We have employed a model system, inspired by SNARE proteins, to facilitate membrane fusion between Giant Unilamellar Vesicles (GUVs) and Large Unilamellar Vesicles (LUVs) under physiological conditions. In this system, two synthetic lipopeptide constructs comprising the coiled-coil heterodimer-forming peptides K4, (KIAALKE)4, or E4, (EIAALEK)4, a PEG spacer of variable length, and a cholesterol moiety to anchor the peptides into the liposome membrane replace the natural SNARE proteins. GUVs are functionalized with one of the lipopeptide constructs and the fusion process is triggered by adding LUVs bearing the complementary lipopeptide. Dual-colour time lapse fluorescence microscopy was used to visualize lipid- and content-mixing. Using conventional confocal microscopy, lipid mixing was observed on the lipid bilayer of individual GUVs. In addition to lipid-mixing, content-mixing assays showed a low efficiency due to clustering of K4-functionalized LUVs on the GUVs target membranes. We showed that, through the use of the non-ionic surfactant Tween 20, content-mixing between GUVs and LUVs could be improved, meaning this system has the potential to be employed for drug delivery in biological systems.

J Heyrovský Institute of Physical Chemistry Academy of Sciences of the Czech Republic v v i Dolejškova 2155 3 182 23 Prague 8 Czech Republic

Macromolecular Biochemistry Leiden Institute of Chemistry Leiden University P O Box 9502 2300 RA Leiden The Netherlands

Supramolecular and Biomaterials Chemistry Leiden Institute of Chemistry Leiden University P O Box 9502 2300 RA Leiden The Netherlands

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Ma M, Paredes A, Bong D. Intra- and intermembrane pairwise molecular recognition between synthetic hydrogen-bonding phospholipids. J. Am. Chem. Soc. 2008;130:14456–14458. doi: 10.1021/ja806954u. PubMed DOI

Chan YHM, van Lengerich B, Boxer SG. Lipid-anchored DNA mediates vesicle fusion as observed by lipid and content mixing. Biointerphases. 2008;3:FA17–FA21. doi: 10.1116/1.2889062. PubMed DOI

Jumeaux C, et al. MicroRNA Detection by DNA-Mediated Liposome Fusion. Chembiochem. 2018;19:434–438. doi: 10.1002/cbic.201700592. PubMed DOI PMC

Meng ZJ, et al. Efficient Fusion of Liposomes by Nucleobase Quadruple-Anchored DNA. Chem. Eur. J. 2017;23:9391–9396. doi: 10.1002/chem.201701379. PubMed DOI

Ries O, Loffler PMG, Rabe A, Malavan JJ, Vogel S. Efficient liposome fusion mediated by lipid-nucleic acid conjugates. Org. Biomol. Chem. 2017;15:8936–8945. doi: 10.1039/C7OB01939D. PubMed DOI

Stengel G, Zahn R, Hook F. DNA-induced programmable fusion of phospholipid vesicles. J. Am. Chem. Soc. 2007;129:9584–9585. doi: 10.1021/ja073200k. PubMed DOI

Chan YHM, van Lengerich B, Boxer SG. Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides. P. Natl. Acad. Sci. USA. 2009;106:979–984. doi: 10.1073/pnas.0812356106. PubMed DOI PMC

Flavier KM, Boxer SG. Vesicle Fusion Mediated by Solanesol-Anchored DNA. Biophysical Journal. 2017;113:1260–1268. doi: 10.1016/j.bpj.2017.05.034. PubMed DOI PMC

Loffler PMG, et al. A DNA-Programmed Liposome Fusion Cascade. Angew. Chem. Int. Ed. 2017;56:13228–13231. doi: 10.1002/anie.201703243. PubMed DOI

Xu W, Wang J, Rothman JE, Pincet F. Accelerating SNARE-Mediated Membrane Fusion by DNA–Lipid Tethers. Angew. Chem. Int. Ed. 2015;54:14388–14392. doi: 10.1002/anie.201506844. PubMed DOI PMC

Noonan PS, Mohan P, Goodwin AP, Schwartz DK. DNA Hybridization-Mediated Liposome Fusion at the Aqueous Liquid Crystal Interface. Adv. Funct. Mater. 2014;24:3206–3212. doi: 10.1002/adfm.201303885. PubMed DOI PMC

