Calibrated gas-filled microvesicles with ligand

Abstract

Formulations of calibrated gas-filled microvesicles comprising a ligand, which may advantageously be used in methods for separating cells or biological materials The formulations comprise a phospholipid and a suitable mixture of a pegylated phospholipid and of a pegylated phospholipid comprising a ligand.

Description

[0001] CALIBRATED GAS-FILLED MICROVESICLES WITH LIGAND

[0002] Technical field

[0003] The invention relates to new formulations of calibrated gas-filled microvesicles comprising a ligand, which may advantageously be used in a method for separating cells or biological materials.

[0004] Background of the invention

[0005] Isolating a specific cell type from a mixture of cells is typically the first step in cell analysis and examination. The use of cell isolation tools is fundamental in several biomedical fields. In one application, in the field of cell and gene therapy (CGT), cells are harvested from a patient, treated to express the desired gene, expanded and administered back to the patient. Another possible application is for the separation of circulating tumor cells (CTCs) or circulating biomarkers (e.g. liquid biopsy).

[0006] Above all the various cell isolation methods, the antibody-binding methodology relies on the antigen-antibody recognition system of cell-surface biomarkers, and therefore provides precise sorting, such as in fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS) and buoyancy-activated cell sorting (BACS).

[0007] BACS is an isolation method based on the buoyancy of microvesicles or microparticles, reported to be a simple and gentle way to isolate specific cells wherein the microvesicle / cell interaction is also performed using antibodies or other molecules (see e.g WO2020 / 127816). Conventional BACS methods commonly rely on gas-filled microbubbles stabilized by materials such as polymers, lipids, or proteins. These microbubbles are typically produced as polydisperse compositions, exhibiting large size distribution, which may negatively impact their cell sorting efficiency. Therefore, ensuring a narrow size distribution is critical to maximize the cell sorting efficiency of future formulations suitable for said application.

[0008] Monosize microbubbles (MSB) are a new generation of gaseous microbubbles having a narrow calibrated and controlled size distribution compared to commercially available polydisperse microbubble ultrasound contrast-agents (USCA). Monosize microbubbles, known also as calibrated or monodisperse microvesicles, are for instance described in WO2018041906A1, WO2019170606A1, W02020260420A1 and WO2020260423A1.

[0009] Up to now, according to Applicant's knowledge, such calibrated gas-filled microvesicles have not been applied yet in buoyancy-activated cell sorting applications.

[0010] It has now been found that formulations comprising monosize microbubbles stabilized by a phospholipid and a suitable mixture of a pegylated phospholipid and of a pegylated phospholipid comprising a ligand may be advantageously used in a method for separating cells or biological material with a higher efficiency compared to standard polydisperse microbubbles.

[0011] Summary of the invention

[0012] A first 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: a phospholipid less than 4% by moles of a first pegylated phospholipid comprising a reactive moiety, at least a portion of said first pegylated phospholipid being bound to a ligand through said reactive moiety, and at least 15% by moles of a second pegylated phospholipid not comprising a reactive moiety wherein said suspension of calibrated gas-filled microvesicles has a geometric standard deviation (GSD) lower than 1.2.

[0013] In an embodiment, said outer layer comprises a total amount of pegylated phospholipids of at least 16%, more preferably at least 18%, up to 22%, preferably to 20%.

[0014] In a further embodiment, said outer layer comprises at least 3%, preferably at least 2%, more preferably at least 1%, still more preferably at least 0.5%, down to 0.25% by moles of said first pegylated phospholipid.

[0015] In another embodiment, said outer layer comprises at least 16% or higher, preferably at least 17% or higher, more preferably at least 18% or higher, still more preferably at least 19% or higher, still more preferably at least 20% or higher, up to 21% by moles of said second pegylated phospholipid.

[0016] In a further embodiment, said first or said second pegylated phospholipid is a phospholipid covalently linked to a polyethylene glycol having a number average molecular weight of from 1000 to 8000 g / mol, preferably from 1500 to 3000 g / mol, still more preferably said second pegylated phospholipid has a molecular weight of 2000 g / mol + / - 5%. In another embodiment, said ligand is a biomolecule-binding moiety, preferably a biotin-binding protein selected from the group consisting of avidin, neutravidin and streptavidin.

[0017] In a further embodiment, said ligand has a density on the surface of the outer layer at least 1500 molecules / pm2, preferably of at least 1700 molecules / pm2, more preferably of at least 1900 molecules / pm2and even more preferably of at least 2200 molecules / pm2, up to e.g. 3000 molecules / pm2. In another embodiment, the molar amount of phospholipid is from 60% to 95%, preferably from 70% to 90%.

[0018] In another embodiment, said phospholipid is selected from dimyristoylphosphatidylcholine (DMPC), dipalmitoyl-phosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidoyl-phosphatidylcholine (DAPC), dipalmitoyl phosphatidic acid (DMPA), dipalmitoyl phosphatidic acid (DPPA), distearoyl phosphatidic acid (DSPA), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylserine (DPPS) and distearoylphosphatidylserine (DSPS).

[0019] Preferably said first or second pegylated phospholipid is a pegylated phosphatidylethanolamine (PE- PEG).

[0020] A further aspect of the invention relates to the use of the suspension as above defined for cell separation.

[0021] Another aspect of the present invention relates to a method for preparing a suspension of calibrated gas-filled microvesicles with ligand as above defined which comprises:

[0022] A. providing (i) a gaseous flow and (ii) an aqueous liquid flow comprising providing (I) a gaseous flow and (ii) an aqueous liquid flow comprising: a phospholipid; less than 4% by moles of a first pegylated phospholipid comprising a reactive moiety, and at least 15% by moles of a second pegylated phospholipid not comprising a reactive moiety;

[0023] B. directing said gaseous flow and said liquid flow through respective inlet channels towards a contact zone;

[0024] 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;

[0025] D. collecting said suspension comprising said microvesicles from an outlet channel;

[0026] E. adding a ligand capable of reacting with said reactive moiety to said collected suspension;

[0027] F. coupling said first pegylated phospholipid with said ligand; and

[0028] G. collecting a suspension of calibrated gas-filled microvesicles with ligand.

[0029] According to an embodiment, after step D) said method comprises an optional step (D') of washing said collected suspension of calibrated gas-filled microvesicles. According to a further embodiment, after the coupling step F) said method comprises an optional step F') of washing said obtained suspension of calibrated gas-filled microvesicles with ligand.

[0030] Another aspect of the invention, relates to a method for manufacturing a lyophilized precursor for the preparation of a suspension of calibrated gas-filled microvesicles as defined above, comprising: i. preparing a first suspension of calibrated gas-filled microvesicles, said suspension comprising: a phospholipid; less than 4% by moles of a first pegylated phospholipid comprising a reactive moiety, and at least 15% by moles of a second pegylated phospholipid not comprising a reactive moiety; and a freeze-drying protecting component; ii. adding a ligand to said first suspension capable of reacting with said reactive moiety; iii. coupling said pegylated phospholipid with said ligand; iv. lyophilizing said emulsion to obtain a freeze-dried residue.

