Freeze-fracture planes of methanogen membranes correlate with the content of tetraether lipids.

Methanospirillum hungatei GP1 contained 50% of its ether core lipids (polar lipids less head groups) as tetraether lipids, and its plasma membrane failed to fracture along its hydrophobic domain during freeze-etching. The membrane of Methanosaeta ("Methanothrix") concilii did not contain tetraether lipids and easily fractured to reveal typical intramembranous particles. Methanococcus jannaschii grown at 50 degrees C contained 20% tetraether core lipids, which increased to 45% when cells were grown at 70 degrees C. The frequency of membrane fracture was reduced as the membrane-spanning tetraether lipids approached 45%. As the tetraether lipid content increased, and while fracture was still possible, the particle density in the membrane increased; these added particles could be tetraether lipid complexes torn from the opposing membrane face. The diether membrane (no tetraether lipid) of Methanococcus voltae easily fractured, and the intramembranous particle density was low. Protein-free liposomes containing tetraether core lipids (ca. 45%) also did not fracture, whereas those made up exclusively of diether lipids did split, indicating that tetraether lipids add considerable vertical stability to the membrane. At tetraether lipid concentrations below 45%, liposome bilayers fractured to reveal small intramembranous particles which we interpret to be tetraether lipid complexes.     

Functional reconstitution of membrane proteins in monolayer liposomes from bipolar lipids of Sulfolobus acidocaldarius.

Membranes of Sulfolobus acidocaldarius, an extreme thermophilic archaebacterium, are composed of unusual bipolar lipids. They consist of macrocyclic tetraethers with two polar heads linked by two hydrophobic C40 phytanyl chains which are thought to be arranged as a monolayer in the cytoplasmic membrane. Fractionation of a total lipid-extract from S. acidocaldarius yielded a lipid fraction which forms closed and stable unilamellar liposomes in aqueous media. Beef heart cytochrome c-oxidase could be functionally reconstituted in these liposomes. In the presence of reduced cytochrome c, a protonmotive force (delta p) across the liposomal membrane was generated of up to -92 mV. Upon fusion of these proteoliposomes with membrane vesicles of Lactococcus lactis, the delta p generated by cytochrome c-oxidase activity was capable to drive uphill transport of leucine. Electron microscopic analysis indicated that the tetraether lipids form a single monolayer liposome. The results demonstrate that tetraether lipids of archaebacteria can form a suitable matrix for the function of exogenous membrane proteins originating from a regular lipid bilayer.     

Effect of nicotinic acid on microviscosity in mixed liposomal system of lecithin and sphingomyelin

In rat liver plasma membrane, the molar ratio of sphingomyelin and phospholipid is approximately 1:4, whereas, the molar ratio of phospholipid and cholesterol is 3:1. Considering this ratio to be typical for a real biological membrane, we have studied the effect of anticholesterol and the vasodialatory drug nicotinic acid (NA) on the fluidity profile of a liposomal system of lipids mixed in this ratio using the fluorescence polarization probe 1,6-diphenyl-1-1,3,5-hexatriene. The study reveals that when NA is added to the aqueous dispersion of the mixed lipid system (molar ratio of lipid:NA, 1:1) it creates a more fluid environment for the probe molecule and modifies the fluidity profile of the cholesterol-incorporated liposomal system by eliminating the effect of cholesterol to some extent. The drug also affects the activation energy of diffusion of this system. These results on fluidity have been compared with those in cases of liposomes of individual lipids. The effect of NA on fluidity may be attributed to a mechanical interaction of the drug molecules with the lipid molecules.     

Enhancement of apparent substrate selectivity of proteinase K encapsulated in liposomes through a cholate-induced alteration of the bilayer permeability

