Microbubble comprising a fluorinated polymer or copolymer and a fluorinated gas

Abstract

The invention belongs to the field of pathologies affecting the central nervous system: in particular, severe cerebral pathologies, more particularly 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 (Huntington's, myopathies, Leigh syndrome, Rett syndrome), but also to the field of cancers, musculoskeletal and immunological disorders, vascular diseases (thrombus) 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, to the use thereof and also to the polymer or copolymer intermediate compounds.

Description

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

[0001] technical field

[0002] 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).

[0003] 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.

[0004] State of the art

[0005] The effectiveness and development 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 impede 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. These can be controlled by modifying the ultrasonic parameters. Typically, pulsed ultrasound is transmitted to the focal point for a few minutes.

[0006] Since 2015, several clinical trials have been conducted to evaluate the safety and validate the benefit of sonopermeabilization for local treatment (a few cm 3 Treatments for cancer or neurodegenerative diseases are conducted. However, in the case 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.

[0007] To date, the SonoCloud-9 implant (Carthera) offers expanded clinical treatment options by increasing the surface area of ​​the ultrasound devices. However, this invasive approach remains limited by the size of the therapeutic implant. Transcranial ultrasound can be successively focused on multiple points to enlarge the treated area. This significantly increases treatment time (several minutes per point), potentially lasting several hours. This option is incompatible with the low stability of the microbubbles in the blood (typically 5 minutes). One proposed solution involves repeatedly reinjecting microbubbles intravenously (every 3 to 5 minutes). However, the duration and efficacy of the treatment remain severely limited by the total volume of injectable ultrasound agents in the body.

[0008] A major problem in ultrasound therapy is the limited lifespan of microbubbles in the body. While this does not pose a problem 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 centimeters. 3 Indeed, the gas contained in the ultrasonic agent (microbubble) escapes within seconds of 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 sufficiently sensitive to ultrasound to induce a biological effect, maintaining its stability in the blood through slow gas diffusion across the shell [1].

[0009] Therefore, there is a need to improve these treatments, particularly with ultrasonic agents, in the form of microbubbles, with significantly improved stability. in order to facilitate the penetration of drugs into the brain, or other organs, over large volumes.

[0010] This issue 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 is then required 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.

[0011] Description of the invention

[0012] 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, allow for the transient permeabilization of the blood-brain barrier and facilitate the penetration of drugs into the brain over large volumes.

[0013] The microbubbles according to the invention are ultrasonic contrast agents with a shell made of 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.

[0014] 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 at least three times longer than with conventionally used agents.

[0015] The permeabilization of biological barriers can be achieved by combining ultrasound with the administration of microbubbles according to the invention. This This method makes it possible, in particular, to increase the delivery of medication and improve the therapeutic effect.

[0016] 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 ultrasonic contrast agents in the field of molecular imaging.

[0017] 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.

[0018] The term “fluorinated polymer or copolymer” means a polymer or copolymer comprising at least one fluorinated alkyl or aryl group.

[0019] 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).

[0020] Advantageously, the perfluorocarbon can be selected from perfluorobutane (decafluorobutane), perfluoropropane (octafluoropropane), perfluoroethane, perfluoropentane, perfluorohexane, and mixtures thereof, preferably perfluorobutane (decafluorobutane) and perfluoropropane (octafluoropropane).

[0021] Advantageously, the microbubble according to the invention may further comprise at least one additional gas. The additional gas may be selected from air, oxygen, nitrogen, nitric oxide, and mixtures thereof.

[0022] 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.

[0023] Advantageously, in the microbubble according to the invention, the fluorinated polymer or copolymer can be chosen from copolymers of formula I: [Chem 1] 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 R 1 independently represents H or CH3, each R 2 independently represents either a linear or branched C2-C20 fluorinated alkyl group, optionally bearing a fluorinated C5-C7 aryl group, or a C5-C7 fluorinated aryl group; R 3and R 4 independently represent H or CH3; and X represents a halogenated group, preferably chosen from Br, Cl and I.

[0024] A "fluorinated alkyl group" or "fluorinated aryl group" is an alkyl or aryl group in which all or some of the hydrogen atoms have been replaced by fluorine atoms. For example, a "fluorinated ethyl group" can be represented by -CH2CF3 or -CF2CF3, and a "fluorinated phenyl group" can be represented by -C6H2F3 or -CeFs.

[0025] Advantageously, the microbubble according to the invention can be a microbubble in which the polymer has formula I, and in which the R group 2can be chosen 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, the R group 2 is chosen from 2,2,2-trifluoroethyl, 2,2,3,3,3-pentafluoropropyl, and hexafluoroisopropyl.

[0026] 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 wherein 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.

