Polysaccharide functionalization reduces lipid vesicle stiffness

Abstract

The biophysical properties of lipid vesicles are important for their stability and integrity, key parameters that control the performance when these vesicles are used for drug delivery. The vesicle properties are determined by the composition of lipids used to form the vesicle. However, for a given lipid composition, they can also be tailored by tethering polymers to the membrane. Typically, synthetic polymers like polyethyleneglycol are used to increase vesicle stability, but the use of polysaccharides in this context is much less explored. Here, we report a general method for functionalizing lipid vesicles with polysaccharides by binding them to cholesterol. We incorporate the polysaccharides on the outer membrane leaflet of giant unilamellar vesicles (GUVs) and investigate their effect on membrane mechanics using micropipette aspiration. We find that the presence of the glycolipid functionalization produces an unexpected softening of GUVs with fluid-like membranes. By contrast, the functionalization of GUVs with polyethylene glycol does not reduce their stretching modulus. This work provides the potential means to study membrane-bound meshworks of polysaccharides similar to the cellular glycocalyx; moreover, it can be used for tuning the mechanical properties of drug delivery vehicles.

Keywords: cholesterol; giant unilamellar lipid vesicles; membrane biophysics; micropipette aspiration; polysaccharides.

Fig. 1.

Glycolipid functionalization of GUV. (A) Schematic representation of amine-chondroitin-sulfate-FITC bound to cholesterol-NHS (Chol-CS), which incorporates in the outer leaflet of the GUV membrane. (B) Confocal image of GUVs functionalized with Chol-CS (orange, FITC-labeled, ${\lambda }_{\mathit{ex}}=$ 488 nm). (Scale bar, 50 $\mathrm{\mu }$m.) (C) Confocal images of Chol-CS-functionalized GUVs (orange, FITC-labeled, ${\lambda }_{\mathit{ex}}=$ 488 nm) before bleach (i), after bleach (ii), and after recovery (iii). The bleaching spot is indicated with dashed white lines. (Scale bar, 10 $\mathrm{\mu }$m.) (D) Chol-CS intensity at the GUV periphery I_peri normalized to the background intensity I_out over Chol-CS concentration. (E) Negative-stain transmission electron microscopy micrographs of unmodified and Chol-CS-functionalized small unilamellar vesicles (SUVs). (Scale bars, 150 and 100 nm, respectively).

Fig. 2.

Cholesterol-modified glycopolymers reduce lipid diffusion. (A) Schematic representation of the three experimentally tested lipid bilayer models: DOPC, DOPC with asymmetric cholesterol deposition (DOPC-Chol-asy.) and DOPC with asymmetric cholesterol-functionalized chondroitin sulfate (DOPC-Chol-CS). (B) Confocal images of the GUV (green, Atto633-labeled, ${\lambda }_{\mathit{ex}}=$ 640nm) top plane prebleach, after bleach and after recovery. The bleaching spot is indicated with dashed white lines. (Scale bar, 10$\mathrm{\mu }$m.) (C) Lipid diffusion coefficient for DOPC, DOPC-Chol-asymmetric, and DOPC-Chol-CS GUVs. Mean $\pm$ SD, n$=$10 to 11.

Fig. 3.

Micropipette aspiration reveals softening of Chol-CS-functionalized GUVs. (A) Confocal time series of a GUV (green, Atto633-labeled, ${\lambda }_{\mathit{ex}}=$ 640nm) being aspirated into a micropipette at different suction pressures. (Scale bar, 5$\mathrm{\mu }$m.) (B) Stretching modulus of DOPC, DOPC-Chol, DOPC-Chol-asymmetric, and DOPC-Chol-CS GUVs. Mean $\pm$ SD, n$=$7 to 22.

Fig. 4.

Impact of glycopolymers on GUV properties is unique. (A) Confocal image a GUV functionalized with Cholesterol-PEG (cyan, FITC-labeled, ${\lambda }_{\mathit{ex}}=$ 488nm). (Scale bar, 10$\mathrm{\mu }$m.) (B) Stretching modulus for DOPC, DOPC-Chol-PEG, and DOPC-Chol-CS GUVs. Mean $\pm$ SD, n$=$19 to 22. (C–E) Representative confocal images of deflated ($c_{\text{out}}/c_{\text{in}}=$1.5) GUVs (green, Atto633-labeled, ${\lambda }_{\mathit{ex}}=$ 640nm) without surface functionalization (C), with Chol-PEG (D) and Chol-CS (E) functionalization. Lipid nanotubes form due to deflation and are indicated with white arrows. (Scale bar, 10$\mathrm{\mu }$m).