Proteinase K-containing liposomes with highly selective membrane permeability properties were prepared. The selectivity obtained was with respect to the two substrate molecules added to the external aqueous phase of the liposomes: acetyl-L-Ala-Ala-Ala-p-nitroanilide (Ac-AAA-pNA) and succinyl-L-Ala-Ala-Ala-p-nitroanilide (Suc-AAA-pNA). The liposome-forming lipid used was POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and modulation of the membrane permeability was achieved using the detergent cholate. Proteinase K-containing mixed liposomes (PKCL) were prepared by adding cholate to preformed proteinase K-containing POPC liposomes (PKL) at a defined effective cholate/POPC molar ratio in the liposomal bilayer membrane R(e). Proteinase K was kept inside PKCL with a negligible amount of leakage into the bulk aqueous phase at R(e) < or = 0.30. At higher R(e), leakage of proteinase K was pronounced, even under conditions where POPC/cholate mixed liposomes seemed to be still intact (0.30 < R(e) < or = 0.39). At R(e) < or = 0.30, the reactivity of proteinase K in the PKCL measured with the externally added substrate Ac-AAA-pNA increased with increasing R(e), while the reactivity measured with Suc-AAA-pNA remained low, regardless of the R(e) value. This showed that externally added Ac-AAA-pNA molecules permeated the liposomal membrane more easily than Suc-AAA-pNA by modulating the membrane with cholate. Consequently, Ac-AAA-pNA was hydrolyzed in PKCL with considerably higher apparent substrate selectivity in comparison with the cases of proteinase K in PKL and free proteinase K (without liposomal encapsulation). The results obtained clearly demonstrate that the prepared PKCL can be utilized as a kind of nano-scaled bioreactor system which can take up a particular target substrate with high apparent substrate selectively from the external phase of the liposomes. Inside the liposomes, the target substrate is then converted into the corresponding products.     

Cholesterol superlattice modulates CA4P release from liposomes and CA4P cytotoxicity on mammary cancer cells

Liposomal drugs are a useful alternative to conventional drugs and hold great promise for targeted delivery in the treatment of many diseases. Most of the liposomal drugs on the market or under clinical trials include cholesterol as a membrane stabilizing agent. Here, we used liposomal CA4P, an antivascular drug, to demonstrate that cholesterol content can actually modulate the release and cytotoxicity of liposomal drugs in a delicate and predictable manner. We found that both the rate of the CA4P release from the interior aqueous compartment of the liposomes to the bulk aqueous phase and the extent of the drug's cytotoxicity undergo a biphasic variation, as large as 50%, with liposomal cholesterol content at the theoretically predicted C(r), e.g., 22.0, 22.2, 25.0, 33.3, 40.0, and 50.0 mol % cholesterol for maximal superlattice formation. It appears that at C(r), CA4P can be released from the liposomes more readily than at non-C(r), probably due to the increased domain boundaries between superlattice and nonsuperlattice regions, which consequently results in increased cytotoxicity. The idea that the increased domain boundaries at C(r) would facilitate the escape of molecules from membranes was further supported by the data of dehydroergosterol transfer from liposomes to MβCD. These results together show that the functional importance of sterol superlattice formation in liposomes can be propagated to distal targeted cells and reveal a new, to our knowledge, mechanism for how sterol content and membrane lateral organization can control the release of entrapped or embedded molecules in membranes.     

Nebulization of liposomal rh-Cu/Zn-SOD with a novel vibrating membrane nebulizer

Liposomes are potential drug carriers for pulmonary drug delivery: They can be prepared from phospholipids, which are endogenous to the respiratory tract as a component of pulmonary surfactant, and at an appropriate dose liposomes do not pose a toxicological risk to this organ. Among the various categories of drug that benefit from liposomal entrapment is the anti-inflammatory enzyme superoxide dismutase, thus prolonging its biological half-life. The delivery of liposomes by nebulization is hampered by stability problems, like physical and chemical changes that may lead to chemical degradation and leakage of the encapsulated drug. Here we present data of liposomes aerosolized with a novel electronic nebulizer based on a vibrating membrane technology (PARI eFlow), which amends drawbacks like liposomes degradation and product release. The data acquisition included aerosol properties such as aerodynamic particle size, nebulization efficiency, and liposome leakage upon nebulization. In conclusion, this study shows the ability of the PARI eFlow to nebulize high amounts of liposomal recombinant human superoxide dismutase with reduced vesicle disruption tested in an enclosing experimental protocol.     

