Use of glutathione-coated and lithium-functionalized gold nanoparticles (LiG-AuNPs) for modulation of glycogen synthase kinase-3 activity

Abstract

The present invention relates to a method for producing glutathione and Li + This paper relates to a method for producing ion-coated gold nanoparticles (AuNPs) (hereinafter referred to as LiG-AuNPs), a method for preparing aggregates of the nanoparticles, and the use of the nanoparticles, aggregates, or combinations thereof for therapeutic use, where LiG-AuNPs are an effective means of maintaining extracellular lithium levels below the systemic toxic threshold (1.5 mEq / L) while simultaneously inhibiting GSK-3 and its downstream molecular targets and exerting antioxidant effects through the glutathione present on their surface.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a compound comprising glutathione and lithium cations (I) (Li + The present invention relates to a method for producing gold nanoparticles (AuNPs) coated with LiG (hereinafter referred to as LiG-AuNPs), a method for preparing aggregates of said nanoparticles, and the use of said nanoparticles, their aggregates, or compositions comprising them for therapeutic use. [Background technology]

[0002] Lithium is an alkali metal that was first identified in 1817, and already a few years after its discovery, in its inorganic form as chloride, carbonate, acetate, citrate, or sulfate, found use as a psychotropic drug for the treatment of mania. Although lithium can act on a variety of molecular targets, its main cellular targets are thought to be i) magnesium-dependent inositol polyphosphate 1 phosphatases and phosphomonoesterases; ii) Na + / K + ATPase (NKA) pumps; and iii) glycogen synthase kinase 3 (GSK-3) enzymes. All three of these classes of molecules require magnesium ions (Mg 2+ ) dependent. What is interesting to note is that Li + The ionic radius of Mg 2+ is similar to the ionic radius of Li, which determines the known diagonal relationships between the elements of the periodic table. + Inositol polyphosphate monophosphate 1-phosphatase binds Ca(II) from intracellular stores. 2+ NKA is a molecule involved in the production of inositol triphosphate (IP3), an important intracellular mediator involved in the release of Na. +Finally, GSK-3 is a key serine / threonine kinase involved in several intracellular signaling pathways, regulating various cellular processes, including, but not limited to, cell proliferation and differentiation, apoptosis, and immune response. GSK-3 is also highly implicated in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease (AD) and other tauopathies, because GSK-3 is the primary kinase responsible for the hyperphosphorylation of tau protein and is also involved in the proteolytic cleavage of amyloid precursor protein (APP) to form the β-amyloid peptide, a hallmark sign of AD. In addition, other studies have demonstrated a role for GSK-3 in the phosphorylation of proteins key to the entry and replication of various viruses in cells, including herpes simplex virus (HSV)-1 (see Murru et al. 2020 and references cited therein), and various coronaviruses, e.g., SARS-CoV-1 and SARS-CoV-2 (the agent responsible for CoViD-19 - Liu et al. 2021).

[0003] GSK-3 is thought to be the most important target among various molecular targets of lithium. GSK-3 has two isoforms, α and β, and is a constitutively active kinase. The isoforms α and β are phosphorylated at the 21st and 9th amino acid residues, respectively (pGSK-3 Ser9 / 21) represents the main inhibitory and regulatory mechanism of GSK-3 activity. Under physiological conditions, such phosphorylation is mediated by protein kinase B, also called Akt, which is involved in the intracellular signaling pathway PI3K-mTOR-Akt. However, GSK-3 can be further activated by tyrosine phosphorylation at positions 279 / 216 for the isoforms α / β, respectively, which is dependent on the tyrosine kinase Fyn. Several studies (Snitow et al. 2021 and references therein) have shown that lithium regulates GSK-3 activity at the intracellular level by inducing phosphorylation of serine 9 / 21. Once activated, some transporters, such as Na + -Li + Exchange transporters, or nonselective ion channels, e.g., Na + Once inside the cell, Li + exerts its inhibitory effect on GSK-3 by competing with magnesium(II) for the magnesium binding site (direct action) or by activating Akt (indirect action).

[0004] Lithium has several therapeutic uses, including: i) the treatment of pathologies that present activation of GSK-3 (especially isoform β) as a primary molecular mechanism, among them mood and personality disorders (bipolar disorder); ii) the treatment of neurodegenerative diseases (e.g. Alzheimer's disease and various tauopathies, Parkinson's disease, and Huntington's disease, all of which depend on GSK-3-mediated phosphorylation of various disease-key proteins); iii) the treatment of neoplastic diseases and viral infections, such as those caused by herpes simplex virus and respiratory viruses, such as coronaviruses.

[0005] Lithium-based drugs, primarily in tablet formulations of lithium carbonate, have long been used for the treatment of mood disorders and / or cluster headaches. However, the toxicity of lithium at concentrations effective for neuropsychiatric and neurodegenerative disorders remains an unresolved issue. This necessitates constant monitoring of plasma lithium levels, which must be maintained within the range of 0.8–1.2 mEq / L in patients treated with these drugs. This aspect is particularly limiting when considering the impossibility of off-label use in the treatment of all serious conditions (e.g., neurodegenerative diseases and viral infections) that require therapeutic concentrations of lithium far above the toxic limit. It is important to note that many of these conditions are also characterized by severe oxidative stress and often require combined treatment with antioxidants.

[0006] Therefore, the development of new lithium-based drugs that are highly effective at concentrations below the toxic levels of this metal is highly desirable in the treatment of many human and animal disease states.

[0007] As already emphasized above, the pharmaceutical use of lithium is strongly limited by its high toxicity. + Drugs that determine the concentration of lithium actually cause significant functional abnormalities, especially at the thyroid and renal system level, and even at low concentrations, undesirable effects have been observed in people with predispositions to dysfunction or in subjects who are weak due to other pathologies. Lithium-based drugs (e.g., lithium carbonate in 300 mg tablets), which are primarily used for mood disorders, are generally taken by the oral route and are administered in large doses. + The ion is distributed throughout the body by systemic routes, reaching all organs and tissues, including the kidneys and thyroid gland, which are particularly affected by the toxic effects of this metal cation. Therefore, the dosage regimen of such drugs must be constantly monitored with the aim of maintaining plasma lithium concentrations in the range of 0.4-1.2 mEq / L and ensuring an individual benefit / harm ratio. Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, it has become important to develop an administration system (drug delivery) that ensures the distribution of lithium ions in a site-specific (non-systemic) manner and at the same time ensures pharmaceutical efficacy by lowering the effective concentration below the toxic threshold. [Means for solving the problem]

[0009] The authors of the present invention have reported that glutathione and Li + Ion-coated gold nanoparticles, dispersed in a suitable solvent, form aggregates (within seconds) having diameters of approximately 100-300 nm, also referred to herein as LiG-AuNP (lithium glutathione-gold nanoparticle) aggregates, producing gold nanoparticles that are sufficiently stable over time and at high temperatures, particularly between -20 and 120°C. Only after approximately 30 days do the colloidal aggregates reach a size of approximately 1000 nm in diameter (see Figure 1), which functions equally well for the purposes described and claimed below. The authors of the present invention surprisingly found that the aggregates are rapidly internalized in vitro in cells of human origin (e.g., neuroblastoma or hepatocellular carcinoma cells) or animal origin (e.g., astrocytes or mouse neurons) in a time range between 1 hour and 24 hours (see Buonerba et al., Scientific Reports 2020 and Figure 2), and that during this internalization process, the aggregates induce Li+ transport in the cytoplasm and / or in the organelles they reach. + They found that it can be broken down into single nanoparticles while releasing ions.

