Lipid Microbubbles for Targeted Delivery of Active Ingredients

Abstract

The present invention relates to optimized microbubbles that can be used in the prevention and treatment of diseases or for marking, in particular, lipid microbubbles. The present invention relates in particular to lipid microbubbles comprising at least one cationic compound selected from lipophosphoramidates, histidyl polyethyleneimine, and any mixture thereof. The microbubbles preferably further comprise at least one agent selected from therapeutic agents, targeting agents, marking agents, and any combination thereof. The present invention further relates to a method for producing such microbubbles. The present invention further relates to a composition, in particular a pharmaceutical composition, and a kit comprising at least one of these microbubbles. The present invention further relates to the use of these microbubbles, compositions, kits as pharmaceuticals or marking agents.

Description

Technical Field

[0001] The present invention belongs to the field of targeted delivery of active ingredients for treatment and / or labeling, and the field of microbubbles, particularly functionalized microbubbles.

[0002] The present invention particularly relates to optimized microbubbles, particularly lipid microbubbles, which can be used for disease prevention, treatment, and marking.

[0003] The present invention particularly relates to lipid microbubbles containing at least one cationic compound selected from lipophosphoramidate, histidyl polyethyleneimine, and any mixture thereof. The microbubbles preferably further contain at least one agent selected from therapeutic agents, targeting agents, marking agents, and any combination thereof.

[0004] The present invention further relates to a method for producing these microbubbles.

[0005] The present invention further relates to a composition, particularly a pharmaceutical composition, and a kit containing at least one of these microbubbles. The present invention further relates to the use of these microbubbles, compositions, and kits as pharmaceuticals or marking agents.

Background Art

[0006] Microbubbles (MBs), initially developed for diagnostic purposes, are also used for therapeutic purposes. It can be used as a vector for. In fact, thanks to the wide variety of molecules that can form envelopes, it is possible to encapsulate drugs or complex nucleic acids. Generally, the MBs used as vectors have a functional lipid envelope, and it is possible to embed therapeutic molecules between the lipids or dissolve them in the oil droplets within the envelope. Furthermore, using cationic lipids, nucleic acids can be complexed by electrostatic interactions. Other lipids can also be used to attach nanoparticles or antibodies using streptavidin-biotin interactions or can be functionalized for chemical click applications.

[0007] Several studies have led to the production of MBs as vectors for the molecule of interest. Zhu et al., Scientific Reports, 2016, described MBs loaded with a chemotherapeutic agent (paclitaxel). Fan et al., Biomaterials, 2013, reported the development of microbubbles loaded with an antitumor agent (1,3-bis(2-chloroethyl)-1-nitrosourea or BCNU). However, the drugs that form complexes with these MBs are limited to anticancer chemical compounds. In particular, the possibility of delivering nucleic acids using these microbubbles has not been demonstrated. However, an increasing number of new therapies, particularly anticancer therapies, are using nucleic acids. These molecules provide a flexibility unmatched by compounds and enable, for example, personalized therapies.

[0008] Delalande et al., Bioscience Reports, 2017, described cationic MBs complexed with plasmid DNA. However, the nucleic acid delivery efficiency and target specificity of these MBs remain limited. These aspects are particularly important in the treatment of brain pathologies. Incidentally, one of the major obstacles to treatment relates to crossing the Blood-Brain Barrier (BBB). For these reasons, for the central nervous system The molecules to be delivered have traditionally been selected for their ability to passively cross the BBB. This means low nucleic acid delivery efficiency and substantially no targeting, which can cause many harmful side effects.

[0009] Therefore, it is essential to develop new, effective, and specific methods for delivering therapeutic agents, particularly nucleic acids, which can pass through the BBB without degradation, to the brain.

[0010] In this regard, microbubbles are a promising system. For example, Fan et al. (2016) produced folate-containing MBs capable of complexing nucleic acids for targeted brain transfection (transduction).

[0011] These cationic microbubbles use trimethylammonium functional groups as cationic charges for complex formation based on the use of DSTAP lipids or DPTAP lipids. The main drawback of these formulations is that they are not sufficiently effective for use in gene therapy protocols. Moreover, these formulations have been shown to be toxic.

[0012] Therefore, there is still a need to develop microbubbles capable of efficiently and specifically delivering therapeutic agents such as nucleic acids to specific organs or even cells.

[0013] The present invention makes it possible to meet this need by describing microbubbles based on a novel cationic formulation having the following advantages: - better nucleic acid compaction (embryonic cell compaction), and - lower toxicity, and - use of an endosomal escape system, the "proton sponge effect", to increase transfection and avoid the lysosomal pathway, and - improved transfection efficiency.

[0014] Thus, the microbubbles developed by the inventors of the present invention have the ability to stably transport drugs, particularly nucleic acids, into the bloodstream, actively cross the blood-brain barrier, and deliver drugs in a targeted manner, particularly to a target antigen. The technology developed herein enables the temporary opening of the BBB and the efficient delivery of nucleic acid-based active ingredients across it. It further enables the permeabilization of blood vessels so that the molecule of interest can be delivered.

Summary of the Invention

[0015] In the context of the present invention, the inventors have developed innovative microbubbles, particularly innovative lipid microbubbles, capable of stably transporting drugs, particularly nucleic acids, into the bloodstream, actively crossing the blood-brain barrier (BBB), and delivering drugs in a targeted manner, particularly to a target antigen.

[0016] In particular, the inventors have surprisingly shown that the lipid microbubbles thus developed have significantly improved stability, unlike the microbubbles described in the prior art. Notably, the data further reveals that these optimized microbubbles are capable of delivering the drug of interest, particularly nucleic acids, more efficiently than the microbubbles described in the prior art. In particular, these microbubbles are capable of actively crossing blood vessels as well as the BBB or the tumor microenvironment. The inventors have further demonstrated that the local application of ultrasound enables these optimized microbubbles to be very precisely targeted to the area to be treated, including very difficult-to-access areas such as the central nervous system, blood vessels, and tumor microenvironment. Thus, these data reveal the therapeutic potential of these lipid microbubbles for treating a wide range of lesions, including central nervous system lesions, vascular lesions, tumors, and cancers, in a targeted manner. The data further shows that these optimized microbubbles are detection and imaging tools.

[0017] Accordingly, the present invention provides both a powerful and broad-spectrum treatment method for medical conditions, as well as an efficient and reliable diagnostic method.

DETAILED DESCRIPTION OF THE INVENTION

[0018] (SUMMARY OF THE INVENTION) The present invention relates to lipid microbubbles that can be used, in particular, in both the prevention and treatment of medical conditions, as well as in detection and imaging, especially in medicine.

[0019] The present invention relates in particular to lipid microbubbles comprising at least one cationic compound selected from lipophosphoramidate, histidyl polyethyleneimine, and any mixture thereof.

[0020] The present invention further relates to a pharmaceutical composition comprising at least one microbubble as defined above and optionally a pharmaceutically acceptable excipient, wherein the concentration of microbubbles in the composition is preferably 10 6 ~10 14 microbubbles / ml, more preferably 10 7 ~10 13 microbubbles / ml, more preferably 10 8 ~10 12 microbubbles / ml, more preferably 10 9 ~10 11 microbubbles / ml, and more preferably the concentration of microbubbles in the composition is about 10 10 microbubbles / ml, and relates to a pharmaceutical composition.

[0021] The present invention further relates to a kit comprising a) at least one microbubble as defined above in a first container, and b) at least one therapeutic agent in a second container, and c) optionally, at least one targeting agent in a third container, and d) optionally, at least one marking agent in a fourth container, and e) Optionally, instructions for preparation and / or use, The therapeutic agent and / or the targeting agent and / or the marking agent are preferably 1) a nucleic acid, 2) a lipophilic active ingredient, 3) a chemotherapeutic agent such as a cytotoxic agent and / or a cytostatic agent, 4) an antibody, 5) a protein, 6) an antigen, 7) a toxin, 8) a receptor, 9) an enzyme, 10) a hormone, 11) a ligand, 12) a viral vector, 13) a nanoparticle, preferably a nanoparticle containing at least one agent selected from a therapeutic agent, a targeting agent, a marking agent, and any combination thereof, 14) a derivative of any of 1) to 13), preferably a functional derivative of any of these, 15) a fragment of any of 1) to 14), preferably a functional fragment of any of these, 16) any combination of 1) to 15), relates to a kit. More preferably, it is from a nucleic acid, a lipophilic active ingredient, a chemotherapeutic agent, an antibody, an antibody derivative, a functional fragment of an antibody or its derivative, a protein, a protein fragment, a nanoparticle, and any combination thereof.

[0022] According to another aspect, the present invention relates to the microbubbles, pharmaceutical compositions, or kits defined above for use as a medicament or as a marking agent.

[0023] According to another aspect, the present invention relates to a method for producing at least one microbubble defined above, the method comprising the following steps: a) a step of a cationic compound selected from lipophosphoramidate, histidyl polyethyleneimine, and any mixture thereof, ethanol, and optionally at least one agent selected from a therapeutic agent, a targeting agent, a marking agent, and any combination thereof, in a mixture in a container b) a step of evaporating the mixture obtained in step a) to obtain a lipid film, and rehydrating the lipid film to form a liposome suspension (this entire step can further be performed using microfluidics); c) a step of lyophilizing the liposome suspension obtained in step b); d) replacing the air contained in the container containing the lyophilized product obtained in step c) with a biocompatible gas preferably selected from perfluorobutane (C4F10), perfluoropropane (C3F8), dinitrogen (N2), sulfur hexafluoride (SF6), nitrogen oxide (NO), hydrogen, dioxygen, helium, xenon, argon, nitrous oxide (N2O), and any mixture thereof; e) a step of rehydrating the lyophilized product from step d) to obtain a solution; f) a step of stirring the solution obtained in step d) to form microbubbles; g) optionally, a step of functionalizing the microbubbles with at least one agent selected from a therapeutic agent, a targeting agent, a marking agent, and any combination thereof, and / or a step of functionalizing the microbubbles by adding at least one functional group enabling binding to at least one agent selected from a therapeutic agent, a targeting agent, a marking agent, and any combination thereof.

[0024] Definitions The term "at least one" is considered herein to be synonymous with the term "one or more". ​

[0025] As used herein, the term "microbubble" refers to a bubble having a diameter of about 1 micrometer to several tens of micrometers (up to about 100 micrometers), typically in the range of about 1 micrometer to about 10 micrometers. Microbubbles include an envelope surrounding a filling material. The envelope may consist of lipids, proteins, sugars, ionic compounds, or mixtures thereof. In the case of an envelope made of or essentially made of lipids, the inventors describe lipid microbubbles.

[0026] Compounds that can be used to form the microbubble envelope according to the present invention include, in particular, the following: - Cationic compounds such as lipophosphoramidates, histidylated polyethyleneimines, and mixtures thereof, and / or - Lipids, in particular, dimyristoyl-glycero-phosphocholine, distearoyl-glycero-phosphocholine, dimyristoyl-glycero-phosphoethanolamine-polyethylene glycol, distearoyl-glycero-phosphoethanolamine-polyethylene glycol 2000, distearoyl-glycero-phosphoethanolamine-[biotinyl(polyethylene glycol)], cholesterol, β-sitosterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), 1-oleoyl-2-[6-[(7-nitro-2-1,3- benzoxadiazol-4-yl)amino]hexanoyl]-3-trimethylammonium propane (1-oleoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-3-trimethylammonium propane, DOTAP), 1,2-dimyristoyl-rac-glycero -3-Methoxypolyethylene glycol-2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, DMG-PEG2000), Clickable Lipid DB A lipid selected from the group consisting of CO, tetrazine, methyltetrazine, NHS(DSPE-PEG2000-X), and any combination thereof; - and any combination thereof.

[0027] The filling material of the microbubbles can be any type of gas. The filling gas is advantageously biocompatible (i.e. well tolerated by the living body). In particular, the filling gas can be selected from perfluorobutane (C4F10), perfluoropropane (C3F8), dinitrogen (N2), sulfur hexafluoride (SF6), nitrogen oxide (NO), hydrogen, dioxygen, helium, xenon, argon, nitrous oxide (N2O), and any mixture thereof.

[0028] According to the present inventors, the term "lipid microbubble" refers to the It is meant that the envelope is essentially made up of lipids or is made up of lipids. The term "envelope essentially made up of lipids" is used herein to mean microbubbles having an envelope made up of 55% or more lipids, preferably 60% or more lipids. By this is meant an envelope comprising lipids, preferably 70% or more lipids, preferably 80% or more lipids, preferably 90% or more lipids, preferably 91% or more lipids, preferably 92% or more lipids, preferably 93% or more lipids, preferably 94% or more lipids, preferably 95% or more lipids, preferably 96% or more lipids, preferably 97% or more lipids, preferably 98% or more lipids, even more preferably 99% or more lipids, the percentage being expressed as lipid mass relative to the total mass of the envelope.

[0029] Advantageously, the lipid microbubbles comprise at least one cationic compound, in particular a lipid bound to a cationic lipid or at least one cationic polymer.

[0030] The envelope of the lipid microbubbles may comprise a lipid bound to a cationic lipid or at least one cationic polymer, or may consist essentially of a lipid bound to a cationic lipid or at least one cationic polymer, or may consist of a lipid bound to a cationic lipid and / or at least one cationic polymer.

[0031] Advantageously, the envelope comprises a lipid bound to a cationic lipid and / or at least one cationic polymer, and a fusogenic lipid.

[0032] According to an embodiment, the lipid envelope of the microbubbles comprises 2 to 50% cationic lipid, preferably at least 2% cationic lipid, preferably at least 10% cationic lipid, preferably at least 20% cationic lipid, preferably at least 30% cationic lipid, preferably at least 40% cationic lipid, preferably 50% cationic lipid, the percentage being expressed as the number of moles of cationic lipid relative to the number of moles of total lipid constituting the envelope.

[0033] According to the inventors, "cationic compound" means a compound containing at least one cation and having a net positive charge in solution. Examples of cationic compounds (cationic lipids) that can be used to form the microbubble envelope include, but are not limited to, lipophosphoramidates, histidylated polyethyleneimines, and any mixtures thereof. According to the inventors, "cationic lipid" means a lipid containing at least one cation and having a net positive charge in solution.

[0034] According to the inventors, "lipophosphoramidate" means a bio-inspired amphiphilic phospholipid having a cationic charge in its polar head. It means a bio-inspired amphiphilic phospholipid having a cationic charge in its polar head.

[0035] Examples of lipophosphoramidates include dimyristoyl phosphoramidate (such as dimyristoyl bromide phosphoramidate and dimyristoyl histamine phosphoramidate), dioleoyl phosphoramidate (such as dioleoyl methylimidazolium phosphoramidate), dipalmitoyl phosphoramidate, and distearoyl phosphoramidate.

[0036] "Polyethylenimine" or "polyethyleneimine" or "PEI (polyethyleneimine, polyethyleneimine)" or "polyaziridine" means an organic polymer with the chemical formula H[CH2-CH2-NH-] n H. "Histidylated polyethylenimine" means a polyethyleneimine containing at least one histidyl group. Advantageously, the histidylated polyethyleneimine used to form microbubbles is bound to a fatty acid. Examples of fatty acids that can be bound to histidylated polyethyleneimine include stearic acid, myristic acid, palmitic acid, oleic acid, and any combination thereof.

[0037] "Compound / agent exposed to the microbubble surface" means a compound or agent that is conjugated / coupled / bound to the outer surface of the microbubble (e.g., conjugated / coupled / bound to the microbubble envelope) and is in contact with the medium outside the microbubble. It means a compound or agent that is conjugated / coupled / bound to the outer surface of the microbubble (e.g., conjugated / coupled / bound to the microbubble envelope) and is in contact with the medium outside the microbubble.

[0038] "Compound / agent embedded in the lipid envelope of the microbubble" means a compound or agent that is partially or fully integrated / incorporated into the layer of the compound (especially lipid) forming the microbubble envelope. Thus, a compound or agent that is fully integrated / incorporated into the envelope is in contact only with the compound (especially lipid) forming the microbubble envelope: in this case, the compound or agent is not in contact with either the external environment or the internal environment outside the microbubble. On the other hand, a compound or agent that is partially incorporated / taken up into the envelope can be in contact with the external medium of the microbubble, or the internal medium of the microbubble, or both the external and internal media of the microbubble ("through-compound").

[0039] "Compound / agent incorporated inside the microbubble" means a compound or agent located in the internal medium of the microbubble (i.e., in the medium / cavity formed by the microbubble envelope). This compound or agent may be in contact with the inner surface of the microbubble (e.g., the inner surface of the microbubble envelope). In particular, it may be conjugated / coupled / bound to the inner surface of the microbubble (e.g., the inner surface of the microbubble envelope).

[0040] "Therapeutic agent" or "therapeutic compound" means It refers to any drug, compound, or molecule presented as having curative or preventive properties with respect to the medical condition or disease of a human or animal body. Thus, a therapeutic agent or compound includes any drug or compound that can be used or administered to a human or animal for the purpose of establishing a medical diagnosis or for restoring, correcting, or modifying their physiological functions by exerting pharmacological, immunological, and / or metabolic effects. Thus, a therapeutic agent may be a drug.

[0041] The therapeutic agent or compound can be of any nature or type and is not dependent on its origin. The therapeutic agent may be chemically synthesized, naturally occurring, recombinantly produced (and optionally purified), or synthetically designed and produced. In particular, it may be a small molecule, nucleic acid, peptide (including post-translationally modified peptides), polypeptide (including post-translationally modified polypeptides), protein (including post-translationally modified proteins), chemical compound, cancer chemotherapeutic agent (such as a cytostatic agent or a cytological agent), antibody, toxin, antigen, hormone, enzyme, ligand, receptor, antiviral compound, antibiotic compound, antifungal compound, antibacterial compound, nanoparticle, or any fragment thereof (preferably a functionally active fragment), or any derivative thereof (preferably a functionally active derivative), for example, inter alia, a peptidomimetic, an antibody mimetic, a chemical derivative. The therapeutic agent or compound may include, for example, nucleic acids, lipophilic active substances, chemotherapeutic agents (such as cytotoxic agents and / or cytostatic agents), antibodies, antibody derivatives, functional fragments of antibodies or antibody derivatives, proteins (including post-translationally modified proteins), protein fragments (such as peptides, antigens, epitopes, functional protein domains, and any combination thereof (including post-translationally modified protein fragments)), nanoparticles, etc., or may essentially consist of them, or may consist of them.