Visualization of bioavailable liposomal doxorubicin using a non-perturbing confocal imaging technique

Commonly employed tissue processing techniques can significantly alter tissue drug distribution patterns for liposomal encapsulated drugs by virtue of drug leakage via loss of membrane integrity. We report here a method that has been developed to determine the fluorescence of bioavailable doxorubicin (DOX) in tissues after administration of liposomal DOX formulations. A non-perturbing confocal fluorescence microscopy (CFM) technique with image processing analysis was used with unprocessed fresh tissues. This method takes advantage of the fact that considerable quenching occurs when DOX is within liposomes, leading to the selective visualization of the fluorescence due to DOX released from liposomes. We demonstrate that fresh tissue confocal imaging can be applied to provide detailed drug distribution information with improved accuracy and is a superior method for analyzing tissue distribution of liposome entrapped fluorescent agents.     

Anticancer double lipid prodrugs: liposomal preparation and characterization

The escape of encapsulated anticancer drugs from liposomes by passive diffusion often leads to suboptimal drug concentrations in the cancer tissue, therefore calling for effective trigger mechanisms to release the drug at the target. We investigated mixtures of lipid components that not only form stable liposomes, but also can be turned into active drugs by secretory phospholipase A? (sPLA?), an enzyme that is upregulated in various cancer cells, without the necessity for conventional liposome drug loading. The liposomes are composed of a novel lipid-based retinoid prodrug premixed with saturated phospholipids. The prodrug is found to be miscible with phospholipids, and the lipid mixtures are shown to form liposomes with the desired size distribution. The preparation procedure, phase behavior, and physicochemical properties of the formed liposomes are described as a function of lipid composition. We show that the premixing of the prodrug with phospholipids can be used to modify the physicochemical properties of liposomal formulations. The results should prove useful for further exploration of the potential for using these novel lipid prodrugs in liposomal formulations for cancer treatment.     

Physiochemical characterization of the nisin-membrane interaction with liposomes derived from Listeria monocytogenes.

Mechanistic information about the bacteriocin nisin was obtained by examining the efflux of 5(6)-carboxy-fluorescein from Listeria monocytogenes-derived liposomes. The initial leakage rate (percentage of efflux per minute) of the entrapped dye was dependent on both nisin and lipid concentrations. At all nisin concentrations tested, 5(6)-carboxyfluorescein efflux plateaued before all of the 5(6)-carboxyfluorescein was released (suggesting that pore formation was transient), but efflux resumed when more nisin was added. Isotherms for the binding of nisin to liposomes constructed on the basis of the Langmuir isotherm gave an apparent binding constant of 6.2 x 10(5)M(-1) at pH 6.0. The critical number of nisin molecules required to induce efflux from liposomes at pH 6.0 was approximately 7,000 molecules per liposome. The pH affected the 5(6)-carboxyfluorescein leakage rates, with higher pH values resulting in higher leakage rates. The increased leakage rate observed at higher pH values was not due to an increase in the binding affinity of the nisin molecules towards the liposomal membrane. Rather, the critical number of nisin molecules required to induce activity was decreased (approximately 1,000 nisin molecules per liposome at pH 7.0). These data are consistent with a poration mechanism in which the ionization state of histidine residues in nisin plays an important role in membrane permeabilization.     

Nebulization of Liposomal rh-Cu/Zn–SOD with a Novel Vibrating Membrane Nebulizer

Liposomes are potential drug carriers for pulmonary drug delivery: They can be prepared from phospholipids, which are endogenous to the respiratory tract as a component of pulmonary surfactant, and at an appropriate dose liposomes do not pose a toxicological risk to this organ. Among the various categories of drug that benefit from liposomal entrapment is the anti-inflammatory enzyme superoxide dismutase, thus prolonging its biological half-life. The delivery of liposomes by nebulization is hampered by stability problems, like physical and chemical changes that may lead to chemical degradation and leakage of the encapsulated drug. Here we present data of liposomes aerosolized with a novel electronic nebulizer based on a vibrating membrane technology (PARI eFlow?), which amends drawbacks like liposomes degradation and product release. The data acquisition included aerosol properties such as aerodynamic particle size, nebulization efficiency, and liposome leakage upon nebulization. In conclusion, this study shows the ability of the PARI eFlow? to nebulize high amounts of liposomal recombinant human superoxide dismutase with reduced vesicle disruption tested in an enclosing experimental protocol.     