[0010] Therefore, treatment with the aggregates described above provides pharmacologically effective Li over a shorter period of time and at lower concentrations than commonly used lithium salts. + Allows for controlled release of ions.

[0011] Without wishing to be bound by theory, the authors believe that LiG-AuNP aggregates and nanoparticles are the cause of Li + The mechanism of cation release is believed to be the exchange of this cation with other cations, such as sodium and / or potassium, which are present in high concentrations extracellularly or intracellularly. To support this mechanism hypothesis, when the LiG-AuNPs decomposed after sonication were dispersed in Dulbecco's modified Eagle's medium (DMEM, a standard medium for cell culture), approximately 83.5% of the Li was released in 1 hour. + It was observed that cations were released into the medium, reaching a value of 91.4% at 24 h. The remaining Li still present in the LiG-AuNPs (presumably inside the aggregates) + The cations are internalized into the cells along with the aggregates and then decomposed inside the cells, which may determine a more effective intracellular accumulation of lithium than methods based solely on internalization via non-selective transporters or ion channels (see operation scheme, Figure 11).

[0012] First, the authors evaluated the potential in vitro cytotoxicity of LiG-AuNPs. To this end, VERO cells (renal epithelial cells isolated from African green chlorocebus monkeys) and SH-SY5Y cells (human neuroblastoma) were exposed for 24 hours to increasing concentrations (0.1 mg / mL to 10 mg / mL) of LiG-AuNP aggregates dispersed in the cell culture medium. For both cell models, LiG-AuNPs did not cause significant cell death (>85% viability) at concentrations below 2 mg / mL, above which cell viability decreased. The CC50, the concentration at which half of the cells die, was estimated to be approximately 4.5–5.0 mg / mL in vitro (Figure 3).

[0013] Next, the authors investigated the Li + We decided to evaluate the effectiveness of LiG-AuNPs in vehiculating ions. For this purpose, SH-SY5Y cells were treated with a non-toxic concentration of LiG-AuNP aggregates (1 mg / mL, Li + The cells were treated with 3 mEq / L of Li for 24 hours.+ Ion concentrations were determined via ICP-OES. As a comparative example, other SH-SY5Y cells were treated with LiCl to remove extracellular Li. + The cells were treated with LiG-AuNP aggregates at an equal concentration (3 mEq / L) in the culture medium. + The concentrations were observed to be approximately 26 times greater than those measured in similar treatments using lithium chloride, specifically, 0.154–0.3375 pg in cells treated with LiG-AuNPs. Li / cell and 0.007–0.0375 pg in LiCl-treated cells Li Extracellular lithium is generally expressed as Na + It is emphasized that the ATP enters the cell nonspecifically using the channels for ATP.

[0014] The authors of the present invention have demonstrated that LiG-AuNP aggregates and nanoparticles inhibit the inhibitory phosphorylation of GSK-3β (pGSK-3β) under in vitro experimental conditions. Ser9 It was further found that the LiG-AuNP aggregates (Li) at a concentration of 1 mg / mL were particularly effective in inducing a significant increase in the LiG-AuNP aggregates (Li) for 1 hour or 24 hours. + SH-SY5Y human neuroblastoma cells treated with 3 mEq / L of 6 mM lithium chloride (Li + corresponds to 6 mEq / L, which is the minimal concentration usually used for in vitro studies of GSK-3 inhibition. Ser9 The ratio of pGSK-3β to pGSK-3β is shown in Figure 4. Ser9 To confirm the effectiveness of LiG-AuNPs in determining a significant increase in extracellular Li, SH-SY5Y cells were incubated for 24 hours in a manner that was largely ineffective. + Concentration (0.05 mg / mL, Li + The pGSK-3β cells were treated with LiG-AuNP aggregates (corresponding to 0.15 mEq / L). Ser9 The ratio of GSK-3β / GSK-3β was significantly higher than that found in vehicle-treated cells (+54% ± 12%; p < 1 × 10 vs. vehicle).-3 ), and is higher than the ratio found in cells treated with 0.15 nM LiCl for the same time (Figure 4). Finally, to confirm the specificity of the effect for lithium, some experiments were repeated treating SH-SY5Y cells for 24 hours with similar nanoparticles functionalized with sodium instead of lithium, NaG-AuNPs (1 mg / mL concentration). Such treatment significantly increased the expression of pGSK-3β. Ser9 / GSK-3β ratio (Fig. 4).

[0015] The LiG-AuNP aggregates and nanoparticles were synthesized using the above-mentioned pGSK-3β Ser9 In addition to regulating the above kinases, the demonstrated efficacy in inhibiting their downstream targets, particularly tau protein phosphorylation and herpes simplex virus type 1 (HSV-1) replication, two events related to these in neuronal cells, is also demonstrated (De Chiara et al., Plos Pathogens, 2019). HSV-1 replication is well known to be sensitive to the effects of lithium and depends on oxidative stress caused by viral invasion. In particular, LiG-AuNP aggregates and nanoparticles were shown to inhibit both HSV-1 infection and late-induced tau phosphorylation in vitro at a concentration of 0.05 mg / mL (extracellular Li + It has been demonstrated that even concentrations of 0.15 mEq are effective, which is much lower than the concentrations usually used for this type of treatment (> 10 mM), and especially Li + (See Examples and Figures 5-7). It is important to emphasize that, advantageously, in parallel with the action of lithium, the presence of glutathione on the outer crown of the nanoparticles antagonizes the oxidative stress induced by viral invasion (See Examples and Figure 8).

[0016] Finally, when administered in a mouse model, it was demonstrated that LiG-AuNPs readily reach the brain (as measured by the presence of gold / lithium), especially when administered by the intranasal route, and that once loco, LiG-AuNPs are able to modulate GSK-3 activity, especially at the hippocampal level, without establishing side effects (e.g., gliosis; see section in the Examples and Figures 9 and 10).

[0017] As detailed hereafter, the synthesis, purification, and recovery procedures for LiG-AuNPs are simple, inexpensive, and do not require the use of sophisticated and expensive equipment. LiG-AuNPs are isolated in a solid and sterile form, allowing them to be redispersed in aqueous and organic media to form aggregates useful for internalization into target cells. The by-products and waste products produced do not pose toxicity or environmental compatibility issues. Finally, the synthesis method is potentially readily scalable to the quantities required in the pharmaceutical industry.

[0018] The LiG-AuNPs and their aggregates according to the present invention find application in all biological phenomena that are regulated by GSK-3 and / or determined by changes in the intracellular redox state, such as, for example, neuropsychiatric diseases (e.g., mood disorders), neurodegenerative diseases (e.g., tauopathies), viral infections (e.g., HSV-1 or Sars-CoV) or neoplastic pathologies.

