Ligands for trapping microvesicles and uses thereof - Patents.com

Abstract

The present invention relates to ligands capable of binding to microvesicles, which comprise a plurality of metal(II)-dipicolylamine moieties, and their use in the diagnosis or prognosis of disorders in a subject.

Description

[Technical field]

[0001] The present invention relates to synthetic ligands for capturing microvesicles and their use in the diagnosis and prognosis of disease in a subject. [Background technology]

[0002] Microvesicles (also known as cellular microparticles) are submicron extracellular vesicles found both in the interstitial space between cells and in body fluids. Microvesicles are released from the surface of cells or from the plasma membrane by outward budding and fission of the plasma membrane. The budding process involves multiple signaling pathways, including elevation of intracellular calcium and rearrangement of the structural scaffolding of the cell. Budding occurs at unique sites on the cell membrane that are enriched with specific lipids and proteins reflecting their cellular origin. At these sites, proteins, lipids and nucleic acids are selectively incorporated into the microvesicles and released into the surrounding environment. Microvesicles play a role in cell-cell communication and can transport molecules such as mRNA, miRNA, mitochondrial DNA and proteins between cells.

[0003] The microvesicles are released during cell activation or apoptosis, during a variety of pathologies including cancer, inflammation or various diseases such as diabetes, Parkinson's disease, cardiovascular diseases with increased risk of thrombosis or even neurovascular ischemic conditions.

[0004] Microvesicles have a high density of phosphatidylserine exposed on the outer leaflet. Microvesicles also expose various cell surface proteins according to their cellular origin and the type of pathological or physiological event that induces their release. Moreover, it has been shown that an increase in microvesicles can correlate with the onset and presence of disease in patients. For example, increased levels of leukocyte-derived microvesicles have been reported in the blood of patients with sepsis or high atherothrombotic risk. Microvesicles are also believed to play an important role in regulating cell-cell communication, inflammatory responses and vascular function.

[0005] It has therefore been proposed to detect, quantify and / or analyse microvesicles present in body fluids in order to diagnose and assess the risk of developing various diseases and related disorders in patients, in particular thrombotic, inflammatory and / or metabolic disorders.

[0006] Methods for detecting and quantifying microparticles have been described in the prior art. Flow cytometry is the most widely used. This technique allows the measurement and identification of the phenotype of microvesicles by using (CD) specific antibodies. Due to the high affinity of annexin A5 for phosphatidylserine molecules present in the outer layer of microvesicles, it is also possible to use A5 as a probe to mark microvesicles.

[0007] Methods for capturing / quantifying microparticles have also been proposed and are based on the use of solid supports such as microplates or magnetic beads that have ligands thereon that can specifically capture microvesicles. These ligands include annexin 5, antibodies and synthetic ligands.

[0008] For example, Cointe et al. (J Extracell vesicles, 2018, 7(1):1494482) describe a bioassay for capturing leukocyte-derived microvesicles and measuring their plasmin generating capacity, e.g., for the prognosis of sepsis outcome in patients.

[0009] International Patent Application No. WO2012 / 127175 describes binuclear metal complexes that can be used as ligands for the specific capture of microvesicles and their use in the diagnosis of disease. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Patent Application No. WO2012 / 127175 [Non-patent literature]

[0011] [Non-Patent Document 1] Cointe et al. (J Extracell vesicles, 2018, 7(1):1494482) [Non-Patent Document 2] Koniev, O., Wagner, A., Chem. Soc. Rev., 44, 5495 (2015) [Non-Patent Document 3] Kolb et al., Angew. Chem. Int. Ed. 2001, 40, 2004~2021 [Non-Patent Document 4] Rudolf et al., Current opinion in Chemical Biology, 2013, 17:110~117 [Non-Patent Document 5] Calmet et al. (Sci Rep, 2016. 6:36181 [Non-Patent Document 6] Chollet, C. et al., Biomol Eng, 2007. 24(5): pp. 477-82 Summary of the Invention [Problem to be solved by the invention]

[0012] However, there remains a need for alternative methods for capturing microvesicles from biological samples, particularly in the context of diagnosing disease. [Means for solving the problem]

[0013] The present invention relates to a compound of formula (I): [ka] [In the formula, M is Zn 2+ , Mn 2+ , Co 2+ , Ni 2+ , Cu 2+ and Fe 2+ is a metal cation preferably selected from the group consisting of The present invention relates to a molecule capable of binding to microvesicles, comprising at least one, and preferably two, moieties:

[0014] In some embodiments, the molecule of the invention has formula (Ia): [ka] may include at least one portion of

[0015] For example, a molecule of the invention may have the formula (Ib): [ka] and The moiety of formula (Ia) or (Ib) - each X1 is independently O, S or NH; - each X2, if present, is independently O, S or NH; - M is Zn 2+ , Co 2+ , Cu 2+ and Fe 2+ Preferably, Zn 2+ is It's like that.

[0016] In some embodiments, the molecules of the invention are dendrimers comprising a branched core carrying a plurality of moieties of formula (I), said branched core preferably comprising groups selected from 3,5-di(hydroxymethyl)phenol, 3,5-di(thiomethyl)phenol, 3,5-di(thiomethyl)thiophenol, 3,5-dialkylphenol, 3,5-di(aminomethyl)phenol and 3,5-di(aminomethyl)phenylamine as building blocks, more preferably 3,5-di(hydroxymethyl)phenol as building block. In some alternative or additional embodiments, the molecules of the invention may further comprise means for immobilization on a support attached at the ends of the spacer chains.

[0017] For example, the molecule of the invention may have the formula (II) [ka] [In the formula, - n is an integer from 1 to 10, preferably from 2 to 6, more preferably 2 or 4, m, p and o are independently 0 or 1; M is a divalent metal cation; - [the core] carries at least one moiety of formula (I) and has a molecular weight of at most 25000 g.mol -1 of 10000 or 5000 g.mol -1 is a chemical entity having a molecular weight of - [IMM] is a means for covalent or non-covalent immobilization on a support, - [Spacer] is selected from the group consisting of peptides, polypeptides, oligo- or polysaccharides, saturated or unsaturated hydrocarbon chains, polymers including homopolymers, copolymers and block polymers, optionally interrupted by one or more heteroatoms (e.g., S, O or NH), optionally bearing heteroatoms such as S, O and NH on at least one of its termini, and optionally substituted by one or more substituents such as hydroxyl, halogen, C1-C3 alcoxy, -CN, -CF3 or C1-C3 alkyl, and combinations thereof. It may be of the following type.

[0018] The molecule has the following features: m, p and o are 1; - M is Zn 2+ , Co 2+ , Cu 2+ and Fe 2+ Preferably, Zn 2+ and - [Spacer] is an optionally substituted C2-C 20 They are selected from saturated or unsaturated hydrocarbon chains, polyamide chains containing from 2 to 20 monomers, polyester chains and polyether chains such as polyethylene glycol, and combinations thereof. [IMM] comprises or consists of a moiety selected from the group consisting of an amino group, preferably -NH2, OH, -COOH, an activated carboxylic acid, -SH, an iodoacetyl group, a carbonyl, a hydrazide group, an azide and a strained alkyne. - [Core] is a chemical moiety made of at least one 3,5-di(hydroxymethyl)phenol. The present invention may be further characterized by one, some or all of the following:

[0019] In particular, the molecules of the invention are compounds of formula (IIa), (IIb) or (IIc): [ka] [In the formula, - M, m, p, [IMM] and [spacer] are as defined above for formula (II), - each X1 and X2, if present, is selected from the group consisting of O, NH and S; - X3 is -O-, C(=O) -OC(O)-, -C(O)O-, -OC(O)O-, -S-, -SS-, -SC(O)-, -OC(S)-, NR1-, -NR1C(O)-, -C(O)NR1-, NR1C(S)-, C(S)NR1-, -OC(O)S-, -OC(S)O-, -SC(O)O-, -OC(S)S-, -SC(O)S-, -SC(S)O-, -SC(S)S-, OC(O)NR1-, -OC(S)NR1-, -NR1C(S)O-, -NR1C(O)S-, NR1C(O)NR2-, -NR1C(S)NR2-, -SC(O)S-, -SC(S)O-, -S(O)-, -S(O)2-, - -P(O)(R1)-, -P(O)(OR1)-, P(O)(R1)O-, OP(O)(OR1), OP(O)(R1)O-, NRP(O)(R2)-, -NRP(O)(OR2)-, NRP(O)(R2)O-, OP(O)(OR1) and OP(O)(R1)O-, wherein R1 and R2 are independently H or CH3. It may be.

[0020] For example, the molecule of formula (IIa), (IIb) or (IIc) may be m and p are 1, - M is Zn 2+ , Co 2+ , Cu 2+ or Fe 2+ , preferably Zn 2+ and - X1 and X2, if present, are O; X3 is O, NH, NHCO, CONH, O(C=O), (O=C)O, O(C=O)O or NHCONH, - [IMM]-[spacer] is NH2-(CH2) r - or NH2-[(CH2)2-O] r where r is an integer from 2 to 10, - the molecule comprises one or more counter anions selected from the group consisting of perchlorate, tosylate, nitrate, sulfate, sulfonate, thiosulfate, halide, hexafluorophosphate, tetraphenylborate, carbonate, and tetrafluoroborate. It may be characterized as follows.

[0021] Preferably, the molecule is a compound of formula (IIb) or (IIc).

[0022] Examples of molecules of the invention are ligands of formula (IIIb) and (IIIc): [ka] It is.

[0023] The present invention also relates to a support for trapping microvesicles comprising a molecule as defined above on its surface and to a support comprising such a surface.

[0024] The present invention also relates to an in vitro method for capturing microvesicles from a sample, comprising the step of contacting the sample with a molecule or a support as defined above. - providing a sample susceptible to containing microvesicles of interest from a bodily fluid of a subject; and / or - washing the support after the step of contacting the support with the sample, and / or - detecting the formation of complexes between the microvesicles of interest and said support or molecule of the invention, and / or - recovering the complexes formed between the microvesicles and the molecules of the invention, and / or - quantification of said microvesicles of interest, and / or - characterizing the captured microvesicles, for example by detecting and / or quantifying biomarkers present on the surface or within the microvesicles; and / or - releasing the microvesicles from the complex; and / or - recovering the desired microvesicles; The method may further include at least one step selected from the following:

[0025] The present invention provides an in vitro method for the diagnosis, differential diagnosis, prognosis, risk assessment, and / or monitoring of a disorder in a subject, comprising: (a) providing a support or molecule of the present invention; (a) contacting said support or said molecule with a sample obtained from a subject under conditions suitable for the formation of complexes between the molecules of the invention and microvesicles possibly present in the sample; (b) detecting, quantifying and / or characterizing the microvesicles captured in step (b) by complex formation; The present invention also relates to an in vitro method comprising:

[0026] Typically the disorder may be selected from the group consisting of cancer, parasitic diseases, diabetes and related disorders such as diabetic nephropathy, diabetic neuropathy, diabetic retinopathy and diabetic foot syndrome, multiple sclerosis, cancer, Alzheimer's disease, Parkinson's disease, aneurysm, cerebrovascular spasm, stroke and coronary artery disease and / or the sample may be or be derived from blood, plasma and urine.

[0027] A further object of the invention is the use of a molecule or support as defined above for capturing microvesicles from a sample from a subject, preferably as a means for the diagnosis, prognosis or monitoring in a subject of diseases such as cancer, parasitic diseases, diabetes and related disorders including diabetic nephropathy, diabetic neuropathy, diabetic retinopathy and diabetic foot syndrome, multiple sclerosis, cancer, Alzheimer's disease, Parkinson's disease, aneurysm, cerebral vasospasm, stroke and coronary artery disease. [Brief description of the drawings]

[0028] [Figure 1A] Figure 1. PWR titration of different complex solutions into PSPC membranes. A, B: Fitting of Cplx1-PSPC membrane titration in s and p polarized light, respectively; C, D: Fitting of Cplx2-PSPC membrane titration in s and p polarized light, respectively; E, F: Fitting of Cplx4-PSPC membrane titration in s and p polarized light, respectively. PSPC membrane composition: 1 equivalent of PS, 5 equivalents of PC. Fitting curves were obtained using GraphPad Prism 7. [Figure 1B] Figure 1. PWR titration of different complex solutions into PSPC membranes. A, B: Fitting of Cplx1-PSPC membrane titration in s and p polarized light, respectively; C, D: Fitting of Cplx2-PSPC membrane titration in s and p polarized light, respectively; E, F: Fitting of Cplx4-PSPC membrane titration in s and p polarized light, respectively. PSPC membrane composition: 1 equivalent of PS, 5 equivalents of PC. Fitting curves were obtained using GraphPad Prism 7. [Figure 1C] Figure 1. PWR titration of different complex solutions into PSPC membranes. A, B: Fitting of Cplx1-PSPC membrane titration in s and p polarized light, respectively; C, D: Fitting of Cplx2-PSPC membrane titration in s and p polarized light, respectively; E, F: Fitting of Cplx4-PSPC membrane titration in s and p polarized light, respectively. PSPC membrane composition: 1 equivalent of PS, 5 equivalents of PC. Fitting curves were obtained using GraphPad Prism 7. [Figure 1D]Figure 1. PWR titration of different complex solutions into PSPC membranes. A, B: Fitting of Cplx1-PSPC membrane titration in s and p polarized light, respectively; C, D: Fitting of Cplx2-PSPC membrane titration in s and p polarized light, respectively; E, F: Fitting of Cplx4-PSPC membrane titration in s and p polarized light, respectively. PSPC membrane composition: 1 equivalent of PS, 5 equivalents of PC. Fitting curves were obtained using GraphPad Prism 7. [Figure 1E] Figure 1. PWR titration of different complex solutions into PSPC membranes. A, B: Fitting of Cplx1-PSPC membrane titration in s and p polarized light, respectively; C, D: Fitting of Cplx2-PSPC membrane titration in s and p polarized light, respectively; E, F: Fitting of Cplx4-PSPC membrane titration in s and p polarized light, respectively. PSPC membrane composition: 1 equivalent of PS, 5 equivalents of PC. Fitting curves were obtained using GraphPad Prism 7. [Figure 1F] Figure 1. PWR titration of different complex solutions into PSPC membranes. A, B: Fitting of Cplx1-PSPC membrane titration in s and p polarized light, respectively; C, D: Fitting of Cplx2-PSPC membrane titration in s and p polarized light, respectively; E, F: Fitting of Cplx4-PSPC membrane titration in s and p polarized light, respectively. PSPC membrane composition: 1 equivalent of PS, 5 equivalents of PC. Fitting curves were obtained using GraphPad Prism 7. [Diagram 2] FIG. 1 shows a TBO calibration curve of carboxyl group density plotted against solution absorbance at 633 nm. [Diagram 3] FIG. 2 shows the carboxyl group density on treated PET surfaces (unit: pmol / mm2) determined by TBO experiments. [Figure 4] Figure 1 shows Cplx1-grafted PET surfaces incubated with microvesicles, A: Fluorescence micrograph, B: Cryo-SEM micrograph. Scale bars are provided in the images. [Diagram 5] Cplx2-grafted PET surface incubated with microvesicles. A: Fluorescence micrograph, B: Cryo-SEM micrograph. Scale bars are provided in the images. [Figure 6] Cplx4-grafted PET surface incubated with microvesicles. A: Fluorescence micrograph, B: Cryo-SEM micrograph. Scale bars are provided in the images. [Figure 7] PET surface grafted with cplx8 incubated with microvesicles. A: Fluorescence micrograph, B: Cryo-SEM micrograph. Scale bars are provided in the images. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present inventors have conceived of new synthetic ligands capable of selectively binding with high affinity to microvesicles. These ligands are metal complexes that contain multiple metal(II)-bis(dipicolylamine) (M-BDPA) moieties that can interact with phosphatidylserine present in the outer leaflet of the microvesicles. Without being bound by any theory, the present inventors are of the opinion that the number of M-BDPAs and their proximity in the ligands of the present invention allows for optimizing binding with phospholipid-enriched microvesicles with high specificity.

