Coated nanoparticles and their use for delivery of therapeutic and diagnostic agents

Abstract

Nanoconstructs consisting of a core and a lipid coating, suitable for conveying therapeutic and diagnostic (or "theranostic") agents to target tissues or organs, delivering them to the cells of interest where the nanoparticle load is released, are described. A further aspect of the invention concerns diagnostic, therapeutic or theranostic compositions comprising coated nanoparticles together with pharmaceutically acceptable vehicles or excipients.

Description

[0001] COATED NANOPARTICLES AND THEIR USE FOR DELIVERY OF THERAPEUTIC AND DIAGNOSTIC AGENTS

[0002] The present invention concerns coated nanoparticles capable of mimicking the function of the extracellular vesicles and of delivering therapeutic or diagnostic agents to target cells or tissues, and of releasing them intracellularly.

[0003] A further aspect of the invention concerns diagnostic, therapeutic or theranostic compositions comprising coated nanoparticles together with pharmaceutically acceptable vehicles or excipients.

[0004] BACKGROUND OF THE INVENTION

[0005] Extracellular vesicles (EVs) play an important role in the communication between human and animal body cells, being responsible for the transport and delivery of countless biomolecules, including proteins and nucleic acids. They are of fundamental interest as they naturally show a high efficiency and specificity in the delivery of their content. For these reasons, EVs are usually isolated from various biological fluids, tissues, cell cultures, as well as foodstuffs such as fruits and vegetables to be then used as disease diagnostic systems or for the development of drug delivery systems. However, the growing interest in large-scale production of EVs is limited by the difficult, time-consuming, and expensive practices of isolating and purifying EVs themselves, as well as the difficulty of obtaining high-purity, standardized clinical-grade populations. Moreover, isolating a particular subpopulation of EVs with specific size and molecular characteristics is even more difficult. Consequently, the development of EV-based nanovehicles is still an open challenge due to the lack of a standard and clear characterization to ensure reproducibility, reliability and safety from a pharmaceutical and clinical point of view of the currently produced EVs.

[0006] To date, a medical branch that seeks to exploit the potential of the EVs is the diagnostic and therapeutic branch in the tumour field. Since one of the most challenging issues related to cancer therapy is the complexity and heterogeneity of tumour biology, and therefore the peculiar nano-bio interaction that occurs between tumours and nanomedicine, a precise control of the chemical-physical properties of the EVs, or more generally of the nanoparticles, must be a priority. As far as purely inorganic nanoparticles (NPs) are concerned, one of the main drawbacks of their use is linked to their aggregation in the biological fluids, and therefore their short life in the bloodstream, which hinders their effective localisation in the target organs and triggers a rapid counter-immune response. A possible solution to exploit them in the biological environment may be to have an effective and compact coating, such as the one with phospholipids. This coating may be able to mimic cell membranes and therefore be the simplest strategy to give these NPs mimicking properties with respect to the immune system and beyond. Furthermore, by introducing targeting strategies in the form of surface functionalizations with proteins, peptides, aptamers etc., NPs could also exploit an active delivery and targeting towards the target organ, tissue or cell, generally improving their toxicity profile in the body. In fact, we must bear in mind the natural ability of natural EVs to avoid rapid clearance by the immune system. Finally, some in vitro studies indicate that natural EVs possess an intrinsic tropism and can selectively biodistribute in particular organs and tissues, thanks to the peculiar molecular (lipid and protein) composition of the progenitor cell that produced them. For this reason, to date, we aim to use natural EVs as a coating of artificial NPs, thus constituting therapeutic and diagnostic (or ‘theranostic’) nano-constructs. This would represent an improvement over the use of more conventional liposomes, thanks to the aforementioned biological properties. Liposomes, however, being made up of commercially available lipids, make high reproducibility and scale-up of liposome-coated inorganic NPs systems possible. On the other hand, the aforementioned intrinsic capabilities of EVs are often completely lost when try to coat nanoparticles, introducing disadvantages such as low biodistribution and rapid clearance, as well as the lack of specific targeting.

[0007] One solution could be to determine the composition of lipids and membrane proteins and then create bioartificial nanoparticles capable of maintaining the advantages of natural EVs. These bioartificial nanoparticles can consist of

[0008] - an inorganic (core) capable of delivering a cargo molecule for imaging or therapy; and

[0009] - a coating (shell) derived from fragments of EVs or consisting of completely synthetic lipidic formulations; able to biomimic the properties of EVs, but containing only the key elements necessary for their specific purpose; thus providing a high and specific therapeutic and diagnostic efficacy.

[0010] The latter is a requirement not met either by natural EVs, due to the variety and complexity of their composition, nor by commercially available liposomes.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention makes available coated nanoparticles suitable for delivering therapeutic and diagnostic (or “theranostic”) agents to target tissues or organs, delivering them to the cells of interest where the nanoparticle load is released.

[0013] The nanoparticles subject matter of the invention consist of a core consisting of organic or inorganic material in the solid or semi solid state, and a lipid coating of the core, consisting of charged lipids, neutral lipids, cholesterol, PEG-functionalized lipids, mono- and bifunctional lipids, lipids conjugated with peptides or proteins, phospholipids and sphingomyelin, combined in such a way as to optimize the delivery capacity and selectivity of the nanoparticle.

[0014] The material constituting the core of the nanoparticle may be per se endowed with biological activity, or it may be loaded with therapeutic or diagnostic agents to be released into the target cell. Furthermore, the lipids constituting the lipid coating may be functionalized with peptides or antibodies to facilitate targeting the nanoparticle towards the target tissue.

[0015] A further object of the invention is the use of the coated nanoparticles for conveying therapeutic or diagnostic agents towards target cells, tissues or organs, and a diagnostic, therapeutic or theranostic composition comprising a coated nanoparticle according to the invention, together with pharmaceutically acceptable vehicles or excipients.

[0016] DESCRIPTION OF THE DRAWINGS

[0017] Figure 1. 10 nm OSCs characterization. A) Electron microscopy images B) DLS analysis of ssOSCs 10 nm, C) FTIR spectra in which the S-S fingerprinting bands within the silica structure can be observed, D) TGA demonstrating complete elimination of the surfactant and E) TGA comparing OSCs and ssOSCs 10 nm.

[0018] Figure 2. 20 nm OSCs characterization. A) Electron microscopy images B) DLS analysis of ssOSCs 20 nm, C) FTIR spectra in which the S-S bands in the infrared region defined as "fingerprint" within the silica structure can be observed, D) TGA demonstrating complete elimination of the surfactant and E) TGA comparing OSCs and ssOSCs 20 nm.

[0019] Figure 3. 20 nm ssMSN characterization. A) Electron microscope images B) DLS analysis C) FTIR spectra D) and E) TGA.

[0020] Figure 4. Characterization of functionalized degradable nanocapsules. A) Electron microscope images of naked, PEG-coated, NFL-functionalized NCs. B) DLS analysis C) fluorescence emission spectra and D) zeta potential.

[0021] Figure 5. Cyclic silane used for the positive functionalization of NCs.

[0022] Figure 6. Characterization of degradable NCs functionalized with the cyclic positive silane. A) DLS analysis B) zeta potential surface charge evaluation, C) FTIR spectra and D) TGA analysis. Figure 7. Characterization of zinc oxide nanoparticles, naked and coated with 3C- formulation in terms of dimensional distribution made by DLS (A), NTA (B) and zeta potential (C).

[0023] Figure 8. A) DLS (Dynalmic Light Scattering) analysis and B) Measurement of the Zeta potential of unfunctionalized organosilica nanocages (ssOSCs), functionalized with PEG (ssOSCs@PEG) and with NH2 (ssOSCs @ NH2) groups as such and coated by 3C- formulation. C) DLS analysis of organosilica nanocages functionalized with NH2 (ssOSCs- NH2) groups as such and coated by control 2Cn and 3C- formulation. D) NTA (Nano Tracking Analysis) analysis of the sample ssOSCs-NEE, coated by 3C- formulation. E) FTIR analysis of ssOSCs-NEE and ssOSCs-NEE coated by 3C- formulation: in the coated sample, the characteristic peaks of lipids are clearly visible, together with those of silica. F) thermogravimetric evaluation (TGA) of ssOSCs- NH2 and ssOSCs-NFE coated by 3C- formulation. The recorded weight loss corresponds to the amount of lipids added to the nanoparticles for coating, i.e. 50% by mass with respect to the nanoparticles.

[0024] Figure 9. A) DLS (Dynalmic Light Scattering) analysis and B) Measurement of the Zeta potential of unfunctionalized organosilica nanocapsules (NCs), functionalized with PEG (NCs@PEG) and with NH2 (NCs @ NH2) groups as such and coated by 3C+ formulation.

[0025] Figure 10. Characterization of polymeric nanoparticles as such and coated by lipids. A) Zeta potential measurements of particles of PLGA, chitosan-functionalized PLGA (PLGA-CS) and gelatin as such and coated by 3C+ or 3C- formulations depending on the starting Zeta potential. B) DLS (Dynamic Light Scattering) analysis of PLGA particles as such and coated by the 3C+ formulation. C) DLS (Dynamic Light Scattering) analysis of Gelatin particles as such and coated by the 3C- formulation. D) DLS (Dynamic Light Scattering) analysis of PLGA particles functionalized with chitosan (PLGA-CS) and coated by the 3C- formulation.

[0026] Figure 11. Characterisation of degradable nanocapsules functionalized with NH2 groups (NCs) as such and coated by MIMIC 1 and MIMIC3 formulation. A) DLS analysis. B) Zeta potential. C) NTA (Nano Tracking Analysis) analysis NCs coated by MIMIC1 formulation.

[0027] Figure 12. A) TEM analysis of ssOSCs@Calcein, B) Hydrodynamic diameter of loaded NPs, C) FTIR spectra, D) UV-VIS absorption graph and E) emission spectra of calcein-loaded NPs after excitation at 480 nm.

[0028] Figure 13. A) FTIR spectrum and B) DLS and zeta potential of ssOSCSCalcein@ NH2coated with lipidic 3C- formulation.

[0029] Figure 14. TEM analysis of ssOSCs @D0X, B) Hydrodynamic diameter of loaded NPs, C) FTIR spectra and D) UV-VIS absorption graph.

[0030] Figure 15. A) FTIR spectrum and B) DLS and zeta potential value of ssOSCs@DOX@ NH2coated with lipidic 3C- formulation.

[0031] Figure 16. Internalization of the zinc oxide nanoparticles coated by 3C- formulation, with and without the CKAAKN peptide and in the presence of the FITC fluorophore, in terms of A) positive events expressed as a percentage compared to untreated control cells, and B) histograms of fluorescence intensities analysed by cytofluorimetry.

[0032] Figure 17. Evaluation on HT-29 cells (colorectal cancer line) of: A) cytotoxicity of the zinc oxide particles as such and coated by 3C- formulation and bioconjugated with the targeting peptide YSAby WST1 assay, B) Internalization of the zinc oxide particles coated by 3C- formulation, bioconjugated or not with the targeting peptide YSA. The evaluation was carried out by cytofluorimetry. C) Hemocompatibility on human plasma sample of zinc oxide nanoparticles as such and coated by 3C- formulation, bioconjugated or not with the targeting peptide YSA. The evaluation was carried out by calculating the coagulation time of the citrate plasma, obtained from the UV-visible emission absorption curves.

