Microfluidic preparation of biphasic nanodroplets with fluorinated compounds

Abstract

The present invention relates to calibrated biphasic nanodroplets comprising an outer layer and an inner core, the outer layer comprising a biocompatible fluorinated surfactant, and the inner core comprising a fluorinated compound and a biocompatible oil. The invention further relates to a method for preparing said calibrated biphasic nanodroplets by microfluidic technology and to their use for in vivo or in vitro diagnosis and / or for therapy.

Description

Technical Field

[0001] The present invention generally relates to calibrated biphasic nanodroplets stabilized by biocompatible fluorinated surfactants and having a core comprising a fluorinated compound and a biocompatible oil and methods for their preparation by microfluidics. The present invention also relates to the use of such calibrated nanodroplets for in vitro or in vivo diagnosis and / or for therapy. Background Art

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

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

[0004] In the last decade, the possibility of additionally adding an oil phase in the composition of nanodroplets, precisely into their inner core, has been investigated, benefiting from combining the intrinsic properties of perfluorocarbon-filled nanodroplets with those conferred by the presence of oil in the core of such nanodroplets, like encapsulating hydrophobic molecules for drug delivery applications.

[0005] For example, Astafyeva et al. reported perfluorocarbon nanodroplets stabilized by a biocompatible fluorinated surfactant called "FTAC" and containing an oil phase (i.e., triacetin) in their core in 2015, and they investigated perfluorocarbon emulsions as theranostic agents. In this work, ultrasonic homogenization was used to produce perfluorocarbon nanoemulsions, followed by centrifugation and washing steps.

[0006] WO2016185425 teaches the synthesis of DendriTAC and its use as a stabilizer in the preparation of perfluorocarbon nanoemulsions. Standard preparation methods are proposed for emulsion preparation, such as vortexing, sonication, and microfluidizers (high-pressure homogenizers). The possibility of using DendriTAC to stabilize nanodroplets that also contain an oil phase is also mentioned.

[0007] Al Rifai et al. disclosed DendriTAC-stabilized nanodroplets that also contained an oil phase (i.e., tributyl O-acetyl citrate) in a perfluorocarbon core for the delivery of the hydrophobic drug paclitaxel in 2020. For their preparation, standard emulsification techniques combined with a centrifugation step were used.

[0008] The main limitation of nanodroplets is their relatively limited physicochemical stability over time, which may affect their use in diagnostic and therapeutic applications.

[0009] As reported in the literature, a specific washing procedure is usually required at the end of the preparation of biphasic nanodroplets in order to improve their size and size distribution profile.

[0010] Both the size and size distribution of nanodroplets are important quality attributes that determine the vaporization threshold, which corresponds to the value of ultrasound pressure required to transform a liquid nucleus droplet into a gaseous bubble. In a polydisperse suspension, characterized by particles of different sizes, nanodroplets with larger sizes that require less energy to vaporize than smaller nanodroplets affect the vaporization of the nanodroplet suspension.

[0011] Conversely, in a monodisperse system containing particles of relatively uniform size, having a similar and uniform acoustic response to ultrasound exposure, it is possible to apply the lowest acoustic pressure to achieve the highest vaporization efficiency.

[0012] Recently, microfluidics (MF) technology, also known as “lab-on-a-chip,” has emerged as a powerful and scalable alternative for the consistent preparation of a wide range of size-controlled nanoparticles.

[0013] Melich et al. (2020) reported the use of rapid and controlled microfluidic mixing to fabricate PFC-NDs.

[0014] To the best of the applicant's knowledge, perfluorocarbon emulsions stabilized by biocompatible fluorinated surfactants and comprising biocompatible oils have not been prepared so far by microfluidic technology.

[0015] The Applicant has now developed a novel composition comprising calibrated biphasic nanodroplets, stabilized by a biocompatible fluorinated surfactant and having a core comprising a fluorinated compound and a biocompatible oil, said nanodroplets being obtained by microfluidics.

[0016] Often, in the art, the term "calibrated" is also denoted as "size controlled", "uniformly sized droplets", "monodisperse(d)" or "single size(d)".

[0017] Applicants have observed that the presence of biocompatible oils in the core of calibrated nanodroplets can affect the properties of calibrated nanodroplets manufactured according to microfluidic techniques.

[0018] In fact, the inventors have surprisingly found that improved stability properties of calibrated biphasic NDs can be obtained when adding biocompatible oils in the core of the nanodroplets compared to conventional preparation methods, which are usually based on multi-step and tedious procedures. Summary of the invention

[0019] One aspect of the present invention relates to a nanodroplet comprising an outer layer and an inner core, the outer layer comprising a biocompatible fluorinated surfactant, and the inner core comprising a fluorinated compound and a triglyceride having a logP value greater than 5, wherein the biocompatible fluorinated surfactant is selected from:

[0020] (A) Amphiphilic dendrimer (Dendri-TAC);

[0021] (B) Amphiphilic linear oligomer (F-TAC)

[0022] or a mixture thereof.

[0023] In one embodiment, the fluorinated compound is a perfluorocarbon.

[0024] In a preferred embodiment, the triglycerides have a logP value higher than 7, still more preferably higher than 9, up to 25 for example.

[0025] In one embodiment, the triglyceride is selected from tricaprylin, trilaurin, triolein, trilinolein or a mixture thereof, preferably tricaprylin.

[0026] Another aspect relates to an aqueous suspension comprising a plurality of nanodroplets as defined above, wherein the nanodroplets have a polydispersity index (PDI) lower than 0.25, preferably lower than 0.20, more preferably lower than 0.15, even more preferably lower than 0.10, and a z-average diameter between 100 nm and 1000 nm, preferably between 120 nm and 800 nm, more preferably between 150 nm and 400 nm.

[0027] Yet another aspect relates to a method for preparing an aqueous suspension comprising a plurality of nanodroplets, said nanodroplets comprising an outer layer and an inner core, said outer layer comprising a biocompatible fluorinated surfactant as defined above, and said inner core comprising a fluorinated compound and a biocompatible oil having a logP value higher than 5, said method comprising the steps of:

[0028] a) preparing an aqueous phase;

[0029] b) preparing an organic phase,

[0030] in

[0031] i) the aqueous phase comprises a biocompatible fluorinated surfactant selected from Dendri-TAC, F-TAC or mixtures thereof, and the organic phase comprises a fluorinated compound and a biocompatible oil; or

[0032] ii) the organic phase comprises a biocompatible fluorinated surfactant selected from Dendri-TAC, F-TAC or mixtures thereof, a fluorinated compound and a biocompatible oil;

[0033] c) injecting the aqueous phase into a first inlet of a microfluidic cartridge, and injecting the organic phase into a second inlet of a microfluidic cartridge, thereby mixing the aqueous phase and the organic phase in a mixing device of the microfluidic cartridge, wherein the working pressure into the microfluidic cartridge is lower than 7000 kPa, to obtain the aqueous phase; and

[0034] d) collecting the aqueous suspension from the outlet channel of the microfluidic cartridge.

[0035] Preferably, the biocompatible oil is a triglyceride.

[0036] More preferably, the triglycerides have a logP value higher than 7, still more preferably higher than 9, up to, for example, 25.

[0037] Still more preferably, the triglyceride is selected from glyceryl tricaprylate, glyceryl trilaurate, glyceryl trioleate, glyceryl trilinoleate or a mixture thereof, preferably glyceryl tricaprylate.

[0038] According to a preferred embodiment, the aqueous phase comprises a biocompatible fluorinated surfactant selected from Dendri-TAC, F-TAC or mixtures thereof, and the organic phase comprises a fluorinated compound and a biocompatible oil.

[0039] Preferably, the aqueous suspension comprises a plurality of nanodroplets as defined above, having a z-average diameter between 100 nm and 1000 nm and a polydispersity lower than 0.25.

[0040] Optionally, after step d), the collected aqueous suspension is diluted.

[0041] Another aspect of the invention relates to a method for preparing an aqueous suspension comprising a plurality of nanodroplets, said nanodroplets comprising an outer layer and an inner core, said outer layer comprising a biocompatible fluorinated surfactant, and said inner core comprising a fluorinated compound and a biocompatible oil having a logP value greater than 5, said method comprising the steps of:

[0042] a) preparing an aqueous phase comprising a biocompatible fluorinated surfactant;

[0043] b) preparing an organic phase comprising a fluorinated compound and a biocompatible oil having a logP value greater than 5;

[0044] c) injecting the aqueous phase into a first inlet of a microfluidic cartridge and injecting the organic phase into a second inlet of a microfluidic cartridge, thereby mixing the aqueous phase and the organic phase in a mixing device of the microfluidic cartridge, wherein the working pressure into the microfluidic cartridge is lower than 7000 kPa, to obtain the aqueous suspension; and

[0045] d) collecting the aqueous suspension from the outlet channel of the microfluidic cartridge.

[0046] Yet another aspect relates to the aqueous suspension according to the invention for use in diagnostic and / or therapeutic treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is a schematic diagram of the core portion of the microfluidic cartridge.

[0048] Figure 2 A cross-sectional schematic of the staggered herringbone mixer (SHM) design is shown. DETAILED DESCRIPTION

[0049] The present invention relates to novel compositions comprising calibrated biphasic nanodroplets stabilized by biocompatible fluorinated surfactants and having a core comprising a fluorinated compound and a biocompatible oil, preferably obtained by microfluidics. The calibrated nanodroplets are suitable as contrast agents in ultrasound imaging techniques known as contrast enhanced ultrasound (CEUS) imaging or in therapeutic applications such as thermal ablation or for ultrasound mediated drug delivery.

[0050] Biphasic nanodroplets

[0051] In the present invention, the expression "biphasic nanodroplets" refers to nanodroplets stabilized by a biocompatible fluorinated surfactant and having a core comprising a fluorinated compound and a biocompatible oil, said nanodroplets being preferably obtained by microfluidics.

[0052] The term "calibrated" refers to the distribution of the two-phase nanodroplets and indicates the polydispersity of a population of nanodroplets (e.g., having a z-average diameter between 100 nm and 1000 nm), whose polydispersity index (PDI) is lower than 0.25, preferably lower than 0.2, more preferably lower than 0.15, and even more preferably lower than 0.1.

