Anionic gas-filled microvesicles with calibrated sizes

Abstract

The invention relates to new calibrated-size gas-filled microvesicles bearing an overall negative charge, to their method of manufacturing and to their use. Said gas-filled microvesicles have a geometric standard deviation (GSD) value of at least 1.20 or lower and a stabilizing envelope comprising a phospholipid selected from phosphatidic acid, phosphatidylserine or a mixture thereof and a pegylated phospholipid.

Description

[0001] ANIONIC GAS-FILLED MICROVESICLES WITH CALIBRATED SIZES

[0002] Technical field

[0003] The invention relates to new calibrated gas-filled microvesicles bearing an overall negative charge, to their method of manufacturing and to their use.

[0004] Background of the invention

[0005] Calibrated-size gas-filled microvesicles (in short "calibrated microvesicles" or "CMV") are a new generation of gas-filled microvesicles having a relatively narrow and controlled size distribution (with mean diameter sizes which can be selected to range between 2 and 8 pm), as compared to commercially available polydisperse microbubble ultrasound contrast-agents (USCA). Calibrated microvesicles are in particular designed to enhance imaging sensitivity and improve efficiency in therapeutic treatments, e.g. in delivering drugs or genes to specific organs through different techniques, including, for instance, BBB (Blood Brain Barrier) disruption, thermal ablation, sonopermeabilisation or sonothrombolysis. Calibrated microvesicles can be produced using various techniques: decantation, mechanical filtration, centrifugation, bubble sorting and flow-focusing.

[0006] Particularly, the flow-focusing technology allows the manufacturing of calibrated microvesicles (characterized by a relatively low value of geometric standard deviation (GSD), e.g. down to about 1.05-1.08) in a highly reproducible way at a reasonable production rate (~106bubbles per second), with acceptable concentrations of suspended microvesicles (e.g. up to 4xl08CMV / mL) for subsequent uses. Calibrated microvesicles prepared by flow-focusing technology are for instance described in WO2018041906A1, WO2019 170606A1, W02020260420A1, WO2020260423A1 and

[0007] WO2023285628A1.

[0008] Employing flow-focusing techniques to produce calibrated microbubbles stabilized by phospholipids presents a significant challenge, largely due to the inherent instability that affects these freshly formed microbubbles, e.g. coalescence of monodisperse bubbles that results in a lower bubble concentration and in a broader microbubble size distribution, leading to a less efficient contrast agent (Segers, 2019).

[0009] Microvesicles bearing an overall negative net charge are generally known in the art for a variety of clinical applications to specifically image and diagnose organs, for instance for imaging of endothelium in capillaries, late after injection of contrast agents, thanks to the retention via complement-mediated attachment to endothelium (Fisher, 2002).

[0010] Up to now, according to Applicant's knowledge, anionic gas-filled microvesicles have not been prepared yet with calibrated sizes, in particular using flow-focusing techniques.

[0011] Although polyethylene glycol (PEG) is often added to the lipid coating of contrast microbubbles to prevent coalescence and improve circulation, the repeated injection of PEGylated microbubbles can cause subsequent injections to be rapidly eliminated from circulation, a phenomenon called « accelerated blood clearance (ABC) effect » (Ref. Fix, 2018).

[0012] There is thus the need to substantially reduce the amount of PEGylated compounds in the shell of microbubbles.

[0013] The Applicant has observed that the incorporation of negatively charged phospholipids in the shell of calibrated microvesicles prepared by flow-focusing method may substantially reduce the amount of PEGylated lipids in the stabilizing layer while preserving the stability of the formulations, e.g. substantially reducing the coalescence of freshly formed microvesicles.

[0014] Therefore, the present invention provides novel compositions of anionic gas-filled microvesicles with calibrated sizes obtained by flow-focusing microfluidic method, characterized by a reduced amount of pegylated (PEGylated) phospholipids providing enhanced stability and biocompatibility.

[0015] Summary of the invention

[0016] An aspect of the invention relates to a suspension of calibrated gas-filled microvesicles, said microvesicles comprising an inner core and an outer layer, said inner core comprising a physiologically acceptable gas and a said outer layer comprising:

[0017] 55% by moles or more of a phospholipid selected from phosphatidic acid, phosphatidylserine, or a mixture thereof and

[0018] 7.5% by moles or less, down to 0.5% of a pegylated phospholipid wherein said suspension of calibrated gas-filled microvesicles has a geometric standard deviation (GSD) of at least 1.20 or lower.

[0019] In a preferred embodiment said phospholipid is selected from the group consisting of l,2-Dilauroyl-sn-glycero-3-phosphate (DLPA), l,2-Dimyristoyl-sn-glycero-3-phosphate (DMPA), l,2-Dipalmitoyl-sn-glycero-3-phosphate (DPPA), l,2-Distearoyl-sn-glycero-3- phosphate (DSPA), l,2-dilauroyl-sn-glycero-3-phospho-L-serine (DLPS), 1,2-Dimyristoyl- sn-glycero-3-phospho-L-serine (DM PS), l,2-Dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), l,2-Distearoyl-sn-glycero-3-phospho-L-serine (DSPS) or mixtures thereof.

[0020] In a further preferred embodiment, said phospholipid is 1,2-Dimyristoyl-sn-glycero- 3-phosphate (DMPA) or l,2-Dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS).

[0021] In an embodiment, said pegylated phospholipid is a phospholipid covalently linked to a polyethylene glycol having average molecular weight of from 1000 to 8000 g / mol.

[0022] In an embodiment, said outer layer further comprises a neutral phospholipid.

[0023] In a further embodiment, said suspension of gas-filled microvesicles has a Z- potential of - 5 mV or lower. In another embodiment, said pegylated phospholipid comprises a pegylated phospholipid comprising a ligand.

[0024] Preferably said ligand is selected from the group consisting of avidin, neutravidin and streptavidin.

[0025] Another aspect of the invention relates to the use of the suspension as above defined for cell separation.

[0026] A further aspect of the invention relates to a method for preparing a suspension of calibrated gas-filled microvesicles as above defined, said method comprising the following steps: a) providing (I) a gaseous flow and (ii) an aqueous liquid flow comprising:

[0027] - 55% by moles or more of a phospholipid selected from phosphatidic acid, phosphatidylserine or a mixture thereof and

[0028] -7.5% by moles or lower of a pegylated phospholipid b) directing said gaseous flow and said liquid flow through respective inlet channels towards a contact zone; c) directing said gaseous flow and said liquid flow from the contact zone through a calibrated orifice to obtain an aqueous suspension comprising said gas-filled microvesicles; d) collecting a suspension of calibrated gas-filled microvesicles, wherein said suspension has a geometric standard deviation (GSD) of at least 1.20 or lower.

[0029] Another aspect relates to a suspension of calibrated gas-filed microvesicles as above defined obtainable by the above defined manufacturing method.

[0030] Another aspect relates to a suspension of calibrated gas-filled microvesicles as above defined for use in diagnostic and / or therapeutic treatment.

[0031] Detailed description of the invention

[0032] It has now been found that calibrated gas-filled microvesicles stabilized by suitable anionic phospholipids may be advantageously obtained by flow-focusing microfluidic method with a reduced amount of pegylated phospholipids preserving the stability of the freshly formed microvesicles.

[0033] The Applicant unexpectedly observed that using a suitable amount of anionic phospholipids selected from phosphatidic acid or phosphatidylserine as stabilizing materials allowed to significantly reduce, e.g. to halve, the amount of pegylated phospholipids in the outer layer of the calibrated microvesicles without affecting the coalescence stability of the freshly formed CMV, i.e. by limiting (or substantially reducing) their coalescence at the end of their preparation. The Applicant has further surprisingly observed that such novel compositions of anionic monosize microvesicles, when properly functionalized, possess an improved cell separation efficiency and may thus be used in methods for collecting / recovering cells or biological material displaying.

[0034] In this description and claims, the term "calibrated" is used interchangeably with "size-controlled", "monodispersed" or "monosize(d)" microvesicles.

[0035] Calibrated gas-filled microvesicles (CMV) are preferably produced by using a microfluidic flow-focusing technology, where a gas thread is focused between two liquid flows in a flow-focusing device and phospholipid-stabilized calibrated microvesicles form and are collected in the outlet channel. Through this approach calibrated microvesicles are manufactured in a highly reproducible way at a reasonable production rate (~60 million bubbles per minute) (as described for instance in WO2018041906A1, WO2019 170606A1, W02020260420A1, WO2020260423A1 and

[0036] WO2023285628A1 which are hereby incorporated by reference).

[0037] Depending on the parameters of the manufacturing process and device, the calibrated microvesicles may be obtained with relatively narrow size distribution around any desired mean diameter, e.g. at least 3 pm.

[0038] In a preferred embodiment, the mean diameter of said calibrated gas filled microvesicles is of at least 3 pm, preferably at least 4 pm, more preferably at least 5 pm, still more preferably at least 6 pm, still more preferably at least 7 pm, still more preferably at least 8 pm, still more preferably at least 9 pm, up to e.g. 10 pm.

[0039] The size distribution of said calibrated microvesicles is typically characterized by a geometric standard deviation (GSD) value of at least 1.20 or lower, preferably of at least 1.15 or lower, down to e.g. 1.05.

[0040] The calibrated microvesicles concentration (particularly upon production with microfluidic flow-focusing) is preferably not lower than 3xl08CMV / mL, preferably at least 4xl08CMV / mL.

[0041] The "geometric standard deviation" (GSD) generally provides a suitable value for characterizing the breath of the size distribution in a population of particles (gas-filled microvesicles in the specific case). A population of particles with a broad range of sizes will thus have a larger GSD value than one in which the particles sizes are narrowly distributed around a mean value (i.e. relatively similar in size).