[0027] The rate of fluorine group incorporation is defined as the proportion of fluorine groups covalently bonded to the units of poly(malic acid). Rate = number of moles of bonded fluorine group / total number of moles of malic acid (x100 for the percentage). The rate of incorporation is determined by NMR. The number n represents the number of alpha units of the poly(malic acid). The number m represents the number of beta units of the poly(malic acid). The number y represents the number of alpha units of the poly(malic acid) substituted by a fluorine group. The number z represents the number of beta units of the poly(malic acid) modified by a fluorine group.

[0028] The PEG group grafting rate refers to the proportion of PEG groups covalently bonded to poly(malic acid) units. Rate = Number of moles of bonded PEG group / total number of moles of malic acid (x100 for the percentage). The grafting rate is determined by NMR. The number n represents the number of alpha units of PMA. The number m represents the number of beta units of PMA. The number y represents the number of alpha units of PMA substituted by a PEG group. The number z represents the number of beta units of PMA modified by a PEG group.

[0029] Advantageously, when the polymer is of formula II, the rate of grafting of fluorinated groups can be in a range of 1 to 30%, preferably 5 to 25%.

[0030] 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%.

[0031] 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 1H,1H,2H,2H-heptadecafluoro-1-decyl, 1H-heptafluoro-1-butyl and perfluoro-tert-butyl.

[0032] Advantageously, the microbubble according to the invention may further comprise a lipid.

[0033] The term "lipid" is understood to mean an amphiphilic molecule comprising a hydrophilic fraction (e.g., 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 (e.g., 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 preferably 14 to 22 carbon atoms. If the lipid comprises more than one, e.g., two or three, fatty acid groups or their derivatives or analogs, these groups Fatty acids, their derivatives, or analogs may be identical or different. The term "lipid" includes both cationic and non-cationic lipids, i.e., neutral or anionic lipids. Lipids may include phospholipids or their derivatives, glycerolipids or their derivatives, sphingolipids (e.g., sphingomyelin) or their derivatives, or sterol lipids (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, may be glycerophospholipids. Lipids may 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., suitable as excipients or as components of drug delivery formulations.

[0034] 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 salt) (DPPS), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,dalmitoyl-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.

[0035] Advantageously, the microbubble according to the invention can be a microbubble in which the mass ratio of lipid(s) / fluorinated polymer or copolymer is within a range of 0.1 to 0.9, preferably from 0.5 to 0.9.

[0036] The invention also relates to a composition comprising microbubbles according to the invention and an aqueous solution.

[0037] Advantageously, the concentration of polymer or copolymer in the composition according to the invention can be between 0.01 and 10 mg / mL.

[0038] Advantageously, the aqueous solution of the composition according to the invention may comprise a buffer, glucose and / or physiological saline.

[0039] Advantageously, the concentration of microbubbles in the composition according to the invention can be between 10 5 and 10 14 microbubbles per mL of aqueous solution, preferably 10 6 and 10 12 microbubbles per mL.

[0040] 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.

[0041] The invention also relates to a microbubble or a composition according to the invention for use in ultrasound therapy. Ultrasound therapy Ultrasound can be used in the treatment of diffuse brain diseases, in chemotherapy, immunotherapy, targeted therapy, for glial cell activation and gene therapy, preferably chosen from gliomas, brain metastases, neurodegenerative diseases (e.g. Alzheimer's, Parkinson's, ALS), and genetic diseases (e.g. Huntington's, Leigh's, myopathies, Leigh syndrome or Rett syndrome).

[0042] The invention also relates to a microbubble or a composition according to the invention for use in ultrasound therapy for the treatment of organ pathologies (e.g., liver, kidney, muscle) such as thrombi, or cancers of the prostate, breast, pancreas, bladder, melanoma, or colon, and in combination with other complementary therapies. These complementary therapies may include chemotherapy, immunotherapy, targeted therapy, and gene therapy.

[0043] 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 a peptide can be bound to the bubble to target a receptor of interest.

[0044] The invention relates to a copolymer of formula I: [Chem 3] Formula I in which, PEG represents a polyethylene glycol group with a molar mass in the range of 2000 to 50000 g / mol, preferably from 2000 to 5000 g / mol, p is an integer in the range of 5 to 50, preferably from 5 to 15, each R 1 independently represents H or CH3, each R 2 independently represents either a linear or branched fluorinated alkyl group at C2 to C20, optionally bearing a fluorinated aryl group at C5 to C7, or a fluorinated aryl group at C5 to C7 R 3 and R 4 independently represent H or CH3; and X represents a halogenated group, preferably chosen from Br, Cl and I.

[0045] The invention relates to a polymer of formula II: [Chem 4] Formula II wherein 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. 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 in the 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 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 B1), at a polymer or copolymer concentration within a 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. 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).