[0031] Detailed description of the invention

[0032] This invention provides novel compositions of calibrated gas-filled microvesicles characterized by enhanced stability (lack of coalescence), which may be advantageously used in methods for collecting / recovering cells or biological material, with an improved cell separation efficiency as compared to standard polydisperse microbubbles.

[0033] Producing functionalized calibrated microbubbles e.g. by employing flow-focusing techniques present significant challenges, largely due to the inherent instability that affects these freshly formed microbubbles, in particular resulting in coalescence of monodisperse bubbles.

[0034] The Applicant unexpectedly observed that by using suitable amounts of pegylated phospholipids as stabilizing materials significantly improved the stability of the calibrated microvesicles by limiting their coalescence at the end of their preparation as compared to conventional amounts of pegylated phospholipids known in the literature.

[0035] Moreover, the Applicant has further observed that such novel compositions of calibrated gas-filled microvesicles can generally be employed in cell sorting procedures at lower amounts than those typically used with standard polydisperse microbubbles (e.g. by 40-times factor) without affecting the high cell recovery Utilizing lower amounts of microbubbles in cell sorting application may significantly reduce material costs, thereby enhancing the overall economic efficiency of the sorting procedure.

[0036] The expression "gas-filled microvesicles" generally refers to bubbles of gas bounded, at the gas / liqu id interface, by a very thin envelope (film) involving a stabilizing amphiphilic material, typically a phospholipid, disposed at the gas to liquid interface. Said calibrated gas-filled microvesicles are suitable as contrast agents in ultrasound imaging techniques, known as Contrast-Enhanced Ultrasound (CEUS) Imaging, or in therapeutic applications, e.g. in combination with ultrasound mediated drug delivery.

[0037] These stabilized gas bubbles (dispersed in a suitable physiological solution) are generally referred to in the art with various terminologies, depending typically on the stabilizing material employed for their preparation; these terms include, for instance, "microspheres", "microbubbles", "microcapsules" or "microballoons", globally referred to here as "gas-filled microvesicles" (or "microvesicles" in short).

[0038] The term "calibrated" (when referred to gas-filled microvesicles) specifically refers to microvesicles suspensions with highly calibrated microvesicles (CMV) of micrometric sizes, characterized by a size distribution having a geometric standard deviation (GSD) of at least 1.2 or lower, preferably of at least 1.1, down to e.g. 1.05.

[0039] Considering the application in the BACS method, as observed by the Applicant, the separation efficiency of gas-filled microvesicles largely depends on their sizes: larger gas- filled microvesicles are typically able to provide higher cell recovery than smaller ones (compared at the same concentration per cell (e.g. microvesicles / cells)).

[0040] Furthermore, at lower amounts of microvesicles per cell (e.g. 3 CMV / cell), the calibrated gas-filled microvesicles characterized by larger sizes (e.g. 7.5 pm) were found to remarkably increase the separation efficiency in comparison to smaller microvesicles (e.g. 4.3 pm).

[0041] In this description and claims, the term "calibrated" is used interchangeably with "size-controlled", "monodispersed" or "monosize(d)" microvesicles.

[0042] Calibrated gas-filled microvesicles are preferably produced by using a microfluidic flow-focusing technology, where a gas thread is focused between two liquid flows in a flowfocusing 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, WO2019170606A1,

[0043] W02020260420A1 and WO2020260423A1 which are hereby incorporated by reference). 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.

[0044] 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.

[0045] 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, down to e.g. 1.05.

[0046] The calibrated microvesicles concentration (particularly upon production with microfluidic flow-focusing) is preferably not lower than 3xl08CMV / mL, preferably at least 4xl08CMV / mL.

[0047] 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).

[0048] 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).

[0049] 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:

[0050] Eq.l GSD = Where: n, = percentage of volume of gas (with respect to the total one) entrapped in the microvesicles measured for the ith channel

[0051] = volume of the microvesicles in the ithchannel, where

[0052] Eq. 1.1. Xi = di3.n / 6

[0053] (di = diameter of the microvesicle in the ith channel center) x= geometric mean of the volume of the microvesicles in the selected range, where:

[0054] 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.

[0055] 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.

[0056] 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.

[0057] Components of the outer shell of the gas-filled microvesicles

[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] Examples of naturally occurring phospholipids are natural lecithins (phosphatidylcholine (PC) derivatives) such as, typically, soya bean or egg yolk lecithins.

[0062] Examples of semisynthetic phospholipids are the partially or fully hydrogenated derivatives of the naturally occurring lecithins. Preferred phospholipids are fatty acids diesters of phosphatidylcholine, phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) or of sphingomyelin.

[0063] Examples of preferred phospholipids are, for instance, dilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidylcholine (DMPC), dipalmitoyl-phosphatidylcholine (DPPC), distearoyl-phosphatidylcholine (DSPC), diarachidoyl-phosphatidylcholine (DAPC), 1,2- dibehenoyl-sn-glycero-3-phosphocholine (DBPC), dioleoyl-phosphatidylcholine (DOPC), d i pentadeca noyl-phosphatidylcholine (DPDPC), l-myristoyl-2-palmitoyl- phosphatidylcholine (MPPC), l-palmitoyl-2-myristoyl-phosphatidylcholine (PMPC), 1- palmitoyl-2-stearoyl-phosphatidylcholine (PSPC), l-stearoyl-2-palmitoyl- phosphatidylcholine (SPPC), l-palmitoyl-2-oleylphosphatidylcholine (POPC), l-oleyl-2- palmitoyl-phosphatidylcholine (OPPC), dilauroyl-phosphatidylglycerol (DLPG) and its alkali metal salts, diarachidoylphosphatidyl-glycerol (DAPG) and its alkali metal salts, di myristoyl phosphatidylglycerol (DMPG) and its alkali metal salts, dipalmitoylphosphatidylglycerol (DPPG) and its alkali metal salts, distea roylphosphatidylglycerol (DSPG) and its alkali 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), diarachidoylphosphatidic acid (DAPA) and its alkali metal salts, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoyl phosphatidyl-ethanolamine (DSPE), dioleylphosphatidyl-ethanolamine (DOPE), diarachidoylphosphatidylethanolamine (DAPE), dilinoleylphosphatidylethanolamine (DLPE), dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), diarachidoyl phosphatidylserine (DAPS), dioleoylphosphatidylserine (DOPS), dipalmitoyl sphingomyelin (DPSP), and distearoylsphingomyelin (DSSP), dilauroylphosphatidylinositol (DLPI), diarachidoylphosphatidylinositol (DAPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dioleoyl-phosphatidylinositol (DOPI).

