Cross-linked vesicles with low-boiling point fluorinated compound

Abstract

The invention relates to a crosslinked vesicle comprising an outer layer and an inner core, said outer layer comprising a cationic amphiphilic peptide and said inner core comprising a liquid fluorinated compound having a boiling point lower than 25°C, wherein said amphiphilic peptide is a compound of formula (I) HB – CL – HP (I), wherein HB is a fluorinated hydrophobic block, CL is a cross-linking motif, HP is a cationic hydrophilic amino acid sequence. The invention further relates to a method for the preparation of an aqueous suspensions comprising a plurality of said cross-linked vesicles.

Description

[0001] CROSS-LINKED VESICLES WITH LOW-BOILING POINT FLUORINATED COMPOUND

[0002] Technical field

[0003] The invention generally relates to cross-linked vesicles having a liquid inner core comprising a fluorinated compound having a boiling point lower than 25°C at atmospheric pressure and stabilized by an amphiphilic peptide. The invention further relates to a method for the preparation of an aqueous suspension of said cross-linked vesicles and its use in ultrasound mediated diagnostic and / or therapeutic treatments.

[0004] Background of the invention

[0005] Phase-change contrast agents (PCCAs), also known as acoustically activated nanodroplets, are receiving increased popularity in both ultrasound diagnostic and therapeutic delivery. Except for the core, often consisting of liquid perfluorocarbons, nanodroplets display similar composition to commercially available gas-filled microbubbles. Owing to Acoustic Droplet Vaporization (ADV) process, encapsulated droplets are converted into gas bubbles upon exposure to ultrasound energy beyond a vaporization threshold. In fact, ultrasounds act as a remote trigger to promote the vaporization of the droplets in a controllable, non-invasive and localized manner. Thanks to their liquid core and smaller size compared to conventional microbubbles, nanodroplets display prolonged in vivo circulation and deep penetration into the tissues via the extravascular space. Moreover, below vaporization threshold, they are ultrasonically stable with low acoustic attenuation and can be acoustically vaporized at the location of interest.

[0006] Perfluorocarbon nanodroplets (PFC-NDs) present a real potential as an extravascular ultrasound contrast agent in numerous diagnostic and therapeutic applications including sonopermeabilization, blood brain barrier (BBB) disruption, multimodal imaging modalities and to allow passive (due to the enhanced permeability and retention (EPR) effect in the tumor tissues) or active targeting (by incorporating targeted ligands) for localized delivery of therapeutic drugs or genes. Another potentially valuable characteristic of PFC-NDs is their possible application for novel imaging strategies such as UltraSound Super-Resolution Imaging since these agents can be activated and deactivated on demand by applying intermittent acoustic pulses.

[0007] PCCAs are usually composed of an outer stabilizing shell, consisting of lipids, surfactants, polymers or proteins, and an inner core comprising a fluorinated compound. Depending on the encapsulated fluorinated compound, it is possible to distinguish between PCCAs comprising fluorinated compound with boiling points above room temperature (RT; 25 °C), i.e. fluorinated compounds which are in a liquid form at Standard Ambient Temperature and Pressure (SATP), namely at 25°C and 1 atm (101.325 kPa), and low boiling point PCCAs, comprising fluorinated compounds with boiling points below room temperature, i.e. fluorinated compound which are a gas at SATP.

[0008] Recently, low boiling point PCCAs have attracted much attention due to their intrinsic characteristics. Once prepared, the core of nanodroplets comprising low boiling point fluorinated compound remains metastable in liquid form even at temperatures well above the bulk boiling point of the core comprising a fluorinated compound, due to homogenous nucleation and fluorinated compound intermolecular forces. For this reason, nanodroplets comprising low boiling point fluorinated compound can undergo ADV at much lower acoustic pressures than those required to vaporize liquid PCCAs, enabling their use in a wider variety of biomedical applications, reducing negative bioeffects related to the use of high activation energy (Durham and Dayton, 2021).

[0009] The preparation of monodispersed PCCAs filled with a liquid fluorinated compound having a low boiling point (e.g. lower than 25°C) represents an area of growing interest due to their advantages, such as a higher vaporization efficiency compared to broadly distributed nanodroplets.

[0010] WO2019023706 discloses PFC-NDs (i.e. nanopeptisomes) having a core filled with a perfluorocarbon liquid (present in a liquid state at ambient temperature of about 25°C) containing a cargo, e.g. a therapeutically active agent, and a plurality of amphiphilic peptides surrounding the perfluorocarbon core, wherein said amphiphilic peptides comprise a fluorinated hydrophobic block, such as a fluorinated hydrophobic amino acid sequence, a cross-linking motif, and a hydrophilic amino acid sequence, such as a targeting motif. The disclosed nanopeptisomes are characterized by a zeta potential lower than 15 mV. Nanopeptisomes are obtained through a solvent-exchange procedure in which water is slowly added to an organic emulsion of peptide and PFC, ultimately leading to the spontaneous assembly of the peptide at the surface of PFC nanodroplets.

[0011] WO2023084048 discloses the preparation through microfluidic technique of an aqueous suspension of monodispersed cross-linked vesicles filled with high-boiling point fluorinated compounds (b.p. higher than 25°C) and stabilized by cross-linkable amphiphilic peptides.

[0012] Up to now, according to Applicants' knowledge, such vesicles stabilized by said amphiphilic peptides have not been obtained yet with a core filled with a low boiling point fluorinated compound.

[0013] Furthermore, the Applicant has observed that the preparation methods disclosed in the literature are not suitable for the preparation of cross-linked vesicles having a liquid core filled with a fluorinated compound with a low-boiling point (e.g. lower than 25°C) and stabilized by an amphiphilic peptide, the initial properties of which were found negatively affected at the end of the existing procedures.

[0014] The present invention aims at providing a novel method for the preparation of an aqueous suspension of cross-linked vesicles, wherein said vesicles comprise an inner core and an outer layer, said inner core comprising a fluorinated compound having a boiling point lower than 25°C) and said outer shell comprising an amphiphilic peptide.

[0015] Summary of the invention

[0016] An aspect of the invention relates to a cross-linked vesicle comprising an outer layer and an inner core, said outer layer comprising a cationic amphiphilic peptide and said inner core comprising a liquid fluorinated compound having a boiling point lower than 25°C, wherein said amphiphilic peptide is a compound of formula (I)

[0017] HB - CL - HP (I) wherein

[0018] HB is a fluorinated hydrophobic block,

[0019] CL is a cross-linking motif, and

[0020] HP is a cationic hydrophilic amino acid sequence having an alpha-helix structure and comprising at least 1 positive charge, preferably at least 2, more preferably at least 3, more preferably at least 4, still more preferably at least 5 positive charges, up to 40.

[0021] In a preferred embodiment the boiling point of said fluorinated compound is 22°C or lower, more preferably is 18°C or lower, still more preferably is 10°C or lower, still more preferably is 5°C or lower, still more preferably is 0°C or lower. Preferably, said boiling point is higher than -70°C, more preferably is -50°C or higher, still more preferably is -45°C or higher, still more preferably is -40°C or higher, still more preferably is -30°C or higher, still more preferably is -20°C or higher, still more preferably is -10°C or higher, still more preferably is -5°C or higher.

[0022] In a further embodiment, said fluorinated compound is a perfluorocarbon, selected from perfluorobutane, perfluoropropane, or a mixture thereof.

[0023] In a further preferred embodiment, said outer layer further comprises an additional amphiphilic peptide having formula V

[0024] HB - CL - HP'(V) wherein

[0025] HB is a fluorinated hydrophobic block,

[0026] CL is a cross-linking motif and

[0027] HP' is a hydrophilic amino acid sequence. In a preferred embodiment, HP' has a randomly-organized secondary structure.

[0028] In another embodiment, said additional amphiphilic peptide is a cationic amphiphilic peptide, wherein preferably HP' is a cationic hydrophilic amino acid sequence comprising at least one positive charge.

[0029] A further aspect of the invention relates to an aqueous suspension comprising a plurality of cross-linked vesicles as above defined.

[0030] Another aspect relates to a method for the preparation of an aqueous suspension comprising a plurality of cross-linked vesicles as defined above, comprising the steps of: a) Preparing an aqueous phase comprising an amphiphilic peptide, wherein the pH of said aqueous phase is of 4 or lower; b) Preparing an organic phase, comprising a fluorinated compound having a boiling point lower than 25°C; c) Injecting said aqueous phase in a first inlet and said organic phase in a second inlet of a microfluidic cartridge, thereby mixing said aqueous phase and said organic phase in a mixing portion of the microfluidic cartridge, wherein the operating pressure into said microfluidic cartridge is lower than 7000 kPa, to obtain an aqueous suspension of cross-linkable vesicles; d) Collecting the aqueous suspension of cross-linkable vesicles from an exit channel of the microfluidic cartridge; e) Diluting the aqueous suspension of cross-linkable vesicles, and f) Contacting said aqueous suspension of cross-linkable vesicles with an oxidizing source able to induce the cross-linking of the cross-linkable amphiphilic peptides to obtain an aqueous suspension of cross-linked vesicles, wherein said oxidizing source does not comprise DMSO.

[0031] Preferably, said oxidizing source is an oxidizing solution comprising disulfiram.

[0032] In a further embodiment, the method of the invention may comprise optional step g), which comprises washing the aqueous suspension of cross-linked vesicles after step e) and / or after step f).

[0033] A further aspect relates to an aqueous suspension comprising a plurality of crosslinked vesicles as above defined for use in a diagnostic and / or therapeutic treatment.

[0034] Figures

[0035] Figure 1: secondary structure prediction obtained using ColabFold (ColabFold vl.5.2-patch). Panels a) shows an example of amino acid sequence arranged in an alpha helix secondary structure, namely Pres2 (SEQ ID NO: 1; PLSSIFSRIGDP). Panels b) and c) show two examples of amino acid sequences arranged in a randomly organized secondary structure, namely TAT (SEQ ID NO: 2; YGRKKRRQRRR) and SV40 (SEQ ID NO: 3; PKKKRKV).

[0036] Detailed description of the invention

[0037] The Applicant has observed that existing methods for the preparation of crosslinked vesicles are not suitable for obtaining cross-linked vesicles comprising in their inner core a fluorinated compound with a boiling point lower than 25°C, such as perfluorobutane. In particular, it was found that the initial properties of the freshly- prepared cross-linkable vesicles filled with a liquid fluorinated compound having a boiling point lower than 25°C, such as sizes and monodispersity, were not preserved when performing standard cross-linking procedures disclosed in the literature, e.g. oxidation reaction with DMSO, to obtain a final suspension of cross-linked vesicles.

[0038] The Applicant observed that using preparation method comprising alternative oxidizing procedure, e.g. by using alternative oxidizing source different from DMSO, unexpectedly improved the quality of said cross-linked vesicles by substantially preserving their initial sizes (e.g. Size %Evol lower than 20%) and polydispersity profile.

[0039] Definitions

[0040] The term "vesicle" indicates an assembly comprising an outer layer and a liquid inner core, said outer layer comprising a cross-linkable amphiphilic peptide and said inner core comprising a fluorinated compound in liquid form having a boiling point lower than 25°C e.g. a perfluorocarbon. In said vesicles, the cross-linkable amphiphilic peptide molecules are oriented in such a way that the hydrophobic portions of the peptides are located at a surface of the fluorinated compound of the inner core.

[0041] According to the present invention, said vesicle can be a cationic vesicle bearing positive charges, i.e. a cross-linked vesicle having a zeta potential of at least 20 mV.

[0042] The expression "low boiling point cross-linked vesicle" indicates an assembly comprising an outer layer and an inner core, said outer layer comprising an amphiphilic peptide and said inner core comprising a liquid fluorinated compound having a boiling point lower than 25°C at atmospheric pressure.

[0043] The expression "low boiling point fluorinated compound" indicates a highly volatile fluorinated compound, in particular a gas having a boiling point lower than 25°C, i.e. characterized by being a gas at Standard Ambient Temperature and Pressure (SATP), namely at 25°C and 1 atm (101.325 kPa).

[0044] The expression "acoustic droplet vaporization" (ADV) refers to the phenomenon wherein said low boiling point cross-linked vesicles can be converted in bubbles by exposure to ultrasonic energy, with the corresponding increase in size, e.g. from nanometric to larger size echogenic bubbles.

[0045] The term "bubble" as used herein refers to an assembly comprising an outer layer and an inner core, said outer layer comprising an amphiphilic component as defined above and said inner core comprising a gaseous low boiling point fluorinated compound. Preferably said bubbles are microbubbles.

