The effect of cholesterol in small amounts on lipid-bilayer softness in the region of the main phase transition

The temperature dependence of the small-angle neutron scattering from aqueous multilammellar DMPC lipid bilayers, containing small amounts of cholesterol, is analyzed near the main phase transition by means of a simple geometric model which yields the lamellar repeat distance, the hydrophobic thickness of the bilayer, the interlamellar aqueous spacing, as well as fluctuation parameters. The observation of anomalous swelling behavior in the transition region is interpreted as an indication of bilayer softening and thermally reduced bending rigidity. Our results indicate that the effect of small amounts of cholesterol, ≲3 mole%, is a softening of the bilayers in the transition region, whereas cholesterol contents above this range lead to the well-known effect of rigidification. The possible biological relevance of this result is discussed. Received: 24 October 1996 / Accepted: 9 December 1996     

Temperature dependence of the ripple structure in dimyristoylphosphatidylcholine studied by synchrotron X-ray small-angle diffraction

The ripple structure of 1,2-dimyristoyl-L-phosphatidylcholine (DMPC) multibilayer containing excess water (60 wt%) was studied by synchrotron X-ray small-angle diffraction. The (0,1) spacing which corresponds to the ripple repeat distance depends on temperature: At 13 degrees C the (0,1) spacing is 14.15 nm, the spacing decreases at higher temperatures and reaches 12.1 nm at 23.5 degrees C, just below the main transition temperature. The spacing is in good agreement between heating process and cooling process except for the supercooling region. The result suggests that the rearrangement of the ripple structure takes place during temperature change successively. The Landau-de Gennes free energy equation explains well the temperature dependence of the ripple repeat distance.     

The rippled structure in bilayer membranes of phosphatidylcholine and binary mixtures of phosphatidylcholine and cholesterol

Freeze-fracture electron microscopy is used to study the rippled texture in pure dimyristoyl and dipalmitoyl phosphatidylcholine membranes and in mixtures of dimyristoyl phosphatidylcholine and cholesterol. Evidence is presented that the apparent phase transition properties of multilamellar liposomes may be dependent on the manner in which liposomes are prepared. Under certain conditions the ripple structures as visualized by freeze-fracture electron microscopy for the pure phosphatidylcholines are observed to be temperature dependent in the vicinity of the pretransition. Thus the transition can sometimes appear to be a gradual transition rather than a sharp, first-order phase transition. In mixtures of dimyristoyl phosphatidylcholine and cholesterol, the ripple repeat distance is found to increase as the cholesterol concentration is increased between 0 and 20 mol%. Above 20 mol%, no rippling is observed. A simple theory is presented for the dependence of ripple repeat spacing on cholesterol concentration in the range 0–20 mol%. This theory accounts for the otherwise inexplicable abrupt increase in the lateral diffusion coefficients of fluorescent lipids in binary mixtures of phosphatidylcholine and cholesterol when the cholesterol concentration is increased above 20 mol%.     

Thermocontrol of solute permeation across polymer membrane composed of poly(N,N-dimethylaminoethyl methacrylate) and its copolymers

Polymer membranes composed ofN,N-dimethylaminoethyl methacrylate (DMAEMA) and acrylamide (AAm) (or ethyl acrylamide (EAAm)) were prepared to demonstrate the thermocontrol of solute permeation. Poly DMEMA has a lower critical solution temperature (LCST) at around 50°C in water. With the copolymerization of DMAEMA with AAm (or EAAm), a shift in the LCST to a lower temperature was observed, probably due to the formation of hydrogen bonds between the amide andN,N-dimethylamino groups. However, the temperature-induced phase transition of poly (DMAEMA-co-EAAm) did not show a similar trend to that of poly (DMAEMA-co-AAm) in the gel state. The hydrogen bonds in poly (DMAEMA-co-EAAm) were significantly disrupted with the formation of a gel network, which led to a difference in the swelling behavior of polymer gels in response to temperature. To apply these polymers to temperature-sensitive solute permeation, polymer membranes were prepared. The permeation pattern of hydrocortisone, used as the model solute, was explained based on the temperature-sensitive swelling behavior of the polymer membranes.     