[0046] Brief description of the figures

[0047] [Fig. 1] Figure 1 represents an optical microscopy observation of micrometric microbubbles stabilized by a fluorinated polymer 1.

[0048] [Fig.2] Figure 2 represents a diagram of the different microbubbles: SonoVue, lipid or lipid-fluorinated copolymer mixture 1 as well as their size distribution.

[0049] [Fig. 3] Figure 3 shows the evolution of microbubbles over time. A: Distribution of microbubble size over time. B: Microbubble concentration as a function of time.

[0050] [Fig. 4] Figure 4 represents the harmonic response of the microbubbles. A: Acoustic response in the frequency domain for each bubble type 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).

[0051] [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 the ultraharmonics plotted as a function of the applied pressure for SonoVue, lipid microbubbles (LIP), and microbubbles stabilized by a fluorinated polymer (POL).

[0052] [Fig. 6] Figure 6 represents the experimental protocol for permeabilization of the blood-brain barrier by ultrasound.

[0053] [Fig. 7] Figure 7 illustrates blood-brain barrier permeabilization by ultrasound in mice using microbubbles. From left to right, top to bottom: Schematic representation of the ultrasound beam trajectory and parameters. 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.

[0054] [Fig. 8] Figure 8 shows the time it takes for microbubbles to become effective after injection for opening the BBB. SonoVue microbubbles take 5 minutes, lipid microbubbles LIP 7 min 30 sec, and fluoropolymer-stabilized microbubbles 1 POL 15 min for 2 x 10⁻¹ injections. 7 bubbles (n=3).

[0055] [Fig. 9] Figure 9 shows, under an optical microscope in white light, 2 P15 microbubbles and a C4F / air mixture according to the invention, at 20X magnification, for a light exposure time of 10 seconds, dilution x6 vials (photographs A and B correspond to 2 separate tests, and show the reproducibility of the process of forming 2 microbubbles according to the invention).

[0056] [Fig. 10] Figure 10 shows the long-term efficacy, at the liver level, of microbubbles incorporating fluorinated polymers 2, compared with commercial microbubbles (SonoVue). From top to bottom: SonoVue microbubbles (A), 2 P15 microbubbles (B), and 2 P20 microbubbles (C).

[0057] [Fig. 11] Figure 11 represents the hepatic ultrasound intensity as a function of time of microbubbles 2 P15 and P20) compared with commercial microbubbles (SonoVue), illustrating the effectiveness at the liver level of the different microbubbles tested.

[0058] [Fig. 12] Figure 12 is a diagram representing both the half-life and mean transit time (MTT) of microbubbles 2 P15 and P20, compared with commercial microbubbles (SonoVue), illustrating the liver efficacy of the different microbubbles tested.

[0059] [Fig. 13] Figure 13 represents the cerebral microvasculature of a spleen into which 2P15 microbubbles have been injected, this image having been obtained by localized ultrasonic microscopy (ULM).

[0060] [Fig. 14] Figure 14 represents the evolution of the Doppler signal as a function of time of 2 P15 microbubbles, compared with commercial microbubbles (SonoVue), illustrating the effectiveness at the brain level of the tested microbubbles.

[0061] [Fig. 15] Figure 15 is a diagram representing both the half-life and mean transit time (MTT) of 2 P15 microbubbles, compared with commercial microbubbles (SonoVue), illustrating the brain-level efficacy of the different microbubbles tested.

[0062] [Fig. 16] Figure 16 is a set of diagrams and photographs representing BBB opening in mice with C4F10 microbubbles stabilized by polymer 2, compared with SonoVue® microbubbles. Diagram A) outlines the experimental protocol; diagram B) illustrates the ultrasound sequence used; photographs C) to G) are T1-weighted MRI images obtained for the different mice injected with 2P10 microbubbles (C), 2P15 microbubbles (D), 2P20 microbubbles (E), SonoVue® microbubbles (F), and a saline solution without microbubbles (G).

[0063] Examples

[0064] Example 1: Synthesis of fluorinated polymers and copolymers and formulation of ultrasonic agents.

[0065] 1.a. Copolymer Synthesis 1

[0066] 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],

[0067] 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 solvent THF 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. [Chem 5] Diagram 1: Synthesis of fluorinated copolymer 1.

[0068] 1.b. Copolymer synthesis 2

[0069] Step 1: Synthesis of α-P-PMA (poly(malic acid))

[0070] α-p-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.

[0071] Step 2: Grafting of fluorinated chains onto PMA by esterification, fluorinated PMA (fPMA)

[0072] Different grafting rates with fluorinated alcohols were tested (10, 15 and 20). The synthesis is described for a theoretical grafting rate of 15%.

