Freeze-dried nanodroplets with fluorinated compounds

Abstract

The present invention relates generally to the field of ultrasound contrast agents (USCA). Specifically, the present invention relates to freeze-dried compositions comprising an amphiphilic lipid compound comprising a phospholipid, a fluorinated compound in liquid form, and a freeze-drying protective component, which can be reconstituted to prepare a suspension of nanodroplets useful for diagnostic or therapeutic applications. The invention further relates to a method for preparing such a freeze-dried composition.

Description

Technical Field

[0001] The present invention relates generally to the field of ultrasound contrast agents (USCA). In particular, the present invention relates to a freeze-dried composition comprising an amphiphilic lipid compound including a phospholipid, a fluorinated compound in liquid form and a freeze-drying protective component, the freeze-dried composition can be reconstituted to prepare a suspension of nanodroplets filled with the fluorinated compound and stabilized by the amphiphilic lipid compound, which can be used for diagnostic or therapeutic applications. The present invention further relates to a method for preparing such a freeze-dried composition. Background Art

[0002] Phase change contrast agents (PCCA) or acoustically activated nanodroplets are becoming more and more popular in ultrasound diagnosis and treatment delivery. In addition to the core usually composed of liquid perfluorocarbons, nanodroplets show a composition similar to commercially available gas-filled microbubbles. Due to the acoustic droplet vaporization (ADV, Acoustic Droplet Vaporization) process, the encapsulated droplets are converted into bubbles when exposed to ultrasonic energy exceeding the vaporization threshold. In fact, ultrasound acts as a remote trigger and promotes the vaporization of droplets in a controllable, non-invasive and local manner. Due to its smaller size compared to conventional microbubbles, nanodroplets show extended in vivo circulation and penetrate into tissues via the extravascular space depth. In addition, below the vaporization threshold, they are ultrasonically stable, have low acoustic attenuation, and can be acoustically vaporized at the location of interest.

[0003] Perfluorocarbon nanodroplets ("PFC-NDs") present real potential as extravascular ultrasound contrast agents in many diagnostic and therapeutic applications, including sonoperation, thermal ablation, blood-brain barrier (BBB) ​​disruption, multimodal imaging modalities, and allow passive (due to enhanced permeability and retention (EPR) effect in tumor tissue) or active targeting (by introduction of targeting ligands) for local delivery of therapeutic drugs or genes. Another potentially valuable property of PFC-NDs is their possible application in new imaging strategies, such as ultrasound super-resolution imaging (also known as ultrasound localization microscopy), as these agents can be activated and deactivated on demand by applying intermittent acoustic pulses.

[0004] PFC-NDs are usually composed of an encapsulating shell and a core filled with liquid perfluorocarbon (PFC). Currently, commonly used shell materials include soft shell materials such as lipids and fluorinated surfactants, or hard shell materials such as proteins and polymers (Zhang, 2022).

[0005] Although lipids have been successfully employed in the fabrication of ultrasound-responsive contrast agents such as microbubbles and droplets due to their properties such as elasticity and acoustic behavior, lipid-shell nanodroplets have been reported to lack stability after storage, possibly due to vesicle aggregation.

[0006] Melich, 2020 reported the use of rapid and controlled microfluidic mixing to fabricate various types of PFC-NDs with different stability shells. In this study, it was demonstrated that polymer-coated nanodroplets (PLGA) showed higher stability than lipid-shelled (DPPC:DSPE-PEG2000).

[0007] Therefore, there is a need to develop methods for long-term storage of lipid-shelled nanodroplets. Freeze drying, also known as lyophilization, is a complex and challenging method widely used in the pharmaceutical industry, which has the advantage of preserving pharmaceutical products in dry form for several months. In fact, freeze-dried products show greater storage stability, extended shelf life and can be easily transported.

[0008] To date, to the best of the applicant's knowledge, such techniques have not been applied to prepare freeze-dried compositions from suspensions of nanodroplets stabilized by a lipid shell having a core comprising a fluorinated compound in liquid form.

[0009] As observed by the Applicants, the main challenges in preparing freeze-dried compositions of nanodroplets filled with fluorinated compounds are related to the need to avoid the removal of volatile fluorinated compounds during the freeze-drying process and to substantially preserve the properties of the initial suspension of nanodroplets, such as average size, size distribution, amount of fluorinated compound and nanodroplet concentration.

[0010] The applicant has now found that such initial properties can be retained to an acceptable extent after the freeze-drying process.

[0011] Furthermore, the Applicant has demonstrated that by simply adding an aqueous solution to the freeze-dried composition without any further gas instillation, a reconstituted suspension of lipid-stabilized nanodroplets can be obtained, characterized by a substantially unchanged amount of fluorinated compound relative to the amount before freeze-drying. Summary of the invention

[0012] One aspect of the present invention relates to a freeze-dried composition comprising

[0013] i) an amphiphilic lipid compound comprising a phospholipid,

[0014] ii) a fluorinated compound in liquid form, and

[0015] iii) freeze-dried protective components,

[0016] The composition, when reconstituted with a pharmaceutically acceptable liquid carrier, provides a suspension of nanodroplets comprising an inner core and an outer layer, wherein the inner core comprises the fluorinated compound in liquid form and the outer layer comprises the amphiphilic lipid compound.

[0017] In a preferred embodiment, the weight ratio between the freeze-drying protection component and the fluorinated compound is greater than 10, preferably greater than 20, preferably greater than 30, preferably greater than 50, preferably greater than 60, preferably greater than 100, and up to 250.

[0018] In a preferred embodiment, the fluorinated compound has a boiling point between -70°C and 160°C, preferably between -50°C and 100°C, more preferably between -40°C and 60°C.

[0019] Still more preferably, the fluorinated compound is a perfluorocarbon selected from perfluoropentane, perfluorohexane, perfluorobutane, perfluoropropane or mixtures thereof.

[0020] In another embodiment, the freeze-drying protective component is a sugar, preferably a disaccharide, selected from trehalose, sucrose, maltose or a mixture thereof, preferably trehalose.

[0021] According to another aspect of the present invention, it relates to a method for preparing a freeze-dried composition as defined above, comprising the following steps:

[0022] a) preparing an initial suspension, the initial suspension comprising

[0023] i) a plurality of nanodroplets comprising an amphiphilic lipid compound including a phospholipid and a fluorinated compound in liquid form, and

[0024] ii) a freeze-drying protection component; and

[0025] b) freeze drying the initial suspension

[0026] wherein the freeze-dried composition, when reconstituted with a pharmaceutically acceptable liquid carrier, provides a suspension of nanodroplets as defined above, wherein the amount of the fluorinated compound is 50% to 100% of the amount of the fluorinated compound contained in the initial suspension of step a).

[0027] In a preferred embodiment, the reconstitution does not include an additional infusion of a fluorinated compound.

[0028] In another aspect, the present invention relates to a method for preparing a reconstituted suspension of nanodroplets comprising an inner core and an outer layer, wherein the inner core comprises a fluorinated compound in liquid form and the outer layer comprises an amphiphilic lipid compound comprising a phospholipid, the method comprising the following steps:

[0029] a) preparing an initial suspension, the initial suspension comprising

[0030] i) a plurality of nanodroplets comprising an amphiphilic lipid compound including a phospholipid and a fluorinated compound in liquid form, and

[0031] ii) freeze-drying protection component;

[0032] b) freeze-drying the initial suspension to obtain a freeze-dried composition, and

[0033] c) reconstituting the freeze-dried composition with a pharmaceutically acceptable liquid carrier to provide a suspension of the nanodroplets, wherein the amount of the fluorinated compound is 50% to 100% of the amount of the fluorinated compound contained in the initial suspension of step a).

[0034] In a preferred embodiment, in step c), the reconstitution of the freeze-dried composition does not comprise an additional infusion of a fluorinated compound.

[0035] Another aspect relates to a freeze-dried composition as defined above for use in diagnostic and / or therapeutic treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is the DLS size distribution of composition 3T before (dashed line) and after (solid line) the freeze-drying process. DETAILED DESCRIPTION

[0037] The present invention generally relates to freeze-dried compositions comprising an amphiphilic lipid compound including a phospholipid, a fluorinated compound in liquid form, and a freeze-drying protective component, which, when reconstituted with a pharmaceutically acceptable liquid carrier, provides a suspension of nanodroplets whose properties (i.e., size, size distribution, amount of fluorinated compound, and ND concentration) result in substantial changes relative to the characteristics of the suspension prior to freeze-drying.

[0038] The nanodroplets are suitable as contrast agents in ultrasound imaging techniques (known as Contrast-Enhanced Ultrasound Imaging (CEUS) imaging), or for ultrasound-mediated therapeutic applications, such as thermal ablation or for ultrasound-mediated drug / gene delivery.

[0039] As observed by the applicant, the main properties of the nanodroplets, such as size, size distribution, amount of fluorinated compound and ND concentration, may not be maintained and may gradually deteriorate after storage for a period of time in the form of a suspension. The shelf life of such suspensions of nanodroplets containing fluorinated compounds in liquid form is therefore relatively short for pharmaceutical products and there is a need to develop long-term storage procedures capable of retaining their initial properties, such as size, size distribution, amount of fluorinated compound and ND concentration, for a longer period of time, such as months or years. The freeze-drying method is a suitable way to obtain a dry form of nanodroplets filled with fluorinated compounds, which has an increased stability over time and retained initial properties. It has now been surprisingly found that the initial properties can be substantially retained after the freeze-drying process.

[0040] definition

[0041] The term "nanodroplet" refers to an assembly comprising an outer layer comprising an amphiphilic lipid compound including phospholipids and an inner core comprising a fluorinated compound in liquid form. In the nanodroplet, the amphiphilic lipid compound is oriented in such a way that the hydrophobic part of the amphiphilic lipid is located at the surface of the fluorinated compound of the inner core.

[0042] According to the present invention, the formulation of nanodroplets has a z-average diameter between 100 nm and 1000 nm, preferably between 120 nm and 800 nm, more preferably between 150 nm and 400 nm. In certain embodiments, the nanodroplets are "calibrated nanodroplets", which indicates a nanodroplet formulation having a population of nanodroplets with a z-average as defined above and a polydispersity of less than 0.25, preferably less than 0.20, more preferably less than 0.15, even more preferably less than 0.10.

[0043] The "calibrated nanodroplets" are preferably obtained by microfluidics technology.

[0044] As used herein, the expression "freeze-dried composition" refers to any dry dosage form for long-term storage of the nanodroplets filled with the fluorinated compound in liquid form obtained by freeze-drying method. The freeze-dried composition may comprise one or more active ingredients and a freeze-drying protective component. As used herein, the expression "active ingredient" includes the nanodroplet stabilizing material, i.e., the amphiphilic lipid compound, and the core material, i.e., the fluorinated compound.

[0045] The freeze-dried composition typically comprises the fluorinated compound entrapped in liquid form therein, particularly in the core of the freeze-dried nanodroplets.

[0046] As used herein, the expression "freshly prepared nanodroplets" refers to (a suspension of) nanodroplets filled with the fluorinated compound in liquid form at the end of their preparation, for example within a few minutes (eg 5 minutes) from the last step of the preparation method.

[0047] As used herein, the expression "initial suspension of nanodroplets" refers to an aqueous suspension of nanodroplets that additionally comprises a freeze-drying protective component. Typically, the initial suspension of nanodroplets can be obtained by mixing a freeze-drying protective component in the form of a suitable aqueous solution to the suspension of nanodroplets, after obtaining a freshly prepared suspension of nanodroplets by any suitable preparation method (as described in detail below). The initial suspension is then subjected to a freeze-drying process to prepare a freeze-dried composition as defined above. The freeze-dried composition is then reconstituted into a liquid form prior to in vivo administration.