[0042] The "targeting agent" or "targeting compound" means , means any agent, compound, or molecule that is presented as being capable of recognizing and / or binding, preferably in a specific manner, another molecule (well-known as the "target molecule"). Thus, these terms further include "binding agent" or "binding compound". The term "binding agent" or "binding compound" refers to any agent or compound or molecule that is capable of binding to another molecule (well-known as the "target molecule"), and the binding is preferably specific binding.

[0043] The targeting / binding agent recognizes a defined site, domain, region, pocket, epitope, conformation, chemical group, or any combination thereof of the target molecule. The targeting / binding agent can be of any nature or type and is not dependent on its origin. The targeting / binding agent can be chemically synthesized, can occur naturally, can be recombinantly produced (and purified if necessary), or can be synthetically designed and produced. In particular, it can be a small molecule, nucleic acid, peptide (including post-translationally modified peptides), polypeptide (including post-translationally modified polypeptides), protein (including post-translationally modified proteins), compound, antibody, toxin, antigen, epitope, hormone, enzyme, ligand, receptor, nanoparticle, or any fragment thereof (preferably a functionally active fragment), or any derivative thereof (preferably a functionally active derivative), such as, inter alia, peptidomimetics, mimetic antibodies, chemical derivatives, etc. The targeting / binding agent can include, for example, nucleic acids, lipophilic active substances, chemotherapeutic agents (such as cytotoxic agents and / or cell growth inhibitors), antibodies, antibody derivatives, functional fragments of antibodies or antibody derivatives, proteins (including post-translationally modified proteins), protein fragments (such as peptides, antigens, epitopes, functional protein domains, and any combination thereof (including post-translationally modified protein fragments), nanoparticles, etc., or can consist essentially of them, or can consist of them.

[0044] The target molecule can be of any nature or type, regardless of its origin. In particular, the target molecule can be a pathogen (such as a virus, bacterium, parasite, etc.) or a fragment thereof (e.g., a nucleic acid, a protein, a polypeptide, a peptide, an epitope, a lipid, a sugar, etc.).

[0045] A "marking agent" or "marker" is a molecule that is attached to another molecule ( Marking refers to any agent or compound, or molecule, that can be marked (preferably in a specific way) and presented as detectable by any means. Means for detecting marking agents are well known to those skilled in the art, and the skilled person is entirely capable of selecting the appropriate technique depending on the marking agent used. Means for detecting marking agents include, but are not limited to, optical detection techniques (e.g., fluorescence, absorbance, diffraction, light scattering, interferometry, reflectometry, ellipsometry, Surface Plasmon Resonance (SPR), spectroscopy, techniques using magnetic particles, etc.), mechanical detection techniques (e.g., fluorescence, absorbance, diffraction, light scattering, interferometry, reflectometry, ellipsometry, surface plasmon resonance (SPR), spectroscopy, techniques using magnetic particles, etc.), mechanical detection techniques (e.g., fluorescence, absorbance, diffraction, light scattering, interferometry, interferometry, reflectometry, ellipsometry, surface plasmon resonance (SPR), spectroscopy, techniques using magnetic particles, etc.), mechanical detection techniques (e.g., fluorescence, absorbance, diffraction, light scattering, interferometry, interferometry, reflectometry, ellipsometry, interferometry, techniques using magnetic particles, etc.), mechanical detection techniques (e.g., fluorescence, absorbance, diffraction, light scattering, interferometry ... Mechanical detection techniques (eg optical detection of the marking agent), electrical detection techniques (eg techniques using electrodes, electrical sensors, etc.) Thus, the marking agent may be a contrast agent.

[0046] The targeting / linking agent can be of any nature or type and is not dependent on its origin. The targeting / linking agent can be chemically synthesized, can occur naturally, can be recombinantly produced (and purified if necessary), or can be synthetically designed and produced. In particular, it can be a small molecule, nucleic acid, peptide (including post-translationally modified peptides), polypeptide (including post-translationally modified polypeptides), protein (including post-translationally modified proteins), compound, antibody, toxin, antigen, epitope, hormone, enzyme, ligand, receptor, nanoparticle, or any fragment thereof (preferably a functionally active fragment), or any derivative thereof (preferably a functionally active derivative), such as, among others, peptidomimetics, mimetic antibodies, chemical derivatives, etc. The labeling agent can include, for example, nucleic acids, lipophilic active substances, chemotherapeutic agents (e.g., cytotoxic agents and / or cell growth inhibitors), antibodies, antibody derivatives, functional fragments of antibodies or antibody derivatives, proteins (including post-translationally modified proteins), protein fragments (e.g., peptides, antigens, epitopes, functional protein domains, and any combination thereof (including post-translationally modified protein fragments)), nanoparticles, etc., or can consist essentially of them, or can consist of them.

[0047] The labeling agent can include, for example, fluorophores (fluorescent dye molecules) (e.g., fluorescein or luciferase), fluorescent proteins / polypeptides / peptides (e.g., GFP and its variants, RFP, CFP, YFP, etc.), radioisotopes (particularly suitable for scintigraphy, e.g., 99m Tc), antibody-recognizable labels (e.g., c-Myc protein or polyhistidine label), affinity labels (e.g., biotin, streptavidin, etc.), enzymes (e.g., horseradish peroxidase), contrast agents, peptide tags, etc., or can consist essentially of them.

[0048] The term "derivative" is generally used to denote a component or species (protein, antibody, protein fragment, polypeptide, polynucleotide, oligonucleotide, nucleoside, nucleotide, vector, virus, etc.) having one or more modifications as compared to a reference component (e.g., the initially identified wild-type component found in nature, i.e., the corresponding "original" component known as the original component). A derivative may in particular be a fragment, part, variant, mutant, (e.g., especially one produced in vitro) synthetic variant, mimetic, or a combination thereof of the original component or species. The terms "variant" or "mutant" may be used interchangeably to generally refer to a component or species (protein, antibody, protein fragment, polypeptide, polynucleotide, oligonucleotide, nucleoside, nucleotide, vector, virus, etc.) presenting one or more modifications with respect to a reference component ( e.g., the initially identified wild-type component found in nature, i.e., the corresponding "original" component referred to as the original component). Nucleotide or nucleoside variants may have modified bases and / or modified sugars and / or modified linkages. For polypeptide, polynucleotide, and antibody variants, any modification including substitution, insertion, deletion, and any combination thereof of one or more nucleotide / amino acid residues may be envisaged. Variants may be of natural or artificial origin (e.g., mutated and / or engineered).

[0049] When several mutations are envisaged, they may be in consecutive and / or non-consecutive residues May be relevant. Variants that maintain a sequence identity of at least 80% with a reference component (e.g., each corresponding "original" protein, corresponding "original" protein fragment, corresponding "original" polynucleotide) (e.g., protein variants, peptide variants, antibody variants, virus variants, etc.) are preferred. By way of example, "at least 80% identity" means 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, at least 80% identity also encompasses 100% identity.

[0050] According to the inventors, a "functional fragment" or "functionally active fragment" means any fragment of a molecule, agent, species, or compound that exhibits at least one of the original functions of the molecule, agent, species, or compound from which the fragment is derived. Preferably, the functional fragment performs the function with an efficiency equal to at least 30%, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85% of the peptide or protein, but preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, preferably at least 100% of the effectiveness of the molecule, agent, species, compound from which the fragment is derived.

[0051] "Identity" or "sequence identity" refers to two poly It means an exact sequence match between peptides or amino acids, or between two nucleic acid molecules or oligonucleotides. The percentage of identity referred to in the present invention is determined after the best optimal global alignment (global alignment) of the sequences to be compared, and thus it may include one or more additions, deletions, truncations and / or substitutions. This percentage of identity can be calculated by any sequence analysis method well-known to those skilled in the art. The percentage of identity is determined over their entire length as a whole after the global alignment of the sequences to be compared. In addition to manual methods, global sequence alignment can further be determined using the algorithm of Needleman and Wunsch (1970).

[0052] In particular, for nucleotide sequences, sequence comparison can be carried out using any software well-known to those skilled in the art, such as Needle software. The parameters used may include "Gap Open" equal to 10.0, "Gap Extend" equal to 0.5 and the EDNAFULL matrix (EMBOSS version of NCBI NUC4.4).

[0053] Amino acid sequences can be compared using any software program well-known to those skilled in the art, such as Needle. The parameters used may include "Gap Open" equal to 10.0, "Gap Extend" equal to 0.5 and the BLOSUM62 matrix.

[0054] By way of example, "at least 80% sequence identity" as used herein particularly represents 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0055] The terms "polynucleotide", "nucleic acid molecule", and "nucleic acid" are used interchangeably herein and refer to polymeric macromolecules or oligomers composed of nucleotide monomers (preferably at least 5 nucleotide monomers, also known as nucleotide residues). Nucleotide monomers are composed of a nucleobase, a pentose sugar (e.g., ribose or 2'-deoxyribose, but not limited thereto), and one to three phosphate groups. Typically, polynucleotides are formed by phosphodiester bonds between individual nucleotide monomers. Examples of nucleic acid molecules include ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixtures thereof, such as RNA -DNA hybrids (mixed polyribo-polydeoxyribonucleotides), but are not limited thereto. These terms include single-stranded or double-stranded, linear or circular, natural or synthetic, their unmodified or modified variants (e.g., genetically modified polynucleotides, optimized polynucleotides), sense polynucleotides or antisense polynucleotides, chimeric mixtures (e.g., RNA-DNA hybrids). Furthermore, polynucleotides may contain nucleotides of non-natural origin and may be interrupted by non-nucleotide constituents. Examples of DNA nucleic acids include complementary DNA (cDNA), genomic DNA, plasmid DNA, DNA vectors, Viral DNA (e.g., viral genome, viral vector), oligonucleotide, probe, primer, satellite DNA, microsatellite DNA, coding DNA, non-coding DNA, antisense DNA, and any mixture thereof, although not limited thereto. Exemplary RNA nucleic acids include messenger RNA (mRNA), precursor messenger RNA (pre-mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), RNA vector, viral RNA, guide RNA (gRNA), antisense RNA, coding RNA, non-coding RNA, antisense RNA, satellite RNA, small cytoplasmic RNA, small nuclear RNA, etc., although not limited thereto. The polynucleotides described herein can be synthesized, for example, using an automated DNA synthesizer (such as those commercially available from Biosearch, Applied Biosystems, etc.) by standard methods well known in the art, or obtained from natural sources (e.g., genome, cDNA, etc.) or artificial sources (e.g., commercially available libraries, plasmids, etc.) using molecular biology techniques well known in the art (such as cloning, PCR, etc.). Nucleic acids can be chemically synthesized, for example, according to the phosphotriester method (see, e.g., Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584). pin RNA (shRNA), microRNA (miRN A), RNA vector, viral RNA, guide RNA (gRNA), ant isense RNA, coding RNA, non-coding RNA, antisense RNA, satellite RNA, small cytoplasmic RNA, small nuclear RNA, etc., although not limited thereto. The polynucleotides described herein can be synthesized, for example, using an automated DNA synthesizer (such as those commercially available from Biosearch, Applied Biosystems, etc.) by standard methods well known in the art, or obtained from natural sources (e.g., genome, cDNA, etc.) or artificial sources (e.g., commercially available libraries, plasmids, etc.) using molecular biology techniques well known in the art (such as cloning, PCR, etc.). Nucleic acids can be chemically synthesized, for example, according to the phosphotriester method (see, e.g., Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584). from commercially available ones, etc.) by standard methods well known in the art, or obtained from natural sources (e.g., genome, cDNA, etc.) or artificial sources (e.g., commercially available libraries, plasmids, etc.) using molecular biology techniques well known in the art (such as cloning, PCR, etc.). Nucleic acids can be chemically synthesized, for example, according to the phosphotriester method (see, e.g., Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584).

[0056] The nucleic acid may contain at least one modified nucleotide (i.e., a nucleotide that is not a nucleotide of natural DNA or RNA). In particular, these modified nucleotides can be used to increase the resistance of the nucleic acid to degradation by nucleases. This is particularly advantageous for RNA, which is generally more sensitive to nucleases than DNA aptamers. RNA containing at least one modified nucleotide is called modified RNA. DNA containing at least one modified nucleotide is called modified DNA.

[0057] In addition to the nucleotides that make up its nucleic acid sequence, the nucleic acid may further contain at least one additional group. In this way, the nucleic acid can be linked to at least one additional group.

[0058] "Modified DNA" means DNA containing at least one modified nucleotide. Modified DNA may in particular be DNA in which the nucleic acid backbone is modified in whole or in part, in particular DNA that is resistant to hydrolysis by the action of nucleases. The DNA can be modified in its entirety (i.e., all the nucleotides that make it up are modified), or in part (i.e., only some of the nucleotides that make it up are modified). When the DNA is partially modified, one can choose to modify all or part of the purines and / or all or part of the pyrimidines.

[0059] "Modified RNA" means RNA containing at least one modified nucleotide. Modified RNA may in particular be RNA in which the nucleic acid backbone is modified in whole or in part, in particular RNA that is resistant to hydrolysis by the action of nucleases. The RNA can be modified in its entirety (i.e., all the nucleotides that make it up are modified), or in part (i.e., only some of the nucleotides that make it up are modified). When the RNA is partially modified, one can choose to modify all or part of the purines and / or all or part of the pyrimidines.

[0060] Modifications of DNA or RNA (and / or nucleotides) are well known to those skilled in the art and can be selected from the following: modification of the OH functional group on the 2'-carbon of ribose by methylation, substitution of the OH functional group on the 2'-carbon of ribose by an O-methoxyethyl group, substitution of the OH functional group on the 2'-carbon of ribose by an amino group, substitution of the OH functional group on the 2'-carbon of ribose by a halogen (especially fluorine), substitution of phosphodiester (PO) by a phosphorothioate (PS) group (referred to as a phosphorothioate backbone), use of a Locked Nucleic Acid (LNA) structure, i.e., formation of a methylene bridge to fix ribose in the C3'-endo (N-type) conformation, use of a Peptide Nucleic Acid (PNA) structure, i.e., substitution of the sugar-phosphate backbone by a peptide-type backbone, and any combination of these. That is, formation of a methylene bridge to fix ribose in the C3'-endo (N-type) conformation, use of a Peptide Nucleic Acid (PNA) structure, i.e., substitution of the sugar-phosphate backbone by a peptide-type backbone, and any combination of these.

[0061] According to the inventors, "vector" means a vehicle, preferably a nucleic acid molecule or a virus particle, which contains the necessary elements to enable the administration, propagation, and / or expression of one or more nucleic acid molecules in a host cell or organism.

[0062] From a functional perspective, this term encompasses vectors for maintenance (cloning vectors), vectors for expression in various host cells or organisms (expression vectors), extrachromosomal vectors (e.g., multicopy plasmids) or integration vectors (e.g., designed to be integrated into the genome of a host cell and produce additional copies of the nucleic acid molecule it contains when the host cell replicates). This term further encompasses shuttle vectors (e.g., those that operate in both prokaryotic and / or eukaryotic hosts) and transfer vectors (e.g., for transferring nucleic acid molecule(s) into the genome of a host cell).

[0063] From a structural point of view, a vector can be of natural, synthetic or artificial genetic origin, or a combination of natural and artificial genetic elements.

[0064] Thus, in the context of the present invention, the term "vector" should be understood broadly to include plasmid vectors and viral vectors.

[0065] As used herein, "plasmid" refers to a replicable DNA construct. Typically, a plasmid vector contains a selectable marker gene that enables a host cell carrying the plasmid to be positively or negatively identified and / or selected in the presence of a compound corresponding to the selectable marker. Various positive or negative selectable marker genes are well known in the art. By way of example, an antibiotic resistance gene can be used as a positive selectable marker gene for selecting host cells in the presence of the corresponding antibiotic.

[0066] As used herein, the term "viral vector" refers to a nucleic acid vector that contains at least one element of a viral genome and can be packaged into viral particles or viral-like particles. A viral vector can be replication-competent or selective (e.g., designed to replicate better or selectively in a particular host cell), or can be genetically inactivated to be replication-deficient or defective.

[0067] "Polypeptide", "protein", "protein fragment" and "peptide" are linked by peptide bonds It means a polymer of amino acid residues containing at least 9 amino acids. The polymer may be linear, branched, or cyclic. The polymer may contain natural amino acids and / or amino acid analogs and may be interrupted by non-amino acid residues. As a general guideline, but not limited to this specification, when an amino acid polymer contains more than 50 amino acid residues, it is preferably called a polypeptide or a protein, while when the polymer consists of 50 or fewer amino acids, it is preferably called a "peptide". The reading and writing directions of the amino acid sequences of polypeptides, proteins, and peptides used in this specification are the conventional reading and writing directions. For the reading and writing conventions of the amino acid sequences of polypeptides, proteins, and peptides, the amine terminus is placed on the left side, and then the sequence is written and read from the amine terminus (N-terminus) to the carboxyl terminus (C-terminus), from left to right.

[0068] Amino acids constituting polypeptides, proteins, and peptides include "standard" amino acids (also known as "natural amino acids", and a non-exhaustive list of which is shown in Table 1 below), as well as non-standard amino acids (also known as "rare amino acids", for example, pyrrolysine (represented by the letter O), selenocysteine (represented by the letter U), alloisoleucine, allothreonine, ornithine, etc.).

[0069]

Table 1

[0070] "Peptide or protein fragment" or "peptide or protein part" refers to a part of a peptide or a protein , that is, it means a part of the continuous amino acid sequence constituting the peptide or protein (referred to as the peptide or protein from which the fragment is derived). The peptide or protein fragment preferably contains at least 10 consecutive amino acids of the peptide or protein from which it is derived, more preferably at least 12 consecutive amino acids of the peptide or protein from which it is derived, even more preferably at least 15 consecutive amino acids, even more preferably at least 20 consecutive amino acids, and even more preferably at least 30 consecutive amino acids. The peptide or protein fragment preferably has a three-dimensional structure under non-denaturing conditions (for example, in the absence of denaturing agents and / or chaotropic agents, conditions that are usually non-denaturing for proteins).

