IT202300015396A1 - COATED NANOPARTICLES AND THEIR USE FOR THERAPEUTIC AND DIAGNOSTIC DELIVERY - Google Patents

Description

Description of the patent for an industrial invention entitled:

COATED NANOPARTICLES AND THEIR USE FOR THERAPEUTIC AND DIAGNOSTIC DELIVERY?

The present invention relates to coated nanoparticles capable of mimicking the function of extracellular vesicles and of transporting therapeutic or diagnostic agents to target cells or tissues, and of releasing them intracellularly.

A further aspect of the invention relates to diagnostic, therapeutic or theranostic compositions comprising the coated nanoparticles together with pharmaceutically acceptable carriers or excipients.

BACKGROUND OF THE INVENTION

Extracellular vesicles (EVs) play an important role in cell-cell communication in humans and animals, being responsible for the transport and delivery of countless biomolecules, including proteins and nucleic acids. They are of fundamental interest because they naturally exhibit high efficiency and specificity in delivering their contents. For these reasons, EVs are commonly isolated from various biological fluids, tissues, cell cultures, as well as foods such as fruits and vegetables to be used as disease diagnostics or for the development of drug delivery systems. However, the growing interest in large-scale EV production is limited by the difficult, time-consuming, and expensive practices of EV isolation and purification, as well as the difficulty of obtaining high-purity, standardized clinical-grade populations. Furthermore, it is even more difficult to isolate a particular subpopulation of EVs with specific size and molecular characteristics. Consequently, the development of EV-based nanocarriers is challenging. 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 EVs currently produced.

To date, one medical branch seeking to exploit the potential of EVs is the diagnostic and therapeutic field in cancer. Since one of the most challenging issues related to cancer therapy is the complexity and heterogeneity of tumor biology, and therefore the peculiar nano-bio interaction that occurs between tumors and nanomedicine, precise control of the chemical-physical properties of EVs, or more generally of nanoparticles, must be a priority. As for purely inorganic nanoparticles (NPs), one of the main drawbacks of their use is related to their aggregation in biological fluids, and therefore to their short lifespan in the bloodstream, which hinders their effective localization in target organs and triggers a rapid adverse immune response. A possible solution to exploit them in a biological environment could be to have an effective and compact coating, such as one with phospholipids. This coating may be able to mimic cell membranes and therefore be the most effective strategy. It is simple to give these NPs camouflage 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., the NPs could also exploit active transport and targeting towards the target organ, tissue, or cell, generally improving their toxicity profile in the organism. Indeed, it is necessary to keep 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 composition (lipid and protein) of the progenitor cell that produced them. For this reason, to date, the aim is to use natural EVs as a coating on artificial NPs, thus constituting therapeutic and diagnostic (or "theranostic"?) nanoconstructs. This would represent an improvement over the use of more complex nanoparticles. These EVs are highly effective against conventional liposomes, thanks to the aforementioned biological properties. Liposomes, however, being made from commercially available lipids, enable high reproducibility and scale-up of liposome-coated inorganic NP systems. However, the aforementioned intrinsic capabilities of EVs are completely lost, introducing disadvantages such as poor biodistribution and rapid clearance, as well as the lack of specific targeting.

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

- an inorganic core capable of carrying a cargo molecule for imaging or therapy; and

- a coating (shell) derived from EV fragments or made of fully synthetic lipid formulations; capable of biomimicking the properties of EVs, but containing only the key elements necessary for their specific purpose, thus providing high and specific therapeutic and diagnostic efficacy.

This latter requirement is not met by natural EVs, due to the variety and complexity of their composition, nor by commercially available liposomes.

SUMMARY OF THE INVENTION

The present invention provides coated nanoparticles suitable for delivering therapeutic and diagnostic (or ?theranostic?) agents to target tissues or organs, transporting them to the cells of interest where the nanoparticle cargo is released.

The nanoparticles of the invention consist of a core made of organic or inorganic material in the solid or semi-solid state, and a lipid coating of the core, made 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.

The material constituting the nanoparticle's core can be biologically active in itself, or it can be loaded with therapeutic or diagnostic agents for release into the target cell. Furthermore, the lipids constituting the lipid coating can be functionalized with peptides or antibodies to facilitate the nanoparticle's targeting to the target tissue.

A further object of the invention is the use of the coated nanoparticle to deliver therapeutic or diagnostic agents to target cells, tissues or organs, and a diagnostic, therapeutic or theranostic composition comprising a coated nanoparticle according to the invention, together with pharmaceutically acceptable carriers or excipients.

DESCRIPTION OF THE FIGURES

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

Figure 2. Characterization of 20 nm OSCs. A) Electron microscopy images, B) DLS analysis of 20 nm ssOSCs, C) FTIR spectra showing S-S bands in the infrared region called the ?fingerprint? within the silica structure, D) TGA demonstrating complete elimination of the surfactant, and E) TGA comparing 20 nm OSCs and 20 nm ssOSCs.

Figure 3. Characterization of 20 nm ssMSNs. A) Electron microscope images B) DLS analysis C) FTIR spectra D) and E) TGA.

Figure 4. Characterization of functionalized degradable nanocapsules. A) Electron microscope images of bare, PEG-coated, and NH2-functionalized NCs. B) DLS analysis, C) fluorescence emission spectra, and D) zeta potential.

Figure 5. Cyclic silane used for the positive functionalization of NC.

Figure 6. Characterization of degradable NCs functionalized with cyclic positive silane. A) DLS analysis, B) surface charge evaluation of zeta potential, C) FTIR spectra, and D) TGA analysis.

Figure 7. Characterization of bare and coated zinc oxide nanoparticles with the 3C<- > formulation in terms of size distribution performed by DLS (A) and NTA (B) and zeta potential (C).

Figure 8. A) Dynamic Light Scattering (DLS) analysis and B) Zeta potential measurement of unfunctionalized organosilica nanocages (ssOSCs), functionalized with PEG (ssOSCs@PEG) and with NH2 groups (ssOSCs@NH2) as is and coated with 2Cn and 3C- formulations. C) Dynamic Light Scattering (DLS) analysis of organosilica nanocages functionalized with NH2 groups (ssOSCs-NH2) as is and coated with 2Cn and 3C- formulations. D) Nano Tracking Analysis (NTA) analysis of the ssOSCs-NH2 sample, coated with 3C- formulation. E) FTIR analysis of ssOSCs-NH2 and ssOSCs-NH2 coated with 3C- formulation: in the coated sample, the characteristic peaks of the lipids are clearly visible, together with those of the silica. F) Thermogravimetric evaluation (TGA) of ssOSCs-NH2 and ssOSCs-NH2 coated with 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.