[0046] Due to their nanometric size, after in vivo administration said low boiling point cross-linked vesicles can extravasate, for instance into the interstitial space of a solid tumor, and provide sufficient contrast for ultrasound imaging after their conversion into bubbles upon exposure to ultrasound energy beyond a vaporization threshold.

[0047] Preferably, for the extravasation the mean diameter of said low boiling point vesicles is lower than 400 nm, e.g. between 100 nm and 300 nm.

[0048] Said vesicles having a liquid core filled with a low boiling fluorinated compound are referred to as "metastable", because they are stable as droplets at room conditions (i.e. SATP) and physiological conditions (typically a temperature from 36.5 to 37.5 °C and a pressure of 120 / 80 mm / Hg), meaning that they do not spontaneously expand into gas bubbles without being submitted to an external acoustic energy. For instance, after their administration, the sole exposition of said vesicles filled with a low boiling fluorinated compound to the body temperature and physiological pressure do not cause their activation and conversion in microbubbles. Additional energy is thus required to trigger this phenomenon after administration, such as ultrasound stimulus provided by a medical device.

[0049] The presence of a low boiling point fluorinated compound in the inner core makes the resulting droplets acoustically activatable with substantially less energy than other phase-change contrast agents, e.g. similarly-sized nanodroplets comprising higher boiling point fluorinated compound, endowing to considerable advantages for their applications in diagnostics, therapeutics and other treatments.

[0050] In other words, the core of the vesicles of this invention is characterized by the ability to remain in a liquid state despite the low boiling point of the core substance. Upon exposure to an external stimulus, such as ultrasound, said vesicles can be activated to transition to a gaseous state, resulting in a phase change that can be utilized for diagnostic or therapeutic applications.

[0051] Preferably the vesicles of the present invention are calibrated cross-linked nanodroplets.

[0052] The expression "calibrated" (or "monodisperse") refers to a population of vesicles as above defined, wherein said vesicles have a z-average diameter comprised between 100 nm and 1000 nm and a polydispersity lower than 0.3. Said calibrated vesicles are typically dispersed or suspended in an aqueous carrier and preferably obtained through microfluidic technique.

[0053] Generally in the state of the art, the term "calibrated" is also indicated as "size- controlled", "uniform-sized droplets", "monodisperse(d)" or "monosize(d)".

[0054] The expression "cross-linkable amphiphilic peptide" refers to any amphiphilic peptide comprising cross-linkable moieties that can potentially be covalently linked to each other through a cross-linking reaction. Suitable examples of cross-linkable moieties are cross-linkable aminoacids such as cysteine residues, that can be intermolecularly connected to an adjacent cysteine residue via disulfide cross-linking groups (-S-S-).

[0055] In a preferred embodiment, said cross-linkable moieties are cross-linkable amino acids, preferred being cysteine amino acids comprised in adjacent cross-linkable amphiphilic peptides.

[0056] According to the present invention, said cross-linkable amphiphilic peptides can be cationic amphiphilic peptides, comprising in their sequence at least one positive charge.

[0057] In the present description and claims, the expression "cross-linking reaction" indicates the process of forming covalent bonds between cross-linkable moieties comprised in adjacent cross-linkable amphiphilic peptides in order to bind cross-linkable amphiphilic peptides molecules together.

[0058] The term "cross-linkable vesicle" indicates an assembly comprising an outer layer and an inner core, said outer layer comprising a cross-linkable amphiphilic peptide and said inner core comprising a fluorinated compound having a boiling lower than 25°C at atmospheric pressure, wherein said cross-linkable amphiphilic peptide is not bonded intermolecularly to an adjacent amphiphilic peptide. As stated above, the cross-linkable amphiphilic peptides forming the outer layer of the cross-linkable vesicles comprise cross-linkable moieties that can potentially be covalently linked to each other.

[0059] A suspension of cross-linkable vesicles has not been yet submitted to any procedure aiming at inducing the cross-linking of the cross-linkable amphiphilic peptides forming the outer layer of the vesicles.

[0060] The term "cross-linked vesicle" indicates an assembly comprising an outer layer and an inner core, said outer layer comprising a cross-linkable amphiphilic peptide and said inner core comprising a fluorinated compound having a boiling point lower than 25°C at atmospheric pressure, wherein said cross-linkable amphiphilic peptide is covalently linked to an adjacent cross-linkable amphiphilic peptide through the crosslinking of the cross-linkable moieties. For instance, when the cross-linkable amphiphilic peptide includes a cysteine residue, said cysteine residue may be intermolecularly connected to an adjacent cysteine residue via disulfide cross-linking groups (-S-S-). The expression "calibrated cross-linked vesicles" indicates an aqueous suspension of calibrated cross-linked vesicles as above defined, said suspension being preferably obtained through microfluidic technique. After the collection from the microfluidic cartridge, e.g. within 30 minutes to 60 minutes, an aqueous suspension of calibrated cross-linkable vesicles can be submitted to a procedure aiming at inducing the crosslinking of the cross-linkable amphiphilic peptides forming the outer layer of the vesicles. As result, an aqueous suspension of calibrated cross-linked vesicles is obtained.

[0061] As mentioned above, according to the present invention said vesicle can be a cationic vesicle either as cross-linkable vesicle or as cross-linked vesicle.

[0062] The parameter "Size %Evolution" (%Evol), refers to the quantitative measurement of the changes in the dimensions or volume of vesicles over a specified period or under specific conditions and can be calculated according to the following equation (Eq.l) :

[0063] %oEvol=( D final — D initial ) / D initial X 100

[0064] Eq.l wherein:

[0065] D final is the z-average diameter of the vesicles after a certain time from the end of the cross-linking step (e.g. within minutes), or from the end of the preparation process (e.g. after 60 minutes from the end of preparation process or after a storage period (e.g. one week) at different conditions (different temperatures, pressure, etc.)); and

[0066] D initial is the z-average diameter of the vesicles immediately (e.g. within minutes) at the end of its preparation process.

[0067] In the present invention, a value of %Evol close to 0 (either positive or negative) indicates a higher stability of the vesicles suspension, whereby the nanodroplets in the suspension substantially maintain over time their initial mean dimensions.

[0068] According to the present invention, the %Evol of suspension of cross-linked vesicles is preferably lower than ±50%, more preferably lower than ±30%, and still more preferably is lower than ±20%.

[0069] Amphiphilic peptide

[0070] The expression "amphiphilic peptide" refers to a cross-linkable amphiphilic peptide of formula I

[0071] HB-CL-HP (I) wherein HB is a fluorinated hydrophobic polymer, CL is a cross-linking motif and HP is a hydrophilic amino acid sequence.

[0072] The cross-linkable amphiphilic peptide has a molecular weight in the range of about 1000 - 5000 daltons, wherein the cross-linkable amphiphilic peptide comprises from 5 to 50 amino acids residues, preferably from 5 to 40, more preferably from 5 to 35, wherein at least two of the amino acid residues are consecutively linked to each other in a chain by a peptide bond.

[0073] Cross-linkable amphiphilic peptides can be synthesized using techniques known to one of ordinary skill in the art, such as, but not limited to, solid-phase synthesis, recombinant methodologies polymerization, and conjugation methods.

[0074] As used herein, the term "fluorinated hydrophobic polymers" refers to a covalently linked chain of monomer residues forming a fluorinated hydrophobic homopolymer or copolymer. The monomeric units which form the fluorinated hydrophobic polymer may each be fluorinated according to embodiments, or some, or one, of the monomeric units is fluorinated such that at least one or more of the monomer residues of the fluorinated hydrophobic polymer is fluorinated.

[0075] Typically, said cross-linkable amphiphilic peptide does not include lipids.

[0076] According to an embodiment, the fluorinated hydrophobic polymer includes a hydrophobic amino acid sequence wherein the amino acids of the hydrophobic amino acid sequence have non-polar side chains. Preferably, the fluorinated hydrophobic polymer includes one or more synthetic non-amino acid monomeric units wherein at least one of the monomeric units is fluorinated such that at least one of the monomer residues of the fluorinated hydrophobic polymer is fluorinated. Non limiting examples of synthetic monomeric units which can be fluorinated and reacted to form a fluorinated hydrophobic polymer include methyl methacrylate, lactic acid, glycolic acid and olefins such as ethylene, propylene, styrene.

[0077] In said embodiment, the cross-linkable amphiphilic peptide is a compound of Formula (II):

[0078] HB'-CL-HP (II) wherein HB' is a fluorinated hydrophobic amino acid sequence; CL is a cross-linking motif; and HP is a hydrophilic amino acid sequence.

[0079] Preferably HP is a hydrophilic amino acid sequence wherein the C-terminal amino acid is amidated or hydroxylated (-OH), still more preferably the C-terminal amino acid is amidated(-NH2).

[0080] As used herein, the term "hydrophobic amino acid sequence" refers to a sequence of hydrophobic amino acids having non-polar side chains or a combination of a hydrophobic polymer and a sequence of hydrophobic amino acids having non-polar side chains.

[0081] Hydrophobic amino acids may be naturally occurring or non-natural (artificially produced). Examples of the naturally occurring hydrophobic amino acids include, but are not limited to, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, cysteine, and methionine. Examples of non-natural hydrophobic amino acids may include D amino acids, as well as specific non-natural amino acids such as selenocysteine, pyrrolysine, and the like.

[0082] In the cross-linkable amphiphilic peptide, the fluorinated hydrophobic amino acid sequence may include one to ten fluorinated hydrophobic amino acids consecutively connected by peptide bonds, which may be unsubstituted or substituted with a substituent selected from -F, -Cl, -Br, -I, a C1-C30 alkyl group, a C2-C30 alkenyl group, a C2-C30 alkynyl group, a C3-C30 cycloalkyl group, a C3-C30 cycloalkenyl group, a C6-C30 aryl group, a C7-C30 arylalkyl group, but are not limited thereto. Fluorinated hydrophobic amino acids include, for example, fluorinated alanine, fluorinated valine, fluorinated leucine, fluorinated isoleucine, fluorinated proline, fluorinated phenylalanine, fluorinated tryptophan, fluorinated cysteine, fluorinated methionine, fluorinated selenocysteine and fluorinated pyrrolysine. The fluorinated hydrophobic amino acids can be D or L amino acids and can be fluorinated at any suitable position, typically replacing a hydrogen atom.

[0083] In a preferred embodiment, the fluorinated hydrophobic amino acids sequence may include pentafluoro-phenylalanine (2,3,4,5,6-pentafluoro-L-phenylalanine and / or 2,3,4,5,6-pentafluoro-D-phenylalanine) at a terminal thereof.

[0084] In another embodiment, the fluorinated hydrophobic amino acid sequence may comprise one to ten consecutively connected pentafluoro-phenylalanine residues at a terminal thereof.

[0085] Still more preferably, the fluorinated hydrophobic amino acid sequence HB' comprises three consecutively connected pentafluoro-phenylalanine residues at a terminal thereof and is a compound of formula III

[0086] Formula III Suitable fluorinated amino acids are those described for instance in WO2019023707.

[0087] A combination of a hydrophobic polymer and a sequence of hydrophobic amino acids having non-polar side chains can be included in the fluorinated hydrophobic polymer wherein at least one of the monomer residues of the fluorinated hydrophobic polymer is fluorinated and / or at least one of the amino acid residues is fluorinated.

[0088] As used herein, the term "hydrophilic amino acid sequence" refers to a sequence of hydrophilic amino acids consecutively connected by peptide bonds, wherein the hydrophilic amino acids have a polar side chain, wherein the polar side chain includes a group capable of forming a hydrogen bond with molecules of water.

[0089] The hydrophilic amino acid sequence HP can comprise both hydrophilic and hydrophobic amino acids.

[0090] Hydrophilic and hydrophobic amino acids may be naturally occurring or nonnatural (artificially produced) and can be D or L amino acids.

[0091] Examples of naturally occurring hydrophilic amino acids include, but are not limited to, serine, threonine, asparagine, glutamine, histidine, arginine, lysine, aspartic acid and glutamic acid. Examples of non-natural hydrophilic amino acids include amino acids having various heterocyclic groups as a part of the side chain.

[0092] Examples of the naturally occurring and non-natural hydrophobic amino acids are those mentioned above. In the cross-linkable amphiphilic peptide, the hydrophilic amino acid sequence HP may include from 3 to 40 hydrophilic amino acids, preferably from 3 to 30, more preferably from 3 to 20, consecutively connected by peptide bonds.