[0042] W02020260420A1 (Figure 1) shows an example of a size distribution graph (by volume) of a population of gas-filled microvesicles which can be obtained with a commercial particle analyser instrument (e.g. Coulter Counter Multisizer 3, equipped with the Multisizer 3 software). Typically, the geometric standard deviation of a suspension of gas-filled microvesicles can be determined by: i) measuring the number of calibrated gas-filled microvesicles, their respective mode diameter in volume, and volume distribution in a selected size range (e.g. between 3 pm and 6 pm for a 4.5 pm CMV mean diameter) using a commercial particle analyzer instrument, (such as a Coulter Counter Multisizer 3 equipped with Multisizer 3 software, with incremental diameters of e.g. 0.1 microns); ii) configuring the particle analyzer instrument preferences to select the Geometric Statistic Type (instead of Arithmetic Statistic type used for polydisperse suspensions); iii) calculating the GSD of a CMV distribution using the particle analyser instrument, by appying the following Equation 1:

[0043] Eq.l GSD =

[0044] Where: n, = percentage of volume of gas (with respect to the total one) entrapped in the microvesicles measured for the ith channel

[0045] = volume of the microvesicles in the ithchannel, where

[0046] Eq. 1.1. Xi = di3.n / 6

[0047] (di = diameter of the microvesicle in the ith channel center) x= geometric mean of the volume of the microvesicles in the selected range, where: rZ(nflof) 1

[0048] Eq.1.2. x = 101 ni ]

[0049] Among the various commercially available analytical instruments, the Coulter Counter Multisizer 3, equipped with the Multisizer 3 software, can calculate and provide such GSD value as defined above.

[0050] For instance, a GSD value of 1.2 indicates that about the 50% of CMV are calibrated between 2.5 and 5 pm, for a mean diameter of 4 pm; a GSD of 1.05-1.08 (< 1.1) indicates that about the 90-95% of CMV have sizes comprised between 2.5 and 5 pm.

[0051] According to the present invention, the geometric standard deviation of the disclosed suspension of calibrated gas-filled microvesicles is measured using the method described above. The expression "microvesicles concentration "as used herein refers to the number of CMV in a volume unit, determined using a Coulter Counter apparatus, i.e. number of CMV / mL.

[0052] As used herein, the term "anionic calibrated microvesicles" refers to calibrated gas- filled vesicles as defined above that possess a net negative electrical charge. These microvesicles typically comprise a gas core encapsulated by a stabilizing shell comprising anionic compounds, e.g. anionic phospholipids, that confer a negative charge to the surface. The anionic nature of these microbubbles allows them to interact favorably with positively charged entities, such as certain biomolecules or cellular structures, in various applications including diagnostic and / or therapeutic procedures.

[0053] Typically, a suspension of anionic calibrated gas-filled microvesicles has a Z-potential of at least - 5 mV or lower, preferably -10 mV or lower, more preferably -20 mV, more preferably -30 mV or lower, more preferably -40 or lower, down to -50 mV.

[0054] Components of the outer shell of the gas-filled microvesicles

[0055] According to the invention, the outer shell of said anionic calibrated gas-filled microvesicles can be stabilized by using a formulation comprising an anionic phospholipid, selected from phosphatidic acid (PA) or phosphatidylserine (PS), and a pegylated phospholipid.

[0056] The microvesicles may further comprise neutral phospholipids and / or fatty acids, in admixture with the above anionic phospholipid and pegylated compound.

[0057] Furthermore, the disclosed anionic calibrated microvesicles may be advantageously functionalized with suitable ligands for applications in cell sorting techniques.

[0058] Phospholipids

[0059] The term "phospholipid(s)" as used herein include esters of glycerol with one or preferably two (equal or different) residues of a fatty acid and with a phosphoric acid residue, wherein the phosphoric acid residue is in turn bound to a hydrophilic group, such as, for instance, choline (phosphatidylcholines - PC), serine (phosphatidylserines - PS), glycerol (phosphatidylglycerols - PG), ethanolamine (phosphatidylethanolamines - PE), inositol (phosphatidylinositol). Esters of phospholipids with only one residue of fatty acid are generally referred to in the art as the "lyso" forms of the phospholipid or "lysophospholipids". Fatty acids residues present in the phospholipids are in general long chain aliphatic acids, typically containing from 12 to 24 carbon atoms, preferably from 14 to 22; the aliphatic chain may contain one or more unsaturations or is preferably completely saturated. Examples of suitable fatty acids included in the phospholipids are, for instance, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, linoleic acid, and linolenic acid. Preferably, saturated fatty acids such as myristic acid, palmitic acid, stearic acid and arachidic acid are employed.

[0060] As used herein, the term phospholipids include either naturally occurring, semisynthetic or synthetically prepared products that can be employed either singularly or as mixtures.

[0061] Phospholipids can be further categorized based on their charge properties into negatively charged (anionic) and positively charged (cationic) types.

[0062] Anionic phospholipids, as utilized herein, denote a class of phospholipids characterized by the presence of phosphate groups which confer a negative charge to the molecule. These phospholipids possess a net negative charge at physiological pH levels (approximately 7.35 to 7.45), rendering them capable of interacting with positively charged molecules, proteins, or surfaces, influencing various physiological processes.

[0063] According to the present invention, highly stable compositions of calibrated gas-filled microvesicles characterized by a reduced amount of pegylated phospholipids can be obtained by incorporating in the outer shell of said microvesicles an (anionic) phospholipid selected from phosphatidic acid (PA), phosphatidylserine (PS) or a mixture thereof.

[0064] According to an embodiment, said anionic phospholipid is preferably selected from the group consisting of l,2-Dilauroyl-sn-glycero-3-phosphate (DLPA), 1,2-Dimyristoyl-sn- glycero-3-phosphate (DMPA), l,2-Dipalmitoyl-sn-glycero-3-phosphate (DPPA), 1,2- Distearoyl-sn-glycero-3- phosphate (DS PA), l,2-dilauroyl-sn-glycero-3-phospho-L-serine (DLPS), l,2-Dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), 1,2-Dipalmitoyl-sn- glycero-3-phospho-L-serine (DPPS), l,2-Distearoyl-sn-glycero-3-phospho-L-serine (DSPS) or mixtures thereof.

[0065] Preferably said anionic phospholipid is phosphatidic acid (PA), more preferably is 1,2- Dimyristoyl-sn-glycero-3-phosphate (DMPA).

[0066] In another embodiment, the outer shell may further comprise an anionic phospholipid selected from naturally occurring anionic phospholipids (e.g. soya bean or egg yolk derived), semisynthetic (e.g. partially or fully hydrogenated), phosphatidylglycerol (PG), phosphatidylinositol (PI) and its phosphorylated derivatives (e.g. the phosphoinositides phosphatidylinositol-4-phosphate (PI4P) and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), and cardiolipin (CL) phospholipids.

[0067] Anionic phospholipids can generally be employed as salts with suitable positive counterions interacting electrostatically with the negatively charged phosphate group of the anionic phospholipid. Suitable examples of counterions are inorganic (monovalent) cations, including the group of alkali ions, such as potassium (K+) and sodium (Na+), and the ammonium ion (NH4+). For instance, when the anionic phospholipid is phosphatidic acid (PA) or phosphatidylserine (PS), the molecule may thus be in the form of a corresponding salt with sodium.

[0068] In addition, the outer layer of said anionic calibrated gas-filled microvesicles may further comprise a lipid, said lipid being preferably a neutral phospholipid and / or a fatty acid.

[0069] As used herein, the terms "neutral phospholipid" refers to a type of phospholipid molecule that possesses a net neutral charge at physiological pH (approximately 7.35 to 7.45). Neutral phospholipids are characterized by having both a hydrophilic phosphate head group and hydrophobic fatty acid tails, but unlike anionic or cationic phospholipids, their head group does not contribute a net positive or negative charge. Neutral phospholipids remain electrically neutral under standard biological conditions.

[0070] Neutral phospholipids may include, for instance, C12-C24, preferably C14-C20, fatty acid derivatives of phosphatidylcholine or of phosphatidylethanolamine, such as dilauroyl- sn-glycero-3-phosphocholine (DLPC), dimyristoyl-sn-glycero-3-phosphocholine (DMPC), di pa Im itoyl-sn-glycero-3- phosphocholine (DPPC), distearoyl-sn-glycero-3-phosphocholine (DSPC), diarachidoyl-sn-glycero-3-phosphocholine (DAPC), l,2-dibehenoyl-sn-glycero-3- phosphocholine (DBPC) dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipentadecanoyl- sn-glycero-3-phosphocholine (DPDPC), l-myristoyl-2-palmitoyl-sn-glycero-3- phosphocholine (MPPC), l-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), 1- palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), l-stearoyl-2-palmitoyl-sn- glycero-3-phosphocholine (SPPC), l-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC), l-oleyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC), dilauroyl-sn-glycero-3- phosphoethanolamine (DLPE), dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), di pa Im itoyl-sn-glycero-3- phosphoethanolamine (DPPE), diarachidoyl-sn-glycero-3- phosphoethanolamine (DAPE), distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), di pentadeca noy l-sn-glycero-3- phosphoethanolamine (DPDPE), l-myristoyl-2-palmitoyl-sn-glycero-3- phosphoethanolamine (MPPE), l-palmitoyl-2-myristoyl-sn-glycero-3- phosphoethanolamine (PM PE), l-palmitoyl-2-stearoyl-sn-glycero-3-phosphoethanolamine (PSPE), l-stearoyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine (SPPE), 1-palmitoyl- 2-oleyl-sn-glycero-3-phosphoethanolamine (POPE), l-oleyl-2-palmitoyl-sn-glycero-3- phosphoethanolamine (OPPE).