[0064] Particularly preferred phospholipids are DMPC, DPPC, DSPC, DAPC, DMPA, DPPA, DSPA, DMPG, DPPG, DSPG, DMPS, DPPS and DSPS. Most preferred are DMPC, DPPC, DSPC and DAPC.

[0065] Mixtures of phospholipids can also be used, such as, for instance, mixtures of DPPE and / or DSPE, DPPC, DSPC and / or DAPC with DSPS, DPPS, DSPA, DPPA, DSPG and DPPG.

[0066] Pegylated phospholipids

[0067] 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.

[0068] 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%).

[0069] 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.

[0070] According to a preferred embodiment, said pegylated phospholipid(s) are those comprising a PEG residue having an average molecular weight of from 1500 to 3000 g / mol, preferably said PEG residue has an average molecular weight of 2000 g / mol, (i.e. PEG2000).

[0071] Preferably the PEG is covalently bound to a phosphatidylethanolamine ("PE") residue bearing a respective lipid chain, e.g. myristoyl, palmitoyl or steaoryl.

[0072] 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.

[0073] Where necessary, 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. a biomolecule-binding moiety, such as avidin, neutravidin or streptavidin moiety). Suitable reactive moieties include, for instance, biotin, NHS (N- hydroxy-succiminide), amino, sulfhydryl, maleimide, azide or DBCO (dibenzocyclooctyne). 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 -pyridy Ith io 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).

[0074] 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.

[0075] According to the present description and claims, the expression "first pegylated phospholipid comprising 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 (e.g. DSPE-PEG2000-Biotin) capable of reacting with a corresponding reactive moiety on a functionalized ligand (e.g. a biomolecule-binding moiety, such as avidin, neutravidin or streptavidin moiety).

[0076] According to the present description and claims, the expression "second pegylated phospholipid not comprising a reactive moiety" indicates a compound comprising a polyethylene glycol residue ("PEG") covalently bound to a phospholipid residue, as above defined, which is not further functionalized with a reactive moiety and therefore is not able to couple a ligand (e.g. DSPE-PEG2000).

[0077] Ligand

[0078] 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. The term "ligand" refers to a moiety capable of specifically recognizing and attaching to a target biomolecule, facilitating interactions that are crucial for various therapeutic and diagnostic applications.

[0079] In particular, the ligand is preferably a biomolecule-binding moiety, where biomolecule refers to any target molecule that can be specifically recognized and bound by these moieties. Target molecules include, but is not limited to, small molecules, peptides, proteins, and complex macromolecules present on or bound to cell surfaces or within biological systems.

[0080] Examples of biomolecule-binding moieties (and respective binding pairs) include for instance biotin-binding proteins, such as avidin, neutravidin and streptavidin, which can form a binding pair with biotin, acting as target biomolecule (e.g. biotinylated antibody bound to the cell). Streptavidin is preferred for the present invention.

[0081] In one embodiment, said ligands can be bound to the microvesicles shell, i.e. to the reactive moiety on the functionalized pegylated phospholipid, in their native forms. For instance, 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.

[0082] In an alternative embodiment, ligands can be chemically modified, i.e. 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, the biomoleculebinding moiety (e.g. a peptide or an antibody) may comprise a thiol moiety to allow the maleimide / thiol coupling. Alternatively, when a DBCO functionalized lipid is used the biomolecule-binding moiety (e.g. a peptide or an antibody) may comprise an azide moiety to allow click chemistry coupling.

[0083] Inner core

[0084] 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. 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, perfluorotrimethylcyclobutanes, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclopentanes, perfluorocyclohexane, perfluoromethylcyclohexane and perfluorocycloheptane). Preferred saturated perfluorocarbons include, for example, CF4, C2F6, C3F8, C4F8, C4F10, C5F12 anc| C6F14.

[0085] 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.

[0086] Particularly preferred gases are those which are in gaseous form at room temperature, including SFe, C3F8 and C4F10.

[0087] Aqueous suspension of gas-filled microvesicles

[0088] This invention provides novel compositions of monosize gas-filled microvesicles suitable for cell sorting application, characterized by enhanced stability and by an improved cell sorting efficiency compared to existing polydisperse compositions.

[0089] As observed by the Applicant, it is advantageous according to the invention to obtain a suspension of calibrated gas-filled microvesicles stabilized by a mixture of a first pegylated phospholipid comprising a reactive moiety and of a second pegylated phospholipid without said reactive moiety, wherein 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, down to e.g. 1.05.

[0090] In an aspect of the invention, the outer shell of the disclosed calibrated gas-filled microvesicles comprises: a phospholipid, less than 4% by moles of a first pegylated phospholipid comprising a reactive moiety, at least a portion of said first pegylated phospholipid being bound to a ligand through said reactive moiety, and at least 15% by moles of a second pegylated phospholipid not comprising a reactive moiety. Typically, the phospholipid represents the larger portion of the outer layer of the calibrated gas-filled microvesicles components, e.g. up to 98% mol / mol. In certain embodiments, the molar amount of phospholipid may range from 60% to 95%, preferably from 70% to 90%.

[0091] In addition, the outer layer of the gas-filled microvesicles may further comprise a lipid, preferably a fatty acid such as, for instance palmitic acid, stearic acid, arachidonic acid or oleic acid.

[0092] The optional lipid (in particular fatty acid) may be present in a molar amount of for instance from 10% to 30%, more preferably from 15% to 25%.

[0093] According to an embodiment, the first pegylated phospholipid comprising a ligand is in a molar amount lower than 4%, preferably lower than 3%, more preferably lower than 2%, still more preferably lower than 1%, still more preferably lower than 0.5%, down to 0.25%.

[0094] According to another embodiment, the second pegylated phospholipid not comprising a reactive moiety) is in a molar amount of at least 15%, preferably at least 16% or higher, still more preferably at least 17% or higher, still more preferably at least 18% or higher, still more preferably at least 19% or higher, still more preferably at least 20% or higher, up to 21%.

[0095] According to a further embodiment the total molar amount of pegylated phospholipids is preferably at least 16%, more preferably at least 18%, up to 22%, preferably to 20%.

[0096] The expression "total amount of pegylated phospholipids" indicates the sum (e.g. molar %) of the molar amount of the first pegylated phospholipid with ligand and of the molar amount of the second pegylated phospholipid not comprising a reactive moiety (%).

[0097] For instance, the total amount of pegylated phospholipids comprised in the outer shell of a calibrated gas-filled microvesicle comprising 1% of a first pegylated phospholipid comprising a ligand and 18% of a second pegylated phospholipid, is of 19%.

[0098] Said first and second pegylated phospholipid ("PE-PEG"), which can be the same or different, 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).

