Functionalized nanoparticles (NPS)

Abstract

The present invention relates to the field of medicine, in particular to functionalized nanoparticles (NPs) to be used in diagnosis, to pharmaceutical compositions or nanodevices that comprise them, and also to the method for obtaining said functionalized nanoparticles.

Description

[0001] Functionalized nanoparticles (NPs)

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention relates to the field of medicine, in particular to functionalized nanoparticles (NPs) to be used in diagnosis, to pharmaceutical compositions or nanodevices that comprise them, and also to the method for obtaining said functionalized nanoparticles.

[0004] BACKGROUND ART

[0005] In recent years, extracellular vesicles (EVs), and particularly a subpopulation called exosomes, have gained prominence in the field of biomarkers and diagnostics. All living cells secrete these membrane-derived nanoscopic vesicles; EVs are naturally occurring particles surrounded by a lipid bilayer that carry transmembrane proteins on their surface, as well as other proteins and nucleic acids in their lumen, such as DNA, mRNA, mRNA, and other small RNAs. These vesicles are key in intercellular communication, transferring complex molecular information from a cell of origin to other cells throughout the body, associated with a variety of physiological functions and pathological disease states.

[0006] Living cells secrete heterogeneous populations of EVs, including exosomes, microvesicles, and apoptotic bodies. Exosomes are the best-known type of EVs and are formed by the folding of intracellular membranes into endosomes that generate intraluminal vesicles (ILVs) and are secreted after the fusion of multivesicular bodies (MVBs) with the plasma membrane. Due to their intraluminal origin, these particles are usually between 50 and 150 nm in size. Microvesicles are formed by budding and direct fission of the plasma membrane to the outside, and usually have a size between 100- 1000nm. Apoptotic bodies are formed during cell apoptosis and contain multiple cell fragments and DNA, giving them heterogeneous characteristics in terms of size, morphology, and content. Due to the cellular processes associated with the biogenesis of EVs, different content can be selectively incorporated into each type of EV. To distinguish EVs derived from plasma membrane or intracellular membrane compartments, tetraspanins such as CD63, CD81 and CD9 are frequently used. CD63 has been suggested to be a key component for EVs originating from the plasma membrane, as it is largely found on exosomes along with other tetraspanins. Therefore, tetraspanin CD63 has been used to identify exosomes in many studies.

[0007] Exosomes are secreted around the cells of origin and released into biofluids, such as blood, urine, cerebrospinal fluid, and lymph, thus reaching distant locations in the body. Since exosomes derived from many cells in the body can be found in biofluids, identification of their cell of origin, monitoring of their content and biodistribution, and determination of their fate in recipient cells remain key issues that still need to be addressed, in this field. Knowing the content of EVs and their capacity to transport biomolecules will allow exploring their potential as a biomarker to monitor the state of a disease in response to therapy, particularly in hard-to-reach places where biopsies are extremely difficult or not possible to obtain.

[0008] The fact that exosomes are very stable in body fluids makes them perfect reservoirs for disease biomarkers and, in particular, tumour exosomes released into the blood are a great diagnostic source. Cancer cell-derived exosomes that are introduced into the blood sample are stored for a long period of time, making them an excellent tool for biomarker analysis.

[0009] The isolation of exosomes or other subtypes of EVs from biofluids remains challenging due to their heterogeneity, the current lack of specific surface markers for isolation, and the understanding of their cellular origin. The current purification methods that isolate the largest number of extracellular materials are mainly polymer precipitation kits and those based on serial ultracentrifugation (UC). These approaches are not capable of differentiating specific populations of exosomes from different cell origins or other subtypes of EVs or free proteins, necessary for the study of specific tissue-derived exosome biomarkers. Indeed, bulk measurement of a mixture of vesicle populations could potentially mask essential biomarkers, severely impairing investigations of associated pathological mechanisms, making non-specific exosomes one of the main contaminants of preparations. Being a highly enriched source of biomarkers, the use of exosomes would allow the identification of specific multiomics molecular information of their pathogenesis.

[0010] There is a wide variety of commercial precipitation and affinity kits for isolating EVs from biofluids, such as polymeric precipitation, tetraspanin (TSN) affinity, or transferrin kits. Polymer-based precipitation (typically using polyethylene glycol (PEG)) is commonly used to isolate EVs by creating a hydrophobic microenvironment through the interaction of highly hydrophilic polymers and the water molecules surrounding the EVs, which precipitate them as a result of depletion, of its solubility. Some of these kits are Exo-Prep (HansaBioMed, Tallinn, Estonia), Total Exosome Isolation Reagent (ThermoFisher, Waltham, MA, USA), and ExoQuick (System Biosciences, Palo Alto, CA, USA). Despite having a high isolation yield, the samples are characterized by their low purity due to the coprecipitation of proteins, nucleic acids and lipoproteins. Contamination negatively impacts the identification of exosome-associated proteins in multiomics assays, due to non-specific interactions causing false positives or specific signal blocking in both conventional immunoassays and emerging detection platforms.

[0011] Another commonly used method is isolation based on immunoaffinity with the surface proteins of exosomes. This method offers unique advantages for the isolation of exosomes from complex fluids, in terms of higher efficiency and specificity in exosome capture, as well as higher integrity of the isolated vesicles. Unlike precipitation-based techniques, these methods are more specific in terms of exosome capture, with high efficiency in recovering highly intact exosomes from complex biofluids, while reducing costs and labour time. Isolation by immunoaffinity specifically selects for a subpopulation of vesicles that co-express one or more surface markers that reveal their cell of origin and their specific function, including diagnostic markers for disease. Among them, immunomagnetic particles are the most widely used. The exosomes bind to the antibody on the magnetic particle and are separated from the other EVs by applying an external magnetic field. Some examples are exosome isolation / detection with human CD63 (Invitrogen, Waltham, MA, USA), CD81 / CD63 exosome isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and Exo-Flow™ Selective Exosome Capture (System Biosciences). However, the reproducibility between samples and biofluids is not very high, which affects the subsequent multiomics analysis and the conclusions related to the biomarker profile. This variability is due to the lack of specificity of the antibody epitope, non-specific adsorption on the magnetic particle surface, the presence of contaminants in the biofluids, and poor pre-treatment of the starting material.

[0012] To counteract these limitations, some nanomaterials have been used in exosome isolation methods, due to their unique properties and versatility. For example, nanorods, metal-organic frameworks (MOFs), and nanostructured graphene oxide (GO)Zpolypropylene (PDA) were used as platforms to increase the binding surface area of antibodies and thus increase the sensitivity of the assays. In otherstudies, they coated polystyrene particles with zwitterionic MPC polymers to reduce nonspecific protein binding. On the other hand, the ExoCAs-2 methodology allows capturing and releasing exosomes using buffers with different pH, since it is a chitosan-based material that contains magnetic particles functionalized with polycationic polymers that change with a simple pH adjustment.

[0013] BRIEF DESCRIPTION OF THE INVENTON

[0014] The present invention refers to a nanoparticle conjugate (nanoparticle conjugate of the invention) of formula A-X-B-C, wherein: a. A is a nanoparticle preferably an amino nanoparticle such as an amino polystyrene nanoparticle (as an amino methyl nanoparticle), b. X is a linker that is cleaved when exposed to an ultraviolet wavelength; c. B is one or more amino acids or analogues thereof, preferably a lysine having the N-a-amino and N-8 groups thereof optionally protected by orthogonal protecting groups such as Dde and Fmoc, preferably Fmoc- Lys (Dde); and d. C is a bioactive molecule such as an antibody that binds specifically to a target molecule, in particular to an extracellular vesicle (preferably an exosome), wherein each of A and X, X and B, and B and C are directly linked to each other, optionally through a linker such as a PEG (Polyethylene glycol).

[0015] An embodiment refers to the nanoparticle conjugate of the invention, wherein the nanoparticle (A) is capable of being functionalized with (a) at least one imaging agent (T), and at least one bioactive molecule (D), and said conjugate is characterized by a. the nanoparticle A being directly linked, preferably via an amide bond, optionally through a linker, to the cleavable spacer; b. the cleavable spacer (X) being further directly linked, preferably via an amide bond, optionally through a linker such as PEG, to one or more amino acids or analogues thereof (B); and c. the one or more amino acids or analogues thereof (B) being further directly linked, preferably via an amide bond, optionally through a linker such as PEG, to a bioactive molecule or, also optionally through the linker such as PEG, to a suitable molecule capable of binding a bioactive molecule.

[0016] A further embodiment refers to the nanoparticle conjugate of the invention, wherein the the cleavable spacer is amino-3-2-nitrophenyl propionic acid.

[0017] A further embodiment refers to the nanoparticle conjugate of the invention, wherein the suitable molecule capable of binding a bioactive molecule is such as 3-[(2- hydroxyethyl)dithio]propanoic acid.

[0018] A further embodiment refers to the nanoparticle conjugate of the invention, wherein the bioactive molecule is an extracellular vesicle (preferably exosome) binding agent such as an antibody or fragment thereof (such as antiCD63, antiCD81 and / or antiCD9 antibody or fragment thereof).

[0019] A further embodiment refers to the nanoparticle conjugate of the invention, wherein the nanoparticle conjugate is functionalized with (a) at least one imaging agent (T), wherein the T is bonded to the N-a-amino and / or N-8 groups once deprotected.

[0020] A further embodiment refers to the nanoparticle conjugate of the invention, wherein the imaging agent (T) is a fluorophore, preferably a far-red cyanine derivative (Cy7) or a metal such as a chloride hexahydrate lanthanide salt.

[0021] The present invention further refers to a method (method of the invention) for producing nanoparticles (NP) (nanoparticle conjugate of the invention) that can be functionalised, comprising the following steps : a. conjugating the NPs to a cleavable spacer to provide cleavable NPs; b. conjugating the resultant cleavable NPs of step a) to one or more amino acids or analogues thereof characterized by comprising orthogonal protecting groups such as Dde and Fmoc, wherein the conjugation step b) is optionally followed or preceded by one or more PEGylation steps; and c. conjugating the product resultant from step b), optionally followed or preceded by one or more PEGylation steps, with a chemical group capable of binding to a binding agent such as an extracellular vesicle binding agent, such as an antibody.

[0022] An embodiment refers to the method of the invention, wherein the nanoparticles are polystyrene nanoparticles (NPs), or amino NPs or preferably polystyrene amino NPs.

[0023] A further embodiment refers to the method of the invention, wherein the the cleavable spacer is amino-3-2-nitrophenyl propionic acid.

[0024] A further embodiment refers to the method of the invention, wherein the suitable molecule capable of binding a bioactive molecule is such as 3-[(2-hydroxyethyl)dithio]propanoic acid.

[0025] A further embodiment refers to the method of the invention, wherein the bioactive molecule is an extracellular vesicle (preferably exosome) binding agent such as an antibody or fragment thereof (such as antiCD63, antiCD81 and / or antiCD9 antibody or fragment thereof).

[0026] A further embodiment refers to the method of the invention, wherein the method comprises starting with the product of the method of the invention: a. removing the orthogonal protecting group, such as Dde, for conjugation of the nanoparticle to a tracking molecule such as a fluorophore or a metal such as a chloride hexahydrate lanthanide salt; and / or b. further conjugating the product of step a) to a binding agent such as an extracellular vesicle binding agent, such as an antibody.

[0027] The present invention further refers to a nanodevice comprising the nanoparticle conjugate of the invention as well as uses of such nanodevice for isolating extracellular vesicles, preferably exosomes.

[0028] BRIEF DESCRIPTION OF THE FIGURES

[0029] Figure 1. Exosomes biomarkers, receptors and content.

[0030] Figure 2. Core of principle.

[0031] Figure 3. a) Histograms show hydrodynamic diameter values of developed nanodevice with and without antibody, determined by DLS. Inset image is a representative TEM image of developed nanodevice; b) ^-potential values of the developed nanodevice and its controls.

[0032] Figure 4. Signal of coordinated lanthanide to the developed nanodevices analyzed by a) mass cytometry and b) HRTEM-EDX.

[0033] Figure 5. Immunofluorescence of CD63-Tb-NPs (30) (dark red) and its negative control, Tb-NPs (29) (pink).

[0034] Figure 6. Evaluation of CD63-Tb-NPs (30) cargo release. Nanoparticles were incubated with a rabbit anti-mouse IgG secondary antibody conjugated with Alexa Fluor® 488 solution prior to UV exposure.

[0035] Figure 7. Analysis of the size of isolated exosomes by UC by NTA. Inset is a representative image of HRTEM.

[0036] Figure 8. Dot plots of110Cd vs159Tb signal of a) isolated exosomes incubated with110Cd- antiCD63 (1 :100), b) Tb-NPs (29) and CD63-Tb-NPs (30) in absence of exosomes, c) Tb-NPs (29) and CD63-Tb-NPs (30) incubated with exosomes and stained with110Cd- antiCD63 (1 :100), and d) supernatants of samples in c) after 5 min of UV light exposure.

[0037] Figure 9. Evaluation of exosomes capture and release efficiency of CD63-Tb-NPs (30) analyzed by HRTEM-EDX. a) CD63-Tb-NPs (30) incubated with exosomes prior to UV cleavage, and b) supernatant after exosomes capture and release by the nanodevice.

[0038] Figure 10. 1 H NMR (400 MHz, D2O) of Photocleavable-DOTA-linker, compound 20.

[0039] Figure 11. HRMS-ESI of Photocleavable-DOTA-linker, compound 20.

[0040] Figure 12. HPLC of Photocleavable-DOTA-linker, compound 20.

