Osmotic Pressure Enables High-Yield Assembly of Giant Vesicles in Solutions of Physiological Ionic Strengths

Abstract

Giant unilamellar vesicles (GUVs) are micrometer-scale minimal cellular mimics that are useful for bottom-up synthetic biology and drug delivery. Unlike assembly in low-salt solutions, assembly of GUVs in solutions with ionic concentrations of 100–150 mM Na/KCl (salty solutions) is challenging. Chemical compounds deposited on the substrate or incorporated into the lipid mixture could assist in the assembly of GUVs. Here, we investigate quantitatively the effects of temperature and chemical identity of six polymeric compounds and one small molecule compound on the molar yields of GUVs composed of three different lipid mixtures using high-resolution confocal microscopy and large data set image analysis. All the polymers moderately increased the yields of GUVs either at 22 or 37 °C, whereas the small molecule compound was ineffective. Low-gelling temperature agarose is the singular compound that consistently produces yields of GUVs of greater than 10%. We propose a free energy model of budding to explain the effects of polymers in assisting the assembly of GUVs. The osmotic pressure exerted on the membranes by the dissolved polymer balances the increased adhesion between the membranes, thus reducing the free energy for bud formation. Data obtained by modulating the ionic strength and ion valency of the solution shows that the evolution of the yield of GUVs supports our model’s prediction. In addition, polymer-specific interactions with the substrate and the lipid mixture affects yields. The uncovered mechanistic insights provide a quantitative experimental and theoretical framework to guide future studies. Additionally, this work shows a facile means for obtaining GUVs in solutions of physiological ionic strengths.

Ultrahigh yields of giant vesicles obtained through mesophase evolution and breakup

“…Each bar is an average of N=3 independent repeats and the error bars are one standard deviation from the mean. Following previous convention 12,13,16 , we The mean yield of GUVs obtained using the PAPYRUS method is 33 ± 2 % and 35 ± 2 % for pure DOPC and DOPC + 3 mol % PEG2000-DSPE respectively (Fig. 1a).…”

“…Lipid mixtures were prepared as previously described with minor adaptations 12,13,16 . We created working solutions of DOPC with 0, 0.…”

“…[1][2][3] Additionally, GUVs show promise for use in biomedical applications [4][5][6][7][8] and for bottom-up synthetic biology. [9][10][11] GUVs are routinely obtained by hydrating dry thin lipid films with low ionic strength aqueous solutions, [12][13][14][15] yet the pathways of assembly of GUVs remains to be fully understood.…”

“…Recent experimental advances have allowed the interrogation of the pathways of assembly of GUVs upon hydration of thin lipid films. By correlating the evolution of the film on the surface with quantitative measurements of the yields and the distribution of sizes of populations of GUVs, we studied the effects of physicochemical parameters such as the geometry and chemical composition of the substrate used to support the lipid film 13 , the lipid composition 12 , the identity of assisting polymeric compounds, 12 the duration 16 and the temperature 12 of incubation, and the ionic strength of the hydrating solution 12 . Depending on the conditions, GUV yields ranged from low, 0.3 -4.9 %, moderate, 5 -19.9 %, and high, 20 -40 %.…”

“…A similar free energy argument shows that the dissolution of partially soluble polymers exerts an osmotic pressure that can balance the increased adhesion between bilayers due to the screening of electrostatic charges in salty solutions. The osmotic pressure contribution lowers the free energy of budding, thus resulting in high yields of GUVs in salty solutions 12 .…”