[0099] In a preferred embodiment, said first pegylated phospholipid comprising a reactive moiety is a phospholipid covalently linked to a polyethylene glycol (PEG) having an average molecular weight of from 1500 to 3000 g / mol. In a further preferred embodiment, said second pegylated phospholipid not comprising a reactive moiety is a phospholipid covalently linked to a polyethylene glycol (PEG) having an average molecular weight of from 1000 to 8000 g / mol.

[0100] 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 second pegylated phospholipid is a PE-PEG2000 (e.g. DSPE- PEG2000).

[0101] The use of stabilizing materials in the amounts disclosed in the prior art for the manufacturing of functionalized gas-filled microbubbles with the flow-focusing methods led to significant challenges, largely due to the inherent instability that affected the freshly formed microbubbles, resulting in the coalescence of monodisperse bubbles, with consequent reduction of monodispersity.

[0102] The Applicant unexpectedly observed that by suitably tuning the respective amounts of pegylated phospholipids significantly improved the stability of the calibrated microvesicles 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).

[0103] 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.

[0104] 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:

[0105] - 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

[0106] - 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.

[0107] The percentage of coalescence can thus be calculated by normalizing the total number of coalesced microparticles by the total number of produced microparticles,

[0108] Equation 2

[0109] In general, a coalescence percentage of about 1 or less is desirable, down to coalescence percentages of e.g. 0.01%.

[0110] As discovered by the Applicant, such undesired coalescence phenomenon may be substantially reduced by preparing calibrated gas-filled microvesicles stabilized by an amount of second PE-PEG of 15% or higher. Improved results in terms of coalescence reduction were reached by gas-filled microvesicles stabilized by an outer layer comprising a molar amount of a second pegylated phospholipid (i.e. not bearing a reactive moiety) at 15% or higher and a molar amount of first pegylated phospholipid bearing a reactive moiety of at least 1%.

[0111] A significant reduction of the coalescence effect can be obtained by preparing calibrated gas-filled microvesicles stabilized by an outer layer comprising a total amount (molar %) of pegylated phospholipids preferably of at least 16%, more preferably at least 18%, up to 22%, preferably to 20%.

[0112] Furthermore, the Applicant found that the disclosed calibrated gas-filled microvesicles may be advantageously used in methods for sorting biological materials such as cells with a higher efficiency compared to standard polydisperse microbubbles, due to their higher and controllable size.

[0113] 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.

[0114] 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.

[0115] 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%).

[0116] Surprisingly it was observed that the cell recovery (%) can be significantly increased by using calibrated gas-filled microvesicles stabilized by a mixture of pegylated phospholipids combined in specific molar amounts.

[0117] As discovered by the Applicant, the efficiency of a cell separation process can be considerably improved by using lower molar amounts of a first pegylated phospholipid with a ligand to stabilize the calibrated microvesicles. In fact, an increased recovery of cells (e.g. about 15-times higher) was observed when using formulations of gas-filled microvesicles stabilized by less than 4% of a first pegylated phospholipid with a ligand as compared to formulations having higher molar amounts (e.g. higher than 4%) of a first pegylated phospholipid with a ligand in the presence of equal molar amounts of a second pegylated phospholipid.

[0118] Furthermore, the Applicant observed that relatively high cell recovery percentages, e.g. about 90%, are preserved by significantly lowering the amount of calibrated gas- microvesicles per cell (CMV / cell) in the mixture undergoing separation (e.g. down to 3 CMV / cell).

[0119] According to the invention the amount of 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.

[0120] Microvesicles according to the present invention typically have a ligand density of at least 1500 molecules / pm2, preferably of at least 1700 molecules / pm2, more preferably of at least 1900 molecules / pm2and even more preferably of at least 2200 molecules / pm2, up to e.g. 3000 molecules / pm2.

[0121] The relative molar ratio between the first pegylated phospholipid comprising a reactive moiety and the second pegylated phospholipid not comprising a reactive moiety is preferably from 1 :5 to 1 :40, preferably from 1 :7.5 to 1 :30 and even more preferably from 1:9 to 1:20.

[0122] According to another embodiment of the invention, the first pegylated phospholipid comprising a reactive moiety is a functionalized PE-PEG2000, e.g. DSPE-PEG2000-biotin or DSPE-PEG2000-maleimide, while the second pegylated phospholipid is a PE-PEG2000, e.g. DSPE-PEG2000.

[0123] As observed by the Applicant, the amount of pegylated phospholipids and the molecular weight of the PEG-chains contained in said pegylated phospholipids may be correlated in such a way to allow a suitable selection of the two, in order to provide the desired suspension of microvesicles.

[0124] Said correlation can be advantageously expressed by a number identified herein as "NPEG" defined as follows:

[0125] NPEG = MWl*mol%l + MW2*mol%2 where MW1 and mol%l respectively refer to the molecular weight and to molar % of the PEG chain contained in the pegylated phospholipid comprising a reactive moiety, while MW2 and mol%2 respectively refer to the molecular weight and molar % of the PEG chain contained in the pegylated phospholipid without a reactive moiety.

[0126] For instance, if the composition comprises 2.5% of functionalized PE-PEG2000 and 1% of PE-PEG5000, the NPEG number is:

[0127] NPEG= 2000*0.025 + 5000*0.01 = 100

[0128] As observed by the Applicant, in order to obtain a stable formulation (e.g. characterized by substantially reduced coalescence), the number N shall preferably be of from 315 to 450. Preferably MW1 is similar to MW2, more preferably both being about 2000 (+ / - 5%). Preferably mol%l is less than 0.04. Preferably mol%2 is at least 0.15.

[0129] According to an embodiment of the invention, the pegylated phospholipid comprising a reactive moiety is a functionalized PE-PEG2000 (e.g. DSPE-PEG2000-Mal) while the second pegylated phospholipid is a PE-PEG2000 (e.g. DSPE-PE2000). The relative molar ratio between the two respective PE-PEGs is preferably from 1: 1 to 1:8, preferably from 1: 1.5 to 1:5 and even more preferably from 1:2 to 1:4.

[0130] Method of preparation

[0131] Another aspect of the present invention relates to a method for preparing a suspension of calibrated gas-filled microvesicles with a ligand as above defined, which comprises: a) preparing an aqueous suspension of calibrated gas-filled microvesicles comprising an inner core and an outer layer, said inner core comprising a physiologically acceptable gas and a said outer layer comprising a phospholipid; less than 4% by moles of a first pegylated phospholipid comprising a reactive moiety, and at least 15% by molee of a second pegylated phospholipid not comprising a reactive moiety wherein said suspension of calibrated gas-filled microvesicles has a geometric standard deviation (GSD) lower than 1.2; b) adding a ligand capable of reacting with said reactive moiety; c) coupling said first pegylated phospholipid with said ligand; d) collecting a suspension of calibrated gas-filled microvesicles with ligand, wherein said suspension has a geometric standard deviation (GSD) lower than 1.2.

