MICROBUBBLE COMPRISING A FLUORIDED POLYMER OR COPOLYMER AND A FLUORIDE GAS

Abstract

The invention relates to the field of pathologies affecting the central nervous system, particularly severe cerebral pathologies, especially those restricted by the presence of the blood-brain barrier (BBB): gliomas, brain metastases, neurodegenerative diseases (e.g., Alzheimer's, Parkinson's, or ALS), genetic diseases (Huntington's, myopathies, Leigh syndrome, Rett syndrome), but also to the field of cancers, musculoskeletal and immunological disorders, vascular diseases (thrombosis) in numerous organs (e.g., liver, kidney, or muscle) and in combination with numerous therapeutic approaches (e.g., chemotherapy, immunotherapy, targeted therapy, or gene therapy). The invention relates to a microbubble comprising a fluorinated polymer or copolymer and a fluorinated gas, its use, and intermediate polymer or copolymer compounds. Abbreviated figure: 0

Description

Title of the invention: MICROBUBBLE COMPRISING A FLUORIDED POLYMER OR COPOLYMER AND A FLUORESCENT GAS technical field

[0001] The invention belongs to the field of pathologies impacting the central nervous system, in particular severe cerebral pathologies, especially those restricted by the presence of the blood-brain barrier (BBB): Gliomas, cerebral metastases, neurodegenerative diseases (e.g., Alzheimer's, Parkinson's or ALS), genetic diseases (e.g., Huntington's, myopathies, Leigh syndrome or Rett syndrome), but also to the field of cancers, musculoskeletal and immunological disorders, vascular diseases (thrombus) at the level of many organs (e.g., brain, liver, kidney or muscle) and in combination with many therapeutic approaches (e.g., chemotherapy, immunotherapy, targeted therapy or gene therapy).

[0002] The invention relates to a microbubble comprising a fluorinated polymer or copolymer and a fluorinated gas, its use for the treatment of the aforementioned pathologies, as well as the intermediate polymer or copolymer compounds included in the microbubble. Prior art

[0003] The efficacy and emergence of innovative treatments for severe brain diseases are limited by the presence of the blood-brain barrier (BBB), which blocks the passage of more than 95% of therapeutic molecules from the blood into diseased cells. The BBB is composed of endothelial cells connected by tight junctions that block the diffusion of therapeutic agents. Several studies have demonstrated that focused ultrasound can be used through the skull to deliver molecules into the brain. Combined with the intravenous injection of microbubbles (ultrasonic agents), ultrasound can induce a localized and reversible opening of the BBB through sonopermeabilization. Indeed, the oscillations of the bubbles induced by ultrasound weaken the tight junctions present in the cerebral vascular endothelium and facilitate the passage of molecules into the extravascular tissue.The duration and effectiveness of BBB opening can be controlled by modifying the ultrasound parameters. Typically, pulsed ultrasound is transmitted to the focal point for a few minutes.

[0004] Since 2015, several clinical trials evaluating the safety and validating the benefit of sonopermeabilization for the local treatment (a few cm3) of cancer or neurodegenerative diseases have been conducted. However, in the In cases of diffuse brain pathologies (e.g., cancer metastases, Alzheimer's, genetic diseases), it becomes necessary to permeabilize the blood-brain barrier over large volumes, or even the entire cerebral parenchyma. This generalized treatment represents a major clinical challenge.

[0005] To date, the SonoCloud-9 implant (Carthera) offers expanded clinical treatment by increasing the surface area of ​​the ultrasound devices. This invasive approach, however, remains limited by the size of the therapeutic implant. Transcranially, ultrasound can be focused successively on several points to enlarge the treated area. The treatment time is then considerably increased (several minutes per point), potentially lasting up to several hours. This option is incompatible with the low stability of the bubbles in the blood (typically 5 minutes). One of the solutions considered involves repeatedly reinjecting microbubbles intravenously (every 3 to 5 minutes). However, the duration and effectiveness of the treatment remain severely limited by the total volume of injectable ultrasound agents in the body.

[0006] A major problem in ultrasound therapy is the limited lifespan of microbubbles in the body. While this does not pose a difficulty for treatment in rodents (small volume to be treated), the rapid dissolution of ultrasonic agents in the body becomes problematic as soon as the volume to be treated reaches a few cm³. Indeed, the gas contained in the ultrasonic agent (microbubble) escapes within the first few seconds after administration, rendering the agent unstable and considerably reducing its lifespan in the blood (half-life on the order of a few minutes). It therefore becomes necessary to have an agent that is sufficiently sensitive to ultrasound to induce a biological effect, while maintaining its stability in the blood through slow gas diffusion across the shell [1].

[0007] There is therefore a need to improve these treatments, in particular with ultrasonic agents, in the form of microbubbles, with greatly improved stability, in order to promote the penetration of drugs into the brain, or into other organs, over large volumes.

[0008] This problem of microbubble stability is also a limitation in contrast-enhanced molecular ultrasound. In this imaging modality, the injected functionalized agents target a specific vascular receptor that is overexpressed in pathology (e.g., cancer, inflammation). A certain amount of time must then be allowed for the circulating microbubbles to reach their targets and bind to the receptors. This delay is generally limited to 10 minutes because beyond this time, the injected microbubbles are completely dissolved in the body. The use of more stable agents would therefore promote the binding of microbubbles to the targeted receptors and lead to signal enhancement in molecular imaging. Description of the invention

[0009] The invention proposes a new formulation of ultrasonic agents in the form of microbubbles, said microbubbles comprising a perfluorocarbon (PFC) core and a shell based on fluorinated polymers or copolymers. The ultrasonic agents, in the form of microbubbles, according to the invention, make it possible to transiently permeabilize the blood-brain barrier and to promote the penetration of drugs into the brain over large volumes.

[0010] The microbubbles according to the invention are ultrasonic contrast agents having a shell based on fluorinated polymers or copolymers that improve the stability of these agents in the blood and their sensitivity to ultrasound. These more stable agents should thus allow for prolonged treatment, making it possible to treat a larger volume of the brain.

[0011] In particular, it has been possible to obtain agents with improved stability through the use of amphiphilic polymers or copolymers bearing fluorinated chains to promote the anchoring of the polymers or copolymers at the fluorine / blood interface. The administration of microbubbles based on fluorinated polymers or copolymers according to the invention substantially improves the duration of the ultrasound treatment, which can be applied for at least three times longer than with conventionally used agents.