[0071] As used herein, the term "post-translational modification" refers to chemical or enzymatic modifications that occur naturally or non-naturally on a protein or protein fragment after or simultaneously with protein translation (for example, using cellular machinery, such as biological synthesis or biochemical synthesis), or after or simultaneously with protein synthesis (for example, artificial synthesis and / or chemical synthesis). This means that at least one of the natural amino acids of the protein or protein fragment is modified by the addition and / or modification of at least one chemical group of a natural amino acid (including but not limited to removal). Examples of such chemical or enzymatic modifications include, but are not limited to, glycosylation, phosphorylation, acylation, carboxylation, acetylation, biotinylation, hydroxylation, lipoylation, amidation, ubiquitination, SUMOylation, deamination, etc. A "post-translationally modified protein" refers to a protein having at least one post-translational modification. Means a protein. "Post-translationally modified protein fragment" means a protein fragment having at least one post-translational modification.

[0072] "Liposoluble active ingredient" means any drug, compound or molecule that can be solubilized in a fatty substance and has biological activity, particularly therapeutic activity, pharmacological activity, targeting activity or marking activity, as defined above with respect to therapeutic agents, targeting agents or marking agents. Liposoluble active ingredients include those that can be solubilized in lipids or their derivatives, such as oils, butters, oily esters, and other components containing lipids or their derivatives. "Liposoluble active ingredient" means any drug, compound or molecule that can be solubilized in a fatty substance and has biological activity, particularly therapeutic activity, pharmacological activity, targeting activity or marking activity, as defined above with respect to therapeutic agents, targeting agents or marking agents. Liposoluble active ingredients include those that can be solubilized in lipids or their derivatives, such as oils, butters, oily esters, and other components containing lipids or their derivatives.

[0073] "Chemotherapeutic agent" means any drug, compound or molecule having chemotherapeutic activity. Chemotherapeutic agents include drugs having anti-cancer activity (particularly drugs capable of eliminating cancer cells and / or tumors and / or inducing / stimulating the elimination of cancer cells and / or tumors), as well as drugs having anti-autoimmune disease activity. Thus, chemotherapeutic agents may be cytotoxic agents and / or cell growth inhibitors. Examples of chemotherapeutic agents include, but are not limited to, paclitaxel, doxorubicin, gemcitabine (e.g., Gemzar), temozolomide, etc.

[0074] "Antibody" means a protein or glycoprotein belonging to the immunoglobulin superfamily. The terms antibody and immunoglobulin are used interchangeably. In mammals, antibodies are mainly secreted from cells derived from B lymphocytes, i.e., plasma cells. In particular, they are used by the immune system to specifically detect and neutralize foreign substances (particularly pathogens such as bacteria, viruses, parasites, etc.). Antibodies further include autoantibodies (e.g., those produced in autoimmune diseases). Antibodies recognize specific portions of their antigens, which are foreign targets.

[0075] Antibodies consist of four polypeptide chains (150,000 amu or daltons): two identical heavy chains (H (Heavy) for "heavy", each 50,000 amu) and two identical light chains (H (Heavy) for "heavy", each 50,000 amu). A structure consisting of a chain (L (Light), 25,000 amu each) Each light chain has a constant domain (CL) and a variable domain (VL), which are linked by a variable number of disulfide bridges that ensure the binding of the molecules. These chains form a Y structure (one half of each light chain constitutes an arm of the Y) and are composed of immunoglobulin domains of up to 110 amino acids. Each light chain is composed of a constant domain (called CL) and a variable domain (called VL). The heavy chain is composed of a variable domain (called VH) and three or four constant domains, called CH1, CH2, CH3, (CH4), respectively, depending on the isotype. For a given antibody, the two heavy chains are identical and the two light chains are also identical. The constant domains are characterized by amino acid sequences that are very similar from one antibody to the next, characteristic of the species and isotype. The constant domains are generally not involved in antigen recognition, but are involved in the activation of the complement system and in the clearance of immune complexes (antibodies bound to their antigens) by immune cells that bear the constant fragment receptor (cFR). Antibodies are composed of two "arms ( Each antibody has four variable domains located at the end of a pair of “arms.” The variable domain is carried by a heavy chain (VH) and the adjacent variable domain is carried by a light chain (VL). The pairing with the variable domain constitutes the antigen recognition site (or paratope). Thus, an immunoglobulin molecule has two antigen-binding sites, one at the end of each arm. These two sites are identical (but are intended for different epitopes), enabling binding to two antigen molecules per antibody. The antigen recognition site (or paratope) contains six regions known as Complementarity-Determining Regions (CDRs). Each VH has three CDRs, and each VL also has three.

[0076] Specific enzymatic cleavage allows different fragments to be isolated: -Fc fragment (fragment crystallizable). This is the basis for the biological properties of immunoglobulins, particularly the ability to be recognized by immune effectors or to activate complement. It is composed of the constant fragment of the heavy chain (CH2) beyond the hinge region. It generally does not recognize antigens: -Fv fragment (variable fragment). This is the smallest fragment of an immunoglobulin that retains the properties of an antibody. It consists only of the VL and VH variable regions, binds to antigens with the same affinity as the full antibody, and is monovalent: -Fab fragment (fragment antigen-binding). This fragment has the same affinity for antigens as the full antibody. The Fab fragment is composed of the entire light chain (VL + CL) and a part of the heavy chain (VH + CH1). It is monovalent: -F(ab’)2 fragment. It corresponds to the pairing of two Fab fragments linked by a small part of the constant part of the heavy chain, the hinge region. It has the same affinity for antigens as the antibody and is divalent.

[0077] As used herein, the term antibody encompasses natural antibodies and their functional derivatives, provided that such derivatives are capable of specifically binding to an antigen (referred to as a "functional antibody derivative"). As used herein, the term "antibody derivative" The term "antibody" also includes antibody fragments. Preferably, an "antibody fragment" is capable of specifically binding to an antigen (referred to as a "functional antibody fragment"). and is capable of specifically binding to an antigen (referred to as a "functional antibody fragment").

[0078] Antibodies can be produced by various systems well known to those skilled in the art. Examples of antibody production systems include, for example, animal systems (such as rodents, camels, etc.), hybridoma systems, mammalian cell lines (especially CHO cell lines (Chinese hamster ovary cells, such as CHO-K1, CHO-DG44, etc.), mouse myeloma cell lines (such as NS0), baby hamster kidney cell lines (such as baby hamster kidney cell line, BHK), human embryonic kidney cell lines (such as human embryonic kidney cell line, HEK293), etc.), yeast systems (including glycosylation-enhanced yeasts) , insect cell lines (including glycosylation-enhanced insect cell lines), plant cell lines (including glycosylation-enhanced plant cell lines), and the like.

[0079] Preferably, the antibody is an animal antibody, preferably a mammalian antibody, more preferably a human antibody or a humanized antibody. Advantageously, the antibody is humanized.

[0080] The term "antibody" preferably includes natural antibodies and their derivatives (such as mutated and / or modified antibodies and mimetic antibodies), provided that these derivatives are capable of specifically binding to an antigen.

[0081] Various categories of antibodies and their production methods are well known to those skilled in the art. Those skilled in the art are particularly referred to the references in the art (such as Thomas D. Pollard, W William C. Earnshaw, Jennifer Lippincott-Schwartz, Graham Johnson Cell Biology E-Book, Elsevier Health Sciences, November 1, 2016, Mohammed Zourob, Recognition Receptors in Biosensors, DOI 10.1007 / 978-1-4419-0919-0, Springer-Verlag New York 2010, Abbas, Lichtman, Pillai, Cellular and Molecular Immunology E-Book, Elsevier Health Sciences, August 22, 2014, Bayer V., An overview of monoclonal antibodies. Semin Oncol Nurs. September 2019, 2:150927, Wang W, Wang EQ, Balthasar JP. Pharmacokinetics and pharmacodynamics of monoclonal antibodies. Clin Pharmacol Ther. November 2008, 84(5):548-58). It is possible to refer to

[0082] As used herein, the term "functional antibody fragment" refers to one or more portions and / or fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed by the term "functional antibody fragment" include antigen-binding fragment (Fab), Fab' fragment, F(ab')2 fragment, variable fragment (Fv), single chain variable fragment (scFv) (VH and V fused by a linker peptide (corresponding to the L region), dsFv fragment ("disulfide bond-stabilized Fv"), ds-scFv fragment ("disulfide bond-stabilized scFv"), VH domain, VL domain, di-scFv (bivalent scFv, consisting of the pairing of two scFvs), diabody (consisting of the covalent or non-covalent binding of two scFvs), single-chain diabody, triple body, minibody (consisting of the VL-VH-CH3 fragment), nanobody, single-domain antibody (single-domain antibody, sdAb), single-chain antibody fragment (single-chain antibody fragment, SCab ), heavy chain antibody (heavy chain antibody, HCAb), VHH, variable new antigen receptor (variable new antigen receptor, VNAR), immunoglobulin new antigen receptor (immunoglobulin new antigen receptor, IgNAR), bispecific T-cell engager (bispecific T-cell engager, BiTE), dual affinity retargeting molecule, DART), and any combination thereof (e.g., their fusion proteins ), but are not limited thereto.

[0083] As used herein, the term "single-domain antibody" or "sdAb" refers to an antibody fragment consisting of a single monomeric variable domain of an antibody. These antibodies specifically include only the monomeric variable regions of heavy chain antibodies produced by camels or cartilaginous fish. Due to their different origins, they are further referred to as VHH fragments (camels) or VNARs (variable new antigen receptors, cartilaginous fish). Single-domain antibodies are also well known as nanobodies. Single-domain antibodies can also be obtained by monomerizing the variable domains of conventional mouse or human antibodies by genetic manipulation. They have a molecular weight of about 12-15 kDa and are thus the smallest antibody fragments capable of recognizing antigens.

[0084] As used herein, the term "diabody" refers to a fusion protein or bivalent antibody that can bind to different antigens. A diabody is composed of two distinct protein chains that are antibody fragments, i.e., contain variable fragments. A diabody contains a variable heavy chain (VH) domain linked to a variable light chain (VL) domain on the same polypeptide chain (VH-VL, or VL-VH). Using a short peptide that links the two variable domains, the domains are paired with the complementary domains of another chain to create two antigen-binding sites. A diabody can target the same antigen (monospecific) or different antigens (bispecific).

[0085] As used herein, the term "mimetic antibody" or "antibody's mimetic" refers to a compound that can bind specifically to an antigen in an antibody-like manner but is not structurally related to an antibody. Typically, an antibody mimetic is a peptide or modified protein having a molar mass of about 3 to 20 kDa that contains one, two, or more exposed antigen-specific binding domains. Examples of antibody mimetics include LACI-D1 (lipoprotein-associated coagulation inhibitor), affilins such as human ubiquitin or human γB-crystallin, cystatin, Sac7D from Sulfolobus acidocaldarius, lipocalin and lipocalin-derived anticalins, DARPins (designed ankyrin repeat proteins), the SH3 domain of Fyn, the K unit domain of protease inhibitors, monobodies such as the 10th type III domain of fibronectin, adnectin, knottins (cysteine knot miniproteins), trimers, evibodies, affibodies such as the 3-helix bundle of the Z domain of protein A from Staphylococcus aureus, and trinectins. ​​​​Examples of the body include, but are not limited to, human transferrin, tetranectin, such as the monomeric or trimeric domain of human C-type lectin, microbody, such as trypsin-II inhibitor, and armadillo repeat protein. Nucleic acids and small molecules can be further regarded as antibody mimetics (e.g., aptamers), but cannot be regarded as artificial antibodies, antibody fragments, and fusion proteins composed thereof. Common advantages over antibodies include better solubility, tissue penetration, thermal and enzymatic stability, and relatively low manufacturing costs.

[0086] The term "DARPin" or "Designed Ankyrin Repeat Protein" refers herein to a genetically engineered antibody mimetic protein that generally exhibits high affinity binding to a target protein. They are derived from naturally occurring ankyrin repeat proteins, which are one of the most common classes of natural binding proteins and are involved in diverse functions such as cell signaling, regulation, and structural integrity. A DARPin contains, consists essentially of, or consists of at least three repeat motifs or modules, and most of its N- and C-terminal modules are called "caps" because they protect the hydrophobic core of the protein. The number of internal modules is indicated by a number (e.g., N1C, N2C, N3C,...), and the caps are indicated by "N" or "C", respectively.

[0087] An "antigen" is a substance that, when recognized by the antibodies or cells of an organism's immune system, It refers to natural or synthetic molecules capable of inducing an immune response. In this way, any foreign substance or microorganism introduced into the body can act as an antigen and induce the production of specific proteins - antibodies - that neutralize the harmful effects of the foreign substance. Antigens are generally peptides, proteins, sugars (such as polysaccharides or polyosides), and their lipid derivatives (lipids). Antigens can further be nucleic acids or haptens (i.e., antigen fragments). Antigens, as markers of foreign factors, form the basis of the adaptive immune response. Activating specific immunity is the recognition of an antigen by immune cells, either directly or via antigen-presenting cells (APCs). In the case of protein antigens, the part of the antigen recognized by antibodies or lymphocyte receptors is called an "epitope" or "antigenic determinant". The same antigen may contain several (identical or different) epitopes and thus induce various immune responses. There are continuous epitopes corresponding to the amino acid sequence and conformational epitopes linked to the structure of the protein and thus sensitive to denaturation. Antigen recognition by lymphocytes depends on the nature of the epitope. B lymphocytes bind directly to conformational epitopes via their membrane immunoglobulins. T lymphocytes recognize continuous epitopes presented by antigen-presenting cells. Antigens can be exogenous, i.e., foreign to the individual and may be (in which case the antigen may be allogeneic: derived from individuals of the same species, or xenogeneic: derived from other species), or endogenous, i.e., antigens specific to the host (self-antigens). Antigens are preferably microorganisms, plants, algae, microalgae, bacteria, viruses, parasites, yeasts, fungi, insects, animals, or tumor antigens, preferably pathogens or cancer antigens of eukaryotes or prokaryotes, preferably bacterial, viral, parasitic, yeast, fungal, or tumor proteins, lipids, or sugars, antigens.

[0088] The term antigen includes natural antigens and their derivatives (e.g., mutant and / or modified antigens), preferably provided that these derivatives can be targets of the immune response.

[0089] The various categories of antigens are well known to those skilled in the art, and those skilled in the art can refer to references in the relevant technical field (for example, G.J.V. Nossal, G.L. Ada, Antigens, Lymphoid Cells and the Immune Response, Academic Press, 1971, Marc H.V. Van Regernortel, Structure of Antigens, Volume 3, CRC Press, December 20, 1995, Edouard Drouhet, Garry T. Cole, Louis De Repentigny, Jean Latge, Fungal Antigens: Isolation, Purification, and Detection, Springer Science & Business Media, November 11, 2013, Graziano D.F., Finn O.J. (2005) Tumor Antigens and Tumor Antigen Discovery. In: Khleif S.N. (eds) Tumor Immunology and Cancer Vaccines. Cancer Treatment and Research, vol 123. Springer, Boston, MA, Wang M, Claesson MH, Methods Mol Biol. 2014, 1184:309-17. Classification of human leukocyte antigen (HLA) supertypes, and, Galperin, Fernande z-Suarez, Rigden, The 24th annual Nucleic Acids Research database issue: a look back and upcoming changes, NAR, Volume 45, Issue D1, January 2017, pp. D1-D11, such as specialized databases as described in, in particular, the PMAPP database, the database of human autoantigens (in particular, available at aagatlas.ncpsb.org).

[0090] As used herein, "antigen fragment" means any part of an antigen, and preferably, this fragment / part can be a target of an immune response (e.g., epitope, immunogenic domain, etc.). In the case of a protein antigen, the antigenic fragment preferably contains at least 6 consecutive amino acid residues of the antigen (preferably at least 8 consecutive amino acid residues of the antigen, preferably at least 10, preferably at least 15, preferably at least 20, preferably at least 30 amino acid residues of the antigen).

[0091] "Toxin" means a substance that is toxic to one or more organisms. Toxins are , typically synthesized by living organisms (bacteria, poisonous fungi, poisonous insects or snakes) and conferring pathogenicity to them. Toxins produced by bacteria are called bacteriotoxins, toxins produced by fungi are called mycotoxins, toxins produced by plants are called phytotoxins, toxins produced by algae are called phycotoxins, and toxins produced by animals are called animal toxins. Toxins can be chemical molecules, peptides, proteins, glycoproteins, sugars, osides, lipids, nucleic acids, or any combination thereof. Some families of bacteria secrete biotoxins (exotoxins) into the tissues where they form colonies. Other bacteria (gram-negative) retain most of the toxic compounds within themselves and release them only during cell lysis by chemical, physical or mechanical means (endotoxin). Poisonous plants have their secondary protein, sugar, oside, lipid, nucleic acid, or any combination thereof. Some families of bacteria secrete biotoxins (exotoxins) into the tissues where they form colonies. Other bacteria (gram-negative) retain most of the toxic compounds within themselves and release them only during cell lysis by chemical, physical or mechanical means (endotoxin). Poisonous plants have their secondary secrete biotoxins (exotoxins) into the tissues where they form colonies. Other bacteria (gram-negative) retain most of the toxic compounds within themselves and release them only during cell lysis by chemical, physical or mechanical means (endotoxin). Poisonous plants have their secondary substances that are toxic to one or more organisms. Toxins are typically synthesized by living organisms (bacteria, poisonous fungi, poisonous insects or snakes) and confer pathogenicity to them. Toxins produced by bacteria are called bacteriotoxins, toxins produced by fungi are called mycotoxins, toxins produced by plants are called phytotoxins, toxins produced by algae are called phycotoxins, and toxins produced by animals are called animal toxins. Toxins can be chemical molecules, peptides, proteins, glycoproteins, sugars, osides, lipids, nucleic acids, or any combination thereof. Some families of bacteria secrete biotoxins (exotoxins) into the tissues where they form colonies. Other bacteria (gram-negative) retain most of the toxic compounds within themselves and release them only during cell lysis by chemical, physical or mechanical means (endotoxin). Poisonous plants have their secondary Producing toxins via metabolites: These are molecules (i.e., primary metabolites) produced outside the metabolic pathways necessary for survival, unlike primary toxins (such as proteins, lipids, carbohydrates, amino acids, etc.). Phytotoxins can be classified into three groups: phenols, nitrogen compounds, and terpenes. Toxins can be neurotoxins (neurotoxins) (toxins that act on the nervous system), myotoxins (myotoxins) (acting on muscle contraction, especially cardiotoxins of the heart and others like strychnine of respiratory muscles), hemotoxins (acting on the blood), cytotoxins (acting on cells), dermotoxins (acting on the skin and mucous membranes), hepatotoxins (acting on the liver), nephrotoxins (acting on the kidneys), enterotoxins (acting on the gastrointestinal tract), etc. Toxins can also be anatoxins, i.e., toxins treated in such a way that they retain their antigenicity and lose their toxicity. Toxins are preferably toxins of microorganisms, plants, algae, microalgae, bacteria, viruses, parasites, yeasts, fungi, insects, animals, or tumors. Preferably, they are toxins of eukaryotic pathogens or prokaryotic pathogens, or toxins of cancer.