[0073] 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 argon flow, 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 DMAP (molar ratio nDMAP = 0.1 nMA) are purged under argon flow 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 is carried out overnight at room temperature, then the solution is filtered and the product condensed by evaporating the solvent. The product was dissolved in THF and purified by precipitation in a mixture of diethyl ether: petroleum ether then dialyzed. The fluorinated polymer was finally lyophilized. [Chem 6] 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.

[0074] Example 2: Formulation of microbubbles

[0075] The formulation processes for conventional lipid bubbles and fluorinated polymer-based bubbles are slightly different. 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.

[0076] 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 5mg / mL (lipid suspension). The resulting mixture is then aliquoted into 3mL glass vials by adding 1.5mL of lipid suspension.

[0077] 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 previously described and rehydrated with HEPES at 70°C before aliquoting the solution to a lipid concentration of 5 mg / mL. The thin-film hydration method is shown to generate bubbles that exhibit greater stability, particularly in their response to ultrasound, than the direct dissolution of lipids / polymers in aqueous solution.

[0078] 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 (1 µM, 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.

[0079] Before use, the vials undergo gas exchange: the vial is placed under vacuum (air is removed from the vial) before being filled with PFC (CsFs or C4F10). Microbubbles are then formed using a standard agitation technique with a Vialmix (Bristol-Myers-Squibb) shaker for 45 seconds at a speed of 4500 rpm.

[0080] Example 3: Characterization of microbubbles 1

[0081] 3. a. Size and concentration of microbubbles 1

[0082] 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.

[0083] The size distribution and concentration (number of microbubbles per mL of aqueous solution) of Microbubbles 1 were characterized using bright-field optical microscopy (Figure 1). The microbubble suspension was diluted in Milli-Q water to obtain approximately 1000 microbubbles per image. After which, 1 OPL of the dilution is deposited onto a cell counting chamber slide (Thoma Brand Blaubrand) which is placed under the microscope objective. The light intensity is adjusted according to the following fixed parameters: 20X magnification and 10 ms exposure. The optical microscope used has a resolution limit of 0.45 µm.

[0084] To accurately characterize the microbubbles, several positions on the slide are captured for each vial (typically between 10 and 20). A minimum microbubble count threshold has been determined to ensure reliable characterization.

[0085] Three formulations of polydisperse microbubbles (SonoVue, commercial; LIP: CE microbubbles with a purely lipid shell; POL: microbubbles with a lipid shell incorporating fluorinated polymers) were studied. The mean size and mean concentration of the different microbubbles are presented in Table 1, and the size distribution of each of the measured vials is presented in Figure 2. All the data for each formulation were pooled to plot the cumulative frequency plot (Figure 2) and calculate the first (d) and ninth (dg0) percentiles, as well as the median (dg0) and range of the size distribution (Table 1). The SonoVue and POL microbubbles have a concentration of the same order of magnitude (approximately 5 × 10⁻³). 8 bubbles / mL), while LIPs are approximately 100 times more concentrated ((7.3 ± 2.3) x 10 10bubbles / mL). The average size of SonoVue and POL is slightly larger (approximately 2.5-2.7 pm) than that of LIP (1.9 pm). [Table 1] SonoVue, LIP and POL microbubbles measured by optical microscopy. * : n=8 vials for POL and n=3 vials for LIP and SonoVue; AND: standard deviation between vials; d: first percentile; dso: median; dgo: ninth percentile; d, dso, dgo and the range (span in English) are indicated.

[0086] 3. b. Storage stability of microbubbles 1

[0087] The storage stability of microbubbles over time was evaluated for the three formulations presented above. After activation, the bubbles were stored at 4°C between measurements. The evolution of the average microbubble size over time is shown in Figure 3-A. Overall, the microbubble volume increased over time. The interquartile range for SonoVue and LIP did not exceed 6 µm over 28 days, whereas this threshold was 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 Figure 3-B. The microbubble concentration decreased rapidly during the first few hours or even days after activation before stabilizing. The evolution of the concentration of SonoVue, LIP, and POL over time was linear during the first 24 hours for SonoVue (r 2= 1, the regression being plotted for only 2 points), 72h for LIP (r 2 = 0.96) and 48h for POLs (r 2 = 0.9). Using the linear equation that models this evolution, we determined the half-life in solution of these microbubbles, which is 17h for SonoVue, 41 h for LIP and 24h for POL when stored at 4°C after their activation.

[0088] 3.c. Acoustic characterization of microbubbles 1

[0089] An acoustic characterization step is essential to verify the ultrasonic sensitivity of the microbubbles according to the invention (activation threshold, vibration mode, acoustic stability). From the analysis of the nonlinear response, it is thus possible to determine the cavitation regime of the microbubbles as a function of the applied acoustic pressure. Indeed, the acoustic signature of the microbubbles is primarily characterized by their harmonic and sub / ultraharmonic responses. The presence of this intrinsic nonlinear signal of the microbubbles is linked to the applied ultrasonic parameters, and in particular to the acoustic pressure.

