Focused ultrasound thermal ablation with thermal enhancers

Abstract

Use of selected gas-filled vesicles as thermal enhancers in thermal ablation treatments with focused ultrasounds. The gas / filled vesicles are selected to have resistance to relatively high values of mechanical index. In preferred embodiments, said gas-filled vesicles are microcapsules with a rigid envelope of stabilizing material comprising tripalmitin and DSPG or polystyrene and DSPG.

Description

[0001] FOCUSED ULTRASOUND THERMAL ABLATION WITH THERMAL ENHANCERS

[0002] Technical field

[0003] The invention relates to a certain gas-filled vesicles for use as thermal enhancers in thermal ablation treatments with focused ultrasounds.

[0004] Background of the invention

[0005] Thermal ablation treatments are medical treatments including localized generation of high temperatures, by the mean of radiofrequency electrical current, microwave radiation, laser, light, and ultrasound, within the aim of thermally destroy a pathological tissue.

[0006] Focused ultrasound's thermal ablation (FUS-TA) is based on the use of specific ultrasound transducers, namely Focused Ultrasound (FUS) transducers, for generating such high temperatures in a focused manner. The technique is also known as High Intensity Focused Ultrasounds (HIFU) thermal ablation. Briefly, acoustic pressure of the ultrasound emitted by the FUS transducer increases from the surface of the transducer towards its geometrical focus as shown in Figure 1. In general, ultrasound waves are attenuated by the tissues in the body, resulting in the conversion of the acoustic energy into heat; such conversion of acoustic energy into heat allows to increase the temperature of tissue above 60°C at the focus. Generation of such high temperatures may induce, among others, protein denaturation, coagulative necrosis and ultimately tissue destruction. The thermal lesion generated by a FUS-TA treatment has typically the form and size of a rice grain between the geometrical focus of the transducer and the transducer (see Fig. 2).

[0007] FUS-TA can be used in many clinical applications for treating several diseases including, for instance, prostatic tumors, uterine fibroids, rectal endometriosis etc.

[0008] To improve the efficacy of the treatment, the use of thermal enhancer materials such as gas-filled vesicles ("Gfv") has been proposed. For instance, Peng et al. (Peng, S., Xiong, Y., Li, K., He, M., Deng, Y., Chen, L., Zou, M., Chen, W., Wang, Z., He, J., & Zhang, L. (2012). Clinical utility of a microbubble-enhancing contrast ("SonoVue") in treatment of uterine fibroids with high intensity focused ultrasound: a retrospective study. European journal of radiology, 81(12), 3832-3838) have reported that injection of Sonovue during a FUS-TA procedure allows decreasing the sonication time needed to obtain ablation of about 1 cm3of tissue.

[0009] Suspensions of gas-filled vesicles (typically dispersed in a suitable physiologically acceptable solution) are known since decades for contrast-enhanced ultrasound imaging (CEUS) and more recently also for therapeutic treatments. The gas is typically entrapped or encapsulated in a layer of stabilizing material comprising, for instance, emulsifiers, oils, thickeners or sugars. Gas-filled vesicles are generally referred to in the art with various terminologies, depending typically on the stabilizing material employed for their preparation; these terms include, for instance, "microspheres", "microbubbles", "nanobubbles", "microcapsules" or "microballoons", globally referred to herein as "gas- filled vesicles" (in brief: "Gfv"). Microbubbles typically include aqueous suspensions in which bubbles of gas are stabilized at the gas / liqu id interface by a very thin envelope (typically less than 5 nm) comprising a stabilizing amphiphilic material, typically a phospholipid. On the other hand, microcapsules (or microballoons) include suspensions in which the bubbles of gas are surrounded by a rigid envelope of a stabilizing material, typically of water-insoluble lipid or of natural or synthetic polymers. The thickness of microcapsules envelope may vary from few tenths to few hundreds of nanometers.

[0010] In experiments conducted with a commercial formulation of microbubbles (Sonazoid), Clark et al. (Alicia Clark, Sierra Bonilla, Dingjie Suo, Yeruham Shapira, Michalakis Averkiou. Microbubble-Enhanced Heating: Exploring the Effect of Microbubble Concentration and Pressure Amplitude on High-Intensity Focused Ultrasound Treatments, Ultrasound in Medicine & Biology, Volume 47, Issue 8, 2021) have however observed that "if the concentration of microbubbles is too high, the targeting of the treatment area will be misplaced because of non-linear attenuation by the prefocal microbubbles. This has important clinical implications because it is imperative that clinicians know exactly where in the body heating will be introduced. For example, if the microbubble concentration is too high, the HIFU could ablate the prefocal region, instead of the intended location at the focus, resulting in unsuccessful treatment". To limit the occurrence of the pre-focal shift, Clarke et al. suggests to proceed with "local bubble injections in the tissue only in the vicinity of the treatment area. In this way, the alteration of the HIFU pressure field caused by bubble attenuation would be eliminated and the lesions would naturally occur at the focus."

[0011] As local bubbles injection is however a cumbersome procedure and not always feasible, injection of microbubbles suspensions in the whole body is more generally practiced. In this case, relatively high concentration of microbubbles can however easily be achieved in a highly perfused organ; if such highly perfused has to undergo the FUS- TA treatment, or if such highly perfused organ is placed between the transducer surface and the respective geometrical focal point in the region to be treated, the risk of generating an undesired pre-focal lesion in the patient is highly probable.

[0012] WO 2014 / 052911 discloses the use of hollow nanoparticles, having a porous silica- based shell filled with perfluoropentane, for thermal ablation. Because of increasing environmental concerns, use of perfluorinated gases shall however be limited or preferably avoided. Furthermore, preparation of silica-based nanoshells requires a relatively cumbersome method of manufacturing, which is difficultly to scale-up at industrial level. Applicant has now found that by suitably selecting the type of gas-filled vesicles, it is possible to substantially limit or avoid such pre-focal phenomenon.

[0013] The Applicant has observed in particular that gas-filled vesicles having a resistance to Mechanical Index (MI) higher than 0.25 are capable of substantially reducing or avoiding such phenomenon.

[0014] Summary of the invention

[0015] An aspect of the invention relates to a gas-filled vesicle having a resistance to MI of 0.25 or higher for use as a thermal enhancer in a focused ultrasound thermal ablation treatment, said gas-filled vesicle having a gaseous core comprising a gas selected from air; nitrogen; oxygen; carbon dioxide; nitrous oxide; a noble gas; a radioactive gas; a hyperpolarized noble gas; or mixtures thereof. Preferably, said resistance to MI is of 0.30 or higher, more preferably of at least 0.35 or higher and even more preferably of at least 0.40 or higher.

[0016] According to an embodiment, said gas-filled vesicles are microcapsules with a rigid envelope of stabilizing material. Typically, the stabilizing material is solid at room temperature. Preferably, the thickness of said envelope is of at least 30 nm, more preferably of at least 50 nm. The thickness may be up to few hundreds nm, e.g. 500 nm, preferably less than 400 nm, more preferably less than 300 nm.