[0041] Figure 13. Schematic representation of synthesis and functionalization of Exo-Hunter NPs. Figure 14. Exo-Hunter Nanoparticles characterization (CD63-Tb-NPs (8), CD81-Eu-NPs (10) and CD9-Gd-NPs (12)). (a) Schematic formulation of NPs; (b) hydrodynamic diameter values of NCB-NPs (1), Metal-NPs and Exo- Hunter- NPs by DLS; (c) zeta potential values of NCB-NPs (1), Metal-NPs and Exo- Hunter- NPs; (d) Metal count of Metal-NPs; (e) immunofluorescence of NCB-NPs (1) and Exo- Hunter- NPs; (f) Metal-NPs morphology by HR-TEM and metals signal of C (blue), Tb (pink), Eu (red) and Gd (green) by EDX.

[0042] Figure 15. Schematic representation of synthesis and functionalization of Exo-Hunter- Nanospheres.

[0043] Figure 16. Exo-Hunter Nanospheres characterization (CD63-Tb-Nanospheres (24), CD81-Eu-Nanospheres (26) and CD9-Gd-Nanospheres (28)) (a) Schematic formulation of NPs; (b) size values of Nanosphero (21), Metal-Nanospheres and Exo-Hunter- Nanospheres by DLS; (c) zeta potential values of Nanosphero (21), Metal-Nanospheres and Exo-Hunter-Nanospheres; (d) Metal count of Metal-Nanospheres; (e) immunofluorescence of Nanosphero (21) and Exo-Hunter-Nanospheres.

[0044] Figure 17. Exosomes characterization, (a) size values of exosomes derived of MDA-MB- 231 y MDA-MB-468 by DLS; (b) concentration values of exosomes samples by BCA (mg / ml) and flow cytometry (particles / ml); (c) WB analysis of MDA-MB-231 , MDA-MB- 468 and A549 cell lines and the exosomes isolated with and without lysis treatment.

[0045] Figure 18. Mass cytometry analysis of the capture and release of Exo-Hunter NPs: (a) histogram representing Cd labelled exosomes, (b), (f) and (j) histograms representing metal count for each NP labelled with Tb, Eu and Gd respectively; (c), (g) and (k) dot plots representing Cd count corresponding to negative controls without specific antibody for recognition; (d), (h) and (I) dot plots representing Cd count corresponding to selective capture and release of exosomes; (e), (i) and (m) histograms comparing Cd signal of CD63-Tb-NPs (8); CD81-Eu-NPs (10) and CD9-Gd-NPs (12) respectively with their negative controls.

[0046] Figure 19. Selective exosomes capture of CD63-Tb-NPs (8) by HR-TEM labelling Tb signal and U signal.

[0047] Figure 20. Mass cytometry analysis of the capture and release of Exo-Hunter Nanospheres: (a) histogram representing Cd labelled exosomes, (b), (f) and (j) histograms representing metal count for each Nanospheres labelled with Tb, Eu and Gd respectively; (c), (g) and (k) dot plots representing Cd count corresponding to negative controls without specific antibody for recognition; (d), (h) and (I) dot plots representing Cd count corresponding to selective capture and release of exosomes; (e), (i) and (m) histograms comparing Cd signal of CD63-Tb-Nanospheres (24); CD81-Eu-Nanospheres (26) and CD9-Gd-Nanospheres (28) respectively with their negative controls.

[0048] Figure 21. Terbium signal in Tb-NPs (7) spectrum.

[0049] Figure 22. Europium signal in Eu-NPs (9) spectrum.

[0050] Figure 23. Gadolinium signal in Gd-NPs (11) spectrum.

[0051] DESCRIPTION

[0052] In the present invention we have developed a nanodevice for the detection of extracellular vesicles (EVs) using monoclonal antibody specific recognition applied to flow and mass cytometry. Specifically, we have implemented a covalent conjugation approach to attach exosome-specific antibodies for CD63 and CD81 tetraspanins. This strategy allows for easy and reproducible recognition of exosomes. Previous studies have reported the use of nanoparticles for exosomes detection by immune affinity, but they required the use of high-tech equipment for their analysis3537-39.

[0053] The developed nanodevices demonstrated clear functionality and recognition of exosomes, as evidenced by flow cytometry, BCA assay, and HRTEM-EDX analysis, as other reported nanomaterials employed for EVs immune-capture, such as nanowires38, metal organic frameworks (MOFs)21and nanostructured graphene oxide (GO)Zpolydopamine (PDA)39. In comparison to other methods, the nanodevices developed in this invention offer several advantages, such as greater versatility in terms of conjugation, they are easy to handle, and do not require any sophisticated equipment. The results of our study demonstrated the successful detection and capture of fluorophore-labelled exosomes in the media using the developed nanodevice, with particularly higher capture efficiency observed with the CD81-Cy5-NPs (25B). However, it is important to note that a minimal level of non-specific adsorption of exosomes to the nanodevices was observed in the negative control. This is a common challenge encountered when using exosome immune-capture nanodevices and it can be attributed to various factors such as protein contaminants, non-targeted extracellular vesicles (EVs), or the inherent lipidic nature of exosomes, which can interact with the nanomaterial21’32’36’37’41. In the subsequent stage of this invention, a novel nanodevice has been developed for the direct labeling of exosomes during their isolation from biological fluids. A new approach using a UV photocleavable linker has been employed to conjugate exosome- specific antibodies (CD63 as a proof of concept) enabling efficient recognition and capture of exosomes. This unique design allows for the tagging and selective release of the captured exosomes in a straightforward and reproducible manner. To further enhance the applicability of this nanodevice in mass cytometry, a lanthanide (Tb) has been successfully conjugated for specific labeling of the captured exosomes.

[0054] The nanotechnology-based approach developed in this chapter for immune-capture and release of tagged exosomes has shown promising results. Although previous methods, such as ExoCAS-2, have employed pH-adjustable buffers and chitosan-based materials with polycationic polymer-functionalized magnetic beads for capturing and releasing exosomes41.

[0055] To the best of our knowledge, this is the first instance of directly labelling immune- captured exosomes for subsequent release and analysis using mass cytometry. While many immune-capture nanodevices rely on magnetic nanomaterials363841, these are not compatible with mass cytometry analysis, making our approach a novel advancement in the field providing a comprehensive dataset of information from the sample in a single step, offering a valuable tool for studying exosomes.

[0056] Further development could be achieved by utilizing other exosomes specific tetraspanins such as CD81 and CD9 and other specific antibodies to isolated tumor-derived exosomes such as TS10147. Each of these nanodevices could achieve the labelling of different exosomes with specific metals like europium and gadolinium.

[0057] The uniqueness of this platform lies in its capability to analyze individual exosomes. As each exosome passes through the nebulizer of the mass cytometer, it is individualized, resulting in a unique mass spectrum for each exosome. Consequently, specific single exosome profiling can be achieved for each developed nanodevice.

[0058] Exosomes are a relevant field of study due to their capacity to act as messengers, transporting their cargo between cells

[0025] , It is widely known that exosomes play an important role in physiological and pathological processes. Currently, there is a lack of standardized techniques for the reliable isolation of exosomes, and the commonly used methods present diverse limitations, particularly in terms of achieving both high yield and purity of the obtained samples

[0026] , To address these limitations, in this study we propose a double-metal-labeled nanosystem to be employed in mass cytometry offering the advantages of the multiple labelling and more sensitive analysis in a single step

[0027] , This nanosystem combines a metal-labeled nanodevice and an additional metal-labeled antibody. This nanodevice consists on nanoparticles containing antibodies against CD63, CD81 , and CD9, which specifically recognize exosome receptors [3], and three different lanthanides, Tb, Eu and Gd, which allow the label and measurement by mass cytometry, a novel detection technique for exosome isolation.

[0059] We developed two different nanodevices following two diverse synthetic routes. The Exo- Hunter-NP nanodevice involved the solid-phase protocol (Figure 1), which has been widely proven for efficiency in NanoChemBio group [13,15], In contrast, Exo-Hunter- Nanospheres employed Rink Amide resin synthesis (Figure 3), which enhances loading capacity and facilitates step-by-step characterization. Besides, this approach enabled the conjugation in a single step of the compound with the nanoparticle, avoiding the loss of loading in various couplings steps.

[0060] On the other hand, exosome isolation protocols commonly employ ultracentrifugation methods. However, it is necessary to note its limitations. Since this method relies on sizebased separation and considering that various types of EVs present similar sizes, the samples obtained may exhibit a relatively low level of purity. Therefore, it is crucial to confirm that what’s being captured are indeed exosomes. For that reason, and as shown in example 2, the second part of the nanosystem of the present invention includes the metal-label antibody. In this case, the antibody used was against CD63, labelled with another different metal (Cd) to enable dual labelling of the system.

[0061] Furthermore, and in contrast with other immuno-capture techniques, the nanosystem of the present invention enables the direct isolation of exosomes from cell culture and facilitates the easy release of these vesicles from the nanoparticles without membrane damage. Moreover, for this invention, we implemented the utilization of polystyrene nanoparticles (compatible with mass cytometry analysis) and their functionalization with the photocleavable linker (Photocleavable-DOTA-linker). The incorporation of this photolabile compound (R)-3-(Fmoc-amino)-3-(2-nitrophenyl)propionic greatly enhanced the efficiency and simplicity of the release process compared to the dilution methods that require magnetic beads, which do not maintain the structural integrity of exosomes.

[0062] The characterization of the developed nanodevices demonstrated the correct functionalization of both metal and antibodies. This was confirmed through mass cytometry in combination with EDX-TEM as well as BCA and flow cytometry analysis. Furthermore, DLS analysis and PDI values demonstrated that the nanoparticles solutions were monodisperses and not aggregated.

[0063] Moreover, as shown in example 2, both Exo-Hunter NPs and Exo-Hunter Nanospheres nanodevices exhibit clear functionality and effective recognition and release of exosomes, as evidenced by mass cytometry analysis. We observed a significant higher release efficiency, especially with CD63-Tb-NPs (8) and CD81-Eu-NPs (10).

[0064] Additionally, the nanosystem of the present invention offers the possibility of different antibodies and metal labelling, expanding its applications. As example, the secondary labelling can be enhanced by using a mixture of antibodies to overcome the limitations of immunocapture when different expression levels are present in exosomes samples. It can also be conjugated with other specific antibodies that recognize different membrane surface receptor molecules, such as ALIX or Annexin

[0038] , as well as different lanthanides.

[0065] In conclusion, for the present invention, we have successfully developed an effective nanosystem that isolates exosomes based on their membrane protein expression employing two different chemical strategies. We confirmed that the nanosystem of the present invention improves the commonly used exosome isolation techniques that are included in the MISEV 2022 guidelines for the definition of extracellular vesicles. It incorporates up-to-date techniques for both releases using a novel chemical strategy of the photolabile linker and analysis by mass cytometry.

[0066] Method of the invention

[0067] Therefore, a first aspect of the present invention relates to a method (method of the invention) for producing nanoparticles (NP) that can be bi-functionalised, comprising the following steps: a) conjugating the NPs to a cleavable spacer such as one or more amino-3-2- nitrophenyl propionic acids to provide cleavable NPs; b) conjugating the resultant cleavable NPs of step a) to one or more amino acids or analogues thereof characterized by comprising orthogonal protecting groups such as Dde and Fmoc, wherein the conjugation step b) is optionally followed or preceded by one or more PEGylation steps; and c) conjugating the product resultant from step b), optionally followed or preceded by one or more PEGylation steps, with a chemical group such as 3-[(2- hydroxyethyl)dithio]propanoic acid capable of binding to a binding agent such as an extracellular vesicle binding agent, such as an antibody.

[0068] A "cleavable spacer” or “cleavable linker” refers to a chemical compound that separates two other chemical moieties, and that may be cleaved at least at one site under exposition to a MALDI laser. Most MALDI lasers usually have an ultraviolet (UV: inferior to 500 nm) wavelength, usually between 300 and 500 nm. For instance, many UV-MALDI analyzers have a pulsed nitrogen laser with a wavelength of 337 nm. Thus, a linker that is photocleavable at the wavelength of a MALDI laser may be efficiently cleaved at least at one site under exposition to a wavelength of 250 to 500 nm, preferably a wavelength of 320 to 360 nm or 320 to 350 nm, more preferably a wavelength of 337 nm, so that the UV-MALDI laser acts both to cleave the linker and to ionize the sample. Other MALDI analyser display an infrared (IR: superior to 770 nm) laser. For instance, a Nd:YAG laser (wavelength = 1060 nm), Er:YAG laser (wavelength = 2940 nm), a mid-infrared optical parametric oscillator (OPO) (wavelength = 2940 nm) or a TEA-CO2 laser (wavelength = 10600 nm) may be used as IR-MALDI lasers. A linker that is photocleavable at the wavelength of a MALDI laser may thus be efficiently cleaved at least one site under exposition to a wavelength of 1000 to 1100 nm, preferably a wavelength of 1060 nm, or a wavelength of 2900 to 3000 nm, preferably a wavelength of 2940 nm, or a wavelength of 10500 to 10700 nm, preferably a wavelength of 10600 nm, so that respectively a Nd:YAG, a Er:YAG , or a TEA-CO2 IR-MALDI laser acts both to cleave the linker and to ionize the sample.

[0069] A linker molecule X that is photocleavable at the wavelength of a MALDI laser may comprise a moiety chosen in the group constituted of:

[0070] wherein R is a C1-C6 alkyl group and m is an integer comprised between 1 and 4.

