A protein-coated gas vesicle

Abstract

The present invention is directed to a protein-coated gas vesicle comprising a class II type hydrophobin HFBI. According to the present invention, there is also provided a protein-coated gas vesicle comprising class II type hydrophobin HFBI for use in medical applications. According to the present invention, there is also provided an ultrasound contrast agent comprising a plurality of vesicles comprising class II type hydrophobin HFBI.

Description

[0001]A protein-coated gas vesicle FIELD This invention relates to the interfacial mechanics of highly stable protein-coated bubbles, especially used as contrast agents for non-invasive ultrasound imaging. BACKGROUND Gas vesicles (GV) that are produced in bacteria to achieve cellular buoyancy, have opened new avenues in medicine, both for medical imaging and tumor treatment. They are air-filled protein nanostructures, hundreds of nanometers in size, which have been genetically encoded in cells and bacteria to serve as contrast agents for ultrasound imaging and as cell-killing agents by inertia-induced cavitation (1). If small unilamellar vesicles are used for drug delivery, all experiments to investigate the physics of membranes have been performed using giant unilamellar vesicles. Similarly, we study here the mechanical properties of giant gas vesicles (GGV) of sizes ranging from 10 to about 100 microns, formed by coating microbubbles with surface-active proteins adsorbed on their surface. Ultrasound imaging is a well-established diagnostic method, and its use has grown rapidly over the past few decades due to several advantages, such as its safety, portability, real-time imaging capability, and cost-efficiency (2). It has become the second most commonly used method after X-ray imaging (3). Ultrasound imaging is based on the exposure of a body to high-frequency sound waves, which are reflected at different interfaces. By calculating the propagating time and amplitude of the reflected wave (echo), an image can be constructed (4). As a mechanical wave, the echo of the ultrasound wave mainly depends on the physical properties of the medium and interfaces. To create a clear image using ultrasound, there should be clear differences in the mechanical properties between the investigated body parts. This could be achieved by using ultrasound enhanced contrast agents (UECA), which are substances with predesigned mechanical properties, mainly micro and nano GV (5, 6). Currently, GV play a fundamental role in therapeutic techniques and diagnosis. They can be used as drug or oxygen cargo in therapy and as contrast agents in ultrasound repetition imaging (7, 8). The main challenge in the UECA development is finding the balance between the half-life of the GV and their echogenicity, which is the ability of a substance to reflect or transmit ultrasound waves to the surrounding medium (9). This balance depends on the interfacial properties of the shell and the nature of the gas core. Therefore, getting insight into the shell properties is of fundamental importance for developing new contrast-enhanced agents. As cargos in therapy or as contrast agents in diagnostic, GV need to be injected intravenously. In this case, they must be robust, as it leads to a significant increase in the pressure of the gas inside, the pressure being the sum of Laplace pressure and blood pressure. The stability of GV is related to the physical properties of their shell, the nature (e.g., molecular size and polarity) of the gas core, and the viscosity of the ambient liquid. On the other hand, the echogenicity of GV is highly related to the mechanical properties of the shell. For instance, bubbles stabilized with phospholipids reflect ultrasound waves better than bubbles stabilized with polymers (5, 10), yet are more fragile than protein coated bubbles. The lysis tension ranges between 2 and 15 mN m-1for liposomes (11) whereas in our case GV are subjected to tensions up to 55 mN m-1without rupture. SUMMARY The invention is defined by the features of the independent claims. Some specific embodiments are defined in the dependent claims. According to a first aspect of the present invention, there is provided a protein- coated gas vesicle comprising class II type hydrophobin HFBI. According to a second aspect of the present invention, there is provided a protein- coated gas vesicle comprising class II type hydrophobin HFBI for use in medical applications. According to a third aspect of the present invention, there is provided an ultrasound contrast agent comprising a plurality of gas vesicles comprising class II type hydrophobin HFBI. According to a fourth aspect of the present invention, there is provided a use of the protein-coated gas vesicle comprising a surface layer of class II type hydrophobin HFBI as an ultrasound contrast agent. BRIEF DESCRIPTION OF THE DRAWINGS Figure 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 fM acting on the hatched zone of the tongue. L(t) is the penetration length for ΔP > ΔPc. The HFBI deformable coating membrane is represented by the light grey 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), ^^^ 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 line corresponds 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. Figure 2. Structure of the protein coating at the gas-liquid interface of gas vesicles. (A) 3D