[0030] As illustrated in the examples, ligands of formulae (IIIa), (IIIb) and (IIIc) exhibited 10 -6 These ligands have dissociation constants (Kd) in the M range. When immobilized on a support, these ligands were able to capture microvesicles. In contrast, supports grafted with ligands of formula (IIId), which contain only one dipicolylamine moiety, were shown to have a very limited ability to capture microvesicles.

[0031] The ligands of the present invention have been shown to be effective ligands for capturing microvesicles on a support, allowing for the subsequent detection, quantification and characterization of the microvesicles. Of note, the multivalent ligand of formula (IIIb) (also referred to as Cplx4 in the Examples section) is capable of capturing microvesicles while maintaining their shape, allowing the detection and quantification of biomarkers of interest present within the membrane of the microvesicles, but also present inside the microvesicles. 31 P NMR analysis confirmed that Cplx4 was the complex with the highest interaction with POPS among the tested compounds. Notably, the interaction was higher for Cplx4 than for Cplx8, which showed a negative dendritic effect for the large ligand.

[0032] The possibility of detecting biomarkers within the membrane and / or inside the microvesicles is of high interest in the design of diagnostic methods, since the microvesicle proteome has been shown to reflect the pathological state of the cell of origin.

[0033] The ligands of the invention therefore find use in the in vitro diagnosis and prognosis of a variety of disorders.

[0034] - a ligand according to the invention In a first aspect, the present invention relates to a ligand that comprises a plurality of metal(II)-bis(dipicolylamine) (M-BDPA) moieties and that is associated with microvesicles, preferably phosphatidylserine-enriched microvesicles.

[0035] More precisely, the present invention relates to a compound of formula (I): [ka] wherein M is a metal cation, preferably a divalent metal cation. The present invention relates to a molecule capable of binding to a microvesicle comprising at least one portion (e.g., at least two, three or four portions) of:

[0036] In some embodiments, M is Zn 2+, Mn 2+ , Co 2+ , Ni 2+ , Cu 2+ and Fe 2+ , preferably Zn 2+ , Fe 2+ , Co 2+ and Cu 2+ and more preferably Zn 2+ It is.

[0037] In a preferred embodiment, the molecule of the invention comprises at least two moieties of formula (I), for example, two, three or four moieties of formula (I).

[0038] Typically, the molecules of the invention have a molecular weight of up to 25000 g.mol -1 of 10000, preferably up to 20000, for example up to 15000, 10000, 7000, 5000 or 3000 g.mol -1 The molecular weight of

[0039] As explained in the Background, "microvesicles" (also known as cellular microparticles) refer to submicron extracellular vesicles found both in the interstitial space between cells and in bodily fluids. Microvesicles are released from the surface of cells or from the plasma membrane by outward budding and fission of the plasma membrane. Microvesicles play a role in intercellular communication and can transport molecules such as mRNA, miRNA and proteins between cells.

[0040] Phosphatidylserine (PS) accounts for approximately 2-10% of total cellular phospholipids in a typical mammalian cell plasma membrane. In healthy cells, PS is almost exclusively sequestered in the inner leaflet of the cell. Under certain conditions, particularly during cell death pathways, PS appears within the outer leaflet of the plasma membrane due to attenuation of active transport mechanisms that maintain the asymmetric transmembrane distribution of PS. Microvesicles have been shown to exhibit phosphatidylserine in the outer leaflet of their membrane.

[0041] As used herein, "phosphatidylserine-enriched microvesicles or phosphatodylserine-rich microvesicles" refers to microvesicles in which PS can be found in the outer leaflet of the vesicle's membrane. In other words, "phosphatidylserine-enriched microvesicles" refers to microvesicles with PS present in the outer leaflet of the bilayer lipid membrane. For example, such microvesicles may be derived from cells in which cell death pathways have been activated. Synthetic vesicles made of POPC (1-palmitoyl-2-oleoyl-sn-glyco-3 phosphocholine) and POPS (1-palmitoyl-2-oleoyl-sn-glyco-3 phosphoserine) in a mass ratio of 5:1 (w / w) can be used as a model of "PS-enriched microvesicles" to evaluate in vitro the ability of molecules to bind to the microvesicles.

[0042] As used herein, "a molecule binds to a microvesicle" means that the molecule is capable of binding to a microvesicle, preferably a phosphatidylserine-enriched microvesicle. Such molecules are also referred to herein as "ligands of the microvesicles" or "ligands".

[0043] Preferably, a molecule binds to the microvesicles with high affinity, e.g., up to 10 -4 or 10 -5 M, etc., up to 10 -3 It refers to a molecule that can bind with a dissociation constant (Kd) of M.

[0044] Kd is preferably determined by plasmon waveguide resonance spectroscopy, as exemplified in Example 2. Such an assay is performed by using vesicles prepared by mixing POPC with POPS in a mass ratio of 5:1 (w / w) and 1× Dulbecco's phosphate buffered saline (pH 7.1-7.5) as the medium.

[0045] Alternatively, it is possible to identify whether a molecule can bind to microvesicles by a capture assay using, for example, cryo-scanning electron microscopy (cryo-SEM), as illustrated in Example 4. Typically, the molecule to be evaluated is immobilized on a support and then incubated with a solution containing microvesicles in a phosphate saline buffer. After washing and freezing, the surface of the support is analyzed by cryo-SEM to determine whether microvesicles are immobilized thereon.

[0046] In some embodiments, the molecules of the invention bind with specificity to microvesicles, meaning that their affinity for microvesicles (i.e., phosphatidylserine-enriched microvesicles) is higher than for other cellular particles such as exosomes, and more generally for cellular particles that do not exhibit PS in their membrane outleaflets. For example, the molecules of the invention may have a Kd for microvesicles that is at least 5-fold lower, preferably at least 10-, 20-, 30-, 40-, 50-, 100-, 200-, 500-, or 1000-fold lower than its Kd for other cellular particles such as exosomes. In some embodiments, the molecules of the invention do not cross-react with cellular particles other than microvesicles, i.e., have relatively little detectable reactivity with other molecules that may be present in the sample.

[0047] The ligands of the present invention may include one or more counter anions to balance the positive charge of the moiety of formula (I). The counter anions may be selected from the group consisting of perchlorate, tosylate, nitrate, sulfate, sulfonate, thiosulfate, halides, hexafluorophosphate, tetraphenylborate, carbonate, and tetrafluoroborate, in particular from perchlorate, nitrate, sulfate, halide, and carbonate anions. In some preferred embodiments, the counter anions are selected from nitrate and perchlorate.

[0048] As shown in Example 3, the ligand of formula (IIIb) (referred to in the Examples section as Cplx4) is particularly effective for capturing microvesicles while maintaining their integrity. Indeed, Figure 6 shows that captured microvesicles uniformly covered the Cplx4-functionalized surface without any significant fusion or aggregation between them, thereby maintaining the integrity of the microvesicles.

[0049] It is worth noting that the ligand of formula (IIIb) bears two moieties of formula (I).

[0050] Thus, in a preferred embodiment, the molecules of the invention comprise at least two moieties of formula (I).

[0051] For example, the ligand of the present invention may have the formula (Ia): [ka] [In the formula, M is a divalent metal cation. M is Zn 2+ , Mn 2+ , Co 2+ , Ni 2+ , Cu 2+ and Fe 2+ , preferably Zn 2+ , Fe 2+ , Co 2+ and Cu 2+ and more preferably selected from the group consisting of Zn 2+ and - [ChEn] is up to 20000 g.mol -1 of 10000, 7000, 5000, 3000 or 2000 g.mol -1[ChEn] is a chemical entity having a molecular weight of 1 to 60 carbon atoms. [ChEn] may contain acyclic and / or cyclic moieties. For example, [ChEn] may be selected from among bifunctional, trifunctional and tetrafunctional linkers having a backbone of 1 to 60 carbon atoms and at least two heteroatoms independently selected from N, S and O. Alternatively or additionally, [ChEn] may contain one or more cyclic groups (e.g., at least 2, 3, 4, 5 or 6) preferably selected from pyrrole, furan, thiophene, pyrazole, imidazole, oxazole, triazole, triazine, phenyl, naphthalene, pyridine, piperidine, pyridazine, pyrimidine, pyrazine, oxazine, dioxine, piperazine, morpholine and thiazine. The compound includes at least one portion (e.g., 1, 2, 3, or 4 portions) of

[0052] In some embodiments, "ChEn" has the formula: [ka] In such a case, the ligand of the present invention may be of formula (Ib): [ka] [In the formula, - each X1 is independently selected from S, O and NH. Preferably, all X1 are the same, preferably O; M is as defined above, preferably Zn 2+ is] Contains at least one portion of.

[0053] In another embodiment, the ligand of the present invention comprises two moieties of formula (1b). For example, the ligand of the present invention comprises a moiety of formula (Ic): [ka] [In the formula, - M is as defined above, X1 is as defined above, - each X2 is independently O, NH or S. Includes parts of.

[0054] In some embodiments, all X1 and X2 are the same, for example, S, O, or NH, preferably O.

[0055] In a particular embodiment, the ligand of the invention is a dendrimer comprising a branched core carrying multiple moieties of formula (I), preferably at least 2, 4, 6 or 8 moieties of formula (I). The dendrimer comprises from 1 to 8, preferably from 1 to 4, e.g. 1, 2 or 3 layers of molecules radiating from a central core. The outermost layer is the layer carrying the moieties of formula (I). The building blocks of the dendrimer may be selected from the group consisting of 3,5-di(hydroxymethyl)phenol, 3,5-di(thiomethyl)phenol, 3,5-di(thiomethyl)thiophenol, 3,5-dialkylphenol, 3,5-di(aminomethyl)phenol, 3,5-dialkylphenol and 3,5-di(aminomethyl)phenylamine.

[0056] In some embodiments, the dendrimer building block is 3,5-di(hydroxymethyl)phenol. Thus, the ligands of the present invention comprise the repeat unit: [ka] The branched core may be based on

[0057] As mentioned above, the molecules of the present invention find use in the detection, quantification and analysis of microvesicles.As a result, the molecules of the present invention may contain one or more additional moieties that allow the complex formed between the molecules of the present invention and the microvesicles to be removed and / or detected.For example, the molecules of the present invention may contain one or more moieties selected from the group consisting of reporter moieties and / or means for immobilization on a support.

[0058] Means for immobilization on the support are further described below (see definitions given for [IMM]). On the other hand, the reporting moiety may be selected from the group consisting of fluorescent molecules, dyes, biotin, radioactive substances, quantum dots, enzymes such as horseradish peroxidase, and the like.

[0059] In a particular embodiment, the molecules of the invention will be immobilized on a support. In that case, the molecules of the invention may further comprise a means for immobilization attached on the spacer chain. More precisely, the molecules of the invention have the formula (II): [ka] [In the formula, - n is an integer from 1 to 10, preferably from 2 to 6, more preferably 2, 3 or 4; m, p and o are independently 0 or 1; M is a divalent metal cation as defined above, - [Core] is a maximum of 25000g.mol -1 is a chemical entity having a molecular weight of - [IMM] is a means for covalent or non-covalent immobilization onto a support. - [Spacer] is selected from the group consisting of peptides, polypeptides, oligo- or polysaccharides, saturated or unsaturated hydrocarbon chains, polymers including homopolymers, copolymers and block polymers, optionally interrupted by one or more heteroatoms (e.g., S, O or NH), optionally having at least one of its termini a heteroatom such as S, O and NH, and optionally substituted by one or more substituents such as hydroxyl, halogen, C1-C3 alkoxy, -CN, -CF3 or C1-C3 alkyl, and combinations thereof. It may comprise or be of the formula:

[0060] "m is 0" means that the IMM is not present, whereas "m is 1" means that the IMM is present.

[0061] "p is 0" means that the spacer is absent, whereas "p is 1" means that the spacer is present.

[0062] "o is 0" means that the core is not present, whereas "o is 1" means that the core is present.

[0063] The molecules of the present invention have a maximum molecular weight of 25000 g.mol -1 of 10000, preferably up to 20000, for example up to 15000, 10000, 7000, 5000 or 3000 g.mol -1 The molecular weight of the compound may be 1 to 200 nm.

[0064] As mentioned above, the molecule may contain one or more counter anions to balance the positive charge of the metal complex. The counter anions are as defined above and are preferably selected from the group consisting of perchlorate, tosylate, nitrate, sulfate, sulfonate, thiosulfate, halides, hexafluorophosphate, tetraphenylborate, carbonate and tetrafluoroborate, in particular from perchlorate, nitrate, sulfate, halide and carbonate anions. In some preferred embodiments, the counter anions are selected from nitrate and perchlorate.

[0065] The core is typically at most 20000 g.mol, preferably at most 10000, 7000, 5000, 3000 or 2000 g.mol -1The core is a chemical moiety having a molecular weight of 0. The core may include acyclic and / or cyclic moieties. For example, the core may be selected from O, NH, S, and multifunctional linkers such as bifunctional, trifunctional, tetrafunctional, pentafunctional, octafunctional, and even decafunctional linkers having a backbone of 1 to 60 carbon atoms and at least two heteroatoms independently selected from N, S, and O. Alternatively or additionally, the core may include one or more cyclic groups (e.g., at least 2, 3, 4, 5, or 6) preferably selected from pyrrole, furan, thiophene, pyrazole, imidazole, oxazole, triazole, triazine, phenyl, naphthalene, pyridine, piperidine, pyridazine, pyrimidine, pyrazine, oxazine, dioxine, piperazine, morpholine, and thiazine.