In vitro characterization of a novel polymeric-based pH-sensitive liposome system

This study demonstrates rapid and pH-sensitive release of a highly water-soluble fluorescent aqueous content marker, pyranine, from egg phosphatidylcholine liposomes following incorporation of N-isopropylacrylamide (NIPA) copolymers in liposomal membranes. The pH-sensitivity of this system correlates with the precipitation of the copolymers at acidic pH. In vitro release can be significantly improved by increasing the percentage of anchor in the copolymer and thus favoring its binding to the liposomal bilayer. In the case of liposomes containing a poly(ethylene glycol)-phospholipid conjugate, the insertion of the pH-sensitive copolymer in the liposomal membrane appears to be sterically inhibited. Dye release from these formulations at acidic pH can still be achieved by varying the anchor molar ratio and/or molecular mass of the polymers or by including the latter during the liposome preparation procedure. Removal of unbound polymer results in decreased leakage only when the copolymer is inserted by incubation with preformed liposomes, but can be overcome by preparing liposomes in the presence of polymer. Aqueous content and lipid mixing assays suggest contents release can occur without membrane fusion. The results of this study indicate that the addition of pH-sensitive copolymers of NIPA represents promising strategy for improving liposomal drug delivery.     

Investigation of parameters influencing incorporation,retention and cellular cytotoxicity in liposomal formulations of poorly soluble camptothecin

Abstract

Improving tumor delivery of lipophilic drugs through identifying advanced drug carrier systems with efficient carrier potency is of high importance. We have performed an investigative approach to identify parameters that affect liposomes’ ability to effectively deliver lipophilic camptothecin (CPT) to target cells. CPT is a potent anticancer drug, but its undesired physiological properties are impairing its therapeutic use. In this study, we have identified parameters influencing incorporation and retention of lipophilic CPT in liposomes, evaluating the effect of lipid composition, lipid chemical structure (head and tail group variations, polymer inclusion), zeta potential and anisotropy. Polyethyleneglycol (PEG) surface decoration was included to avoid liposome fusing and increase the potential for prolonged in vivo circulation time. The in vitro effect of the different carrier formulations on cell cytotoxicity was compared and the effect of active targeting of one of the formulations was evaluated. We found that a combination of liposome surface charge, lipid headgroup and carbon chain unsaturation affect CPT incorporation. Retention in liposomes was highly dependent on the liposomal surroundings and liposome zeta potential. Inclusion of lipid tethered PEG provided stability and prevented liposome fusing. PEGylation negatively affected CPT incorporation while improving retention. In vitro cell culture testing demonstrated that all formulations increased CPT potency compared to free CPT, while cationic formulations proved significantly more toxic to cancer cells that healthy cells. Finally, antibody mediated targeting of one liposome formulation further enhanced the selectivity towards targeted cancer cells, rendering normal cells fully viable after 1 hour exposure to targeted liposomes.     

The potential of pectin as a stabilizer for liposomal drug delivery systems

The aim of the present study was to investigate the potential of different types of pectin as stabilizers for liposomal drug delivery systems. Positively charged liposomes were coated with commercially available and purified low-methoxylated (LM), high-methoxylated (HM) and amidated (AM) pectins. The samples were stored for up to 12 weeks at 4°C, at room temperature and at 35°C. The change in liposomal size and size distribution, zeta potential, pH, leakage of encapsulated carboxyfluorescein (CF), and lipid degradation were studied. All the types of pectin were found to protect the liposomes against aggregation during storage. The pectin coat did not affect the permeability of the liposome membrane. HM and LM pectin seemed to be the most promising types of pectin due to minimal changes in the zeta potentials during storage for these samples and no detectable lipid degradation. It is concluded that pectin may be used for stabilizing liposomal drug delivery systems.     

Bipolar tetraether archaeosomes exhibit unusual stability against autoclaving as studied by dynamic light scattering and electron microscopy