structure of an HFBI protein. The hydrophilic part and the hydrophobic part are shown. (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 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). Figure 3. Solid-like behavior of gas vesicles. (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 are 20 μm. Figure 4. Main characteristics of the liquid-like and glassy gas vesicles calculated using the pipette aspiration technique. (A) Elastic modulus (n = 15 for liquid-like GV and n = 6 for solid-like GV), (B) surface viscosity ^s(n = 15 and 6), (C) sealing parameter Q (n = 11 and 6), and (D) permeability PM (n = 11 and 6). Figure 5. Suspension of C4F10bubbles stabilized by HFBI. After 15 min, the suspension appears homogenous , while after 75 min, the lower part of the vial is less dense, meaning that the larger bubbles have already migrated to the surface. Figure 6. Microbubbles under high pressure. (A) shows the changes in the projected surface area of the microbubbles under external pressures between 0 and 250 mbar with consecutive steps of 50 mbar, wherein the projected surface area lowers while the pressure is increased. (B) shows the changes in the projected area during the relaxation time (the pressure is decreased from 250 to 0 mbar). (C) shows the bubble shape and size under the same pressures as in (A). The bubbles survived under the high pressure and returned to their original size as the pressure decreased to 0. EMBODIMENTS Hydrophobins are a group of highly surface-active proteins produced by filamentous fungi. They have a common structural feature, which is a characteristic sequence of eight cysteine residues in conserved order in their primary structure (12). These cysteine residues create four disulfide bridges (S-S bond) that bind hydrophobin protein leading to a globular shape. In addition, a hydrophobin molecule can be pictured as a natural Janus nanoparticle due to its non-centrosymmetric amphiphilic structure (13). Hydrophobins are grouped into two classes: hydrophobins I and II (14). Even though both classes share the main features such as surface activity and surface adhesion, the main functional difference between them is their solubility. The aggregates of class I hydrophobins are highly insoluble in buffer solution, while class II hydrophobins aggregates dissolve more easily (15). In addition, Linder et al. have reported that there are also differences related to the interfacial structures of class II hydrophobins (17, 18) HFBI and HFBII, which are small-sized proteins (7.5 and 7.2 kDa, respectively), produced by Trichoderma reesei fungi (18). Because they are produced by the same fungi and share almost the same amphiphilic structure, HFBI and HFBII have different interfacial properties. The clearest difference between these two hydrophobins is that even if both create a viscoelastic layer at gas-liquid interfaces, HFBI layers are more glassy (19). This difference in interfacial behavior is supposed to be related to the hierarchical structure of the hydrophobin film (20). The dilatational mechanical properties, such as storage and loss modulus, give an insight into the compressibility of molecules adsorbed at the interface, which is relevant to ultrasound imaging applications. The surface shear rheological properties: elastic (G’) and viscous (G’’) moduli, which tell more about the interaction between the molecules at the interface, are reported (about 700 mN m-1 and 200 mN m-1 respectively) (15) contrary to the compressibility of the HFBI coating. Due to their unique physical and chemical properties, perfluorocarbons (PFCs) are confidently used in medical applications for diagnostics as well as therapy. PFCs are among the most inert and stable chemicals. Therefore, perfluoro-n-butane gas (C4F10) is one of the most commonly used gas for injectable ultrasound contrast agents (21). In this study, the mechanical properties of HFBI hydrophobin layer assembled at the gas-liquid (C4F10-water) bubble interface are measured using micropipette technique as an alternative to the traditional surface dilatational rheology technique, which failed to measure the storage and loss modulus due to glassy nature of the layer. Accordingly, the present invention is directed to a protein-coated gas vesicle comprising class II type hydrophobin HFBI. In a preferred embodiment, said class II type hydrophobin HFBI is a 7.5 kDa protein originating from Trichoderma reesei. In a preferred embodiment, the present invention is directed to a protein-coated gas vesicle comprising a surface layer of class II type hydrophobin HFBI. In another preferred embodiment, said gas vesicle is formed by a mechanical agitation method. In an alternative embodiment, said gas vesicle is formed by using microfluidics. In a preferred embodiment, said gas vesicle is filled with perfluoro-n-butane gas (C4F10). In an alternative embodiment, said gas vesicle is filled with any fluoro- containing gas. In further alternative embodiments, said gas vesicle is filled with a gas selected from the group consisting of: N2, Ar, Ne, C2F6, CF4, C4H10, C3H8, C2H6, CH4, SF6, C3F8, C4F10, CF3H, CF2H2, CFH3, C2F4H2, C2F5H, C3F7H, C3F6H2, C3F5H3, C3F4H4, C3F3H5, and mixtures thereof. In another preferred embodiment, stability of said gas vesicle is based on the adsorption of the HFBI proteins at the liquid / gas interface of said gas vesicle. In