[0090] In therapy, and more specifically in the brain, the cavitation dose must be calculated in order to determine the vibration regime of the microbubbles. The in situ dosimetric indicator must be precise because the difference between effective dose and injury dose is small (on the order of a hundred kPa). By convention, the stable cavitation dose is determined by the harmonic signal of the microbubbles: (n+1) fO (where n is N* and fO is the ultrasound emission frequency), while 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 inertial cavitation. Locally violent physical phenomena induced by the sudden implosion of microbubbles under the effect of ultrasound can lead to irreversible tissue or blood vessel damage.

[0091] There is also an intermediate regime corresponding to a destabilization of the microbubble shell (subharmonic and ultraharmonic radiation). Over time, the gas contained within the microbubble diffuses into the surrounding medium, resulting in a buckling state of its envelope (e.g., excess lipid on the bubble's envelope). This state is associated with the appearance of specific frequencies (subharmonic and ultraharmonic: ((2n+1)) / 2 fO, where ne is the emitted frequency of the ultrasound). 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, preventing any risk to the patient.

[0092] 2x10 7Microbubbles diluted in 40 mL of degassed Milli-Q water are agitated in a custom-made tank with Mylar walls. The tank is fixed in the middle of a larger reservoir of degassed Milli-Q water. The 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 transmitting transducer itself, which is also positioned 4.6 cm from the tank. An ultrasonic-absorbing material is placed on the opposite side of the transmitting 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 - Area. 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 (AUCR) ratios between the microbubble signal and the signal emitted by the mylar cuvette filled only with water (reference signal) are 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.

[0093] Figure 4 shows 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.

[0094] Figure 5 shows the AUCR of the ultraharmonic 1.5f0 as a function of the applied pressure for each type of microbubble, 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 microbubble 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]).

[0095] 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.

[0096] Example 4: In vivo validation for blood-brain barrier permeabilization 11

[0097] 4. a. In vivo protocol

[0098] The experiments were performed on C57BL / 6 mice (17 to 49 g). The animals were anesthetized with 1.5% isoflurane in a 50 / 50 (v / v) oxygen / air mixture. A bolus containing 100 pL of Dotarem® (Gd-DOTA, Guerbet) and 2 x 10 7Microbubbles (SonoVue, LIP, or POL) are 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 (Île-de-France No. 044) and by the French Ministry of Agriculture (APAFIS #34522-2022010412087915 v1).

[0099] 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 an 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 traced the contours of a 5 mm square by transmitting ultrasound in near-continuous mode (91% duty cycle). The ultrasound emission was stopped at each change of direction of the motors. The sequence diagram is illustrated in Figure 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.

[0100] 4. b. Opening threshold, immediate permeabilization of the BBB, and protocol security

[0101] This experiment is conducted to validate the ability of these microbubbles 1 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 injection. Intravenous administration (within < 3 seconds) of the MRI contrast agent and microbubbles is performed. This protocol allows testing the efficacy of the bubbles immediately after their administration into the body. Figure 7 shows a T1-weighted 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, provided the blood-brain barrier (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 between mice. However, in the same mouse, it is possible to determine whether or not permeabilization has occurred and to detect an opening threshold through 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.

[0102] 4.c. Estimation of the temporal threshold of bubble effectiveness for BBB permeabilization

[0103] 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 time points after injection. This is particularly important for scaling up the technology to larger models and for volumetric treatments in these subjects. Figure 8 presents the results obtained at different times after the 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 (using the same protocol as before), a 10-minute delay was initially applied between intravenous administration of the bubbles and the 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 (Le., stability of bubbles in the blood). Conversely, if... The opening is still visible 10 minutes post-injection; the time frame is then increased by 5 minutes to determine the maximum efficacy time of the microbubbles. All the experiments performed are summarized in Table 2, which specifies whether, for each condition tested, blood-brain barrier (BBB) ​​permeabilization was visible on 5-weighted 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 an opening of the BBB 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. [Table 2] 5 experiments performed for an injection of 2x10 7 Bubbles. Columns: time between microbubble injection and FUS sequence launch. Rows: experiments performed according to bubble type, yes: BBB permeabilization was observed, no: no BBB permeabilization visible on T1-weighted MRI images.

[0104] In view of these results, it is evident that the addition of fluorinated polymers 1 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.