[0017] According to a preferred embodiment the gas-filled vesicles have a stabilizing envelope comprising a water-insoluble lipid. Preferably said water-insoluble lipid is a glyceride (i.e. a glycerol derivative with a fatty acid), more preferably a diglyceride or triglyceride.

[0018] According to another aspect, the invention relates to a method of thermal ablation which comprises: a. Administering an aqueous suspension of gas-filled vesicles to a patient; b. directing a focused ultrasound beam to a region of interest in said patient, in the presence of said gas-filled vesicles, to generate a thermal lesion of a tissue located in said region of interest; wherein said gas-filled vesicle have a resistance to Mechanical Index of 0.25 or higher and have a gaseous core comprising a gas selected from air; nitrogen; oxygen; carbon dioxide; nitrous oxide; a noble gas; a radioactive gas; a hyperpolarized noble gas; or mixtures thereof.

[0019] Figures

[0020] Figure 1 illustrates a schematic example of an experimental setup for conducting in vitro FUS-TA experiments.

[0021] Figure 2 illustrates schematic examples of thermal lesions. Detailed description of the invention

[0022] Gas-filled vesicles suitable for use as thermal enhancers as described herein have a resistance to MI of 0.25 or higher.

[0023] The mechanical index (MI) as used herein is dimensionless, being defined as: wherein P is the peak negative acoustic pressure (in MPa, measured in water), f the frequency (in MHz) of the applied ultrasound wave and CMI is a normalization constant which equals 1 MPa MHz1 / 2.

[0024] The term "resistance to MI" means that Gfv (e.g. microcapsules) for use in a method of thermal ablation as described herein can withstand a predetermined threshold value of acoustic pressure applied at a predetermined frequency without suffering substantial damages. In particular, it is considered that a population of Gfvs is not "substantially damaged", when subjected to a sonication at a predetermined MI, if less than 40% of the initial volume of gas contained in the Gfvs preparation is released upon sonication at said predetermined MI; preferably less than 30%, more preferably less than 25% and even more preferably less than 20% of the initial volume of gas is released upon sonication at said predetermined MI. For instance, when Gfv for use in a method as described herein are subjected to an ultrasound beam at a MI of 0.25 (and up to 0.35), more than 95% of the initial volume of gas contained in the Gfv is maintained. Also, when the MI is increased to 0.40, more than 90% of the initial volume of gas is maintained while when the MI is increased up to 0.50, still more than 80% of the initial volume of gas is maintained. To the contrary, soft-shell Gfv (e.g. microbubbles) have much lower resistances to MI. Typically, already at an MI of 0.25, more than 40% of the gas is released upon sonication at said predetermined MI.

[0025] Preferably, Gfv for use in a method as described herein have a resistance to a mechanical index of at least 0.30, more preferably of at least 0.35 and even more preferably of at least 0.40, up to e.g. 1.0.

[0026] The resistance to MI can be determined, for instance, by submitting the gas-filled vesicles to an ultrasound beam at a frequency of 1.61 MHz, preferably with a pulse of 1ms every 100ms, for 20 seconds.

[0027] For instance, said Gfv show a resistance to an acoustic pressure of at least about 300 kPa, preferably at least 400 kPa, more preferably at least 500 KPa at the above indicated frequency, pulse interval and duration. Gas-filled vesicles composition

[0028] Gas-filled vesicles suitable as thermal enhancers as described herein include microcapsules with a rigid envelope.

[0029] In general, the envelope of microcapsules has a thickness of at least 30 nm, preferably at least 50 nm (up to, e.g., 500 nm, preferably up to 400 nm, more preferably up to 300 nm). On the other hand, soft-shell microbubbles are stabilized by thin films of material (e.g. phospholipids), typically in the form of a mono-molecular stabilizing layer with a corresponding thickness of less than 10 nm, typically 2-5 nm.

[0030] According to an embodiment, the envelope of the gas-filled vesicles comprises a biodegradable water-insoluble lipid. As used herein, the term water-insoluble refers to lipid having a solubility in water of IO-5mol / L or less, preferably of 1010mol / L or less and even more preferably 1015mol / L or less, down to e.g. IO-20mol / L.

[0031] Biodegradable water-insoluble lipids useful for forming the rigid envelope of microcapsules comprise, for instance, water insoluble glycerides, preferably solid at room temperature (about 20°C). Glycerides (also known as "acylglycerols" or "glycerol acylates") are ester derivatives of glycerol with one, two or three fatty acid residues, also known as mono-, di- or tri-glycerides or glycerol mono-, di- or tri-acylates. Fatty acids useful for forming glycerides are saturated and unsaturated fatty acids having at least 12 carbon atoms in their alkyl chain (e.g. up to 30 carbon atoms) including, for instance, lauric, myristic, palmitic, stearic, arachidic, behenic, cerotic, melissic, palmitoleic, oleic, linoleic, linolenic, gondoic and erucic acid. Preferred glycerides are di- or triglycerides, more preferably triglycerides. Di- and tri-glycerides may include the same or different fatty acid derivatives. Examples of di- and tri-glycerides include, for instance 1,2- or 1,3-dilauroyl glycerol, 1,2- or 1,3-dimyristoyl glycerol, 1,2- or 1,3- dipalmitoyl glycerol, 1,2- or 1,3-distearoyl glycerol, 1,2- or 1,3-diarachidoyl glycerol, 1,2- or 1,3-dibehenoyl glycerol, 1,2-or 1,3-dipalmitoleoyl glycerol, 1,2- or 1,3-dioleoyl glycerol, 1,2- or 1,3-dilinolenoyl glycerol, 1,2- or 1,3-dilinoleoyl glycerol, l-palmitoyl-3- stearoylglycerol, l-oleoyl-2-palmitoyl glycerol, l-oleoyl-3-palmitoyl glycerol, 1- palmitoyl-2-oleoyl glycerol, l-oleoyl-2-linoleoyl glycerol, l-oleoyl-3-linoleoyl glycerol,

[0032] 1.2.3-trilauroyl glycerol (trilaurin), 1,2,3-trimyristoyl glycerol (trimyristin), 1,2,3- tripalmitoyl glycerol (tripalmitin), 1,2,3-tristearoyl glycerol (tristearin), 1,2,3- triarachidoyl glycerol (triarachidin), 1,2,3-tribehenoyl glycerol (tribehenin), 1,2,3- trilinolenoyl glycerol, 1,2,3-linoleoyl glycerol, l,3-dipalmitoyl-2-stearoylglycerol, 1,2- dipalmitoyl-3-oleoyl glycerol, l,3-dipalmitoyl-2-oleoyl glycerol, l,2-dioleoyl-3-palmitoyl glycerol, l,3-dioleoyl-2-palmitoyl glycerol, l,3-dioleoyl-2-stearoylglycerol, 1-Linoleoyl-

[0033] 2.3-dilinolenoylglycerol, l-palmitoyl-2-oleoyl-3-stearoylglycerol and mixtures thereof; tripalmitin and tristearin are preferred. The stabilizing envelope preferably comprises at least 30% (by moles) of a biodegradable water-insoluble lipid as above defined.