[0066] In some embodiments, the [core] is composed of one or more 3,5-dimethoxyphenol moieties.

[0067] In another embodiment, the [core] is a branched core, preferably a dendritic core, made of 3.5-di(hydroxymethyl)phenol, 3,5-di(thiomethyl)phenol, 3,5-di(thiomethyl)thiophenol, 3.5-dialkylphenols, 3,5-di(aminomethyl)phenol and 3,5-di(aminomethyl)phenylamine, and all related oxo, thio and amino derivatives as building blocks.

[0068] As used herein, "combination" means that the [spacer] (hereinafter "spacer") may comprise several hydrocarbon, oligomeric or polymeric chains (e.g., 2, 3, 4, 5 or 6), linked by any suitable groups, such as -O-, -S-, -NHC(O)-, -OC(O)-, -NH-, -NH-CO-NH-, -O-CO-NH-, phosphodiester or phosphorothioate groups.

[0069] Typically, the spacer may comprise from 2 to 200 carbon atoms, preferably from 2 to 40, for example from 2 to 20 carbon atoms. The length and chemical nature of the spacer may be optimized depending on the support to which the molecule is immobilized and / or to optimize the interaction between the molecule and the microvesicles.

[0070] In some embodiments, the spacer may be selected from the group consisting of polyethers such as polyethylene glycol (PEG) or polypropylene glycol, polyesters such as polyvinyl alcohol, polylacte, polyamides such as polyacrylates polymethacrylates, polysilicones, polycaprolactones, unsaturated or saturated, branched or unbranched hydrocarbon chains, optionally having heteroatoms such as O, NH and N at at least one terminus, and combinations thereof.

[0071] In some preferred embodiments, the spacer is a C2-C aryl group optionally having a heteroatom such as O, NH, or N at at least one end. 20 It is selected from saturated or unsaturated hydrocarbon chains, polyether chains containing from 2 to 20 monomers, and combinations thereof.

[0072] For example, the spacer comprises at least one polyethylene glycol moiety comprising from 2 to 20 monomers. By way of example only, the spacer may comprise from 2 to 10 triethylene glycol blocks linked together by a linker. As another example, the spacer may comprise at least one polyethylene glycol moiety comprising from 2 to 20 monomers. 12 Alternatively, the spacer may be a hydrophilic triethylene glycol ethylamine derivative. 40 Hydrocarbon chains, especially C 10 ~C 20 C2-C alkyl chain or C6 alkyl chain 10 It may be an alkyl chain, which may have a group such as NH, S or O at at least one end.

[0073] In certain embodiments, the spacer is a linear or branched C-C20 In certain embodiments, the spacer is selected from polymers such as polyethylene glycols, polyesters or polyamides containing alkyl chains and from 2 to 10 monomers. 20 It is selected from an alkyl chain and a polyethylene glycol having from 2 to 10 monomers. The alkyl chain may have a group such as NH, S or O on at least one of its termini.

[0074] The [IMM] (hereinafter "means for immobilization") will be selected depending on the support for immobilization and the type of bond (covalent or non-covalent bond) desired. For example, the [IMM] may contain a moiety that allows non-covalent binding to the support. For example, the [IMM] may contain biotin while the support may contain streptavidin or avidin thereon, or vice versa. The [IMM] may contain a ligand moiety, such as, for example, one to six sugars, peptides, protein fragments, in particular short oligosaccharides containing antigen fragments, while the support has a binding moiety capable of specifically binding to said ligand, such as antibodies, antibody fragments, or constructs, for example as Fab, Fc fragments or ScFv, aptamers, or vice versa.

[0075] Preferably, the [IMM] comprises a chemically reactive group for immobilizing the molecule of the invention by a covalent bond on the support. The chemical reactivity is typically selected to react with the chemical entity present on the support of interest under conditions that do not alter the binding specificity of the molecule of the invention, in particular the ability of the molecule to specifically bind to the microvesicles of interest. In other words, the [IMM] and the chemical entity present on the support may be selected to react together under conditions that are unlikely to alter the structure and / or conformation of the ligand of the invention. For example, the [IMM] may comprise an amino group, preferably a primary amino group (-NH2) or a primary hydroxyl group, and the support may comprise a chemically reactive group capable of forming a covalent bond with said amino or hydroxyl group, or vice versa. Chemically reactive groups capable of forming a covalent bond with an amino or hydroxyl group include, but are not limited to, a carboxylic acid group, an activated carboxylic acid group, a lactone group, and an imidazole carbamate.

[0076] The term "activated carboxylic acid group" is intended to mean a chemical functionality derived from a "carboxylic acid" group that can react with a nucleophile, such as a primary amino group. "Activated carboxylic acid" groups are well known to those skilled in the art and include acid chlorides, mixed anhydrides, and esters.

[0077] In some embodiments, the activated carboxylic acid group is in the form of an ester. This ester may result from the reaction of the carboxylic acid group with a compound selected from 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt) and N-hydroxysuccinimide or a derivative thereof, preferably N-hydroxysuccinimide or a derivative thereof such as sulfo-NHS or N-bromosuccinimide. In a particular embodiment, the activated carboxylic acid group has the formula [ka] is the N-hydroxysuccinimidyl ester of

[0078] In some other embodiments, the [IMM] comprises a sulfhydryl group (-SH) and the chemical entity of the support comprises a chemically reactive group capable of forming a covalent bond with said sulfhydryl group, such as an iodoacetyl group, or vice versa. The resulting bond is a thioether bond.

[0079] In another embodiment, [IMM] comprises a carbonyl group (-C(=O)-H) and the chemical entity of the support comprises a chemically reactive group capable of forming a covalent bond with said carbonyl group, such as a hydrazide group, or vice versa. The resulting bond formed is a hydrazine bond.

[0080] In some embodiments, the [IMM] may comprise a chemically reactive moiety suitable for creating a covalent bond by click chemistry or by a bioconjugation reaction.

[0081] Bioconjugation reactions involve reactions between amino acids such as lysine, cysteine ​​or tyrosine and reactive groups as detailed in Koniev, O., Wagner, A, Chem. Soc. Rev., 44, 5495 (2015). For example, the functional moiety may include a maleimide group or a squalane moiety, which can react with cysteine ​​or tyrosine residues, respectively. The maleimide or squalane moiety may be present in the [IMM] while the amino acid residue may be present on the support, or vice versa. ... [ka] These include, but are not limited to:

[0082] On the other hand, "click reaction" or "click chemistry" refers to chemical reactions characterized by high yields, high chemical selectivity, which are easy to carry out and produce harmless by-products. Click reactions are typically used to create covalent heteroatom linkages (CXC) between two entities of interest. Reviews of click chemistry can be found in Kolb et al., Angew. Chem. Int. Ed. 2001, 40, 2004-2021 and Rudolf et al., Current opinion in Chemical Biology, 2013, 17:110-117.

[0083] Examples of click reactions include, but are not limited to, copper-catalyzed azide-alkyne dipolar cycloaddition (CuAAC), strain-promoted alkyne-azide cycloaddition (SPAAC), Diels-Alder reaction with tetrazine and strained alkyne or alkene, tetrazine-isonitrile cycloaddition, thiol-alkene click reactions such as maleimide-cysteine ​​cycloaddition, Staudinger azide-triarylphosphine conjugation, and sydnone-alkyne cycloaddition.

[0084] In some embodiments, the chemical reactive group of [IMM] and the chemical entity of the support can be selected to promote the free metal click reaction. A preferred free metal click reaction is strain-promoted alkyne-azide 1,3-dipolar cycloaddition (SPAAC). This reaction refers to the reaction between an azide group and a strained alkyne moiety, which leads to the formation of a triazole moiety. The azide group can be present in [IMM] and the strained alkyne moiety can be present on the support, or vice versa.

[0085] Preferred strained alkynes are C6-C aryl alkynes, in which the triple bond is sterically strained, especially in the cyclooctyne scaffold. 30 Alkynes, preferably C8-C 20The strained alkyne may comprise a cyclooctyne scaffold, optionally substituted with one or several substituents, such as halogens, and / or fused with one or several rings, including heterocycles. For example, the strained alkyne may comprise the following cyclooctyne scaffolds (a) to (f): [ka] may include one of the following:

[0086] In some other embodiments, [IMM] may contain photoreactive groups. Photoreactive groups, also called photoreactive crosslinkers, refer to chemically inert compounds that become reactive when exposed to ultraviolet or visible light. Photoreactive groups include, but are not limited to, aryl azides, azido-methyl-coumarins, benzophenones, anthraquinones, diazo compounds, diazirines, and psoralen derivatives. Examples of photoreactive groups include phenyl azides, ortho-hydroxyphenyl azides, meta-hydroxyphenyl azides, tetrafluorophenyl azides, diazirines, azido-methylcourmarins, benzophenones, and psoralens. Photoreactive groups can react with many amino acids. Thus, when [IMM] contains photoreactive groups, the chemical moieties on the support can be amino acids, and vice versa. In certain embodiments, [IMM] comprises or consists of a moiety selected from amino, preferably an amino group, -NH2, -COOH, OH, an activated carboxylic acid, -SH, an iodoacetyl group, a carbonyl, a hydrazide group, an azide, and a strained alkyne. For example, [IMM] comprises or consists of a moiety selected from -NH2-COOH and an activated carboxylic acid group, such as a hydroxy-succinimidyle ester.

[0087] In a further embodiment, the molecule of the invention has formula (II): - n is an integer from 1 to 6, preferably from 2 to 4, m and p are 1; [Spacer] and [IMM] are as defined above, - [core] is a chemical moiety made of at least one (e.g., at least 2, 3, 4, 5, or 6) 3,5-di(hydroxymethyl)phenol moiety] It is of the following.

[0088] In certain embodiments, the molecules of the invention are [ka] [In the formula, - M, m, p, [IMM] and [spacer] are as defined above for formula (II), - each X1 and X2, if present, is independently selected from the group consisting of O, NH or S. Preferably, all X1 and X2 are the same, e.g., O; - X3 is -O-, C(=O) -OC(O)-, -C(O)O-, -OC(O)O-, -S-, -SS-, -SC(O)-, -OC(S)-, NR1-, -NR1C(O)-, -C(O)NR1-, NR1C(S)-, C(S)NR1-, -OC(O)S-, -OC(S)O-, -SC(O)O-, -OC(S)S-, -SC(O)S-, -SC(S)O-, -SC(S)S-, OC(O)NR1-, -OC(S)NR1-, -NR1C(S)O-, -NR1C(O)S-, NR1C(O)NR2-, -NR1C(S)NR2-, -SC(O)S-, -SC(S)O-, -S(O)-, -S(O)2-, - -P(O)(R1)-, -P(O)(OR1)-, P(O)(R1)O-, OP(O)(OR1)-, OP(O)(R1)O-, NRP(O)(R2)-, -NRP(O)(OR2)-, NRP(O)(R2)O-, OP(O)(OR1)- and OP(O)(R1)O-, where R1 and R2 are independently H or CH3, preferably H. Preferably, X3 is O, NH, NHCO, CONH, O(C=O), (O=C)O, O(C=O)O or NHCONH. is selected from the group consisting of:

[0089] In some embodiments, the molecule of the invention is a molecule of formula (IIa), (IIb) or (IIc), m and p are 1; - M is Fe 2+ , Cu 2+ or Zn 2+ , preferably Zn 2+ and - X1 and X2 are O; X3 is O, NH, NHCO, CONH, O(C=O), (O=C)O, O(C=O)O or NHCONH, - [Spacer] is unsaturated or saturated C2-C 20 Hydrocarbon chains, optionally substituted, selected from the group consisting of polyamides, polyesters, polyethers, such as polyethylene glycols (PEG) or polypropylene glycols, polyvinyl alcohols, polyacrylates, polymethacrylates, polysilicones, unsaturated or saturated, branched or unbranched, hydrocarbon chains, and combinations thereof; - [IMM] comprises or consists of a moiety selected from an amino group, preferably -NH2, -SH, -OH, an activated carboxylic acid, preferably an N-hydroxysuccinimidyl ester, -SH, an azide, and a strained alkyne. It is.

[0090] In some embodiments, the [spacer] is selected from polyethylene glycol (PEG) or polypropylene glycol containing from 2 to 20 monomers, preferably from 3 to 10 monomers.

[0091] In a preferred embodiment, the spacer is a C2-C aryl group optionally having a heteroatom such as O, NH or S on at least one of its termini. 20 It is selected from saturated or unsaturated hydrocarbon chains, polyether chains containing from 2 to 20 monomers, and combinations thereof.

[0092] In additional embodiments, the molecule of the invention is a molecule of formula (IIa), (IIb) or (IIc), m and p are 1; - M is Fe 2+ , Cu 2+ or Zn 2+ , preferably Zn 2+ and - X1 and X2 are O; X3 is O, NH, NHCO, CONH, O(C=O), (O=C)O, O(C=O)O or NHCONH, - [IMM]-[Spacer] is NH2-(CH2) r -, where r is an integer from 2 to 10. Alternatively, [IMM]-[spacer] is or NH2-[(CH2)2-O] r -May be] It is.

[0093] The counter anions present in the molecules of formula (IIa), (IIb) and (IIc) are preferably selected from perchlorate, nitrate, sulfate, halide and carbonate.

[0094] In certain embodiments, the molecules of the invention are [ka] (also referred to herein as Cplx2) [ka] (also referred to herein as Cplx4) and [ka] (also referred to herein as Cplx8) is selected from.

[0095] Preferably, the molecules of the invention are of formula (IIIb) or (IIIc), more preferably of formula (IIIb).

[0096] It goes without saying that the counter anions shown in formulas (IIIa), (IIIb) and (IIIc) can be replaced by any other biocompatible anion, in particular from perchlorate, nitrate, sulfate, halide and carbonate anions, such as perchlorate, tosylate, nitrate, sulfate, sulfonate, thiosulfate, halide, hexafluorophosphate, tetraphenylborate, carbonate and tetrafluoroborate. In some preferred embodiments, the counter anion is selected from nitrate and perchlorate.

[0097] The molecules of the invention can be synthesized by conventional chemical reactions and can be adapted from the synthesis of molecules of formulae (IIIa), (IIIb) and (IIIc) as shown in Example 1.

[0098] Supports and devices comprising the ligands of the present invention As mentioned above, the molecules of the invention may be immobilized on a support. The invention therefore also relates to a support having thereon a ligand as defined above and to a device comprising such a support.