[0092] The various categories of toxins are well known to those skilled in the art, who are particularly referred to references in the art (e.g., Michael W. Parker, Protein Toxin Structure, Springer Science & Business Media, June 29, 2013, Michael R. Dobbs, Clinical Neurotoxicology E-Book: Syndromes, Substances, Environments, Elsevier Health Sciences, July 22, 2009, Walker AA, Robinson SD, Yeates DK, Jin J, Baumann K, Dobson J, Fry BG, King GF. Venomics: The evolution, biology and biochemistry of insect venoms. Toxicon. November 2018, 154:15 - 27, Vilarino N, Louzao MC, Abal P, Cagide E, Carrera C, Vieytes MR, Botana LM. Human Poisoning from Marine Toxins: Unknowns for Optimal Consumer Protection. Toxins (Basel). August 9, 2018, 10(8), and, Galperin, Fernandez - Suarez, Rigden, The 24th annual Nucleic Acids Research database issue: a look back and upcoming changes, NAR, Volume 45, Issue D1, January 2017, pp. D1 - D11, such as specialized databases described in, Davis, Grondin Murphy, Johnson, Lay, Lennon - Hopkins, Saraceni - Richards, Sciaky, King, Rosenstein, Wiegers, Mattingly, The Comparative Toxicogenomics Database: 2013 update, NAR, Volume 41, Issue D1, January 1, 2013, pp. D1104 - D1114 (available especially at ctdbase.org), it is possible to refer to the Comparative Toxicogenomics Database described in it.

[0093] As used herein, a "toxin fragment" is preferably any part of a toxin, provided that such fragment / portion can be toxic to an organism and / or cell. In the case of a protein toxin, the toxin fragment preferably comprises at least 6 contiguous amino acid residues of the toxin (preferably at least 8 contiguous amino acid residues of the toxin, preferably at least 10, preferably at least 15, preferably at least 20, preferably at least 30 amino acid residues).

[0094] The term "receptor" refers to a molecule in the cell membrane, cytoplasm, or nucleus that specifically binds to a particular agent (a ligand such as a neurotransmitter, hormone or other substance) and induces a cellular response to that ligand. Ligand-induced changes in receptor behavior lead to physiological modifications that constitute the "biological effect" of the ligand. Receptors are It may contain at least a peptide, protein, glycoprotein, sugar, oside, lipid, nucleic acid, or any combination thereof. A receptor is generally a protein or a hybrid protein (which is paired with a modified protein and / or another molecule). The receptor may be a receptor on the outer portion of the plasma membrane, a transmembrane receptor embedded in the lipid bilayer of the cell membrane (usually a transmembrane protein, for example, acting as a receptor for hormones and neurotransmitters, and these receptors bind to G proteins, or have enzyme or ion channel activity that enables activation of a metabolic signaling pathway in response to ligand binding), or an intracellular receptor (these receptors may enter the cell nucleus and regulate the expression of specific genes in response to ligand activation). The receptor is preferably a microbial, plant, algal, microalgal, bacterial, viral, parasitic, yeast, fungal, insect, animal, or tumor receptor, preferably a pathogen or cancer receptor of eukaryotes or prokaryotes, preferably a bacterial, viral, parasitic, yeast, fungal, or tumor protein, or a glycoprotein receptor.Receptors of various categories are well-known to those skilled in the art. Those skilled in the art are particularly referred to references in the art (for example, Thomas D. Pollard, William C. Earnshaw, Jennifer Lippincott-Schwartz, Graham Johnson Cell Biology E-Book, Elsevier Health Sciences, November 1, 2016, Mohammed Zourob, Recognition Receptors in Biosensors, DOI 10.1007 / 978-1-4419-0919-0, Springer-Verlag New York 2010, Abbas, Lichtman, Pillai, Cellular and Molecular Immunology E-Book, Elsevier Health Sciences, August 22, 2014, and specialized databases as described in Galperin, Fernandez-Suarez, Rigden, The 24th annual Nucleic Acids Research database issue: a look back and upcoming changes, NAR, Vol. 45, No. D1, January 2017, pp. D1 - D11, particularly, Isberg V., Mordalski S., Munk C., Rataj K., Harpsoe K., Hauser A.S., Vroling B., Bojarski A.J., Vriend. G., Gloriam D.E. described in the GPCRdb database..GPCRdb: an information system for G protein-coupled receptors. Nucleic Acids Res. 2016, 44: D356 - D364 (particularly available at gpcrdb.org)) can be referred to.

[0095] As used herein, "receptor fragment" refers to a fragment of a receptor It is an arbitrary part, preferably on the condition that this fragment / part can specifically bind to a specific factor (e.g., a ligand, a hormone or other substance). In the case of a protein receptor, the receptor fragment preferably comprises at least 6 consecutive amino acid residues of the receptor (preferably at least 8 consecutive amino acid residues of the receptor, preferably at least 10, preferably at least 15, preferably at least 20, preferably at least 30 amino acid residues).

[0096] "Enzyme" means a protein having catalytic properties. Substantially all biomolecules capable of catalyzing chemical reactions within a cell are enzymes. However, some catalytic biomolecules are composed of RNA and are thus different from enzymes: these are ribozymes. Enzymes act by lowering the activation energy of a chemical reaction and thereby increasing the reaction rate. Enzymes are not modified during the reaction. The initial molecules are enzyme substrates, and the molecules formed from these substrates are reaction products. Enzymes are characterized by their very high specificity. Furthermore, enzymes are reusable.

[0097] Enzymes are generally globular proteins that act alone or as complexes of several enzymes or subunits. Like all proteins, enzymes are composed of one or more polypeptide chains folded to form a three-dimensional structure corresponding to their native state.

[0098] Enzymes are molecules much larger than their substrates. Their size can vary from about 50 residues to over 2000 residues. Only a very small part of an enzyme - usually 2 - 4 residues, sometimes more - is directly involved in catalysis and is the so-called catalytic site (or catalytic domain). The latter may be located near one or more binding sites to which the substrate(s) bind and are oriented to catalyze a chemical reaction. The catalytic site and the binding site form the enzyme active site.

[0099] Enzymes perform a wide range of functions in living organisms. For example, they are involved in cell processes, movement generation, active metabolism, digestion, transmembrane transport, the immune system, nucleic acid digestion, nucleic acid cleavage, and nucleic acid production (referred to herein as "nucleic acid enzyme"), as well as signal conversion and regulation of prodrug conversion ( conversion from prodrug to drug). Enzymes are preferably prokaryotic, eukaryotic or viral enzymes, most preferably enzymes derived from animals, plants, algae, microalgae, insects, microorganisms, bacteria, parasites, yeast, fungi or viruses, and most preferably mammalian enzymes such as human enzymes. Various categories of enzymes are well known to those skilled in the art. Those skilled in the art are particularly aware of references in the relevant technical field (for example, Schomburg D., Schomburg I., Springer Handbook of Enzymes. 2 edn. Heidelberg: Springer, 2001 - 2009, Liebecq C., Joint Commission on Biochemical Nomenclature (JCBN) of IUPAC - IUBMB and Nomenclature Committee of IUBMB (NC - IUBMB). Biochem. Mol. Biol. Int. 1997, 4 3:1151-1156, IUBMB (1992), Enzyme Nomenclature 1992, Academic Press, San Diego, and Schomburg D, Schomburg I. Methods Mol Biol. 2010, 609:113-28. Enzyme database, in particular, BRENDA database (available in particular at brenda-enzymes.org), for example, Chang A, Schomburg I, Placzek S, Jeske L, Ulbrich M, Xiao M, Sensen CW, Schomburg D, Nucleic Acids Res. January 2015, 43. Epub November 5, 2014. BRENDA 2015: exciting developments in its 25th year of existence) can be referred to.

[0100] As used herein, "enzyme fragment" preferably is any part of an enzyme, provided that this fragment / portion can have enzyme activity. In the case of a protein enzyme, the enzyme fragment preferably comprises at least 6 consecutive amino acid residues of the enzyme (preferably at least 8 consecutive amino acid residues of the enzyme, preferably at least 10, preferably at least 15, preferably at least 20, preferably at least 30 amino acid residues) (preferably the catalytic site of the enzyme).

[0101] The "enzymatic activity" or "catalytic activity" or "activity" of an enzyme means the efficiency of the enzyme in converting a substrate into a product in a given environment. Enzyme efficiency takes into account the rate at which the enzyme converts the substrate into the product and the rate at which the enzyme converts the substrate into the product. The "rate of conversion of substrate to product by the enzyme" means the ratio of the amount of the final product obtained to the initial amount of the substrate relative to a specified amount of the enzyme. For example, the enzyme activity in the context of the present invention can be expressed as the amount of phloroglucinol produced (g / L) in a given volume.

[0102] According to the present inventors, "hormone" means a biologically active chemical substance, which is generally synthesized by glandular cells (usually following stimulation) and secreted into the internal environment in which it circulates (via blood, lymph or sap). It transmits a message in chemical form (generally by acting on specific receptors on target cells) and thus acts as a messenger in the body. It is capable of acting at very low doses. Hormones are preferably plant hormones or animal hormones. Plant hormones are also well known as plant hormones or growth factors. Their functions often ensure plant growth or morphogenesis. Animal hormones are mostly produced by the endocrine system (endocrine glands or tissues).

[0103] Advantageously, the hormone is a vertebrate hormone and is preferably selected from the following chemical classes:

[0104] - Amino-derived hormones consisting of a single amino acid (tyrosine or tryptophan) but in derivative form. - Peptide hormones are chains of amino acids (proteins), and the shorter ones are called peptides. ​- Steroid hormones, which are steroids derived from cholesterol. - Hormones of the lipid and phospholipid systems.

[0105] Hormones are preferably selected from peptide or protein hormones, amine-derived hormones, steroid hormones, and lipid hormones. Hormones are preferably animal hormones or plant hormones, preferably mammalian hormones, and most preferably human hormones. The various categories of hormones are well known to those skilled in the art, and those skilled in the art can refer, in particular, to references in the art (e.g., Davies P.J. (2010) The Plant Hormones: Their Nature, Occurrence, and Functions. Davies P.J. (eds) Plant Hormones. Springer, Dordrecht, AW Norman, G Litwack, Hormones, Academic Press, 1997, Kastin, Handbook of biologically active peptides, Academic Press, 2013).

[0106] As used herein, a "hormone fragment" is preferably any part of a hormone provided that this fragment / portion can stimulate and / or inhibit a biological process. In the case of protein hormones, the hormone fragment preferably contains at least 6 consecutive amino acid residues of the hormone (preferably at least 8 consecutive amino acid residues of the hormone, preferably at least 10, preferably at least 15, preferably at least 20, preferably at least 30 amino acid residues of the hormone).

[0107] The term "ligand" generally refers to a substance that binds to a cell receptor and induces a biological signal. In particular, the term ligand encompasses the terms "addressing or targeting or transport signal", "signaling molecule", "signal", and "cellular signal". Examples of ligands include peptide and protein addressing sequences, oligosaccharides, molecules that enable cell transport and / or internalization, neurotransmitters, receptor ligands (where the receptor is as defined above), and cell recognition molecules such as Toll Like receptor ligands or C-type lectin receptor ligands. An addressing sequence is a short amino acid sequence, generally located at the N-terminus of a protein, that specifies the protein to be addressed and is used to indicate their destination. Thus, an addressing or targeting or transport signal can be an addressing or targeting or transport signal to / from the nucleus, an addressing or targeting or transport signal to / from the cytoplasm, an addressing or targeting or transport signal to / from the cytosol, an addressing or targeting or transport signal to / from the cell membrane, an addressing or targeting or transport signal to / from the mitochondria, an addressing or targeting or transport signal to / from the peroxisome, an addressing or targeting or transport signal to / from the lysosome, an addressing or targeting or transport signal to / from the endoplasmic reticulum, an addressing or targeting or transport signal to / from the secretory pathway, and can be a ligand for a membrane receptor or transmembrane receptor, preferably a receptor selected from the cell membrane, extracellular membrane, cytoplasmic membrane, or nuclear membrane. An addressing or targeting or transport signal may comprise at least a peptide, protein, glycoprotein, sugar, oside, lipid, nucleic acid, or any combination thereof. Preferably, the addressing or targeting or transport signal comprises at least one peptide, protein, glycoprotein, or nucleic acid.The signal is preferably a prokaryotic, eukaryotic or viral signal, most preferably a signal of animal, plant, algae, microalgae, microorganism, bacteria, parasite, yeast, fungus, insect, virus or cancer origin, even more preferably a mammalian signal, such as a human signal. The various categories of signals are well known to those skilled in the art, who can refer in particular to references in this field (e.g. Thomas D. Pollard, William C. Earnshaw, Jennifer Lippincott-Schwartz, Graham Johnson Cell Biology E-Book, Elsevier Health Sciences, November 1, 2016; Mohammed Zourob, Recognition Receptors in Biosensors, DOI 10.1007 / 978-1-4419-0919-0, Springer-Verlag New York 2010; Abbas, Lichtman, Pillai, Cellular and Molecular Immunology E-Book, Elsevier Health Sciences, August 22, 2014).

[0108] A "ligand fragment" preferably refers to a fragment / portion of a ligand that binds to a cell receptor. A ligand fragment is any portion of a ligand, provided that it is capable of binding to and inducing a biological signal. In the case of a protein ligand, a ligand fragment preferably comprises at least 6 consecutive amino acid residues of the ligand (preferably at least 8 consecutive amino acid residues of the ligand, preferably at least 10, preferably at least 15, preferably at least 20, preferably at least 30 amino acid residues of the ligand).

[0109] "Nanoparticle" means an object whose three dimensions are on the nanometer scale, ie, a particle having a nominal diameter of less than about 100 nm (eg, as defined by ISO standard TS / 27687).

[0110] "Functional group" or "functional group" means a reactive group, i.e., a group capable of forming at least one chemical reaction, biological reaction, biochemical reaction, or enzymatic reaction, or any combination thereof, with another molecule. "Agent-binding functional group" means a functional group having the ability to form at least one chemical reaction, biological reaction, biochemical reaction, or enzymatic reaction, or any combination thereof, with an agent (especially selected from therapeutic agents, targeting agents, marking agents, and any combination thereof).

[0111] Functional groups may include, for example, peptide tags, chemical groups (such as clickable functional groups, cross-linking groups, and any combination thereof), antibodies, antibody derivatives, functional fragment-binding tags of antibodies or their derivatives (such as biotin, streptavidin, chitin-binding protein (CBP), maltose-binding protein (MBP), Strep tag, glutathione-S-transferase ( , GST), poly(His) tag, etc.), or may consist essentially of them, or may consist of them.

[0112] "Clickable function", or "chemistry-click" or "fast bio-orthogonal chemistry" means a chemical group that can react with another chemical group in the absence of a solvent at physiological pH without forming residues or by-products. Examples of clickable functional groups include, but are not limited to, azide groups, alkyne groups (such as acetylene), and any combination thereof. In particular, clickable functional groups may be selected from N-hydroxysuccinimide (NHS), dibenzocyclooctyne (DBCO), tetrazine, methyl-tetrazine groups, and any combination thereof.

[0113] As used herein, the term "peptide label" or "peptide tag" refers to a peptide sequence of 6 to 400 amino acids (preferably 8 to 300 amino acids, more preferably 10 to 200 amino acids, more preferably 12 to 82 amino acids). Examples of peptide labels include binding peptide labels, solubilizing peptide labels, chromatographic peptide labels, epitope peptide labels, fluorescent peptide labels, etc. Affinity peptide tags are generally added to proteins so that they can be purified from their crude biological sources using affinity techniques. Examples include chitin-binding protein (CBP), maltose-binding protein (MBP), Strep tag, glutathione-S-transferase (GST), poly(His) tag, etc. Solubilizing peptide tags are used particularly for proteins expressed in chaperone-deficient species (e.g., Escherichia coli, E. coli) to assist in the correct protein folding and prevent precipitation. These include thioredoxin It contains TRX and poly(NANP). Some affinity peptide labels have a dual role as solubilizing agents (e.g., MBP and GST). Chromatography tags are used to modify the chromatographic properties of proteins to enable different separations in specific separation techniques. They often consist of polyanionic amino acids such as the FLAG tag. Epitope tags are short peptide sequences selected so that high-affinity antibodies can be reliably produced in many different species. They generally originate from viral genes. Examples of epitope tags include the ALFA tag, V5 tag, Myc tag, HA tag, Spot tag, T7 tag, NE tag, etc. Fluorescent tags are used especially to provide a visual readout of proteins. GFP and its variants are the most commonly used fluorescent tags. Peptide tags can enable specific enzymatic modifications (such as biotinylation by biotin ligase) or chemical modifications (such as reaction with FlAsH-EDT2 for fluorescence imaging). Peptide tags can be combined especially to link proteins to some other components and can be combined to link proteins to some other components. Peptide tags further include covalent peptide tags. Examples of covalent peptide tags include, but are not limited to, the following: - Isopeptag (covalently binds to pyrin - C protein), - SpyTag (covalently binds to SpyCatcher protein), - SnoopTag (covalently binds to SnoopCatcher protein), - SnoopTagJr (covalently binds to SnoopCatcher protein or DogTag protein (mediated by SnoopLigase) ), - DogTag (covalently binds to SnoopTagJr protein, mediated by SnoopLigase), - SpyTag (covalently binds to SdyCatcher protein), - All of their variants.

[0114] Marker peptide variants are well documented in the literature and are available to those skilled in the art and need not be described in detail herein.

[0115] "Illness" or "disease" or "disorder" or "pathology" (these terms are considered synonymous herein) refers to a change in the function or health of the body. This includes both diseases that refer to all changes in health and illnesses that refer to specific entities characterized by their own causes, symptoms, course, and treatability.