another preferred embodiment, the HFBI proteins form a monolayer. In another preferred embodiment, the HFBI proteins provide an amphiphilic shell around the gas vesicle. In another preferred embodiment, the size of said gas vesicle is in the range of 10 to 100 microns. In an alternative embodiment, the size of said gas vesicle is in the range of 100 nm to 100 µm. The present invention is also directed to a protein-coated gas vesicle as defined herein for use in medical applications including drug delivery. In an alternative embodiment, the present invention is directed to a protein-coated gas vesicle as defined herein for use as a medicine. In a preferred embodiment, the protein-coated gas vesicle for said use is to be administered intravenously. It is also notable that it would be obvious for a person skilled in the art reading this disclosure that the protein-coated gas vesicle as defined herein has many other uses in medical diagnostics, delivery of therapeutics, and delivery of theranostics. The present invention is also directed to an ultrasound contrast agent comprising a plurality of the protein-coated gas vesicles as defined herein. In a preferred embodiment, said ultrasound contrast agent is for intravenous administration. The present invention is also directed to a use of the protein-coated gas vesicle of the present invention as an ultrasound contrast agent. During the last few decades, one of the most utilized methods for measuring interfacial mechanics is surface dilatational rheology based on axisymmetric drop shape analysis (ADSA) (22–24). There are various advantages that make ADSA a cornerstone technique in many laboratories doing interfacial research. In comparison with other methods, such as Langmuir trough and Wilhelmy plate, ADSA is easy to handle, needs a relatively small sample (that makes it very popular especially if the sample is expensive), and can be done in challenging experimental conditions such as high pressure and temperature. The theory and the applications of the ADSA method are explained in a number of review articles (25–27). This technique has been used previously to approximate the surface tension of HFBI films at the air / water interface. Also, the viscoelastic properties of HFBI and HFBII films, and HFBII macroscopic liquid droplets have been reported using surface shear rheology (15) and ADSA (28), respectively. Still, a challenge is to find a way to measure the properties of glassy membrane-like HFBI layer stabilizing microbubble. The pipette aspiration technique has been developed to characterize mechanical properties of GUV (11), in particular the membrane tension, the dilatation elastic modulus and the membrane viscosity. Its use has been extended to a wide variety of soft matter objects from living systems such as single cells (29–31) or cellular aggregates (32), to artificial systems such as liposomes (11, 32–34), emulsions (35–39) or polymersomes (40, 41). Micropipette aspiration has previously been used to measure the water porosity of liposomes (42, 43). A liposome initially in an observation chamber at low osmolarity is aspirated in a micropipette and then transferred in another one with a higher osmolarity. The tongue length of the liposome in the micropipette is monitored. Because of the osmolarity change, the reduction of the volume of the vesicle gives rise to an excess area, which is incorporated in the tube, and the length of the aspirated tongue increases. For GV, the leaking out is induced by the Laplace pressure acting on the sucked bubble. The mechanical parameters of the bubbles can be deduced from the micropipette aspiration method developed by Evans (33). A pressure difference ΔP is imposed between a liquid medium and a pipette, and the capsule penetrates the pipette forming a tongue of length L. We describe the statics and the dynamics of suction for four cases: 1) incompressible, 2) compressible, 3) porous GV (Figure 1A-C), and 4) ultra-rigid GV (Figure 1D-F). Aspiration of ultra-rigid gas vesicles: gas extraction regime The rigidity of the coating depends on the incubation time. As shown in Figure 4, glassy regimes are observed for bubbles below a radius ^∗ ^ depending on the incubation time. These GV are characterized by a yield stress σy. If ∆^ < ^^, the coating cannot expand and σ remains equal to the initial tension σ0. We show here ^^ that a tongue of gas can form if ∆^ ≳ where ^ is the surface tension of the medium equal to 64.5 mN m-1for a glycerol solution (50% w / w in distilled water) -1^^ and 72 mN m for pure water. We study the regime ^^> ∆^ > ^^illustrated in Figure 1D-F. In this case, σ is constant (σ = σ0) and the driving force on the tongue is given by ^^ As soon as ∆^ > a tongue of gas is formed and expands. The gas pressure decreases and may lead to a deflation of the capsule. The tongue ultimately stops when all the gas is sucked in the pipette. We have ~18 kPa for Rp = 7 μm and γ = 64.5 mN m-1. The dynamics of the tongue is extremely fast. fM is balanced by the friction force ^^= 8^^^^^^̇, due to the viscous flow inside the pipette induced by the expansion of the tongue of gas, where Lt is the length of the micropipette and ^^the medium viscosity. It leads to Considering R p = 7 μm, ηL = 6.510-3Pa s, Lt = 1 cm, and ^^ − ~ 2 kPa, it leads to an aspiration velocity ^̇ = 200 μm s-1. This value is in agreement with the