[0053] The liquid core of the biphasic nanodroplet is typically partitioned into two distinct phases: a first phase comprising the fluorinated compound(s) and a second phase comprising the oil(s), which are substantially immiscible with each other.

[0054] This separation is related to the inherent properties of fluorinated compounds, which are significantly more hydrophobic than hydrocarbon compounds, such as biocompatible oils, and also more lipophobic, repelling both water and lipids.

[0055] Biocompatible fluorinated surfactants

[0056] As used herein, the term "biocompatible" refers to compounds and / or compositions that have substantial compatibility with living tissues or systems, are not toxic, injurious, or physiologically reactive, and typically do not induce immunological rejection.

[0057] In the present description and claims, the expression "surfactant" has its conventional meaning in the chemical field and refers to a compound suitable for forming a stabilization layer of nanodroplets.

[0058] The expression "fluorinated surfactant" refers to an amphiphilic organic compound suitable for forming a stabilizing layer for nanodroplets, which compound comprises a hydrophilic part and a hydrophobic part, said hydrophobic part comprising fluorine atoms (ie a fluorinated part).

[0059] The nanodroplets of the invention are preferably dispersed in an aqueous solvent and are stabilized by a layer composed of a biocompatible fluorinated surfactant, which advantageously exhibits a high affinity for both the inner core comprising a fluorinated compound and a biocompatible oil and the surrounding water.

[0060] In the present specification and claims, the term Dendri-TAC refers to an amphiphilic dendrimer of generation n comprising:

[0061] - a divalent or trivalent hydrophobic central core;

[0062] - chains of generations connected to a central core and branching around it; and

[0063] - hydrophilic terminal groups at the end of each generation chain;

[0064] in

[0065] n is an integer from 0 to 12;

[0066] Hydrophilic end groups include:

[0067] - monosaccharide, oligosaccharide or polysaccharide residues,

[0068] - cyclodextrin residues,

[0069] - peptide residues,

[0070] -Tris(hydroxymethyl)aminomethane (Tris), or

[0071] -2-amino-2-methylpropane-1,3-diol;

[0072] The hydrophobic core is a group of formula (Ia) or (Ib):

[0073]

[0074] in:

[0075] W is R F or a group selected from W0, W1, W2 or W3:

[0076]

[0077] R F C4-C 10 Perfluoroalkyl,

[0078] R H C1-C 24 Alkyl groups,

[0079] p is 0, 1, 2, 3 or 4;

[0080] q is 0, 1, 2, 3, or 4;

[0081] L is a straight chain or branched C1-C 12 an alkylene group, optionally interrupted by one or more -O-, -S-,

[0082] Z is C(=O)NH or NHC(=O),

[0083] R is a C1-C6 alkyl group, and

[0084] e is independently selected from 0, 1, 2, 3 or 4 at each occurrence.

[0085] In one embodiment, R F C4-C 10 perfluoroalkyl, and R H C1-C 24In this case, the hydrophobic central core of the amphiphilic dendrimer does contain perfluorinated alkyl groups and the dendrimer is referred to herein as a fluorinated amphiphilic dendrimer.

[0086] As used herein, "m-valence of the central core" refers to the number of generations of chains connected to the central core, as shown in Scheme 1 below:

[0087] Solution 1

[0088]

[0089] As used herein, a dendrimer of generation n=0 means that the m-generation chain is connected to the central core via the first branching point (G0), corresponding to the valence state of the central core. A dendrimer of generation n=1 means that each of the m-generation chains branches once on itself, more specifically at the branching point G1 (see Scheme 2).

[0090] Solution 2

[0091]

[0092] According to a preferred embodiment, n is 0, 1 or 2, more preferably n is 0.

[0093] Each generation of the amphiphilic dendrimers according to the invention ends with a hydrophilic end group.

[0094] In this respect, the monosaccharide, oligosaccharide or polysaccharide residue may be, inter alia, glucose, galactose, mannose, arabinose, ribose, maltose, lactose, hyaluronic acid.

[0095] The cyclodextrin residue may be selected from α, β or γ-cyclodextrin.

[0096] The peptide residues may be selected from linear or cyclic peptides containing the sequence arginine-glycine-aspartic acid (RGD).

[0097] In another embodiment, dendrimers are included wherein the generation chains are linked to a central core via the following groups:

[0098] Group (a):

[0099]

[0100] Group (b):

[0101]

[0102] in

[0103] Z is C(=O)NH or NHC(=O) and is attached to the central core,

[0104] R is a C1-C6 alkyl group, and

[0105] e is independently selected from 0, 1, 2, 3 or 4 at each occurrence.

[0106] In a further embodiment, dendrimers are included wherein the central core is a group of formula (Ia) or (Ib):

[0107]

[0108] in:

[0109] W is R F or a group selected from W0, W1, W2 or W3:

[0110]

[0111] R F C4-C 10 Perfluoroalkyl,

[0112] R H C1-C 24 Alkyl groups,

[0113] p is 0, 1, 2, 3 or 4;

[0114] q is 0, 1, 2, 3, or 4;

[0115] L is a straight or branched C1-C 12 an alkylene group, which is optionally interrupted by one or more -O-, -S-,

[0116] In yet other embodiments, include dendrimers wherein WL is a group selected from:

[0117]

[0118] In yet another embodiment, dendrimers are included wherein each chain generation (n) is branched n times by groups (a) or groups (b) as defined above.

[0119] In another embodiment, dendrimers are included wherein the terminal groups comprise the following hydrophilic moieties:

[0120]

[0121] In certain embodiments, dendrimers having the formula:

[0122] in:

[0123] W is R F or a group selected from the following:

[0124]

[0125] R F C4-C 10 Perfluoroalkyl and R H C1-C 24 Alkyl groups,

[0126] p is 0, 1, 2, 3 or 4;

[0127] q is 0, 1, 2, 3, or 4;

[0128] Z is (CO)NH or NH(CO);

[0129] R1, R2, R3 are H, or a group selected from (c) or (d):

[0130]

[0131] The conditions are:

[0132] R1, R2, R3 are the same and are selected from group (c) or (d)

[0133] or:

[0134] One of R1, R2 and R3 is H, and the other two are the same and are selected from group (c) or (d);

[0135] When j is 1, X is X a , and when j is 0, X is X b ;

[0136] X a is independently selected at each occurrence from -OC(=O)CH2-NH-, -OC(=O)CH2-O-CH2-, -O(CH2) r C(=O)-NH-, -O(CH2) r C(=O)-O-CH2, OC(=O)NH-, -C(=O)-, -NH- and -OCH2-;

[0137] Y a for

[0138]

[0139] X b for

[0140]

[0141] Y b Independently selected from:

[0142]

[0143] V is:

[0144]

[0145] R4 and R6 are each independently selected from H, C1-C6 alkyl or CH2OR 10 ;

[0146] R5 is a monosaccharide, oligosaccharide, polysaccharide or cyclodextrin residue;

[0147] R7 and R8 are each independently a peptide residue;

[0148] R 10 is H or a monosaccharide selected from glucose, galactose or mannose;

[0149] i is 0 or 1;

[0150] j is 0 or 1;

[0151] e is 0, 1, 2, 3, or 4;

[0152] k is an integer from 1 to 12, preferably from 1 to 5;

[0153] r is an integer from 1 to 10;

[0154] u is 0, 1, 2, 3, or 4;

[0155] v is 1, 2, or 3;

[0156] w is an integer from 1 to 20, preferably from 1 to 10;

[0157] x and y are each independently an integer of 1 to 6.

[0158] In certain embodiments, dendrimers having the formula:

[0159]

[0160] in:

[0161] W is R F or a group selected from the following:

[0162]

[0163] R F C1-C 24 A perfluoroalkyl group, and R H C1-C 24 Alkyl groups,

[0164] p is 0, 1, 2, 3 or 4;

[0165] q is 0, 1, 2, 3, or 4;

[0166] Z is (CO)NH or NH(CO);

[0167] R1, R2, R3 are H or a group selected from (c) or (d):

[0168]

[0169] The conditions are:

[0170] R1, R2, R3 are the same and are selected from group (c) or (d)

[0171] or:

[0172] One of R1, R2 and R3 is H, and the other two are the same and are selected from group (c) or (d);

[0173] When j is 1, X is X a , and when j is 0, X is X b ;

[0174] X a is independently selected at each occurrence from -OC(=O)CH2-NH-, -OC(=O)CH2-O-CH2-, -O(CH2) r C(=O)-NH-, -O(CH2) r C(=O)-O-CH2, OC(=O)NH-, -C(=O)-, -NH- and -OCH2-;

[0175] Y a for

[0176]

[0177] X b for

[0178]

[0179] Y b Independently selected from:

[0180]

[0181] V is:

[0182]

[0183] R4 and R6 are each independently selected from H, C1-C6 alkyl or CH2OR 10 ;

[0184] R5 is a monosaccharide, oligosaccharide, polysaccharide or cyclodextrin residue;

[0185] R7 and R8 are each independently a peptide residue;

[0186] R 10 is H or a monosaccharide selected from glucose, galactose or mannose;

[0187] i is 0 or 1;

[0188] j is 0 or 1;

[0189] e is 0, 1, 2, 3, or 4;

[0190] k is an integer from 1 to 12, preferably from 1 to 5;

[0191] r is an integer from 1 to 10;

[0192] u is 0, 1, 2, 3, or 4;

[0193] v is 1, 2, or 3;

[0194] w is an integer from 1 to 20, preferably from 1 to 10;

[0195] x and y are each independently an integer of 1 to 6.

[0196] In a particular embodiment, the hydrophilic end group of the surfactant defined above has the formula:

[0197]

[0198] Among them, R6, R 10 , v and w are as defined above, v in particular being equal to 3.

[0199] In a particular embodiment, the hydrophobic end group of the surfactant defined above has the formula:

[0200]

[0201] wherein v and w are as defined above, v being in particular equal to 3.