[0071] Examples of fatty acids, preferably employed in combination with any of the above neutral phospholipids, can be selected among those C12-C24 fatty acids previously listed, preferably palmitic or stearic acid.

[0072] Mixtures of neutral phospholipids can also be used. Pegylated phospholipids

[0073] The expression "pegylated phospholipid(s)" as used herein includes within its meaning any polyethylene glycol residue ("PEG") covalently bound to a phospholipid residue, such as those illustrated above.

[0074] Polyethylene glycols are typically identified by means of their average molecular weight ("AMW", e.g. number average molecular weight "Mn"); for instance, as used herein, PEG2000 identifies a polyethylene glycol with an AMW of about 2000 g / mol (typically + / - 5%).

[0075] Suitable pegylated phospholipid(s) are those comprising a PEG residue having an average molecular weight of from about 1000 g / mol (i.e. PEG1000) to about 8000 g / mol (i.e PEG8000). Specific examples of PEG polymers useful for forming the pegylated phospholipids as defined above include PEG750, PEG1000, PEG2000, PEG3400, PEG4000, PEG5000, PEG6000, PEG7000 and PEG8000.

[0076] According to a preferred embodiment, said pegylated phospholipid(s) are those comprising a PEG residue having an average molecular weight of from 1000 to 8000 g / mol, preferably from 1500 to 6000 g / mol, still more preferably has a molecular weight of 2000 g / mol + / - 5 (i.e. PEG2000) or of 5000 g / mol + / - 5 (i.e. PEG5000). Preferably, said pegylated phospholipid comprise a PEG residue having an average molecular weight of 5000 g / mol + / - 5 (i.e. PEG5000).

[0077] Preferably the PEG is covalently bound to a phosphatidylethanolamine ("PE") residue bearing a respective lipid chain, e.g. myristoyl, palmitoyl or steaoryl.

[0078] Examples of suitable pegylated phospholipids are for instance DMPE-PEG, DPPE-PEG and DSPE-PEG, which are generally commercially available as pegylated phospholipids with the PEG having the above indicated average molecular weights, e.g. as DMPE- PEG2000, DMPE-PEG3400, DMPE-PEG5000, DPPE-PEG2000, DPPE-PEG3400, DPPE- PEG5000, DSPE-PEG2000, DSPE-PEG3400 or DSPE-PEG5000.

[0079] According to an embodiment, said pegylated phospholipid is a PE-PEG2000 or PE- PEG5000, preferably is DSPE-PEG2000 or DPPE-PEG5000, still more preferably is DPPE- PEG5000.

[0080] The anionic calibrated microvesicles of the invention can be advantageously functionalized with suitable ligand to allow different applications, such as in cell sorting techniques.

[0081] According to an embodiment, said pegylated phospholipid comprises a pegylated phospholipid comprising a ligand through a reactive moiety.

[0082] According to the present description and claims, the expression "pegylated phospholipid bound to a ligand through a reactive moiety", indicates a compound comprising a polyethylene glycol residue ("PEG") covalently bound to a phospholipid residue, as above defined, further functionalized with a reactive moiety capable of reacting with a corresponding reactive moiety on a functionalized ligand.

[0083] According to said embodiment, said calibrated microvesicles functionalized with a ligand are thus composed by an outer shell comprising a pegylated phospholipid comprising a ligand (e.g. DSPE-PEG2000-STV) and a pegylated phospholipid not coupled to a ligand (e.g. DSPE-PEG2000).

[0084] For this purpose, the pegylated phospholipid may be suitably functionalized with a reactive moiety, particularly one capable of reacting with a corresponding reactive moiety on a functionalized ligand (e.g. an avidin, neutravidin or streptavidin moiety).

[0085] Suitable reactive moieties include, for instance, NHS (N-hydroxy-succinimiide), amino, sulfhydryl, maleimide, azide or DBCO (dibenzocyclooctyne).

[0086] For instance, if one of the two reacting components includes a reactive amino group, it can be reacted with the other component containing a suitable corresponding reactive moiety, such as an isothiocyanate group (to form a thiourea bond), a reactive ester (to form an amide bond), or an aldehyde group (to form an imine bond, which may be reduced to an alkylamine bond). Alternatively, if one of the two reacting components includes a reactive thiol group, suitable complementary reactive moieties on the other component may include haloacetyl derivatives, maleimides (to form a thioether bond) or a mixed disulfide comprising a sulphide in the form of a 2-pyridylthio group which upon reaction with a thiol derived from the thiol-bearing component results in the formation of a stable disulfide bond between the two components. Furthermore, if one of the two reacting components includes a reactive carboxylic group, suitable reactive moieties on the other component can be amines and hydrazides (to form amide or N-acyl, N'-alkylhydrazide functions).

[0087] Suitable examples of said pegylated phospholipids can be a maleimide-derivatized pegylated phospholipid (e.g. PE-PEG2000-Mal), obtained by reaction with a ligand bearing a thiol (-SH) reactive moiety, introduced on the ligand e.g. by reaction with Sulfo-LC-SPDP (sulfosuccinimidyl 6-(3’-(2-pyridyldithio)propionamido)hexanoate), or a biotin-derivatized pegylated phospholipid (e.g. PE-PEG2000-Biot) may be reacted directly with a ligand, e.g. avidin, neutravidin or streptavidin, as a result of their natural and irreversible affinity.

[0088] The ligand bound to the pegylated phospholipid and incorporated into the microvesicle's envelope is a ligand forming a specific "binding pair" with another respective molecule. Examples of ligands (and respective binding pairs) include for instance avidin, neutravidin and streptavidin, which can form a binding pair with biotin (e.g. biotinylated antibody bound to the cell). Streptavidin is preferred for the present invention. In one embodiment a biotinylated pegylated phospholipid (i.e. a pegylated phospholipid functionalized with biotin (PE-PEG-Biot)) can be used, to which the ligand, e.g. streptavidin, can be directly bound due to their natural affinity. When using such biotinylated pegylated phospholipid no reactive moiety needs to be further added to biotin.

[0089] In an alternative embodiment the ligand can be suitably derivatized to introduce a reactive moiety capable of covalently reacting with a respective reactive moiety on the functionalized pegylated phospholipid. For instance, when a maleimide functionalized pegylated phospholipid (PE-PEG-Mal) is used, a thiol moiety is introduced in the ligand to allow coupling maleimide / thiol. When DBCO functionalized lipids were used, an azide moiety was introduced in streptavidin to allow click chemistry coupling.

[0090] Inner core

[0091] Suitable gases comprise biocompatible fluorinated gases, preferably perfluorinated gases. Fluorinated gases include materials which contain at least one fluorine atom such as, for instance, fluorinated hydrocarbons (organic compounds containing one or more carbon atoms and fluorine); sulfur hexafluoride; fluorinated, preferably perfluorinated, ketones such as perfluoroacetone; and fluorinated, preferably perfluorinated, ethers such as perfluorodiethyl ether. Preferred compounds are perfluorinated gases, such as SFe or perfluorocarbons (perfluorinated hydrocarbons), i.e. hydrocarbons where all the hydrogen atoms are replaced by fluorine atoms, which are known to form particularly stable gas- filled microvesicles suspensions.

[0092] The term "perfluorocarbon" includes saturated, unsaturated, and cyclic perfluorocarbons. Examples of biocompatible, physiologically acceptable perfluorocarbons are: perfluoroalkanes, such as perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutanes (e.g. perfluoro-n-butane, optionally in admixture with other isomers such as perfluoro-isobutane), perfluoropentanes, perfluorohexanes or perfluoroheptanes; perfluoroalkenes, such as perfluoropropene, perfluorobutenes (e.g. perfluorobut-2ene) or perfluorobutadiene; perfluoroalkynes (e.g. perfluorobut-2-yne); and perfluorocycloalkanes (e.g. perfluorocyclobutane, perfluoromethylcyclobutane, perfluorodimethylcyclobutanes, perfluorotrim ethylcyclobutanes, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclopentanes, perfluorocyclohexane, perfluoromethylcyclohexane and perfluorocycloheptane). Preferred saturated perfluorocarbons include, for example, CF4, C2F6, C3F8, C4F8, C4F10, C5F12 and CeFi4.

[0093] In a further embodiment said gaseous fluorinated compound is a perfluoro olefin, selected from C4-C6 perfluoro olefins, preferably C4-C5, more preferably C5 perfluoro olefins. Specific examples include perfluoro-2-butene, perfluoro-l-pentene, perfluoro-2- pentene, or mixtures thereof. More preferably the perfluoro olefin is perfluoro-2-pentene. Particularly preferred gases are those which are in gaseous form at room temperature, including SFe, CsFs and C4F10.

[0094] Aqueous suspension of calibrated gas-filled microvesicles

[0095] According to the invention, it is advantageous to obtain a suspension of anionic calibrated gas-filled microvesicles stabilized by a mixture of an anionic phospholipid selected from phosphatidic acid (PA), phosphatidylserine (PS) or a mixture thereof and a pegylated phospholipid, wherein the size distribution of said anionic calibrated microvesicles is typically characterized by a geometric standard deviation (GSD) value of at least 1.20 or lower, preferably of at least 1.15 or lower, down to e.g. 1.05.

[0096] The Applicant unexpectedly observed that by using suitable amounts of phosphatidic acid or phosphatidylserine allowed to significantly reduce the amount of pegylated phospholipids preserving the coalescence stability of the freshly-formed calibrated microvesicles, i.e. by limiting the coalescence at the end of their preparation (e.g. at the collection from the outlet channel of the microfluidic device or within few hours (e.g. 2 hours) from the collection).