[0071] A linker molecule X that is photocleavable at the wavelength of a MALDI laser may alternatively comprise a moiety chosen in the group constituted of:

[0072] In a preferred embodiment of the first aspect of the invention, the nanoparticles are polystyrene nanoparticles (NPs), or amino NPs or preferably polystyrene amino NPs.

[0073] In another preferred embodiment of the first aspect of the invention, step a) is performed by introducing the NPs in a suitable medium, preferably dimethylformamide (DMF), in which a cleavable spacer such as amino-3-2-nitrophenyl propionic acid is preferably dissolved and activated in the medium or activated before being dissolved in the medium, for a period of time sufficient for coupling the cleavable spacer protected with Fmoc to the nanoparticles, preferably to the amino nanoparticles. Preferably, step a) is carried out by introducing the nanoparticles, preferably amino nanoparticles such as aminomethyl nanoparticles, in a suitable medium, preferably dimethylformamide (DMF), and separately a cleavable spacer such as Fmoc-amino-3-2-nitrophenyl propionic acid is dissolved in a suitable medium, preferably dimethylformamide (DMF), then a suitable reagent for amide coupling such as oxyma and / or N,N-diisopropylcarbodiimide is added to the solution comprising the cleavable spacer, then the solution is mixed with the medium comprising the nanoparticles for a period of time sufficient for coupling the cleavable spacer protected with Fmoc to the nanoparticles, preferably to the amino nanoparticles. More preferably, aminomethyl nanoparticles are suspended in N,N- dimethylformamide (DMF), separately, a cleavable spacer such as Fmoc-amino-3-2- nitrophenyl propionic acid is dissolved in DMF, then oxyma is added and the solution mixture mixed before the addition of DIC. The solution mixture is then added to the nanoparticles, preferably amino-nanoparticles, and suspension mixed to provide cleavable NPs.

[0074] In another preferred embodiment of the first aspect of the invention, step b) is performed by conjugating the resultant cleavable NPs of step a) to one or more amino acids or analogues thereof, preferably one or more lysines having the N-a-amino and N-8 groups thereof protected by orthogonal protecting groups such as Dde and Fmoc, preferably Fmoc-Lys (Dde), wherein the conjugation step b) is optionally followed or preceded by one or more PEGylation steps. Preferably, step b) is performed by conjugating the cleavable nanoparticles with one or more lysines having the N-a-amino and N-8 groups thereof protected by orthogonal protecting groups such as Dde and Fmoc, preferably Fmoc-Lys (Dde), wherein the conjugation step b) is optionally followed or preceded by one or more PEGylation steps.

[0075] It is noted that the one or more PEGylation steps can be carried out by, for example, dissolving Fmoc-PEG in a suitable medium such as DMF together with a suitable reagent for amide coupling such as oxyma and / or N,N-diisopropylcarbodiimide, and the solution mixture mixed. The solution mixture can then be added to the nanoparticles and mixed to provide the one or more PEGylation steps.

[0076] In another preferred embodiment of the first aspect of the invention, step c) can be carried out by conjugating a suitable molecule such as 3-[(2-hydroxyethyl)dithio]propanoic acid, preferably dissolved in a suitable medium such as DMF, together with a suitable reagent for amide coupling such as oxyma and / or N,N-diisopropylcarbodiimide, and the solution mixture mixed. The solution mixture is then added to nanoparticles and mixed to provide nanoparticles (NP) that can be bi-functionalised.

[0077] A non-limiting specific manner of carrying out the steps indicated in the first aspect of the invention to provide nanoparticles (NP) that can be bi-functionalised detailed herein below:

[0078] Aminomethyl nanoparticles (1 mL, 2% SC, 54 pmol / g, 1 pmol, 1 eq) were washed in DMF (1 mL x 3 times) and suspended in N,N-dimethylformamide (DMF) (1 mL). Separately, the cleavable spacer Fmoc-amino-3-2-nitrophenyl propionic acid (15 eq) was dissolved in DMF (1 mL), then oxyma (15 eq) was added and the solution mixture mixed for 4 minutes at room temperature before the addition of DIC (15 eq) and mixed for 8-10 minutes at room temperature. The solution mixture was then added to amino-nanoparticles and suspension mixed on the Thermomixer at 1400 rpm for 2 hours at 60°C. The resulted nanoparticles were washed three times in DMF (1 mL x 3 times).

[0079] Then a capping process was achieved by the incubation of the conjugated cleavable nanoparticles with a solution of acetic anhydride / N,N- diidopropylethylamine (50 eq) in DMF as organic solvent for 30 minutes at 1400 rpm and room temperature. The resulted nanoparticles were washed three times in DMF (1 mL x 3 times).

[0080] Fmoc deprotection was achieved by treating nanoparticles with 20% piperidine / DMF for 20 min at room temperature and 1400 rpm three times. The resulted nanoparticles were washed three times in DMF (1 mL x 3 times).

[0081] Then, Fmoc-Lys(Dde)OH (15 eq) was dissolved in DMF (1 mL), then oxyma (15 eq) was added and the solution mixture mixed for 4 minutes at room temperature before the addition of DIC (15 eq) and mixed for 8 minutes at room temperature. The solution mixture was then added to nanoparticles and suspension mixed on the Thermomixer at 1400 rpm for 2 hours at 60°C. The resulted nanoparticles were washed three times in DMF (1 mL x 3 times).

[0082] Then Fmoc deprotection step was repeated to conjugate one unit of Fmoc-PEG spacer. For that purpose, Fmoc-PEG (15 eq) was dissolved in DMF (1 mL) together with oxyma (15 eq), and the solution mixture mixed for 4 minutes at room temperature before the addition of DIC (15 eq) and mixed for 8 minutes at room temperature at 1400 rpm. The solution mixture was then added to amino- nanoparticles and suspension mixed on the Thermomixer at 1400 rpm for 2 hours at 60°C.

[0083] Then Fmoc deprotection step was repeated to conjugate 3-[(2- hydroxyethyl)dithio]propanoic acid. For that purpose, 3-[(2- hydroxyethyl)dithio]propanoic acid (10 eq) was dissolved in DMF (1 mL) together with oxyma (10 eq), and the solution mixture mixed for 4 minutes at room temperature before the addition of DIC (10 eq) and mixed for 8 minutes at room temperature at 1400 rpm. The solution mixture was then added to amino- nanoparticles and suspension mixed on the Thermomixer at 1400 rpm for 2 hours at 60°C.

[0084] On the other hand, to provide bi-functionalised nanoparticles (NP), the orthogonal protecting group, such as Dde, is removed for conjugation of the nanoparticle to a tracking molecule such as a fluorophore or a metal such as a chloride hexahydrate lanthanide salt. This lanthanide salt can be, as examples, europium(lll) (Eu), gadolinium(lll) (Gd), holmium(lll) (Ho), palladium(ll) (Pd) or terbium(lll) (Tb).

[0085] Furthermore, the nanoparticle is further conjugated to a binding agent such as an extracellular vesicle binding agent, such as an antibody. For that purpose, labelled nanoparticles are conjugated to a desired binding agent such as an extracellular vesicle binding agent as an antibody (such as antiCD63, antiCD81 or antiCD9).

[0086] Therefore, a second aspect of the invention refers to the functionalization of the nanoparticles (NP) that can be bi-functionalised obtained by the method of the invention, wherein said functionalization is carried by a method comprising the following steps: a) removing the orthogonal protecting group for conjugation of the nanoparticle to a tracking molecule such as a fluorophore or a metal such as a chloride hexahydrate lanthanide salt; and / or b) conjugating the nanoparticle to a binding agent such as an extracellular vesicle binding agent, such as an antibody. For that purpose, nanoparticles, preferably labelled nanoparticles according to step a), are conjugated to a desired binding agent such as an extracellular vesicle binding agent as an antibody (such as antiCD63, antiCD81 or antiCD9).

[0087] Non limiting methods of providing the to provide bi-functionalised nanoparticles (NP) are detailed below:

[0088] Orthogonal protecting group Dde was removed by treating resulted nanoparticles with 2% hydrazine solution (1 mL) for 7 minutes at 1400 rpm at room temperature three times. Then nanoparticles were subsequently washed with DMF (3 x 1 m).

[0089] Then labelling of nanoparticles was carried out. Nanoparticles (1 mL, 1 eq.) were washed (2 x 1 mL) and resuspended in anhydrous DMF (1 mL). DOTA tris(acid)- amido-dPEG4 TFP ester (5 eq) and N,N-diidopropylethylamine (5 eq) was dissolved in anhydrous DMF (1 mL), and suspension mixed with nanoparticles on the Thermomixer at 1400 rpm for 15 hours at room temperature. Nanoparticles were washed then. A solution of a chloride hexahydrate lanthanide salt (1 eq) was incubated with nanoparticles for 15 hours at 1400 rpm for coordination to DOTA molecules. This lanthanide salt can be, as examples, europium(lll) (Eu), gadolinium(lll) (Gd), holmium(lll) (Ho), palladium^ I) (Pd) or terbium(lll) (Tb). For Eu or Tb coordination, it requires to be dissolved in a 2:3 45 mM triethylamine (pH 9-10) / water solution at to be incubated with nanoparticles at room temperature. In the case of Gd or Ho coordination, metal must be dissolved in citrate buffer (pH 6) and incubated with nanoparticles at 50°C. After that, resulted nanoparticles were washed in deionized water (3 x 1 mL).

[0090] Finally, antibody conjugation was achieved. For that purpose, labelled nanoparticles were treated with a 1 M solution of dithiothreitol, mixing for 3 hours at 1400 rpm and room temperature in order to reduce the thiol group of the conjugated 3-[(2-hydroxyethyl)dithio]propanoic acid. Then, nanoparticles were washed with phosphate buffer saline (PBS) (3 x 1 mL).

[0091] Separately, 2.5 pM of desired antibody (such as antiCD63, antiCD81 or antiCD9) was incubated with a 50 mM solution of sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-1 -carboxylate (sulfo-SMCC) for 30 minutes at room temperature. After this time of incubation, the excess of sulfo-SMCC was removed using centrifugal filters of 30 kDa (MWCO) by centrifugation at 14000 x g for 20 minutes, and then, in a new tube with the filter upside down, another centrifugation at 1000 x g for 2 minutes was done to obtain sulfo-functionalized purified antibodies.

[0092] In the second embodiment of the present invention, the nanoparticle is at least bifunctionalised with (a) at least one imaging agent (T), and at least one bioactive molecule (D), preferably a binding agent such as an extracellular vesicle (preferably exosome) binding agent as an antibody (such as antiCD63, antiCD81 and / or antiCD9).

[0093] In an embodiment of the present invention, the imaging agent (T) is a fluorophore, preferably a far-red cyanine derivative (Cy7) or a metal such as a chloride hexahydrate lanthanide salt. Nanoparticles of the invention

[0094] A third aspect of the present invention relates to a nanoparticle, preferably an amino nanoparticle such as an amino polystyrene nanoparticle (nanoparticle of the invention), capable of being functionalized with (a) at least one imaging agent (T), and at least one bioactive molecule (D), wherein said nanoparticle is characterized by a. the nanoparticle, preferably the amino group of an amino nanoparticle such as an amino polystyrene nanoparticle (as an amino methyl nanoparticle), being directly linked, preferably via an amide bond, optionally through a linker, to a cleavable spacer such as Fmoc-amino-3-2-nitrophenyl propionic acid; b. the cleavable spacer being further directly linked, preferably via an amide bond, optionally through a linker such as PEG, to one or more amino acids or analogues thereof, preferably a lysine having the N-a-amino and N-8 groups thereof optionally protected by orthogonal protecting groups such as Dde and Fmoc, preferably Fmoc-Lys (Dde); c. the one or more amino acids or analogues thereof being further directly linked, preferably via an amide bond, optionally through a linker such as PEG, to a bioactive molecule or, also optionally through the linker such as PEG, to a suitable molecule such as 3-[(2-hydroxyethyl)dithio]propanoic acid capable of binding a bioactive molecule.

[0095] The nanoparticle of the invention can be thus defined as a conjugate of formula A-X-B- C, wherein:

[0096] - A is a nanoparticle preferably an amino nanoparticle such as an amino polystyrene nanoparticle (as an amino methyl nanoparticle),

[0097] - X is a linker that is cleaved according to the invention,

[0098] - B is one or more amino acids or analogues thereof, preferably a lysine having the N-a-amino and N-8 groups thereof optionally protected by orthogonal protecting groups such as Dde and Fmoc, preferably Fmoc-Lys (Dde); and - C is an antibody that binds specifically to a target molecule, in particular to an extracellular vesicle (preferably an exosome).

[0099] Preferably, A is optionally linked to X directly or via a linker such as PEG, X is optionally linked to B directly or via a linker such as PEG, and B is optionally linked to C directly or via a linker such as PEG or via a suitable molecule such as 3-[(2- hydroxyethyl)dithio]propanoic acid capable of binding the bioactive molecule.

[0100] A non-limiting example of a conjugate of formula A-X-B-C, is:

[0101] Preferably, the bioactive molecule is an extracellular vesicle (preferably exosome) binding agent as an antibody (such as antiCD63, antiCD81 and / or antiCD9).

[0102] Preferably, the nanoparticle, preferably an amino nanoparticle such as an amino polystyrene nanoparticle (nanoparticle of the invention), of the third aspect of the invention is functionalized with (a) at least one imaging agent (T), and at least one bioactive molecule (D), wherein the T is bonded to the N-a-amino and / or N-8 groups once deprotected; and wherein the D is bonded to the one or more amino acids or analogues thereof, preferably via an amide bond, optionally through a linker such as PEG, or to a suitable molecule such as 3-[(2-hydroxyethyl)dithio]propanoic acid bound, optionally through a linker, to the said one or more amino acids or analogues thereof.

