Calibrated gas-filled microvesicles with ligands

Abstract

Formulations of calibrated gas-filled microvesicles comprising a ligand can be advantageously used in methods for isolating cells or biological material. The formulations comprise a phospholipid and a suitable mixture of a pegylated phospholipid and a pegylated phospholipid comprising a ligand.

Description

Technical Field

[0001] This invention relates to novel formulations of calibrated aerated microbubbles containing ligands, which can be advantageously used in methods for separating cells or biological materials. Background Technology

[0002] 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, processed to express a desired gene, amplified, and then administered back to the patient. Another possible application is for isolating circulating tumor cells (CTCs) or circulating biomarkers (e.g., liquid biopsy).

[0003] Among all the various cell separation methods, antibody-binding methods rely on antigen-antibody recognition systems based on cell-surface biomarkers, and thus provide precise sorting, such as in fluorescence-activated cell sorting (FACS), magnetically activated cell sorting (MACS), and buoyancy-activated cell sorting (BACS).

[0004] BACS (Bipolar Interval Separation Method) is a buoyancy-based separation method based on microbubbles or microparticles, reported as a simple and gentle way to separate specific cells, where antibodies or other molecules are also used to achieve microbubble / cell interactions (see, for example, WO 2020 / 127816). Conventional BACS methods typically rely on aerated microbubbles stabilized by materials such as polymers, lipids, or proteins. These microbubbles are typically generated as polydisperse compositions exhibiting a large size distribution, which can negatively affect their cell sorting efficiency. Therefore, ensuring a narrow size distribution is crucial for maximizing the cell sorting efficiency of future formulations suitable for the application in question.

[0005] Single-size microbubbles (MSBs) are a new generation of gaseous microbubbles that offer a narrower calibration and controlled size distribution compared to commercially available polydisperse microbubble ultrasound contrast agents (USCAs). Single-size microbubbles, also known as calibrated or monodisperse microbubbles, are described, for example, in WO2018041906A1, WO2019170606A1, WO2020260420A1, and WO2020260423A1.

[0006] To date, to the applicant’s knowledge, such calibrated inflatable microbubbles have not been used in buoyancy-activated cell sorting applications.

[0007] It has been found that formulations containing monodisperse microbubbles stabilized by phospholipids can be used advantageously in methods for separating cells or biological materials with higher efficiency than standard polydisperse microbubbles. Summary of the Invention

[0008] A first aspect of the invention relates to a calibrated suspension of aerated microbubbles, the microbubbles comprising a core and an outer layer, the core comprising a physiologically acceptable gas, and the outer layer comprising:

[0009] - Phospholipids

[0010] - Less than 4 mol% of a first polyethylene glycol-modified phospholipid containing a reactive structural moiety, wherein at least a portion of the first polyethylene glycol-modified phospholipid binds to a ligand via the reactive structural moiety, and

[0011] - At least 15 mol% of a second polyethylene glycol-modified phospholipid that does not contain reactive structural moieties.

[0012] The calibrated suspension of aerated microbubbles has a geometric standard deviation (GSD) of less than 1.2.

[0013] In one embodiment, the outer layer comprises at least 16%, more preferably at least 18%, at most 22%, and preferably at most 20% of a total amount of polyethylene glycol-modified phospholipid.

[0014] In a further embodiment, the outer layer contains at least 3 mol%, preferably at least 2 mol%, more preferably at least 1 mol%, and even more preferably at least 0.5 mol%, as low as 0.25 mol% of the first polyethylene glycol phospholipid.

[0015] In another embodiment, the outer layer comprises at least 16 mol% or higher, preferably at least 17 mol% or higher, more preferably at least 18 mol% or higher, even more preferably at least 19 mol% or higher, even more preferably at least 20 mol% or higher, and at most 21 mol% of the second polyethylene glycol phospholipid.

[0016] In a further embodiment, the first or second polyethylene glycol-modified phospholipid is a phospholipid covalently linked to polyethylene glycol with a number-average molecular weight of 1000-8000 g / mol, preferably 1500-3000 g / mol, and more preferably, the second polyethylene glycol-modified phospholipid has a molecular weight of 2000 g / mol ± 5%. In another embodiment, the ligand is a biomolecule-binding structural portion, preferably a biotin-binding protein selected from the group consisting of: avidin, neutral avidin, and streptavidin.

[0017] In another embodiment, the ligand has a concentration of at least 1500 molecules / µm. 2 Preferably at least 1700 molecules / µm 2 More preferably at least 1900 molecules / µm 2 And even more preferably, at least 2200 molecules / µm 2 At most, for example, 3000 molecules / µm 2 The density of on the surface of the outer layer.

[0018] In another embodiment, the molar amount of phospholipid is 60% to 95%, preferably 70% to 90%.

[0019] In another embodiment, the phospholipid is selected from dimyristoyl-phosphatidic acid (DMPC), dipalmitoyl-phosphatidic acid (DPPC), distearyl-phosphatidic acid (DSPC), arachidoyl-phosphatidic acid (DAPC), dimyristoyl phosphatidic acid (DMPA), dipalmitoyl phosphatidic acid (DPPA), distearyl phosphatidic acid (DSPA), dimyristoyl phosphatidic glycerol (DMPG), dipalmitoyl phosphatidic glycerol (DPPG), distearyl phosphatidic glycerol (DSPG), dimyristoyl phosphatidic serine (DMPS), dipalmitoyl phosphatidic serine (DPPS), and distearyl phosphatidic serine (DSPS).

[0020] Preferably, the first or second polyethylene glycol phospholipid is polyethylene glycol phosphatidylethanolamine (PE-PEG).

[0021] Another aspect of the invention relates to the use of suspensions as defined above for cell separation.

[0022] Another aspect of the invention relates to a method for preparing a suspension of calibrated aerated microbubbles having ligands as defined above, the method comprising:

[0023] A. Provide (i) a gas stream and (ii) an aqueous liquid stream, the aqueous liquid stream comprising:

[0024] - Phospholipids;

[0025] - Less than 4 mol% of the first polyethylene glycol-modified phospholipid containing the reactive structural moiety, and

[0026] - At least 15 mol% of a second polyethylene glycol-modified phospholipid that does not contain reactive structural moieties;

[0027] B. Guide the gas flow and the liquid flow toward the contact area through their respective inlet channels;

[0028] C. Guide the gas flow and the liquid flow from the contact area through the calibration orifice to obtain an aqueous suspension containing the aerated microbubbles;

[0029] D. Collect the suspension containing the microbubbles from the outlet channel;

[0030] E. Add a ligand capable of reacting with the reactive structural portion to the collected suspension;

[0031] F. Couple the first polyethylene glycol-modified phospholipid to the ligand; and

[0032] G. Collect a suspension of calibrated aerated microbubbles containing ligands.

[0033] According to one embodiment, after step D), the method includes an optional step (D'): washing the collected suspension of calibrated aerated microbubbles.

[0034] According to another embodiment, after the coupling step F), the method includes an optional step F'): washing the obtained suspension of calibrated aerated microbubbles with ligands.

[0035] Another aspect of the invention relates to a method for manufacturing a lyophilized precursor for preparing a suspension of calibrated aerated microbubbles as defined above, the method comprising:

[0036] i. Preparing a first suspension of calibrated aerated microbubbles, the suspension comprising:

[0037] - Phospholipids;

[0038] - Less than 4 mol% of the first polyethylene glycol-modified phospholipid containing the reactive structural moiety, and

[0039] - At least 15 mol% of a second polyethylene glycol-modified phospholipid that does not contain reactive structural moieties; and

[0040] - Freeze-dried protective components;

[0041] ii. Add a ligand capable of reacting with the reactive structural portion to the first suspension;

[0042] iii. Couple the polyethylene glycol-modified phospholipid to the ligand;

[0043] iv. Freeze-dry the emulsion to obtain a freeze-dried residue. Detailed Implementation

[0044] The present invention provides a novel composition of calibrated aerated microbubbles characterized by enhanced stability (lack of coalescence), which can be advantageously used in methods for collecting / recovering cells or biological materials, with improved cell separation efficiency compared to standard polydisperse microbubbles.

[0045] Generating functionalized calibration microbubbles (e.g., by using flow focusing techniques) presents significant challenges, primarily due to inherent instabilities that affect these newly formed microbubbles, particularly leading to the aggregation of monodisperse bubbles.

[0046] The applicant unexpectedly observed that, compared with conventional amounts of polyethylene glycol phospholipids known in the literature, the stability of calibrated microbubbles was significantly improved by limiting their aggregation at the end of microbubble preparation by using an appropriate amount of polyethylene glycol phospholipids as a stabilizing material.

[0047] Furthermore, the applicant has observed that this novel composition of calibrated aerated microbubbles can typically be used in cell sorting processes at a lower amount (e.g., 40-fold factor) than that typically used with standard polydisperse microbubbles without compromising high cell recovery rates.

[0048] In cell sorting applications, the use of a lower amount of microbubbles can significantly reduce material costs, thereby improving the overall economic efficiency of the sorting process.

[0049] The term "aerosolized microbubble" generally refers to a gas bubble enclosed by a very thin membrane at the gas / liquid interface, the membrane containing a stable amphiphilic material, typically phospholipids, disposed at the gas-liquid interface. Such calibrated aerosolized microbubbles are suitable as contrast agents in ultrasound imaging techniques (known as contrast-enhanced ultrasound (CEUS) imaging) or therapeutic applications (e.g., in combination with ultrasound-mediated drug delivery).