Lygina AS, Meyenberg K, Jahn R, Diederichsen U. Transmembrane Domain Peptide/Peptide Nucleic Acid Hybrid as a Model of a SNARE Protein in Vesicle Fusion. Angew. Chem. Int. Ed. 2011;50:8597–8601. doi: 10.1002/anie.201101951. PubMed DOI

Rabe A, Loffler PMG, Ries O, Vogel S. Programmable fusion of liposomes mediated by lipidated PNA. Chem. Commun. 2017;53:11921–11924. doi: 10.1039/C7CC06058K. PubMed DOI

Muheeb S, Daniel B, Dragomir M, Reinhard J, Ulf D. Distance Regulated Vesicle Fusion and Docking Mediated by β-Peptide Nucleic Acid SNARE Protein Analogues. Chem. Bio. Chem. 2016;17:479–485. doi: 10.1002/cbic.201500517. PubMed DOI

Ayumi K, Kiyomi M, Toshihisa M, Toshiki T. Construction of a pH-Responsive Artificial Membrane Fusion System by Using Designed Coiled-Coil Polypeptides. Chem-Eur. J. 2008;14:7343–7350. doi: 10.1002/chem.200701726. PubMed DOI

Kashiwada A, et al. Design and Characterization of Endosomal-pH-Responsive Coiled Coils for Constructing an Artificial Membrane Fusion System. Chem-Eur. J. 2011;17:6179–6186. doi: 10.1002/chem.201003392. PubMed DOI

Meyenberg K, Lygina AS, van den Bogaart G, Jahn R, Diederichsen U. SNARE derived peptide mimic inducing membrane fusion. Chem. Commun. 2011;47:9405–9407. doi: 10.1039/c1cc12879e. PubMed DOI

Skyttner C, Enander K, Aronsson C, Aili D. Tuning Liposome Membrane Permeability by Competitive Coiled Coil Heterodimerization and Heterodimer Exchange. Langmuir. 2018;34:6529–6537. doi: 10.1021/acs.langmuir.8b00592. PubMed DOI

Gong Y, Luo YM, Bong D. Membrane activation: Selective vesicle fusion via small molecule recognition. J. Am. Chem. Soc. 2006;128:14430–14431. doi: 10.1021/ja0644576. PubMed DOI

Kashiwada, A., Tsuboi, M. & Matsuda, K. Target-selective vesicle fusion induced by molecular recognition on lipid bilayers. Chem. Commun. 695–697, 10.1039/b815688c (2009). PubMed

Kashiwada A, Yamane I, Tsuboi M, Ando S, Matsuda K. Design, Construction, and Characterization of High-Performance Membrane Fusion Devices with Target-Selectivity. Langmuir. 2012;28:2299–2305. doi: 10.1021/la2038075. PubMed DOI

Whitehead SA, et al. Artificial Membrane Fusion Triggered by Strain-Promoted Alkyne-Azide Cycloaddition. Bioconjug. Chem. 2017;28:923–932. doi: 10.1021/acs.bioconjchem.6600578. PubMed DOI PMC

Valérie, M.-A. et al. Selective Adhesion, Lipid Exchange and Membrane-Fusion Processes between Vesicles of Various Sizes Bearing Complementary Molecular Recognition Groups. ChemPhysChem2, 367–376, doi:10.1002/1439-7641(20010618)2:6<367::AID-CPHC367>3.0.CO;2-# (2001). PubMed

Litowski JR, Hodges RS. Designing heterodimeric two-stranded alpha-helical coiled-coils: the effect of chain length on protein folding, stability and specificity. J. Pept. Res. 2001;58:477–492. doi: 10.1034/j.1399-3011.2001.10972.x. PubMed DOI

Marsden HR, Elbers NA, Bomans PHH, Sommerdijk N, Kros A. A Reduced SNARE Model for Membrane Fusion. Angew. Chem. Int. Ed. 2009;48:2330–2333. doi: 10.1002/anie.200804493. PubMed DOI

Zheng TT, et al. A non-zipper-like tetrameric coiled coil promotes membrane fusion. RSC Advances. 2016;6:7990–7998. doi: 10.1039/c5ra26175a. DOI

Zheng TT, et al. Controlling the rate of coiled coil driven membrane fusion. Chem. Commun. 2013;49:3649–3651. doi: 10.1039/c3cc38926j. PubMed DOI

Daudey GA, Zope HR, Voskuhl J, Kros A, Boyle AL. Membrane-Fusogen Distance Is Critical for Efficient Coiled-Coil-Peptide-Mediated Liposome Fusion. Langmuir. 2017;33:12443–12452. doi: 10.1021/acs.langmuir.7b02931. PubMed DOI PMC

