One-Pot Assembly of Complex Giant Unilamellar Vesicle-Based Synthetic Cells

Abstract

Here, we introduce a one-pot method for the bottom-up assembly of complex single- and multicompartment synthetic cells. Cellular components are enclosed within giant unilamellar vesicles (GUVs), produced at the milliliter scale directly from small unilamellar vesicles (SUVs) or proteoliposomes with only basic laboratory equipment within minutes. Toward this end, we layer an aqueous solution, containing SUVs and all biocomponents, on top of an oil-surfactant mix. Manual shaking induces the spontaneous formation of surfactant-stabilized water-in-oil droplets with a spherical supported lipid bilayer at their periphery. Finally, to release GUV-based synthetic cells from the oil and the surfactant shell into the physiological environment, we add an aqueous buffer and a droplet-destabilizing agent. We prove that the obtained GUVs are unilamellar by reconstituting the pore-forming membrane protein α-hemolysin and assess the membrane quality with cryotransmission electron microscopy (cryoTEM), fluorescence recovery after photobleaching (FRAP), and zeta-potential measurements as well as confocal fluorescence imaging. We further demonstrate that our GUV formation method overcomes key challenges of standard techniques, offering high volumes, a flexible choice of lipid compositions and buffer conditions, straightforward coreconstitution of proteins, and a high encapsulation efficiency of biomolecules and even large cargo including cells. We thereby provide a simple, robust, and broadly applicable strategy to mass-produce complex multicomponent GUVs for high-throughput testing in synthetic biology and biomedicine, which can directly be implemented in laboratories around the world.

Keywords: bottom-up assembly; giant unilamellar vesicles (GUVs); proteoliposomes; protocells; synthetic cells; water-in-oil droplets.

Figure 1

(A) Schematic illustration of the 3-step “shaking” strategy for the formation of GUV-based synthetic cells. Step 1: An aqueous solution containing the SUVs and/or proteo-liposomes and the species for entrapment is layered on top of fluorinated oil supplemented with PEG-based fluorosurfactants. Step 2: Manual shaking or vortexing induces the formation of surfactant-stabilized water-in-oil droplets. SUVs and proteo-liposomes fuse to form a spherical supported lipid bilayer at the droplet interface (termed droplet-stabilized GUVs, dsGUVs). Step 3: Upon addition of an aqueous buffer and a droplet destabilizing agent, GUVs are released from the surfactant shell and the oil phase into the aqueous buffer. (B) Confocal fluorescence images and schematics illustrating the conditions for the charge-mediated formation of dsGUVs. In the absence of Mg²⁺, negatively charged SUVs (here, 30% DOPG, green) remain homogeneously distributed inside the droplet (i), while dsGUVs are formed in the presence of Mg²⁺ (10 mM, ii). The opposite is true for positively charged SUVs (here, 30% DOTAP, red, iii and iv). Multicompartment GUVs can be formed from a mixture of positively (red) and negatively (green) charged SUVs (v). Note that SUVs are smaller than the diffraction limit and that the droplet interface is negatively charged due to the presence of Krytox (10.5 mM, light green in illustration). Scale bars: 10 μm.

Figure 2

Formation of free-standing GUVs via the shaking method. (A) Confocal fluorescence imaging of droplet-stabilized GUVs obtained after encapsulation of SUVs into water-in-oil droplets via the shaking method. Oil phase: 1.4 wt % PEG-based fluorosurfactant, 10.5 mM Krytox in HFE. Aqueous phase: 1.2 mM lipidmix (SUVs made of 30% DOPG, 15% cholesterol, 27.25% DOPC, 27.25% POPC, 0.5% Atto488-labeled DOPE) in 10 mM MgCl₂, 30 mM Tris, pH 7.4. (B) Free-standing GUVs after release. (C) Histogram showing the size distribution of the droplet-stabilized GUVs produced by the shaking method before release. (D) Histogram of the size distribution of free-standing GUVs after release. The size distribution was analyzed with ImageJ using manual thresholding and the particle analysis tool. (E) Confocal fluorescence imaging (left, scale bars, 10 μm) and zeta potential measurements (right) of free-standing GUVs produced from SUVs with different mol % of charged lipids: up to 50% negatively charged lipids (green, DOPG in 10 mM MgCl₂, 30 mM Tris), neutral lipids (yellow, 50% DOPC, 50% POPC in 100 mM KCl, 30 mM Tris), and up to 50% positively charged lipids (red, DOTAP in 30 mM Tris). 0.5 mol % Atto488-labeled DOPE or LissRhod-PE was added to the lipid mixture for visualization purposes.

Figure 3

Confirmation of unilamellarity of GUVs produced via the shaking method. (A) FRAP measurements provide a diffusion coefficient of 2.30 ± 0.25 μm²/s. Error bars correspond to the standard deviation of five independent measurements. (B) CryoTEM micrograph of a GUV (20% EggPG, 79% EggPC, 1% LissRhod-PE in PBS, 10 mM MgCl₂) showing the unilamellarity of the lipid bilayer. (C) Dye (fluorescein) influx measurements performed after addition of 10.7 nM heptameric α-hemolysin nanopores (75 nM monomers). Left: The mean intensity inside GUVs over the intensity outside of the GUVs (N = 25) is plotted as a function of time. The mean and the standard deviation are shown. Right: Representative confocal fluorescence images of a GUV at the beginning and the end of the measurement. (D) Dye influx measurements performed without addition of α-hemolysin nanopores. N = 28. Error bars correspond to the standard deviation.

Figure 4

Confocal fluorescence images showing the diverse possibilities for encapsulation and reconstitution into the GUVs produced by the shaking method. Free-standing GUVs with (A) reconstituted TAMARA-labeled α_IIbβ₃ integrin; (B) Cy3-labeled membrane-adhering cholesterol-tagged DNA; (C) 100 nM encapsulated pyranine; (D) SYBR Green I-stained mRNA; (E) multifluorescent polystyrene beads; (F) mitochondria isolated from HeLa cells and stained with MitoTracker Green; (G) GFP-labeled E. coli; (H) GUVs; and (I) lipid budding and tube formation in osmotically deflated GUVs. Scale bars: 10 μm.