[0012] The permeabilization of biological barriers can be achieved by combining ultrasound with the administration of microbubbles according to the invention. This method makes it possible, in particular, to increase drug delivery and improve the therapeutic effect.

[0013] One of the primary applications of the microbubbles according to the invention is thus the opening of the blood-brain barrier for drug delivery to the brain. This approach is versatile and can also be used to treat various pathologies in numerous organs (e.g., liver, kidney, muscle) and in combination with many therapeutic approaches (e.g., chemotherapy, immunotherapy, targeted therapy, gene therapy). The microbubbles according to the invention can also be used as ultrasound contrast agents in the field of molecular imaging.

[0014] The invention thus relates to an ultrasonic contrast agent, in the form of a microbubble comprising a fluorinated polymer or copolymer, and a fluorinated gas, preferably chosen from perfluorocarbons and sulfur hexafluoride.

[0015] The term “fluorinated polymer or copolymer” means a polymer or copolymer comprising at least one fluorinated alkyl or aryl group.

[0016] Advantageously, the microbubble according to the invention can have a diameter within a range of 1 to 10 micrometers, preferably from 1 to 5 micrometers (measured by optical or granulometric measurement).

[0017] Advantageously, the perfluorocarbon can be selected from perfluorobutane (decafluorobutane), perfluoropropane (octafluoropropane), perfluoroethane, perfluoropentane, perfluorohexane, and mixtures thereof, preferably perfluorobutane (decafluorobutane) and perfluoropropane (octafluoropropane).

[0018] Advantageously, the microbubble according to the invention may further comprise at least one additional gas. The additional gas may be chosen from air, oxygen, nitrogen, nitric oxide, and mixtures thereof.

[0019] Advantageously, the volume ratio between the fluorinated gas and at least one additional gas, in the microbubble according to the invention, can be within a range from 90:10 to 50:50, preferably from 80:20 to 60:40.

[0020] Advantageously, in the microbubble according to the invention, the fluorinated polymer or copolymer can be chosen from copolymers of formula I: [Chem 1] in which, PEG represents a polyethylene glycol group with a molar mass in the range of 2000 to 50000 g / mol, preferably 2000 to 5000 g / mol, p is an integer in the range of 5 to 50, preferably 5 to 15, each R1 independently represents H or CH3, each R2 independently represents either a linear or branched C2 to C20 fluorinated alkyl group, optionally bearing a C5 to C7 fluorinated aryl group, or a C5 to C7 fluorinated aryl group; R3 and R4 independently represent H or CH3; and X represents a halogenated group, preferably chosen from Br, Cl and I.

[0021] A "fluorinated alkyl group" or "fluorinated aryl group" is understood to be an alkyl or aryl group in which all or part of the hydrogen atoms have been replaced by fluorine atoms. By way of illustration, a "fluorinated ethyl group" may be represented, for example, by -CH2CF3 or -CF2CF3 and a "fluorinated phenyl group" may be represented, for example, by -C6H2F3 or -C6F5.

[0022] Advantageously, the microbubble according to the invention can be a microbubble in which the polymer is of formula I, and in which the group R2 can be selected from 2,2,2-trifluoroethyl, 2,2,3,3,3-pentafluoropropyl, 2,2,3,3,4,4,4-heptaafluorobutyl, 1H,1H,2H,2H-tridecafluoro-n-octyl, 1H,1H,2H,2H-heptadecafluorodecyl, 2,2,3,4,4,4-hexafluorobutyl, 2,2,3,3-tetrafluoropropyl, 1H,1H,2H,2H-nonafluorohexyl, 1H,1H,5H-octafluoropentyl, 2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptyl, hexafluoroisopropyl, pentafluorobenzyl and pentafluorophenyl. Preferably, group R2 is selected from 2,2,2-trifluoroethyl, 2,2,3,3,3-pentafluoropropyl, and hexafluoroisopropyl.

[0023] Advantageously, in the microbubble according to the invention, the fluorinated polymer or copolymer can be chosen from polymers of formula II: [Chem 2] Formula II in which, n and m are positive integers in the range of 10 to 100, y is a positive integer less than or equal to n, z is a positive integer less than or equal to m, each R group independently represents a linear or branched C2-C20 fluorinated alkyl group, or a polyethylene glycol group with a molar mass in the range of 500 to 2000 g / mol, at least one of the R groups being a fluorinated alkyl group.

[0024] The term "grafting rate of fluorinated groups" refers to the proportion of fluorinated groups covalently bonded to the poly(malic acid) units. Rate = number of moles of bonded fluorinated group / total number of moles of malic acid (1000 for the percentage). The grafting rate is determined by NMR. The number n represents the number of alpha units of the PMA. The number m represents the number of beta units of the PMA. The number y represents the number of alpha units of the PMA substituted by a fluorine group. The number z represents the number of beta units of the PMA modified by a fluorine group.

[0025] The PEG group grafting rate is defined as the proportion of PEG groups covalently bonded to the poly(malic acid) units. Rate = number of moles of bonded PEG group / total number of moles of malic acid (1000 for the percentage). The grafting rate is determined by NMR. The number n represents the number of alpha units of the PMA. The number m represents the number of beta units of the PMA. The number y represents the number of alpha units of the PMA substituted by a PEG group. The number z represents the number of beta units of the PMA modified by a PEG group.

[0026] Advantageously, when the polymer is of formula II, the rate of grafting of fluorinated groups can be within a range of 1 to 30%, preferably from 5 to 25%.

[0027] Advantageously, when the polymer is of formula II, the rate of grafting of PEG groups can be within a range of 0 to 50%, preferably from 0 to 30%.

[0028] Advantageously, the microbubble according to the invention can be a microbubble in which the polymer is of formula II, and in which the R group can be selected from 1H,1H,2H,2H-heptadecafluoro-1-decyl, 2,2,3,3,4,4,4-heptafluoro-1-butyl, 2,2,2-trifluoroethyl, 1H,1H-perfluoro-1-heptyl, 1H,1H-heptadecafluoro-1-nonyl, 1H,1H-perfluorododecan-1-ol, 1H,1H-perfluoro-1-tetradecyl, perfluoro-tert-butyl, hexafluoro-2-4-bis(trifluoromethyl)-1-pentyl. Preferably R is chosen from among 1H,1H,2H,2H-heptadecafluoro-1-decyl, 1'-heptafluoro-1-butyl and perfluoro-tert-butyl.