Figure 9. A) DLS (Dynalmic Light Scattering) analysis and B) Zeta potential measurement of unfunctionalized organosilica nanocapsules (NCs), functionalized with PEG (NCs@PEG) and with NH2 groups (NCs@NH2) as is and covered with 2Cn and 3C<+> formulation.

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

Figure 11. Characterization of degradable nanocapsules functionalized with NH2 groups (NCs) as is and coated with Formulation 3C-, MIMIC1, MIMIC2 or MIMIC3. A) DLS analysis. B) Zeta potential. C) NTA analysis (Nano Tracking Analysis) NCs coated with Formulation MIMIC1.

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

Figure 13. A) FTIR spectrum and B) DLS and zeta potential of ssOSCSCalcein@NH2 coated with the 3C- lipid formulation.

Figure 14. TEM analysis of ssOSCs@DOX, B) Hydrodynamic diameter of loaded NPs, C) FTIR spectra, and D) UV-VIS absorption plot.

Figure 15. A) FTIR spectrum and B) DLS and zeta potential value of ssOSCs@DOX@NH2 coated with the 3C- lipid formulation.

Figure 16. Internalization of zinc oxide nanoparticles coated with the 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 analyzed by flow cytometry.

Figure 17. Evaluation on HT-29 cells (colorectal tumor cell line) of: A) cytotoxicity of zinc oxide particles as is and coated with 3C<- > formulation and bioconjugated with the YSA targeting peptide by WST1 assay, B) Internalization of zinc oxide particles coated with 3C- formulation, bioconjugated or not with the YSA targeting peptide. The evaluation was carried out by flow cytometry. C) Hemocompatibility on a human plasma sample of zinc oxide nanoparticles as is and coated with 3C- formulation, bioconjugated or not with the YSA targeting peptide. The evaluation was carried out by calculating the clotting time of citrated plasma, obtained from the UV-visible emission absorption curves.

DETAILED DESCRIPTION OF THE INVENTION

The object of the present invention are coated nanoparticles loaded with diagnostic or therapeutic agents, capable of reaching target cells, inside which they release their contents (cargo).

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

(i) formulation containing 45.0 to 65.0% 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP+) or 1,2-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; from 1.0 to 10% of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000 (DSPE-PEG(2000)-amine) and optionally from 0.1 to 0.6% of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000 (DSPE-PEG(2000)-maleimide);

(ii) formulation containing 10.0 to 35.0% cholesterol; 8.0 to 32% sphingomyelin (SM); 11.0 to 21.0% DSPE-PEG(2000)-amine; 18.0 to 34.0% DOPC; 15.0 to 35.0% DOPA;

(iii) formulation containing 10.0 to 35.0% cholesterol; 8.0 to 32% SM; 11.0 to 21.0% DSPE-PEG (PEG(2000)-amine); 18.0 to 34.0% DOPC; 15.0 to 35.0% PS;

(iv) 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);

where 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.

The use of the coated nanoparticles of the invention, compared to natural EVs alone or the more common liposomes, provides high biomolecule loading efficiency, targeted delivery of the cargo to the target cell, complete dissolution and subsequent release, triggered by endogenous stimuli already present in the recipient cell, as well as lower complexity than their natural counterparts.

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

(a) formulation (i) containing DOPA(-) 57.3%; DOPC 12.5%; cholesterol 23.6%; DSPE-PEG(2000)-amine 6.6%;

(b) formulation (ii) containing DOTAP(+) 56.5%; DOPC 12.7%; cholesterol 24%; DSPE-PEG(2000)-amine 6.7%;

(c) formulation (iii) containing cholesterol 13%; SM 20%; DSPE-PEG(2000)-amine 16%; DOPC 26%; DOPA 25%;

(d) formulation (iv) containing cholesterol 30%; SM 16%; DSPE-PEG(2000)-amine 13%; DOPC 21%; DOPA 20%;

(e) formulation (v) containing cholesterol 13%; SM 20%; DSPE-PEG(2000)-amine 16%; DOPC 26%; PS 25%;

(f) formulation (vi) containing cholesterol 30%; SM 16%; DSPE-PEG(2000)-amine 13%; DOPC 21%; PS 20%;

(g) formulation (vii) containing cholesterol 13%; SM 20%; DSPE-PEG(2000)-amine 10%; PE 6%; DOPC 26%; PS 25%;

(h) formulation (viii) containing cholesterol 30%; SM 16.0%; DSPE-PEG(2000)-amine 8%; PE 5%; DOPC 21%; PS 20%.

An advantage of these formulations is the dual function of the lipid with a PEG group, namely: (i) stabilizing the nanoparticle coating in an aqueous and physiological environment, ensuring greater circulation and consequently maximizing the probability of reaching the target tissue while protecting the contents from degradation; (ii) allowing for grafting through simple chemical reactions of peptides and antibody fragments, permitting a highly varied customization of the shell depending on the use; (iii) conferring the so-called "stealth" property to the nanoparticles, that is, avoiding their immediate recognition by the immune system and therefore the foreign body response.

In a preferred embodiment, the lipid coating of the nanoparticle is linked to one or more molecules for the recognition of target cells, tissues, or organs, such as proteins, antibodies, antibody fragments, peptide sequences, carbohydrate molecules, sugars, aptamers, or other nucleic acid sequences.

The invention also provides a method for preparing a lipid formulation suitable for coating nanoparticles, which comprises the following steps:

(i) acquisition of the natural composition of fatty acids and polar head groups of lipids, by methods including lipidomics, belonging to an extracellular vesicle, biovesicle, nanovesicle, ectosome, derived from a specific tissue, organ, biological fluid, primary cell, immortalized cell line;

(ii) the families of phospholipids present, whose sum in mass quantity must constitute 100%, are divided into two groups: the first according to the nature of the polar heads and the second according to the nature of the fatty acids that constitute the hydrophobic chains of the phospholipids;

(iii) taking the total mass quantity of fatty acids present divided by class as 100, quantities of fatty acids less than 1.5% by mass percentage are ignored;

(iv) the percentages of the remaining fatty acids are rebalanced by redistributing the percentage corresponding to the total of the neglected phospholipids to the percentage of the selected phospholipids in a weighted manner (in relation to their initial percentage), and brought to the total sum of 100% by mass; this formulation is then considered the reference formulation;

(v) each family of phospholipids (of the first group) present in the said reference formulation is assigned the closest commercially available phospholipid specimens, 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;

(vi) additional components are added, such as monounsaturated phosphatidylethanolamine, present in the PEGylated DSPE-PEG form with an amino and/or maleimide functional group, in a maximum quantity of 1.5% by mass, 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, by 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 lipid mass;

(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 coating the nanoparticles.

