Membrane permeability changes at early stages of influenza hemagglutinin-mediated fusion

While biological membrane fusion is classically defined as the leak-free merger of membranes and contents, leakage is a finding in both experimental and theoretical studies. The fusion stages, if any, that allow membrane permeation are uncharted. In this study we monitored membrane ionic permeability at early stages of fusion mediated by the fusogenic protein influenza hemagglutinin (HA). HAb2 cells, expressing HA on their plasma membrane, fused with human red blood cells, cultured liver cells PLC/PRF/5, or planar phospholipid bilayer membranes. With a probability that depended upon the target membrane, an increase of the electrical conductance of the fusing membranes (leakage) by up to several nS was generally detected. This leakage was recorded at the initial stages of fusion, when fusion pores formed. This leakage usually accompanied the "flickering" stage of the early fusion pore development. As the pore widened, the leakage reduced; concomitantly, the lipid exchange between the fusing membranes accelerated. We conclude that during fusion pore formation, HA locally and temporarily increases the permeability of fusing membranes. Subsequent rearrangement in the fusion complex leads to the resealing of the leaky membranes and enlargement of the pore.     

Membrane mechanics can account for fusion pore dilation in stages.

Once formed, fusion pores rapidly enlarge to semi-stable conductance values. The membranes lining the fusion pore are continuous bilayer structures, so variations of conductance in time reflect bending and stretching of membranes. We therefore modeled the evolution of fusion pores using the theory of the mechanics of deforming homogeneous membranes. We calculated the changes in length and width of theoretical fusion pores according to standard dynamical equations of motion. Theoretical fusion pores quickly achieve semi-stable dimensions, which correspond to energy minima located in a canyon between energy barriers. The height of the barrier preventing pore expansion diminishes along the dimensions of length and width. The bottom of the canyon slopes gently downward along increasing length. As a consequence, theoretical fusion pores slowly lengthen and widen as the dimensions migrate along the bottom of the canyon, until the barrier vanishes and the pore rapidly enlarges. The dynamics of growth is sensitive to tension, spontaneous curvature, bending elasticity, and mobilities. This sensitivity can account for the quantitative differences in pore evolution observed in two experimental systems: HA-expressing cells fusing to planar bilayer membranes and beige mouse mast cell degranulation. We conclude that the mechanics of membranes could cause the phenomenon of stagewise growth of fusion pores.     

The Pathway of Membrane Fusion Catalyzed by Influenza Hemagglutinin: Restriction of Lipids, Hemifusion, and Lipidic Fusion Pore Formation

The mechanism of bilayer unification in biological fusion is unclear. We reversibly arrested hemagglutinin (HA)-mediated cell–cell fusion right before fusion pore opening. A low-pH conformation of HA was required to form this intermediate and to ensure fusion beyond it. We present evidence indicating that outer monolayers of the fusing membranes were merged and continuous in this intermediate, but HA restricted lipid mixing. Depending on the surface density of HA and the membrane lipid composition, this restricted hemifusion intermediate either transformed into a fusion pore or expanded into an unrestricted hemifusion, without pores but with unrestricted lipid mixing. Our results suggest that restriction of lipid flux by a ring of activated HA is necessary for successful fusion, during which a lipidic fusion pore develops in a local and transient hemifusion diaphragm.     

Lipid flow through fusion pores connecting membranes of different tensions.

When two membranes fuse, their components mix; this is usually described as a purely diffusional process. However, if the membranes are under different tensions, the material will spread predominantly by convection. We use standard fluid mechanics to rigorously calculate the steady-state convective flux of lipids. A fusion pore is modeled as a toroid shape, connecting two planar membranes. Each of the membrane monolayers is considered separately as incompressible viscous media with the same shear viscosity, etas. The two monolayers interact by sliding past each other, described by an intermonolayer viscosity, etar. Combining a continuity equation with an equation that balances the work provided by the tension difference, Deltasigma, against the energy dissipated by flow in the viscous membrane, yields expressions for lipid velocity, upsilon, and area of lipid flux, Phi. These expressions for upsilon and Phi depend on Deltasigma, etas, etar, and geometrical aspects of a toroidal pore, but the general features of the theory hold for any fusion pore that has a roughly hourglass shape. These expressions are readily applicable to data from any experiments that monitor movement of lipid dye between fused membranes under different tensions. Lipid velocity increases nonlinearly from a small value for small pore radii, rp, to a saturating value at large rp. As a result of velocity saturation, the flux increases linearly with pore radius for large pores. The calculated lipid flux is in agreement with available experimental data for both large and transient fusion pores.     

