Cationic cross-linked vesicles

Abstract

The present invention relates to crosslinked vesicles comprising an outer layer and an inner core, said outer layer comprising a cationic amphiphilic peptide and said inner core comprising a fluorinated compound in liquid form wherein said cationic amphiphilic peptide is a compound of formula (I) HB-CL-HP (I) wherein HB is a fluorinated hydrophobic block, CL is a crosslinking-type motif, HP is a cationic hydrophilic amino acid sequence, the vesicle has an alpha-helical structure and contains at least one positive charge, and the vesicle has a zeta potential of at least (20) mV.

Description

Technical Field

[0001] The present invention generally relates to cationic cross-linked vesicles. The present invention also relates to methods for preparing aqueous suspensions of said vesicles and their use in ultrasound-mediated diagnostic and / or therapeutic treatments. Background Art

[0002] Ultrasound (US) imaging has become one of the most frequently performed clinical imaging procedures. To improve imaging performance, contrast-enhanced US (CEUS) agents, such as gas-filled microbubbles (MBs), are injected intravenously into patients. These MBs are typically composed of perfluorocarbon gases encapsulated in a lipid shell. When US frequencies are applied to these bubbles, they resonate, generating strong scattering and, consequently, a contrast signal. By using US at higher pressures, the bubbles rupture, generating shock waves that can create pores and even disrupt cell membranes. This phenomenon, known as the sonoporation effect, has opened up new potential areas for the delivery of therapeutic agents.

[0003] Despite these advantages, MBs have a short lifespan in vivo and fail to accumulate in tissues due to their large size (typically between 1 and 10 μm).

[0004] Perfluorocarbon (PFC) nanodroplets (NDs) can overcome these challenges due to their submicron size and enhanced stability. These phase-change contrast agents (PCCAs) or perfluorocarbon nanodroplets are traditionally stabilized by surfactants, lipids, proteins, or polymers and can be evaporated into echogenic MBs by focused US irradiation.

[0005] WO2019 / 023706 discloses PFC-ND (i.e., nanopeptisomes), which have a perfluorocarbon liquid core containing a cargo, such as a therapeutically active agent, and a plurality of amphiphilic peptides surrounding the perfluorocarbon core, wherein the amphiphilic peptides comprise a fluorinated hydrophobic block such as a fluorinated hydrophobic amino acid sequence, a cross-linked motif, and a hydrophilic amino acid sequence such as a targeting motif. The disclosed nanopeptisomes are characterized in that the zeta potential is less than 15 mV. Nanopeptisomes are obtained by a solvent exchange procedure, in which water is slowly added to an organic emulsion of peptides and PFCs, ultimately leading to the spontaneous assembly of peptides at the surface of PFC nanodroplets.

[0006] To date, to the applicant's knowledge, no such PFC-NDs stabilized by said amphiphilic peptides have been obtained with a total positive charge higher than 20 mV.

[0007] The applicant has now prepared cationic cross-linked vesicles comprising an inner core comprising a fluorinated compound in liquid form and an outer shell comprising a cationic amphiphilic peptide having specific structural properties and a positive charge, and wherein the cross-linked vesicles have a zeta potential higher than 20 mV.

[0008] Furthermore, applicants have prepared cationic cross-linked vesicles stabilized by a mixture of amphiphilic peptides that unexpectedly impart significantly high zeta potentials. Summary of the Invention

[0009] One aspect of the present invention relates to a cross-linked vesicle comprising an outer layer and an inner core, wherein the outer layer comprises a cationic amphiphilic peptide and the inner core comprises a fluorinated compound in liquid form, wherein the cationic amphiphilic peptide is a compound of formula (I)

[0010] HB-CL-HP(I)

[0011] in

[0012] HB is a fluorinated hydrophobic block,

[0013] CL is a cross-linking motif,

[0014] HP is a cationic hydrophilic amino acid sequence having an α-helical structure and containing at least one positive charge, and

[0015] wherein the vesicles have a zeta potential of at least 20 mV.

[0016] Preferably, HP comprises at least 2 positive charges, more preferably at least 3 positive charges, still more preferably at least 4 positive charges, still more preferably at least 5 positive charges, up to 40.

[0017] In a preferred embodiment, HB is a fluorinated hydrophobic amino acid sequence HB'.

[0018] Preferably, HB' comprises three consecutively linked pentafluoro-phenylalanine residues at its terminus and is a compound of formula IV

[0019]

[0020] In another preferred embodiment, the cross-linking motif CL comprises cysteine.

[0021] Preferably, CL comprises the amino acid sequence GGGCCGG.

[0022] In another preferred embodiment, the cationic amphiphilic peptide is selected from the group consisting of

[0023] H2N-F F F F F FGGGCCGGKGPLSSIFSRIGDP-NH2, or

[0024] H2N-F F F F F F In another embodiment, the outer layer further comprises an additional amphiphilic peptide HB-CL-HP' (VI) having the formula VI

[0025] in,

[0026] -HB is a fluorinated hydrophobic block,

[0027] -CL is a cross-linking motif, and

[0028] -HP' is a hydrophilic amino acid sequence.

[0029] Preferably, the additional amphiphilic peptide is a cationic amphiphilic peptide, wherein HP' is a cationic hydrophilic amino acid sequence comprising at least one positive charge.

[0030] In a preferred embodiment, the additional amphiphilic peptide is selected from the group consisting of:

[0031] H2N-F F F F F F GGGCCGGKGYGRKKRRQRRR-NH2,

[0032] H2N-F F F F F F GGGCCGGKGPKKKRKV-NH2, or

[0033] H2N-F F F F F F GGGCCGKGAGA-NH2.

[0034] In another embodiment, the fluorinated compound is a perfluorocarbon compound selected from perfluoropentane, perfluorohexane, or mixtures thereof.

[0035] Preferably, the vesicles have a zeta potential of at least 30 mV.

[0036] Another aspect relates to an aqueous suspension comprising a plurality of cross-linked vesicles as defined above.

[0037] Preferably, the vesicles are calibrated cross-linked vesicles having a z-average diameter between 100 nm and 1000 nm and a polydispersity below 0.2.

[0038] Another aspect relates to a method for preparing an aqueous suspension comprising a plurality of cross-linked vesicles as defined above, the method comprising the steps of:

[0039] a) preparing an aqueous phase comprising a cationic amphiphilic peptide, wherein the pH of the aqueous phase is below 4;

[0040] b) preparing an organic phase comprising a fluorinated compound;

[0041] c) injecting the aqueous phase into a first inlet of a microfluidic cartridge and injecting the organic phase into a second inlet of the microfluidic cartridge, thereby mixing the aqueous phase and the organic phase in a mixing portion of the microfluidic cartridge, wherein the operating pressure entering the microfluidic cartridge is less than 7000 kPa, to obtain an aqueous suspension of vesicles;

[0042] d) collecting the aqueous suspension of cross-linkable vesicles from the outlet channel of the microfluidic cartridge;

[0043] f) diluting the aqueous suspension of cross-linkable vesicles, and

[0044] e) cross-linking the cationic amphiphilic peptide to obtain an aqueous suspension of cross-linked vesicles.

[0045] Another aspect relates to an aqueous suspension for diagnostic and / or therapeutic treatment comprising a plurality of cross-linked vesicles as defined above.

[0046] Another aspect relates to an assembly comprising a cross-linked vesicle as defined above and a cargo molecule, wherein the cargo molecule is electrostatically bound to the outer layer of the cross-linked vesicle.

[0047] Another aspect relates to cationic amphiphilic peptides of formula (I)

[0048] HB-CL-HP(I)

[0049] in

[0050] -HB is a fluorinated hydrophobic block,

[0051] -CL is a cross-linking motif, and

[0052] -HP is a cell-penetrating peptide or nuclear localization sequence, which has an α-helical structure and contains at least one positive charge. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1Secondary structure predictions obtained using ColabFold (ColabFold v1.5.2-patch). Panels a) and b) show two examples of amino acid sequences arranged in an α-helical secondary structure, namely Pres2 (PLSSIFSRIGDP) and Pepfect14 (AGYLLGKLLOOLAAAALOOLL). Panels c) and d) show two examples of amino acid sequences arranged in a randomly organized secondary structure, namely TAT (YGRKKRRQRRR) and SV40 (PKKKRKV). DETAILED DESCRIPTION

[0054] The present invention relates generally to cationic cross-linked vesicles having an inner core comprising a fluorinated compound in liquid form and an outer shell comprising a cationic amphiphilic peptide of formula (I),

[0055] HB-CL-HP(I)

[0056] in

[0057] -HB is a fluorinated hydrophobic block,

[0058] -CL is a cross-linking motif,

[0059] -HP is a cationic hydrophilic amino acid sequence having an α-helical structure and comprising at least one positive charge, preferably at least five positive charges, and

[0060] The vesicles have a zeta potential of at least 20 mV, preferably at least 30 mV, more preferably at least 40 mV, and at most 100 mV.

[0061] Applicants have observed that the use of cationic amphiphilic peptides comprising cationic hydrophilic amino acid sequences characterized by specific structural properties, namely an α-helical structure combined with at least a positive charge, unexpectedly allows obtaining cross-linked vesicles with a very high overall positive charge, e.g. zeta potential > 20 mV.

[0062] Furthermore, cross-linked vesicles having an outer shell stabilized by a mixture of two or more cationic amphiphilic peptides, the mixture comprising at least one cationic hydrophilic amino acid sequence characterized by an α-helical structure and comprising at least one positively charged cationic hydrophilic amino acid sequence, surprisingly exhibit a significantly higher zeta potential than cross-linked vesicles stabilized by a single cationic amphiphilic peptide. Furthermore, the Applicant observed that this increased zeta potential correlated with improved adsorption of the cargo molecule onto the outer shell.

[0063] definition

[0064] The term "vesicle" refers to an assembly comprising an outer layer comprising a cross-linkable amphiphilic peptide and an inner core comprising a fluorinated compound, such as a perfluorocarbon, in liquid form. In the vesicle, the cross-linkable amphiphilic peptide molecules are oriented in such a way that the hydrophobic portion of the peptide is located at the surface of the fluorinated compound of the inner core.

[0065] According to the present invention, the vesicles are cationic vesicles with positive charges.

[0066] The expression "cross-linkable amphiphilic peptide" refers to any amphiphilic peptide comprising a cross-linkable moiety that is capable of being covalently linked to one another potentially via a cross-linking reaction. Suitable examples of cross-linkable moieties are cross-linkable amino acids, such as cysteine ​​residues, which can be intermolecularly linked to adjacent cysteine ​​residues via disulfide cross-linking groups (-SS-).

[0067] In a preferred embodiment, the cross-linkable moiety is a cross-linkable amino acid, preferably a cysteine ​​amino acid contained in an adjacent cross-linkable amphiphilic peptide.

[0068] According to the present invention, the cross-linkable amphiphilic peptides are preferably cationic amphiphilic peptides, comprising at least one positive charge in their sequence.