[0019] Thus, the present invention relates to the following:

[0020] The method comprises the steps of: + Preparation of gold nanoparticles coated with: i) a gold precursor at a concentration of 0.0001 M to 10 M, preferably 0.001 M to 0.1 M, more preferably 0.010 M to 0.020 M, even more preferably 0.017 M; at least one polar solvent; glutathione in a molar ratio to the gold precursor of 0.1:1 to 100:0.1, preferably 1:1 to 1:0.1; a basic lithium compound in a molar ratio relative to the gold precursor of 0.01:10 to 10:0.01, preferably 1:1 to 1:0.1, more preferably 1:0.05 to 1:0.15, and even more preferably 1:0.09; preparing the mixture at a temperature of -20°C to +120°C, preferably at room temperature, in an environment equipped with a stirring and shaking system to obtain a colorless and transparent solution; ii) diluting the solution obtained in step i) with at least one polar solvent in the range of 1:0 to 1:1000, preferably 1:5 to 1:50, more preferably 1:5 to 1:30, even more preferably 1:20; iii) adding a reducing agent for the gold precursor to the diluted solution of step ii) in a molar ratio relative to the gold precursor of 1:1 to 100:0.1, preferably 1:1 to 1:0.1, more preferably 1:0.1 to 1:0.3, even more preferably 1:0.22, at a temperature of -20°C to +120°C, preferably at room temperature, in an environment equipped with a stirring and shaking system, to obtain a colloidal suspension of gold nanoparticles; iv) adding a lithium salt to the gold precursor in a molar ratio of 0.1:1 to 100:0.001, preferably 1:0.001 to 1:1, more preferably 1:0.001 to 1:0.005, and even more preferably 1:0.002; v) diluting the solution obtained in step iv) with at least one polar solvent in the range of 1:0 to 1:1000, preferably 1:5 to 1:50, more preferably 1:5 to 1:30, even more preferably 1:20; vi) purifying the nanoparticles obtained in steps iv)-v). (Here, if the dilution is 1:0, then step ii) above is not performed.)

[0021] Glutathione and Li + wherein the nanoparticles have a diameter of 0.1 to 100 nm.

[0022] A method for preparing aggregates of LiG-AuNPs comprising the aforementioned steps (i-iv) of the method according to the present invention and an additional step for dispersing the particles in a solvent selected from deionized water, alcohol, an aqueous alcoholic solution, or a pharmaceutically acceptable solution or suspension.

[0023] An aggregate of nanoparticles as defined herein and in the claims, wherein the aggregate has a diameter of 0.1 to 5000 nm.

[0024] A pharmaceutical composition comprising an aggregate of nanoparticles as defined herein and in the claims, in a solvent selected from deionized water, alcohol or an aqueous alcoholic solution, or a solution or suspension, and at least one pharmaceutically acceptable excipient and / or carrier.

[0025] A kit comprising a plurality of the nanoparticles of the present invention as defined herein and in the claims, and one or more aliquots of deionized water, alcohol or hydroalcoholic solution, or a pharmaceutically acceptable solution or suspension.

[0026] A gold nanoparticle, or nanoparticle aggregate, or composition, or kit as defined herein for use in therapeutic treatment.

[0027] Additional advantages and / or embodiments of the present invention may become apparent from the following detailed description. [Brief explanation of the drawings]