[0097] The term coalescence refers to the process by which two or more particles, such as microvesicles, merge during contact to form a single larger particle.

[0098] The percentage of coalescence can be determined by calculating the total number of coalesced microparticles from the peaks of the size distribution from the images obtained with the optical microscope at the outlet channel of the flow-focusing device, e.g. by: multiplying the total number of microparticles with a volume equal to two times the volume of the initially formed microparticles Vi (second peak of the size distribution) by a factor two (since the coalesced microparticles originated from two microparticles); and adding this number to the total number of microparticles with three times the initial volume (third peak of the size distribution) multiplied by a factor three, and so on up to the nthpeak in the measured size distribution.

[0099] The percentage of coalescence can thus be calculated by normalizing the total number of coalesced microparticles by the total number of produced microparticles :

[0100] Equation 2

[0101] In general, a coalescence percentage of about 1 or less is desirable, down to coalescence percentages of e.g. 0.01%.

[0102] As showed in the Examples, the reduction of the amount of pegylated phospholipids in calibrated gas-filled microvesicles obtained by flow-focusing microfluidic method and stabilized by a mixture of neutral phospholipids (e.g. DSPC) and pegylated phospholipids, leads to undesired coalescence phenomenon.

[0103] The Applicant unexpectedly observed that the amount of pegylated phospholipids may be substantially reduced without incurring in undesired coalescence phenomena by stabilizing the shell of said calibrated gas-filled microvesicles with a suitable amount of an anionic phospholipid selected from phosphatidic acid, phosphatidylserine or a mixture thereof.

[0104] Preservation of the stability of the gas-filled microvesicles (i.e. limited coalescence) while halving the amount of pegylated phospholipids to 7.5% mol / mol was obtained by stabilizing the CMV outer layer with a molar amount of phosphatidic acid, phosphatidylserine or a mixture thereof of at least 55% or higher.

[0105] In an aspect of the invention, the outer shell of the disclosed calibrated gas-filled microvesicles comprises:

[0106] - 55% by moles or more of a phospholipid selected from phosphatidic acid, phosphatidylserine or a mixture thereof and

[0107] -7.5% by moles or less of a pegylated phospholipid.

[0108] Typically, the anionic phospholipid represents the larger portion of the outer layer of the calibrated gas-filled microvesicles components, e.g. up to 99.5% mol / mol.

[0109] In certain embodiments, the molar amount of said phospholipid may range from 55% to 99.5%, preferably the molar amount of said phospholipid is 60% or higher, more preferably 65% or higher, more preferably 70% or higher, more preferably 80% or higher, more preferably 90% or higher, more preferably 92% or higher, more preferably 94% or higher, more preferably 96% or higher, more preferably 98% or higher, up to 99.5% mol / mol.

[0110] According to an embodiment, said phospholipid is preferably selected from the group consisting of DLPA, DMPA, DPPA, DSPA, DLPS, DMPS, DPPS or DSPS.

[0111] According to a preferred embodiment, said phospholipid is phosphatidic acid, more preferably it is selected from the group of DLPA, DMPA, DSPA or DPPA, being preferred DMPA.

[0112] According to an alternative embodiment, said phospholipid is phosphatidylserine, preferably it is selected from the group of DLPS, DMPS, DPPS or DSPS, being preferred DPPS.

[0113] Said pegylated phospholipid ("PE-PEG") is a phospholipid covalently linked to a polyethylene glycol (PEG) having an average molecular weight of from 1000 to 8000 g / mol, preferably of from 1500 to 6000 g / mol, preferably said PEG residue has an average molecular weight of 2000 g / mol (i.e. PEG2000) or of 5000 g / mol (i.e. PEG5000), being preferred of 5000 g / mol. According to an embodiment, the molar amount of said pegylated phospholipid is 6% by moles or lower, 5% or lower, 4% or lower, 3% or lower, 2% or lower, 1% or lower, down to 0.5%.

[0114] In another embodiment, the outer layer of the anionic calibrated gas-filled microvesicles may further comprise a lipid, said lipid being preferably a neutral phospholipid and / or a fatty acid such as, for instance palmitic acid, stearic acid, arachidonic acid or oleic acid.

[0115] The optional neutral phospholipid may be present in a molar amount of for instance 37.5% or lower, 35% or lower, 30% or lower, 25% or lower, 20% or lower, 15% or lower, 10% or lower, 5% or lower, down to 0% mol / mol.

[0116] The optional fatty acid may be present in a molar amount of for instance 10% or higher, 20% or higher, 30% or higher, more preferably from 15% to 25%.

[0117] In a preferred embodiment the outer layer of the disclosed calibrated gas-filled microvesicles comprises:

[0118] - 90% by moles or higher of DMPA and

[0119] - 7.5% by moles or lower of a pegylated phospholipid.

[0120] More preferably the molar amount of DMPA is comprised between 92.7% and 99.5% by moles and molar amount of pegylated phospholipids is comprised between 6% and 0.5%.

[0121] Still more preferably the molar amount of DMPA is 95% by moles or higher and the molar amount of pegylated phospholipid is 5% by moles or lower.

[0122] Still more preferably the molar amount of DMPA is 99% by moles or higher, up to 99.5%, and the molar amount of pegylated phospholipid is 1% by moles or lower, down to 0.5%.

[0123] In a preferred embodiment, said suspension of anionic calibrated gas-filled microvesicles has a Z-potential of - 5 mV or lower, preferably -10 mV or lower, more preferably -20 mV, more preferably -30 mV or lower, more preferably -40 or lower, up to -50 mV.

[0124] In addition to the reduced coalescence, the Applicant further observed that the disclosed formulations were characterized by a substantially high long-term stability over time.

[0125] The term "long-term stability" is defined as the ability of the suspension of calibrated gas-filled microvesicles to preserve their initial characteristics (e.g. size, polydispersity and concentration) over a specified period under given storage conditions (e.g. four weeks at RT (25°C)). After four weeks, the disclosed compositions were found to be characterized by substantially similar values of sizes and polydispersity. Furthermore, also the % of remaining microvesicles was found to be higher than 90% after one month. As used herein, the term "% of remaining microvesicles" refers to the amount of gas-filled microvesicles that maintain their structural integrity after a defined period of time at given conditions (e.g. one month at RT). This percentage is calculated by comparing the number of gas-filled microvesicles present at the end of the test period to the number initially present at the start of the test. A higher percentage, e.g. higher than 80%, preferably higher than 85%, still more preferably higher than 90%, indicates greater stability of the gas-filled microvesicles formulation.

[0126] Furthermore, the Applicant found that the disclosed calibrated gas-filled microvesicles may be used in methods for sorting cells biological materials with a higher efficiency compared to standard polydisperse microbubbles, due to their higher and controllable size.

[0127] The efficiency of a cell separation method can be determined by different parameters, such as the cell recovery. "Cell recovery" measures the effectiveness of a cell separation assay and refers to the proportion of desired cells isolated during the separation process compared to the number of desired cells available in the starting sample.

[0128] This parameter is generally reported as "cell recovery percentage", which describes the percentage of cells that are obtained post sorting compared to the number of total cells or target cells in the original suspension. A high cell recovery percentage, e.g. 80% or higher, indicates an effective cell separation.

[0129] According to the present invention, a cell recovery percentage of 80%, preferably of 85%, more preferably of 90% and even more preferably of 95% is desirable, up to a cell recovery percentage of about 100% (e.g. 99%).

[0130] Surprisingly it was observed that a significantly high cell recovery (%), e.g. higher than 90%, can be obtained by using anionic calibrated gas-filled microvesicles of the invention.

[0131] In certain embodiments, the pegylated phospholipid of the outer shell may comprise a pegylated phospholipid comprising a ligand.

[0132] According to the present invention, the total molar amount of pegylated phospholipids is preferably 7.5% or less.

[0133] The expression "total amount of pegylated phospholipids" indicates the sum (e.g. molar %) of the molar amount of the pegylated phospholipid with ligand and / or of the molar amount of the pegylated phospholipid without ligand (%).

[0134] Preferably, the pegylated phospholipid of the outer shell comprise a mixture of pegylated phospholipid comprising a ligand (PE-PEG-LIGAND) and a pegylated phospholipid without ligand (PE-PEG). Said pegylated phospholipid ("PE-PEG”) is a phospholipid covalently linked to a polyethylene glycol (PEG) having an average molecular weight of from 1000 to 8000 g / mol, preferably of from 1500 to 3000 g / mol, preferably said PEG residue has an average molecular weight of 2000 g / mol, (i.e. PEG2000).

[0135] According to a preferred embodiment of the invention, the pegylated phospholipid comprising a reactive moiety is a functionalized PE-PEG2000 (e.g. DSPE-PEG2000-Biot or DSPE-PEG2000-Mal) while the pegylated phospholipid without ligand is a PE-PEG2000 (e.g. DSPE-PEG2000).

[0136] Overall charge of microvesicles

[0137] The overall charge on the gas-filled microvesicles in the suspension can be expressed in terms of Zeta potential (or ^-potential). The Zeta potential of microparticles can be determined, for instance, by using laser Doppler electrophoresis, e.g. by using a Nano ZSP Zetasizer (Malvern Ltd., Malvern, UK). In short, the charge or (zeta-potential) of particles is determined by measuring their velocity while they are moving due to electrophoresis. Particles and molecules that have a non-zero zeta potential will migrate towards an electrode because of the applied electric field. The particle's mobility is proportional to the field strength and its zeta-potential. By knowing the field strength, and by measuring the speed of the particles (by using laser Doppler electrophoresis), the zeta-potential of the particle can be determined (See e.g. Ja'Affar et al., "Surface Charge Measurement of SonoVue™, Definity™ and Optison™: a comparison of Laser Doppler Electrophoresis and Micro-Electrophoresis", Nov 2015, 41(11): 2990-3000).

