thermotropic parameters of DPPC vesicles, whereas trimethyl-β-cyclodextrin (TM-β-CyD) at molar fractions higher than 0.12 caused broadening of the transition peak due to a possible interaction with the hydrophobic part of the bilayers. Experiments on DPPC–cholesterol (10 mol%) vesicles showed the capability of β-CyD and TM-β-CyD to extract cholesterol from the ordered bilayer structures, triggering an alteration in the lipid constituents of the membranes. HP-β-CyD caused no variation in the thermotropic parameters of the DPPC–cholesterol (10 mol%) vesicles. The findings show that HP-β-CyD seems the most suitable molecular drug carrier forin vivoadministration.     

Lipid Nanoparticles Containing siRNA Synthesized by Microfluidic Mixing Exhibit an Electron-Dense Nanostructured Core

Lipid nanoparticles (LNP) containing ionizable cationic lipids are the leading systems for enabling therapeutic applications of siRNA; however, the structure of these systems has not been defined. Here we examine the structure of LNP siRNA systems containing DLinKC2-DMA(an ionizable cationic lipid), phospholipid, cholesterol and a polyethylene glycol (PEG) lipid formed using a rapid microfluidic mixing process. Techniques employed include cryo-transmission electron microscopy, (31)P NMR, membrane fusion assays, density measurements, and molecular modeling. The experimental results indicate that these LNP siRNA systems have an interior lipid core containing siRNA duplexes complexed to cationic lipid and that the interior core also contains phospholipid and cholesterol. Consistent with experimental observations, molecular modeling calculations indicate that the interior of LNP siRNA systems exhibits a periodic structure of aqueous compartments, where some compartments contain siRNA. It is concluded that LNP siRNA systems formulated by rapid mixing of an ethanol solution of lipid with an aqueous medium containing siRNA exhibit a nanostructured core. The results give insight into the mechanism whereby LNP siRNA systems are formed, providing an understanding of the high encapsulation efficiencies that can be achieved and information on methods of constructing more sophisticated LNP systems.     

A facile approach for assembling lipid bilayer membranes on template-stripped gold

Lipid vesicles are designed with functional chemical groups to promote vesicle fusion on template-stripped gold (TS Au) surfaces that does not spontaneously occur on unfunctionalized Au surfaces. Three types of vesicles were exposed to TS Au surfaces: (1) vesicles composed of only 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids; (2) vesicles composed of lipid mixtures of 2.5 mol % of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-2000-N-[3-(2-pyridyldithio)propionate] (DSPE-PEG-PDP) and 97.5 mol % of POPC; and (3) vesicles composed of 2.5 mol % of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (DSPE-PEG) and 97.5 mol % POPC. Atomic force microscopy (AFM) topography and force spectroscopy measurements acquired in a fluid environment confirmed tethered lipid bilayer membrane (tLBM) formation only for vesicles composed of 2.5 mol % DSPE-PEG-PDP/97.5 mol % POPC, thus indicating that the sulfur-containing PDP group is necessary to achieve tLBM formation on TS Au via Au-thiolate bonds. Analysis of force-distance curves for 2.5 mol % DSPE-PEG-PDP/97.5 mol % POPC tLBMs on TS Au yielded a breakthrough distance of 4.8 ± 0.4 nm, which is about 1.7 nm thicker than that of POPC lipid bilayer membrane formed on mica. Thus, the PEG group serves as a spacer layer between the tLBM and the TS Au surface. Fluorescence microscopy results indicate that these tLBMs also have greater mechanical stability than solid-supported lipid bilayer membranes made from the same vesicles on mica. The described process for assembling stable tLBMs on Au surfaces is compatible with microdispensing used in array fabrication.     

Characterizing the structural transition of cationic DPPC liposomes from the approach of TEM, SAXS and AFM measurements

The structural transition of L-alpha-dipalmitoylphosphatidylcholine (DPPC) liposomes, caused by the addition of a small amount of stearylamine (SA), has been characterized. It has been reported that the shape of DPPC liposomes changes from multilamellar vesicles to large-unilamellar vesicles at the molar concentration ratio of DPPC/SA=9.5/0.5, however, the possible diving factors for this phenomenon have not so far been presented. Flat lipid membranes consisting of DPPC and SA, prepared by the quasi-Bangham method or the Langmuir-Blodgett (LB) technique, are employed in this study when considering the molecular interaction in and between lipid membranes, which should play a key role for determining the liposome shape. The colloid probe atomic force microscopy reveals that the addition of SA results in an inter-film electrosteric repulsion. This repulsive interaction causes a significant increase in the inter-film distance, which is confirmed with freeze-fracture transmission electron microscopy (FF-TEM) and small-angle X-ray scattering (SAXS), and thereby, the large-unilamellar vesicles are formed for reducing the inter- and intra-firm repulsive forces. Taking the molecular structures into consideration, it seems that the shape transition of DPPC liposomes results from such electrostatic interactions as well as packing geometry of the two components.     