[0028] [Figure 1A-E]Microscopic features and dynamic light scattering (DLS) of LiG-AuNPs. (A,B) Representative images of LiG-AuNPs obtained in a classical transmission electron microscope (TEM) using two different magnifications. (C-E) Representative images obtained in a classical transmission electron microscope (STEM) in scanning mode showing an annular dark-field image (HAADF, panel C), the presence of sodium ions (Na+, panel D), and chloride ions (Cl-, panel E) in the outer crown of the nanoparticles. [Figure 1F-a] (Fa-d) Dynamic light scattering (DLS) analysis of LiG-AuNPs left in deionized water at +4 °C for a) 0 days (analyzed immediately after ultrasonic deagglomeration); c) 7 days; c) 15 days; d) 30 days, showing the presence of a) dissociated nanoparticles (hydrodynamic diameter of approximately 3.8 nm), b) small aggregates (approximately 250 nm), c) aggregates with an average size (approximately 300 nm) with the formation of larger sized aggregates, and d) large aggregates (> 1 μm). [Figure 1F-b] (Fa-d) Dynamic light scattering (DLS) analysis of LiG-AuNPs left in deionized water at +4 °C for a) 0 days (analyzed immediately after ultrasonic deagglomeration); c) 7 days; c) 15 days; d) 30 days, showing the presence of a) dissociated nanoparticles (hydrodynamic diameter of approximately 3.8 nm), b) small aggregates (approximately 250 nm), c) aggregates with an average size (approximately 300 nm) with the formation of larger sized aggregates, and d) large aggregates (> 1 μm). [Figure 1F-c] (Fa-d) Dynamic light scattering (DLS) analysis of LiG-AuNPs left in deionized water at +4 °C for a) 0 days (analyzed immediately after ultrasonic deagglomeration); c) 7 days; c) 15 days; d) 30 days, showing the presence of a) dissociated nanoparticles (hydrodynamic diameter of approximately 3.8 nm), b) small aggregates (approximately 250 nm), c) aggregates with an average size (approximately 300 nm) with the formation of larger sized aggregates, and d) large aggregates (> 1 μm). [Figure 1F-d](Fa-d) Dynamic light scattering (DLS) analysis of LiG-AuNPs left in deionized water at +4 °C for a) 0 days (analyzed immediately after ultrasonic deagglomeration); c) 7 days; c) 15 days; d) 30 days, showing the presence of a) dissociated nanoparticles (hydrodynamic diameter of approximately 3.8 nm), b) small aggregates (approximately 250 nm), c) aggregates with an average size (approximately 300 nm) with the formation of larger sized aggregates, and d) large aggregates (> 1 μm). [Figure 1-1] Microscopic features and dynamic light scattering (DLS) of G-AuNPs. [Figure 2] Internalization of LiG-AuNPs in SH-SY5Y human neuroblastoma cells. (A,B) Representative images of SH-SY5Y human neuroblastoma cells treated with vehicle (deionized water; panel A) or 1 mg / mL LiG-AuNPs for 24 h, acquired by transmission laser microscopy (λex: 514 nm). Arrows indicate dark spots, which are areas of gold where light could not pass through. [Figure 3] Evaluation of the cytotoxic effect of LiG-AuNPs. Graph showing the percentage of cell viability at various tested concentrations of LiG-AuNPs. LiG-AuNP aggregates at extracellular concentrations of 2 mg / mL or higher induce a decrease in cell viability in VERO and SH-SY5Y cells. The cytotoxic concentration at which half of the cells die (CC50) was estimated to be 4.3-5.0 mg / mL. ns: No significant difference compared to vehicle (= 0 mg / mL). [Figure 4]LiG-AuNP aggregates induce phosphorylation of serine 9 in GSK-3β, even at low concentrations of lithium: (A) Representative Western blots of pGSK-3βS9 in SH-SY5Y neuroblastoma cells treated for 1 or 24 hours with LiCl (6 mM, equivalent to 6 mEq / L lithium) or 1 mg / mL (3 mEq / L Li) and 0.05 mg / mL (0.15 mEq / L) LiG-AuNPs aggregated in deionized water for 1–7 days. Vehicle treatment with deionized water is used as a control. GAPDH was used as a loading control. (B) Bar graph showing the ratio of phosphorylated GSK-3β (serine 9) expression to total GSK-3β expression under the various experimental conditions shown in (A). Treatment with NaG-AuNPs was used as a control for 24 hours of LiG-AuNPs. Bars represent the mean of n=5-10 independent experiments per condition. Points indicate the values ​​obtained in each experiment. **p<0.01 (vs. vehicle); ***p<0.001 (vs. vehicle); ns, not significantly different from vehicle. [Figure 5]LiG-AuNPs inhibit HSV-1 infection in mouse cortical astrocytes (as determined by immunofluorescence analysis of ICP4 viral protein expression). (A-D) Representative images obtained by confocal microscopy of mouse cortical astrocytes infected with HSV-1 (multiplicity of infection = 1) and immunotreated for ICP4 viral protein (green) 24 h postinfection. Glial fibrillary acidic protein (GFAP, red) was used to stain astrocytes. Cell nuclei were stained blue with DAPI. Panel (A) shows mock-infected cells. Panel (B) shows cells infected with HSV-1 without additional treatment. Increased red color (GFAP) indicates the presence of astrocytes. In panels C and D, infected cells were treated with LiG-AuNPs and NaG-AuNPs (1 mg / mL), respectively, throughout the entire postinfection period. (E) Bar graph quantifying the percentage of ICP4-positive (infected) cells under the conditions depicted in panels A-D. LiCl condition (6 mEq / L lithium) was added as an additional control. A significant decrease in the percentage expression of ICP4 protein expression is observed only in cells treated with LiG-AuNPs and LiCl. **p<0.001. ns: not statistically significant. [Figure 6-1]LiG-AuNPs inhibit HSV-1 infection (by plaque assay and molecular biology / Western blot). (A) Quantitative evaluation of virus titer in terms of plaque-forming units (PLU) / mL by plaque standard assay on supernatants of VERO cells infected with HSV-1 (1 MOI) in the absence (vehicle) and presence (1 and 2 mg / mL) of LiG-AuNPs, assessed 24 hours postinfection. (B) Western blot analysis of lysates obtained from VERO cells infected with HSV-1 (1 MOI) and treated with vehicle or LiG-AuNPs (1 and 2 mg / mL) during adsorption (ADS) or postadsorption (pi), analyzed 24 hours postinfection. Infection is quantified in terms of immunoreactivity for the viral envelope gB glycoprotein or the early viral protein ICP0. Actin is used as a loading internal standard. (C) Bar graph quantifying the optical density of the bands represented in panel B. White bars are for gB, while gray bars are for ICP0. (D) Representative Western blot analysis of lysates from SH-SY5Y human neuroblastoma cells infected with HSV-1 (1 MOI) and treated with vehicle (double distilled water) or a low concentration of LiG-AuNPs (0.05 mg / mL, corresponding to 0.15 mEq / L of lithium) during the adsorption phase (Ads), the entire postinfection period (pi), or during all stages of infection (all stages: Ads + pi), assessed at the end of 24 hours pi. Lysates were probed with direct antibodies against various proteins of the HSV-1 virus (panHSV-1). GAPDH was used as a loading control. (E) Bar graph showing quantification of the bands depicted in panel D, HSV-1 (white bars) and ICP0 (gray bars, WB not shown). *p<0.05 and **p<0.001 vs. vehicle. [Figure 6-2] LiG-AuNPs inhibit HSV-1 infection (by plaque assay and molecular biology / Western blot). [Figure 7]Treatment with LiG-AuNPs reduces tau phosphorylation at threonine 205 in HSV-1-infected cells. (A-C) Representative confocal microscopy images of mock-infected SH-SY5Y cells or cells infected with HSV-1 (1 MOI) treated with vehicle (A), 0.05 mg / mL (B), or 1 mg / mL (C) LiG-AuNPs, fixed 8 hours postinfection, and then immunotreated for pTauT205. (D) Bar graph quantifying immunoreactivity for pTauT205 under the above conditions and in SH-SY5Y cells represented in panels A-C. White bars represent mock-infected (mock) cells, while HSV-1-infected cells are colored. **p<0.001 for linear regression analysis. [Figure 8-1]LiG-AuNPs exert antioxidant effects. (A-C) Representative images obtained by confocal microscopy of SH-SY5Y human neuroblastoma cells treated with lipopolysaccharide (LPS, 5 μg / mL) for 24 hours, vehicle (deionized water) or LiG-AuNPs (1 mg / mL), followed by fixation with PFA (4%) and treatment with dihydroethidium (DHE), a fluorescent label for superoxide. (D) Bar graph showing quantification of the experiments depicted in panels A-C. (E) Bar graph quantifying the levels of NADPH oxidase 4 (NOX4) enzyme in VERO cells infected with HSV-1 (1 MOI) and treated with or without LiG-AuNPs (1 mg / mL), as assessed by Western blot analysis (not shown). (F) Representative Western blot analysis of lysates from VERO cells infected with HSV-1 (1 MOI) or not (mock-infected) and subsequently treated with vehicle or LiG-AuNPs (1 or 2 mg / mL, either only during the viral adsorption phase—Ads—or for the entire postinfection period—pi—) labeled with direct antibodies against the immature / inactive form of the proinflammatory cytokine IL-1β (IL-1β precursor, 31 KDa) or the active (cleaved) form of IL-1β (IL-1β, 17 KDa). Actin was used as a loading internal control. (G) Bar graph showing quantification of the experiment depicted in panel F. White bars refer to IL-1β precursor, while gray bars refer to IL-1β. **p<0.001; ***p<0.0001. [Figure 8-2] LiG-AuNPs exhibit antioxidant properties. [Figure 9]Intranasal administration of LiG-AuNPs induces phosphorylation of serine 9 of GSK-3β in the mouse hippocampus. (A) Western blot analysis of pGSK-3β Ser9 and GSK-3 (total) in hippocampal lysates from C57Bl / 6 mice treated intranasally (3 μL per nostril, bilaterally) with vehicle (left band) or LiG-AuNPs (1, 10, and 100 mg / mL) for 5 consecutive days and dissected 6 hours after the last administration. GAPDH was used as a loading internal standard. (B) Bar graph quantifying the optical density values ​​of pGSK-3β Ser9 (light gray), GSK-3β "total" (dark gray), and their ratio (red bars / very dark gray) from the WB analysis shown in panel A. The dotted line represents the mean level of the control (vehicle: 0 mg / mL) value. (C-D) Bar graphs quantifying the optical density values ​​of pGSK-3βSer9 (light gray), GSK-3β "total" (dark gray), and their ratio (green bars / very dark gray) in lysates extracted from the cortex (panel C) and olfactory bulb (panel D) of mice treated as in A. *p<0.05; **p<0.01 ***p<0.001; ns: not significant. [Figure 10] Intranasal administration of LiG-AuNPs does not induce a glial response in the hippocampus of treated mice. (A) Western blot analysis of GFAP in hippocampal lysates from C57Bl / 6 mice treated intranasally (3 μL per nostril bilaterally) with vehicle (left band) or LiG-AuNPs (1, 10, and 100 mg / mL) for 5 consecutive days and dissected 6 hours after the last administration. GAPDH was used as a loading internal standard. (B) Bar graph quantifying the optical density values ​​for GFAP. ns, no significant difference. [Figure 11]Representative Operation Scheme of LiG-AuNPs: 1) When LiG-AuNPs are anhydrous, the nanoparticles are separated from each other. 2) When LiG-AuNPs are placed in a deionized aqueous environment, they tend to form aggregates in a time-dependent manner (see Figure 1). 3) When the aggregates are placed in a solution containing monovalent cations (e.g., Na+, K+), such as cell culture medium, the lithium present on the outer edge of the aggregates is released by exchange with the cations, but the majority of the "inner" lithium ions remain protected from the external environment and cannot be released. 4) When LiG-AuNP aggregates enter cells, they disaggregate into single nanoparticles (see Buonerba et al., Scientific Reports 2020), and thus the attached lithium is released according to the normal mechanism of cation(s) exchange. The decomposition of LiG-AuNPs at the intracellular stage determines the increase in Li+ ion concentration in the cytoplasm and / or intracellular organelles, thus effectively contributing to the increase in intracellular lithium levels and its therapeutic action. The lithium thus released into the cytoplasm has the potential to directly interact with GSK-3, determining its inhibition and all its associated downstream effects. DETAILED DESCRIPTION OF THE INVENTION