[0093] The hydrophilic amino acid sequence may comprise a targeting agent that interacts (e.g. through covalent or non-covalent binding) with a specific moiety, such as a receptor expressed by a target cell, resulting in the binding of the vesicle to said moiety or molecule.

[0094] The target cells can be cells of any organism, such as, but not limited to, a mammal, bird, fish, or bacterial cell. According to an embodiment, the target cell is a human cell or a bacterial cell within a human body.

[0095] The targeting agent may include a minimal targeting motif peptide and optionally includes one or more hydrophilic amino acids attached to the N-terminus or C-terminus of the minimal targeting motif peptide by peptide bonds.

[0096] Typically, amino acids of the targeting motif peptide are L-amino acids but these may include one or more D-amino acids so long as the targeting motif still correctly mediates binding with the receptor. The one or more hydrophilic amino acids attached to the N-terminus or C-terminus of the minimal targeting motif peptide by peptide bonds can be D or L amino acids.

[0097] Examples of suitable targeting motifs and examples of hydrophilic amino acid sequences including a targeting motif that can be comprised in the cross-linkable amphiphilic peptides are described, for instance, in WO2019023707.

[0098] According to present invention, said cross-linkable amphiphilic peptide is a cationic amphiphilic peptide, wherein HP is a cationic hydrophilic amino acid sequence having an alpha (a) -helix structure and comprising at least 1 positive charge, preferably at least 2, more preferably at least 3, more preferably at least 4, still more preferably at least 5 positive charges, up to 40.

[0099] Preferably, HP is a cationic hydrophilic amino acid sequence wherein the C-terminal amino acid is amidated or hydroxylated (-OH), still more preferably the C-terminal amino acid is amidated (-NH2).

[0100] Preferably, at least 50% of the amino acid residues of said cationic hydrophilic amino acid sequence are arranged in an alpha helix.

[0101] The likelihood of a specific amino acid sequence forming an a-helical structure can be reasonably predicted by identifying certain structural features within the peptide's amino acid sequence.

[0102] Said predominant structural features known to favor a-helix formation are described in several studies, including (Branden, 1998), (Errington, 2006), (Wang, 2003), and (Pace, 1998). Examples of such structural features are, but not limited to, the amino acid composition, the average length of the sequence and side chain interactions.

[0103] In general, certain amino acids are more likely to be found in a-helices due to their structural properties and ability to participate in the formation of the helical structure. The most recurrent amino acids in a-helix structures include Alanine (Ala, A), Leucine (Leu, L), Glutamate (Glu, E), Lysine (Lys, K), Methionine (Met, M), Glutamine (Gin, Q), Arginine (Arg, R), Histidine (His, H). Moreover, specific sequence patterns and periodicity can indicate a propensity for a-helix formation.

[0104] The average length of an a-helix in proteins typically ranges from 4 to 40 amino acids, preferably from 8 to 30, still more preferably from 15 to 25. In terms of structural measurements, 10 amino acids correspond to about 15 A (angstroms) in length. The average length can vary depending on the protein and its specific function, as some a- helices can be shorter or considerably longer.

[0105] Additional details about said predominant structural features are extensively described in the literature, as reported above. Suitable examples of cationic hydrophilic amino acid sequences having an alphahelix structure and comprising at least 1 positive charge are Pres2 (SEQ ID PLSSIFSRIGDP), PepFectl4 (SEQ ID NO: 4; AGYLLGKLLOOLAAAALOOLL), RALA (SEQ ID NO: 5; WEARLARALARALARHLARALARALRACEA), Penetratin (SEQ ID NO: 6; RQIKIWFQNRRMKWKK), MPG (SEQ ID NO: 7; GALFLGFLGAAGSTMGAWSQPKKKRKV), gH 625 (SEQ ID NO: 8; HGLASTLTRWAHYNALIRAF), Bim (SEQ ID NO: 9; EIWIAQELRRIGDEFNAYYARLLC), GALA (SEQ ID NO: 10;

[0106] WEAALAEALAEALAEHLAEALAEALEALAA), BP100 (SEQ ID NO: 11; KKLFKKILKYL), DPV3 (SEQ ID NO: 12; RKKRRRESRKKRRRES) and Scl8 (SEQ ID NO: 13; GLRKRLRKFRNKIKEK).

[0107] Typically, after evaluating the presence of such key structural features, it is possible to use secondary structure prediction tools, such as computational tools and algorithms, to analyze a specific amino acid sequence for predicting the secondary structure.

[0108] The secondary structure of an amino acid sequence can be essentially predicted from its primary structure by using suitable bioinformatics methods, such as ColabFold (Mirdita, 2022), e.g. ColabFold vl.5.5-patch.

[0109] Typically, the secondary structure of an amino acid sequence can be, but not limited to, alpha helix, n helix, beta-sheet or beta turn. Further examples of suitable secondary structure may be found in Branden, 1998.

[0110] The expression secondary structure has its conventional meaning and refers to regular, recurring arrangements in space of adjacent amino acid residues in a polypeptide chain. It is maintained by hydrogen bonds between amide hydrogens and carbonyl oxygens of the peptide backbone. The major secondary structures are a-helices and p-structures.

[0111] Alternatively, an amino acid sequence can have a randomly-organized secondary structure, e.g. a random coil structure (Smith, 1996) meaning that said sequence do not form any regular secondary structure, as defined above, and are characterized by a disordered arrangement.

[0112] In other words, by using said bioinformatic methods is possible to predict whether a given amino acid sequence tend to form a defined secondary structure, such as alpha helices and beta sheets, or whether it will tend to arrange in a randomly-organized secondary structure.

[0113] Suitable examples of secondary structure prediction obtained by using ColabFold are given in Figure 1. Figure la displays a cationic hydrophilic amino acid sequence, as defined in this invention, namely Pres2, arranged in an alpha helix structure, while Figures lb and lc show two examples of hydrophilic amino acid sequences, namely TAT and SV40, arranged in a randomly-organized structure.

[0114] In an embodiment, said cationic hydrophilic amino acid sequence is selected from a cell penetrating peptide (CPP) or a nuclear localization sequence (NLS).

[0115] The expression cell penetrating peptide indicates a class of short peptides with 5- 30 amino acids long, generally positively charged, that can penetrate biological membrane and deliver a wide variety of cargos into cells. A comprehensive review about CPP can be found in Derakhshankhah, 2018.

[0116] Suitable examples of CPP can be found in the database CPPsite 2.0 (http: / / crdd.osdd.net / raghava / cppsite / ).

[0117] The expression nuclear localization sequence indicates a class of short peptides based on lysine-, arginine- or proline-rich motifs that can be transported to the nucleus through the nuclear pore complex, which is a multimeric complex containing 50-100 different proteins. NLSs can be further divided into monopartite and bipartite signals, which consist, respectively, of one or two clusters of four or more basic amino acids (Lu, 2021).

[0118] In the present description and claims, the expression "cross-linking motif" refers to a cross-linkable moiety, comprised in the cross-linkable amphiphilic peptide, that can potentially be covalently linked to another cross-linkable moiety through a cross-linking reaction. Examples of cross-linking motif are sulfhydryl cross-linkers, UV cross-linkers, aza-benzenes, photosensitive cross-linkers, such as azides or benzophenones, nitriles, pH-sensitive cross-linkers, or enzymatic cross-linkers and click-chemistry-based crosslinkers (e.g. a cross-linking motif comprising an azide group able to react with an alkyne-containing cross-linking motif via click chemistry reactions, such as the copper- catalyzed azide alkyne cycloaddition).

[0119] In a preferred embodiment, the "cross-linking motif" is an amino acids sequence comprising at least one cross-linkable amino acid residues that can potentially be covalently linked to corresponding cross-linkable amino acid residues through a crosslinking reaction. The cross-linking motif may comprise from 1 to about 40 amino acid residues, preferably from 3 to 30, more preferably from 3 to 20.

[0120] In the cross-linking motif said cross-linkable amino acid residues may be at any position in the sequence and can be naturally occurring amino acids and / or non-naturally occurring amino acids.

[0121] An example of a naturally occurring amino acid able to cross-link with a corresponding cross-linkable amino acid residue is cysteine.

[0122] Non-naturally occurring amino acids may be obtained through structure functionalization of naturally occurring amino acids providing the ability to bind to a naturally occurring or non-naturally occurring amino acid in the crosslinking motif of an adjacent cross-linkable residue.

[0123] In an embodiment, the cross-linking motif comprises a cysteine.

[0124] In a further embodiment, the cross-linking motif comprises a cysteine and a glycine.

[0125] In a preferred embodiment, the cross-linking motif comprises the amino acid sequence GGGCCGG (SEQ ID NO: 14), wherein G is glycine and C is cysteine.

[0126] In an embodiment, the degree of cross-linking of the cross-linkable amphiphilic peptide molecules is higher than 80%, preferably at least 85%, more preferably at least 90%, still more preferably at least 95%. Preferably the degree of cross-linking is 100%.

[0127] The expression "degree of cross-linking" refers to the total number of cross-linked amino acid residues, e.g. cysteines, that interconnect adjacent cross-linkable amphiphilic peptides. The degree of cross-linking is generally expressed in percent and can be measured using a colorimetric disulfide formation assay.

[0128] In a preferred embodiment said cross-linkable amphiphilic peptide is a cationic amphiphilic peptide of formula IV

[0129] HB' - CL - HP (IV) wherein

[0130] HB' is a fluorinated hydrophobic amino acid sequence comprising a pentafluoro-phenylalanine,

[0131] CL is a cross-linking motif comprising GGGCCGG, where G is glycine and C is cysteine and

[0132] HP is a cationic hydrophilic amino acid sequence, having an alpha-helix structure and comprising at least 1 positive charge, preferably at least 2, more preferably at least 3, more preferably at least 4, still more preferably at least 5 positive charges, up to 40.

[0133] In a further embodiment, the cationic amphiphilic peptide is H2N- FFFFFFGGGCCGGKGPLSSIFSRIGDP-NH2(AP-PRES2) (SEQ ID NO: 15), wherein each modified amino acid Ff is pentafluoro phenylalanine (2,3,4,5,6-pentafluoro-L- phenylalanine).

[0134] In this description and claims, the amino acid sequences are reported following the conventional one letter code, wherein for instance G is Glycine, C is Cysteine, K is Lysine, P is Proline, L is Leucine, S is Serine, F is Phenylalanine, D is Aspartic Acid, I is Isoleucine, R is Arginine O is Ornithine, A is Alanine and Y is Tyrosine.

[0135] Mixture of cationic amphiphilic peptides In a preferred embodiment, the invention relates to a cross-linked vesicle as above defined, wherein said outer layer further comprises an additional amphiphilic peptide having formula V:

[0136] HB - CL - HP'(V) wherein

[0137] HB is a fluorinated hydrophobic block,

[0138] CL is a cross-linking motif and

[0139] HP' is a hydrophilic amino acid sequence.

[0140] According to this invention, said hydrophilic amino acid sequence HP' can be any hydrophilic amino acid sequence as defined above.

[0141] The secondary structure of HP' can be any suitable secondary structure, such as alpha helix, n- helix, beta-sheet, beta turn or a mixture thereof as defined above. Alternatively, HP' has a randomly-organized secondary structure.

[0142] At least 50% of the amino acid residues of HP' are arranged in a secondary structure, as defined above.

[0143] In a preferred embodiment, HP' has a randomly-organized secondary structure.

[0144] Preferably HP' is a cationic hydrophilic amino acid sequence comprising at least one positive charge, more preferably 2 or more positive charges, up to 40.

[0145] Preferably HP' is TAT (SEQ ID NO:2; YGRKKRRQRRR) or SV40 (SEQ ID NO: 3; PKKKRKV).

[0146] Preferably in said additional amphiphilic peptide said HB is HB', which is a fluorinated hydrophobic amino acid sequence comprising a pentafluoro-phenylalanine as defined above. Still more preferably, the fluorinated hydrophobic amino acid sequence HB' comprises three consecutively connected pentafluoro-phenylalanine residues at a terminal thereof and is a compound of formula III.

[0147] Preferably CL is a cross-linking motif comprising GGGCCGG, where G is glycine and C is cysteine.

[0148] In a further embodiment, the additional amphiphilic peptide is H2N- FFFFFFGGGCCGGKGYGRKKRRQRRR-NH2(SEQ ID NO: 16; AP-TAT) or H2N- FFFFFFGGGCCGGKGPKKKRKV-NH2(SEQ ID NO: 17; AP-SV40).

