Membrane Fusion Of Liposomes Research Articles

Lipid Selectivity of Membrane Action of the Fragments of Fusion Peptides of Marburg and Ebola Viruses.

The life cycle of Ebola and Marburg viruses includes a step of the virion envelope fusion with the cell membrane. Here, we analyzed whether the fusion of liposome membranes under the action of fragments of fusion peptides of Ebola and Marburg viruses depends on the composition of lipid vesicles. A fluorescence assay and electron microscopy were used to quantify the fusogenic activity of the virus fusion peptides and to identify the lipid determinants affecting membrane merging. Differential scanning calorimetry of lipid phase transitions revealed alterations in the physical properties of the lipid matrix produced by virus fusion peptides. Additionally, we found that plant polyphenols, quercetin, and myricetin inhibited vesicle fusion induced by the Marburg virus fusion peptide.

Prevention of liposome cryoaggregation by a radish (Raphanus sativus L.) vacuolar calcium-binding protein (RVCaB)

Previously we found that the soluble fraction of radish (Raphanus sativus L.) taproots contained a large amount of an intrinsically disordered protein, which we identified as radish vacuolar calcium binding protein (RVCaB). Although RVCaB is known to inhibit cryo-damage to freeze-sensitive enzyme lactate dehydrogenase, there is no information on its cryoprotection of other types of macromolecules, such as biomembranes. In this study we found that RVCaB inhibited the cryoaggregation of phospholipid liposomes to a greater extent than common cryoprotectants, such as trehalose, glycine betaine, proline, bovine serum albumin (BSA), and polyethylene glycol (PEG). RVCaB inhibited the freeze/thaw-induced membrane fusion of liposomes. It became structurally disordered in solution, and this disorder was maintained in the presence of liposomes. Finally, the mechanism underlying RVCaB's inhibition of liposome cryoaggregation was discussed.

The N-Terminal Region of Soybean PM1 Protein Protects Liposomes during Freeze-Thaw

Late embryogenesis abundant (LEA) group 1 (LEA_1) proteins are intrinsically disordered proteins (IDPs) that play important roles in protecting plants from abiotic stress. Their protective function, at a molecular level, has not yet been fully elucidated, but several studies suggest their involvement in membrane stabilization under stress conditions. In this paper, the soybean LEA_1 protein PM1 and its truncated forms (PM1-N: N-terminal half; PM1-C: C-terminal half) were tested for the ability to protect liposomes against damage induced by freeze-thaw stress. Turbidity measurement and light microscopy showed that full-length PM1 and PM1-N, but not PM1-C, can prevent freeze-thaw-induced aggregation of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes and native thylakoid membranes, isolated from spinach leaves (Spinacia oleracea). Particle size distribution analysis by dynamic light scattering (DLS) further confirmed that PM1 and PM1-N can prevent liposome aggregation during freeze-thaw. Furthermore, PM1 or PM1-N could significantly inhibit membrane fusion of liposomes, but not reduce the leakage of their contents following freezing stress. The results of proteolytic digestion and circular dichroism experiments suggest that PM1 and PM1-N proteins bind mainly on the surface of the POPC liposome. We propose that, through its N-terminal region, PM1 functions as a membrane-stabilizing protein during abiotic stress, and might inhibit membrane fusion and aggregation of vesicles or other endomembrane structures within the plant cell.

Near-infrared light-activated membrane fusion for cancer cell therapeutic applications.