[0116] "Prevention" or "prevention of a disease" or "prevention of the onset of a disease" means reducing the risk of the onset, progression, or amplification of a disease, the cause of the disease, the symptoms of the disease, the effects (or consequences, preferably adverse effects / consequences) of the disease, or any combination thereof, and / or delaying the onset, progression, or amplification of a disease, the cause of the disease, the symptoms of the disease, the effects (or consequences, preferably adverse effects / consequences) of the disease, or any combination thereof. Prevention includes, in particular, prophylactic measures.

[0117] "Treatment" or "treatment of a disease" means reducing, inhibiting, and / or eliminating a disease, the cause of the disease, the symptoms of the disease, the effects (or consequences, preferably adverse, toxic effects / consequences) of the disease, or any combination thereof. Treatment is preferably curative.

[0118] "Treatment" or "therapy" includes one or more molecules and / or drugs (chemical (including any type of molecule or agent, such as a compound or biological compound, antibody, antigen, gene therapy, cell therapy, immunotherapy, chemotherapy, combinations thereof, etc.), and / or other treatments (radiation therapy, immunotherapy, chemotherapy, surgery, endoscopy, interventional radiology, physical oncology, phototherapy, etc.), and / or other treatments (radiation therapy, immunotherapy, chemotherapy, surgery, endoscopy, interventional radiology, physical oncology, phototherapy, ultrasound therapy, hyperthermia, cryotherapy, electrotherapy, electroconvulsive therapy, oxygen therapy, assisted ventilation, hydrotherapy massage, organ / tissue / fluid transplantation, transplantation, and any combination thereof, etc.), but are not limited thereto. Treatments can be delivered by different modes of administration. Those skilled in the art are well aware of the method of selecting the most appropriate mode(s) of administration according to the treatment, disease, and subject being treated. For example, modes of administration include oral administration, intravenous (intravenous, IV), intramuscular ( intramuscular, IM), administration by injection into the space around the spinal cord (intrathecal), subcutaneous (subcutaneous, sc), sublingual administration, buccal administration, rectal administration, vaginal administration, ocular route, otic route, nasal administration, administration by inhalation, administration by spraying, dermal, topical or systemic administration, transdermal administration, etc., but are not limited thereto.

[0119] "Medicament" or "drug" means any substance or composition represented as having therapeutic or prophylactic properties with respect to human or animal diseases. Thus, a medicament includes any substance or composition that can be used or administered to a human or animal for the purpose of making a medical diagnosis or for the purpose of restoring, correcting, or modifying their physiological functions by exerting a pharmacological, immunological, or metabolic effect. The term medicament includes, in particular, vaccines.

[0120] The terms "therapeutic" or "therapeutic use" in the context of the present invention encompass the use of "prevention", "treatment", and "vaccine". Thereof.

[0121] A "therapeutically effective amount" is an amount of each active ingredient sufficient to produce a beneficial health outcome. An "immunologically effective quantity" corresponds to an amount of each active entity sufficient to produce a detectable immune response. An active entity is, for example, an active ingredient, therapeutic agent, targeting agent, labeling agent, or any combination thereof. As used herein, a "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" is intended to include all carriers, solvents, diluents, excipients, adjuvants, vehicles, dispersion media, coatings, antibacterial and antifungal agents, absorption delaying agents, etc. that are compatible with administration in any subject, particularly animals including humans. Carriers suitable for use herein are well known in the art (see, e.g., Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams & Wilkins, latest edition). Non-limiting examples of excipients include water, NaCl, saline, sugar solutions (e.g., glucose, trehalose, sucrose, dextrose, etc.), lactated Ringer's solution, alcohol, oil, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, etc. In particular, sugars such as lactose, sucrose, trehalose, sorbitol, glucose, raffinose or mannitol, preferably swelling agents such as lactose, sucrose, trehalose, glucose or mannitol, amino acids such as arginine, glycine or histidine, preferably glycine, or dextran or polyethylene glycol type polymers, or mixtures thereof can be used as adjuvants.

[0122] As used herein, carrier)"

[0123] "Subject" or "patient" means a human individual or an animal other than a human. A subject is, for example, a human or animal body that is likely to have a disease, is likely to be affected by a disease, or has a disease. The subject is preferably a human. The subject may be a child (human subject under 16 years old) or an adult (human subject over 16 years old). "Healthy subject" means a subject who does not have the disease in question. In the context of the present invention, a healthy subject is preferably a subject who does not have any disease. "Reference subject" means a subject who has a well-known disease at a well-known disease stage.

[0124] "Biological sample" or "specimen" derived from a subject, according to the inventors, means an entire organ or tissue obtained from such a subject, or a part of such an organ or tissue, such a fluid or fraction of such a fluid, a cell or cell component, and a homogenate, lysate or extract prepared therefrom. In particular, a "biological sample" or "specimen" is preferably any tissue (preferably a part or fraction thereof) that can be used to detect a disease, and examples include plasma, blood, lymph, serum, urine, mucus, saliva, central nervous system (CNS, e.g., brain sample or spinal cord sample, etc.), airway sample (e.g., lung sample, etc.), salivary gland sample, nasopharyngeal sample, oral pharyngeal sample, digestive system sample (e.g., colon, intestine, etc.), skin sample, organ sample (e.g., liver, kidney, spleen, etc.), etc., but are not limited thereto.

[0125] ​​​​Biological samples may have been previously obtained by any technique well known in the art. Such techniques include, for example, swabbing, needle, or syringe sampling, surgery (such as stereotactic brain surgery), puncture, explant, excision, and biopsy. "Excision" means a surgical procedure consisting of cutting (excising) a tissue, preferably a more or less extensive or deep portion of a tissue abnormality or growth. Excision may be performed to remove and / or analyze a cancerous tumor or a suspected tumor. As used herein, the term "biopsy" refers to a sample of cells or tissue taken for analysis. Several types of biopsy procedures are known and practiced in the art. The most common types include (1) incisional biopsy, in which only a sample of the tissue is removed, (2) excisional biopsy (or surgical biopsy) in which the tumor mass can be completely removed for treatment and diagnostic procedures, and (3) needle biopsy in which a sample of the tissue is removed using a large or thin needle. There are other types of biopsies, such as smear or scraping, that can also be used to obtain samples. As a result, the sample can be, for example, an extract, an excised specimen, a biopsy specimen, etc. The sample is preferably obtained by minimally invasive surgery such as stereotactic surgery.

[0126] In the following detailed description, embodiments may be employed singly or in any suitable combination by those skilled in the art, and the above definitions apply to all embodiments described below and to combinations thereof.

[0127] Lipid microbubbles In the context of the present invention, the inventors have developed innovative microbubbles capable of delivering a drug of interest in a precisely targeted manner in the body.

[0128] In particular, the inventors have surprisingly shown that the lipid microbubbles developed in this way have significantly improved stability, unlike the microbubbles described in the prior art. Notably, the data further reveals that these optimized microbubbles can more effectively deliver different types of therapeutic agents, targeting agents, and / or labeling agents containing nucleic acids than the microbubbles described in the prior art. In particular, these microbubbles can actively pass through blood vessels, the blood-brain barrier (BBB), or the tumor microenvironment. The inventors have further demonstrated that local application of ultrasound can very accurately target these optimized microbubbles to the area to be treated. Thus, these data demonstrate the therapeutic potential of these lipid microbubbles for treating a wide range of lesions, including central nervous system lesions, vascular lesions, tumors, and cancers, in a targeted manner.

[0129] The data further shows that these optimized microbubbles can be used as marking, detection, and imaging tools.

[0130] This high degree of flexibility is due in particular to the original formulation of the microbubbles using a mixture of cationic molecules such as lipophosphoramidates and / or histidylated polyethyleneamines.

[0131] Thus, the present invention relates to lipophosphoramidates, histidylated polyethyleneimine, Relates to lipid microbubbles comprising, consisting essentially of, or consisting of at least one cationic compound selected from these and any mixtures thereof. Advantageously, the envelope of the lipid microbubble comprises, consists essentially of, or consists of at least one cationic compound selected from lipophosphoramidates, histidylated polyethyleneimines, and any mixtures thereof. Thus, according to an advantageous embodiment, the invention relates to lipid microbubbles having an envelope comprising, consisting essentially of, or consisting of at least one cationic compound selected from lipophosphoramidates, histidylated polyethyleneimines, and any mixtures thereof.

[0132] The lipid microbubbles according to the invention can in particular be ultrasonic contrast agents.

[0133] According to a preferred embodiment, the microbubbles according to the invention further comprise at least one agent selected from therapeutic agents / pharmacological agents, targeting agents, marking agents, and any combinations thereof.

[0134] Therapeutic agents / pharmacological agents, targeting agents, labeling agents, and combinations thereof are preferably i. exposed on the surface of the microbubble, or ii. embedded in the lipid envelope of the microbubble, or iii. incorporated into the interior of the microbubble, or iv. any combination of i - iii.

[0135] Therapeutic agents / pharmacological agents, targeting agents and labeling agents are advantageously as defined in the "definition" section above.

[0136] According to one embodiment, the at least one agent is 1) a nucleic acid, and 2) a lipophilic active ingredient, and 3) a chemotherapeutic agent such as a cytotoxic agent and / or a cytostatic agent, and 4) an antibody, 5) a protein, 6) an antigen, 7) a toxin, 8) a receptor, 9) an enzyme, 10) a hormone, 11) a ligand, 12) a viral vector, 13) a nanoparticle, preferably a nanoparticle comprising at least one agent selected from a therapeutic agent, a targeting agent, a marking agent, and any combination thereof, 14) a derivative of any of 1) to 13), preferably a functional derivative of any of these, 15) a fragment of any of 1) to 14), preferably a functional fragment of any of these, 16) any combination of 1) to 15), and comprising, or consisting essentially of, or consisting of, or selected from (plural available).

[0137] In particular, according to a preferred embodiment, the at least one agent is a) a nucleic acid, b) a lipophilic active ingredient, c) a chemotherapeutic agent such as a cytotoxic agent and / or a cell growth inhibitor, d) an antibody, e) an antibody derivative, f) a functional fragment of an antibody or an antibody derivative, g) a protein, h) a protein fragment such as a peptide (e.g., a cell-penetrating peptide (CPP), an antigen, an epitope, a functional protein domain, etc.), i) preferably a nanoparticle comprising at least one agent selected from a therapeutic agent, a targeting agent, a marking agent, and any combination thereof (including hydrophilic agents / non-lipophilic agents) (and may further comprise), j) any combination of a) to i), and comprising, or consisting essentially of, or consisting of, or selected from (plural available).

[0138] The various types of agents 1) to 16) and a) to i) listed above are preferably as defined in the above section "Definitions".

[0139] Preferably, the nucleic acid 1) and / or a) comprises, consists essentially of, or consists of a DNA encoding a plasmid, vector, complementary DNA (cDNA), single-stranded DNA, double-stranded DNA, gene or gene fragment, a DNA, gene or gene fragment (preferably a functional fragment) encoding a gene or gene fragment, RNA, double-stranded RNA, messenger RNA, non-coding RNA, small RNA, etc.

[0140] Preferably, the chemotherapeutic agent 3) or c) is selected from cytotoxic agents, cell growth inhibitors, and combinations thereof. In particular, the chemotherapeutic agent can be selected from, but is not limited to, paclitaxel, doxorubicin, gemcitabine (e.g., Gemzar), temozolomide, etc.

[0141] The targeting agent may in particular comprise, consist essentially of, or consist of an antibody, antibody derivative, functional fragment of an antibody or its derivative, protein, protein fragment (e.g., peptide, antigen, epitope, protein functional domain, etc.), nanoparticle (which may further comprise at least one therapeutic agent, in particular a hydrophilic therapeutic agent), and any combination thereof.

[0142] According to an advantageous embodiment, the microbubbles according to the invention further comprise at least one functional group enabling the binding to at least one agent selected from at least one therapeutic agent, targeting agent, marking agent, and any combination thereof. Said group is preferably i. exposed on the surface of the microbubble, or ii. embedded in the lipid envelope of the microbubble, or iii. incorporated into the interior of the microbubble, or iv. any combination of i to iii.

[0143] The functional group is preferably as defined in the above "Definition" section.

[0144] Preferably, the functional group is a) a peptide label, and b) a chemical group, wherein the chemical group is preferably selected from a clickable functional group, a coupling group, and any combination thereof, c) an antibody, and d) an antibody derivative, and e) a functional fragment of an antibody or an antibody derivative, and f) a binding tag (e.g., biotin, streptavidin, chitin-binding protein (C BP), maltose-binding protein (MBP), Strep tag, glutathione-S-transferase (GST), poly(His) tag, etc.), and g) any combination of a) to f), or consists essentially of, or consists of, or is selected from.

[0145] According to one embodiment, the lipophosphoramidate is selected from dimyristoylphosphoramidate (preferably dimyristoylphosphoramidate bromide (preferably O,O-dimyristoyl-N-[3N-(N-methylimidazolium bromide)propylene]phosphoramidate (Compound KLN27)), dimyristoylhistamine phosphoramidate (preferably O,O-dimyristoyl(-N-(histamine)phosphoramidate (Compound MM30)), and any combination thereof), dioleoylphosphoramidate (preferably dioleoylmethylimidazolium phosphoramidate (preferably O,O-dioleoyl-N-(3N-(N-methylimidazolium iodide)propylene)phosphoramidate (Compound KLN25)), dipalmitoylphosphoramidate, distearoylphosphoramidate, and any mixture thereof, or consists essentially of them, or consists of them, or is selected from the group consisting of them.

[0146] In one embodiment, the histidyl polyethyleneimine is preferably bound to a fatty acid selected from stearic acid, myristic acid, palmitic acid, oleic acid, and any combination thereof.

[0147] Advantageously, the microbubbles further comprise (in particular, the microbubble envelope further comprises) additional lipids selected from dimyristoyl-glycero-phosphocholine (preferably 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)), distearoyl-glycero-phosphocholine (preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)), dimyristoyl-glycero-phosphoethanolamine-(polyethylene glycol) (preferably 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG2000)), distearoyl-glycero-phosphoethanolamine-(polyethylene glycol) (preferably 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000)), and distearoyl-glycero-phosphoethanolamine-[biotinyl(polyethylene glycol)] (preferably 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG2000-biotin)), cholesterol, β-sitosterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-oleoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-3-trimethylammonium propane (DOTAP), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), and any combination thereof.

[0148] In a preferred embodiment, the microbubbles also contain a biocompatible gas. The biocompatible gas is preferably contained by the microbubble envelope (i.e., it is inside the microbubbles within the medium / cavity formed by the envelope). The biocompatible gas is preferably selected from perfluorobutane (C4F10), perfluoropropane (C3F8), dinitrogen (N2), sulfur hexafluoride (SF6), nitrogen oxides (NO), hydrogen, dioxygen, helium, xenon, argon, nitrous oxide (N2O), and any mixture thereof, and is preferably selected from perfluorobutane (C4F10), perfluoropropane (C3F8), dinitrogen (N2), sulfur hexafluoride (SF6), oxygen, nitrous oxide (N2O), and mixtures thereof. In particular, preferably, the gas is selected from the perfluorobutane group (C4F10).

[0149] Data obtained by the inventors demonstrate that the microbubbles according to the present invention as defined above are significantly capable of actively passing through blood vessels, the blood-brain barrier (BBB), and / or the tumor microenvironment (especially during local application of ultrasound). The inventors further demonstrated that local application of ultrasound enables these microbubbles to be very precisely targeted to the area to be treated, including difficult-to-access areas such as the central nervous system, blood vessels, and the tumor microenvironment. Furthermore, the data further show that these optimized microbubbles are capable of efficiently delivering different types of therapeutic agents, targeting agents, and / or labeling agents containing nucleic acids to these difficult-to-reach areas.

[0150] Thus, according to an advantageous embodiment, the microbubbles according to the present invention are characterized in that they are capable of actively passing through the blood-brain barrier and / or the tumor microenvironment (especially during local application of ultrasound).

[0151] Method for producing lipid microbubbles The present invention further relates to a method for producing at least one microbubble according to the present invention as described above, which comprises, consists essentially of, or consists of the following steps: a) A step of a mixture in a container of a cationic compound selected from lipophosphoramidates, histidyl polyethyleneimine, and any mixture thereof, ethanol, and optionally at least one agent selected from therapeutic agents, targeting agents, marking agents, and any combination thereof; b) A step of evaporating the mixture obtained in step a) to obtain a lipid film and rehydrating the lipid film to form a liposome suspension. (This entire step can also be performed using microfluidics); c) A step of lyophilizing the liposome suspension obtained in step b); d) A step of replacing the air contained in the container containing the lyophilizate obtained in step c) with a biocompatible gas, the biocompatible gas preferably being selected from perfluorobutane (C4F10), perfluoropropane (C3F8), dinitrogen (N2), sulfur hexafluoride (SF6), nitrogen oxides (NO), hydrogen, dioxygen, helium, xenon, argon, nitrous oxide (N2O), and any mixture thereof; e) A step of rehydrating the lyophilizate from step d) to obtain a solution; f) A step of stirring the solution obtained in step d) to form microbubbles; g) Optionally, a step of functionalizing the microbubbles with at least one agent selected from therapeutic agents, targeting agents, marking agents, and any combination thereof, and / or a step of functionalizing the microbubbles by adding at least one functional group that enables binding to at least one agent selected from therapeutic agents, targeting agents, marking agents, and any combination thereof.

[0152] Lipophosphoramidate, histidyl polyethyleneimine, histidyl polyethyleneimine, therapeutic agent, targeting agent, marking agent, biocompatible gas are advantageously as described in the preceding paragraphs (the paragraphs of "Definitions" and / or "Lipid Microbubbles").

[0153] The present invention further relates to microbubbles that can be obtained, or are obtained, or are directly obtained by the above-described production method.

[0154] Compositions and Kits The present invention further relates to a kit comprising or consisting essentially of or consisting of at least one microbubble according to the invention as defined above.

[0155] The present invention further relates to a composition comprising or consisting essentially of or consisting of at least one microbubble according to the invention as defined above and optionally an excipient.

[0156] The present invention particularly relates to a pharmaceutical composition comprising or consisting essentially of or consisting of at least one microbubble according to the invention as defined above and optionally a pharmaceutically acceptable excipient.