[0202] Suitable examples of amphiphilic dendrimers (Dendri-TAC) and their preparation are described in WO2016185425, including F6DiTAC 11 、F6DiTAC6、F6DiTAC 15 , F8DiTAC5, DiF6DiTAC7, DiF6DiTAC 15 、DiF8DiTAC5、DiF8DiTAC 11 , with the following formula:

[0203]

[0204]

[0205]

[0206]

[0207]

[0208] In a preferred embodiment of the present invention, the amphiphilic dendrimer Dendri-TAC is a compound of formula IA

[0209]

[0210] The compound of formula IA is DiF6DiTAC7.

[0211] As used herein, the term "F-TAC" includes a hydrophilic portion comprising a polyTRIS type oligomer and a hydrophobic portion comprising a linear fluorinated alkyl chain.

[0212] In the present specification and claims, the term F-TAC refers to a linear fluorinated surfactant having formula II:

[0213]

[0214] in:

[0215] - n is the number of repeating Tris units (n = DPn is the average degree of polymerization), wherein the term Tris denotes a tris(hydroxymethyl)aminomethane unit, and

[0216] -i is the number of carbon atoms in the fluoroalkyl chain.

[0217] In this specification and claims, the compound of formula II may be represented interchangeably as F i TAC n ,in:

[0218] -n is the number of repeating Tris units (n = DPn is the average degree of polymerization), and

[0219] -i is the number of carbon atoms in the fluoroalkyl chain.

[0220] According to one embodiment, i is between 4 and 12, preferably between 6 and 10.

[0221] According to another embodiment, when i is between 6 and 10, n is between 1 and 40, preferably between 4 and 30, still more preferably between 5 and 20.

[0222] According to yet another embodiment, when i is 8, n is between 1 and 40, such as between 4 and 30.

[0223] For example, Astafyeva, 2015, discloses suitable examples of amphiphilic linear oligomers F-TAC, including F8TAC7, F8TAC 18 、F8TAC 13 and F6TAC8, having the following formula:

[0224]

[0225]

[0226] In one embodiment of the present invention, the amphiphilic linear oligomer (F-TAC) is a compound of the following formula IIA

[0227]

[0228] Wherein, the compound of formula IIA is F8TAC 18 .

[0229] Applicants have now discovered that the physicochemical properties of the biocompatible fluorinated surfactants of the present invention can influence the size of the disclosed (P)FC-NDs.

[0230] Table 1 shows selected physicochemical properties of preferred biocompatible fluorinated surfactants. In particular, values ​​for surface tension, critical micelle concentration and molecular weight have been reported.

[0231] Table 1 Physicochemical properties of biocompatible fluorinated surfactants

[0232]

[0233] ST: surface tension at 25°C; CMC: critical micelle concentration; MW: molecular weight

[0234] For example, it has been observed that, in general, the lower the surface tension value associated with a biocompatible fluorinated surfactant, the smaller the ND size.

[0235] In the present description and claims, the expression "surface tension" has its ordinary meaning in the chemical field and indicates the tendency of the surface of a liquid to shrink as much as possible to the smallest surface area. Surfactants, such as the disclosed biocompatible fluorinated surfactants, are compounds that reduce the surface tension between two liquids, between a gas and a liquid, or between a liquid and a solid.

[0236] For example, the surface tension can be measured using the Wilhelmy plate technique, for example at an air / water interface using a K100 tensiometer (Kruss, Hamburg, Germany) at (25.0 ± 0.5) °C.

[0237] According to one embodiment, preferred biocompatible fluorinated surfactants are characterized by surface tension values ​​lower than 70 mN / m, more preferably lower than 50 mN / m.

[0238] Fluorinated compounds

[0239] In the present description and claims, the term "fluorinated compounds" refers to a group of fluorine-containing compounds derived from (optionally substituted) hydrocarbons, wherein hydrogen atoms have been partially or completely replaced by fluorine atoms, the fluorinated compounds being liquid at room temperature.

[0240] The expression "optionally substituted" refers to the presence of functional groups such as amines, ethers and halogen-containing groups.

[0241] Preferably, the fluorinated compound is a perfluorocarbon (PFC), ie a fluorinated hydrocarbon in which all hydrogen atoms are replaced by fluorine atoms.

[0242] The liquid fluorinated compound is characterized by a boiling point between 25 and 160° C. In the present invention, the fluorinated compound is preferably characterized by a boiling point between 25 and 100° C., still more preferably between 27 and 60° C.

[0243] Suitable examples of fluorinated compounds are 1-fluorobutane, 2-fluorobutane, 2,2-difluorobutane, 2,2,3,3-tetrafluorobutane, 1,1,1,3,3-pentafluorobutane, 1,1,1,4,4,4-hexafluorobutane, 1,1,1,2,4,4,4-heptafluorobutane, 1,1,2,2,3,3,4,4-octafluorobutane, 1,1,1,2,2-pentafluoropentane, 1,1,1,2,2,3,3,4-octafluoropentane, 1,1,1,2,2,3,4,5,5,5-decafluoropentane, 1,1,2,2,3,3,4,4,5,5,6,6-dodecafluorohexane.

[0244] Suitable examples of perfluorocarbons are perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluorononane, perfluorodecalin, perfluorooctane bromide (PFOB), perfluoro-15-crown-5-ether (PFCE), perfluorodichlorooctane (PFDCO), perfluorotributylamine (PFTBA), perfluorononane (PFN) and 1,1,1-tris(perfluoro-tert-butoxymethyl)ethane (TPFBME) or mixtures thereof.

[0245] In one embodiment, the perfluorocarbon is preferably perfluoropentane (PFP) (boiling point 29° C.), perfluorohexane (PFH) (boiling point 57° C.), or perfluorooctyl bromide (PFOB) (boiling point 142° C.).

[0246] oil phase

[0247] In the present invention, the calibrated nanodroplets are characterized by an inner liquid core comprising a fluorinated compound and oil.

[0248] The oil may be a single oil or a mixture of different oils.

[0249] The term "oil" has its usual meaning in the chemical field and refers to an organic compound that is liquid at room temperature, selected from fatty acids, fatty acid esters or hydrocarbons. In the present invention, the oil is a biocompatible oil (see the above definition of biocompatible).

[0250] Examples of biocompatible oils include mono-, di- or triesters of glycerol, glycerol derivatives, mono-, di-, triesters or tetra-esters of citric acid, citric acid derivatives, fatty acid monoesters, sterol esters, sphingolipids, glycerophospholipids, glycolipids, terpenes, lipid derivatives of prenol or mixtures thereof.

[0251] The biocompatible oil may be a fatty acid ester, derived from a combination of fatty acids and alcohols. Examples of fatty acid esters are monoglycerides, diglycerides and triglycerides, which consist of a glycerol backbone esterified once, twice or three times with fatty acids on the hydroxyl groups of the glycerol molecules, respectively.

[0252] Preferably, the biocompatible oil is a triglyceride.

[0253] Based on the length of the fatty acid unit, triglycerides can be classified as short-chain fatty acids of less than 6 carbon atoms (e.g., triacetin and tributyrin), medium-chain fatty acids containing 6 to 12 carbon atoms (e.g., tricaprylin), long-chain fatty acids containing 13 to 21 carbon atoms (e.g., triolein), and very long-chain fatty acids of 22 or more carbon atoms (e.g., tridocosahexaenoic acid).

[0254] Triglycerides can also be classified as saturated and unsaturated, with the unsaturated triglycerides tending to have lower melting points than the saturated analogs; thus, unsaturated triglycerides are generally liquid at room temperature.

[0255] In a preferred embodiment, the triglyceride is selected from triglycerides containing medium-chain, unsaturated long-chain, unsaturated very long-chain fatty acids or mixtures thereof.

[0256] Preferably, the triglycerides comprise medium chain fatty acids.

[0257] Still more preferably, the triglyceride is selected from glyceryl tricaprylate, glyceryl trilaurate, glyceryl trioleate, glyceryl trilinoleate or a mixture thereof, preferably glyceryl tricaprylate.

[0258] According to the present invention, other suitable examples of biocompatible oils may be acetyl trihexyl citrate, myristoleic acid, palmitoleic acid, oleic acid, gadoleic acid, erucic acid, ricinoleic acid, linoleic acid, eicosadienoic acid, α-linolenic acid, γ-linolenic acid, arachidonic acid, coconut oil, soybean oil, castor oil, isopropyl palmitate, isopropyl myristate, squalene, squalane, vitamin E acetate and DL-α-tocopherol.

[0259] Applicants have observed that the lipophilicity of the biocompatible oils, particularly triglycerides, used to prepare the disclosed biphasic nanodroplets plays a role in the stabilization of the nanodroplets.

[0260] One of the most common ways to define the lipophilic character of a compound is the partition coefficient P or its decimal logarithm logP.

[0261] The "logP value" is a parameter representing the partition coefficient (P), which describes the tendency of a neutral (uncharged) compound to dissolve in an immiscible two-phase system of lipids (e.g., fats, oils, organic solvents) and water. In other words, it measures how much the aqueous portion of the solute dissolves compared to the organic portion. Solutes that dissolve primarily in the aqueous layer are called hydrophilic, while solutes that dissolve primarily in lipids are called lipophilic.

[0262] The logP value is a constant defined in Equation 1 below:

[0263] LogP = log10 ([organic] / [water]) Equation 1

[0264] where P represents the partition coefficient, and [] represents the concentration of the solute in the organic and aqueous fractions.

[0265] A negative value of logP means that the compound has a higher affinity for the aqueous phase (more hydrophilic); when logP = 0, the compound partitions equally between the lipid and aqueous phases; a positive value of logP indicates a higher concentration in the lipid phase (ie, the compound is more lipophilic).

[0266] LogP values ​​can be determined experimentally or by calculation. Different well-established methods are available for experimental determination of LogP, such as shake flasks and high performance liquid chromatography (HPLC).

[0267] The logarithm of the partition coefficient (logP) can also be calculated and estimated by several methods.

[0268] In the present invention, the logP value is based on the calculation method XLOGP3, which predicts the logP value of a query compound by using the known logP value of a reference compound as a starting point. Further details on the calculation method XLOGP3 can be found in Cheng et al., 2007.

[0269] Applicants have observed that the use of triglycerides characterized by a lower lipophilicity (eg logP below 5) results in a significantly reduced stability over time (eg higher % evol values).