[0050] Typically, depending on the stabilizing material used to prepare them, these stable bubbles (dispersed in a suitable physiological solution) are generally referred to in the art by various terms; these terms include, for example, “microspheres,” “microbubbles,” “microcapsules,” or “microspheres,” and are collectively referred to herein as “aerated microbubbles” (or simply “microbubbles”).

[0051] The term “calibrated” (when referring to aerated microbubbles) specifically refers to a microbubble suspension with height-calibrated microbubbles (CMV) of micrometer size, characterized by a size distribution with a geometric standard deviation (GSD) of at least 1.2 or lower, preferably at least 1.1, for example as low as 1.05.

[0052] Given the application of BACS methods, as observed by the applicant, the separation efficiency of aerated microbubbles depends largely on their size: larger aerated microbubbles typically provide higher cell recovery rates compared to smaller aerated microbubbles (at the same concentration / cell (e.g., microbubble / cell)).

[0053] Furthermore, at lower amounts of microbubbles / cells (e.g., 3 CMV / cell), calibrated aerated microbubbles with larger sizes (e.g., 7.5 µm) were found to significantly increase separation efficiency compared to smaller microbubbles (e.g., 4.3 µm).

[0054] In this specification and claims, the terms “calibrated” and “size-controlled”, “monodispersive” or “single-size” microbubbles are used interchangeably.

[0055] The calibrated aerated microbubbles are preferably generated using microfluidic flow focusing technology, in which a gas line is focused between two liquid streams in a flow focusing device, forming phospholipid-stabilized calibrated microbubbles, which are then collected in an outlet channel. This method allows for the production of calibrated microbubbles in a highly repeatable manner at a reasonable productivity (approximately 60 million bubbles / minute) (e.g., as described in WO2018041906A1, WO2019170606A1, WO2020260420A1, and WO2020260423A1, which are incorporated herein by reference).

[0056] Depending on the parameters of the manufacturing process and apparatus, calibrated microbubbles with a relatively narrow size distribution near any desired average diameter (e.g., at least 3 µm) can be obtained.

[0057] In a preferred embodiment, the average diameter of the calibrated inflatable microbubbles is at least 3µm, preferably at least 4µm, more preferably at least 5µm, even more preferably at least 6µm, even more preferably at least 7µm, even more preferably at least 8µm, even more preferably at least 9µm, for example at most 10µm.

[0058] The size distribution of the calibrated microbubbles is typically characterized by a geometric standard deviation (GSD) value of at least 1.20 or lower, preferably at least 1.15, for example as low as 1.05.

[0059] The calibrated microbubble concentration (especially when using microfluidic flow focusing) is preferably not less than 3 × 10⁻⁶. 8 CMV / mL, preferably at least 4 × 10⁻⁶ 8 CMV / mL.

[0060] The “geometric standard deviation” (GSD) typically provides a suitable value for characterizing the width of the size distribution in a particle swarm (in specific cases, aerated microbubbles). Therefore, a particle swarm with a wide size range will have a larger GSD value compared to a particle swarm with a narrow distribution of particle size around the mean (i.e., relatively similar sizes).

[0061] WO2020260420A1 (Figure 1) shows an example of a size distribution map (by volume) of an aerated microbubble cluster that can be obtained using a commercial particle analysis instrument, such as the Coulter Counter Multisizer 3 equipped with Multisizer 3 software.

[0062] Typically, the geometric standard deviation of a suspension of aerated microbubbles can be determined as follows:

[0063] i) Measure the number of calibrated aerated microbubbles, their individual diameters in volume mode, and their volume distribution within a selected size range (e.g., between 3µm and 6µm for a 4.5µm CMV average diameter) using a commercial particle analyzer instrument (such as the Coulter CounterMultisizer 3 equipped with Multisizer 3 software, with an incremental diameter of, for example, 0.1µm).

[0064] ii) Configure the particle analyzer instrument preferences to select the geometric statistics type (instead of the arithmetic statistics type for polydisperse suspensions);

[0065] iii) Calculate the GSD of the CMV distribution using a particle analysis instrument by applying the following Equation 1:

[0066] Equation 1

[0067] in:

[0068] n i = The percentage of gas trapped in the microbubble in the i-th channel (relative to the total volume).

[0069] x i = the volume of the microbubbles in the i-th channel, where

[0070] Equation 1.1

[0071] (d) i = Diameter of the microbubble in the center of the i-th channel)

[0072]

[0073] Equation 1.2

[0074] Among the various commercially available analytical instruments, the Coulter CounterMultisizer 3, equipped with Multisizer 3 software, can calculate and provide the GSD value as defined above.

[0075] For example, a GSD value of 1.2 indicates that approximately 50% of CMVs are calibrated between 2.5 and 5 µm for an average diameter of 4 µm; a GSD of 1.05–1.08 (<1.1) indicates that approximately 90%–95% of CMVs have dimensions between 2.5 and 5 µm.

[0076] As used in this article, “microbubble concentration” refers to the number of CMVs per unit volume determined using a Coulter Counter apparatus, i.e., the number of CMVs / mL.

[0077] Components of the shell of inflatable microbubbles

[0078] Phospholipids

[0079] As used herein, the term "one or more phospholipids" includes esters of glycerol having one or preferably two (identical or different) fatty acid residues and a phosphate residue, wherein the phosphate residue is further bound to a hydrophilic group such as choline (phosphatidylcholine-PC), serine (phosphatidylserine-PS), glycerol (phosphatidylglycerol-PG), ethanolamine (phosphatidylethanolamine-PE), or inositol (phosphatidylinositol). Esters of phospholipids having only one fatty acid residue are commonly referred to in the art as the "hemolytic" form of phospholipids or "lysophospholipids." The fatty acid residues present in phospholipids are typically long-chain fatty acids, typically containing 12-24 carbon atoms, preferably 14-22 carbon atoms; the fatty chain may contain one or more degrees of unsaturation or is preferably fully saturated. Examples of suitable fatty acids included in phospholipids are, for example, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, linoleic acid, and linolenic acid. Preferably, saturated fatty acids such as myristic acid, palmitic acid, stearic acid, and arachidic acid are used.

[0080] As used herein, the term phospholipids includes naturally occurring, semi-synthetic, or synthetically prepared products that can be used alone or as mixtures.

[0081] Examples of naturally occurring phospholipids are natural lecithin (phosphatidylcholine (PC) derivatives), such as soybean or egg yolk lecithin.

[0082] Examples of semi-synthetic phospholipids are partially or fully hydrogenated derivatives of naturally occurring lecithin. Preferred phospholipids are fatty acid diesters of phosphatidylcholine, phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), or sphingomyelin.

[0083] Examples of preferred phospholipids are, for example, dilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidylcholine (DMPC), dipalmitoyl-phosphatidylcholine (DPPC), distearatel-phosphatidylcholine (DSPC), arachidoyl-phosphatidylcholine (DAPC), 1,2-Dibenzyl-sn-glycerol-3-phosphatidylcholine (DBPC), dioleoyl-phosphatidylcholine (DOPC), octadecanoyl-phosphatidylcholine (DPDPC), 1-myristoyl-2-palmitoyl-phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl-phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl-phosphatidylcholine (SPPC), 1-palmitoyl-2-oleylphosphatidylcholine (POP) C) 1-oleyl-2-palmitoyl-phosphatidylcholine (OPPC), dilauroyl-phosphatidylglycerol (DLPG) and its alkali metal salts, diarachidoyl-phosphatidylglycerol (DAPG) and its alkali metal salts, dimyristoyl-phosphatidylglycerol (DMPG) and its alkali metal salts, dipalmitoyl-phosphatidylglycerol (DPPG) and its alkali metal salts, distearyl-phosphatidylglycerol (DSPG) and its alkali metal salts, dioleoyl-phosphatidylglycerol (DOPG) and its alkali metal salts, dimyristoyl-phosphatidic acid (DMPA) and its alkali metal salts. Dipalmitoylphosphatidic acid (DPPA) and its alkali metal salts, distearylphosphatidic acid (DSPA), arachidoylphosphatidic acid (DAPA) and its alkali metal salts, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearylphosphatidylethanolamine (DSPE), dioleylphosphatidylethanolamine (DOPE), diarachidoylphosphatidylethanolamine (DAPE), dilinoleylphosphatidylethanolamine (D... LPE, dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylserine (DPPS), distearyl phosphatidylserine (DSPS), arachidoyl phosphatidylserine (DAPS), dioleoyl phosphatidylserine (DOPS), dipalmitoyl sphingomyelin (DPSP), and distearyl sphingomyelin (DSSP), dilauroyl phosphatidylinositol (DLPI), diarachidoyl phosphatidylinositol (DAPI), dimyristoyl phosphatidylinositol (DMPI), dipalmitoyl phosphatidylinositol (DPPI), distearyl phosphatidylinositol (DSPI), and dioleoyl phosphatidylinositol (DOPI).

[0084] 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.

[0085] Mixtures of phospholipids may also be used, such as mixtures of DPPE and / or DSPE, DPPC, DSPC and / or DAPC with DSPS, DPPS, DSPA, DPPA, DSPG and DPPG.

[0086] Polyethylene glycol-modified phospholipids

[0087] As used herein, the expression “one or more polyethylene glycol-modified phospholipids” includes, within its meaning, any polyethylene glycol residue (“PEG”) covalently bonded to those phospholipid residues as described above.

[0088] Polyethylene glycols are typically identified by their average molecular weight (“AMW”, e.g., number-average molecular weight “Mn”); for example, as used herein, PEG2000 is identified as a polyethylene glycol with an AMW of about 2000 g / mol (typically ± 5%).