The spatiotemporal stimulation of liposome-liposome or liposome-membrane fusion processes attracts growing interest as a means to mimic cell-cell interactions in nature and for using these processes for biomedical applications. We report the use of o-nitrobenzyl phosphate functionalized-cholesterol tethered nucleic acid-modified liposomes as functional photoresponsive units for inducing, by NIR-irradiation, spatiotemporal liposome-liposome or liposome-membrane fusion processes. The liposomes are loaded with upconversion nanoparticles (UCNPs) and their NIR irradiation (λ = 980 nm) yields luminescence at λ = 365 nm, providing a localized light-source to deprotect the o-nitrobenzyl phosphate groups and resulting in the fragmentation of the nucleic acid structures. In one system, the NIR-triggered fusion of two liposomes, L1 and L2, is exemplified. Liposome L1 is loaded with UCNPs and Tb3+ ions, and the liposome boundary is functionalized with a cholesterol-tethered, o-nitrobenzyl phosphate caged hairpin nucleic acid structure. Liposome L2 is loaded with 2,6-pyridinedicarboxylic acid, DPA, and its boundary is modified with a cholesterol-tethered nucleic acid, complementary to a part of the caged hairpin, associated with L1. NIR-irradiation of the L1/L2 mixture resulted in the photocleavage of the hairpin structure, associated with L1, and the resulting fragmented nucleic acid associated with L1 hybridized with the nucleic acid linked to L2, leading to the fusion of the two liposomes. The fusion process was followed by dynamic light scattering, and by monitoring the fluorescence of the Tb3+-DPA complex generated upon the fusion of the liposomes and their exchange of contents (fusion efficiency 30%). In a second system, the fusion of the liposomes L1, loaded with UCNPs and doxorubicin (DOX), with HeLa cancer cells functionalized with nucleic acid tethers, complementary to the hairpin units associated with the boundary of L1, and linked to the MUC-1 receptor sites associated with the HeLa cells, through a MUC-1 aptamer unit is exemplified. The effect of DOX-loaded L1/HeLa cell fusion on the cytotoxicity towards HeLa cells is addressed. The NIR UCNP-stimulated cleavage of the o-nitrobenzyl phosphate caged hairpin units associated with L1 leads to the fragmentation of the hairpin units and the resulting nucleic acid tethers hybridize with the nucleic acid-modified HeLa cells, resulting in the liposome-HeLa cell fusion and the release of DOX into the HeLa cells. Selective spatiotemporal cytotoxicity towards HeLa cells is demonstrated (ca. 40% cell killing within two days). The study presents a comprehensive stepwise set of experiments directed towards the development of NIR-driven liposome-liposome or liposome-membrane fusion processes.

Influence of Membrane-Fusogen Distance on the Secondary Structure of Fusogenic Coiled Coil Peptides.

Liposomal membrane fusion is an important tool to study complex biological fusion mechanisms. We use lipidated derivatives of the specific heterodimeric coiled coil pair E: (EIAALEK)3 and K: (KIAALKE)3 to study and control the fusion of liposomes. In this model system, peptides are tethered to their liposomes via a poly(ethylene glycol) (PEG) spacer and a lipid anchor. The efficiency of the fusion mechanism and function of the peptides is highly affected by the PEG-spacer length and the lipid anchor type. Here, the influence of membrane–fusogen distance on the peptide–membrane interactions and the peptide secondary structures is studied with Langmuir film balance and infrared reflection absorption spectroscopy. We found that the introduction of a spacer to monolayer-tethered peptide E changes its conformation from solvated random coils to homo-oligomers. In contrast, the described peptide–monolayer interaction of peptide K is not affected by the PEG-spacer length. Furthermore, the coexistence of different conformations when both lipopeptides E and K are present at the membrane surface is demonstrated empirically, which has many implications for the design of effective fusogenic recognition units and the field of artificial membrane fusion.

Liposomal membrane fusion induced by extrusion method

Objective To study the application of extrusion method in inducing liposome membrane fusion or membrane component mixing, and to investigate the effect of different extrusion conditions on liposome membrane fusion rate. Methods N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) phosphatidylethanolamine (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl) phosphatidylethanolamine (N-Rh-PE) labeled 1, 2-dioleoyl lecithin (DOPC) liposomes and non-fluorescently labeled DOPC monolayer liposomes were mixed and extruded. The fluorescence changes before and after the extrusion of the mixed liposomes were observed using laser scanning confocal microscope, and the membrane fusion rate of the mixed liposomes was calculated by fluorescence resonance energy transfer method. Besides, the effects of extrusion times, extrusion pressure and temperature on the fusion rate of liposome membrane were studied. Results The results of laser scanning confocal microscopy showed that the distribution density and intensity of the green fluorescence of N-NBD-PE increased significantly after the extrusion of fluorescently labeled and non-fluorescent labeled DOPC liposomes, which confirmed membrane fusion. After 75 times of extrusion treatments, the liposome membrane fusion rate can reach 26%. The number of extrusions, extrusion pressure and temperature had a significant effect on the fusion rate of the liposome membrane. The higher the number of the extrusions, the smaller the extrusion pressure and the higher the efficiency of the liposome membrane fusion were at physiological temperature. Conclusions Extrusion method can induce liposome membrane fusion and membrane component mixing, and the prepared liposome has a narrower particle size distribution, which is expected to be a new method to induce the bilayer membrane fusion of liposome or lipid vesicle. Key words: Extrusion; Liposome; Lipid bilayer fusion; Fluorescence resonance energy transfer