[0069] In the present specification and claims, the expression "cross-linking reaction" refers to a process involving the formation of covalent bonds between cross-linkable moieties in adjacent cross-linkable amphiphilic peptides, thereby binding the cross-linkable amphiphilic peptide molecules together.

[0070] The term "cross-linkable vesicle" refers to an assembly comprising an outer layer comprising a cross-linkable amphiphilic peptide and an inner core comprising a fluorinated compound in liquid form, wherein the cross-linkable amphiphilic peptide does not intermolecularly bind to adjacent amphiphilic peptides. As described above, the cross-linkable amphiphilic peptides forming the outer layer of the cross-linkable vesicle comprise cross-linkable moieties that can potentially be covalently linked to each other.

[0071] The term "cross-linked vesicle" refers to an assembly comprising an outer layer and an inner core, wherein the outer layer comprises a cross-linkable amphiphilic peptide and the inner core comprises a fluorinated compound in liquid form, wherein the cross-linkable amphiphilic peptide is covalently linked to adjacent cross-linkable amphiphilic peptides via cross-linking of cross-linkable moieties. For example, when the cross-linkable amphiphilic peptide comprises cysteine ​​residues, the cysteine ​​residues can be intermolecularly linked to adjacent cysteine ​​residues via disulfide cross-linking groups (-SS-).

[0072] As described above, according to the present invention, the vesicles are cationic vesicles as cross-linkable vesicles or as cross-linked vesicles.

[0073] Preferably, the vesicles of the present invention are cationic cross-linked nanodroplets, wherein the term "nanodroplets" refers to vesicles having a z-average diameter between 100 nm and 1000 nm.

[0074] The expression "calibrated" refers to a population of (cross-linkable or cross-linked) cationic (per)fluorocarbon-filled vesicles as defined above, wherein the vesicles have a z-average diameter between 100 nm and 1000 nm and a polydispersity lower than 0.25.

[0075] Often, in the prior art, the term "calibrated" is also expressed as "size-controlled", "uniformly sized droplets", "microdispersed", or "single-size".

[0076] In the present invention, the calibrated cationic cross-linked vesicles are preferably obtained by microfluidics technology.

[0077] The expression "calibrated cationic cross-linked vesicles" refers to an aqueous suspension comprising a plurality of calibrated (per)fluorocarbon-filled cross-linkable vesicles as defined above, wherein said vesicles have a zeta potential of at least 20 mV, preferably obtained by microfluidics. The suspension of calibrated (per)fluorocarbon cross-linkable vesicles has not been subjected to any procedure aimed at inducing cross-linking of the cross-linkable amphiphilic peptides that form the outer layer of the vesicles.

[0078] The expression "calibrated (per)fluorocarbon cross-linked vesicles" refers to an aqueous suspension of calibrated (per)fluorocarbon-filled cross-linked vesicles as defined above, obtained by microfluidics. After collection from the microfluidic cartridge, for example within 5 minutes, the aqueous suspension of calibrated (per)fluorocarbon cross-linkable vesicles can be subjected to a procedure aimed at inducing cross-linking of the cross-linkable amphiphilic peptides that form the outer layer of the vesicles. As a result, an aqueous suspension of calibrated (per)fluorocarbon cross-linked vesicles is obtained.

[0079] Cationic amphiphilic peptides

[0080] The expression "cationic amphiphilic peptide" refers to a cross-linkable amphiphilic peptide, as defined above, which comprises at least one positive charge in its sequence.

[0081] The amphiphilic peptides are capable of assembling at the surface of the liquid fluorinated compound to form cross-linked vesicles.

[0082] According to the present invention, the expression "cationic amphiphilic peptide" refers to a peptide of formula I

[0083] HB-CL-HP(I)

[0084] Among them, HB is a fluorinated hydrophobic block, CL is a cross-linking motif and HP is a cationic hydrophilic amino acid sequence.

[0085] The cationic amphiphilic peptide has a molecular weight in the range of about 1000-5000 Daltons, wherein the cationic amphiphilic peptide comprises 5 to 50 amino acid residues, preferably 5 to 40, more preferably 5 to 35, wherein at least two of the amino acid residues are continuously linked to each other in the chain by a peptide bond.

[0086] Cationic amphiphilic peptides can be synthesized using techniques known to those of ordinary skill in the art, such as, but not limited to, solid phase synthesis, liquid phase synthesis, recombinant methods, polymerization, and conjugation methods.

[0087] As used herein, the term "fluorinated hydrophobic block" refers to a covalently linked chain of monomer residues that forms a fluorinated hydrophobic homopolymer or copolymer. According to an embodiment, the monomer units that form the fluorinated hydrophobic polymer may be individually fluorinated, or some or one of the monomer units may be fluorinated such that at least one or more monomer residues of the fluorinated hydrophobic polymer are fluorinated.

[0088] According to one embodiment, the cross-linkable amphiphilic peptide does not comprise a lipid.

[0089] According to another embodiment, the fluorinated hydrophobic polymer comprises a hydrophobic amino acid sequence, wherein the amino acids of the hydrophobic amino acid sequence have non-polar side chains.

[0090] According to one embodiment, the fluorinated hydrophobic polymer comprises one or more synthetic non-amino acid monomer units, wherein at least one monomer unit is fluorinated such that at least one of the monomer residues of the fluorinated hydrophobic polymer is fluorinated. Non-limiting examples of synthetic monomer units that can be fluorinated and reacted to form the fluorinated hydrophobic polymer include methyl methacrylate, lactic acid, glycolic acid, and olefins such as ethylene, propylene, styrene.

[0091] In one embodiment, the cross-linkable amphiphilic peptide is a compound of formula (II):

[0092] HB'-CL-HP(II)

[0093] Wherein, HB' is a fluorinated hydrophobic amino acid sequence; CL is a cross-linking motif; and HP is a hydrophilic amino acid sequence.

[0094] Preferably, HP is a hydrophilic amino acid sequence in which the C-terminal amino acid is amidated (—NH 2 ) or hydroxylated (—OH).

[0095] In another embodiment, the cross-linkable amphiphilic peptide is a compound of formula (III):

[0096] HB'-CL-HP-NH2(III)

[0097] Wherein, HB' is a fluorinated hydrophobic amino acid sequence; CL is a cross-linking motif; and HP is a hydrophilic amino acid sequence, wherein the C-terminal amino acid is amidated.

[0098] 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.

[0099] Hydrophobic amino acids can be naturally occurring or non-natural (artificially produced). Examples of 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 can include D amino acids, as well as specific non-natural amino acids such as selenocysteine, pyrrolysine, and the like.

[0100] In the cross-linkable amphiphilic peptide, the fluorinated hydrophobic amino acid sequence may comprise 1 to 10 fluorinated hydrophobic amino acids consecutively linked by peptide bonds, which may be unsubstituted or substituted with a substituent selected from the group consisting of -F, -Cl, -Br, -I, Cl-C 30 Alkyl groups, C2-C 30 Alkenyl groups, C2-C 30 Alkynyl group, C3-C 30 Cycloalkyl groups, C3-C 30 Cycloalkenyl groups, C6-C 30 Aryl group, C7-C 30 Arylalkyl groups include, but are not limited to, fluorinated hydrophobic amino acids. 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.

[0101] In a preferred embodiment, the fluorinated hydrophobic amino acid sequence may include pentafluoro-phenylalanine (2,3,4,5,6-pentafluoro-L-phenylalanine and / or 2,3,4,5,6-pentafluoro-D-phenylalanine) at its terminus.

[0102] In another embodiment, the fluorinated hydrophobic amino acid sequence may comprise one to ten consecutively linked pentafluoro-phenylalanine residues at its terminus.

[0103] Still more preferably, the fluorinated hydrophobic amino acid sequence HB' comprises three consecutively linked pentafluoro-phenylalanine residues at its termini and is a compound of formula IV

[0104]

[0105] Suitable fluorinated amino acids are those described, for example, in WO 2019 / 023707.

[0106] Combinations of hydrophobic polymers and sequences of hydrophobic amino acids with non-polar side chains may 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.

[0107] As used herein, the term "cationic hydrophilic amino acid sequence" refers to a sequence of amino acids consecutively linked by peptide bonds, wherein the hydrophilic amino acids have polar side chains, wherein the polar side chains include groups capable of forming hydrogen bonds with water molecules.

[0108] In the present invention, the cationic hydrophilic amino acid sequence HP may contain both hydrophilic and hydrophobic amino acids.

[0109] The hydrophilic and hydrophobic amino acids may be naturally occurring or non-natural (artificially produced) and may be D or L amino acids.

[0110] 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 part of the side chain.

[0111] Examples of naturally occurring hydrophobic amino acids and non-natural hydrophobic amino acids are those mentioned above. In the cationic amphiphilic peptide, the cationic hydrophilic amino acid sequence HP may comprise 3 to 40, preferably 3 to 30, more preferably 3 to 20 hydrophilic amino acids consecutively linked by peptide bonds.

[0112] According to the present invention, the cationic hydrophilic amino acid sequence contains at least one positive charge, preferably at least 2 positive charges, more preferably at least 3 positive charges, even more preferably at least 4 positive charges, even more preferably at least 5 positive charges, and up to 40 positive charges.

[0113] The secondary structure of the cationic hydrophilic amino acid sequence can be any suitable secondary structure, such as, but not limited to, an α-helix, a π-helix, a β-sheet or a β-turn. Additional examples of suitable secondary structures can be found in Branden, 1998.

[0114] The term "secondary structure" has its conventional meaning and refers to the regular, repeating arrangement of adjacent amino acid residues in space within a polypeptide chain. It is maintained by hydrogen bonds between the amide hydrogens and carbonyl oxygens of the peptide backbone. The main secondary structures are α-helices and β-structures.

[0115] According to the present invention, the cationic hydrophilic amino acid sequence has a secondary structure selected from the group consisting of α-helix, π-helix, β-sheet, β-turn or a mixture thereof.

[0116] Typically, at least 50% of the amino acid residues of the cationic hydrophilic amino acid sequence are arranged in a secondary structure as defined above.

[0117] Alternatively, the cationic hydrophilic amino acid sequence has a randomly organized secondary structure, such as a random coil structure (Smith, 1996), which means that the sequence does not form any regular secondary structure as defined above and is characterized by a disordered arrangement.

[0118] In a preferred embodiment, the cationic hydrophilic amino acid sequence has an α-helical structure and comprises 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 a maximum of 40. Preferably, at least 50% of the amino acid residues of the cationic hydrophilic amino acid sequence are arranged in an α-helix.

[0119] In a preferred embodiment, the cationic hydrophilic amino acid sequence HP is selected from Pres2 (PLSSIFSRIGDP) and PepFect14 (AGYLLGKLLOOLAAAALOOLL).

[0120] The secondary structure of an amino acid sequence can essentially be predicted from its primary structure using appropriate bioinformatics methods, such as ColabFold (Mirdita, 2022).

[0121] In other words, by using said bioinformatics methods it is possible to predict whether a given amino acid sequence tends to form defined secondary structures, such as alpha helices and beta sheets, or whether it will tend to arrange in randomly organized secondary structures.