[0138] In a preferred embodiment, said suspension of anionic calibrated gas-filled microvesicles has a Z-potential of - 5 mV or lower, preferably -10 mV or lower, more preferably -20 mV, more preferably -30 mV or lower, more preferably -40 or lower.

[0139] While there is in principle no upper limit for the z-potential measured on the microvesicles of the invention, it is in general not necessary to use microvesicles with a z- potential lower than -50 mV.

[0140] Method of preparation

[0141] The microvesicles of the invention can be advantageously prepared by microfluidic technique, according to the manufacturing method disclosed in WO2018041906A1 and WO2019170606A1, hereby incorporated by reference.

[0142] A further aspect of the present invention thus relates to a method for preparing a suspension of calibrated gas-filled microvesicles as above defined, wherein said suspension of calibrated gas-filled microvesicles has a geometric standard deviation (GSD) of at least 1.2 or lower, said method comprising: a) providing (I) a gaseous flow and (ii) an aqueous liquid flow comprising: - more than 55% by moles of an anionic phospholipid selected from phosphatidic acid, phosphatidylserine or a mixture thereof and -7.5% by moles or less, down to 0.5% of a pegylated phospholipid b) directing said gaseous flow and said liquid flow through respective inlet channels towards a contact zone; c) directing said gaseous flow and said liquid flow from the contact zone through a calibrated orifice to obtain an aqueous suspension comprising said gas-filled microvesicles; d) collecting a suspension of anionic calibrated gas-filled microvesicles, wherein said suspension has a geometric standard deviation (GSD) of at least 1.2 or lower.

[0143] Liquid flow

[0144] The aqueous liquid flow for preparing the calibrated gas-filled microvesicles according to the invention comprises the mixture of amphiphilic materials as above defined (i.e. a (anionic) phospholipid, a pegylated phospholipid and optionally a neutral phospholipid and / or fatty acid) at a concentration of e.g. from 5.0 to 20 mg / mL, preferably from 7.5 to 15 mg / mL, dispersed in an aqueous carrier.

[0145] The type, respective relative molar amounts and molar ratios of the amphiphilic materials in the liquid flow are as indicated above.

[0146] Suitable aqueous carriers, which are preferably physiologically acceptable, comprise water (preferably sterile water), aqueous solutions such as saline (which may advantageously be balanced so that the final product for injection is not hypotonic), or solutions of one or more tonicity adjusting substances. Tonicity adjusting substances comprise salts or sugars, sugar alcohols, glycols or other non-ionic polyol materials (e.g. glucose, sucrose, sorbitol, mannitol, glycerol, polyethylene glycols, propylene glycols and the like), chitosan derivatives, such as carboxymethyl chitosan, trimethyl chitosan or gelifying compounds, such as carboxymethylcellulose, hydroxyethyl starch or dextran.

[0147] In an alternative embodiment, an additional oil phase may be added for incorporating therapeutic hydrophobic substances into the microvesicles. To this end, two additional conduits (channels) may be provided in the device for supplying the desired oil phase, as described for instance by WO2018041906A1 or WO2019170606A1. The formed gas-filled microvesicles will thus have a film of oil disposed at the interface between gas and the stabilizing layer of amphiphilic material, which can be loaded with a desired therapeutic agent. Suitable oils may include any biocompatible oil which is liquid at room temperature including, for instance, mono-, di- or tri-esters of glycerol with saturated or unsaturated (C2-C18) alkyl chains (including homo- or hetero-allkylesters), such as glycerol monobutyrin, glycerol monolinoleate, 1,2-dihexanoyl glycerol, 1,2 dioctanoyl glycerol, 1,2- dioleyl-sn-glycerol, triacetin, tributyrin, tricaproin, tricaprylin, tricaprin, and mixtures thereof; or natural oils such as soya oil, olive oil, safflower seed oil, sunflower seed oil, peanut oil and mixtures thereof.

[0148] Gas flow

[0149] The freshly formed microvesicles comprise a gas selected among those previously indicated. Preferably the gas is a mixture of a gas highly soluble in water ("HS gas") and of a gas with low solubility in water ("LS gas"), as described in WO2019170606A1.

[0150] Examples of HS gases include nitrogen, air, and carbon dioxide, this latter being particularly preferred because of its higher solubility in water.

[0151] Suitable LS gases are fluorinated gases, preferably perfluorinated gases, such as. those previously illustrated.

[0152] In a preferred embodiment of the invention, gas-filled microvesicles comprising CO2 / C4F10 in a volume ratio of from 80 / 20 to 90 / 10, e.g. 85 / 15 can be prepared with a gas-mixing device similar to the one schematically illustrated in WO2019170606A1.

[0153] After obtaining the suspension of calibrated gas-filled microvesicles, i.e. after collecting it from a microfluidic apparatus, it is highly recommended to treat said suspension using suitable washing techniques, in order to remove not-assembled amphiphilic material and possible additive compounds.

[0154] According to an embodiment, said methods for preparing a suspension of anionic calibrated gas-filled microvesicles comprises an optional step (d') of washing said obtained suspension of anionic calibrated gas-filled microvesicles.

[0155] For instance, said optional step may be performed after step d) of collecting said aqueous suspension of anionic calibrated gas filled microvesicles from the exit channel of the microfluidic apparatus.

[0156] In the present description, the term "washing" indicates any operation carried out on the freshly prepared microvesicles suspension, finalized to remove (or substantially reduce the amount of) not-assembled amphiphilic material and additive compounds.

[0157] According to this description, suitable washing techniques comprise centrifugation, filtration, bubble sorting and decantation, preferred is centrifugation.

[0158] In the present description, the expression "not-assembled amphiphilic material" indicates amphiphilic molecules that, at the end of the preparation process, are present in the calibrated microvesicles suspension, but are not forming the stabilizing layer of the gas-filled microvesicles. Examples of amphiphilic materials are phospholipids and pegylated phospholipids used to stabilize the outer shell of the calibrated gas-filled microvesicles.

[0159] In the present description "additive compounds" indicate any possible substance that can be added to the suspension during the microvesicles preparation, such as tonicity adjusters like for example salts or sugars, sugar alcohols, glycols or other non-ionic polyol materials (e.g. glucose, sucrose, sorbitol, mannitol, glycerol, polyethylene glycols, propylene glycols and the like), chitosan derivatives, such as carboxymethyl chitosan, trimethyl chitosan or gelifying compounds, such as carboxymethylcellulose, hydroxyethyl starch or dextran. For instance, additive compounds can be added to the suspension of gas-filled microvesicles after the optional washing performed after the coupling reaction between the ligand and the reactive moiety bound to the pegylated phospholipid.

[0160] Therefore, at the end of said optional washing step d') the aqueous suspension of anionic calibrated gas-filled microvesicles is a composition substantially free from "not- assembled amphiphilic materials", indicating that all (or the most part of) the present amphiphilic materials result to be involved in forming the outer shells of said microvesicles. In other words, a washed aqueous suspension of gas-filled microvesicles essentially does not comprise free amphiphilic materials in suspension.

[0161] The term "washed" means after being treated with a suitable washing technique.

[0162] The anionic calibrated microvesicles of the invention can be advantageously used for cell sorting applications.

[0163] For this purpose, in certain embodiments said microvesicles are submitted to functionalization step, as described in EP23216400, which is hereby incorporated by reference. For this purpose, at the end of the preparation of the suspension of the anionic calibrated gas-filled microvesicles, e.g. after collecting said suspension from the outlet channel of the microfluidic apparatus and / or washing the obtained suspension with a suitable washing technique, it is possible to add a ligand capable of reacting with said pegylated phospholipid through a reactive moiety.

[0164] In an embodiment, at step a) of the above method, said pegylated phospholipid comprises a pegylated phospholipid being bound to a reactive moiety.

[0165] In another embodiment, said method further comprises the following steps: e) adding a functionalized ligand capable of reacting with said reactive moiety to said collected suspension; f) coupling said pegylated phospholipid with said ligand; and collecting a suspension of calibrated gas-filled microvesicles with ligand, wherein said suspension has a geometric standard deviation (GSD) lower than 1.2. According to the invention the ligand is preferably added in molar ratio of from 1:2 to 2: 1 with respect to the amount of pegylated phospholipid bearing the corresponding reactive moiety. More preferably said molar ratio is 1: 1.

[0166] At the end of the coupling reaction between the ligand and the reactive moiety linked to the pegylated phospholipid, the obtained suspension of anionic calibrated gas-filled microvesicles with ligand can be advantageously further washed as described above, in order to remove (or substantially reduce the amount of) not-assembled ligand.

[0167] According to an embodiment, after the coupling step said method may comprise an optional step f') of washing said obtained suspension of calibrated gas-filled microvesicles with ligand.

[0168] Therefore, at the end of this further optional washing step the aqueous suspension of calibrated gas-filled microvesicles is a composition substantially free from "not- assembled ligand", indicating that all (or the most part of) the present ligand results incorporated in the final stabilizing envelope of said microvesicles. In other words, a washed aqueous suspension of anionic calibrated gas-filled microvesicles with ligand essentially does not comprise free ligand.