Interplay between membrane dynamics, diffusion and swelling pressure governs individual vesicular exocytotic events during release of adrenaline by chromaffin cells

Release of adrenaline by chromaffin cells occurs through a process involving docking and then fusion of a secretory vesicle to the cytoplasmic membrane of the cell. Fusion proceeds in two main stages. The first one leads to the creation of a stable fusion pore passing through the two membranes and which gives a constant release flux of neurotransmitter (pore-release stage). After a few milliseconds, this initial stage which is not investigated here proceeds through a sudden enlargement of the initial pore (full-fusion stage) up to the complete incorporation of the vesicle membrane into that of the cell and total exposure of the initial matrix vesicle core to the extracellular fluid. The precise time-resolved dynamics of the release and of the vesicle membrane during the full-fusion phase can be extracted with a precision never achieved so far by de-convolution of experimental chronoamperometric currents monitored during individual exocytotic secretion events. The peculiar dynamics of the vesicle membrane proves that exocytotic events are powered by the swelling of the matrix polyelectrolyte core of the vesicle, although they are kinetically regulated by diffusion in the matrix and by the dynamics of the vesicle and cell membranes. Two simple theoretical models based on the dynamics of pores are developed to account for these dynamics and are shown to predict behaviors which are essentially identical to the experimental ones. This offers a new view of the kinetic grounds which control the full-fusion stage, and therefore provides a new interpretation of the sudden transition between the pore-release and the full-fusion stages. This transition occurs when the increasing membrane surface tension energy due to the refrained internal swelling pressure overcomes the edge energy of the pore, so that the initial fusion pore becomes unstable and is disrupted. This new view predicts that secretory vesicles which contain matrixes energetically similar to those of the adrenal cells investigated here can be separated into two classes according to their radius and catecholamine content. Small vesicles (less than ca. 25 nm radius, and containing less than ca. 20000 molecules) should always release through pores. Larger vesicles should always end into fusing except if another mechanism closes the pore before ca. 10000 molecules of catecholamines have been released.     

The Mechanism of a Nuclear Pore Assembly: A Molecular Biophysics View

The basic problem of nuclear pore assembly is the big perinuclear space that must be overcome for nuclear membrane fusion and pore creation. Our investigations of ternary complexes: DNA–PC liposomes–Mg²⁺, and modern conceptions of nuclear pore structure allowed us to introduce a new mechanism of nuclear pore assembly. DNA-induced fusion of liposomes (membrane vesicles) with a single-lipid bilayer or two closely located nuclear membranes is considered. After such fusion on the lipid bilayer surface, traces of a complex of ssDNA with lipids were revealed. At fusion of two identical small liposomes (membrane vesicles) <100 nm in diameter, a “big” liposome (vesicle) with ssDNA on the vesicle equator is formed. ssDNA occurrence on liposome surface gives a biphasic character to the fusion kinetics. The “big” membrane vesicle surrounded by ssDNA is the base of nuclear pore assembly. Its contact with the nuclear envelope leads to fast fusion of half of the vesicles with one nuclear membrane; then ensues a fusion delay when ssDNA reaches the membrane. The next step is to turn inside out the second vesicle half and its fusion to other nuclear membrane. A hole is formed between the two membranes, and nucleoporins begin pore complex assembly around the ssDNA. The surface tension of vesicles and nuclear membranes along with the kinetic energy of a liquid inside a vesicle play the main roles in this process. Special cases of nuclear pore formation are considered: pore formation on both nuclear envelope sides, the difference of pores formed in various cell-cycle phases and linear nuclear pore clusters.     

