WO2017025826A1 - Bulles hfb - Google Patents

Bulles hfb Download PDF

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Publication number
WO2017025826A1
WO2017025826A1 PCT/IB2016/054181 IB2016054181W WO2017025826A1 WO 2017025826 A1 WO2017025826 A1 WO 2017025826A1 IB 2016054181 W IB2016054181 W IB 2016054181W WO 2017025826 A1 WO2017025826 A1 WO 2017025826A1
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WO
WIPO (PCT)
Prior art keywords
microbubbles
hfbii
hexane
hydrophobin
air
Prior art date
Application number
PCT/IB2016/054181
Other languages
English (en)
Inventor
Pierangelo Metrangolo
Giuseppe Resnati
Roberto Milani
Marie Pierre KRAFFT
Original Assignee
Politecnico Di Milano
Centre National De La Recherche Scientifique
Vtt-Technical Research Centre Of Finland Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by Politecnico Di Milano, Centre National De La Recherche Scientifique, Vtt-Technical Research Centre Of Finland Ltd. filed Critical Politecnico Di Milano
Publication of WO2017025826A1 publication Critical patent/WO2017025826A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
    • A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres

Definitions

  • microbubbles One important problem in the development of microbubbles for medical applications is to prepare small and narrowly dispersed microbubbles that are both stable and echogenic by using compounds that are biologically inert. The same problem is at least partially present also for microbubbles to be used in food technology or in building technology and construction.
  • Ultrasound contrast agents strongly improve the diagnostic capabilities of ultrasound (US) imaging by enhancing signals from small and/or deep-lying vessels, and increasing the contrast between normal and abnormal tissues in organs such as liver and spleen.
  • Aqueous dispersions of microbubbles have excellent potential as UCA, as well as drug and gene delivery vehicles and metabolic gas transport.
  • innovative components that form the microbubbles' shell.
  • Phospholipid- stabilized microbubbles have a thin (molecular-thick, tens of nm) shell and are therefore highly echogenic, but they have to be prepared from freeze-dried extracts just prior use.
  • polymer-stabilized microbubbles are stable for months, but their shell is much thicker (several hundreds of nm) , which dampens the US waves and considerably decreases their echogenicity .
  • Hydrophobins are small fungal amphiphilic proteins with remarkable surface activity (M. Linder, Curr. Opin. Colloid In. 2009, 14:356-363) .
  • a protein of this class, Trichoderma reesei hydrophobin, HFBII has been extensively studied and it is known to form ordered monolayers at interfaces.
  • HFBII was reported to stabilize inorganic and polymer nanoparticles and fluorocarbon emulsions in water (A. Schulz et al . Mater. Chem. 2011, 21:9731- 9736; R. Milani et al . Soft Matter 2013, 9:6505-6514; C. Pigliacelli et al . J. Fluorine Chem. 2015, 177:62-69) .
  • This invention describes microbubbles that are particularly stable and echogenic.
  • Figure 1 morphology of aggregates formed in aqueous dispersions of HFBII under air (a) and F-hexane saturated air (b) by optical microscopy on shear-mixed samples and by TEM (negative staining) on samples before mixing (c) and (d) .
  • Figure 2 effect of F-hexane on adsorption of HFBII at the air/water interface.
  • Figure 3 HFBI I-stabilised air microbubble dispersions.
  • Figure 4 F-hexane-saturated air microbubbles stabilized by HFBII.
  • Figure 5 FTIR spectra of HFBII, F-hexane and the bubble shell materials retrieved after centrifugation/washing/drying of microbubble dispersion samples.
  • Figure 6 Variation of the attenuation coefficient a with time for various frequency values for (a) air-only microbubbles and (c) F- hexane saturated air microbubbles. Variation of a as a function of frequency f at initial time (i.e. 32 s after injection of the microbubbles in the cell) for (b) air-only microbubbles and (d) F- Hexane saturated air microbubbles.
  • Figure 7 Time evolution of HFBII microbubbles prepared with F- hexane, as assessed by static light scattering.
  • hydrophobin use as stabilizers of microbubbles (particularly containing perfluorocarbon, preferably perfluorohexane, perfluoropentane and/or perfluorobutane, perfluoroalkyl ether or sulphur hexafluoride gases) .
  • perfluorocarbon preferably perfluorohexane, perfluoropentane and/or perfluorobutane, perfluoroalkyl ether or sulphur hexafluoride gases
  • Microbubble dispersions of air saturated with fluorocarbon vapors and stabilized with a hydrophobin monolayer have been prepared. More preferably, said microbubble dispersions of air saturated with F-hexane vapors, and stabilized with a monolayer of the hydrophobin HFBII have been prepared. This small amphiphilic protein forms monolayer films at the air/water interface. However, the state-of-the-art shows that these monolayers are in a solid, rigid state (N. A. Alexandrov et al . Colloid Interf. Sci . 2012, 376:296-306) . As a consequence, preparing air microbubbles stabilized by HFBII leads to highly heterogeneous populations of elongated (non-spherical) bubbles.