The stability of liposomes made of the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius against autoclaving has been studied by using dynamic light scattering and transmission electron microscopy. PLFE lipids have structures distinctly different from those derived from eukaryotes and prokaryotes. PLFE lipids are bipolar tetraether molecules and may contain up to four cyclopentane rings in each of the two dibiphytanyl chains. In the pH range 4-10, PLFE-based archaeosomes, with and without polyethyleneglycol- and maleimide-lipids, are able to retain vesicle size, size distribution, and morphology through at least six autoclaving cycles. The cell growth temperature (65 °C vs. 78 °C), hence the number of cyclopentane rings in the hydrocarbon chains, does not affect this general conclusion. By contrast, at the same pH range, most conventional liposomes made of monopolar diester lipids and cholesterol or pegylated lipids cannot withhold vesicle size and size distribution against just one cycle of autoclaving. At pH < 4, the particle size and polydispersity of PLFE-based archaeosomes increase with autoclaving cycles, suggesting that aggregation or membrane disruption may have occurred at extreme acidic conditions during heat sterilization. Under high salt conditions, dye leakage from PLFE archaeosomes due to autoclaving is significantly less than that from pegylated liposomes composed of conventional lipids. The ability to maintain vesicle integrity after multiple autoclaving cycles indicates the potential usefulness of utilizing PLFE-based archaeosomes as autoclavable and durable drug (including genes, peptides, vaccines, siRNA) delivery vehicles.     

Macrophage specific drug delivery in experimental leishmaniasis

Macrophage-specific delivery systems are the subject of much interest nowadays, because of the fact that macrophages act as host cells for many parasites and bacteria, which give rise to outbreak of so many deadly diseases(eg. leishmaniasis, tuberculosis etc.) in humans. To combat these deadly diseases initially macrophage specific liposomal delivery system were thought of and tested in vivo against experimental leishmaniasis in hamsters using a series of indigenous or synthetic antileishmanial compounds and the results were critically discussed. In vitro testing was also done against macrophages infected with Leishmania donovani, the causative agent for visceral leishmaniasis. The common problem of liposome therapy being their larger size, stability and storage, non-ionic surfactant vesicles, niosomes were prepared, for their different drug distribution and release characteristics compared to liposomes. When tested in vivo, the retention capacity of niosomes was found to be higher than that of liposomes due to the absence of lipid molecules and their smaller size. Thus the therapeutic efficacy of certain antileishmanial compounds was found to be better than that in the liposomal form. The niosomes, being cheaper, less toxic, biodegradable and non-immunogenic, were considered for sometime as suitable alternatives to liposomes as drug carriers. Besides the advent of other classical drugs carriers(e.g. neoglycoproteins), the biggest challenge came from polymeric delivery vehicles, specially the polymeric nanoparticles which were made of cost effective biodegradable polymers and different natural polymers. Because of very small size and highly stable nature, use of nanoparticles as effective drug carriers has been explored in experimental leishmaniasis using a series of antileishmanial compounds, both of indigenous and synthetic origin. The feasibility of application in vivo, when tested for biological as well as for other physicochemical parameters, the polymeric nanoparticles have turned out to be the best and thus may be projected for effective use in the clinics.     

Factors affecting the pharmacokinetics and pharmacodynamics of liposomal drugs

Various attempts to increase the therapeutic index of the drug while minimizing side effects have been made in drug delivery systems. Among several promising strategies, liposomes represent an advanced technology to target active molecules to the site of action. Rapid clearance of circulating liposomal drugs administered intravenously has been a critical issue because circulation time in the blood affects drug exposure at the target site. The clinical use of liposomal drugs is complicated by large intra- and interindividual variability in their pharmacokinetics (PK) and pharmacodynamics (PD). Thus, it is important to understand the factors affecting the PK/PD of the liposomal formulation of drugs and to elucidate the mechanisms underlying the variability in the PK/PD of liposomal drugs. In this review article, we describe the characteristics of liposome formulations and discuss the effects of various factors, including liposome-associated factors, host-associated factors, and treatment on the PK/PD of liposomal agents.     

Factors affecting the pharmacokinetics and pharmacodynamics of liposomal drugs

Various attempts to increase the therapeutic index of the drug while minimizing side effects have been made in drug delivery systems. Among several promising strategies, liposomes represent an advanced technology to target active molecules to the site of action. Rapid clearance of circulating liposomal drugs administered intravenously has been a critical issue because circulation time in the blood affects drug exposure at the target site. The clinical use of liposomal drugs is complicated by large intra- and interindividual variability in their pharmacokinetics (PK) and pharmacodynamics (PD). Thus, it is important to understand the factors affecting the PK/PD of the liposomal formulation of drugs and to elucidate the mechanisms underlying the variability in the PK/PD of liposomal drugs. In this review article, we describe the characteristics of liposome formulations and discuss the effects of various factors, including liposome-associated factors, host-associated factors, and treatment on the PK/PD of liposomal agents.     