The effect of dolichol on the structure and phase behaviour of phospholipid model membranes

The effect of dolichol C(95) on the structure and thermotropic phase behaviour of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine and stearoyloleoylphosphatidylethanolamine has been examined by synchrotron X-ray diffraction and differential scanning calorimetry. The presence of dolichol C(95) had no detectable effects on the temperature of either the gel to ripple or the ripple to liquid-crystal phase transition of dipalmitoylphosphatidylcholine. A proportionate increase of a few degrees in the temperature of the gel to lamellar liquid-crystal phase transition is observed in dispersions of dipalmitoylphosphatidylethanolamine and significantly there is a decrease in the temperature of the lamellar to non-lamellar phase transition of stearoyloleoylphosphatidylethanolamine. There was no significant change in the bilayer repeat spacing of all three mixed dispersions in gel phase in the presence of up to 20 mol% dolichol C(95). Electron density calculations showed that there was no change of bilayer thickness of dipalmitoylphosphatidylcholine with incorporation of up to 7.5 mol% dolichol C(95). These data suggest that effect of dolichol on the phospholipid model membranes depend on both the head group and the hydrocarbon chains of the phospholipid molecules. The presence of dolichol in phosphatidylcholine bilayers conforms to a model in which the polyisoprene compound is phase separated into a central domain sandwiched between the two monolayers in gel phase. In bilayers of phosphatidylethanolamines dolichol tends to stabilize the bilayers in gel phase at low temperatures and destabilize the bilayers in lamellar disordered structure at high temperatures. Non-lamellar structures coexist with lamellar disordered phase over a wide temperature range suggesting that dolichol is enriched in domains of non-lamellar structure and depleted from lamellar phase. These findings are useful to understand the function of dolichol in cell membranes.     

Lateral phase separations and structural integrity of the inner membrane of rat-liver mitochondria: Effect of compression. Implications in the centrifugation of these organelles

When maintained in the vicinity of the lower transition temperature of their membrane lipids, rat-liver mitochondria undergo lysis as shown by the release of malate dehydrogenase, (an enzyme located within the mitochondrial matrix), in the surrounding medium.Structural changes take place in the membranes of mitochondria subjected to increasing pressure at 0°C, when the pressure reaches 750 kg/cm². Freeze-fracture electron microscopy shows the appearance of smooth areas devoid of particles in fracture faces of mitochondrial membranes, together with zones, where aggregated particles can be seen. Concurrently, a suppression of the malate dehydrogenase structure-linked latency is observed. These structural changes can be prevented by increasing the temperature at which compression is performed. The freeze-etching observations suggest that lateral phase separations occur in mitochondrial membranes subjected to high pressure. This can be explained by supposing that pressure promotes the gel-phase appearance in a lipid system and raises the transition temperature since the transition liquid crystal → gel is accompanied by a decrease in volume. The deterioration of mitochondria subjected to high pressure is interpreted as a result of the lateral phase separation induced by compression in the membranes.These results are discussed with respect to our interpretation of the damaging effects that hydrostatic pressure, generated by centrifugation, exerts on rat-liver mitochondria.     

Properties of bilayer membranes in the phase transition or phase separation region

The increase in passive permeability of bilayer membranes near the phase transition temperature is usually explained as caused by either the increase in the amount of ‘boundary lipid’ present in the membrane, or by the increase in lateral compressibility of the membrane. Since both the amount of ‘boundary lipid’ and the lateral compressibility show a similar anomaly near the transition temperature, it is difficult to distinguish experimentally between the two proposed mechanisms.We have examined some details of both of the proposed pictures. The fluid-solid boundary energy, neglected in previous work, has been computed as a function of the domain size. For a single component uncharged lipid bilayer, the results rule out the existence of even loosely defined solid domains in a fluid phase, or vice versa. Thermodynamic fluctuations, which are responsible for anomalous behaviour near the phase transition temperature, are not intense enough to approximate the formation of a domain of the opposite phase.Turning next to lateral compressibility of bilayer membranes we have considered two-component mixtures in the phase separation region. We present the first calculation of lateral compressibility for such systems. The behaviour shows interesting anomalies, which should correlate with existing and future data on transport across membranes.     

Partial purification, from Xenopus laevis oocytes, of an ATP-dependent activity required for nucleosome spacing in vitro.