[0157] Advantageously, the composition, particularly the pharmaceutical composition, comprises a therapeutically effective amount of microbubbles. The concentration of microbubbles in the composition, particularly the pharmaceutical composition, is preferably 10 6 ~10 14 microbubbles / ml, even more preferably 10 7 ~10 13 microbubbles / ml, even more preferably 10 8 ~10 12 microbubbles / ml, even more preferably 10 9 ~10 11 microbubbles / ml, and even more preferably the concentration of microbubbles in the composition is about 10 10 microbubbles / ml.

[0158] According to one embodiment, a composition, particularly a pharmaceutical composition, contains, in the composition, a pharmaceutically acceptable excipient in an amount in the range of 5% to 99% by weight, preferably 10% to 97% by weight, preferably 20% to 95% by weight, preferably 30% to 90% by weight, preferably 40% to 85% by weight, preferably 50% to 80% by weight, preferably 60% to 70% by weight, based on the total weight of the composition.

[0159] According to one embodiment, the kit a) at least one (as defined above) microbubble according to the invention, or a composition as defined above, particularly a pharmaceutical composition as defined above, in a first container, and b) at least one therapeutic agent in a second container, and c) optionally, at least one targeting agent in a third container, and d) optionally, at least one marking agent in a fourth container, and e) optionally, instructions for preparation and / or use, and comprises, or consists essentially of, or consists of.

[0160] Advantageously, the kit further comprises means suitable for detecting the presence or absence of the marking agent in the sample and / or the subject.

[0161] The microbubbles and their components, such as lipophosphoramidate, histidyl polyethyleneimine, therapeutic agent, targeting agent, marking agent, biocompatible gas, are advantageously as described in the preceding paragraphs (the section of "Definitions", and / or "Lipid microbubble", and / or the section of "Methods of producing a lipid microbubble").

[0162] Thus, preferably, the therapeutic agent and / or the targeting agent and / or the labeling agent 1) a nucleic acid, and 2) a fat-soluble active ingredient, and 3) Chemotherapeutic agents, such as cytotoxic agents and / or cell growth inhibitors, and 4) antibodies, 5) proteins, 6) antigens, 7) toxins, 8) receptors, 9) enzymes, 10) hormones, 11) ligands, 12) viral vectors, 13) nanoparticles, preferably nanoparticles containing at least one agent selected from therapeutic agents, targeting agents, marking agents, and any combination thereof, 14) derivatives of any of 1) to 13), preferably functional derivatives of any of these, 15) fragments of any of 1) to 14), preferably functional fragments of any of these, 16) any combination of 1) to 15), and are included, consisting essentially of, consisting of, or selected from them. More preferably, they are from nucleic acids, lipid-soluble active ingredients, chemotherapeutic agents, antibodies, antibody derivatives, functional fragments of antibodies or their derivatives, proteins, protein fragments, nanoparticles, and any combination thereof.

[0163] Therapeutic Uses and Methods The inventors have shown, in particular, and surprisingly, that the innovative microbubbles developed herein have the ability to deliver a drug of interest in an accurate and targeted manner into the body, including very difficult-to-access areas such as the central nervous system, blood vessels, and tumor microenvironment. In fact, the inventors have shown, surprisingly, that the lipid microbubbles are significantly more stable than prior art microbubbles. In particular, they are able to actively pass through blood vessels, the blood-brain barrier (BBB), and the tumor microenvironment. The inventors have further demonstrated that local application of ultrasound enables these optimized microbubbles to be very precisely targeted to the area to be treated. Thus, these data reveal the therapeutic potential of these lipid microbubbles for treating a wide range of conditions, including central nervous system pathologies, in a targeted manner.

[0164] The data further show that these optimized microbubbles can be used as marking, detection, and imaging tools.

[0165] Accordingly, the present invention provides both a powerful and broad-spectrum treatment method for a condition, as well as a marking method, in particular a marking method for medical imaging.

[0166] Accordingly, the present invention relates to a pharmaceutical composition or kit comprising, or consisting essentially of, a microbubble according to the invention (as defined above), or at least one microbubble according to the invention (as defined above), or a pharmaceutical composition as defined above, or a kit as defined above, or any combination thereof, which are for use as a medicament.

[0167] The present invention further relates to a pharmaceutical composition or kit comprising, or consisting essentially of, microbubbles according to the present invention (as defined above), or at least one microbubble according to the present invention (as defined above), or a pharmaceutical composition as defined above, or a kit as defined above, or any combination thereof, which are for use as a marking agent, in particular as a contrast agent.

[0168] The present invention further relates to the use of microbubbles according to the present invention (as defined above), or a pharmaceutical composition or kit comprising, or consisting essentially of, at least one microbubble according to the present invention (as defined above), or a pharmaceutical composition as defined above, or a kit as defined above, or any combination thereof, which are for use as a medicament.

[0169] The present invention further relates to the use of microbubbles according to the present invention (as defined above), or a pharmaceutical composition or kit comprising, or consisting essentially of, at least one microbubble according to the present invention (as defined above), or a pharmaceutical composition as defined above, or a kit as defined above, or any combination thereof, which are for use as a marking agent, in particular as a contrast agent.

[0170] The present invention further relates to the use of microbubbles according to the present invention (as defined above), or a pharmaceutical composition or kit comprising, or consisting essentially of, at least one microbubble according to the present invention (as defined above), or a pharmaceutical composition as defined above, or a kit as defined above, or any combination thereof, which are for use in the manufacture of a medicament.

[0171] The present invention further relates to the use of microbubbles according to the present invention (as defined above), or a pharmaceutical composition or kit comprising, or consisting essentially of, at least one microbubble according to the present invention (as defined above), or a pharmaceutical composition as defined above, or a kit as defined above, or any combination thereof, which are for use in the manufacture of a marking agent, in particular a contrast agent.

[0172] The present invention further relates to a treatment method comprising administering microbubbles according to the present invention (as defined above), or a pharmaceutical composition or kit comprising or consisting essentially of at least one microbubble according to the present invention (as defined above), or a pharmaceutical composition as defined above, or a kit as defined above, or any combination thereof, which comprises administering these to a subject (preferably a subject in need thereof).

[0173] The present invention further relates to a marking method comprising administering microbubbles according to the present invention (as defined above), or a pharmaceutical composition or kit comprising or consisting essentially of at least one microbubble according to the present invention (as defined above), or a pharmaceutical composition as defined above, or a kit as defined above, or any combination thereof, which comprises administering these to a subject (preferably a subject in need thereof).

[0174] Microbubbles and their components, such as lipophosphoramidate, histidyl polyethyleneimine, therapeutic agents, targeting agents, marking agents, biocompatible gases, are preferably as described in the preceding paragraphs (the "Definitions" and / or the paragraphs of "Lipid microbubble" and / or "Methods of producing a lipid microbubble").

[0175] Preferably, the pharmaceutical composition and / or kit are as described above in the paragraph of "Compositions and kit".

[0176] According to a preferred embodiment, the microbubbles, compositions, kits, or any combination thereof are administered to a subject in need thereof, preferably in a therapeutically effective amount.

[0177] (In the context of the foregoing methods of use and treatment) The medical conditions treated by administration of microbubbles, compositions, kits, or any combination thereof can be of any type. In particular, the medical condition may be selected from vascular conditions, central nervous system conditions, tumors, cancers, and any combination thereof.

[0178] (In the context of the foregoing methods of use and treatment) The regions marked by administration of microbubbles, compositions, kits, or any combination thereof can be of any kind. The region marked can be any part of the subject's body being treated and / or marked. In particular, the region marked can be selected from vascular regions, central nervous system regions, tumor microenvironment regions, and any combination thereof.

[0179] Thus, the region being treated and / or the region being marked can advantageously be located in blood vessels, the central nervous system, the tumor microenvironment, or any combination thereof (preferably the central nervous system).

[0180] Microbubbles, compositions, kits, or any combination thereof are preferably formulated to be administered one or more times by the same or different routes. All conventional routes of administration, including oral, parenteral, and topical, are applicable in the context of the present invention.

[0181] The parenteral route is intended for administration by injection or infusion and includes systemic as well as local routes. Preferably, microbubbles, compositions, kits, or any combination thereof are administered one or more times by parenteral administration, preferably intravenous (into a vein), intravascular (in a blood vessel), intra-arterial (in an artery), intradermal (in the dermis), subcutaneous (under the skin), intramuscular (in the muscle). in the artery), intradermal (in the dermis), subcutaneous (under the skin), intramuscular (in the muscle) in (in muscle), intraperitoneal (in the peritoneum), or for administration intratumorally (in a tumor), it is formulated. It can be administered as a single bolus dose or via a continuous infusion pump.

[0182] Preferably, the microbubbles, compositions, kits, or any combination thereof are formulated for administration by intravenous injection.

[0183] Administration can be by conventional syringe and needle (e.g., Quadrafuse injection needle), or any compound or device available in the art that can facilitate or enhance the delivery of microbubbles to a subject (e.g., electroporation for intramuscular administration). An alternative is the use of a needleless injection device (e.g., Biojector™ device). Transdermal patches may also be considered.

[0184] Multiple doses within the indicated range may be administered to a subject. In the case of repeated administration over several days, the treatment is generally maintained until the appearance of an observable clinical benefit. These doses may be administered intermittently, e.g., daily, every 2 or 3 days, weekly, every 2 weeks, every 3 weeks, or monthly (e.g., such that the subject receives about 2 to about 20 doses of the composition). The doses can further be adapted for each administration (e.g., one or more higher initial doses followed by one or more lower doses).

[0185] In one embodiment, the microbubbles, compositions, kits, or any combination thereof are administered according to a "prime boost" approach that includes sequential administration of one or more priming compositions and one or more boosting compositions. Typically, the priming The composition and the boosting composition may use the same active agent (i.e., microbubbles, composition, kit, or any combination thereof), or may use different active agents (i.e., microbubbles, composition, kit, or any combination thereof). Further, the initial administration composition and the boosting composition may be administered to the same region or different regions of the body by the same administration route or different administration routes. Preferred initial administration (priming) approaches and boosting approaches include a first injection (e.g., subcutaneous, intramuscular, intradermal, intratumoral, or intravenous) (priming), followed by a second injection (e.g., subcutaneous, intramuscular, intradermal, intratumoral, or intravenous) after an optimal period. The present invention encompasses one or more administrations of the initial administration composition and / or the boosting composition (s), preferably via subcutaneous, intramuscular, intradermal, intratumoral, intranasal, and intravenous routes. The period between the initial administration and the boosting administration varies from 1 week to 6 months, preferably from 1 week to 1 month, and more preferably from 1 to 2 weeks.

[0186] The microbubbles, composition, kit, or any combination thereof are preferably administered in combination with the application of ultrasound, preferably local application of ultrasound, preferably local application of ultrasound on and / or to the area to be treated and / or marked. The ultrasound is preferably administered at a frequency in the range of 1 kHz to 10 MHz, preferably 10 kHz to 9 MHz, preferably 50 kHz to 8 MHz, preferably 100 kHz to 7 MHz, preferably 150 kHz to 6 MHz, preferably 200 kHz to 5 MHz, preferably 300 kHz to 4 MHz, preferably 400 kHz to 3 MHz, preferably 500 kHz to 2 MHz, preferably 750 kHz to 1.5 MHz, preferably 0.8 MHz to 1.2 MHz, and even more preferably at a frequency of about 1 MHz.

[0187] The ultrasonic waves are preferably pulsed at a frequency in the range of 1 Hz to 10 kHz, preferably 10 Hz to 9 kHz, preferably 50 Hz to 8 kHz, preferably 100 Hz to 7 kHz, preferably 150 Hz to 6 kHz, preferably 200 Hz to 5 kHz, preferably 300 Hz to 4 kHz, preferably 400 Hz to 3 kHz, preferably 500 Hz to 2 kHz, preferably 750 Hz to 1.5 kHz, and more preferably at a frequency of about 1 kHz.

[0188] The ultrasonic waves are preferably administered at a sound pressure in the range of 100 to 800 kPa (negative peak), preferably 200 to 700 kPa, more preferably 300 to 600 kPa, and even more preferably 400 to 500 kPa.

[0189] The ultrasonic waves are preferably administered for 30 to 300 seconds, preferably 40 to 250 seconds, preferably 50 to 200 seconds, preferably 60 to 180 seconds, preferably 80 to 150 seconds, and preferably 90 to 120 seconds.

[0190] The region to be treated and / or marked can be any part of the body of the subject to be treated and / or marked. The region is preferably selected from difficult-to-access zones such as the central nervous system, blood vessels, and tumor microenvironment. In fact, the inventors have shown, particularly surprisingly, that the lipid microbubbles according to the present invention are capable of actively passing through blood vessels, the blood-brain barrier (BBB), or even the tumor microenvironment. The inventors have further demonstrated that the local application of ultrasonic waves enables these optimized microbubbles to be very accurately targeted to the region to be treated. Thus, according to an advantageous embodiment, the microbubbles, compositions, kits, or any combination thereof are administered in combination with the local application of ultrasonic waves to the region(s) to be treated and / or mark the central nervous system, blood vessels, tumor microenvironment, or any combination thereof.

[0191] Advantageously, a method of administering microbubbles, a composition, a kit, or any combination thereof comprises, consists essentially of, or consists of the following steps: a) administering to a subject a microbubble, a composition, a kit, or any combination thereof by a suitable mode of administration (in particular, as described above, preferably parenterally, more preferably intravenously); b) applying ultrasound to the treatment and / or marked area (preferably according to the above frequency, pulse, sound pressure, and duration conditions).

[0192] The administration method may further include an additional step for preparing a microbubble, a composition, a kit, or any combination thereof before the administration step a), which, in particular, when the micro bubble, composition, kit, or any combination thereof is in lyophilized form, the additional steps are as follows: (i) optionally, a step of suspending the lyophilized product; (ii) optionally, a step of activating the microbubbles; (iii) optionally, mixing the microbubbles with an excipient (in particular, a diluent) and incubating for 0.5 to 15 minutes, preferably 1 to 10 minutes, preferably 1.5 to 8 minutes, preferably 2 to 6 minutes, preferably 1 to 5 minutes, preferably 1 to 3 minutes; (iv) optionally, contacting the microbubbles with a therapeutic agent, a targeting agent, a marking agent, or any combination thereof (the therapeutic agent, the targeting agent, the marking agent, or any combination thereof may be pre-mixed with an excipient (in particular, a diluent)).

[0193] According to one embodiment, the microbubbles are activated using a mechanical stirrer, for example, stirred at 2000 to 5000 revolutions per minute (rpm), preferably 4000 rpm, and the period is 10 to 120 seconds, preferably 45 seconds.

[0194] According to one embodiment, when microbubbles, compositions, kits, or any combination thereof are used for marking purposes, the method further includes a step of detecting the presence or absence of a marking agent.

[0195] In Vitro Applications and Methods The data demonstrate that the microbubbles according to the present invention can be effectively used as marking, detection, and imaging tools.

[0196] Accordingly, the present invention provides an efficient and reliable diagnostic method. In particular, it enables the diagnosis, prognosis, stratification, or monitoring of diseases, or the evaluation of treatment effectiveness.

[0197] Accordingly, the present invention relates to the in vitro use of at least one microbubble according to the present invention, or a composition or kit (especially a pharmaceutical) comprising, or consisting essentially of, at least one microbubble (as defined above) according to the present invention, or a (especially a pharmaceutical) composition as defined above, or a kit as defined above, or any combination thereof, which are in the following cases: i. Delivery of a drug (a therapeutic agent, a targeting agent, a marking agent, or any combination thereof) into a cell or a biological sample, ii. Marking of a cell or a biological sample, iii. Diagnosis of a disease in a subject who may be suffering from the disease, iv. Follow-up care for a subject suffering from a disease, v. Stratification of a subject suffering from a disease, vi. Evaluating the effectiveness of a (especially curative) treatment administered to a subject suffering from a disease, vii. Detecting the presence or absence of at least one microbubble in a sample, especially a biological sample, viii. Determining the presence or amount of at least one marking agent (especially a contrast agent) in a sample, especially a biological sample, ix. Screening for a compound / molecule that is effective in the prevention, treatment, marking, or any combination thereof of a disease, or Any combination of i. to ix.

[0198] The present invention particularly relates to the in vitro use of at least one microbubble according to the present invention, or a composition or kit comprising or consisting essentially of (in particular, a pharmaceutical) at least one microbubble (as defined above) according to the present invention, or a ( In particular, a pharmaceutical) composition, or a kit as defined above, or any combination thereof, preferably relating to the delivery of a drug into the interior of at least one cell or biological sample, preferably by sonoporation, The drug is preferably selected from therapeutic agents, targeting agents, marking agents, and any combination thereof. The cells are preferably selected from animal cells, plant cells, microbial cells, and any combination thereof.

[0199] The microbubble, composition, kit, or any combination thereof is preferably brought into contact with (in particular, administered to) the cell or biological sample in combination with the application of ultrasound, preferably local application of ultrasound, preferably local application of ultrasound over and / or to the region to be treated and / or marked. The ultrasound is preferably applied at a frequency in the range of 1 kHz to 10 MHz, preferably 10 kHz to 9 MHz, preferably 50 kHz to 8 MHz, preferably 100 kHz to 7 MHz, preferably 150 kHz to 6 MHz, preferably 200 kHz to 5 MHz, preferably 300 kHz to 4 MHz, preferably 400 kHz to 3 MHz, preferably 500 kHz to 2 MHz, preferably 750 kHz to 1.5 MHz, preferably 0.8 MHz to 1.2 MHz, and even more preferably at a frequency of about 1 MHz.

[0200] The ultrasonic waves are preferably pulsed at a frequency in the range of 1 Hz to 10 kHz, preferably 10 Hz to 9 kHz, preferably 50 Hz to 8 kHz, preferably 100 Hz to 7 kHz, preferably 150 Hz to 6 kHz, preferably 200 Hz to 5 kHz, preferably 300 Hz to 4 kHz, preferably 400 Hz to 3 kHz, preferably 500 Hz to 2 kHz, preferably 750 Hz to 1.5 kHz, more preferably about 1 kHz.

[0201] The ultrasonic waves are preferably administered at a sound pressure in the range of 100 to 800 kPa (negative peak), preferably 200 to 700 kPa, more preferably 300 to 600 kPa, even more preferably 400 to 500 kPa.