[0132] Preferably, 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.

[0133] A further aspect of the present invention thus relates to a method for preparing a suspension of calibrated gas-filled microvesicles with ligand as above defined, wherein said suspension of calibrated gas-filled microvesicles has a geometric standard deviation (GSD) lower than 1.2, said method comprising:

[0134] A. providing (I) a gaseous flow and (ii) an aqueous liquid flow comprising : a phospholipid; less than 4% by moles of a first pegylated phospholipid comprising a reactive moiety, and at least 15% by moles of a second pegylated phospholipid not comprising a reactive moiety; B. directing said gaseous flow and said liquid flow through respective inlet channels towards a contact zone;

[0135] 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;

[0136] D. collecting said suspension comprising said microvesicles from an outlet channel;

[0137] E. adding a functionalized ligand capable of reacting with said reactive moiety to said suspension;

[0138] F. coupling said first pegylated phospholipid with said ligand, and

[0139] G. collecting a suspension of calibrated gas-filled microvesicles with ligand, wherein said suspension has a geometric standard deviation (GSD) lower than 1.2.

[0140] At step a) or step A), suitable phospholipids, pegylated phospholipids, reactive moiety and ligands can be any of those previously listed.

[0141] Moreover, suitable molar amounts of each component are those indicated above.

[0142] After obtaining the suspension of calibrated gas-filled microvesicles, e.g. after step a) or after step D), 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.

[0143] According to an embodiment, said methods for preparing a suspension of calibrated gas-filled microvesicles with ligand comprises an optional step (D') of washing said obtained suspension of calibrated gas-filled microvesicles.

[0144] For instance, said optional step may be performed after step a) or after step D) of collecting said aqueous suspension of calibrated gas filled microvesicles from the exit channel of the microfluidic apparatus.

[0145] 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.

[0146] According to this description, suitable washing techniques comprise centrifugation, filtration, bubble sorting and decantation, preferred is centrifugation.

[0147] 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.

[0148] 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.

[0149] Therefore, at the end of said optional washing step D') the aqueous suspension of 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.

[0150] The term "washed" means after being treated with a suitable washing technique.

[0151] At the end of the preparation of the suspension of the calibrated gas-filled microvesicles, e.g. after collecting the suspension from the microfluidic apparatus and / or washing the obtained suspension with a suitable washing technique, a functionalized ligand capable of reacting with the reactive moiety linked to the first pegylated phospholipid is added to said suspension.

[0152] 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.

[0153] At the end of the coupling reaction between the ligand and the reactive moiety linked to the first pegylated phospholipid, the obtained suspension of 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.

[0154] According to an embodiment, after the coupling step said method comprises an optional step F') of washing said obtained suspension of calibrated gas-filled microvesicles with ligand.

[0155] 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 calibrated gas-filled microvesicles with ligand essentially does not comprise free ligand.

[0156] 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 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.

[0157] 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.

[0158] In the present description and claims, the expression "at least a portion of said first pegylated phospholipids being bound to a ligand through said reactive moiety" 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".

[0159] In the present description and claims, the term "unreacted" means not coupled to the ligand.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] Freeze-dried product

[0164] 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.

[0165] An aspect of the present invention relates to a freeze-dried composition comprising a phospholipid, a first pegylated phospholipid comprising a reactive moiety, a second pegylated phospholipid not comprising a reactive moiety and a freeze-drying protecting component which, upon reconstitution with a pharmaceutically acceptable solution in the presence of a biocompatible gas, provides a suspension of calibrated gas-filled microvesicles, wherein said reconstituted suspension of microvesicles have a geometric standard deviation (GSD) value of at least 1.22 or lower.

[0166] The number, dimensions and size distribution of the microvesicles obtained after reconstitution are substantially comparable with the number, dimensions and size distribution of the microvesicles in the preparation emulsion.

[0167] Suitable examples of freeze-drying protecting component are polymers, preferred being hydrophilic polymers, more preferably polyglycols, still more preferably polyethylene glycol (PEG).

[0168] Alternatively said freeze-drying protecting component is a mixture of freeze-drying protecting components comprising a polymer, preferably a hydrophilic polymer, more preferably a polyglycol, such a PEG, and a polyol or a saccharide, such as sorbitol and sucrose.

[0169] Another aspect of the invention, relates to a method for manufacturing a lyophilized precursor for the preparation of a suspension of calibrated gas-filled microvesicles as defined above, comprising: i. preparing a first suspension of calibrated gas-filled microvesicles, said suspension comprising: a phospholipid; less than 4% by moles of a first pegylated phospholipid comprising a reactive moiety, and at least 15% by moles of a second pegylated phospholipid not comprising a reactive moiety; and a freeze-drying protecting component; ii. adding a ligand to said first suspension capable of reacting with said reactive moiety; iii. coupling said pegylated phospholipid with said ligand; iv. lyophilizing said emulsion to obtain a freeze-dried residue.

[0170] Preferably, at step i) the gas-filled microvesicles are prepared by using a microfluidic flow-focusing technique.

[0171] Suitable phospholipids are those indicated above. In addition, said first suspension of gas-filled calibrated microvesicles may further comprise a lipid, preferably a fatty acid such as, for instance palmitic acid, stearic acid, arachidonic acid or oleic acid.

[0172] The functionalized pegylated phospholipid can be any of the pegylated phospholipids previously listed, suitably functionalized as discussed before.

[0173] Similarly, the functionalized ligand to be bound to the functionalized pegylated phospholipid can be selected among those previously listed.

[0174] Preferably said freeze-drying protecting component comprises a polymer, preferred being a hydrophilic polymer, more preferably a polyglycol, still more preferably a polyethylene glycol (PEG).

[0175] Alternatively, said freeze-drying protecting component further comprises a polyol or a saccharide, such as sorbitol and sucrose.

[0176] Methods of use

[0177] An aspect of the present invention relates to the disclosed gas-filled microvesicles for use in biotechnological applications. In particular, they may be advantageously used in a method for separating cells in the context of cells therapy manufacturing, 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. Particularly preferred are formulations of calibrated gas-filled microvesicles comprising a first pegylated phospholipid which is a functionalized PE-PEG2000 (e.g. DSPE-PEG2000-Biot or DSPE-PEG2000-Mal) and a second pegylated phospholipid is a PE- PEG2000 (e.g. DSPE-PE2000). Preferably the first pegylated phospholipid is present in a molar amount lower than 4%, more preferably lower than 3%, still more preferably lower than 2%, still more preferably lower than 1%, down to 0.25%; the second pegylated phospholipid is preferably in a molar amount of 15% or higher, more preferably 16% or higher, still more preferably 17% or higher, still more preferably 18% or higher, still more preferably 19% or higher, still more preferably 20% or higher, up to 21%.

[0178] The following examples will help to further illustrate the invention.