A critical feature of chromatin with regard to structure and function is the regular spacing of nucleosomes. In vivo, spacing of nucleosomes occurs in at least two steps, but the mechanism is not understood. In this report, we have mimicked the two-step process in vitro. A novel spacing activity has been partially purified from Xenopus laevis ovaries. When this activity is added, either at the beginning or at the end of a nucleosomal assembly reaction, it can convert a DNA template consisting of irregularly spaced nucleosomes into a chromatin structure made up of regularly spaced nucleosomes with a repeat length of about 165 base pairs. The reaction requires ATP. Histone H1 is able to increase the nucleosomal repeat from 165 to 190 base pairs. This two-step increase in nucleosomal repeat length suggests that both the spacing activity and histone H1 contribute to generating repeat lengths of greater than 165 base pairs and that their contributions may be additive. Alternatively, the critical step in the spacing reaction may not be the formation of the 165-base pair repeat but may be the sliding of nucleosomes or the reorganization of the octamer structure induced by the spacing activity.     

The effect of dolichol on the structure and phase behaviour of phospholipid model membranes

The effect of dolichol C₉₅ on the structure and thermotropic phase behaviour of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine and stearoyloleoylphosphatidylethanolamine has been examined by synchrotron X-ray diffraction and differential scanning calorimetry. The presence of dolichol C₉₅ had no detectible effects on the temperature of either the gel to ripple or the ripple to liquid-crystal phase transition of dipalmitoylphosphatidylcholine. A proportionate increase of a few degrees in the temperature of the gel to lamellar liquid-crystal phase transition is observed in dispersions of dipalmitoylphosphatidylethanolamine and significantly there is a decrease in the temperature of the lamellar to non-lamellar phase transition of stearoyloleoylphosphatidylethanolamine. There was no significant change in the bilayer repeat spacing of all three mixed dispersions in gel phase in the presence of up to 20 mol% dolichol C₉₅. Electron density calculations showed that there was no change of bilayer thickness of dipalmitoylphosphatidylcholine with incorporation of up to 7.5 mol% dolichol C₉₅. These data suggest that effect of dolichol on the phospholipid model membranes depend on both the head group and the hydrocarbon chains of the phospholipid molecules. The presence of dolichol in phosphatidylcholine bilayers conforms to a model in which the polyisoprene compound is phase separated into a central domain sandwiched between the two monolayers in gel phase. In bilayers of phosphatidylethanolamines dolichol tends to stabilize the bilayers in gel phase at low temperatures and destabilize the bilayers in lamellar disordered structure at high temperatures. Non-lamellar structures coexist with lamellar disordered phase over a wide temperature range suggesting that dolichol is enriched in domains of non-lamellar structure and depleted from lamellar phase. These findings are useful to understand the function of dolichol in cell membranes.     

Transition processes in stratum corneum model lipid membranes with a mixture of free fatty acids

The hydration of model membranes based on ceramide 6 with a mixture of free fatty acids most commonly encountered in the native lipid matrix of stratum corneum, the outermost layer of the mammalian skin, has been studied by neutron diffraction. Membrane hydration with water vapor at a temperature of 25°C is characterized by a small increase in the repeat distance Δd ₀ = 1.0 Å, which is comparable with membrane swelling in the presence of excess water. The kinetics of changes in the repeat distance, connected with an increase of the water layer between bilayers during hydration, and water exchange during the processes of hydration and H-D isotopic substitution, consists of a fast initial and a subsequent slow stage and is well described by exponentials with two characteristic times lying in the range from a few tens of minutes to several hundreds of minutes. During hydration at a temperature of 57°C, the repeat distance increases by Δd ₀ = 1.6 Å, after which the membrane irreversibly separates into two phases. One of the phases is formed mainly by long-chain free fatty acids and is characterized by a large decrease in the repeat distance Δd _ph = 8.3 Å on dehydration. The investigation of the structure of model membranes in the temperature range 20–72°C indicated that the system with 20% (w) of cholesterol in the range of 63–67°C undergoes a structural phase transition caused by the melting of hydrocarbon chains of lipids. In the system with a smaller content of cholesterol, no phase transition was observed up to a temperature of 72°C.     

Alcohol interaction with high entropy states of macromolecules: critical temperature hypothesis for anesthesia cutoff.