[0029] Additional advantages and / or embodiments of the present invention may become apparent from the following detailed description.

[0030] Glossary The LiG-AuNPs (lithium glutathione-gold nanoparticles) in the present invention are coated with glutathione and functionalized with lithium (I) ions (i.e., where some functional groups of glutathione are Li + The present application relates to spherical gold nanoparticles (bound to ions) having diameters in the range of 0.1 to 10 nm, and which are believed to have an average diameter of 2 nm based on the synthesis procedure developed and claimed in the present application.

[0031] The LiG-AuNP aggregates according to the present description are aggregates of the nanoparticles obtainable by dispersing the nanoparticles in water, alcohol or an aqueous alcoholic solution, or a pharmaceutically acceptable solution or suspension for 1 to 7 days, preferably 7 days, and such aggregates have a diameter of about 100 to 300 nm and are stable over time at temperatures in the range of +4 to +43°C for a period of 25 to 35 days, after which they form aggregates with a diameter of about 1000 nm.

[0032] The gold precursor is an inorganic or organic gold compound, as well as preformed gold nanoparticles. The gold compound chosen for the synthesis of LiG-AuNPs is tetrachloroauric acid (HAuCl4; CAS number: 16903-35-8) in the anhydrous form or tetrachloroauric acid (HAuCl4·3H2O; CAS number: 16961-25-4) in the trihydrate form.

[0033] Reduced glutathione according to the invention relates to the tripeptide having the following formula and more precisely the CAS number: 70-18-8: [ka]

[0034] The basic lithium compound in the present invention lithiates glutathione, i.e., connects the proton of the carboxylic acid with Li + Any inorganic, organic, or metallic lithium compound capable of chemically replacing the cation. Lithium hydroxide (anhydrous) (CAS number: 1310-65-2) or its hydrated form (CAS number: 1310-66-3) is the basic lithium compound of choice for the synthesis of LiG-AuNPs.

[0035] Lithium salts are inorganic or organic ionic lithium compounds whose function is to facilitate the formation of particle aggregates and, in the case of aqueous alcoholic solutions, the precipitation of the particles themselves. Lithium chloride (CAS number: 7447-41-8) is the lithium salt chosen for the synthesis of LiG-AuNPs.

[0036] The reducing agent for the gold precursor is any compound that has a reducing power under standard conditions that is lower than that of gold under the same conditions. Sodium borohydride, lithium borohydride, and lithium aluminum hydride are some examples of compounds that can be used to obtain LiG-AuNPs.

[0037] Suitable solutions for administration The solutions described in this invention may be in a pharmaceutically acceptable form and therefore suitable for administration to a patient in need thereof.

[0038] In the present description, GSK-3 has the meaning widely accepted in the scientific literature and refers to glycogen synthase kinase 3, which exists in two equally abundant isoforms (α and β), with β being associated with degenerative diseases. Regulation of kinase activity depends on the phosphorylation site, which differs depending on the isoform: serine 21 for isoform α and serine 9 for isoform β. pGSK-3 means that the protein GSK-3 is phosphorylated, and a superscript after the protein name (the isoform in question (α / β)) may indicate that one or more of its amino acids are phosphorylated, e.g., pGSK-3β Ser9 indicates that the protein (isoform β) is phosphorylated at serine 9, and pGSK-3α Ser21 indicates that phosphorylation occurs at serine 21 in isoform α.

[0039] It is noteworthy that the human and mouse proteins share the same regulatory mechanism. Below are the SwissProt codes for both proteins: -P49841 (GSK3B_human) -P49840 (GSK3A_human) -Q9WV60 (GSK3B_mouse) -Q2NL51 (GSK3A_mouse)

[0040] In any part of this description and claims, the phrase "glutathione and Li + "Gold nanoparticles coated with the lithium salt of glutathione" can be replaced by any one of the following expressions: "gold nanoparticles coated with the lithium salt of glutathione", "gold nanoparticles coated with glutathione and lithium cations", or "gold nanoparticles coated with glutathione and Li(I)".

[0041] In any part of this description and claims, the term "comprising" may be replaced by "consisting of."

[0042] The term Li(I) in the description and chemical nomenclature of this specification refers to the term Li + is a synonym for

[0043] Detailed Description of the Invention The present invention provides a method for producing glutathione and Li, comprising the steps of: + Regarding the preparation method of gold nanoparticles coated with: i) a gold precursor at a concentration of 0.0001 M to 10 M, preferably 0.001 M to 0.1 M, more preferably 0.010 M to 0.020 M, even more preferably 0.017 M; at least one polar solvent; glutathione in a molar ratio to the gold precursor of 0.1:1 to 100:1, preferably 1:1 to 1:0.1; a basic lithium compound in a molar ratio relative to the gold precursor of 0.01:10 to 10:0.01, preferably 1:1 to 1:0.1, more preferably 1:0.05 to 1:0.15, and even more preferably 1:0.09; preparing the mixture at a temperature ranging from -20° to 120°C, preferably at room temperature, in an environment equipped with a stirring and shaking system to obtain a colorless and transparent solution; ii) diluting the solution obtained in step i) with at least one polar solvent in the range of 1:0 to 1:1000, preferably 1:5 to 1:50, more preferably 1:5 to 1:30, even more preferably 1:20; iii) adding a reducing agent for the gold precursor to the diluted solution of step ii) in a molar ratio relative to the gold precursor of 1:1 to 100:0.1, preferably 1:1 to 1:0.1, more preferably 1:0.1 to 1:0.3, even more preferably 1:0.22, at a temperature in the range of −20° to 120° C., preferably at room temperature, in an environment equipped with a stirring and shaking system, to obtain a colloidal suspension of gold nanoparticles; iv) adding a lithium salt to the gold precursor in a molar ratio of 0.1:1 to 100:0.001, preferably 1:0.001 to 1:1, more preferably 1:0.001 to 1:0.005, and even more preferably 1:0.002; v) diluting the solution obtained in step i) with at least one polar solvent in the range of 1:0 to 1:1000, preferably 1:5 to 1:50, more preferably 1:5 to 1:30, even more preferably 1:20; vi) purifying the nanoparticles obtained in steps iv)-v).