Segregation of lipid and polymer in emulsion droplets captured by confocal laser scanning microscopy

The phase behaviors of the subsystems of the ethyl acetate (EtAc)/monoolein/polyethylene glycol-poly(D,L-lactide-co-glycolide) (PLG)/water system have been determined. EtAc simultaneously solves MO and PLG in a liquid phase, denoted L. Lipid/polymer composite particles have here been formed by emulsification of such an L phase into aqueous solutions. Characterization, by means of confocal laser scanning microscopy, revealed that distinctive lipid domains appear inside the particles. In aqueous solutions, these lipid domains swell and finally leave the concentrated polymer matrix. The system exhibits a suitable phase behavior in order to form lipid/polymer composite particles. These composite particles may be interesting for drug delivery applications.     

Asymmetric distribution of anionic phospholipids in supported lipid bilayers

Lipid bilayers with a controlled content of anionic lipids are a prerequisite for the quantitative study of hydrophobic-electrostatic interactions of proteins with lipid bilayers. Here, the asymmetric distribution of zwitterionic and anionic lipids in supported lipid bilayers is studied by neutron reflectometry. We prepare POPC/POPS (3:1) unilamellar vesicles in a high-salt-concentration buffer. Initially, no fusion of the vesicles to a SiO(2) surface is observed over hours and days. Once the isotonic buffer is exchanged with hypotonic buffer, vesicle fusion and bilayer formation occur by osmotic shock. Neutron reflectivity on the bilayers formed this way reveals the presence of anionic lipids (d(31)-POPS) in the outer bilayer leaflet only, and no POPS is observed in the leaflet facing the SiO(2) substrate. We argue that this asymmetric distribution of POPS is induced by the electrostatic repulsion of the phosphatidylserines from the negatively charged hydroxy surface groups of the silicon block. Such bilayers with controlled and high contents of anionic lipids in the outer leaflet are versatile platforms for studying anionic lipid protein interactions that are key elements in signal transduction pathways in the cytoplasmic leaflet of eukaryotic cells.     

Tethered bilayer lipid membranes studied by simultaneous attenuated total reflectance infrared spectroscopy and electrochemical impedance spectroscopy

The formation of tethered lipid bilayer membranes (tBLMs) from unilamelar vesicles of egg yolk phosphatidylcholine (EggPC) on mixed self-assembled monolayers (SAMs) from varying ratios of 6-mercaptohexanol and EO(3)Cholesteryl on gold has been monitored by simultaneous attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS). The influence of the lipid orientation (and hence the anisotropy) of lipids on a gold film on the dichroic ratio was studied by simulations of spectra with a matrix method for anisotropic layers. It is shown that for certain tilt angles of the dielectric tensor of the adsorbed anisotropic layer dispersive and negative absorption bands are possible. The experimental data indicate that the structure of the assemblies obtained varies with varying SAM composition. On SAMs with a high content of EO(3)Cholesteryl, tBLMs with reduced fluidity are formed. For SAMs with a high content of 6-mercaptohexanol, the results are consistent with the adsorption of flattened vesicles, and spherical vesicles have been found in a small range of surface compositions. The kinetics of the adsorption process is consistent with the assumption of spherical vesicles as long-living intermediates for surfaces of a high 6-mercaptohexanol content. No long-living spherical vesicles have been detected for surfaces with a large fraction of EO(3)Cholesteryl tethers. The observed differences between the surfaces suggest that for the formation of tBLMs (unlike supported BLMs) no critical surface coverage of vesicles is needed prior to lipid bilayer formation.     

Modulation of the physicochemical state of interior agents to prepare controlled release liposomes

To prepare controlled release liposomes, freeze-drying of double emulsions (FDE) was employed to entrap a model drug, topotecan, which has been reported to tend to leak rapidly from vesicles. In addition, hydrogenated soy phosphatidylcholine (HSPC), N-(carbonyl-methoxypolyethyleneglycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-PE), and cholesterol (3:1:1, mass ratio) were used as the vesicle lipid. Different inner aqueous phases (W1) containing topotecan together with a variety of chemicals, such as citrate, sulfate, and divalent copper ions, were used to prepare W1/O/W2 double emulsions, which were then freeze-dried to obtain dry products. Upon rehydration, the dry products formed topotecan-entrapping unilamelar liposomes with an encapsulation efficiency of 80% and a mean diameter of less than 200 nm. The in vitro release experiments demonstrated that the drug release of topotecan-entrapping FDE liposomes could be successfully controlled through altering the state of the incorporated drug by means of protonation, precipitation, or forming a transition metal-complex. Specifically, the formulation of 300 mM CuSO₄ had a drug release half-life of 36 h. This novel method is a promising way to prepare controlled release liposomes that are more suitable for therapeutic application. In addition, the liposome formation mechanism was discussed based on micrographs and the size of the double emulsions and vesicles, as well as the small angle X-ray scattering pattern and transmission electron microscopy images.     