[0033] Figure 18 A) Cryo-Electron Microscopy (CryoEM) image of lipid-coated Zinc Oxide Nanocrystals (L-ZnO), (lipidic formulation is the 3C ) depicting the ZnO nanocrystals surrounded by a visible lipid bilayer, evidenced by the black arrows. Scale bar is 50 nm. B) Dynamic Light Scattering (DLS) of uncoated (ZnO) and lipid-coated (L-ZnO, YSA-L-ZnO) in water and C) in cell culture media; D) Z-Potential of uncoated (ZnO) and coated (L-ZnO, YSA-L-ZnO) in water; E) Hemocompatibility studies comparing the clotting time of human plasma after calcium chloride addition in absence and in presence of ZnO, L-ZnO or YSA-L-ZnO.

[0034] Figure 19 - A) and B) Results of cytotoxicity of HT-29 CRC malignant cells and CCD-I8C0 fibroblast-like, healthy colorectal cells, treated with 30 -coated ZnO (L-ZnO) and L-ZnO decorated with the targeting peptide (YSA-L-ZnO). The viability was measured trough WST-1 metabolic assay after 48 and 96h of cells exposure to the nanoparticles, respectively. Cells were treated with different concentrations of L-ZnO and YSA-L-ZnO (15, 3, 50, 75 and 100 pg / mL). Flow cytometry results of internalization tests on CCD- I8C0 healthy cells and HT-29 CRC malignant cells at 5h, 24h and 48h after nanoparticles administration, respectively.

[0035] Figure 20 - A) Cytotoxicity test of 3C lipid coated ZnO (L-ZnO) on HT-29 spheroids. Spheroids were left untreated (control) or treated with different concentrations of targeted YSA-L-ZnO or with the untargeted control NPs (L-ZnO). After 24, 48, 72 and 96h from the exposure, spheroids were disaggregated, dead cells were stained with propidium iodide, and the viability was measured through flow cytometry. Data are normalized by the control sample, which is considered to be 100% vital. (B) Live-cell fluorescence microscopy images of the spheroids treated with YSA-L-ZnO at different concentrations and time steps. Spheroids were stained with Calcein AM (green channel) and Propidium Iodide (red channel) to mark the live and the dead cells respectively C) Flow cytometry results of internalization test on HT-29 spheroids.

[0036] Figure 21 - Ex-vivo quantification at 3h (full dots) and 24 h (empty dots) of different organs collected from NOD-SCID tumor xenograft mice model receiving intravenous (IV) and intratumor (IT) administration of the test samples or NT: control media. The ZnO nanocrystal coated with the 3C- lipidic formulation were administered at doses D4: 20 mg / kg and D5: 30 mg / kg). Mean values ± SEM are shown (n=3 / group).

[0037] Figure 22 - Characterization of Mimic-coated silica nanocapsules (NCs) A) DLS measurements of pristine NCs, functionalized with amine groups (NCs@NH2), coated with Mimic 1 or 3 formulations or a wrong formulation called ScrambleO. B) DLS measurements of NCs@NH2 coated with cholesterol-enhanced (CE) Mimic formulations or CE ScrambleO. C) Summary of DLS results obtained for the lipid-coated NCs@NH2. D) Zeta potential results. E) Results of super resolution confocal microscopy colocalization test. The percentages refer to the number of NCs signals which are superimposed to a signal from the lipids with respect to the total of NCs signals. F) Single-molecule localization microscopy results in terms of polarity profile. This was obtained considering the emission of Nile red, employed to create a transient hydrophobic binding in the lipid bilayer of the nanoconstruct or natural PC3-derived EVs, used as reference. The obtained spectra are also presented as box plot. G) Nano Tracking Analysis (NTA) result example of CE-Mimicl- coated NCs and H) NTA result of PC3-derived EVs.

[0038] Figure 23 - In vivo biodistribution of Cytochorme C-loaded silica nanocapsules, coated by MIMIC 1 and MIMIC 3 (G3 and G4 respectively), and for comparison nude silica nanocapsules (G2) and the cargo only, Cytochorme C (Gl).

[0039] Figure 24 - Characterization of ZnO coated with SCRAMBLE formulations. A) table with the molar composition of formulations SCRAMBLE 1 SCRAMBLE2 (with 15% of cholesterol) and SCRAMBLES (with 30% of cholesterol). B) Zeta potential measurements, C) DLS results in terms of average and D) Polidispersity index (PDI) of ZnO nanocrystals coated with SCRAMBLE 1, 2, or 3 lipidic formulations. E) table with the molar composition of formulations SCRAMBLE4 (with 50% of anionic phospholipid DOPA) and SCRAMBLES (with 50% of cationic phospholipid DOTAP), in comparison with SCRAMBLE 1. F) Zeta potential measurements, G) DLS results in terms of average and H) Poly dispersity index (PDI) of ZnO nanocrystals coated with SCRAMBLE 1, 4, or 5 lipidic formulations.

[0040] DETAILED DESCRIPTION OF THE INVENTION

[0041] Object of the present invention are coated nanoparticles loaded with diagnostic or therapeutic agents, capable of reaching the target cells, within which they release their content (cargo).

[0042] The coated nanoparticles for delivery of therapeutic or diagnostic agents, according to the invention, consist of a core and a lipid coating, wherein said core is a nanoparticle of organic or inorganic material in the solid or semisolid state and said lipid coating is a formulation of charged lipids, neutral lipids, cholesterol, PEG-functionalized lipids, phospholipids and / or sphingomyelin, wherein said formulation is selected from the group comprising:

[0043] (i) formulation containing 10.0 to 35.0% cholesterol; 8.0 to 32% sphingomyelin (SM); 11.0 to 21.0% of DSPE-PEG(2000)-amine; 18.0 to 34.0% DOPC; 15.0 to 35.0% DOPA; and 0.1 to 0.6% l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy (polyethylene glycol)-2000 N-hydroxysuccinimide ester (DSPE-PEG(2000) Carboxy NHS ester);

[0044] (ii) formulation containing 45.0 to 65.0% l,2-dioleoyl-3 -trimethylammonium - propane (DOTAP+) or l,2-dioleoyl-sn-glycero-3 -phosphate (DOPA-); 7.0 to 15.0% 1,2- dioleoyl-sn-glycero-3 -phosphocholine (DOPC); 15.0 to 35.0% cholesterol; 1.0 to 10% 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000 (DSPE-PEG(2000)-amine) and 0.1 to 0.6% l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[maleimide(poly ethylene glycol)-2000 (DSPE-PEG(2000)- maleimide) or l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy (polyethylene glycol)-2000 N-hydroxysuccinimide ester (DSPE-PEG(2000) Carboxy NHS ester);

[0045] (iii) formulation containing 10.0 to 35.0% cholesterol; 8.0 to 32% sphingomyelin (SM); 5.0 to 15.0% DSPE-PEG(2000)-amine; 1.0 to 11.0% phosphatidylethanolamine (PE); 18.0 to 34.0% DOPC; 15.0 to 35.0% phosphatidylserine (PS) and 0.1 to 0.6% (DSPE- PEG(2000)-maleimide) or l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy (polyethylene glycol)-2000 N-hydroxysuccinimide ester (DSPE-PEG(2000) Carboxy NHS ester), wherein said percentages refer to the total weight of the lipid coating, and wherein the core of said coated nanoparticle or a component of the core possesses therapeutic or diagnostic activity per se or the core of said coated nanoparticle is loaded with a diagnostic or therapeutic agent.

[0046] The use of the coated nanoparticles of the invention, compared to the natural EVs alone or the more common liposomes, gives high loading efficiency of the biomolecules, a targeted delivery of the load to the target cell, a complete dissolution and the consequent release, triggered by endogenous stimuli already present in the recipient cell, as well as a lower complexity compared to their natural counterpart.

[0047] In preferred embodiments of the invention, said coated nanoparticle has a lipid coating selected from the following formulations:

[0048] (a) formulation (ii) containing DOPA(-) 57.3%; DOPC 12.5%; cholesterol 23.6%; DSPE-PEG(2000)-amine 6.5% and (DSPE-PEG(2000)-maleimide) 0.1%;

[0049] (b) formulation (ii) containing DOTAP(+) 56.5%; DOPC 12.7%; cholesterol 24%; DSPE-PEG(2000)-amine 6.6% and (DSP-PEG(2000)-maleimide) 0.1%;

[0050] (c) formulation (i) containing cholesterol 13%; SM 20%; DSPE-PEG(2000)- amine 15.9%; DOPC 26%; DOPA25%; (DSPE-PEG(2000)-maleimide 0.1%

[0051] (d) formulation (i) containing cholesterol 30%; SM 16%; DSPE-PEG(2000)- amine 12.9%; DOPC 21%; DOPA 20%; (DSPE-PEG(2000)-maleimide 0.1%

[0052] (e) formulation (iii) containing cholesterol 13%; SM 20%; DSPE-PEG(2000)- amine 9.9%; PE 6%; DOPC 26%; PS 25% and (DSPE-PEG(2000)-maleimide) 0.1%;

[0053] (f) formulation (iii) containing cholesterol 30%; SM 16.0%; DSPE-PEG(2000)- amine 7.9%; PE 5%; DOPC 21%; PS 20% and (DSPE-PEG(2000)-maleimide) 0.1%.

[0054] An advantage of these formulations is the triple function of the lipid with PEG group, that is of: (i) stabilizing the coating of the nanoparticle in an aqueous and physiological environment, ensuring greater circulation and consequently maximizing the probability of reaching the target tissue protecting the content from degradation; (ii) allowing the bond through simple chemical reactions of peptides and antibody fragments, allowing a very varied customization of the shell depending on the use; (iii) conferring the so-called “stealth” property to the nanoparticles, i.e. avoiding immediate recognition by the immune system and therefore the foreign body response. In a preferred aspect, the lipid coating of the nanoparticle is linked to one or more molecules for recognition of target tissue cells or organs, such as proteins, antibodies, antibody fragments, peptide sequences, carbohydrate molecules, sugars, aptamers, or other nucleic acid sequences.