[0048] As used herein, the expression "reconstituted suspension of nanodroplets" refers to a suspension of nanodroplets comprising an inner core and an outer layer, wherein the inner core comprises a fluorinated compound in liquid form and the outer layer comprises an amphiphilic lipid compound comprising phospholipids, the suspension being obtained by reconstituting the freeze-dried composition with a pharmaceutically suitable liquid carrier (as defined below).

[0049] According to the present invention, by using a suitable freeze-drying protective component, the initial properties of the initial suspension of nanodroplets, such as size, size distribution, amount of fluorinated compound and ND concentration, can be substantially retained after the freeze-drying process. It can be seen that the properties of the reconstituted suspension of the nanodroplets filled with the fluorinated compound are substantially unchanged relative to the characteristics characterizing the initial suspension.

[0050] Freeze-dried protective components

[0051] When freeze-dried protective components are used, the original properties of the nanodroplets filled with the fluorinated compound in liquid form and stabilized by the amphiphilic lipid compound are particularly retained.

[0052] As used herein, the expression "freeze drying protective component" refers to a component suitable for freeze drying, which is included in the suspension of nanodroplets prior to its freeze drying process. The term "freeze drying protective component" refers to any compound added to protect the active ingredient during any stage of the freeze drying process. Examples of suitable freeze drying protective components are polyethylene glycol (PEG), polyols, sugars, surfactants, buffers, amino acids, chelate complexes, inorganic salts and mixtures thereof. Preferably, the freeze drying protective component comprises polyethylene glycol (PEG), polyols, sugars or mixtures thereof.

[0053] According to one embodiment of the present invention, the freeze-drying protective component comprises sugar.

[0054] The term "sugar" has its standard meaning in the field of chemistry. Sugars, also known as carbohydrates, are molecular compounds composed of only three elements: carbon, hydrogen, and oxygen. The simplest sugars are called monosaccharides, which are the building blocks of larger sugars such as disaccharides, trisaccharides, and polysaccharides.

[0055] Preferably, the sugar is a disaccharide.

[0056] Disaccharide (C 12 H 22 O 11 ) is a sugar consisting of two monosaccharide units connected by a glycosidic bond. The latter is a covalent bond formed by the reaction of the anomeric carbon of one cyclic monosaccharide with the OH group of a second monosaccharide. Disaccharides differ from each other in their monosaccharide components and the specific type of glycosidic linker that connects them. Examples of disaccharides include trehalose, sucrose, maltose, and lactose.

[0057] In the present invention, the freeze-drying protective component is preferably a disaccharide selected from trehalose, sucrose, maltose or a mixture thereof. Trehalose is particularly preferred.

[0058] Amphiphilic lipid compounds

[0059] In the present specification and claims, the expression "amphiphilic lipid compound" has its conventional meaning in the chemical field and refers to an organic lipid compound comprising a hydrophilic structure part and a hydrophobic structure part suitable for forming a stabilizing layer (i.e., outer layer) of a nanodroplet in which the amphiphilic lipid compound molecules are oriented in such a way that the hydrophobic part is located at the surface of the liquid fluorinated compound placed in the inner core.

[0060] The disclosed nanodroplets are generally stabilized by one or more amphiphilic lipid compounds including phospholipids. Other suitable amphiphilic lipid compounds include, for example, lysophospholipids; fatty acids such as palmitic acid, stearic acid, arachidonic acid or oleic acid; lipids carrying polymers such as chitin, hyaluronic acid, polyvinyl pyrrolidone or polyethylene glycol (PEG), also known as "pegylated lipids"; lipids with sulfonated mono-, di-, oligo- or polysaccharides; cholesterol, cholesterol sulfate or cholesterol hemisuccinate; tocopherol hemisuccinate; lipids with ether or ester-linked fatty acids; diacetyl phosphate; dicetyl phosphate; ceramides; polyoxyethylene fatty acid esters (such as polyoxyethylene fatty acid stearate), polyoxyethylene fatty alcohols, polyoxyethylene esters of sugars and fatty acids, including sucrose laurate, fructose laurate, sucrose palmitate, sucrose stearate, glycerol or monoesters of glycerol and fatty acids, including glycerol monopalmitate, glycerol monostearate, glycerol monomyristate or glycerol monolaurate; long-chain alcohols, including n-decyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol or n-octadecyl alcohol; 1,2-dioleyl-sn-glycerol; 1,2-dipalmitoyl-sn-3-succinylglycerol; 1,3-dipalmitoyl-2-succinylglycerol; 1-hexadecyl-2-palmitoyl glycerophosphoethanolamine; containing at least one (C 10 -C 20 ), preferably (C 14 -C 18 )alkyl chain, such as N-stearylamine, N,N'-distearylamine, N-hexadecylamine, N,N'-di(hexadecyl)amine, N-stearylammonium chloride, N,N'-distearylammonium chloride, N-hexadecylammonium chloride, N,N'-di(hexadecyl)ammonium chloride, dimethyldi(octadecyl)ammonium bromide (DDAB), hexadecyltrimethylammonium bromide (CTAB); one or preferably two (C3-C6)alkylene bridges connected to the N atom 10 -C 20 ), preferably (C 14 -C 18) tertiary or quaternary ammonium salts of acyl chains, such as 1,2-distearoyl-3-trimethylammonium-propane (DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), 1,2-oleoyl-3-trimethylammonium-propane (DOTAP), 1,2-distearoyl-3-trimethylammonium-propane (DSTAP), (N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane- 1-amine (DOPAQ), (6Z,9Z,28Z,31Z)-triacontriacontria-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA) or 2-[2,2-bis[(9Z,12Z)-octadec-9,12-dienyl]-1,3-dioxolan-4-yl]-N,N-dimethylethylamine (DLin-KC2-DMA); and mixtures or combinations thereof.

[0061] The term "phospholipid" is intended to encompass any amphiphilic phospholipid compound, the molecules of which can form a stabilizing membrane at the boundary interface between the aqueous phase and the fluorinated compound in the final nanodroplet suspension.

[0062] Examples of suitable phospholipids include esters of glycerol with one or preferably two (same or different) fatty acid residues and with phosphoric acid, wherein the phosphoric acid residue in turn is bound to a hydrophilic group, such as choline (phosphatidylcholine-PC), serine (phosphatidylserine-PS), glycerol (phosphatidylglycerol-PG), ethanolamine (phosphatidylethanolamine-PE), inositol (phosphatidylinositol). Esters of phospholipids with only one fatty acid residue are generally referred to in the art as "lyso" forms of phospholipids or "lysophospholipids". The fatty acid residues present in phospholipids are generally long-chain aliphatic acids, typically containing 12 to 24 carbon atoms, preferably 14 to 22; the aliphatic chain may contain one or more degrees of unsaturation, or preferably is fully saturated. Examples of suitable fatty acids included in phospholipids are, for example, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, linoleic acid and linolenic acid. Saturated fatty acids such as myristic acid, palmitic acid, stearic acid and arachidic acid are preferably used.

[0063] Further examples of phospholipids are phosphatidic acid, i.e. a diester of glycerol-phosphoric acid with fatty acids; sphingolipids such as sphingomyelin, i.e. those phosphatidylcholine analogs in which the residues of the diglyceride and fatty acids are replaced by ceramide chains; cardiolipin, i.e. an ester of 1,3-diphosphatidylglycerol with fatty acids; glycolipids such as ganglioside GM1 (or GM2) or cerebrosides; glycolipids; sulfatides and glycosphingolipids.

[0064] As used herein, the term "phospholipid" includes naturally occurring, semisynthetic or synthetically prepared products, which may be used alone or as a mixture.

[0065] Examples of naturally occurring phospholipids are natural phosphatidylcholine (phosphatidylcholine (PC) derivatives), such as typically soy or egg yolk lecithin.

[0066] Examples of semisynthetic phospholipids are partially or fully hydrogenated derivatives of naturally occurring phosphatidylcholine. Preferred phospholipids are fatty acid diesters of phosphatidylcholine, ethylphosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol or sphingomyelin.

[0067] Examples of preferred phospholipids are, for example, dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), dioleoyl-phosphatidylcholine (DOPC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (ethyl-DSPC), dipentadecanoyl-phosphatidylcholine (DPDPC), 1-myristoyl-2-palmitoyl-phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl-phosphatidylcholine (P MPC), 1-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl-phosphatidylcholine (SPPC), 1-palmitoyl-2-oleylphosphatidylcholine (POPC), 1-oleyl-2-palmitoyl-phosphatidylcholine (OPPC), dilauroyl-phosphatidylglycerol (DLPG) and its alkali metal salts, diarachidoylphosphatidylglycerol (DAPG) and its alkali metal salts, dimyristoylphosphatidylglycerol (DMPG) and its alkali metal salts, dipalmitoylphosphatidylglycerol (DPPG) and its alkali metal salts, distearoylphosphatidylglycerol (DSPG) and its alkali metal salts. metal salts, dioleoyl-phosphatidylglycerol (DOPG) and its alkali metal salts, dimyristoyl phosphatidic acid (DMPA) and its alkali metal salts, dipalmitoyl phosphatidic acid (DPPA) and its alkali metal salts, distearoyl phosphatidic acid (DSPA), diarachidoyl phosphatidic acid (DAPA) and its alkali metal salts, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), distearoyl phosphatidylethanolamine (DSPE), dioleyl phosphatidylethanolamine (DOPE), diarachidoyl phosphatidylethanolamine (DAPE), dilinoleyl phosphatidylethanolamine (DLPE), dimyristoyl phosphatidylserine (DMPS), diarachidoylphosphatidylserine (DAPS), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), dioleoylphosphatidylserine (DOPS), dipalmitoylsphingomyelin (DPSP), distearoylsphingomyelin (DSSP), dilauroyl-phosphatidylinositol (DLPI), diarachidoylphosphatidylinositol (DAPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dioleoyl-phosphatidylinositol (DOPI).

[0068] Suitable phospholipids further include phospholipids modified by attaching a hydrophilic polymer such as polyethylene glycol (PEG) or polypropylene glycol (PPG) thereto. Preferred polymer-modified phospholipids include "PEGylated phospholipids", i.e., phospholipids bound to PEG polymers. Examples of PEGylated phospholipids are PEGylated phosphatidylethanolamines (abbreviated as "PE-PEG"), i.e., phosphatidylethanolamines in which the hydrophilic ethanolamine moiety is attached to a PEG molecule of variable molecular weight (e.g., 300 to 20,000 daltons, preferably 500 to 5,000 daltons), such as DPPE-PEG (or DSPE-PEG, DMPE-PEG, DAPE-PEG, or DOPE-PEG). For example, DPPE-PEG2000 refers to a DPPE to which a PEG polymer having an average molecular weight of about 2,000 is attached.

[0069] Particularly preferred phospholipids are DMPC, DPPC, DSPC, DMPG, DPPG, DMPA, DPPA, DMPS, DPPS and ethyl-DPPC. Most preferred are DPPC, DMPA and DPPG.

[0070] Mixtures of phospholipids may also be used, for example mixtures of DPPE and / or DSPE (including pegylated derivatives), DPPC with DMPA, DPPA, DMPG, DPPG or ethyl-DPPC.

[0071] For example, the mixture of phospholipids can include phosphatidylcholine derivatives, phosphatidic acid derivatives and pegylated phosphatidylethanolamine, such as DPPC / DMPA / DPPE-PEG, DPPC / DPPA / DSPE-PEG, DPPC / DPPG / DPPE-PEG, DPPC / DPPG / DSPE-PEG.