[0122] pass Figure 1 A suitable example of secondary structure prediction obtained by using ColabFold (ColabFold v1.5.2-patch is given. Figure 1 a and 1b show two cationic hydrophilic amino acid sequences as defined in the present invention, namely Pres2 and PepFect14, arranged in an α-helical structure, while Figure 1 c and 1d show two examples of hydrophilic amino acid sequences arranged in randomly organized structures, namely TAT and SV40.

[0123] In a preferred embodiment, the cationic hydrophilic amino acid sequence is selected from a cell penetrating peptide (CPP) or a nuclear localization sequence (NLS).

[0124] The expression "cell penetrating peptide" refers to a class of short peptides with a length of 5-30 amino acids, preferably positively charged, which can penetrate biological membranes and deliver a variety of cargoes into cells. A comprehensive review of CPPs can be found in Derakhshankhah, 2018.

[0125] Suitable examples of CPPs can be found in the database CPPsite 2.0 (http: / / crdd.osdd.net / raghava / cppsite / ).

[0126] The term “nuclear localization sequence” refers to a class of short peptides based on a lysine-, arginine-, or proline-rich motif that can be transported to the cell nucleus through the nuclear pore complex, a multi-subunit complex containing 50–100 different proteins. NLSs can be further divided into monopartite and bipartite signals, each consisting of one or two clusters of four or more basic amino acids (Lu, 2021).

[0127] In the present specification and claims, the expression "cross-linking motif" refers to a cross-linkable moiety contained in a cross-linkable amphiphilic peptide that can potentially be covalently linked to another cross-linkable moiety via a cross-linking reaction. Examples of cross-linking motifs are sulfhydryl cross-linkers, UV cross-linkers, azabenzenes, photosensitive cross-linkers such as azides or benzophenones, nitriles, pH-sensitive cross-linkers or enzymatic cross-linkers and click-chemistry-based cross-linkers (e.g., a cross-linking motif comprising an azide group that can react with an alkyne-containing cross-linking motif via a click chemistry reaction such as copper-catalyzed azide-alkyne cycloaddition).

[0128] In a preferred embodiment, a "cross-linkable motif" is an amino acid sequence comprising at least one cross-linkable amino acid residue that can potentially covalently link to a corresponding cross-linkable amino acid residue via a cross-linking reaction. A cross-linkable motif may comprise from 1 to about 40 amino acid residues, preferably from 3 to 30, more preferably from 3 to 20.

[0129] In a cross-linkable motif, the cross-linkable amino acid residue may be at any position in the sequence and may be a naturally occurring amino acid and / or a non-naturally occurring amino acid.

[0130] An example of a naturally occurring amino acid that can be cross-linked to a corresponding cross-linkable amino acid residue is cysteine.

[0131] Non-naturally occurring amino acids can be obtained by functionalizing the structure of naturally occurring amino acids, which provide the ability to bind to naturally occurring or non-naturally occurring amino acids in a cross-linking motif of adjacent cross-linkable residues.

[0132] In one embodiment, the cross-linking motif comprises cysteine.

[0133] In another embodiment, the cross-linking motif comprises cysteine ​​and glycine.

[0134] In a preferred embodiment, the cross-linking motif comprises the amino acid sequence GGGCCGG, wherein G is glycine and C is cysteine.

[0135] In one 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%, even more preferably at least 95%.Preferably, the degree of cross-linking is 100%.

[0136] The expression "degree of cross-linking" refers to the total number of cross-linked amino acid residues (eg, cysteines) interconnecting adjacent cross-linkable amphiphilic peptides. The degree of cross-linking is usually expressed as a percentage and can be measured using a colorimetric disulfide formation assay.

[0137] In the present invention, it is preferred that the amino acid sequence comprises three pentafluoro-phenylalanine amino acids (F f The C-terminus of the fluorinated domain is a cysteine-containing cross-linking motif (GGGCCGG).

[0138] In a preferred embodiment, the cationic amphiphilic peptide is a compound of formula V

[0139] HB'-CL-HP(V)

[0140] in

[0141] -HB' is a fluorinated hydrophobic amino acid sequence comprising pentafluoro-phenylalanine,

[0142] -CL is a cross-linked motif comprising GGGCCGG, where G is glycine and C is cysteine, and

[0143] -HP is a cationic hydrophilic amino acid sequence that has an α-helical structure and contains at least one positive charge.

[0144] In another embodiment, the cationic amphiphilic peptide is selected from the group consisting of:

[0145] H2N-F F F F F F GGGCCGGKGPLSSIFSRIGDP-NH2(AP-PRES2), or

[0146] H2N-F F F F F FGGGCCGGKGAGYLLGKLLOOLAAAALOOLL-NH2(AP-PEPFECT14).

[0147] In this specification and claims, amino acid sequences are reported according to the conventional single-letter code, where, for example, 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.

[0148] In the present specification and claims, each modified amino acid Ff is pentafluorophenylalanine (2,3,4,5,6-pentafluoro-L-phenylalanine).

[0149] Mixture of cationic amphiphilic peptides

[0150] Advantageously, the Applicant has observed that the use of a mixture comprising two or more amphiphilic peptides, comprising at least one cationic hydrophilic amino acid sequence characterized by an α-helical structure and comprising at least a positive charge, surprisingly leads to cross-linked vesicles characterized by a significantly high zeta potential. Furthermore, the Applicant has observed that this increased zeta potential is associated with improved adsorption of the load molecule on the shell.

[0151] In a preferred embodiment, the present invention relates to a cross-linked vesicle as defined above, wherein said outer layer additionally comprises an additional amphiphilic peptide having formula VI

[0152] HB-CL-HP'(VI)

[0153] in,

[0154] -HB is a fluorinated hydrophobic block,

[0155] -CL is a cross-linking motif, and

[0156] -HP' is a hydrophilic amino acid sequence.

[0157] Preferably, HB and CL may be as defined above.

[0158] Preferably, the additional amphiphilic peptide is a cationic amphiphilic peptide.

[0159] According to the present invention, the additional cationic amphiphilic peptide may be any suitable cationic amphiphilic peptide as defined above.

[0160] According to the present invention, the hydrophilic amino acid sequence HP' may be any hydrophilic amino acid sequence as defined above.

[0161] For example, HP' may be selected from the group of amino acid sequences consisting of AGA, TAT (YGRKKRRQRRR), or SV40 (PKKKRKV).

[0162] Preferably, HP' is a cationic hydrophilic amino acid sequence comprising at least one positive charge, more preferably more than 2 positive charges, and up to 40 positive charges.

[0163] The secondary structure of HP' may be any suitable secondary structure, such as an α-helix, a π-helix, a β-sheet or a β-turn, as defined above.

[0164] According to the present invention, HP' has a secondary structure selected from the group consisting of an α-helix, a π-helix, a β-sheet, a β-turn, or a mixture thereof.

[0165] Instead, HP' has a randomly organized secondary structure.

[0166] In another embodiment, at least 50% of the amino acid residues of HP' are arranged in a secondary structure, as defined above.

[0167] In another embodiment, the additional amphiphilic peptide is selected from the group consisting of:

[0168] H2N-F F F F F F GGGCCGGKGYGRKKRRQRRR-NH2(AP-TAT),

[0169] H2N-F F F F F F GGGCCGGKGPKKKRKV-NH2(AP-SV40), or

[0170] H2N-F F F F F F GGGCCGKGAGA-NH2(AP-AGA).

[0171] In a preferred embodiment, HP' has a randomly organized secondary structure and contains at least 5 positive charges.

[0172] Fluorinated compounds

[0173] As described above, "cross-linked vesicles" refer to an assembly comprising an outer layer comprising a cross-linkable amphiphilic peptide and an inner core comprising a fluorinated compound in liquid form, which allows activation of the vesicles upon application of ultrasound (US).

[0174] In the present specification and claims, the term "fluorinated compound" refers to the group of fluorine-containing compounds derived from hydrocarbons by partial or complete substitution of hydrogen atoms with fluorine atoms, which are liquid at room temperature. Preferably, the fluorinated compound is a perfluorocarbon (PFC), i.e., a fluorinated hydrocarbon in which all hydrogen atoms are substituted with fluorine atoms.

[0175] The fluorinated compound is selected from fluorinated compounds having a high boiling point, ie above room temperature (RT; 25°C), which are in liquid form at standard ambient temperature and pressure (SATP), ie at 25°C and 1 atm (101.325 kPa).

[0176] The liquid fluorinated compound is characterized by a boiling point between 25 and 160° C. In the present invention, the fluorinated compound is preferably characterized by a boiling point between 25 and 100° C., still more preferably between 27 and 60° C.

[0177] Suitable examples of fluorinated compounds include C4-C6 fluorinated compounds such as 1-fluorobutane, 2-fluorobutane, 2,2-difluorobutane, 2,2,3,3-tetrafluorobutane, 1,1,1,3,3-pentafluorobutane, 1,1,1,4,4,4-hexafluorobutane, 1,1,1,2,4,4,4-heptafluorobutane, 1,1,2,2,3,3,4,4-octafluorobutane, 1,1,1,2,2-pentafluoropentane, 1,1,1,2,2,3,3,4-octafluoropentane, 1,1,1,2,2,3,4,5,5,5-decafluoropentane, 1,1,2,2,3,3,4,4,5,5,6,6-dodecafluorohexane.

[0178] Suitable examples of perfluorocarbons are perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluorononane, perfluorodecalin, perfluorooctane bromide (PFOB), perfluoro-15-crown-5-ether (PFCE), perfluorodichlorooctane (PFDCO), perfluorotributylamine (PFTBA), perfluorononane (PFN) and 1,1,1-tris(perfluoro-tert-butoxymethyl)ethane (TPFBME) or mixtures thereof.

[0179] In one embodiment, the perfluorocarbon compound is preferably perfluoropentane (PFP) (boiling point 29° C.), perfluorohexane (PFH) (boiling point 57° C.) or a mixture thereof.

[0180] Preparation of cationic cross-linked vesicles

[0181] According to the present invention, the disclosed cationic cross-linked vesicles can be prepared by using any preparation technique suitable for producing calibrated nanodroplets having a Z-average diameter comprised between 100 nm and 1000 nm, preferably between 120 nm and 800 nm, and more preferably between 150 nm and 400 nm.

[0182] Examples of preparation techniques include sonication, homogenization, extrusion, microfluidics, and microbubble condensation.

[0183] In a preferred embodiment, the cationic cross-linked vesicles are prepared using a preparation technique suitable for producing calibrated nanodroplets having a polydispersity index (PDI) lower than 0.25, preferably lower than 0.20, more preferably lower than 0.15, even more preferably lower than 0.10, and a Z-average diameter comprised between 100 nm and 1000 nm, preferably between 120 nm and 800 nm, more preferably between 150 nm and 400 nm.

[0184] For example, the preparation technology is selected from ultrasonic treatment, microbubble condensation or microfluidics, and preferably the preparation technology is microfluidics.

[0185] In this specification and claims, the expression "microfluidics" refers to a technology for manufacturing calibrated nanodroplets, such as the cationic cross-linked vesicles of the present invention, by means of a microfluidics chip designed to manipulate fluids in channels at a microscale.