[0103] In an embodiment of the present invention, the imaging agent (T) is a fluorophore, preferably a far-red cyanine derivative (Cy7) or a metal such as a chloride hexahydrate lanthanide salt. This lanthanide salt can be, as examples, europium(lll) (Eu), gadolinium(lll) (Gd), holmium(lll) (Ho), palladium^ I) (Pd) or terbium(lll) (Tb).

[0104] In the third preferred embodiment of the nanoparticle of the invention, the size range of the nanoparticle is from 100 nm to 2000 nm. Preferably, the nanoparticle of the invention has a size of about 200 nm.

[0105] Nanodevice of the invention

[0106] The fourth embodiment of the present invention relates to a nanodevice, particularly a diagnostic nanodevice, comprising any of the nanoparticles of the invention described in the third aspect of the invention or a nanoparticle obtained by means of the method of the invention.

[0107] Uses of the nanoparticles of the invention

[0108] The fifth aspect of the present invention relates to the nanoparticle, the nanodevice, or the nanoparticle obtained by means of the method of the invention, for use in the diagnosis of cancer, or in the control of the treatment of cancer. In particular, such nanoparticle, nanodevice, or nanoparticle obtained by means of the method of the invention is used to capture extracellular vesicles such as exosomes. In particular, this can be achieved by incorporating the cleavable linker that releases the EVs such as exosomes captured due to antibody specific recognition when it is exposed to UV light. The use of mass cytometry for detecting the isolated exosomes is also encompass within the present invention.

[0109] EXAMPLES

[0110] MATERIALS AND METHODS

[0111] Synthesis of CD63-Tb-NPs (30)

[0112] NK-NPs (1) (1 mL, 1 eq) were washed in A / ,A / -dimethylformamide (DMF) (1 mL x 3 times) and suspended in DMF (1 mL). Separately, the cleavable spacer Fmoc-amino-3-2- nitrophenyl propionic acid (15 eq) was dissolved in DMF (1 mL), then oxyma (15 eq) was added and the solution mixture mixed for 4 minutes at room temperature before the addition of DIC (15 eq) and mixed for 8-10 minutes at room temperature. The solution mixture was then added to amino-NPs and suspension mixed on the Thermomixer at 1 ,400 rpm for 2 hours at 60°C. The resulted NPs were washed three times in DMF (1 mL x 3 times).

[0113] Then a capping process was achieved by the incubation of the conjugated cleavable NPs with a solution of acetic anhydride / / V, / V-diidopropylethylamine (50 eq) in DMF as organic solvent for 30 minutes at 1 ,400 rpm and room temperature. The resulted NPs were washed three times in DMF (1 mL x 3 times).

[0114] Then, Fmoc deprotection was achieved as previously described prior to the conjugation of Fmoc-Lys(Dde)OH (15 eq) followed by a PEGylation step.

[0115] Then Fmoc deprotection step was repeated to conjugate 3-[(2- hydroxyethyl)dithio]propanoic acid. For that purpose, 3-[(2-hydroxyethyl)dithio]propanoic acid (10 eq) was dissolved in DMF (1 mL) together with oxyma (10 eq), and the solution mixture mixed for 4 minutes at room temperature before the addition of DIC (10 eq) and mixed for 8 minutes at room temperature at 1 ,400 rpm. The solution mixture was then added to amino-NPs and suspension mixed on the Thermomixer at 1 ,400 rpm for 2 hours at 60°C.

[0116] Orthogonal protecting group Dde was removed by treating resulted NPs with 2% hydrazine solution (1 mL).

[0117] Then, labelling of NPs was carried out. Resulting NPs (1 mL, 1 eq) were washed twice with DMF (1 mL) and resuspended in anhydrous DMF (1 mL). DOTA tris(acid)-amido- dPEG4 TFP ester (5 eq) and A / ,A / -diidopropylethylamine (5 eq) was dissolved in anhydrous DMF (1 mL), and suspension mixed with NPs on the Thermomixer at 1 ,400 rpm for 15 hours at room temperature. NPs were washed and a solution of a terbium chloride hexahydrate (1 eq) was incubated with NPs for 15 hours at 1400 rpm for coordination to DOTA molecules. For that purpose, TbCh ■ 6 H2O requires to be dissolved in a 2:3 45 mM triethylamine (pH 9-10) / water solution. After that, resulted NPs were washed in deionized water (3 x 1 mL).

[0118] Finally, antibody conjugation was achieved by the treating them with a 1 M solution of dithiothreitol, mixing for 3 hours at 1400 rpm and room temperature in order to reduce the thiol group of the conjugated 3-[(2-hydroxyethyl)dithio]propanoic acid. Then, NPs were washed with phosphate buffer saline (PBS) (3 x 1 mL).

[0119] Separately, 2.5 pM of antiCD63 antibody was functionalized. The conjugation of reduced labelled-NPs with sulfo-antibodies was optimized incubating both of them together in PBS and suspension mixed on the Thermomixer at 1 ,000 rpm for 15 hours at room temperature. Finally, resulted NPs were washed with sterile PBS, and CD63-Tb-NPs (30) were obtained.

[0120] UV cleavage

[0121] 100 pL of sample were placed in a 96-well plate. Then, they were placed for 5 minutes in darkness on a Visi-Blue™ UV Transilluminator (VB-26, P / N 95-0461-02 of 220-230 V ~ 50-60 Hz and 0.2 Amps) (UVP).

[0122] Exosomes isolation and purification

[0123] To obtain exosomes for testing the developed nanodevices, cells were cultured at standard anchorage-dependent culture conditions with fresh medium supplemented with 10% exosome-depleted FBS, until 80% confluence. FBS was depleted of bovine exosomes by ultracentrifugation (UC) at 100,000 xg for 70 min. Supernatant fractions collected from 72 h cell cultures were centrifuged at 500 xg for 10 min to remove cell debris. Exosomes were purified by sequential centrifugation as previously described48with minor modifications. Briefly, to remove any possible apoptotic bodies, dead cells and large cell debris, the supernatants were first spun at 10,000 xg for 40 min at 4°C. Exosomes were collected by ultracentrifugation at 100,000 xg for 80 min at 4°C (Beckman SW28 rotor). Exosome pellets were washed with DPBS and pelleted again by ultracentrifugation at 100,000 xg for 80 min at 4°C. The final pellet was resuspended in 100 pL of DPBS and stored frozen at -80°C.

[0124] Exosomes analysis by NTA, DLS, HRTEM and BCA

[0125] NTA analyses were performed on NanoSightNS500 instruments (Malvern Instruments, UK). The instrument was equipped with a 488 nm laser, a high sensitivity CMOS camera and a syringe pump. The measurements were analyzed using the NTA2.3 software (Malvern) after capture 3 videos of 60 sec. For DLS, HRTEM and BCA analysis, protocols were described above.

[0126] For all these methods, exosomes samples were diluted 1 :1000 in DPBS buffer to obtain a concentration range (1-10 x 108particles / mL).

[0127] Exosomes recognition by NPs by HRTEM-EDX

[0128] Ratio exosomes:NPs was the same as before. However, the samples with exosomes had a negative staining with uranyl acetate in order to be able to see them (done by the Samples Preparation Unit). It was necessary just 100 pL of exosomes-NPs solution. The HRTEM-EDX equipment used was the one mentioned in Section Error! Reference source not found..

[0129] Detection of captured and released exosomes by mass cytometry

[0130] Isolated exosomes were incubated with 1 pL of CD63-Tb-NP (30) for 30 minutes in darkness and 25°C. To remove the non-captured exosomes, a series of centrifugations at 14,000 rpm was carried out, resuspending the pellet with DPBS. Then, samples were incubated with110Cd-antiCD63 (1 :100) for 30 min, and after washing steps in DPBS, they were exposed to UV light as previously described in a 96-well plate. Samples were analyzed using mass cytometry measuring110Cd and159Tb intensities.

[0131] Example 1 . Development of a nanodevice for the detection of extracellular vesicles (EVs) based on monoclonal antibody specific recognition applied to flow and mass cytometry 1.1. Liquid Biopsy and EVs

[0132] Liquid biopsy has emerged as an alternative to conventional tissue biopsy for the diagnosis of cancer due to the (i) minimal invasiveness, (ii) higher sensitivity than standard tissue biopsies and (iii) faster sample extraction. In addition, the procedure presents minimal side effects, it is painless and has the ability to diagnose early and real time monitoring, the main disadvantages ofthe tissue biopsy. Liquid biopsies may include extracellular vesicles (EVs), circulating tumor cells (CTCs), and cell-free circulating tumor DNA (ctDNA)1. This emerging technique allows the detection of biomarkers of specific pathologies with difficult access for tissue biopsy, allowing an early diagnosis and monitoring the disease progression2.

[0133] Living cells secrete heterogeneous populations of membrane vesicles, including (i) exosomes, 50-150 nm-sized EV formed from intracellular membranes, specifically intraluminal vesicles within endosomes, which are secreted upon fusion with the plasma membrane3-9; (ii) microvesicles or ectosomes 100-1000 nm-sized EV formed by outward budding from the plasma membrane that contain cytosolic proteins and exhibit common biochemical characteristics, including high levels of phosphatidyl serine and shared surface markers (CD40 ligands, integrins, and selectins)10 11, and (iii) apoptotic bodies, 50-5000 nm-sized EV heterogeneous in size, morphology, and content, as they contain multiple cellular fragments and DNAs35 10 12.

[0134] Due to the cellular processes associated with EVs biogenesis, different components can be selectively incorporated into each type of EVs. To distinguish EVs derived from the plasma membrane or intracellular membrane compartments, tetraspanins such as CD63, CD81 and CD9 are frequently used13-15. CD63 has been suggested to be a key determinator for MVB originating EVs as it is highly retrieved in exosomes along with other tetraspanins16-19. In recent years, EVs, particularly the exosomes subpopulation, are rising attention in the biomarkers and diagnostic field. All living cells secrete these nanoscopic membrane-derived vesicle, containing produced particles surrounded by a lipid bilayer which transport transmembrane proteins at their surface, as well as other proteins and nucleic acids in their lumen, such as DNAs, mRNAs, miRNAs and other small RNAs3-5. These vesicles are important mediators of intercellular communication, transferring complex molecular information from one source cell to other cells throughout the body being associated with a variety of physiological functions and pathological disease states67.

[0135] In particular, exosomes are secreted to the surrounding area of the cells of origin and released to biofluids, such as blood, urine, cerebral spinal fluid, and lymph, thereby reaching distant locations in the body20. Since exosomes derived from many cells in the body can be retrieved in biofluids, tracing their cell of origin, monitoring their cargo and biodistribution and determining their fate in recipient cells remain essential questions still to be addressed in the field8921. A better understanding of EVs content and biomolecule carrier capacity will allow to explore their potential as biomarkers to monitor disease status in response to therapy, particularly in difficult access compartments where biopsies are extremely difficult or not possible to obtain22

[0136] Liquid biopsies have traditionally been the dominant methodology to study biomarkers. The profile of nucleic acids presents in circulating exosomes isolated from patients in glioblastoma, bladder, liver, colorectal, lung and prostate cancers typically contains double-stranded DNA, tumor-specific mutations and miRNAs dysregulations that allows distinguishing between disease and non-disease conditions22-25. In addition, the protein profile of exosomes has been found dysregulated in several tumors compared with healthy donors2627.

[0137] The fact that exosomes are highly stable in body fluids makes them perfect reservoirs for disease biomarkers and, in particular, tumoral exosomes released into the blood are a great diagnostic source. Cancer cell-derived exosomes spiked into blood sample has been stored for a long period of time making them an excellent tool for biomarker analysis28.

[0138] 1.2. Methods for isolation of exosomes

[0139] Despite having several developed protocols in the biomarker field (Table 1)1’28-30, isolating exosomes from biofluids is still a challenge due to their heterogeneity, current lack of specific surrogate surface markers for isolation and understanding of their cellular origin14.

[0140] Current purification methods that recover the highest number of extracellular materials are mainly the precipitation polymer kits and lengthy ultracentrifugation (UC) based approach31. Such isolation methods cannot distinguish specific exosome populations or separate them from other EV subtypes and free proteins. This limitation hampers the study of biomarkers from tissue-specific exosomes and masks important biosignatures, making unspecified exosomes a major contaminant in preparations32 33. As the perfectly enriched biomarker sources, using exosomes for mapping multi-omic molecular information specific to their pathogenesis in biomarker identification is still extraordinarily challenging.

[0141] Polymer-based precipitation, commonly using PEG, is a popular method for isolating vesicles. It creates a hydrophobic environment that causes vesicles to precipitate due to decreased solubility. However, this technique results in low purity due to the coprecipitation of proteins, nucleic acids, and lipoproteins. This contamination hinders the accurate identification of exosome-associated proteins and can lead to false positives or interference in immunoassays and advanced detection platforms1 2831.

[0142]

[0143] Another approach commonly used is isolation based on immune affinity interactions with exosome surface proteins34-38. Immunoaffinity isolation is advantageous for recovering exosomes from complex fluids. It provides increased efficiency, specificity, and integrity of captured exosomes. Unlike precipitation-based techniques, immunoaffinity methods offer higher purity and selectivity, reducing costs and hands-on time. These methods target specific surface markers to identify exosome origin and function, including disease markers. Immunomagnetic beads are commonly used in this approach3638. Exosomes are isolated using magnetic beads with attached antibodies, allowing their separation from other EVs. However, variability in samples and biofluids hampers reproducibility and impacts multi-omic analysis of biomarkers. This is due to issues with antibody specificity, nonspecific adsorption on bead surfaces, high levels of contaminants in biofluids, and inadequate pre-treatment of starting materials.