[0270] On the other hand, triglycerides with higher lipophilicity (eg, logP above 5) values ​​impart a more stable composition to the biphasic nanodroplets.

[0271] In one embodiment, the biocompatible oil, in particular triglycerides, has a logP value higher than 5, more preferably higher than 7, still more preferably higher than 9, up to 25.

[0272] In another embodiment, the biocompatible oil is a mixture of oils.

[0273] In one embodiment, the mixture comprises at least one oil having a logP value above 5, and at least one oil having a logP value below 5.

[0274] Preferably, the amount of said oil having a logP higher than 5 is higher than 75 mol%, more preferably higher than 90%.

[0275] Preferably, the amount of said oil having a logP above 5 is at most 25 mol%, more preferably at most 10%.

[0276] In certain embodiments, the oil(s) having a logP value greater than 5 comprises 100 mol% of the biocompatible oil.

[0277] In one embodiment, the oil may further comprise bioactive molecules (e.g., therapeutic agents or contrast agents) dispersed or dissolved therein. Bioactive molecules include any molecule, compound, formulation or material that can be combined with biphasic nanodroplets for ultrasound-mediated therapeutic treatment and can produce a biological or therapeutically active effect on the area or organ to be treated.

[0278] Preferably, the bioactive molecule is a hydrophobic drug.The bioactive molecule may advantageously be dissolved in the inner core of the biphasic nanodroplet, in particular in the phase comprising the biocompatible oil(s).

[0279] Molar ratio of BFS / fluorinated compound

[0280] In the present specification and claims, the expression "BFS / fluorinated compound molar ratio" (Nr) represents the ratio of the biocompatible fluorinated surfactant and the fluorinated compound used to prepare the disclosed biphasic nanodroplets. The molar ratio can be calculated by using the following formula:

[0281]

[0282] in:

[0283] - the expression "total number of moles of biocompatible fluorinated surfactant" means the molar amount of biocompatible fluorinated surfactant used to prepare the nanodroplet suspension, and

[0284] - The expression "total moles of fluorinated compounds" denotes the molar amount of fluorinated compounds used for the preparation of the nanodroplet suspension.

[0285] The molar amounts represent the amount of each component contained in the aqueous phase and the organic phase, respectively, used to prepare the calibrated biphasic nanodroplets.

[0286] In one embodiment, the molar ratio between the biocompatible fluorinated surfactant and the fluorinated compound is between 0.002 and 0.250, preferably between 0.005 and 0.100.

[0287] Biocompatible oil / fluorinated compound molar ratio

[0288] In the present specification and claims, the expression "biocompatible oil / fluorinated compound molar ratio" (Nr) means the molar ratio of the biocompatible oil to the fluorinated compound used to prepare the disclosed biphasic nanodroplets. The molar ratio can be calculated by using the following formula:

[0289]

[0290] in:

[0291] - the expression "total moles of biocompatible oil" means the molar amount of biocompatible oil used to prepare the nanodroplet suspension, and

[0292] - The expression "total number of moles of fluorinated compounds" denotes the molar amount of (per)fluorinated compounds forming the inner core of the ND.

[0293] The molar amounts represent the amount of each component contained in the aqueous phase and the organic phase, respectively, used to prepare the calibrated biphasic nanodroplets.

[0294] In one embodiment, the molar ratio between the biocompatible oil and the fluorinated compound is between 0.05 and 0.500, preferably between 0.07 and 0.400.

[0295] Biocompatible oil / fluorinated compound volume ratio (v / v%)

[0296] In the present specification and claims, "biocompatible oil / fluorinated compound volume ratio" (v / v %) means the volume ratio of the biocompatible oil to the fluorinated compound used to prepare the disclosed biphasic nanodroplets. The volume ratio can be calculated by using the following formula:

[0297]

[0298] The percentage volume ratio indicates the volume of the nanodroplet occupied by the biocompatible oil. For example, a volume ratio of 50% means that 50% of the droplet core volume is occupied by the biocompatible oil.

[0299] The applicant has observed that by using the disclosed microfluidic method it is possible to obtain biphasic nanodroplets having a biocompatible oil / fluorinated compound volume ratio higher than 10%, preferably higher than 15%, more preferably higher than 20%, even more preferably higher than 30%, even more preferably higher than 40%, up to 50%.

[0300] Calibrated biphasic nanodroplets

[0301] Another aspect relates to an aqueous suspension comprising nanodroplets as defined above.

[0302] Another aspect relates to an aqueous suspension comprising a plurality of biphasic nanodroplets as defined above, wherein the nanodroplets have a z-average diameter between 100 nm and 1000 nm and a polydispersity lower than 0.25.

[0303] In the present specification and claims, the term "plurality of biphasic nanodroplets" refers to a population of nanodroplets characterized by a calibrated distribution, meaning that substantially all nanodroplets have substantially similar sizes.

[0304] The expression "calibrated distribution" denotes a polydispersity of a population of nanodroplets (e.g. having a z-average diameter between 100 and 1000 nm) having a polydispersity index (PDI) below 0.25, preferably below 0.2, more preferably below 0.15, even more preferably below 0.1.

[0305] The skilled person is aware that the above values ​​are subject to experimental variations, which may limit their accuracy. For this reason, in the present invention, the error associated with the experimental values ​​is set to ±15%.

[0306] The term "polydispersity" (PDI) refers to a dimensionless measure of the breadth of the size distribution calculated from the cumulant analysis defined in the international standards for dynamic light scattering ISO 13321 (1996) and ISO 22412 (2008), which gives an estimate of the median particle size (z-average) and the breadth of the distribution (polydispersity index).

[0307] For example, a polydispersity above 0.7 indicates a very broad distribution of particle sizes, whereas a value below 0.08 indicates an almost monodisperse sample characterized by a unimodal distribution. The polydispersity can be measured by using, for example, a Malvern Zetasizer Nano-ZS instrument (Malvern Instruments Ltd., UK) employing the dynamic light scattering technique (DLS).

[0308] The "z-average diameter (ZD)" is defined as the intensity-weighted mean diameter derived from cumulant analysis. In other words, it relates to the average of the calibrated nanodroplet sizes dispersed in an aqueous suspension measured by dynamic light scattering (DLS).

[0309] In the present invention, the z-average diameter is between 100 nm and 1000 nm, preferably between 120 and 800, more preferably between 150 and 400.

[0310] Suitable aqueous carriers for aqueous suspensions of the present invention are preferably physiologically acceptable and include water (preferably sterile water), aqueous solutions such as saline (which may be advantageously balanced so that the final product for injection is not hypotonic) or solutions of one or more tonicity-adjusting substances. Tonicity-adjusting substances include salts or sugars, sugar alcohols, glycols or other nonionic polyol substances (e.g., glucose, sucrose, trehalose, 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.

[0311] In the present invention, calibrated two-phase nanodroplets are preferably produced by using microfluidics technology.

[0312] stability

[0313] The Applicant has now surprisingly found that the stability of the disclosed calibrated biphasic nanodroplets can be substantially improved by adding an oil as defined above to the core of said nanodroplets.

[0314] In the present specification and claims, the term "stability" refers to the property of a nanodroplet composition to substantially maintain its initial ND size over time, and preferably also maintain its initial monodisperse distribution.

[0315] Initial ND size and initial monodisperse distribution refer to the values ​​of ND size and monodispersity of the calibrated ND composition at the end of the preparation process.

[0316] For the sake of clarity, the expression "end of the preparation process" refers to i) collecting the calibrated NDs from the outlet channel of the microfluidic cartridge or ii) collecting the calibrated NDs and then performing a dilution step (ie, step e).

[0317] Both alternative final steps were performed prior to any storage period of the calibrated NDs.

[0318] The expression "shelf life" denotes a period of time, for example expressed in hours, days or weeks, during which a microfluidically prepared aqueous suspension of calibrated NDs is maintained under certain temperature conditions after the preparation process has been completed.

[0319] The stability of the calibrated nanodroplets can be calculated by using the ND size evolution (%Evol) parameter according to the following equation (Eq. 3):

[0320]

[0321] in:

[0322] Dfinal is the z-average diameter of the calibrated NDs after a period of time from the end of the manufacturing process, for example 60 minutes from the end of the manufacturing process or after a storage period (e.g. one week) under different conditions (different temperature, pressure, etc.); and

[0323] - Dinitial is the z-average diameter of the NDs calibrated immediately (eg, within a few minutes) at the end of their preparation process.

[0324] In the present invention, %Evol values ​​(positive or negative) close to 0 indicate a higher stability of the calibrated ND suspension, so that the nanodroplets in the suspension substantially maintain their initial median size over time.

[0325] According to the present invention, the % Evol of the calibrated ND suspension is preferably below ±50%, more preferably below ±30%, and still more preferably below ±20%.

[0326] Applicants have surprisingly discovered that the presence of oil into the core of the nanodroplets allows to reduce the %Evol and therefore improves the stability of the calibrated NDs stabilized by BFS.

[0327] Applicants have found that the %Evol parameter is significantly correlated with the presence of biocompatible oils in the core composition of the nanodroplets. Improved stability can be achieved by using biocompatible oils with a logP above 5, preferably above 7, even more preferably above 9, up to 25.

[0328] Furthermore, it has been observed that using a suitable microfluidic preparation method it is possible to obtain ready-to-use suspensions of calibrated biphasic nanodroplets characterized by good PDI values ​​and improved stability over time without the need to additionally submit the collected preparation to further treatments such as centrifugation.

[0329] Microfluidics Box

[0330] The calibrated biphasic NDs of the present invention are preferably produced by a bottom-up approach using microfluidics technology.

[0331] In this specification and claims, the expression "microfluidics" refers to a technique for manufacturing nanodroplets by means of a microfluidics cassette designed to manipulate fluids in channels on a microscale.

[0332] The microfluidics technique is a bottom-up approach, that is, nanodroplets are obtained by assembling molecules (such as BFS, biocompatible oils and fluorinated compounds) into larger nanostructures (ie, biphasic nanodroplets).