[0072] According to the present invention, phospholipids can be conveniently used in any combination with the amphipathic compounds listed above. Therefore, for example, lipids such as cholesterol, ergosterol, phytosterol, sitosterol, lanosterol, tocopherol, propyl gallate or ascorbyl palmitate, fatty acids such as myristic acid, palmitic acid, stearic acid, arachidic acid and derivatives thereof or butylated hydroxytoluene and / or other non-phospholipid compounds can be optionally added to one or more of the aforementioned phospholipids. For example, the mixture of the amphipathic lipid compound comprising phospholipids and fatty acids can be advantageously used, including DPPC / DPPE-PEG / palmitic acid, DSPC / DPPE-PEG / palmitic acid, DPPC / DSPE-PEG / palmitic acid, DPPC / DPPE-PEG / stearic acid or DPPC / DSPE-PEG / stearic acid.

[0073] In one embodiment, the amphiphilic lipid compound is a mixture of amphiphilic lipid compounds comprising phospholipids. Still more preferably, the mixture additionally comprises a lipid with a polymer, preferably a PEGylated phospholipid, as defined above.

[0074] According to another embodiment, the mixture additionally comprises a fatty acid, preferably palmitic acid.

[0075] Fluorinated compounds

[0076] As described above, the disclosed nanodroplets comprise an outer layer comprising an amphiphilic lipid compound including phospholipids and an inner core comprising a fluorinated compound in liquid form that allows activation of the vesicles upon application of ultrasound (US).

[0077] In the present specification and claims, the term "fluorinated compounds" refers to a group of fluorine-containing compounds derived from (optionally substituted) hydrocarbons in which hydrogen atoms have been partially or completely replaced by fluorine atoms, which are liquid or gaseous at room temperature.

[0078] The expression "optionally substituted" refers to the presence of functional groups such as amines, ethers and halogen-containing groups.

[0079] Suitable examples of fluorinated compounds are saturated and unsaturated hydrofluorocarbons, perfluorocarbons, fluorinated ethers, fluorinated ketones or perfluorinated nitriles. Preferably, the fluorinated compound is a perfluorocarbon (PFC), ie a fluorinated hydrocarbon in which all hydrogen atoms are replaced by fluorine atoms.

[0080] The fluorinated compound can be selected from fluorinated compounds with a high boiling point (i.e., higher than room temperature (RT; 25°C)) that are in liquid form at standard ambient temperature and pressure (SATP, Standard Ambient Temperature and Pressure), i.e., at 25°C and 1 atm (101.325 kPa), or fluorinated compounds with a low boiling point (i.e., lower than room temperature), referring to fluorinated compounds that are gaseous at SATP (i.e., highly volatile fluorocarbons).

[0081] In one embodiment, the fluorinated compound has a boiling point between -70°C and 160°C, preferably between -50°C and 100°C, more preferably between -40°C and 60°C.

[0082] In a preferred embodiment, the fluorinated compound is a perfluorocarbon.

[0083] The liquid fluorinated compound is characterized by a boiling point higher than 25°C, for example between 25°C and 160°C. In the present invention, the fluorinated compound is preferably characterized by a boiling point between 25°C and 100°C, still more preferably between 27°C and 60°C.

[0084] Suitable examples of liquid fluorinated compounds are 1-fluorobutane, 2-fluorobutane, 2,2-difluorobutane, 2,2,3,3-tetrafluorobutane, 1,1,1,3,3-pentafluorobutane, 1,1,1,4,4,4-hexafluorobutane, 1,1,1,2,4,4,4-heptafluorobutane, 1,1,2,2,3,3,4,4-octafluorobutane, 1,1,1,2,2-pentafluoropentane, 1,1,1,2,2,3,3,4-octafluoropentane, 1,1,1,2,2,3,4,5,5,5-decafluoropentane, 1,1,2,2,3,3,4,4,5,5,6,6-dodecafluorohexane or mixtures thereof.

[0085] Suitable examples of liquid perfluorocarbons are perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluorononane, perfluorodecalin, perfluorooctyl bromide (PFOB), perfluoro-15-crown-5-ether (PFCE), perfluorodichlorooctane (PFDCO), perfluorononane (PFN) and 1,1,1-tris(perfluoro-tert-butoxymethyl)ethane (TPFBME) or mixtures thereof.

[0086] In one embodiment, the perfluorocarbon is preferably perfluoropentane (PFP) (boiling point 29° C.) or perfluorohexane (PFH) (boiling point 57° C.).

[0087] The gaseous fluorinated compound is characterized by a boiling point below 25° C. at ambient pressure, for example between -70° C. and 25° C. In the present invention, the fluorinated compound is preferably characterized by a boiling point between -50° C. and 22° C., more preferably between -45° C. and 18° C., more preferably between -40° C. and 5° C., still more preferably between -5° C. and 5° C.

[0088] Suitable examples of gaseous fluorinated compounds are 1,1,1,2,3,3,3-heptafluoropropane, 1,1,1,2,2,3-hexafluoropropane, 1,1,1,2,3,3-hexafluoropropane, 1,1,1,3,3,3-hexafluoropropane, 1,1,1,2,2,3,3,4,4-nonafluorobutane, 1,1,1,3,3,3-hexafluoro-2-(trifluoromethyl)propane, 1,1,1,2,2,3,3,4-octafluorobutane or mixtures thereof.

[0089] Suitable examples of gaseous perfluorocarbons are perfluorocyclopropane, perfluoropropane, perfluorocyclobutane, perfluorobutane, perfluoroisobutane or mixtures thereof.

[0090] In one embodiment, the gaseous perfluorocarbon is preferably perfluorobutane (boiling point -2°C) or perfluoropropane (boiling point -37°C).

[0091] In another embodiment, the perfluorocarbon comprises a mixture of a liquid perfluorocarbon and a gaseous perfluorocarbon.

[0092] In a preferred embodiment, the perfluorocarbon is selected from perfluorohexane, perfluoropentane, perfluorobutane, perfluoropropane or mixtures thereof.

[0093] Preparation of freeze-dried compositions

[0094] Another aspect of the invention relates to a method for preparing a freeze-dried composition as defined above, comprising the following steps:

[0095] a) preparing an initial suspension, the initial suspension comprising

[0096] i) a plurality of nanodroplets comprising an amphiphilic lipid compound including a phospholipid and a fluorinated compound in liquid form, and

[0097] ii) a freeze-drying protection component; and

[0098] b) freeze drying the initial suspension

[0099] The freeze-dried composition, when reconstituted with a pharmaceutically acceptable liquid carrier, provides a suspension of nanodroplets, wherein the amount of the fluorinated compound is 50% to 100% of the amount of the fluorinated compound contained in the initial suspension of step a).

[0100] Step a) Preparation of an initial suspension of nanodroplets filled with a fluorinated compound

[0101] According to the invention, in step a), the initial suspension is prepared using any preparation technique suitable for producing nanodroplets having a Z-average diameter between 100 and 1000 nm, preferably between 120 and 800 nm, more preferably between 150 and 400 nm.

[0102] Examples of preparation techniques include sonication, homogenization, extrusion, microfluidics, and microbubble coagulation.

[0103] In a preferred embodiment, in step a), the suspension is preferably prepared by using a preparation technique suitable for producing calibrated nanodroplets having a polydispersity index (PDI) lower than 0.25, preferably lower than 0.20, more preferably lower than 0.15, even more preferably lower than 0.10 and a Z-average diameter between 100 nm and 1000 nm, preferably between 120 and 800 nm, more preferably between 150 and 400 nm.

[0104] For example, the preparation technique is selected from sonication, microbubble coagulation or microfluidics.

[0105] In the present specification and claims, the expression "sonication" refers to a technique based on making nanodroplets by using ultrasonic emulsification of the components of the nanodroplets (i.e., lipid shell material and fluorinated compound) in a continuous aqueous phase. For example, the technique can be performed using a sonic bath or a probe sonicator.

[0106] In one embodiment, in step a), the initial suspension is prepared by using a sonication technique.

[0107] Typically, sonication techniques include the following steps:

[0108] a i ) preparing an aqueous mixture comprising an amphiphilic lipid compound including a phospholipid and a fluorinated compound;

[0109] b ii ) subjecting the mixture to ultrasound, and

[0110] c iii ) obtaining an aqueous suspension of nanodroplets comprising an outer shell comprising an amphiphilic lipid compound and an inner core comprising a fluorinated compound in liquid form.

[0111] At the end of the sonication process, the suspension of nanodroplets can be mixed directly to a solution of a freeze-dried protective component, as described above, to obtain an initial suspension of nanodroplets as defined in the present invention.

[0112] In the present description and claims, the expression "microbubble condensation" refers to a technique for making nanodroplets based on the generation of microbubbles filled with gaseous perfluorocarbons and then condensing this gaseous precursor into liquid core nanodroplets by cooling and / or applying pressure.

[0113] In another embodiment, in step a), the initial suspension is prepared by using a microbubble coagulation method.

[0114] Typically, the microbubble coagulation method comprises the following steps:

[0115] a*) preparing a suspension of gas-filled vesicle microbubbles having an outer shell comprising an amphiphilic lipid compound including phospholipids and an inner core comprising a fluorinated compound that is in gaseous form at room temperature and atmospheric pressure;

[0116] b*) condensing the gaseous fluorinated compound into a liquid fluorinated compound, and

[0117] c*) obtaining a suspension of nanodroplets comprising an outer shell comprising an amphiphilic lipid compound including a phospholipid and an inner core comprising the fluorinated compound in liquid form.

[0118] After condensation, the inner core of the obtained nanodroplets consists of the fluorinated compound in liquid form, independently of the temperature at which they are subsequently stored.

[0119] In other words, even when stored at a temperature higher than the boiling point of the fluorinated compound used to manufacture the ND, the fluorinated compound remains in a liquid state due to its incorporation into the outer stability shell.

[0120] In one embodiment, the fluorinated compound comprises a gaseous fluorinated compound as defined above having a boiling point below room temperature (25°C).

[0121] Said suspension of gas-filled microvesicles can advantageously be prepared by using any method known in the literature, for example by reconstituting a freeze-dried product with a suitable pharmaceutically acceptable solution to obtain a suspension of gas-filled microvesicles.

[0122] In one embodiment, step b*) of the disclosed microfluidic condensation method comprises cooling a suspension of gas-filled microvesicles under pressure until the encapsulated gas condenses into a liquid phase.

[0123] Suitable examples of temperature and pressure to be applied in step b*) can be found in US20220000790.

[0124] In the present specification and claims, the expression "microfluidics technology" refers to a technology for manufacturing nanodroplets by a microfluidics cartridge designed to manipulate a fluid in a channel on a microscale.

[0125] The microfluidics technique is a bottom-up approach, that is, nanodroplets are obtained by assembling molecules (such as amphiphilic lipid compounds and fluorinated compounds) into larger nanostructures (ie, calibrated nanodroplets).

[0126] In a preferred embodiment, in step a), the initial suspension is prepared by using microfluidics technology.

[0127] More preferably, the microfluidics technique is performed by using a mixing device, such as a microfluidics cartridge equipped with a staggered herringbone micromixer or a toroidal mixer.

[0128] A description of a microfluidic cartridge suitable for the microfluidic method of the present invention is described in WO2022101365A1 (BraccoSuisse SA).