[0144] To counteract these limitations, some nanomaterials were applied in exosome isolation methods, owing to their unique properties and versatile functionalities. For example, nanowires38, metal organic frameworks (MOFs)21and nanostructured graphene oxide (GO)Zpolydopamine (PDA)39were used as platforms to increase the surface area to immobilize antibodies and then increase the sensitivity of the assays. Polystyrene beads were coated with Zwitterionic MPC polymers to reduce nonspecific protein binding40. The ExoCAS-2 methodology allows the capture and release of exosomes using buffers with different pH, since it is a chitosan-based material that contains polycationic polymer- functionalized magnetic beads that switches by simple adjusting pH41.

[0145] Given the challenges associated with labelling isolated exosomes for characterization and analysis, it is imperative to develop a method that enables labelling of exosomes with specific tags during their isolation from biological fluids. This would address the complexity of handling exosomes separately for labelling and fulfil the actual need for a streamlined approach to facilitate exosome analysis.

[0146] To tackle these challenges, the present invention focuses on the development and adapting novel and existing chemistries to enable the efficient isolation of single exosomes derived from tumor cells. This innovative approach will allow to overcome the limitations of current methods.

[0147] For that purpose, a bifunctionalized nanodevice has been developed, conjugation a tracking molecule (fluorophore or metal) and a monoclonal antibody for exosome capture. Firstly, the isolation of exosomes using the developed nanodevice was evaluated prior to an optimisation and development of a novel bifunctionalized nanodevice that release the captured cargo for a further analysis. To do so, a novel strategy has been designed to conjugate exosome-specific antibodies targeting CD63, CD81 and CD9 tetraspanins through a covalent approach, maleimide-thiol bond. This innovative method allows for efficient and precise labelling of exosomes, enabling robust characterization and analysis of these small vesicles with improved specificity and accuracy.

[0148] Finally, the validation of the novel nanodevice for the selective capture, labelling with fluorescent or metal tags for the consequent and innocuous release of exosomes from biological fluids to further analyzed by flow mass cytometry for cancer diagnosis.

[0149] 1.3 DEVELOPMENT OF A NANODEVICE FOR THE IMMUNE-CAPTURE AND RELEASE OF EXOSOMES BASED ON MONOCLONAL ANTIBODY SPECIFIC RECOGNITION

[0150] As previously described, in order to overcome the challenges associated with labelling isolated exosomes for characterization and analysis, it is crucial to develop a method that allows for labeling of exosomes with specific tags during their isolation from biological fluids. This approach would address the complexity of handling exosomes separately for labeling and fulfill the practical need for a streamlined approach to facilitate exosome analysis. By enabling direct labeling of exosomes during the isolation process, this method would offer a more efficient and convenient way to study exosomes, eliminating the need for additional handling steps and minimizing potential artifacts or loss of exosome integrity. Such a streamlined approach would greatly enhance the accuracy and reliability of exosome characterization and analysis, leading to a deeper understanding of their biological functions and potential applications in various fields, including diagnostics, therapeutics, and biomarker discovery.

[0151] To overcome these exosomes analysis issues, the development of a novel strategy to capture exosomes using nanotechnology was achieved, incorporating a cleavable linker to release the exosomes captured due to antibody specific recognition when it is exposed to UV light. In this case, as a proof of concept, a nanodevice was developed combining a monoclonal antibody against CD63, and a metal (terbium), as tracking molecule, in order to be analyzed by mass cytometry (Figure 2). The use of mass cytometry for detecting exosomes would enable the simultaneous labelling of multiple biomarkers and their analysis at the single-cell level. This approach would provide a comprehensive dataset of information from the sample in a single step, offering a valuable tool for studying exosomes. Exosomes were obtained from TNC breast cancer cells. Results

[0152] Preparation of nanodevice for immune-capture and release of exosomes

[0153] In this case, larger cross-linked aminomethyl nanoparticles were synthesized via dispersion polymerization42in an inert atmosphere (argon), using an ethanol / water mixture (86:14) as the dispersion medium. Poly(N-vinylpyrrolidone) (PVP) with a molecular weight of 29,000 g / mol served as the stabilizer, while VBAH was employed as the amino-functionalized monomer and 2,2'-azobisisobutyronitrile (AIBN) as the radical initiator. The reaction was conducted at a controlled temperature of 65°C, stirring at 350 rpm under an inert atmosphere of argon. Once the reaction was complete, the nanoparticle suspension was allowed to cool for 30 minutes, followed by purification through washing with methanol and water, centrifugation, and dispersion cycles (Scheme 1). The resulting nanoparticles were of 580 nm of diameter.

[0154] Scheme 1. Synthesis of NK-NPs (1) by dispersion polymerization. Reagents and conditions: (i) PVP, AIBN, ethanol / water, 65°C, 350 rpm, Argon atm, 16 h.

[0155] To prepare the cleavable nanodevice designed for immune-capture and release of exosomes, aminomethyl nanoparticles (NK-NPs (1)) were conjugated with a widely-used UV photocleavable linker, amino-3-2-nitrophenyl propionic acid43-46. The aromatic photolabile group nitrophenyl would enable selective release of the exosomes. In addition, a tracking molecule, such as a lanthanide, was conjugated to label the exosomes for subsequent analysis. Finally, an antibody targeting an exosomal biomarker was conjugated to the nanoparticles to enable specific capture of exosome (Scheme). Briefly, NK-NPs (1) were conjugated with the photocleavable spacer, prior to a capping process was achieved by incubating the conjugated nanoparticles with a solution of acetic anhydride / N,N-diidopropylethylamine in DMF, obtaining ANPPA-NPs (26). To ensure that subsequent conjugations take place exclusively on the photocleavable spacer -NH2 groups, the capping step is performed to remove any remaining free -NH2 groups present on the surface of the nanoparticles. Fmoc-Lys(Dde)OH was conjugated to the nanoparticles and after another Fmoc deprotection, Fmoc-PEGs spacer was conjugated. To introduce a thiol functionality on the nanoparticle to be reactive with the maleimide functionalized antibody, 3-[(2-hydroxyethyl)dithio] propanoic acid was conjugated. Labelling of nanoparticles was carried out by incubating the nanoparticles with DOTA tris(acid)-amido-dPEG4™ TFP ester and N,N-diidopropylethylamine. The nanoparticles were washed and then incubated with a solution of terbium(lll) chloride hexahydrate (1 eq), obtaining Tb-NPs (29).

[0156] Finally, antibody conjugation was achieved by treating the labeled nanoparticles with a solution of dithiothreitol to reduce the thiol group of the conjugated 3-[(2- hydroxyethyl)dithio]propanoic acid. Parallelly, the antibody against CD63 was functionalized with sulfo-SMCC 50 mM and conjugated to NPs to obtain CD63-Tb-NPs (30).

[0157]

[0158] Scheme 2. Schematic representation of the chemical synthesis of CD63-Tb-NPs (30).

[0159] Reagents and conditions: (i) Fmoc-amino-3-2-nitrophenyl propionic acid (15 eq), Oxyma (15 eq), DIC (15 eq), DMF, 2 h, 60° C; (ii) acetic anhydride (50 eq), DIPEA (50 eq), DMF, 30 min, 25°C; (iii) 20% piperidine / DMF, 3 x 20 min; (iv) Fmoc-Lys-Dde (OH) (15 eq), Oxyma (15 eq), DIC (15 eq), DMF, 2 h, 60°C; (v) Fmoc-PEG (15 eq), Oxyma (15 eq), DIC (15 eq), DMF, 2 h, 60°C; (vi) 3-[(2-hydroxyethyl)dithio]propanoic acid (10 eq), Oxyma (10 eq), DIC (10 eq), 2 h, 60°C; (vii) 2% hydrazine / DMF, 3 x 7 min, 25°C; (viii) DOTA tris(acid)-amido-dPEG4 TFP ester (3 eq), DIPEA (3 eq), 15 h, 25°C; (ix) TbCI3-6H2O (1 eq), TEA 45 mM, 15 h, 25°C or 50°C; (x) DTT 1 M, H2O, 3 h, 25°C; (xi) CD-SMCC, PBS, 15h.

[0160] Physical-chemical characterization of developed nanodevices

[0161] The developed nanodevices for EVs immune-capture and release underwent a thorough physical-chemical characterization. Their size distribution was determined using Dynamic Light Scattering (DLS), which revealed a homogeneous size of 583.2 nm for CD63-Tb-NPs (30), and a low polydispersity (PDI of 0.095), as shown in Figure 3. Further evidence of the size of different nanodevices was provided by HRTEM, which showed no aggregation (insets in Figure 3). The ^-potential value for CD63-Tb-NPs (30) was -27.4 mV (Figure 3), and it was a more negative value than non-conjugated nanoparticles (NK- NPs (1)), demonstrating the successful conjugation of the different molecules.

[0162] The successful conjugation of the lanthanide was confirmed by mass cytometry (Figure 4a) and HRTEM-EDX analysis (Figure 4b).

[0163] The effectiveness of antibody conjugation was assessed by immunofluorescence (Figure 5). CD63-Tb-NPs (30) was tested, while the nanoparticles with no antibody conjugated were used as a negative control. The qualitative analysis of the results confirmed the presence of the antibody conjugated to the NPs.

[0164] The efficiency of conjugation and loading capacity (LC) of the nanoparticles with antibodies were estimated and detailed in Table 2, with detailed calculations provided in Section 6.2.3.12.

[0165] Table 2. Characterization of NPs

[0166] C.E.= Conjugation efficiency; L.C.= Loading capacity; Molec.= Molecules

[0167] Evaluation of the functionality of the developed nanodevices

[0168] Before assessing the efficacy of the newly developed nanodevices in capturing and releasing exosomes, it was necessary to optimize the protocol for the release process. To evaluate the release efficacy, CD63-Tb-NPs (30) were incubated with a fluorescent labelled secondary antibody for 30 minutes, and then exposed to UV light for 5 minutes. After undergoing appropriate washing steps, the resulting nanoparticles were analyzed using flow cytometry. As observed in Figure 6, there is a significant release of nanoparticles’ cargo.

[0169] Regarding to exosomes samples, in this case they were obtained from cell culture of TNC breast cancer cell line MDA-MB-468. Size and integrity of exosomes was analyzed by NTA and HRTEM, obtaining most of particles’ population with a size around 250 nm (Figure 7). The isolated exosomes presented a regular morphology after ultracentrifugation steps, and their size were corroborated by HRTEM, after being negatively stained with uranyl acetate to visualize them (inset in Figure 7Error! Reference source not found.).

[0170] To quantify the isolated exosomes, samples were analyzed by NTA and BCA protein assay. In Table 3, concentration of particles per mL and concentration of protein are detailed.

[0171] Table 3. Quantification of exosomes

[0172] NTA = Nanoparticle Tracking Analysis;

[0173] BCA = Bicinchoninic acid assay

[0174] The capture and release efficiency of exosomes by each nanodevice was evaluated using two methods: (1) mass cytometry, and (2) HRTEM-EDX.

[0175] In the first method, exosomes were tagged with a secondary metal-tag antibody. Specifically, an antibody against CD63 was selected and labeled with110Cd using the Maxpar MCP9 Antibody Labeling Kit from Standard Biotools™ (Figure 8).

[0176] CD63-Tb-NPs (30) were then incubated with exosomes to assess the capture and release efficiency of these nanodevices, while Tb-NPs (29) were used as a negative control. After 30 minutes of incubation at room temperature, samples were stained with110Cd-antiCD63 (1 :100) for 30 min, and then, the experiments were divided into two sets: one set involved exposing half of the CD63-Tb-NPs (30) to UV light to facilitate the release of exosomes, while the other half was not exposed to UV light. The samples were analyzed using mass cytometry (Figure 8Error! Reference source not found. a-d). Despite showing a similar110Cd signal in the lanthanide positive population (which corresponds to the tracking molecule of the nanodevice) (Figure 8c), there was a110Cd+ signal 34.50% higher in the supernatant of the CD63-Tb-NPs (30) sample compared to the Tb-NPs (29) after UV exposure (Figure 8d). This means that despite there is some adsorption of the exosomes on the nanodevice surface, only the exosomes capture by the antibody of the nanodevice are released.

[0177] Finally, the exosomes capture and release efficiency of CD63-Tb-NPs (30) was also evaluated by HRTEM-EDX analysis. After the incubation of the nanodevice with the exosomes and the UV cleavage, samples were negatively stained with uranyl acetate to visualize exosomes. As shown in Figure 9, exosomes signal was detected by EDX according to the uranium signal. In Figure 9Error! Reference source not found. a (samples with CD63-Tb-NPs (30) prior to UV cleavage), small low-electronic enriched objects could be seen all over the nanodevices surfaces. Further analysis by EDX technique showed that these small particles had high uranium signal, which corresponds to exosomes. In Figure 9b, the supernatant with the cargo release after exosomes capture was analyzed, and exosomes could be found in the sample.

[0178] In summary, we have successfully optimized the previously developed nanodevice for immune-capture of extracellular vesicles (EVs). This innovative nanodevice enables the labeling and controlled release of isolated exosomes, which can be tracked using mass cytometry. By utilizing this technique, we can obtain a comprehensive proteomic profile of the exosomes. The release mechanism of the exosomes is facilitated by the conjugation of a UV photolabile linker. We have demonstrated the functionality of this nanodevice by effectively isolating exosomes from a TNC cell line. To establish its versatility, further development is required, including targeting other exosomal biomarkers.