[0034] Glycerides as defined above may be advantageously admixed with other components, such as sterols, phospholipids or fatty acids.

[0035] Suitable fatty acids include those listed above.

[0036] As used herein, the term phospholipids include either naturally occurring, semisynthetic or synthetically prepared products that can be employed either singularly or as mixtures.

[0037] Examples of naturally occurring phospholipids are natural lecithins (phosphatidylcholine (PC) derivatives) such as, typically, soya bean or egg yolk lecithins.

[0038] Examples of semisynthetic phospholipids are the partially or fully hydrogenated derivatives of the naturally occurring lecithins. Preferred phospholipids are fatty acids diesters of phosphatidylcholine, phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) or of sphingomyelin.

[0039] Examples of preferred phospholipids are, for instance, dilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidylcholine (DMPC), dipalmitoyl-phosphatidylcholine (DPPC), distearoyl-phosphatidylcholine (DSPC), diarachidoyl-phosphatidylcholine (DAPC), l,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC), dioleoylphosphatidylcholine (DOPC), dipentadecanoyl-phosphatidylcholine (DPDPC), 1- myristoyl-2-palmitoyl-phosphatidylcholine (MPPC), l-palmitoyl-2-myristoyl- phosphatidylcholine (PMPC), l-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC), 1- stearoyl-2-palmitoyl-phosphatidylcholine (SPPC), l-palmitoyl-2-oleylphosphatidylcholine (POPC), l-oleyl-2-palmitoyl-phosphatidylcholine (OPPC), dilauroyl-phosphatidylglycerol (DLPG) and its alkali metal salts, diarachidoylphosphatidyl-glycerol (DAPG) and its alkali metal salts, dimyristoylphosphatidylglycerol (DMPG) and its alkali metal salts, dipalmitoylphosphatidylglycerol (DPPG) and its alkali metal salts, distearoylphosphatidylglycerol (DSPG) and its alkali metal salts, dioleoylphosphatidylglycerol (DOPG) and its alkali metal salts, dimyristoyl phosphatidic acid (DMPA) and its alkali metal salts, dipalmitoyl phosphatidic acid (DPPA) and its alkali metal salts, distearoyl phosphatidic acid (DSPA), diarachidoylphosphatidic acid (DAPA) and its alkali metal salts, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoyl phosphatidyl-ethanolamine (DSPE), dioleylphosphatidyl-ethanolamine (DOPE), diarachidoylphosphatidylethanolamine (DAPE), dil inoleyl phosphatidylethanolamine (DLPE), dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), diarachidoyl phosphatidylserine (DAPS), dioleoylphosphatidylserine (DOPS), dipalmitoyl sphingomyelin (DPSP), and distearoylsphingomyelin (DSSP), dilauroyl-phosphatidylinositol (DLPI), diarachidoylphosphatidylinositol (DAPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dioleoylphosphatidylinositol (DOPI).

[0040] Particularly preferred phospholipids are DPPC, DSPC, DPPA, DSPA, DPPG, DSPG, DPPS and DSPS. Most preferred are DSPG, DSPC, DPPC, DPPG and DAPC.

[0041] Mixtures of phospholipids can also be used, such as, for instance, mixtures of DPPE and / or DSPE, DPPC, DSPC or DAPC with DSPS, DPPS, DSPA, DPPA, DSPG and DPPG.

[0042] According to certain embodiments, the stabilizing envelope comprises a mixture of a biodegradable water-insoluble lipid, preferably a glyceride, and of a phospholipid. Preferably the water insoluble lipid and the phospholipid are in a molar ration of from 30 / 70 to 70 / 30, preferably from 33 / 67 to 60 / 40.

[0043] Examples of suitable sterols include, for instance, cholesterol, phytosterol, lanosterol, ergosterol, etc. and esters of the sterols with the above mentioned fatty acids; cholesterol is preferred.

[0044] Biodegradable lipid containing microcapsules can be prepared, for instance, by dispersing a mixture of one or more of the water-insoluble lipids in an organic solvent and emulsifying said organic solution with an aqueous phase, so as to produce an oil-in- water emulsion, as described for instance in US 6,333,021 herein incorporated by reference. The aqueous phase may contain an effective amount of an additional amphiphilic material, such as a phospholipid. Alternatively, relatively poorly soluble additional compounds (e.g. fatty acids or sterols) may be dissolved in the organic phase.

[0045] Typically, a redispersing agent and / or a cryoprotecting agent can then be added to the emulsion, and the emulsion is subjected to freeze-drying. Redispersing and / or cryoprotecting agents include, for instance albumin, gelatine, polyvinyl pyrolidone (PVP), polyvinyl alcohol (PVA), amino-acids such as glycine; carbohydrates, including sugars such as sucrose, mannitol, maltose, trehalose, glucose, lactose or cyclodextrin, or polysaccharides such as dextran; polyglycols such as polyethylene glycol (PEG) or ethyleneoxide-propyleneoxide block copolymer (e.g. Pluronic®, or Synperonic®), and mixtures thereof.

[0046] According to an alternative embodiment, the envelope of the gas-filled vesicles comprises a water-insoluble polymer, preferably a biodegradable polymer.

[0047] Polymers forming the envelope of the injectable microcapsules are preferably hydrophilic, biodegradable physiologically compatible polymers. Examples of microvesicles with water-insoluble polymeric envelopes are disclosed for instance in US 5,711,933, US or US 5,837,221, together with their respective preparation methods.

[0048] Examples of suitable polymers, which may be natural or synthetic, include substantially insoluble polysaccharides (e.g. chitosan or chitin), polycyanoacrylates, polylactides and polyglycolides and their copolymers, copolymers of lactides and lactones such as g-caprolactone or d-valerolactone, copolymers of ethyleneoxide and lactides, polyethyleneimines, polypeptides, and proteins such as gelatin, collagen, globulins or albumins. Other suitable polymers include poly-(ortho)esters, polylactic and polyglycolic acid and their copolymers (e.g. DEXON®, Davis & Geek, Montreal, Canada); poly(DL-lactide-co-y-ca prolactone), poly(DL-lactide-co-6-valerolactone), poly(DL-lactide- co-y-butyrolactone), polyalkylcyanoacrylates; polyamides, polyhydroxybutyrate; polydioxanone; poly-B-aminoketones; polyphosphazenes; and polyanhydrides. Polyamino-acids such as polyglutamic and polyaspartic acids can also be used, as well as their derivatives, such as partial esters with lower alcohols or glycols. Copolymers with other amino acids such as methionine, leucine, valine, proline, glycine, alanine, etc. can also be used. Derivatives of polyglutamic and polyaspartic acid with controlled biodegradability (such as those described in WO87 / 03891, US 4,888,398 or EP 130935,) can also be used. These polymers (and copolymers with other amino-acids) have formulae of the following type: -(NH-CHA-CO)w -(NH-CHX-CO)y- where X designates the side chain of an amino acid residue (e.g. methyl, isopropyl, isobutyl, or benzyl); A is a group of formula -(CH2)n COOR1 R2 -OCOR, -(CH2)n COO-CHR1COOR, -(CH2)n CO(NH-CHX-CO)m NH-CH(COOH)-(CH2)p COOH, or the respective anhydrides thereof, wherein R1 and R2 represent H or lower alkyls, and R represents alkyl or aryl; or R and R1 are connected together by a substituted or unsubstituted linking member to provide 5- or 6- membered rings; n, m and p are lower integers, not exceeding 5; and w and y are integers selected for having molecular weights not below 5000.