  • F-hexane perfluorohexane
  • F-hexane acts as a co-surfactant by being inserted in between the HFBII molecules, thereby reducing their lateral interactions (reduction of the viscoelastic modulus of about 20-30%) .
  • F- hexane acts synergistically at the interface, resulting in an acceleration of the adsorption of HFBII at the gas/water interface and a significant reduction of HFBII surface tension at equilibrium, thus facilitating formation of micron-sized microbubbles .
  • microbubbles are both stable and highly echogenic. Another asset in the here described solution is the fact that both components are expected to be biologically inert.
  • the stabilization of aqueous microbubble dispersions can be achieved in two ways: (i) decreasing the water solubility of the gas contained in the bubble by incorporating a tiny amount of a fluorocarbon gas, and (ii) assembling a protective layer around the bubble (most commonly phospholipids or polymers, although use of proteins such as albumin is reported) , which decreases the surface tension and provides a physical barrier towards gas dissolution in water and bubble coalescence.
  • dispersions were prepared making use of air saturated with F-hexane, which has very low water solubility, and a monolayer of the hydrophobin HFBII, which is known to be surface-active and self-assemble at aqueous/gas interfaces rapidly and efficiently, forming a strong and elastic layer.
  • fluorinated gases are commonly employed in microbubble formulations for imaging due to their low water solubility, and that they are biologically inert.
  • Hydrophobins are known to be non-toxic (D.J.Ebbole Trends Microbiol. 1997, 5:405-408), and available data strongly suggest their non-immunogenicity (M . Sarparanta et al . J.
  • hydrophobin coatings effectively shield antigens in the cell wall of fungal aerial structures and thus protect them from responses by the immune system (V.Aimanianda et al . Nature 2009, 460:1117-1121; S.Bruns et al . PLoS Pathog. 2010, 6:el000873) . While hydrophobins have been widely used as foaming agents, their use for the elaboration of UCA (US imaging) and with perfluorocarbon gases is new.
  • the dispersions are prepared simply by treating an aqueous HFBII solution with a homogenizer for short durations (about 2 minutes) under an atmosphere of air saturated with F-hexane.
  • the here described bubbles have potential uses in several applications. Their unique characteristics make them suitable as ultrasound contrast agents.
  • the here claimed microbubbles are suitable for applications in the building but also in the food industry. They are the component of choice in any situation where it is necessary to deal with foam materials.
  • HFBII was produced using recombinant strains of T. reesei, purified by RP-HPLC, as described previously and lyophilized before use (M. J. Bailey et al . Appl . Microbiol. Biotechnol. 2002, 58:721; A. Paananen et al . Biochemistry 2003, 42:5253; M. B. Linder et al . Biomacromolecules 2001, 2:511) . HFBII purity was 99% or 90%, depending on the experiments. Experiment replicates showed that the self-assembly behavior of the protein did not vary. F- hexane was supplied by Fluorochem (95%) . Water was obtained from a MilliQ (Millipore) system ( ⁇ : 71.7 ⁇ 0.5 mN nf 1 at 25°C; resistivity 18.2 ⁇ cm) .
  • Milli-Q water (20 mL) was added to HFBII (90%, 4 mg) in a glass vial and the resulting dispersion (0.2 mg m!T 1 ) was gently hand shaken, avoiding foam and bubbles formation, and treated in a sonicating bath for 15 min. The dispersion was split into two 10 ml aliquots.
  • HFBII dispersions (90%, 10 mL) were prepared as described above, with their atmosphere being saturated, or not, with F-hexane, and placed in glass tubes.
  • the dispersions were then mechanically stirred using an Ultra-Turrax mixer (IKA T10) during two 45 s periods (using settings 4 then 5, successively) .
  • the dispersions were gently mixed 3 times at room temperature before taking aliquots (100 ⁇ ) exactly half-way through the tube after allowing a flotation time of 5 min. All these procedures were achieved by maintaining the dispersions under air or under F-hexane-saturated air .
  • microbubble samples were centrifuged (300 rpm for 1 min; Sigma 4K10 centrifuge, Bioblock Scientific) and the resulting foam was collected and washed twice in order to remove the non-encapsulated materials. After freeze-drying in vacuo, the resulting powder was analyzed by FTIR (Vertex 70, Bruker) . Absorptions are reported in wavenumbers ( v , cnf 1 ) and only peaks of interest are reported.
  • the method is based on the attenuation, that is the reduction in amplitude of an acoustical wave that propagates through an aqueous dispersion of bubbles.
