In Situ Modification of Plain Liposomes with Lipidated Coiled Coil Forming Peptides Induces Membrane Fusion

Abstract

Complementary coiled coil forming lipidated peptides embedded in liposomal membranes are able to induce rapid, controlled, and targeted membrane fusion. Traditionally, such fusogenic liposomes are prepared by mixing lipids and lipidated peptides in organic solvent (e.g., chloroform). Here we prepared fusogenic liposomes in situ, i.e., by addition of a lipidated peptide solution to plain liposomes. As the lipid anchor is vital for the correct insertion of lipidated peptides into liposomal membranes, a small library of lipidated coiled coil forming peptides was designed in which the lipid structure was varied. The fusogenicity was screened using lipid and content mixing assays showing that cholesterol modified coiled coil peptides induced the most efficient fusion of membranes. Importantly, both lipid and content mixing experiments demonstrated that the in situ modification of plain liposomes with the cholesterol modified peptides yielded highly fusogenic liposomes. This work shows that existing membranes can be activated with lipidated coiled coil forming peptides, which might lead to highly potent applications such as the fusion of liposomes with cells.

A Coiled-Coil Peptide Shaping Lipid Bilayers upon Fusion

“…Identity of the peptides was determined by liquid chromatography-mass spectrometry. The lipopeptides were synthesized and purified as described elsewhere (7,9). Peptide stock solutions in PBS were prepared~2 mg/ml and the concentration was determined by ultraviolet absorbance at 280 nm, for peptides without tryptophan the concentration was based on the mass.…”

“…Fig. 2 A displays the change of fluorescence intensity upon varying the lipid to peptide ratio by addition of vesicles of the composition DOPC:DOPE:cholesterol (molar ratio: 2:1:1), as used in recent vesicle fusion studies (5)(6)(7)(8)(9)(10)16,44,45). For comparative purposes, the total concentration of the tryptophan bearing peptide (X*, X ¼ K or E, respectively) was kept constant.…”

“…A simple model system that has been used in numerous membrane fusion studies was inspired by the SNARE proteins (6)(7)(8)(9)(10)(11)(12)(13). It is based on two complementary coiled-coil forming peptides with the sequences (EIAALEK) 3 and (KIAALKE) 3 named E and K, respectively (Fig.…”

Geometry of the Contact Zone between Fused Membrane-Coated Beads Mimicking Cell-Cell Fusion

“…The lipopeptides were analyzed by electrospray-ionization mass spectrometry (E3Cys-MCCDOPE: 3882 m z À1 [Mþ], K3Cys-MCCDOPE: 3827 m z À1 [Mþ]). Additionally, cholesterol-PEG12-E4 and cholesterol-PEG12-K4 were used to induce membrane fusion between the silica beads (10)(11)(12)(13). some experiments one fraction contained a different fluorescent dye lipid such as Oregon Green 488 DPPE, Atto488-DOPE, or ATTO390DOPE (1 mol %).…”

Membrane Fusion Mediated Intracellular Delivery of Lipid Bilayer Coated Mesoporous Silica Nanoparticles

“…In order to enhance the intracellular delivery of MSNs/cytC, the nanoparticles were coated with a fusogenic lipid bilayer containing 1 mol% CP 4 E 4 (MSNs/cytC@CPE-LBs). To induce nanoparticle uptake via membrane fusion, cells were pretreated with CP 4 K 4 [29] and subsequently MSNs/cytC@CPE-LBs were added to the medium. Lipopeptides and lipid bilayer coated MSNs with or without CP 4 E 4 were well tolerated by HeLa cells as no signs of toxicity were observed ( Figure S2, Supporting Information).…”