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 frameworks (MOFs), covalent organic frameworks (COFs), carbon-based nanoparticles, in particular nanoonions and fullerenes, natural or synthetic polymers, in particular gelatin and PLGA, and hydrogel; preferably said core is an organosilica or a metal oxide, where said metal oxide is preferably zinc oxide.

The nucleus itself may function as a therapeutic, diagnostic, or theranostic agent, as in the case of a zinc oxide nanoparticle capable of inducing a toxic effect through ultrasound activation. Alternatively, the nucleus may contain and transport a therapeutic, diagnostic, or theranostic agent (cargo), which is released through an exogenous or endogenous mechanism, usually through the disintegration of the nucleus itself.

In a preferred embodiment, the core is a porous organosilica nanoparticle chosen from mesoporous silica, organosilica nanocage, and organosilica nanocapsule.

Porous organosilica exhibits several useful properties: i) it has high porosity, ii) a large surface area, iii) the pores are uniform and easily adjustable in diameter; finally, iv) it is highly biocompatible.

In a further preferred aspect, the organosilica incorporates -S-S- disulfide groups, which are cleaved in the presence of enzymes or reducing agents, thereby allowing the delivery of the therapeutic or diagnostic payload.

Furthermore, the organosilica can be functionalized with linear or cyclic alkyl silanes, which by increasing the zeta potential of the organosilica favor the subsequent attack of the lipid formulation, in particular the following cyclooctane silane of formula (I):

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-inflammatory drugs, hormones, proteins, enzymes, nucleic acids, peptides; or with a diagnostic agent selected from organic fluorescent dyes, nuclear magnetic resonance contrast agents, and isotopes for PET (Positron Emission Tomography).

Some examples of drugs particularly suitable for transport within the core of the nanoparticle include anticancer drugs, antibiotics, anti-inflammatory drugs, corticosteroids, inhibitors, biomolecules, proteins, enzymes, nucleic acids.

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 to a subject requiring such treatment.

Diseases that can be treated therapeutically or diagnostically according to the invention include cancer, particularly colorectal cancer, pancreatic (PDAC-Pancreatic Ductal Adenocarcinoma), cervical cancer, osteosarcoma, leukemia, lymphoma, multiple myeloma, breast cancer, and melanoma; and neurological diseases, such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, and Huntington's disease.

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, which comprises a nanoparticle according to the invention.

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 carriers or excipients.

EXAMPLES

Example 1 - 10 nm Organosilica Nanocages (OSCs)

The nanomaterial was synthesised by adding 0.23 mmol CTAB and 2 ml of 0.02 M ammonium hydroxide aqueous solution to 8 ml of distilled water (dH2O). The solution was then stirred at 30°C for 30 minutes until the CTAB was completely dissolved. Subsequently, 0.43 mmol TMOS and 0.215 mmol BDTS (in case of degradable NPs due 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 stirred at 80°C for 24 hours. The solution was dialyzed in 100 mL of acidic solution (a mixture of ethanol, dH2O, and acetic acid with a volume ratio of 1:1:0.007) for 24 hours to eliminate 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 smaller than 10 nm. Transmission electron microscopy (TEM) images revealed the presence of a single pore in nanoparticles smaller than 10 nm (Figure 1A). According to TEM results, the nanoparticles exhibit a diameter of 9.3 nm, a hydrodynamic diameter of 12 as analyzed by DLS, and a negative zeta potential (Figure 1B). In order to verify the correct elimination of the surfactant used during the synthesis strategy and the presence of S-S bonds that provide the NPS with their unique properties, a 3D model was performed. To determine degradability, FTIR analysis (Figure 1C) and TGA analysis (Figure 1D) were performed. The ssOSCs showed a typical OSC spectrum with Si-OH (3445 cm<-1>), Si-O-Si (1077 cm<-1>), Si-OH (947 cm<-1>), and C-H (805 cm<-1>) peaks characteristic of the organosilica structure. The presence of 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<-1 >were observed in the FTIR spectra (Figure 1C). TGA analysis indicates that all CTAB used in the synthesis was completely eliminated and allows quantifying the amount of degradable groups introduced into the organosilica structure (about 20%).

Example 2 - 20 nm Organosilica Nanocages (OSCs)

In a 100 ml round-bottom glass flask equipped with a magnetic stirrer, CTAB (408 mg, 1.12 mmol) was dissolved in deionized water (50 ml) at 50°C, stirring the solution for 30 min at 250 rpm. Ammonia (12.5 μL, 28% in water) was added and the stirring speed was increased to 750 rpm. Subsequently, TEOS solution (448.1 μL, 2 mmol) was added and the mixture was stirred for 20 h at 50°C, at 750 rpm. The particle dispersion was observed. was purified by dialysis in an EtOH/H2O/AcOH mixture (1:1:0.007 v/v/v) for 24 h and finally in an EtOH/H2O mixture (1:1 v/v) for another 24 h where the dialysis solution was replaced every 2 h, when possible. To obtain particles degradable via S-S bonding, BTDS (102.8 μL, 0.223 mmol) was also added to the synthesis. 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 was approximately 18 nm when analyzed by DLS (hydrodynamic diameter, Figure 2B) and a negative zeta potential of -10.98 mV. In order to verify the correct elimination of the surfactant used during the synthesis strategy and the presence of S-S bonds that provide OSCs with degradable properties, FTIR analysis (Figure 2C) and TGA analysis (Figure 2D) were performed. The ssOSCs showed a typical characteristic spectrum with Si-OH (3445 cm<-1>), Si-O-Si (1077 cm<-1>), Si-OH (947 cm<-1>), and C-H (805 cm<-1>) peaks 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<-1 >were observed in the FTIR spectra (Figure 2C). TGA analysis indicates that all the CTAB used in the synthesis approach was present in the silica structure. has been completely eliminated and allows to quantify the amount of degradable groups introduced into the silica structure (again, about 20%, Figure 2E).