[0049] Non-biodegradable polymers can also be used (e.g. for making microcapsules to be used in the digestive tract), optionally in combination with the above biodegradable polymers. These can be selected from most water-insoluble, physiologically acceptable, bioresistant polymers including, for example, polyolefins (polystyrene), acrylic resins (polyacrylates, polyacrylonitrile), polyesters (polycarbonate), polyurethanes, polyurea and their copolymers. ABS (acryl-butadiene-styrene) is a preferred copolymer.

[0050] Advantageously, ionic polymers (i.e. polymers bearing ionic moieties in their structure), preferably biodegradable ionic polymers, can also be used to form the stabilizing envelope of the microcapsules, thus conferring an overall net charge thereto. Ionic polymers can be used as main components of the stabilizing envelope or they can be admixed in various amounts (e.g. from 2 to 80% by weight) with non ionic polymers. Suitable ionic polymers are, for instance, polymers comprising a quaternized nitrogen atom, such as quaternized amines or polymers comprising carboxylic, sulphate, sulphonate or phosphonate moieties. Examples of suitable ionic polymers include, without limitation, poly(diallyldimethylammonium chloride), poly{bis(2- chloroethyl)ether-a It- l,3-bis[3-(di methylamino) propyl] urea} quaternized (Polyquaternium®-2), poly(4-vinylpyridinium tribromide), hydroxyethylcellulose ethoxylate quaternized (Polyquaternium®-4, poly(p-xylene tetrahydrothiophenium chloride), poly(L-lysine), chitin, diethyleneaminoethyl dextran, poly(acrylic acid), poly(methacrylic acid), poly(styrene-alt-maleic acid), poly(amino acids), alginic acid, poly(uridylic acid) , hyaluronic acid, i.e. poly(!3-glucuronic acid-alt-B-N- acetylclucosamide), poly(galacturonic acid), poly(vinyl acetate-co-crotonic acid), poly(3, 3', 4, 4' -benzophenonetetracarboxylic dianhydride-co-4,4'-oxydianiline), poly(isoprene-graft- maleic acid monomethyl ether), copolymer of glutamic acid with alkyl glutamate, heparin, poly(styrene sulphonate), sulfonated poly(isophthalic acid), poly(vinyl sulphonate, potassium salt), poly(vinyl sulphate, potassium salt), chondroitin sulfate A, dextran sulfate, fucoidan, polyphosphoric acid, sodium polyphosphate, sodium polyvinylphosphonate, chitosan, chitosan sulfate, sodium alginate, alginic acid and ligninsulfonate.

[0051] Because of the relative gaseous impermeability of the envelopes of the Gfv according to the invention, the gas contained in the core does not necessarily need to be a gas with very low solubility in water, such as fluorinated gases.

[0052] While any biocompatible gas, gas precursor or mixture thereof may be employed to fill the microcapsules illustrated above, the microvesicles of the invention comprise at least a gas selected from air; nitrogen; oxygen; carbon dioxide; nitrous oxide; a noble or inert gas such as helium, argon, xenon or krypton; a radioactive gas such as Xel33 or Kr81; a hyperpolarized noble gas such as hyperpolarized helium, hyperpolarized xenon or hyperpolarized neon; or mixtures thereof. Advantageously, the gas contained in the Gfv's core is selected from the group consisting of air, nitrogen, oxygen, carbon dioxide or mixtures thereof. Optionally, the gaseous core may further comprise, in a volume amount of less than 50%, preferably less than 40%, more preferably less than 30%, in admixture with any of the previous gases, an halogenated gase, preferably fluorinated gas, such as or halogenated, fluorinated or perfluorinated low molecular weight hydrocarbons (e.g. containing up to 7 carbon atoms); or a mixture of any of the foregoing. Fluorinated gases include materials which contain at least one fluorine atom such as, for instance fluorinated hydrocarbons (organic compounds containing one or more carbon atoms and fluorine); sulfur hexafluoride; fluorinated, preferably perfluorinated, ketones such as perfluoroacetone; and fluorinated, preferably perfluorinated, ethers such as perfluorodiethyl ether. Examples of perfluorinated gases are SF6 or perfluorocarbons (perfluorinated hydrocarbons). Perfluorocarbon includes saturated, unsaturated, and cyclic perfluorocarbons. Examples of biocompatible, physiologically acceptable perfluorocarbons are: perfluoroalkanes, such as perfluoromethane, perfluoroethane, perfluoropropanes, perfluorobutanes (e.g. perfluoro- n-butane, optionally in admixture with other isomers such as perfluoro-isobutane), perfluoropentanes, perfluorohexanes or perfluoroheptanes; perfluoroalkenes, such as perfluoropropene, perfluorobutenes (e.g. perfluoro-2-butene), perfluorobutadiene or perfluoropentene (e.g. perfluoro-2-pentene); perfluoroalkynes (e.g. perfluoro-2-butyne); and perfluorocycloalkanes (e.g. perfluorocyclobutane, perfluoromethylcyclobutane, perfluorodimethylcyclobutanes, perfluorotrimethylcyclobutanes, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclopentanes, perfluorocyclohexane, perfluoromethylcyclohexane and perfluorocycloheptane).

[0053] Effects of FUS-TA with thermal enhancers

[0054] Figure 1 schematically illustrates a laboratory setup for in vitro FUS-TA experiments with various types of gas-filled vesicles. The setup includes a focused ultrasound transducer (101), which generates a corresponding focused ultrasound beam (102); the geometrical focus (103) of the US beam is indicated by X. The acoustic pressure (AP) of the US beam increases from the transducer towards the focal point (same amount of energy applied on progressively reducing surface).

[0055] A thermochromic tissue-mimicking phantom (TMP), in the form of a rectangular prism (104), is sonicated with the focused US beam 102. TMP is a gel suitable for embedding gas-filled vesicles and can be used in in vitro experiments for reproducing thermal lesions generated by focused ultrasounds. The gel comprises a thermochromic material (i.e. a component that changes color upon temperature increase), such as egg white. When temperature rises to approximately 60°C (the typical temperature applied in FUS-TA treatments for causing thermal lesions), the thermochromic material, originally translucid, becomes opaque, simulating a thermal lesion in a tissue.