[0333] Figure 1 A schematic diagram of the core part 100 of a microfluidic cartridge that can be used in the method of the present invention is shown. The cartridge comprises a first inlet 101 for supplying an aqueous phase 101' and a second inlet 102 for supplying an organic phase 102'. The aqueous and organic phases are directed to a mixing device 103, e.g. Figure 2 The staggered herringbone micromixer 203 shown in FIG. 2 is used to mix the micromixers (e.g., Figure 2 In the case of a micromixer, ND is formed by laminar mixing.

[0334] The calibrated fluorinated compounds ND are then directed to the outlet channel 104 from where they are collected in a suitable container (eg a vial).

[0335] Alternatively, the microfluidic cartridge may be equipped with an additional channel, for example placed between the mixing device 103 and the outlet channel 104, intended to dilute the calibrated fluorinated compound ND suspension with a suitable solvent before directing them to the outlet channel 104 (ie in-line dilution).

[0336] The mixing device 103 is generally characterized by a suitable geometry that enhances the microfluidic mixing performance. In fact, the mixing process takes place in the special microchannel geometry of the mixing device, which causes the fluid streams to mix together on the way out of the microfluidic cartridge.

[0337] Different types of mixing devices are available in different shapes or microstructures. Suitable examples of mixing devices can be classified into passive micromixers, such as T- and Y-shaped mixers (e.g., staggered herringbone micromixers or annular mixers) and mixers using flow focusing; and active micromixers, such as mixers using pressure field perturbations, electrodynamic active micromixers, and ultrasonic active micromixers.

[0338] The present invention preferably uses a staggered herringbone micro mixer ( Figure 2 ), in which the mixing of the two liquid phases is controlled by laminar mixing or annular micromixers.

[0339] During the mixing phase, biphasic nanodroplets are formed and directed to the outlet channel of the microfluidic cartridge, or alternatively, directed to another channel to dilute the nanodroplets before directing them to the outlet channel.

[0340] In the present specification and claims, the expression "exit / outlet channel" refers to the end portion of the microfluidic box, to which the newly formed nanodroplets are guided from the mixing device and from which the formed nanodroplet suspension can be collected into a suitable container (e.g., a vial).

[0341] Typically, the operating pressure into the microfluidic box is less than 1000psi (about 7000kPa), preferably less than 500psi (about 3500kPa), more preferably less than 300psi (about 2000kPa), and more preferably less than 100psi (about 700kPa), for example between 10psi and 90psi.

[0342] An example of a microfluidic cartridge is the commercially available NxGen cartridge (with or without in-line dilution) from Precision Nanosystems (Vancouver, Canada). These microfluidic cartridges may include staggered herringbone or annular micromixers, both of which operate under non-turbulent conditions. For the manufacturing process, the microfluidic cartridge is mounted on a microfluidic instrument, which is typically equipped with a cartridge adapter to receive the microfluidic cartridge and has a container (e.g., a syringe or vial for continuous flow injection) directly connected to the microfluidic cartridge inlet and specially designed to pump the liquid phase into the inlet. An example of a microfluidic instrument is the Nano Benchtop automated instrument (Precision Nanosystems (Vancouver, Canada)).

[0343] One aspect of the invention relates to a method for preparing an aqueous suspension comprising a plurality of nanodroplets, the nanodroplets comprising an outer layer and an inner core, the outer layer comprising a biocompatible fluorinated surfactant as defined above, and the inner core comprising a fluorinated compound and a biocompatible oil having a logP value higher than 5, the method comprising the following steps:

[0344] a) preparing an aqueous phase;

[0345] b) preparing an organic phase,

[0346] in

[0347] i) the aqueous phase comprises a biocompatible fluorinated surfactant as defined above or a mixture thereof, and the organic phase comprises a fluorinated compound and a biocompatible oil having a logP value greater than 5, or

[0348] ii) the organic phase comprises a biocompatible fluorinated surfactant as defined above or a mixture thereof, a fluorinated compound and a biocompatible oil having a logP value higher than 5;

[0349] c) injecting the aqueous phase into a first inlet of a microfluidic cartridge and injecting the organic phase into a second inlet of a microfluidic cartridge, thereby mixing the aqueous phase and the organic phase in a mixing device of the microfluidic cartridge, wherein the working pressure into the microfluidic cartridge is lower than 7000 kPa, to obtain the aqueous suspension, and

[0350] d) collecting the aqueous suspension from the outlet channel of the microfluidic cartridge.

[0351] According to a preferred embodiment, the aqueous phase comprises a biocompatible fluorinated surfactant selected from Dendri-TAC, F-TAC or mixtures thereof, and the organic phase comprises a fluorinated compound and a biocompatible oil having a logP value higher than 5.

[0352] Preferably, the fluorinated compound is a perfluorocarbon.

[0353] Preferably, the biocompatible oil is a triglyceride having a logP value greater than 5.

[0354] Preferably, the triglycerides have a logP value higher than 7, still more preferably higher than 9, up to 25.

[0355] Preferably, the triglyceride is selected from glyceryl tricaprylate, glyceryl trilaurate, glyceryl trioleate, glyceryl trilinoleate or a mixture thereof, preferably glyceryl tricaprylate.

[0356] Preferably, the aqueous suspension comprises a plurality of nanodroplets as defined above, wherein the nanodroplets have a z-average diameter between 100 nm and 1000 nm and a polydispersity below 0.25, preferably below 0.20, more preferably below 0.15, even more preferably below 0.1.

[0357] In another embodiment, the biocompatible oil is a mixture of oils.

[0358] In one embodiment, the mixture comprises at least one oil having a logP value above 5, and at least one oil having a logP value below 5.

[0359] Preferably, the amount of oil having a logP value higher than 5 is higher than 75 mol%, more preferably higher than 90%.

[0360] Preferably, the amount of oil having a logP value below 5 is at most 25 mol%, more preferably at most 10%.

[0361] Typically, the injection of the aqueous phase and the injection of the organic phase are carried out simultaneously in step c).

[0362] The expression "simultaneously" means that the aqueous phase and the organic phase are injected into the microfluidic box at the same time (ie, co-injected), that is, the aqueous phase and the organic phase are injected into two separate inlets of the microfluidic box at the same time or substantially the same time (eg, within a few seconds).

[0363] According to the disclosed method, a calibrated aqueous suspension of biphasic nanodroplets can be obtained by a single pass of the liquid phase through a dual-channel microfluidic system.

[0364] In one embodiment, the method for preparing an aqueous suspension of calibrated two-phase nanodroplets is microfluidics, wherein the calibrated nanodroplets (z-average diameter between 100 and 1000 nm) have a polydispersity index (PDI) below 0.25, preferably below 0.20, more preferably below 0.15, and even more preferably below 0.1.

[0365] Advantageously, the novel method can be used to prepare aqueous suspensions of calibrated nanodroplets stabilized by any other biocompatible surfactants besides Dendri-TAC and FTAC, in particular biocompatible fluorinated surfactants, when the surfactant is dissolved in the aqueous phase.

[0366] As mentioned above, the expression "biocompatible fluorinated surfactant" refers to an amphiphilic organic compound suitable for forming a nanodroplet stabilization layer, comprising a hydrophilic portion and a hydrophobic portion. The amphiphilic organic compound has substantial compatibility with living tissues or living systems, has no toxicity, harmfulness or physiological reactivity, and typically does not cause immune rejection.

[0367] Another aspect of the present invention relates to a method for preparing an aqueous suspension comprising a plurality of nanodroplets, the nanodroplets comprising an outer layer and an inner core, the outer layer comprising a biocompatible fluorinated surfactant, and the inner core comprising a fluorinated compound and a biocompatible oil having a logP value greater than 5, the method comprising the steps of:

[0368] a) preparing an aqueous phase comprising a biocompatible fluorinated surfactant;

[0369] b) preparing an organic phase comprising a fluorinated compound and a biocompatible oil having a logP value greater than 5;

[0370] c) injecting the aqueous phase into a first inlet of a microfluidic cartridge, and injecting the organic phase into a second inlet of a microfluidic cartridge, thereby mixing the aqueous phase and the organic phase in a mixing device of the microfluidic cartridge, wherein the operating pressure into the microfluidic cartridge is lower than 7000 kPa, to obtain the aqueous suspension; and

[0371] d) collecting the aqueous suspension from the outlet channel of the microfluidic cartridge.

[0372] Preferably, the aqueous suspension comprises a plurality of nanodroplets as defined above, wherein the nanodroplets have a z-average diameter between 100 nm and 1000 nm and a polydispersity of less than 0.25, preferably less than 0.20, more preferably less than 0.15, even more preferably less than 0.1.

[0373] Water Phase

[0374] The expression "aqueous phase" refers to a liquid comprising an aqueous liquid component, including, for example, water, an aqueous buffer solution, an aqueous isotonic solution or a mixture thereof. Preferably, the aqueous phase is water.

[0375] According to a preferred embodiment, the aqueous phase comprises a biocompatible fluorinated surfactant chosen from the amphiphilic linear oligomers F-TAC and the amphiphilic dendrimers Dendri-TAC as defined above, or mixtures thereof.

[0376] For example, a biocompatible fluorinated surfactant can be mixed with an aqueous component by conventional techniques (eg, stirring) to prepare an aqueous phase to be injected into a first inlet of a microfluidic cartridge.

[0377] At step a), the aqueous phase comprises the biocompatible fluorinated surfactant in a concentration range between 0.0006 mmol / mL and 0.006 mmol / mL, more preferably between 0.0001 mmol / mL and 0.015 mmol / mL, still more preferably between 0.001 mmol / mL and 0.01 mmol / mL.

[0378] Organic phase

[0379] The expression "organic phase" refers to a liquid comprising an organic solvent miscible with water, including methanol, ethanol, isopropanol, acetonitrile and acetone. Preferably, the organic phase is ethanol.

[0380] According to a preferred embodiment, the organic phase comprises:

[0381] - a fluorinated compound or a mixture of different fluorinated compounds, and

[0382] - a biocompatible oil or a mixture of different biocompatible oils having a logP higher than 5,

[0383] wherein the biocompatible oil is substantially insoluble in the fluorinated compound.

[0384] Due to its solubility properties, the biocompatible oil forms a separate phase in the inner core of the nanodroplet, which is thus divided into two distinct phases: a liquid phase containing the fluorinated compound and an oil phase.