[0099] In other words, the present invention relates to supports functionalized with, for example grafted with, ligands as defined above. Preferred ligands are those shown in formulae (II), (IIa), (IIb), (IIc), (IIIa), (IIIb) and (IIIc). In some embodiments, the supports of the present invention comprise at least one ligand of formula (IIb) or (IIIb).

[0100] The ligands of the invention may be bound to the support either covalently or non-covalently.

[0101] The support may be of any type, provided that the ligand may be immobilized thereon, either covalently or non-covalently. The support may be pre-functionalized, e.g. chemically pretreated or functionalized, with a specific binding entity to allow for the immobilization of the ligand of the invention.

[0102] The solid support components can be of any type, including glass, metals, such as steel, gold, silver, aluminum or copper, ceramics, hydroxyapatite, silica, bentonite, polysaccharides, such as cellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, diethylaminocellulose (DEAE), dextran, cross-linked dextran agarose, cross-linked agarose, starch, alginates, chitosan and derivatives thereof, plastics and polymers, such as polyethylene, polypropylene, polyamides, polyvinylidene fluoride, polyacrylamide, and the like. Examples of such substrates include amides, polyesters, for example, polyethylene terephthalate (PET), polyglycolic acid (PLA), polycaprolactone (PCL), polyethylene adipate (PEA), polybutylene terephthalate (PBT), or poly(3-hydroxybutyrate-co-3-hydroxyvalerate), commonly known as PHBV, acrylic acid based polymers and copolymers and derivatives thereof, polyamides, polystyrene (PS), organopolysiloxanes, polyacrylates, polyvinylpolyacrilin, derivatives or combinations thereof. Such substrates are commercially available.

[0103] In some embodiments, the support is a polymeric support, preferably a support made of polyvinyl chloride (PVC), polystyrene (PS) or polyethylene terephthalate (PET).

[0104] By way of example only, when the ligands of the invention contain primary amine groups as a means for immobilization, the support can be, for example, a polystyrene support pre-functionalized with N-oxysuccinimide (NOS) or pre-functionalized PET. PET can be pre-functionalized by a treatment that includes hydrolysis of ester groups present on the surface of the support, increasing the COOH surface density by oxidation (e.g., using potassium permanganate) and activation of the COOH groups, for example, using N-hydroxysuccinimide as shown in Example 3.

[0105] A device comprising such a support may be of any type and in any form, for example the device may be a film, a strip, a sheet, a chip, a microchip, a sensor, a reactor, e.g. a microreactor, a microfluidic chamber or channel, a cartridge, a plate or a microplate, a chromatography column resin or gel, a support for a lateral flow immunochromatographic assay, a support for an ELISA type assay, a filtration membrane, a filter, a bead such as a polymeric bead or a magnetic bead.

[0106] Methods and uses according to the invention Typically, the molecules, supports and devices of the invention can be used to capture, immobilize, detect, quantitate and / or analyze microvesicles present in a sample. More generally, the invention relates to the use of the molecules, supports and devices as described above to capture microvesicles present in a sample for in vitro diagnosis of a pathological condition in a subject.

[0107] In certain aspects, the present invention provides a method for capturing microvesicles from a sample, comprising the steps of: - providing a support or a molecule of the invention; - contacting said support or said molecule with a sample susceptible to containing the microvesicles of interest under conditions suitable for the formation of a complex between the molecule of the invention and the microvesicles, whereby the microvesicles are captured; The present invention relates to a method comprising the steps of:

[0108] A sample may be any composition potentially containing microvesicles of interest. Typically, a sample may be or may be derived from a bodily fluid. Bodily fluids are typically removed from a human, preferably a human suffering from or at risk of developing a disease as further described below.

[0109] As used herein, the term "biological fluid" refers to any extractable or removable bodily fluid, including, for example, blood, plasma, cerebrospinal fluid, bronchoalveolar lavage fluid, urine, synovial fluid, breast milk, saliva, tears, semen, peritoneal fluid, amniotic fluid, and exudates (pleural or otherwise). In the context of the present invention, preferred biological fluids are blood, plasma, and urine.

[0110] As used herein, "a sample is derived from a body fluid" means that the sample is obtained from said body fluid by subjecting said body fluid to one or more processing steps, for example to remove contaminants and / or separate microvesicles from certain components of said body fluid. For example, the body fluid may be subjected to one or more processes such as a precipitation process, for example salting-out precipitation, cryoprecipitation or flocculation, a filtration process, for example depth filtration or ultrafiltration, centrifugation, clarification, chromatography, an extraction process, for example liquid-liquid or solid-liquid extraction, viral inactivation, pasteurization, concentration, dialysis, freeze / thaw process, etc.

[0111] The sample typically contains contaminants from which the microvesicles of interest are to be isolated. The contaminants may be of any type and will depend on the nature of the starting composition. Contaminants include proteins, salts, hormones, vitamins, nutrients, lipids, cells, cellular debris such as cell membrane fragments, etc.

[0112] In some embodiments, the methods of the invention are performed on untreated bodily fluids, preferably on untreated urine or plasmatic blood.

[0113] In some embodiments, the method comprises: - preparing a sample from a bodily fluid of a subject which is likely to contain microvesicles of interest; and / or - washing the support after the step of contacting the support with the sample, for example to remove contaminants; and / or - detecting the formation of complexes between the microvesicles of interest and said support or molecule of the invention, and / or - recovering the complexes formed between the microvesicles and the molecules of the invention, and / or - quantification of said microvesicles of interest, and / or - detecting said microvesicles, and / or - characterizing the captured microvesicles, for example by detecting and / or quantifying biomarkers present on the surface or within the microvesicles; and / or - releasing the microvesicles from the complex; and / or - recovering the desired microvesicles; The method may further comprise one or more steps selected from the following:

[0114] As mentioned above, the molecules, supports and devices of the invention find use in in vitro diagnostics.

[0115] Indeed, the detection, quantification and / or characterization of microvesicles in a sample may be useful in the context of the diagnosis and prognosis of disease in a patient: the levels of microvesicles in bodily fluids and their composition in terms of lipids, proteins and genetic material such as mRNA and mitochondrial RNA are indicative of the pathological state of the subject.

[0116] Besides, the determination of the microvesicle proteome may make it possible to determine the physiological and / or pathological state of the cells from which they originate and may therefore represent a tool of choice for the early detection of pathological conditions.

[0117] Thus, detection, quantification and / or characterization of captured microvesicles may enable the diagnosis of a medical condition, the assessment of the risk of developing a medical condition, the prognosis of a medical condition, the differential diagnosis of a medical condition, tracking the evolution of a medical condition, and / or monitoring the efficacy of a treatment in a patient.

[0118] Thus, in a further aspect, the present invention relates to the use of a molecule, support or device of the invention in an in vitro method for the diagnosis, differential diagnosis, prognosis, risk assessment and / or monitoring of the evolution of a disorder in a subject.

[0119] The present invention also relates to the use of a molecule, a support or a device according to the invention in an in vitro method for monitoring the efficacy of a therapeutic treatment in a patient.

[0120] The disease or disorder of interest may be of any type, including thrombotic, inflammatory and / or metabolic disorders, as well as cardiovascular or neurovascular diseases. For example, disorders of interest include diabetes and related disorders such as diabetic nephropathy, diabetic neuropathy, diabetic retinopathy and diabetic foot syndrome, multiple sclerosis, cancer, Alzheimer's disease, Parkinson's disease, aneurysm, cerebrovascular spasm, stroke, coronary artery disease, parasitic diseases, cancer, and other conditions.

[0121] In certain embodiments, the present invention provides an in vitro method for the diagnosis, differential diagnosis, prognosis, risk assessment, and / or monitoring of a disorder in a subject, comprising: (a) providing a support or molecule of the present invention; (b) contacting said support or said molecule with a sample obtained from a subject under conditions suitable for the formation of complexes between the molecules of the invention and microvesicles possibly present in the sample; (c) detecting, quantifying and / or characterizing the microvesicles captured in step (b) by complex formation; The present invention relates to an in vitro method comprising:

[0122] As mentioned above, preferred disorders include thrombotic, inflammatory and / or metabolic disorders, and cardiovascular or neurovascular diseases. In a preferred embodiment, the disorder is selected from the group consisting of diabetes and related disorders such as diabetic nephropathy, diabetic neuropathy, diabetic retinopathy and diabetic foot syndrome, multiple sclerosis, cancer, Alzheimer's disease, Parkinson's disease, aneurysm, cerebrovascular spasm, stroke, coronary artery disease, parasitic diseases, cancer, and other pathologies.

[0123] The method may further comprise the step of comparing the results obtained in step (c) with those obtained under similar conditions from one or more control samples.

[0124] The control sample may be obtained from a healthy subject or from a subject suffering from the disorder to be diagnosed. Alternatively, the sample is from the same subject but obtained earlier. Comparison with earlier samples from the same subject may allow to monitor the evolution of the disease and / or the efficacy of treatment and / or to predict the outcome of the disease or treatment in the subject.

[0125] Detection, quantification and / or characterization of microvesicles may be performed by standard methods known to those of skill in the art.

[0126] As used herein, "characterization" of a microvesicle refers to the detection, determination of the presence or absence, and / or quantification of at least one biomarker that is likely to be present in the microvesicle and that correlates with the disorder being diagnosed.

[0127] A biomarker can be any molecule of interest, such as a protein, lipid, hormone, mitochondrial DNA, miRNA or mRNA, etc. Such biomarkers can be detected and / or quantified by methods well known to those of skill in the art.

[0128] For example, proteins can be detected and quantified by standard immunological assays, such as ELISA or Western blot techniques. Nucleic acid detection and characterization can include specific nucleic acid detection methods such as PCR / qPCR, RT-PCR / RT-qPCR and sequencing.

[0129] In some embodiments, the method comprises a step of normalization of the results obtained in step (c). For example, said normalization may be performed based on the quantification of markers present on or in the microvesicles, such as Annexin-A5 and beta-actin.

[0130] Thus, in certain embodiments, step (c) of the method comprises: - quantifying the biomarker of interest in the captured microvesicles; - quantifying the normalization biomarker in the captured microvesicles; - normalizing the amount detected for the biomarker by the amount obtained for the normalization biomarker (e.g., by calculating the ratio of the amount of the biomarker to that of the normalization biomarker); - optionally comparing the obtained normalized amount of the biomarker of interest with that obtained under similar conditions for a control sample; may include.

[0131] Comparison with normalized amounts obtained for a control sample, e.g., a sample obtained from a healthy subject or a sample obtained from a subject suffering from the disorder to be diagnosed, may allow one to determine a diagnosis or prognosis of the disease in the subject.

[0132] It will be appreciated that in the methods of the invention, the biomarkers for detecting or quantitating microvesicles will be selected depending on the disorder of interest.

[0133] For illustration purposes only: For the diagnosis, particularly the early diagnosis, of nephropathy in a subject, the biomarker may be podocalyxin. - For the diagnosis of Parkinson's disease, particularly for early diagnosis, the biomarker may be alpha-synuclein.

[0134] - a kit according to the invention The invention also relates to kits comprising the molecules, supports or devices according to the invention. Such kits are typically specialized for at least partially implementing the methods of the invention, such as methods for capturing microvesicles from a sample and / or in vitro methods for diagnosis, differential diagnosis, prognosis, risk assessment and / or monitoring of a disorder in a subject.

[0135] Typically, the kit comprises a molecule of the invention, e.g. as shown in any one of formula II, (IIa), (IIb), (IIc), (IIIa), (IIIb) or (IIIc), or a support or device having said molecule thereon as described above, and at least one additional means useful for implementing the method of the invention.

[0136] For example, the at least one additional means may include: - means for preparing the sample to be analysed, e.g. means for pre-treating a body fluid obtained from a subject; - buffers for carrying out the method of the invention, such as suspension buffers, washing buffers, elution buffers (for example for dissociation of the complex form between the microvesicles and the molecules of the invention), - a means for detection of captured microvesicles; - means for characterization of the captured microvesicles, e.g. means for detecting and / or quantifying at least one biomarker possibly present in the microvesicles; - means for detecting and / or quantifying at least one normalizing biomarker in the captured microvesicles, such as Annexin V or beta-actin; - a control sample for normalization of the biomarkers quantified in the microvesicles; - Control samples, such as positive or negative control samples, for validation purposes can be selected from:

[0137] As used herein, "means for detection or quantification of a biomarker" refers to any means known by a person skilled in the art for detecting or quantifying said biomarker. Said means depends on the nature of the biomarker. For example, protein biomarkers can be detected / quantified by immunological techniques (especially ELISA and Western blot), while nucleic acid biomarkers can be detected by specific amplification techniques that can be qualitative or quantitative.

[0138] Moreover, kits according to the invention may contain instructions providing a user with instructions for utilizing the kit to implement methods according to the invention.

[0139] Further aspects and advantages of the present invention are disclosed in the following experimental section, which should be considered as illustrative and not limiting the scope of the present application. EXAMPLES

[0140] Example 1 Synthesis of Ligands According to the Invention A. Synthesis of tert-butyl (6-iodohexyl)carbamate (intermediate)

[0141] [ka]

[0142] tert-Butyl (6-hydroxyhexyl)carbamate: 3.72 g (17 mmol) of di-tert-butyl dicarbonate was added to a solution of 2 g (17 mmol) of 6-amino-1-hexanol in 40 mL of tetrahydrofuran with stirring at 0° C. The reacting mixture was allowed to warm slowly to room temperature. After 24 h, the solvent was evaporated. The residue was dissolved in 50 mL of dichloromethane and washed three times with 50 mL of water. The organic phase was collected and dried over sodium sulfate, and the solvent was then removed on a rotary evaporator. The product was a colorless oil with a yield of 91%. 1 H NMR (CDCl3, 300 MHz, 25℃): δ(ppm) 3.60 (t, 2H), 3.08 (t, 2H), 1.59-1.28 (m, 17H).

[0143] tert-Butyl (6-bromohexyl) carbamate: To a solution of 1 g (4.6 mmol) of tert-butyl (6-hydroxyhexyl) carbamate and 2.1 g (8.1 mmol) of triphenylphosphine in 12 ml of tetrahydrofuran, a solution of 2.7 g (8.1 mmol) of tetrabromomethane in 8 mL of tetrahydrofuran was added dropwise with stirring at 0° C. The reacting mixture was allowed to warm slowly to room temperature. After 24 hours of reaction, the solvent was removed. The residue was added to a solvent mixture of 50 mL of petroleum ether and 10 mL of ethyl acetate. A precipitate formed instantly and was then filtered off while the filtrate was collected and evaporated to leave a colorless oil. The oil was passed through a silica flash column using an eluent of petroleum ether:ethyl acetate=10:1. The solvent was removed on a rotary evaporator. The product was a colorless oil with a yield of 93%. 1 H NMR (CDCl3, 300 MHz, 25℃): δ(ppm) 3.44 (t, 2H), 3.14 (t, 2H), 1.91-1.86 (m, 2H), 1.52-1.33 (m, 15H).