experimental data, where the very fast aspiration of the tongue can be seen in the plot in Figure 1F. The verbs “to comprise” and “to include” are used in this document as open limitations that neither exclude nor require the existence of also un-recited features. The features recited in depending claims are mutually freely combinable unless otherwise explicitly stated. Furthermore, it is to be understood that the use of "a" or "an", i.e. a singular form, throughout this document does not exclude a plurality. While the forgoing examples are illustrative of the principles of the present invention in one or more particular applications, it will be apparent to those of ordinary skill in the art that numerous modifications in form, usage and details of implementation can be made without the exercise of inventive faculty, and without departing from the principles and concepts of the invention. Accordingly, it is not intended that the invention be limited, except as by the claims set forth below. EXPERIMENTAL SECTION Results and Discussion Formation of gas vesicles The gas vesicles are produced by mechanical agitation.15 min after their formation, a single white phase is observed. Afterward, they migrate according to their size and we observe the formation of a very diffuse layer on the top containing the larger GV, while the rest of the sample, less diffusive and opaque, contains the smaller ones. Structure of gas vesicles Due to its amphiphilic nature (Figure 2D) and its high affinity to self-assemble at hydrophobic-hydrophilic interfaces, HFBI creates an amphiphilic shell around C4F10bubbles, resulting in spherical GV of nanometer to micrometer size. The structure of these GV studied by cryo-TEM (Figure 2A) shows that for all sizes encountered (up to 1 µm), the membrane thickness is 2.7 ± 0.4 nm (n = 8). As this value is comparable to the size of an HFBI protein (19), we can deduce that the bubbles are covered by a protein monolayer. We are not able to measure larger GV by cryo-TEM, but we assume that their membrane thickness also corresponds to a protein monolayer. The AFM image of the HFBI layer at the air-water interface (Figure 2C) shows a crystalline arrangement of HFBI molecules with a regular roughly hexagonal pattern of holes. These nanosized holes represent the structural porosity of the shell. The large dark areas in the image are empty and most probably are sample preparation defects. The layer structure in this image matches the structure reported in the literature (19, 44). Even though the molecular size and polarity of C4F10and air differ, we assume that the HFBI layer structure could be the same because both gases are hydrophobic. Surface tension ^^of gas vesicles A non-polar gas bubble (C4F10) is initiated on the tip of a hooked needle inside a solution consisting of 0.01 mg ml-1of HFBI. The surface tension vs time plot shows the three phases of the self-assembly process. First, the HFBI molecules diffuse to the bubble surface. Second, the molecules adsorb at the interface, and lastly, the surface reaches the saturation state, whose surface tension value represents the static surface tension of the HFBI solution at the gas surface. The value is about ^^= 55 mN m-1, which is significantly lower than the surface tension of pure water (72 mN m-1). It means that we have a layer of HFBI adsorbed at the interface. For comparison, surface tensions ranging from 30 mN m-1to 50 mN m-1were found performing equilibrium surface tensiometry experiments for similar HFBI concentrations (15) and shape analysis of deflated capsules (28). Also, the determination of the surface tension for high concentrations has to be taken with caution due to the formation of the glassy film of HFBI (15). The difficulty in determining the surface tension of HFBI layers is confirmed by Szilvay et al.(19) who failed to determine it using the ADSA method due to the unexpected deformation of the drops. Micropipette aspiration of gas vesicles To study the elastic and viscous properties of the GV, we use the micropipette aspiration technique. As we will see, it is necessary to apply high pressures to aspirate these GV. It is then cumbersome to use a classical pipette setup in which the pressure is controlled by modifying the height between the observation chamber and the water reservoir. Instead, we use here a piezoelectric pressure controller allowing to easily exceed 10 kPa, otherwise requiring classically a displacement of the water tank higher than 1 m. GV range in size from the nanoscale to the micrometer scale. Since we are limited by the minimum radius of the micropipettes that can be prepared, we consider GV with R ≥ 15 μm when their dynamics of aspiration is studied, and R ≥ 10 μm when only the observation of their behavior is needed (i.e. without parameter determination). We apply a pressure ΔP between 12 and 20 kPa, which is ^^ extremely large ( ^^~0.1 − 0.2), meaning that the gas compression will be an important factor. We aspirate a GV for about 10 to 60 s so that the length of the tongue in the pipette is at least 3 times the pipette radius (Figure 3A-D). The parameters are chosen individually for each GV to apply sufficient pressure to aspirate the vesicles and sufficient time so that we observe both the initial elastic deformation followed by a steady flow in the pipette. As the GV is aspirated, its shape becomes