[0385] Preferably, the fluorinated compound is a perfluorocarbon.

[0386] Preferably, the biocompatible oil has a logP value higher than 5, more preferably higher than 7, even more preferably higher than 9, up to 25. Even more preferably, the biocompatible oil is a triglyceride.

[0387] Suitable examples of fluorinated compounds and biocompatible oils are those mentioned above.

[0388] As an example, the fluorinated compound and the biocompatible oil can be mixed with an organic solvent by conventional techniques (eg, stirring) to prepare an organic phase to be injected into the second inlet of the microfluidic cartridge.

[0389] In one embodiment, in step b), the organic phase comprises the fluorinated compound in a concentration range between 0.003 mmol / mL and 0.142 mmol / mL, more preferably between 0.011 mmol / mL and 0.085 mmol / mL, still more preferably between 0.013 mmol / mL and 0.057 mmol / mL.

[0390] In another embodiment, in step b), the organic phase comprises the biocompatible oil in a concentration range between 0.1 μmole / mL and 40 μmole / mL, more preferably between 0.5 μmole / mL and 25 μmole / mL, still more preferably between 1 μmole / mL and 15 μmole / mL.

[0391] In an alternative embodiment, the organic phase comprises:

[0392] - a biocompatible fluorinated surfactant as defined above,

[0393] - fluorinated compounds, and

[0394] - Biocompatible oil.

[0395] For example, the organic phase comprises

[0396] i) a biocompatible fluorinated surfactant in a concentration range between 0.0006 mmol / mL and 0.006 mmol / mL, more preferably between 0.0001 mmol / mL and 0.015 mmol / mL, still more preferably between 0.001 mmol / mL and 0.01 mmol / mL;

[0397] ii) a fluorinated compound in a concentration range between 0.003 mmol / mL and 0.142 mmol / mL, more preferably between 0.011 mmol / mL and 0.085 mmol / mL, still more preferably between 0.013 mmol / mL and 0.057 mmol / mL; and

[0398] iii) a biocompatible oil having a concentration range between 0.1 μmole / mL and 40 μmole / mL, more preferably between 0.5 μmole / mL and 25 μmole / mL, still more preferably between 1 μmole / mL and 15 μmole / mL.

[0399] In yet another embodiment of the present invention, both the aqueous phase and the organic phase are preferably injected into the microfluidic cartridge at a temperature below room temperature (eg, from about 4°C to 20°C) to avoid vaporization of fluorinated compounds having a boiling point close to room temperature.

[0400] Another aspect relates to an aqueous suspension comprising a plurality of nanodroplets, said nanodroplets comprising an outer layer and an inner core, said outer layer comprising a biocompatible fluorinated surfactant as defined above, said inner core comprising a fluorinated compound and a biocompatible oil having a logP value greater than 5, said aqueous suspension being obtainable by a preparation method comprising the following steps:

[0401] a) preparing an aqueous phase;

[0402] b) preparing an organic phase,

[0403] wherein i) the aqueous phase comprises a biocompatible fluorinated surfactant selected from Dendri-TAC, F-TAC or mixtures thereof, and the organic phase comprises a fluorinated compound and a triglyceride having a logP value greater than 5, or

[0404] ii) the organic phase comprises a biocompatible fluorinated surfactant selected from Dendri-TAC, F-TAC or a mixture thereof, a fluorinated compound and a triglyceride having a logP value higher than 5.

[0405] c) injecting the aqueous phase into a first inlet of a microfluidic cartridge, and injecting the organic phase into a second inlet of a microfluidic cartridge, thereby mixing the aqueous phase and the organic phase in a mixing device of the microfluidic cartridge, wherein the operating pressure into the microfluidic cartridge is lower than 7000 kPa, to obtain the aqueous suspension; and

[0406] d) collecting the aqueous suspension from an outlet channel of the microfluidic cartridge; and

[0407] Preferably, the triglycerides have a logP value higher than 7, still more preferably higher than 9, up to 25.

[0408] In a preferred embodiment, the triglyceride is selected from tricaprylin, trilaurin, triolein, trilinolein or a mixture thereof, preferably tricaprylin.

[0409] In a preferred embodiment, the aqueous phase comprises a biocompatible fluorinated surfactant selected from Dendri-TAC, F-TAC or mixtures thereof, and the organic phase comprises a fluorinated compound (preferably a perfluorocarbon) and a triglyceride having a logP value higher than 5.

[0410] In one embodiment, the organic phase further comprises a hydrophobic drug.

[0411] Preferably, the aqueous suspension comprises a plurality of nanodroplets as defined above, wherein the nanodroplets have a z-average diameter between 100 nm and 1000 nm and a polydispersity below 0.25, preferably below 0.20, more preferably below 0.15, even more preferably below 0.1.

[0412] Total Flow Rate (TFR) and Flow Rate Ratio (FRR)

[0413] The method of the present invention allows the properties of the two-phase nanodroplets to be controlled by varying two process parameters: the total flow rate and the flow rate ratio.

[0414] Expression " Total Flow Rate (TFR) " refers to the total flow rate of two fluid streams, namely the aqueous phase and the organic phase, pumped through two separate inlets of the microfluidic cartridge. The unit of measurement of TFR is mL / min.

[0415] According to one embodiment, the TFR is preferably between 2 mL / min and 18 mL / min, more preferably between 5 mL / min and 16 mL / min, still more preferably, the TFR is 10 mL / min.

[0416] The expression "flow rate ratio (FRR)" refers to the ratio between the amount of aqueous phase flowing into the microfluidic cartridge and the amount of organic phase, according to Equation 4:

[0417]

[0418] The volumes of the aqueous and organic phases may be expressed, for example, in mL.

[0419] In a preferred embodiment, the FRR (volume of aqueous phase to volume of organic phase) is between 1:1 and 5:1, preferably between 1:1 and 3:1, more preferably the FRR is 1:1.

[0420] In the present invention, the respective concentrations of the biocompatible fluorinated surfactant, the fluorinated compound and the biocompatible oil can be purposefully adjusted to obtain specific molar ratios between the biocompatible fluorinated surfactant and the fluorinated compound and between the biocompatible oil and the fluorinated compound.

[0421] Advantageously, at the end of the disclosed method, a ready-to-use composition of calibrated biphasic nanodroplets can be successfully obtained, characterized by an improved stability over time.

[0422] Applicants have demonstrated that stable calibrated biphasic nanodroplets can be generated through a single entry into a microfluidic cartridge without the need for additional purification processes such as multiple centrifugation treatments reported in the prior art.

[0423] The calibrated biphasic nanodroplets obtained by the disclosed microfluidic method exhibit advantages over similar preparations obtained by conventional preparation methods, such as increased monodispersity and improved stability. In addition, the disclosed microfluidic method provides for the collection of the monodisperse and stable calibrated biphasic nanodroplets directly from the microfluidic cartridge without any purification steps, such as centrifugation procedures.

[0424] Optional step e) dilution

[0425] According to the invention, the preparation method further comprises an optional step e) which consists in diluting the collected aqueous suspension of calibrated biphasic nanodroplets.

[0426] In this specification and claims, the term "dilution" refers to the process of reducing the concentration of calibrated nanodroplets in a suspension by adding a suitable amount of an aqueous liquid, including water or an aqueous solution.

[0427] A suitable amount of aqueous liquid corresponds to the amount of aqueous liquid required to reduce the concentration of calibrated nanodroplets in the aqueous suspension by a factor of 2 to 10.

[0428] In a preferred embodiment, optional step e) of the method comprises diluting the collected aqueous suspension of calibrated nanodroplets 1 to 20 times, preferably 3 to 8 times, still more preferably diluting the collected aqueous suspension 5 times.

[0429] An additional effect of dilution is to reduce the relative amount of organic solvent in the suspension.

[0430] In another preferred embodiment, step e) of the method comprises diluting the collected suspension of calibrated nanodroplets with water.

[0431] As mentioned above, due to the additional channels (e.g. Figure 1 The dilution step can be optionally performed within the microfluidic cartridge (placed between the mixing device 103 and the outlet channel 104) is intended to dilute the calibrated ND suspensions before they are directed to the outlet channel.

[0432] In this case, step e) of the method comprises diluting the suspension of calibrated nanodroplets 1 to 20 times, preferably 3 to 8 times, and even more preferably diluting the collected aqueous suspension 5 times before collection from the microfluidic cartridge.

[0433] Sonoinduced droplet vaporization

[0434] The expression "acoustic droplet vaporization (ADV)" refers to the phase transition of the inner core of a nanodroplet comprising a fluorinated compound as defined above from the liquid state to the gaseous state due to the application of ultrasonic energy exceeding the vaporization threshold.

[0435] The ultrasound acts as an external stimulus to promote vaporization of droplets in a controlled, non-invasive and localized manner.

[0436] Below the vaporization threshold, nanodroplets are ultrasonically stable, have low acoustic attenuation, and can be sonicated at locations of interest. Due to their smaller size and volume compared to conventional microbubbles, nanodroplets exhibit prolonged in vivo circulation, penetrating deeply into tissues via the extravascular space (Helfield et al., 2020).

[0437] use

[0438] Calibrated biphasic nanodroplets can replace gaseous microbubbles for medical (therapeutic) ultrasound applications. Upon application of ultrasound energy, the droplets can selectively vaporize to form microbubbles in the area of ​​interest. After activation, the calibrated biphasic nanodroplets can be used in essentially the same manner as conventional contrast-enhanced ultrasound (CEUS).

[0439] One aspect relates to an aqueous suspension obtainable according to the method defined above for use in diagnostic and / or therapeutic treatment.

[0440] Diagnostic examinations include any method that allows enhanced visualization of a part or region of an animal (including human) body using biphasic nanodroplets, including imaging for preclinical and clinical studies. Suitable examples of diagnostic applications are molecular and perfusion imaging, tumor imaging (EPR effect), multimodal imaging (MR-guided tumor ablation, fluorescence, photoacoustic activation).