[0186] The microfluidics technique is a bottom-up approach, in other words, nanodroplets are obtained by assembling molecules (such as amphiphilic lipid compounds and (per)fluorocarbons) into larger nanostructures (ie, aligned nanodroplets).

[0187] More preferably, the microfluidics technique is performed by using a mixing device such as a microfluidic cartridge equipped with a staggered herringbone micromixer or a ring mixer.

[0188] A detailed description of a microfluidic cartridge suitable for the microfluidic method of the present invention is described in WO2022101365A1 (Bracco Suisse SA).

[0189] One aspect of the present invention relates to a method for preparing an aqueous suspension comprising a plurality of cross-linked vesicles as defined above, said method comprising the steps of:

[0190] a) preparing an aqueous phase comprising a cationic amphiphilic peptide, wherein the pH of the aqueous phase is below 4;

[0191] b) preparing an organic phase comprising a fluorinated compound;

[0192] c) injecting the aqueous phase into a first inlet of a microfluidic cartridge and injecting the organic phase into a second inlet of a microfluidic cartridge, thereby mixing the aqueous phase and the organic phase in a mixing portion of the microfluidic cartridge, wherein the operating pressure entering the microfluidic cartridge is less than 7000 kPa, to obtain an aqueous suspension of vesicles;

[0193] d) collecting the aqueous suspension of cross-linkable vesicles from the outlet channel of the microfluidic cartridge;

[0194] e) diluting the collected aqueous suspension of cross-linkable vesicles, and

[0195] f) cross-linking the cationic amphiphilic peptide to obtain an aqueous suspension of cross-linked vesicles.

[0196] According to a preferred embodiment, the aqueous phase further comprises an additional amphiphilic peptide, preferably an additional cationic amphiphilic peptide.

[0197] According to the disclosed method, a calibrated aqueous suspension of cross-linkable vesicles can be obtained by passing the liquid phase through the mixing section of a microfluidic cartridge in a single pass.

[0198] Step a) Preparation of aqueous phase

[0199] The "aqueous phase" typically comprises an aqueous liquid component, including, for example, water, an aqueous buffer solution, or an aqueous isotonic solution. The aqueous phase is in liquid form.

[0200] Suitable examples of aqueous buffer solutions are phosphate-buffered saline (ie, PBS buffer), sodium acetate buffer, tris(hydroxymethyl)aminomethane buffer (ie, TRIS buffer), or mixtures thereof.

[0201] Suitable examples of isotonic solutions are Ringer's solution, lactated Ringer's solution, saline, oral rehydration salt solution or mixtures thereof.

[0202] Preferably, the aqueous liquid component has a pH below 7.0, more preferably below 6.5, still more preferably below 6.0, more preferably below 5.5, more preferably below 5, more preferably below 4.5, yet more preferably below 4.0, up to e.g. 0, preferably 1.0.

[0203] Preferably, the aqueous liquid component is a sodium acetate buffer.

[0204] For example, the cationic amphiphilic peptide can be mixed with the aqueous components by conventional techniques (eg, stirring) to prepare the aqueous phase to be injected into the first inlet of the microfluidic cartridge.

[0205] According to a preferred embodiment, the aqueous phase additionally comprises a further amphiphilic peptide, preferably a further cationic amphiphilic peptide.

[0206] In step a), the aqueous phase contains the cross-linkable cationic amphiphilic peptide, preferably at a concentration in the range of 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.

[0207] In one embodiment, during step a), the temperature of the aqueous phase is typically below 25° C., preferably below 10° C., more preferably at a temperature of about 5±2° C. The temperature is preferably not below 2° C.

[0208] Step b) Preparation of organic phase

[0209] The "organic phase" typically comprises an organic solvent, preferably miscible with water, including, for example, methanol, ethanol, isopropanol, acetonitrile, DMF, DMSO and acetone. Preferably, the organic solvent is ethanol. The organic phase is in liquid form.

[0210] In the present invention, the expression "water-miscible organic solvent" means an organic solvent that can be mixed with water in any proportion (eg, any concentration) without separation of two phases, ie, forms a homogeneous solution.

[0211] For example, C1-C3 alcohols such as methanol, ethanol, and propanol are very soluble in water due to hydrogen bonding between the hydroxyl groups in the alcohol and water molecules. However, as the length of the hydrocarbon chain increases, the solubility in water decreases, resulting in low miscibility of the two liquids, and if mixed, two immiscible layers will form.

[0212] According to a preferred embodiment, the organic phase comprises a fluorinated compound or a mixture of different fluorinated compounds dispersed in an organic solvent. Preferably, the fluorinated compound is a perfluorocarbon compound.

[0213] Suitable examples of fluorinated compounds are those mentioned above.

[0214] As an example, the liquid organic phase to be injected into the second inlet of the microfluidic cartridge can be prepared by mixing the fluorinated compound in liquid form with the organic solvent in liquid form by conventional techniques (eg, stirring).

[0215] In another embodiment, at step b), the organic phase comprises the fluorinated compound in a concentration ranging from 0.003 mmol / mL to 0.142 mmol / mL, more preferably from 0.011 mmol / mL to 0.085 mmol / mL, still more preferably from 0.013 mmol / mL to 0.057 mmol / mL.

[0216] In one embodiment, the organic solvent is a polar organic solvent.

[0217] 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 is characterized by a relative polarity of 1. In contrast, non-polar solvents have low relative polarity values, such as dimethylformamide (DMF) which has a relative polarity value of 0.386.

[0218] Applicants have observed that low or medium-low polarity of the organic solvent can negatively affect the monodispersity of the final suspension of cross-linked vesicles. For example, the use of organic solvents with relatively low polarity, such as dimethylformamide (rp 0.386) or isopropanol (rp 0.546), can result in vesicle size distributions with relatively high PDI values ​​(e.g., greater than 0.25). Conversely, the use of organic solvents with relatively high polarity, such as methanol (rp 0.762) or ethanol (rp 0.654), generally allows for the acquisition of particle size distributions with lower PDI values, indicating good monodispersity of the sample.

[0219] Furthermore, the size of cross-linked vesicles can also be affected by the polarity of the organic phase. In particular, the size is inversely proportional to the polarity of the organic phase: high-polarity solvents, such as methanol and ethanol, result in smaller sizes than low- and medium-polarity solvents, such as DMF and isopropanol.

[0220] In a preferred embodiment, the organic solvent is a polar organic solvent, said solvent having a polarity between 0.60 and 0.80, preferably between 0.63 and 0.78, still more preferably between 0.65 and 0.77.

[0221] In another embodiment, the organic solvent is selected from methanol, ethanol and mixtures thereof. Preferably, the organic phase is ethanol.

[0222] In step b), the temperature of the organic phase is preferably below room temperature (25°C), for example 4°C, to avoid vaporization of the fluorinated compound having a boiling point close to 25°C.

[0223] In one embodiment, during step b), the temperature of the organic phase is typically below 25° C., preferably below 10° C., more preferably the temperature is about 5±2° C. The temperature is preferably not less than 2° C.

[0224] Step c) Injection into the microfluidic box

[0225] Typically, in step c), the injection of the aqueous phase and the injection of the organic phase are carried out simultaneously.

[0226] The expression "simultaneously" means that the aqueous phase and the organic phase are injected (ie co-injected) into the microfluidic cartridge at the same time, that is, the aqueous phase and the organic phase are injected into two separate inlets of the microfluidic cartridge at the same time or substantially at the same time (eg within a few seconds).

[0227] In a preferred embodiment, both the aqueous phase and the organic phase are injected into the microfluidic cartridge at a temperature suitable to avoid or substantially limit evaporation of the fluorinated compound. For example, after their respective preparation (i.e., step a) and step a)), both the aqueous phase and the organic phase can be stored in an ice bath (about 4°C) before being injected into separate inlets of the microfluidic cartridge (e.g., for 5 minutes) to limit the temperature increase during the time between step a), step b) and the subsequent step c).

[0228] According to the invention, after their injection, the aqueous and organic phases are directed to a mixing device in which they are mixed (for example by laminar mixing in the case of staggered herringbone micromixers), giving rise to the formation of NDs.

[0229] Typically, the operating pressure entering the microfluidic box is less than 1000psi (about 7000kPa), preferably less than 500psi (about 3500kPa), more preferably less than 300psi (about 2000kPa), and more preferably less than 100psi (about 700kPa), for example between 10 and 90psi.

[0230] The temperature of the mixing section, where the mixing process takes place in the special microchannel geometry of the mixing section, may be comprised between 0°C and 25°C, preferably comprised between 0°C and 15°C, more preferably between 0°C and 5°C.

[0231] For example, the microfluidic cartridge can be stored in a refrigerator (eg, 4° C.) for a suitable period of time to reach the desired temperature.

[0232] Total flow rate (TFR) and flow rate ratio (FRR)

[0233] The method of the present invention allows controlling the properties of (per)fluorocarbon cross-linked vesicles by varying two process parameters: the total flow rate and the flow rate ratio.

[0234] Expression " Total flow rate (TFR) "TFR" refers to the total flow rate of two fluid streams, namely the aqueous phase and the organic phase, pumped through two separate inlets of the microfluidic cartridge. The unit of measurement of TFR is mL / min.

[0235] According to one embodiment, the TFR is preferably 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.

[0236] 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 Equation 1:

[0237]

[0238] The volumes of the aqueous and organic phases may be expressed, for example, in mL.

[0239] In a preferred embodiment, the FRR (volume of aqueous phase to volume of organic phase) is between 1:1 and 5:1, preferably between 1:1 and 3:1, more preferably the FRR is 2:1.

[0240] In the present invention, the respective concentrations of the cross-linkable amphiphilic peptide and the (per)fluorocarbon compound and the FRR can be purposefully adjusted to obtain a molar ratio between the cross-linkable amphiphilic peptide and the (per)fluorocarbon compound that is suitable for ensuring the stability of the cross-linked vesicles (i.e., certain values ​​of size and PDI). The molar ratio between the cross-linkable amphiphilic peptide and the (per)fluorocarbon compound is preferably 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, and even more preferably not higher than 1.500. More preferably, the molar ratio is not lower than 0.001, not lower than 0.004, and even more preferably not lower than 0.050.

[0241] Step e) dilution

[0242] In one embodiment, the preparation method further comprises a step e) comprising diluting the calibrated aqueous suspension of cross-linkable vesicles.

[0243] In one embodiment, step e) is performed sequentially with step d). For example, step e) can be performed between step e) and step f), in particular after step d), for example within 5 minutes of collecting the sample from the microfluidic cartridge and before starting the cross-linking phase.

[0244] Applicants have unexpectedly observed that a dilution step performed after preparing calibrated cross-linkable vesicles using a microfluidic cartridge has a favorable effect on initial size and initial monodispersity. In fact, in the absence of dilution, the cross-linkable vesicle size is larger than in the dilution case.