[0202] The ultrasonic waves are preferably applied for 30 to 300 seconds, preferably 40 to 250 seconds, preferably 50 to 200 seconds, preferably 60 to 180 seconds, preferably 80 to 150 seconds, preferably 90 to 120 seconds, preferably 60 seconds.

[0203] Advantageously, the method of administering microbubbles, a composition, a kit, or any combination thereof comprises, consists essentially of, or consists of the following steps: a) contacting the microbubbles, composition, kit, or any combination thereof with a cell or biological sample, or administering the microbubbles, composition, kit, or any combination thereof, the biological sample, by an appropriate mode of administration (such as those described above in the section "Therapeutic Uses and Methods"); b) applying ultrasonic waves to the treatment and / or marked area (preferably according to the above frequency, pulse, sound pressure, and duration conditions).

[0204] The administration method may further include an additional step for preparing microbubbles, a composition, a kit, or any combination thereof, before the administration step a), provided that, particularly when the microbubbles, the composition, the kit, or any combination thereof is in lyophilized form, the additional step is as follows: (i) Optionally, a step of suspending the lyophilized product, (ii) Optionally, a step of activating the microbubbles, (iii) Optionally, mixing the microbubbles with an excipient (particularly a diluent) and incubating for 0. 5 to 15 minutes, preferably 1 to 10 minutes, preferably 1.5 to 8 minutes, preferably 2 to 6 minutes, preferably 1 to 5 minutes, preferably 1 to 3 minutes, (iv) Optionally, contacting the microbubbles with a therapeutic agent, a targeting agent, a marking agent, or any combination thereof (the therapeutic agent, the targeting agent, the marking agent, or any combination thereof may be pre-mixed with an excipient (particularly a diluent)).

[0205] According to one embodiment, when the microbubbles, the composition, the kit, or any combination thereof is used for marking purposes, the method further includes a step of detecting the presence or absence of a marking agent in cells and / or biological samples.

[0206] Lipophosphoramidate, histidyl polyethyleneimine, histidyl polyethyleneimine, therapeutic agent, targeting agent, marking agent, biocompatible gas are advantageously as described in the preceding paragraphs (the paragraphs of "Definitions" and / or "Lipid Microbubbles" and / or "Method for Producing Lipid Microbubbles").

[0207] Advantageously, the composition and / or the kit are as described above in the paragraph of "Compositions and Kits".

[0208] The following examples are intended to illustrate the invention and should not be regarded as limiting.

Brief Description of the Drawings

[0209]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Example

[0210] Example: Design, Development, and Characterization of Microbubbles for Targeted Delivery of Active Ingredients Introduction In connection with the present invention, new formulations of gas microbubbles have been developed. These microbubbles can encapsulate different types of active ingredients, such as nucleic acids, and can deliver them locally after activation by focused ultrasound, which is particularly advantageous (Figure 1). New devices have also been developed that enable ultrasound to be delivered to the mouse brain in a targeted manner. This system is coupled to these original gas microbubbles for encapsulation and delivery of the active ingredient. The formulation of the gas microbubbles can be activated and then the active ingredient can be present in the formulation using a stirrer or added after the formulation has been activated. The device is electrified and positioned at coordinates corresponding to the desired delivery site of the user. The original microbubbles developed here are capable of passing through the BBB. In this way, the positioning system developed can be implemented using a brain atlas to locate the brain structure to be treated. The gas microbubbles are injected systemically, followed by ultrasound. This device can be equipped with a passive cavitation detection system for real-time monitoring of the ultrasonic activation of the microbubbles.

[0211] Microbubble gas formulations can be used to encapsulate or co-encapsulate various active ingredients: (i) In particular, lipophilic active ingredients such as chemotherapeutic agents (e.g., paclitaxel) on the surface of the microbubbles and / or within the envelope, (ii) Anionic active substances such as nucleic acids (plasmid DNA, small RNAs, messenger RNAs, etc., e.g., for gene therapy), where, for example, cationic lipids are used, (iii) Antibodies using, for example, streptavidin-biotin couples, or chemical clicks, or lipids coupled to methyltetrazine groups, (iv) Proteins or peptides (such as cell-penetrating peptides (CPPs)), using, for example, lipids coupled to streptavidin-biotin, chemical clicks or methyltetrazine groups.

[0212] This high degree of flexibility is due, in particular, to the original formulation of the microbubbles using a mixture of cationic molecules such as lipophosphoramidate and / or histidyl polyethyleneamine.

[0213] In fact, two types of cationic microbubbles have been developed: one based on lipophosphoramidate and the other based on histidyl polyethyleneamine conjugated to fatty acids.

[0214] These microbubbles have already shown their in vitro and in vivo effectiveness as ultrasonic contrast agents (for therapy and imaging). The blood-brain barrier is transiently permeabilized without causing danger to the animal, as verified by MRI imaging and histology. The expression of the luciferase transgene was detected after the sonoporation protocol.

[0215] Example 1: Microbubbles Containing Lipophosphoramidate 1.1 Materials and Methods 1.1.1. Production of Microbubbles (MB) According to the Invention The first step in MB production is to mix different lipids according to the type of microbubble required.

[0216] 1.1.1.1. MB with KLN27 KLN27 and MM30 lipids (Figure 2) are derived from a joint study with the University of Berchel. These are phosphoramidate lipids, where lipid 1 (KLN27) is essentially cationic, while lipid 2 (MM30) is used as a fusogenic lipid (a lipid capable of fusing with membranes, particularly used to promote endosomal escape). All other lipids used (DMPC, DSPC, DMPE-PEG2000, DSPE-PEG2000, DSPE-PEG2000 biot) were supplied by Avanti Polar Lipids (Alabaster, Alabama, USA). The various lipids are mixed in a flask in the presence of absolute ethanol (purity 99.96%). During this step, PTX solubilized in absolute ethanol (10 mM, manufactured by Merck, Germany) is added according to the formulation (Table 2).

[0217]

Table 2

[0218] Next, a Rotavapor (manufactured by Buchi, Schwaebach, Germany) is used to evaporate the mixture at 20 rpm and 60 °C for 30 minutes. After this stage, a lipid film is formed in the flask. The formed lipid membrane is taken up in 2 mL of HEPES (10 mM, pH 7.4) and sonicated for 5 minutes to obtain a homogeneous lipid solution (Figure 11). Next, the solution is distributed equally into four crimp vials (manufactured by VWR International, Radnor, Pennsylvania, USA) and stored at -80 °C for 1 hour. Finally, the vials are placed in a freeze dryer (manufactured by Bioblock Scientific, Illkirch, France) overnight. Once the lyophilized product is recovered, the vials are crimped by hand and stored at 4 °C before activation. Activation is achieved by overpressurizing the air in the flask with perfluorobutane (C4F10, manufactured by F2 Chemical, UK Replace with (s), and then rehydrate the lyophilized product with 500 μl of 10 mM HEPES solution. Stir the flask for 45 seconds using VIALMIX (manufactured by Bristol Myers Squibb, USA). This step forms microbubbles in the vial, but it is necessary to wait for 5 minutes after stirring before sampling. Once activated, the vial can be stored at 4°C for several days.

[0219] 1.1.1.2. MB with KLN25 MB containing phosphoramidate lipid KLN25 is also prepared as described above (paragraph 1.1.1.1).

[0220] The ratios (mol%) of the various lipids constituting the envelope are as follows with respect to the total molar amount of lipids: - 47.5% of KLN25, - 47.5% of MM27, and - 5% of DSPE-PEG(5000).

[0221] 1.1.1.3. MB containing dipalmitoylphosphoramidate MB containing dipalmitoylphosphoramidate lipid is also prepared as described above (paragraph 1.1.1.1).

[0222] The ratios (mol%) of the various lipids constituting the envelope are as follows with respect to the total molar amount of lipids: - 47.5% of dipalmitoylphosphoramidate, - 47.5% of MM27, and - 5% of DSPE-PEG(5000).

[0223] 1.1.1.4. MB containing distearoylphosphoramidate MB containing distearoylphosphoramidate lipid is also prepared as described above (paragraph 1.1.1.1).

[0224] The ratios (mol%) of the various lipids constituting the envelope are as follows with respect to the total molar amount of lipids: - 47.5% distearoyl phosphatamidate, - 47.5% MM27, and - 5% DSPE-PEG(5000).

[0225] 1.1.2. Anionic MB (comparative example) Anionic microbubbles (MBa) are generated using the method described in paragraph 2.1.1 below.

[0226] 1.1.3. Characterization of microbubbles 1.1.3.1. Concentration and size Microbubbles are observed using an inverted microscope (Nikon Diaphot 300 invert) connected to a computer. A FASTCAM SA7 camera (manufactured by Photron, USA) and ICcapture (registered trademark) software are used to take pictures. Pictures are taken with different lenses (×10, ×20, ×40) and then processed by ImageJ (registered trademark). The processing consists of 8-bit image conversion followed by thresholding to detect the MB contour. Next, particle analysis is used to count the number of particles and obtain their sizes. Microbubbles are diluted 1 / 10 or 1 / 100 in HEPES (10 mM, pH 7.4) and then placed on a Malassez slide.

[0227] 1.1.3.2. Measurement of zeta potential (ζ) The ζ potential corresponds to the total charge of the particles at the shear plane (particle surface). It is measured using a Nano partica SZ-100 (Horiba, Japan). To perform the measurement, 30 μL of MB is diluted with 970 μL of 10 mM HEPES pH 7.4. The analysis is carried out at 25°C.

[0228] 1.2 Results 1.2.1. Characterization of Microbubbles According to the Present Invention and Comparison with Anionic Microbubbles The size and concentration of the microbubbles prepared according to this example (MB according to the present invention prepared as described in paragraph 1.1.1 above) are evaluated by optical imaging.

[0229] 1.2.1.1. MB with KLN27 Figure 3 shows the results obtained for the different formulations in Table 2. The size information collected shows an average diameter of 1.41 μm for MBc and 1.41 μm for MBc-PTX, but 1.55 μm for MBc-t. MBc-tPTX has an average diameter of 1.98 μm. For anionic microbubbles, anionic MB (MBa) has an average size of 1.41 μm. The concentrations of the different formulations are shown in Table 3. The size and concentration distributions remain homogeneous between the two main categories of microbubbles (cationic and anionic). Generally speaking, anionic MB (similar to commercially available MB) is similar in size to cationic MB but has a higher concentration. MBc functionalized with PTX is slightly smaller than the basic formulation (MBc) and appears to be at a higher concentration. MBc-tPTX, which has both biotinylated lipid and PTX, has the largest size and the lowest concentration. However, the size distribution of the MB remains below 10 μm, enabling injection in vivo.

[0230] [Table 3]

[0231] Figure 4 shows the ζ-potential measurements of the developed formulations. These results show that there is no significant change in the overall charge when MBc is simultaneously functionalized with PTX, biotin, or both. These remain positive (average MBc: +28.8 mV). The MBa formulation, which does not have KLN27 (cationic lipid), serves as a control with a charge of -23.4 mV.

[0232] 1.2.1.2. MB with KLN25 Figure 5 shows the results obtained for the KLN25-containing formulation, confirming the possibility of forming KLN25-containing MBs. The size and concentration distributions of the MBs containing KLN25 are comparable to those of the MBs containing KLN27.

[0233] 1.2.1.3. MBs Containing Dipalmitoylphosphoramidate or Distearoylphosphoramidate The results obtained for the formulations containing dipalmitoylphosphoramidate or distearoylphosphoramidate are equivalent to those obtained for the formulations containing KLN27 or KLN25. The size and concentration distributions of the MBs containing dipalmitoylphosphoramidate or distearoylphosphoramidate are comparable to those of the MBs containing KLN27 or KLN25. These data confirm the possibility of forming MBs from dipalmitoylphosphoramidate or distearoylphosphoramidate.

[0234] 1.2.2. Complexation of Nucleic Acids The ability of microbubbles to vectorize nucleic acids is evaluated by gel retardation. Figure 6 shows the complex formation ability of MBc microbubbles (having the cationic lipid KLN27) in the presence of 1 μg of plasmid DNA (plasmid pLuc). In the absence of microbubbles, a band migrating at approximately 3 kilobases appears after UV visualization. This band corresponds to non-complexed plasmid DNA. As the MB concentration increases, the intensity of the band at 3 kb decreases, which corresponds to the complex formation of pDNA and microbubbles. When pDNA is complexed, it no longer migrates and remains in the deposition well. When 10 μL of MBs are used, all of the pDNA is complexed, the 3 kb band is no longer visible, and instead is marked in the well.

[0235] This complex formation ability was also confirmed by confocal fluorescence microscopy (Figure 7). MBc-tPTX is complexed with FITC-conjugated CpG oligonucleotide for 2 minutes at a ratio of 1 μg of nucleic acid to 10 μL of microbubble. Fluorescence around the microbubble confirms nucleic acid complex formation. Fluorescent debris is seen around the microbubble, presumably indicating the formation of lipoplexes from fragmented microbubbles that have burst or free lipids in solution.

[0236] 1.2.3. Evaluation of functionalized MB targeting To evaluate the ability of MB to target the VEGF receptor, hCMEC / D3 cells stimulated to produce VEGFR are cultured in Ibidi® and subsequently flow analysis of MB is performed using an optical microscope. Seed 18,000 cells / channel in a 6-channel flow culture plate (Ibidi® μ-Slide VI0.4, Clinisciences). Once the cells have adhered to the bottom of the channel, fill the wells with culture medium supplemented (or not) with 5 ng / ml of TGFβ and incubate the cells at 37 °C in a 5% CO2 atmosphere for 24 or 48 hours. The optimal TGFβ concentration for increasing the number of VEGFR2 receptors on the endothelial cell surface is determined by flow cytometry.

[0237] Next, place the Ibidi® plate on an inverted microscope equipped with a camera and connect it to a computer. The settings used throughout the experiment are shown in Figure 8.

[0238] To analyze microbubble attachment, a field of view is selected. Different types of microbubbles are used (described in Table 2 above). After flow cytometry analysis, some microbubbles are functionalized with an antibody against the VEGF receptor (VEGFR2). For this purpose, 1.23 μL of streptavidin (15 mM) is incubated in the presence of 0.3465 μL of anti-VEGFR 2-Biot antibody (anti-mouse CD309, 0.5 mg / mL, eBioscience) to obtain a ratio of 2.5 moles of antibody per mole of streptavidin. Next, 20 μL of the MB solution is incubated for 10 minutes to functionalize the MBs and prevent the formation of microbubble aggregates by streptavidin-biotin binding. At the end of 10 minutes, a solution of 10 mM HEPES pH 7.4 is added to reach a final volume of 1 mL.

[0239] The remainder of the experiment consists of a series of washes of the cultured cells. First, a 10-minute wash with PBS is performed at a flow rate of 0.137 mL / min (0.25 dyn / cm 2 ). Next, the MBs contained in 1 mL are injected at the same flow rate for 15 minutes, followed by a first rinse with PBS for 10 minutes, then a second rinse at 0.275 mL / min (0.5 dyn / cm 2 ) for 10 minutes, and finally a third rinse at 0.550 mL / min (1 dyn / cm 2 ) for 10 minutes, followed by a final rinse at 1.1 mL / min (2 dyn / cm 2 ). Photographs of the channel are taken every minute after injection. Next, a series of photographs are processed with ImageJ® to obtain a count of the number of MBs per minute.

[0240] The ability of microbubbles to target the VEGF receptor (VEGFR-2) was evaluated in real time in vitro using a flow-through culture chamber. The cells used in this analysis were hCMEC / D3 endothelial cells that were either stimulated or not stimulated with TGFβ. Different formulations were tested on these cells to evaluate the effect of different components on microbubble attachment ability (Figure 9).

[0241] Figure 9A shows the results obtained for the anionic formulation MBa. The volume of MB injected is the same for each condition tested. This formulation is tested on cells stimulated for 24 hours without antibody (dashed line), on unstimulated cells with antibody (dotted line), and on cells stimulated for 24 hours with antibody (solid line). At t = 15 minutes, the first wash begins. The results show a very small number of bound MBs on the dashed curve corresponding to non-specific interactions of these MBs on the cells, and 42 MBa bound at t = 15 minutes. This number drops to 27 at the end of the wash. For the dotted curve corresponding to specific binding of MB to the cells, the number of bound MBs is 112 at t = 15 minutes. This number decreases to 33 at the end of the wash. The solid curve corresponding to cells stimulated to produce VEGFR2 in the presence of the target MB shows a number of bound MBs of 102 at t = 15 minutes. This number decreases to 52 at the end of the wash. Thus, this curve records the highest number of attachments over time.

[0242] Figures 9B and 9C show the results obtained for the cationic formulation MBc-tPTX (incorporating PTX and biotinylated lipid). This formulation is tested on cells stimulated at 24 hours and 48 hours. The injection and wash rates are halved compared to Figure 9A so as not to stress the cells too quickly. When the cationic microbubbles are functionalized with the anti-VEGFR 2 antibody, they bind more than the microbubbles not functionalized with the antibody (Figures 9B, 9C). In the absence of nucleic acid complexation, the number of antibody-fixed MBs is approximately 110% higher than the number of antibody-free MBs. Complex formation of CpG oligonucleotides (Figure 9C) by the microbubbles decreases the receptor-binding ability. When the microbubbles contain both antibody and nucleic acid, the number of bound MBs is approximately 60% higher than the antibody-free control.

[0243] 1.2.4. In vivo experiments 1.2.4.1. In vivo sonoporation To perform MB and USF transfection, the hair on the mouse's skull must be removed to allow for complete US transmission between the skin and the ultrasonic probe. Once the mouse is prepared, it is anesthetized throughout the experiment with a 1.5% isoflurane (Vetflurane, France) oxygen / air mixture. A catheter with a 26G needle is inserted into the tail vein for intravenous injection. An injection of Evans blue (5%, 1 mL / kg saline solution) is performed. Evans blue is a dye that binds to circulating albumin and does not naturally cross the BBB. However, its extravasation is visible after opening via MB+USF and allows us to obtain information regarding the location of the regions targeted by our protocol when the brain is removed from the skull.