Nerve excitation generates heat and decreases the entropy (review by Ritchie and Keynes (1985) Q. Rev. Biophys. 18, 451-476). The data suggest the existence of at least two thermodynamically identifiable states: resting and excited, with a thermotropic transition between the two. We envision that nerve excitation is a transition between the two states of the excitation machinery consisting of proteins and lipids, rather than the sodium channel protein alone. Presumably, both proteins and lipids change their conformation at excitation. We proposed (Kaminoh et al. (1991) Ann. N.Y. Acad. Sci. 625, 315-317) that anesthesia occurs when compounds have a higher affinity to the resting state than to the excited state of excitable membranes, and that there is a critical temperature above which the affinity to the excited state becomes greater than to the resting state. When the temperature exceeds this critical level, compounds lose their anesthetic potency. We used thermotropic phase-transition of macromolecules as a model for the excitation process. Anesthetic alcohols decreased the main transition temperature of dipalmitoylphosphatidylcholine (DPPC) membranes and also the temperature of the alpha-helix to beta-sheet transition of poly(L-lysine). The affinity of alcohols to the high- and low-temperature states of the DPPC membranes were separately estimated. The difference in the affinity of n-alcohols to the liquid (high-temperature) and solid (low-temperature) states correlated with their anesthetic potency. It is not the total number of bound anesthetic molecules that determines the anesthesia, rather, the difference in the affinity between the higher and lower entropy states determines the effects. The critical temperatures of the long-chain alcohols were found to be lower than those of the short-chain alcohols. Cutoff occurs when the critical temperature of long-chain alcohols is below the physiological temperature, such that the anesthetic potency is not manifested in the experimental temperature range.     

Phase transitions of phospholipid vesicles under osmotic stress and in the presence of ethylene glycol.

The effects of poly(ethylene glycol) (PEG) on the phase transition of phospholipid multilamellar vesicles (MLVs) were investigated by using differential scanning calorimetry (DSC). Main transition temperature (Tm) and the pre-transition temperature (Tp) of neutral phospholipid-, DMPC-1, DPPC- and DSPC-MLVs increased with an increase in PEG concentration. The subtransition temperature of DPPC-MLV also increased with an increase in PEG concentration. These results could be qualitatively explained by enhancement of the lateral packing on the basis of the osmoelastic coupling theory. The pretransition temperature increased faster than the main transition temperature did with an increase in PEG concentration. The increment of Tm depended on the hydrocarbon chain length, the shorter the hydrocarbon chain length was, the larger the increment was. The transition width in the DSC peak was broadened with an increase in PEG concentration. These three above-mentioned effects are the main differences between the effects of the osmotic stress on the phase transition of MLVs and those of hydrostatic pressure. On the other hand, ethylene glycol (EG), which is the monomer of PEG, had a biphasic effect on transition temperature of DPPC-, DSPC-, and DMPC-MLV, reducing Tm and Tp at low concentrations, but increasing Tm and extinguishing pretransition at high concentrations. This is explained by the induction of an interdigitated gel phase at high concentrations of EG, which indicates that EG can easily penetrate into the head group region of the lipid, in contrast with PEG 6K, because EG is small. Temperature-EG concentration phase diagrams for the various PC-MLVs were determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anomaly of transport of dipicrylamine anions across the central barrier of planar dipalmitoylphosphatidylcholine bilayer membranes at the phase transition

The rate of translocation of the hydrophobic ion dipicrylamine across planar lipid membranes formed from dipalmitoyllecithin in n-decane was determined by voltage jump relaxation experiments. The activation energy of the rate constant shows a change from a positive to a negative value at about 42°C near the main phase transition temperature of this lipid. Below this temperature, the rate constant was found to increase with decreasing temperature. This anomalous behaviour extends over a temperature range of at least 10 K and may be formally interpreted as an enhanced mobility of dipicrylamine in the solid state of the membrane.     