[0105] Example 5: Microbubbles 2 comprising the fluorinated polymer 2 at 5 different grafting rates

[0106] 5. a. Formulation of microbubbles 2 comprising the fluorinated polymer 2 at 10%, 15% and 20% grafting rates respectively, and C4F10

[0107] Fluorinated polymer 2 was able to be synthesized with three different grafting rates, according to the procedure of example 1.b. [Table 3] Table 3: Variation in the grafting rate of fluorinated polymer 2.

[0108] Microbubbles comprising fluorinated polymer 2 at 10%, 15% and 20% grafting rates respectively (microbubbles 2 P10, microbubbles 2 P15 and microbubbles 2 P20) and C4F10 were formulated, at a concentration of 1 mg / mL, according to the procedure described in Example 2 (“Microbubbles 2”).

[0109] 5. b. Formulation of microbubbles 2 comprising the fluorinated polymer 2 and an air / C4F mixture

[0110] Microbubbles comprising the fluorinated polymer 2 at 15% grafting rate and an air / C4F mixture were formulated.

[0111] For this purpose, the f-PMA polymer (Polymer 2) with a grafting rate of 15% is dissolved in HEPES buffer (10 mM, 0.2 µm filtered, pH 7.4) to obtain a concentration of 1 mg / mL. The resulting mixture is then aliquoted into 3 mL glass bottles by adding 1.5 mL of the polymer solution.

[0112] Before use, the vials undergo gas exchange: the vial is placed under vacuum (air is removed) before being filled with PFC (C4F10). The vials are degassed, and then, using a syringe, 500 µL of air are added to the vials (C4F:air mixture ratio 70:30). After a 45-minute waiting period to allow for better homogenization, microbubbles are then formed using a standard agitation technique with a Vialmix shaker (Bristol-Myers-Squibb) for 45 seconds at a speed of 4500 rpm.

[0113] 5.c. Characterization of microbubbles 2

[0114] The size distribution and concentration of microbubbles 2 were characterized using bright-field optical microscopy, according to the procedure of example 3. a. The optical microscope used has a resolution limit of 0.25 pm.

[0115] In order to accurately characterize the microbubbles, several positions on the slide are captured for each vial (typically between 10 and 20).

[0116] The average size and average concentration of the different microbubbles 2 (P10, P15 and P20) are presented in Table 4, and the images obtained by optical microscopy in Figure 9. The air / C4F microbubbles 2 have a size and concentration of the same order of magnitude as the microbubbles 2 formulations with C4F10. [Table 4] Table 4: Comparison of the main characteristics of microbubbles 2 for formulations with C4F10 and with an air / C4F mixture. At least 3 vials were analyzed for each formulation.

[0117] These microbubbles 2 according to the invention, incorporating fluorinated polymers 2, were then implemented in in vivo experiments to study their stability and their ability to open / permeabilize the BBB.

[0118] 5.d. In vivo validation of microbubbles 2 in the liver

[0119] The experiments were performed on healthy female C57BL / 6 mice (20–27 g). The mice were anesthetized with 1.5% isoflurane in a 50 / 50 (v / v) oxygen / air mixture. The experiments were conducted in accordance with the recommendations of the European Community (2010 / 63 / EU) and the French National Committee (Law 2013-118) for the care and use of laboratory animals. The experimental protocol was approved by a local ethics committee. Animal experimentation (Ethics Committee for Animal Experimentation, EC No. 44) and by the French Ministry of Agriculture (APAFIS #48237-2024032011265575 v2).

[0120] The mice's abdomens were then shaved. The ultrasound probe (UF29X) was positioned to image the liver using a VEVO F2 ultrasound scanner. Approximately 2 x 10 7SonoVue microbubbles, 2P15 microbubbles, and 2P20 microbubbles, respectively, were injected intravenously, and contrast-enhanced ultrasound imaging was performed on the liver for 10 minutes. Ultrasound images over time (at T0, T=30 seconds, T=2 minutes, and T=5 minutes) are shown in Figure 10 (A, B, C).

[0121] For this experiment, a region of interest (ROI) was drawn in the liver, avoiding shadowed areas, to measure hepatic luminosity over time (intensity-time curve, shown in Figure 11), and fitted to extract pharmacokinetic parameters. Microbubble perfusion in the mouse liver was modeled using a first-order pharmacokinetic model [5,6]. The vascular system was considered a single compartment, with microbubble uptake and clearance assumed to follow first-order kinetics. Consequently, the contrast signal C was fitted using MATLAB software according to the following model: in which Co represents the initial contrast, and ki and k2 represent the first-order velocity constants characterizing respectively the influx and efflux of microbubbles from the vascular compartment.

[0122] The mean transit time (MTT), reflecting the average duration of microbubbles circulating in the liver, was calculated as the ratio of the area under the intensity-time curve (curve in Figure 11) to the maximum signal intensity. The microbubbles' half-life was also determined as the time it takes for the signal intensity to drop to 50% of its maximum value. These two parameters (MTT and half-life) are illustrated in the diagram in Figure 12.