[0179] Example 2.

[0180] Materials and Methods

[0181] General Methods

[0182] All solvents and chemicals were purchased from Sigma-Aldrich and Thermo Fisher. Fmoc-rink amide was purchased by Novabiochem. Amino-Sphero-NPs APX-20-10 was purchased from Spherotech. Mass cytometry experiments were performed using a CyTOF (DVS Sciences, Standard BioTools) and flow cytometry experiments were carried out on a FACSCanto II system (Becton Dickinson & Co., NJ, USA) using the Flowjo® 10 software for analysis. Transmission electron microscopy (TEM) was performed on LIBRA 120 PLUS de Carl Zeiss SMT (Oberkochen, Germany). The1H spectra were recorded with Varian Direct Drive 400 MHz spectrometer for solutions in CDCh or CD3OD (internal Me4Si). Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet, and br., broad. Mass spectra were recorded with a High-Resolution Mass Spectrometer LCT-TOF Premier XE, Micromass Technology and Bruker QTOF Compact. HPLC analyses were performed on an Agilent 1260 infinity. Method A: Analytical HPLC analyses were performed with an Agilent Poroshell 120® EC-C18, 2.7 pm, 50 x 4.6 mm column. Detection was by UV absorbance at 260 nm. The following eluents were used: (A) H2O + 0.1 % TFA; (B) MeCN + 0.1 % TFA. HPLC grade eluents were employed, at a flow rate of 1 mL / min and filtered prior to injection. The following HPLC method was used: 5% to 95% B over 4 min, 95% B during 3 min, 95% to 5% B over 1 min, then 5% B over 1 min. Method B: Semipreparative HPLC runs were done with an Agilent Eclipse XDB-C18, 5 pm, 250 x 9.4 mm column. HPLC grade eluents were employed, at a flow rate of 2.5 mL / min. The following eluents were used: (A) H2O + 0.1 % TFA; (B) MeCN + 0.1 % TFA; The following HPLC method was used: 5% to 95% B over 18 min, 95% to 5% B over 1 min, then 5% B over 1 min. All synthetic reactions in which the photocleavable linker is already present are carried out in the absence of light.

[0183] Synthesis of ‘’Exo-Hunter Nanoparticles”

[0184] Amino-methyl cross-linked polystyrene nanoparticles (NCB-NPs (1)) were synthesized by NanoChemBio group and functionalized using conventional amide coupling conditions, employing oxime / DIC as chemical reagents. The NPs used in the present work have a diameter of 500 nm, 0,0015 mmol / eq of loading and a solid content of 3% p / v.

[0185] Synthesis of DOTA-NPs (6)

[0186] NCB-NPs (1) were conditioned in N,N-Dimethylformamide (DMF) and the first conjugation was done. Oxyma (15 eq.) and 1-etil-3-(3-dimetilaminopropil)carbodiimide (DIC, 15 eq.) were added to a solution of (R)-3-(Fmoc-amino)-3-(2-nitrophenyl)propionic acid (15 eq.) in DMF. The mixture was shaken for 8 minutes and was added to NCB-NPs (1) under overnight (o / n) and room temperature (RT) conditions. Then, after the reagents were removed by centrifugation, capping solution (DIPEA (50 eq.) and acetic anhydride (50 eq.) in DMF, was added to the nanoparticles and was left stirring for 30 min at 25 °C. Following that, resulted NPs (2) were washed three times with DMF and Fmoc group deprotection was performed. For that, three successive incubations of the nanoparticles in a solution of 20% piperidine / DMF for 20 minutes at 25 °C was done. Subsequently, nanoparticles were washed in DMF and conjugated as previously described with Fmoc- Lys(Dde)-OH (15 eq) in DMF, stirring for 15 h at 25 °C to obtain NPs (3). Next, another Fmoc deprotection and coupling step was carried out to obtain NPs (4). In this case, nanoparticles were treated with 25 equivalents of activated Fmoc-PEG-OH in DMF. The Fmoc groups were released and the group 3-[(2-hydroxyethyl)dithio]propanoic acid (15 eq.) was incorporated to the nanoparticles in order to obtain NPs (5). Finally, after Dde deprotection by incubations in 2% Hydrazine / DMF (3 x 20 min), tris(acid)-amido-dPEG4 TFP ester (DOTA) (3,5 eq) activated with DIPEA (3,5 eq.) was incorporated to nanoparticles giving rise to DOTA-NPs (6) (see the Scheme below).

[0187] Scheme Al. Synthesis of DOTA-NPs (6). Reagents and conditions: (i) (R)-3-(Fmoc-amino)-3-(2- nitrophenyljpropionic acid (15 eq), oxyma (15 eq), DIC (15 eq), DMF, 15 h, 25 °C; (ii) DIPEA (50 eq), Acetic Anhydride (50 eq), DMF, 30 min, 25 °C; (ill) 20% piperidine / DMF, 3 x 20 min , 25 °C; (iv) Fmoc-Lys-Dde(OH) (25 eq), oxyma (25 eq), DIC (25 eq), DMF, 15 h, 25 °C; (v) Fmoc-PEG-OH (25 eq), oxyma (25 eq), DIC (25 eq), DMF, 15 h, 25 °C; (vi) Coupling 3-[(2-hydroxyethyl)dithio]propanoic acid (10 eq), oxyma (10 eq), DIC (10 eq), DMF, 15 h, 25 °C; (vii) 2% Hydrazine / DMF, 3 x 20 min , 25 °C; (viii) Tris(acid)-amido-dPEG4 TFP ester (3,5 eq), DIPEA (3,5 eq) DMF, 15 h, 25 °C.

[0188] Synthesis of Metal-NPs

[0189] Tb-NPs (7), Eu-NPs (9) and Gd-NPs (11) were functionalized from DOTA-NPs (6). Tb- NPs (7) and Eu-NPs (9) were washed three times in TEA buffer (45 mM, pH 9-10) and added to a TEA solution of Terbium (III) and Europium (III) chloride hexahydrate (2 eq), mixing during 15h at 25 °C. In the case of Gd-NPs (12), after three washes in citrate buffer (pH 6), nanoparticles were added to a citrate solution of Gadolinium (III) chloride hexahydrate (2 eq) and mixed o / n at 50 °C.

[0190] Synthesis of Exo-Hunter-NPs

[0191] CD63-Tb-NPs (8), CD81 -Eu-NPs (10) and CD9-Gd-NPs (12) were functionalized from Tb-NPs (7), Eu-NPs (9) and Gd-NPs (12) respectively. After Milli-Q water washed, nanoparticles were activated by adding 3,5 pl of DTT 1 M in Milli-Q water and left stirring during 3h at 25 °C. Separately, antibody was activated. 23 pg of antibody (CD63 (10628D), CD81 (10630D) and CD9 (10626D)) was mixed with 250 pl of sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1 -carboxylate (sulfo-SMCC, 50mM) in PBS during 30 min at 25 °C. After that, excess of sulfo-SMCC was removed using a 10 KDa MWCO purification column by a 20 min centrifugation at 14.000 RCF. The activated antibody was recovered by a 2 min centrifugation at 1 .000 RCF and added to the activated nanoparticles. The mixture was left o / n at 25 °C. After that, the functionalized nanoparticles were washed three times with water to remove unbound antibody and to get the corresponding Exo-Hunter-NPs (see Scheme below). Scheme A2. Synthesis of Exo-Hunter-NPs. (i) Terbium(lll) chloride hexahydrate (2 eq), pH=9, 15 h, 25 °C; (ii) Europium(lll) chloride hexahydrate (2 eq), TEA buffer (pH=9), 15 h, 25 °C; (iii) Gadolinium(lll) chloride hexahydrate (2 eq), citrate buffer (pH=6), 15 h, 50 °C; (iv) DTT (IM), 3 h, 25 °C; (v) CD63-MCC, PBS, 15h, 25 °C; (vi) CD81-MCC, PBS, 15h, 25 °C; (vii) CD9-MCC, PBS, 15h, 25 °C.

[0192] Synthesis of Photocleavable-DOTA-linker, compound 20.

[0193] Preparation of pre-loaded rink Amide resin and checking loading

[0194] Fmoc-rink Amide resin 13 (0,3 g, 0,5 mmol / g) (Scheme below) was swelled with DMF stirring vigorously during 1 hour at RT into a PolyPrep chromatography column. Fmoc deprotection and loading calculation was done by treatment of the resin with 20% piperidine in DMF (3 x 5 min) at 25 °C followed by washing with DMF (3 x 1 min). For the resin cargo determination, as close to 1 mg of dried resin was weighed and add it to 2 mL 20% piperidine in DMF for 5 minutes. The adduct of dibenzofulvene-piperidine liberated during the deprotection has been measured by UVA / is reading at 302 nm against the 20% piperidine blank (Equation 1).

[0195] Equation 1 . Loading

[0196] A302 = absorbance measure at 302 nm E = molar extinction coefficient (7800 M-1cm-1) V = supernatant volume (ml) w = resin mass weigh (mg) d = cuvette diameter (cm)

[0197] Equation 1. Amino loading calculation by Fmoc test.

[0198] First amino acid coupling

[0199] Oxyma (5 eq) and DIC (5 eq) were added to a solution of Fmoc-Glu(OtBu)-OH (5 eq) in DMF. The mixture was shaken for 8 minutes and was added to unprotected resin under o / n and RT conditions to obtain compound 14. Then, after the reagents were removed by suction, the resin was washed with DMF (3 x 1 min).

[0200] Capping resin

[0201] Capping solution (acetic anhydride:pyridine 3:2) was added to the resin 14 and was left stirring for 30 min at 25°C. After that, the mixture was eliminated by suction and the resin was washed with DMF (3 x 1 min). Compound elongation

[0202] Compound chain elongation by peptide bonds, was carried out by cycles of deprotection and coupling steps incorporating different protected Fmoc-amino acids compounds (3,5 eq). Compound 15 was loaded onto resin by added 3,5 eq of (R)-3-(Fmoc-amino)-3-(2- nitrophenyl)propionic acid into DMF during 15 h at 25 °C using oxyma and DIC for the acid activation. Then, and after Fmoc deprotection, the resin was treated with a solution of Fmoc-Lys(Dde)-OH (3,5 eq) in DMF, activated with oxyma and DIC, and stirring for 15 h at 25 °C to obtain 16. The result resin was then treated with 3,5 eq of activated Fmoc- PEG-OH in DMF (17). The peptide chain was next elongated using 3,5 eq of 3-[(2- hydroxyethyl)dithio]propanoic acid (18). Finally, after Dde deprotection (2% hydrazine in DMF, 2 x 15 min, 25 °C), tris(acid)-amido-dPEG4 TFP ester (DOTA) (3,5 eq) activated with DIPEA was incorporated to the resin to form compound 19 (see the Scheme below). Scheme A3. Synthesis of Photodeavable-DOTA-Linker compound. Reagents and conditions: (i) 20% piperidine / DMF 3 x 20 min , 25 °C; (ii) Fmoc-Glu(OtBu)-OH (5 eq), oxyma (5 eq), DIC (5 eq), DMF, 15 h, 25 °C; (ill) acetic anhydride:pyridine (3:2 v / v), 30 min, 25 °C; (iv) (R)-3-(Fmoc-amino)-3-(2-nitrophenyl)propionic acid (3,5 eq), oxyma (3,5 eq), DIC (3,5 eq), DMF, 15 h, 25 °C; (v) Fmoc-Lys-Dde(OH) (3,5 eq), oxyma (3,5 eq), DIC (3,5 eq), DMF, 15 h, 25 °C; (vi) Fmoc-PEG-OH (3,5 eq), oxyma (3,5 eq), DIC (3,5 eq), DMF, 15 h, 25 °C; (vii) 3-[(2-hydroxyethyl)dithio]propanoic acid (3,5 eq), oxyma (3,5 eq), DIC (3,5 eq), DMF, 15 h, 25 °C; (viii) 2% Hidrazine / DMF, 3 x 20 min , 25 °C; (ix) Tris(acid)-amido-dPEG4 TFP ester (3,5 eq), DIPEA (3,5 eq.), DMF, 15 h, 25 °C; (x) TFA:H2O:TIPS (18:1:1), lh, 25 °C.

[0203] Cleavage from resin

[0204] The cleavage mix consist of 3 mL of TFA / TIPS / H2O (18:1 :1) was added to the construct and left stirring during 1 h at 25 °C. The result solution was collected in a 250 mL round bottom flask, precipitated with cold ether, and the impure compound 20 was decanted from the ether.

[0205] Purification and characterization of Photocleavable-DOTA-linker compound 20.

[0206] The final product defined as Photocleavable-DOTA-linker, compound 20, was purified by semipreparative HPLC using method B. The purity of compound 20 was analyzed by analytical HPLC using method A. The HRMS spectrum and1H-RMN were consistent with the structure of 20 (Figures 10, 11 and 12).

[0207] Synthesis of ‘’Exo-Hunter Nanospheres”

[0208] Nanosphero (21) were functionalized using solid-phase strategy. These nanospheres have a diameter of around 1-2 pm and a solid content of 1 % p / v. Sphero nanoparticles were conditioned in DMF and added to a DMF solution containing the compound 20 activated with oxyma and DIC. The mixture is stirred under o / n and RT conditions. Then, after the reagents were removed by centrifugation, and the Nanosphero (21) were washed three times with DMF, the three different metals and the three different antibodies were conjugated to the nanoparticles following the same protocol described above yielding the correspondent Exo-Hunter-Nanospheres, CD63-Tb-Nanospheres (24), CD81-Eu-Nanospheres (26) and CD9-Gd-Nanospheres (28) (see the Scheme below).