[0029] Advantageously, the microbubble according to the invention may further comprise a lipid.

[0030] The term "lipid" means an amphiphilic molecule comprising a hydrophilic fraction (for example, a polar head group) and a lipophilic or hydrophobic fraction. The lipophilic or hydrophobic fraction may comprise at least one branched or linear fatty acid fraction, saturated or unsaturated, or a derivative or analog thereof (for example, a fluorocarbon). A fatty acid fraction essentially consists of a hydrocarbon fraction / chain, in particular an acyl chain. Preferably, the fatty acid fraction or its derivative or analog has a length of 10 to 30, preferably 12 to 25, and more preferably 14 to 22 carbon atoms. If the lipid comprises more than one, for example two or three, fatty acid groups or their derivatives or analogs, these fatty acid groups or their derivatives or analogs may be identical or different.The term "lipid" includes cationic lipids and non-cationic lipids, i.e., neutral or . Anionic lipids can include phospholipids or their derivatives, glycerolipids or their derivatives, sphingolipids (e.g., sphingomyelin) or their derivatives, or lipid sterols (e.g., cholesterol) or their derivatives. Glycerolipids are composed of glycerols mono-, di-, or tri-substituted with fatty acid groups. Phospholipids, whose hydrophilic portion includes a phosphate group, can be glycerophospholipids. Lipids can also be functionalized / modified, for example, with (oligo)peptides, polymers (e.g., PEG), or other functional groups.In an aqueous medium, lipids can also be organized in a supramolecular manner, for example, as lipid-based particles or lyotropic phases, such as liposomes, lamellar phases, hexagonal and inverted hexagonal phases, cubic phases, micelles, and inverted micelles composed of monolayers. The stabilizing effect according to the present invention applies to all types of supramolecular lipid organization. Preferably, the lipids used in the invention are pharmaceutically acceptable, i.e., they are suitable as excipients or as components of drug delivery formulations.

[0031] Advantageously, the microbubble according to the invention can be a microbubble in which the lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000 (DPPE-PEG5000), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (DPPE-PEG2000), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 (DPPE-PEG1000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000 (DSPE-PEG5000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-PEG2000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 (DSPE-PEG1000), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol)-5000 (DMPE-PEG5000), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol)-2000 (DMPE-PEG2000), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol)-1000 (DMPE-PEG1000), 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA), 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA), 1,2-distearoyl-sn-glycero-3-phosphate (DSPA), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium sait) (DPPS), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dipalmitoyl-sn-glycero-3-pho, Phosphatidylamine (DPPE), L,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), L,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), L,dahnitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), egg phosphatidylcholine, soy phosphatidylcholine, and mixtures thereof. Preferably, the lipid is selected from DSPC, DPPC, DMPC, DSPE-PEG2000, DPPE-PEG2000, DMPE-PEG2000, and mixtures thereof.

[0032] Advantageously, the microbubble according to the invention can be a microbubble in which the mass ratio lipid(s) / fluorinated polymer or copolymer is within a range of 0.1 to 0.9, preferably from 0.5 to 0.9.

[0033] The invention also relates to a composition comprising microbubbles according to the invention and an aqueous solution.

[0034] Advantageously, the concentration of polymer or copolymer in the composition according to the invention can be between 0.01 and 10 mg / mL.

[0035] Advantageously, the aqueous solution of the composition according to the invention may comprise a buffer, glucose and / or physiological saline.

[0036] Advantageously, the concentration of microbubbles in the composition according to the invention can be between 105 and 1014 microbubbles per mL of aqueous solution, preferably between 106 and 1012 microbubbles per mL.

[0037] The invention also relates to a microbubble or a composition according to the invention for its use in the permeabilization of the blood-brain barrier.

[0038] The invention also relates to a microbubble or a composition according to the invention for its use in ultrasound therapy. Ultrasound therapy can be implemented in the treatment of diffuse pathologies in the brain, in chemotherapy, immunotherapy, targeted therapy, for the activation of glial cells and in gene therapy, preferably chosen from among gliomas, brain metastases, neurodegenerative diseases (e.g. Alzheimer's, Parkinson's, ALS), and genetic diseases (e.g. Huntington's, Leigh, myopathies, Leigh syndrome or Rett syndrome).

[0039] The invention also relates to a microbubble or a composition according to the invention for its use in ultrasound therapy for the treatment of organ pathologies (e.g., liver, kidney, muscle) such as, for example, thrombi, or cancers of the prostate, breast, pancreas, bladder, melanoma, or colon, and in combination in a complementary therapeutic approach. This complementary therapeutic approach may be chosen from chemotherapy, immunotherapy, targeted therapy, or gene therapy.

[0040] The invention also relates to a microbubble or a composition according to the invention for its use as an ultrasonic contrast agent in molecular imaging. According to this use, an antibody or peptide can be linked to the bubble to target a receptor of interest.

[0041] The invention relates to a copolymer of formula I: [Chem 3] in which, PEG represents a polyethylene glycol group with a molar mass in the range of 2000 to 50000 g / mol, preferably 2000 to 5000 g / mol; p is an integer in the range of 5 to 50, preferably 5 to 15; each R1 independently represents H or CH3; each R2 independently represents either a linear or branched C2-C20 fluorinated alkyl group, optionally bearing a C5-C7 fluorinated aryl group, or a C5-C7 fluorinated aryl group. R3 and R4 independently represent H or CH3; and X represents a halogenated group, preferably chosen from Br, Cl and I.

[0042] The invention relates to a polymer of formula II: [Chem 4] COÔH œœ II in which, n and m are positive integers within an interval ranging from 10 to 100. y is a positive integer less than or equal to n, z is a positive integer less than or equal to m, Each R group independently represents a linear or branched C2-C20 fluorinated alkyl group, or a polyethylene glycol group with a molar mass in the range of 500 to 2000 g / mol, at least one of the R groups being a fluorinated alkyl group.