[0055] The invention also makes available a method for preparing a lipidic formulation suitable for coating nanoparticles, which comprises the following steps:

[0056] (i) acquisition of the natural composition of the fatty acids and of the polar heads of the lipids, with methods comprising lipidomics, belonging to an extracellular vesicle, biovesicle, nanovesicle, ectosome, deriving from a specific tissue, organ, biological liquid, primary cell, immortalized cell line;

[0057] (ii) the families of phospholipids present, the sum of which by mass quantity must constitute 100%, are subdivided into two groups: the first depending on the nature of the polar heads and the second based on the nature of the fatty acids that constitute the hydrophobic chains of the phospholipids;

[0058] (iii) made 100 the total mass quantity of fatty acids present divided by classes, the quantities of fatty acids lower than 1.5% by mass percentage are neglected;

[0059] (iv) the percentages of the remaining fatty acids are rebalanced, redistributing the percentage corresponding to the total of the neglected phospholipids to the percentage of the phospholipids selected in a weighed manner (in relation to their initial percentage), and brought to the total sum of 100% by mass; said formulation is therefore considered the reference formulation, '

[0060] (v) the closest commercially available examples of phospholipids are attributed to each family of phospholipids (of the first group) present in said reference formulation, respecting where possible the presence of saturated, mono- and polyunsaturated fatty acid chains, the length of the fatty acid chains, the polarity and charge of the polar heads;

[0061] (vi) further components such as monounsaturated phosphatidyl-ethanolamine, present in the PEGylated form DSPE-PEG with amino and maleimide functional group or -NHS ester, are added, to the detriment of other components of the same phospholipid family, if present, so as to maintain the total percentage of phospholipids equal to 100%; alternatively, these additional components can be added, rebalancing all the other species, removing in a weighted manner (in relation to their initial percentage) from each component the corresponding part of the percentage of the additional component to obtain 100% of total mass in lipids;

[0062] (vii) cholesterol is added to the formulation, in an amount ranging from 13 to 30%, thus obtaining the final composition of the formulation considered the biomimetic formulation, which is used for the coating of the nanoparticles.

[0063] In one aspect of the invention, the core of the coated nanoparticle according to the invention is selected from silica nanoparticles, organosilica, metal oxide, nanozyme, semiconductor, perovskite, quantum dots, semiconductor material, metal organic framework (MOF), covalent organic frameworks (COFs), carbon-based nanoparticle, in particular nanoonion and fullerene, natural or synthetic polymer, in particular gelatin and PLGA and hydrogel; preferably said core is an organosilica or a metal oxide, wherein said metal oxide is preferably zinc oxide.

[0064] The core can itself perform the function of a therapeutic, diagnostic or theranostic agent, as in the case of a nanoparticle formed by zinc oxide, capable of causing a toxic effect by activation with ultrasound. Alternatively, the core may contain and deliver a therapeutic, diagnostic or theranostic agent (cargo) which, following an exogenous or endogenous mechanism, is released, usually through disintegration of the core itself.

[0065] In a preferred aspect, the core is a porous organosilica nanoparticle selected from mesoporous silica, organosilica nanocage, and organosilica nanocapsule.

[0066] The porous organosilica has several useful properties: i) it has a high porosity, ii) a large surface, iii) the pores are uniform and easily modifiable in terms of diameter; finally, iv) it is highly biocompatible.

[0067] In a further preferred aspect, the organosilica incorporates disulfide groups -S-S-, which are cleaved in the presence of enzymes or reducing agents, thus allowing the therapeutic or diagnostic load to be relinquished.

[0068] In addition, organosilica can be functionalized with linear or cyclic alkyl silanes, which by increasing the zeta potential of the organosilica favour the subsequent attack of the lipidic formulation, in particular the following cyclooctane silane of formula (I):

[0069] Preferably, the core of the coated nanoparticle according to the invention has a hydrodynamic diameter ranging from 20 to 200 nm and a positive z-potential. In a further aspect of the invention, the core of the coated nanoparticle according to the invention is loaded with a therapeutic agent selected from anticancer drugs, antibiotics, anti-inflammatories, hormones, proteins, enzymes, nucleic acids, peptides; or with a diagnostic agent selected from fluorescent organic dyes, nuclear magnetic resonance contrast agents and isotopes for PET (Positron Emission Tomography).

[0070] Some examples of drugs particularly suitable for being delivered within the core of the nanoparticle comprise anticancer agents, antibiotics, anti-inflammatories, corticosteroids, inhibitors, biomolecules, proteins, enzymes, nucleic acids.

[0071] The coated nanoparticle according to the invention, loaded with the therapeutic or diagnostic agent, can be used in a therapeutic or diagnostic treatment method involving the administration of said nanoparticle into a subject in need of such treatment.

[0072] Diseases that can be treated in a therapeutical or diagnostical manner according to the invention include tumour disease, in particular tumours of the colorectum, pancreas (PDAC- Pancreatic Ductal AdenoCarcinoma), cervix, osteosarcoma, leukaemia, lymphoma, multiple myeloma, breast, melanoma; neurological disease, such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, Huntington's disease.

[0073] A further object of the invention is a system for the delivery of a therapeutic or diagnostic agent to a target cell, tissue or organ, comprising a nanoparticle according to the invention.

[0074] A further object of the invention is a diagnostic, therapeutic or theranostic composition comprising a coated nanoparticle according to the invention, together with pharmaceutically acceptable vehicles or excipients.

[0075] EXAMPLES

[0076] Example 1 - Organosilica nanocages (OSCs) 10 nm

[0077] The synthesis of the nanomaterial took place by adding 0.23 mmol of CTAB and 2 ml of 0.02 M aqueous ammonium hydroxide solution in 8 ml of distilled water (dH2O). Then the solution was stirred at 30°C for 30 minutes until complete dissolution of the CTAB. Subsequently, 0.43 mmol of TMOS and 0.215 mmol of BDTS (in case of NPs degradable thanks to the presence of S-S bonds) were added to the solution under vigorous stirring and the solution was further stirred at 30°C for 24 hours. In the next step, the temperature was increased from 30°C to 80°C and then stirred at 80°C for another 24 hours. Subsequently, the solution was cooled to room temperature and then dialyzed into 100 ml of acid solution (mixture of ethanol, dH2O and acetic acid with the volume ratio 1 : 1 :0.007) for 24 hours to remove CTAB from the particle pores. This process was repeated three times. The solution was then dialyzed in 2 L of dH2O for another 24 hours. Figure 1 includes the characterization of OSCs with a size less than 10 nm. Transmission electron microscope (TEM) images revealed the presence of a single pore in nanoparticles of less than 10 nm (Figure 1 A). According to the TEM results, the nanoparticles show a diameter of 9.3 nm, a hydrodynamic diameter of 12 analysed by DLS and a negative zeta potential (Figure IB). In order to verify the correct elimination of the surfactant used during the synthesis strategy and the presence of the S-S bonds that provide the NPS with degradability properties, FTIR analysis (Figure 1C) and TGA analysis (Figure ID) were performed. The ssOSCs showed a typical spectrum characteristic of OSCs with the peaks Si-OH (3445 cm'1) Si-O-Si (1077 cm'1) Si-OH (947 cm'1) and C-H (805 cm'1) characteristic of the organosilica structure. The presence of the S-S bonds in the silica structure was confirmed by FTIR and TGA analysis. When S-S was incorporated into the silica structure, peaks around 2900 and 1500 cm‘l were observed in the FTIR spectra (Figure 1C). The TGA analysis indicates that all the CTAB used in the synthesis was completely eliminated and allows to quantify the amount of degradable groups introduced into the organosilica structure (about 20%).

[0078] Example 2 - Organosilica nanocages (OSCs) 20 nm

[0079] In a 100 ml round-bottomed glass flask equipped with a magnetic stir bar, CTAB (408 mg, 1.12 mmol) was dissolved in deonized water (50 ml) at 50°C, stirring the solution for 30 minutes at 250 rpm. Ammonia (12.5 pL, 28% in water) was added and the stirring speed was increased to 750 rpm. A solution of TEOS (448.1 pL, 2 mmol) was then added and the mixture was stirred for 20 hours at 50°C, at 750 rpm. The particle dispersion was purified by dialysis in an EtOH / H2O / AcOH (1 : 1 :0.007 v / v / v) mixture for 24 hours and finally in an EtOH / H2O (1 : 1 v / v) mixture for another 24 hours in which the dialysis solution was replaced every 2 hours, when possible. BTDS (102.8 pL, 0.223 mmol) was also added in order to have particles degradable through S-S groups. Transmission electron microscopy (TEM) images revealed the presence of a single pore in the 20 nm cage-shaped nanoparticles (Figure 2A). According to the TEM results, the nanoparticles showed a diameter of 21.3 nm, while this is around 18 nm if analysed by DLS (hydrodynamic diameter, Figure 2B) and a negative zeta potential -10.98 mV. In order to verify the correct elimination of the surfactant used during the synthesis strategy and the presence of the S- S bonds that provide the OSCs with degradability properties, FTIR analyses (Figure 2C) and TGA analyses (Figure 2D) were performed. The ssOSCs showed a typical characteristic spectrum with peaks Si-OH (3445 Si-OH (947 representative of the organosilica structure. The presence of S- S bonds in the silica structure was confirmed by FTIR and TGA. When S-S was incorporated into the silica structure, peaks around 2900 and 1500 cm'l were observed in the FTIR spectra (Figure 2C). TGA analysis indicates that all CTAB employed in the synthesis approach was completely eliminated and allows to quantify the amount of degradable groups introduced into the silica structure (again, about 20%, Figure 2E).

[0080] Example 3 - 20 nm meso porous organosilica (ssMSN)

[0081] In a round-bottomed flask (250 ml), the CTAB (816 mg) was dissolved in a solution of dH2O (100 ml), the solution was heated to 50°C and stirred (between 500-750 rpm). for 30 min. Then, 25 pL of 28% NH40H, followed by 1 mL of TEOS were added consecutively into the solution. After 12 h of reaction at 50°C (with flask closed) the solution was cooled to room temperature and then transferred to a tubular dialysis membrane. The solution was dialyzed in 1 L of acidic solution (mixture of dH2O, ethanol and acetic acid with volume ratio 1 : 1 :0.007) for 24 hours to extract the CTAB from the pores of the particles. The solution was then dialyzed in 2 L of dH2O for another 24 hours. This process was repeated again three times. BTDS (241 pL, 0.526 mmol) was also added in order to have particles degradable through S-S groups. TEM analysis confirmed the synthesis of 20 nm ssMSN (Figure 3A). DLS allowed to detect a mean hydrodynamic diameter of 21 nm with a Gaussian distribution confirming the good colloidal stability of the particle suspension. The zeta potential measurements were recorded in water, obtaining a negative zeta potential characteristic of silica (-8 mV). The obtained hydrodynamic diameter confirms the expected size of 20 nm for ssMSN (Figure 3B). From the FTIR analysis (Figure 3C) the characteristic bands of Si-OH (3445 Si-OH (947 were observed and it can be confirmed that all the CTAB (surfactant used during the synthesis) was completely eliminated. TGA analysis (Figure 3D) revealed that almost 100% of the mass of the MSNs correspond to inorganic compounds, and therefore to silica. This result confirmed once again the complete elimination of the surfactant used during the synthesis. Like the previous samples, MSNs were synthesized both in the presence and absence of disulfide bonds within their structure. The presence of S-S bonds in the silica structure was confirmed by FTIR and TGA (Figure 3D and E). When S-S was incorporated into the silica structure, peaks around 2900 and 1500 cm"! were observed in the FTIR spectra (Figure 3C). In addition, TGA results demonstrated that when S-S was incorporated into MSNs, a decrease in ssMSN sample weight around 400°C was observed, corresponding to about 15% of the sample mass. This decrease was not attributed to the CTAB, which has a lower combustion temperature, but to the presence of the S-S disulfide groups (grey line in the graph of Figure 3E).