less and less spherical. At that point, we stop the applied pressure and observe two types of behavior: either a liquid-like behavior where the tongue retracts completely, and the bubble recovers completely with spherical shape as before the aspiration or a glassy behavior where the tongue does not relax and retains the sleeve shape, while the vesicle keeps a pear-like shape (Figure 3E). The tongue remains permanent after expulsion from the pipette. We also successively aspirated the GV, changing the position of the micropipette on the surface of the same vesicles between several consecutive aspirations. For liquid-like GV that showed no deformation during the first aspiration, there was still no change after several successive aspirations. On the other hand, for the more rigid GV that were moderately deformed after the first aspiration, the deformation is increasingly pronounced. Finally, for glassy GV with a strong remanent deformation after the first aspiration, we observe the aspiration of a new tongue accompanied simultaneously by the retraction of the first one (Figure 3E-H). This feature has also been observed for glassy lipid vesicles and glassy polymersomes, which flow above a threshold pressure named the yield stress (40). The remanent deformation disappears when we apply a pressure higher than the yield stress, which ranges from from 8.1 to 13.1 kPa, n = 8, with a mean value 10.5 kPa. These behaviors differ greatly from what we would have obtained with uncoated bubbles. They would have been completely aspirated as soon as ΔP is larger than ^ ^ a critical pressure ∆^^= 2^ ^ ^^− ^^, related to the surface tension γ of the liquid, which is constant. In the presence of the hydrophobin coating of the bubbles, γ is replaced by the membrane tension σ, which increases when the vesicle is sucked into the pipette. Materials and Methods Materials HFBI hydrophobin protein is produced and purified as described in Linder et al.(18) C4F1098% (CAS 355-25-9) was purchased from ABCR (Karlsruhe, Germany). Water was purified using a Milli-Q system (Millipore) with resistivity around 18.2 MΩ cm. Formation of bubbles The bubbles used in this study were created by a mechanical agitation method. The desired amount of HFBI (0.02, 0.067, or 0.2 mg) were solubilized in 1 ml of phosphate buffer saline (1x) placed inside a 2.5 ml glass vial. The vial was sealed using an aluminum cap and a septum, and the air inside was replaced with C4F10using a 50 ml Hamilton syringe. The vial was placed in a VialMix shaker (Lantheus Medical imaging In) and agitated for 45 s, forming a white colored suspension consisting of micro and nanobubbles (Figure 5). Cryo-Transmission Electron Microscopy (Cryo-TEM) A 5 μl droplet of nanosized bubble dispersion was deposited onto a plasma- treated lacey carbon-coated copper grid (Electron Microscopy Sciences). The droplet was blotted with filter paper, plunged into liquid ethane (–170 °C) using an automatic plunge freezer (EM GP2, Leica), and stored in liquid nitrogen. The samples were inspected using a JEM-3200FSC (JEOL) microscope with an accelerating voltage of 300 kV and images were acquired using Digital Micrograph software (Gatan, version 1.83.842). Atomic Force Microscopy (AFM) The AFM measurements were carried out using a Dimension Icon AFM (Bruker AXS, France; formerly Veeco) with ScanAsyst-air cantilevers (sharp silicon nitride tips with a nominal radius of 2 nm for PeakForce Tapping in air). The scan size was set to 100 nm × 100 nm with a resolution of 256 pix × 256 pix, and the scanning was performed with a scan rate of 1 kHz. ScanAsyst Auto control was set to “individual” for the sample with PeakForce Amplitude of 170 nm. The spring constant and peak force frequency were 0.4 N m−1and 2 kHz for all samples. Individual scans for each sample were taken at multiple locations on the surface. Sample preparation for AFM A 100 ml droplet of HFBI solution (0.2 mg ml-1) was placed on a parafilm substrate for about one hour to get a flattened area on the top of the droplet and confirm that we had a monolayer at the interface. After that, a highly ordered pyrolytic graphite (HOPG) substrate was brought into contact with the top of the droplet. The substrate with the hydrophobin monolayer was gently washed with 200 ml of Milli- Q water. The sample was placed on the AFM stage and we waited for about 30 min to ensure that the sample was dry. Finally, the sample was imaged. Surface tension and surface dilatational rheology Measurements of the surface tension and mechanical properties of the HFBI adsorbed layer were carried out using an optical tensiometer (Theta tensiometer, Biolin Scientific, Finland) and the obtained data were analyzed using the instrument software (OneAttension). A hooked needle filled with C4F10 using a Hamilton syringe was immersed in a solution of HFBI (0.01 mg ml-1). Then, a 10 μl gas bubble was created on the needle tip and the bubble was monitored for about 3 h until the surface tension curvature reached a steady state. At that point, the static surface tension was measured by applying a sinusoidal volume disruption using an integrated pulsated droplet module (PD-200, Biolin Scientific, Finland). The change in bubble volume was monitored for 40 s. Density and viscosity measurements Density measurements of distilled water (as a reference) and a glycerol solution (50% w / w in