Example 3 - 20 nm mesoporous organosilicates (ssMSNs)

In a round-bottom flask (250 mL), CTAB (816 mg) was dissolved in dH2O solution (100 mL), the solution was heated to 50°C and stirred (500–750 rpm) for 30 min. Then, 25 mL of 28% NH4OH, followed by 1 mL of TEOS were added consecutively into the solution. After 12 h of reaction at 50°C (with the 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 the volume ratio 1:1:0.007) for 24 h to extract CTAB from the pores of the particles. The solution was The ssMSN was then dialyzed in <2 L of dH>2<O for another 24 h. This process was repeated three times. To obtain particles degradable by S-S groups, BTDS (241 μL, 0.526 mmol) was also added. TEM analysis confirmed the synthesis of 20 nm ssMSNs (Figure 3A). DLS revealed an average hydrodynamic diameter of 21 nm with a Gaussian distribution confirming the good colloidal stability of the particle suspension. Zeta potential measurements were recorded in water, yielding a negative zeta potential characteristic of silica (-8 mV). The obtained hydrodynamic diameter confirms the expected size of 20 nm for ssMSNs (Figure 3B). FTIR analysis (Figure 3C) revealed the characteristic bands of Si-OH (3445 cm<-1>), Si-O-Si (1077 cm<-1>), Si-OH (947 cm<-1>), and C-H (805 cm<-1>) and confirmed that all CTAB (surfactant used during the synthesis) was completely eliminated. TGA analysis (Figure 3D) revealed that almost 100% of the mass of the MSNs corresponded to inorganic compounds, and therefore to silica. This result once again confirmed the complete elimination of the surfactant used during the synthesis. Like the previous samples, the 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 the S-S was present, the MSNs were synthesized in the presence and absence of disulfide bonds. When S-S was incorporated into the silica structure, peaks around 2900 and 1500 cm<-1 > were observed in the FTIR spectra (Figure 3C). Furthermore, TGA results showed that when S-S was incorporated into MSNs, a decrease in the weight of the ssMSN sample was observed around 400°C, corresponding to approximately 15% of the sample mass. This decrease was not attributed to CTAB, which has a lower combustion temperature, but rather to the presence of the S-S disulfide groups (gray line in the graph of Figure 3E).

Example 4 - Nanocapsules (NCs)

Degradable hybrid organosilica nanocapsules were synthesized using the St?ber 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-bottom flask and stirring on a magnetic stirrer for 15 min. Separately, 600 μL of water was mixed with 40 μL of TEOS and 60 μL of BTDS. After stirring, this mixture was added to the organic solution. The hydrolysis of TEOS was initiated by adding 50 μL of 28% aq.NH3, and the mixture was stirred at room temperature for 5 h.

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

Example 5 - Functionalization of nanocapsules (NCs)

Considering the negative zeta potential value of the nanocapsules@NH2, even coated with amino groups, a new functionalization was tested, using a cyclic silane group (Figure 5), which has a higher number of amino groups and a greater steric hindrance 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 by DLS demonstrated the functionalization of the NCs with the cyclic silane (an increase in the hydrodynamic diameter and a change in the zeta potential of the NCs from negative to positive was clearly observed). Furthermore, by FTIR it was possible to observe the functionalization of the NCs with the cyclic silane. The presence of amino groups from the cyclic silane was observed: 1523 cm<-1 >NH2, 1626 cm<-1 >NH2, 2600-3650 cm<-1 >NH2 (Figure 6C). By TGA it was possible to quantify the amount of <S-S >groups present in both the bare and functionalized NCs, which turns out to be 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% mass loss attributed to the presence of silacyclooctane.

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

The synthesis of zinc oxide nanocrystals was carried out by a wet chemical method, using a sol-gel solution with a zinc precursor and oleic acid as a stabilizing agent. The reagent zinc acetate dihydrate (526 mg, ACS Reagent, Sigma-Aldrich) was dissolved in 40 mL of ethanol and heated to 70°C. To the solution were added double-distilled water (bd, 1 mL, produced by a Direct Q3 system, Millipore, Burlington), oleic acid (140 μL, Sigma-Aldrich), and tetramethylammonium hydroxide (1.044 mg, TMAH, Sigma-Aldrich) previously dissolved in 1.052 mL of bd water and 10 mL of ethanol. After 10 min, ZnO nanocrystals (NCs) were recovered by centrifugation at 14,000 g for 10 min and resuspended in ethanol. This washing procedure was repeated three times. The surface of the ZnO NCs was then decorated with aminopropyl functional groups by adding 10 mol% 3-aminopropyltrimethoxysilane (APTMS, Sigma-Aldrich). More specifically, the ZnO NCs were dispersed in ethanol at a concentration of 2.5 mg/mL and heated to 70°C under nitrogen and reflux conditions. APTMS was added to the solution, and the reaction was carried out for 6 h at 70°C. At the end of the procedure, the NCs were collected and washed three times by centrifugation and redispersion in fresh ethanol and stored as colloidal ethanol suspensions.

To characterize as-is and aminopropyl-functionalized ZnO NCs, Dynamic Light Scattering (DLS) and Z-Potential measurements were performed with the Zetasizer Nano ZS90 instrument (Malvern Instruments) in dry water at a concentration of 100 μg/mL. Nanoparticle Tracking Analysis (NTA) measurements were performed on functionalized ZnO NCs with a NanoSight NS300 (Malvern Panalytical). To prepare the sample, 6 μL of 1 mg/mL NCs solution was diluted to 1 mL in double-distilled water. For each sample, three 60-s videos of the samples flowing into the instrument chamber were captured and analyzed with NTA 3.4 software (Malvern Panalytical).

The functionalized ZnO nanocrystals were characterized by transmission electron microscopy (TEM) by dispersing them in water at a concentration of 50 μg/mL. Then, 10 μL of the solution was deposited on a Lacey carbon support film (300 mesh, Cu, Ted Pella Inc.) and allowed to dry. Measurements were performed with a Thermo Scientific Talos® F200X G2 S(TEM) microscope at an operating voltage of 200 kV.

Example 7 - Realization of 2Cn, 3C<+ >and 3C<- >lipid formulations for lipid coating

The 2Cn, 3C<+ >and 3C<- >formulations were designed to create a highly customizable lipid shell and, in the case of the 3C<+ >and 3C- formulations, electrostatically charged with the aim of acting as a coating for nanoparticles, as a drug delivery system, precisely by virtue of an electrostatic attraction between the lipid shell and the internal load.

The approach followed to design the formulation therefore consisted in using permanently charged lipids (negatively charged in the 3C<- > formulation, positively charged in the 3C<+ > formulation, while the 2Cn formulation is neutral as a control), and maintaining a majority ratio of these lipids with respect to the other individual components of the lipid shell, chosen from neutral lipids, cholesterol and lipids containing a polyethylene glycol (PEG) group, obtaining a molar ratio of 50:10:38.5:1.5 respectively.