Liposome-Membrane Fusion Rates Altered by Dose and Location of Short-Chain Alcohols

Liposome-Membrane Fusion Rates Altered by Dose and Location of Short-Chain Alcohols

Drunken Membranes: Short-Chain Alcohols Alter Fusion of Liposomes to Planar Lipid Bilayers

Although the effects of ethanol on protein receptors and lipid membranes have been studied extensively, ethanol’s effect on vesicles fusing to lipid bilayers is not known. To determine the effect of alcohols on fusion rates, we utilized the nystatin/ergosterol fusion assay to measure fusion of liposomes to a planar lipid bilayer (BLM). The addition of ethanol excited fusion when applied on the cis (vesicle) side, and inhibited fusion on the trans side. Other short-chain alcohols followed a similar pattern. In general, the inhibitory effect of alcohols (trans) occurs at lower doses than the excitatory (cis) effect, with a decrease of 29% in fusion rates at the legal driving limit of 0.08% (w/v) ethanol (IC50 = 0.2% v/v, 34 mM). Similar inhibitory effects were observed with methanol, propanol, and butanol, with ethanol being the most potent. Significant variability was observed with different alcohols when applied to the cis side. Ethanol and propanol enhanced fusion, butanol also enhanced fusion but was less potent, and low doses of methanol mildly inhibited fusion. The inhibition by trans addition of alcohols implies that they alter the planar membrane structure and thereby increase the activation energy required for fusion, likely through an increase in membrane fluidity. The cis data are likely a combination of the above effect and a proportionally greater lowering of the vesicle lysis tension and hydration repulsive pressure that combine to enhance fusion. Alternate hypotheses are also discussed. The inhibitory effect of ethanol on liposome-membrane fusion is large enough to provide a possible biophysical explanation of compromised neuronal behavior.

Calmodulin EF-hand peptides as Ca2+ -switchable recognition tags.

Calmodulin is a representative calcium-binding protein comprised of four Ca2+ -binding motifs with a helix-loop-helix structure (EF-hands). In this study, we clarified the potential of peptide segments derived from the third and fourth EF-hands (EF3 and EF4) to act as recognition tags. Through an analysis of the mode of disulfide formation among cysteines inserted at the N- or C-terminus of these peptide segments, EF3 and EF4 peptides were suggested to form a heterodimer with a topology similar to that in the wild-type protein. Heterodimer formation was shown to be a function of the Ca2+ concentration, suggesting that these structures may be used as Ca2+ -switchable recognition tags. An example of an "EF-tag" system involving the membrane fusion of liposomes decorated with EF3 and EF4 peptides is presented. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2016.

Development of flexible nanocarriers for siRNA delivery into tumor tissue

Various non-viral delivery systems for small interfering RNAs (siRNA) have been developed. Such delivery systems generally exhibit tightly formed spherical structures. While such carriers have demonstrated good transfection activity in mono-layered cell systems, effects against solid tumors are often less apparent and difficult to demonstrate, likely due to the rigid structures of the carriers, which may prevent penetration to deeper regions within tumor tissue. Herein, we developed a flexible nanocarrier (FNC) system that is able to penetrate to deeper regions within tumor tissue. Specifically, we employed previously found flexible polyplexes comprised of siRNA and poly-l-lysine as wick structures for the preparation of FNCs. FNCs were constructed by coating the wick structures with lipids using a liposomal membrane fusion method. The diameters of the resulting FNCs were ca. 170nm, and the shapes were non-spherical. Lipid coating was confirmed using a nuclease resistance assay. Furthermore, FNCs showed significant RNA interference effects, comparable to Lipofectamine 2000, in a mono-layered cell system. To accelerate tumor penetration, the FNC surface was modified with polyethylene glycol (PEG) and the tight junction opener peptide AT1002. Surface-modified FNCs demonstrated effective penetrability into a cancer spheroid. Thus, we developed a novel and unique tumor-penetrable siRNA FNC system.