[0144] tert-Butyl (6-iodohexyl)carbamate: 1.5 g (5.4 mmol) of tert-butyl (6-bromohexyl)carbamate was dissolved in 15 mL of acetonitrile. 4.02 g (26.8 mmol) of sodium iodide was added to the solution. The mixture was stirred at room temperature and kept in the dark for 48 hours. The solvent was removed under vacuum and the residue was extracted with 50 mL of dichloromethane and 50 mL of water. The organic phase was washed with a saturated solution of sodium thiosulfate and then dried over sodium sulfate. The solvent was removed on a rotary evaporator. The product was a colorless oil with a yield of 96%. 1 H NMR (CDCl3, 300 MHz, 25℃): δ(ppm) 3.21 (t, 2H), 1.90-1.80 (m, 2H), 1.56-1.30 (m, 15H).

[0145] B. Synthesis of the complex of formula (IIId) (Comparative - Cplxl)

[0146] [ka]

[0147] 4-(Chloromethyl)phenylpropionate: 30 mL of propionyl chloride was cooled to 0° C. while stirring. 5.1 g (41 mmol) of 4-(hydroxymethyl)phenol was slowly added to the flask to control the evolution of hydrogen chloride at a moderate rate. After the addition, the mixture was allowed to warm slowly to room temperature. After 12 hours, the mixture was poured into 500 mL of ice-cold water and then neutralized with sodium bicarbonate. The mixture was then extracted three times with 50 mL of diethyl ether. The organic phases were combined and washed three times with 30 mL of water. The solution was then dried over sodium sulfate and the solvent was removed using a rotary evaporator. The product was a pale yellow oil with a yield of 97%. 1 H NMR (CDCl3, 300 MHz, 25℃): δ(ppm) 7.42 (d, 2H), 7.12 (d, 2H), 4.60 (s, 2H), 2.62 (q, 2H), 1.30 (t, 3H).

[0148] Ligand 1-OH: 199 mg (1 mmol) of 4-(chloromethyl)phenylpropionate and 199 mg (1 mmol) of di-(2-picolyl)amine were dissolved in 10 mL of dimethylsulfoxide with stirring. After adding 414.6 mg (3 mmol) of potassium carbonate, the mixture was then heated to 60° C. After 24 hours, the reaction was cooled to room temperature and then a solution of 0.6 g of potassium carbonate in 3 mL of water was added at room temperature. After another 24 hours, the reaction mixture was dissolved in 30 mL of dichloromethane and then washed with water (30 mL, 3 times) and dried over sodium sulfate. The solvent was removed under vacuum to give the product as a white solid. The yield was 74%. 1H NMR (DMSO, 300 MHz, 25℃): δ(ppm) 9.31 (s, 1H), 8.51-8.49 (m, 2H), 7.83-7.77 (m, 2H), 7.58 (d, 2H), 7.28-7.24 (m, 2H), 7.20 (d, 2H), 6.73 (d, 2H), 3.68 (s, 4H), 3.51 (s, 2H); 13 C NMR (DMSO, 75MHz, 25℃): δ(ppm) 159.80, 156.87, 149.26, 137.08, 130.38, 128.98, 122.87, 122.59, 115.48, 59.33, 57.35.

[0149] Ligand 1-Boc: 305 mg (1 mmol) of Ligand 1-OH was dissolved in 4 mL of dimethylformamide with stirring. 112 mg (2 mmol) of potassium hydroxide was added to the solution at room temperature. After 1 h, the mixture was cooled to -20°C, and then 392 mg (1.2 mmol) of tert-butyl (6-iodohexyl) carbamate was added to the mixture. The reaction was allowed to warm slowly to room temperature. After 3 h, the reaction was quenched with water. The mixture was extracted three times with 20 mL of dichloromethane, and then the combined organic phase was washed twice with 20 mL of brine and dried over sodium sulfate. The solvent was removed under vacuum to give a brown oil. The crude product was then purified by silica gel column chromatography using an eluent of ethyl acetate:acetonitrile (5:1). The product is obtained as a pale yellow solid in 75% yield. 1 H NMR (MeOD, 300 MHz, 25℃): δ(ppm) 8.43 (m, 2H), 7.86-7.71 (m, 2H), 7.70 (d, 2H), 7.32-7.28 (m, 4H), 6.89 (d, 2H), 3.99 (t, 2H), 3.77 (s, 4H), 3.61 (s, 2H), 3.06 (t, 2H), 1.81-1.76 (m, 2H), 1.54-1.44 (m, 15H); 13C NMR (MeOD, 75MHz, 25℃): δ(ppm) 159.34, 154.18, 147.94, 137.34, 129.86, 123.88, 123.32, 122.39, 113.99, 67.45, 66.43, 59.12, 57.81, 28.94, 27.39, 25.48.

[0150] Ligand 1-NH2: 802.2 mg (1.6 mmol) of ligand 1-Boc was dissolved in 15 mL of tetrahydrofuran. Trifluoroacetic acid was then slowly added into the solution at room temperature. Five minutes after the addition of trifluoroacetic acid, the solution was heated to 60°C. After 30 hours, the solution was cooled to room temperature and then poured into ice-cold sodium bicarbonate solution. The product was extracted with dichloromethane (30 mL) under vacuum, and the compound was obtained in 85% yield. 1 H NMR (MeOD, 300 MHz, 25℃): δ(ppm) 8.54 (d, 2H), 7.71-7.66 (m, 2H), 7.60 (d, 2H), 7.19-7.14 (m, 2H), 6.87 (d, 2H), 3.96 (t, 2H), 3.82 (s, 4H), 3.64 (s, 2H), 2.73 (t, 2H), 1.83-1.77 (m, 2H), 1.53-1.41 (m, 6H); 13 C NMR (MeOD, 75MHz, 25℃): δ(ppm) 159.94, 158.25, 148.96, 136.44, 130.69, 130.06, 122.81, 121.94, 114.31, 67.86, 59.84, 57.88, 41.88, 33.24, 29.27, 26.66, 25.94;

[0151] Complex of formula (IIId) - Comparative - also referred to herein as Cplx1

[0152] [ka]

[0153] 108 mg (0.27 mmol) of the ligand 1-NH2 was dissolved in 10 mL of methanol with stirring at room temperature. 79.4 mg (0.27 mmol) of zinc nitrate hexahydrate was dissolved in 5 mL of methanol and added dropwise to the solution of the ligand. After 12 h, the solvent was removed under vacuum to quantitatively obtain the final product as a pale yellow powder (100% yield). 1 H NMR (DMSO, 300 MHz, 25℃): δ(ppm) 8.69 (d, 2H), 8.12 (t, 2H), 7.66 (m, 4H), 7.30 (d, 2H), 7.02 (d, 2H), 4.25 (d, 2H), 4.02 (t, 2H), 3.71 (t, 4H), 2.80 (t, 2H), 1.78-1.73 (m, 2H), 1.60-1.56 (m, 2H), 1.48-1.39 (m, 6H); 13 C NMR (MeOD, 75MHz, 25℃): δ(ppm) 159.34, 154.81, 148.40, 141.26, 133.32, 125.33, 125.19, 123.98, 114.90, 67.88, 56.40, 55.68, 29.22, 29.01, 28.09, 26.10;

[0154] C. Synthesis of the complex of formula (IIIa) (hereinafter, Cplx2)

[0155] [ka]

[0156] 5-Hydroxyphenyl-1,3-dimethanol: To a dispersion of 1.3 g (34.3 mmol) of lithium aluminum hydride in 40 mL of anhydrous tetrahydrofuran at 0° C., a solution of 3 g (14.3 mmol) of dimethyl 5-hydroxyisophthalate in 60 mL of anhydrous tetrahydrofuran was added dropwise. The reaction was allowed to warm to room temperature during 3 hours. After 12 hours, the mixture was cooled again to 0° C. and quenched with 10% hydrochloric acid. The solvent was removed under reduced pressure. The residue was diluted in 50 mL of brine and the product was extracted from the aqueous solution (6×50 mL) using ethyl acetate. The organic layers were combined, dried over sodium sulfate, and the solvent was removed under vacuum. The product is obtained as a white solid in 94% yield. 1 H NMR (CDCl3, 300 MHz, 25°C): δ(ppm) 6.69 (s, 1H), 6.61 (s, 2H), 4.41 (s, 4H). 3.5-Bis(bromomethyl)phenol

[0157] 3.5-Bis(bromomethyl)phenol: 8.2 mL of 33% hydrogen bromide solution was added dropwise to a solution of 2 g (13 mmol) of 5-hydroxyphenyl)-1,3-dimethanol in 20 mL of acetic acid at 0° C. The mixture was extracted with 3×70 mL of dichloromethane. The combined organic phases were washed with 2×100 mL of water, then with 2×100 mL of saturated sodium bicarbonate solution and again with 100 mL of water. The organic phase was dried over sodium sulfate, filtered and concentrated. The crude product was purified by flash column silica chrommtography using a mixture of petroleum ether:ethyl acetate (9:1) to give a white solid (90% yield). 1 H NMR (CDCl3, 300 MHz, 25℃): δ(ppm) 3.21 (t, 2H), 1.90-1.80(m, 2H), 1.56-1.30 (m, 15H).

[0158] Ligand 2-OH: 672 mg (2.4 mmol) of 3,5-bis(bromomethyl)phenol, 1196 mg (6 mmol) of N,N-dipyricolylamine and 398 mg (2.88 mmol) of potassium carbonate were added in 10 mL of dimethylformamide under nitrogen protection. The mixture was stirred at room temperature. After 3 hours, 50 mL of water was added to the mixture and extracted with 2×50 mL of dichloromethane. The organic layers were combined, washed with 2×50 mL of water, dried over sodium sulfate, filtered and evaporated using a rotary evaporator. The residue was purified using silica gel column chromatography to obtain the final product as a white solid (yield 80%). 1 H NMR (CDCl3, 300 MHz, 25℃): δ(ppm) 8.48-8.46 (m, 4H), 7.60-7.58 (m, 8H), 7.11 (q, 4H), 6.96 (s, 1H), 6.86 (s, 2H), 3.79 (s, 8H), 3.57 (s, 4H);

[0159] Ligand 2-Boc: 520 mg (1 mmol) of Ligand 2-OH was dissolved in 4 mL of dimethylformamide with stirring. 120 mg (2 mmol) of potassium hydroxide was added to the solution at room temperature. After 1 h, the mixture was cooled to -20°C, and then 400 mg (1.2 mmol) of tert-butyl (6-iodohexyl) carbamate was added to the mixture. The reaction was allowed to warm slowly to room temperature. After 3 h, the reaction was quenched with water. The mixture was extracted three times with 20 mL of dichloromethane, and then the combined organic phase was washed twice with 20 mL of brine and dried over sodium sulfate. The solvent was removed under vacuum to give a brown oil. The crude product was then purified by silica gel column chromatography using an eluent of ethyl acetate:acetonitrile (5:1). The product is a pale yellow solid in 75% yield. 1H NMR (DMSO, 300 MHz, 25℃): δ(ppm) 8.43 (d, 4H), 7.79 (ddd, 4H), 7.28 (tt, 4H), 7.04 (s, 1H), 3.97 (t, 2H), 3.80 (s, 8H), 3.65 (s, 4H), 3.07 (t, 2H), 1.79 (t, 2H), 1.58-1.48 (m, 15H);

[0160] Ligand 2-NH2: 1350 mg (1.89 mmol) of Ligand 2-Boc was dissolved in 15 mL of tetrahydrofuran. Then, 7.2 mL (94.3 mmol) of trifluoroacetic acid was slowly added into the solution at room temperature. 5 minutes after the addition of trifluoroacetic acid, the solution was heated to 60°C. After 30 hours, the solution was cooled to room temperature and then poured into ice-cold sodium bicarbonate solution. The product was extracted with dichloromethane (3 x 30 mL). The organic phase was then dried over sodium sulfate and the solvent was removed using a rotary evaporator. The yield was 85%. 1 H NMR (DMSO, 300 MHz, 25℃): δ(ppm) 8.49 (d, 4H), 7.74 (ddd, 4H), 7.57 (d, 4H), 7.25 (dd, 4H), 7.07 (s, 1H), 6.82 (s, 2H), 3.93 (t, 2H), 3.71 (s, 8H), 3.60 (s, 4H), 3.32 (b, 4H), 1.69 (t, 2H), 1.42-1.34 (m, 6H); 13 C NMR (MeOD, 75MHz, 25℃): δ(ppm) 159.79, 159.20, 148.97, 140.49, 136.42, 122.76, 121.95, 121.48, 113.56, 67.85, 60.09, 58.63, 41.31, 29.07, 26.81, 26.58, 26.13.

[0161] Complex of formula (IIIa) - (hereinafter, Cplx2)

[0162] [ka]

[0163] 1110 mg (1.8 mmol) of the ligand 2-NH2 was dissolved in 10 mL of methanol at room temperature with stirring. 1072 mg (3.6 mmol) of zinc nitrate hexahydrate was dissolved in 5 mL of methanol and added dropwise to the solution of the ligand. After 12 hours, the solvent in the mixture was removed under vacuum to give the final product as a pale yellow powder (100% yield). 1 H NMR (DMSO, 300 MHz, 25℃): δ(ppm) 8.71 (b, 4H), 8.11 (t, 4H), 7.67 (t, 4H), 7.60 (d, 4H), 7.01 (b, 2H), 6.89 (s, 1H), 4.35 (d, 2H), 4.07 (b, 2H), 3.81 (d, 8H), 2.84 (t, 2H), 1.79 (b, 2H), 1.65-1.46 (m, 6H); 13 C NMR (MeOD, 75MHz, 25℃): δ(ppm) 162.85, 159.27, 155.04, 148.50, 141.37, 134.32, 127.12, 125.32, 117.96, 68.03, 57.51, 56.15, 39.86, 36.26, 31.24, 29.05, 27.79, 26.11, 25.62.