Molecular dynamics simulations of hydrophilic pores in lipid bilayers

Hydrophilic pores are formed in peptide free lipid bilayers under mechanical stress. It has been proposed that the transport of ionic species across such membranes is largely determined by the existence of such meta-stable hydrophilic pores. To study the properties of these structures and understand the mechanism by which pore expansion leads to membrane rupture, a series of molecular dynamics simulations of a dipalmitoylphosphatidylcholine (DPPC) bilayer have been conducted. The system was simulated in two different states; first, as a bilayer containing a meta-stable pore and second, as an equilibrated bilayer without a pore. Surface tension in both cases was applied to study the formation and stability of hydrophilic pores inside the bilayers. It is observed that below a critical threshold tension of approximately 38 mN/m the pores are stabilized. The minimum radius at which a pore can be stabilized is 0.7 nm. Based on the critical threshold tension the line tension of the bilayer was estimated to be approximately 3 x 10(-11) N, in good agreement with experimental measurements. The flux of water molecules through these stabilized pores was analyzed, and the structure and size of the pores characterized. When the lateral pressure exceeds the threshold tension, the pores become unstable and start to expand causing the rupture of the membrane. In the simulations the mechanical threshold tension necessary to cause rupture of the membrane on a nanosecond timescale is much higher in the case of the equilibrated bilayers, as compared with membranes containing preexisting pores.     

Dynamically stabilized pores in bilayer membranes.

Zhelev and Needham have recently created large, quasistable pores in artificial lipid bilayer vesicles. Initially created by electroporation, the pores remain open for up to several seconds before quickly snapping shut. This result is surprising, in light of the large line tension for holes in bilayer membranes and the rapid time scale for closure of large pores. We show how pores can be dynamically stabilized via a new feedback mechanism. We also explain quantitatively the observed sudden pore closure as a tangent bifurcation. Finally, we show how Zhelev and Needham's experiment can be used to measure accurately the pore line tension, an important material parameter. For their stearoyloleoylphosphatidylcholine/cholesterol mixture we obtain a line tension of 2.6 x 10(-6) dyn.     

Influenza hemagglutinin-mediated fusion pores connecting cells to planar membranes: flickering to final expansion

We have studied the fusion between voltage-clamped planar lipid bilayers and influenza virus infected MDCK cells, adhered to one side of the bilayer, using measurements of electrical admittance and fluorescence. The changes in currents in-phase and 90 degrees out-of- phase with respect to the applied sinusoidal voltage were used to monitor the addition of the cell membrane capacitance to that of the lipid bilayer through a fusion pore connecting the two membranes. When ethidium bromide was included in the solution of the cell-free side of the bilayer, increases in cell fluorescence accompanied tee admittance changes, independently confirming that these changes were due to formation of a fusion pore. Fusion required acidic pH on the cell- containing side and depended on temperature. For fusion to occur, the influenza hemagglutinin (HA) had to be cleaved into HA1 and HA2 subunits. The incorporation of gangliosides into the planar bilayers greatly augmented fusion. Fusion pores developed in four distinct stages after acidification: (a) a pre-pore, electrically quiescent stage; (b) a flickering stage, with 1-2 nS pores opening and closing repetitively; (c) an irreversibly opened stage, in which pore conductances varied between 2 and 100 nS and exhibited diverse kinetics; (d) a fully opened stage, initiated by an instantaneous, time- resolution limited, increase in conductance leveling at approximately 500 nS. The expansion of pores by stages has also been shown to occur during exocytosis in mast cells and fusion of HA-expressing cells and erythrocytes. We conclude that essential features of fusion pores are produced with proteins in just one of the two fusing membranes.     

The hemifusion intermediate and its conversion to complete fusion: regulation by membrane composition.

To fuse, membranes must bend. The energy of each lipid monolayer with respect to bending is minimized at the spontaneous curvature of the monolayer. Two lipids known to promote opposite spontaneous curvatures, lysophosphatidylcholine and arachidonic acid, were added to different sides of planar phospholipid membranes. Lysophosphatidylcholine added to the contacting monolayers of fusing membranes inhibited the hemifusion we observed between lipid vesicles and planar membranes. In contrast, fusion pore formation depended upon the distal monolayer of the planar membrane; lysophosphatidylcholine promoted and arachidonic acid inhibited. Thus, the intermediates of hemifusion and fusion pores in phospholipid membranes involve different membrane monolayers and may have opposite net curvatures, Biological fusion may proceed through similar intermediates.     