Development of paclitaxel-loaded liposomal nanocarrier stabilized by triglyceride incorporation.

Studies have highlighted the challenge of developing injectable liposomes as a paclitaxel (PTX) carrier, a challenge attributable to the limitations in liposomal stability caused by PTX loading. Poor stability of PTX-loaded liposomes is caused by PTX-triggered aggregation or fusion of liposomal membranes and is exacerbated in the presence of PEGylated lipid. In the present study, the effect of triglyceride incorporation on the stability of PTX-loaded/PEGylated liposomes was explored. Incorporation of a medium chain triglyceride Captex 300 into saturated phosphatidylcholine (PC)-based liposomes (1,2-dimyristoyl-sn-glycero-3-phosphocholine [DMPC]:cholesterol [CHOL]:N-(Carbonyl-methoxypolyethyleneglycol 2000)-1, 2-distearoyl-sn-glycero-3-phospho-ethanolamine [PE-PEG]), produced a fine, homogeneous, and membrane-filterable PTX-loaded liposomes fulfilling the requirement of an injectable lipid formulation. Triglyceride incorporation also greatly inhibited the time-dependent leakage of PTX from saturated PC-based liposomes, which appears to be mediated by the inhibition of liposome fusion. In contrast, triglyceride incorporation induced the destabilization and PTX leakage of unsaturated PC-based liposomes, indicating the opposite effect of triglyceride depending on the fluidity status of PC constituting the liposomal membrane. PTX release profile and the in vitro and in vivo anticancer efficacy of triglyceride-incorporated DMPC:CHOL:PE-PEG liposomes were similar to Taxol® while the toxicity of liposomal PTX was significantly lower than that of Taxol. Taken together, triglyceride incorporation provided an injectable PTX formulation by functioning as a formulation stabilizer of PEGylated/saturated PC-based liposomes.

Application of Coiled Coil Peptides in Liposomal Anticancer Drug Delivery Using a Zebrafish Xenograft Model.

The complementary coiled coil forming peptides E4 [(EIAALEK)4] and K4 [(KIAALKE)4] are known to trigger liposomal membrane fusion when tethered to lipid vesicles in the form of lipopeptides. In this study, we examined whether these coiled coil forming peptides can be used for drug delivery applications. First, we prepared E4 peptide modified liposomes containing the far-red fluorescent dye TO-PRO-3 iodide (E4-Lipo-TP3) and confirmed that E4-liposomes could deliver TP3 into HeLa cells expressing K4 peptide on the membrane (HeLa-K) under cell culture conditions in a selective manner. Next, we prepared doxorubicin-containing E4-liposomes (E4-Lipo-DOX) and confirmed that E4-liposomes could also deliver DOX into HeLa-K cells. Moreover, E4-Lipo-DOX showed enhanced cytotoxicity toward HeLa-K cells compared to free doxorubicin. To prove the suitability of E4/K4 coiled coil formation for in vivo drug delivery, we injected E4-Lipo-TP3 or E4-Lipo-DOX into zebrafish xenografts of HeLa-K. As a result, E4-liposomes delivered TP3 to the implanted HeLa-K cells, and E4-Lipo-DOX could suppress cancer proliferation in the xenograft when compared to nontargeted conditions (i.e., zebrafish xenograft with free DOX injection). These data demonstrate that coiled coil formation enables drug selectivity and efficacy in vivo. It is envisaged that these findings are a step forward toward biorthogonal targeting systems as a tool for clinical drug delivery.