[0089] Suitable one or more polyethylene glycol phospholipids are those containing PEG residues having an average molecular weight of about 1000 g / mol (i.e., PEG1000) to about 8000 g / mol (i.e., PEG8000). Specific examples of PEG polymers available for forming polyethylene glycol phospholipids as defined above include PEG750, PEG1000, PEG2000, PEG3400, PEG4000, PEG5000, PEG6000, PEG7000, and PEG8000.

[0090] According to a preferred embodiment, the one or more polyethylene glycol phospholipids are those containing PEG residues having an average molecular weight of 1500-3000 g / mol, preferably the PEG residues having an average molecular weight of 2000 g / mol (i.e., PEG2000).

[0091] Preferably, PEG is covalently bound to phosphatidylethanolamine (“PE”) residues with the corresponding lipid chains, such as myristoyl, palmitoyl, or stearoyl.

[0092] Suitable examples of polyethylene glycol-modified phospholipids are, for example, DMPE-PEG, DPPE-PEG, and DSPE-PEG, which are generally commercially available as polyethylene glycol-modified phospholipids, wherein the PEG has the average molecular weight indicated above, for example as DMPE-PEG2000, DMPE-PEG3400, DMPE-PEG5000, DPPE-PEG2000, DPPE-PEG3400, DPPE-PEG5000, DSPE-PEG2000, DSPE-PEG3400, or DSPE-PEG5000.

[0093] Where necessary, the polyethylene glycol-modified phospholipid can be appropriately functionalized with reactive structural moieties, particularly reactive structural moieties capable of reacting with corresponding reactive structural moieties on functionalized ligands (e.g., biomolecule-binding structural moieties, such as avidin, neutral avidin, or streptavidin structural moieties). Suitable reactive structural moieties include, for example, biotin, NHS (N-hydroxysuccinimide), amino, thiol, maleimide, azide, or DBCO (dibenzocyclooctylene).

[0094] For example, if one of the two reacting components includes a reactive amino group, it can react with another component containing a suitable corresponding reactive structural 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 can be reduced to an alkylamine bond). Alternatively, if one of the two reacting components includes a reactive thiol group, a suitable complementary reactive structural moiety on the other component can include a haloacetyl derivative, a maleimide (to form a thioether bond), or a mixed disulfide containing a sulfide in the form of a 2-pyridylthio group, which, upon reaction with a thiol derived from the thiol-containing 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 carboxyl group, a suitable reactive structural moiety on the other component can be an amine and an acylhydrazine (to form an amide or N-acyl, N'-alkylhydrazine functional group).

[0095] Suitable examples of the PEGylated phospholipids may be maleimide-derived PEGylated phospholipids (e.g., PE-PEG2000-Mal), which are obtained by reacting with a ligand having a thiol (-SH) reactive moiety, which is introduced onto the ligand, for example, by reacting with Sulfo-LC-SPDP (sulfosuccinimide-6-(3'-(2-pyridyldithio)propamido)hexanoate); or biotin-derived PEGylated phospholipids (e.g., PE-PEG2000-Biot) that can react directly with ligands (e.g., avidin, neutral avidin, or streptavidin) due to their natural and irreversible affinity.

[0096] According to this specification and claims, the expression "first polyethylene glycol phospholipid comprising a reactive structural portion" means a compound comprising polyethylene glycol residues ("PEG") covalently bound to phospholipid residues as defined above, which is further functionalized with a reactive structural portion (e.g., DSPE-PEG2000-Biotin) capable of reacting with a corresponding reactive structural portion on a functionalized ligand (e.g., a biomolecular binding structural portion such as avidin, neutral avidin, or streptavidin structural portion).

[0097] According to this specification and claims, the expression "second polyethylene glycol phospholipid not containing a reactive structural portion" means a compound containing polyethylene glycol residues ("PEG") covalently bound to phospholipid residues as defined above, which is not further functionalized with a reactive structural portion and therefore cannot be coupled with ligands (e.g., DSPE-PEG2000).

[0098] ligands

[0099] The ligands that bind to polyethylene glycol phospholipids and are incorporated into the microbubble membrane are ligands that form specific "binding pairs" with other corresponding molecules.

[0100] The term "ligand" refers to a structural part that can specifically recognize and attach to a target biomolecule, thereby promoting an interaction that is crucial for a variety of therapeutic and diagnostic applications.

[0101] Specifically, the ligand is preferably a biomolecule binding structural moiety, where a biomolecule refers to any target molecule that can be specifically recognized and bound by these structural moieties. Target molecules include, but are not limited to, small molecules, peptides, proteins, and complex macromolecules that are present on or bound to the cell surface or within a biological system.

[0102] Examples of biomolecule binding structural portions (and corresponding binding pairs) include, for example, biotin-binding proteins such as avidin, neutral avidin, and streptavidin, which can form binding pairs with biotin to act as target biomolecules (e.g., biotinylated antibodies bound to cells). Streptavidin is preferred for this invention.

[0103] In one embodiment, the ligand can be bound to the microbubble shell in its natural form, i.e., to the reactive structural portion of the functionalized polyethylene glycol phospholipid. For example, biotinylated polyethylene glycol phospholipids (i.e., biotin-functionalized polyethylene glycol phospholipids (PE-PEG-Biot)) can be used, and ligands (e.g., streptavidin) can be directly bound thereto due to their natural affinity.

[0104] In alternative embodiments, the ligand may be chemically modified (i.e., appropriately derivatized) to introduce a reactive structural portion capable of covalently reacting with a corresponding reactive structural portion on the functionalized polyethylene glycol phospholipid. For example, when using maleimide-functionalized polyethylene glycol phospholipids (PE-PEG-Mal), the biomolecular binding structural portion (e.g., a peptide or antibody) may include a thiol portion to allow maleimide / thiol coupling. Alternatively, when using DBCO-functionalized lipids, the biomolecular binding structural portion (e.g., a peptide or antibody) may include an azide structural portion to allow click chemical coupling.

[0105] inner core

[0106] Suitable gases include biocompatible fluorinated gases, preferably perfluorinated gases. Fluorinated gases include materials containing at least one fluorine atom, such as 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 SF6, or perfluorinated carbons (perfluorinated hydrocarbons), i.e., hydrocarbons in which all hydrogen atoms are replaced by fluorine atoms, which are known to form particularly stable aerated microbubble suspensions.

[0107] The term "perfluorinated carbon" includes saturated, unsaturated, and cyclic perfluorinated carbons. Examples of biocompatible and physiologically acceptable perfluorinated carbons are: perfluoroalkanes such as perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane (e.g., perfluoron-butane, optionally mixed with other isomers such as perfluoroisobutane), perfluoropentane, perfluorohexane, or perfluoroheptane; perfluoroolefins such as perfluoropropylene, perfluorobutene (e.g., perfluorobut-2-ene), or perfluorobutadiene; perfluoroalkynes (e.g., perfluorobut-2-yne); and perfluorocycloalkanes (e.g., perfluorocyclobutane, perfluoromethylcyclobutane, perfluorodimethylcyclobutane, perfluorotrimethylcyclobutane, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclopentane, perfluorocyclohexane, perfluoromethylcyclohexane, and perfluorocycloheptane). Preferred saturated perfluorinated carbons include, for example, CF4, C2F6, C3F8, C4F8, and C4F6. 10 C5F 12 and C6F 14 .

[0108] In another embodiment, the gaseous fluorinated compound is a perfluoroolefin, selected from C4-C6 perfluoroolefins, preferably C4-C5, and more preferably C5 perfluoroolefins. Specific examples include perfluoro-2-butene, perfluoro-1-pentene, perfluoro-2-pentene, or mixtures thereof. More preferably, the perfluoroolefin is perfluoro-2-pentene.

[0109] Particularly preferred gases are those that are in gaseous form at room temperature, including SF6, C3F8, and C4F. 10 .

[0110] Aqueous suspension of aerated microbubbles

[0111] This invention provides a novel composition of monodisperse aerated microbubbles suitable for cell sorting applications, characterized by enhanced stability and improved cell sorting efficiency compared to existing polydisperse compositions.

[0112] As observed by the applicant, it is advantageous according to the invention to obtain a suspension of calibrated aerated microbubbles stabilized by a mixture of a first polyethylene glycol phospholipid containing a reactive structural portion and a second polyethylene glycol phospholipid not having said reactive structural portion, wherein the size distribution of said calibrated microbubbles is typically characterized by a geometric standard deviation (GSD) value of at least 1.20 or lower, preferably at least 1.15, for example as low as 1.05.

[0113] In one aspect of the invention, the disclosed calibrated inflatable microbubble shell comprises:

[0114] -phospholipids,

[0115] - Less than 4 mol% of a first polyethylene glycol-modified phospholipid containing a reactive structural portion, at least a portion of the first polyethylene glycol-modified phospholipid binding to a ligand via the reactive structural portion, and

[0116] - At least 15 mol% of a second polyethylene glycol-modified phospholipid that does not contain reactive structural moieties.

[0117] Typically, phospholipids constitute a significant portion of the outer layer of the calibrated aerated microbubble component, for example, up to 98% mol / mol. In some embodiments, the molar amount of phospholipids can range from 60% to 95%, preferably from 70% to 90%.

[0118] In addition, the outer layer of the inflatable microbubbles may further contain lipids, preferably fatty acids such as palmitic acid, stearic acid, arachidonic acid or oleic acid.