[0179] EXAMPLES

[0180] Materials: Methods

[0181] Microvesicles size and concentration : 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. Background noise measurements were performed prior to each microbubble measurement.

[0182] 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. CMV. MVC stands for the mean volume concentration or the volume of microvesicles in a sample volume.

[0183] The "geometric standard deviation" (GSD) of the tested formulations was determined according to Equation 1, as described above.

[0184] Coalescence measurement

[0185] 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:

[0186] - 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

[0187] - 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.

[0188] The percentage of coalescence can thus be calculated by normalizing the total number of coalesced microparticles by the total number of produced microparticles :

[0189] Equation 2

[0190] In general, a coalescence percentage of about 1 or less is desirable, down to coalescence percentages of e.g. 0.01%.

[0191] Streptavidin concentration determination :

[0192] The streptavidin (STV) content was determined in washed microvesicles suspension using the 4-biotin fluorescein assay. Briefly, after coulter measurement of washed microvesicles, they are collapsed in an ultrasound tank (Branson 5200 - 3 x 2 min) until obtaining a clear solution. The solution was then sampled in lOOuL portions in eight 5mL-glass tubes. The appropriate volume of PBS and the appropriate volume of 4-Biotin fluorescein (4-BF) solution (3050 pmoles / mL) were calculated to each sample to have 0.1, 0.2, 0.4, 0.75, 1.5, 2, 2.5 and 3-fold the theoretical biotin capacity. The PBS was added to the sample solution and then the corresponding 4-BF solution.

[0193] Then, the solution was mixed (vortex) and incubated 30 min at room temperature in the dark. Each mixture was then sampled in a 96-well plate (100 uL / well and 2 wells per condition) and the fluorescence was read using the Cytation 5 reader {Xexc 480 nm - Xem 525 nm). The curve fluorescence as a function of the 4-BF concentration was drawn for the four first points (low 4-BF) using a second order polynomial fit and for the four last points (high 4-BF) using a linear fit - The intersection was determined between the two curves and the STV concentration was determined using the standard curve (from free streptavidin solutions).

[0194] The STV density on microvesicles was calculated by dividing the STV amount as determined above by the Total CMV surface determined by the Coulter counter measurement (in molecules / pm2).

[0195] Streptavidin coupling yield determination :

[0196] The coupling yield was calculated by dividing the streptavidin density determined on washed microvesicles suspension by the streptavidin density determined on native microvesicles suspension (as a certain percentage of microvesicles can be removed during washing).

[0197] Cell recovery tests:

[0198] The cell recovery test protocol was performed as described in WO2020 / 127816.

[0199] 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.

[0200] The cell suspension (1 mL, about 5xl06cells) was transferred in a 2mL-low binding Eppendorf and 160 pL of Biotinylated mouse anti-human CD45 antibody (#555481 - BD Pharmigen) 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).

[0201] The microvesicles suspension (CMV volume depends on the CMV / Cell ratio, e.g. from 30 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.

[0202] The gas-filled microvesicles were then collapsed (by applying positive pressure) and cells were counted in both supernatant and infranatant fractions using a hemacytometer.

[0203] The amount of cell recovery was determined as follows:

[0204] Cells supernatant

[0205] Cell recovery (%) = — - - — — — - -x 100

[0206] (Cells supernatant + Cells infranatant)

[0207] Equation 3

[0208] The test is considered validated only if the cell balance is between 90 and 110%, wherein cell balance is determined by the following equation:

[0209] (Cells supernatant + Cells infranatant)

[0210] Cell balance (%) = - i ■ni ■ti ■a ,l — ce tll; -s xlOO

[0211] Equation 4

[0212] Example 1

[0213] Preparation of aqueous dispersions of amphiphilic materials

[0214] Dispersions of amphiphilic materials were prepared according to WO2018041906 (Example 1). The materials were added with the molar ratios shown in Tables la, lb and 1c, at a concentration of 20 mg / mL to a 2: 1 (volume ratio) chloroform / methanol mixture under stirring at 60°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, as detailed in the part "preparation of microvesicles") in saline or saline buffered with phosphate at pH 6.4 for CMV-maleimide at 60 °C under stirring for 30 minutes. The dispersion was then sonicated by using a tip sonicator (Branson Sonifier 250) to homogenously disperse the material. The preparations were then filtered using a polycarbonate filter (0.45 piq pore size), cooled down to room temperature and degassed.

[0215] The specific type and amounts of amphiphilic materials used are summarized in Tables la, lb, 1c. Table la Aqueous dispersions comprising DSPC:DSPE-PEG2000:DSPE- PEG2000-BIOTIN (molar amount (%))

[0216] Table lb Aqueous dispersions comprising DSPC:DSPE-PEG2000:DSPE- PEG2000-M ALEIMIDE (molar amounts (%))

[0217] Table 1c Aqueous dispersions comprising pegylated phospholipids with different average molecular weight Example 2

[0218] Preparation of calibrated gas-filled microvesicles functionalized with STV

[0219] 2.1 Preparation of native suspensions of calibrated gas-filled microvesicles

[0220] The above listed dispersions of amphiphilic lipids were used to prepare calibrated gas-filled microvesicles characterized by various amounts of pegylated phospholipids.

[0221] Calibrated gas-filled microvesicles were synthesized according to WO2018041906 (Example 2) using a commercially available microfluidic flow- focusing device (Dolomite microfluidics, small droplet chip, 14 pM etch depth, part no. 3200136), mounted in a commercially available chip holder (Dolomite microfluidics, part numbers: 3000024, 3000109, 3000021) 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 that was mounted on an inverted microscope equipped with a 20 times magnification objective (Olympus, LMPLAN 20x) and a CCD camera (Lumenera, LM156M). The liquid co-flow rate was controlled using a syringe pump (Harvard PHD4400). The gas (N2) was pressure controlled using a pressure regulator (Omega, PRG101-25) connected to a pressure sensor (Omega, DPG1000B-30G). Individual microvesicles were automatically detected from the recorded optical images to measure their sizes offline on a PC using Matlab software (The Mathworks Inc., Natick, MA. Liquid co-flow rates were set at 120 pL / min, 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%).

[0222] Table 2a reports the characterization of native suspensions of CMV, obtained as described above. The expression "native suspension" refers to suspensions of calibrated gas-filled microvesicles comprising a phospholipid, a first pegylated phospholipid comprising a reactive moiety and a second pegylated phospholipid, 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 a native 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 native suspension has not yet been subjected to a washing procedure. The characterization was performed using the Coulter counter protocol reported in the previous method section. Table 2a Native suspensions after their preparation

[0223] 2.2. Functionalization of calibrated microvesicles comprising PE-PEG-BIOTIN with STV After their collection, suspensions of calibrated microvesicles comprising PE-PEG- BIOTIN (CMV-BIOT) were washed twice by centrifugation (at 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 minutes. CMV-BIOT-STV were then washed again twice by centrifugation (at 6' / 600 RPM) and resuspended in saline 0.9%. Coulter Counter measurement was performed to determine CMV volume to add to cells to have 30 CMV / cell.