[0129] In a still more preferred embodiment, the preparation method of step a) comprises the following steps:

[0130] A) preparing an aqueous phase;

[0131] B) preparing an organic phase;

[0132] in:

[0133] - the aqueous phase comprises an amphiphilic lipid compound including phospholipids and the organic phase comprises a fluorinated compound; or

[0134] - the organic phase comprises an amphiphilic lipid compound including a phospholipid and a fluorinated compound;

[0135] C) injecting the aqueous phase into a first inlet of a microfluidic cartridge and injecting the organic phase into a second inlet of the microfluidic cartridge, thereby mixing the aqueous phase and the organic phase in a mixing device of the microfluidic cartridge, wherein the operating pressure entering the microfluidic cartridge is lower than 7000 kPa, to obtain an aqueous suspension of calibrated nanodroplets;

[0136] D) collecting the calibrated aqueous suspension of nanodroplets from the outlet channel of the microfluidic cartridge, and

[0137] E) Dilution of the collected aqueous suspension of calibrated nanodroplets.

[0138] In one embodiment, the method further comprises a step F) of washing the obtained aqueous suspension of calibrated nanodroplets.

[0139] The expression "calibrated nanodroplets" relates to a formulation of nanodroplets having a population of nanodroplets having a z-average diameter of between 100 nm and 1000 nm, preferably between 120 nm and 800 nm, more preferably between 150 nm and 400 nm, and a polydispersity of less than 0.25, preferably less than 0.20, more preferably less than 0.15, even more preferably less than 0.10.

[0140] According to the disclosed microfluidic method, an aqueous suspension of calibrated nanodroplets can be obtained by a single pass of the liquid phase through a dual-channel microfluidic system.

[0141] According to a preferred embodiment, the organic phase comprises an amphiphilic lipid compound including a phospholipid and a fluorinated compound.

[0142] Preferably, the fluorinated compound is a perfluorocarbon.

[0143] The fluorinated compounds are those described above in the present specification.

[0144] In one embodiment, the amphiphilic lipid compound comprising a phospholipid is a mixture of amphiphilic lipid compounds.

[0145] Still more preferably, the mixture additionally comprises a lipid carrying a polymer, preferably a PEGylated phospholipid, as defined above.

[0146] According to another embodiment, the mixture additionally comprises a fatty acid, preferably palmitic acid.

[0147] The amphiphilic lipid compounds are those described hereinabove in the present specification.

[0148] Step A) Preparation of aqueous phase

[0149] The expression "aqueous phase" refers to a liquid comprising an aqueous liquid component, including, for example, water, an aqueous buffer solution, an aqueous isotonic solution or a mixture thereof. Preferably, the aqueous phase is water.

[0150] According to an alternative embodiment, the aqueous phase comprises an amphiphilic lipid compound as defined above.

[0151] Examples of suitable amphiphilic lipid compounds to be dissolved in the aqueous phase are pegylated phospholipids.

[0152] For example, the amphiphilic lipid compound can be mixed with an aqueous component by conventional techniques (eg, stirring) to prepare an aqueous phase to be injected into the first inlet of the microfluidic cartridge.

[0153] The aqueous phase may additionally comprise further amphiphilic components, for example polyethylene oxide derivatives, such as polyethylene oxide sorbitan esters (ie polysorbates) or polyethylene oxide-polypropylene oxide block copolymers (ie poloxamers).

[0154] Suitable examples of polyoxyethylene sorbitan are polyoxyethylene sorbitan laurate, palmitate, stearate or oleate, such as polysorbate 20 (monolaurate), polysorbate 40 (monopalmitate), polysorbate 60 (monostearate), polysorbate 65 (tricestearate) or polysorbate 80 (oleate).

[0155] A suitable example of a polyethylene oxide-polypropylene oxide block copolymer is Pluronic F68.

[0156] Step B) Preparation of organic phase

[0157] The expression "organic phase" refers to an organic solution comprising an organic solvent miscible with water, including methanol, ethanol, propanol-1, isopropanol, acetonitrile and acetone. Preferably, the organic phase is ethanol.

[0158] In the present invention, the expression "water-miscible organic solvent" means an organic solvent that can be mixed with water in any ratio (eg, any concentration) without two-phase separation, that is, forms a uniform solution.

[0159] In a preferred embodiment, the organic phase comprises an amphiphilic lipid compound including phospholipids. For example, the amphiphilic lipid compound can be mixed with an organic solvent by conventional techniques (eg, stirring) to prepare an organic solution to be mixed with the fluorinated compound.

[0160] In one embodiment, the amphiphilic lipid compound comprising phospholipids is selected from lipids with polymers, fatty acids or mixtures thereof.

[0161] In a preferred embodiment, the amphiphilic lipid compound comprising phospholipids is a mixture of amphiphilic lipid compounds.

[0162] Still more preferably, the mixture additionally comprises a lipid carrying a polymer, preferably a PEGylated phospholipid, as defined above.

[0163] According to another embodiment, the mixture additionally comprises a fatty acid, preferably palmitic acid.

[0164] In one embodiment, the concentration of the mixture in the organic phase is higher than 0.5 mg / mL, preferably at least 2 mg / mL or more, preferably 4 mg / mL or more, more preferably higher than 5 mg / mL, up to, for example, 10 mg / mL, preferably up to 8 mg / mL.

[0165] As disclosed herein, the organic phase further comprises a fluorinated compound having a boiling point between -70°C and 160°C, preferably between -50°C and 100°C, more preferably between -40°C and 60°C.

[0166] Preferably, the fluorinated compound is a perfluorocarbon.

[0167] Suitable examples of fluorinated compounds are those mentioned above.

[0168] According to the disclosed method, in step B), the preparation of the organic phase to be injected into the second inlet of the microfluidic cartridge can be performed by alternative methods depending on the nature of the fluorinated compound.

[0169] The liquid fluorinated compound can be mixed with an organic solvent by conventional techniques (eg, stirring) to prepare an organic solution.

[0170] The gaseous fluorinated compound (having a boiling point between -70°C and 25°C at atmospheric pressure) can be added to the organic solvent as a liquid (eg by dissolving into the organic solvent) or as a gas (eg by bubbling into the organic solvent).

[0171] A fluorinated compound having a boiling point below room temperature (i.e., 25° C.) is mixed as a liquid phase

[0172] According to one embodiment, step B) comprises preparing an organic phase by adding as liquid a fluorinated compound having a boiling point between -70°C and 25°C at atmospheric pressure to an organic solvent.

[0173] In one embodiment, step B) relates to a process for preparing an organic phase comprising a fluorinated compound having a boiling point between -70°C and 25°C at atmospheric pressure, comprising the following steps:

[0174] B.1) condensing the fluorinated compound to obtain a liquid fluorinated compound;

[0175] B.2) preparing a liquid organic solution comprising an organic solvent;

[0176] B.3) cooling the liquid organic solution to a temperature below the boiling point of the liquid fluorinated compound;

[0177] B.4) mixing the liquid fluorinated compound and the liquid organic solution to obtain an organic phase comprising the liquid fluorinated compound.

[0178] In a preferred embodiment, step B.1) is performed by cooling the fluorinated compound to a temperature below the boiling point of the low-boiling fluorinated compound.

[0179] In another embodiment, in step B.4), the mixing is carried out at a temperature below the boiling point of the low-boiling fluorinated compound.

[0180] Preferably, the temperature in step B.4) is the same as the temperature in step B.3).

[0181] Still more preferably, the temperatures in step B.1), step B.3) and step B.4) are suitable for avoiding or substantially limiting evaporation of the liquid fluorinated compound having a boiling point between -70°C and 25°C at atmospheric pressure until the organic phase is injected into the microfluidic box (i.e. step C).

[0182] The temperature in step B.1), step B.3) and step B.4) is below the boiling point of the fluorinated compound, preferably at least 5°C lower than the boiling point of the fluorinated compound, more preferably at least 10°C lower, even more preferably at least 20°C lower, and at most for example 50°C lower.

[0183] Thus, the temperature of the organic phase obtained at the end of step B.4) is below the boiling point of the fluorinated compound, preferably at least 5°C below, more preferably at least 10°C below, even more preferably at least 20°C below, for example at most 50°C below.

[0184] In step B.4), the mixing is carried out for a suitable time to dissolve the liquid fluorinated compound into the liquid organic solution, for example within 5 minutes. At the end of the mixing, the organic phase is obtained in the form of a homogeneous solution. The organic phase should be injected into the microfluidic cartridge within a time suitable for avoiding or substantially limiting the evaporation of the fluorinated compound from the organic phase, for example within 5 minutes of the end of step B).

[0185] In another embodiment, in step B.4), the organic phase comprises the fluorinated compound in a concentration range between 1 and 100 μL / mL, preferably between 10 and 50 μL / mL, still more preferably between 15 and 30 μL / mL.

[0186] A fluorinated compound having a boiling point below room temperature (i.e., 25° C.) is mixed as a gas phase

[0187] According to an alternative embodiment, step B) comprises preparing an organic phase by adding a fluorinated compound having a boiling point between -70°C and 25°C at atmospheric pressure as a gas phase, ie in gaseous form, to an organic solvent.

[0188] In one embodiment, step B) relates to a process for preparing an organic phase comprising a fluorinated compound having a boiling point between -70°C and 25°C at atmospheric pressure, the process comprising the steps of:

[0189] Bi) preparing a liquid organic solution comprising an organic solvent, and

[0190] B.ii) mixing a gaseous fluorinated compound having a boiling point between -70°C and 25°C at atmospheric pressure to the organic liquid solution.

[0191] Preferably, in step B.ii), the mixing is carried out by bubbling the gaseous fluorinated compound into the organic solution.

[0192] According to a preferred embodiment, the temperature of the organic solution is set above the boiling point of the gaseous fluorinated compound prior to the mixing in step B.ii). Temperatures above the boiling point of the fluorinated compound are generally preferred to avoid or substantially limit the phase shift of the fluorinated compound from gas to liquid, which may lead to solubility problems of the condensed fluorinated compound into the organic solvent (e.g., formation of droplets having a liquid core filled with the fluorinated compound suspended in the organic solvent).

[0193] The temperature of the organic phase obtained at the end of step B.ii) is above the boiling point of the gaseous fluorinated compound.

[0194] Preferably, the temperature is between the boiling point and RT, more preferably at most 15°C above the boiling point, even more preferably at most 10°C above the boiling point.

[0195] In step B.ii), the concentration of the fluorinated compound entering the organic phase can be any concentration, preferably up to the saturation concentration. Preferably, the concentration of the fluorinated compound is the saturation concentration.

[0196] The saturation concentration of the fluorinated compound depends on the solvent used and the temperature. For example, considering ethanol as a solvent, the saturation concentration of perfluorobutane is 2.5% by volume and the saturation concentration of perfluoropropane is 2.7% by volume (US2019307908A1).

[0197] Preferably, in step B.ii), the mixing is carried out for a time suitable to achieve a saturated concentration of the gaseous fluorinated compound into the organic liquid solution, for example 2 minutes.

[0198] At the end of mixing, an organic phase consisting of a homogeneous solution was obtained.

[0199] In another embodiment, at the end of its preparation process, the organic phase can be diluted by adding a suitable amount of organic solvent to reduce the concentration of nanodroplets in the organic phase.

[0200] The expression "appropriate amount of organic solvent" means the amount of organic solvent (eg, in mL) necessary to reduce the initial concentration (eg, saturation concentration) of nanodroplets entering the organic phase. The organic phase is then injected into the microfluidic cartridge.

[0201] Step C) Injection into the microfluidic cartridge

[0202] Typically, in step C), the injection of the aqueous phase and the injection of the organic phase are carried out simultaneously.