Tissue distribution of EDTA encapsulated within liposomes of varying surface properties

Liposomes containing ethylenediaminetetraacetic acid (EDTA) were prepared with different surface properties by varying the liposomal lipid constituents. Positively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and stearylamine. Negatively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and phosphatidylserine. Neutral liposomes were prepared with phosphatidylcholine alone, dipalmitoyl phosphatidylcholine alone, or with a mixture of phosphatidylcholine and cholesterol. Distributions of ¹⁴C-labeled EDTA were determined in mouse tissues from 5 min to 24 h after a single intravenous injection of liposome preparation. Differences in tissue distribution were produced by the different liposomal lipid compositions. Uptake of EDTA by spleen and marrow was highest from negatively charged liposomes. Uptake of EDTA by lungs was highest from positively charged liposomes; lungs and brain retained relatively high levels of EDTA from these liposomes between 1 and 6 h after injection. Liver uptake of EDTA from positively or negatively charged liposomes was similar; the highest EDTA uptake by liver was from the neutral liposomes composed of a mixture of phosphatidylcholine and cholesterol. Liposomes composed of dipalmitoyl phosphatidylcholine produced the lowest liposomal EDTA uptake observed in liver and marrow but modrate uptake by lungs. Tissue uptake and retention of EDTA from all of the liposome preparations were greater than those of non-encapsulated EDTA. The results presented demonstrate that the tissue distribution of a molecule can be modified by encapsulation of that substance into liposomes of different surface properties. Selective delivery of liposome-encapsulated drugs to specific tissues could be effectively used in chemotherapy and membrane biochemistry.     

A kinetic method for measuring functional delivery of amphotericin B by drug delivery systems.

The human toxicity of amphotericin B can be considerably reduced by associating the drug with liposomes of varying lipid compositions. Some lipid compositions are much more effective than others. We show that a simple kinetic fluorescence assay using pyranine as an indirect probe of amphotericin-induced K+ currents may be used to study different liposomal drug delivery systems in vitro. We find that lipid mixtures composed of DMPC/DMPG/amphotericin at a 7:3:1 mole ratio show very slow functional delivery with a preference for ergosterol over cholesterol-containing membrane vesicles. On the other hand, amphotericin delivered from egg phosphatidylcholine liposomes lead to 100-fold increases in K+ leakage at one-fifth the amphotericin concentration of the 7:3:1 system. The egg phosphatidylcholine system as well as micellar amphotericin also show a slight selectivity towards cholesterol-containing vesicles over ergosterol. These results are consistent with previous clinical and in vitro cellular studies and this technique may prove valuable in screening of other delivery systems.     

Cell-penetrating peptide induces leaky fusion of liposomes containing late endosome-specific anionic lipid

Cationic cell-penetrating peptides (CPPs) are a promising vehicle for the delivery of macromolecular drugs. Although many studies have indicated that CPPs enter cells by endocytosis, the mechanisms by which they cross endosomal membranes remain elusive. On the basis of experiments with liposomes, we propose that CPP escape into the cytosol is based on leaky fusion (i.e., fusion associated with the permeabilization of membranes) of the bis(monoacylglycero)phosphate (BMP)-enriched membranes of late endosomes. In our experiments, prototypic CPP HIV-1 TAT peptide did not interact with liposomes mimicking the outer leaflet of the plasma membrane, but it did induce lipid mixing and membrane leakage as it translocated into liposomes mimicking the lipid composition of late endosome. Both membrane leakage and lipid mixing depended on the BMP content and were promoted at acidic pH, which is characteristic of late endosomes. Substitution of BMP with its structural isomer, phosphatidylglycerol (PG), significantly reduced both leakage of the aqueous probe from liposomes and lipid mixing between liposomes. Although affinity of binding to TAT was similar for BMP and PG, BMP exhibited a higher tendency to support the inverted hexagonal phase than PG. Finally, membrane leakage and peptide translocation were both inhibited by inhibitors of lipid mixing, further substantiating the hypothesis that cationic peptides cross BMP-enriched membranes by inducing leaky fusion between them.