[0056] The whole setup is placed in a container filled with water at 37°C Figure 2a schematically illustrates the typical shape, size and position of a focal thermal lesion (corresponding to an opacification of a region in the TMP prism), generated with the experimental setup described above, while figures 2b and 2c schematically illustrate two examples of pre-focal lesions. As illustrated in figure 2a, a focal lesion has a typical rice grain shape (201), with a length (along the direction of the applied acoustic radiation) of few centimetres, for instance of about 1 to 3 cm (for a sonication frequency of about 0.5 MHz). The position of the focal lesion is typically slightly before (i.e. closer to the transducer) with respect to the geometrical focal point of the focused ultrasound beam. On the other hand, pre-focal lesion (originated by the presence of high concentrations of gas-filled microbubbles) result in lesions much farther from the geometrical focal point and / or of larger size. For instance, figure 2b illustrates a larger cone-shaped pre-focal thermal lesion (202), while figure 2c illustrates a larger disc-like thermal lesion (203) generated in the proximity of the transducer; figure 2d illustrated the front view of the disc-like lesion (203) of figure 2c. In this latter case, opacification of a substantial portion of the TMP prism in the vicinity of the transducer generates a screen which prevents the further passage of ultrasound radiation.

[0057] As observed by the Applicant, use of selected gas-filled vesicles allows to substantially reduce or avoid the risk of generating pre-focal lesion during a treatment comprising a thermal ablation of a tissue by application of focused ultrasounds in the presence of relatively high concentration of gas-filled vesicles in (or in the proximity of) the sonicated region. As illustrated in the experimental part, Gfv with a resistance to MI of at least 0.25 allow in fact to obtain a rice grain shaped focal lesion at any tested concentration. On the other hand, increasing concentrations of gas-filled microbubbles result in pre-focal shifting of the thermal lesion, with larger dimensions of the lesion.

[0058] Methods of treatment

[0059] Gas-filled vesicles as defined above can advantageously be used in any focused ultrasound thermal ablation method, for enhancing the efficacy of the treatment and / or reducing the acoustic energy necessary for the treatment.

[0060] Gfv are typically administered as aqueous suspensions of Gfv in a pharmaceutically acceptable aqueous carrier; suitable aqueous carriers include, for instance, water, saline and glucose solutions. The suspension of Gfv is administered, typically intravenously, before the treatment. In general, Gfv suspensions contain from about 1.0-108to about 1.0-1010of Gfv per mL of suspension. In terms of gas volumes, the Gfv suspension may contain from about 1 to about 150 pL of gas per mL of suspension, preferably 5-20 |iL / mL. The effective dosage of Gfv depends on a variety of factors, such as, for instance, the Gfv employed, the type of pathology to treat, location, size and degree of vascularization of the tissue / organ to treat, weight of patient. For instance, the Gfv dosage can vary from 0.05 to 30 ml / kg, preferably from 0.1 to 20 ml / kg, more preferably from to 1 to 10 ml / kg, injected either as a single bolus or a continuous infusion.

[0061] Any focused ultrasound probe can be used for the thermal ablation treatment. Typically, the skilled person is capable of setting the suitable sonication conditions, depending on the location, size and type of the tissue or organ undergoing the thermal ablation. For instance, the frequency of the ultrasound beam can vary from about 20 MHz (for treatments of a skin, e.g. at about 0.17 cm from the ultrasound probe) up to about 0.25 MHz (for treatments of deeper body parts, e.g. at about 20 cm from of the ultrasound probe).

[0062] Thermal ablation of a tissue (e.g. a tumor) in a patient is achieved by subjecting the tissue to be treated to a controlled focused sonication in the presence of gas-filled vesicles as described above. Gas-filled vesicles are preferably administered to the patient before sonication. The focused sonication is preferably performed on multiple single spots of limited volumes, for better precision. The number of single spots is determined by the size of tissue to ablate and the size of the focal spot. Typically, parameters such as acoustic pressure and / or pulse length can be modulated in order to optimize the thermal treatment on a single spot. For instance, acoustic pressure at the geometrid focal point may vary from 500 and 15500 Kpa; pulse length may vary from 0,05 ms to 180 000 ms. If needed, multiple runs of treatment could be performed to achieve thermal ablation of the whole volume.

[0063] The following examples will help to further illustrate the invention.

[0064] EXAMPLES

[0065] Materials

[0066] The gas-filled vesicles prepared according to the following examples were characterized by means of a coulter counter device (Beckman Coulter, Multisizer 4e, 10 or 30 |im aperture, as specified in the specific examples)

[0067] Example 1

[0068] Preparation of comparative formulations of gas-filled microbubbles (Formulations CompOl and Comp02)

[0069] Preparation of CompOl

[0070] 25 mg of lipid blend (DSPC / palmitic acid 8 / 2 n / n ratio) were dissolved in cyclooctane (4 mL) at 80°C. 16.7 mg of DPPE-PEG5000 were dissolved in a 10%(w / w) PEG4000 solution in water (50 mL).

[0071] The organic phase was emulsified with the 50 mL aqueous phase using a MEGATRON® homogenizer (10000 rpm) for about 3.5 min.

[0072] The obtained emulsion was transferred to a 100 mL glass vessel and placed in a bath at 80°C during lh.

[0073] The emulsion was cooled to room temperature and diluted by two with a 10%(w / w) PEG4000 solution in water. It was aliquoted to DIN20R vials (3 mL / vial) and freeze-dried.

[0074] After freeze-drying, the vials are filled with C4F10 / N2 (35:65) at ambient pressure and then capped before being placed in an oven at 38°C for 16h. Before use, the freeze- dried cake is resuspended in 5 ml of 0.9% saline.

[0075] Preparation of Comp02

[0076] Comp02 formulation was prepared as described in the above cited paper of Clark et al. (2021), with reference to the preparation of "Definity-like (Bubble-G)" microbubbles (page 2298, 1stcolumn of the paper); in particular, Bubble-G microbubbles, composed of l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG) in a 95:5 molar ratio, were prepared as previously described in De Cock et al. (De Cock I, Zagato E, Braeckmans K, Luan Y, de Jong N, De SmedtSC, Lentacker I. Ultrasound and microbubble mediated drug delivery: Acoustic pressure as determinant for uptake via membrane pores or endocytosis. J Control Release 2015; 197:20-28.).

[0077] Example 2

[0078] Preparation of gas-filled microcapsules with envelope comprising a water-insoluble lipid (Prep01-03)

[0079] 60 mg of tripalmitin were dissolved in cyclohexane (0.6 ml) at 50°C.

[0080] 100 mg of DSPG.Na were dispersed in distilled water (75 ml) at 70°C for 30 minutes and the solution cooled to about 48°C.