[0123] Thus, in a completely innovative and advantageous way, the inventors have demonstrated that the microbubbles 2 P15 and P20 according to the invention exhibit a time of half-life and MTT five times greater than those of commercial SonoVue microbubbles: 2 P15 microbubbles and 2 P20 microbubbles both have a half-life and MTT of about 300 seconds, while Sonovue microbubbles have a half-life and MTT of only about 50 seconds.

[0124] 5.e. In vivo validation of microbubbles 2 in the brain

[0125] The experiments were performed on a female Sprague Dawley rat (375 g, 10 weeks old) and aimed to evaluate the circulation of microbubbles in vivo in the brain using localized ultrasonic microscopy (ULM). The experiments were conducted according to the European Community guidelines (2010 / 63 / EU) and validated by the APAFIS protocol #21064, approved by the French National Committee.

[0126] The spleen underwent surgery to replace part of the skull with an ultrasound-permeable prosthesis, under ketamine-xylazine anesthesia (80 / 12 mg / kg) and buprenorphine analgesia (0.05 mg / kg). This surgery involved incising the skin above the skull, then drilling through the parietal and frontal bones from Bregma -7 to Bregma +3 mm to expose an area of ​​approximately 1 cm 2The bones were carefully removed so as not to damage the dura mater, and the exposed brain was covered with saline solution and then with a polymethylpentene plastic sheet (PMP, 125 µm thick, Goodfellow, Huntington UK), fixed using an acrylic resin (GC Unifast Trad) ([7]).

[0127] After one week of recovery, the spleen was anesthetized with 2–2.5% isoflurane in a 50 / 50 (v / v) oxygen / air mixture, and a catheter was placed in the caudal vein for microbubbles 2 administration. The animal was positioned in a stereotaxic frame with a heating pad maintaining body temperature at 36.5°C. Ultrasound gel was applied between the prosthesis and the ultrasound probe. Three doses of microbubbles 2 P15 (each dose containing 4 x 10⁻¹⁰) were administered. 7Microbubbles (approximately 50 pL) from three different vials were administered, with an interval of at least 10 minutes between each administration. Ultralight imaging began immediately after each injection and continued for at least 10 minutes. At the end of the experiment, the spleen was removed from the stereotaxic frame and kept warm until it awoke before being returned to its cage.

[0128] Two days later, the same preparation was repeated for the administration of Sonovue microbubbles (2 doses of 4x10 7 microbubbles, approximately 100 pL each).

[0129] Ultrasound imaging was performed using a linear probe composed of 256 transducers operating at a center frequency of 20 MHz (UHF 29x, Vevo), connected to an ultrafast ultrasound scanner (Vevo 2, Fujifilm Visualsonics, Toronto, Canada). Only 128 central transducers were used for acquisitions. A 2D ULM image was obtained from the accumulation of 42 acquisitions, spaced approximately 12 seconds apart, for a total duration of approximately 9 minutes. Each acquisition consisted of 1024 images taken at a rate of 1 kHz, corresponding to a duration of 1.024 seconds. Each image resulted from the combination of three plane waves emitted at inclination angles of -5°, 0°, and +5°. Post-processing was performed using MATLAB software.

[0130] ULM processing was performed as described by Heiles [8]. The 2D ULM images were processed using an open-source tool, publicly available online (https: / / github.com / AChavignon / PALA). Microbubbles were detected and localized using a 2D radial symmetry algorithm. Figure 13 illustrates the cerebral microvasculature of the spleen, obtained by ULM.

[0131] The Doppler signal, corresponding to the echogenic particles in motion, was calculated for each 2D ultrasound image via processing on the MATLAB software, as previously described in prior art work [8]. The evolution of the Doppler signal (in dB), for respectively microbubbles 2 according to the invention and commercial SonoVue microbubbles, was plotted over time, and is shown in Figure 14.

[0132] The mean transit time (MTT) and the half-life of microbubbles in the brain of the spleen were calculated by applying the same models as those used for in vivo measurements in the liver, in Example 5.d. These two parameters are illustrated in the diagram in Figure 15.

[0133] Thus, similar to the efficacy results of microbubbles 2 according to the invention in the liver, the inventors have demonstrated, unexpectedly and advantageously, that microbubbles 2 P15 have a half-life and MTT significantly higher than those of commercial SonoVue microbubbles: the microbubbles 2 P15 exhibit a half-life and MTT of approximately 100 to 150 seconds, while Sonovue microbubbles exhibit a half-life and MTT of approximately 30 to 50 seconds.

