Controlled Peptide-Mediated Vesicle Fusion Assessed by Simultaneous Dual-Colour Time-Lapsed Fluorescence Microscopy

Abstract

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 heterodimerforming peptides K 4 , (KiAALKe) 4 , or e 4 , (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 K 4-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.

Molecular Simulations of PEGylated Biomolecules, Liposomes, and Nanoparticles for Drug Delivery Applications

“…In addition, coiled coils can self-assemble to mechanically rigid protein fibers and thus have been synthesized for drug delivery applications as templates to promote the assembly of other molecules [ 87 , 88 ]. To increase their solubility and stability, PEGylated coiled coils have been experimentally synthesized by the Klok group [ 89 , 90 , 91 , 92 , 93 ], the Kros group [ 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 ], and the Xu group [ 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 ], showing membrane fusion and micelle assembly modulated by peptide sequence, PEG size and density, which have been theoretically complemented by simulations.…”

Synthetic Cell as a Platform for Understanding Membrane-Membrane Interactions

“…These oligopeptides form a stable heterodimer with a dissociation constant of ~10 −7 M [129]. A recent study, reported by the same group, demonstrated membrane fusion between GUVs with peptide K (KIAALKE) 4 and LUVs with peptide E (EIAALEK) 4 (Figure 3C) [122].…”

“…Controlling fusion by using DNA-tethered vesicles provides exquisite specificity and expands the opportunities to control spatiotemporal dynamics of CFE reactions. Reproduced from Reference [122] with permission from Springer Nature, copyright 2020.…”

“…Appearance of florescence signals after 30 min of incubation confirmed lipid mixing, while in content mixing, release of an encapsulated dye in the lumen of GUVs was observed (yellow arrows). Reproduced from Reference [122] with permission from Springer Nature, copyright 2020.…”

Liposome fusion with orthogonal coiled coil peptides as fusogens: the efficacy of roleplaying peptides

“…Previously, we found that the efficiency of these fusogens strongly depends on the concerted influences of membrane anchor type, 29 PEGspacer length, 30 and peptide length and orientation, [31][32][33][34] with cholesterol -PEG 4 -E 4 /K 4 being very efficient both in vitro (fusion of liposomes with GUVs) and in vivo (liposome -cell fusion). [35][36][37][38] We identified defined, separate roles of E 3 and K 3 which are crucial for efficient fusion; Peptide K 3 promotes the formation of lipid protrusions by spontaneous membrane insertion, while peptide E 3 acts as a handle to enable the docking of opposing liposomes. [39][40][41][42][43] Most model systems, including ours, are based on a single complementary recognition unit, 44 and are thus only a limited mimic of the targeted specificity of the SNARE protein family.…”