[0209]

[0210] Scheme A4. Synthesis of Exo-Hunter-Nanospheres. (i) Photocleavable-DOTA-linker compound (20) (5 eq.), oxyma (5 eq.), DIC (5 eq.), DMF, 15 h, 25 °C; (ii) Terbi um{ 111) chloride hexahydrate (2 eq), TEA buffer pH=9, 15 h, 25 °C; (iii) Eu ropiu m( 111 ) chloride hexahydrate (2 eq), TEA buffer (pH =9), 15 h, 25 °C; (iv)

[0211] Gadoliniu m(l 11) chloride hexahydrate (2 eq), citrate buffer ( pH =6), 15 h, 50 °C; (v) DTT (IM), 3 h, 25 °C; (vi) CD63-MCC, PBS, 15h, 25 °C; (vii) CD81-MCC, PBS, 15h, 25 °C; (viii) CD9-MCC, PBS, 15h, 25 °C.

[0212] Characterization of Nanosystems Hydrodynamic diameter, size distribution and zeta potential values of NPs was measured by DLS. Sample preparation consist of 1 pl of NPs resuspended in 999 pl of Milli-Q water. Shape, morphology, and metal present were determined by HR-TEM and EDX: 1 pl of the different NPs resuspended in 999 pl of absolute ethanol. Metal count was done by mass cytometry by measuring a 1 :10.000 dilution of a NPs solution in PBS. Immunofluorescence analysis was performed by mixing a blocking solution (0,3% BSA, 10% serum, 0,1 % tween in PBS) to 1 pl of NPs for two hours at 4 °C. After two washed of the NPs in PBS, a 1 :100 dilution of Rabbit anti-Mouse IgG secondary antibody Alexa488 (A-11059, Thermo Fisher, Waltham, MA, USA) was added. This suspension remained incubating at 4 °C for 30 min and finally, NPs was washed in PBS twice. The correct functionalization was measured by flow cytometry in FITC channel.

[0213] Cancer cell lines culture and exosomes isolation

[0214] Cell culture

[0215] Human breast carcinoma MDA-MB-231 and MDA-MB-468 cells lines (provided by the Cell Bank the Center of Scientific Instrumentation of the University of Granada) were cultured in DMEM supplemented with 10% (v / v) Fetal Bovine Serum (FBS, previously conditioned), 2 mM of L-glutamine (2 mM), and 1 % (v / v) penicillin / streptomycin and incubated in a tissue culture incubator at 37 °C, 5% CO2 and 95% relative humidity. This step of FBS conditioning consist of ultracentrifugate the commercial FBS (Sorvall VX, Thermo Scientific, Waltham, MA, USA) at 10.000 RCF during 90 min at 4°C, in order to eliminate the possible own exosomes from the serum. After that, pellet was removed, and supernatant was recollected to be used in cell culture (free exosomes-FBS). Next, cell lines were expanded in growth medium and subcultured twice a week until 40% and 90-100% confluency respectively. Each time, culture medium was collected and stored at -80 °C for further processing.

[0216] Exosomes isolation

[0217] Isolation of EVs was performed by ultracentrifugation. Cell culture supernatants were centrifuged at 500 RCF during 5 min at RT conditions to remove cell debris. The remaining supernatant was transferred into an ultracentrifugation tube and it was centrifuged three times at different speeds and times. To remove dead cells, the supernatant was centrifuged at 2.000 RCF during 15 min at 4 °C. Subsequently, supernatant was transferred to a new and clean tube and was centrifuged at 10.000 RCF for 30 min at 4 °C. Later, the supernatant was removed carefully for another centrifugation and the resultant pellets were resuspended in PBS to obtain MVs.

[0218] To obtain exosomes, these pellets are then subjected to another centrifugation (100.000 RCF, 90 min, 4 °C). The resulting pellet from this final centrifugation was pooled, resuspended in PBS and the final volume was stored at -80 °C until use. If there was a need to prioritize a higher enrichment of exosome protein over preserving their integrity (for example for western blot analyses), a filtration step (0,2 pm filter) was applied to pellets before this last centrifugation. Exosomes characterization and biological analysis

[0219] Size and morphology

[0220] Particle size distribution was determined by DLS by measuring 1 pl of exosomes extracted in 999 pl of PBS. Exosomes morphology was evaluated using negative staining methods forTEM. This protocol involves contrasting and embedding the EVs in a mixture of uranyl compounds and methylcellulose to preserve their bilayer morphology.

[0221] Concentration

[0222] Total protein concentrations were measured with a BCA Protein Assay (ThermoFisher, Waltham, MA, USA) and total samples particles were determined by light scattering by measuring on Flow NanoAnalyzer (Nanofcm, Nottingham, UK).

[0223] Western blot

[0224] MDA-MB-231 and MDA-MB-468 cell lines and the exosomes isolated from them were subjected to protein analyses. Secreted protein derived from human breast carcinoma were resolved over SDS-PAGE and transferred to nitrocellulose membrane.

[0225] First, samples were lysed with 500 pL RIPA buffer containing 100 pM leupeptin, 10 pg / mL aprotinin and 1 mM PMSF during 15 min and centrifuged at 12.000 RCF at 4°C during 15 min. Supernatants were collected and quantified with Pierce BCA Protein Assay Kit and measurement in an Infinite 200 PRO NanoQuant reader (Tecan, Mannedorf, Switzerland).

[0226] Quantified protein was heated to 100 °C for 5 min in Laemmli buffer (1610747, Bio-Rad, Hercules, CA, USA). Proteins were resolved over SDS-PAGE and transferred to PVDF membrane (Bio-Rad, Hercules, CA, USA). Membranes were stained with Red Ponceau solution, washed with deionized water, and blocked with 5% milk in 1xPBS-0,5% Tween. Consecutively, membranes were incubated overnight at 4°C with anti-CD81 (10630D, ThermoFisher, Waltham, MA, USA, 1 :1000), anti-human HSC70 (sc-7298, Santa Cruz Biotechnology, Dallas, TX, USA, 1 :1000) and anti-betta actin (8H10D10, Cell Signaling Technology, Danvers, MA, USA, 1 :1000) antibodies diluted in 5% milk in 1xPBS-0,5% Tween. After 3 washing steps with PBS-0,5%Tween, membranes were incubated during 1 h at RT conditions with peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (62-6520, ThermoFisher, Waltham, MA, USA, 1 :5000). Proteins were visualized using ImageQuant LAS400 (GE Healthcare Life Sciences, Marlborough, MA, USA) with ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences, Marlborough, MA, USA). Exosomes isolation by Exo-Hunter-Nanosystems and mass cytometry analysis.

[0227] Exosomes nanodevices (Exo-Hunter NPs and Exo-Hunter Nanospheres) were incubated with exosomes recently isolated by ultracentrifugation under o / n and without agitation conditions. Subsequently, supernatants were removed to eliminate any excess of unbound exosomes and the nanoparticles were washed with PBS.

[0228] The second labeling step involved the addition of a PBS solution of the CD63-Cd (1 :100) to the nanoparticle samples followed by an incubation of 30 min. Afterwards, the excess solution was removed, and the nanoparticles were washed twice in PBS. Next, the liberation process was done by a 5 min UV light exposure (UVP Stainless Steel M-20V Model Transilluminator) of the samples. The nanoparticles were then centrifuged, and supernatants containing the liberated fraction were collected for mass cytometry analysis (1 :1000 in PBS).

[0229] Results

[0230] Functionalization of ‘’Exo-Hunter Nanoparticles”

[0231] The synthetic route of Exo-Hunter Nanoparticles that involved amide bond formation was carried out by standard protocols of solid phase synthesis on amino-methyl cross-linked polystyrene nanoparticles synthetized by NanoChemBio group (NCB-NPs (1)).

[0232] Firstly, a linker with photolabile moieties, based on the structure which include a nitrophenyl group, was selected. This choice was due to its light-responsive cytocompatibility and the observed in vitro and in vivo compatibility of its cleavage products when bound to polymeric materials. These properties are vital in designing materials for biological applications [21 ,22], Specifically, Fmoc-amino-3-2-nitrophenyl propionic acid was chosen to be incorporated onto the the NCB-NPs (1) using DIC / Oxyma to active carboxylic (Figure 13, step a). Metal addition allows labeling and tracking of nanoparticles by mass cytometry. For that purpose, dodecane tetraacetic acid (DOTA) was conjugated leading to DOTA-NPs (6). This metal chelator allows the effective coordination of the different lanthanide ions (Tb, Eu and Gd, in this case) through the formation of coordination bonds between the carboxylic acid groups and nitrogen groups of DOTA moiety and the metal ions (Figure 13, step b and Scheme A1). Finally, the conjugation of antibodies that enables the selective capture of exosomes was carried out following a maleimide-thiol strategy through the conjugation of 3-[(2- hydroxyethyl)dithio]propanoic acid. The thiol group resulted from the treatment of Metal- NPs (7,9,11) with dithiothreitol (DTT), which cleaved the disulfide bond, reacts with the different functionalized antibodies with the maleimide group (Ab-MMC), obtaining the developed Exo-Hunter NPs (Figure 13, step c). Antibody and metal nanoparticles were functionalized according to the Scheme A2 obtaining three different Exo-Hunter Nanoparticles from DOTA-NPs (6): CD63-Tb-NPs (8), CD81-Eu-NPs (10) and CD9-Gd- NPs (12).

[0233] For the building of the central skeleton of the Exo-Hunter NPs, Fmoc-Lys(Dde)-OH and Fmoc-PEG-OH groups were also linked to the nanoparticles. The bifunctionalization of the nanoparticles is achieved with the introduction of the aminoacid lysine, that introduces two arms, while the addition of a PEG3 spacer aims to increase the distance between groups. The efficiency of each step of coupling was monitored by ninhydrin colorimetric test through the unbound amino group’s detection. It is based on the chemical reaction of ninhydrin, an oxidizing agent with free amino groups. When the reaction occurs, an intense blue color is observed, indicating the presence of free amino groups. Conversely, if there are no free amino groups to react with, the color remains yellow.

[0234] Characterization of ‘’Exo-Hunter Nanoparticles”

[0235] A complete characterization of the Exo-Hunter NPs (CD63-Tb-NPs (8), CD81-Eu-NPs (10) and CD9-Gd-NPs (12)) was performed (Figure 14).

[0236] Chemical structure of Exo-Hunter Nanoparticles has been represented in Figure 14a. Hydrodynamic diameter and polydispersity index (PDI) values of the results nanoparticles were measured by Dynamic Light Scattering (DLS) (Figure 14b), in contrast with NCB- NPs (1) and their equivalents NPs formulation without antibodies (Meta I- NPs). DLS is a method that can be used to measure the diameter of particles in diluted aqueous dispersions and indicates if the particles present in the sample are sufficiently monodisperse. The hydrodynamic values obtained were 496,89 nm, 447,87 nm, and 467,78 nm for the CD63-Tb-NPs (8), CD81-Eu-NPs (10) and CD9-Gd-NPs (12) respectively. The PDI values obtained were 0,483, 0,005 and 0,301 , respectively. These values shown the nanoparticles homogenous sizes and monodisperse populations.

[0237] The results of zeta potential measurements (Figure 14c) show negatives values for Exo- Hunter-NPs (-22,47; -17,09; -35,54) in contrast with positive values for NCB-NPs (1) of 36,90. These findings validate the capacity of zeta potential measurements for monitoring chemical reactions conducted on nanoparticles.

[0238] Mass cytometry analysis (Figure 14d) checked the presence of Tb, Eu and Gd in the three different Metal-NPs, as well as their intensity and concentration. The efficacy of primary antibody conjugation (CD63, CD81 , and CD9) was corroborated by immunofluorescence (Figure 14e) using NCB-NPs (1) as negative control without antibody conjugation. In this case, the signal of the secondary antibody in the FITC channel was indicative not only of the presence of the primary antibody, but also the correct position of it in the nanoparticles. Then, the conjugation efficiency (CE) and loading capacity (LC) of the different antibodies were determined by BCA assay (see table 2).

[0239] Finally, microscopy images have been represented in Figure 14f: Energy Dispersive X- ray (EDX) images confirm the presence of metal signal in their surface, and High- Resolution Transmission Electron Microscopy (HR-TEM) images corroborate their morphology and size. Also, each chromatogram from EDX analysis, that quantify the metals signal for each nanoparticle, are represented in Figure 21 , 22 and 23.

[0240] Synthesis of Photocleavable-DOTA-linker (compound 20) and functionalization of “’ExoHunter Nanospheres”

[0241] In order to test if the capture and release of exosomes can be improved by increasing the size of the nanoparticles, commercially available 2 pm polystyrene nanoparticles, called Nanosphero (21), were used. In this case, a different chemical strategy was employed (Scheme A3), involving the solid-phase synthesis of compound 20, which contains the photocleavable group and DOTA in its structure, using Rink Amide AM resin as the solid support. Once compound 20 was synthesized and characterized, the coupling of this compound to the Nanosphero (21) was performed following amide bond formation (Figure 15, step a). Subsequently, after the coordination of the corresponding metals (Figure 15, step b) and the coupling of the corresponding Ab-MMC using the maleimide-thiol strategy (Figure 15, step c) as described earlier, the corresponding Exo- Hunter-Nanospheres were obtained (CD63-Tb-Nanospheres (24), CD81-Eu- Nanospheres (26) and CD9-Gd-Nanospheres (28)) according to Scheme A4.