[0149] Fluorinated compounds

[0150] The disclosed cross-linked vesicles are characterized by a liquid inner core that allows for activation of the vesicles upon activation of ultrasound, said inner core comprising a liquid fluorinated compound having a boiling point lower than 25°C. In the present description and claims the term "fluorinated compound" refers to a group of fluorine-containing compounds derived from (optionally substituted) hydrocarbons by partial or complete substitution of hydrogen atoms with fluorine atoms, which are gas (i.e. gaseous fluorinated compounds) at SATP conditions, namely at 25 °C and 1 atm (101.325 kPa), (i.e. highly volatile fluorinated compounds).

[0151] The expression "optionally substituted" refers to presence of functional groups, such as amines, ethers and halogen-containing groups.

[0152] Suitable examples of fluorinated compounds are hydrofluorocarbons, both saturated and unsaturated, perfluorocarbons, fluorinated ethers, fluorinated ketones or perfluorinated nitrile. Preferably the fluorinated compound is a perfluorocarbon (PFC), i.e. a fluorinated hydrocarbon where all the hydrogen atoms are substituted with fluorine atoms.

[0153] Gaseous fluorinated compounds are characterized by a boiling point lower than 25°C, preferably said boiling point is 22°C or lower, more preferably is 18°C or lower, still more preferably is 5°C or lower, still more preferably is 0°C or lower. Preferably, said boiling point is higher than -70°C, more preferably is -50°C or higher, still more preferably is -45°C or higher, still more preferably is -40°C or higher, still more preferably is -30°C or higher, still more preferably is -20°C or higher, still more preferably is -10°C or higher, still more preferably is -5°C or higher.

[0154] Suitable examples of fluorinated compounds include C3-C4 fluorinate compounds, such as 1,1, 1,2, 3, 3, 3 heptafluoropropane, 1,1,1,2,2,3-Hexafluoropropane, 1,1, 1,2, 3, 3- Hexafluoropropane, 1,1,1,3,3,3-Hexafluoropropane, 1,1, 1,2, 2, 3, 3, 4, 4

[0155] Nonafluorobutane, l,l,l,3,3,3-Hexafluoro-2-(trifluoromethyl)propane, 1,1, 1,2, 2, 3, 3, 4 Octafluorobutane or a mixture thereof.

[0156] Suitable examples of perfluorocarbons are perfluorocyclopropane, perfluoropropane, perfluorocyclobutane, perfluorobutane, perfluoroisobutane or a mixture thereof.

[0157] In an embodiment said perfluorocarbon is preferably perfluorobutane (boiling point -2°C).

[0158] Preparation of cross-linked vesicles

[0159] According to the present invention, the disclosed cross-linked vesicles filled with a low boiling point fluorinated compound in liquid form can be prepared by using any preparation technique suitable to manufacture nanodroplets having a Z-average diameter comprised between 100 nm and 1000 nm, preferably between 120 and 800 nm, more preferably between 150 and 400 nm with relatively narrow size distribution (e.g. PDI lower than 0.3). Example of preparation techniques include sonication, homogenization, extrusion, microfluidic and microbubble condensation.

[0160] In a preferred embodiment, said cross-linked vesicles is prepared by using a preparation technique suitable to manufacture nanodroplets having a polydispersity index (PDI) lower than 0.30, preferably lower than 0.25, more preferably lower than 0.20, even more preferably lower than 0.15, even more preferably lower than 0.10, and a Z-average diameter comprised between 100 nm and 1000 nm, preferably between 120 and 800 nm, more preferably between 150 and 400 nm.

[0161] For instance, said preparation technique may be selected from sonication, microbubble condensation or microfluidic, preferably said preparation technique is the micro fluidic.

[0162] In the present description and claims the expression "microfluidic technique" refers to a technology of manufacturing calibrated nanodroplets, such as the cross-linked vesicle of this invention, through a microfluidic cartridge designed to manipulate fluids in channels at the microscale.

[0163] Said microfluidic technique is a bottom-up approach, that is to say that the nanodroplets are obtained by assembling molecules (e.g. amphiphilic lipid compounds and fluorinated compounds) into larger nanostructures (i.e. calibrated nanodroplets).

[0164] More preferably said microfluidic technique is carried out by using a mixing device such as a microfluidic cartridge equipped by a staggered herringbone micromixer or toroidal mixer.

[0165] A detailed description of a microfluidic cartridge suitable for the microfluidic process of the present invention is described in WO2023084048 (Bracco Suisse SA), which is here incorporated by reference.

[0166] An aspect of the invention relates to a method for the preparation of an aqueous suspension comprising a plurality of cross-linked vesicles as defined above, said method comprising the steps of: a) Preparing an aqueous phase comprising an amphiphilic peptide, wherein the pH of said aqueous phase is of 4 or lower; b) Preparing an organic phase, comprising a fluorinated compound having a boiling point lower than 25°C; c) Injecting said aqueous phase in a first inlet and said organic phase in a second inlet of a microfluidic cartridge, thereby mixing said aqueous phase and said organic phase in a mixing portion of the microfluidic cartridge, wherein the operating pressure into said microfluidic cartridge is lower than 7000 kPa, to obtain an aqueous suspension of said vesicles; d) Collecting the aqueous suspension of cross-linkable vesicles from an exit channel of the microfluidic cartridge; e) Diluting the collected aqueous suspension of cross-linkable vesicles, and f) Contacting said aqueous suspension of cross-linkable vesicles with an oxidizing source able to induce the cross-linking of the amphiphilic peptides to obtain an aqueous suspension of cross-linked vesicles, wherein said oxidizing source does not comprise DMSO.

[0167] Preferably, said oxidizing source is an oxidizing solution comprising disulfiram.

[0168] In a further embodiment, the method of the invention comprises optional step g), which comprises washing the aqueous suspension of cross-linked vesicles after step e) and / or after step f).

[0169] According to an embodiment, said aqueous suspension of cross-linked vesicles has a polydispersity index (PDI) lower than 0.30, preferably lower than 0.25, more preferably lower than 0.20, even more preferably lower than 0.15, even more preferably lower than 0.10, and a Z-average diameter comprised between 100 nm and 1000 nm, preferably between 120 and 800 nm, more preferably between 150 and 400 nm.

[0170] According to the disclosed method, it is possible to obtain an aqueous suspension of calibrated cross-linkable vesicles by a single passage of the liquid phases through the microfluidic cartridge mixing portion. a) Preparing an aqueous phase

[0171] Step a) relates to the preparation of an aqueous phase comprising an amphiphilic peptide, wherein the pH of said aqueous phase is of 4 or lower.

[0172] The "aqueous phase" typically comprises an aqueous liquid component, including, for instance, water, aqueous buffered solutions or aqueous isotonic solutions.

[0173] Suitable examples of aqueous buffered solutions are phosphate-buffered saline (i.e. PBS buffer), sodium acetate buffer, tris(hydroxymethyl)aminomethane buffer (i.e. TRIS buffer) or a mixture thereof.

[0174] Suitable examples of isotonic solutions are Ringer solution, Ringer's lactate solution, saline, oral rehydration solution or a mixture thereof.

[0175] Preferably the aqueous liquid component has a pH lower than 7.0, more preferably lower than 6.5, still more preferably lower than 6.0, more preferably lower than 5.5., more preferably lower than 5, more preferably lower than 4.5, still more preferably lower than 4.0, up to e.g. 0, preferably 1.0.

[0176] Preferably the aqueous liquid component is sodium acetate buffer. For instance, an amphiphilic peptide can be admixed with an aqueous component through traditional techniques (e.g. stirring) in order to prepare the aqueous phase to be injected into the first inlet of the microfluidic cartridge.

[0177] Preferably the amphiphilic peptide is a compound of formula (I)

[0178] HB - CL - HP (I) wherein

[0179] HB is a fluorinated hydrophobic block,

[0180] CL is a cross-linking motif, and

[0181] HP is a hydrophilic amino acid sequence.

[0182] Preferably said amphiphilic peptide is a cationic amphiphilic peptide, wherein HP is a cationic hydrophilic amino acid sequence having an alpha-helix structure and comprising at least 1 positive charge, preferably at least 2, more preferably at least 3, more preferably at least 4, still more preferably at least 5 positive charges, up to 40.

[0183] According to another embodiment, said aqueous phase further comprises an additional amphiphilic peptide, having formula V

[0184] HB - CL - HP'

[0185] (V) wherein

[0186] HB is a fluorinated hydrophobic block,

[0187] CL is a cross-linking motif and

[0188] HP' is a hydrophilic amino acid sequence.

[0189] Preferably said additional amphiphilic peptide is a cationic amphiphilic peptide as defined above.

[0190] At step a) the aqueous phase comprises a cross-linkable amphiphilic peptide preferably at a concentration ranging between 0.0003 mmol / mL and 0.006 mmol / mL, more preferably between 0.0006 mmol / mL and 0.004 mmol / mL, still more preferably between 0.001 mmol / mL and 0.003 mmol / mL.

[0191] Suitable examples of amphiphilic peptides and addition amphiphilic peptides are those mentioned above.

[0192] In an embodiment, at the step a) the temperature of the aqueous phase is typically lower than 25°C, preferably lower than 20°C, more preferably lower than 15°C, still more preferably lower than 10°C, still more preferably the temperature is about 5 ± 2°C. Said temperature is preferably not lower than 2°C. b) Preparing an organic phase Step b) relates to the preparation of an organic phase, comprising a fluorinated compound having a boiling point lower than 25°C.

[0193] The "organic phase" typically comprises an organic solvent, preferably miscible with water, including, for instance, methanol, ethanol, isopropanol, acetonitrile, DMF, DMSO and acetone. Preferably the organic solvent is ethanol.

[0194] In the present invention the expression "organic solvent miscible with water" indicates an organic solvent capable of mixing in any ratio (e.g. any concentration) with water without separation of the two phases, i.e. forming a homogeneous solution.

[0195] For instance, C1-C3 alcohols, such as methanol, ethanol and propanol, are very soluble in water due to the hydrogen bonding engaging the hydroxyl groups in the alcohol molecules and the water molecules. However, as the length of the hydrocarbon chain increases, the solubility in water decreases leading to a low miscibility of the two liquids that, if mixed, will form two immiscible layers.

[0196] In an embodiment, the organic solvent is a polar organic solvent.

[0197] The expression "polar organic solvent" has its conventional meaning in the chemical field. Solvents can be classified by their relative polarity (rp): for example, water is the most polar solvent and it is characterized by a relative polarity of 1. On the contrary non-polar solvents have low value of relative polarity, such as dimethylformamide (DMF) with a relative polarity value of 0.386.

[0198] In a preferred embodiment, the organic solvent is a polar organic solvent, said solvent having a polarity comprised between 0.60 and 0.80, preferably comprised between 0.63 and 0.78, still more preferably comprised between 0.65 and 0.77.

[0199] In a further embodiment, said organic solvent is selected from methanol, ethanol and mixture thereof. Preferably the organic phase is ethanol.

[0200] According to the invention, said organic phase comprises a fluorinated compound having a boiling point lower than 25°C at atmospheric pressure or a mixture of different fluorinated compounds having a boiling point lower than 25°C at atmospheric pressure dispersed in the organic solvent. Preferably the boiling point of said fluorinated compound is 22°C or lower, more preferably is 18°C or lower, still more preferably is 10°C or lower, still more preferably is 5°C or lower, still more preferably is 0°C or lower. Preferably, said boiling point is higher than -70°C, more preferably is -50°C or higher, still more preferably is -45°C or higher, still more preferably is -40°C or higher, still more preferably is -30°C or higher, still more preferably is -20°C or higher, still more preferably is -10°C or higher, still more preferably is -5°C or higher.

[0201] In a preferred embodiment, said fluorinated compound is a perfluorocarbon, selected from the group consisting of perfluorobutane, perfluoropropane, or a mixture thereof, preferred being perfluorobutane Admixing of fluorinated compound having a boiling point below room temperature (i.e. 25°C) as liquid phase

[0202] According to an embodiment, step b) comprises preparing an organic phase by the addition of a fluorinated compound having a boiling point lower than 25°C at atmospheric pressure as a liquid into an organic solvent.

[0203] In an embodiment, said step b) relates to a method for the preparation of an organic phase comprising a fluorinated compound having a boiling point lower than 25°C at atmospheric pressure, said method comprising the steps of: b.l) Condensing said fluorinated compound to obtain a liquid fluorinated compound; b.2) Preparing a liquid organic solution comprising an organic solvent; b.3) Cooling said liquid organic solution to a temperature below the boiling point of said liquid fluorinated compound; b.4) Mixing said liquid fluorinated compound and said liquid organic solution to obtain an organic phase comprising a liquid fluorinated compound.