[0081] The organic phase was emulsified with the 30 mL aqueous phase using a POLYTRON® homogenizer (9000 rpm) for about 1 min.

[0082] The obtained emulsion was transferred to a 500 mL glass vessel containing 5 ml of PVA solution in distilled water (4% w / w). After mixing, the resulting emulsion was rapidly frozen at -45°C and lyophilized (Christ Beta 1-8K).

[0083] The obtained cake was dispersed in 20 mL of distilled water in a rotary evaporator, (Rotavapor®) for two hours. The suspension was transferred to a 50 mL flask together with 5 mL of distilled water. After centrifugation (800g, 10 min) the infranatant was removed and 20 mL of a 10% maltose solution containing 0.05% Pluronic® F68 were added thereto. Centrifugal washing was repeated and the infranatant removed; Maltose / Pluronic solution was added to a final volume of 15 mL.

[0084] The suspension is placed in a 20 mL syringe (eccentric tip). The syringe is held horizontally for 8 min and the infra-natant (~14.5 mL) is collected in order to remove any floating aggregates.

[0085] The suspension was analyzed by coulter counter (10 jim aperture).

[0086] The suspension (PrepOl) can be used as such for subsequent experiments or can be subjected to a second freeze-drying in case of long-term storage. In this case, it is aliquoted into DIN8R vials (ImL / vial) for the additional freeze-drying step. After freeze- drying, the vials are filled with air at ambient pressure and then capped. Before use, the freeze-dried cake is resuspended in 1 ml of distilled water.

[0087] The molar ratio of tripalmitin / DSPG forming the layer of microcapsules of PrepOl was of 60 / 40.

[0088] The above preparation was repeated, by modifying the amount of tripalmitin dispersed in the organic phase, in particular 30 mg (Prep02) or 20 mg (Prep03) for a respective tripalmitin / DSPG molar ratio of 43 / 57 and 33 / 67.

[0089] The following table 1 summarizes the composition and main characteristics of the above preparations.

[0090] Table 1: characterization and composition of gas-filled vesicles prepared in Example 2

[0091] Example 3

[0092] Preparation of microcapsules with an envelope comprising a polymer (PrepQ4)

[0093] 100 mg of 280 kDa MW polystyrene (Average MW 280,000, Merck) were dissolved in cyclohexane (1 ml). Distearoyl phosphatidylglycerol, sodium salt (DSPG.Na - 40 mg) was dispersed in distilled water (30 ml) at 70°C for 20 minutes and cooled to 45°C. The organic phase was emulsified in the aqueous phase using a POLYTRON® homogeniser (16'500 rpm, Kinematica, Switzerland).

[0094] The emulsion was added to a 500 ml glass vessel containing 200 mg of polyvinyl alcohol (PVA - MW 9000 - from Aldrich) dissolved in 5 ml of distilled water. After mixing, the resulting emulsion was rapidly frozen at -40°C and lyophilised.

[0095] The freeze-dried residue was redispersed with 20 mL of milliQ water. The suspension was then centrifuged at 800g for 10 minutes and the supernatant collected. The microcapsules were suspended in 20 mL of 10% maltose solution containing 0.05% Pluronic F68 and the centrifugation repeated. The microcapsules were collected and suspended in 15 mL of 10% maltose solution containing 0.05% Pluronic F68. The suspension was distributed in 10 mL vials (1 mL / vial) and freeze-dried. 1 mL of milliQ water was added to each vial before analysis with a Coulter Counter III and use of the microcapsules.

[0096] Table la below shows the characteristics of the microcapsules (mean of three preparations).

[0097] Table la: characterization of polymeric microcapsules (Prep04)

[0098] Example 4

[0099] Measurement of resistance to MI

[0100] The gas-filled vesicles prepared according to the above examples were tested for their resistance to Mechanical Index.

[0101] Briefly, the setup was composed as follows: a removable test cell with a 7mL volume for receiving the suspension to be tested; and a rigid Plexiglas frame with the transducer encased within, allowing the operator to precisely align the transducer focus with the center of the test cell.

[0102] Aliquots of gas-filled vesicles suspensions prepared as above were diluted 100 times with distilled water and 7mL of the diluted suspension were transferred in the test cell. The cell was placed at the transducer focus and a magnetic agitator was placed below the Plexiglas frame to keep the suspension under constant gentle agitation. The transducer was then activated to transmit pulses of 1ms every 100ms, for 20 seconds at a predetermined MI and at a frequency of 1.61 MHz. After the 20 seconds the cell is removed from the transducer focus, the solution is collected with a syringe and characterized with a coulter counter (30 jim aperture) to determine the volume of gas in the suspension subjected to sonication. Subsequent measurements at increasing MI were performed on other aliquots of the same preparation. The results are expressed as % of remaining gas volume with respect to the volume of gas in a same suspension not subjected to sonication in table 2 below. Table 2: Resistance to MI of various preparations of gas-filled vesicles

[0103] As inferable from the above results, rigid-shell Gfv (PrepOl and Prep04) show a substantial resistance to a mechanical index of at least 0.35 (more than 95% of volume of gas remaining), independently from the material forming the envelope. Also at a MI of 0.75, the residual gas volume remains higher than 80%. On the other hand, soft-shell microbubbles show a substantially lower resistance to MI: for instance, less than 60% of the initial volume of gas remains after sonication at a MI of 0.25, while less than 50% remains after sonication at MI of 0.30; the percentage falls below 30% for an applied MI of 0.40.

[0104] Example 5

[0105] Focused Ultrasound Thermal Ablation with gas-filled vesicles

[0106] To evaluate the effect of the FUS-TA treatment in the presence of different gas- filled vesicles as thermal enhancers, a thermochromic tissue-mimicking phantom (TMP) suitable for embedding the gas-filled vesicles was prepared.

[0107] TMP samples was prepared as described by Tung et al. by mixing egg white, degassed milIQwater, glycerol, acrylamide / bis 40% (19: 1), suspension of Gfv, ammonium persulfate (10 wt %) and tetramethylethylenediamine.

[0108] Suspension of Gfv were incorporated in the TMP preparation mixture at different concentrations (number of Gfv / mL of suspension.), namely 1.33-104, 1.33-105or 1.33-106of GfV / mL. After completing the mixing, the mixture polymerizes in about 15 minutes. The polymerized TMP sample (a rectangular prism of about 3.5 x 3.5 x 8 cm cm) was placed on a support in a deionized degassed water tank at 37°C. A focused ultrasound transducer (H-107, Sonic concepts, Bothel, WA, USA), with a geometrical focus at 6 cm, was also placed in the water tank at 1 cm from the TMP sample.

[0109] Sonication started upon reaching a temperature of 37°C inside the TMP sample. Sonication parameters were as follows:

[0110] Frequency: 0.5 MHz

[0111] Acoustic pressure: 2 MPa

[0112] Pulse duration (continuous wave) : 60s.