[0244] Next, the mouse is placed in the cradle of the in vivo sonoporation platform (Figure 10). USF is applied using a single-element focused ultrasonic transducer (Precision Acoustics, UK) with a diameter of 54 mM. The transducer is placed within a sealed cylinder filled with degassed water and sealed with a membrane. Air bubbles should be avoided to prevent inadequate US propagation. The transducer is positioned on an electric control platform connected to a computer and it is possible to move the transducer using Repetier software. The transfection zone is visually targeted using a pointer located on the transducer. The selected zone corresponds to half of the eye-ear distance in the left hemisphere (zone 2). Ultrasonic conducting gel is applied to the mouse's skull to enable US transmission. After aiming, the transducer is positioned over the aiming zone and then lowered to contact the gel.

[0245] Next, a solution of 30 μg of pDNA diluted in 60 μL of 10 mM HEPES pH 7.4 and 20 μL of 20% sucrose is prepared. This solution is incubated for 2 minutes in the presence of 120 μL of MBc and then intravenously injected into the mouse. Ten seconds after the end of the injection, US is sent for 60 seconds (1 MHz, 5% duty cycle, pulse duration 1 second). Two sound pressures were tested: 109 kPa and 145 kPa. After US application, the mouse was awakened.

[0246] 1.2.4.2. Measurement of luciferase activity (RLU assay) Twenty-four hours after sonoporation, mice transfected with the plasmid encoding luciferase are euthanized and their brains are harvested. The brains are divided into six parts: zones 1, 2, and 3 correspond to the left side of the brain, and zones 4, 5, and 6 correspond to the right side. The transfection zone targeted by our protocol is located in zone 2 and is visualized by Evans blue overflow (Figure 11).

[0247] Next, each section is placed in an Eppendorf (registered trademark) tube and immersed in liquid nitrogen. Next, the sections are manually ground in a mortar in the presence of liquid nitrogen and then the ground material is solubilized in 500 μL of CCLR lysis buffer for 1 hour 30 minutes. Centrifugation is performed (5 minutes, 12,000 RPM, 4 °C) and 150 μL of the supernatant is collected for reading with a Lumat LB9507 luminometer (manufactured by Berthold, Germany). Next, the instrument is loaded with a solution of LAR (luciferase assay reagent) to measure luciferase activity. Next, the remaining lysate is used to determine the protein concentration contained in each zone. This quantification is performed using the BCA assay. Next, the obtained values are normalized with the luciferase activity value (RLU / mg protein).

[0248] The efficiency of nucleic acid delivery was tested by measuring the activity of a reporter gene encoding luciferase. This gene was transfected into the brain using cationic microbubbles (MBc) and focused ultrasound. Two different sound pressures, 109 kPa (n = 8) and 145 kPa (n = 5), were tested.

[0249] The results shown in Figure 12 show strong luciferase activity (between 5.5 × 10 3 RLU / mg protein and 1.05 × 10 4 RLU / mg protein) in zones 2 and 3 of the mouse brain for the two ultrasound outputs used. Luciferase expression was significantly different between zones 2 and 4, and between zones 2 and 5 at 109 kPa. The results obtained at 145 kPa were not significantly different from those obtained at 109 kPa. Luciferase activity was low (less than 10 3 RLU / mg protein) in brain zones 1, 4, 5, and 6. It should be noted that these values are close to the background values of non-transfected tissue. Since targeting is not localization and zoning is inaccurate, zones 1 and 3 can show luciferase activity. Thus, high luciferase expression was mainly measured in the left hemisphere (where targeting occurs) and not in the right, confirming the possibility of using focused ultrasound combined with cationic microbubbles for nucleic acid delivery.

[0250] 1.2.4.3 Immunohistochemistry Brain sections from mice transfected with different plasmids were cut to study the regions of transfection and the cell types involved. The transfection zone was determined by observing the extravasation of Evans blue on the sections.

[0251] Example 2: Microbubbles Containing Histidylated Polyethyleneimine (MBPEI) 2.1. Materials and Methods 2.1.1. Production of Anionic Microbubbles (Comparative Example) The production of anionic microbubbles (MBa) involves several steps. The first involves mixing phospholipids in a 50 mL flask. For this formulation of MB, distearoylphosphatidylcholine (DSPC) is mixed with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE) in a ratio of 90% - 10% - 87% - 13% (molar ratio) according to the batch of MB in 99.9% ethanol with a final volume of 2 mL. Next, this solution is heated to 60 °C under vacuum in a Rotavapor (Büchi, Schwaebach, Germany) until a lipid film is formed in the flask. Next, this film is resuspended in 2 mL of HEPES (10 mM, pH 7.4, filtered through a 0.2 μm filter) and sonicated in a sonication bath (35 kHz) for 5 minutes. Next, this solution is dispensed into four glass vials and stored at -80 °C for 1 hour. Finally, the vials are lyophilized for at least 12 hours (Bioblock Scientific, Illkirch, France). Next, the vials are crimped and sealed and stored at 4 °C until activation.

[0252] The vials are activated by replacing the air inside with a high molecular weight gas, perfluorobutane (C4F10, F2 Chemicals, UK), and then 500 μL of HEPES (10 mM, pH 7.4, filtered through a 0.2 μm filter) is added to the vials using a syringe. Next, the vials are shaken for 45 seconds using VIALMIX (Bristol Myers Squibb, USA). The MB thus produced is called MB1.

[0253] 2.1.2. Production of MBPEI microbubbles according to the present invention Cationic MBs containing histidyl polyethyleneimine are produced by a process involving steps similar to those described in paragraph 2.1.1 (MB1 production) above. However, here, PEI (polyethyleneimine + stearic acid) lipids conjugated to histidine are used to obtain a positively charged envelope. The method of activating these microbubbles is the same as in the case of MB1s. The MBs thus produced are called MBPEIhis.

[0254] 2.1.3. Characterization of Microbubbles 2.1.3.1. Concentration and Size Microbubbles are observed using an inverted microscope (Nikon Diaphot 300 invert) connected to a computer. Photographs are taken using a FASTCAM SA7 camera (manufactured by Photron, USA) and ICCapture (registered trademark) software. Photographs are taken with different lenses (×10, ×20, ×40) and then processed by ImageJ (registered trademark). The processing consists of 8-bit image conversion followed by thresholding to detect the MB contours. Next, particle analysis is used to count the number of particles and obtain their sizes. Microbubbles are diluted 1 / 10 or 1 / 100 in HEPES (10 mM, pH 7.4) and then placed on a Malassez slide.

[0255] 2.1.3.2. Evaluation of In Vitro Nucleic Acid Delivery In vitro sonoporation experiments were performed on HeLa and HepG2 cell lines at 20,000 cells / well (HeLa) and 30,000 cells / well (HepG2) using plasmid DNA encoding GFP protein. To evaluate the transfection ability of MBs in the presence or absence of US, each sonoporation condition was performed in duplicate on a 48-well culture plate. Sonoporation transfection was performed in 190 μL of Opti-MEMTM medium containing 10 μL of MB / pDNA complex solution It was carried out. Each transfection condition in the presence of US was treated for 1 minute. After transfection, the culture plate was cultured in Opti-MEMTM medium at 37 °C under 5% CO2 for 30 minutes, and then cultured in a conventional culture medium at 37 °C under 5% CO2 for 48 hours. The transfection results were analyzed by a fluorescence microscope 24 hours after sonoporation.

[0256] 2.2. Results 2.2.1. Characterization of MB according to the present invention and comparison with MBa The size and concentration of the MB (MBPEIhis according to the present invention prepared as described in paragraph 2.1.2 above) prepared according to this example were evaluated by optical imaging. Figure 13 shows the results obtained for different formulations. The size information collected shows an average diameter of 1.55 μm for MBa and 1.31 μm for MBPEIhis. Therefore, the size distribution of MBPEIhis is less than 10 μm, enabling them to be injected in vivo.

[0257] The average concentration is 2.18×10 10 MB / mL for MBa and 8.51×10 9 MB / mL at 1.31 μm for MBPEIhis.

[0258] 2.2.2. Evaluation of in vitro nucleic acid delivery The in vitro nucleic acid delivery efficiency was measured using the eGFP reporter gene. This gene was transfected into the HeLa cell line using MBPEIs or MB1 in the presence or absence of US. Different MB:pDNA ratios and sound pressures were tested. Next, the results of this transfection were analyzed by a fluorescence microscope, showing the sonoporation transfection efficiency of MBPEIs microbubbles (Figure 14).

[0259] Discussion Collectively, these data indicate that the innovative microbubbles developed by the inventors are capable of stably transporting different types of drugs containing nucleic acids in the bloodstream, which includes the nucleic acids being stably present in the bloodstream, passing through the blood-brain barrier (BBB), and delivering the drugs in a targeted manner, particularly to the antigen of interest.

[0260] In particular, the inventors have surprisingly shown that the lipid microbubbles thus developed have significantly improved stability, unlike the microbubbles described in the prior art. Notably, the data further reveals that these optimized microbubbles are capable of delivering different types of drugs of interest containing nucleic acids more efficiently than the microbubbles described in the prior art. In particular, interestingly, these microbubbles are capable of passing through blood vessels as well as the BBB. The inventors have further demonstrated that the local application of ultrasound enables these optimized microbubbles to be very precisely targeted to the area to be treated. Therefore, these data reveal the therapeutic potential of these lipid microbubbles for treating a wide range of lesions, including central nervous system lesions, vascular lesions, cancers, and tumors, in a targeted manner.

[0261] The data further shows that these optimized microbubbles are effective detection and imaging tools.

[0262] Therefore, the present invention provides both a powerful and broad-spectrum treatment method for medical conditions and an efficient and reliable diagnostic method.

[0263] References Zhu X,Guo J,He C,Geng H,Yu G,Li J,Zheng H,Ji X,Yan F. Ultrasound triggered image-guided drug delivery for inhibiting vascular remodeling by paclitaxel-loaded microbubbles delivery to inhibit vascular reconstruction via paclitaxel-loaded microbubbles ).Sci Rep.2016 / 2 / 22 6:21683.doi:10.1038 / srep21683.PMID:26899550, PMCID:PMC4761943. Fan CH, Ting CY, Liu HL, Huang CY, Hsieh HY, Yen TC, Wei KC, Yeh CK. Antiangiogenic-targeting drug-loaded microbubbles combined with focused ultrasound for the treatment of glioma. microbubbles combined with focused ultrasound for glioma treatment).Biom aterials.March 2013, 34(8):2142-55.doi:10.1016 / j.biomaterials.2012.11.048.Epub December 14, 2012. PMID:23246066. Delalande A, Bastie C, Pigeon L, Manta S, Lebertre M, Mignet N, Midoux P, Pichon C. Cationic gas-filled microbubbles for ultrasound-based nucleic acids delivery. Biosci Rep. 2017 Dec 22,37(6):BSR20160619. doi:10.1042 / BSR20160619. PMID:29180378, PMCID:PMC5741830. Fan CH, Chang EL, Ting CY, Lin YC, Liao EC, Huang CY, Chang YC, Chan HL, Wei KC, Yeh CK. Folate-conjugated gene-carrying microbubbles with focused ultrasound for concurrent blood-brain barrier opening and local gene delivery . Biomaterials. November 2016, 106:46-57. doi:10.1016 / j.biomaterials.2016.08.017. Epub August 12, 2016. PMID:27544926.

Claims

1. A lipid microbubble comprising at least one cationic compound selected from lipophosphoramidate, histidyl polyethyleneimine, and any mixture thereof.

2. Further comprising at least one agent selected from a therapeutic agent, a targeting agent, a marking agent, and any combination thereof, wherein the at least one agent is preferably i. exposed on the surface of the microbubble, or ii. embedded in the lipid envelope of the microbubble, or iii. incorporated into the interior of the microbubble, or iv. any combination of i to iii. The microbubble according to claim 1.

3. The at least one agent is 1) a nucleic acid, 2) a lipid-soluble active ingredient, 3) a chemotherapeutic agent such as a cytotoxic agent and / or a cell growth inhibitor, 4) an antibody, 5) a protein, 6) an antigen, 7) a toxin, 8) a receptor, 9) an enzyme, 10) a hormone, 11) a ligand, 12) a viral vector, 13) a nanoparticle, preferably a nanoparticle containing at least one agent selected from a therapeutic agent, a targeting agent, a marking agent, and any combination thereof, 14) a derivative of any of 1) to 13), preferably a functional derivative of any of these, 15) a fragment of any of 1) to 14), preferably a functional fragment of any of these, 16) any combination of 1) to 15). The microbubble according to claim 1 or claim 2, characterized in that it is selected from

4. Further comprising at least one functional group that enables binding to at least one agent selected from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof, wherein the group is preferably i. exposed on the surface of the microbubble, or ii. embedded in the lipid envelope of the microbubble, or iii. incorporated into the interior of the microbubble, or iv. any combination of i to iii. The microbubble according to any one of claims 1 to 3.

5. The functional group is - a peptide label, - a chemical group, preferably a chemical group selected from a clickable functional group, a coupling group, and any combination thereof, - an antibody, - an antibody derivative, - a functional fragment of an antibody or an antibody derivative, - an affinity label, - The microbubble according to claim 4, selected from any combination of these.

6. The lipophosphoramidate is selected from the group consisting of dimyristoylphosphoramidate, preferably dimyristoyl bromide phosphoramidate, dimyristoyl histamine phosphoramidate, and any combination thereof, preferably dioleylmethylimidazolium phosphoramidate and any combination thereof, dioleylphosphoramidate, dipalmitoylphosphoramidate, distearoylphosphoramidate, and any mixture thereof, characterized in that the microbubble according to any one of claims 1 to 5 is selected therefrom.

7. The histidyl polyethyleneimine is coupled to a fatty acid, and the fatty acid is preferably selected from stearic acid, myristic acid, palmitic acid, oleic acid, and any combination thereof, characterized in that the microbubble according to any one of claims 1 to 6 is selected therefrom.

8. Dimyristoyl-glycero-phosphocholine, distearoyl-glycero-phosphocholine, dimyristoyl-glycero-phosphoethanolamine-polyethylene glycol, distearoyl-glycero-phosphoethanolamine-polyethylene glycol, distearoyl-glycero-phosphoethanolamine-[biotinyl(polyethylene glycol)], cholesterol, β-sitosterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE ), 1-oleoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-3-trimethylammonium propane (DOTAP), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), clickable lipid DBCO, tetrazine, methyltetrazine, NHS (DSPE-PEG2000-X), and additional lipids selected from the group consisting of any combination thereof. The microbubble according to any one of claims 1 to 7, further comprising The microbubble according to any one of claims 1 to 7, further comprising an additional lipid selected from the group consisting of DOTAP, DMG-PEG2000, clickable lipid DBCO, tetrazine, methyltetrazine, NHS (DSPE-PEG2000-X), and any combination thereof. **Claim 9** A biocompatible gas, wherein the gas is preferably selected from perfluorobutane (C4F10), perfluoropropane (C3F8), dinitrogen (N2), sulfur hexafluoride (SF6), nitrogen oxides (NO), hydrogen, dioxygen, helium, xenon, argon, nitrous oxide (N2O), and any mixture thereof. The microbubble according to any one of claims 1 to 8. **Claim 10** The concentration of the microbubbles in the composition is preferably 10 ６ to 10 １４ microbubbles / ml, more preferably 10 ７ to 10 １３ microbubbles / ml, more preferably 10 ８ to 10 １２ microbubbles / ml, more preferably 10 ９ to 10 １１ microbubbles / ml, and more preferably the concentration of the microbubbles in the composition is about 10 １０ microbubbles / ml, and a pharmaceutical composition comprising at least one kind of microbubbles according to any one of claims 1 to 9, and optionally a pharmaceutically acceptable excipient. **Claim 11** A kit, wherein the kit a) at least one microbubble according to any one of claims 1 to 9 in a first container; b) at least one therapeutic agent in a second container; c) optionally, at least one targeting agent in a third container; d) optionally, at least one marking agent in a fourth container; e) optionally, instructions for preparation and / or use, and comprising The therapeutic agent and / or the targeting agent and / or the marking agent are preferably 1) nucleic acid; 2) a lipophilic active ingredient; 3) a chemotherapeutic agent such as a cytotoxic agent and / or a cell growth inhibitor; 4) an antibody; 5) a protein; 6) an antigen; 7) a toxin; 8) a receptor; 9) an enzyme; 10) a hormone; 11) a ligand; 12) a viral vector; 13) a nanoparticle, preferably a nanoparticle comprising at least one agent selected from a therapeutic agent, a targeting agent, a marking agent, and any combination thereof. 14) A derivative of any one of 1) to 13), preferably a functional derivative of any one of these, and 15) A fragment of any one of 1) to 14), preferably a functional fragment of any one of these, and 16) Any combination of 1) to 15), selected from More preferably, a kit selected from nucleic acids, lipophilic active ingredients, chemotherapeutic agents, antibodies, antibody derivatives, functional fragments of antibodies or their derivatives, proteins, protein fragments, nanoparticles, and any combination thereof.

12. The microbubble according to any one of claims 1 to 9, the pharmaceutical composition according to claim 10, or the kit according to claim 11, for use as a pharmaceutical or as a marking agent.

13. The following steps a) A step of a mixture in a container of a cationic compound selected from lipophosphoramidate, histidyl polyethyleneimine, and any mixture thereof, ethanol, and at least one agent selected from a therapeutic agent, a targeting agent, a marking agent, and any combination thereof. b) Evaporating the mixture obtained in step a) to obtain a lipid film, and rehydrating the lipid film to form a liposome suspension. c) A step of lyophilizing the liposome suspension obtained in step b). d) A step of replacing the air contained in the container containing the lyophilized product obtained in step c) with a biocompatible gas, wherein the biocompatible gas is preferably selected from perfluorobutane (C4F10), perfluoropropane (C3F8), dinitrogen (N2), sulfur hexafluoride (SF6), nitrogen oxides (NO), hydrogen, dioxygen, helium, xenon, argon, nitrous oxide (N2O), and any mixture thereof. e) A step of rehydrating the lyophilized product from step d) to obtain a solution. f) A step of stirring the solution obtained in step d) to form microbubbles. g) Optionally, a step of functionalizing the microbubbles with at least one agent selected from a therapeutic agent, a targeting agent, a marking agent, and any combination thereof, and / or a step of functionalizing the microbubbles by adding at least one functional group that enables binding to at least one agent selected from a therapeutic agent, a targeting agent, a marking agent, and any combination thereof. A method for generating at least one kind of microbubble according to any one of claims 1 to 9, including

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