[0119] Optional lipids (especially fatty acids) may be present in, for example, 10% to 30%, more preferably 15% to 25% in molar amounts.

[0120] According to one embodiment, the molar amount of the first polyethylene glycol phospholipid containing the ligand is less than 4%, preferably less than 3%, more preferably less than 2%, even more preferably less than 1%, even more preferably less than 0.5%, and as low as 0.25%.

[0121] According to another embodiment, the molar amount of the second polyethylene glycol phospholipid, which does not contain the reactive structural portion, is at least 15%, preferably at least 16% or higher, even more preferably at least 17% or higher, even more preferably at least 18% or higher, even more preferably at least 19% or higher, even more preferably at least 20% or higher, and at most 21%.

[0122] According to another embodiment, the total molar amount of polyethylene glycol phospholipid is preferably at least 16%, more preferably at least 18%, at most 22%, and most preferably at most 20%.

[0123] The expression "total amount of polyethylene glycol phospholipids" refers to the sum (e.g., mol%) of the molar amount of the first polyethylene glycol phospholipid containing ligands and the second polyethylene glycol phospholipid not containing reactive structural moieties.

[0124] For example, the total amount of polyethylene glycol phospholipids contained in the shell of the calibrated inflatable microbubble is 19%, which includes 1% of a first polyethylene glycol phospholipid containing ligands and 18% of a second polyethylene glycol phospholipid.

[0125] The first and second polyethylene glycol phospholipids (“PE-PEG”) (which may be the same or different) are phospholipids covalently linked to polyethylene glycol (PEG) having an average molecular weight of 1000-8000 g / mol, preferably 1500-3000 g / mol, and preferably the PEG residues having an average molecular weight of 2000 g / mol (i.e., PEG2000).

[0126] In a preferred embodiment, the first polyethylene glycol phospholipid containing the reactive structural portion is a phospholipid covalently linked to polyethylene glycol (PEG) with an average molecular weight of 1500-3000 g / mol.

[0127] In a further preferred embodiment, the second polyethylene glycol phospholipid that does not contain a reactive structural portion is a phospholipid covalently linked to polyethylene glycol (PEG) with an average molecular weight of 1000-8000 g / mol.

[0128] According to a preferred embodiment of the invention, the polyethylene glycol phospholipid containing the reactive structural portion is a functionalized PE-PEG2000 (e.g., DSPE-PEG2000-Biot or DSPE-PEG2000-Mal) and the second polyethylene glycol phospholipid is PE-PEG2000 (e.g., DSPE-PEG2000).

[0129] Using existing methods to fabricate functionalized aerated microbubbles using stabilizing materials in quantities disclosed in the prior art presents significant challenges, primarily due to inherent instabilities that affect newly formed microbubbles, leading to the aggregation of monodisperse bubbles and consequently reducing monodispersity.

[0130] The applicant unexpectedly observed that by appropriately tuning the corresponding amount of polyethylene glycol-modified phospholipid, the stability of the calibrated microbubbles was significantly improved by limiting agglomeration at the end of microbubble preparation (e.g., when collected from the outlet channel of the microfluidic device or within a few hours - e.g., 2 hours from collection).

[0131] The term coalescence refers to the process by which two or more particles, such as microbubbles, merge during contact to form a single, larger particle.

[0132] The percentage of aggregates can be determined by calculating the total number of aggregated microparticles from the peaks of the size distribution in an image obtained using an optical microscope at the outlet channel of a flow focusing device. For example, it can be determined as follows:

[0133] - Multiply the total number of particles whose volume is twice the initial particle volume Vi (the second peak of the size distribution) by a factor of two (because aggregated particles originate from two particles); and

[0134] Add this number to the number obtained by multiplying the total number of particles in three times the initial volume (the third peak of the size distribution) by a factor of three, and so on, until the nth peak in the measured size distribution.

[0135] The percentage of coalescence can therefore be calculated by normalizing the total number of coalesced particles to the total number of particles produced.

[0136] Equation 2

[0137] Typically, a coalescence percentage of about 1% or less is desirable, such as a coalescence percentage as low as 0.01%.

[0138] As the applicant has discovered, such undesirable agglomeration can be significantly reduced by preparing calibrated aerated microbubbles stabilized by 15% or more of a second PE-PEG.

[0139] The improvement in terms of reducing agglomeration is achieved by an outer layer of stable aerated microbubbles containing 15 mol% or more of a second polyethylene glycol phospholipid (i.e., without reactive structural moieties) and at least 1 mol% of a first polyethylene glycol phospholipid with reactive structural moieties.

[0140] A significant reduction in agglomeration can be achieved by preparing calibrated aerated microbubbles with an outer layer that is stable by comprising, preferably at least 16%, more preferably at least 18%, at most 22%, and preferably at most 20% of a total amount (mol%) of polyethylene glycol-modified phospholipids.

[0141] Furthermore, the applicant has discovered that the calibrated aerated microbubbles of this disclosure can be advantageously used in methods for sorting biological materials (such as cells) with higher efficiency compared to standard polydisperse microbubbles, due to their larger and more controllable size.

[0142] The efficiency of cell separation methods can be determined by various parameters, such as cell recovery rate. "Cell recovery rate" measures the effectiveness of cell separation assays and refers to the ratio of the desired cells separated during the separation process to the number of desired cells available in the starting sample.

[0143] This parameter is typically recorded as "cell recovery percentage," which describes the percentage of cells obtained after sorting compared to the total number of cells or target cells in the original suspension. A high cell recovery percentage, such as 80% or higher, indicates effective cell separation.

[0144] According to the invention, a cell recovery percentage of 80%, preferably 85%, more preferably 90%, and even more preferably 95% is desirable, up to about 100% (e.g., 99%).

[0145] Surprisingly, it was observed that cell recovery (%) could be significantly increased by using calibrated aerated microbubbles stabilized by a mixture of polyethylene glycol-modified phospholipids in specific molar amounts.

[0146] As the applicant has discovered, the efficiency of the cell separation process can be significantly improved by stabilizing calibrated microbubbles with a lower molar amount of ligand-containing first polyethylene glycol phospholipid. In fact, in the presence of an equimolar amount of second polyethylene glycol phospholipid, compared with formulations having a higher molar amount (e.g., more than 4%) of ligand-containing first polyethylene glycol phospholipid, an increased cell recovery rate (e.g., approximately 15-fold higher) was observed when using an aerated microbubble formulation stabilized by less than 4% of ligand-containing first polyethylene glycol phospholipid.

[0147] Furthermore, the applicant observed that a relatively high percentage of cell recovery (e.g., about 90%) was maintained by significantly reducing the amount of calibrated gas-microbubbles / cells (CMV / cell) in the mixture undergoing separation (e.g., as low as 3 CMV / cell).

[0148] According to the present invention, the amount of ligand is preferably added in a molar ratio of 1:2 to 2:1 relative to the amount of polyethylene glycol-modified phospholipid having the corresponding reactive structural moiety. More preferably, the molar ratio is 1:1.

[0149] The microbubbles according to the present invention typically have at least 1500 molecules / µm 2 Preferably at least 1700 molecules / µm 2 More preferably at least 1900 molecules / µm 2 And even more preferably, at least 2200 molecules / µm 2 For example, up to 3000 molecules / µm 2 ligand density.

[0150] The relative molar ratio between the first polyethylene glycol phospholipid containing the reactive structural portion and the second polyethylene glycol phospholipid not containing the reactive structural portion is preferably 1:5 to 1:40, more preferably 1:7.5 to 1:30, and even more preferably 1:9 to 1:20.

[0151] According to another embodiment of the present invention, the first polyethylene glycol phospholipid containing the reactive structural portion is a functionalized PE-PEG2000, such as DSPE-PEG2000-Biotin or DSPE-PEG2000-Maleimide, while the second polyethylene glycol phospholipid is PE-PEG2000, such as DSPE-PEG2000.

[0152] As observed by the applicant, the amount of polyethylene glycol phospholipid and the molecular weight of the PEG chains contained in the polyethylene glycol phospholipid can be correlated in a manner that allows for appropriate selection of both in order to provide a desired suspension of microbubbles.

[0153] The correlation can be advantageously expressed by a number defined herein as “NPEG”:

[0154] NPEG = MW1 mol%1 + MW2 mol%2

[0155] MW1 and mol%1 refer to the molecular weight and mol% of the PEG chain contained in the polyethylene glycol phospholipid containing the reactive structural part, respectively, while MW2 and mol%2 refer to the molecular weight and mol% of the PEG chain contained in the polyethylene glycol phospholipid without the reactive structural part, respectively.

[0156] For example, if the composition contains 2.5% functionalized PE-PEG2000 and 1% PE-PEG5000, then the NPEG number is:

[0157] NPEG = 2000 0.025 + 5000 0.01 = 100

[0158] As observed by the applicant, in order to obtain a stable formulation (e.g., characterized by substantially reduced aggregation), the number N should preferably be 315-450. Preferably, MW1 is similar to MW2, more preferably both are about 2000 (± 5%). Preferably, mol%1 is less than 0.04. Preferably, mol%2 is at least 0.15.

[0159] According to embodiments of the present invention, the polyethylene glycol-modified phospholipid comprising the reactive structural portion is a functionalized PE-PEG2000 (e.g., DSPE-PEG2000-Mal), and the second polyethylene glycol-modified phospholipid is PE-PEG2000 (e.g., DSPE-PE2000). The relative molar ratio between the two corresponding PE-PEGs is preferably 1:1 to 1:8, more preferably 1:1.5 to 1:5, and even more preferably 1:2 to 1:4.