In order to be able to use only commercial lipids whose use was already well established, this proportion was used in the 3C- formulation, specifically composed of 3 lipids: DOPA (18:1 PA, 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt), in chloroform solution), DOPC (18:1 (?9-Cis) PC (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine, in chloroform solution), DSPE-PEG(2000) Amine (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene-glycol)-2000] (ammonium salt) and Cholesterol in chloroform.

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

As regards the presence of the PEG group, the lipid containing it has a dual function, that of:

(i) stabilize the shell in an aqueous and, in future applications, physiological environment, ensuring greater circulation and consequently maximizing the probability of reaching the tissue under study while protecting the contents from degradation;

(ii) allow for the grafting of peptides and antibody fragments through simple chemical reactions, allowing for a very varied customization of the shell depending on future use;

(iii) confer the so-called "stealth" property to nanoparticles, that is, avoid their immediate recognition by the immune system and therefore the foreign body response.

To this end, the lipid with a 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 a "functional lipid", is added in a molar fraction varying between 0.1% and 0.5% to replace part of the DSPE-PEG(2000)-Amine lipid.

Below is a table (Table 1) summarizing the mass fraction ranges, and one of the same ranges in the preferred version (Table 2). In Table 1, it should be noted that the sum of the values used in the mole fraction in all their possible combinations must always be 100%.

Table 1

Table 2

The 3C<- >? formulation is considered the most relevant of these three formulations and was designed to interact electrostatically with positively charged nanoparticles, ensuring stable coverage over time and reducing the risks associated with the use of highly positively charged biomaterials in the body. These materials have a well-established clinical and materials science-based tendency to promote blood clotting and protein adsorption to their surfaces. The markedly negative charge of the constructs coated with the 3C- formulation, combined with the presence of the anti-fouling PEG group, ensures prolonged circulation and stability in biological environments, making them ideal for future clinical uses. This offers a high degree of customization thanks to the potential surface functionalizations enabled by the presence of DSPE-PEG(2000) Maleimide.

The amount of PEG inserted in the shell is the right compromise to allow customization of the lipid shell without inducing adverse reactions resulting from the massive presence of PEG in the body, recently identified as a risk factor for allergies common to 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 quantities gives the lipid nanoparticles the right degree of both fluidity and structural solidity.

Once obtained, the lipid formulations were characterized and the characterization results are presented in Table 3:

Table 3

Example 8 - Creation of MIMIC 1, 2, and 3 lipid formulations for lipid coating

To develop MIMIC 1, 2, and 3 formulations, the starting point was a study of the state-of-the-art composition of natural EVs from the PC3 prostate cancer cell line. This cell line produces extracellular vesicles that have a natural tropism for another tissue, specifically the skeletal system. Therefore, with the aim of reaching bone tissue for therapeutic purposes and with the understanding that lipid composition plays a fundamental role in the chemical and physical properties of vesicles, and that these in turn play a role in the recognition and internalization of a particular cell species, we attempted to create artificial lipid formulations that would increasingly resemble those of natural vesicles.

Among the articles found in the literature on the composition of EVs derived from PC3 prostate cancer cells, one in particular was selected:

"The n-10 fatty acids family in the lipidome of human prostatic adenocarcinoma cell membranes and extracellular vesicles," vol. 12, no. 4, pp. 1-16, 2020, doi: 10.3390/cancers12040900, which reports the mass percentages of both the lipid families that make up this type of natural vesicle and the distribution of fatty acids, which constitute the hydrophobic part of the lipid bilayer. While the composition of 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 related fatty acids, which constitute their characteristic hydrophobic tails, is unknown.

Therefore, we sought to develop artificial compositions that primarily satisfied the mass ratios between the hydrophilic heads, considered to be the most biologically relevant, as they are ideally exposed at the forefront of the lipid bilayer. At the same time, we sought a compromise that would also satisfy the balance between fatty acids as closely as possible, with particular attention to the balance between the three most well-known categories of fatty acids: saturated, monounsaturated, and polyunsaturated. Finally, the three formulations represent an evolution from the 3C- formulation, used as a starting point, to the reference composition, which is a simplification of the natural composition of EVs reported in Ferrari et al.

To obtain the reference composition, the natural composition of fatty acids was simplified, while that of the lipid families was maintained. In particular, fatty acids present in a mass percentage lower than 1.5% were neglected (this cutoff represents only slightly more than 5% of the total lipid mass). 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 the 3C<- > formulation to arrive, in consecutive steps, at a commercial lipid composition as close as possible to the reference, while still maintaining a low number of components for practicality.

The MIMIC 1 composition is given by the simple rebalancing of the lipid families present in the 3C- formulation with the inclusion of sphingomyelins. In this way, all the lipid families of the reference are present, with the exception of phosphatidylserines, whose percentage by mass has been assigned to the anionic phospholipid DOPA. The latter is in fact the cornerstone of the 3C- 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 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 the 3C- formulation. The only notable addition is the addition of a new saturated fatty acid, C16:0, and a monounsaturated one, C18:1, thanks to the presence of sphingomyelins.

The MIMIC 2 composition is based on the previous one; it is in fact identical to the MIMIC 1 formulation, but in this case the percentage of DOPA is replaced with phosphatidylserine (PS C16:0). In this way, all types of hydrophilic heads of the reference formulation are present and it is possible to evaluate whether the presence of the anionic phospholipid is essential for effective and stable coating of the nanoparticles. As for the fatty acids, this commercial phospholipid, PS C16:0, was chosen to increase the percentage of saturated acids, which in the MIMIC 1 formulation was too unbalanced in favor of monounsaturated ones.

The MIMIC 3 composition includes a new type of fatty acid compared to the previous two formulations, while maintaining the same ratio of hydrophilic heads compared to the MIMIC 2 formulation, which was already identical to the reference formulation. The new category is that of polyunsaturated fatty acids by replacing part of the percentage of monounsaturated phosphatidylethanolamine, present in the PEGylated DSPE-PEG form, with PE 18:2. This allows for the study of the effect of the presence of polyunsaturated lipids on the behavior of the lipid bilayer. With this addition and considering the presence of the errors reported in the original natural composition, it can be stated that the MIMIC 3 formulation, with a good approximation, achieves the correct balance between the families of hydrophilic heads and the categories of fatty acids.

The following tables show two preferred forms for MIMIC compositions 1, 2, and 3: both contain the same proportions of lipids, but in case 1 there is 13% cholesterol by mass, while in case 2 there is 30% cholesterol by mass. The sum of all components is always equal to 100%. The % range of values that the compositions can have is shown on the side.