[0441] Therapeutic treatment includes any method of treating a patient. In preferred embodiments, the treatment includes the combined use of ultrasound and biphasic nanodroplets themselves (e.g., in ultrasound-mediated thrombolysis, high-intensity focused ultrasound ablation, blood-brain barrier permeabilization, immunomodulation, neuromodulation, radiosensitization) or in combination with therapeutic agents (i.e., ultrasound-mediated delivery, such as delivery of drugs or biologically active compounds to selected sites or tissues, such as in tumor therapy, gene therapy, infectious disease therapy, metabolic disease therapy, chronic disease therapy, degenerative disease therapy, inflammatory disease therapy, immune or autoimmune disease therapy or as a vaccine), wherein the presence of the nanodroplets itself can provide a therapeutic effect or can enhance the therapeutic effect of the applied ultrasound, such as by applying or being responsible for applying a biological effect in vitro and / or in vivo, whether by itself or specifically activated by various physical methods (including, for example, ultrasound-mediated delivery).

[0442] The following examples will help to further explain the invention.

[0443] Example

[0444] Materials and methods

[0445] The following materials were used in the subsequent examples:

[0446]

[0447] Example 1

[0448] Fabrication of biphasic nanodroplets using a microfluidic platform

[0449] Using Nano Assemblr from Precision Nanosystems (Vancouver, Canada) TMBiphasic nanodroplets were formulated using a benchtop automated instrument equipped with a staggered herringbone micromixer (SHM) that allows size-controlled self-assembly. Briefly, an aqueous phase containing a biocompatible fluorinated surfactant (BFS) was injected into the first inlet, while an organic phase consisting of a perfluorocarbon (PFC) and a biocompatible oil (both dissolved in ethanol) was injected into the second inlet of the microfluidic cartridge ( Figure 1 ). Prior to ND formulation, the two phases were placed in an ice bath at approximately 4°C. The microscopic properties of the channel were designed to induce accelerated mixing of the two fluid streams in a controlled manner. The microfluidic process settings, namely the total flow rate (TFR, in mL / min) and the flow rate ratio (FRR), were varied to control the ND properties. The ND suspension was collected from the outlet channel into a Falcon bottle (15 mL) and diluted 5-fold with ultrapure water.

[0450] Example 2

[0451] Effect of the presence of biocompatible oil in the core of the calibrated nanodroplets

[0452] The effect of the presence of a biocompatible oil in the inner core of nanodroplets obtained in a microfluidic manner was investigated.

[0453] For this purpose, several compositions comprising nanodroplet suspensions stabilized by the Dendri-TAC surfactant DiF6DiTAC7 and having a core comprising perfluoropentane as PFC were prepared and characterized with and without the addition of tricaprylin as biocompatible oil.

[0454] The compositions were prepared as described in Example 1 and the process parameters were set to obtain different molar ratios between BFS and PFC for each composition (from low to high). For this purpose, three different FRRs were tested: 3:1, 2:1 and 1:1. The TFR was 10 mL / min. The concentration of BFS in the aqueous phase was 1 mg / mL and the concentration of perfluoropentane in the organic phase was 10 μL / mL.

[0455] For formulations containing biocompatible oils, three different concentrations of tricaprylin in the organic phase, 2 μL / mL, 5 μL / mL and 10 μL / mL, were tested and compared to formulations without oil. As described in Example 1, to obtain calibrated biphasic nanodroplets, the biocompatible oil was added to the organic phase together with the selected perfluorocarbon before injection into the microfluidic cartridge.

[0456] A summary of all the compositions obtained is listed in Table 2, where the amount of biocompatible oil, the BFS / PFC molar ratio and the respective FRR are shown in the first left column.

[0457] The obtained formulations were characterized using a Malvern Zetasizer Nano-ZS instrument (Malvern Instruments Ltd., UK) to measure size (z-average) and polydispersity (PDI) over time after storage at 4°C for one week.

[0458] Each measurement was performed at room temperature (ie 25°C).

[0459] result

[0460] Table 2a Effect of the presence of biocompatible oil in the core of the calibrated nanodroplets

[0461]

[0462] The overall results of the study of the effect of adding biocompatible oils in the core of calibrated nanodroplets are presented in Table 2, which shows the comparison between the size and PDI characterization of different compositions obtained by microfluidics.

[0463] In particular, each composition was prepared by adding tricaprylic acid (compositions 1B-D, 2B-D and 3B-D) or not (compositions 1A, 2A and 3A) to the organic phase to observe any effect originating from the presence of oil in the inner core of the nanodroplets. In addition, three different amounts of oil (2, 5 and 10 μL / mL) were tested to study the effect of the amount of oil on the properties of the nanodroplets.

[0464] The change in ND size over time is expressed as % Evol, calculated according to Equation 4, as described in the specification section.

[0465] %Evol values ​​close to 0 indicate a high stability of the calibrated ND suspension, whereby the nanodroplets in the suspension substantially maintain their initial median size over time.

[0466] The results show that the addition of biocompatible oils imparted improved ND stability over time (ie, lower % Evol) in all compositions tested compared to compositions containing only perfluorocarbons in the core.

[0467] It was particularly observed that despite different molar ratios between BFS and PFC and different amounts of biocompatible oil, similar stability was achieved for all compositions comprising biocompatible oil.

[0468] In addition, the biocompatible oil / fluorinated surfactant volume ratio of Composition 1 was determined (Table 2b).

[0469] From the results it can be inferred that the volume ratio between the biocompatible oil and the fluorinated compound is quite high, with the biocompatible oil occupying up to 50% of the core volume.

[0470] Table 2b Molar and volume ratios between biocompatible oils and fluorinated compounds

[0471]

[0472] Example 3

[0473] Effects of biocompatible fluorinated surfactant properties on the stability of calibrated biphasic nanodroplets

[0474] To investigate the effect of biocompatible fluorinated surfactants on ND size and size distribution, different compositions containing different BFS were tested, namely:

[0475] - Compositions 1-6: comprising a suspension of nanodroplets stabilized by the Dendri-TAC surfactant DiF6DiTAC7 and having a core comprising perfluoropentane as PFC and tricaprylin as biocompatible oil, and

[0476] - Compositions 7-12: comprising F-TAC surfactant F8TAC 18 Stable nanodroplet suspensions with a core comprising perfluoropentane as the PFC and tricaprylin as the biocompatible oil.

[0477] All the compositions studied were prepared as described in Example 1 and the processing parameters were set to obtain different molar ratios between BFS and PFC for each composition (from low to high). For this purpose, three different FRRs were tested: 3:1, 2:1 and 1:1. The TFR was 10 ml / min. For all compositions 1-12, the concentrations of perfluoropentane and tricaprylin in the organic phase were 10 μL / mL and 5 μL / mL, respectively. Two different concentrations of BFS in the aqueous phase were tested, namely 1 mg / mL (to obtain a lower molar ratio BFS / PFC) and 5 mg / ml (to obtain a higher molar ratio BFS / PFC).

[0478] A summary of all compositions obtained is listed in Table 3 (compositions 1-6) and Table 4 (compositions 7-12), wherein the amount of biocompatible oil, the BPF / PFC molar ratio and the respective FRR are shown in the first left column.

[0479] The resulting formulations were characterized using a Malvern Zetasizer Nano-ZS instrument (Malvern Instruments Ltd., 10 UK) to measure size (z-average) and polydispersity (PDI) over time after storage at 4°C for one week.

[0480] Each measurement was performed at room temperature (ie 25°C).

[0481] result

[0482] Tables 3 and 4 report the results obtained from the characterization of compositions 1-12 prepared in a microfluidic manner.

[0483] To observe any effects arising from the presence of oil in the inner core of the nanodroplets, each composition was prepared with or without the addition of tricaprylin (compositions 1-6C) to the organic phase.

[0484] It was observed that using both DendriTAC and FTAC as BFS to stabilize the shell of calibrated biphasic nanodroplets allowed obtaining monodisperse ND suspensions characterized by uniform nanodroplets with a size range of approximately 150-700 nm.

[0485] The change in ND size over time is expressed as % Evol, calculated according to Equation 4, as described in the specification section.

[0486] %Evol values ​​close to 0 indicate a high stability of the calibrated ND suspension, whereby the nanodroplets in the suspension substantially maintain their initial median size over time.

[0487] The results show that the addition of biocompatible oils imparted improved ND stability over time in all compositions tested (ie, low % Evol) compared to compositions containing only perfluorocarbons in the core (higher % Evol values).

[0488] In particular, it was observed that, despite different molar ratios between BFS and PFC and different amounts of biocompatible oil, similar stability was achieved for all compositions stabilized by different biocompatible fluorinated surfactants and comprising a biocompatible oil.

[0489] Table 3 Effect of the oil phase on the composition of DendriTAC stabilized calibrated nanodroplets

[0490]

[0491] Table 4 Effect of the oil phase on the composition of FTAC-stabilized calibrated nanodroplets

[0492]

[0493]

[0494] Example 4

[0495] Effects of biocompatible oil properties on the stability of calibrated biphasic nanodroplets

[0496] In order to study the influence of the properties of biocompatible oils on the stability of NDs, several compositions of biphasic nanodroplets containing different biocompatible oils were studied. For this purpose, two oils characterized by different lipophilic properties were selected, namely tricaprylic acid glyceryl (XLogP3-AA = 8.9, calculated by XLogP3 3.0 (PubChem 2021.05.07 release)) and tributyrin (XLogP3-AA = 2.4, calculated by XLogP3 3.0 (PubChem 2021.05.07 release)).

[0497] The following compositions were compared:

[0498] - Compositions 7C, 9C and 11C: containing F-TAC surfactant F8TAC 18 Stable nanodroplet suspensions with a core containing perfluoropentane as PFC and tricaprylin (5 μL / mL) as a biocompatible oil;

[0499] - Compositions 13, 14 and 15: comprising F-TAC surfactant F8TAC 18 Stable nanodroplet suspension with a core containing perfluoropentane as PFC and tributyrin (5 μL / mL) as biocompatible oil.

[0500] All the above compositions were prepared as described in Example 1 and the processing parameters were set to obtain different molar ratios (from low to high) between BFS and PFC for each composition (i.e. 0.005, 0.010 and 0.015). Three different FRRs were tested: 3:1, 2:1 and 1:1.