The exocytotic fusion pore interface: a model of the site of neurotransmitter release

infrastructurel techniques have shown that an early event in the exocytotic fusion of a secretory vesicle is the formation of a narrow, water-filled pore spanning both the vesicle and plasma membranes and connecting the lumen of the secretory vesicle to the extracellular environment. Smaller precursors of the exocytotic fusion pore have been detected using electrophysio-logical techniques, which reveal a dynamic fusion pore that quickly expands to the size of the pores seen with electron microscopy. While it is clear that in the latter stages of expansion, when the size of the fusion pore is several orders of magnitude bigger than any known macromolecule, the fusion pore must be mainly made of lipids, the structure of the smaller precursors is unknown. Patch-clamp measurements of the activity of individual fusion pores in mast cells have shown that the fusion pore has some unusual and unexpected properties, namely that there is a large flux of lipid through the pore and the rate of pore closure has a discontinuous temperature dependency, suggesting a purely lipidic fusion pore. Moreover, comparisons of experimental data with theoretical fusion pores and with breakdown pores support the view that the fusion pore is initially a pore through a single bilayer, as would be expected for membrane fusion proceeding through a hemifusion mechanism. Based on these observations we present a model where the fusion pore is initially a pore through a single bilayer. Fusion pore formation is regulated by a macromolecular scaffold of proteins that is responsible for bringing the plasma membrane into a highly curved dimple very close to a tense secretory granule membrane, creating the architecture where the strongly attractive hydrophobic force causes the membranes to form a ‘hemifusion’ intermediate. Membrane fusion is completed by the formation of an aqueous pore after rupture of the shared bilayer. We also propose that the microenvironment of the interface when the pore first opens, dominated by the charged groups on the secretory vesicle matrix and phospholipids, will greatly influence the release of secretory products.     

Hemifusion between cells expressing hemagglutinin of influenza virus and planar membranes can precede the formation of fusion pores that subsequently fully enlarge

The chronological relation between the establishment of lipid continuity and fusion pore formation has been investigated for fusion of cells expressing hemagglutinin (HA) of influenza virus to planar bilayer membranes. Self-quenching concentrations of lipid dye were placed in the planar membrane to monitor lipid mixing, and time-resolved admittance measurements were used to measure fusion pores. For rhodamine-PE, fusion pores always occurred before a detectable amount of dye moved into an HA-expressing cell. However, with DiI in the planar membrane, the relationship was reversed: the spread of dye preceded formation of small pores. In other words, by using DiI as probe, hemifusion was clearly observed to occur before pore formation. For hemifused cells, a small pore could form and subsequently fully enlarge. In contrast, for cells that express a glycosylphosphatidylinositol-anchored ectodomain of HA, hemifusion occurred, but no fully enlarged pores were observed. Therefore, the transmembrane domain of HA is required for the formation of fully enlarging pores. Thus, with the planar bilayer membranes as target, hemifusion can precede pore formation, and the occurrence of lipid dye spread does not preclude formation of pores that can enlarge fully.     

Minimum Membrane Bending Energies of Fusion Pores

Membranes fuse by forming highly curved intermediates, culminating in structures described as fusion pores. These hourglass-like figures that join two fusing membranes have high bending energies, which can be estimated using continuum elasticity models. Fusion pore bending energies depend strongly on shape, and the present study developed a method for determining the shape that minimizes bending energy. This was first applied to a fusion pore modeled as a single surface and then extended to a more realistic model treating a bilayer as two monolayers. For the two-monolayer model, fusion pores were found to have metastable states with energy minima at particular values of the pore diameter and bilayer separation. Fusion pore energies were relatively insensitive to membrane thickness but highly sensitive to spontaneous curvature and membrane asymmetry. With symmetrical bilayers and monolayer spontaneous curvatures of ?0.1 nm^?1 (a typical value) separated by 6 nm (closest distance determined by repulsive hydration forces), fusion pore formation required 43–65 kT. The pore radius of ~2.25 nm fell within the range estimated from conductance measurements. With bilayer separation >6 nm, fusion pore formation required less energy, suggesting that protein scaffolds can promote fusion by bending membranes toward one another. With nonzero spontaneous monolayer curvature, the shape that minimized the energy change during fusion pore formation differed from the shape that minimized its energy after it formed. Thus, a nascent fusion pore will relax spontaneously to a new shape, consistent with the experimentally observed expansion of nascent fusion pores during viral fusion.     