[0203] The expression "simultaneously" means that the aqueous phase and the organic phase are injected (ie co-injected) into the microfluidic cartridge simultaneously, that is, the aqueous phase and the organic phase are injected into two separate inlets of the microfluidic cartridge simultaneously or substantially simultaneously (eg within a few seconds).

[0204] In a preferred embodiment, both the aqueous phase and the organic phase are injected into the microfluidic cartridge at a temperature suitable to avoid or substantially limit the evaporation of the fluorinated compound. For example, after their respective preparation (i.e., step A) and step B), the aqueous phase and the organic phase may be stored in an ice bath (about 4°C) before injecting both into separate inlets of the microfluidic cartridge (e.g., 5 minutes) to limit the temperature rise during the time between step a), step b) and the subsequent step C).

[0205] According to the invention, after their injection, the aqueous and organic phases are directed to a mixing device where they are mixed, for example by laminar mixing in the case of a staggered herringbone micromixer (see FIG. 2 ), thereby forming a ND.

[0206] Typically, the operating pressure entering the microfluidic box is less than 1000psi (about 7000kPa), preferably less than 500psi (about 3500kPa), more preferably less than 300psi (about 2000kPa), and more preferably less than 100psi (about 700kPa), for example between 10 and 90psi.

[0207] The temperature of the mixing section, where the mixing process takes place in the special microchannel geometry of the mixing section, may be between 0°C and 25°C, preferably between 0°C and 15°C, more preferably between 0°C and 5°C.

[0208] For example, the microfluidic cartridge may be stored in a refrigerator (eg, 4° C.) for a suitable time to reach the desired temperature.

[0209] Total Flow Rate (TFR) and Flow Rate Ratio (FRR)

[0210] The method of the present invention allows the control of nanodroplet characteristics by varying two process parameters: total flow rate and flow rate ratio.

[0211] The expression "total flow rate (TFR)" refers to the total flow rate of the two fluid streams, ie the aqueous phase and the organic phase, pumped through the two separate inlets of the microfluidic cartridge. The unit of measurement of TFR is mL / min.

[0212] According to one embodiment, the TFR is preferably between 2 mL / min and 200 mL / min, preferably between 2 mL / min and 18 mL / min, more preferably between 5 mL / min and 16 mL / min, still more preferably the TFR is 10 mL / min.

[0213] The expression "flow rate ratio (FRR)" refers to the ratio between the amount of aqueous phase and the amount of organic phase flowing into the microfluidic cartridge according to Equation 1:

[0214]

[0215] The volumes of the aqueous and organic phases may be expressed, for example, in mL.

[0216] In a preferred embodiment, the FRR (volume of aqueous phase to volume of organic phase) is between 1:1 and 5:1, preferably between 1:1 and 3:1.

[0217] Step D) Collecting the Aqueous Suspension of Calibrated Nanodroplets

[0218] According to step D) of the disclosed method, at the end of the mixing process, an aqueous suspension of calibrated nanodroplets comprising an inner core and an outer layer is collected from the outlet channel of the microfluidic cartridge, wherein the inner core comprises a fluorinated compound in liquid form and the outer layer comprises an amphiphilic lipid compound comprising a phospholipid.

[0219] After their collection, the inner cores of the microfluidically prepared nanodroplets consist of liquid fluorinated compounds, regardless of the temperature at which they are collected and subsequently stored, and regardless of the nature of the fluorinated compound added to the organic phase (i.e., gaseous or liquid under SATP conditions).

[0220] In other words, even if the collection step (and subsequent storage) is carried out at a temperature above the boiling point of the fluorinated compound used to make the calibrated NDs, the fluorinated compound remains liquid due to its incorporation into the outer stability shell comprising the amphiphilic component. However, it is preferred that such temperature is not too high to limit possible partial phase changes of the entrapped fluorinated compound.

[0221] According to one embodiment, the collecting step may be carried out at a temperature between 0°C and 25°C, preferably between 0°C and 15°C, more preferably between 0°C and 5°C.

[0222] Preferably, the collected aqueous suspension of calibrated nanodroplets has a temperature between 0°C and 25°C, preferably between 0°C and 15°C, more preferably between 0°C and 5°C.

[0223] For example, after collection, the aqueous suspension can be stored in a refrigerator at 4°C.

[0224] Step E) Dilution

[0225] Step E) comprises diluting the collected aqueous suspension of calibrated nanodroplets.

[0226] As described above, the expressions "initial monodisperse distribution" and "initial ND size" refer to the values ​​of monodispersity and ND size of the calibrated ND composition at the end of its preparation process, wherein the end of the preparation process refers to i) collecting the calibrated NDs from the outlet channel of the microfluidic box, or ii) collecting the calibrated NDs and then performing a dilution step (i.e., step E).

[0227] In the present specification and claims, the term "dilution" refers to the process of reducing the concentration of calibrated nanodroplets in a suspension by adding an appropriate amount of water or an aqueous solution.

[0228] Suitable examples of aqueous solutions are saline (which may advantageously be balanced so that the final product for infusion is not hypotonic), or a solution of one or more tonicity-adjusting substances.

[0229] A suitable amount of water or aqueous solution corresponds to the amount of aqueous solution required to reduce the concentration of calibrated nanodroplets in the aqueous suspension by a factor of 2 to 10.

[0230] In a preferred embodiment, step E) of the method comprises diluting the collected aqueous suspension of calibrated nanodroplets 1 to 20 times, preferably 3 to 8 times, and even more preferably diluting the collected aqueous suspension 5 times.

[0231] An additional effect of dilution is to reduce the relative amount of organic solvent in the suspension, thereby reducing the size of the nanodroplets.

[0232] Preferably, step E) is carried out at a temperature suitable for obtaining a diluted aqueous suspension of calibrated nanodroplets, the temperature of the diluted aqueous suspension of calibrated nanodroplets being between 0°C and 25°C, preferably between 0°C and 15°C, more preferably between 0°C and 5°C.

[0233] The dilution step may alternatively be performed inside the microfluidic cartridge with the aid of an additional channel (eg placed between the mixing device and the outlet channel) which is intended to dilute the calibrated fluorocarbon ND suspensions before they are directed to the outlet channel.

[0234] In this case, step E) of the method comprises diluting the suspension of calibrated fluorocarbon-filled nanodroplets 1 to 20 times, preferably 3 to 8 times, and more preferably diluting the collected aqueous suspension 5 times, before collecting them from the microfluidic cartridge.

[0235] Optional step F) washing

[0236] After the dilution step, the calibrated nanodroplets may optionally be treated using a suitable washing technique.

[0237] In this specification, the term "separation technique" refers to any manipulation performed on a suspension of freshly prepared nanodroplets that ultimately results in the removal of (or substantially reducing the amount of) the organic solvent (e.g., ethanol) used to prepare the nanodroplets and by the removal of (or substantially reducing the amount of) unassembled amphiphilic lipid compounds.

[0238] In the present description, the expression "unassembled amphiphilic lipid compounds" refers to amphiphilic lipid molecules present in the calibrated nanodroplet suspension at the end of the microfluidic preparation process but which do not form a stabilizing shell of said nanodroplets.

[0239] Suitable separation techniques include, for example, centrifugation and filtration (eg tangential flow filtration), with centrifugation being preferred.

[0240] Centrifugation parameters such as duration, temperature, and rotation can be varied based on the initial characteristics of the nanodroplets.

[0241] For example, the separation phase can be performed sequentially with the dilution phase (step E) of the disclosed microfluidic method, for example within 5 minutes.

[0242] At the end of the separation procedure, two separate phases are obtained: a liquid supernatant phase comprising the organic solvent and a solid phase comprising the freshly prepared calibrated nanodroplets.

[0243] According to the invention, the solid phase is then mixed to an aqueous solution comprising a freeze-drying protective component, as defined above.

[0244] According to the present invention, after obtaining a freshly prepared nanodroplet suspension by using any suitable preparation method as detailed above, said freshly prepared nanodroplets are then mixed to a freeze-drying protective component to obtain the initial suspension of step a) of the disclosed method.

[0245] Prior to freeze drying (ie step b)), the freeze drying protection component is added in the form of a suitable aqueous solution to the freshly prepared suspension of nanodroplets.

[0246] By "suitable aqueous solution" is meant an aqueous solution of the freeze-drying protective component which, upon mixing into a freshly prepared suspension of nanodroplets, provides an initial suspension of nanodroplets comprising the freeze-drying protective component having a concentration between 1 and 50%, preferably between 5 and 20%, and still more preferably a total concentration between 8 and 12% (w / v%).

[0247] Therefore, before mixing into a freshly prepared suspension of nanodroplets, the concentration of the freeze-drying protective component in the suitable aqueous solution may be adjusted, e.g. depending on the initial volume of the suspension, with the aim of obtaining a specific concentration of the freeze-drying protective component after the mixing step.

[0248] In a preferred embodiment of the invention, prior to the freeze-drying process, the initial suspension of nanodroplets comprises a freeze-drying protective component in a concentration between 1 and 50%, preferably between 5 and 20% and even more preferably in a total concentration between 8 and 12% (w / v%).

[0249] The lyophilization-protective component represents the major amount of the final lyophilized composition, wherein it is typically at least 75%, preferably above 80%, more preferably above 85%, up to e.g. 99.5% (w / w).

[0250] After the freeze-drying process, the freeze-dried composition is reconstituted with a pharmaceutically acceptable liquid carrier to obtain a reconstituted suspension of nanodroplets. The concentration of the freeze-drying protection component in the reconstituted suspension can vary depending on the volume of the pharmaceutically acceptable liquid carrier used in the reconstitution stage.

[0251] The reconstitution with the pharmaceutically acceptable liquid carrier does not include additional infusion of the fluorinated compound.

[0252] Suitable pharmaceutically acceptable (aqueous) liquid carriers may be water, typically sterile, pyrogen-free water (to prevent as much as possible contamination in the final reconstituted product), an aqueous solution such as saline (which may advantageously be balanced so that the final product for injection is not hypotonic), or an aqueous solution of one or more tonicity adjusting substances such as salts or sugars, sugar alcohols, glycols or other non-ionic polyol materials.

[0253] Freeze-dried compositions are typically reconstituted with a volume of liquid carrier similar to the volume of the suspension undergoing the freeze-drying process.

[0254] Thus, the concentration of the lyoprotectant component in the reconstituted suspension is substantially the same as in the initial suspension.

[0255] Alternatively, the volume of the pharmaceutically acceptable liquid carrier is higher than the volume of the initial suspension of the nanodroplets. In the latter embodiment, the concentration of the freeze-drying protective component in the reconstituted suspension of the nanodroplets results in a concentration lower than that contained in the initial suspension of the fluorocarbon-filled nanodroplets before the freeze-drying process. For example, the volume of the reconstituted liquid carrier can be twice the volume of the initial suspension, resulting in a reconstituted suspension characterized by a freeze-drying protective component concentration that is halved.

[0256] The freeze-drying protective components are those described above in the present specification.

[0257] According to one embodiment, the freeze-drying protective component contained in the initial suspension of the nanodroplets comprises a sugar, preferably a disaccharide selected from trehalose, sucrose, maltose or a mixture thereof, more preferably the freeze-drying protective component is trehalose.

[0258] According to another embodiment, the amphiphilic lipid compound comprising phospholipids in the nanodroplet initial suspension is a mixture of amphiphilic lipid compounds.

[0259] Still more preferably, the mixture additionally comprises a lipid carrying a polymer, preferably a PEGylated phospholipid, as defined above.

[0260] According to another embodiment, the mixture additionally comprises a fatty acid, preferably palmitic acid.

