Ionic Combisomes: A New Class of Biomimetic Vesicles to Fuse with Life

Abstract

The construction of biomembranes that faithfully capture the properties and dynamic functions of cell membranes remains a challenge in the development of synthetic cells and their application. Here a new concept for synthetic cell membranes based on the self-assembly of amphiphilic comb polymers into vesicles, termed ionic combisomes (i-combisomes) is introduced. These combs consist of a polyzwitterionic backbone to which hydrophobic tails are linked by electrostatic interactions. Using a range of microscopies and molecular simulations, the self-assembly of a library of combs in water is screened. It is discovered that the hydrophobic tails form the membrane's core and force the backbone into a rod conformation with nematic-like ordering confined to the interface with water. This particular organization resulted in membranes that combine the stability of classic polymersomes with the biomimetic thickness, flexibility, and lateral mobility of liposomes. Such unparalleled matching of biophysical properties and the ability to locally reconfigure the molecular topology of its constituents enable the harboring of functional components of natural membranes and fusion with living bacteria to "hijack" their periphery. This provides an almost inexhaustible palette to design the chemical and biological makeup of the i-combisomes membrane resulting in a powerful platform for fundamental studies and technological applications.

Keywords: amphiphilic comb polymers; bottom-up synthetic biology; hybrid vesicles; polyelectrolyte-surfactant complexes; polymersomes; synthetic biomembranes; vesicle fusion.

Figure 1

i‐Combisomes concept: iCPs self‐assembled into biomimetic unilamellar vesicles where the backbone is a rod confined at the water interface. a) The chemical structure of iCP shows the hydrophilic backbone of poly(CBAA‐co‐DMAPAA) to which the hydrophobic DDP is complexed. In water, the iCPs form bilayers with biomimetic thickness and high flexibility. b) Biofunctionalization of the i‐combisome membrane with lipids and generation of raft‐like domains. c) Formation of a pore by the insertion of pore‐forming peptides. d) Facile fusion with liposomes. e) Formation of hybrid protocells by fusion with living bacteria.

Figure 2

Self‐assembly of a library of iCPs with DP  =  85 in water studied by CLSM. The assembly was studied as a function of ρ _DDP which controls the packing parameter of an equivalent repeating unit depicted below the respective confocal images. ρ _DDP was adjusted by varying N and DS. The insets show an image of a representative i‐combisome. Scale bars are 30 µm for overview images and 10 µm for insets.

Figure 3

Molecular organization of iCPs at the i‐combisome membrane. a) 3D reconstruction of 70 confocal scans showing a bisected i‐combisome labeled with nile red (0.1 mol%). Scale bar: 10 µm. b) AFM height image of a deposited i‐combisome (DP₈₅N₄₃DS₁₀₀) on mica at 55% RH. The bilayer height was analyzed along the arrow yielding a membrane thickness of 4.4 nm. Scale bar: 500 nm. c) Cryo‐TEM of i‐combisomes (DP₈₅N₄₃DS₁₀₀) formed by the injection method with the respective membrane thickness. The inset shows an example of a faceted vesicle. Scale bar: 200 nm. d) The thickness of i‐combisome membranes was determined on multiple cryo‐TEM images and presented as the average of n = 15. e,f) Snapshots of simulated bilayers of DP₃₀N₅₀DS₈₀ and DP₃₀N₇₀DS₁₀₀. Top view images show the backbones (no explicit side groups) organized at the interface with water. The side views (cross‐sections along the normal to the membrane with and without visualization of the side groups) display the organization of the membrane with the backbone restricted to the interface with water. g) Density profile of DDP (green), polymer backbone (blue), and water (black) in i‐combisome DP₃₀N₅₀DS₈₀. h) The simulated thickness of the DDP zone (green) and the total bilayer (blue). i) Orientational order parameter of the backbone (S ^backbone, blue) and of DDP (S ^DDP, green). j) Deuterium order parameter (S _CD) for three different i‐combisomes.

Figure 4

a) Boxplots for the diffusion coefficients of Rhod‐PE in supported bilayers of DP₈₅N₄₃DS₁₀₀ and for the Rhod‐labeled backbone of DP₈₅N₄₃DS₁₀₀. Boxes were generated from ten data points and contain the 25th to the 75th percentile of each data set. The line represents the median, while an open rectangle indicates the average. The whiskers show the standard deviation, while the outliers are displayed outside of the whiskers. b) Angular fluctuation of radii (Δr(φ)’) after subtracting the first two harmonics of the cosine decomposition and distribution of the fluctuations (left). BDEO: polymersome from poly(BD₈₇‐b‐EO₇₂), DLPC: liposomes. c–e) Thermal stability assay: i‐combisomes (DP₈₅N₄₃DS₁₀₀) and liposomes were formed in a calcein solution. The external calcein was quenched by the addition of a solution of Co²⁺. Subsequently, the temperature of the dispersion was risen to 80 °C for 1 h and cooled down before measurement. The i‐combisomes remained intact d), while liposomes underwent breakage and aggregation upon thermal treatment e). Scale bars: 10 µm.

Figure 5

Coassembly with lipids a–d) and insertion of pore‐forming peptides e). a) Hybrid lipid—i‐combisomes containing 40 mol% Rhod‐PE (red). Scale bars: 20 and 5 µm. b) Hybrid lipid—i‐combisomes with rhod‐labeled iCP (red) and 16:0‐NBD‐PE lipid (cyan). Left: red channel (rhod), middle: cyan channel (NBD), right: merge. Scale bar: 5 µm. c,d) Coassembly with 20 mol% of lipids with different lengths of the hydrophobic tails: DLPC (C12), DPPC (C16), and DSPC (C18) labeled with Laurdan. c) Merged CLSM images of Laurdan emission detected at λ  =  415–445 nm (red) and λ  =  490–530 nm (cyan). Scale bars: 5 µm d) Distribution of the GP of Laurdan in lipid–i‐combisomes hybrid. e) Scheme depicting the insertion of α‐hemolysin and quenching of calcein fluorescence by Co²⁺ (top). i‐Combisomes labeled with nile red (membrane) and calcein (lumen) are contacted with Co²⁺. Spiking α‐hemolysin results in pore formation and the diffusion of Co²⁺ into the i‐combisome's lumen, quenching calcein. Scale bar: 5 µm. All i‐combisomes in this figure were formed from DP₈₅N₄₃DS₁₀₀.

Figure 6

a) Electrostatically‐driven fusion of anionic liposomes (DLPC‐DLPG, 8:2) and cationic i‐combisomes (DP₈₅N₄₃DS₇₀). The liposomes and i‐combisomes are labeled with NBD‐PG and Rhod‐PE, respectively. CLSM of representative vesicles immediately after mixing and after 6 h. Left: red channel, middle: cyan channel, right: merge. b) Fusion of i‐combisomes (DP₈₅N₄₃DS₁₀₀) and liposomes (DLPC) assembled from electroneutral components labeled with Rhod‐PE and NBD‐PC. CLSM images of representative vesicles immediately after mixing and after 6 h. At time zero it was possible to observe the hemifusion diaphragm. Left: red channel, middle: green channel, right: merge. Scale bars: 5 µm. c) Scheme of the model of fusion between lipids (top bilayer) and i‐combisome (bottom bilayer) highlighting the steps.

Figure 7

Fusion of i‐combisomes with living E. coli. a) Scheme demonstrating the adhesion and fusion process. b) i‐Combisomes before contact with bacteria. c) Adhesion of E. coli after 2 min. d) Formation of patches after fusion. e) Z‐scan of a hybrid vesicle showing elliptical patches. Scale bar: 5 µm.