[0501] The samples were characterized using a Malvern Zetasizer Nano-ZS instrument to determine ND size (z-average) and polydispersity (PDI) over time after storage at 4°C for one week.

[0502] Each measurement was performed at room temperature (ie 25°C).

[0503] Table 5 shows the range of BFS / PFC molar ratios evaluated as a function of the selected FRR.

[0504] result

[0505] Table 5 Effect of biocompatible oil properties on calibration stability

[0506]

[0507] Table 5 reports the results obtained by characterizing different calibrated biphasic nanodroplet compositions containing different biocompatible oils.

[0508] In particular, the effect of oil on the stability of the nanodroplets over time was studied and the results are expressed as % Evol, calculated according to Equation 3, as described in the description section.

[0509] The overall findings indicate that the presence of a biocompatible oil with a higher lipophilic character, such as tricaprylin (XLogP3-AA=8.9), confers significantly improved stability over time compared to a less lipophilic oil, such as tributyrin (XLogP3-AA=2.4).

[0510] In fact, compositions 7C, 9C and 11C comprising tricaprylin are characterized by %Evol values ​​closer to 0 than those measured for compositions 13, 14 and 15 comprising tributyrin, indicating a higher stability of the calibrated ND suspension, whereby the nanodroplets in the suspension substantially maintain their initial median size over time.

[0511] Example 5

[0512] Encapsulation of dyes into calibrated biphasic nanodroplets

[0513] The ability of the disclosed calibrated biphasic nanodroplets to encapsulate dyes into the inner core was evaluated.

[0514] For this purpose, composition 4-6C was selected to load the fluorescent DIO dye. Composition 4-6C was prepared as described in Example 1, and DIO dye was additionally added to the organic phase at a concentration of 0.0625 mg / mL, which was then injected into the microfluidic box. The processing parameters were set to obtain different molar ratios between BFS and PFC for each composition (from low to high).

[0515] Three different FRRs were tested: 3:1, 2:1, and 1:1.

[0516] At the end of the microfluidic preparation, the nanodroplet composition was centrifuged at 5000 g for 12 min to separate the particles containing nanodroplets encapsulating DIO from the nanodroplet-free supernatant containing non-encapsulated DIO.

[0517] Both the pellet and the supernatant were then analyzed in order to assess the amount of DIO encapsulated into the nanodroplets using the fluorometer Cytation 5 (Cell Imaging Multimode Reader)

[0518] result

[0519] Table 6 shows the overall results obtained by determining the amount of DIO encapsulated in the calibrated biphasic nanodroplets.

[0520] All investigated compositions were able to incorporate DIO into the particles, confirming the encapsulation capability of the disclosed calibrated biphasic nanodroplets to form particles.

[0521] Table 6 Amount of DIO encapsulated into calibrated biphasic nanodroplets

[0522] FRR Molar ratio BFS / PFC Amount of encapsulated dye nr ％ Composition 4C 1:1 0.005 48 Composition 5C 2:1 0.010 30 Composition 6C 3:1 0.015 28

[0523] In addition, the amount of DIO dye encapsulated into the nanodroplets (particles) was evaluated to be between 28% and 48% of the amount of DIO added during nanodroplet preparation. This amount is a significant improvement compared to the work of Al Rifai et al., 2020, indicating that the use of the disclosed microfluidic method can reduce the amount of dye lost during nanodroplet preparation, which is usually caused by the centrifugation process.

[0524] Example 6

[0525] Measurement of acoustic droplet vaporization (ADV)

[0526] The expression "acoustic droplet vaporization (ADV) threshold" refers to the minimum acoustic pressure required to transform nanodroplets into echogenic microbubbles.

[0527] The acoustic droplet vaporization (ADV) threshold of the calibrated biphasic ND prepared according to the aforementioned embodiment can be determined according to conventional methodology using B-mode imaging methods. For example, a suspension of NDs can be vaporized when passing through the focal zone of the transducer, and the acoustic pressure is increased by about 0.2 MPa every 5 seconds until ND vaporization is observed.

[0528] Nanodroplet activation was performed by focused ultrasound at five aligned focal points, allowing activation only in the region of interest where the acoustic pressure was highest. Pulses were fired in burst mode at a frequency of 6 MHz, with 20 cycles per pulse and a pulse repetition frequency (PRF) of 1 Hz.

[0529] The acoustic pressure was increased every 5 s until vaporization of ND was observed.

[0530] To determine ADV, two different compositions were tested: Composition 1A, containing no biocompatible oil, and Composition ID, containing tricaprylin.For all experiments, 0.4 mL of each ND suspension was diluted in 40 mL of water at 37°C.

[0531] result

[0532] The overall results obtained from the determination of the ADV threshold are reported in Table 7. Each value is the average of three consecutive determinations.

[0533] Table 7: Acoustic Droplet Vaporization (ADV) Threshold as a Function of the Presence of Biocompatible Oils

[0534] Composition Oil MPa Composition 1A none 7.93 Composition 1D 10μL / mL 8.14

[0535] As can be seen from the comparison, the two tested compositions have similar ADV thresholds, confirming their similar acoustic responses.

[0536] References:

[0537] Astafyeva et al,J.Mater.Chem.B,3,2015,2892-2907

[0538] WO2016185425

[0539] Al Rifai et al,J.Mater.Chem.B,8,2020,1640-1648

[0540] Melich et al.,International Journal of Pharmaceutics,587,2020,119651

[0541] Cheng et al,J.Chem.Inf.Model.2007,47,2140-2148.

[0542] Helfield et al., Ultrasound in Medicine & Biology, 46, 10, 2020, 2861-2870.

Claims

1. A nanodroplet comprising an outer layer and an inner core, wherein the outer layer comprises a biocompatible fluorinated surfactant, and the inner core comprises a fluorinated compound and a triglyceride having a logP value greater than 5, wherein the biocompatible fluorinated surfactant is selected from: (A) An amphiphilic dendrimer of generation n, said dendrimer comprising: - a divalent or trivalent hydrophobic central core; - chains of generations connected to a central core and branching around it; and - hydrophilic terminal groups at the end of each generation chain; in n is an integer from 0 to 12; The hydrophilic end groups include: - monosaccharide, oligosaccharide or polysaccharide residues, - cyclodextrin residues, - peptide residues, -Tris(hydroxymethyl)aminomethane (Tris), or -2-amino-2-methylpropane-1,3-diol; The hydrophobic central core is a group of formula (Ia) or (Ib): in: W is R F or a group selected from W0, W1, W2 or W3: R F C4-C 10 Perfluoroalkyl, R H C1-C 24 Alkyl groups, p is 0, 1, 2, 3 or 4; q is 0, 1, 2, 3, or 4; L is a straight or branched C1-C 12 an alkylene group, optionally interrupted by one or more -O-, -S-, Z is C(=O)NH or NHC(=O), R is a C1-C6 alkyl group, and e is independently selected at each occurrence from 0, 1, 2, 3 or 4; (B) Amphiphilic linear oligomer of formula II in: - n is the number of repeating Tris units (n = DPn is the average degree of polymerization), wherein the term Tris denotes a tris(hydroxymethyl)aminomethane unit, and -i is the number of carbon atoms in the fluoroalkyl chain or a mixture thereof.

2. The nanodroplet according to claim 1, wherein the fluorinated compound is a perfluorocarbon.

3. The nanodroplet according to any one of the preceding claims, wherein the triglyceride has a logP value higher than 7, still more preferably higher than 9, up to for example 25.

4. The nanodroplet according to any one of the preceding claims, wherein the triglyceride is selected from tricaprylin, trilaurin, triolein, trilinolein or a mixture thereof.

5. An aqueous suspension comprising a plurality of nanodroplets according to claims 1-4, wherein the nanodroplets have a z-average diameter between 100 nm and 1000 nm and a polydispersity lower than 0.

25.

6. A method for preparing an aqueous suspension comprising a plurality of nanodroplets, said nanodroplets comprising an outer layer and an inner core, said outer layer comprising a biocompatible fluorinated surfactant as defined in claim 1, and said inner core comprising a fluorinated compound and a biocompatible oil having a logP value higher than 5, said method comprising the steps of: a) preparing an aqueous phase; b) preparing an organic phase, in i) the aqueous phase comprises a biocompatible fluorinated surfactant as defined in claim 1 or a mixture thereof, and the organic phase comprises a fluorinated compound and a biocompatible oil having a logP value higher than 5; or ii) the organic phase comprises a biocompatible fluorinated surfactant as defined in claim 1 or a mixture thereof, a fluorinated compound and a biocompatible oil having a logP value higher than 5; c) injecting the aqueous phase into a first inlet of a microfluidic cartridge and injecting the organic phase into a second inlet of the microfluidic cartridge, thereby mixing the aqueous phase and the organic phase in a mixing device of the microfluidic cartridge, wherein the working pressure into the microfluidic cartridge is lower than 7000 kPa, to obtain the aqueous suspension; and d) collecting the aqueous suspension from the outlet channel of the microfluidic cartridge.

7. The method according to claim 6, wherein the aqueous phase comprises the biocompatible fluorinated surfactant, and the organic phase comprises a fluorinated compound and a biocompatible oil having a LogP value higher than 5.

8. The method of claim 6 or 7, wherein the fluorinated compound is a perfluorocarbon.

9. The method according to any one of claims 6 to 8, wherein the biocompatible oil has a logP value higher than 7, still more preferably higher than 9, up to for example 25.

10. The method according to any one of claims 6 to 9, wherein the biocompatible oil is a triglyceride.

11. The method according to claim 10, wherein the triglyceride is selected from the group consisting of tricaprylin, trilaurin, triolein, trilinolein or a mixture thereof.

12. The process according to any one of claims 6 to 11, wherein the ratio between the volume of the aqueous phase and the volume of the organic phase is between 1:1 and 5:

1.

13. The method according to any one of claims 6 to 12, further comprising an additional step e) wherein the collected aqueous suspension is diluted, preferably with water.

14. The method of any one of claims 6 to 13, wherein the aqueous suspension comprises a plurality of nanodroplets having a z-average diameter between 100 nm and 1000 nm and a polydispersity below 0.

25.

15. The aqueous suspension according to claim 5, for use in diagnostic and / or therapeutic treatment.

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