[0082] Example 4 - Nanocapsules (NCs)

[0083] Organosilica degradable hybrid nanocapsules were synthesized using the Stober process in a water / oil (W / O) microemulsion prepared by mixing 1.77 mL of TRITON X- 100, 7.5 mL of cyclohexane and 1.8 mL of n-hexanol in a 100 mL round-bottomed flask and stirring on a magnetic stirrer for 15 minutes. Separately, 600 pL of water were mixed with 40 pL of TEOS and 60 pL of BTDS. After stirring, this mixture was added to the organic solution. TEOS hydrolysis was initiated by addition of 50 pL of 28% aq.NH3and the mixture was stirred at room temperature for 5 hours.

[0084] Subsequently 20 mL of pure acetone were added to precipitate the NPs and the material was recovered by centrifugation. Figure 4A includes TEM images of naked nanocapsules, coated with PEG and functionalized with NH2. All nanocapsules samples showed a diameter of between 30 and 50 nm. These diameters are in agreement with the hydrodynamic diameter obtained from the DLS analysis (50- 60 nm) shown in Figure 4B. Figure 4D shows the zeta potential of nanocapsules, demonstrating that the presence of PEG does not affect the surface charge and that, conversely, the presence of NH2produces a zeta potential value less negative and closer to neutrality. Figure 4C shows the fluorescence emission spectra of the nanocapsules conjugated to the fluorescent dye Cy5.

[0085] Example 5 - Functionalization of nanocapsules (NCs)

[0086] In consideration of the negative value of the Zeta potential of the nanocapsules @NH2, also coated by amino groups, a new functionalization was tested, using a cyclic silane group (Figure 5), which has a greater number of amino groups and a greater steric bulk on the organosilica surface. In this case, 10% of the cyclic silane was incubated with the NCs overnight at room temperature using toluene as solvent. Subsequently, the functionalized NCs were washed with dH2O for further characterization. Both the hydrodynamic diameter (Figure 6A) and the zeta potential (Figure 6B) measured at DLS demonstrated the functionalization of NCs with cyclic silane (an increase in hydrodynamic diameter and a change in zeta potential of the NCs from negative to positive was clearly observed). In addition, the presence of the amino groups from the cyclic silane was observed by FTIR: 1523 cm NH2, 1626 cm NH2, 2600-3650 cm NH2(Figure 6C). Using TGA, it was possible to quantify the amount of S-S groups present in both naked NCs and functionalized NCs, which is approximately 30% of the sample mass (Figure 6D). In the functionalized NCs, another weight decay was observed at a higher temperature corresponding to a 15% of mass loss attributed to the presence of silacyclooctane.

[0087] Example 6 - Synthesis of zinc oxide (ZnO) nanocrystals and their functionalization

[0088] The synthesis of zinc oxide nanocrystals took place by wet chemical method, exploiting a sol-gel solution with a zinc precursor and oleic acid as a stabilising agent. The zinc acetate dihydrate reagent (526 mg, ACS Reagent, Sigma-Aldrich) was dissolved in 40 mL of ethanol and heated to 70°C. Bidistilled water (bd, 1 mL, produced by a Direct Q3 system, Millipore, Burlington) and oleic acid (140 pL, Sigma-Aldrich), tetramethylammonium hydroxide (1.044 mg, TMAH, Sigma-Aldrich) previously dissolved in 1052 mL of bd water and 10 mL of ethanol were added to the solution. After 10 minutes, ZnO nanocrystals (NCs) were recovered by centrifugation at 14000 g for 10 minutes and resuspended in ethanol. This washing procedure was repeated three times. The surface of the ZnO NCs was then decorated with amino-propyl functional groups by adding 10 mol% of 3-aminopropyletrimethoxysilane (APTMS, Sigma- Aldrich). More in detail, the ZnO NCs were dispersed in ethanol at a concentration of 2.5 mg / mL and heated up to 70°C under nitrogen atmosphere and reflux conditions. The APTMS was added to the solution, and the reaction was conducted for 6 hours at 70°C. At the end of the procedure, the NCs were collected and washed three times through a process of centrifugation and redispersion in fresh ethanol and stored as ethanol colloidal suspensions.

[0089] For the characterization of the ZnO NCs as such and functionalized with aminopropyl groups (Figure 7), Dynamic Light Scattering (DLS) (Figure 7A) and Z-Potential (Figure 7C) measurements were carried out with the Zetasizer Nano ZS90 instrument (Malvern Instruments) in bd water at a concentration of 100 pg / mL. NTA (Nanoparticles Tracking Analysis, Figure 7B) measurements were performed on ZnO NCs functionalized with a NanoSight NS300 (Malvern Panalytical). To prepare the sample, 6 pL of 1 mg / mL NCs solution was diluted up to 1 mL in bidistilled water. For each sample, three 60-sec videos of the samples flowing in the instrument chamber were captured and analysed with NTA 3.4 (Malvern Panalytical) software.

[0090] Example 7 - Realization of lipidic formulations, 3C+and 3C~ for lipid coating The 3C+and 30 formulations were realized to create a highly customizable lipid shell that is electrostatically charged with the aim of acting as a nanoparticle coating, as a drug delivery system, precisely by virtue of an electrostatic attraction between lipid shell and internal load.

[0091] The approach followed to devise the formulation therefore consisted of using permanently charged lipids (in the case of the 30 formulation negatively charged, for the 30 formulation positively charged) and maintaining their majority ratio with respect to the other individual components of the lipid shell, selected from neutral lipids, cholesterol and lipids containing a polyethylene glycol (PEG) group, obtaining a molar ratio of 50: 10:38.5: 1.5, respectively.

[0092] For the purpose of a comparison between the charges of these formulations, use was made of a control formulation called 2Cn with neutral charge (consisting as follows: DOPC 71.2%; cholesterol 22.5%, DSPE-PEG(2000)amine 5.8%; DSPE_PEG(2000) maleimide 0.1% ),

[0093] In order to be able to use exclusively commercial lipids and whose use was already abundantly established, this proportion was used in 30 formulation, specifically composed of 3 lipids: DOPA (18: 1 PA, l,2-dioleoyl-sn-glycero-3 -phosphate (sodium salt), in chloroform solution), DOPC (18: 1 (A9-Cis) PC (DOPC), l,2-dioleoyl-sn-glycero-3- phosphocholine, in chloroform solution), DSPE-PEG(2000) Amines (1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[amino(polyethylene-glycol)-2000] (ammonium salt) and from Cholesterol in chloroform.

[0094] Similarly, the 3C+formulation (with 3 lipids and cholesterol) maintains the same molar ratios as 30 but replaces the negatively charged lipid (DOPA) with a positively charged one the DOTAP (18:1 TAP, l,2-dioleoyl-3-trimethylammonium-propane (chloride salt). Finally, as a completely neutral control formulation, only DOPC neutral lipid is employed in the 2Cn formulation, as described above.

[0095] With regards to the presence of the PEG group, the lipid that contains it has a triple function, that of:

[0096] (i) stabilizing the shell in an aqueous environment and, in future applications, physiological one, ensuring greater circulation and consequently maximizing the probability of reaching the tissue under study protecting the content from degradation;

[0097] (ii) allowing the bond through simple chemical reactions of peptides and antibody fragments, allowing a very varied customization of the shell depending on future use;

[0098] (iii) conferring the so-called “stealth” property to the nanoparticles, i.e. avoiding immediate recognition thereof by the immune system and therefore the foreign body response.

[0099] To this end, the lipid with PEG group used is DSPE-PEG(2000) Maleimide (1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (ammonium salt), whose cysteine of the maleimide group can, in fact, react, under certain conditions, with amino acid groups of peptides or antibody fragments. This lipid, defined as “functional lipid”, is added in a molar fraction ranging between 0.1% and 0.5% in replacement of part of the lipid DSPE-PEG(2000)-Amine.

[0100] In addition, the lipid with PEG group used is the DSPE-PEG(2000) carboxy NHS ester, l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy (polyethylene glycol)- 2000 N-hydroxysuccinimide ester, whose NHS group can, in fact, react, under certain conditions, with amino groups of peptides or proteins, including antibodies. This lipid, also defined as “functional lipid”, is added in a molar fraction ranging between 0.1% and 0.5% in replacement of part of the DSPE-PEG(2000)- Amine lipid.

[0101] Below, a table (Table 1) summarizing the ranges of the mass fraction, and one of them in the preferred version (Table 2). In Table 1 it should be borne in mind that the summation of values used in the molar fraction in all their possible combinations must always be 100%.

[0102] Table 1 Table 2

[0103] 3C formulation is considered the most relevant and was designed to interact electrostatically with positively charged nanoparticles, ensuring stable coating over time and reducing the risks related to the use of highly positively charged biomaterials in the body, which have the tendency, widely demonstrated in clinical and materials science, to favour blood coagulation and protein absorption on their surface. The markedly negative charge of the constructs coated by the 30 formulation, combined with the presence of the PEG group with anti-fouling effect, guarantee prolonged circulation and stability in biological environments that lends itself perfectly to future clinical uses, offering a very high degree of customisation thanks to the possible surface functionalizations allowed by the presence of the DSPE-PEG(2000) Maleimide.

[0104] The amount of PEG inserted in the shell is the right compromise to allow the customization of the lipidic shell without arising adverse reactions deriving from the massive presence of PEG in the body, recently identified as an allergy risk factor that unites nanotechnologies as well as cosmetic and medical products used in everyday life. Finally, the molar proportions of the various components of the shell are able to maximize cargo packing thanks to the massive presence of charged lipids, while the presence of cholesterol in these amounts gives the lipid nanoparticles a fair degree of both fluidity and structural solidity.

[0105] Once obtained, the lipidic formulations were characterized and the results of the characterization are presented in Table 3: Table 3

[0106] Example 8 - Realization of MIMIC 1 and 3 lipidic formulations for lipid coating

[0107] For the realization of the MIMIC 1 and 3 formulations, the starting point was the study of the state of the art with regard to the composition of natural EVs from the PC3 prostate cancer line. The aforesaid line produces in fact extracellular vesicles that have a natural tropism towards another tissue, in this specific case the skeletal system. Therefore, in order to reach the bone tissue with therapeutic purposes and with the awareness that the lipid composition plays a fundamental role in the chemical-physical properties of the vesicles, and that the latter in turn play a role during the recognition and internalization of a particular cell species, we tried to create artificial lipidic formulations that gradually became more and more similar to that of natural vesicles.

[0108] Among the articles found in the literature on the composition of EVs derived from PC3 prostate cancer cells, one in particular by Ferreri et al. (C. Ferreri et al., “The n-10 fatty acids family in the lipidome of human prostatic adenocarcinoma cell membranes and extracellular vesicles, ” Cancers (Basel)., vol. 12, no. 4, pp. 1-16, 2020, doi: 10.3390 / cancersl2040900) was selected which reports the mass percentages of both the lipid families that make up this type of natural vesicles, and the distribution of fatty acids, which make up the hydrophobic part of the lipid bilayer. While the composition concerning the lipid families is easily reproducible and consists of cholesterol and 4 other types of phospholipids (phosphatidyl-serine, phosphatidyl-choline and phosphatidyl-ethanolamine, sphingomyelins), that of the fatty acids is rather complex. Furthermore, the connection and distribution between the hydrophilic heads of phospholipids and sphingomyelins and the relative fatty acids, which constitute their characteristic hydrophobic tails, is not known.