[0164] D. Synthesis of 2-(trimethylsilyl)ethyl(6-iodohexyl)carbamate (intermediate)

[0165] [ka]

[0166] 2-(Trimethylsilyl)ethyl (6-hydroxyhexyl)carbamate: 352 mg (3 mmol) of 6-amino-1-hexanol was dissolved in 5 mL of dichloromethane. 0.9 mL (6 mmol) of triethylamine was added to the solution, then 850 mg (3 mmol) of 4-nitrophenyl 2-(trimethylsilyl)ethyl carbonate in 1.4 mL of dichloromethane was also added to the mixture with stirring at room temperature. After 24 h, the solvent was evaporated. The residue was dissolved in 50 mL of dichloromethane and washed with 3 x 50 mL of saturated NaHCO3 solution, then with 3 x 50 mL of NAOH (2 M) solution. The organic phase was collected, dried over sodium sulfate and the solvent was removed on a rotary evaporator. The product is obtained as a colorless oil in 98% yield. 1 H NMR (CDCl3, 300 MHz, 25℃): δ(ppm) 4.64 (b, 1H), 4.17 (t, 2H), 3.67 (t, 2H), 3.20 (q, 2H), 1.68-1.34 (m, 8H), 1.00 (t, 2H), 0.07 (s, 9H);

[0167] 2-(Trimethylsilyl)ethyl(6-bromohexyl)carbamate: To a solution of 748 mg (2.86 mmol) of 2-(trimethylsilyl)ethyl(6-hydroxyhexyl)carbamate and 1311 mg (5 mmol) of triphenylphosphine in 12 mL of tetrahydrofuran, a solution of 1.66 g (5 mmol) of tetrabromomethane in 8 mL of tetrahydrofuran was added dropwise with stirring at 0° C. The reacting mixture was allowed to warm slowly to room temperature. After 24 hours of reaction, the solvent was removed. The residue was added to a solvent mixture of 50 mL of petroleum ether and 10 mL of ethyl acetate. A precipitate formed instantly and was filtered off. The filtrate was collected and evaporated to give a colorless oil. The oil was passed through a flash chromatography column of silica gel using an eluent of petroleum ether:ethyl acetate (10:1). The solvent was removed under vacuum and a colorless oil was obtained in 84% yield. 1H NMR (CDCl3, 300 MHz, 25℃): δ(ppm) 4.61 (b, 1H), 4.18 (t, 2H), 3.43 (t, 2H), 3.19 (q, 2H), 1.94-1.84 (m, 2H), 1.62-1.34 (m, 6H), 1.00 (t, 2H), 0.07 (s, 9H);

[0168] 2-(Trimethylsilyl)ethyl(6-iodohexyl)carbamate: 846 mg (2.61 mmol) of 2-(trimethylsilyl)ethyl(6-bromohexyl)carbamate was dissolved in 15 mL of acetonitrile. 2 g (13 mmol) of sodium iodide was added into the solution. The mixture was stirred at room temperature and kept in the dark for 48 hours. The solvent was removed under vacuum and the residue was extracted with 50 mL of dichloromethane and 50 mL of water. The organic phase was washed with a saturated solution of sodium thiosulfate and then dried over sodium sulfate. The solvent was removed under vacuum and the product was obtained as a colorless oil in 96% yield. 1 H NMR (CDCl3, 300 MHz, 25℃): δ(ppm) 4.61 (b, 1H), 4.18 (t, 2H), 3.24-3.16 (m, 4H), 1.89-1.80 (m, 2H), 1.58-1.33 (m, 6H), 1.00 (t, 2H), 0.07 (s, 9H);

[0169] E. Synthesis of the complex of formula (IIIb) (hereinafter, Cplx4)

[0170] [ka]

[0171] 3.5-Bis(iodomethyl)phenylpropionate: 1.15 mL (13 mmol) of propionyl chloride was dissolved in 30 mL of dichloromethane. At 0° C., 2.8 g (10 mmol) of 3,5-bis(bromomethyl)phenol dissolved in 10 mL of dichloromethane was added to the solution with stirring. After 5 min, 3 mL (13 mmol) of triethylamine in 10 mL of dichloromethane was also added to the solution and the cold bath was removed. 2 h after the addition of triethylamine, the reaction was quenched with 10 mL of water. The mixture was extracted twice with 50 mL of dichloromethane, the organic phases were combined and washed first with 15 mL of saturated sodium bicarbonate solution and then with 15 mL of brine. The organic phase was dried over sodium sulfate and then the solvent was removed under vacuum. The product was dissolved in 15 mL of acetonitrile. 7.5 g (50 mmol) of sodium iodide was added into the solution. The mixture was stirred at room temperature and kept in the dark for 48 hours. The solvent was removed under vacuum and the residue was extracted between 50 mL of dichloromethane and 50 mL of water. After washing the organic phase with a saturated solution of sodium thiosulfate, the organic phase was collected and dried over sodium sulfate. The solvent was removed on a rotary evaporator. The product is a colorless solid (96%). 1 H NMR (CDCl3, 300 MHz, 25℃): δ(ppm) 7.27 (t, 1H), 7.04 (d, 2H), 4.41 (s, 4H), 2.62 (q, 2H), 1.30 (t, 3H);

[0172] Ligand 4-OH: 1284 mg (2.5 mmol) of ligand 2-OH was dissolved in 4 mL of dimethylformamide with stirring. 126 mg (2.5 mmol) of potassium hydroxide was added to the solution at room temperature. After 1 h, the mixture was cooled to -20°C, and then 520 mg (1.2 mmol) of 3,5-bis(iodomethyl)phenylpropionate was added to the mixture. The reaction was allowed to warm slowly to room temperature. After 3 h, the reaction was quenched with water. The mixture was extracted three times with 20 mL of dichloromethane, and then the combined organic phase was washed with 2 x 20 mL of brine and dried over sodium sulfate. The solvent was removed on a rotary evaporator to give a brown oil. The crude product was then purified by silica gel column chromatography using an eluent of ethyl acetate:acetonitrile = (5:1). The product is a pale yellow solid. The yield was 75%. 1 H NMR (DMSO, 300 MHz, 25℃): δ(ppm) 8.47 (tt, 8H), 7.71 (ddd, 8H), 7.54-7.51 (m, 8H), 7.20 (qq, 8H), 7.06 (s, 2H), 6.96 (s, 1H), 6.90 (s, 4H), 6.83 (s, 2H), 5.01(s, 4H), 3.70 (s, 16H), 3.57 (s, 8H); 13 C NMR (CDCl3, 75MHz, 25℃): δ(ppm) 159.68, 158.63, 157.79, 148.87, 140.61, 139.88, 138.99, 136.64, 122.84, 122.49, 122.05, 117.89, 114.94, 115.18, 69.96, 59.87, 58.33.

[0173] Ligand 4-Teoc: 930 mg (0.8 mmol) of Ligand 4-OH was dissolved in 10 mL of dimethylformamide with stirring. 90.5 mg (1.6 mmol) of potassium hydroxide was added to the solution at room temperature. After 1 h, the mixture was cooled to -20°C, and then 360 mg (0.97 mmol) of 2-(trimethylsilyl)ethyl(6-iodohexyl)carbamate was added into the mixture. The reaction was allowed to warm slowly to room temperature. After 3 h, the reaction was quenched with water. The mixture was extracted with 3 x 20 mL of dichloromethane, and then the combined organic phase was washed with 2 x 20 mL of brine and dried over sodium sulfate. The solvent was removed on a rotary evaporator to give a brown oil. The crude product was then purified by silica gel column chromatography using an eluent of ethyl acetate:acetonitrile (5:1). The product is a pale yellow solid. The yield was 75%. 1 H NMR (CDCl3, 300 MHz, 25℃): δ(ppm) 8.54-8.51 (m, 8H), 7.65-7.55 (m, 16H), 7.16-7.11 (m, 11H), 7.00-6.98 (m, 6H), 5.05 (s, 4H), 4.17 (t, 2H), 3.97 (t, 2H), 3.82 (s, 16H), 3.68(s, 8H), 3.18 (q, 2H), 1.83-1.72 (m, 2H), 1.54-1.34 (m, 6H), 0.99 (t, 2H), 0.06 (s, 9H); 13 C NMR (CDCl3, 75MHz, 25℃): δ(ppm) 162.55, 159.75, 159.67, 159.02, 148.99, 140.73, 139.09, 136.42, 122.73, 121.96, 121.88, 118.52, 113.82, 113.11, 69.91, 67.92, 60.10, 58.57, 40.86, 36.49, 30.94, 30.05, 29.19, 26.54, 25.79, 17.80, -1.44;

[0174] Ligand 4-NH2: 900 mg (0.65 mmol) of ligand 4-Teoc was dissolved in 10 mL of tetrahydrofuran with stirring. Then 6.5 mL (6.5 mmol) of 1 M tetrabutylammonium fluoride solution was added at room temperature. The reaction mixture was then kept at 60° C. overnight. After cooling to room temperature, the solvent was removed in vacuum. The residue was dissolved in dichloromethane and then washed once with 50 mL of 0.5 M NaOH solution and twice with 50 mL of saturated NaHCO3 solution. The organic phase was dried over sodium sulfate. The solvent was removed on a rotary evaporator to give a brown solid in 90% yield. 1 H NMR (DMSO, 300 MHz, 25℃): δ(ppm) 8.48-8.45 (t, 8H), 7.70 (ddd, 8H), 7.51 (d, 8H), 7.22 (ddd, 8H), 7.11 (s, 1H), 7.06 (s, 2H), 6.97 (s, 2H), 6.92 (s, 4H), 5.07 (s, 4H), 3.89 (t, 2H), 3.69 (s, 16H), 3.57 (s, 8H), 1.61 (t, 2H), 1.37-1.19 (m, 6H); 13 C NMR (DMSO, 75MHz, 25℃): δ(ppm) 159.61, 159.29, 158.79, 149.79, 149.26, 140.72, 139.49, 136.95, 122.81, 122.57, 121.69, 114.10, 113.30, 69.48, 59.66, 57.94, 55.39, 41.52, 32.60, 29.05, 26.48, 25.80.

[0175] 1) Complex of formula IIIb - (hereinafter, Cplx4)

[0176] [ka]

[0177] 125 mg (0.1 mmol) of the ligand 4-NH2 was dissolved in 10 mL of methanol with stirring at room temperature. 119 mg (0.4 mmol) of zinc nitrate hexahydrate was dissolved in 5 mL of methanol and added dropwise to the solution of the ligand. After 12 hours, the solvent in the mixture was removed under vacuum to quantitatively obtain the final product as a brown powder (100% yield). 1 H NMR (DMSO, 300 MHz, 25℃): δ(ppm) 8.70 (d, 8H), 8.10 (t, 8H), 7.67 (t, 8H), 7.58 (d, 8H), 7.27 (s, 1H), 7.17 (s, 4H), 7.06 (s, 1H), 6.96 (s, 2H), 5.27 (s, 4H), 4.38 (d, 8H), 4.10 (q, 2H), 3.83 (t, 16H), 2.81 (t, 2H), 2.22 (t, 2H), 1.55 (t, 2H), 1.44-1.32 (m, 4H); 13 C NMR (DMSO, 75MHz, 25℃): δ(ppm) 159.44, 159.08, 154.85, 148.45, 141.27, 139.20, 134.29, 127.73, 125.32, 125.13, 118.35, 114.09, 69.86, 68.05, 57.42, 56.14, 49.09, 29.27, 26.05, 25.55, 20.34, 19.72;

[0178] F. Synthesis of the complex of formula (IIIc) (hereinafter, Cplx8)

[0179] [ka]

[0180] Ligand 8-OH: 1151 mg (1 mmol) of ligand 4-OH was dissolved in 4 mL of dimethylformamide with stirring. 126 mg (2.25 mmol) of potassium hydroxide was added to the solution at room temperature. After 1 h, the mixture was cooled to -20°C, and then 220 mg (0.51 mmol) of 3,5-bis(iodomethyl)phenylpropionate was added to the mixture. The reaction was allowed to warm slowly to room temperature. After 3 h, the reaction was quenched with water. The mixture was extracted with 3 x 20 mL of dichloromethane, and the combined organic phase was washed with 2 x 20 mL of brine and dried over sodium sulfate. The solvent was removed on a rotary evaporator to give a brown oil. The crude product was then purified by silica column chromatography using an eluent of ethyl acetate:acetonitrile = 5:1. The product is obtained as a pale yellow solid in 75% yield. 1 H NMR (DMSO, 300 MHz, 25℃): δ(ppm) 8.48-8.44 (m, 16H), 7.74-7.64 (m, 16H), 7.54-7.51 (m, 16H), 7.22-7.14 (m, 16H), 7.07 (d, 6H), 6.96 (s, 1H), 6.90 (d, 8H), 6.83 (s, 2H), 6.79 (s, 1H), 6.75 (s, 1H), 6.71 (s, 1H), 6.66 (s, 1H), 5.05-4.97(m, 12H), 3.69 (d, 32H), 3.57 (d, 16H); 13 C NMR (CDCl3, 75MHz, 25℃): δ(ppm) 159.68, 158.63, 157.79, 148.87, 140.61, 139.88, 138.99, 136.64, 122.84, 122.49, 122.05, 117.89, 114.94, 115.18, 69.96, 59.87, 58.33.