[0204] In a preferred embodiment, step b.l) is performed by cooling said fluorinated compound down to a temperature below the boiling point of said low boiling fluorinated compound.

[0205] In a further embodiment, at step b.4) the temperature at which the mixing is performed is a temperature below the boiling point of said low boiling fluorinated compound.

[0206] Preferably, the temperature at step b.4) is the same temperature of step b.3).

[0207] Still more preferably the temperature at step b.l), step b.3) and step b.4) is suitable to avoid or substantially limit the evaporation of said liquid low boiling fluorinated compound having a boiling point comprised between -70°C and 25°C at atmospheric pressure until to the injection of said organic phase into the microfluidic cartridge (i.e. step c).

[0208] The temperature at step b.l), step b.3) and step b.4) is lower than the boiling point of said fluorinated compound, preferably it is at least 5°C lower than the boiling point of said fluorinated compound, more preferably at least 10°C lower, even more preferably at least 20°C lower, up to e.g. 50°C lower.

[0209] The temperature of the obtained organic phase at the end of step b.4) is thus lower than the boiling point of said fluorinated compound , preferably it is at least 5°C lower than the boiling point of said fluorinated compound, more preferably at least 10°C lower, even more preferably at least 20°C lower, up to e.g. 50°C lower. At step b.4) said mixing is performed for a time suitable to allow the dissolution of said liquid fluorinated compound into said liquid organic solution, e.g. within 5 minutes. At the end of said mixing, an organic phase is obtained in the form of a homogenous solution. Said organic phase shall be injected into the microfluidic cartridge within a time suitable for avoiding or substantially limiting the evaporation of the fluorinated compound from the organic phase, e.g. within 5 minutes from the end of step b).

[0210] In a further embodiment, at step b.4) the organic phase comprises a fluorinated compound at a concentration ranging between 1 and 100 pL / mL, preferably between 10 and 50 pL / mL, still more preferably between 15 and 30 pL / mL.

[0211] Admixing a fluorinated compound having a boiling point below room temperature (i.e. 25°C) as a gaseous phase

[0212] Alternatively, step b) may comprise preparing an organic phase by the addition of a fluorinated compound having a boiling point lower than 25°C at atmospheric pressure as gaseous phase (i.e in gas form) into an organic solvent.

[0213] Accordingly, said step b) may relate to a method for the preparation of an organic phase comprising a fluorinated compound having a boiling point lower than 25°C at atmospheric pressure, said method comprising the steps of: b.i) Preparing a liquid organic solution comprising an organic solvent, and b.ii) Admixing a gaseous fluorinated compound having a boiling point comprised lower than 25°C at atmospheric pressure to said organic liquid solution.

[0214] Preferably at step b.ii) the admixing is performed by bubbling said gaseous fluorinated compound into the organic solution.

[0215] Preferably, before the admixing of step b.ii) the temperature of the organic solution is set above the boiling point of said gaseous fluorinated compound. A temperature above the boiling point of the fluorinated compound is generally preferred in order to avoid or substantially limit the fluorinated compound phase-shift from gas to liquid, which could lead to solubility issue of the condensed fluorinated compound into the organic solvent (e.g. formation of droplets having a liquid core filled with a fluorinated compound suspended in the organic solvent).

[0216] The temperature of the obtained organic phase at the end of step b.ii) is above the boiling point of said gaseous fluorinated compound.

[0217] Preferably, said temperature is between the boiling point and RT, more preferably up to 15 °C higher than the boiling point, even more preferably up to 10°C higher.

[0218] At step b.ii) the concentration of said fluorinated compound into the organic phase can be any concentration, preferably up to the saturation concentration. Preferably the concentration of said fluorinated compound is the saturation concentration. The saturation concentration of a fluorinated compound depends on the solvent used and on the temperature. For instance, considering ethanol as solvent, the saturation concentration of perfluorobutane is 2.5% by volume and the saturation concentration of perfluoropropane is 2.7% by volume (US2019307908A1).

[0219] Preferably, at step b.ii) said admixing is performed for a time suitable to reach the saturation concentration of the gaseous fluorinated compound into the organic liquid solution, e.g. for 2 minutes.

[0220] At the end of the admixing, an organic phase consisting of a homogenous solution is obtained.

[0221] Furthermore, at the end of its preparation process, said organic phase can be diluted by adding a suitable amount of organic solvent in order to reduce the concentration of the nanodroplets into the organic phase.

[0222] The expression "suitable amount of organic solvent" indicates the quantity of organic solvent (e.g. in mL) necessary to reduce the initial concentration of nanodroplets into the organic phase (e.g. the saturation concentration).

[0223] Said organic phase is then injected into the microfluidic cartridge.

[0224] At step b) the temperature of the organic phase is preferably lower than room temperature (25°C), e.g. about 4°C, to avoid vaporization of fluorinated compounds having a boiling point close to 25°C.

[0225] In an embodiment, at the step b) the temperature of the organic phase is typically lower than 25°C, preferably lower than 10°C, more preferably the temperature is about 5 ± 2°C. Said temperature is preferably not lower than 2°C.

[0226] Step c) Injection into the microfluidic cartridge

[0227] Typically, at step c) the injection of the aqueous phase and the injection of the organic phase are carried out simultaneously.

[0228] The expression "simultaneously" indicates the simultaneous injection (i.e. coinjection) of the aqueous phase and the organic phase into the microfluidic cartridge, that is to say that the aqueous phase and organic phase are injected into two separate inlets of the microfluidic cartridge at the same time or at substantially the same time (e.g. within few seconds).

[0229] In a preferred embodiment, both aqueous and organic phases are injected into the microfluidic cartridge at a temperature suitable to avoid or substantially limit the evaporation of the fluorinated compound. For instance, after their respective preparations (i.e. step a) and step b)) both the aqueous phase and the organic phase can be stored in an ice bath (about 4°C) before their injection into the separate inlets of the microfluidic cartridge (e.g. for 5 minutes), in order to limit the temperature increase during the time between step a), step b) and the subsequent step c).

[0230] According to the present invention, after their injections, the aqueous phase and the organic phase are directed towards a mixing device, wherein they are mixed (e.g. through laminar mixing in the case of a staggered herringbone micromixer (see Figure 2)) endowing to the formation of NDs.

[0231] Typically, the operating pressure into the microfluidic cartridge is lower than 1000 psi (about 7000 kPa), preferably lower than 500 psi (about 3500 kPa), still more preferably lower than 300 psi (about 2000 kPa), still more preferably lower than 100 psi, (about 700 kPa), e.g. between 10 and 90 psi.

[0232] The temperature of the mixing portion, wherein the mixing process takes place into the peculiar micro-channel geometry of the mixing portion, can be comprised between 0°C and 25°C, preferably comprised between 0°C and 15°C, more preferably between 0°C and 5°C.

[0233] For instance, the microfluidic cartridge can be stored in the fridge (e.g. 4°C), for a suitable time able to reach the desired temperature.

[0234] Total Flow Rate (TFR) and Flow Rate Ratio (FRR)

[0235] The method of the present invention allows controlling the cross-linkable vesicles characteristics by varying two process parameters: the Total Flow Rate and the Flow Rate Ratio.

[0236] The expression "Total Flow Rate (TFR)" refers to the total flow of both fluid streams, namely the aqueous phase and the organic phase, being pumped through the two separate inlets of the microfluidic cartridge. The unit of measurement of the TFR is mL / min.

[0237] According to an embodiment, the TFR is preferably comprised between 2 mL / min and 200 mL / min, preferably 2 and 18 mL / min, more preferably between 5 mL / min and 16 mL / min, still more preferably the TFR is 10 mL / min.

[0238] The expression "Flow Rate Ratio (FRR)" refers to the ratio between the amount of aqueous phase and the amount of organic phase flowing into the microfluidic cartridge, according to the Equation 2: volume of aqueous phase

[0239] Flow rate ratio = — - - — - — - volume of or game phase

[0240] Eq.2

[0241] The volume of aqueous and organic phases can be expressed as e.g. mL. In a preferred embodiment, the FRR (volume of aqueous phase vs. volume of organic phase) is between 1: 1 to 5: 1, preferably between 1: 1 and 3: 1, more preferably the FRR is 2: 1.

[0242] In the present invention, the respective concentrations of both cross-linkable amphiphilic peptide and fluorinated compound and the FRR can be purposely tuned in order to obtain a molar ratio between said cross-linkable amphiphilic peptide and said fluorinated compound suitable to assure the stability of the cross-linked vesicle (i.e certain values of sizes and PDI). The molar ratio between said cross-linkable amphiphilic peptide and said fluorinated compound is preferably comprised between 0.002 and 7.000. More preferably the ratio is not higher than 6.000, not higher than 5.000, not higher than 4.000, not higher than 3.000, not higher than 2.000, even more preferably not higher than 1.500. More preferably the molar ratio is not lower than 0.001, not lower than 0.004, even more preferably not lower than 0.050.

[0243] Step d) Collection of the aqueous suspension of calibrated nanodroplets

[0244] According to the step d) of the disclosed method, at the end of the mixing process, an aqueous suspension of cross-linkable vesicles comprising a liquidinner core and an outer layer, wherein said inner core comprises a fluorinated compound having a boiling point lower than 25°C at atmospheric pressure, and said outer layer comprises an amphiphilic peptide as above defined is collected from the exit channel of the microfluidic cartridge.

[0245] Preferably, at the end of step d) the suspension of cross-linkable vesicles is characterized by a polydispersity index (PDI) lower than 0.30, preferably lower than 0.25, more preferably lower than 0.20, even more preferably lower than 0.15, even more preferably lower than 0.10, and a Z-average diameter comprised between 100 nm and 1000 nm, preferably between 120 and 800 nm, more preferably between 150 and 400 nm.

[0246] After their collection, the inner core of said microfluidically prepared vesicles is composed by a fluorinated compound in liquid form independently from the temperature at which they are collected and subsequently stored and independently from the nature of fluorinated compound added to the organic phase (i.e. gaseous or liquid at SATP conditions).

[0247] In other words, even if the collection step is performed (and the subsequent storage is affected) at a temperature above the boiling point of the fluorinated compound used for the manufacturing of the cross-linkable vesicles, said fluorinated compound remains in a liquid state due to its incorporation into the outer stabilizing shell comprising an amphiphilic component. Nevertheless, it is preferable that such temperature is not excessively high, in order to limit possible partial phase transition of the entrapped fluorinated compound.

[0248] According to an embodiment, the temperature at which said collection step is performed can be comprised between 0°C and 25°C, preferably comprised between 0°C and 15°C, more preferably between 0°C and 5°C.

[0249] Preferably, the collected aqueous suspension of cross-linkable vesicles has a temperature comprised between 0°C and 25°C, preferably comprised between 0°C and 15°C, more preferably between 0°C and 5°C.

[0250] For instance, after the collection, said aqueous suspension can be stored at 4°C in a fridge.

[0251] Step el Dilution

[0252] In an embodiment, the method of preparation further comprises a step e), which comprises diluting the aqueous suspension of collected cross-linkable vesicles.

[0253] In an embodiment, the step e) is carried out sequentially to step d). For example, the step e) can be performed between step d) and step f), in particular after the step d), for instance within 5 minutes from collecting the sample from the microfluidic cartridge, and before starting the cross-linking phase.

[0254] As indicated above, the expressions "initial monodispersed distribution" and "initial sizes" (also defined as "initial characteristics" or "initial properties")refer to the values of monodispersity and sizes of the calibrated cross-linkable vesicles composition collected from the exit channel of the microfluidic cartridge at the step d) of the disclosed method of preparation.

[0255] In the present description and claims the term "dilution" refers to the process of reducing the concentration of calibrated cross-linkable vesicles in the suspension, by adding a suitable amount of aqueous component.

[0256] A suitable amount of aqueous component corresponds to the quantity of aqueous solution necessary to reduce the concentration of the calibrated vesicles in the aqueous suspension from 2 to 10- folds.

[0257] In a preferred embodiment, the optional step e) of the present method comprises diluting the aqueous suspension of calibrated vesicles from 1 to 20-folds, preferably from 2 to 10-folds, still more preferably from 3- to 8-folds e.g. about 5-fold.

[0258] Suitable aqueous components are water, aqueous solutions or oxidizing solutions as described below.