[0160] Preparation method

[0161] Another aspect of the present invention relates to a method for preparing a suspension of calibrated aerated microbubbles having ligands as defined above, the method comprising:

[0162] a) Prepare an aqueous suspension of calibrated aerated microbubbles comprising a core and an outer layer, wherein the core comprises a physiologically acceptable gas and the outer layer comprises:

[0163] - Phospholipids;

[0164] - Less than 4 mol% of the first polyethylene glycol-modified phospholipid containing the reactive structural moiety, and

[0165] - At least 15 mol% of a second polyethylene glycol-modified phospholipid that does not contain reactive structural moieties.

[0166] The calibrated suspension of aerated microbubbles has a geometric standard deviation (GSD) of less than 1.2.

[0167] b) Adding a ligand capable of reacting with the said reactive structural moiety;

[0168] c) Couple the first polyethylene glycol-modified phospholipid to the ligand;

[0169] d) Collect a suspension of calibrated aerated microbubbles with ligands, wherein the suspension has a geometric standard deviation (GSD) of less than 1.2.

[0170] Preferably, the microbubbles of the present invention can be advantageously prepared by microfluidic technology according to the manufacturing methods disclosed in WO2018041906A1 and WO2019170606A1 (incorporated by reference herein).

[0171] Therefore, another aspect of the present invention relates to a method for preparing a suspension of calibrated aerated microbubbles having ligands as defined above, wherein the suspension of calibrated aerated microbubbles has a geometric standard deviation (GSD) of less than 1.2, the method comprising:

[0172] A. Provide (i) a gas stream and (ii) an aqueous liquid stream, the aqueous liquid stream comprising:

[0173] - Phospholipids;

[0174] - Less than 4 mol% of the first polyethylene glycol-modified phospholipid containing the reactive structural moiety, and

[0175] - At least 15 mol% of a second polyethylene glycol-modified phospholipid that does not contain reactive structural moieties;

[0176] B. Guide the gas flow and the liquid flow toward the contact area through their respective inlet channels;

[0177] C. Guide the gas flow and the liquid flow from the contact area through the calibration orifice to obtain an aqueous suspension containing the aerated microbubbles;

[0178] D. Collect the suspension containing the microbubbles from the outlet channel;

[0179] E. Add a functionalized ligand capable of reacting with the reactive structural portion to the suspension;

[0180] F. Couple the first polyethylene glycol-modified phospholipid to the ligand, and

[0181] G. Collect a suspension of calibrated aerated microbubbles with ligands, wherein the suspension has a geometric standard deviation (GSD) of less than 1.2.

[0182] In step a) or step A), the suitable phospholipid, polyethylene glycol phospholipid, reactive structural moiety, and ligand can be any of those previously listed.

[0183] In addition, the appropriate molar amounts of each component are those mentioned above.

[0184] After obtaining a suspension of calibrated aerated microbubbles, such as after step a) or step D) (i.e. after collecting it from the microfluidic device), it is highly recommended to treat the suspension with a suitable washing technique to remove unassembled amphiphilic materials and possible additive compounds.

[0185] According to one embodiment, the method for preparing a suspension of calibrated aerated microbubbles with ligands includes an optional step (D'): washing the obtained suspension of calibrated aerated microbubbles.

[0186] For example, the optional step may be performed after step a) or after step D) of collecting the aqueous suspension of calibrated aerated microbubbles from the outlet channel of the microfluidic device.

[0187] In this specification, the term "washing" refers to any operation performed on a freshly prepared microbubble suspension with the ultimate goal of removing (or substantially reducing) unassembled amphiphilic materials and additive compounds.

[0188] According to this specification, suitable washing techniques include centrifugation, filtration, bubble separation, and decantation, with centrifugation being preferred.

[0189] In this specification, the term "unassembled amphiphilic material" refers to amphiphilic molecules that are present in a calibrated microbubble suspension at the end of the preparation process but do not form a stable layer of aerated microbubbles.

[0190] Examples of amphiphilic materials are phospholipids and polyethylene glycol-modified phospholipids, which are used to stabilize the shells of calibrated inflatable microbubbles.

[0191] In this specification, "additive compound" refers to any possible substance that can be added to the suspension during the microbubble preparation process, such as tension modifiers like salts or sugars, sugar alcohols, glycols, or other nonionic polyol materials (e.g., glucose, sucrose, sorbitol, mannitol, glycerol, polyethylene glycol, propylene glycol, etc.), chitosan derivatives such as carboxymethyl chitosan, trimethyl chitosan, or gelling compounds such as carboxymethyl cellulose, hydroxyethyl starch, or dextran. For example, the additive compound can be added to the suspension of aerated microbubbles after an optional washing following a coupling reaction between the ligand and the reactive structural portion bound to the polyethylene glycol-modified phospholipid.

[0192] Therefore, at the end of the optional washing step D'), the calibrated aqueous suspension of aerated microbubbles is a composition substantially free of "unassembled amphiphilic material," indicating that all (or most) of the amphiphilic material of the present invention participates in the formation of the microbubble shell. In other words, the washed aqueous suspension of aerated microbubbles contains substantially no free amphiphilic substances in the suspension.

[0193] The term "washed / washed" means after being treated with appropriate washing techniques.

[0194] At the end of the preparation of the calibrated suspension of aerated microbubbles, for example after collecting the suspension from the microfluidic device and / or washing the obtained suspension with a suitable washing technique, a functionalized ligand capable of reacting with the reactive structural portion attached to the first polyethylene glycol phospholipid is added to the suspension.

[0195] According to the present invention, the ligand is preferably added in a molar ratio of 1:2 to 2:1 relative to the amount of polyethylene glycol-modified phospholipid having the corresponding reactive structural moiety. More preferably, the molar ratio is 1:1.

[0196] At the end of the coupling reaction between the ligand and the reactive structural portion attached to the first polyethylene glycol phospholipid, the resulting suspension of calibrated aerated microbubbles with the ligand can be advantageously further washed as described above in order to remove unassembled ligands (or substantially reduce their amount).

[0197] According to one embodiment, after the coupling step, the method includes an optional step F'): washing the obtained suspension of calibrated aerated microbubbles with ligands.

[0198] Therefore, at the end of this additional optional washing step, the aqueous suspension of calibrated aerated microbubbles is a composition substantially free of "unassembled ligands," indicating that all (or most) of the ligand results of the present invention are incorporated into the final stabilized coating of the microbubbles. In other words, the washed aqueous suspension of calibrated aerated microbubbles with ligands substantially does not contain free ligands.

[0199] At the end of the coupling procedure (e.g., after the coupling reaction between the added ligand and the reactive structural portion introduced into the microbubble shell and / or after a further washing procedure following the coupling), the aqueous suspension of the calibrated aerated microbubbles with the ligand may contain unreacted structural portions on PE-PEG, depending on the molar ratio between the ligand and the first polyethylene glycol-modified phospholipid with the reactive structural portion.

[0200] For example, when the molar amount of the added ligand is less than the molar amount of the polyethylene glycol phospholipid to which the ligand must be coupled (e.g., when the molar ratio between the ligand and the polyethylene glycol phospholipid with the corresponding reactive structural portion is less than 1), the final aqueous suspension of the calibrated aerated microbubbles with the ligand contains unreacted reactive structural portions bound to the polyethylene glycol phospholipid in the shell. Therefore, in this case, the stabilizing layer of the microbubble comprises unreacted polyethylene glycol phospholipid and polyethylene glycol phospholipid covalently bonded to the ligand.

[0201] In this specification and claims, the statement "at least a portion of the first polyethylene glycol phospholipid is bound to the ligand via the reactive structural portion" means that at least a portion of the total amount of the reactive structural portion bound to the stable layer of the formulation of this disclosure is bound to the ligand, while the remaining portion of the total amount is not bound to the ligand and remains in an "unreacted form".

[0202] In this specification and claims, the term "unreacted" means not coupled to a ligand.

[0203] For example, considering a polyethylene glycol-modified phospholipid (PE-PEG-Biot) with biotin as a reactive structural moiety, its unreacted form corresponds to the compound PE-PEG-Biot, where biotin does not bind to any ligand such as streptavidin (STV). In this embodiment, the reactive form (or coupled form) corresponds to PE-PEG-Biot-STV, where the reactive structural moiety of biotin is coupled to the ligand streptavidin.

[0204] Therefore, in this invention, the outer layer of the calibrated inflatable microbubble of this disclosure is formed by at least a portion of the reacted polyethylene glycol phospholipid with reactive structural portions and the remaining portion of the unreacted polyethylene glycol phospholipid with reactive structural portions.

[0205] The unreacted reactive structural portion on the polyethylene glycol-modified phospholipid can then be "deactivated" by reacting it with a suitable corresponding deactivating structural portion. For example, if the reactive structural portion on the polyethylene glycol-modified phospholipid is maleimide (PE-PEG-Mal), it can be deactivated by reacting it with cysteine. Alternatively, the reactive structural portion can undergo a natural deactivation process, such as hydrolysis, without the need for the addition of a specific deactivating structural portion.

[0206] freeze-dried products

[0207] Advantageously, the calibrated suspension of aerated microbubbles of this disclosure can be freeze-dried, for example as described in WO2020260420A1 and WO2020260423A1, which are incorporated herein by reference.