[0181] Ligand 8-Teoc: 605 mg (0.25 mmol) of Ligand 8-OH was dissolved in 10 mL of dimethylformamide with stirring. 30 mg (0.54 mmol) of potassium hydroxide was added to the solution at room temperature. After 1 h, the mixture was cooled to -20°C, and then 95 mg (0.256 mmol) of 2-(trimethylsilyl)ethyl(6-iodohexyl)carbamate was added to the mixture. The reaction was allowed to warm slowly to room temperature. After 3 h, the reaction was quenched with water. The mixture was extracted with 3 x 20 mL of dichloromethane, and the combined organic phase was washed with 2 x 20 mL of brine and dried over sodium sulfate. The solvent was removed on a rotary evaporator to give a brown oil. The crude product was then purified by silica gel column chromatography using an eluent of ethyl acetate:acetonitrile (5:1). The product is a pale yellow solid obtained in 73% yield. 1 H NMR (DMSO, 300 MHz, 25℃): δ(ppm) 8.47-8.43 (m, 16H), 7.72-7.63 (m, 16H), 7.53-7.47 (m, 16H), 7.23-7.13 (m, 16H), 7.00 (b, 2H), 6.93-6.88 (m, 9H), 6.81 (s, 1H), 6.78 (s, 1H), 5.13-4.99 (m, 12H), 4.05-3.98 (m, 2H), 3.67 (d, 32H), 3.55 (d, 16H), 1.71-1.58 (m, 2H), 1.43-1.20 (m, 8H), 0.94-0.84 (m, 2H), 0.00(s, 9H); 13 C NMR (CDCl3, 75MHz, 25℃): δ(ppm) 162.55, 159.75, 159.67, 159.02, 148.99, 140.73, 139.09, 136.42, 122.73, 121.96, 121.88, 118.52, 113.82, 113.11, 69.91, 67.92, 60.10, 58.57, 40.86, 36.49, 30.94, 30.05, 29.19, 26.54, 25.79, 17.80, -1.44;

[0182] Ligand 8-NH2: 400 mg (0.15 mmol) of ligand 8-Teoc was dissolved in 10 mL of tetrahydrofuran with stirring. Then 1.5 mL of 1 M tetrabutylammonium fluoride solution was added at room temperature. The reaction mixture was then kept at 60° C. overnight. After cooling to room temperature, the solvent was removed in vacuum. The residue was dissolved in dichloromethane and then washed once with 50 mL of 0.5 M NaOH solution and twice with 50 mL of saturated NaHCO3 solution. The organic phase was dried over sodium sulfate. The solvent was removed on a rotary evaporator to give a brown solid. The yield was 90%. 1 H NMR (DMSO, 300 MHz, 25℃): δ(ppm) 8.45-8.43 (m, 16H), 7.73-7.58 (m, 16H), 7.56-7.45 (m, 16H), 7.24-7.11 (m, 18H), 7.10-6.97 (m, 10H), 6.93-6.88 (m, 9H), 5.05-4.99 (m, 12H), 3.91-3.84 (m, 2H), 3.67 (b, 32H), 3.55 (b, 16H), 1.64-1.56 (m, 2H), 1.37-1.23 (m, 8H); 13 C NMR (DMSO, 75MHz, 25℃): δ(ppm) 159.61, 159.29, 158.79, 149.79, 149.26, 140.72, 139.49, 136.95, 122.81, 122.57, 121.69, 114.10, 113.30, 69.48, 59.66, 57.94, 55.39, 41.52, 32.60, 29.05, 26.48, 25.80;

[0183] Complex 8-NH2 (molecule of formula IIIc - hereafter Cplx8):

[0184] [ka]

[0185] 250 mg (0.1 mmol) of the ligand 8-NH2 was dissolved in 10 mL of methanol with stirring at room temperature. 30 mg (1 mmol) of zinc nitrate hexahydrate was dissolved in 5 mL of methanol and added dropwise to the solution of the ligand. After 12 h, the solvent was removed under vacuum to give the final product as a brown powder (100% yield). 1 H NMR (DMSO, 300 MHz, 25℃): δ(ppm) 8.45 (b, 16H), 7.67 (b, 16H), 7.51 (b, 16H), 7.20 (b, 18H), 7.07 (b, 9H), 6.90 (b, 10H), 5.05 (b, 12H), 3.90 (b, 2H), 3.67 (b, 32H), 3.56 (b, 16H), 1.58 (b, 2H), 1.25 (b, 8H); 13 C NMR (DMSO, 75MHz, 25℃): δ(ppm) 159.44, 159.08, 154.85, 148.45, 141.27, 139.20, 134.29, 127.73, 125.32, 125.13, 118.35, 114.09, 69.86, 68.05, 57.42, 56.14, 49.09, 29.27, 26.05, 25.55, 20.34, 19.72.

[0186] Example 2 Ligand-PS-enriched microvesicle interactions studied by PWR spectroscopy The detailed configuration of the PWR and lipid bilayer structure used herein is described in Calmet et al. (Sci Rep, 2016. 6:36181).

[0187] After the formation of a POPC:POPS = 5:1 (w / w) bilayer on the prism surface, titration experiments are performed for each complex. Briefly, each complex was dissolved in PBS buffer and then titrated into a Teflon sample chamber. Once equilibrium was reached for each addition, the complex concentration and the spectral shift of s-polarized light (parallel to the membrane surface) were recorded. The titration was stopped when the addition of the complex solution did not induce any further change in the spectral position. The complex concentration was then plotted against the resonance angle and the plot was fitted using GraghPad Prism 5. The obtained K d The values ​​are shown below in Table 1. Figure 1 shows the titration curves for each of the ligands tested.

[0188] [Table 1]

[0189] Example 3 Immobilization and characterization of ligands on PET films Immobilization of the ligand of the present invention 10cm 2 A PET film of was first cleaned with ethanol and then immersed in hydrolysis solution (20 mL water, 20 mL acetonitrile and 0.2 g NaOH). Hydrolysis was kept at 60°C for 18 hours. The film was cleaned with water and then immersed in oxidation solution (38.4 ml fresh Milli-Q water, 1.6 ml H2SO4, 2 g KMnO4). Oxidation was kept at 60°C for 1 hour. The oxidized film was first washed once with 50% HCl and then three times with water. The oxidized film was immersed in activation solution (MES hydrate 390.5 mg / 2 mmol, ethylcarbodiimide hydrochloride (EDC) 766.8 mg / 4 mmol, N-hydroxysuccinimide (NHS) 230.18 mg / 2 mmol) and then kept at room temperature for 1 hour. The film was washed with water. The activated film was then immersed in a 1 mM solution of the complex in 20 mL of DMSO at room temperature. After 24 h, the film was removed from the solution and washed extensively with water.

[0190] - TBO characterization The functionalization of the PET surface is always accompanied by a change in the carboxyl group density on the material surface; hydrolysis of ester bonds and oxidation of alcohol groups increase the carboxyl density, while EDC / NHS activation and immobilization of complexes continuously decrease the carboxyl density. The absolute amount of complexes immobilized on the PET surface is difficult to determine using established analytical methods, whereas the change in carboxyl group density can be easily determined by the Toluidine Blue-o (TBO) adsorption test. Thus, the TBO test can provide an indirect assessment of the complex density on the PET surface. Briefly, the carboxyl groups on the material surface were first deprotonated in a basic medium, followed by adsorption of TBO cations. The areal density of carboxyl groups could be determined by measuring the amount of adsorbed TBO molecules, provided that TBO reacts with the carboxyl groups in a 1:1 stoichiometry. The adsorbed TBO molecules can be easily washed off with acid, and their concentration in acid can be determined by UV-Vis absorption.

[0191] The TBO test was performed according to a reported procedure (Chollet, C. et al., Biomol Eng, 2007. 24(5): 477-82).

[0192] First, a linear correlation between TBO concentration in 50% AcOH and the absorbance of the solution at 633 nm (200 μL of solution in each well of a clear 96-well plate) was established using a series of TBO solutions shown in Tab. 3. Future TBO testing will involve 1 cm TBO using 5 mL of 50% acetic acid. 2 This would remove the TBO dye adsorbed onto the squares and 200 μL of each solution would be added to each individual well of a clear 96-well plate for absorbance testing. Therefore, the TBO concentrations in Table 2 can then be further converted to molecular density on the PET surface. The plot was then fitted linearly as shown in Figure 2.

[0193] [Table 2]

[0194] After obtaining the calibration curve, the TBO test was performed: 5 x 10 of Toluidine Blue-O solution -4 The M solution was prepared by first dissolving NaOH in Milli-Q water to pH 10, then dissolving Toluidine Blue-o to reach the desired concentration. A 1 cm x 1 cm PET square was immersed in 10 mL of TBO solution and then kept shaking at room temperature in the dark. After 6 hours, the supernatant was removed from the tube and the stained PET surface was washed once with 10 mL of NaOH solution (pH 10) and 2 x 10 mL of pure water. The stained film was then immersed in 5 mL of 50% acetic acid to remove the adsorbed TBO dye for 10 minutes. 200 μL of the washed solution from each sample was loaded into a 96-well plate and the absorbance at 633 nm was measured with a 96-well plate reader. The absorbance was then converted to areal density of carboxyl groups and summarized in Figure 3 using the calibration curve shown in Figure 4. After the activation step, the COOH density of 43.06 pmol / mm 2 resulting from the replacement of hydrogen at COOH by NHS was 1.03 pmol / mm 2 . 2 After the complexes are grafted onto the surface, a further drop in COOH density can be observed. This drop can result from two events: first, there is a reaction of amines and COOH in the presence of the complexes, which further eliminates carboxyl groups on the PET surface; second, when grafted onto the PET surface, the positively charged complexes repel the TBO+ ions, preventing them from adsorbing onto the PET.

[0195] - X-ray photoelectron spectroscopy (XPS) A VG Scientific ESCALAB photoelectron spectrometer was used for surface analysis with a 100W non-monochromated MgK1253.6 eV source. The area of ​​the analytical X-ray spot on the sample surface is about 250 microns. We used an insertion angle of 45 degrees, which corresponds to an analysis depth of 3-5 nm. A flood gun was used for charge compensation. Acquisition of high-resolution spectra was performed with a constant pass energy of 20 eV. Fitting was then realized with software provided by VG Scientific, and each spectrum was referenced to a carbon contamination at 284.8 eV. Binding energy values ​​are listed with an accuracy of ±0.2 eV.

[0196] [Table 3]

[0197] The peak areas after fitting of the high-resolution spectra are summarized in Tab. 4 (Table 4). The XPS results correspond well to each treatment of the PET surface: after the hydrolysis and oxidation steps, the ester bonds were hydrolyzed and then oxidized to carboxyl groups; therefore, the highest value of the COO signal is observed among all samples. In the activation step, the NHS molecules replaced the hydrogen of the carboxyl groups. In the process, the number of surface carboxyl groups remains the same, while NHS transfers nitrogen to the surface, leading to a significant increase in the N1s and NC=O signals and an unchanged -COO signal intensity. After functionalization with the complex, several changes in the XPS spectrum can be observed. First, the appearance of Zn indicates the successful attachment of the complex onto the material surface. Second, the significant increase in C1s NC=O / C1s COO and N1s 399.9 / O1s O=C can be interpreted as the successful replacement of NHS by the complex, further confirming the covalent attachment of the complex to the PET surface. Thirdly, comparing the Zn content among the grated surfaces, Cplx1 has the highest abundance on the surface, while the other complexes have more and more difficulty in binding to the material surface, whereas the comparison of the N1s 399.9 signal shows that Cplx1, Cplx2 and Cplx4 should have almost the same abundance on the material surface, and Cplx8 had the highest abundance on the material surface. The two conclusions seem to cause arguments against each other, but taking into account solubility and stability can explain them: Cplx1, Cplx2 and Cplx4 are water-soluble, and therefore non-covalently bound molecules can be easily washed away in the washing process, while Cplx8, on the other hand, is insoluble in water, and the adsorbed molecules would be much more difficult to remove by water. During washing, water behaves as a complexing agent, removing the Zn(II) ions from the complexes, leaving only the ligand on the surface. This effect may become more pronounced with increasing dendritic branching as the Zn(II) cations move closer to each other in space and experience more repulsive forces from each other.

[0198] Regarding the impurities appearing in the XPS spectrum, Si most likely originates from the glass container as we processed all the PET sheets, S is from the manufacturing of the material as its concentration is relatively stable throughout the implantation procedure, and finally, Cl in the oxidized and activated surface originates from the hydrochloric acid used in the surface cleaning of the PET.

[0199] Example 4 Microvesicle capture and characterization using cryo-SEM - Microvesicle preparation Microvesicles were collected from mesenchymal stem cells. After 6 passages, TNFα was introduced into the incubation medium at a concentration of 100ng / ml. After 36 hours, the supernatant was collected and then purified in three steps: 1) Removal of cell debris: The incubation supernatant was centrifuged at 1500g at 4°C for 15 minutes, the supernatant was removed and centrifuged at 13000g at 4°C for another 2 minutes. 2) Concentration of microvesicles: The supernatant was again removed and centrifuged at 20000g at 4°C for 90 minutes, then the pellet was removed. 3) Washing of microvesicles: The pellet was redispersed in 500μL of 1×PBS at 4°C and centrifuged at 20000g at 4°C for 90 minutes. Remove 400 μL of the supernatant without disturbing the pellet, then add another 400 μL of fresh 1×PBS at 4° C., redisperse again, and centrifuge at 20,000 g for 90 min at 4° C. Remove 450 μL of the supernatant without disturbing the pellet, then add 50 μL of 1×PBS at 4° C. to redisperse the pellet. When not in use, the microvesicles were stored at −80° C.

[0200] - Microvesicle staining The microvesicle stock was warmed to room temperature, and then 950 μL of 1×PBS was used to dilute the suspension. 1 μL of CellMask™ Deep Red plasma membrane stain was added to the diluted suspension to stain the microvesicles at room temperature. After 15 minutes, the stained suspension was centrifuged at 20000 g for 90 minutes at 4° C. Then, 950 μL of the supernatant was removed without disturbing the pellet, and another 950 μL of fresh PBS was added to redisperse the pellet. The centrifugation-redispersion step was repeated twice to remove free CellMask™ Deep Red molecules from the PBS buffer.

[0201] - Microvesicle characterization by fluorescence microscopy The stained microvesicles were visible under a fluorescence microscope (Leica microsystem DM5500B, microscope with motorized programmable stage using a CoolSnap HQ camera controlled by Metamorph 7.6). Fluorescent dots of different sizes were observed. The dots with larger diameters were suspected to be aggregates of microvesicles, while the smaller ones should be monodispersed microvesicles. Apparently, it was not possible to perform a morphological study of the microvesicles, since their diameters were usually below the resolution of such instruments.

[0202] - Microvesicle characterization using Nanoparticle Tracking Analysis (NTA): NTA experiments of the prepared microvesicles were carried out using a NanoSight NS300 instrument. The analysis was carried out at 22° C. using a 532 nm laser beam as the light source. The instrument was calibrated using a standard nanoparticle dispersion provided by the manufacturer prior to testing.

[0203] For testing, the microvesicle stock was warmed to room temperature and then the suspension was diluted to 1 mL using 1×PBS. The suspension was then vortexed to achieve an even distribution of the microvesicles within the diluent.

[0204] NTA experiments were performed by five 30 second video recordings of the microvesicle dispersion flowing through the sample chamber at a syringe pump speed of 70 (AU).

[0205] The images were simultaneously analyzed by the software NTA3.2 development build 3.2.16, where the microvesicle concentration and size distribution per image frame were recorded.

[0206] The concentration of microvesicles in the five images was 1.87 × 10 8 ±4.69×10 6 ml -1 The homogeneity of the microvesicle suspension was evidenced by the small standard deviation across the five images. Multiple subpopulations of particles of different sizes were found in the microvesicle suspension. The mean diameter of all particles was 139.9±1.1 nm, which is in good agreement with previously published results, while the mean diameter of 2.45×10 5 Only a small population of exosomes was found with a size below 50 nm, and these were suspected to be exosomes.