Crone Niek, Minnee Dirk, Kros Alexander, Boyle Aimee. Peptide-Mediated Liposome Fusion: The Effect of Anchor Positioning. International Journal of Molecular Sciences. 2018;19(1):211. doi: 10.3390/ijms19010211. PubMed DOI PMC

Versluis F, Dominguez J, Voskuhl J, Kros A. Coiled-coil driven membrane fusion: zipper-like vs. non-zipper-like peptide orientation. Faraday Discuss. 2013;166:349–359. doi: 10.1039/c3fd00061c. PubMed DOI

Versluis F, et al. In Situ Modification of Plain Liposomes with Lipidated Coiled Coil Forming Peptides Induces Membrane Fusion. J. Am. Chem. Soc. 2013;135:8057–8062. doi: 10.1021/ja4031227. PubMed DOI

Mora NL, et al. Targeted anion transporter delivery by coiled-coil driven membrane fusion. Chem. Sci. 2016;7:1768–1772. doi: 10.1039/c5sc04282h. PubMed DOI PMC

Yang J, et al. Drug Delivery via Cell Membrane Fusion Using Lipopeptide Modified Liposomes. ACS Cent. Sci. 2016;2:621–630. doi: 10.1021/acscentsci.6b00172. PubMed DOI PMC

Yang J, et al. Application of Coiled Coil Peptides in Liposomal Anticancer Drug Delivery Using a Zebrafish Xenograft Model. ACS Nano. 2016;10:7428–7435. doi: 10.1021/acsnano.6b01410. PubMed DOI

Kong L, Askes SH, Bonnet S, Kros A, Campbell F. Temporal Control of Membrane Fusion through Photolabile PEGylation of Liposome Membranes. Angew. Chem. Int. Ed. 2016;55:1396–1400. doi: 10.1002/anie.201509673. PubMed DOI

Yoon TY, Okumus B, Zhang F, Shin YK, Ha T. Multiple intermediates in SNARE-induced membrane fusion. Proc. Natl. Acad. Sci. USA. 2006;103:19731–19736. doi: 10.1073/pnas.0606032103. PubMed DOI PMC

Karatekin E, et al. A fast, single-vesicle fusion assay mimics physiological SNARE requirements. Proc. Natl. Acad. Sci. USA. 2010;107:3517–3521. doi: 10.1073/pnas.0914723107. PubMed DOI PMC

Kyoung M, et al. In vitro system capable of differentiating fast Ca2+-triggered content mixing from lipid exchange for mechanistic studies of neurotransmitter release. Proc. Natl. Acad. Sci. USA. 2011;108:E304–313. doi: 10.1073/pnas.1107900108. PubMed DOI PMC

Bowen ME, Weninger K, Brunger AT, Chu S. Single molecule observation of liposome-bilayer fusion thermally induced by soluble N-ethyl maleimide sensitive-factor attachment protein receptors (SNAREs) Biophys. J. 2004;87:3569–3584. doi: 10.1529/biophysj.104.048637. PubMed DOI PMC

Tareste D, Shen J, Melia TJ, Rothman JE. SNAREpin/Munc18 promotes adhesion and fusion of large vesicles to giant membranes. Proc. Natl. Acad. Sci. USA. 2008;105:2380–2385. doi: 10.1073/pnas.0712125105. PubMed DOI PMC

Witkowska A, Jahn R. Rapid SNARE-Mediated Fusion of Liposomes and Chromaffin Granules with Giant Unilamellar Vesicles. Biophys. J. 2017;113:1251–1259. doi: 10.1016/j.bpj.2017.03.010. PubMed DOI PMC

van den Bogaart G, et al. Membrane protein sequestering by ionic protein–lipid interactions. Nature. 2011;479:552. doi: 10.1038/nature10545. PubMed DOI PMC

Kuhlmann JW, Junius M, Diederichsen U, Steinem C. SNARE-Mediated Single-Vesicle Fusion Events with Supported and Freestanding Lipid Membranes. Biophys. J. 2017;112:2348–2356. doi: 10.1016/j.bpj.2017.04.032. PubMed DOI PMC

Etzerodt TP, Trier S, Henriksen JR, Andresen TL. A GALA lipopeptide mediates pH- and membrane charge dependent fusion with stable giant unilamellar vesicles. Soft Matter. 2012;8:5933–5939. doi: 10.1039/c2sm25075f. DOI