Membrane Bending Energy and Fusion Pore Kinetics in Ca-Triggered Exocytosis

A fusion pore composed of lipid is an obligatory kinetic intermediate of membrane fusion, and its formation requires energy to bend membranes into highly curved shapes. The energetics of such deformations in viral fusion is well established, but the role of membrane bending in Ca²⁺-triggered exocytosis remains largely untested. Amperometry recording showed that during exocytosis in chromaffin and PC12 cells, fusion pores formed by smaller vesicles dilated more rapidly than fusion pores formed by larger vesicles. The logarithm of 1/(fusion pore lifetime) varied linearly with vesicle curvature. The vesicle size dependence of fusion pore lifetime quantitatively accounted for the nonexponential fusion pore lifetime distribution. Experimentally manipulating vesicle size failed to alter the size dependence of fusion pore lifetime. Manipulations of membrane spontaneous curvature altered this dependence, and applying the curvature perturbants to the opposite side of the membrane reversed their effects. These effects of curvature perturbants were opposite to those seen in viral fusion. These results indicate that during Ca²⁺-triggered exocytosis membrane bending opposes fusion pore dilation rather than fusion pore formation. Ca²⁺-triggered exocytosis begins with a proteinaceous fusion pore with less stressed membrane, and becomes lipidic as it dilates, bending membrane into a highly curved shape.     

Pore formation in a lipid bilayer under a tension ramp: modeling the distribution of rupture tensions

The rupture of fluid membrane vesicles with a steady ramp of micropipette suction has been shown to produce a distribution of breakage tensions, with a mean that rises rapidly with tension rate. Starting from a lattice model that incorporates the essential features of the lipid bilayers held together with hydrophobic forces, and developing it to handle varying tension rates, we reproduce the main features of the experimental results. In essence, we show that the rupture kinetics are driven by the nucleation and growth of pores, with two limiting kinetics-growth-limited and nucleation-limited. The model has been extended to address the role of peptides in solution that can adsorb and insert themselves into the bilayer. At concentrations below those required to spontaneously rupture the membrane, the effect of the peptides is to lower the rupture tensions systematically for all tension rates.     

The exocytotic fusion pore modeled as a lipidic pore.

Freeze-fracture electron micrographs from degranulating cells show that the lumen of the secretory granule is connected to the extracellular compartment via large (20 to 150 nm diameter) aqueous pores. These exocytotic fusion pores appear to be made up of a highly curved bilayer that spans the plasma and granule membranes. Conductance measurements, using the patch-clamp technique, have been used to study the fusion pore from the instant it conducts ions. These measurements reveal the presence of early fusion pores that are much smaller than those observed in electron micrographs. Early fusion pores open abruptly, fluctuate, and then either expand irreversibly or close. The molecular structure of these early fusion pores is unknown. In the simplest extremes, these early fusion pores could be either ion channel like protein pores or lipidic pores. Here, we explored the latter possibility, namely that of the early exocytotic fusion pore modeled as a lipid-lined pore whose free energy was composed of curvature elastic energy and work done by tension. Like early exocytotic fusion pores, we found that these lipidic pores could open abruptly, fluctuate, and expand irreversibly. Closure of these lipidic pores could be caused by slight changes in lipid composition. Conductance distributions for stable lipidic pores matched those of exocytotic fusion pores. These findings demonstrate that lipidic pores can exhibit the properties of exocytotic fusion pores, thus providing an alternate framework with which to understand and interpret exocytotic fusion pore data.     

Sterols and sphingolipids strongly affect the growth of fusion pores induced by the hemagglutinin of influenza virus

Cells expressing the hemagglutinin (HA) of influenza virus were fused to planar phospholipid bilayer membranes to evaluate the effects of sterols and sphingolipids in the target bilayer membranes on properties of fusion pores. Typically, in the absence of sterol, flickering pores are observed, followed by a successful pore (i.e., a pore that fully opens). The incorporation of cholesterol into the lipid bilayer had a marked effect: it greatly decreased the number of flickers, and the first pore formed was usually successful. Similar effects were produced by the sterols epicholesterol and 5beta-cholestanol. In contrast, the sterols cholesteryl acetate, coprostanol, and stanolone did not affect pore flickering, and a successful pore was observed to follow the typical number of flickers. 5alpha-cholestanol gave intermediate results. From these results, it follows that the 3-OH of cholesterol is essential to reduce flickering, but it does not matter if the 3-OH is in an alpha or beta configuration. The double bond is also not critical for the actions of cholesterol nor is the fact that it is a flat molecule. The sphingolipids sphingomyelin, lactosyl cerebroside, and glucosyl cerebroside tended to inhibit full pore enlargement, prolonging the stage of pore flickering. If a sphingolipid and a sterol that strongly interact were both included in the planar membrane, the pattern of flickering was the same as if neither had been included in the bilayer. However, if a sphingolipid and sterol that do not interact with each other were included in the bilayer, the reduced flickering characteristic of the sterol was observed.     