[0245] As mentioned above, the expressions "initial monodisperse distribution" and "initial size" refer to the values ​​of monodispersity and size of the calibrated (per)fluorocarbon cross-linkable vesicle composition collected from the outlet channel of the microfluidic cartridge at step d) of the disclosed preparation method.

[0246] In this specification and claims, the term "dilution" refers to the process of reducing the concentration of calibrated cross-linkable vesicles in a suspension by adding an appropriate amount of an aqueous component.

[0247] A suitable amount of the aqueous component corresponds to the amount of aqueous solution required to reduce the concentration of calibrated vesicles in the aqueous suspension by 2 to 10 fold.

[0248] In a preferred embodiment, optional step e) of the method comprises diluting the aqueous suspension of calibrated fluorocarbon vesicles 1 to 20 times, preferably 2 to 10 times, even more preferably 3 to 8 times, such as about 5 times.

[0249] Suitable aqueous components are water or an oxidizing solution as described below.

[0250] Preferably, the aqueous component is an oxidizing solution, preferably an aqueous solution of DMSO.

[0251] In a preferred embodiment, step e) of the method comprises diluting the suspension of calibrated cross-linkable vesicles with an aqueous DMSO solution.

[0252] In a preferred embodiment, the concentration of DMSO in the aqueous solution is typically at least 0.1%, preferably at least 1%, more preferably at least 1.5%. The concentration of DMSO is typically no more than 10%, preferably less than 5%, more preferably less than 3.5%.

[0253] Alternatively, the dilution step can be carried out by means of an additional channel (e.g. placed in the Figure 1 The dilution step is performed directly inside the microfluidic cartridge (i.e., in-line dilution) between the mixing section 103 and the outlet channel 104 in the flow cell), the additional channel being suitable for diluting the calibrated fluorocarbon-cross-linkable vesicle suspension with the desired aqueous component before it is directed to the outlet channel and before the cross-linking step e).

[0254] Step f) Cross-linking step

[0255] Step f) of the method of the present invention comprises cross-linking the cross-linkable amphiphilic peptide to obtain an aqueous suspension of calibrated fluorocarbon-cross-linked vesicles.

[0256] In this specification and claims, the expression "cross-linking" refers to the process of forming covalent bonds between cross-linkable moieties contained in adjacent cross-linkable amphiphilic peptides, thereby binding the cross-linkable amphiphilic peptide molecules together. Preferably, the cross-linkable moieties are cross-linkable amino acids contained in adjacent cross-linkable amphiphilic peptides.

[0257] In a preferred embodiment, the cross-linkable amino acids are cysteine ​​amino acids contained in adjacent cross-linkable amphiphilic peptides. The cysteine ​​amino acids are cross-linked via disulfide cross-linking groups (-SS-).

[0258] In a preferred embodiment, step f) comprises contacting the aqueous suspension of calibrated cross-linkable vesicles with an oxidative source capable of inducing cross-linking of the cross-linkable amphiphilic peptides to obtain an aqueous suspension of calibrated cross-linked vesicles.

[0259] An oxidative source is contacted with the freshly prepared aqueous suspension of calibrated cross-linkable vesicles for a time sufficient to induce cross-linking of the cross-linkable amphiphilic peptides contained in the outer layer of the vesicles.

[0260] In one embodiment, the suitable time is typically at least 1 minute, preferably at least 30 minutes, more preferably at least 1 hour. The time is typically no more than 24 hours, preferably less than 18 hours, more preferably less than 15 hours.

[0261] According to another embodiment, the oxidative source is contacted with the freshly prepared aqueous suspension of cross-linkable vesicles for a time sufficient to induce a degree of cross-linking of greater than 80%, preferably at least 85%, more preferably at least 90% of the cross-linkable amphiphilic peptide molecules. Preferably, the degree of cross-linking is 100%.

[0262] Suitable examples of oxidizing sources are oxidizing solutions, oxidizing gases or mixtures thereof.

[0263] The term "oxidative solution" refers to any aqueous solution that can cause or help induce the formation of covalent bonds between cross-linkable amino acids, such as between thiol groups of cysteine ​​amino acids contained in adjacent cross-linkable amphiphilic peptides.

[0264] Suitable examples of oxidizing solutions are water, aqueous DMSO solutions, alkaline solutions comprising halogens, aqueous hydrogen peroxide solutions comprising iodine, and catalysts based on selenides in the presence of air.

[0265] In one embodiment, the oxidizing solution is preferably an aqueous solution of DMSO, wherein the concentration of the DMSO is typically at least 0.1%, preferably at least 1%, more preferably at least 1.5%. The concentration of the DMSO is typically no more than 10%, preferably less than 5%, more preferably less than 3.5%.

[0266] In a preferred embodiment, the oxidizing solution is preferably a 2.5% DMSO solution in water.

[0267] The term "oxidizing gas" refers to any gas that can induce or facilitate the formation of covalent bonds between cross-linkable amino acids, such as between thiol groups of cysteine ​​amino acids contained in adjacent cross-linkable amphiphilic peptides.

[0268] Suitable examples of oxidizing gases are oxygen, air or suitable gas mixtures comprising oxygen.

[0269] In a preferred embodiment, the oxidizing gas is air.

[0270] The oxidizing gas is contacted with the freshly prepared calibrated aqueous suspension of cross-linkable vesicles for a period of at least 1 minute, preferably at least 30 minutes, more preferably at least 1 hour. This period typically does not exceed 24 hours, preferably less than 10 hours, more preferably less than 3 hours.

[0271] For example, at the end of the microfluidic process, an oxidizing gas such as air can be injected by bubbling into the oxidizing solution comprising a dialysis device comprising a calibrated aqueous suspension of PFC-crosslinkable vesicles.

[0272] In yet another more preferred embodiment, the oxidizing source comprises a mixture of an oxidizing solution and an oxidizing gas.

[0273] Preferably, the oxidizing source comprises a mixture of an oxidizing aqueous solution and air.

[0274] In a preferred embodiment, step f) is a dialysis procedure comprising contacting the aqueous suspension of calibrated cross-linkable vesicles with an oxidative source capable of inducing cross-linking of the cross-linkable amphiphilic peptides to obtain an aqueous suspension of calibrated fluorocarbon cross-linked vesicles.

[0275] Preferably, the oxidizing source is an oxidizing solution as described above.

[0276] Alternatively, the oxidizing source comprises a mixture of an oxidizing solution and an oxidizing gas as described above.

[0277] In this specification, the expression "dialysis" denotes a procedure that promotes the cross-linking of a freshly prepared aqueous suspension of calibrated (per)fluorocarbon-cross-linkable vesicles by the mechanism described above.

[0278] According to this embodiment, after collection from the microfluidic cartridge, the calibrated aqueous suspension of cross-linkable vesicles is loaded into a dialysis device comprising a semipermeable membrane in contact with an oxidative source, for example by suspending the dialysis device in a large volume of an oxidative solution, as defined above, while injecting an oxidative gas therein.

[0279] Suitable examples of dialysis devices comprising a semipermeable membrane can be conventional dialysis tubing or advanced dialysis devices such as dialysis cassettes, dialysis bottles, or dialysis plates. The permeability of the semipermeable membrane allows the oxidizing solution to contact the calibrated (per)fluorocarbon cross-linkable vesicles loaded in the dialysis device, thereby inducing their cross-linking. The dialysis procedure allows for efficient recovery of an aqueous suspension of the calibrated (per)fluorocarbon cross-linked vesicles.

[0280] In a preferred embodiment, step f) is a dialysis procedure comprising the following steps:

[0281] f i ) loading the calibrated aqueous suspension of cross-linkable vesicles into a dialysis device comprising a semipermeable membrane;

[0282] f ii ) contacting the loaded dialysis device with an oxidizing solution, and

[0283] f iii ) Collect the calibrated aqueous suspension of cross-linked vesicles from the device.

[0284] In one embodiment, in step f ii ), the oxidizing solution is a 2.5% DMSO aqueous solution.

[0285] In another embodiment, step f ii ) comprising contacting the dialysis device with a mixture of an oxidizing solution and an oxidizing gas. Preferably, the mixture comprises a 2.5% DMSO solution in water and air.

[0286] Preferably, step f ii ) comprises contacting the dialysis device with a 2.5% aqueous solution of DMSO for 12 hours and with air for the first hour.

[0287] For example, an aqueous suspension of calibrated cross-linkable vesicles can be loaded into a dialysis device comprising a semipermeable membrane, and the device can then be contacted with a 2.5% DMSO solution in water for 12 hours, with the first hour under constant air bubbling.

[0288] In yet another embodiment, step f ii ) Repeat 1 to 5 times, preferably up to 3 times, more preferably step f ii ) Repeat 2 times.

[0289] At each step f ii ) is completed, the oxidizing solution can be replaced with fresh oxidizing solution or, alternatively, with a different oxidizing solution.

[0290] In one embodiment, each step can be performed by contacting the dialysis device with the same oxidizing solution, such as a 2.5% DMSO solution in water. ii ). The oxidizing solution can be replaced with a freshly prepared solution at the beginning of each repeated step.

[0291] Alternatively, each step can be performed by contacting the dialysis device with a different oxidizing solution. ii For example, an aqueous suspension of calibrated cross-linkable vesicles can be loaded into a dialysis device comprising a semipermeable membrane, and the device can then be contacted with a 2.5% DMSO solution in water for 12 hours, with the first hour under constant air bubbling and the contact with water can continue for 2 hours.

[0292] In some embodiments, in step f ii ), DMSO was replaced with water to reduce the amount of DMSO in the final aqueous suspension of calibrated cross-linked vesicles.

[0293] As observed by the applicant, following the microfluidic preparation of cross-linkable vesicles and their cross-linking as described above, the degree of cross-linking is unexpectedly high, typically above 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95%. Preferably, the degree of cross-linking is 100%.

[0294] Optional step g: washing

[0295] In another embodiment, the method of the present invention may comprise an optional step g) comprising washing the obtained aqueous suspension of calibrated cross-linked vesicles.

[0296] According to this embodiment, following the cross-linking stage, the calibrated cross-linked vesicles are treated using suitable washing techniques.

[0297] In this specification, the term "washing" refers to any operation performed on a freshly prepared cross-linked vesicle suspension to ultimately remove (or substantially reduce the amount of) assemblies that do not contain fluorinated compounds, or alternatively, the washing can be done to replace the aqueous solution in which the cross-linked vesicles are suspended at the end of the dialysis step f).

[0298] In this specification, the expression "assembly without fluorinated compounds" refers to an assembly comprising cross-linkable amphiphilic peptides that spontaneously assemble in particles due to hydrophobic interactions. Such assemblies can be formed during the microfluidics process and can be present in the aqueous suspension of calibrated cross-linked vesicles at the end of the preparation process, but due to their smaller size, they contribute to the formation of the second particle population, thereby conferring a higher PDI value.

[0299] For example, assemblies lacking fluorinated compounds are characterized by sizes between 50 nm and 150 nm.

[0300] The Applicant observed that a washing procedure of freshly prepared calibrated cross-linked vesicle suspensions resulted in lower PDI values, increasing the stability of the compositions obtained by microfluidic means.