[0242] Characterization of “’Exo-Hunter Nanospheres”

[0243] A comprehensive characterization was conducted for the functionalized nanospheres CD63-Tb-Nanospheres (24), CD81-Eu-Nanospheres (26) and CD9-Gd-Nanospheres (28) (Figure 16).

[0244] The schematic representation of the chemical structure of each Exo-Hunter Nanospheres has been represented in Figure 16a. Size distribution and PDI were also measured by DLS (Figure 16b). The obtained hydrodynamic diameter values were 1165,61 nm, 1203,17 nm, and 1355,58 nm respectively. The PDI values obtained were 0,367, 0,133 and 0,463 respectively. This result also demonstrates the homogeneity and monodispersity of the functionalized nanospheres.

[0245] The zeta potential measurements (Figure 16c) exhibit negative values for Exo-Hunter- Nanospheres (-23,26; -38,32; -40,31 , respectively), while Nanosphero (21) display it positive, with a value of 29,56. In the same way as NCB-NPs (1) values, this result confirms the effective conjugation of the formulations.

[0246] The presence, intensity and concentration of metals (Tb, Eu and Gd) were measured by mass cytometry analysis. The results of this assessments are shown in Figure 16d.

[0247] The primary antibody (CD63, CD81 and CD9) correct conjugation of each nanosphere was corroborated by immunofluorescence (Figure 16e), in which we can also evaluated the correct orientation of the antibody. FITC signal can be seen in each Exo-Hunter- Nanospheres (24, 26 and 28) in contrast to Nanosphero (21). Similarly to Exo-Hunter- NPs, CE and LC of the different antibodies conjugated were calculated (Table 2) by BCA assay.

[0248] Table 2. Determination of conjugation efficiency and loading capacity of CD63, CD81 and CD9 for Exo-Hunter NPs and Exo-Hunter Nanospheres.

[0249] CD63- CD81-

[0250] CD63- CD81- CD9-Gd-

[0251] CD9-Gd- Tb- Eu- Tb-NPs Eu-NPs Sphero-

[0252] NPs (12) Sphero- Sphero-

[0253] (8) (10) NPs (28) NPs (24) NPs (26) . . . . . .

[0254] (Molecules / NPs)

[0255] Exosomes characterization and biological analysis

[0256] In the present invention, exosomes obtained from the ultracentrifugation process and prior capture and release from two different tumour cells lines (MDA-MB-231 and MDA- MB-468), were analysed employing two different characterization methods. These methods included physical characterization, which involved the analysis of morphology, concentration, and particle size using techniques such as DLS, flow cytometry, and microscopy; and compositional characterization, which included the analysis of membrane proteins and lipid content using techniques such as western blot (WB) (Figure 17).

[0257] Regarding hydrodynamic diameter study we observed that EVs samples may not have monodisperse size distribution and this is the reason why we obtained a width distribution (Figure 17a).

[0258] Samples concentration after isolation process was evaluated using two different procedures: BCA quantification that show the total protein concentration, obtaining values around 0,3 mg / ml for both samples; and light scattering that show the total concentration of specific exosomes in particles / ml (Figure 17b).

[0259] Western blotting was carried out to verify the presence of typically exosome-associated proteins in our extract (Figure 17c). HSC70 and CD81 protein levels were evaluated, and p-Actin was used as positive control. Since lysis step could affect the integrity of the exosomes, we decided to compare samples that had been lysed with those that not. Furthermore, cell lysate samples from two different cell lines were loaded as positive controls: the cell line corresponding to each exosome sample's origin and the A549 lung cancer line, which is known to exhibit high levels of expression of these proteins (data from Cancer Cell Line Encyclopedia - cbioportal).

[0260] HSC70 was present in all the evaluated samples except the lysed MDA-MB-231 exosomes. However, the absence also of detectable p-Actin in this sample suggests that the lysis step might be too aggressive for this sample. CD81 was present in all our samples except MDA-MB-231 exosomes (with and without lysis), suggesting that this particular type of exosomes might not express this protein.

[0261] Hence, the variations observed in the presence of different proteins such as HSC70 and CD81 , depending on the cell lines suggest that exosomes originating from various sources might display varying expression levels, as well as the difference in terms of size that we also observed (Figure 17a).

[0262] Exosomes capture by Exo-Hunter Nanosystems and release by UV light.

[0263] Prior to assessing the functionality of the developed nanosystems, exosomes were labeled with a metal-labeled secondary antibody. Specifically, the anti-CD63 antibody was labeled with110Cd using the Standard Biotools™ Maxpar MCP9 Antibody Labeling Kit, resulting in a robust signal for Cd as shown in Figure 18a. Additionally, the presence of the metals (Tb, Eu, and Gd) in the developed Exo-Hunter NPs was verified (Figure 18b, f, j). Subsequently, the capture and release capacity of the Exo-Hunter Nanoparticles was evaluated, and nanoparticles Tb-NPs (7), Eu-NPs (9), and Gd-NPs (11), functionalized following the same route of synthesis but without antibody conjugation, were used as negative controls.

[0264] First, the process of capturing exosomes was carried out by incubating the developed nanoparticles and the negative controls with exosomes extracted from the MDA-MB-231 and MDA-MB-468 cells lines. Next, UV light-mediated release was performed after 5 minutes of exposure, and the supernatants containing the liberated fraction recuperated after centrifugation of the nanoparticles, were analyzed using mass cytometry.

[0265] The capture and release of exosomes by the Exo-Hunter NPs provide dual labeling: Cd from the exosomes, and the metals Tb, Eu, and Gd from the described NPs, as previously explained. Consequently, a high Cd positive signal in the samples will be indicative of the specific capture and liberation of exosomes. However, for the negative samples, while the release of the nanoparticle would still take place, resulting in the detection of the nanoparticle's labeled metal signal (Tb, Eu and Gd), the selective capture should not occur because of the absence of capture antibody. In this case, Cd positive signal should not appear, and we should observe only a single metal signal corresponding to the labeled nanoparticle.

[0266] The negative controls show a signal for the metals Tb (Figure 18c), Eu (Figure 18g), and Gd (Figure 18k), confirming that the release by the cleavable moiety of the nanosystem has been successfully carried out. However, we can observe a small percentage of Cd signal from Exo-Hunter NPs 8 and 10, which corresponds to nonspecific adsorption of these nanosystems.

[0267] The capture and release efficiency of the developed Exo-Hunter NPs is highly successful, as demonstrated in Figure 18. Specifically, in Figure 18d we can find the dual labeling of exosomes, Cd and Tb, in the supernatant after the release of CD63-Tb-NPs (8). The increase of the double positive signal and the Tb signal is indicative of the liberated portion containing both, exosomes (Cd-labeled) and a liberated portion without specific exosomes but with the hunter compound (Photocleavable-DOTA-linker) (Tb-labeled). A comparison of the Cd positive signals between the negative control and Exo-Hunter NPs (8) shows an increase in the Cd signal for the nanosystem release, providing evidence of selective capture for CD63-Tb-NPs (8) (Figure 18e). These results have been corroborated by HR-TEM (Figure 19), showing both metals signal: Tb for labelling of the nanoparticle and Uranium (U) that labels the exosomes surface. A similar behavior can be observed for CD81-Eu-NPs (10), with an increase in the Cd and Eu signals (Figure 18h). Furthermore, we find that the Cd signal significantly increases when compared to its negative control (Figure 18g). The different intensity in the Cd positive signal between the two samples are the represented in Figure 18i.

[0268] Regarding the CD9-Gd-NPs (12), we can observe a tendency toward the double positive signal with a low increase in the Cd signal. This suggests that even though the metal signal marking the exosome is not particularly strong, selective capture and release have been successful (Figure 181). Furthermore, when compared to its negative control (Figure 18k), without nonspecific adsorption, we clearly observe this increase in the Cd signal originating from the exosomes (Figure 18m).

[0269] At the same way, Exo-Hunter-Nanospheres CD63-Tb-Nanospheres (24), CD81-Eu- Nanospheres (26) and CD9-Gd-Nanospheres (28) with their corresponding negatives controls were measured (Figure 20).

[0270] First, the confirmation whether the different labeling systems (nanospheres and exosomes) has taken place successfully was carried out by measuring the signal intensity of each metal: Cd-antibody in Figure 20b and the different metal-nanospheres in Figures 20b, f, j.

[0271] The positive metal signal from the negative controls for Tb (Figure 20c) and Eu (Figure 20g), confirms the successful release from the nanosystem. Nevertheless, in Figure 20k, we can observe a very low Gd count, indicating the failed conjugation of the metal.

[0272] Regarding capture and release study and focusing on the Cd positive signal, in the study of CD63-Tb-Nanospheres (24), we can find this residual signal presented in all negative controls, which corresponds to nonspecific binding (Figure 20c). However, the expected increase in the antibody-nanosphere sample in this signal is not observed (Figure 20d). Following with the Eu-labeled nanospheres (26), we can find a significative increase in the Cd positive signal in the antibody-nanospheres sample (Figure 20h) compared to the negative control (Figure 20g). These quantitative results are depicted in Figure 20i.

[0273] For the CD9-Gd-Nanospheres (28), we cannot detect Gd positive signal in any of the samples. The only noticeable difference is the Cd signal in the positive control (Figure 20I), corresponding to the remaining of antibody-Cd labeled.

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Claims

CLAIMS1 . A nanoparticle conjugate of formula A-X-B-C, wherein: a. A is a nanoparticle preferably an amino nanoparticle such as an amino polystyrene nanoparticle (as an amino methyl nanoparticle), b. X is a linker that is cleaved when exposed to an ultraviolet wavelength; c. B is one or more amino acids or analogues thereof, preferably a lysine having the N-a-amino and N-8 groups thereof optionally protected by orthogonal protecting groups such as Dde and Fmoc, preferably Fmoc- Lys (Dde); and d. C is a bioactive molecule such as an antibody that binds specifically to a target molecule, in particular to an extracellular vesicle (preferably an exosome), wherein each of A and X, X and B, and B and C are directly linked to each other, optionally through a linker.

2. The nanoparticle conjugate of claim 1 , wherein the nanoparticle (A) is capable of being functionalized with (a) at least one imaging agent (T), and at least one bioactive molecule (D), and said conjugate is characterized by a. the nanoparticle A being directly linked, preferably via an amide bond, optionally through a linker, to the cleavable spacer; b. the cleavable spacer (X) being further directly linked, preferably via an amide bond, optionally through a linker such as PEG, to one or more amino acids or analogues thereof (B); and c. the one or more amino acids or analogues thereof (B) being further directly linked, preferably via an amide bond, optionally through a linker such as PEG, to a bioactive molecule or, also optionally through the linker such as PEG, to a suitable molecule capable of binding a bioactive molecule.

3. The nanoparticle conjugate of any of claims 1 or 2, wherein the the cleavable spacer is amino-3-2-nitrophenyl propionic acid.

4. The nanoparticle conjugate of any of claims 1 to 3, wherein the suitable molecule capable of binding a bioactive molecule is such as 3-[(2- hydroxyethyl)dithio]propanoic acid.

5. The nanoparticle conjugate of any of claims 1 to 4, wherein the bioactive molecule is an extracellular vesicle (preferably exosome) binding agent such as an antibody or fragment thereof (such as antiCD63, antiCD81 and / or antiCD9 antibody or fragment thereof).

6. The nanoparticle conjugate of any of claims 1 to 5, wherein the nanoparticle conjugate is functionalized with (a) at least one imaging agent (T), wherein the T is bonded to the N-a-amino and / or N-8 groups once deprotected.

7. The nanoparticle conjugate of claim 6, wherein the imaging agent (T) is a fluorophore, preferably a far-red cyanine derivative (Cy7) or a metal such as a chloride hexahydrate lanthanide salt.

8. A method for producing nanoparticles (NP) that can be bi-functionalised, comprising the following steps: a. conjugating the NPs to a cleavable spacer to provide cleavable NPs; b. conjugating the resultant cleavable NPs of step a) to one or more amino acids or analogues thereof characterized by comprising orthogonal protecting groups such as Dde and Fmoc, wherein the conjugation step b) is optionally followed or preceded by one or more PEGylation steps; and c. conjugating the product resultant from step b), optionally followed or preceded by one or more PEGylation steps, with a chemical group capable of binding to a binding agent such as an extracellular vesicle binding agent, such as an antibody.

9. The method of claim 8, wherein the nanoparticles are polystyrene nanoparticles (NPs), or amino NPs or preferably polystyrene amino NPs.

10. The method of anyone of claims 8 to 9, wherein the the cleavable spacer is amino- 3-2-nitrophenyl propionic acid.11 . The method of anyone of claims 8 to 10, wherein the suitable molecule capable of binding a bioactive molecule is such as 3-[(2-hydroxyethyl)dithio]propanoic acid.

12. The method of anyone of claims 8 to 11 , wherein the bioactive molecule is an extracellular vesicle (preferably exosome) binding agent such as an antibody or fragment thereof (such as antiCD63, antiCD81 and / or antiCD9 antibody or fragment thereof).

13. A method of producing functionalised nanoparticles (NP), wherein the method comprises starting with the product of the method of any one of claims 8 to 12: a. removing the orthogonal protecting group, such as Dde, for conjugation of the nanoparticle to a tracking molecule such as a fluorophore or a metal such as a chloride hexahydrate lanthanide salt; and / or b. further conjugating the product of step a) to a binding agent such as an extracellular vesicle binding agent, such as an antibody.

14. A nanodevice comprising the nanoparticle conjugate of any one of claims 1 to 7.

15. In vitro use of the nanodevice of claim 14 for isolating extracellular vesicles, preferably exosomes.

Images