[0044] The concentration of the gold precursor in step i) of the method according to the invention can be any precise value up to three decimal points between 0.0001M and 10M, but in a preferred embodiment the ratio is between 0.010M and 0.0030M, preferably between 0.010M and 0.020M, and even more preferably 0.011M, 0.012M, 0.013M, 0.014M, 0.015M, 0.016M, 0.017M, 0.018M or 0.019M.

[0045] In step i) of the method of the present invention, the molar ratio of glutathione to gold precursor may be any ratio lying within the above range of 0.1:1 to 100:0.01, and in one embodiment, the ratio is preferably 1:0.6 to 1:0.3, and even more preferably 1:0.55, 1:0.50, 1:0.45, 1:0.40, or 1:0.35.

[0046] In step i) of the method of the present invention, the molar ratio of the basic lithium compound to the gold precursor may be any ratio lying within the above-mentioned range of 0.01:10 to 10:0.01, and in one embodiment, the ratio is preferably 1:0.15 to 1:0.05, and even more preferably 1:0.14, 1:0.13, 1:0.12, 1:0.11, 1:0.10, 1:0.09, 1:0.08, 1:0.07, or 1:0.06.

[0047] In a preferred embodiment, in step i) of the method according to the present invention, the concentration of the gold precursor is selected from 0.010 M to 0.020 M, even more preferably 0.011 M, 0.012 M, 0.013 M, 0.014 M, 0.015 M, 0.016 M, 0.017 M, 0.018 M, or 0.019 M, the molar ratio of glutathione to gold precursor is selected from 1:0.55, 1:0.50, 1:0.45, 1:0.40, or 1:0.35, and the molar ratio of basic lithium compound to gold precursor is selected from 1:0.14, 1:0.13, 1:0.12, 1:0.11, 1:0.10, 1:0.09, 1:0.08, 1:0.07, or 1:0.06.

[0048] In an even more preferred embodiment, in step i) of the method according to the invention, the concentration of the gold precursor is between 0.010 M and 0.020 M, in particular 0.017 M, the molar ratio of glutathione to gold precursor is 1:0.45, and the molar ratio of basic lithium compound to gold precursor is 1:0.09. As described herein, the temperature in step i) of the method according to any embodiment of the invention is preferably between 10° C. and 50° C., even more preferably room temperature (room temperature is usually considered to be a temperature of approximately 25° C.).

[0049] The dilution in steps ii) and v) of the method of the present invention can be any dilution of the dilution obtained in step i) and step iv) with at least one polar solvent, respectively, from 1:0 to 1:1000, preferably from 1:5 to 1:30, even more preferably 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, or 1:25. In the most preferred embodiment, the dilution is 1:20. It is clear that when the dilution is 1:0, one or more dilution steps of the method can be omitted.

[0050] In a preferred embodiment, in step i) of the method according to the present invention, the concentration of the gold precursor is 0.017 M, the molar ratio of glutathione to the gold precursor is 1:0.45, and the molar ratio of the basic lithium compound to the gold precursor is 1:0.09, the temperature is room temperature, and the dilution in step ii) is a 1:20 dilution.

[0051] According to the present invention, the molar ratio of reducing agent to gold precursor in step iii) of the method is any ratio between 1:1 and 100:0.10. Preferably, the ratio is any ratio between 1:0.30 and 1:0.10, and even more preferably, the ratio is 1:0.25, 1:0.24, 1:0.23, 1:0.22, 1:0.21, or 1:0.20. In an even more preferred embodiment, the ratio is 1:0.22.

[0052] In a preferred embodiment, in step i) of the method according to the present invention, the concentration of the gold precursor is 0.017 M, the molar ratio of glutathione to the gold precursor is 1:0.45, and the molar ratio of the basic lithium compound to the gold precursor is 1:0.09, the temperature is room temperature, the dilution in step ii) is a 1:20 dilution, and the molar ratio of the reducing agent to the gold precursor in step iii) is 1:0.22.

[0053] As described herein, the temperature in step iii) of the method according to any embodiment of the present invention is preferably between −20° C. and 120° C., even more preferably room temperature (typically, room temperature is considered to be a temperature of approximately 25° C.).

[0054] In a preferred embodiment, in step i) of the method according to the present invention, the concentration of the gold precursor is 0.017 M, the molar ratio of glutathione to the gold precursor is 1:0.45, and the molar ratio of the basic lithium compound to the gold precursor is 1:0.09, the dilution in step ii) is a 1:20 dilution, the molar ratio of the reducing agent to the gold precursor in step iii) is 1:0.22, and the temperature in steps i) and iii) is room temperature.

[0055] According to the present invention, the lithium salt added in step iv) of the method is in any molar ratio to the gold precursor between 0.1:1 and 100:0.001, preferably between 1.005 and 1:0.001, and even more preferably, the ratio is 1:0.004, 1:0.003, or 1:0.002. In a preferred embodiment, the ratio is 1:0.002.

[0056] In a preferred embodiment, in step i) of the method according to the present invention, the concentration of the gold precursor is 0.017 M, the molar ratio of glutathione to the gold precursor is 1:0.45, and the molar ratio of the basic lithium compound to the gold precursor is 1:0.09, the molar ratio of the reducing agent to the gold precursor in step iii) is 1:0.22, the temperature in steps i) and iii) is room temperature, the ratio of the lithium salt to the precursor in step iv) is 1:0.002, and the dilution in step ii) is a 1:20 dilution.

[0057] According to the present invention, it is clear that no dilution is performed when the dilution ratio in steps ii) and / or v) is 1:0. Therefore, the method of the present invention also includes embodiments in which the dilution steps ii) and / or v) are not performed. In such embodiments, the method of the present invention is represented by the following steps: i) a gold precursor at a concentration of 0.0001 M to 10 M, preferably 0.001 M to 0.1 M, more preferably 0.010 M to 0.020 M, even more preferably 0.017 M; at least one polar solvent; glutathione in a molar ratio to the gold precursor of 0.1:1 to 100:0.1, preferably 1:1 to 1:0.1; a basic lithium compound in a molar ratio relative to the gold precursor of 0.01:10 to 10:0.01, preferably 1:1 to 1:0.1, more preferably 1:0.05 to 1:0.15, and even more preferably 1:0.09; preparing the mixture at a temperature of -20°C to +120°C, preferably at room temperature, in an environment equipped with a stirring and shaking system to obtain a colorless and transparent solution; Optionally, [ii) diluting the solution obtained in step i) with at least one polar solvent in a range of 1:0 to 1:1000, preferably 1:5 to 1:50, more preferably 1:5 to 1:30, even more preferably 1:20. iii) adding a reducing agent for the gold precursor to the diluted solution of step ii) in a molar ratio relative to the gold precursor of 1:1 to 100:0.1, preferably 1:1 to 1:0.1, more preferably 1:0.1 to 1:0.3, even more preferably 1:0.22, at a temperature of -20°C to +120°C, preferably at room temperature, in an environment equipped with a stirring and shaking system, to obtain a colloidal suspension of gold nanoparticles; iv) adding a lithium salt to the gold precursor in a molar ratio of 0.1:1 to 100:0.001, preferably 1:0.001 to 1:1, more preferably 1:0.001 to 1:0.005, and even more preferably 1:0.002; Optionally, [v) diluting the solution obtained in step i) with at least one polar solvent in a range of 1:0 to 1:1000, preferably 1:5 to 1:50, more preferably 1:5 to 1:30, even more preferably 1:20. vi) purifying the nanoparticles obtained in steps iv)-v).