[0224] 2.3. Functionalization of calibrated microvesicles comprising PE-PEG-MALEIMIDE with STV After their collection, suspensions of calibrated microvesicles comprising PE-PEG- MALEIMIDE (CMV-MAL) were washed twice by centrifugation (at 6' / 600 RPM) with saline 0.9%. A solution of thiolated-streptavidin (STV-SH) was prepared according to the protocol described in WO2020127816A1 (Streptavidin (STV) derivatization). Briefly a STV solution was prepared by dissolving 9 mg of lyophilized streptavidin (0.85 mg of STV / mg of powder (from IBA) - 143 nmol) in 0,2 mL of distilled water and 150 pL of buffer A (50 mM phosphate, 150 mM saline pH 7.4). A clear solution was obtained (concentration ~22 mg / mL), Sulfo-LC-SPDP (2.5 mg) was dissolved in 250 pL of milliQ water. This solution (19 pmoles / mL) was freshly prepared just before the experiment.

[0225] A sample of the sulfo-LC-SPDP solution (26 pL - 0.5 pmoles - 3.5 equivalents) was added to the STV solution. The obtained solution was mixed (vortex) and incubated at room temperature for 40 min (stirred using vortex every 5 min).

[0226] Two 2mL-Zeba columns were equilibrated using buffer B (5 mM phosphate pH 7.4). After the incubation, the STV-SPDP solution was purified on the first Zeba column. TCEP (4 mg) was dissolved in 185 pL of Buffer C (Tris 500 mM, EDTA 50mM pH: 7) to obtain a 75 mM solution. A sample of TCEP solution (19 pL - ~10 equivalents) was added to the STV-SPDP solution. After mixing (e.g. with vortex), the solution was incubated at room temperature for 30 min, to deprotect STV-SDPD and obtain the thiolated STV (STV-SFI). One 2 mL-Zeba column was equilibrated using buffer B. After the incubation, the STV-SH solution was purified on the 2 mL-Zeba column The volume of the recovered solution was of about 0.39 mL, with a concentration of STV-SH of about 300 nmoles / ml .

[0227] The STV-SH-containing solution was used for subsequent coupling reactions with CMV-MAL to obtain calibrated microvesicles comprising PE-PEG-MALEIMIDE functionalized with streptavidin (CMV-MAL-STV).

[0228] Table 2b reports the characterization of suspensions of CMV functionalized with STV, obtained as described above. The characterization was performed using the Coulter counter protocol reported in the previous method section.

[0229] Table 2b Characterization of functionalized suspensions

[0230] Example 3

[0231] Influence of the composition of calibrated STV microvesicles Calibrated gas-filled microvesicles functionalized with STV were prepared according to Example 2.

[0232] Different preparations characterized by various molar amounts of DSPC / PE-PEG / PE- PEG-2000-reactive moiety were compared by determining the coalescence percentage as previously reported (see Method section). For this study, two different reactive moieties (RM) were tested, namely biotin and maleimide.

[0233] Results

[0234] Table 3a reports the results obtained by testing the formulation comprising DSPC / PE-PEG / PE-PEG-biot, while Table 3b reports the results obtained by testing the formulation comprising DSPC / PE-PEG / PE-PEG-mal.

[0235] T a b I e 3a Coalescence ° / o by varying the amount of PE-PEG and PE-PEG-biot

[0236] Table 3b Coalescence % by varying the amount of PE-PEG and PE-PEG- maleimide

[0237] As inferable from the above results, the molar amounts of PE-PEG and PE-PEG-RM (e.g. biotin or maleimide) affect the stability of the calibrated gas-filled microvesicles.

[0238] A significant reduction of the coalescence effect can be obtained by using total amounts (molar %) of pegylated phospholipids comprised between 16% and 22% as compared to higher coalescence measured for the preparations having lower or higher total amount of pegylated phospholipids. Moreover, by using a molar amount of PE-PEG of 15% or higher in combination with at least 1% of PE-PEG-biot endowed to a coalescence less than 1%.

[0239] Example 4

[0240] Preparation of calibrated STV microvesicles using PE-PEG2000 with various amounts of PE-PEG2000-biot

[0241] Calibrated gas-filled microvesicles functionalized with STV were prepared according to Example 2.

[0242] In order to evaluate the influence of the amount of PE-PEG-RM on the ability of calibrated STV microvesicles to collect cells, different compositions characterized by increasing molar percentages of PE-PEG-RM were compared.

[0243] For this purpose, cell recovery tests were performed using CCL-119 cells (30 MB / cell) as previously described.

[0244] Streptavidin (STV) was added to 1 mL of washed microvesicles comprising PE- PEG2000-biot (e.g. in molar amounts comprised from 0.5% to 7.5%) with a STV / BIOT molar ratio of 1.

[0245] Results

[0246] Table 4 Cell recovery (%) of formulations comprising PE-PEG2000 15% at increasing amounts of PE-PEG2000-biot (1, 2, 4, 6, 7.5%) Table 5 Cell recovery( %) of formulations comprising PE-PEG2000 18% at increasing amounts of PE-PEG2000-biot (0.5, 1, 2, 4%)

[0247] As inferable from the above results, with equal molar amount of PE-PEG, the presence of lower molar amounts of PE-PEG-biot, i.e. between 0.5% and 2%, provided an increased recovery of cells (about 3-times higher) with respect to formulations with higher molar amounts (i.e. PE-PEG-biot 4%-7.5%).

[0248] A similar trend was confirmed by testing the formulation CMV-Mal-2 (DSPC / DSPE- PEG2000 / DSPE-PEG2000-MAL 80 / 18 / 2) at 30 CMV / cell (CCL119), for which the cell recovery % was found to be 97%.

[0249] Similar results were obtained using the above compositions in cell recovery tests at different STV / Biot molar ratios, namely 0.5 and 2.

[0250] Example 5

[0251] Influence of the molecular weight of the pegylated phospholipids

[0252] Calibrated gas-filled microvesicles derivatized with STV were prepared according to Example 2. In order to evaluate the influence of the nature of pegylated phospholipids on the ability of calibrated gas-filled MV in collecting cells, composition MSB-9 (DSPC / DSPE- PEG2K / DSPE-PEG2K-BIOT (81 / 15 / 4)) was compared with two different compositions, i.e. Cl and C2, composed by pegylated phospholipids with number average molecular weights higher than 2000 g / mol.