distilled water) were performed in triplicate using a handheld density meter (Mettler Toledo, DensitoPro model). The viscosity of these solutions was then measured using a home-made setup consisting of a piezoelectric pressure controller (OB1 Mk3, Elveflow) connected to a balance via a reservoir. The reservoir was filled with the solution to be characterized and connected to the balance via a tubing inside a glass vial placed on the balance. The flow rate Q of the liquid was deduced by observing the increase in mass of the vial after a 30 s increase in pressure in the reservoir and using the density of the solution. Pressures of 125 were applied for distilled water and 500 Pa the glycerol solution using the software instrument (ESI v3.06.05). Measurements were performed at room temperature (T = 23 ± 1 °C) and were repeated 15 times for each solution. The flow rate was then used to determine the viscosity of the solution using the Hagen-Poiseuille equation where ΔP is the applied pressure, r is the radius of the tubing and L is the length of the tubing. Pipette aspiration setup and microscopy Micropipettes were prepared by pulling borosilicate capillaries (WPI, 1 mm / 0.5 mm outer / inner diameter) using a puller (PN-31, Narishige). Afterward, the micropipettes were sized to a few micrometers in diameter and bent using a microforge (MF-900, Narishige) to introduce the pipette horizontally in the observation chamber. The pipette was connected through tubing to a water tank attached to the piezoelectric pressure controller mentioned previously and then filled with water. The observation chamber was made of two glass coverslips separated with a few layers of parafilm and was filled with the solution containing the bubbles. The micropipette was brought into contact with a GGV (HFBI coated microbubble) and a negative pressure was applied, resulting in the suction of the microbubble with the formation of a tongue of length L(t). The experiments were performed at room temperature (T = 23 ± 1 °C) and aspirated microbubbles were visualized with an inverted microscope (Nikon Eclipse Ti). Bright-field images were recorded with a sCMOS camera (Zyla-4.2-CL10, Andor) at a time interval between 0.2 and 1 s, and operated using μManager (2.0 beta) open-source microscopy software. Conclusion The mechanical properties and stability of gas vesicles are crucial as they play a fundamental role in therapeutic and diagnostic techniques as contrast agents for ultrasound imaging. In addition, they can be used as a drug and oxygen cargo for therapy. It has also been shown that they can serve as agents for cell destruction and tissue disruption through ultrasound-induced inertial cavitation (45). This property is enabled by GV produced by bacteria, which are protein nanostructures filled with gas to achieve cellular buoyancy. They have given rise to bacterial therapies since the genes that encode GV can be expressed in mammalian cells. The giant gas vesicles studied here are not protein nanostructures but microstructures, which allows to study their mechanical properties. We have demonstrated that the hydrophobin-coated bubbles formed here have improved mechanical properties, which will enable them to be used for echogenic applications. Their stability is ensured by the adsorption of proteins at the liquid / gas interface. This surface film can be in the liquid or solid state depending on the amount of hydrophobin adsorbed at the interface. In the first case, the surface tension can be measured by the hanging drop technique by analyzing its shape. Alternatively, the response of the surface tension σ (V, ω) to a periodic modulation of the volume of the bubble can be measured. This allows to highlight the viscoelastic properties of the surface film from the phase shift between the volume change and the response of the protein film tension. We show here that the micropipette aspiration technique is the most efficient because it allows the study of GV in both liquid and glassy states. If the film is glassy, it presents a transition to a liquid state if a stress higher than a yield stress is applied to it. It corresponds to a threshold suction pressure above which the GV enters the pipette in a liquid state, forming a sleeve that adapts the shape of the pipette. In all cases, the vesicles penetrates only if ∆^ is larger than the Laplace threshold ∆^^(^^). Contrary to the suction of simple bubbles or liquid drops, the GV penetrates only partially because the membrane tension increases with the size of the sleeve, and the penetration stops when ∆^ = ∆^^(^^). In the glassy case, the tongue penetration is achieved if both conditions (σ larger than the yield stress and the aspiration Laplace pressure threshold) are met. When the aspiration is stopped, the sleeve becomes glassy again and keeps its shape. It can be resorbed by sucking the deformed vesicle beyond the yield stress. We have discovered a new regime where a very long tongue is formed at ultra-fast velocity. It corresponds to the aspiration of gas from the glassy vesicles. In this case, the stress σ acting on the protein shell is smaller than the yield stress and larger than the Laplace pressure to aspirate a bubble of gas. This extraction of gas from solid GV may have applications in ultrasound-induced inertial cavitation. 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Claims

