Glucosomes: Glycosylated Vesicle-in-Vesicle Aggregates in Water from pH-Responsive Microbial Glycolipid

Abstract

Vesicle-in-vesicle self-assembled containers, or vesosomes, are promising alternatives to liposomes because of their possible hierarchical encapsulation and high stability. We report herein the first example of sugar-based vesicles-in-vesicles, which we baptize glucosomes. These were prepared by using a natural microbial glycolipid (branched C22 sophorolipid) extracted from the culture medium of the yeast Pseudohyphozyma bogoriensis. Glucosomes spontaneously formed in water between pH 6 and pH 4 at room temperature, without the requirement of any additive. By means of pH-resolved in situ small angle X-ray scattering, we provided direct evidence for the vesicle-formation mechanism. Statistical treatment of the vesicle radii distribution measured by cryo-tansmission electron microscopy by using a derived form of the Helfrich bending free-energy expression provided an order of magnitude for the effective bending constant (the sum of the curvature and the saddle-splay moduli) of the lipid membrane to K=(0.4±0.1) k_BT. This value is in agreement with the bending constant measured for hydrocarbon-based vesicles membranes.

Keywords: bolaforms; glycolipids; self-assembly; surfactants; vesosomes.

Figure 1

Chemical formula of aSL‐C22:0₁₃, a branched sophorolipid bearing a hydroxy behenic acid (13‐hydroxydocosanoic acid).

Figure 2

a) pH‐resolved in situ SAXS at room temperature on the aSL‐C22:0₁₃ compound at 0.5 wt % in water showing the characteristic signal of its self‐assembly as a function of pH. The evaluation of the micelle‐to‐vesicle ratio [x parameter in Eq. (2)] as a function of pH obtained after fitting the SAXS data [Eq. (2)] is presented in panel b. The pH evolution of the typical micellar radius (R) and hydrophilic shell thickness (ST), as well as the vesicle bilayer length (L) and face thickness (FT), as illustrated by the cartoon, are given in panel c. The micelles (core–shell ellipsoid of revolution) form factor model was used to fit SAXS data in the 12<pH<6 interval, whereas a core–shell bilayer form factor was used to fit SAXS data in the 6<pH<4 interval. The pH evolution of the shell scattering length density (SLD) is presented in panel d. The SLD, which is a parameter of the fit and an equivalent of the electron density of the shell, is useful to identify the hydration/dehydration phenomena of the hydrophilic shell.

Figure 3

a–c) A series of three cryo‐TEM images of glucosomes of aSL‐C22:0₁₃ (0.5 w %) recorded at pH 4.9 and room temperature. d) Close‐up image of the black box in panel c. Images show the large number of vesicle‐in‐vesicle systems.

Figure 4

Cryo‐TEM images of glucosomes of aSL‐C22:0₁₃ (0.5 w %) recorded at pH 9.4 and room temperature and showing a micellar environment (arrows 1) surrounding vesicular objects of different sizes (arrows 2 and 3). These data agree with the SAXS signal in the basic pH medium.

Figure 5

a–e) Series of additional cryo‐TEM images of aSL‐C22:0₁₃ (0.5 wt %) recorded at pH 4.9 and room temperature showing the detail of the glucosome surface roughness and structure: arrows point at invagination (negative curvature) and exvagination (positive curvature) sites visible at the membrane surface. f) Distribution (binning size: 2.5 nm; count≈350 vesicles) of the vesicle radii measured from cryo‐TEM images. The data were fitted by using a log‐normal distribution (dotted line) and Equation (4) (continuous line). The r₀ value, the mean of the distribution, is defined in Equation (5), whereas K refers to the effective bending constant of the vesicle membrane defined in Equation (3).

Figure 6

Scheme of the pH‐driven formation of glucosomes by using aSL‐C22:0₁₃ branched sophorolipid. According to the in situ SAXS data, the system is composed of a majority of micelles and a minority of vesicles from basic to neutral pH. In the transition pH region, at around pH 6, micelles seem to act as reservoirs of matter for glucosome formation. Below pH 4, a lamellar phase forms.