[0259] In a preferred embodiment, the step e) of the present method comprises diluting the suspension of calibrated cross-linkable vesicles with water. Alternatively, the diluting step can be performed directly inside the microfluidic cartridge (i.e. in-line dilution), by way of an additional channel (e.g. placed between the mixing portion 103 and the exit channel 104 in Fig. 1 of WO20234084048) suitable for diluting the calibrated cross-linkable vesicles suspension with the desired aqueous component before their direction to the exit channel and before the cross-linking step e).

[0260] Step f) Cross-linking step

[0261] The step f) of the method of the present invention comprises cross-linking the cross-linkable amphiphilic peptides to obtain an aqueous suspension of fluorocarbon- filled cross-linked vesicles, by contacting said aqueous suspension of cross-linkable vesicles with an oxidizing source able to induce the cross-linking of the cross-linkable amphiphilic peptides to obtain an aqueous suspension of cross-linked vesicles, wherein said oxidizing source does not comprise dimethyl sulfoxide, i.e. DMSO.

[0262] In the present description and claims, the expression "cross-linking" indicates the process of forming covalent bonds between cross-linkable moieties comprised in adjacent cross-linkable amphiphilic peptides in order to bind cross-linkable amphiphilic peptides molecules together. Preferably said cross-linkable moieties are cross-linkable amino acids comprised in adjacent cross-linkable amphiphilic peptides.

[0263] In a preferred embodiment, said cross-linkable amino acids are cysteine amino acids comprised in adjacent cross-linkable amphiphilic peptides. Said cysteine amino acids are cross-linked via disulfide cross-linking groups (-S-S-).

[0264] In the present description and claims, suitable examples of oxidizing sources are oxidizing solutions, oxidizing gas or a mixture thereof.

[0265] The term "oxidizing solution" indicates an aqueous solution which may cause or contribute to induce the formation of covalent bonds between cross-linkable amino acids, e.g. between the thiol groups of cysteine amino acids comprised in adjacent crosslinkable amphiphilic peptides. According to this invention, said oxidizing solution does not comprise DMSO.

[0266] Suitable examples of oxidizing solutions are water, basic solutions comprising halogens, aqueous hydrogen peroxide comprising iodine and selenide-based catalyst in presence of air.

[0267] The Applicant has observed that the initial properties (e.g. initial monodispersed distribution" and "initial sizes) of freshly-prepared cross-linkable vesicles filled with a liquid fluorinated compound having a boiling point lower than 25°C, e.g. vesicles collected from the microfluidic apparatus (step d) and not submitted yet to any crosslinking procedure, were not preserved when using the standard cross-linking procedures disclosed in the literature for the preparation of similar nanodroplets comprising high- boiling point perfluorocarbon (i.e. b.p. higher than 25°C, such as perfluoropentane), such as crosslinking procedures using as oxidizing source an aqueous solution of dimethylsulfoxide (DMSO).

[0268] Unexpectedly, performing the step f) by using an oxidizing solution comprising disulfiram led to the substantial preservation of the initial sizes of said cross-linked vesicles (e.g. %Evol lower than ±20%) and to polydisperisity index lower than 0.3. In other words, the initial sizes and polydispersity profile characterizing the suspension of cross-linkable vesicles were not negatively affected by using an oxidizing source comprising disulfiram (DSF) at the cross-linking step.

[0269] The term disulfiram (DSF) indicates the bis(diethylthiocarbamoyl)disulfide (C10H20N2S4), a dithiocarbamate drug of formula:

[0270] The oxidizing solution comprising disulfiram is contacted with the freshly prepared aqueous suspension of calibrated cross-linkable vesicles for a time sufficient to induce the cross-linking of the cross-linkable amphiphilic peptides comprised in the outer layer of the vesicles.

[0271] According to this invention, the aqueous solution comprising DFS has a pH preferably lower than 7, more preferably lower than 6.5, still more preferably lower than 6, even more preferably lower than 5.5, even more preferably lower than 5, even more preferably lower than 4.5, down to 4.

[0272] The aqueous oxidizing solution comprising DFS can advantageously further comprise a compound able to enhance the cross-linking step, for instance by regulating the pH as above described and by reducing the total time of the cross-linking reaction, e.g. the time required to induce a degree of cross-linking of the cross-linkable amphiphilic peptide molecules of higher than 80%, preferably at least 85%, more preferably at least 90%. Preferably the degree of cross-linking is 100%.

[0273] Suitable examples of compounds able to enhance the cross-linking step are glucose, guanidine HCI, or a mixture thereof, preferred being glucose.

[0274] For example, the oxidizing source is contacted with the freshly prepared aqueous suspension of cross-linkable vesicles for a time sufficient to induce a degree of crosslinking of the cross-linkable amphiphilic peptide molecules of higher than 80%, preferably at least 85%, more preferably at least 90%. Preferably the degree of crosslinking is 100%.

[0275] Alternatively, the oxidizing source can be an oxiding gas. The term "oxidizing gas" indicates any gas which may induce or contribute to the formation of covalent bonds between cross-linkable amino acids, e.g. between the thiol groups of cysteine amino acids comprised in adjacent cross-linkable amphiphilic peptides.

[0276] Suitable examples of oxidizing gas are oxygen, air or suitable mixtures of gas comprising oxygen, preferred being air.

[0277] The oxidizing gas is contacted with the freshly prepared aqueous suspension of calibrated cross-linkable vesicles for a time of at least one minute, preferably at least 30 minutes, more preferably at least one hour. The time typically does not exceed 24 hours, preferably less than 10 hours, more preferably less than 3 hours.

[0278] For instance, at the end of the microfluidic process, an oxidizing gas, e.g. air, can be injected by bubbling into the oxidizing solution contacted with the calibrated crosslinkable vesicles.

[0279] Said oxidizing source may comprise a mixture of an oxidizing solution and an oxidizing gas, for instance it may comprise a mixture of an aqueous oxidizing solution and air.

[0280] The step f) may also be a dialysis procedure comprising contacting an aqueous suspension of calibrated cross-linkable vesicles with an oxidizing source able to induce the cross-linking of the cross-linkable amphiphilic peptides to obtain an aqueous suspension of calibrated fluorocarbon cross-linked vesicles, wherein said oxidizing source does not comprise DMSO.

[0281] In the present description, the expression "dialysis" indicates a procedure for promoting the cross-linking of the freshly prepared aqueous suspension of cross-linkable vesicles, through the mechanisms described above.

[0282] Examples of oxidizing sources suitable for a dialysis procedure are those described above.

[0283] The dialysis procedure may for instance be performed after the collection from the microfluidic cartridge, by loading the aqueous suspension of calibrated cross-linkable vesicles inside a dialysis device comprising a semi-permeable membrane which is contacted with an oxidizing source, for instance by suspending said dialysis device in a large volume of an oxidizing solution, as defined above, into which is simultaneously injected an oxidizing gas.

[0284] Suitable examples of dialysis devices comprising a semi-permeable membrane can be traditional dialysis tubings or advanced dialysis devices, such as dialysis cassettes, dialysis flasks or dialysis plates. The permeability of said semi-permeable membrane is such that the oxidizing solution can contact the calibrated (per)fluorocarbon cross-linkable vesicles, loaded inside the dialysis device, consequently inducing their cross-linking. Said dialysis procedure allows efficient recovery of an aqueous suspension of calibrated (per)fluorocarbon cross-linked vesicles.

[0285] Following the microfluidic preparation of the cross-linkable vesicles and their cross-linking as above described, the degree of cross-linking is typically higher than 80%, preferably at least 85%, more preferably at least 90%, still more preferably at least 95%. Preferably the degree of cross-linking is 100%.

[0286] In a preferred embodiment, step f) is performed at a temperature higher than 5 (± 2°C), preferably higher than 10°C, more preferably higher than 15°C, more preferably higher than 20°, more preferably higher than 25°C, more preferably higher than 30, more preferably higher than 35, up to 40°C.

[0287] Optional step g: Washing

[0288] In a further embodiment, the method of the invention may comprise optional step g), which comprises washing the aqueous suspension of vesicles after step e) and / or after step f).

[0289] According to this embodiment, suitable washing techniques may be used i) after step e) on the diluted cross-linkable vesicles (e.g. before being submitted to the crosslinking treatment); ii) after step f) on the cross-linked vesicles; or ii) after both step e) and step f).

[0290] In the present description, the term "washing" indicates any operation carried out on the freshly diluted cross-linkable vesicles (after step e) or on the freshly prepared cross-linked vesicles suspensions (after step f), finalized to remove (or substantially reduce the amount of) fluorinated compound-free assemblies, or alternatively, finalized to replace the aqueous solution in which the cross-linked vesicles are suspended at the end of the step f), e.g. to remove (or substantially reduce the amount of) the excess of oxidizing source

[0291] In the present description, the expression "fluorinated compound-free assembly" indicates an assembly comprising cross-linkable amphiphilic peptides spontaneously assembled in a particle due to hydrophobic interactions. Said assemblies may be formed during the microfluidic process and may be present in the aqueous suspension of calibrated vesicles at the end of the preparation process, but due to their lower sizes, they contribute to forming a second population of particles, endowing to a higher value of PDI.

[0292] For example, the fluorinated compound-free assemblies are characterized by sizes comprised between 50 nm and 150 nm. The washing in step g) can be carried out from 1 time to 10 times, preferably is performed from 2 times to 5 times, still more preferably is performed 3 times.

[0293] For example, when the washing step g) is performed after step e) on the diluted cross-linkable vesicles, it is possible to remove (or substantially reduce the amount of) said fluorinate compound-free assemblies from the suspension of cross-linkable vesicles and subsequently adding an oxidizing source in order to perform step f) of cross-linking.

[0294] Alternatively, when the washing step is performed after step f) on the freshly cross-linked vesicles, after removing (or substantially reducing the amount of) oxidizing source, it is possible to add an aqueous to obtain an aqueous suspension of calibrated cross-linked vesicles.

[0295] The aqueous solution is preferably physiologically acceptable, comprising water (preferably sterile water), aqueous solutions such as saline (which may advantageously be balanced so that the final product for injection is not hypotonic), or solutions of one or more pharmaceutical excipients. Suitable examples of pharmaceutical excipients are tonicity adjusting substances. Tonicity adjusting substances comprise salts or sugars, sugar alcohols, glycols or other non-ionic polyol materials (e.g. glucose, sucrose, trehalose, sorbitol, mannitol, glycerol, polyethylene glycols, propylene glycols and the like), chitosan derivatives, such as carboxymethyl chitosan, trimethyl chitosan or jellifying compounds, such as carboxymethylcellulose, hydroxyethyl starch, hydrolyzed collagen, or dextran.

[0296] Suitable washing techniques comprise, for instance, centrifugation, ultracentrifugation, filtration and decantation.

[0297] Preferably said washing technique is centrifugation.

[0298] The duration of the centrifugation is of at least 1 minute, preferably at least 3 minutes, more preferably at least 4 minutes. The duration of the centrifugation is typically lower than 20 minutes, preferably lower than 10 minutes, more preferably the duration is 5 minutes.

[0299] The temperature at which the centrifugation is performed is typically of at least 1°C, preferably at least 2°C, more preferably at least 3 °C. Said temperature does not exceed 25°C, preferably is lower than 20°C, more preferably is lower than 10 °C.

[0300] The rotation at which is performed the centrifugation is comprised between 1000 g and 10000 g, preferably between 2000 g and 6000 g, still more preferably the rotation is 5000 g.

[0301] The aqueous solution is preferably physiologically acceptable, comprising water (preferably sterile water), aqueous solutions such as saline (which may advantageously be balanced so that the final product for injection is not hypotonic), or solutions of one or more pharmaceutical excipients. Suitable examples of pharmaceutical excipients are tonicity adjusting substances. Tonicity adjusting substances comprise salts or sugars, sugar alcohols, glycols or other non-ionic polyol materials (e.g. glucose, sucrose, trehalose, sorbitol, mannitol, glycerol, polyethylene glycols, propylene glycols and the like), chitosan derivatives, such as carboxymethyl chitosan, trimethyl chitosan or jellifying compounds, such as carboxymethylcellulose, hydroxyethyl starch, hydrolyzed collagen, or dextran.

[0302] Preferred is a glucose solution. The glucose aqueous solution typically comprises glucose at a concentration of at least 1%, preferably at least 2.5%, more preferably at least 3%. The glucose concentration typically does not exceed 20%, preferably is less than 10%, more preferably is less than 7%.