CLAIMS 1. A protein-coated gas vesicle comprising class II type hydrophobin HFBI.

2. The protein-coated gas vesicle according to claim 1, wherein said class II type hydrophobin HFBI is a 7.5 kDa protein originating from Trichoderma reesei.

3. The protein-coated gas vesicle according to claim 1 or 2 comprising a surface layer of class II type hydrophobin HFBI.

4. The protein-coated gas vesicle according to any of claims 1-3, wherein said gas vesicle is formed by a mechanical agitation method.

5. The protein-coated gas vesicle according to any of claims 1-4, wherein said gas vesicle is filled with perfluoro-n-butane gas (C4F10).

6. The protein-coated gas vesicle according to any of claims 1-5, wherein stability of said gas vesicle is based on the adsorption of the HFBI proteins at the liquid / gas interface of said gas vesicle.

7. The protein-coated gas vesicle according to any of claims 1-6, wherein the HFBI proteins form a monolayer.

8. The protein-coated gas vesicle according to any of claims 1-7, wherein the HFBI proteins provide an amphiphilic shell around the gas vesicle.

9. The protein-coated gas vesicle according to any of claims 1-8, wherein the size of said gas vesicle is in the range of 100 nm to 100 µm.

10. The protein-coated gas vesicle according to claim 9, wherein the size of said gas vesicle is in the range of 10 to 100 µm.

11. A protein-coated gas vesicle according to any of claims 1-10 for use in medical applications including drug delivery.

12. The protein-coated gas vesicle for use according to claim 11, wherein said gas vesicle is to be administered intravenously.

13. An ultrasound contrast agent comprising a plurality of the protein-coated gas vesicles according to any of claims 1-10.

14. The ultrasound contrast agent according to claim 13, wherein said ultrasound contrast agent is for intravenous administration.

15. Use of the protein-coated gas vesicle according to any of claims 1-10 as an ultrasound contrast agent.

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