[0208] One aspect of the present invention relates to a lyophilized composition comprising a phospholipid, a first polyethylene glycol phospholipid containing a reactive structural portion, a second polyethylene glycol phospholipid not containing a reactive structural portion, and a lyophilization protective component, wherein the lyophilized composition, after being reconstituted / reconstructed with a pharmaceutically acceptable solution in the presence of a biocompatible gas, provides a calibrated suspension of aerated microbubbles, wherein the reconstituted / reconstructed suspension of microbubbles has a geometric standard deviation (GSD) value of at least 1.22 or lower.

[0209] The number, size, and size distribution of microbubbles obtained after reconstitution / reconstruction are essentially equivalent to those of microbubbles in the prepared emulsion.

[0210] Suitable examples of freeze-drying protective components are polymers, preferably hydrophilic polymers, more preferably polyglycols, and even more preferably polyethylene glycol (PEG).

[0211] Alternatively, the freeze-drying protective component is a mixture of a polymer, preferably a hydrophilic polymer, more preferably a polydiol such as PEG, and a polyol or sugar such as sorbitol and sucrose.

[0212] Another aspect of the invention relates to a method for manufacturing a lyophilized precursor for preparing a suspension of calibrated aerated microbubbles as defined above, the method comprising:

[0213] i. Preparing a first suspension of calibrated aerated microbubbles, the suspension comprising:

[0214] - Phospholipids;

[0215] - Less than 4 mol% of the first polyethylene glycol-modified phospholipid containing the reactive structural moiety, and

[0216] - At least 15 mol% of a second polyethylene glycol-modified phospholipid that does not contain reactive structural moieties; and

[0217] - Freeze-dried protective components;

[0218] ii. Add a ligand capable of reacting with the reactive structural portion to the first suspension;

[0219] iii. Couple the polyethylene glycol-modified phospholipid to the ligand;

[0220] iv. Freeze-dry the emulsion to obtain a freeze-dried residue.

[0221] Preferably, in step i), inflatable microbubbles are prepared by using microfluidic flow focusing technology.

[0222] Suitable phospholipids are those mentioned above. Furthermore, the first suspension of the aerated, calibrated microbubbles may also contain lipids, preferably fatty acids such as palmitic acid, stearic acid, arachidonic acid, or oleic acid.

[0223] Functionalized polyethylene glycol phospholipids can be any polyethylene glycol phospholipids that are appropriately functionalized as previously listed and discussed.

[0224] Similarly, the functionalized ligands to be bound to the functionalized polyethylene glycol phospholipids can be selected from those previously listed.

[0225] Preferably, the freeze-drying protective component comprises a polymer, preferably a hydrophilic polymer, more preferably a polyethylene glycol, and even more preferably polyethylene glycol (PEG).

[0226] Alternatively, the freeze-dried protective component may also contain polyols or sugars, such as sorbitol and sucrose.

[0227] How to use

[0228] One aspect of the invention relates to the use of the aerated microbubbles of this disclosure in biotechnological applications. Specifically, they can be advantageously used in methods for separating cells within the context of cell therapy manufacturing (typically via buoyancy (also known as buoyancy-activated cell sorting, "BACS")). This method can be used to separate cells of a desired type from other cells in physiological fluids (e.g., blood or plasma). In particular, the separation method involves labeling the desired cells to be separated with a suitable labeled antibody capable of binding to specific (and selective) receptors on the cells. The microbubbles of the invention are then added to a suspension of cells to be separated (including those with labeled antibodies); the microbubbles of the invention then associate with labeled residues bound to the antibody / cell construct via ligands, thereby allowing cell separation by buoyancy (see, for example, WO2017117349). In a preferred embodiment, the labeled antibody is a biotinylated antibody, wherein biotin residues are capable of associating with corresponding structural moieties, such as avidin, neutral avidin, or streptavidin residues on calibrated aerated microbubbles. Therefore, the microbubbles of the present invention can be used to separate large numbers of cells from physiological fluids, provided that such cells can be appropriately labeled with corresponding marker (biotinylated) antibodies.

[0229] Particularly preferred are formulations comprising calibrated aerated microbubbles containing a first polyethylene glycol phospholipid as a functionalized PE-PEG2000 (e.g., DSPE-PEG2000-Biot or DSPE-PEG2000-Mal) and a second polyethylene glycol phospholipid as a PE-PEG2000 (e.g., DSPE-PE2000). Preferably, the first polyethylene glycol phospholipid is present in a molar amount of less than 4%, more preferably less than 3%, even more preferably less than 2%, even more preferably less than 1%, and as low as 0.25%; the second polyethylene glycol phospholipid is preferably present in a molar amount of 15% or higher, more preferably 16% or higher, even more preferably 17% or higher, even more preferably 18% or higher, even more preferably 19% or higher, even more preferably 20% or higher, and at most 21%.

[0230] The following examples will help to further illustrate the present invention.

[0231] Example

[0232] Material:

[0233]

[0234] method

[0235] Microbubble size and concentration:

[0236] Size distribution and microbubble concentration were measured using a Coulter Counter Multisizer 3 (Beckman Coulter, Fullerton, CA) with a 30 μm diameter tube (allowing a measurable size range of 0.7–18 μm). Fifty (50) μL of the bubble suspension was diluted in 100 mL of 0.9% saline (analytical volume = 100 μL), and microbubble parameters were measured over a 30-second time interval. Background noise was measured prior to each microbubble measurement.

[0237] Dv mode refers to the diameter at the peak of the microbubble distribution in volumetric terms. CMV concentration represents the number of calibrated microbubbles in the sample volume. MVC represents the average volume concentration or volume of microbubbles in the sample volume.

[0238] The "geometric standard deviation" (GSD) of the test formulation is determined according to Equation 1 as described above.

[0239] coalescence measurement

[0240] The percentage of aggregates can be determined by calculating the total number of aggregated particles from the peaks of the size distribution in an image obtained using an optical microscope at the exit channel of a flow focusing device. For example, it can be determined as follows:

[0241] - Multiply the total number of particles whose volume is twice the initial particle volume Vi (the second peak of the size distribution) by a factor of two (because aggregated particles originate from two particles); and

[0242] - Add this number to the total number of particles with three times the initial volume (the third peak of the size distribution) multiplied by a factor of three, and so on, until the nth peak in the measured size distribution.

[0243] The percentage of coalescence can therefore be calculated by normalizing the total number of coalesced particles to the total number of particles produced:

[0244] Equation 2

[0245] Typically, a coalescence percentage of about 1 or less is desirable, for example, a coalescence percentage as low as 0.01%.

[0246] Streptavidin concentration determination:

[0247] Streptavidin (STV) content was determined in washed microbubble suspensions using 4-biotin-fluorescein assay.

[0248] Briefly, after washing the microbubbles and performing Coulter measurements, they were ruptured in an ultrasonic jar (Branson 5200 - 3 × 2 min) until a clear solution was obtained. This solution was then sampled in eight 5 mL glass tubes at 100 μL aliquots. For each sample, the appropriate volume of PBS and the appropriate volume of 4-biotin-fluorescein (4-BF) solution (3050 picomoles / mL) were calculated to be 0.1, 0.2, 0.4, 0.75, 1.5, 2, 2.5, and 3 times the theoretical biotin capacity. PBS was added to the sample solution, followed by the corresponding 4-BF solution.

[0249] The solutions were then mixed (vortexed) and incubated in the dark at room temperature for 30 min. Each mixture was then sampled into 96-well plates (100 μL / well and 2 wells per condition), and fluorescence was read using a Cytation 5 reader {Xexc 480 nm - Xem 525 nm}. Second-order polynomial fitting was used for the four first points (low 4-BF), and linear fitting was used for the four last points (high 4-BF), plotting fluorescence as a function of 4-BF concentration—intersection was determined between the two curves, and STV concentration was determined using a standard curve (from a free streptavidin solution).

[0250] The STV density on the microbubbles (in molecules / µm) is calculated by dividing the STV amount determined above by the total CMV surface area determined by Coulter counter. 2 (unit)

[0251] Streptavidin conjugation yield determination:

[0252] The coupling yield is calculated as follows: divide the streptavidin density measured for the washed microbubble suspension by the streptavidin density measured for the natural microbubble suspension (because a certain percentage of microbubbles can be removed during washing).

[0253] Cell recovery test:

[0254] Perform cell recovery testing as described in WO2020 / 127816.

[0255] That is, first, CCL119 (CCRF-CEM cells) or MCF-7 cells (from ATTC) are cultured and expanded according to the protocol provided by the supplier. Just before testing, the cells are cultured at a ratio depending on the cell type (i.e., 5 × 10⁶ for CCL119). 6 10 cells / mL and for MCF-7 cells 1×10 6The concentration of cells / mL was resuspended in BSA / EDTA buffer (1% BSA and 2 mM EDTA in PBS, w / o Ca / Mg).

[0256] Prepare the cell suspension (1 mL, approximately 5 × 10⁻⁶ cells / mL). 6 Transfer 100 cells to 2 mL of low-binding Eppendorf solution and add 160 µL of biotinylated mouse anti-human CD45 antibody (#555481 - BD Pharmigen) to these cells. Incubate the mixture at room temperature on a rotary mixer for 30 min, then wash the cells by centrifugation (400 g / 5 min); discard the supernatant and resuspend the cells in 1 mL of BSA / EDTA buffer (mix on a rotary mixer for 5 min).

[0257] The microbubble suspension (CMV volume depending on the CMV / cell ratio, e.g., 30 CMV / cell to 3 CMV / cell) was then added to the cell suspension, and the mixture was incubated at room temperature on a rotary mixer for 20 minutes. The mixture was then centrifuged (400 g / 5 min), and the supernatant (cell / microbubble complex) was recovered by manual pipetting along the meniscus of the liquid.