[0109] Therefore, an attempt was made to obtain artificial compositions that would satisfy first of all the massive ratios between the hydrophilic heads, considered biologically more relevant, as they are ideally exposed in the first line in the lipid bilayer. At the same time, a compromise was sought that would satisfy also the balancing between fatty acids as much as possible, with particular attention to the balance between the 3 most well-known categories of fatty acids: saturated, monounsaturated, and polyunsaturated. Finally, the three formulations constitute an evolution from 30 formulation, used as a starting point, up to reaching the reference composition, which is a simplification of the natural composition of the EVs reported in Ferrari et al.

[0110] To obtain the reference composition, the natural composition of the fatty acids was simplified, while that of the lipid families was maintained. In particular, fatty acids present in a mass percentage of less than 1.5% have been neglected (this cut represents only slightly more than 5% with respect to the total mass of lipids). The percentages of the remaining fatty acids were rebalanced accordingly, respecting the ratios between the initial masses. Once the reference formulation was obtained, we started from 30 formulation and arrived through consecutive steps at a commercial lipid composition as close as possible to the reference, but maintaining a low number of components for practicality.

[0111] The MIMIC 1 composition is given by the simple rebalancing of the lipid families present in 30 formulation and with the insertion of sphingomyelins. In this way, all lipid families of the reference are present, with the exception of phosphatidyl-serines, whose mass percentage was assigned to the anionic phospholipid DOPA. The latter is in fact the cornerstone of the 30 formulation, which bases its functioning on the electrostatic interaction between the positively charged surface of the nanoparticles and the net negative charge conferred on the lipid mix precisely thanks to the presence of this particular phospholipid. In this case, less attention was paid to the composition of the fatty acids, and the result is a natural rebalancing of the elements already present in 30 formulation. Noteworthy is only the insertion of a new saturated fatty acid, C16:0 and a monounsaturated one, Cl 8: 1, thanks to the presence of sphingomyelins.

[0112] The MIMIC 3 composition, besides the addition of phosphatidylserine (PS C16:0), involves the insertion of a new type of fatty acids compared to the previous formulation. The category inserted is the one of the polyunsaturated fatty acids by replacing part of the percentage of monounsaturated phosphatidyl-ethanolamine, present in the PEGylated form DSPE-PEG, with PE 18:2. In this way it is possible to study the effect of the presence of polyunsaturated lipids in the behaviour of the lipid bilayer. With this addition and considering the presence of the errors reported in the starting natural composition, it can be stated that the MIMIC 3 formulation, with good approximation, reaches the correct balance between the families of hydrophilic heads and the categories of fatty acids.

[0113] The following tables report two preferred forms for the MIMIC 1 and 3 compositions: they both contain the same proportions of lipids, but in case 1 there is 13% by mass of cholesterol, whereas in case 2 there is 30% by mass of cholesterol. The sum of all components is always equal to 100%. On the side, the % range of values that the compositions can have is reported.

[0114] Table 4. Compositions of the MIMIC 1 and 3 formulations, in their two preferred forms (case 1 and case 2) and with the variation range% of the components (mass %). The 30 formulations are also reported, as a starting point and comparison for MIMIC formulations and the formulation of the EVs from PC3 cells (prostate cancer) as an end arrival point. Table 5. Summary table of the mass % between the components of the different formulations (only for the preferred case 1), compared with the reference composition of the EVs extracted from PC3. The table is subdivided into three parts representing: the polar heads (top) the fatty acids (middle) and the three categories of fatty acids (bottom).

[0115] Example 9 - Method for coating nanoparticles

[0116] Solvent exchange method

[0117] This technique consists in preparing a formulation of different phospholipids and cholesterol, taking them from their stock chloroform solution and rehydrating them with a 40% v ethanol and 60% v water solution. The nanoparticles are then initially mixed with the lipid solution and then the water is added in excess. The rapid increase in water content is fundamental to this method, as it drives the self-assembly of phospholipids on the surface of the nanoparticles. In short, after allowing the desired lipidic formulation to dry overnight under vacuum, these were hydrated with a solution composed of ethanol (99%, Sigma Aldrich) and bidistilled water in a volumetric proportion of 40%-60%. This ratio was carefully designed to avoid any unwanted lipid self-assembly, and in this regard, ethanol was added as the first solvent. The resulting dispersion of lipids, whose colour turns whitish after the addition of water, is stable and stored at 4°C for the subsequent steps.

[0118] The coating process consists of taking a certain amount (defined later for the optimized NPs coating protocol) of nanoparticles from the ethanol solution by centrifugation at 14000 g for 10 minutes and removing the supernatant. Subsequently, addition is made of an amount of lipid solution to the nanoparticle pellet with a weight ratio of 2: 1 (preferred option) or a 1 : 1 nanoparticle with respect to the lipids, and a first sonication step (3 minutes) is carried out using an ultrasonic bath (59 kHz, Branson 3800 CPXH, Branson Ultrasonics Corporation), to allow a good dispersion of the nanoparticles and lipids. Subsequently, to self-assemble the lipid bilayer on the surface of the nanoparticles, excess bidistilled water is added in relation to the mixture, until an ethanol / water volume ratio of 1 :9 is reached. The final suspension is subjected to a second sonication cycle (5 minutes) to homogenize the system and to aid in the formation of a homogeneous self-assembled lipid bilayer enclosing the nanoparticles.

[0119] Example 10 - Characterization of the lipid-coated nanoparticles

[0120] To characterize the dimensional distribution and colloidal stability of the coated nanoparticles, DLS and Z-Potential (Zetasizer Nano ZS90 from Malvern Instruments) and Nanoparticles Tracking Analysis (NT A, NanoSight NS300 from Malvern Panalytical) were performed in deionized water and at room temperature. All measurements were conducted 3 times and then averaged. For the analyses, an amount corresponding to 100 pg of NPs was taken from the suspension and added to 900 pL of bidistilled water and analysed. For NTA analysis, for each sample, three 60-second videos of the samples flowing through the instrument chamber were recorded and analysed with Malvern Panalytical's NTA 3.4 software. The coating with 30 and 3C+formulations was tested on different types of nanoparticles, which were coated depending on the zeta potential value measured on the particles as such (the lipidic formulation with opposite charge is used to exploit the electrostatic interaction with the nanoparticle and ensure a correct coating, or the neutral charge formulation as a control) and subsequently characterized. Below are some examples of characterization of coated nanoparticles: zinc oxide nanoparticles (Figure 7), organosilica nanocages (Figure 8), organosilica nanocapsules (Figure 9) and polymeric nanoparticles (Figure 10). Similarly, the coating of the particles with MIMIC 1 and 3 formulations was tested and characterized and compared with 30 formulation, of which the MIMIC formulations represent an evolution. Some exemplary results are reported in Figure 11.

[0121] In detail, Figure 7 shows the characterization of the zinc oxide (ZnO) nanoparticles coated with 30 formulation and compares the data with the same naked nanoparticles. In terms of dimensional distribution, the coated ZnO nanoparticles of 30 formulation are monodisperse and with a hydrodynamic diameter around 100 nm, as detected by the analyses carried out by DLS (Figure 7A) and NTA (Figure 7B). In contrast, the naked zinc oxide NPs show aggregation to NTA (black curve in Figure 7B). The Z-potential and zeta potential of the naked ZnO NPs is positive, about +25 mV, as these NPs are functionalized with aminopropyl groups. Once coated by 30 formulation, the Z potential turns out to be negative, equal to -20 mV, to demonstrate the efficacy of the coating (Figure 7C).

[0122] Figure 8 shows the data of degradable organosilica nanocages that are unfunctionalized (ssOSCs), functionalized with PEG (ssOSCs @PEG) and with NH2 (ssOSCs @NH2) groups as such and coated by 2Cn and 30 formulation. The analyses of DLS (Dynalmic Light Scattering) in Figure 8A and of the Zeta potential (Figure 8B) show that the ssOSCs as such have a size equal to 60 nm and a neutral Z potential: when they are coated with the 2Cn formulation or with the 30 formulation, their dimensional distribution remains unchanged and the Z potential changes a lot, which becomes very positive (about +60mV) with the 2Cn formulation and very negative (-40m V) using the 30 formulation. When PEG functionalization is present on ssOSCs, they are smaller (about 30 nm from DLS, Figure 8A) and weakly negative (-15 mV of Z potential, Figure 8B) and can be coated with the neutral 2Cn formulation, demonstrating an increase in hydrodynamic diameter (60 nm, Figure 8A) and a Z potential close to neutrality (+8 mV). Finally, analyses on organosilica nanocages functionalized with NH2 (ssOSCs-NFE) groups as such show a size in the DLS of approximately 20 nm (Figure 8A in DLS and Figure 8C in NTA) and positive charge (+50 mV, Figure 8B). When they are coated by 2Cn formulation or 30 formulation their size increases respectively to 80 nm and 120 nm (Figures 8A and 8C) and the Z potential varies depending on the charge of the lipidic formulation: with the 2Cn formulation it becomes more neutral (+ 30 mV) and with the 30 formulation it becomes very negative (-50 mV) to demonstrate the effective coating of NPs with lipids. NTA analysis (Nanoparticle Tracking Analysis, Figure 8D) was done on the ssOSCs-NFE sample, coated by 30, formulation which demonstrates good monodispersity and a peak at 126 nm. FTIR analysis of ssOSCs-NFE and ssOSCs-NH 2 coated by 30 formulation (Figure 8E) show that in the coated sample, the characteristic peaks of the lipids in the area from 3200 cm'l to 1500 cm‘l, along with those of silica, are clearly visible. Thermogravimetric evaluation (TGA, Figure 8F) of ssOSCs-NFE and SSOSCS-NH2 coated by 30 formulation shows that the recorded weight loss corresponds to the amount of lipids added to the nanoparticles for coating, i.e. 50% by mass with respect to the nanoparticles.

[0123] Figure 9 reports the DLS analysis (Figure 9A) and Zeta potential measurement (Figure 9B) of organosilica nanocapsules that are unfunctionalized (NCs), functionalized with PEG (NCs@PEG) and with NH2 (NCs@NH2) groups as such and coated by 3C+formulation and as a control with neutral 2Cn formulation. It can be clearly noted that when NCs are coated with 2Cn and 3C+lipids, their hydrodynamic diameter increases compared to NCs as such and that the Z potential varies greatly depending on the charge of the formulation used. In particular, NCs are originally with negative Z potential (about -22 mV) and the coating with the 3C+formulation brings the Z potential to clearly positive values (about +30 mV).

[0124] Figure 10 shows the characterization of polymeric nanoparticles as such and coated by lipids. The measure of the Zeta potential of particles of PLGA, chitosan-functionalized PLGA (PLGA-CS) and gelatin as such changes considerably once they are coated by the 3C+or 30 formulations depending on the starting Zeta potential (Figure 10 A) as the electrostatic interaction between the polymer surface and the lipidic formulation is exploited. DLS analyses of the PLGA particles as such show an increase in size when they are coated by the 3 C+formulation (Figure 10B) and similarly this happens for the Gelatin particles as such and coated by the 30 formulation and of chitosan-functionalized PLGA (PLGA-CS) as such and coated by the 30 formulation.