[0043] The invention also relates to a process A for forming microbubbles according to the invention, comprising the steps: a1) direct dissolution of the polymer or copolymer of formula I or II in an aqueous solution at a polymer or copolymer concentration within a range of 0.01 to 10 mg / mL of aqueous solution; A2) gas exchange of the solution obtained in step A1) with a fluorinated gas, under stirring; and formation of microbubbles; or of a process B for forming microbubbles according to the invention, comprising the steps: B1) formation of a thin film by contacting a polymer or copolymer of formula I or II and optionally at least one lipid in an organic solvent, preferably chloroform, B2) put an aqueous solution of the thin film obtained in step Bl), at a polymer or copolymer concentration in the range of 0.01 to 10 mg / mL of aqueous solution; B3) gas exchange of the solution obtained in step B2) with a fluorinated gas, under agitation; and obtaining microbubbles.

[0044] Advantageously, steps A1), A2), B1), B2) and B3) of processes A or B according to the invention can be carried out at room temperature (from 15 to 25°C). Brief description of the figures

[0045] [Fig-1] Fig. 1 represents an optical microscopy observation of microbubbles micrometric particles stabilized by a fluorinated polymer.

[0046] [Fig.2] Fig.2 represents a diagram of the different microbubbles: SonoVue, lipids or mixtures of lipids-fluorinated copolymers as well as their size distribution.

[0047] [Fig. 3] Figure 3 represents the evolution of the microbubbles over time. A: Microbubble size distribution over time. B: Microbubble concentration as a function of time.

[0048] [Fig. 4] [Fig. 4] represents the harmonic response of the microbubbles. A: Response Acoustics in the frequency domain for each type of bubble at an applied pressure of 90 kPa. B: Measurement of the second harmonic plotted as a function of the applied pressure for SonoVue, lipid microbubbles LIP and microbubbles stabilized by a fluorinated polymer POL.

[0049] [Fig. 5] Figure 5 represents the ultraharmonic response of the microbubbles. A: Plot of the frequency response for each type of bubble at an applied pressure of 220 kPa. B: Measurement of ultraharmonic plots as a function of applied pressure for SonoVue, lipid microbubbles LIP and microbubbles stabilized by a fluorinated polymer POL.

[0050] [Fig.6] Fig.6 represents the experimental protocol for permeabilizing the Blood-brain barrier by ultrasound.

[0051] [Fig.7] Fig.7 represents the permeabilization of the blood-brain barrier Ultrasound in mice using microbubbles. From left to right, top to bottom: Schematic representation of the trajectory and parameters of the ultrasound beam. Ti-weighted MRI image of the control without microbubble injection. Ti-weighted MRI images for each type of microbubbles used (n=3 per condition). T2-weighted MRI images 48 hours post-treatment.

[0052] [Fig.8] Fig.8 represents the time to effectiveness of microbubbles after their injection for opening the BBB. SonoVue 5 min, lipid microbubbles LIP 7 min 30, and microbubbles stabilized by a fluorinated polymer POL 15 min for injection 2 x 0.7 bubbles (n=3).

[0053] Examples

[0054] Example 1: Synthesis of fluorinated polymers and copolymers and formulation of ultrasonic agents.

[0055] The. Copolymer Synthesis 1

[0056] PEG-Br was synthesized by esterification of poly(ethylene glycol) monomethyl ether (PEG-OCH3, Fluka Chemika) with α-bromoisobutyryl bromide (Sigma Aldrich) as described in the literature [2].

[0057] In a second step, the polymer was obtained by ATRP as described in the literature [3]. PEG 2000-Br (500 mg, 0.238 mmol), copper(I) bromide (CuBr), N,N,N',N”,N”-pentamethyldiethylenetriamine (PMDETA), and 2,2,2-trifluoroethyl methacrylate (TFEMA) were loaded into a Schlenk tube equipped with a magnetic stir bar at a ratio of 1:1:1:X, where X = 5 to 20. The mixture was dissolved in anhydrous THF and then degassed. Polymerization was carried out at 90°C for 20 hours. The product was then diluted with THF and passed through a neutral aluminum oxide column to remove the catalyst (CuBr). The THF solvent is removed under vacuum, and purification is carried out by two successive precipitations in a diethyl ether / petroleum ether mixture (1:1, v / v). The final product is then dried under vacuum.

[0058] [Chem.5] LBt .¾ $ o A Ô „ JO, A CwBr, PMDSTA .................** k'A- x ii OA ..................>- H^C ; ' 'u O .' you TM.W ' / < X

[0059] Scheme 1: Synthesis of fluorinated copolymer 1.

[0060] lb Copolymer Synthesis 2

[0061] Step 1: synthesis of a-P-PMA (poly(malic acid))

[0062] α-[]-PMA is synthesized by direct polycondensation of L-malic acid as described previously [4]. 10 g of L-malic acid are placed in a Schlenk tube and heated at 110°C with stirring for 72 h under 1-5 mmHg. The resulting polyester is dissolved in tetrahydrofuran (THF) and precipitated twice in a mixture of diethyl ether and petroleum ether, then lyophilized.

[0063] Step 2: grafting of fluorinated chains onto PMA by esterification, fluorinated PMA (fPMA)

[0064] Different grafting rates in fluorinated alcohols were tested (10, 15 and 20). The synthesis is described for a theoretical grafting rate of 15%.

[0065] In one flask, 1000 mg of PMA and 600 mg of 1H,1H,2H,2H-heptadecafluoro-1-decanol (molar ratio: nf-OH = 0.15 nMA) are added, purged under a flow of argon, and dissolved in 10 mL of anhydrous THF with magnetic stirring. In another flask, 400 mg of DCC (molar ratio nDCC = 1.5 nf-OH) and 105 mg of DM AP (molar ratio nDMAP = 0.1nMA) are purged under a flow of argon and dissolved in 10 mL of anhydrous THF with magnetic stirring. After complete dissolution, the DCC:DMAP solution is added dropwise to the PMA:f-OH solution at 0°C for 30 min. The reaction took place overnight at room temperature, after which the solution was filtered and the product condensed by evaporating the solvent. The product was dissolved in THF and purified by precipitation in a diethyl ether / petroleum ether mixture, then dialyzed. The fluorinated polymer was finally lyophilized.

[0066] [Chem.6] HAS) L-fA ssîdc wàiKpc swiiï (i'MÀt read ■PMA Ai>:<wi Jbrë y«> Syœêf« IM sv«« «A^iutwtfagsrSe !>«< <!--?<; sAAy<br-->

[0067] Scheme 2: Synthesis strategy of fluorinated polymer 2 (fPMA) A) polycondensation of L-(-)- malic acid B) esterification by a fluorinated alcohol (R-OH) of the pendant carboxylic groups.