Identification of the Fusion Peptide-Containing Region in Betacoronavirus Spike Glycoproteins.

The fusion peptides (FP) play an essential role in fusion of viral envelope with cellular membranes. The location and properties of the FPs in the spike (S) glycoproteins of different coronaviruses (CoV) have not yet been determined. Through amino acid sequence analysis of S proteins of representative CoVs, we identified a common region as a possible FP (pFP) that shares the characteristics of FPs of class I viral fusion proteins, including high Ala/Gly content, intermediate hydrophobicity, and few charged residues. To test the hypothesis that this region contains the CoV FP, we systemically mutated every residue in the pFP of Middle East respiratory syndrome betacoronavirus (MERS-CoV) and found that 11 of the 22 residues in the pFP (from G953 to L964, except for A956) were essential for S protein-mediated cell-cell fusion and virus entry. The synthetic MERS-CoV pFP core peptide (955IAGVGWTAGL964) induced extensive fusion of liposome membranes, while mutant peptide failed to induce any lipid mixing. We also selectively mutated residues in pFPs of two other β-CoVs, severe acute respiratory syndrome coronavirus (SARS-CoV) and mouse hepatitis virus (MHV). Although the amino acid sequences of these two pFPs differed significantly from that of MERS-CoV and each other, most of the pFP mutants of SARS-CoV and MHV also failed to mediate membrane fusion, suggesting that these pFPs are also the functional FPs. Thus, the FPs of 3 different lineages of β-CoVs are conserved in location within the S glycoproteins and in their functions, although their amino acid sequences have diverged significantly during CoV evolution. Within the class I viral fusion proteins of many enveloped viruses, the FP is the critical mediator of fusion of the viral envelope with host cell membranes leading to virus infection. FPs from within a virus family, like influenza viruses or human immunodeficiency viruses (HIV), tend to share high amino acid sequence identity. In this study, we determined the location and amino acid sequences of the FPs of S glycoproteins of 3 β-CoVs, MERS-CoV, SARS-CoV, and MHV, and demonstrated that they were essential for mediating cell-cell fusion and virus entry. Interestingly, in marked contrast to the FPs of influenza and HIV, the primary amino acid sequences of the FPs of β-CoVs in 3 different lineages differed significantly. Thus, during evolution the FPs of β-CoVs have diverged significantly in their primary sequences while maintaining the same essential biological functions. Our findings identify a potential new target for development of drugs against CoVs.

A non-zipper-like tetrameric coiled coil promotes membrane fusion

A parallel heterodimeric coiled coil can be mutated to an antiparallel tetrameric species by reversing the sequences of one of the peptides. This tetramer is capable of facilitating fast, efficient, membrane fusion of liposomes.

Effect of Complexin II on Membrane Fusion between Liposomes Containing Mast Cell SNARE Proteins.

Mast cells are involved in allergic responses and undergo exocytotic release of inflammatory mediators in response to antigen stimulation. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are involved in this membrane fusion process; some SNARE-binding proteins regulate SNARE-dependent liposome membrane fusion. SNARE-binding protein complexin II is expressed in mast cells, where it positively regulates exocytotic release after antigen stimulation. We found that complexin II suppressed SNARE-dependent membrane fusion between mast cell SNARE-containing liposomes. This inhibitory effect of complexin II was abolished when we used a structurally divergent mutant (R59H) complexin II, where Arg59 is substituted with histidine. These results suggest that complexin II negatively regulates SNARE-dependent exocytotic membrane fusion in mast cells, and this inhibitory effect is dependent upon Arg59.

Temporal Control of Membrane Fusion through Photolabile PEGylation of Liposome Membranes

AbstractMembrane fusion results in the transport and mixing of (bio)molecules across otherwise impermeable barriers. In this communication, we describe the temporal control of targeted liposome–liposome membrane fusion and contents mixing using light as an external trigger. Our method relies on steric shielding and rapid, photoinduced deshielding of complementary fusogenic peptides tethered to opposing liposomal membranes. In an analogous approach, we were also able to demonstrate precise spatiotemporal control of liposome accumulation at cellular membranes in vitro.