[0058] It is clear here that the dilution step ii) and / or step v) are optional steps of the method of the present invention.

[0059] According to the present invention, the gold precursor is selected from gold halides, gold chalcogens, gold pycnogens, gold crystallogens, or gold complexes and clusters (I or III), or organogold compounds, or mixtures thereof. Preferably, the gold precursor is tetrachloroauric acid in the trihydrate form.

[0060] According to the present invention, the polar solvent can be selected from aprotic polar solvents such as acetone, acetonitrile, tetrahydrofuran, dioxane, dimethyl sulfoxide, dimethylformamide, peralkylated ureas, e.g., tetramethylurea and 1,3-dimethyl-2-imidazolidinone, and hexamethylphosphoramide, or the polar solvent can be a protic polar solvent, e.g., alcohol, aqueous alcoholic solution, carboxylic acid, amine, or sulfonated or nitrated compound, or a mixture thereof. Preferably, the polar solvent selected for the synthesis of LiG-AuNPs is methanol in a mixture with one or more additional solvents, preferably an aqueous alcoholic solution of methanol, which is a mixture containing water and methanol (e.g., 20%-25%, 20%-30% aqueous alcoholic solution of methanol).

[0061] In an even more preferred embodiment, the polar solvent is a 25% aqueous alcoholic solution of methanol.

[0062] In one embodiment, the glutathione is the reduced form of glutathione (CAS number: 70-18-8).

[0063] In additional embodiments, the basic lithium compound is selected from lithium hydroxide (anhydrous) or lithium hydroxide monohydrate, lithium oxide, lithium hydride, lithium alkoxide, lithium amide, lithium carbonate, lithium bicarbonate, lithiated Zintl compound, metallic lithium, lithium / ammonia solution, lithium amalgam, lithiated anion resin, lithium phosphate, lithium sulfate, lithium carboxyl compound, lithium tetraborate, lithiated borate, lithium fluoride, lithium hypochlorite, lithium chlorite, lithium oxyanion, organolytic compound, or mixtures thereof. Preferably, the basic lithium compound is lithium hydroxide monohydrate.

[0064] In one embodiment, the reducing agent for the gold precursor is selected from sodium borohydride, lithium borohydride, and lithium aluminum hydride. Preferably, the reducing agent is sodium borohydride.

[0065] In one embodiment, the lithium salt is an inorganic or organic ionic lithium compound, preferably selected from lithium chloride, lithium iodide, lithium fluoride, lithium bromide, lithium oxide, lithium hydroxide, lithium sulfide, and preferably lithium chloride.

[0066] In one embodiment of the method of the present invention, the gold precursor is tetrachloroauric acid trihydrate, the polar solvent is a mixture containing methanol and water, the glutathione is reduced glutathione (GSH), the basic lithium compound is lithium hydroxide monohydrate, the reducing agent of the gold precursor is sodium borohydride, and the lithium salt is lithium chloride.

[0067] In one embodiment, step (iv) is carried out by stirring for a period ranging from 1 to 120 hours, preferably 48 hours. The constant stirring may be carried out using a stirring system known to those skilled in the art, such as a magnetic anchor and a magnetic stirrer.

[0068] In a preferred embodiment, step vi) is carried out by removing the supernatant liquid by sedimentation, or centrifugation, or dialysis, filtration or centrifugal ultrafiltration, or a combination thereof, followed by drying in vacuum or under air, or by heating, or by gas flow, or by freeze-drying, or a combination thereof. The purification procedure is carried out according to common techniques known to those skilled in the art. In particular, the precipitation of nanoparticles can be obtained in an Imhoff cone.

[0069] The present invention further provides a method for the preparation of glutathione and Li + The present invention is represented by gold nanoparticles coated with , wherein the coated nanoparticles have a diameter of 0.1 to 100 nm. Preferably, based on the developed procedure claimed in the present application and described in detail in Example 1, the nanoparticles have a spherical shape and an average diameter of 2 nm.

[0070] In one embodiment of the nanoparticles of the invention, the gold is present in an amount of 40-60% w / w, the glutathione is present in an amount of 20-30% w / w, and the lithium is present in an amount of 0.1-10% w / w.

[0071] In one embodiment, the nanoparticles are obtainable by the method according to the invention. The subject of the present invention is therefore the above-described glutathione and Li nanoparticles obtainable by the method according to the invention. + The gold nanoparticles are coated with

[0072] The present invention also relates to a method for preparing aggregates of gold nanoparticles as defined herein, comprising dispersing the particles in a solvent selected from deionized water, alcohol or hydroalcoholic solutions, or pharmaceutically acceptable (and therefore suitable for administration) solutions or suspensions at a temperature in the range of +4 to 56°C, preferably 35 to 40°C, in an amount to provide a concentration of the nanoparticles of 0.00001 to 1000 mg / mL, preferably 1 to 100 mg / mL, for at least 1 minute.

[0073] In particular, the dispersion step of the particles is carried out at a temperature of +4°C at a concentration of the nanoparticles of 100 mg / mL for at least 1 to 7 days, preferably at least 7 days, to form aggregates, and an additional dispersion step (at +4 to 43°C, preferably 37°C) is carried out with an amount of nanoparticles such that a concentration of the nanoparticles of 0.01 to 1 mg / mL for in vitro applications and 10 to 100 mg / mL for in vivo applications is obtained.

[0074] In one embodiment, the nanoparticles are in dry form.

[0075] In one embodiment, the solvent is deionized water with a pH of 5.5 to 6.5, preferably 5.8.

[0076] In one embodiment of the method, steps (i to vi) of the method for preparing nanoparticles according to the present invention can be carried out before the dispersion step.

[0077] The method for preparing aggregates of nanoparticles according to the invention also makes it possible to obtain a composition comprising aggregated nanoparticles.

[0078] The present invention also relates to aggregates of gold nanoparticles as defined herein, wherein the aggregates have a diameter of 0.1 to 5000 nm, preferably between 10 and 300 nm, and even more preferably between 100 and 300 nm, and such aggregates have an icosahedral morphology.

[0079] In one embodiment, the aggregates can be obtained by the method for preparing aggregates of gold nanoparticles according to the present invention.

[0080] The present invention also relates to a pharmaceutical composition comprising an aggregate of glutathione and Li+-coated nanoparticles according to claim 18 or 19, a solvent selected from deionized water, alcohol, an aqueous alcoholic solution, a pharmaceutically acceptable solvent or a pharmaceutically acceptable suspending agent, and at least one pharmaceutically acceptable excipient and / or carrier. The skilled person will be aware of possible excipients and carriers useful for the purposes of the present invention.