[0113] After the thermal treatment, the TMP gel prism was removed from the water tank and the thermal lesion generated therein by the focused ultrasound beam was visually evaluated.

[0114] Table 3 below summarizes the results of the sonication tests.

[0115] Table 3: characterization of thermal lesion with different gas-filled vesicles

[0116] As inferable from the above results, even at the highest tested concentration, gas- filled vesicles with a resistance to MI of at least 0.25 are capable of generating focal thermal lesion in the TMP sample of acceptable size and shape. On the other hand, at concentrations of 1.33-105Gfv / mL or higher, microbubbles with lower resistance to MI generate unacceptable pre-focal lesions of larger dimensions and variable shapes.

Claims

CLAIMS1. A gas-filled vesicle having a resistance to Mechanical Index of 0.25 or higher for use as a thermal enhancer in a focused ultrasound thermal ablation treatment, wherein said gas-filled vesicle has a gaseous core comprising a gas selected from : air; nitrogen; oxygen; carbon dioxide; nitrous oxide; a noble gas; a radioactive gas; a hyperpolarized noble gas; or mixtures thereof.

2. The gas-filled vesicle according to claim 1 wherein said resistance to Mechanical Index is of at least 0.30.

3. The gas-filled vesicle according to claim 2, wherein said resistance to Mechanical Index is of at least 0.35.

4. The gas-filled vesicle according to any of the preceding claims wherein said gas-filled vesicle has a stabilizing envelope comprising a biodegradable waterinsoluble lipid or a water-insoluble polymer.

5. The gas-filled vesicle according to claim 4 wherein said biodegradable waterinsoluble lipid is a glyceride.

6. The gas-filled vesicle according to claim 6 wherein said glyceride is a mono-, di- or tri-glyceride.

7. The gas-filled vesicle according to claim 6 wherein said mono-, di- or triglyceride is a mono-di- or tri-ester derivative of glycerol with one or more fatty acid selected from lauric, myristic, palmitic, stearic, arachidic, behenic, cerotic, melissic, palmitoleic, oleic, linoleic, linolenic, gondoic, erucic acid or mixtures thereof.

8. The gas-filled vesicle according to claim 7 wherein said di- or tri-glyceride is selected from 1,2- or 1,3-dilauroyl glycerol, 1,2- or 1,3-dimyristoyl glycerol, 1,2- or 1,3-dipalmitoyl glycerol, 1,2- or 1,3-distearoyl glycerol, 1,2- or 1,3-diarachidoyl glycerol, 1,2- or 1,3-dibehenoyl glycerol, 1,2-or 1,3-dipalmitoleoyl glycerol, 1,2- or 1,3-dioleoyl glycerol, 1,2- or 1,3-dilinolenoyl glycerol, 1,2- or 1 ,3-d i li noleoyl glycerol, l-palmitoyl-3-stearoylglycerol, l-oleoyl-2-palmitoyl glycerol, l-oleoyl-3- palmitoyl glycerol, l-palmitoyl-2-oleoyl glycerol, l-oleoyl-2-linoleoyl glycerol, 1- oleoyl-3-linoleoyl glycerol, 1,2,3-trilauroyl glycerol (trilaurin), 1,2,3-trimyristoyl glycerol (trimyristin), 1,2,3-tripalmitoyl glycerol (tripalmitin), 1,2,3-tristearoylglycerol (tristearin), 1,2,3-triarachidoyl glycerol (triarachidin), 1,2,3-tribehenoyl glycerol (tribehenin), 1,2,3-trilinolenoyl glycerol, 1,2,3-linoleoyl glycerol, 1,3- dipalmitoyl-2-stearoylglycerol, l,2-dipalmitoyl-3-oleoyl glycerol, 1,3-dipalmitoyl- 2-oleoyl glycerol, l,2-dioleoyl-3-palmitoyl glycerol, l,3-dioleoyl-2-palmitoyl glycerol, l,3-dioleoyl-2-stearoylglycerol, l-Linoleoyl-2,3-dilinolenoylglycerol, 1- palmitoyl-2-oleoyl-3-stearoylglycerol and mixtures thereof.

9. The gas-filled vesicle according to any of the preceding claims 4 to 8 wherein the stabilizing envelope comprises said biodegradable water-insoluble lipid in a molar amount of at least 30%.

10. The gas-filled vesicle according to any of the preceding claims 4 to 9, wherein said stabilizing envelope further comprises a sterol, a phospholipid, a fatty acids or mixtures thereof.

11. The gas-filled vesicle according to claim 10 wherein said phospholipid is selected from dilauroyl-phosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoyl-phosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidoyl-phosphatidylcholine (DAPC), 1,2- dibehenoyl-sn-glycero-3-phosphocholine (DBPC), dioleoyl-phosphatidylcholine (DOPC), dipentadecanoyl-phosphatidylcholine (DPDPC), l-myristoyl-2-palmitoyl- phosphatidylcholine (MPPC), l-palmitoyl-2-myristoyl-phosphatidylcholine (PMPC), l-palmitoyl-2-stearoy I- phosphatidylcholine (PSPC), l-stearoyl-2-palmitoyl- phosphatidylcholine (SPPC), l-palmitoyl-2-oleylphosphatidylcholine (POPC), 1- oleyl-2-palmitoyl-phosphatidylcholine (OPPC), dilauroyl-phosphatidylglycerol (DLPG) and its alkali metal salts, diarachidoylphosphatidyl-glycerol (DAPG) and its alkali metal salts, dimyristoylphosphatidylglycerol (DMPG) and its alkali metal salts, dipalmitoylphosphatidylglycerol (DPPG) and its alkali metal salts, distearoylphosphatidylglycerol (DSPG) and its alkali metal salts, dioleoylphosphatidylglycerol (DOPG) and its alkali metal salts, dimyristoyl phosphatidic acid (DMPA) and its alkali metal salts, dipalmitoyl phosphatidic acid (DPPA) and its alkali metal salts, distearoyl phosphatidic acid (DSPA), diarachidoylphosphatidic acid (DAPA) and its alkali metal salts, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoyl phosphatidylethanolamine (DSPE), dioleylphosphatidyl-ethanolamine (DOPE), diarachidoyl phosphatidylethanolamine (DAPE), dilinoleylphosphatidylethanolamine (DLPE), dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylserine(DPPS), distearoylphosphatidylserine (DSPS), diarachidoyl phosphatidylserine (DAPS), dioleoylphosphatidylserine (DOPS), dipalmitoyl sphingomyelin (DPSP), and distearoylsphingomyelin (DSSP), dilauroyl-phosphatidylinositol (DLPI), diarachidoylphosphatidylinositol (DAPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dioleoyl -phosphatidyl inositol (DOPI).

12. The gas-filled vesicle according to any of the preceding claims wherein said treatment comprises: a. administering an effective amount of an aqueous suspension comprising said gas-filled vesicle to a patient; b. directing a focused ultrasound beam to a region of the patient in need of thermal ablation.