[0301] Suitable washing techniques include, for example, centrifugation, ultracentrifugation, filtration and decantation.

[0302] Step f) is followed sequentially by the optional step g), which is carried out, for example, after completion of the cross-linking step, for example within 5 minutes.

[0303] In a preferred embodiment, the washing step g) comprises the following steps:

[0304] g i ) centrifuging the aqueous suspension of calibrated cross-linked vesicles obtained from step e);

[0305] g ii ) separating the supernatant phase comprising the assembly without the fluorinated compound, and

[0306] g iii ) is added to an aqueous solution to obtain an aqueous suspension of calibrated cross-linked vesicles.

[0307] The washing in step g) can be performed 1 to 10 times, preferably 2 to 5 times, and more preferably 3 times.

[0308] Step g i The duration of centrifugation is at least 1 minute, preferably at least 3 minutes, more preferably at least 4 minutes. The duration of centrifugation is typically less than 10 minutes, preferably less than 7 minutes, and more preferably the duration is 5 minutes.

[0309] In step g i The temperature during centrifugation is typically at least 1° C., preferably at least 2° C., more preferably at least 3° C. The temperature does not exceed 25° C., preferably is below 20° C., more preferably is below 10° C.

[0310] In step g i The centrifugation is performed at a spin of between 1000 g and 10000 g, preferably between 2000 g and 6000 g, and more preferably at 5000 g.

[0311] Step g iii The aqueous solution is preferably physiologically acceptable and includes water (preferably sterile water), an aqueous solution such as saline (which may advantageously be balanced so that the final product for injection is not hypotonic), or a solution of one or more pharmaceutical excipients.

[0312] Suitable examples of pharmaceutical excipients are tonicity regulating substances. Tonicity regulating substances include salts or sugars, sugar alcohols, glycols or other nonionic polyol substances (e.g., glucose, sucrose, trehalose, sorbitol, mannitol, glycerol, polyethylene glycol, propylene glycol, etc.), chitosan derivatives such as carboxymethyl chitosan, trimethyl chitosan or gelling compounds such as carboxymethyl cellulose, hydroxyethyl starch, hydrolyzed collagen or dextran.

[0313] Preferred is a glucose solution. A glucose aqueous solution typically contains 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 less than 10%, more preferably less than 7%.

[0314] Assemblies of cross-linked vesicles and cargo molecules

[0315] Advantageously, Applicants have observed that the disclosed cationic cross-linked vesicles are capable of adsorbing substantially large amounts of cargo molecules on their positive outer layer.

[0316] Another aspect of the invention relates to an assembly comprising the cross-linked vesicles as defined above and a load molecule, wherein the load molecule is electrostatically bound to the outer layer of the cross-linked vesicles.

[0317] As used herein, the term "carrier molecule" refers to a negatively charged molecule, such as genetic material or a nucleic acid mimetic, eg, peptide nucleic acid (PNA).

[0318] In a preferred embodiment, the cargo molecule is genetic material.

[0319] According to the present specification and claims, the expression "genetic material" refers to any nucleic acid-based agent, such as but not limited to RNA, DNA, saRNA, oligonucleotides, miRNA, siRNA, shRNA molecules and dsRNA molecules. Preferably, the genetic material comprises a negative charge.

[0320] In the present invention, the disclosed cationic cross-linked vesicles can deliver cargo molecules adsorbed to their shells due to electrostatic interactions between the positive charges contained in the cationic amphiphilic peptides forming the shell and the negative charges contained in the cargo molecules.

[0321] As an example, load molecules can be mixed with an aqueous suspension of cationic cross-linked vesicles by conventional techniques (eg, stirring) to obtain an aqueous suspension of the assembly, ie, an entity formed by cross-linked vesicles bound to the genetic material.

[0322] After electrostatic binding, the zeta potential of the assembly is generally lower than the initial positive charge of the cross-linked vesicles.

[0323] The expression "initial positive charge of the cross-linked vesicles" refers to the charge of the cross-linked vesicles after their preparation, for example as measured by zeta potential measurement. According to the present invention, the zeta potential of the cross-linked vesicles is higher than 20 mV, preferably higher than 30 mV, more preferably higher than 40 mV, and at most 100 mV.

[0324] For example, the zeta potential of the assembly is lower than 20 mV, preferably lower than 10 mV, and even more preferably the zeta potential has a negative value, ie lower than 0 mV and at most -60 mV.

[0325] Advantageously, the cationic vesicles are capable of adsorbing a substantially large amount of load molecules on the outer shell.

[0326] In one embodiment, the aqueous suspension of the assembly comprises at least 35% of the amount of the load molecule mixed into the suspension of cationic crosslinked vesicles to form an assembly as defined above. Preferably, the amount is at least 50%, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, at most 90%, preferably at most 95%, even more preferably at most 100%.

[0327] Applicants unexpectedly observed that the amount of cargo molecules adsorbed on the shell of cross-linked vesicles stabilized by the cationic amphiphilic peptide mixture was higher than on the shell of cross-linked vesicles stabilized by the cationic AP when used alone as single components.

[0328] In particular, a mixture of two or more amphiphilic peptides is used, said mixture comprising at least one cationic hydrophilic amino acid sequence characterized by an α-helical structure and comprising at least a positive charge to stabilize the shell of the disclosed cationic vesicles, said shell allowing the adsorption of up to 4 times the amount of cargo molecules adsorbed by cationic vesicles comprising the same amphiphilic peptide used as a single shell stabilizing material.

[0329] Another aspect of the present invention relates to a method for preparing an aqueous suspension comprising a plurality of assemblies as described above, the method comprising the steps of:

[0330] a) preparing an initial aqueous suspension of cross-linked vesicles as described above;

[0331] b) mixing said initial suspension with a predetermined amount of support molecules, and

[0332] c) obtaining an aqueous suspension of the assembly, wherein the amount of the carrier molecules electrostatically bound to the outer layer of the cross-linked vesicles is at least 35%, preferably at least 50%, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, at most 90%, preferably at most 95%, even more preferably at most 100% of the amount of the genetic material mixed in step b).

[0333] The amount of support molecules in the aqueous suspension of the assembly can be expressed as a percentage of the initial amount of support molecules (CM) in the initial suspension by using the following equation (Equation 2):

[0334]

[0335] in:

[0336] The expression "amount of CM after preparation of the assembly suspension" refers to the weight (eg μg) of the load molecules (CM) contained in 1 ml of the assembly suspension, and

[0337] • The expression "amount of CM mixed with the cationic vesicle suspension" refers to the weight (eg μg) of cargo molecules contained in 1 ml of the initial suspension of cross-linked vesicles.

[0338] Said amount can be measured, for example, by analytical methods, such as photometry (UV / Vis), fluorescence, electrophoresis, the diphenylamine method, quantification by real-time PCR.

[0339] use

[0340] Acoustic droplet vaporization (ADV) is a phenomenon in which cross-linked vesicles can be converted into gas microbubbles when exposed to ultrasonic energy exceeding the vaporization threshold.

[0341] When administered in vivo, the cross-linked vesicles have many advantages over 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 resulting microbubbles can be effectively used for imaging or therapeutic applications using ultrasound, including sonopermeabilization, thermal ablation, blood-brain barrier (BBB) ​​disruption, multimodal imaging modalities, and allow passive (due to enhanced permeability and retention (EPR) effect in tumor tissue) or active targeting (through the introduction of targeting ligands) for local delivery of therapeutic drugs or genes. Another potentially valuable feature of PFC-NDs is their potential application in novel imaging strategies, such as UltraSound Super-Resolution Imaging, because these agents can be activated and deactivated on demand by applying intermittent acoustic pulses.

[0342] Another aspect relates to an aqueous suspension comprising a plurality of calibrated cross-linked vesicles as defined above, for use in diagnostic and / or therapeutic treatments.

[0343] Another aspect relates to an aqueous suspension comprising a plurality of assemblies as defined above, for use in diagnostic and / or therapeutic treatments.

[0344] Diagnostic therapy includes any method in which the use of cross-linked vesicles allows enhanced visualization of a part or portion of the body of an animal (including humans), including imaging for preclinical and clinical studies. Suitable examples of diagnostic applications are molecular and perfusion imaging, tumor imaging (EPR effect), multimodal imaging (MR-guided tumor ablation, fluorescence, acousto-photoacoustic activation), US aberration correction, and super-resolution imaging.

[0345] Therapeutic treatment includes any method of treating a patient. In a preferred embodiment, the treatment comprises the combined use of ultrasound and (per)fluorocarbon 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 delivering drugs or biologically active compounds to selected sites or tissues, such as in tumor therapy, gene therapy, infectious disease therapy, metabolic disease therapy, chronic disease therapy, degenerative disease therapy, inflammatory disease therapy, immunological or autoimmune disease therapy, or in use as a vaccine), whereby the presence of the vesicles themselves can provide a therapeutic effect or can enhance the therapeutic effect of the applied ultrasound, e.g., by exerting or causing a biological effect in vitro and / or in vivo, by themselves or after specific activation by various physical methods (including, e.g., ultrasound-mediated delivery).

[0346] Another aspect of the present invention relates to cationic amphiphilic peptides of formula (I)

[0347] HB-CL-HP(I)

[0348] in

[0349] -HB is a fluorinated hydrophobic block,

[0350] -CL is a cross-linking motif, and

[0351] -HP is a cell penetrating peptide or a nuclear localization sequence, as defined above.

[0352] Preferably, HP comprises at least 2 positive charges, more preferably at least 3 positive charges, still more preferably at least 4 positive charges, still more preferably at least 5 positive charges, up to 40.

[0353] Preferably, HB is a fluorinated hydrophobic amino acid sequence HB' as defined previously.

[0354] Preferably, the cross-linking motif CL comprises cysteine, more preferably comprises the amino acid sequence GGGCCGG.

[0355] The following examples will help to further illustrate the present invention.

[0356] Example

[0357] Materials and methods

[0358] Example 1

[0359] Synthesis of amphiphilic peptides

[0360] Five amphiphilic peptides, namely AP-AGA, AP-TAT, AP-SV40, AP-Pepfect14 and AP-PreS2, were synthesized by FMOC solid-phase peptide synthesis (SPPS) using H-Rink amide. Peptides were synthesized on a PurePep Chorus peptide synthesizer (GyrosProtein Technologies) using 1% hydroxyethyl cyanoacetate (THF) resin, Rink-Amide AM resin, or Rink-Amide AM resin LL2, using ethyl oxime cyanoacetate and DIC as coupling agents. Resin cleavage was performed in a TFA / H2O / EDT / TIS (94:2.5:2.5:1) solution for 5 hours. The crude peptide was then purified by preparative high-performance liquid chromatography (HPLC).

[0361] 1.AP-AGA(H2N-F F F F F F GGGCCGGKGAGA-NH2)

[0362] Purification: Solid phase: Aeris 5 μm Peptide XB-C18 100A 250x21.2 mm. Mobile phase: 95% H2O + 0.1% TFA / 5% ACN + 0.1% TFA to 5% H2O + 0.1% TFA / 95% ACN + 0.1% TFA within 30 min.