Carrier-inside-carrier: polyelectrolyte microcapsules as reservoir for drug-loaded liposomes

Conventional liposomes have a short life-time in blood, unless they are protected by a polymer envelope, most often polyethylene glycol. However, these stabilizing polymers frequently interfere with cellular uptake, impede liposome-membrane fusion and inhibit escape of liposome content from endosomes. To overcome such drawbacks, polymer-based systems as carriers for liposomes are currently developed. Conforming to this approach, we propose a new and convenient method for embedding small size liposomes, 30–100 nm, inside porous calcium carbonate microparticles. These microparticles served as templates for deposition of various polyelectrolytes to form a protective shell. The carbonate particles were then dissolved to yield hollow polyelectrolyte microcapsules. The main advantage of using this method for liposome encapsulation is that carbonate particles can serve as a sacrificial template for deposition of virtually any polyelectrolyte. By carefully choosing the shell composition, bioavailability of the liposomes and of the encapsulated drug can be modulated to respond to biological requirements and to improve drug delivery to the cytoplasm and avoid endosomal escape.

Coiled-coil driven membrane fusion: zipper-like vs. non-zipper-like peptide orientation

Membrane fusion plays a central role in biological processes such as neurotransmission and exocytosis. An important class of proteins that induce membrane fusion are called SNARE (soluble N-ethyl malemeide sensitive factor attachment protein receptors) proteins. To induce membrane fusion, two SNARE proteins embedded in opposing membranes form a four-helix coiled-coil motif together with a third, cytoplasmic, SNARE protein. Coiled-coil formation brings the two membranes into close proximity allowing fusion to occur. Importantly, structural investigations have demonstrated that native membrane fusion only occurs when the orientation of the coiled-coil motif resembles that of a zipper. The zipper orientation arises when parallel coiled-coil formation takes place between peptides that are anchored into apposing membranes at identical termini, thereby forcing the membranes into close contact. Recently, we have designed a synthetic model for membrane fusion, which is based on a set of lipidated coiled-coil forming peptide pairs which are denoted E-K. When incorporated into liposomal membranes, coiled-coil formation between these lipidated peptides induces targeted and efficient membrane fusion of liposomes. Our model system mimics SNARE-driven membrane fusion, as it contains a coiled-coil motif which has a zipper-like orientation, similar to that of the SNARE proteins. Here we investigate whether the zipper-like orientation of the coiled-coil motifs is a prerequisite for membrane fusion in our model system. Our strategy is based on conjugation of the transmembrane anchor to either the N- or the C-terminus of peptides E and K. Whereas the use of a set of complementary peptides with the membrane anchor on identical peptide termini yields the zipper-like orientation of the coiled-coil complex, membrane anchors on opposite peptide termini results in a non-zipper-like coiled-coil orientation. Surprisingly, it was observed that efficient and targeted membrane fusion was induced even when the coiled-coil motif did not form the zipper-like orientation. This demonstrates that for our model system, the zipper model for membrane fusion does not apply.

Controlling the rate of coiled coil driven membrane fusion

Sets of complementary lipidated coiled-coil forming peptides that fuse membrane fusion have been designed. The influence of the coiled-coil motif on the rate of liposome fusion was studied, by varying the number of heptad repeats. We found that an increased coiled-coil stability of complementary peptides translates into increased rates of membrane fusion of liposomes.

1A2-V03 細胞温度計測のための単一蛍光マイクロセンサの細胞内導入(ナノ・マイクロ作業システム)

In this paper, we developed injection of a single fluorescent sensor into a specific cell by using membrane fusion of liposome and optical tweezers for cellular temperature measurement. The single fluorescent temperature sensor is included in the single liposome using spontaneous transfer of phospholipid-coated sensor through an oil/water interface. The sensor is manipulated by optical tweezers and injected into the cell by membrane fusion of the liposome. We also evaluated measurement accuracy of temperature from temperature calibration by fluorescent intensity and the error of measurement point. We demonstrated the fabrication of the liposome containing single sensor, immobilization of the sensor to cellular membrane, and injection of the sensor into the cell cytoplasm and nucleus.