Table 4. Compositions of MIMIC formulations 1, 2, and 3, in their two preferred forms (case 1 and case 2) and with the range of variation in % of the components (% by mass). The 3C- formulations are also shown, as a starting point and comparison for the MIMIC formulations and the PC3 (prostate cancer) EV formulation as the final point of arrival.

Table 5. Summary table of the mass % of the components of the different formulations (for preferred case 1 only), compared with the reference composition of the EVs extracted from PC3. The table is divided into three parts representing: the polar head groups (top), the fatty acids (middle), and the three fatty acid categories (bottom).

Example 9 - Nanoparticle Coating Method

Solvent exchange method

This technique involves preparing a formulation of various phospholipids and cholesterol, removing them from their reserve chloroform solution, and rehydrating them with a solution of 40% ethanol and 60% water. The nanoparticles are then initially mixed with the lipid solution, and water is subsequently added in excess. The rapid increase in water content is critical to this method, as it drives the self-assembly of the phospholipids on the nanoparticle surface.

Briefly, after drying the desired lipid formulation overnight under vacuum, they were hydrated with a solution composed of ethanol (99%) and double-distilled water in a 40%–60% volumetric ratio. This ratio was carefully studied to avoid any unwanted self-assembly of the lipids, and for this purpose, ethanol was added as the first solvent. The resulting lipid dispersion, whose color turns whitish after the addition of water, is stable and stored at 4°C for subsequent steps.

The coating process consists of removing a certain amount (defined later for the optimized NPs coating protocol) of nanoparticles from the ethanolic solution by centrifugation at 14,000 g for 10 minutes and removing the supernatant. A quantity of lipid solution is then added to the nanoparticle pellet at a weight ratio of 2:1 (preferred option) or a 1:1 nanoparticle to lipid ratio, and an initial sonication step (3 minutes) is performed using an ultrasonic bath (59 kHz, Branson 3800 CPXH, ) to allow good dispersion of the nanoparticles and lipids. Subsequently, to self-assemble the lipid bilayer on the nanoparticle surface, excess double-distilled water is added 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 the formation of a homogeneous self-assembled lipid bilayer enclosing the nanoparticles.

Example 10 - Characterization of Lipid-Coated Nanoparticles To characterize the size distribution and colloidal stability of the coated nanoparticles, DLS and Z-Potential (Zetasizer Nano ZS90 by ) and Nanoparticles Tracking Analysis (NTA, NanoSight NS300 by ) were performed in deionized water at room temperature. All measurements were performed 3 times and then averaged. For analyses, an amount corresponding to 100 μg of NPs was removed from the suspension and added to 900 μL of double-distilled water and analyzed. For NTA analysis, for each sample, three 60-second videos of the samples flowing through the instrument chamber were recorded and analyzed using Malvern Analytical's NTA 3.4 software. Coating with the 2Cn, 3C<- >, and 3C<+ > formulations? The study was tested on different types of nanoparticles, which were coated according to the zeta potential value measured on the as-is particles (the neutral lipid formulation or preferably the one with the opposite charge is used to exploit the electrostatic interaction and ensure correct coating) and subsequently characterized. Some examples of the characterization of coated nanoparticles are shown below: zinc oxide nanoparticles (Figure 7), organosilica nanocages (Figure 8), organosilica nanocapsules (Figure 9), and polymeric nanoparticles (Figure 10). Similarly, the coating of particles with MIMIC formulations 1, 2, and 3 was tested and characterized and compared with the 3C- formulation, of which MIMIC formulations represent an evolution. Some exemplary results are shown in Figure 11.

In detail, Figure 7 shows the characterization of zinc oxide (ZnO) nanoparticles coated with the 3C<- > formulation and compares the data with the same bare nanoparticles. In terms of size distribution, ZnO nanoparticles coated with the 3C<- > formulation are monodisperse and have a hydrodynamic diameter around 100 nm, as detected by DLS (Figure 7A) and NTA (Figure 7B) analyses. In contrast, bare zinc oxide NPs show aggregation upon NTA (black curve in Figure 7B). The Z potential and zeta potential of bare ZnO NPs are positive, around 25 mV, as these NPs are functionalized with aminopropyl groups. Once coated with the 3C- formulation, the Z potential is negative, equal to -20 mV, demonstrating the effectiveness of the coating (Figure 7C).

Figure 8 shows data from unfunctionalized, PEG-functionalized (ssOSCs@PEG) and NH2-functionalized (ssOSCs@NH2) degradable organosilica nanocages as-is and coated with 2Cn and 3C- formulations. Dynamic Light Scattering (DLS) analyses in Figure 8A and Zeta potential analyses (Figure 8B) show that the as-is ssOSCs have a size of 60 nm and a neutral Z-potential: when coated with either the 2Cn or 3C- formulation, their size distribution remains unchanged and the Z-potential changes significantly, becoming very positive (about 60 mV) with the 2Cn formulation and very negative (-40 mV) using the 3C- formulation. When PEG-functionalization is present on the ssOSCs, they are more symmetrical. small (about 30 nm from DLS, Figure 8A) and weakly negative (-15 mV 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 groups (ssOSCs-NH2) as such show a size at DLS of about 20 nm (Figure 8A at DLS and Figure 8C at NTA) and a positive charge (+50 mV, Figure 8B). When coated with the 2Cn formulation or the 3C<- > formulation, their size increases to 80 nm and 120 nm respectively (Figures 8A and 8C) and the Z potential varies as a function of the charge of the lipid formulation: with the 2Cn formulation it becomes smaller (Figure 8C at DLS and Figure 8C at NTA). The NPs are neutral (+ 30 mV) and with the 3C<- > formulation it becomes very negative (-50 mV), demonstrating the effective coating of the NPs with lipids. The NTA (Nanoparticle Tracking Analysis, Figure 8D) analysis was performed on the ssOSCs-NH2 sample, coated with the 3C- formulation, which demonstrates good monodispersity and a peak at 126 nm. The FTIR analysis of ssOSCs-NH2 and ssOSCs-NH2 coated with the 3C<- > formulation (Figure 8E) shows that in the coated sample, the characteristic peaks of the lipids in the region from 3200 cm<-1 >to 1500 cm<-1>, together with those of the silica, are clearly visible. Thermogravimetric evaluation (TGA, Figure 8F) of ssOSCs-NH2 and ssOSCs-NH2 coated with 3C<- > 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.