[0068] Example 2: Formulation of microbubbles

[0069] The formulation processes for conventional lipid bubbles and fluorinated polymer-based bubbles are slightly different. Indeed, several modifications to the microbubble formulation protocol were necessary: ​​preparation solvent, mixing temperature (to ensure better polymer solubilization), and elimination of the lipid film formation step by evaporation.

[0070] Microbubble counterexample (CE). For purely lipid-based shell formulations, outside the scope of this invention, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) (molar ratio 9:1) are dissolved in ethanol, and the solvent is evaporated under vacuum in a rotary evaporator. The resulting thin film is rehydrated with HEPES buffer (10 mM, filtered to 0.2 µm, pH 7.2) at room temperature to a lipid concentration of 5 mg / mL (lipid suspension). The resulting mixture is then aliquoted into 3 mL glass vials by adding 1.5 mL of lipid suspension.

[0071] Microbubbles 1. For lipid-shell formulations incorporating fluorinated polymers, according to the invention, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), and the fluorinated polymer (Polymer 1) (molar ratio 65:25:10) are dissolved in chloroform. A thin film is formed as described above and rehydrated with HEPES at 70°C before aliquoting the solution. a lipid concentration of 5mg / mL. The thin film hydration method proves to generate bubbles that exhibit greater stability, particularly in their response to ultrasound, than the direct dissolution of lipids / polymers in an aqueous solution.

[0072] Microbubbles 2. For purely polymeric shell formulations incorporating fluorinated polymers, according to the invention, the f-PMA polymers (Polymer 2) were dissolved in HEPES buffer (10 mM, 0.2 µm filtered, pH 7.4) to obtain a concentration of 1 mg / mL. The resulting mixture was then aliquoted into 3 mL glass bottles by adding 1.5 mL of the polymer solution.

[0073] Before use, the vials undergo gas exchange: the vial is placed under vacuum (air is removed from the vial) before being filled with PFC (C3F8 or C4F10). The microbubbles are then formed by a standard agitation technique using a Vialmix (Bristol-Myers-Squibb) shaker for 45 seconds at a stirring speed of 4500 rpm.

[0074] Example 3: Characterization of microbubbles

[0075] 3.a. Size and concentration of microbubbles

[0076] Once the agents were formed, the size distribution and concentration of the microbubbles were determined by optical microscopy. All bubbles subsequently presented were formulated with C4F10.

[0077] The size distribution and concentration (number of microbubbles per mL of aqueous solution) of Microbubbles 1 were characterized using bright-field optical microscopy ([Fig. 1]). The microbubble suspension was diluted in Milli-Q water to obtain approximately 1000 microbubbles per image, after which 1 OpL of the dilution were deposited onto a cell counting chamber slide (Thoma BRAND Blaubrand) which was placed under the microscope objective. The light intensity was adjusted according to the following fixed parameters: 20X magnification and 10 ms exposure. The optical microscope used had a resolution limit of 0.45 pm.

[0078] In order to accurately characterize the microbubbles, several positions on the slide are captured for each vial (typically between 10 and 20). A minimum threshold of microbubbles to be counted has been determined to ensure reliable characterization.

[0079] Three formulations of polydisperse microbubbles (SonoVue, commercial; LIP: CE microbubbles with a purely lipid shell; POL: microbubbles 1 with a lipid shell incorporating fluorinated polymers 1) were studied. The average size and average concentration of the different microbubbles are presented in Table 1, and the size distribution of each of the measured vials is presented in [Fig. 2]. All the data for each formulation were combined to plot the cumulative frequency graph ([Fig. 2]) and calculate the first (d0) and ninths. (d90) percentiles, as well as the median (d90) and the range of the size distribution (Table 1). SonoVue and POL microbubbles have a concentration of the same order of magnitude (approximately 5 x 10⁸ bubbles / mL), while LIP microbubbles are approximately 100 times more concentrated ((7.3 ± 2.3) x 10¹⁰ bubbles / mL). The average size of SonoVue and POL microbubbles is slightly larger (approximately 2.5–2.7 pm) than that of LIP microbubbles (1.9 pm). [Tables 1] - mean ± ET (pm) dw (pm) d50 (pm) d90 (pm) range concentration + ET (bulbs / mL) SonoVue 2.7 + 0.1 1.1 2.3 4.6 1.5 (4.5 + 1.7) x 10⁸ LIP 1.9 + 0.1 0.8 1.9 3.0 1.2 (7.3 + 2.3) x 10¹⁰ POL* 2.5 + 0.4 0.9 2.1 4.4 1.6 (6.6 + 4.8) x 10⁸ Table 1: Information on the size distribution and concentration of SonoVue, LIP, and POL microbubbles measured by optical microscopy. *: n = 8 vials for POL and n = 3 vials for LIP and SonoVue; SD: standard deviation between vials; di0: first percentile; d50: median; d90: ninth percentile; di0, d50, d90, and the span are indicated.

[0080] 3.b. Storage stability of microbubbles

[0081] The storage stability of the microbubbles over time was evaluated for the three formulations presented above. After activation, the bubbles were stored at 4°C between each measurement. The evolution of the average microbubble size over time is shown in [Fig. 3]-A. Overall, the microbubble volume increases with time. The interquartile range for SonoVue and LIP does not exceed 6 µm over 28 days, whereas this threshold is reached for POL after only 2 days. The evolution of the concentration over time for the different types of microbubbles and the quantification of this evolution are shown in [Fig. 3]-B. The microbubble concentration decreases rapidly during the first hours or even the first few days after activation before stabilizing.The evolution of the concentration of SonoVue, LIP, and POL over time is linear during the first 24 hours for SonoVue (r² = 1, the regression being plotted for only 2 points), 72 hours for LIP (r² = 0.96), and 48 hours for POL (r² = 0.9). Using the linear equation that models this evolution, we determined the half-life in solution of these microbubbles, which is 17 hours for SonoVue, 41 hours for LIP, and 24 hours for POL when stored at 4°C after their activation.

[0082] 3.c. Acoustic characterization of microbubbles

[0083] An acoustic characterization step is essential to verify the ultrasonic sensitivity of the microbubbles according to the invention (activation threshold, mode (vibration, acoustic stability). From the analysis of the nonlinear response, it is thus possible to determine the cavitation regime of microbubbles as a function of the applied acoustic pressure. Indeed, the acoustic signature of microbubbles is primarily characterized by their harmonic and sub / ultraharmonic responses. The presence of this intrinsic nonlinear signal in microbubbles is linked to the applied ultrasonic parameters, and in particular to the acoustic pressure.