[0363] The structure prediction of the hydrophilic amino acid sequence was performed using AlphaFold2 using Google Colab notebook AlphaFold2.ipynb (ColabFold 1.3.0) (https: / / colab.research.google.com / github / sokrypton / ColabFold / blob / main / AlphaFold2.ipynb#scrollTo=33g5IIegij5R). The predicted secondary structure of the hydrophilic amino acid sequence AGA corresponds to a randomly organized secondary structure.

[0364] 2.AP-TAT(H2N-F F F F F F GGGCCGGKGYGRKKRRQRRR-NH2)

[0365] Purification: Solid phase: Aeris 5 μm Peptide XB-C18 100A 250x21.2 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.

[0366] The structure prediction of cationic hydrophilic amino acid sequences was performed using AlphaFold2 using the Google Colab notebook AlphaFold2.ipynb (ColabFold v1.5.2-patch) (https: / / colab.research.google.com / github / sokrypton / ColabFold / blob / main / AlphaFold2.ipynb#scrollTo=33g5IIegij5R).

[0367] The predicted secondary structure of the cationic hydrophilic amino acid sequence TAT (YGRKKRRQRRR) is shown in Figure 1 c and corresponds to a randomly organized secondary structure.

[0368] 3.AP-SV40(H2N-F F F F F F GGGCCGGKGPKKKRKV-NH2)

[0369] Synthesis: Pentafluorophenylalanine 19 was introduced using HATU and DIPEA as coupling agents.

[0370] Purification: Solid phase: Aeris 5 μm Peptide XB-C18 100A 250x21.2 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.

[0371] The secondary structure of the hydrophilic amino acid sequence SV40 (PKKKRKV) predicted using ColabFold (as shown in Example 1, item 2) is shown in Figure 1 d and corresponds to a randomly organized secondary structure.

[0372] 4.AP-Pepfect14(H2N-F F F F F F GGGCCGGKGAGYLLGKLLOOLAAAALOOLL-NH2)

[0373] Purification: Solid phase: Aeris 5 μm Peptide XB-C18 100A 250x21.2 mm. Mobile phase: 57% H2O + 0.1% TFA / 43% ACN + 0.1% TFA to 47% H2O + 0.1% TFA / 53% ACN + 0.1% TFA in 30 min.

[0374] The secondary structure of the hydrophilic amino acid sequence PepFect14 (AGYLLGKLLOOLAAAALOOLL) predicted using ColabFold (as shown in Example 1, Item 2) is shown in Figure 1 b and corresponds to an α-helical structure.

[0375] 5.AP-PreS2(H2N-F F F F F F GGGCCGGKGPLSSIFSRIGDP-NH2)

[0376] Purification: Solid phase: Aeris 5 μm Peptide XB-C18 100A 250x21.2 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.

[0377] The secondary structure of the hydrophilic amino acid sequence Pres2 (PLSSIFSRIGDP) predicted using ColabFold (as shown in Example 1, item 2) is shown in Figure 1 a and corresponds to an α-helical structure.

[0378] Example 2

[0379] General Procedure for Preparing Aqueous Suspensions of Cationic Cross-linked Vesicles Using a Microfluidic Platform

[0380] Using NanoAssemblr from Precision Nanosystems (Vancouver, Canada) TM Perfluorocarbon-filled cross-linked vesicles were prepared using a benchtop automated instrument equipped with a staggered herringbone micromixer (SHM) that allows size-controlled self-assembly. Briefly, a liquid aqueous phase containing a cationic amphiphilic peptide was injected into the first inlet of a microfluidic cartridge, while a liquid organic phase consisting of a PFC dissolved in ethanol was injected into the second inlet of the microfluidic cartridge. Prior to cross-linkable vesicle preparation, both the aqueous and organic phases were placed in an ice bath at approximately 4°C. The microscopic features of the channel were engineered to cause accelerated mixing of the two fluid streams in a controlled manner. Microfluidic process settings, namely the total flow rate (TFR, in mL / min) and the flow rate ratio (FRR), were varied to control cross-linkable vesicle characteristics.

[0381] The aqueous suspension of calibrated cross-linkable vesicles was collected from the outlet channel into a Falcon vial (15 mL) and then diluted 5-fold in 2.5% DMSO aqueous solution before being transferred to a Slide-A-Lyzer from Thermo Fisher Scientific. TM The suspension was then dialyzed against a 2.5% DMSO solution with air bubbling overnight for the first hour. The sample was dialyzed against water for 1 hour and against 5% aqueous glucose for 4 hours.

[0382] At the end of the procedure, an aqueous suspension of calibrated PFC-crosslinked vesicles is obtained.

[0383] The specific types and amounts of materials used in the formulations shown in the following examples are summarized in Table 1, where for all compositions, the solvent for the aqueous phase was sodium acetate and the solvent for the organic phase was ethanol. The TFR was 10 mL / min.

[0384] Table 1 Compositions used in the present invention

[0385]

[0386]

[0387] Example 3

[0388] Effects of the properties of cationic amphiphilic peptides on the characteristics of cross-linked vesicles

[0389] An aqueous suspension of cationic cross-linked vesicles was prepared by microfluidic methods as described in Example 2.

[0390] To investigate the effect of the nature of the cationic amphiphilic peptides on the properties of the cross-linked vesicles (eg, size, PDI, and zeta potential), different compositions were compared.

[0391] At the end of the dialysis step, the calibrated aqueous suspensions of PFC-crosslinked vesicles were characterized using a Malvern Zetasizer Nano-ZS instrument (Malvern Instruments Ltd., UK) to measure size, size distribution (PDI) and zeta potential (ZP). 100 μL of each suspension was diluted 10-fold in 1 mM KCl aqueous solution for zeta potential measurements.

[0392] Characterization was performed immediately after the dialysis step (eg within 5 minutes) and after storage at 4°C for one day.

[0393] result:

[0394] Table 2 Size, PDI, and ZP of compositions S1-S5 and S7 over time

[0395]

[0396] As can be inferred from Table 2, the final properties of the cross-linked vesicles are substantially influenced by the nature of the cationic AP contained in their outer shell.

[0397] Cationic APs comprising a hydrophilic amino acid sequence with an α-helical secondary structure and at least a positive charge, such as AP-Pres2 (e.g., S1) and AP-Pepfect14 (e.g., S2 and S7), are able to form cross-linked vesicles characterized by good size and PDI values ​​and having a zeta potential higher than 20 mV.

[0398] In contrast, cross-linked vesicles stabilized by cationic APs containing hydrophilic amino acid sequences with randomly organized secondary structures, such as AP-SV40 (S3) and AP-TAT (S4 and S5), are characterized by higher sizes and lower zeta potential values ​​(<20 mV).

[0399] Furthermore, characterization performed one day after their preparation further confirmed that cross-linked vesicles stabilized by AP-Pres2 (S1) and AP-Pepfect14 (S2 and S7) were able to substantially maintain their characteristics, namely size, PDI, and zeta potential, over time, indicating higher stability than cross-linked vesicles stabilized by AP-SV40 or AP-TAT.

[0400] Example 4

[0401] Effects of mixtures of different amphiphilic peptides on the properties of cross-linked vesicles

[0402] An aqueous suspension of cationic cross-linked vesicles was prepared by microfluidic methods as described in Example 2.

[0403] To investigate the effect of combinations of different amphiphilic peptides on the properties of cross-linked vesicles (eg, size, PDI, and zeta potential), different compositions characterized by vesicles stabilized by mixtures of different amphiphilic peptides were compared.

[0404] At the end of the dialysis step, the calibrated aqueous suspensions of PFC-crosslinked vesicles were characterized using a Malvern Zetasizer Nano-ZS instrument (Malvern Instruments Ltd., UK) to measure size, size distribution (PDI) and zeta potential (ZP). 100 μL of each suspension was diluted 10-fold in 1 mM KCl aqueous solution for zeta potential measurements.

[0405] Characterization was performed after one day of storage at 4°C.

[0406] Table 3 Size, PDI, ZP of compositions M1-M12 after 1 day storage

[0407]

[0408]

[0409] result:

[0410] Table 3 shows that the use of mixtures of APs unexpectedly leads to cationic cross-linked vesicles characterized by higher zeta potential values ​​than the use of a single cationic AP by itself.

[0411] As can be inferred from the data, the enhanced zeta potential characterizing the cross-linked vesicles of the mixture comprising APs is higher than the zeta potential characterizing the cross-linked vesicles obtained by using said APs alone as single components (eg S1 and S2).

[0412] For example, cross-linked vesicles stabilized by AP-Pres2 itself (S2) were characterized by a zeta potential of 22 mV, whereas cross-linked vesicles stabilized by AP-SV40 (S3) itself were characterized by a low zeta potential of 13 mV.

[0413] Surprisingly, the mixture of AP-PreS2 and AP-SV40 (M1) allows obtaining cationic cross-linked vesicles characterized by much higher zeta potential values, ie above 40 mV.

[0414] This result is even more surprising considering that cationic AP-SV40 (S3) was unable to impart cationic cross-linked vesicles when used by itself (ie, very low ZP) (see Table 2).

[0415] The use of perfluoropentane (bp: 28-30°C) as a component of the core (eg compositions M5 and M6) demonstrated the same synergistic effect on the overall positive charge of the final cross-linked vesicles.

[0416] Example 5

[0417] Effects of the properties of cationic amphiphilic peptides on the adsorption of genetic material onto the surface of cross-linked vesicles

[0418] An aqueous suspension of cationic cross-linked vesicles was prepared by microfluidic methods as described in Example 2.

[0419] To study the influence of the nature of the cationic amphiphilic peptide on the amount of genetic material adsorbed on the shell of the cross-linked vesicles, different compositions were compared. At the end of the preparation, the calibrated aqueous suspension of cross-linked vesicles was mixed with the genetic material as reported below.

[0420] Add 1.5 mL Eppendorf tube without DNAse or RNAse 5.2 μg of plasmid pCPG-hCMVSCEP-LucSH was loaded, 625 μL of the cross-linked suspension was slowly added, and the suspension was then diluted to 650 μL with 5% aqueous glucose solution, mixed gently, and the mixture was incubated at rt for 15 min.

[0421] To measure the amount of pDNA in the composition, the remaining suspension was centrifuged at 4°C (10 min, 5000 rpm), and 20 μL of the supernatant was added to 180 μL of Qubit TM Diluted in 1x dsDNA BR working solution and quantified with Qubit TM 4Analysis.

[0422] result:

[0423] The amount of pDNA adsorbed on the shell of the cross-linked vesicles was determined according to Equation 2.

[0424] The synergistic effect obtained by using a mixture of APs compared to using a single AP as shell component was further confirmed by the results of measuring the amount of pDNA adsorbed on the shell of the relevant cross-linked vesicles.

[0425] As can be inferred from Table 4, the amount of pDNA adsorbed on the shells of cross-linked vesicles stabilized by a mixture of APs is significantly higher and may even be higher than the amount adsorbed on the shells of cross-linked vesicles stabilized by said APs when used alone as single components.