Figure 9 shows the DLS analyses (Figure 9A) and the Zeta potential measurements (Figure 9B) of unfunctionalized organosilica nanocapsules (NCs), functionalized with PEG (NCs@PEG) and with NH2 groups (NCs@NH2) as is and coated with 2Cn and 3C<+> formulations. It is clearly noted that when the NCs are coated with 2Cn and 3C<+> lipids, their hydrodynamic diameter increases compared to the NCs as is and that the Z potential varies significantly as a function of the charge of the formulation used. In particular, the NCs are originally with a negative Z potential (about -22 mV) and the coating with the 3C<+ > formulation brings the Z potential to significantly positive values (about 30 mV).

Figure 10 shows the characterization of as-is and lipid-coated polymeric nanoparticles. The zeta potential measurement of as-is PLGA, chitosan-functionalized PLGA (PLGA-CS), and gelatin particles changes significantly once they are coated with the 3C<+ > or 3C<- > formulations depending on the starting zeta potential (Figure 10A) as the electrostatic interaction between the polymer surface and the lipid formulation is exploited. DLS analyses of as-is PLGA particles show an increase in size when they are coated with the 3C<+ > formulation (Figure 10B), and similarly for gelatin particles as-is and coated with the 3C<- > formulation and for chitosan-functionalized PLGA (PLGA-CS) as-is and coated with the 3C- formulation.

Figure 11 shows the characterization of degradable nanocapsules functionalized with NH2 groups (NCs) as is and coated with Formulation 3C-, MIMIC1, MIMIC2 or MIMIC3. DLS analyses (Figure 11A) show an increase in hydrodynamic diameter when the NCs are coated and a considerable change in Zeta potential from positive for the uncoated NCs to negative for the NCs coated with the various formulations used (Figure 11B). Finally, NTA analyses (Figure 11C) show the monodispersion of the NCs coated with Formulation MIMIC1 with a peak at 112 nm.

Example 11 - Encapsulation of calcein and doxorubicin in 3C-type lipid-coated ssOSC nanoparticle formulation - The encapsulation of calcein in ssOSC particles was achieved as follows: 50 mg of ssOSCs were suspended in 10 mL of EtOH containing 2M CaCl2. In parallel, calcein (50 mg) was also dissolved in 10 mL of 2M EtOH containing 10 mL of CaCl2. 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), they were washed and centrifuged at 40,000 g for 30 min 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 previously, resuspended in dH2O, and kept at 4°C until coated with the 3C- lipid formulation.

The calcein content loaded within the ssOSCs was analyzed by UV-VIS, fluorescence spectroscopy, TEM, FTIR, and DLS (Figure 12). The calcein-loaded ssOSCs showed a hydrodynamic diameter ranging from 20 to 40 nm (comparable to empty NPs). Surface charge analysis of the samples demonstrated that the zeta potential of the OSCs did not change after calcein encapsulation, remaining negative. FTIR analysis demonstrated the presence of silica peaks along with the characteristic calcein bands. The emission spectra and absorption pattern of the nanoparticle upon excitation at 480 nm were recorded. All these data demonstrated that calcein was successfully encapsulated.

Then, the ssOSCs loaded with positively surface-charged calcein were electrostatically coated with the 3C<- >phospholipid formulation as previously described. Figure 13 demonstrates the successful lipid coating of the calcein-loaded NPs. FTIR analysis demonstrated the presence of characteristic bands originating from the silica structure, the calcein molecule, and the lipids on their surfaces. DLS analysis showed a hydrodynamic diameter of approximately 130 nm and a negative surface charge of the calcein-loaded ssOSCs after coating with the 3C- lipid formulation.

Doxorubicin (20 mg) was dissolved in 5 mL of EtOH and sonicated for 10 minutes. Then, nanocages (ssOSCs@DOX) were added, briefly sonicated for another 20 minutes, and stirred for 12 hours at room temperature. Subsequently, to force encapsulation, vacuum was used, and the sample was placed in a rotavapor for a few minutes until the solvent had completely evaporated.

The doxorubicin-loaded nanomaterials were characterized by DLS, zeta potential, FTIR, and absorption spectra. TEM images confirmed that even after doxorubicin encapsulation, the NPs were still monodisperse and had a diameter of approximately 20 nm (Figure 14A). DLS of the ssOSCs@DOX (Figure 14B), corroborated by the TEM results, confirmed that doxorubicin was encapsulated within the pore of the NPs rather than attached to the surface. Furthermore, the zeta potential of the 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 the organosilicon NPs were observed together with the main bands of doxorubicin (the characteristic sharp peak at 1727 cm<-1 >? related to the carbonyl (C=O) of pure doxorubicin). Finally, the absorbance spectrum of the ssOSCs@DOX was also recorded, showing a maximum absorption peak at 470 nm (Figure 14D).

The ssOSCs@DOX were then functionalized with amino-silane and further coated with the negative lipids, as previously described for the calcein-loaded samples. Figure 15A in the SI includes the FTIR spectra of the ssOSCs@DOX-NH2-negative lipids. The presence of characteristic bands from the organosilicon groups, cleavable disulfide bonds, the doxorubicin fingerprint, and the lipid bands can be observed. The hydrodynamic diameter obtained by DLS was approximately 100 nm (Figure 15B), and the zeta potential was −29.2/−2.2 mV, again demonstrating the successful coating of the silica core with the lipid shell.

Example 12 - Conjugation of DSPE-PEG (2000) Maleimide functional lipid with a peptide or antibody to enhance targeting and homing of coated nanoparticles towards tumor cells

One of the key features of the lipid formulations proposed here is their ability to be adapted to the pathology of interest, thanks to the presence of the functional lipid DSPE-PEG(2000) Maleimide. In fact, through simple chemical reactions it is possible to conjugate biomolecules, such as proteins, peptides and antibodies or antibody fragments that expose thiol (-SH) and/or cysteine groups, to the maleimide group.

Conjugation with targeting peptide

For example, to be selective towards the Wnt-2 receptor present among others on cells of the pancreatic cancer cell line (PDAC), such as Bx-PC3, a custom-made peptide called CKAAKN was constructed.

?Development of doped ZnO-based biomimicking and tumor-targeted nanotheranostics to improve pancreatic cancer treatment? CANCER NANOTECHNOLOGY, 2022, Vol. 13, Article n.37, pp. 1-24, ISSN. 1868-6966, doi: 10.1186/s12645-022-00140-z.;

?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 DSPE-PEG(2000) Maleimide lipid using cysteine, as reported below. Similarly, to be selective towards cells that overexpress the EphinA2 protein (such as melanoma, osteosarcoma, colorectal cancer cells, to name a few), the targeting peptide called YSA was constructed ?An ephrin mimetic peptide that selectively targets the EphA2 receptor? J BIOL CHEM. 2002, 277(49), pp.46974-9. doi: 10.1074/jbc.M208495200).