[0084] In therapy, and more specifically in the brain, the cavitation dose must be calculated to determine the vibration regime of the microbubbles. The in situ dosimetric indicator must be precise because the difference between the effective dose and the injury dose is small (on the order of one hundred kPa). By convention, the stable cavitation dose is determined by the harmonic signal of the microbubbles: (n+1) fO (where ne N* and fO is the emission frequency of the ultrasound), whereas the inertial cavitation dose corresponds to the measurement of the signal emitted at all frequencies (broadband signal, outside the harmonics). Adverse events (e.g., edema, hemorrhage) can occur when the bubbles are in the inertial cavitation regime. Locally violent physical phenomena induced by the sudden implosion of the microbubbles under the effect of ultrasound can lead to the irreversible destruction of tissues or vessels.

[0085] There is also an intermediate regime corresponding to a destabilization of the microbubble shell (subharmonic and ultraharmonic radiation). Indeed, over time, the gas contained in the microbubble diffuses into the surrounding medium, resulting in a buckling state of its envelope (e.g., excess lipid on the bubble envelope). This state is associated with the appearance of specific frequencies (subharmonic and ultraharmonic: ((2n+1)) / 2 fO, where ne N and fO are the ultrasound emission frequencies). This state can appear suddenly during ultrasound excitation and can lead to inertial cavitation. The challenge, therefore, is to detect this destabilization immediately in order to stop or adjust the ultrasound sequence as quickly as possible before any risk to the patient.

[0086] 2xl07 microbubbles diluted in 40 mL of degassed milli-Q water are agitated in a A custom-made tank with Mylar walls is attached to the center of a larger reservoir of degassed Milli-Q water. Microbubbles are excited at fO = 1 MHz (repetition frequency = 10 kHz; pulse duration of 40 cycles) using a focused ultrasonic transducer (active diameter 25 mm, V302-SU, Olympus). The acoustic response of the microbubbles is recorded simultaneously with a 2.25 MHz ultrasonic transducer (active diameter 25 mm, V304-SU, Olympus) positioned 4.6 cm from the tank containing the microbubbles, perpendicular to the emitting transducer, which is also positioned 4.6 cm from the tank. An ultrasonic-absorbing material is placed on the opposite side of the emitting transducer to prevent multiple reflections. The signal is then recorded and processed. The signal observed on a digital oscilloscope is transferred to a computer for data analysis. The area under the curve (AUC) of the second harmonic (2 fO) and the first ultraharmonic (1.5 fO) are calculated. The area under the curve ratio (AUCR) between the microbubble signal and the signal emitted by the mylar cuvette filled only with water (reference signal) is also calculated. For each type of microbubble, the signal is recorded at rarefaction pressures ranging from 50 kPa to 400 kPa with a bolus of fresh microbubbles for each acquisition. Each measurement is repeated three times, and the entire experiment is replicated for three different vials of each formulation. The appearance of the 1.5 fO peak is considered a marker of the microbubble destabilization threshold.

[0087] Figure 4 represents the AUCR of the second harmonic 2f0, an indicator of stable microbubble cavitation, as a function of the applied rarefaction pressure for each type of microbubble, thus providing a quantification of the microbubble cavitation intensity. As expected, the stable cavitation of each formulation increases with the applied acoustic pressure.

[0088] Figure 5 represents the AUCR of the ultraharmonic λ,5f0 as a function of the applied pressure for each type of microbubbles, as well as the destabilization threshold interval as a function of the mechanical index (rarefaction pressure / square root of the frequency) resulting from these graphs. The objective is to determine the ultraharmonic appearance threshold in order to determine the microbubbles' destabilization threshold. The ultraharmonic appearance threshold was set at an AUCR of 5 dB because it visually corresponds to the appearance of these peaks on the Fourier Transform plot of the signal. This value depends on the transmitted signal as well as the noise level of the measurement chain, and in particular the sensor sensitivity. The destabilization of LIP occurs at a lower pressure (120 kPa) than for POL microbubbles (150 kPa) according to the invention and SonoVue (200 kPa).Furthermore, the destabilization range is wider for LIP ([120 kPa - 200 kPa]) than for SonoVue ([200 kPa - 250 kPa]) and POL ([150 kPa - 200 kPa]).

[0089] These results demonstrate the sensitivity of the POL bubbles according to the invention to ultrasound, comparable to other formulations, and their potential for imaging and therapy.

[0090] Example 4: In vivo validation for blood-brain barrier permeabilization

[0091] 4.a. In vivo protocol

[0092] The experiments were performed on C57BL / 6 mice (17 to 49 g). The animals were anesthetized with 1.5% isoflurane in an O2 / air mixture (50 / 50, v / v). A bolus containing 100 pL of Dotarem® (Gd-DOTA, Guerbet) and 2x07 microbubbles (SonoVue, LIP or POL) is injected intravenously via the retro-orbital cavity. All animal experiments were conducted in accordance with the recommendations of the European Community (2010 / 63 / EU) and the French national committees (Law 2013-118) for the care and use of laboratory animals. The experimental protocol was approved by a local ethics committee for animal experimentation (Ile-de-France No. 044) and by the French Ministry of Agriculture (APAFIS #34522-2022010412087915 vl).

[0093] Ultrasound was delivered using a spherical focused transducer (active diameter 25 mm, focal depth 20 mm, axial resolution 5 mm, lateral resolution 1 mm, Imasonic) centered at 1.5 MHz and connected to a single-channel programmable generator (Image Guided Therapy). The transducer was mounted on a motorized XYZ-axis stage and positioned above the head of the anesthetized mouse. The device was coupled to the mouse's head using a latex balloon (filled with degassed Milli-Q water) and ultrasound gel. The distance between the transducer and the skull was adjusted to target the center of the brain at the focal distance (i.e., 20 mm). The ultrasound sequence used traced the contours of a 5 mm square by transmitting the ultrasound in a near-continuous manner (91% duty cycle). The ultrasound emission was stopped at each change of direction of the motors. The sequence diagram is illustrated in [Fig.6].A different rarefaction pressure is applied to each side of the square. The square tracing is repeated 52 times for a total sequence duration of 121 seconds.