[0169] At the end of the coupling procedure (e.g. after the coupling reaction between the added ligand and the reactive moiety incorporated in the microvesicles shells and / or after a further washing procedure following said coupling) said aqueous suspension of anionic calibrated gas-filled microvesicles with ligand may comprise unreacted moieties on the PE- PEG depending on the molar ratio between said ligand and said first pegylated phospholipid bearing a reactive moiety.

[0170] For instance, when the molar amount of the added ligand is lower than the molar amount of the pegylated phospholipid comprising the reactive moiety to which said ligand has to be coupled (e.g. when the molar ratio between the ligand and the pegylated phospholipid bearing the corresponding reactive moiety is lower than 1), the final aqueous suspension of calibrated gas-filled microvesicles with ligand comprises unreacted reactive moieties on the pegylated phospholipids incorporated in the outer shell. Therefore, in this case the stabilizing layer of said microvesicles comprises unreacted pegylated phospholipids together with the pegylated phospholipids covalently bound to the ligand.

[0171] In the present description and claims, the expression "at least a portion of said pegylated phospholipid comprising a ligand" indicates that at least a part of the total amount of reactive moieties incorporated in the stabilizing layer of the disclosed formulation is bound to the ligand, while the remaining part of said total amount is not bound to the ligand and remains in an "unreacted form".

[0172] In the present description and claims, the term "unreacted" means not coupled to the ligand. For instance, considering a pegylated phospholipid bearing biotin as reactive moiety (PE- PEG-Biot), its unreacted form corresponds to the compound PE-PEG-Biot, wherein biotin is not bound to any ligand, e.g. streptavidin (STV). In this example, the reacted form (or coupled form) corresponds to PE-PEG-Biot-STV, wherein the reactive moiety biotin is coupled with the ligand streptavidin.

[0173] Therefore, in the present invention the outer layer of the disclosed calibrated gas-filled microvesicles is formed by at least a part of reacted pegylated phospholipids bearing a reactive moiety and by the remaining part of unreacted pegylated phospholipid s bearing a reactive moiety.

[0174] The unreacted reactive moieties on the pegylated phospholipid may then be "inactivated" by reacting it with a suitable corresponding inactivating moiety. For instance, if the reactive moiety on the pegylated phospholipid is maleimide (PE-PEG-Mal) it can be inactivated by reacting it with cysteine. Alternatively, the reactive moiety may undergo natural inactivation processes, e.g. hydrolysis, without need of adding a specific inactivating moiety.

[0175] Advantageously the disclosed suspension of calibrated gas-filled microvesicles can be freeze-dried, as described for instance in W02020260420A1 and WO2020260423A1, which are here incorporated by reference.

[0176] Methods of use

[0177] The microvesicles of the invention may be used in a variety of biomedical diagnostic and / or therapeutic techniques, including ultrasounds imaging and therapeutic applications.

[0178] Suitable applications of negatively charged microbubbles include imaging the capillary endothelium (as referenced in Fisher, 2002) and detecting focal liver and breast lesions, as for instance performed by the strongly anionic contrast agent Sonazoid (by DAIICHI SANKYO and GE Healthcare), which is composed of hydrogenated egg yolk phosphatidylserine (HEPS).

[0179] Diagnostic methods include any method where the use of the gas-filled microvesicles allows enhancing the visualization of a portion or of a part of an animal (including humans) body, including imaging for preclinical and clinical research purposes. A variety of imaging techniques may be employed in ultrasound applications, for example including, for instance, fundamental and harmonic B-mode imaging, pulse or phase inversion imaging, Power modulation Contrast Mode, Pulse Wave Doppler, Color Doppler, Power Doppler, Maximum Intensity Projection, Ultrafast Imaging, Ultrasound Localization Microscopy, Vector Flow Imaging and Destruction Replenishment Quantification; if desired three-dimensional imaging techniques may be used. Microvesicles according to the invention may typically be administered in a concentration of from about 0.01 to about 1.0 pLof gas per kg of patient, depending e.g. on their respective composition, the tissue or organ to be imaged and / or the chosen imaging technique. This general concentration range may of course vary depending on specific imaging applications, e.g. when signals can be observed at very low doses such as in colour Doppler or power pulse inversion.

[0180] In an embodiment, said method of diagnosing comprises

[0181] (i) administering to a patient a suspension of CMV as defined above; and

[0182] (ii) detecting an ultrasound signal from a region of interest in said patient.

[0183] Other possible diagnostic imaging applications include scintigraphy, light imaging, and X-ray imaging, including X-ray phase contrast imaging.

[0184] An embodiment relates to a suspension of anionic calibrated gas-filled microvesicles as defined above for use in a method of diagnosis in vivo. Preferably said method comprises:

[0185] (i) administering to a patient a suspension of CMV as defined above; and

[0186] (ii) detecting an ultrasound signal from a region of interest in said patient.

[0187] Another aspect relates to a suspension of anionic calibrated gas-filled microvesicles as defined above for use as ultrasound contrast agent.

[0188] Another aspect of the invention relates to the use in a method of therapeutic treatment of a suspension CMV as above defined, including in-vitro, ex-vivo and / or in vivo.

[0189] In-vitro or ex-vivo methods include, for instance, loading of genetic material (such as DNA, SiRNA and similar) or cell transfection or DNA transfection, e.g. by sonoporation. In vitro or ex-vivo therapeutic applications can be used, for instance, to treat neurological disorders with a focus on Parkinson's disease, Huntington's disease, Alzheimer's disease, ALS, stroke and in cardiovascular therapy.

[0190] In-vivo therapeutic treatments include any method of treatment which comprises the combined use of ultrasounds and gas-filled microvesicles either as such (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, e.g. for the delivery of a drug or bioactive compound to a selected tissue, organ or region of interest, such as in tumortreatment, gene therapy, infectious diseases therapy, metabolic diseases therapy, chronic diseases therapy, degenerative diseases therapy, inflammatory diseases therapy, immunologic or autoimmune diseases therapy or in the use as vaccine), whereby the presence of the gas-filled microvesicles may provide a therapeutic effect itself or is capable of enhancing the therapeutic effects of the applied ultrasounds, e.g. by exerting or being responsible to exert a biological effect in vitro and / or in vivo, either by itself orupon specific activation by various physical methods (including e.g. ultrasound mediated delivery of therapeutic material).

[0191] Microvesicles according to the invention can typically be administered for therapeutic purposes in a concentration of from about 0.01 to about 5.0 pLof gas per kg of patient, depending e.g. from their respective composition, the type of subject under treatment, the tissue or organ to be treated and / or the therapeutic method applied.

[0192] In an embodiment said method of ultrasound therapeutic treatment comprises:

[0193] (i) administering to a patient a suspension of CMV as defined above;

[0194] (ii) identifying a region of interest in said patient to be submitted to a therapeutic treatment, said region of interest comprising said suspension of CMV; and

[0195] (iii) applying an ultrasound beam for therapeutically treating said region of interest; whereby said ultrasound therapeutic treatment is enhanced by the presence of said suspension of CMV in said region of interest.

[0196] An embodiment relates to a suspension of anionic calibrated gas-filled vesicles (CMV) as above defined for use in a method of ultrasound mediated treatment. Preferably, said method comprises the following steps:

[0197] (i) administering to a patient a suspension of CMV as defined above;

[0198] (ii) identifying a region of interest in said patient to be submitted to a therapeutic treatment, said region of interest comprising said suspension of CMV; and

[0199] (iii) applying an ultrasound beam for therapeutically treating said region of interest;

[0200] As illustrated in the examples, the anionic gas-filled microvesicles of the invention may be advantageously used in a method for separating cells, typically by buoyancy (also known as buoyancy-activated cell sorting, "BACS"). The method can be useful for separating a desired type of cells from other cells in a physiological liquid (e.g. blood or plasma). In particular, the separation method comprises labelling a desired cell to be separated with a suitable labelled antibody capable of binding to a specific (and selective) receptor on said cell. The microvesicles of the invention are then added to the suspension of cells to be separated (including those bearing the labelled antibody); the microvesicles of the invention will then associate through the ligand with the labelling residue bound to antibody / cell construct thus allowing separation of the cells by buoyancy (see e.g. WO2017117349). In a preferred embodiment the labelled antibody is a biotinylated antibody, where the biotin residue is capable of associating with a respective moiety, such as for instance an avidin, neutravidin or streptavidin residue on the calibrated gas-filled microvesicles. Thus, the microvesicles of the invention can be used for separating a wide number of cells from a physiologic liquid, provided such cell can be suitably labelled with a respective labelled (biotinylated) antibody.

[0201] The following examples will help to further illustrate the invention.

[0202] EXAMPLES

[0203] Materials and Methods

[0204] Materials

[0205] Table 1 Materials

[0206] Methods

[0207] Microvesicles size and concentration :

[0208] Size distribution and microvesicles concentration were measured with a Coulter Counter Multisizer 3 (Beckman Coulter, Fullerton, CA) using an aperture tube with a diameter of 30 pm allowing a measurable size range of 0.7-18 pm. Fifty (50) pL of bubble suspension was diluted in 100 mL of Saline 0.9% (analytical volume = 100 pL) and microbubble parameters were measured over a period of 30 seconds.

[0209] Dv mode refers to the diameter at the peak of the distribution of microvesicles in volume. CMV concentration stands for the number of calibrated microvesicles in a sample volume. MVC stands for the mean volume concentration or the volume of microvesicles in a sample volume. The "geometric standard deviation" (GSD) of the tested formulations was determined according to Equation 1, as described above.