  • a method has been developed that fits standard simple-harmonic resonator curves to measured attenuations in order to infer the size of the bubbles (T.G. Leighton The Acoustic Bubble, Academic Press, San Diego, 1994; N. de Jong et al. Ultrasonics 1992, 30:95-103; D. E. Goertz et al . Ultrasound Med. Biol. 2007, 33:1376-1388) .
  • Determination of radii distribution is based on the fact that the attenuation is maximal when the frequency of the acoustic wave is the same as the resonance frequency of the bubble.
  • the half-life of the bubbles is obtained directly, since the number of bubbles is directly proportional to the attenuation coefficient.
  • a low-power emitter was used that avoids altering bubble characteristics and stability (acoustical power 0.1 W cnf 2 ; peak-to-peak acoustical pressure ⁇ 3 x 10 4 Pa) .
  • the injected volume of microbubbles is 2 ml for air-only microbubbles and 200 ⁇ for F-hexane saturated air microbubbles . Each measurement has been repeated 3 times on 3 to 5 different bubble preparations. The mean diameter of the microbubbles was measured after preparation and over time.
  • aqueous HFBII dispersions (5 ⁇ i ) were deposited onto freshly glow discharged carbon covered 400 mesh copper grids. After 2 min of adsorption, the grids were stained with 5 ⁇ i of a 2% aqueous uranyl acetate solution for 1 min, and the excess of liquid removed with filter paper.
  • TEM imaging was carried out using a Technai G2 microscope (FEI) at 200 kV. Images were acquired with an Eagle 2k CCD camera (FEI) .
  • the samples were observed using a Nikon Eclipse 90i microscope. Three to four droplets of bubble dispersion were placed into a concave slide and covered with a glass slide. Rapid image acquisition was achieved using a Lumenera Infinity 2 CCD camera (Lumenera, Ottawa, Canada) . Bubble morphology was investigated and their diameters measured using the ImageJ software on 5 to 10 slides. Objects for which the circularity was found to be >0.90 or ⁇ 0.30 were considered spherical or elongated, respectively.
  • the mean diameter of the microbubbles was measured after preparation and over time using a laser diffraction particle size analyzer Beckman Coulter LS13 320 MW. Results are reported in Figure 7. The measured mean diameter was 3.1 ⁇ after preparation, 5.0 ⁇ after 48h, and 5.5 ⁇ after 2 months.
  • a Malvern Zetasizer Nano ZS instrument was used at 25°C for dynamic light scattering measurements at a scattering angle of 90°.
  • the z-averaged hydrodynamic mean diameters of the nano-sized HFBII aggregates obtained when the HFBII dispersions were exposed to F-hexane were determined by using the Malvern software. 3. HFBII monolayer analysis
  • Adsorption kinetics Axisymmetric bubble shape analysis was applied to a rising gas bubble formed in an aqueous dispersion of hydrophobin HFBII. Time dependence of ⁇ during adsorption at the gas/liquid interface was measured using a Tracker® tensiometer (Teclis, Longumblegne, France) (J. Benjamins et al . Colloids Surf. A 1996, 114:245-254) .
  • the bubble (5 was formed at the end of a steel capillary fixed on a syringe.
  • the inner bubble gas phase was air or air saturated with perfluorohexane vapor.
  • the vapor pressure of F-hexane and concentration at saturation in air at 25°C are 2.9 x 10 4 Pa and 11.66 mol nf 3 , respectively, and its water solubility at 25°C is 2.7 1CT 4 mol nf 3 .
  • Temperature was 25°C, unless stated otherwise.
  • the error made on the fit of the bubble profile determined using the Laplace equation was automatically calculated by the tensiometer.
  • Dilatational rheology adsorption kinetics were run under sinusoidal oscillations at 25°C (period T 10 s; surface variation amplitude ⁇ 15%) . Oscillations were produced by a position- encoded motor and transmitted to the bubble through a piston coupled to the syringe carrying the capillary. All measurements were repeated three to five times.
  • the dilatational viscoelastic modulus E ⁇ / ⁇ is defined as the ratio of the variation in interfacial tension ⁇ to the relative variation of the bubble' s surface area ⁇ . E moduli were determined in the 800-1000 s time period .

Abstract

La présente invention concerne des populations à taille régulée de microbulles sphériques stabilisées par un gaz fluorocarboné ayant une coque d'hydrophobine. L'invention concerne également un procédé pour obtenir lesdites microbulles.
PCT/IB2016/054181 2015-08-07 2016-07-13 Bulles hfb WO2017025826A1 (fr)

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US201562202346P 2015-08-07 2015-08-07
US62/202,346 2015-08-07

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WO2017025826A1 true WO2017025826A1 (fr) 2017-02-16

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014063097A1 (fr) * 2012-10-19 2014-04-24 Danisco Us Inc. Stabilisation de membranes biomimétiques

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014063097A1 (fr) * 2012-10-19 2014-04-24 Danisco Us Inc. Stabilisation de membranes biomimétiques

Non-Patent Citations (26)

* Cited by examiner, † Cited by third party
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