[0125] Figure 11 reports the characterization of degradable nanocapsules functionalized with NH2 (NCs) groups as such and coated by 30, MIMIC 1, MIMIC3 Formulation. The DLS analyses (Figure 11 A) show an increase in hydrodynamic diameter when NCs are coated and a considerable change in Zeta potential from positive for uncoated NCs to negative for NCs coated with the various formulations used (Figure 1 IB). Finally, the NTA analyses (Figure 11C) show the monodispersion of the NCs coated by MIMIC 1 Formulation with a peak at 112 nm.

[0126] Example 11 - Encapsulation of calcein and Doxorubicin in the formulation of ssOSCs nanoparticles with lipid coating of the 3C~ type

[0127] Encapsulation of calcein in the ssOSCs particles was achieved as follows: 50 mg of ssOSCs were suspended in 10 mL EtOH containing 2M of CaC12. In parallel, calcein (50 mg) was also dissolved in 10 mL of 10 mL of 2M EtOH of CaC12. Both suspensions were sonicated for 10 minutes separately. Subsequently, they were mixed and briefly sonicated for another 20 minutes and stirred for 12 hours at room temperature. To recover calcein- loaded ssOSCs (ssOSCs@calcein), these were washed and centrifuged at 40000 g for 30 minutes with EtOH (10 mL x 3 times) and water (10 mL x 3 times). The resulting material was finally functionalized with the positive silane as mentioned above, resuspended in dH2O and maintained at 4°C until coating with the lipidic formulation 30.

[0128] The calcein content loaded into the ssOSCs was analysed by UV-VIS, fluorescence spectroscopy, TEM, FTIR, and DLS (Figure 12). Calcein-loaded ssOSCs showed a hydrodynamic diameter of between 20 and 40 nm (comparable to empty NPs). The surface charge analysis of the samples demonstrated that the zeta potential of the OSCs did not change after the encapsulation of the calcein, being still negative. FTIR analysis demonstrated the presence of silica peaks together with the characteristic bands of calcein. The emission spectra and the absorption pattern of the nanoparticle upon excitation at 480 nm were recorded. All these data demonstrated that calcein was successfully encapsulated.

[0129] Then, the positively surface charged calcein-loaded ssOSCs were electrostatically coated with the phospholipidic formulation 30 as described above. Figure 13 demonstrates the correct lipid coating of the calcein-loaded NPs. FTIR analysis demonstrated the presence of the characteristic bands from the silica structure, of the calcein molecule and of the lipids on their surface. DLS analysis showed a hydrodynamic diameter of about 130 nm and a negative surface charge of calcein-loaded ssOSCs after coating with the lipidic formulation 30.

[0130] Doxorubicin (20 mg) was dissolved in 5 mL of EtOH and sonicated for 10 minutes. Then, nanocages (ssOSCs @D0X) were added, briefly sonicated for another 20 minutes and stirred for 12 hours at room temperature. Subsequently, to force encapsulation, the vacuum was exploited and the sample was placed in the rotary evaporator for a few minutes until complete evaporation of the solvent.

[0131] Doxorubicin-loaded nanomaterials were characterized by DLS, zeta potential, FTIR, and absorption spectrum. TEM images confirmed that even after encapsulation of doxorubicin, the NPs were still monodisperse and with a diameter of about 20 nm (Figure 14A). DLS of ssOSCs @D0X (Figure 14B), corroborated by TEM results, confirmed that doxorubicin was encapsulated within the pore of the NPs rather than attached to the surface. In addition, the zeta potential of ssOSCs@DOX was in the range of empty NPs (-8.3 ± 1.3 mV). In the FTIR spectra of Figure 14C, the characteristic peaks of organosilica NPs were observed together with the major bands of doxorubicin (the characteristic sharp peak at 1727 cm‘l is correlated to the carbonyl (C=O) of pure doxorubicin). Finally, the absorbance spectrum of ssOSCs@DOX showing a maximum absorption peak at 470 nm was also recorded (Figure 14D).

[0132] The ssOSCs@DOX were then functionalized with the amino-silane and further coated with the negative lipids, as previously described for the calcein-loaded samples. Figure 15A in the I S. includes FTIR spectra of ssOSCs@DOX-NH2-negative lipids. It is possible to observe the presence of the characteristic bands coming from the organosilica groups, from the breakable disulfide bonds, from the fingerprint of doxorubicin and from the lipid bands. The hydrodynamic diameter obtained from DLS was about 100 nm (Figure 15B) and the zeta potential was of -29.2 + / - 2.2 mV, demonstrating once again the success of the coating the silica core with the lipid shell.

[0133] Example 12 - Conjugation of the functional lipid DSPE-PEG(2000) Maleimide with a peptide or antibody to increase targeting and homing to cancer cells of the coated nanoparticles

[0134] One of the key characteristics of the lipidic formulations proposed herein is their possibility of being adapted to the pathology of interest, thanks to the presence of the functional lipid DSPE-PEG(2000) Maleimide. In fact, by means of simple chemical reactions it is possible to conjugate to the maleimide group biomolecules, such as proteins, peptides and antibodies or antibody fragments that exhibit thiol (-SH) and / or cysteine groups.

[0135] Conjugation with targeting peptides

[0136] For example, to be selective towards the Wnt-2 receptor present among others on cells of the pancreatic cancer line (PDAC), such as Bx-PC3, a custom-made peptide was constructed, called CKAAKN (S.Barui, N. M. Percivalle, M. Conte, B. Dumontel, L. Racca, M. Carofiglio, V. Cauda “Development of doped ZnO-based biomimicking and tumor-targeted nanotheranostics to improve pancreatic cancer treatment” CANCER NANOTECHNOLOGY, 2022, Vol. 13, Article no. 37, pp. 1-24, ISSN. 1868-6966, doi: 10.1186 / sl2645-022-00140-z.; S. Barui, M. Conte, N.M. Percivalle, R.M. Garcia Montero, L. Racca, M. Allione, V. Cauda_“Dual drug loaded nanotheranostic platforms as a novel synergistic approach to improve pancreatic cancer treatment” PARTICLE & PARTICLE SYSTEMS CHARACTERIZATION, 2023, 2200138, pp.1-16, ISSN: 1521-4117, doi: 10.1002 / ppsc.202200138), and linked to the lipid DSPE-PEG(2000) Mai eimide exploiting cysteine, as reported below. Similarly, to be selective towards cells that overexpress the EphinA2 protein (such as melanoma cells, osteosarcoma, colorectal cancer, to name a few) the targeting peptide called YSA was constructed (M. Koolpe, M. Dail, E.B. Pasquale “An ephrin mimetic peptide that selectively targets the EphA2 receptor” J BIOL CHEM. 2002, 277(49), pp.46974-9, doi: 10.1074 / jbc.M208495200).

[0137] The lipid DSPE-PEG(2000) Maleimide and the peptide of interest were dissolved in N, N-Dimethylformamide (DMF, Sigma Aldrich) in 3 : 1 molar ratio, at the concentration of 37.5 mM and 50 mM, respectively. Then, the peptide solution was diluted in 0.1 M sodium phosphate buffer (PBS, pH 7.4) and the lipid solution DSPE-PEG(2000) Maleimide was then added, obtaining a 1: 1 DMF / PBS mixture and containing 5 mM of peptide and 15 mM of functional lipid. The reaction occurred for 1 hour at room temperatures and the resulting mixture of peptide-conjugated lipid (hence DSPE- PEG(2000)-CKAAKN or DSPE-PEG(2000)-YSA), was frozen at -20°C and stored as stock. Prior to use, the aliquot of interest is diluted 1 : 10 in ethanol and then inserted into the final lipidic formulation to coat the nanoparticles.

[0138] Example 13 - In-cell internalization of the formulations with functional lipid conjugated to targeting agents

[0139] To verify the correct coating of the nanoparticles with the lipidic formulation and the bioconjugation with the targeting functional peptide, the custom-made peptides CKAAKN or YSA were formulated also with a dye with difluorescein isothiocyanate molecules (FITC, from Bio-Fab Research) and therefore linked to the functional lipid DSPE-PEG(2000) maleimide, as described above. The advantage is therefore that the functional lipid bioconjugated with the dyed peptide is visible with UV-Visible spectroscopy and fluorescence techniques, fluorescence microscopy and cytofluorimetry. The FITC dye molecule can in fact be excited at a wavelength of 488 nm and detected at wavelengths of 500-550 nm.

[0140] Example on pancreatic cancer cells

[0141] As reported in Figure 16, ZnO nanoparticles coated with 30 formulation were incubated with Bx-PC3 pancreatic cancer cell cultures for 24 hours. The lipids 30 provide a moderate increase in internalization compared to nanoparticles as such (the figure expresses the% of positive events compared to untreated control cells), indicating that cells treated with Zn0-3O are able to internalize or immobilize nanoparticles on their surface. In the presence of the targeting peptide, in this case CKAAKN, cell internalization is greatly increased. The dyeing of the targeting peptide in this case did not compromise the cell recognition sites, allowing high internalization with both the non-dyed targeting peptide and the one dyed with FITC.

[0142] Example on colorectal cancer cells

[0143] In another use case it was seen how the ZnO NPs coated with the 30 formulation bioconjugated with YSA targeting peptide directed towards colorectal cancer cells (HT-29 line), for which a cytotoxicity study was carried out (Figure 17 A), demonstrating that the coated particles are significantly less cytotoxic than the uncoated nanoparticles up to the highest concentrations. It was subsequently demonstrated that thanks to bioconjugation with the YSA peptide, nanoparticles are able to be more internalized in the cell than particles coated with the same lipidic formulation, but without peptide (Figure 17B). However, it is worth noting the high biomimicry of the lipidic formulation itself, which is already extensively internalized in high% even without the specific peptide. And finally, a hemocompatibility test was carried out, treating the plasma with the particles as such and the particles coated with the 30 formulation, bioconjugated or not with the peptide YSA and measuring the plasma coagulation time following the treatment. Figure 17C reports the results of this experiment and shows how the nanoparticle samples coated, both with and without the peptide, produce a coagulation time that does not differ from that of plasma alone, demonstrating the hemocompatibility of the nanoconstruct and, indirectly, the correct coating of the particles with the lipidic formulation.