13. The gas-filled vesicle according to claim 12, wherein said ultrasound beam has a frequency of from 0.25 to 20 MHz.

14. The gas-filled vesicle according to any of the preceding claims 12 or 13 wherein said focused ultrasound beam has a geometrical focal point and wherein said focused ultrasound beam has an acoustic pressure at said focal point of from 500 and 15500 Kpa.

15. The gas-filled vesicle according to any of the preceding claims 12 to 14 wherein said suspension comprises from 1.0-108to 1.0-1010of gas-filled vesicles per mL of suspension.

16. A method of thermal ablation of a tissue in a patient which comprises: a. Administering an aqueous suspension of gas-filled vesicles to said patient; b. directing a focused ultrasound beam to a region of interest in said patient, to generate a thermal lesion of said tissue located in said region of interest, in the presence of said gas-filled vesicles; wherein said gas-filled vesicles have a resistance to Mechanical Index of 0.25 or higher and have a gaseous core comprising a gas selected from air; nitrogen; oxygen; carbon dioxide; nitrous oxide; a noble gas; a radioactive gas; a hyperpolarized noble gas; or mixtures thereof.

17. The method according to claim 15 wherein said resistance to Mechanical Index is of at least 0.30.

18. The method according to claim 17, wherein said resistance to Mechanical Index is of at least 0.35.

19. The method according to any of the preceding claims wherein said gas-filled vesicles have a stabilizing envelope comprising a biodegradable water-insoluble lipid or a water-insoluble polymer.

20. The method according to claim 19 wherein said biodegradable waterinsoluble lipid is a glyceride.

21. The method according to claim 20 wherein said glyceride is a mono-, di- or tri-glyceride.

22. The method according to claim 21 wherein said mono-, di- or tri-glyceride is a mono-di- or tri-ester derivative of glycerol with one or more fatty acid selected from lauric, myristic, palmitic, stearic, arachidic, behenic, cerotic, melissic, palmitoleic, oleic, linoleic, linolenic, gondoic, erucic acid or mixtures thereof.

23. The method according to claim 22 wherein said di- or tri-glyceride is selected from 1,2- or 1,3-dilauroyl glycerol, 1,2- or 1,3-dimyristoyl glycerol, 1,2- or 1,3-dipalmitoyl glycerol, 1,2- or 1,3-distearoyl glycerol, 1,2- or 1,3-diarachidoyl glycerol, 1,2- or 1,3-dibehenoyl glycerol, 1,2-or 1,3-dipalmitoleoyl glycerol, 1,2- or 1,3-dioleoyl glycerol, 1,2- or 1,3-dilinolenoyl glycerol, 1,2- or 1 ,3-d i li noleoyl glycerol, l-palmitoyl-3-stearoylglycerol, l-oleoyl-2-palmitoyl glycerol, l-oleoyl-3- palmitoyl glycerol, l-palmitoyl-2-oleoyl glycerol, l-oleoyl-2-linoleoyl glycerol, 1- oleoyl-3-linoleoyl glycerol, 1,2,3-trilauroyl glycerol (trilaurin), 1,2,3-trimyristoyl glycerol (trimyristin), 1,2,3-tripalmitoyl glycerol (tripalmitin), 1,2,3-tristearoyl glycerol (tristearin), 1,2,3-triarachidoyl glycerol (triarachidin), 1,2,3-tribehenoyl glycerol (tribehenin), 1,2,3-trilinolenoyl glycerol, 1,2,3-linoleoyl glycerol, 1,3- dipalmitoyl-2-stearoylglycerol, l,2-dipalmitoyl-3-oleoyl glycerol, 1,3-dipalmitoyl- 2-oleoyl glycerol, l,2-dioleoyl-3-palmitoyl glycerol, l,3-dioleoyl-2-palmitoyl glycerol, l,3-dioleoyl-2-stearoylglycerol, l-Linoleoyl-2,3-dilinolenoylglycerol, 1- palmitoyl-2-oleoyl-3-stearoylglycerol and mixtures thereof.

24. The method according to any of the preceding claims 20 to 23 wherein the stabilizing envelope comprises said biodegradable water-insoluble lipid in a molar amount of at least 30%.

25. The method according to any of the preceding claims 20 to 24, wherein said stabilizing envelope further comprises a sterol, a phospholipid, a fatty acids or mixtures thereof.

26. The method according to claim 25 wherein said phospholipid is selected from dilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidylcholine (DMPC), dipalmitoyl-phosphatidylcholine (DPPC), distearoyl-phosphatidylcholine (DSPC), diarachidoyl-phosphatidylcholine (DAPC), l,2-dibehenoyl-sn-glycero-3- phosphocholine (DBPC), dioleoyl-phosphatidylcholine (DOPC), dipentadecanoyl- phosphatidylcholine (DPDPC), l-myristoyl-2-palmitoyl-phosphatidylcholine (MPPC), l-palmitoyl-2-myristoyl-phosphatidylcholine (PMPC), l-palmitoyl-2- stearoyl-phosphatidylcholine (PSPC), l-stearoyl-2-palmitoyl-phosphatidylcholine (SPPC), l-palmitoyl-2-oleylphosphatidylcholine (POPC), l-oleyl-2-palmitoyl- phosphatidylcholine (OPPC), dilauroyl-phosphatidylglycerol (DLPG) and its alkali metal salts, diarachidoylphosphatidyl-glycerol (DAPG) and its alkali metal salts, dimyristoylphosphatidylglycerol (DMPG) and its alkali metal salts, dipalmitoylphosphatidylglycerol (DPPG) and its alkali metal salts, distearoylphosphatidylglycerol (DSPG) and its alkali metal salts, dioleoylphosphatidylglycerol (DOPG) and its alkali metal salts, dimyristoyl phosphatidic acid (DMPA) and its alkali metal salts, dipalmitoyl phosphatidic acid (DPPA) and its alkali metal salts, distearoyl phosphatidic acid (DSPA), diarachidoylphosphatidic acid (DAPA) and its alkali metal salts, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoyl phosphatidylethanolamine (DSPE), dioleylphosphatidyl-ethanolamine (DOPE), diarachidoyl phosphatidylethanolamine (DAPE), dilinoleylphosphatidylethanolamine (DLPE), dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), diarachidoyl phosphatidylserine (DAPS), dioleoylphosphatidylserine (DOPS), dipalmitoyl sphingomyelin (DPSP), and distearoylsphingomyelin (DSSP), dilauroyl-phosphatidylinositol (DLPI), diarachidoylphosphatidylinositol (DAPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dioleoyl -phosphatidyl inositol (DOPI).

27. The method according to any of claims 15 to 26, wherein said ultrasound beam has a frequency of from 0.25 to 20 MHz.

28. The gas-filled vesicle according to claim 27 wherein said focused ultrasound beam has a geometrical focal point and wherein said focused ultrasound beam has an acoustic pressure at said focal point of from 500 and 15500 Kpa.

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