[0207] - Microvesicle capture The same procedure was used to capture microvesicles for all materials: Stock microvesicle (stained with CellMask™ Deep Red for FL microscopy and unstained for cryo-scanning electron microscopy) dispersions were diluted to 2 mL with 1×PBS. 250 μL of the dispersion was added to a functionalized 1 cm2 PET square and incubated at room temperature for 15 min. The PET squares were then washed with Milli-Q water to remove any free vesicles attached to the surface.

[0208] - Observation of trapped microvesicles using FL microscopy After the capture process, fluorescence microscopy of the PET sheets (Leica microsystem DM5500B, microscope with a motorized programmable stage using a CoolSnap HQ camera controlled by Metamorph 7.6) was used to evaluate the capture ability of PET functionalized with different conjugates.

[0209] - Observation of trapped microvesicles using cryo-scanning electron microscopy (cryo-SEM) After the capture process, the PET sheets were attached to the freezing stubs of the preparation chamber Quorum PP3000T specimen shuttle. The whole was pressed into slush nitrogen paste for freezing. After a quick transfer in the preparation chamber under vacuum, the samples were sublimated at -95 °C for 30 min and then coated by platinum sputtering. They were finally transferred into the cryo-SEM Quanta 250FEG chamber and kept at -140 °C with an accelerating voltage of 10 kV for observation.

[0210] - results Figure 4-Cplx1 Figure 4 shows how microvesicles responded to the Cplx1-grafted surface. In the FL micrograph (A), a small amount of donut-shaped red fluorescent spots can be observed. These structures were around 1 μm in size, much larger than the diameter of the microvesicles. In the cryo-SEM image (B), the PET surface is blank and has a smooth morphology. No vesicle structures can be observed. Both experiments imply that the PET surface functionalized with Cplx1 has a very limited ability to capture microvesicles.

[0211] Figure 5-Cplx2 Microvesicle capture using Cplx2-functionalized PET is shown in FIG. 5A. The entire material surface was covered with membrane structures, but non-uniformity was observed across the surface. Large areas of the surface were covered with fluorescent membranes, but areas with less fluorescence also had clear dots of microvesicle size. Very clear membrane aggregates of irregular shapes with sizes over 1 μm could also be observed. FIG. 5B and FIG. 5C illustrate the surface of the same material observed using cryo-SEM. Most of the PET surface was seen as shown in FIG. 8B. While the PET was covered by coalesced membrane structures, at the boundaries of the aggregates holes in the membrane structures and a small amount of vesicles bound to the PET surface could be observed.

[0212] Cplx2-functionalized surfaces were able to capture microvesicles. When in contact with the functionalized surface, the vesicles tend to fuse with each other into membranes. The fused membranes form spider web-like structures in less enriched areas and large aggregates in highly enriched areas. Although the interaction between PS and Cplx2 was strong enough to capture the vesicles, the morphology of the microvesicles was disrupted. It was suspected that the contents within the vesicles were lost during the capture process.

[0213] Figure 6-Cplx4 As shown in Figures 6A and 6B, both FL micrographs and cryo-SEM show that microvesicles uniformly cover the entire surface functionalized with cplx4. After magnification (Figure 9C), microvesicles appear individually attached to the material surface, while no obvious membrane fusion was observed. Cplx4-functionalized surfaces are also capable of capturing microvesicles. With minimal vesicle-vesicle interactions, both vesicle morphology and contents within the microvesicles should be well preserved.

[0214] Figure 7-Cplx8 Both the FL micrographs and cryo-SEM in Figures 7A and 7B show that the particles uniformly cover the entire Cplx8-functionalized surface. In the enlarged cryo-SEM image (C), the particles are found to be the same size as microvesicles but irregular in shape.

[0215] Both FL micrographs and cryo-SEM confirmed that the cplx8-functionalized surface was able to capture particles in microvesicle buffer, and the fluorescence clearly shows that the particles were composed of phospholipids. Magnified cryo-SEM also shows that the particles were of the same size as microvesicles, without the vesicular structure expected from microvesicles. Individual microvesicles were captured by the surface, but the vesicle membrane was disrupted by the capture, leaving only the membrane on the surface, while the vesicle contents were lost.

[0216] Example 5 NMR investigation of DPA-Zn complex-PS interactions The zinc complex-PS interaction was studied in solution to preliminarily evaluate the binding ability of the complex to PS. For this purpose, we employed NMR studies in which peak shape and chemical shifts could be used as evaluation criteria.

[0217] Since the chemical environment of the phosphorus atom in 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) would be most susceptible if the complex binds to the phosphate of POPS, 31P NMR spectroscopy was used to examine the effect on POPS when it binds to the complex. Given the similarity of the binding events (DPA-Zn to POPS), the change in chemical shift can also roughly represent the strength of such interaction (in our study, the observed chemical shift is assumed to be the mole fraction weighted average of the shifts of free and DPA-Zn bound POPS). Indeed, when one equivalent of different complexes is added to one equivalent of POPS in solution, the change in 31P NMR chemical shift varies significantly. Cplx1, Cplx2 and Cplx4 give a signal shift at high field (from 0.381 ppm for free POPS to -1.48 ppm, -2.00 ppm and -2.88 ppm for Cplx1, Cplx2 and Cplx4, respectively), while Cplx8 shows a signal at low field (0.65, 2.57, 3.70 and 6.29). From Cplx1 to Cplx4, the change in chemical shift also increases with increasing molecular dendritic structure, indicating stronger binding ability with more binding sites. With increasing number of DPA-Zn, one obvious difference is the dendritic scaffold to which DPA-Zn is attached. To investigate the effect of molecular scaffold, another experiment was performed, in which the molar ratio of DPA-Zn units to POPS was fixed at 1:1. The resulting 31P NMR spectra (data not shown) show changes that are in full agreement with the assumption that the DPA-Zn complex binds to phosphate groups on POPS, whereas Cplx4 has the strongest ability to bind POPS.

Claims

1. An in vitro method for capturing microvesicles present in a sample, the method comprising the step of contacting the sample with a ligand capable of binding to microvesicles, the ligand having formula (Ia) as follows: 【Chemistry 1】 [In the formula, - each X 1 is independently O, S or NH; - M is Zn 2+ , Co 2+ , Cu 2+ and Fe 2+ Select from carrying at least one structure of In vitro methods.

2. The ligand is represented by the formula (Ib) 【Chemistry 2】 [In the formula, - each X 1 is independently O, S or NH; - each X 2 is independently O, S or NH; - M is Zn 2+ , Co 2+ , Cu 2+ and Fe 2+ Select from The in vitro method of claim 1, wherein the structure of the

3. 3. The in vitro method of claim 1 or 2, wherein the ligand is a dendrimer having a branched core carrying multiple structures of formula (Ia).

4. 4. The in vitro method of claim 3, wherein the dendrimer is composed of building blocks selected from the group consisting of 3,5-di(hydroxymethyl)phenol, 3,5-di(thiomethyl)phenol, 3,5-di(thiomethyl)thiophenol, 3,5-dialkylphenols, 3,5-di(aminomethyl)phenol, 3,5-dialkylphenols and 3,5-di(aminomethyl)phenylamines.

5. 5. An in vitro method according to any one of claims 1 to 4, wherein the ligand further comprises a means for immobilisation on a support attached at the end of a spacer chain.

6. The ligand is represented by formula (IIb) or (IIc) 【Chemistry 3】 [In the formula, m and p are independently 0 or 1; - M is Zn 2+ , Co 2+ , Cu 2+ and Fe 2+ is selected from - each X 1 and X 2 is independently selected from the group consisting of O, NH, and S; -X 3 -O-, C(=O) -OC(O)-, -C(O)O-, -OC(O)O-, -S-, -SS-, -SC(O)-, -OC(S)-, NR 1 -, -NR 1 C(O)-, -C(O)NR 1 -, NR 1 C(S)-, C(S)NR 1 -, -OC(O)S-, -OC(S)O-, -SC(O)O-, -OC(S)S-, -SC(O)S-, -SC(S)O-, -SC(S)S-, OC(O)NR 1 -, -OC(S)NR 1 -, -NR 1 C(S)O-, -NR 1 C(O)S-, NR 1 C(O)NR 2 -, -NR 1 C(S)NR 2 -, -SC(O)S-, -SC(S)O-, -S(O)-, -S(O) 2 -,- -P(O)(R 1 )-, -P(O)(OR1)-, P(O)(R 1 )O-, OP(O)(OR 1 )-, OP(O)(R 1 )O-, NR 1 P(O)(R 2 )-, -NR 1 P(O)(OR 2 )-, NR 1 P(O)(R 2 )O-, OP(O)(OR 1 )- and OP(O)(R 1 )O-, where R 1 and R 2 are independently H or CH 3 and - [IMM] is a means for covalent or non-covalent immobilization on a support, - [Spacer] is Peptides, Polypeptides, Oligo- or polysaccharides, - saturated or unsaturated, substituted or unsubstituted hydrocarbon chains, optionally interrupted by one or more heteroatoms, and ○ Combinations of these selected from the group consisting of The in vitro method according to claim 5,

7. The ligand has the following features: - m and p are 1; and / or - [Spacer] is: ○ Saturated or unsaturated, substituted or unsubstituted, C, which may have a heteroatom on at least one of its termini 2 ~C 20 Hydrocarbon chains; o Polyamide chains containing from 2 to 20 monomers; o Polyester chains containing from 2 to 10 monomers; ○ Polyether chains containing from 2 to 20 monomers, and ○ Combinations of these and / or - [IMM] carries a moiety selected from the group consisting of an amino group, OH, -COOH, an activated carboxylic acid, -SH, an iodoacetyl group, a carbonyl, a hydrazide group, an azide, and a strained alkyne; 7. The in vitro method of claim 6, further characterized by one, several or all of the following:

8. In the ligand of formula (IIb) or (IIc), m and p are 1, -X 1 and X 2 is O, -X 3 is O, NH, NHCO, CONH, O(C═O), (O═C)O, O(C═O)O or NHCONH, - [IMM]-[spacer] is NH 2 -(CH 2 ) r - or NH 2 -[(CH 2 ) 2 -O] r where r is an integer from 2 to 10, - the ligand comprises one or more counter anions selected from the group consisting of perchlorate, tosylate, nitrate, sulfate, sulfonate, thiosulfate, halide, hexafluorophosphate, tetraphenylborate, carbonate and tetrafluoroborate, 8. The in vitro method of claim 7.

9. The ligand is a compound of formula (IIIb) and (IIIc): 【Chemistry 4】 9. The in vitro method of claim 8, wherein the method is selected from the group consisting of:

10. 10. The in vitro method according to claim 1, wherein the ligand is immobilized on a support.

11. - providing a sample from a body fluid of a subject; and / or - washing the support on which the ligand is immobilized after the step of contacting the ligand with the sample, and / or - detecting the formation of complexes between the microvesicles of interest present in the sample and the ligand, and / or - recovering the complexes formed between the microvesicles and the ligand present in the sample; and / or - quantification of said microvesicles; and / or - characterizing the captured microvesicles, and / or - releasing the microvesicles from the complexes, and / or - recovering the microvesicles.

11. The in vitro method according to claim 1, further comprising at least one step selected from the group consisting of:

12. 1. An in vitro method comprising: (a) providing a sample from a subject; (b) capturing the microvesicles present in the sample by carrying out a method according to any one of claims 1 to 11; and (c) detecting and / or quantifying the microvesicles captured in step (b) by complex formation. Including, The in vitro method is for aiding in the diagnosis, differential diagnosis, prognosis, risk assessment, and / or monitoring of a disorder in a subject; In vitro methods.

13. - the disorder is selected from the group consisting of diabetes and related disorders, multiple sclerosis, cancer, Alzheimer's disease, Parkinson's disease, aneurysm, cerebrovascular spasm, stroke, and coronary artery disease; and / or - the sample is or is derived from blood, plasma and urine, 13. The in vitro method of claim 12.

14. The compound may be associated with a microvesicle having the formula (1a): 【Chemistry 5】 [In the formula, - each X 1 is independently O, S or NH; - M is Zn 2+ , Co 2+ , Cu 2+ and Fe 2+ Select from A molecule having at least one of the structures

15. Formula (Ib) 【Chemistry 6】 [In the formula, - each X 1 is independently O, S or NH; - each X 2 is independently O, S or NH; - M is Zn 2+ , Co 2+ , Cu 2+ and Fe 2+ Select from 15. The molecule of claim 14, having the structure:

16. 16. The molecule of claim 14 or 15, which is a dendrimer having a branched core carrying multiple structures of formula (Ia).

17. 17. The molecule of claim 16, wherein the branched core comprises as building blocks groups selected from 3,5-di(hydroxymethyl)phenol, 3,5-di(thiomethyl)phenol, 3,5-di(thiomethyl)thiophenol, 3,5-dialkylphenol, 3,5-di(aminomethyl)phenol, 3,5-dialkylphenol and 3,5-di(aminomethyl)phenylamine.

18. 18. The molecule of any one of claims 14 to 17, further comprising a means for immobilization on a support attached at the end of the spacer chain.

19. Formula (IIb) or (IIc): 【Chemistry 7】 [In the formula, M, m, p, [IMM], [spacer], X 1 , X 2 and X 3 is as defined in claim 6] 20. The molecule of claim 18 having the structure:

20. In the ligand of formula (IIb) or (IIc), m and p are 1, - M is Zn 2+ , Co 2+ , Cu 2+ or Fe 2+ and -X 1 and X 2 is O, -X 3 is O, NH, NHCO, CONH, O(C═O), (O═C)O, O(C═O)O or NHCONH, - [IMM]-[spacer] is NH 2 -(CH 2 ) r - or NH 2 -[(CH 2 ) 2 -O] r where r is an integer from 2 to 10, - the molecule has one or more counter anions selected from the group consisting of perchlorate, tosylate, nitrate, sulfate, sulfonate, thiosulfate, halide, hexafluorophosphate, tetraphenylborate, carbonate, and tetrafluoroborate; 20. The molecule of claim 19.

21. Formulae (IIIb) and (IIIc): 【Chemistry 8】 21. The molecule of claim 20 having a structure selected from:

22. 22. A support having thereon a molecule as defined in any one of claims 14 to 21.

23. 24. Use of a molecule as defined in any one of claims 14 to 21 or a support as defined in claim 22 as a means for capturing microvesicles from a sample of a subject.

24. 22. A kit comprising a molecule as defined in any one of claims 14 to 21 for use in the diagnosis, prognosis or monitoring of a disease in a subject, wherein the disease is selected from cancer, parasitic diseases, diabetes and related disorders including diabetic nephropathy, diabetic neuropathy, diabetic retinopathy and diabetic foot syndrome, multiple sclerosis, cancer, Alzheimer's disease, Parkinson's disease, aneurysm, cerebral vasospasm, stroke and coronary artery disease.

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