[0258] The inflatable microbubbles are then ruptured (by applying positive pressure), and cells are counted in both the supernatant and substrate portions using a hemocytometer.

[0259] The cell recovery rate is determined as follows:

[0260] Equation 3

[0261] The test is considered valid only when cell balance is between 90% and 110%, and cell balance is determined by the following equation:

[0262] Equation 4

[0263] Example 1

[0264] Preparation of aqueous dispersions of amphiphilic materials

[0265] A dispersion of the amphiphilic material was prepared according to WO2018041906 (Example 1). The material was added to a 2:1 (volume ratio) chloroform / methanol mixture at a concentration of 20 mg / mL in the molar ratios shown in Tables 1a, 1b, and 1c, and stirred at 60°C until the amphiphilic material was completely dissolved. The solvent was then evaporated under reduced pressure, and the resulting membrane was dried under reduced pressure overnight. The dried material was then redispersed (at a concentration of 15 mg / mL, as detailed in the "Preparation of Microbubbles" section) in brine or phosphate-bubble buffered brine (pH 6.4, for CMV-maleimide) and stirred at 60°C for 30 minutes. The dispersion was then sonicated using a Branson Sonifier 250 to homogenize the material. The formulation was then filtered using a polycarbonate filter (0.45 μm pore size), cooled to room temperature, and degassed.

[0266] The specific types and quantities of amphiphilic materials used are summarized in Tables 1a, 1b, and 1c.

[0267] Table 1a Aqueous dispersions containing DSPC:DSPE-PEG2000:DSPE-PEG2000-Biotin (molar amount (%))

[0268]

[0269] Table 1b shows the aqueous dispersions containing DSPC:DSPE-PEG2000:DSPE-PEG2000-maleimide (molar amounts (%)).

[0270]

[0271] Table 1c contains aqueous dispersions of polyethylene glycol-modified phospholipids with different average molecular weights.

[0272]

[0273] Example 2

[0274] Preparation of calibrated aerated microbubbles functionalized with STV

[0275] 2.1 Preparation of calibrated aerated microbubbles in natural suspension

[0276] The amphiphilic lipid dispersions listed above were used to prepare calibrated aerated microbubbles characterized by various amounts of polyethylene glycol-modified phospholipids.

[0277] Calibrated gas-filled microbubbles were synthesized according to WO2018041906 (Example 2) using a commercially available microfluidic flow focusing device (Dolomite Microfluidics, small droplet chip, 14 μM etch depth, part number 3200136) mounted in a commercially available chip holder (Dolomite Microfluidics, part numbers: 3000024, 3000109, 3000021) that allows leakage tight connection between the chip and the gas and liquid supply tube (Peek Upchurch, 1 / 16 inch OD, 150 μm I.D.). The microbubble formation channels had a width of 17 μm and a length of 135 μm. The total channel depth was 14 μm. The chip and its holder were placed in an optically transparent, temperature-controlled water bath mounted on an inverted microscope equipped with a 20x magnification objective (Olympus, LMPLAN20×) and a CCD camera (Lumenera, LM156M). The liquid co-flow rate was controlled using a syringe pump (Harvard PHD4400). Gas (N2) pressure was controlled using a pressure regulator (Omega, PRG101-25) connected to a pressure sensor (Omega, DPG1000B-30G). Individual microbubbles were automatically detected from recorded optical images using Matlab software (The Mathworks Inc., Natick, MA) to measure their size offline on a PC. The liquid co-flow rate was set to 120 µL / min, up to a maximum of 180 µL / min. Generally, setting a low liquid flow rate allows for obtaining CMVs with larger sizes. The suspension of natural microbubbles was then collected in a container filled with C4F. 10 (100%) in a sealed vial.

[0278] Table 2a records the characterization of the natural suspension of CMV obtained as described above. The term "natural suspension" refers to a suspension of calibrated aerated microbubbles containing phospholipids, a first polyethylene glycol-modified phospholipid containing reactive structural moieties, and a second polyethylene glycol-modified phospholipid, wherein the reactive structural moieties are not bound to ligands (e.g., aerated microbubbles with reactive structural moieties not yet functionalized with streptavidin). In the natural suspension, the total amount of reactive structural moieties bound to the stable layer of the aerated microbubbles is in the form of unreactive structural moieties. Typically, the natural suspension has not undergone a washing process. Characterization was performed using the Coulter counting scheme described in the previous Methods section.

[0279] Table 2a Natural suspension after its preparation

[0280]

[0281] 2.2. Functionalization of calibrated microbubbles containing PE-PEG-biotin using STV

[0282] Following collection, a suspension of calibrated microbubbles containing PE-PEG-Biotin (CMV-BIOT) was washed twice by centrifugation with 0.9% saline (6' / 600 RPM). Streptavidin (STV) was added to 1 mL of washed CMV-BIOT at the desired STV / Biotin molar ratio, and the mixture was then incubated on a spin wheel at room temperature for 45 min. CMV-BIOT-STV was then washed twice more by centrifugation (6' / 600 RPM) and resuspended in 0.9% saline. Coulter counts were performed to determine if the added CMV volume reached 30 CMV / cell.

[0283] 2.3. STV-functionalized calibrated microbubbles containing PE-PEG-maleimide

[0284] The suspension of calibrated microbubbles (CMV-MAL) containing PE-PEG-maleimide was washed twice by centrifugation with 0.9% saline (6' / 600 RPM) after collection. A solution of thiolized streptavidin (STV-SH) (streptavidin (STV) derivatization) was prepared according to the protocol described in WO2020127816A1. Briefly, the STV solution was prepared by dissolving 9 mg of lyophilized streptavidin (0.85 mg STV / mg powder (from IBA) - 143 nmol) in 0.2 mL of distilled water and 150 µL of buffer A (50 mM phosphate, 150 mM saline, pH 7.4). A clear solution (concentration approximately 22 mg / mL) was obtained, and Sulfo-LC-SPDP (2.5 mg) was dissolved in 250 µL of ultrapure (milliQ) water. The solution (19 μmol / mL) was freshly prepared just before the experiment.

[0285] The sample of Sulfo-LC-SPDP solution (26 µL – 0.5 µmol – 3.5 equivalents) was added to the STV solution. The resulting solutions were mixed (vortexed) and incubated at room temperature for 40 minutes (vortexing every 5 minutes).

[0286] Equilibrate two 2 mL-Zeba columns using buffer B (5 mM phosphate, pH 7.4).

[0287] After incubation, the STV-SPDP solution was purified on the first Zeba column.

[0288] TCEP (4 mg) was dissolved in 185 µL of buffer C (Tris 500 mM, EDTA 50 mM, pH: 7) to obtain a 75 mM solution. A sample of the TCEP solution (19 µL – approximately 10 equivalents) was added to the STV-SPDP solution. After mixing (e.g., by vortexing), the solution was incubated at room temperature for 30 min to deprotect the STV-SDPD and obtain thiolated STV (STV-SFI). A 2 mL-Zeba column was equilibrated using buffer B. After incubation, the STV-SH solution was purified on a 2 mL-Zeba column. The volume of the recovered solution was approximately 0.39 mL, and the STV-SH concentration was approximately 300 nanomoles / mL.

[0289] The solution containing STV-SH was used for a subsequent coupling reaction with CMV-MAL to obtain calibrated microbubbles (CMV-MAL-STV) containing PE-PEG-maleimide, functionalized with streptavidin.

[0290] Table 2b records the characterization of the CMV suspensions functionalized with STV obtained as described above. Characterization was performed using the Coulter counting scheme described in the previous Methods section.

[0291] Table 2b Characterization of functionalized suspensions

[0292]

[0293] Example 3

[0294] The effect of calibrated STV microbubble composition

[0295] According to Example 2, calibrated air-filled microbubbles with STV functionalization were prepared.

[0296] The feature is that different formulations of various molar amounts of the DSPC / PE-PEG / PE-PEG-2000 reactive structural moiety were compared by determining the percentage of aggregation as described above (see Methods section). For this study, two different reactive structural moieties (RMs), namely biotin and maleimide, were tested.

[0297] result

[0298] Table 3a records the results obtained by testing formulations containing DSPC / PE-PEG / PE-PEG-Biotin, while Table 3b records the results obtained by testing formulations containing DSPC / PE-PEG / PE-PEG-Maleimide.

[0299] Table 3a shows the aggregation percentage of PE-PEG and PE-PEG-Biotin by varying the amounts.

[0300]

[0301] Table 3b shows the percentage of polymerization by varying the amounts of PE-PEG and PE-PEG-maleimide.

[0302]

[0303] As can be seen 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 inflatable microbubbles.

[0304] Compared with the higher aggregation measured for formulations with lower or higher total amounts of polyethylene glycol phospholipids, a significant reduction in the aggregation effect can be achieved by using polyethylene glycol phospholipids with a total amount (mol%) between 16% and 22%.

[0305] Furthermore, less than 1% aggregation was achieved by using a combination of 15% or higher molar amounts of PE-PEG with at least 1% PE-PEG-Biotin.

[0306] Example 4

[0307] Calibrated STV microbubbles were prepared using PE-PEG2000 and varying amounts of PE-PEG2000-Biotin.

[0308] According to Example 2, calibrated air-filled microbubbles with STV functionalization were prepared.

[0309] To assess the effect of the amount of PE-PEG-RM on the ability of calibrated STV microbubbles to collect cells, different compositions characterized by an increased molar percentage of PE-PEG-RM were compared.