The pressure-dependence of the size of extruded vesicles

Variations in the size of vesicles formed by extrusion through small pores are discussed in terms of a simple model. Our model predicts that the radius should decrease as the square root of the applied pressure, consistent with data for vesicles extruded under various conditions. The model also predicts dependencies on the pore size used and on the lysis tension of the vesicles being extruded that are consistent with our data. The pore size was varied by using track-etched polycarbonate membranes with average pore diameters ranging from 50 to 200 nm. To vary the lysis tension, vesicles made from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine), mixtures of POPC and cholesterol, and mixtures of POPC and C(16)-ceramide were studied. The lysis tension, as measured by an extrusion-based technique, of POPC:cholesterol vesicles is higher than that of pure POPC vesicles whereas POPC:ceramide vesicles have lower lysis tensions than POPC vesicles.     

Effect of membrane tension on the electric field and dipole potential of lipid bilayer membrane

The dipole potential of lipid bilayer membrane controls the difference in permeability of the membrane to oppositely charged ions. We have combined molecular dynamics (MD) simulations and experimental studies to determine changes in electric field and electrostatic potential of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer in response to applied membrane tension. MD simulations based on CHARMM36 force field showed that electrostatic potential of DOPC bilayer decreases by ~45mV in the physiologically relevant range of membrane tension values (0 to 15dyn/cm). The electrostatic field exhibits a peak (~0.8×10(9)V/m) near the water/lipid interface which shifts by 0.9? towards the bilayer center at 15dyn/cm. Maximum membrane tension of 15dyn/cm caused 6.4% increase in area per lipid, 4.7% decrease in bilayer thickness and 1.4% increase in the volume of the bilayer. Dipole-potential sensitive fluorescent probes were used to detect membrane tension induced changes in DOPC vesicles exposed to osmotic stress. Experiments confirmed that dipole potential of DOPC bilayer decreases at higher membrane tensions. These results are suggestive of a potentially new mechanosensing mechanism by which mechanically induced structural changes in the lipid bilayer membrane could modulate the function of membrane proteins by altering electrostatic interactions and energetics of protein conformational states.     

Ultradeformable lipid vesicles can penetrate the skin and other semi-permeable barriers unfragmented. Evidence from double label CLSM experiments and direct size measurements

The stability of various aggregates in the form of lipid bilayer vesicles was tested by three different methods before and after crossing different semi-permeable barriers. First, polymer membranes with pores significantly smaller than the average aggregate diameter were used as the skin barrier model; dynamic light scattering was employed to monitor vesicle size changes after barrier passage for several lipid mixtures with different bilayer elasticities. This revealed that vesicles must adapt their size and/or shape, dependent on bilayer stability and elasto-mechanics, to overcome an otherwise confining pore. For the mixed lipid aggregates with highly flexible bilayers (Transfersomes®), the change is transient and only involves vesicle shape and volume adaptation. The constancy of ultradeformable vesicle size before and after pores penetration proves this. This is remarkable in light of the very strong aggregate deformation during an enforced barrier passage. Simple phosphatidylcholine vesicles, with less flexible bilayers, lack such capability and stability. Conventional liposomes are therefore fractured during transport through a semi-permeable barrier; as reported by other researchers, liposomes are fragmented to the size of a narrow pore if sufficient pressure is applied across the barrier; otherwise, liposomes clog the pores. The precise outcome depends on trans-barrier flux and/or on relative vesicle vs. pore size. Lipid vesicles applied on the skin behave accordingly. Mixed lipid vesicles penetrate the skin if they are sufficiently deformable. If this is the case, they cross inter-cellular constrictions in the organ without significant composition or size modification. To prove this, we labelled vesicles with two different fluorescent markers and applied the suspension on intact murine skin without occlusion. The confocal laser scanning microscopy (CLSM) of the skin then revealed a practically indistinguishable distribution of both labels in the stratum corneum, corroborating the first assumption. To confirm the second postulate, we compared vesicle size in the starting suspension and in the blood after non-invasive transcutaneous aggregate delivery. Size exclusion chromatograms of sera from the mice that received ultradeformable vesicles on the skin were undistinguishable from the results measured with the original vesicle suspension. Taken together, the results support our previous postulate that ultradeformable vesicles penetrate the skin intact, that is, without permanent disintegration.