[0303] Assembly of cross-linked vesicles and cargo molecule

[0304] The disclosed cross-linked vesicle are cationic cross-linking vesicles having a zeta potential of at least 20 mV, preferably at least 30 mV, more preferably at least 40 mv, up to 100 mV.

[0305] Advantageously, the disclosed cross-linked vesicles can adsorb on their positive outer layer as cargo molecule.

[0306] It is thus possible to obtain an assembly comprising a cross-linked vesicle as defined above, and a cargo molecule, wherein said cargo molecule is electrostatically bound to the outer layer of said cross-linked vesicle.

[0307] As used herein, the term "cargo molecule" indicates a negatively-charged molecule, such as a genetic material or a nucleic acid mimetic, e.g. a peptide nucleic acid (PNAs).

[0308] According to the present description, the expression genetic material indicates any nucleic acid-based agent, such as, but not limited to, RIMA, DNA, saRNA, oligonucleotides, miRNA, siRNA, shRNA molecule and dsRNA molecule. Preferably said genetic material comprises a negative charge.

[0309] In the present invention, the disclosed cationic cross-linked vesicles can deliver a cargo molecule adsorbed to their shell, due to an electrostatic interaction between the positive charges comprised in the cationic amphiphilic peptides forming said shell and the negative charges comprised in the cargo molecule.

[0310] As an example, a cargo molecule can be admixed with an aqueous suspension of cationic cross-linked vesicles through traditional techniques (e.g. stirring) in order to obtain an aqueous suspension of assemblies, i.e. entities formed by a cross-linked vesicles binding a genetic material.

[0311] After the electrostatic binding, the zeta potential of said assemblies is in general lower than the initial positive charge of the cross-linked vesicles. The expression "initial positive charge of the cross-linked vesicles" refers to the charge of the cross-linked vesicle measured after their preparation, for instance by zeta potential determination.

[0312] An aqueous suspension comprising a plurality of assemblies as describe above may be prepared by a method comprising the steps of: a) preparing an initial aqueous suspension of cross-linked vesicles as described above; b) mixing said initial suspension with a predetermined amount of cargo molecule and c) obtaining an aqueous suspension of assemblies, wherein the amount of said cargo molecule electrostatically bound to the outer layer of said cross-linked vesicles is at least 35% of the amount of genetic material admixed in step b), preferably at least 50%, more preferably at least 60%, still more preferably at least 70%, still more preferably at least 80%, up to 90%, preferably up to 95%, still more preferably up to 100%.

[0313] The amount of cargo molecule in the aqueous suspension of assemblies can be expressed as a percentage of the initial amount of cargo molecule (CM) in the initial suspension by using the following equation (Equation 3): amount of CM after preparation of the assemblies suspension

[0314] % CM in the assemblies suspension = - - - — - - - - - - - - ♦ 100 amount of CM admixed to the cationic vesicles suspension

[0315] Eq.3

[0316] Wherein :

[0317] • the expression "amount of CM after preparation of the assemblies suspension" refers to the weight (e.g. pg) of cargo molecule (CM) comprised in 1 ml of suspension of assemblies and

[0318] • the expression "amount of CM admixed to the cationic vesicles suspension" refers to the weight (e.g. pg) of cargo molecule comprised in 1 ml of initial suspension of cross-linked vesicles.

[0319] Said amount can be measured for instance by analytical methods, such as photometry (UV / Vis), fluorescence, electrophoresis, diphenylamine method, quantification by real-time PCR.

[0320] Use Cross-linked vesicles filled with low-boiling point fluorinated compound are a Phase-Change Contrast Agent (PCCA) that can easily be converted into gas-filled microvesicles using ultrasounds.

[0321] The acoustic droplet vaporization (ADV, typically expressed in megapascals (MPa)) is a phenomenon through which cross-linked vesicles can be converted into gas microbubbles upon exposure to ultrasound energy beyond the vaporization threshold.

[0322] The "vaporization threshold" is defined as the minimum energy input required to induce a phase transition from the liquid state within the vesicle to the gaseous state. The vaporization threshold is influenced by factors such as the composition and properties of the core, the characteristics of the encapsulating shell, and the ambient temperature and pressure conditions. It is determined through empirical measurements under controlled experimental conditions.

[0323] Typically, cross-linked vesicles with fluorinated compounds characterized by low boiling points (i.e. highly volatile) play an important role in the diagnostic applications (e.g. ultrasound imaging) because a low boiling point core induces the vesicles to have an earlier vaporization, due to the reduction of the vaporization threshold.

[0324] When administered in-vivo, said cross-linked vesicles present many advantages with respect to traditional microbubbles, such as inertness, relatively low toxicity, relative stability in circulation, immiscibility in water, and low surface tension (Sheeran et al, 2011). Once vaporized, the generated microbubbles can be effectively used in either imaging or therapeutic applications with ultrasound, including sonopermeabilization, thermal ablation, blood brain barrier (BBB) disruption, multimodal imaging modalities and allow passive (due to the enhanced permeability and retention (EPR) effect in the tumor tissues) or active targeting (by incorporating targeted ligands) for localized delivery of therapeutic drugs or genes. Another potentially valuable characteristic of PFC-NDs is their possible application for novel imaging strategies such as UltraSound Super-Resolution Imaging since these agents can be activated and deactivated on demand by applying intermittent acoustic pulses.

[0325] A further aspect relates to an aqueous suspension comprising a plurality of calibrated cross-linked vesicles as above defined for use in a diagnostic and / or therapeutic treatment.

[0326] Another aspect relates to an aqueous suspension comprising a plurality of assemblies as above defined for use in a diagnostic and / or therapeutic treatment.

[0327] Diagnostic treatment includes any method where the use of the cross-linked vesicles allows enhancing the visualization of a portion or of a part of an animal (including humans) body, including imaging for preclinical and clinical research. Suitable examples of diagnostic applications are molecular and perfusion imaging, tumor imaging (EPR effect), multimodal imaging (MR-guided tumor ablation, fluorescence, sonophotoacoustic activation), US aberration correction and super-resolution imaging.

[0328] Therapeutic treatment includes any method of treatment of a patient. In preferred embodiments, the treatment comprises the combined use of ultrasounds and cross-linked vesicles either as such (e.g. in ultrasound-mediated thrombolysis, high intensity focused ultrasound ablation, blood-brain barrier permeabilization, immunomodulation, neuromodulation, radiosensitization) or in combination with a therapeutic agent (i.e. ultrasound-mediated delivery, e.g. for the delivery of a drug or bioactive compound to a selected site or tissue, such as in tumor treatment, gene therapy, infectious diseases therapy, metabolic diseases therapy, chronic diseases therapy, degenerative diseases therapy, inflammatory diseases therapy, immunologic or autoimmune diseases therapy or in the use as vaccine), whereby the presence of the vesicles may provide a therapeutic effect itself or is capable of enhancing the therapeutic effects of the applied ultrasounds, e.g. by exerting or being responsible to exert a biological effect in vitro and / or in vivo, either by itself or upon specific activation by various physical methods (including e.g. ultrasound-mediated delivery).

[0329] An aspect of the invention relates to an aqueous suspension comprising a plurality of cross-linked vesicles as above defined for use in a method of diagnosis in vivo, wherein said method is preferably ultrasound-mediated.

[0330] A further aspect of the invention relates to an aqueous suspension comprising a plurality of cross-linked vesicles as above defined for use as a contrast agent, preferably as ultrasound contrast agent.

[0331] A still further aspect of the invention relates to a use of an aqueous suspension comprising a plurality of cross-linked vesicles as above defined as contrast agent for imaging, preferably for ultrasound imaging.

[0332] An aspect of the invention relates to an aqueous suspension comprising a plurality of cross-linked vesicles as above defined for use as medicament.

[0333] An embodiment relates to said aqueous suspension for use in an ultrasound- mediated treatment.

[0334] Another aspect relates to an assembly comprising a cross-linked vesicle as defined above, and a cargo molecule, wherein said cargo molecule is electrostatically bound to the outer layer of said cross-linked vesicle for use as medicament.

[0335] An embodiment relates to said assembly for use in an ultrasound-mediated treatment. The following examples will help to further illustrate the invention.

[0336] EXAMPLES

[0337] Table 1 Specific type and amounts of materials used in the preparations illustrated in the following examples

[0338] Example 1

[0339] Synthesis of amphiphilic peptides

[0340] Three amphiphilic peptides, namely AP-TAT, AP-SV40 and AP-PreS2 were synthesized by Fmoc solid-phase peptide synthesis (SPPS) using either H-Rink amide ChemMatrix® resin, Rink-Amide AM resin or Rink-Amide AM resin LL, using either a mixture of Oxyma Pure and DIC or a mixture of HATU and DIPEA as coupling agent, on a PurePep Chorus Peptide Synthesizer (Gyros Protein Technologies). Resin cleavage was performed in a TFA / H2O / EDT / TIS (94:2.5:2.5: 1) solution for 5 hours. Crude peptides were then purified by preparative High Performance Liquid Chromatography (HPLC).

[0341] 1. AP-TAT (H2N-FFFFFFGGGCCGGKGYGRKKRRQRRR-NH2)

[0342] Purification: Solid phase: Aeris 5 pm Peptide XB-C18 100 A 250 x 21.2 mm. Mobile phase: 90% H2O + 0.1 % TFA / 10% ACN+ 0.1 % TFA to 30% H2O + 0.1 % TFA / 70% ACN + 0.1 % TFA over 30 min.T he structural predictions of the cationic hydrophilic amino acid sequence were performed by using AlphaFold2, 5 through GoogleColab notebook AlphaFold2.ipynb (ColabFold vl.5.2-patch).

[0343] (https: / / colab.research.google.com / github / sokrypton / ColabFold / blob / main / AlphaFold2.ip ynb#scrollTo=33g5IIegij5R).

[0344] The predicted secondary structure of the cationic hydrophilic amino acid sequence TAT (YGRKKRRQRRR) is showed in Figure lb and corresponds to a randomly-organized secondary structure.

[0345] 2. AP-SV40 (H2N-FFFFFFGGGCCGGKGPKKKRKV-NH2)

[0346] Synthesis: Pentafluorophenylalanine 19 was introduced using HATU and DIPEA as coupling agents.

[0347] Purification: Solid phase: Aeris 5 pm Peptide XB-C18 100 A 250 x 21.2 mm. Mobile phase: 75% H2O + 0.1 % TFA / 25% ACN+ 0.1 % TFA to 50% H2O + 0.1 % TFA / 50% ACN + 0.1 % TFA over 30 min.

[0348] The secondary structure of the hydrophilic amino acid sequence SV40 (PKKKRKV) predicted using ColabFold (as indicated in Example 1 Item 1.) is showed in Figure lc and corresponds to a randomly-organized secondary structure.

[0349] 3. AP-PreS2 ( H2N-FFFFFFGGGCCGGKGPLSSIFSRIGDP-NH2)

[0350] Purification : Solid phase: Aeris 5 pm Peptide XB-C18 100 A 250 x 21.2 mm. Mobile phase: 65% H2O + 0.1 % TFA / 35% ACN+ 0.1 % TFA to 55% H2O + 0.1 % TFA / 45% ACN + 0.1 % TFA over 30 min. The secondary structure of the hydrophilic amino acid sequence Pres2 (PLSSIFSRIGDP) predicted using ColabFold (as indicated in Example 1 Item 1.) is shown in Figure la and corresponds to an alpha helix structure.

[0351] Example 2

[0352] General procedure for the preparation of an aqueous suspension of vesicles using a microfluidic platform

[0353] Vesicles comprising a low boiling point PFC were formulated with a NanoAssemblr™ Benchtop automated instrument from Precision Nanosystems (Vancouver, Canada) equipped with a staggered herringbone micromixer (SHM) allowing size-controlled selfassemblies.

[0354] Firstly, liquid perfluorobutane (C4F10, b.p. -2°C) was obtained by condensation of PFB gas into a syringe at -20°C and was added to an ethanolic solution at a concentration of 10 or 15 pL / mL. The solution was stirred until the total dissolution of liquid perfluorobutane.

[0355] Then, an aqueous phase comprising an amphiphilic peptide dispersed in aqueous sodium acetate buffer at pH 4 was injected into the first inlet whereas the organic phase composed of perfluorobutane dissolved in ethanol was injected into the second inlet of the microfluidic cartridge. Both the aqueous phase and the organic phase were placed into an ice bath at about 4°C before the cross-linkable vesicles formulation. Microscopic characteristics of the channels are engineered to cause an accelerated mixing of the two fluid streams in a controlled fashion. The microfluidic process settings namely the Total Flow Rate (TFR, in mL / min), and the Flow Rate Ratio (FRR), were varied to control the cross-linkable vesicles characteristics.