[0253] For this purpose, cell recovery tests were performed using CCL-119 cells (30 CMV / cell) as previously described. Streptavidin (STV) was added to 1 mL of washed CMV- biotin at a STV / BIOT molar ratio of 1. Results

[0254] As inferable from Table 6, formulations comprising a first pegylated phospholipids (e.g. PE-PEG-biot) with a PEG moiety having a molecular weight higher than 2000 g / mol ± 10% provide a significantly lower recovery of cells with respect to formulations comprising a first pegylated phospholipid with a PEG moiety having a molecular weight of 2000 g / mol ± 10%.

[0255] Table 6 Influence of the PE-PEG molecular weight on the cell recovery %

[0256] Example 6

[0257] Separation efficiency of the calibrated gas-filled microvesicles

[0258] The separation efficiency of the calibrated gas-filled microvesicles was assessed by comparing different volumes of calibrated CMV-BIOT-STV.

[0259] For this purpose, the formulation MSB-10 (PE-PEG2K / PE-PEG2K-BIOT 18 / 1) was selected.

[0260] As described in Example 2, §2.1, after their collection, 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 during 45 min. CMV-BIOT-STV were then washed again twice by centrifugation (6' / 600 RPM) and resuspended in saline 0.9%. Coulter Counter measurement was performed to determine CMV volume to add to cells in order to have 3, 7,5, 15, and 30 CMV / cell, which were found to correspond to volumes of 62.5 pL, 125 pL, 250 pL, and 500 pL added to 5xl06cells respectively. Incubation time was kept at 20 min at room temperature on a rotating mixer for all the tested formulations. Results

[0261] Table 7 Composition MSB-10: influence of the CMV size on the separation efficiency (cell recovery) at different CMV / cell ratios

[0262] Table 8 Composition MSB-10: influence of various CMV / cell ratios on the cell recovery (%) using CCL119 cell line (cell size: 10-15 pm)

[0263] The separation efficiency was expressed as cell recovery, calculated according to Equation 3.

[0264] Table 7 shows the influence of the CMV size on the separating efficiency (expressed as cell recovery percentage) at different CMV per cell (the three tested CMV compositions were characterized by similar GSD values of at least 1.2 or lower).

[0265] It was observed that at lower microvesicles per cell (e.g. 3 CMV / cell), the calibrated gas-filled microvesicles characterized by larger sizes (e.g. 7.5 pm) were found to remarkably increase the separation efficiency in comparison to smaller microvesicles (e.g. 4.3 pm).

[0266] Furthermore, as inferable from Table 8, the recovery of cells provided by the calibrated CMV-STV formulation is similar at any investigated CMV / cell ratio, demonstrating that it is possible to significantly reduce the CMV / cell ratio without negatively affecting the cell recovery ability of the composition.

[0267] In particular, it is demonstrated that halving the CMV / cell ratios from 30 CMV / cell to 15 CMV / cell did not influence the recovery of cell of the calibrated CMV-STV, which was found to be substantially the same. Similar cell recovery percentages were obtained by performing the cell recovery test using the cell line MCF-7 (cell sizes 20-25 pm). For instance, at 30 CMV / cell, the cell sorting percentage was about 93%. References

[0268] 1. WO2020 / 127816

[0269] 2. WO2018041906A1

[0270] 3. WO2019170606A1

[0271] 4. W02020260420A1 5. WO2020260423A1

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: a phospholipid, less than 4% by moles of a first pegylated phospholipid comprising a reactive moiety, at least a portion of said first pegylated phospholipid being bound to a ligand through said reactive moiety, and at least 15% by moles of a second pegylated phospholipid not comprising a reactive moiety wherein said suspension of calibrated gas-filled microvesicles has a geometric standard deviation (GSD) lower than 1.2.

2. The suspension according to claim 1, wherein said outer layer comprises a total amount of pegylated phospholipids of at least 16%, more preferably at least 18%, up to 22%, preferably to 20%.

3. The suspension according to claim 1 or 2, wherein said outer layer comprises at least 3%, preferably at least 2%, more preferably at least 1%, still more preferably at least 0.5%, down to 0.25% by moles of said first pegylated phospholipid.

4. The suspension according to any of the preceding claims, wherein said outer layer comprises at least 16% or higher, preferably at least 17% or higher, more preferably at least 18% or higher, still more preferably at least 19% or higher, still more preferably at least 20% or higher, up to 21% by moles of said second pegylated phospholipid.

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

6. The suspension according to claim 5, wherein said first or said second pegylated phospholipid is a phospholipid covalently linked to a polyethylene glycol having a number average molecular weight of from 1500 to 3000 g / mol.

7. The suspension according to claim 6, wherein said first and said second pegylated phospholipid has a molecular weight of 2000 g / mol + / - 5%.

8. The suspension according to any of the preceding claims, wherein said ligand is a biomolecule-binding structure.

9. The suspension according to claim 8, wherein said biomolecule-binding structure is a biotin-binding protein selected from the group consisting of avidin, neutravidin and streptavidin.

10. The suspension according to any of the preceding claims, wherein said ligand has a density on the surface of the outer layer of at least 1500 molecules / pm2.

11. The suspension according to any of the preceding claims, wherein the molar amount of phospholipid is from 60% to 95%, preferably from 70% to 90%.

12. The suspension according to any of the preceding claims, wherein said phospholipid is selected from dimyristoyl-phosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoyl-phosphatidylcholine (DSPC), diarachidoyl- phosphatidylcholine (DAPC), dipalmitoyl phosphatidic acid (DMPA), dipalmitoyl phosphatidic acid (DPPA), distearoyl phosphatidic acid (DSPA), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylserine (DPPS) and distearoylphosphatidylserine (DSPS).

13. The suspension according to any of the preceding claims, wherein said first or second pegylated phospholipid is a pegylated phosphatidylethanolamine.

14. Use of the suspension as defined in any of the preceding claims for cell separation.

15. A method for preparing a suspension of calibrated gas-filled microvesicles with ligand as defined in claims 1-13, wherein said suspension of calibrated gas-filled microvesicles has a geometric standard deviation (GSD) lower than 1.2, said method comprising:A. providing (I) a gaseous flow and (ii) an aqueous liquid flow comprising: a phospholipid; less than 4% by moles of a first pegylated phospholipid comprising a reactive moiety, and at least 15% by moles of a second pegylated phospholipid not comprising a reacting moiety;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 said suspension comprising said microvesicles from an outlet channel;E. adding a functionalized ligand capable of reacting with said reactive moiety to said collected suspension; F. coupling said first pegylated phospholipid with said ligand; andG. collecting a suspension of calibrated gas-filled microvesicles with ligand, wherein said suspension has a geometric standard deviation (GSD) lower than 1.2.

16. The method according to claim 15, further comprising an optional step D') of washing said obtained suspension of calibrated gas-filled microvesicles.

17. The method according to claims 16-17, comprising after the coupling step F) a step F') of washing said obtained suspension of calibrated gas-filled microvesicles with ligand.

Images