The DSPE-PEG(2000) Maleimide lipid and the peptide of interest were dissolved in N,N-Dimethylformamide (DMF, Sigma Aldrich) in a 3:1 molar ratio, at concentrations 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 DSPE-PEG(2000) Maleimide lipid solution was then added, obtaining a 1:1 DMF/PBS mixture and containing 5 mM peptide and 15 mM functional lipid. The reaction took place for 1 h at room temperature and the resulting lipid-peptide conjugate mixture (i.e. DSPE-PEG(2000)-CKAAKN or DSPE-PEG(2000)-YSA), was obtained. It was frozen at -20°C and stored as a stock. Before use, the aliquot of interest was diluted 1:10 in ethanol and then incorporated into the final lipid formulation to coat the nanoparticles.

Example 13 - Internalization in cells of formulations with functional lipid conjugated to targeting agents

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

Example on pancreatic cancer cells

As reported in Figure 16, ZnO nanoparticles coated with the 3C<- > formulation were incubated with cultures of Bx-PC3 pancreatic cancer cells for 24 hours. The 3C<- >lipids gave a modest increase in internalization compared to the nanoparticles as is (the figure shows the % of positive events compared to untreated control cells), indicating that the cells treated with ZnO-3C<- > were able to internalize or immobilize the nanoparticles on their surface. In the presence of the targeting peptide, in this case CKAAKN, internalization into the cell increased significantly. The staining of the targeting peptide in this case did not compromise the cell recognition sites, allowing for high internalization with both the unstained and FITC-stained targeting peptide.

Example on colorectal cancer cells

In another use case, ZnO NPs coated with the 3C<- > formulation were bioconjugated with the YSA targeting peptide directed towards colorectal cancer cells (HT-29 cell line), for which a cytotoxicity study was carried out (Figure 17A), demonstrating that the coated particles were significantly less cytotoxic than uncoated nanoparticles up to the highest concentrations. It was subsequently demonstrated that thanks to the bioconjugation with the YSA peptide, the nanoparticles were able to be internalized into the cell to a greater extent than particles coated with the same lipid formulation, but without the peptide (Figure 17B). However, it is worth noting the high biomimetic quality of the lipid formulation itself, which was internalized to a high extent even without the specific peptide. Finally, a hemocompatibility test was conducted by treating plasma with the particles as is and the particles coated with the 3C- formulation, bioconjugated or not with the YSA peptide, and measuring the plasma clotting time following treatment. Figure 17C reports the results of this experiment and shows how the samples of coated nanoparticles, both with and without the peptide, produced a clotting time that did not differ from that of plasma alone, demonstrating the hemocompatibility of the nanoconstruct and, indirectly, the correct coating of the particles with the lipid formulation.

Claims (14)

CLAIMS 1. Coated nanoparticle for the 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) a formulation containing 45.0 to 65.0% 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP+) or 1,2-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 optionally 0.1 to 0.6% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG(2000)-maleimide); (ii) formulation containing 10.0 to 35.0% cholesterol; 8.0 to 32% sphingomyelin (SM); 11.0 to 21.0% DSPE-PEG(2000)-amine; 18.0 to 34.0% DOPC; 15.0 to 35.0% DOPA; (iii) formulation containing 10.0 to 35.0% cholesterol; 8.0 to 32% SM; 11.0 to 21.0% DSPE-PEG (PEG(2000)-amine); 18.0 to 34.0% DOPC; 15.0 to 35.0% PS; (iv) 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); where 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. A coated nanoparticle according to claim 1, wherein the lipid coating is selected from the following formulations: (a) formulation (i) containing DOPA(-) 57.3%; DOPC 12.5%; cholesterol 23.6%; DSPE-PEG(2000)-amine 6.6%; (b) formulation (ii) containing DOTAP(+) 56.5%; DOPC 12.7%; cholesterol 24%; DSPE-PEG(2000)-amine 6.7%; (c) formulation (iii) containing cholesterol 13%; SM 20%; DSPE-PEG(2000)-amine 16%; DOPC 26%; DOPA 25%; (d) formulation (iv) containing cholesterol 30%; SM 16%; DSPE-PEG(2000)-amine 13%; DOPC 21%; DOPA 20%; (e) formulation (v) containing cholesterol 13%; SM 20%; DSPE-PEG(2000)-amine 16%; DOPC 26%; PS 25%; (f) formulation (vi) containing cholesterol 30%; SM 16%; DSPE-PEG(2000)-amine 13%; DOPC 21%; PS 20%; (g) formulation (vii) containing cholesterol 13%; SM 20%; DSPE-PEG(2000)-amine 10%; PE 6%; DOPC 26%; PS 25%; (h) formulation (viii) containing cholesterol 30%; SM 16.0%; DSPE-PEG(2000)-amine 8%; PE 5%; DOPC 21%; PS 20%. 3. Coated nanoparticle according to claims 1-2, wherein the coating is linked to one or more molecules capable of localizing on target cells, tissues, or organs, said molecules being anchored to the external surface of at least one coated nanoparticle. 4. Coated nanoparticle according to claim 3, wherein said molecule capable of localizing on the target cell, tissue, or organ of the therapeutic or diagnostic agent is chosen from proteins, antibodies, antibody fragments, peptides, carbohydrates, aptamers, or nucleic acids. 5. A 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 frameworks (MOFs); covalent organic frameworks (COFs); carbon-based nanoparticle, in particular nanoonions and fullerenes; natural or synthetic polymer, in particular gelatin and PLGA; 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, or organosilica nanocapsule. 9. Coated nanoparticle according to claim 8, wherein said organosilica incorporates a disulfide group -S-S- and is functionalized with silanes derivatives, preferably with the cyclooctane silane of formula (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. A coated nanoparticle according to claims 5-10, wherein the core is loaded with a therapeutic agent selected from anticancer, antibiotic, or anti-inflammatory drugs; hormones; proteins; enzymes; nucleic acids and peptides; or with a diagnostic agent selected from organic fluorescent dyes; contrast agents for nuclear magnetic resonance imaging (NMR) and isotopes for PET (Positron Emission Tomography). 12. A coated nanoparticle according to any preceding claim, for use in a therapeutic or diagnostic treatment method involving the administration of said nanoparticle carrying the therapeutic or diagnostic agent to a subject requiring 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 carriers or excipients.