Compressibility and porosity modulate the mechanical properties of giant gas vesicles

Abstract

Gas vesicles used as contrast agents for noninvasive ultrasound imaging must be formulated to be stable, and their mechanical properties must be assessed. We report here the formation of perfluoro-n-butane microbubbles coated with surface-active proteins that are produced by filamentous fungi (hydrophobin HFBI from Trichoderma reesei). Using pendant drop and pipette aspiration techniques, we show that these giant gas vesicles behave like glassy polymersomes, and we discover novel gas extraction regimes. We develop a model to analyze the micropipette aspiration of these compressible gas vesicles and compare them to incompressible liquid-filled vesicles. We introduce a sealing parameter to characterize the leakage of gas under aspiration through the pores of the protein coating. Utilizing this model, we can determine the elastic dilatation modulus, surface viscosity, and porosity of the membrane. These results demonstrate the engineering potential of protein-coated bubbles for echogenic and therapeutic applications and extend the use of the pipette aspiration technique to compressible and porous systems.

Keywords: bubbles; compressibility; gas vesicles; porosity; viscoelasticity.

Fig. 1.

Aspiration of a giant gas vesicle (A–C) in a liquid-like regime and (D–F) in a glassy regime. (A) Schematic of the experiment showing the geometric parameters, pressure field, and driving force f_M acting on the green hatched zone of the tongue. L(t) is the penetration length for ΔP > ΔP_c. The HFBI deformable coating membrane is represented by the light blue line and the bubble contour by the black line. Leak-out flows, during aspiration, through a porous membrane containing n pores of radius a (J₀ being the leak-out flow per pore), ${\phi }_s~\frac{n{a}^2}{R_0^2}$ is the surface fraction of the holes. (B) Bright-field picture of GV aspirated in a micropipette (ΔP = 13 kPa) and (C) corresponding plot of the tongue length L as a function of time. The dashed red line is a fit using Eq. 10, corresponding to the aspiration of a sealed capsule. The porosity of the membrane leads to a progressive deswelling, characterized by the aspiration velocity. (D) Schematic of the experiment of a GV with a non-deformable protein coating. (E) Bright-field picture of a GV aspirated in a micropipette (ΔP = 18 kPa). The inset shows the GV before aspiration (same magnification). (F) Corresponding plot of L(t) demonstrating the large and fast aspiration of the tongue in this regime.

Fig. 2.

Structure of the protein coating at the gas–liquid interface of GV. (A) 3D structure of an HFBI protein. The yellow color shows the hydrophilic part, and the red color shows the hydrophobic part (PDB ID 2FZ6, image created using Chimera software, version chimera 1.15). (B) Cryo-TEM image of GV consisting of C₄F₁₀ stabilized with HFBI. (C) Thickness of the shell measured from the cryo-TEM images using ImageJ software. (D) AFM image of an HFBI layer at the air–water interface. This image shows the crystalline structure and the porosity of the film [the large dark areas could be sample defects, while the small dark areas represent the areas between the HFBI molecules (pores)].

Fig. 3.

Surface tension development of HFBI at the liquid–gas (C₄F₁₀) interface. The blue part of the curve shows the regime where the software fits the GV shape. As shown in the magnified image below the curve, the fit line (light blue) matches the bubble shape perfectly (black area above the green line). In contrast, the red part shows the regime for which the software could not fit the GV shape. The magnified image above the red curve shows the mismatch between the fit line (light blue) and the bubble shape, the mismatched areas being indicated by two red arrows. This leads to an incorrect determination of the surface tension value.

Fig. 4.

Solid-like behavior of GV. (A–D) Snapshots at different times of aspiration with ΔP = 14.5 kPa revealing the glassy behavior of an HFBI-coated microbubble aspirated into a micropipette. While the GV is aspirated in the micropipette, the outer part is less spherical. (E–H) Glassy behavior of an HFBI-coated microbubble. (E) The microbubble released after aspiration exhibits a remanent deformation. (F–H) Aspiration of the GV with ΔP = 13.5 kPa from the other side at different times, leading to the decrease of the free tongue length. It demonstrates that the stress is larger than the yield above which the solid becomes a liquid. (Scale bars, 20 μm.)

Fig. 5.

Behavior of the aspirated GV over time (n = 83). Either they show a liquid-like behavior (no apparent deformation, blue dots, and area), or a glassy behavior (they remain deformed, green rectangles, and area). The brown-dashed line corresponds to the fit Eq. 30.

Fig. 6.

Main characteristics of the liquid-like and glassy GV calculated using the pipette aspiration technique. (A) Elastic modulus $E_d^{*}$ (n = 22 for liquid-like GV and n = 18 for solid-like GV), (B) surface viscosity η_s (n = 22 and 18), (C) sealing parameter Q (n = 18 and 18), and (D) permeability P_M (n = 18 and 18). For the sake of visualization, the two highest P_M points for liquid-like GV are not represented.