[0134] Therefore, in view of all these results, it is evident that the addition of fluorinated polymers 2 in the composition of the microbubbles according to the invention also contributes, like the microbeads 1 incorporating fluorinated polymers 1, to improving their stability in the blood, while preserving their ability to permeabilize the BBB.

[0135] In particular, after injection in animals, it was possible to open the BBB using these microbubbles formulated from fluorinated polymer 2 for rarefaction pressures greater than 300 kPa, as demonstrated in Figure 16.

[0136] These results also demonstrate the sensitivity of microbubbles 2 according to the invention to ultrasound, and therefore their potential for imaging and therapy.

[0137] List of references: [1] Ambre Dauba, Anthony Delalande, Hermes AS Kamimura, Allegra Conti, Benoit Larrat, Nicolas Tsapis, Anthony Novell. Recent advances on ultrasound contrast agents for blood-brain barrier opening with focused ultrasound. Pharmaceutics. 2020. doi: 10.3390 / pharmaceutics12111125. [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 Carneiro, Laurence Moine, Barbara Tessier, Valerie Nicolas, Orlando D.H. dos Santos, and Elias Fattal. 2019. Pyrazinoic Acid-Poly(Malic Acid) Biodegradable Nanoconjugate for Efficient Intracellular Delivery. Precision Nanomedicine 2 (3): 303-17. [5] S. Garg, A.A. Thomas, M.A. Borden, The effect of lipid monolayer in-plane rigidity on in vivo microbubble circulation persistence, Biomaterials 34 (2013) 6862-6870. https: / / doi.Org / 10.1016 / j.biomaterials.2013.05.053. [6] C.C. Chen, S.R. Sirsi, S. Homma, M.A. Borden, Effect of Surface Architecture on In Vivo Ultrasound Contrast Persistence of Targeted Size-Selected Microbubbles, Ultrasound in Medicine & Biology 38 (2012) 492-503. https: / / doi.Org / 10.1016 / j.ultrasmedbio.2011.12.007. [7] L.-A. Sieu, A. Bergel, E. Tiran, T. Deffieux, M. Pernot, J.-L. Gennisson, M. Tanter, I. Cohen, EEG and functional ultrasound imaging in mobile rats, Nat Methods 12 (2015) 831-834. https: / / doi.org / 10.1038 / nmeth.3506. [8] B. Heiles, A. Chavignon, V. Hingot, P. Lopez, E. Teston, O. Couture, Performance benchmarking of microbubble-localization algorithms for ultrasound localization microscopy, Nat. Biomed. Eng 6 (2022) 605-616. https: / / doi.org / 10.1038 / s41551- 021-00824-8.

Claims

Demands

1. 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.

2. Microbubble according to claim 1, further comprising an additional gas selected from air, oxygen, nitrogen, nitric oxide and mixtures thereof.

3. 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.

4. 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 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 R 1 represents independently H or CH3, each R 2 independently represents either a linear or branched C2-C20 fluorinated alkyl group, optionally bearing a fluorinated C5-C7 aryl group, or a C5-C7 fluorinated aryl group; R 3 and R 4 independently represent H or CH3; and X represents a halogenated group, preferably chosen from Br, Cl and I; [Chem 8] Formula II wherein 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.

5. Microbubble according to any one of the preceding claims, wherein the polymer is of formula I, and the group R 2is chosen from 2,2,2-trifluoroethyl, 2,2,3,3,3-pentafluoropropyl, 2, 2, 3, 3, 4,4,4- heptaafluorobutyl, 1 H,1 H,2H,2H-tridecafluoro-n-octyl, 1 H, 1 H,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 R group 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, 1 H, 1 H-perfluorododecan-1-ol, 1 H, 1 H-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, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000, 1,2-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, 1,2-distearoyl-sn-glycero-3-phosphate, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium sait), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine, 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol, egg phosphatidylcholine, soy phosphatidylcholine and mixtures thereof.

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. 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.

13. Microbubble for its use according to claim 1 to 8 or composition according to claim 9, for its use in ultrasound therapy in the treatment of pathologies at the organ levels, preferably in the treatment of thrombus, or cancers of the prostate, breast, pancreas, bladder, melanomas, or colon, and optionally in combination with a complementary therapeutic approach, preferably chosen from chemotherapy, immunotherapy, targeted therapy, gene therapy.

14. Microbubble according to any one of claims 1 to 8 or composition according to claim 9, for its use as an ultrasonic contrast agent in molecular imaging.

15. Polymer of formula I: [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 R 1 independently represents H or CH3, each R 2 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 R 3 and R 4 independently represent H or CH3; and X represents a halogenated group, preferably chosen from Br, Cl and I.

16. Polymer of formula II: [Chem 10] 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.

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