Dimyristoylphosphatidylcholine/C16:0-ceramide binary liposomes studied by differential scanning calorimetry and wide- and small-angle x-ray scattering

Ceramide has recently been established as a central messenger in the signaling cascades controlling cell behavior. Physicochemical studies have revealed a strong tendency of this lipid toward phase separation in mixtures with phosphatidylcholines. The thermal phase behavior and structure of fully hydrated binary membranes composed of dimyristoylphosphatidylcholine (DMPC) and N-palmitoyl-ceramide (C16:0-ceramide, up to a mole fraction X(cer) = 0.35) were resolved in further detail by high-sensitivity differential scanning calorimetry (DSC) and x-ray diffraction. Both methods reveal very strong hysteresis in the thermal phase behavior of ceramide-containing membranes. A partial phase diagram was constructed based on results from a combination of these two methods. DSC heating scans show that with increased X(cer) the pretransition temperature T(p) first increases, whereafter at X(cer) > 0.06 it can no longer be resolved. The main transition enthalpy DeltaH remains practically unaltered while its width increases significantly, and the upper phase boundary temperature of the mixture shifts to approximately 63 degrees C at X(cer) = 0.30. Upon cooling, profound phase separation is evident, and for all of the studied compositions there is an endotherm in the region close to the T(m) for DMPC. At X(cer) >/= 0.03 a second endotherm is evident at higher temperatures, starting at 32.1 degrees C and reaching 54.6 degrees C at X(cer) = 0.30. X-ray small-angle reflection heating scans reveal a lamellar phase within the temperature range of 15-60 degrees C, regardless of composition. The pretransition is observed up to X(cer) < 0.18, together with an increase in T(p). In the gel phase the lamellar repeat distance d increases from approximately 61 A at X(cer) = 0. 03, to 67 A at X(cer) = 0.35. In the fluid phase increasing X(cer) from 0.06 to 0.35 augments d from 61 A to 64 A. An L(beta')/L(alpha) (ripple/fluid) phase coexistence region is observed at high temperatures (from 31 to 56.5 degrees C) when X(cer) > 0.03. With cooling from temperatures above 50 degrees C we observe a slow increase in d as the coexistence region is entered. A sudden solidification into a metastable, modulated gel phase with high d values is observed for all compositions at approximately 24 degrees C. The anomalous swelling for up to X(cer) = 0.30 in the transition region is interpreted as an indication of bilayer softening and thermally reduced bending rigidity.     

NEW EXPERIMENTS ON ANOMALOUS OSMOSIS

1. It is impossible to reproduce Loeb''s observations on anomalous osmosis with membranes prepared from relatively pure brands of collodion, whereas positive effects can be obtained using collodion containing acidic impurities. 2. The inactive (purer) collodion membranes may be activated by oxidation with NaOBr solution. 3. Properly oxidized membranes give much greater anomalous osmotic effects than those described by Loeb.     

Modulation of the bilayer to hexagonal phase transition and solvation of phosphatidylethanolamines in aqueous salt solutions

Several salts affect the temperature of the bilayer to hexagonal phase transition of phosphatidylethanolamines. Their effects are dependent on the anion as well as the cation of the salt. Salt effects on this transition can be explained by preferential hydration and ion binding. Those salts which are excluded from the solvation sphere of the membrane promote hexagonal phase formation. For example, Na2SO4 promotes preferential hydration and is a hexagonal phase promoter while NaSCN does not do this and is a bilayer stabilizer. Unlike amphiphiles and hydrocarbons, salts can shift the bilayer to hexagonal phase transition temperature without altering the cooperativity of the transition. The effect of these salts on the gel to liquid-crystal transition is opposite to their effect on the bilayer to hexagonal phase transition. We also find that MnCl2 markedly raises the gel to liquid-crystal transition temperature. This effect is due to binding of the cation to the membrane surface. The effect is reduced with MnSO4 because of preferential hydration. Our results demonstrate that the nature of the anion as well as the cation can alter the effect of salts on lipid phase transition properties. The observed effects can be explained as resulting from preferential hydration and ion binding.     

X-ray diffraction studies of the corneal stroma.

A low-angle diffraction pattern has been obtained from corneal stroma. This pattern arises both from the arrangement of the collagen fibrils and from the packing of the tropocollagen molecules along the axes of the fibrils. The spacing arising from the packing of the fibrils increases homogeneously on swelling although the tissue as a whole swells only radially referred to the intact eye. The necessary rearrangement of the fibrils for this type of swelling to occur might result in the formation of regions devoid of collagen fibrils and the water not in the lattice of collagen fibrils could be synonymous with the lakes postulated by Benedek (1971) to explain the loss of transparency on swelling.The spacings due to the packing of the tropocollagen molecules are unusual in that, although they index as the third and fifth orders of the well-known 66 nm repeat, the first order of this spacing is absent. Calculation of the Patterson function for corneal collagen leads to peaks in electron density separated by distances of 0.38 and 0.24 of the repeat distance.     