Kahya N, Pecheur EI, de Boeij WP, Wiersma DA, Hoekstra D. Reconstitution of membrane proteins into giant unilamellar vesicles via peptide-induced fusion. Biophys. J. 2001;81:1464–1474. doi: 10.1016/S0006-3495(01)75801-8. PubMed DOI PMC

Mora NL, et al. Preparation of size tunable giant vesicles from cross-linked dextran(ethylene glycol) hydrogels. Chem. Commun. 2014;50:1953–1955. doi: 10.1039/c3cc49144g. PubMed DOI PMC

Koukalova A, et al. Distinct roles of SNARE-mimicking lipopeptides during initial steps of membrane fusion. Nanoscale. 2018;10:19064–19073. doi: 10.1039/c8nr05730c. PubMed DOI

Rabe M, Schwieger C, Zope HR, Versluis F, Kros A. Membrane Interactions of Fusogenic Coiled-Coil Peptides: Implications for Lipopeptide Mediated Vesicle Fusion. Langmuir. 2014;30:7724–7735. doi: 10.1021/la500987c. PubMed DOI

Rabe M, et al. A Coiled-Coil Peptide Shaping Lipid Bilayers upon Fusion. Biophys. J. 2016;111:2162–2175. doi: 10.1016/j.bpj.2016.10.010. PubMed DOI PMC

Walde P, Cosentino K, Engel H, Stano P. Giant Vesicles: Preparations and Applications. Chembiochem. 2010;11:848–865. doi: 10.1002/cbic.201000010. PubMed DOI

Larsen J, Hatzakis NS, Stamou D. Observation of Inhomogeneity in the Lipid Composition of Individual Nanoscale Liposomes. J. Am. Chem. Soc. 2011;133:10685–10687. doi: 10.1021/ja203984j. PubMed DOI

Apellániz B, Nieva JL, Schwille P, García-Sáez AJ. All-or-None versus Graded: Single-Vesicle Analysis Reveals Lipid Composition Effects on Membrane Permeabilization. Biophys. J. 2010;99:3619–3628. doi: 10.1016/j.bpj.2010.09.027. PubMed DOI PMC

Lohse B, Bolinger P-Y, Stamou D. Encapsulation Efficiency Measured on Single Small Unilamellar Vesicles. J. Am. Chem. Soc. 2008;130:14372–14373. doi: 10.1021/ja805030w. PubMed DOI

Mortensen KI, Tassone C, Ehrlich N, Andresen TL, Flyvbjerg H. How To Characterize Individual Nanosize Liposomes with Simple Self-Calibrating Fluorescence Microscopy. Nano Lett. 2018;18:2844–2851. doi: 10.1021/acs.nanolett.7b05312. PubMed DOI

Otten D, Brown MF, Beyer K. Softening of Membrane Bilayers by Detergents Elucidated by Deuterium NMR Spectroscopy. J. Phys. Chem. B. 2000;104:12119–12129. doi: 10.1021/jp001505e. DOI

Krielgaard L, et al. Effect of tween 20 on freeze-thawing- and agitation-induced aggregation of recombinant human factor XIII. J. Pharm. Sci. 1998;87:1597–1603. doi: 10.1021/js980126i. PubMed DOI

Benda A, et al. How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy. Langmuir. 2003;19:4120–4126. doi: 10.1021/la0270136. DOI

Kessel A, Ben-Tal N, May S. Interactions of cholesterol with lipid bilayers: The preferred configuration and fluctuations. Biophys. J. 2001;81:643–658. doi: 10.1016/S0006-3495(01)75729-3. PubMed DOI PMC

Mora NL, et al. Evaluation of dextran(ethylene glycol) hydrogel films for giant unilamellar lipid vesicle production and their application for the encapsulation of polymersomes. Soft Matter. 2017;13:5580–5588. doi: 10.1039/c7sm00551b. PubMed DOI PMC

Struck DK, Hoekstra D, Pagano RE. Use of Resonance Energy-Transfer to Monitor Membrane-Fusion. Biochemistry. 1981;20:4093–4099. doi: 10.1021/bi00517a023. PubMed DOI

Stryer L, Haugland RP. Energy transfer: a spectroscopic ruler. Proc. Natl. Acad. Sci. USA. 1967;58:719–726. doi: 10.1073/pnas.58.2.719. PubMed DOI PMC

Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods. 2012;9:671–675. doi: 10.1038/nmeth.2089. PubMed DOI PMC