[0356] The aqueous suspension of calibrated vesicles was collected from the exit channel in a Falcon vial (15 mL) and then diluted 5-times in in water before being centrifugated (5000g, 12min, 4°C).

[0357] Subsequently, after removal of the supernatant phase, the pellet was redispersed in a cold 6M guanidine hydrochloride aqueous buffer (pH 7) or in a 5% glucose aqueous solution (pH 4.3), followed by the addition of DSF in acetonitrile (10 Eq; 170 mM).

[0358] At the end of the procedure, an aqueous suspension of calibrated cross-linked vesicles comprising PFB was obtained and stored overnight at 4°C.

[0359] The characterization (Z average and PDI) was performed for all compositions using a Malvern Zetasizer Nano-ZS instrument (Malvern Instruments Ltd., UK) to measure the size and size distribution (PDI) after different storage conditions (i.e. after the dilution step before performing the cross-linking, and after the cross-linking treatment), as illustrated in Table 2. Results

[0360] As inferable from the results, the final characteristics of the cross-linked vesicles are substantially influenced by the nature of the cationic amphiphilic peptide(s) (AP(s)) comprised in their outer shell.

[0361] Cationic APs comprising a hydrophilic amino acid sequence having an alpha-helix secondary structure and at least a positive charge, such as AP-Pres2 (e.g. SI), were able to form cross-linkable vesicles characterized by good sizes and PDI values. Said initial sizes were substantially preserved (size % Evol lower than ±20) after the cross-linking treatment with disulfiram with PDI values still indicating a monodisperse profile.

[0362] On the contrary, cross-linkable vesicles stabilized by cationic APs comprising a hydrophilic amino acid sequence with a randomly-organized secondary structure, such as AP-SV40 (S2) and AP-TAT (S3), were characterized by higher sizes and a highly polydisperse profile after the cross-linking step with disulfiram.

[0363] Furthermore, mixture of amphiphilic peptides comprising at least one cationic AP comprising a hydrophilic amino acid sequence having an alpha-helix secondary structure and at least a positive charge, such as AP-Pres2 (e.g. Ml and M2), were able to form cross-linkable vesicles characterized by optimal characteristics after the cross-linking treatment with disulfiram.

[0364] Table 2 Sizes and PDI after cross-linking with disulfiram.

[0365] Example 3

[0366] Determination of Acoustic Droplet Vaporization (ADV)

[0367] The expression "Acoustic Droplet Vaporization (ADV) threshold" indicates the minimal acoustic pressure that is necessary to obtain the nanodroplets conversion into echogenic microbubbles. The Acoustic Droplet Vaporization (ADV) threshold of the NDs prepared according to the previous examples can be determined according to conventional methodologies using B-mode imaging methods. For instance, the suspension of vesicles can be vaporized while passing through the focal zone of a transducer and the acoustic pressure is increased by about 0.2 MPa each 5 s until the NDs vaporization is observed.

[0368] Nanodroplets activation was performed by focused ultrasound waves on five aligned focal points allowing the activation only within the region of interest where the acoustics pressure was highest. Pulses were emitted in burst mode at a frequency of 6 MHz, 30 cycles per pulse and at a pulse-repetition frequency (PRF) of 1 Hz.

[0369] The acoustic pressure was increased every 5 s until the observation of the NDs vaporization.

[0370] For the ADV determination, three different compositions were tested, namely SI,

[0371] Ml and M2, prepared as described in Example 2 (pellet redispersed in a 5% glucose aqueous solution, followed by the addition of DSF in acetonitrile (10 Eq; 170 mM).

[0372] Results

[0373] Table 3 reports the overall results obtained from the determination of the ADV thresholds. Each value is the average of three successive determinations.

[0374] Results confirmed that formulations comprising cross-linked vesicles stabilized by a single amphiphilic peptide, such as Composition SI, and formulations comprising cross-linked vesicles stabilized by a mixture of amphiphilic peptides (Compositions Ml, M2) could be vaporized at similar acoustic pressure.

[0375] Table 3 Determination of acoustic droplet vaporization threshold Comparative Example 4

[0376] Cross-linking step using DMSO 2.5%

[0377] After preparation using the microfluidic apparatus (Example 2), the collected suspension of vesicles was treated as described in WO2023084048. Briefly, it was diluted 5-times in 2.5 % aqueous DMSO before being transferred to a Slide-A-Lyzer™ Dialysis Cassettes from Thermo Fisher Scientific and dialyzed against 2.5 % aqueous DMSO overnight with air bubbles for the first hour. Subsequently, the suspension was further dialyzed against Milli-Q® water for 2 hours and against aqueous glucose 5 % solution for 4 hours.

[0378] At the end of the procedure, the characterization was performed for all compositions using a Malvern Zetasizer Nano-ZS instrument (Malvern Instruments Ltd., UK) to measure the size and size distribution (PDI) after different conditions (i.e. after the dilution step before performing the cross-linking, and after the cross-linking treatment), as illustrated in Table 4.

[0379] Results

[0380] Table 4 Sizes and PDI after cross-linking with DMSO 2.5%

[0381] As inferable from Table 4, the initial sizes and PDI of the suspensions of crosslinked vesicles comprising a perfluorocarbon with a boiling point lower than 25°C, e.g. perfluorobutane, were not preserved after the standard cross-linking treatment with DSMO (2.5%) as showed in the literature for similar nanodroplets filled in their core with perfluorocarbon with higher boiling point (e.g. higher than 25°C, such as perfluoropentane and perfluorohexane).

[0382] Comparative Example 5

[0383] Preparation of cross-linked vesicle with low boiling fluorinated compound using a standard procedure

[0384] A comparative aqueous suspension of cross-linked vesicles stabilized by AP-PRES-2, (2 mg / ml) was prepared according to the procedure disclosed in W02019 / 023706 (see Par. [144-145] "Peptisome and nanopeptisome formation"). The size and size distribution (polydispersity index, PDI) of the resulting formulation were assessed using a Malvern Zetasizer Nano-ZS instrument (Malvern Instruments Ltd., UK) at various stages of the preparation process, namely before and after the cross-linking treatment with a DMSO solution.

[0385] Results

[0386] Table 5 Characterization of vesicles suspensions obtained with preparation method disclosed in WO2019023706.

[0387] As inferable from Table 5, the initial characteristics of the obtained cross-linkable vesicles comprising a low boiling point fluorinated compound (e.g. perfluorobutane) were negatively affected by the cross-linking treatment in DMSO, which led to suspension of vesicles characterized by substantally lower size and high PDI values.

[0388] References

[0389] • Durham, P.G., Dayton P.A, Curr Opin Colloid Interface, 56, 101498, (2021)

[0390] • W02019 / 023706

[0391] • W02023084048

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[0394] • Errington, N. et al (2006). Structure and Stability of the a-Helix. In: Guerois, R., de la Paz, M.L. (eds) Protein Design. Methods in Molecular Biology, vol 340. Humana Press.

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Claims

CLAIMS1. A cross-linked vesicle comprising an outer layer and an inner core, said outer layer comprising a cationic amphiphilic peptide and said inner core comprising a liquid fluorinated compound having a boiling point lower than 25°C, wherein said amphiphilic peptide is a compound of formula (I)HB - CL - HP (I) whereinHB is a fluorinated hydrophobic block,CL is a cross-linking motif, andHP is a cationic hydrophilic amino acid sequence having an alpha-helix structure and comprising at least 1 positive charge.

2. The cross-linked vesicle according to claim 1, wherein the boiling point of said fluorinated compound is 22°C or lower, up to -70°C.

3. The cross-linked vesicle according to claims 1 and 2, wherein said fluorinated compound is a perfluorocarbon, selected from perfluorobutane, perfluoropropane, or a mixture thereof.

4. The cross-linked vesicle according to any of the preceding claims, wherein said cationic hydrophilic amino acid sequence comprises at least 2 positive charges, up to 40.

5. The cross-linked vesicle according to claim 4, wherein said cationic hydrophilic amino acid sequence comprises at least 3 positive charges, up to 40.

6. The cross-linked vesicle according to 5, wherein said cationic hydrophilic amino acid sequence comprises at least 4 positive charges, up to 40.

7. The cross-linked vesicle according to 6, wherein said cationic hydrophilic amino acid sequence comprises at least 5 positive charges, up to 40.

8. The cross-linked vesicle according to any of the preceding claims, wherein said cationic amphiphilic peptide is H2N-FFFFFFGGGCCGGKGPLSSIFSRIGDP-NH2 (SEQ ID NO: 1).44RECTIFIED SHEET (RULE 91) ISA / EP9. The cross-linked vesicle according to any of the preceding claims, wherein HB is a fluorinated hydrophobic amino acid sequence HB'.

10. The cross-linked vesicle according to claim 9, wherein said fluorinated hydrophobic amino acid sequence HB' comprises three consecutively connected pentafluorophenylalanine residues at a terminal thereof and is a compound of formula IIIFormula III11. The cross-linked vesicle according to any of the preceding claims, wherein the cross-linking motif CL comprises a cysteine.

12. The cross-linked vesicle according to claim 11, wherein said cross-linking motif CL comprises the amino acid sequence GGGCCGG.

13. The cross-linked vesicle according to any of the preceding claims, wherein said outer layer further comprises an additional amphiphilic peptide having formula VHB - CL - HP'(V) whereinHB is a fluorinated hydrophobic block,CL is a cross-linking motif andHP' is a hydrophilic amino acid sequence.

14. The cross-linked vesicle according to claim 13, wherein HP' has a secondary structure selected from the group of alpha helix, n- helix, beta-sheet, beta turn or a mixture thereof.RECTIFIED SHEET (RULE 91) ISA / EP15. The cross-linked vesicle according to claim 14, wherein HP' has a randomly- organized secondary structure.

16. The cross-linked vesicle according to claim 15, wherein said additional amphiphilic peptide is a cationic amphiphilic peptide, wherein HP' is a cationic hydrophilic amino acid sequence comprising at least one positive charge.

17. The cross-linked vesicle according to claim 16, wherein HP' is a cationic hydrophilic amino acid sequence comprising 2 or more positive charges.

18. The cross-linked vesicle according to any claim 15-17, wherein said additional amphiphilic peptide is H2N-FFFFFFGGGCCGGKGYGRKKRRQRRR-NH2 (SEQ ID NO:2) or H2N-FFFFFFGGGCCGGKGPKKKRKV-NH2 (SEQ ID NO:3).

19. The cross-linked vesicle according to any of the preceding claims, having a zeta potential of at least 20 mV, up to 100 mV.

20. An aqueous suspension comprising a plurality of cross-linked vesicles as defined in claims 1-19.

21. The aqueous suspension according to claim 20, wherein said vesicles have a z- average diameter comprised between 100 nm and 1000 nm and a polydispersity lower than 0.3.

22. A method for the preparation of an aqueous suspension comprising a plurality of cross-linked vesicles as defined in claims 1-19, comprising the steps of: a) Preparing an aqueous phase comprising an amphiphilic peptide, wherein the pH of said aqueous phase is of 4 or lower; b) Preparing an organic phase, comprising a fluorinated compound having a boiling point lower than 25°C; c) Injecting said aqueous phase in a first inlet and said organic phase in a second inlet of a microfluidic cartridge, thereby mixing said aqueous phase and said organic phase in a mixing portion of the microfluidic cartridge, wherein the operating pressure into said microfluidic cartridge is lower than 7000 kPa, to obtain an aqueous suspension of cross-linkable vesicles;46RECTIFIED SHEET (RULE 91) ISA / EPd) Collecting the aqueous suspension of cross-linkable vesicles from an exit channel of the microfluidic cartridge; e) Diluting the aqueous suspension of cross-linkable vesicles, and f) Contacting said aqueous suspension of cross-linkable vesicles with an oxidizing source able to induce the cross-linking of the amphiphilic peptides to obtain an aqueous suspension of cross-linked vesicles, wherein said oxidizing source does not comprise DMSO.

23. The method according to claim 22, wherein said oxidizing source is an oxidizing solution comprising disulfiram.

24. An aqueous suspension as defined in claims 20-21 obtainable by the method defined in claims 22-23.

25. An aqueous suspension as defined in claims 20-21 for use in a diagnostic and / or therapeutic treatment.47RECTIFIED SHEET (RULE 91) ISA / EP

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