[0261] According to one embodiment, the fluorinated compound contained in the initial suspension of nanodroplets has a boiling point between -70 and 160°C, preferably between -50 and 100°C, more preferably between -40 and 60°C.

[0262] In a preferred embodiment, the fluorinated compound is a perfluorocarbon selected from perfluorohexane, perfluoropentane, perfluorobutane, perfluoropropane or mixtures thereof.

[0263] Step b) freeze drying the initial suspension of nanodroplets

[0264] In this specification and claims, the term freeze drying has its standard meaning in the field of pharmaceutical technology. The freeze drying process involves drying a pre-frozen liquid product under low pressure or vacuum and at low temperatures. The main purpose is to remove liquid from the product to provide a freeze-dried product suitable for long-term storage.

[0265] The freeze drying parameters may be selected according to the prior art, for example the freezing temperature may be between -30°C and -70°C, and the negative pressure applied during the freeze drying process may be below 0.5 mbar.

[0266] The freeze-drying-protective component represents the major amount of the final freeze-dried formulation, wherein it is typically at least 75%, preferably above 80%, more preferably above 85%, at most 99.5% (w / w).

[0267] In another aspect, the present invention relates to a method for preparing a reconstituted suspension of nanodroplets, the nanodroplets comprising an inner core and an outer layer, wherein the inner core comprises a fluorinated compound in liquid form and the outer layer comprises an amphiphilic lipid compound comprising a phospholipid, the method comprising the following steps:

[0268] a) preparing an initial suspension, the initial suspension comprising

[0269] i) a plurality of nanodroplets comprising an amphiphilic lipid compound including a phospholipid and a fluorinated compound in liquid form, and

[0270] ii) freeze-drying protection component;

[0271] b) freeze-drying the initial suspension to obtain a freeze-dried composition, and

[0272] c) reconstituting the freeze-dried composition with a pharmaceutically acceptable liquid carrier to provide a suspension of nanodroplets, wherein the amount of the fluorinated compound is 50% to 100% of the amount of the fluorinated compound contained in the initial suspension of step a).

[0273] In step c), the reconstitution of the freeze-dried composition does not comprise an additional infusion of the fluorinated compound.

[0274] stability

[0275] In this specification and claims, the term "stability" refers to the property that the reconstituted suspension of nanodroplets substantially retains its initial characteristics over time, such as ND size, size distribution, nanodroplet concentration and amount of fluorinated compound.

[0276] In particular, initial ND size, initial size distribution and initial amount of fluorinated compound refer to values ​​characterizing the ND size, size distribution and amount of fluorinated compound of an initial suspension of nanodroplets as described above.

[0277] In other words, the initial properties are those possessed by the initial suspension of the nanodroplets prior to their freeze-drying and subsequent storage period.

[0278] The expression "shelf life" denotes a period of time, for example expressed in hours, days or weeks, during which the freeze-dried composition is kept under certain conditions (for example temperature conditions) after the preparation process has been completed.

[0279] According to one embodiment, the freeze-dried composition is stored at a temperature not higher than 30°C, preferably not higher than 25°C, more preferably not higher than 10°C, still more preferably the temperature is below 4°C, down to -20°C.

[0280] According to the present invention, the stability of the reconstituted suspension of nanodroplets can be improved by freeze drying in the presence of a suitable freeze drying protective component.

[0281] After freeze drying, it was found that the reconstituted suspension of nanodroplets was characterized by substantially the same initial properties as the initial suspension. In particular, the initial properties were substantially retained after the freeze drying process, even after storing the freeze dried composition at 4°C for 3 months. In contrast, the initial suspension of nanodroplets comprising the freeze drying protection component that was not subjected to the freeze drying process was unable to maintain their size, size distribution and amount of fluorinated compound over time.

[0282] In one embodiment, the freeze-dried composition provides a suspension of nanodroplets after reconstitution with a pharmaceutically acceptable liquid carrier, wherein the amount of the fluorinated compound is 50% to 100% of the amount of the fluorinated compound contained in the initial suspension, preferably 60% to 100% of the amount of the fluorinated compound contained in the initial suspension of step a), more preferably 70 to 100%, even more preferably 80 to 100%.

[0283] The Applicant has surprisingly observed that the amount of fluorinated compound contained in the initial suspension of nanodroplets can be substantially retained after the freeze-drying process without any additional infusion of the fluorinated compound.

[0284] Surprisingly, the reconstituted suspension of nanodroplets substantially retains the initial amount of fluorinated compound characterized prior to the freeze-drying process and is therefore suitable for subsequent pharmaceutical use without the need for any additional addition of the fluorinated compound (e.g., neither included in a pharmaceutically acceptable liquid carrier nor infused as a gas into the freeze-dried product prior to the reconstitution step).

[0285] Substantially retaining the amount of fluorinated compound advantageously imparts to the reconstituted suspension of nanodroplets an acoustic response similar to that of the initial suspension prior to freeze drying. In other words, the reconstituted suspension of nanodroplets can be converted into gas-filled bubbles when exposed to substantially similar acoustic pressures as compared to the acoustic pressures required to convert the suspension prior to the freeze drying process.

[0286] The amount of fluorinated compound (FC) in the reconstituted suspension of nanodroplets can be expressed as a percentage of the initial amount of fluorinated compound in the initial suspension of nanodroplets by using the following equation (Equation 2):

[0287]

[0288] in:

[0289] - the expression "amount of fluorinated compound after freeze-drying" refers to the weight (eg mg) of fluorinated compound contained in 1 ml of the reconstituted suspension of nanodroplets contained in the vial after freeze-drying, and

[0290] - The expression "amount of fluorinated compound before freeze drying" refers to the weight (eg mg) of fluorinated compound contained in 1 ml of the initial suspension of nanodroplets contained in the vial before freeze drying.

[0291] The amount can be measured, for example, by analytical methods such as gas chromatography (GC).

[0292] Good retention of the properties of the nanodroplets in terms of size and size distribution is associated with a % amount of fluorinated compound in the reconstituted suspension being higher than 50%, preferably higher than 60%, more preferably higher than 70%, still more preferably higher than 80%, up to 100% of the amount of fluorinated compound in the initial suspension.

[0293] Applicants have discovered that by mixing a suitable lyophilization-protecting component to freshly prepared nanodroplets prior to the freeze-drying process and without any further addition of fluorinated compound to the freeze-dried composition prior to reconstitution with a pharmaceutical liquid carrier, the stated amounts of fluorinated compounds can be obtained in the reconstituted suspension.

[0294] The applicant has demonstrated in particular that reconstituted suspensions of nanodroplets comprising a disaccharide as freeze-drying protective component retain unexpectedly high amounts of fluorinated compounds, up to 100% of the amount of fluorinated compounds in the initial suspension.

[0295] Another aspect relates to a reconstituted suspension of nanodroplets prepared by reconstituting a freeze-dried composition with a pharmaceutically acceptable liquid carrier, wherein the freeze-dried composition is obtained by freeze-drying an initial suspension comprising

[0296] i) a plurality of nanodroplets comprising an amphiphilic lipid compound including a lipid and a fluorinated compound in liquid form, and

[0297] ii) freeze-drying protection component;

[0298] And wherein the amount of the fluorinated compound is 50% to 100% of the amount of the fluorinated compound in liquid form contained in the initial suspension before freeze drying.

[0299] Freeze-dried protective component / fluorinated compound weight ratio

[0300] In the present specification and claims, the expression "weight ratio between freeze-drying protective component and fluorinated compound" means the ratio between the weight amount (e.g. mg) of freeze-drying protective component (FDPC) contained in a suspension of nanodroplets of a given volume (e.g. mL) and the weight amount (e.g. mg) of fluorinated compound contained in the same given volume (e.g. mL) of suspension of nanodroplets. The "weight ratio" corresponds to the ratio used to stabilize the disclosed nanodroplets. The weight ratio can be calculated using the following equation (Equation 3):

[0301]

[0302] in:

[0303] - the expression "total weight of freeze-drying protective components" denotes the weight of freeze-drying protective components contained in 1 ml of the initial suspension of nanodroplets before freeze-drying, expressed in mg, and

[0304] The expression "total weight of (P)FC" denotes the amount by weight of fluorinated compounds contained in 1 ml of the initial suspension of nanodroplets before freeze-drying.

[0305] In one embodiment, the weight ratio between the freeze-drying protection component and the fluorinated compound in the initial suspension of the nanodroplets before freeze-drying is higher than 10, preferably higher than 20, preferably higher than 30, preferably higher than 50, preferably higher than 60, preferably higher than 100, and at most 250.

[0306] According to a preferred embodiment, the weight ratio between the lyophilization protective component and the fluorinated compound remains substantially unchanged after the lyophilization process.

[0307] Therefore, according to the embodiment, in the freeze-dried composition, the weight ratio between the freeze-drying protection component and the fluorinated compound is higher than 10, preferably higher than 20, preferably higher than 30, preferably higher than 50, preferably higher than 60, preferably higher than 100, and at most 250.

[0308] The weight ratios are also substantially the same in the reconstituted suspension of nanodroplets.

[0309] According to one embodiment, in the reconstituted suspension composition, the weight ratio between the freeze-drying protection component and the fluorinated compound is higher than 10, preferably higher than 20, preferably higher than 30, preferably higher than 50, preferably higher than 60, preferably higher than 100, up to 250.

[0310] use

[0311] Acoustic droplet vaporization (ADV) is a phenomenon by which nanodroplets with a liquid core comprising a fluorinated compound can be converted into gas-filled microbubbles when exposed to ultrasound energy exceeding the vaporization threshold.

[0312] When used in vivo, nanodroplets have many advantages relative to traditional microbubbles, such as inertia, relatively low toxicity, relative stability in circulation, immiscibility in water and low surface tension. Once evaporated, the microbubbles produced can be effectively used for imaging or therapeutic applications using ultrasound, including sonofemorization, thermal ablation, blood-brain barrier (BBB) ​​destruction, multimodal imaging mode, and allow passive (due to enhanced permeability and retention (EPR) effect in tumor tissue) or active targeting (by introducing targeting ligands) for local delivery of therapeutic drugs or genes. Another potentially valuable characteristic of PFC-ND is that they may be applied to new imaging strategies, such as ultrasound super-resolution imaging, because these agents can be activated and deactivated on demand by applying intermittent acoustic pulses.

[0313] Another aspect relates to a freeze-dried composition comprising

[0314] i) an amphiphilic lipid compound comprising a phospholipid,

[0315] ii) a fluorinated compound in liquid form, and

[0316] iii) Freeze-dried protective component

[0317] The composition, when reconstituted with a pharmaceutically acceptable liquid carrier, provides a suspension of nanodroplets comprising an inner core and an outer layer, wherein the inner core comprises a fluorinated compound and the outer layer comprises an amphiphilic lipid compound, for diagnostic and / or therapeutic treatments.

[0318] Diagnostic treatments include any method in which the use of nanodroplets allows enhanced visualization of parts or regions of the animal (including human) body, including imaging for preclinical and clinical studies. Suitable examples of diagnostic applications are molecular and perfusion imaging, tumor imaging (EPR effect), multimodal imaging (MR-guided tumor ablation, fluorescence, photoacoustic activation), US aberration correction, and super-resolution imaging.