[0210] Coalescence measurement

[0211] The percentage of coalescence can be determined by calculating the total number of coalesced microparticles from the peaks of the size distribution from the images obtained with the optical microscope at the outlet channel of the flow-focusing device, e.g. by:

[0212] - multiplying the total number of microparticles with a volume equal to two times the volume of the initially formed microparticles Vi (second peak of the size distribution) by a factor two (since the coalesced microparticles originated from two microparticles); and

[0213] - adding this number to the total number of microparticles with three times the initial volume (third peak of the size distribution) multiplied by a factor three, and so on up to the nth peak in the measured size distribution.

[0214] The percentage of coalescence can thus be calculated by normalizing the total number of coalesced microparticles by the total number of produced microparticles:

[0215] Equation 2

[0216] In general, a coalescence percentage of about 1 or less is desirable, down to coalescence percentages of e.g. 0.01%.

[0217] Cell recovery tests:

[0218] The cell recovery test protocol was performed as described in WO2020 / 127816.

[0219] Namely, CCL119 (CCRF-CEM cells) or MCF-7 cells (from ATTC) were first cultured and expanded according to the protocol from the provider. Just before the test, cells were re-suspended in BSA / EDTA buffer (1% BSA and 2 mM EDTA in PBS, w / o Ca / Mg) at a concentration depending on the cells type, i.e. 5xl06cells / mL for CCL119 and lxlO6cell / mL for MCF-7 cells.

[0220] The cell suspension (1 mL, about 5xl06cells) was transferred in a 2 mL-low binding Eppendorf and 160 pL of Biotinylated mouse anti-human CD45 antibody (#30-0459-11100 Tonbo were added to the cells. The mixture was incubated for 30 min at room temperature on a rotating mixer, the cells were then washed by centrifugation (400 g / 5 min); the supernatant was discarded, and the cells were re-suspended in 1 mL BSA / EDTA buffer (mixing 5 min on a rotating mixer). The microvesicles suspension (CMV volume depends on the ratio CMV / Cell, e.g. from 30 CMV / cell to 3 CMV / cell) was then added to the cell suspension and the mixture was incubated for 20 min at room temperature on a rotating mixer. The mixture was then centrifuged (400 g / 5 min) and the supernatant (cell / microbubbles complexes) was recovered by manual pipetting at the liquid's meniscus.

[0221] The gas-filled microvesicles were then collapsed (by applying positive pressure) and cells were counted in the supernatant fraction and in the infranatant fraction using a hemacytometer.

[0222] The amount of cell recovery was determined as follows:

[0223] Cells supernatant

[0224] Cell recovery (%) = — — - , > „ . . -

[0225] (Cells supernatant + Cells infranatant)100

[0226] Equation 3

[0227] The test is considered validated only if the cell balance is between 90 and 110%, wherein cell balance is determined by the following equation :

[0228] (Cells supernatant + Cells infranatant) Cell balance (%) = - - — - - xlOO initial cells

[0229] Equation 4 Example 1

[0230] Preparation of aqueous dispersions of amphiphilic materials

[0231] Dispersions of amphiphilic materials, i.e. phospholipids and pegylated phospholipids used to stabilize the outer shell of the calibrated gas-filled microvesicles, were prepared according to WO2018041906 (Example 1). The materials were added with the molar ratios showed in Tables 2-13, at a concentration of 20 mg / mL to chloroform 100% under stirring at 60-70°C until complete dissolution the amphiphilic material. The solvent was then evaporated under reduced pressure and the obtained film was dried overnight under reduced pressure. The dried material was then redispersed (at a concentration of 15 mg / mL) in saline at 60 °C under stirring for 30 minutes. The dispersion was then sonicated by using a tip sonicator (Branson Sonifier 250) to homogeneously disperse the material. The preparations were then filtered using a polycarbonate filter (0.45 pm pore size), cooled down to room temperature and degassed.

[0232] The specific type and amounts of amphiphilic materials used are summarized in Tables 2-13 in the following Examples. Example 2

[0233] Preparation of calibrated gas-filled microvesicles

[0234] The dispersions of amphiphilic lipids prepared according to Example 1 were used to prepare calibrated gas-filled microvesicles characterized by various amounts of pegylated phospholipids.

[0235] Calibrated gas-filled microvesicles were synthesized according to WO2018041906 (Example 2) using a commercially available microfluidic flow- focusing device Micronit chip (CU4553.007 N30 design from Micronit Technologies (Enschede, The Netherlands) with 8 pm depth between 2 glass wafers that are aligned and bonded together to form a cylindrical channel of 30 pm length and 16 pm depth in which bubbles pinch off, allowing for the leak tight connection of the chip to the gas and liquid supply tubing (Peek Upchurch, 1 / 16 inch O.D, 150 pm I.D.). The microvesicles formation channel had a width of 17 pm and a length of 135 pm. The overall channel depth was 14 pm. The chip and its holder were positioned in an optically transparent temperature-controlled water bath (T° comprised between 50°C and 75°C) that was mounted on an inverted microscope equipped with a 20 times magnification objective (Olympus, LMPLAN 20x) and a CCD camera (Lumenera, LM156M).

[0236] A gas mixer system was implemented in this set up to mix C4F10 and CO2 in a controlled way. CO2 was controlled by an El-Flow select controller (F-200CV-002-RAD-11- K, Bronkhorst, NL) and C4F10 was controlled by a Low AP flow controller (F-200DV-RAD- 11-Z). A check valve (Swagelok, ref SS-2C4-1, Stainless Steel Poppet Check Valve, Fixed Pressure, 1 / 8 in. FNPT, 1 psig, 0.07 bar) is connected at the outlet of the gas mixer to avoid liquid reflow in the two gas flow controllers. Phospholipid solution contained in the liquid tank was connected to N2 (pressure 5 bars) and liquid flow was controlled by a miniCORI-FLOW controller (L13V12-RAD-11-K). Liquid co-flow rates were set at 120 pljmin, up to 180 pL / min. Typically, setting low liquid flow rate allows to obtain CMV with larger sizes. The suspension of native microvesicles was then collected in a sealed vial filled with C4F10 (100%).

[0237] Table 2 reports the characterization of CMV suspensions, obtained as described above. The characterization was performed using the Coulter counter protocol reported in the previous method section. Table 2 Characterization of CMV suspensions after their preparation Example 3

[0238] Comparative formulations of calibrated gas-filled microvesicles stabilized by neutral phospholipids

[0239] Standard formulations of calibrated gas-filled microvesicles stabilized by neutral phospholipids (e.g. DSPC) were prepared according to Example 2.

[0240] The influence of the amount of PE-PEG on the stability of said formulations was evaluated by measuring the coalescence percentage as described in Method section.

[0241] Results

[0242] Table 3 Comparative compositions stabilized by neutral phospholipid (not comprising anionic phospholipids) n / a = coalescence could not be measured according to Eq.2 because the freshly formed microvesicles exhibited high GSD values (greater than 1.2)

[0243] As inferable from Table 3, the coalescence stability of monosize gas-filled microvesicles stabilized by neutral phospholipid (e.g. DSPC) is substantially affected by the amount of pegylated phospholipids (PE-PEG). The reduction of the molar amount of PE-PEG below 15% endowed to a substantial increase of the coalescence phenomenon (e.g. higher than 1%) in standard compositions of CMV stabilized by neutral phospholipid.

[0244] Example 4

[0245] Influence of anionic phospholipids on the stability of calibrated microvesicles

[0246] The influence of anionic phospholipids in stabilizing calibrated gas-filled microvesicles was studied.

[0247] For this purpose, different types of anionic phospholipids including phosphatidic acid (PA), phosphatidylserine (PS) or phosphatidylglycerol (PG) were tested in combination with different types of pegylated phospholipids (PE-PEG) characterized by different molecular lengths (e.g. PE-PEG2000 and PE-PEG5000).

[0248] Calibrated gas-filled microvesicles were prepared according to Example 2.

[0249] The obtained compositions were compared by determining the coalescence percentage as previously reported (see Method section).

[0250] Results

[0251] Tables 4 and 5 report the results obtained by testing the formulations comprising phosphatidylserine (PS).

[0252] Table 4 Coalescence % by varying the amount of DMPS and PE-PEG

[0253] Table 5 Coalescence % by varying the amount of DPPS and PE-PEG

[0254] Tables 6-8 report the results obtained by testing the formulation comprising phosphatidic acid (PA). Table 6 Coalescence % by varying the amount of DLPA and PE-PEG2000 or PE-PEG5000 Table 7 Coalescence % by varying the amount of DMPA and PE-PEG2000 or PE-PEG5000 Table 8 Coalescence % by varying the amount of DPPA and PE-PEG2000 or

[0255] PE-PEG5000

[0256] Tables 9-11 report the results obtained by testing the formulation comprising phosphatidylglycerol (PG).

[0257] Table 9 Coalescence % by varying the amount of DMPG and PE-PEG2000 or PE-PEG5000 Table 10 Coalescence % by varying the amount of DPPG and PE-PEG2000 or PE-PEG5000

[0258] Table 11 Coalescence % by varying the amount of DSPG and PE-PEG2000 or PE-PEG5000

[0259] As inferable from the above results, the nature of the anionic phospholipid affects the coalescence stability of the calibrated gas-filled microvesicles.

[0260] Using anionic phospholipids comprising phosphatidic acid (PA) or phosphatidylserine (PS) allowed to preserve the stability of the calibrated microvesicles by halving the amount (molar %) of PE-PEG in the shell compositions (7.5% w / w).

[0261] Instead, corresponding formulations comprising phosphatidylglycerol (PG) were found to be characterized by substantially high values of coalescence (e.g. higher than 1%) at the same molar amounts of components.