[0144] Example 14 - DSPE-PEG(2000)-maleimide phospholipid is used to anchor a targeting moiety (peptide) and related characterization of the obtained NPs (Fig. 18)

[0145] Zinc Oxide Nanocrystals (ZnO) were coated with the 30 formulation exploiting a solvent exchange method: owing to the electrostatic interaction among the positively charged nanocrystals and the mainly negatively charged lipid mixture, the lipids easily selfassemble upon hydration forming a thin bilayer around clusters of nanocrystals, as visible through Cryogenic electron microscopy (CryoEM). Lipid coated ZnO (L-ZnO) were also decorated with the targeting YSA peptide (YSAYPDSVPMMS) which ends with a cysteine aminoacid, able to react with the maleimide group exposed by the DSPE-PEG (2000)- Maleimide phospholipid. The peptide, for simplicity called YSA, is pre-conjugated to the phospholipid DSPE-PEG (2000)-Maleimide, and added to the lipid mixture prior to vacuum drying, obtaining the so called YSA-L-ZnO nanoparticles. The effectiveness of the coatings is evidenced with respect to uncoated ZnO (average hydrodynamic size: 120 nm, Poly dispersity Index (Pdl): 0.096) by an increase of the hydrodynamic size in water of L- ZnO and YSA-L-ZnO, which measures 188.37 nm (Pdl = 0.26) and 243.3 nm (Pdl = 0.35), respectively and by the shift in the Zeta potential values from positive to negative. DLS measurements were also performed in cell culture medium, testifying a good stability of the lipid-coated ZnO, which show results comparable to the ones obtained in water (L- ZnO: 172.73 nm, and YSA-L-ZnO: 204.2 nm). In contrast, pristine ZnO manifest an increase of size (194.8 nm), which suggests a tendency to aggregate in biological environments. Additionally, uncoated and both lipid-coated ZnO nanoparticles were tested on human plasma citrate for an in vitro recalcification test, evidencing an excellent hemocompatibility of L-ZnO and YSA-L-ZnO. For both samples the coagulation times were comparable to the physiological solution in plasma. On the contrary, pristine ZnO produced an earlier coagulation of the plasma; this indirectly demonstrates the correct assembly of the lipids around the nanocrystals and their role in isolating the ZnO core from the biological environment.

[0146] Example 15 - Targeting unit anchored to the DSPE-PEG(2000)-maleimide tested on cells cultured in 2D (cancer vs normal cells: Fig. 19)

[0147] The cytotoxicity of L-ZnO and their targeted counterpart, YSA-L-ZnO, was evaluated on a human colorectal cancer (CRC) cell line HT-29 cell line and on CCD-18Co, a human cell line exhibiting fibroblast morphology that was isolated from healthy colon tissue. The results clearly indicate a very high biocompatibility of both L-ZnO and YSA- L-ZnO NPs up to 50 pg / mL in both cell lines. At 50 pg / mL and above, a strong dose- and time-dependent toxicity was observed in HT-29 cells after 48h from administration. Remarkably, CCD-18Co healthy cells were much less affected by the nanoconstructs, giving proof of the high biocompatibility of the nanoconstructs toward the healthy tissues. At 48h, a statistically significant difference between the two cell lines was observed, evidencing a higher cytotoxicity on HT-29 cells. At 96h post administration, this trend resulted strengthened and a severe toxicity was produced in HT-29 cells at the highest concentrations, especially if treated with YSA-L-ZnO, demonstrating the strongest effect of the targeted NPs on this cell line. To assess the efficacy of the YSA peptide in targeting CRC cells, an uptake experiment was set up, comparing the cell internalization of fluorescently -labelled L-ZnO and YSA-L-ZnO in both tumoral and healthy cell lines by flow cytometry. In the case of HT-29, the data demonstrate a time-dependent uptake, which tends to considerably increase, in the first 24h after NPs administration, and shows statistically significant differences with respect to the healthy cell line already at 5h post administration. Remarkably, this behaviour resulted enhanced at 24h, when the difference between the two cell lines is substantial. Notably, a higher uptake of YSA-L-ZnO than L- ZnO was observed and this happened only in the HT-29 cancer cell line, with an different of approximately 15% in positive events between targeted vs untargeted NPs. A completely different behaviour was observed for CCD-18Co cell line: the percentage of positive events remains quite stable in time, never exceeding 40% even at the highest doses. Furthermore, no preferential internalization was observed for the YSA-L-ZnO with respect to the L-ZnO. The collected data indicate that YSA peptide plays an important role in the interaction between nanoparticles and HT-29 cell membranes, proving that the targeted nanoconstruct is a good candidate for the selective targeting of CRC cells.

[0148] Example 16 - Targeting unit anchored to the DSPE-PEG(2000)-maleimide tested on cell spheroids cultured in 3D (colorectal cancer: Figure 20)

[0149] With the aim to approach the complexity of a real CRC tumoral mass, the effects of lipid-coated ZnO and the SWT on HT-29 spheroids were also tested. To assess the cytotoxicity of YSA-L-ZnO NPs and of the untargeted L-ZnO, spheroids were grown for 6 days. The 6th day after cell seeding, the NPs were administered and the spheroid viability was evaluated after 24, 48, 72 and 96 hours. Remarkably, the only dose able to produce a statistically significant cytotoxicity in the cell spheroids was 100 pg / ml of YSA-L-ZnO NPs. Consequently, 3D spheroids demonstrated a higher resistance to NPs, with respect to 2D cell monolayer cultures. Our hypothesis is that the 3D structure of spheroids is more difficult to penetrate by the NPs than 2D monolayers due to their three-dimensional architecture. Here, despite the high number of cells, only the superficial layers of cells in the spheroid are directly exposed to NPs, probably, allowing a renewal of damaged cells from the spheroid core. In the 3D scenario, a targeting peptide can start to make the difference. In fact, a slight but consistent tendency of YSA-L-ZnO NPs to affect the spheroids more than L-ZnO is noticeable from 48 hours after the treatment. To further confirm this hypothesis, a live-cell fluorescence microscopy experiment with calcein AM / PI dual staining was set up. A necrotic core, visible as a lighter circle in the middle of the spheroids, is slightly present in all cases and tends to intensify with the increase of NPs dose, reaching its maximum after 72h post-administration, with the highest concentration of YSA-L-ZnO. These data visually confirm what already observed though flow cytometry and suggest that YSA-L-ZnO are able to impact on the core of spheroids, in a dosedependent manner. In order to corroborate the role of YSA targeting peptide in the penetration of NPs in the spheroids volume, a 3D internalization test was also set up. As previously done for the cytotoxicity assessment, fluorescently labelled particles were administered to the spheroids, which were then disaggregated and analysed through flow cytometry. Although no statistically significant difference between L-ZnO and YSA-L- ZnO was found, the tendency of the YSA-L-ZnO to be more taken up by CRC cells was consistently observed.

[0150] Example 17 - In vivo safety and biodistribution of ZnO nanocrystal coated with the 3C- lipidic formulation (Figure 21)

[0151] Safety and biodistribution of ZnO nanocrystal coated with the 3C- lipidic formulation (conjugated to the YSA lipid at the DSPE-PEG-maleimide lipid in the formulation) was also assessed in vivo, on NOD-SCID mice, previously inoculated subcutaneously with HT-29 human colorectal cancer cell line. The tumor xenograft was left to grow for 14 days until reaching a final size of 1 cm2and then the mice underwent to nanoparticles administration. Two types of NPs administration were evaluated: intravenous (IV) and intratumoral (IT) and two doses of nanoparticles were tested: 20 mg / kg (D4) and 30 mg / kg (D5). SpectrumCT In Vivo Imaging System (Perkin Elmer) was employed to evaluate the biodistribution of nanoconstructs in animals at both tumor and organ level (heart, lungs, spleen, liver, kidney) at different time points: 1 hour, 3 hours, 6 hours and 24 hours after inoculation of the nanoconstructs. Animals were also euthanized after 4 or 24h post administration, to study the biodistribution in the extracted organs and showed no signs of toxicity. As result, the in vivo assay confirmed, for both doses and both administration ways, the complete safety and absence of toxicity. Interestingly, the IT administration resulted effective only at the cancer site, avoiding the involvement of the vital organs considered. With IV administration, on the other hand, it was possible to reach both tumor and organs; in fact, the presence of nanoconstructs in all organs was detected and persisted on site after 24 hours from the infusion. More in detail, signal from the nanoparticles was detected in tumors already from 3 and 6h after administration and at 24h the signal was stronger and present in all the extracted tumors.

[0152] Example 18 - Further characterization of MIMIC 1 and 3 formulations, the role of cholesterol, and the inferior performances of ScrambleO (Figure 22)

[0153] EV-mimicking formulations (Mimic 1, and Mimic3) were employed to coat silica nanocapsules functionalized with amine groups (NC@NH2), to confer higher stability to the nanoparticles and build a nonconstruct, capable to host a therapeutic compound in its core, exposing an EV-mimetic surface to the biological environment. These two formulations were compared with a “wrong” formulation (here called SCRAMBLEO) coating the NC@NH2 nanocapsules to show the need to stick to the claimed compositions of Mimic 1 and 3. For this reason, the SCRAMBLED was prepared as follows: cholesterol 13%; SM 20%; DSPE-PEG(2000)-amine 15,9%; DOPC 26%; PS 25% e (DSPE- PEG(2000)-maleimide) 0,1%;

[0154] All the lipid-coated NC@NH2 were firstly characterized by means of DLS (Figure 5 A, B and C) and Zeta potential (Figure 5D) analysis, evidencing, in general, a shift from positive (27.1 ± 6.22 mV) to negative Zeta potential values (ranging from -20 mV to -40 mV approximately). A trend consisting in the enhancement in the nanoparticle’s hydrodynamic radius was registered, suggesting that the NC@NH2 were effectively coated. In particular, all the formulations, allowed to obtain regular size distributions, with an average hydrodynamic diameter around 150-200 nm (Figure 5 A). Also Cholesterol - Enhanced Mimic formulations (CE-Mimic 1 and CE-Mimic 3 and the negative control CE- ScrambleO), in which the mass percentage of cholesterol was set to 30%, were employed to coat the NCs, achieving narrower size distributions (Figure 5B). This can be attributed to the higher amount of cholesterol present, since it is known to enhance the stability of a lipid bilayer, increasing its rigidity. Also in this case, the average dimensions of the nanocontructs were found between 150 and 200 and the Zeta potential values were also in line with the results obtained with the formulations containing 13% cholesterol (Figure 5D). To further characterize and deepen the understanding of the lipid coated nanoconstructs, a co-localization experiment was performed by means of super resulution confocal microscopy (Figure 5E). While nanoparticles were labelled with ATTO-647-NHS dye, Nile Red was employed to create a transient hydrophobic binding in the lipid bilayer. The results visually confirmed the presence of the lipid bilayer onto the large majority of the silica nanocapsules. In fact, in all cases, with the only exception of ScrambleO formulation, the percentage of NCs whose signal coincides with the one of the Nile Red was around or higher than 70%. The same nanoparticles were also evaluated by singlemolecule localization microscopy (SMLM). This analysis is based on the signal of Nile Red which, when bound, gives a detectable fluorescent signal which depends heavily on the local environment. The hydrophobic environment causes a shift in the emission wavelength, which permits to measure the polarity of the lipid bilayers and, combined with SMLM, to generate a polarity map. Such maps were obtained for NCs coated with all formulations and compared to the one obtained from natural PC3 -derived EVs, which are the natural reference for the compositions (Figure 5F). Results show that Mimicl and Mimic3 formulations, especially when 30% cholesterol is present, have a polarity profile remarkably similar to the one of the natural EVs, reaching a cross-correlation superior to 0.9. SCRAMBLED formulation, on the other hand, was found very different to the natural EV’s profile, with a cross-correlation inferior to 0.1. Nano Tracking Analysis (NTA) was also performed to assess the dimensional similarity with natural EVs. NTA confirmed the data obtained by DLS and showed a size distribution of Mimicl (Figure 5H) and Mimic3- coated NCs, substantially similar to the one of PC3-derived EVs (Figure 5G).