[0319] Therapeutic treatment includes any method for treating a patient. In a preferred embodiment, the treatment includes the combined use of ultrasound and nanodroplets themselves (e.g., in ultrasound-mediated thrombolysis, high-intensity focused ultrasound ablation, blood-brain barrier permeabilization, immunomodulation, neuromodulation, radiosensitization) or in combination with a therapeutic agent (i.e., ultrasound-mediated delivery, for example, for delivering drugs or biologically active compounds to selected sites or tissues, such as in tumor therapy, gene therapy, infectious disease therapy, chronic disease therapy, degenerative disease therapy, inflammatory disease therapy, immune or autoimmune disease therapy or as a vaccine), whereby the presence of the droplets themselves can provide a therapeutic effect or can enhance the therapeutic effect of the applied ultrasound, for example, by themselves or by various physical methods (including, for example, ultrasound-mediated delivery) in vitro and / or in vivo to exert or be responsible for exerting a biological effect.

[0320] The following examples will help to further illustrate the present invention.

[0321] Example

[0322] The following materials were used in the examples that follow:

[0323] Table 1 Materials used in the following examples

[0324]

[0325] Example 1

[0326] Fabrication of nanodroplets using a microfluidic platform

[0327] NanoAssemblr from Precision Nanosystems (Vancouver, Canada) TM Calibrated nanodroplets were formulated using a benchtop automated instrument equipped with a staggered herringbone micromixer (SHM) that allows size-controlled self-assembly (NxGen cartridge, no in-line dilution).

[0328] 1.1. Organic phase containing high boiling point fluorinated compounds

[0329] The organic solution was prepared by adding a lipid mixture of DPPC / PA / DPPE-PEG5k (molar ratio of 74.1 / 18.5 / 7.4) to ethanol at 6 mg / mL and cooled to about 4°C for at least 15 minutes. 12 , bp29.2℃) or perfluorohexane (C6F 14 , bp57.1℃)) was added to the lipid mixture at a concentration of 10 μL / mL to obtain the organic phase.

[0330] The aqueous phase consisting of MilliQ water (at 4° C.) was injected into the first inlet of the microfluidic cartridge, while the organic phase was injected into the second inlet of the microfluidic cartridge ( Figure 1 ). The microscopic features of the channel were designed to induce accelerated mixing of the two fluid streams in a controlled manner. The microfluidic process settings, i.e., total flow rate (TFR, in mL / min) and flow rate ratio (FRR), were an FRR of 3-1 (aqueous-organic) and a TFR of 10 mL / min. The ND suspension was collected from the outlet channel into a Falcon vial (15 mL) and diluted 5-fold with ultrapure water.

[0331] 1.2. Organic phase containing low-boiling fluorinated compounds

[0332] An organic solution was prepared by adding a lipid mixture of DPPC / PA / DPPE-PEG5k (molar ratio of 74.1 / 18.5 / 7.4) to ethanol at 6 mg / mL and cooled to about -20°C. Then, liquid perfluorobutane (C4F 10 , bp-2°C) and added to the lipid ethanol solution at a concentration of 15 μL / mL. The solution was stirred until the liquid perfluorobutane was completely dissolved. The same procedure was performed using perfluoropropane (boiling point -37°C) as the perfluorocarbon, except that the lipid mixture in ethanol was cooled to about -80°C and the liquid perfluoropropane was obtained by condensing into a syringe at -80°C.

[0333] The aqueous phase consisting of MilliQ water (at 4° C.) was injected into the first inlet of the microfluidic cartridge, while the organic phase was injected into the second inlet of the microfluidic cartridge ( Figure 1 ). The microscopic properties of the channel were designed to induce accelerated mixing of the two fluid streams in a controlled manner. The microfluidic process settings, i.e., total flow rate (TFR, in mL / min) and flow rate ratio (FRR), were 1-1 (aqueous-organic) FRR and 10 mL / min TFR. The ND suspension was collected from the outlet channel into a Falcon vial (15 mL) and diluted 5-fold with ultrapure water.

[0334] Example 2

[0335] Preparation of perfluorocarbon-filled nanodroplets by sonication

[0336] Organic solution was prepared by adding a lipid mixture of DPPC / PA / DPPE-PEG5k (molar ratio of 74.1 / 18.5 / 7.4) to ethanol at 6 mg / mL. The solution was then heated at 65°C in a 100 mL balloon under stirring. The solvent was removed under vacuum to obtain a lipid film. The lipid blend was dried overnight at 25°C under vacuum (0.2 mBar).

[0337] The lipid blend was redispersed in 10 mL of Tris buffer 20 mM pH 7.5 at 65° C. for 30 minutes under stirring. The obtained liposome solution was placed in an ultrasonic tank for 3 minutes. The suspension was then cooled to 4° C. in an ice bath.

[0338] Add 0.5 mL of perfluoropentane (C5F 12 , bp 29.2°C). The mixture was emulsified using a Branson sonicator 250 (output 20% - 6x (1 min US on - 30 s US off)) equipped with a 5 mm tip. During the whole process, the suspension was cooled in an ice bath. ND size was measured after emulsification.

[0339] Example 3

[0340] Preparation of perfluorocarbon-filled nanodroplets via microbubble condensation method

[0341] Perfluorobutane-filled microbubbles were prepared as described in US2022211850A1 (Example 6).

[0342] After their preparation, reconstitution of lipid-shelled microbubbles (MBs) was performed by adding 5 mL of sterile saline solution (0.9% NaCl) to the vial using a syringe and mixing for about 10 seconds to obtain a white milky homogeneous liquid.

[0343] Subsequently, 4 mL of the MB suspension sample (6 mL headspace) was taken using a 10-mL syringe, then stoppered and placed at about -20°C for about 6 minutes to prevent the suspension from freezing.

[0344] The syringe piston was then pushed down to the 4 mL graduation / mark to increase the pressure inside the syringe and held in this position for about 30 s, and then the syringe piston was slowly released to obtain a clear suspension of nanodroplets characterized by a core comprising liquid perfluorobutane without microbubbles. The liquid core was stable even during subsequent storage of the suspension at temperatures above the boiling point of perfluorobutane.

[0345] Example 4

[0346] Preparation of freeze-dried compositions

[0347] Prior to the freeze-drying process, the suspension of nanodroplets obtained according to the above examples 1, 2 and 3 is mixed with a suitable solution of a freeze-drying protective component.

[0348] To this end, the mixing phase is carried out according to different schemes, depending on the preparation method used, as described in the following paragraphs.

[0349] 22.5 ml of the calibrated nanodroplet suspension obtained by the microfluidics method as described in Example 1 was centrifuged at 5000 g for 12 minutes at 4°C. The supernatant was discarded and the washed nanodroplets (pellet) were redispersed with a solution containing a freeze-dried protective component to obtain 22.5 mL of a suspension as described in the following examples. 1 ml of the perfluorocarbon-filled nanodroplet suspension obtained by the sonication method as described in Example 2 was directly mixed to 9 ml of a solution containing a freeze-dried protective component.

[0350] The suspension of perfluorocarbon-filled nanodroplets obtained by the microbubble coagulation method described in Example 3 was diluted 2-fold with trehalose solution (20%).

[0351] The specific types and amounts of substances used in the suspensions are summarized in Table 2.

[0352] The suspension was then aliquoted into DIN8R glass vials (1 ml suspension / vial), transferred to a freeze dryer rack and frozen at -55°. The freeze drying process was then carried out by a drying step at -20°C and 200 μBar. At the end of freeze drying, the vials were stoppered, crimped and stored at 4°C until redispersion.

[0353] At the end of this process, the freeze-dried composition was obtained as a white homogeneous dry solid. The vials were stoppered, crimped and stored at 4°C until redispersion.

[0354] In the following examples, after the freeze-drying process, each freeze-dried composition was reconstituted with 1 mL of an aqueous liquid carrier to obtain a stable nanodroplet suspension. Thus, the freeze-dried composition was reconstituted with an aqueous liquid carrier in a volume equal to the volume of the suspension that underwent the freeze-drying process.

[0355] Table 2 Compositions used in the present invention: All compositions contain DPPC / PA / DPPE-PEG5000 (molar ratio 74.1 / 18.5 / 7.4) as stabilizing substances, except that composition 5PT contains DSPC / PA / DPPE-PEG5000 (molar ratio 74.1 / 18.5 / 7.4)

[0356]

[0357] Example 5

[0358] Stability of freeze-dried perfluorocarbon-filled nanodroplets

[0359] To evaluate the effect of the freeze-drying process on the initial properties of NDs, different storage conditions of composition 1T were compared.

[0360] As a comparison, aliquots of each sample were stored as an aqueous suspension at 4°C over time prior to the freeze-drying process.

[0361] Nanodroplet suspensions reconstituted after freeze-drying or stored as is were characterized using a Malvern Zetasizer Nano-ZS instrument (Malvern Instruments Ltd., UK) to measure the size and size distribution (PDI) after different storage conditions, as shown in Table 3.

[0362] result

[0363] Overall results show that freeze-drying allows for better retention of ND properties (size and polydispersity index) over time compared to storage of ND suspensions at 4°C.

[0364] Table 3: Changes in size and PDI of freeze-dried products compared to non-freeze-dried suspensions under different storage conditions

[0365]

[0366] Example 6

[0367] Effect of freeze-drying protective component properties on the characteristics of nanodroplets

[0368] Different freeze-drying protective components were selected to evaluate the effects of their properties on the characteristics of the nanodroplets.

[0369] The detailed qualitative / quantitative composition of each formulation characterized in this example is reported in Table 2. All compositions except 4PT and 5PT were characterized (Z-average and PDI) using a Malvern Zetasizer Nano-ZS instrument (Malvern Instruments Ltd., UK) to measure the size and size distribution (PDI) after different storage conditions, as shown in Table 4.

[0370] The characterization of compositions 4PT and 5PT was performed using a NanoSight NS300 (Malvern) instrument.

[0371] The nanodroplet concentration after different storage conditions was measured by NTA (Nanoparticles Tracking Analysis).

[0372] NTA measurements were performed using a NanoSight NS300 (Malvern) instrument according to the manufacturer's recommendations.

[0373] result

[0374] Overall results show that the use of disaccharides (particularly trehalose, sucrose or maltose) as freeze-drying protectants results in substantially good retention of the initial nanodroplet size, PDI and concentration after freeze-drying.

[0375] The use of disaccharides substantially maintains the ND properties, regardless of the properties of the ND core (i.e., fluorinated compounds with high or low boiling points). In fact, positive results in terms of property retention were obtained for PFCs with high boiling points, such as perfluorohexane (bp 57.1°C) (compositions 1T, 1S, and 1Ma) and perfluoropentane (bp 29.2°C) (compositions 3T and 3S), or PFCs with low boiling points, such as perfluorobutane (bp -2°C) (compositions 4T, 4S, and 4Ma) and perfluoropropane (bp -37°C) (composition 5T).

[0376] The nanodroplet concentration was measured by NTA for compositions 1T and 1Ma, showing that the concentration was substantially maintained after freeze drying (composition 1T: 8.64×10 10 NDs / mL, after freeze drying7.49x10 10 NDs / mL; Composition 1Ma: before freeze drying 8.66×10 10 nds / mL, after freeze drying 7.33×10 10 nds / mL).

[0377] The results further demonstrate that the use of a mixture comprising a disaccharide as a lyophilization protectant results in substantially good retention of ND properties, such as size, after freeze drying (see compositions 4PT and 5PT). Both formulations obtained by the microbubble coagulation method are characterized by substantially high polydispersity before and after freeze drying.

[0378] Although the retention of the initial ND size was quite good, mannitol and PEG4000 (used as the sole lyoprotectants) were less efficient in maintaining the initial PDI values ​​after the freeze-drying process. Compositions containing Dextran 15 already showed high PDI values ​​(>0.250) after their preparation, before the freeze-drying procedure. The freeze-dried composition ID was characterized by significantly different sizes and PDI after the freeze-drying procedure, either immediately after the end of the process or after a storage period of 3 months.