Interaction of charged lipid vesicles with planar bilayer lipid membranes: detection by antibiotic membrane probes.

A technique has been developed for monitoring the interaction of charged phospholipid vesicles with planar bilayer lipid membranes (BLM) by use of the antibiotics Valinomycin, Nonactin, and Monazomycin as surface-charge probes. Anionic phosphatidylserine vesicles, when added to one aqueous compartment of a BLM, are shown to impart negative surface charge to zwitterionic phosphatidylcholine and phosphatidylethanolamine bilayers. The surface charge is distributed asymmetrically, mainly on the vesicular side of the BLM, and is not removed by exchange of the vesicular aqueous solution. Possible mechanisms for the vesicle-BLM interactions are discussed.     

Can a catanionic surfactant mixture act as a drug delivery vehicle?

Surfactants can self-assemble in dilute aqueous solutions into a variety of microstructures, including micelles, vesicles, and bilayers. Recently, there has been an increasing interest in unilamellar vesicles, which are composed of a closed bilayer that separates an inner aqueous compartment from the outer aqueous environment. This interest is motivated by their potential to be applied as vehicles for active agents in drug delivery via several routes of administration. Active drug molecules can be encapsulated in the bilayer membrane if they are lipophilic or in the core of the vesicle if they are hydrophilic. Furthermore vesicles formed by mixing of cationic and anionic surfactants (so called ‘catanionic’ systems) can be used as models for biological membranes as they have low critical micelle concentration (cmc) and are highly biocompatible. In this work the formation of amino acid based mixed surfactant vesicles and their stabilization and biocompatibility were studied systematically using several instrumental techniques.     

Making a tool of an artifact: the application of photoinduced Lo domains in giant unilamellar vesicles to the study of Lo/Ld phase spinodal decomposition and its modulation by the ganglioside GM1

Electroformed giant unilamellar vesicles containing liquid-ordered Lo domains are important tools for the modeling of the physicochemical properties and biological functions of lipid rafts. Lo domains are usually imaged using fluorescence microscopy of differentially phase-partionioning membrane-embedded probes. Recently, it has been shown that these probes also have a photosensitizing effect that leads to lipid chemical modification during the fluorescence microscopy experiments. Moreover, the lipid reaction products are able as such to promote Lo microdomain formation, leading to potential artifacts. We show here that this photoinduced effect can also purposely be used as a new approach to study Lo microdomain formation in giant unilamellar vesicles. Photosensitized lipid modification can promote Lo microdomain appearance and growth uniformly and on a faster time scale, thereby yielding new information on such processes. For instance, in egg phosphatidylcholine/egg sphingomyelin/cholesterol 50:30:20 (mol/mol) giant unilamellar vesicles, photoinduced Lo microdomain formation appears to occur by the rarely observed spinodal decomposition process rather than by the common nucleation process usually observed for Lo domain formation in bilayers. Moreover, temperature and the presence of the ganglioside GM1 have a profound effect on the morphological outcome of the photoinduced phase separation, eventually leading to features such as bicontinuous phases, phase percolation inversions, and patterns evoking double phase separations. GM1 also has the effect of destabilizing Lo microdomains. These properties may have consequences for Lo nanodomains stability and therefore for raft dynamics in biomembranes. Our data show that photoinduced Lo microdomains can be used to obtain new data on fast raft-mimicking processes in giant unilamellar vesicles.     

On the effect of the solid support on the interleaflet distribution of lipids in supported lipid bilayers

The adsorption and spreading of lipid vesicles on solid supports has become a popular way to create supported lipid bilayers (SLBs), but little attention has been paid to the possible redistribution of lipid material between the two leaflets of an SLB. We use the technique of quartz crystal microbalance with dissipation monitoring (QCM-D) to follow the adsorption of prothrombin on SLBs formed from sonicated unilamellar vesicles containing mixtures of dioleoylphosphatidylcholine (DOPC) and dioleoylphospatidylserine (DOPS). The specific interaction of prothrombin with negatively charged lipids is quantified and serves as a reporter of the content of accessible DOPS in SLBs. We compare results obtained on silica and mica and find that the underlying support can induce substantial redistribution of lipid material between the two leaflets. In particular, SLBs formed on mica showed a substantially depleted amount of accessible DOPS in the presence of calcium. The mechanisms that lead to the lipid redistribution process are discussed.     

Recombination of nanometric vesicles during freeze-drying

Concentrated dispersions of nanometric lipid vesicles (mean diameter 20 nm) in water/maltose solutions have been freeze-dried and then redispersed in water, yielding again dispersions of lipid vesicles. At each stage of the freeze-drying process, the organization of the vesicles in the dispersion and their size distribution were examined through small-angle neutron scattering and gel permeation chromatography. It was found that the osmotic deswelling of the vesicles caused them to recombine into larger vesicles. A single burst of recombination events occurred when the maltose concentration in the aqueous phase rose above 100 g/L. The final vesicle population was monopopulated, with a central diameter about twice as large as that of the original dispersion.