A solid-solution theory of anesthetic interaction with lipid membranes: temperature span of the main phase transition

Anesthetics (or any other small additives) depress the temperature of the main phase transition of phospholipid bilayers. Certain anesthetics widen the temperature span of the transition, whereas others do not. The widening in a first-order phase transition is intriguing. In this report, the effects of additive molecules on the temperature and its span were explained by the solid-solution theory. By assuming coexistence of the liquid-crystal and solid-gel phases of lipid membranes at phase transition, the phase boundary is determined from the distribution of anesthetic molecules between the liquid-crystal membrane versus water and between the solid-gel membrane versus water. The theory shows that when the lipid concentration is large or when the lipid solubility of the drug is large, the width of the transition temperature increases, and vice versa. Highly lipid-soluble molecules, such as long-chain alkanols and volatile anesthetics, increase the width of the transition temperature when the lipid:water ratio is large, whereas highly water-soluble molecules, such as methanol and ethanol, do not. The aqueous phase serves as the reservoir for anesthetics. Depletion of the additive molecules from the aqueous phase is the cause of the widening. When the reservoir capacity is large, the temperature width does not increase. The theory also predicts asymmetry of the specific heat profile at the transition.     

A neutron diffraction study of the influence of ions on phospholipid membrane interactions.

Neutron diffraction is used to examine the effects of Ca2+ and ClO4- ions on interactions and some structural features of dipalmitoylphosphatidylcholine membranes in both solid and fluid lamellar phases. The results are described within the framework of Derjaguin-Landau-Verwey-Overbeek (DLVO) theory with reference to electrostatic, van der Waals, and hydration components of disjoining pressure. The Hamaker constants are evaluated under equilibrium conditions. Addition of 100 mM CaCl2 to the aqueous phase substantially increases the lamellar repeat spacing (d), which is interpreted in terms of adsorption of Ca2+ ions to bilayers followed by electrostatic repulsion between membranes. The rise of NaClO4 concentration in the presence of 100 mM CaCl2 leads to gradual decrease in d, evidently resulted from the diminution of Ca(2+)-induced positive surface potential by both electrostatic screening and binding of ClO4- ions. In the absence of CaCl2, elevation of NaClO4 concentration to 100-300 mM drastically enhances the repeat spacing and then dramatically decreases d at about 1 M NaClO4. Estimation of the hydration coefficients showed that the pronounced decrease of the repeat spacing at high NaClO4 concentrations was resulted mainly from the (partial) disruption of the structure of intermembrane bound water by chaotropic ClO4- ions and subsequent decrease in hydration repulsive pressure. Moreover, in the case of solid membranes (20 degrees C) high concentrations of ClO4- induced formation of interdigitated phase paralleled with marked reduction in bilayer thickness and corresponding increase in the effective cross-sectional area per lipid molecule.     

Shear stress induced lipid order and permeability changes of giant unilamellar vesicles

BackgroundThe permeability of a lipid bilayer is a function of its phase state and depends non-linearly on thermodynamic variables such as temperature, pressure or pH. We investigated how shear forces influence the phase state of giant unilamellar vesicles and their membrane permeability.MethodsWe determined the permeability of giant unilamellar vesicles composed of different phospholipid species under shear flow in a tube at various temperatures around and far off the melting point by analyzing the release of fluorescently labelled dextran. Furthermore, we quantified phase state changes of these vesicles under shear forces using spectral decomposition of the membrane embedded fluorescent dye Laurdan.ResultsWe observed that the membrane permeability follows a step function with increasing permeability at the transition from the gel to the fluid phase and vice versa. Second, there was an all-or-nothing permeabilization near the main phase transition temperature and a gradual dye release far off the melting transition. Third, the Laurdan phase state analysis suggests that shear forces induce a reversible melting temperature shift in giant unilamellar vesicle membranes.Major conclusionsThe observed effects can be explained best in a scenario in which shear forces directly induce membrane pores that possess relatively long pore lifetimes in proximity to the phase transition.General significanceOur study elucidates the release mechanism of thermo-responsive drug carriers as we found that liposome permeabilization is not continuous but quantized. Furthermore, the shear force induced melting temperature shift must be taken into consideration when thermo-responsive liposomes are designed.