[0426] For example, cross-linked vesicles stabilized by a mixture of AP-PreS2 and AP-SV40 (M1) were able to adsorb approximately twice the amount of pDNA as cross-linked vesicles stabilized by AP-PreS2 as the single component (S1).

[0427] Table 4 Percentage of adsorbed pDNA

[0428]

[0429]

[0430] As mentioned above, this result is even more surprising considering that the cationic AP-SV40 (S3) used as a single component was able to adsorb only 20% of the pDNA in contact with the aqueous suspension of cross-linked vesicles.

[0431] Furthermore, this improved pDNA adsorption was observed in compositions containing perfluorohexanes (eg, M1-M3) or perfluoropentanes (eg, M5, M6, M11, and M12).

[0432] Example 6

[0433] Determination of the degree of cross-linking.

[0434] The composition M1 and M2 were subjected to the cross-linking degree determination. The cross-linking percentage was measured by Ellman's assay. Briefly, a 10 mM Ellman's reagent solution was prepared in 1 mM EDTA, 200 mM phosphate buffer pH = 7.5. Ten microliters (10 μL) of each suspension and 4 μL of the solution containing Ellman's reagent were added to 140 μL of PBS buffer. The mixture was incubated in the dark for 40 min. The absorbance was measured and the concentration was calculated using the Beer-Lambert law (Equation 2):

[0435] A = ε·l·C (Equation 2)

[0436] Where A is the absorbance, l is the length of the sample chamber (cm), ε is the molar absorptivity (M -1 cm -1 ) and C is the concentration.

[0437] In this specific example, ε is 14150M -1 cm -1 .

[0438] Four microliters (4 μL) of Ellman's reagent in 140 μL of PBS buffer was used as a blank.

[0439] result

[0440] It was found that the cross-linking degree of both compositions M1 and M2 was higher than 90%, in particular, M1 was characterized by a cross-linking degree of 98.6% and M2 was 99%, which confirmed that a large number of cross-linkable cysteine ​​residues contained in the shell of the cross-linked vesicles were intermolecularly connected through disulfide cross-linking groups (-SS).

[0441] Example 6

[0442] Measurement of Acoustic Droplet Vaporization (ADV)

[0443] The expression "Acoustic Droplet Vaporization (ADV) Threshold" denotes the minimum acoustic pressure necessary to obtain the conversion of nanodroplets into echogenic microbubbles.

[0444] The acoustic droplet vaporization (ADV) threshold of NDs prepared according to the aforementioned examples can be determined using B-mode imaging according to conventional methodologies. For example, a suspension of NDs can be vaporized as it passes through the focal region of a transducer, and the acoustic pressure can be increased by approximately 0.2 MPa every 5 seconds until ND evaporation is observed.

[0445] Nanodroplet activation was performed by focused ultrasound at five aligned focal points, allowing activation only in the region of interest where the acoustic pressure was highest. Pulses were emitted in burst mode at a frequency of 6 MHz, 20 cycles per pulse, and a pulse repetition frequency (PRF) of 1 Hz.

[0446] The acoustic pressure was increased every 5 seconds until ND vaporization was observed.

[0447] For the ADV assay, six different compositions were tested, namely S1, S2, M1 (containing perfluorohexane), and M5-M6 (containing perfluoropentane).

[0448] result

[0449] The overall results obtained from determining the ADV threshold are reported in Table 5. Each value is the average of three consecutive determinations.

[0450] Table 5 Determination of acoustic droplet vaporization threshold

[0451] No pDNA With pDNA Composition MPa MPa S1 10.27±0.14 - S2 10.15±0.09 10.24±0.03 S3 10.90±0.13 - M1 10.42±0.25 10.20±0.06 M5 7.34±0.03 - M6 7.36±0.06 - M7 10.05±0.08 10.38±0.05 M11 7.46±0.04 7.76±0.24 M12 7.47±0.28 7.56±0.16

[0452] The results demonstrate that formulations containing cross-linked vesicles stabilized by a single amphiphilic peptide, such as compositions S1, S2, and S3, as well as formulations containing cross-linked vesicles stabilized by a mixture of amphiphilic peptides (compositions M1, M5, M6, M7, M11, M12) can be vaporized at similar acoustic pressures. Adding pDNA to the surface of the particles does not affect the ADV threshold.

[0453] Furthermore, compositions M5, M6, M11 and M12 comprising cross-linked vesicles stabilized by a mixture of APs and containing perfluoropentane in their core are characterized by a lower ADV threshold.

[0454] References

[0455] 1WO2019023706A1

[0456] 2Branden, Tooze, Introduction to Protein Structure, Garland Science, 1998

[0457] 3 Smith, Folding and Design, Vol. 1, No. 5, pp. R95-R106 (1996)

[0458] 4Mirdita et al., Nature Methods, Vol. 19, pp. 679-682 (2022)

[0459] 5Derakhshankhah et al., Biomed Pharmacother, Vol. 108, pp. 1090-1096 (2018)

[0460] 6 www.cppdatabase, accessed on December 22, 2022.

[0461] 7Lu et al., Cell Commun Signal.;19:60(2021)

[0462] 8WO2022101365 A1

[0463] 9 Sheeran et al., Langmuir, 27(17), 10412-10420, (2011).

Claims

1. A cross-linked vesicle comprising an outer layer and an inner core, wherein the outer layer comprises a cationic amphiphilic peptide and the inner core comprises a fluorinated compound in liquid form, wherein the cationic amphiphilic peptide is a compound of formula (I) HB-CL-HP(I) in HB is a fluorinated hydrophobic block, CL is a cross-linking motif, HP is a cationic hydrophilic amino acid sequence having an α-helical structure and containing at least one positive charge, and wherein the vesicles have a zeta potential of at least 20 mV.

2. The cross-linked vesicle of claim 1, wherein HP comprises at least 5 positive charges. The cross-linked vesicle according to claim 1 or 2, wherein HB is a fluorinated hydrophobic amino acid sequence HB'.

4. The cross-linked vesicle of claim 3, wherein the fluorinated hydrophobic amino acid sequence HB' comprises three consecutively linked pentafluoro-phenylalanine residues at its terminus and is a compound of formula IV 5. The cross-linked vesicle of any one of the preceding claims, wherein the cross-linking motif CL comprises cysteine.

6. The cross-linked vesicle of claim 5, wherein the cross-linking motif CL comprises the amino acid sequence GGGCCGG.

7. The cross-linked vesicle of any one of the preceding claims, wherein the cationic amphiphilic peptide is selected from the group consisting of H2N-F F F F F F GGGCCGGKGPLSSIFSRIGDP-NH2, or H2N-F F F F F F GGGCCGGKGAGYLLGKLLOOLAAAALOOLL-NH2。 8. The cross-linked vesicle according to any one of the preceding claims, wherein the outer layer further comprises an additional amphiphilic peptide having formula VI HB-CL-HP'(VI) in -HB is a fluorinated hydrophobic block, -CL is a cross-linking motif, and -HP' is a hydrophilic amino acid sequence. 9 . The cross-linked vesicle of claim 8 , wherein HP′ has a secondary structure selected from the group consisting of an α-helix, a π-helix, a β-sheet, a β-turn, or a mixture thereof.

10. The cross-linked vesicle of claim 8, wherein HP' has a randomly organized secondary structure.

11. The cross-linked vesicle of claim 8, wherein the additional amphiphilic peptide is a cationic amphiphilic peptide, wherein HP' is a cationic hydrophilic amino acid sequence comprising at least one positive charge. The cross-linked vesicle according to claim 11 , wherein HP′ is a cationic hydrophilic amino acid sequence containing two or more positive charges.

13. The cross-linked vesicle of claim 8, wherein the additional amphiphilic peptide is selected from the group consisting of H2N-F F F F F F GGGCCGGKGYGRKKRRQRRR-NH2, H2N-F F F F F F GGGCCGGKGPKKKRKV-NH2, or H2N-F F F F F F GGGCCGKGAGA-NH2。 14. The cross-linked vesicle of any one of the preceding claims, wherein HP and HP' are selected from a cell penetrating peptide or a nuclear localization sequence.

15. The cross-linked vesicle of any one of the preceding claims, wherein the fluorinated compound is a perfluorocarbon compound selected from perfluoropentane, perfluorohexane, or a mixture thereof.

16. The cross-linked vesicle according to any one of the preceding claims, having a degree of cross-linking higher than 80%, preferably at least 85%, more preferably at least 90%, still more preferably at least 95%.

17. The cross-linked vesicle of any preceding claim, wherein the vesicle has a zeta potential of at least 30 mV.

18. An aqueous suspension comprising a plurality of cross-linked vesicles according to any one of claims 1 to 17.

19. The aqueous suspension of claim 18, wherein the vesicles are calibrated cross-linked vesicles having a z-average diameter between 100 nm and 1000 nm and a polydispersity below 0.

2.

20. A method for preparing an aqueous suspension comprising a plurality of cross-linked vesicles according to any one of claims 1 to 17, the method comprising the steps of: a) preparing an aqueous phase comprising a cationic amphiphilic peptide, wherein the pH of the aqueous phase is below 4; b) preparing an organic phase comprising a fluorinated compound; c) injecting the aqueous phase into a first inlet of a microfluidic cartridge and injecting the organic phase into a second inlet of a microfluidic cartridge, thereby mixing the aqueous phase and the organic phase in a mixing portion of the microfluidic cartridge, wherein the operating pressure entering the microfluidic cartridge is less than 7000 kPa, to obtain an aqueous suspension of vesicles; d) collecting the aqueous suspension of cross-linkable vesicles from the outlet channel of the microfluidic cartridge; f) diluting the aqueous suspension of cross-linkable vesicles, and e) cross-linking the cationic amphiphilic peptide to obtain an aqueous suspension of cross-linked vesicles.

21. An aqueous suspension for diagnostic and / or therapeutic treatment comprising a plurality of cross-linked vesicles according to any one of claims 1 to 17.

22. An assembly comprising a cross-linked vesicle as defined in any one of claims 1 to 17 and a load molecule, wherein the load molecule is electrostatically bound to the outer layer of the cross-linked vesicle.

23. Cationic amphiphilic peptide of formula (I) HB-CL-HP(I) in -HB is a fluorinated hydrophobic block, -CL is a cross-linking motif, and -HP is a cell-penetrating peptide or nuclear localization sequence, which has an α-helical structure and contains at least one positive charge.

24. The cationic amphiphilic peptide of claim 23, wherein HP comprises at least 5 positive charges.

25. The cationic amphiphilic peptide according to claims 23 and 24, wherein HB is a fluorinated hydrophobic amino acid sequence HB'.

26. The cationic amphiphilic peptide according to claims 23-25, wherein the fluorinated hydrophobic amino acid sequence HB' comprises three consecutively linked pentafluoro-phenylalanine residues at its terminus and is a compound of formula IV 27. The cationic amphiphilic peptide according to claims 23-26, wherein the cross-linking motif CL comprises cysteine.

28. The cationic amphiphilic peptide according to claim 27, wherein the cross-linking motif CL comprises the amino acid sequence GGGCCGG.

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