[0379] Table 4 Effect of the properties of freeze-drying protection components on size and PDI before and after freeze-drying

[0380]

[0381] *Not applicable due to polydisperse nature

[0382] Example 7

[0383] Determination of the amount of perfluorocarbons after freeze drying

[0384] The effect of the freeze-drying procedure on the amount of perfluorocarbon contained in the reconstituted suspension of nanodroplets was evaluated. To this end, the content of fluorocarbons was measured by gas chromatography coupled to a flame ionization detector (GC-FID) before and after freeze-drying. The amount of perfluorocarbons in the reconstituted suspension of perfluorocarbon-filled NDs was calculated according to Eq.

[0385] result

[0386] Table 5 Effect of freeze-drying protective components (nature and amount) on the amount of perfluorocarbons retained after the freeze-drying process

[0387] Composition No. Remaining PFC (%) 3T 100 4T 100 3S 82 3M 8

[0388] The results showed that the reconstituted suspension of PFC-NDs containing disaccharides as freeze-drying protective components retained a higher amount of perfluorocarbons than those using other freeze-drying protective components. In particular, the amount of perfluorocarbons using trehalose, sucrose and maltose was found to be about ten times higher than the amount of perfluorocarbons of the suspension containing mannitol.

[0389] Surprisingly, it was found that the amount of perfluorocarbon remained essentially unchanged, ie close to 100% of the amount of perfluorocarbon in the initial suspension.

[0390] Good retention of the amount of perfluorocarbons was demonstrated with PFCs having high boiling points, perfluoropentane (bp 29.2°C) (compositions 3T and 3S) and perfluorohexane (bp 57.1°C) (composition 1Ma) or PFCs having low boiling points, such as perfluorobutane (bp -2°C) (composition 4T).

[0391] Example 8

[0392] Effect of the Weight Ratio between Protective Component and Perfluorocarbon in Freeze-Drying

[0393] 8.1. Calibrated PFC nanodroplets obtained by microfluidics

[0394] Different lyophilization protective component / PFC weight ratios were investigated to evaluate their effects on the nanodroplet properties before and after freeze drying. To this end, the procedures shown in Examples 1 and 4 were performed by varying the amount of lyophilization protective component reagent added to the washed nanodroplets (pellets) after centrifugation. In this example, the initial lyophilization protective component / PFC weight ratios were 60, 120, 180 and 240, obtained by using aqueous trehalose solutions of 5, 10, 15 and 20% trehalose.

[0395] The weight ratio is determined according to Equation 3 as described above.

[0396] At the end of freeze drying, the vials were stoppered, sealed and stored at 4°C until redispersion.

[0397] The detailed qualitative / quantitative composition of each formulation characterized in this example as well as the process parameters used for their preparation are reported in Table 2.

[0398] Characterization was performed using a Malvern Zetasizer Nano-ZS instrument (Malvern Instruments Ltd., UK) to measure size and size distribution (PDI) before and after freeze drying as shown in Table 6.

[0399] result

[0400] The results demonstrate that in all investigated compositions with different initial freeze-drying protection component / PFC ratios (60 to 240), the initial ND properties in terms of size (Z-average and PDI) are essentially well retained.

[0401] Table 6: Effect of the weight ratio between freeze-drying protection component and perfluorocarbon on size and PDI

[0402]

[0403] 8.2. PFC nanodroplets obtained by sonication

[0404] The PFC nanodroplets were prepared according to the procedure shown in Example 2. After their preparation, the suspension obtained was then diluted 5-fold, 10-fold, 20-fold or 50-fold with an aqueous solution of trehalose solution (final trehalose concentration in the suspension was 10% by weight) to obtain corresponding freeze-drying protection component / PFC weight ratios of 6.25, 12.5 and 25, respectively. The suspension obtained was sampled in DIN8R vials (1 mL / vial) and then freeze-dried according to Example 4. At the end of freeze-drying, the vials were stoppered, sealed and stored at 4°C until redispersion.

[0405] The detailed qualitative-quantitative composition of each formulation characterized in this example as well as the process parameters used for their preparation are reported in Table 2.

[0406] Characterization was performed using a Malvern Zetasizer Nano-ZS instrument (Malvern Instruments Ltd., UK) to measure size and size distribution (PDI) before and after freeze drying as shown in Table 7.

[0407] result

[0408] It can be inferred from Table 7 that compositions characterized by a lower weight ratio between lyoprotectant component / PFC experienced an increase in ND size and PDI after the lyophilization process. Compositions with higher ratios substantially retained both initial size and PDI.

[0409] Table 7: Effect of the weight ratio of lyoprotectant component to perfluorocarbon on size and PDI

[0410]

[0411] Example 9

[0412] Acoustic Droplet Vaporization (ADV) Measurement

[0413] The expression "Acoustic Droplet Vaporization (ADV) Threshold" refers to the minimum acoustic pressure necessary to obtain the conversion of nanodroplets into echoing gas microbubbles.

[0414] The acoustic droplet vaporization (ADV) threshold of the calibrated PFC ND prepared according to the aforementioned embodiment can be determined according to conventional methodology using B-mode imaging methods. For example, a suspension of NDs can be vaporized when passing through the focal zone of the transducer, and the acoustic pressure is increased by about 0.2 MPa every 5 seconds until ND vaporization is observed.

[0415] Nanodroplet activation was performed by focused ultrasound at five aligned focal points, allowing activation only in the region of interest where the acoustic pressure was highest. Pulses were emitted in burst mode at a frequency of 6 MHz, 20 cycles per pulse, and a pulse repetition frequency (PRF) of 1 Hz.

[0416] ADV determinations were performed on five different compositions, differentiated by the PFCs forming their core, namely perfluorohexane, perfluoropentane and perfluorobutane (see Table 8). The ADV of each composition was determined before and after the freeze drying process.

[0417] For all experiments, 0.4 mL of each ND suspension was diluted in 40 mL of water at 37 °C.

[0418] result

[0419] The overall results obtained from the determination of the ADV threshold are reported in Table 9. Each value is the average of three consecutive determinations.

[0420] Table 8 Acoustic droplet vaporization threshold as a function of PFC core

[0421] Before freeze drying After freeze drying Composition No. MPa MPa 1T 10.28 10.33 3T 9.14 9.15 4T 4.59 4.35 4PT 4.20 4.12 5PT 4.50 4.30

[0422] The results showed that all tested compositions had similar ADV thresholds before and after the freeze-drying process, independent of the nature of the PFCs in the core of the NDs, confirming that the acoustic response was also preserved.

[0423] References

[0424] ●Zhang,W.et al,Nanoscale,2022,14,2943-2965

[0425] ●Melich,R.et al.International Journal of Pharmaceutics,2020,587,119651

[0426] ●US20220000790

[0427] ●WO2022101365A1(Bracco Suisse SA)

[0428] ●US20190307908

[0429] ●US2022211850A1

Claims

1. A freeze-dried composition comprising i) an amphiphilic lipid compound comprising a phospholipid, ii) a fluorinated compound in liquid form, and iii) freeze-dried protective components, The composition, when reconstituted with a pharmaceutically acceptable liquid carrier, provides a suspension of nanodroplets comprising an inner core and an outer layer, wherein the inner core comprises the fluorinated compound in liquid form and the outer layer comprises the amphiphilic lipid compound.

2. The freeze-dried composition according to claim 1, wherein the weight ratio between the freeze-drying protection component and the fluorinated compound is greater than 10 and at most 250.

3. The freeze-dried composition of claim 2, wherein the weight ratio is greater than 20.

4. The freeze-dried composition of claim 3, wherein the weight ratio is greater than 50.

5. The freeze-dried composition according to any one of the preceding claims, wherein the fluorinated compound has a boiling point between -70°C and 160°C.

6. The freeze-dried composition of claim 5, wherein the fluorinated compound has a boiling point between -50°C and 100°C.

7. The freeze-dried composition of claims 5-6, wherein the fluorinated compound has a boiling point between -40°C and 60°C.

8. The freeze-dried composition according to any one of the preceding claims, wherein the fluorinated compound is a perfluorocarbon selected from perfluoropentane, perfluorohexane, perfluorobutane, perfluoropropane or mixtures thereof.

9. The freeze-dried composition according to any one of the preceding claims, wherein the freeze-drying protective component is a sugar.

10. The freeze-dried composition according to claim 9, wherein the sugar is a disaccharide selected from trehalose, sucrose, maltose or a mixture thereof.

11. The freeze-dried composition of claim 10, wherein the disaccharide is trehalose.

12. The freeze-dried composition according to any one of the preceding claims, wherein the amount of the freeze-dried component is at least 75% and at most 99.5% (w / w).

13. The freeze-dried composition according to claim 12, wherein the amount of the freeze-dried component is greater than 80%, up to 99.5% (w / w).

14. The freeze-dried composition according to claim 13, wherein the amount of the freeze-dried component is greater than 85%, up to 99.5%.

15. A process for the preparation of a freeze-dried composition as defined in claims 1 to 14, comprising the following steps: a) preparing an initial suspension, the initial suspension comprising i) a plurality of nanodroplets comprising an amphiphilic lipid compound including a phospholipid and a fluorinated compound in liquid form, and ii) a freeze-drying protection component; and b) freeze drying the initial suspension wherein the freeze-dried composition, when reconstituted with a pharmaceutically acceptable liquid carrier, provides a suspension of nanodroplets as defined in claims 1-14, wherein the amount of the fluorinated compound is 50% to 100% of the amount of the fluorinated compound contained in the initial suspension of step a).

16. The method of claim 15, wherein the reconstitution does not include additional infusion of a fluorinated compound.

17. The method according to claims 15-16, wherein in step a) the initial suspension is prepared by a method selected from sonication, microbubble coagulation or microfluidization.

18. The method of claims 15-17, wherein the initial suspension of nanodroplets comprises a freeze-drying protective component at a concentration between 1 and 50% (w / v %).

19. The method of claim 18, wherein the initial suspension of nanodroplets comprises a freeze-drying protective component at a concentration between 5 and 20% (w / v %).

20. The method of claim 19, wherein the initial suspension of nanodroplets comprises a freeze-drying protective component at a concentration between 8 and 12% (w / v %).

21. The method according to claims 15-20, wherein the weight ratio between the freeze-drying protection component and the fluorinated compound in the initial suspension of nanodroplets is greater than 10 and at most 250.

22. The method according to claims 15-21, wherein the amount of the fluorinated compound in the reconstituted suspension is 80 to 100% of the amount of the fluorinated compound contained in the initial suspension of step a).

23. A method for preparing a reconstituted suspension of nanodroplets, the nanodroplets comprising an inner core and an outer layer, wherein the inner core comprises a fluorinated compound in liquid form and the outer layer comprises an amphiphilic lipid compound comprising a phospholipid, the method comprising the steps of: a) preparing an initial suspension, the initial suspension comprising i) a plurality of nanodroplets comprising an amphiphilic lipid compound including a phospholipid and a fluorinated compound in liquid form, and ii) freeze-drying protection component; b) freeze-drying the initial suspension to obtain a freeze-dried composition as defined in claims 1 to 14, and c) reconstituting the freeze-dried composition with a pharmaceutically acceptable liquid carrier to provide a suspension of the nanodroplets, wherein the amount of the fluorinated compound is 50% to 100% of the amount of the fluorinated compound contained in the initial suspension of step a).

24. The method according to claim 23, wherein in step c), the reconstitution of the freeze-dried composition does not comprise an additional infusion of a fluorinated compound.

25. A freeze-dried composition as defined in claims 1 to 14 for use in diagnostic and / or therapeutic treatment.

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