[0094] 3.b. Opening threshold, immediate permeabilization of the BBB and safety of the protocol

[0095] This experiment is conducted to validate the ability of these microbubbles according to the invention to permeabilize the BBB and to estimate the opening threshold for each type of microbubbles as a function of the applied pressure. In these experiments, ultrasound is applied immediately after the intravenous injection (delay < 3 s) of the MRI contrast agent and the microbubbles. This protocol allows testing the effectiveness of the bubbles immediately after their administration into the body. Figure 7 shows a T1 contrast MRI image for each type of microbubbles, as well as the control without microbubble injection for rarefaction pressures applied between 100 and 600 kPa. After injection of the MRI contrast agent, contrast enhancement becomes visible on the MRI image along the path of the ultrasound beam, if the BBB has been successfully permeabilized. These MRI images are not quantitative.The signal intensity depends on several experimental parameters (mouse weight, injection quality, therapeutic transducer positioning) and is difficult to compare from one mouse to another. However, in the same mouse, it is possible to discern whether or not permeabilization has occurred and to detect an opening threshold by means of a [measurement]. Qualitative analysis. Blood-brain barrier (BBB) ​​opening was observed at 400 kPa and 600 kPa, but not at 200 kPa and 100 kPa, for all microbubbles evaluated (n=3 per condition). No opening was observed in the control condition after injection of MRI contrast agents. Figure 7 also shows T2-weighted images 48 hours after ultrasound application. No hemorrhage or edema was observed in these images 48 hours after the experiment.

[0096] 4.c. Estimation of the temporal threshold of bubble effectiveness for permeabilization of the BBE

[0097] The objective of this protocol is to estimate the stability of circulating microbubbles and to evaluate their ability to permeabilize the blood-brain barrier (BBB) ​​at different times after injection. This concept is particularly important for the translation of the technology to larger models and for volumetric treatments in these subjects. Figure 8 presents the results obtained at different times after application of ultrasound following bolus injection of microbubbles for each formulation. An example image obtained from a T1-weighted MRI sequence is shown. After verifying the openings at 0 min for each formulation (same protocol as before), a 10-minute delay was initially applied between intravenous administration of the bubbles and application of ultrasound. The experiment was repeated three times.If no opening is visible at this time, the delay is reduced by 2 minutes 30 seconds until the temporal threshold for opening is reached (i.e., stability of the bubbles in the blood). Conversely, if the opening is still visible 10 minutes post-injection, the delay is then increased by 5 minutes to determine the maximum time of microbubble efficacy. All the experiments performed are summarized in Table 2, which specifies whether, for each condition tested, blood-brain barrier (BBB) ​​permeabilization was visible on T1-weighted MRI scans or not, depending on the time between microbubble injection and the start of the ultrasound sequence. The POL bubbles according to the invention prove to be twice as stable over time as LIP bubbles and three times more stable than SonoVue bubbles. Indeed, they are effective 15 minutes after injection, whereas LIP bubbles are effective 7 minutes 30 seconds and SonoVue bubbles are effective 5 minutes after injection.For all treated mice, when a BBB opening was observed, contrast enhancement was visible for the insonified areas at 400 kPa and 600 kPa each time and never for 200 kPa and 100 kPa. [Tables 2] . - 0 min 5 min 7 min 30 10 min 15 min 20 min SonoVue yes (n=3) yes (n=3) no (n=2) no (n=3) - - LIP yes (n=3) - yes (n=3) no (n=3) - - POL yes (n=3) - - yes (n=3) yes (n=3) Very light (n=1) Table 2: Was BBB permeabilization observed? All experiments performed for an injection of 2x107 bubbles. Columns: time between microbubble injection and FUS sequence initiation. Rows: experiments performed according to bubble type; yes: BBB permeabilization was observed; no: no BBB permeabilization visible on T1-weighted MRI images.

[0098] In view of these results, it is evident that the addition of fluorinated polymers in the composition of the microbubbles according to the invention contributes to improving their stability in the blood, while preserving their acoustic properties and their ability to permeabilize the BBB.

[0099] The fluorinated polymer 2 was able to be synthesized with three different grafting ratios [Tables 3] Polymer 2 Theoretical grafting rate (%) Experimental grafting rate (%) Mn (g / mol) NMR P10 10% 4 1400 + 300 P15 15% 7.5 1600 + 400 P20 20% 12 1800 + 400 Table 3: Variation in the grafting rate of fluorinated polymer 2.

[0100] These polymers alone were formulated as C4FiO microbubbles at a concentration of 1 mg / mL with the following characteristics for microbubbles. [Table 4] - c4f10 - P10 P15 P20 Concentration (bubbles / mL) 10 8 4.3+1.6 8.3+2.0 6.4+1.7 Size (pm) 1.7 +0.9 1.7 +0.8 2.0+1.3 Table 4: Formulation of fluorinated polymer 2 in microbubbles.

[0101] After injection in the animal, it was possible to open the BBB using these microbubbles formulated from the fluorinated polymer 2 for rarefaction pressures greater than 300 kPa.

[0102] Reference list: [1] Ambre Dauba, Anthony Delalande, Hernies AS Kamimura, Allegra Conti, Benoit Larrat, Nicolas Tsapis, Anthony Novell. Recent advances on ultrasound contrast agents for blood-brain barrier opening with focused ultrasound. Pharmaceuticals. 2020. doi: 10.3390 / pharmaceuticsl2111125. [2] Changkui Fu, Shauna Herbst, Cheng Zhang, Andrew K. Whittaker Polymeric 19 F MRI agents responsive to reactive oxygen species, Polym. Chem., 2017; 8:4585 [3] Hong Li, Weiyin Gu, Le Li, Yongming Zhang, Thomas P. Russell, E. Bryan Coughlin, Synthesis of semicrystalline / fluorinated side-chain crystalline block copolymers and their bulk and thin film nanoordering, Macromolecules, 2013; 46:3737. [4] Simone Pinto Cameiro, Laurence Moine, Barbara Tessier, Valérie Nicolas, Orlando D.H. dos Santos, and Elias Fattal. 2019. Pyrazinoic Acid-Poly(Malic Acid) Biodégradable Nanoconjugate for Efficient Intracellular Delivery. Précision Nanomedicine 2 (3): 303-17.

Claims

1.

2.

3.