[0262] In particular, compositions of gas-filled microvesicles stabilized by anionic phospholipids comprising PA or PS and characterized by a fatty acid chains length of 18 carbon atoms or shorter were characterized by a significant reduction of the coalescence effect while reducing the amount of pegylated phospholipids.

[0263] A further noticeable reduction of the amount of pegylated phospholipids in the shell compositions, e.g. down to 1%, was obtained by increasing the molecular length of the PE-PEG from PE-PEG2000 to PE-PEG5000. Calibrated gas-filled microvesicles stabilized by DMPA displayed the best results in terms of reduction of pegylated phospholipids and preservation of the stability (e.g. absent or significantly low coalescence).

[0264] Example 5

[0265] Long-term stability over time of the anionic calibrated gas-filled microvesicles (CMV)

[0266] The long-term stability of the Composition DPPS-11 (DPPS 92.5% / PE-PEG 7.5%) was evaluated by regularly measuring CMV size distribution and concentration with a Coulter Counter Multisizer 3, as detailed in the Method section.

[0267] Over the course of four weeks, measurements were systematically conducted on a weekly basis. During this period, the suspension was consistently maintained at a temperature of 25°C (RT).

[0268] Table 12 Characterization over time (sizes, PDI, concentration and remaining CMV %) of the Composition DPPS-11

[0269] *The term "To" indicates the point in time immediately following the collection of the aforementioned suspension from the outlet channel of the microfluidic apparatus and / or subsequent to the application of an appropriate washing technique to the obtained suspension.

[0270] Results

[0271] The tested formulation was found to be substantially stable over time, e.g. up to one month, preserving its key characteristics such as size, polydispersity, and concentration. After four weeks, the compositions exhibited substantially similar values of sizes and polydispersity. Furthermore, also the % of remaining microvesicles was found to be higher than 90% after one month.

[0272] Example 6

[0273] Separation efficiency of the calibrated gas-filled microvesicles functionalized with STV

[0274] Calibrated gas-filled microvesicles were prepared according to Example 2 and subsequently functionalized with streptavidin STV. For this purpose, a formulation comprising DMPA (98%) / DSPE-PEG2000(l%) / DSPE- PEG2000-BIOT(1%) was selected.

[0275] After their collection, freshly prepared suspensions of calibrated microvesicles comprising PE-PEG-BIOTIN (CMV-BIOT) were washed twice by centrifugation (6' / 600 RPM) with saline 0.9%. Streptavidin (STV) was added to 1 mL of washed CMV-BIOT at the desired molar ratio STV / biot and then incubated on rotating wheel at RT for 45 min. CMV- BIOT-STV were then washed again twice by centrifugation (6' / 600 RPM) and resuspended in saline 0.9%. Cell recovery tests were performed using CCRF-CEM cells (30 MB / cell) with a STV / BIOT molar ratio of 0.5 or 1.

[0276] The expression "freshly prepared suspensions" refers to suspensions of calibrated gas-filled microvesicles at the end of the preparation method, e.g. after collecting said suspension from the outlet channel of the microfluidic apparatus and / or washing the obtained suspension with a suitable washing technique. Said freshly prepared suspension are characterized by microvesicles stabilized by an outer shell comprising a phospholipid, selected from PA, PS or a mixture thereof and a pegylated phospholipid comprising a reactive moiety, wherein said reactive moiety is not bound to a ligand (e.g. gas-filled microvesicles bearing a reactive moiety not yet functionalized with streptavidin). In said freshly prepared suspension, the total amount of reactive moieties incorporated in the stabilizing layer of the gas-filled microvesicles is in the form of unreacted moieties. Typically a freshly prepared suspension has not yet been subjected to a washing procedure.

[0277] Results

[0278] Table 13 Separating efficiency of CMV stabilized by DMPA

[0279] The separation efficiency was expressed as cell recovery, calculated according to Equation 3.

[0280] As inferable from the above results, a substantially high recovery of cells was achieved by using the calibrated CMV-STV formulation at any STV / BIOT molar ratio. Comparable results can be achieved using suspensions of gas-filled microvesicles comprising phosphatidic acid, such as DPPA. Typically, the formulation will comprise DPPA (91.5%) / DSPE-PEG2000(7.5° / o) / DSPE-PEG2000-BIOT (1%) or DPPA (90.5%) / DSPE- PEG2000 (7.5%) / DSPE-PEG2000-BIOT (2%)STV will be added to these CMV-BIOT after washing, with a STV / BIOT molar ratio of 0.5, 1 or 2, and washed again after incubation with STV. Then CMV-BIOT-STV will be used for cell recovery tests as described above. These CMV-BIOT-STV will give a substantially high recovery of cells.

[0281] Similar outcomes can also be obtained with suspensions of gas-filled microvesicles comprising a phosphatidylserine, including DMPS.

[0282] For example a formulation comprising DMPS (91.5%) / DSPE-PEG2000 (7.5%) / DSPE- PEG2000-BIOT (1%), OR DPPS (91.5%) / DSPE-PEG2000 (7.5%) / DSPE-PEG2000-BIOT (1%), will be prepared and processed as described above to obtain CMV-BIOT-STV with STV / BIOT molar ratios of 0.5, 1 or 2. The CMV-BIOT-STV will be successively used for cell recovery tests using CCRF-CEM cells (30 MB / cell), and high recovery of cells will be obtained.

[0283] References:

[0284] - W02018041906A1

[0285] - W02019170606A1

[0286] - W02020260420A1

[0287] - W02020260423A1

[0288] - WO2023285628A1

[0289] Segers et al, Lab Chip, 2019, 19, 158

[0290] Fisher et al, J Am Coll Cardiol., 2002, 40(4)

[0291] Fix et al, Ultrasound Med Biol, 2018, 44, 1266-1280

[0292] - EP23216400

[0293] - WO2017117349

[0294] Ja'Affar et al., Ultrasound Med Biol, 2015, 41(ll):2990-3000.

Claims

CLAIMS1. A suspension of calibrated gas-filled microvesicles, said microvesicles comprising an inner core and an outer layer, said inner core comprising a physiologically acceptable gas and a said outer layer comprising:55% by moles or more of a phospholipid selected from phosphatidic acid, phosphatidylserine, or a mixture thereof and7.5% by moles or less, down to 0.5% of a pegylated phospholipid wherein said suspension of calibrated gas-filled microvesicles has a geometric standard deviation (GSD) of at least 1.20 or lower.

2. The suspension according to claim 1, wherein said phospholipid is selected from the group consisting of l,2-Dilauroyl-sn-glycero-3-phosphate (DLPA), 1,2- Dimyristoyl-sn-glycero-3-phosphate (DM PA), l,2-Dipalmitoyl-sn-glycero-3- phosphate (DPPA), l,2-Distearoyl-sn-glycero-3-phosphate (DSPA), 1,2- d ila uroy l-sn -glycero-3- phospho- L-serine (DLPS), 1,2-Dimyristoyl-sn-glycero- 3-phospho-L-serine (DM PS), 1, 2- Dipa I m itoy l-sn -glycero-3- phospho-L-serine (DPPS), l,2-Distearoyl-sn-glycero-3-phospho-L-serine (DSPS) or mixtures thereof.

3. The suspension according to claim 2, wherein said phospholipid is 1,2- Dimyristoyl-sn-glycero-3-phosphate (DMPA) or l,2-Dipalmitoyl-sn-glycero-3- phospho-L-serine (DPPS).

4. The suspension according to any of the preceding claims, wherein said pegylated phospholipid is a phospholipid covalently linked to a polyethylene glycol having average molecular weight of from 1000 to 8000 g / mol.

5. The suspension according to any of the preceding claims, wherein the molar amount of said phospholipid is 60% or higher, up to 99.5%.

6. The suspension according to any of the preceding claims, wherein the molar amount of said pegylated phospholipid is 6% or lower, down to 0.5%.

7. The suspension according to claim 6, wherein the molar amount of said pegylated phospholipid is 5% or lower, down to 0.5%.

8. The suspension according to claim 7, wherein the molar amount of said pegylated phospholipid is 3% or lower, down to 0.5%.

9. The suspension according to any of the preceding claims wherein the molar amount of said phospholipid is comprised between 92.7% and 99.5% by moles and the molar amount of pegylated phospholipids is comprised between 7.5% and 0.5% by moles.

10. The suspension according to any of the preceding claims, wherein said outer layer further comprises a neutral phospholipid.

11. The suspension according to any of the preceding claims wherein said suspension of gas-filled microvesicles has a Z-potential of - 5 mV or lower, down to -50 mV.

12. The suspension according to any of the preceding claims, wherein said pegylated phospholipid comprises a ligand.

13. The suspension according to claim 12, wherein said ligand is selected from the group consisting of avidin, neutravidin and streptavidin.

14. Use of the suspension as defined in claims 12-13 for cell separation.

15. A method for preparing a suspension of calibrated gas-filled microvesicles as defined in claims 1-13, said method comprising the following steps: a) providing (I) a gaseous flow and (ii) an aqueous liquid flow comprising:• 55% by moles or more of a phospholipid selected from phosphatidic acid, phosphatidylserine or a mixture thereof and• 7.5% by moles or less, down to 0.5% of a pegylated phospholipid; b) directing said gaseous flow and said liquid flow through respective inlet channels towards a contact zone; c) directing said gaseous flow and said liquid flow from the contact zone through a calibrated orifice to obtain an aqueous suspension comprising said gas-filled microvesicles; d) collecting a suspension of calibrated gas-filled microvesicles, wherein said suspension has a geometric standard deviation (GSD) of at least 1.20 or lower.

16. A suspension of calibrated gas-filled microvesicles according to claims 1-13, for use in diagnostic and / or therapeutic treatment.

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