[0310] For this purpose, cell recovery tests were performed using CCL-119 cells (30 MB / cell) as previously described.

[0311] Streptavidin (STV) was added to 1 mL of washed microbubbles containing PE-PEG2000-Biotin (e.g., in a molar amount of 0.5% to 7.5%) at a STV / BIOT molar ratio of 1.

[0312] result

[0313] Table 4. Cell recovery rates (%) of formulations containing 15% PE-PEG2000 at increased amounts of PE-PEG2000-Biotin (1%, 2%, 4%, 6%, 7.5%).

[0314]

[0315] Table 5. Cell recovery (%) of formulations containing 18% PE-PEG2000 at increased amounts of PE-PEG2000-Biotin (0.5%, 1%, 2%, 4%).

[0316]

[0317] As can be seen from the above results, for equimolar amounts of PE-PEG, lower molar amounts of PE-PEG-Biotin (i.e., between 0.5% and 2%) provide increased cell recovery (i.e., about 3 times higher) compared to formulations with higher molar amounts (i.e., PE-PEG-Biotin 4%-7.5%).

[0318] 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), where the cell recovery rate was found to be 97%.

[0319] Similar results were obtained in cell recovery assays using the above composition at different STV / biotin molar ratios (i.e., 0.5 and 2).

[0320] Example 5

[0321] The effect of molecular weight on polyethylene glycol-modified phospholipids

[0322] According to Example 2, calibrated aerated microbubbles derived from STV were prepared. To evaluate the effect of the properties of PEGylated phospholipids on the ability of the calibrated aerated microbubbles to collect cells, the composition MSB-9 (DSPC / DSPE-PEG2K / DSPE-PEG2K-BIOT (81 / 15 / 4)) was compared with two different compositions (i.e., C1 and C2, composed of PEGylated phospholipids with a number average molecular weight greater than 2000 g / mol).

[0323] For this purpose, a cell recovery test was performed using CCL-119 cells (30 CMV / cell) as previously described. Streptavidin (STV) was added to 1 mL of washed CMV-biotin at an STV / BIOT molar ratio of 1.

[0324] result

[0325] As can be seen from Table 6, formulations containing a first polyethylene glycol phospholipid with a molecular weight greater than 2000 g / mol ± 10% (e.g., PE-PEG-Biotin) provide significantly lower cell recovery compared to formulations containing a first polyethylene glycol phospholipid with a PEG structural portion having a molecular weight of 2000 g / mol ± 10%.

[0326] Table 6. Effect of PE-PEG molecular weight on cell recovery rate (%)

[0327]

[0328] Example 6

[0329] The separation efficiency of the calibrated aerated microbubbles

[0330] The separation efficiency of calibrated aerated microbubbles was evaluated by comparing calibrated CMV-BIOT-STV of different volumes.

[0331] For this purpose, formulation MSB-10 (PE-PEG2K / PE-PEG2K-BIOT 18 / 1) was selected.

[0332] As described in Example 2, §2.1, a suspension of calibrated microbubbles containing PE-PEG-Biotin (CMV-BIOT) was washed twice by centrifugation with 0.9% saline (6' / 600 RPM) after collection. Streptavidin (STV) was added to 1 mL of the washed CMV-BIOT at the desired STV / Biotin molar ratio, and then incubated on a rotating wheel at room temperature for 45 min. CMV-BIOT-STV was then washed twice more by centrifugation (6' / 600 RPM) and resuspended in 0.9% saline. Coulter counts were performed to determine the volume of CMV to be added to the cells, reaching 3, 7, 5, 15, and 30 CMV / cell, which were found to correspond to additions of 5 × 10⁻⁶ CMV / cell. 6 Volumes of 62.5 µL, 125 µL, 250 µL, and 500 µL per cell were prepared. For all test formulations, the incubation time was maintained at room temperature on a rotary mixer for 20 minutes.

[0333] result

[0334] Table 7. Composition of MSB-10: Effect of CMV size on separation efficiency (cell recovery rate) at different CMV / cell ratios.

[0335]

[0336] Table 8. Composition of MSB-10: Effect of different CMV / cell ratios on cell recovery (%) using CCL119 cell lines (cell size: 10–15 µm)

[0337]

[0338] The separation efficiency is expressed as the cell recovery rate calculated according to Equation 3.

[0339] Table 7 shows the effect of CMV size on separation efficiency (expressed as percentage of cell recovery) at different CMV / cell ratios (the CMV compositions tested were characterized by similar GSD values ​​of at least 1.2 or lower).

[0340] It was observed that at lower microbubble / cell ratios (e.g., 3 CMV / cell), calibrated aerated microbubbles characterized by larger size (e.g., 7.5 µm) significantly increased separation efficiency compared to smaller microbubbles (e.g., 4.3 µm).

[0341] Furthermore, as can be seen from Table 8, the cell recovery rate provided by the calibrated CMV-STV formulation is similar at any CMV / cell ratio studied, demonstrating that the CMV / cell ratio can be significantly reduced without negatively affecting the cell recovery capability of the composition.

[0342] Specifically, it was demonstrated that halving the CMV / cell ratio from 30 CMV / cell to 15 CMV / cell did not affect the cell recovery rate of the calibrated CMV-STV, and the cell recovery rate was found to be substantially the same.

[0343] A similar cell recovery percentage was obtained by using the MCF-7 cell line (cell size 20–25 µm) for cell recovery testing. For example, at 30 CMV / cell, the cell sorting percentage was approximately 93%.

[0344] References

[0345] 1. WO2020 / 127816

[0346] 2. WO2018041906A1

[0347] 3. WO2019170606A1

[0348] 4. WO2020260420A1

[0349] 5. WO2020260423A1

Claims

1. A calibrated suspension of aerated microbubbles, the microbubbles comprising a core and an outer layer, the core comprising a physiologically acceptable gas, and the outer layer comprising: - Phospholipids, - Less than 4 mol% of a first polyethylene glycol-modified phospholipid containing a reactive structural moiety, wherein at least a portion of the first polyethylene glycol-modified phospholipid binds to a ligand via the reactive structural moiety, and - At least 15 mol% of a second polyethylene glycol-modified phospholipid that does not contain reactive structural moieties. The calibrated suspension of aerated microbubbles has a geometric standard deviation (GSD) of less than 1.

2.

2. The suspension according to claim 1, wherein the outer layer comprises at least 16%, more preferably at least 18%, at most 22%, preferably at most 20% of the total amount of polyethylene glycol-modified phospholipids.

3. The suspension according to claim 1 or 2, wherein the outer layer comprises at least 3 mol%, preferably at least 2 mol%, more preferably at least 1 mol%, even more preferably at least 0.5 mol%, and as low as 0.25 mol% of the first polyethylene glycol phospholipid.

4. The suspension according to any one of the preceding claims, wherein the outer layer comprises at least 16 mol% or higher, preferably at least 17 mol% or higher, more preferably at least 18 mol% or higher, even more preferably at least 19 mol% or higher, even more preferably at least 20 mol% or higher, and at most 21 mol% of the second polyethylene glycol phospholipid.

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

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

7. The suspension according to claim 6, wherein the first polyethylene glycol phospholipid and the second polyethylene glycol phospholipid have a molecular weight of 2000 g / mol ± 5%.

8. The suspension according to any one of the preceding claims, wherein the ligand is a biomolecular binding structure.

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

10. The suspension according to any one of the preceding claims, wherein the ligand has a concentration of at least 1500 molecules / µm 2 The density of on the surface of the outer layer.

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

12. The suspension according to any one of the preceding claims, wherein the phospholipid is selected from dimyristoyl-phosphatidylcholine (DMPC), dipalmitoyl-phosphatidylcholine (DPPC), distearyl-phosphatidylcholine (DSPC), arachidoyl-phosphatidylcholine (DAPC), dipalmitoylphosphatidic acid (DMPA), dipalmitoylphosphatidic acid (DPPA), distearylphosphatidic acid (DSPA), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearylphosphatidylglycerol (DSPG), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS), and distearylphosphatidylserine (DSPS).

13. The suspension according to any one of the preceding claims, wherein the first polyethylene glycol phospholipid or the second polyethylene glycol phospholipid is polyethylene glycol 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 aerated microbubbles having ligands as defined in claims 1-13, wherein the suspension of calibrated aerated microbubbles has a geometrical standard deviation (GSD) of less than 1.2, the method comprising: A. Provide (i) a gas stream and (ii) an aqueous liquid stream, the aqueous liquid stream comprising: - Phospholipids; - Less than 4 mol% of the first polyethylene glycol-modified phospholipid containing the reactive structural moiety, and - At least 15 mol% of a second polyethylene glycol-modified phospholipid that does not contain reactive structural moieties; B. Guide the gas flow and the liquid flow toward the contact area through their respective inlet channels; C. Guide the gas flow and the liquid flow from the contact area through the calibration orifice to obtain an aqueous suspension containing the aerated microbubbles; D. Collect the suspension containing the microbubbles from the outlet channel; E. Add functionalized ligands that can react with the reactive structural portion to the collected suspension; F. Couple the first polyethylene glycol-modified phospholipid to the ligand; as well as G. Collect a suspension of calibrated aerated microbubbles with ligands, wherein the suspension has a geometric standard deviation (GSD) of less than 1.

2.

16. The method of claim 15, further comprising the optional step D'): washing the obtained suspension of calibrated aerated microbubbles.

17. The method according to claims 16-17, comprising step F') after coupling step F): washing the obtained suspension of calibrated aerated microbubbles with ligands.

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