4. Demands Microbubble comprising a fluorinated polymer or copolymer and a fluorinated gas selected from perfluorocarbons and sulfur hexafluoride, the perfluorocarbon preferably being selected from perfluorobutane, perfluoropropane, perfluoroethane, perfluoropentane, perfluorohexane and mixtures thereof. Microbubble according to claim 1, further comprising an additional gas selected from air, oxygen, nitrogen, nitric oxide and mixtures thereof. Microbubble according to the preceding claim wherein the volume ratio between the fluorinated gas and the additional gas is within a range of 90:10 to 50:50, preferably from 80:20 to 60:

40. Microbubble according to any one of the preceding claims, wherein the polymer is selected from polymers or copolymers of formulas I or II: [Chem 7] Formula I wherein PEG represents a polyethylene glycol group having a molar mass in the range of 2000 to 50000 g / mol, preferably 2000 to 5000 g / mol, p is an integer in the range of 5 to 50, preferably 5 to 15, each R1 independently represents H or CH3, each R2 independently represents either a linear or branched C2 to C20 fluorinated alkyl group, optionally bearing a C5 to C7 fluorinated aryl group, or a C5 to C7 fluorinated aryl group; R3 and R4 independently represent H or CH3; and X represents a halogenated group, preferably chosen from Br, Cl and I; [Chem 8] or* OO Formula £ JH w| j yj| y| Hg | 0 \ s / VA / 1 i / V h / 1.. L. - £ " CH? COQH CQOR II in which, n and m are positive integers in the range of 10 to 100, y is a positive integer less than or equal to n, z is a positive integer less than or equal to m, each R group independently represents a linear or branched C2 to C20 fluorinated alkyl group, or a polyethylene glycol group with a molar mass in the range of 500 to 2000 g / mol, at least one of the R groups being a fluorinated alkyl group.

5. Microbubble according to any one of the preceding claims, wherein the polymer is of formula I, and group R2 is selected from 2,2,2-trifluoroethyl, 2,2,3,3,3-pentafluoropropyl, 2,2,3,3,4,4,4-heptaafluorobutyl, 1H,1H,2H,2H-tridecafluoro-n-octyl, 1H,1H,2H,2H-heptadecafluorodecyl, 2,2,3,4,4,4-hexafluorobutyl, 2,2,3,3-tetrafluoropropyl, 1H,1H,2H,2H-nonafluorohexyl, 1H,1H,5H-octafluoropentyl, 2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptyl, hexafluoroisopropyl, pentafluorobenzyl and pentafluorophenyl.

6. Microbubble according to claim 1 or 2, wherein the polymer is of formula II, and the group R is a fluorinated alkyl group selected from 1H,1H,2H,2H-heptadecafluoro-1-decyl, 2,2,3,3,4,4,4-heptafluoro-1-butyl, 2,2,2-trifluoroethyl, 1H,1H-perfluoro-1-heptyl, 1H,1H-heptadecafluoro-1-nonyl, 1H,1H-perfluorododecan-1-ol, 1H,1H-perfluoro-1-tetradecyl, perfluoro-tert-butyl, hexafluoro-2,4-bis(trifluoromethyl)-1-pentyl.

7. Microbubble according to any one of the preceding claims further comprising a lipid, preferably selected from 1,2- distearoyl-sn-glycero-3-phosphoglycerol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, l,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000, l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000, l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000, 1,2-distearoyl -sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000, 1,2- distearoyl -sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000, 1,2-dimyristoyl - sn-glycero- 3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000, 1,2-dimyristoyl -sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000, 1,2-dimyristoyl -sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000, 1,2-Dimyristoyl-sn-glycero-3-phosphate, 1,2-Dipalmitoyl-sn-glycero-3-phosphate, L,2-Distearoyl-sn-glycero-3-phosphate, L,2-Dioleoyl-sn-glycero-3-phosphocholine, 1,2-Dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium sulfate), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine, L,2-Distearoyl-sn-glycero-3-phosphoethanolamine, L,2-Dioleoyl-sn-glycero-3-phosphoethanolamine, L-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol, egg phosphatidylcholine, phosphatidylcholine of soybeans and their mixtures.

8. Microbubble according to claim 7, wherein the mass ratio lipid(s) / fluorinated polymer or copolymer is within a range of 0.1 to 0.9, preferably 0.5 to 0.

9.

9. Composition comprising microbubbles according to any one of the preceding claims and an aqueous solution.

10. Microbubble according to any one of claims 1 to 8 or composition according to claim 9, for its use in the permeabilization of the blood-brain barrier.

11. Microbubble according to any one of claims 1 to 8 or composition according to claim 9, for its use in ultrasound therapy.

12.

13.

14.

15. Microbubble for its use according to claim 1 to 8 or composition according to claim 9, wherein ultrasound therapy is implemented in the treatment of diffuse pathologies in the brain, in chemotherapy, in immunotherapy, in targeted therapy, for glial cell activation and in gene therapy, preferably selected from gliomas, brain metastases, neurodegenerative diseases, and genetic diseases. Microbubble for its use according to claims 1 to 8 or composition according to claim 9, for its use in ultrasound therapy for the treatment of organ-level pathologies, preferably in the treatment of thrombi, or cancers of the prostate, breast, pancreas, bladder, melanoma, or colon, and optionally in combination with a complementary therapeutic approach, preferably chosen from chemotherapy, immunotherapy, targeted therapy, and gene therapy. Microbubble according to any one of claims 1 to 8 or composition according to claim 9, for its use as an ultrasound contrast agent in molecular imaging. Formula I polymer: [Chem 9] Formula I in which, PEG represents a polyethylene glycol group with a molar mass in the range of 2000 to 50000 g / mol, preferably 2000 to 5000 g / mol, p is an integer in the range of 5 to 50, preferably 5 to 15, each R1 independently represents H or CH3, Each R2 independently represents a linear or branched C2-C20 fluorinated alkyl group, optionally bearing a C5-C7 fluorinated aryl group, or a C5-C7 fluorinated aryl group. R3 and R4 independently represent H or CH3; and X represents a halogenated group, preferably chosen from Br, Cl and I.

16. Formula II polymer: [Chem 10] II in which, n and m are positive integers within an interval ranging from 10 to 100. y is a positive integer less than or equal to n, z is a positive integer less than or equal to m, each R group independently represents a linear or branched C2-C20 fluorinated alkyl group, or a polyethylene glycol group with a molar mass in the range of 500 to 2000 g / mol, at least one of the R groups being a fluorinated alkyl group.

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