[0155] Example 19 - In vivo safety and biodistribution of silica nanocapsules coated with the MIMIC1 and MIMIC3 lipidic formulation (Figure 23)

[0156] Silica Nanocapsules, loaded with cytochrome C (simulating a therapeutic agent) and left uncoated or coated with the Mimicl and Mimic3 formulations were tested in vivo on athimic nude mice. It has to be noted here that the MIMIC 1 and 3 did not presented in this case the DSPE-PEG(2000)-maleimide nor any targeting moiety, so no particular effects on the biodistribution are expected. Two experiments were conducted: the first aims to assess if the NPs provoke acute cytotoxicity in mice and the second aims to evaluate the biodistribution in organs of the NPs. In the Acute toxicity experiment, the animals were divided in 5 treatment groups of 4 elements each: GO, untreated, Gl, treated with equivalent dose of cytochrome C (CytC) present in the NPs, G2 Silica nanocapsules loaded with CytC and labelled with Cy5, G3 and G4 same NPs as G2, but coated with the Mimicl or Mimic3 formulation, respectively. For the acute toxicity, 3 doses of nanoparticles were intravenously administered to the animals, corresponding to 3, 10 and 30 mg / kg mice. None of the injected formulations produced signs of toxicity and the animals were euthanized 24 and 48h after the injection for further analysis.

[0157] For the in vivo biodistribution, the highest does considered in the acute toxicity was then administered to 4 groups of 3 mice each, corresponding to the Gl, G2, G3 and G4 groups described above. The fluorescence of the nanoparticles, which were previously labelled with Cyanin5 (Cy5), was measured by In Vivo Imaging System IVIS (640 nm excitation, 670 nm emission). Results show that the highest accumulation of the NPs was registered in the liver and that Mimicl showed the highest fluorescence at 2h, in the bladder, evidencing that the untargeted, lipid-coated NPs are biocompatible and easily expelled by the body. Example 20 - Comparative study: other lipidic formulations (called Scramble “n”), different from the invention ones, are not able to achieve same results in terms of stable and reproducible data (Figure 24)

[0158] To challenge the EV-biomimicking approach, different scramble formulation of lipids and cholesterol were also tested, deviating from the original formulations of 3C-, 3C+, Mimic 1, or Mimic3. The first scramble formulation (SCRAMBLE 1) tested consisted in a mixture of DOPC, sphingomyelins (SM), Phosphatidylserine (PS), and DSPE-PEG (2000)-amine of 62.57:34.48: 1.42: 1.80 molar ratio, respectively, as indicated in Figure 7A. This scramble formulation was then used to coat ZnO nanoparticles. It resulted in nanoparticles with a very unstable Zeta potential (Figure 7B), variable size over time (Figure 7C) and non-homogeneous population (Figure 7D). With the aim to obtain more stable nanoconstructs, other two scramble formulations were tested, in which cholesterol was also added, in a molar percentage equal to 15% (SCRAMBLE2) and 30% (SCRAMBLES), respectively (the original ratio among the other components was maintained, adjusting the molar percentages accordingly with the increment of the cholesterol amount). In both cases, the results were not satisfying. The Zeta potential resulted indeed very variable (Figure 7B) and irregularities in the size measurements (Figure 7C) and poly dispersity index, PDI (Figure 7D) were also observed during time.

[0159] SCRAMBLEl formulation includes a small percentage (inferior to 1,5% molar) of DOPS, which is known to provide negative net charge to the lipid bilayers (see the Table in Figure 7E). This was identified as a possible cause of the instabilities in the Zeta potential. In order to obtain nanoconstructs with a stronger net charge, and similarly for what done in 3C+and 30 formulations, the addition of 50 % molar of a cationic (DOTAP) or anionic (DOPA) phospholipid was tested (giving rise to the SCRAMBLE4 and SCRAMBLES formulations, respectively). While in the case of DOPA addition, the zeta potential of SCRAMBLES remained dramatically unstable, the addition of DOTAP in SCRAMBLE4 seemed to produce a consistently positive Zeta potential (Figure 7F). However, the size values (Figure 7G) resulted very low to be attributed to ZnO nanocrystal properly coated with a lipid bilayer (which is expected to be around 150-200 nm), suggesting instead the presence of uncoated nanocrystals and perhaps lipid micelles. Similarly, the PDI shows huge variations for all the formulations SCRAMBLE4, 5 and 6 (Figure 7H). These unreliable data were confirmed by a fluorescence microscopy colocalization analysis (here not reported), according to which only approximately 33% of the signal from ZnO nanocrystals was superimposed to the one originated by the lipids. As a result, these data clearly show that the proposed EV-mimicking formulations of 3C-, 3C+, 2Cn, MIMIC 1, or MIMIC3, enable to achieve stable and reproducible data, with efficient safety, haemocompatible and targeted delivery results, in contrast to other formulations which requires either further optimization or simply do not achieve the same results.

Claims

CLAIMS1. A coated nanoparticle for delivery of therapeutic or diagnostic agents, said nanoparticle consisting of a core and a lipid coating, wherein said core is a nanoparticle of organic or inorganic material in the solid or semisolid state and said lipid coating is a formulation of charged lipids, neutral lipids, cholesterol, PEG- functionalized lipids, phospholipids and / or sphingomyelin, wherein said formulation is selected from the group comprising:(i) formulation containing 10.0 to 35.0% cholesterol; 8.0 to 32% sphingomyelin (SM); 11.0 to 21.0% of DSPE-PEG(2000)-amine; 18.0 to 34.0% DOPC; 15.0 to 35.0% DOPA; and 0.1 to 0.6% l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide(poly ethylene glycol)-2000 (DSPE-PEG(2000)-mal eimide) or 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy(polyethylene glycol)- 2000-hydroxysuccinimide ester (DSPE-PEG(2000)-NHS);(ii) formulation containing 45.0 to 65.0% l,2-dioleoyl-3-trimethylammonium- propane (DOTAP+) or l,2-dioleoyl-sn-glycero-3 -phosphate (DOPA-); 7.0 to 15.0% l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC); 15.0 to 35.0% cholesterol; 1.0 to 10% l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000 (DSPE-PEG(2000)-amine) and 0.1 to 0.6% (DSPE-PEG(2000)-maleimide) or (DSPE-PEG(2000)-carboxy NHS ester);(iii) formulation containing 10.0 to 35.0% cholesterol; 8.0 to 32% sphingomyelin (SM); 5.0 to 15.0% DSPE-PEG(2000)-amine; 1.0 to 11.0% phosphatidylethanolamine (PE); 18.0 to 34.0% DOPC; 15.0 to 35.0% phosphatidyl-serine (PS) and 0.1 to 0.6% (DSPE-PEG(2000)-maleimide) or (DSPE-PEG(2000)-carboxy NHS ester); wherein said percentages refer to the total weight of the lipid coating, and wherein the core of said coated nanoparticle or a component of the core possesses therapeutic or diagnostic activity per se or the core of said coated nanoparticle is loaded with a diagnostic or therapeutic agent.

2. The coated nanoparticle according to claim 1, wherein the lipid coating is selected from the following formulations:(a) formulation (ii) containing DOPA(-) 57.3%; DOPC 12.5%; cholesterol 23.6%; DSPE-PEG(2000)-amine 6.5% and (DSPE-PEG(2000)-maleimide)(b) formulation (ii) containing DOTAP(+) 56.5%; DOPC 12.7%; cholesterol 24%; DSPE-PEG(2000)-amine 6.6% and (DSPE-PEG(2000)-maleimide) 0.1%;(c) formulation (i) containing cholesterol 13%; SM 20%; DSPE-PEG(2000)- amine 15.9%; DOPC 26%; DOPA 25%, and (DSPE-PEG(2000)-maleimide) 0.1%;(d) formulation (i) containing cholesterol 30%; SM 16%; DSPE-PEG(2000)- amine 12.9%; DOPC 21%; DOPA 20%; and (DSPE-PEG(2000)-maleimide) 0.1%;(e) formulation (iii) containing cholesterol 13%; SM 20%; DSPE-PEG(2000)- amine 9.9%; PE 6%; DOPC 26%; PS 25% and (DSPE-PEG(2000)-maleimide) 0.1%;(f) formulation (iii) containing cholesterol 30%; SM 16.0%; DSPE- PEG(2000)-amine 7.9%; PE 5%; DOPC 21%; PS 20% and (DSPE- PEG(2000)-maleimide) 0.1%.

3. Coated nanoparticle according to claims 1-2, wherein the coating is bound to one or more molecules capable of localizing on target cells, tissues or organs, said molecules being anchored to the outer surface of at least one coated nanoparticle.

4. Coated nanoparticle according to claim 3, wherein said molecule capable of localizing on the cell, tissue or organ targeted by the therapeutic or diagnostic agent is selected from proteins, antibodies, antibody fragments, peptides, carbohydrates, aptamers, nucleic acids.

5. Coated nanoparticle according to claim 1, wherein the core is selected from silica nanoparticles; organosilica; metal oxide; nanozyme; semiconductor; perovskite; quantum dots; semiconductor material; metal organic framework (MOF); covalent organic frameworks (COFs); carbon-based nanoparticle, in particular nanoonion and fullerene; natural or synthetic polymer, particularly gelatin and PLGA; and hydrogel.

6. Coated nanoparticle according to claim 5, wherein said core is an organosilica or metal oxide nanoparticle.

7. Coated nanoparticle according to claim 6, wherein said metal oxide is zinc oxide.

8. Coated nanoparticle according to claim 6, wherein said core is a porous organosilica nanoparticle selected from mesoporous silica, organosilica nanocage,organosilica nanocapsule.

9. Coated nanoparticle according to claim 8, wherein said organosilica incorporates a disulfide group -S-S- and is functionalized with silane derivatives, preferably with the cyclooctane silane of formula (I):(I).

10. Coated nanoparticle according to claims 5-9, wherein the core consists of a nanoparticle having a hydrodynamic diameter ranging from 20 to 200 nm and a positive z-potential.

11. Coated nanoparticle according to claims 5-10, wherein the core is loaded with a therapeutic agent selected from anticancer drugs, antibiotics or anti-inflammatory drugs; hormones; proteins; enzymes; nucleic acids and peptides; or with a diagnostic agent selected from fluorescent organic dyes; nuclear magnetic resonance contrast agents and isotopes for PET (Positron Emission Tomography).

12. Coated nanoparticle according to any of the preceding claims, for use in a therapeutic or diagnostic treatment method involving the administration of said nanoparticle bearing the therapeutic or diagnostic agent into a subject in need of such treatment.

13. A system for delivering a therapeutic or diagnostic agent to a target cell, tissue or organ, comprising a coated nanoparticle according to claims 1 to 11.

14. A diagnostic, therapeutic or theranostic composition comprising a coated nanoparticle according to claims 1 to 11 together with pharmaceutically acceptable vehicles or excipients.

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