[0081] In additional embodiments, the pharmaceutical composition is in a form suitable for administration by oral, systemic, parenteral, injectable, intravenous, aerosol, spray, topical, intranasal, nasopharyngeal and / or oropharyngeal, rectal, or vaginal routes, and is in the form of a cream, ointment, salve, aerosol, solution, suspension, gel, hydrogel, emulsion, soft or hard gelatin capsule, sprayable solution or suspension.

[0082] In one embodiment, the pharmaceutical composition is in a two-component form that is mixed before use and comprises the nanoparticles of the present invention in a dry form suitable for administration and a suitable solvent in a suitable dose, or in a single container with separate compartments or in two different containers.

[0083] The present invention also relates to a kit comprising a plurality of nanoparticles as defined herein and one or more aliquots of a solvent selected from deionized water, alcohol, a pharmaceutically acceptable solvent, a pharmaceutically acceptable suspending agent, or an aqueous alcoholic solution.

[0084] Finally, the present invention relates to a gold nanoparticle, or aggregate of nanoparticles, or composition, or kit according to the present description, as defined herein and in the claims, for use in therapeutic treatment.

[0085] In one embodiment, the gold nanoparticles, or nanoparticle aggregates, or compositions, or kits as defined herein and in the claims are for use in the treatment or as an adjuvant in the treatment of infectious diseases, infections caused by DNA or RNA viruses, neurodegenerative diseases, and mood disorders, all of which are associated with the activation or positive regulation of glycogen synthase kinase-3 (GSK-3). In other words, infectious, neurodegenerative diseases, and mood disorders would benefit from the inhibition of glycogen synthase kinase-3 (GSK-3).

[0086] In particular, the inhibition of GSK-3 is achieved by phosphorylation of the serine at position 9 for the isoform β (pGSK-3β Ser9 ), and for isoform α, phosphorylation of the serine at amino acid position 21 (pGSK-3α Ser21 ) is carried out.

[0087] In one embodiment, the infectious, neurodegenerative disease, and mood disorder correlated with activation or positive regulation of glycogen synthase kinase-3 (GSK-3) activity is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, tauopathies such as FDT (frontotemporal dementia), PSP (progressive supranuclear palsy), and PART (primary age-related tauopathy), and the infectious disease caused by a DNA or RNA virus is selected from infections caused by coronaviruses, orthomyxoviruses, filoviruses, flaviviruses, hepadnaviruses, hepeviruses, herpesviruses, papillomaviruses, pneumoviruses, poxviruses, rhinoviruses, reoviruses, togaviruses, and influenza viruses.

[0088] In a preferred embodiment, the herpes virus is herpes simplex virus type 1 (HSV-1) and the coronavirus is SARS-CoV-1 and SARS-CoV-2.

[0089] In additional embodiments, the nanoparticle aggregates or compositions as defined herein and in the claims are administered in the treatment or in the adjuvant to the treatment by oral, systemic, parenteral, injection, intravenous, aerosol, spray, topical, intranasal, nasopharyngeal and / or oropharyngeal, rectal, or intravaginal routes at a concentration of the nanoparticles between 1 and 100 mg / mL.

[0090] The present invention also relates to therapeutic methods for the treatment of diseases as described above, which methods comprise one or more steps of administering to a subject in need thereof gold nanoparticles, or nanoparticle aggregates, or compositions as defined herein and in the claims, which are administered by oral, systemic, parenteral, injection, intravenous, aerosol, spray, topical, intranasal, nasopharyngeal and / or oropharyngeal, rectal, or intravaginal routes in said treatment or said adjuvant to said treatment.

[0091] In any part of the description and claims of this specification, the term "comprising" may be replaced by the term "consisting of."

[0092] Each embodiment of a feature described herein can be combined with one or more of the other embodiments of various features, and in particular, combinations of the preferred and most preferred embodiments of each feature of the methods, nanoparticles, aggregates, compositions, or kits of the invention described herein are preferred.

[0093] The examples reported below have the purpose of better illustrating the methods disclosed in the present description, and such examples should in no way be considered as limiting the scope of the foregoing description and the following claims.

[0094] The declaration required by Article 170bis of the CPI for biological substances is inserted below:

[0095] Pursuant to Article 170-2, paragraph 2 of the CPI and in accordance with Article 21, Chapter 2 of the Implementing Regulations of the CPI adopted by Ministerial Order No. 33 dated 13 January 2010, I hereby declare as follows: The animal / plant-derived materials underlying the present invention in the above application are derived from mice and humans. Regarding mouse-derived materials, the cells are astrocytes and / or primary neurons obtained from C57Bl / 6 mice (Ministry of Health Approval No.: 594 / 2022-PR). Regarding human-derived cells, they are tumor cells (neuroblastoma SH-SY5Y, cervical cancer HeLa, monkey kidney epithelial cells (VERO), etc.) purchased from ATCC.

[0096] In accordance with Article 170-2, paragraph 4 of the CPI, I hereby declare as follows: With regard to the biological material containing microorganisms or genetically modified organisms that is the subject of or used in this application, the obligations arising from national or Community regulations and in particular the provisions relating to Chapter VI of Legislative Act No. 206 of 12 April 2001 and its amendment No. 224 of 8 July 2003 have been respected. [Example]

[0097] The nanoparticles of the present invention, LiG-AuNP, were synthesized by modifying a literature procedure already described by some of the present inventors for the similar NaG-AuNP system described in Buonerba et al., Scientific Reports, 2020, 10: 11380. As a non-limiting example, the synthesis and application of LiG-AuNP are reported below.

[0098] The average elemental composition of the LiG-AuNPs was Li = 2.0 ± 0.4%, C = 17.3 ± 0.2%, N = 5.4 ± 0.1%, S = 4.4 ± 0.2%, and Au = 53.6 ± 0.3%, corresponding to a molar ratio of approximately Li / C / N / S / Au = 1.1 / 5.3 / 1.4 / 0.5 / 1, where all carboxyl functional groups of glutathione were lithiated. Analysis of the LiG-AuNPs using transmission electron microscopy (TEM) highlighted the icosahedral morphology of the gold clusters with diameters in the 0.5–5 nm range, more precisely 2 nm (Figure 1A, B).

[0099] Dynamic light scattering (DLS) analysis has shown that when redispersed in deionized water, LiG-AuNPs rapidly (within seconds) form aggregates with diameters ranging from 10 to 300 nm. More precisely, when dispersed in deionized water (pH: 5.8, temperature = 37 °C) at concentrations ranging from 0.01 to 100 mg / mL, the formation of aggregates with average diameters centered in the 250-300 nm range is observed. These aggregates are stable in deionized water for up to 30 days. Even within this period, the formation of aggregates with larger diameters is observed (Figure 1F).

[0100] Determination of lithium release Buonerba et al., Scientific Reports 2020, presents TEM microscopy images of hepatocellular carcinoma tumor cells (HepG2) incubated for 1 hour in culture medium containing NaG-AuNP aggregates at concentrations of 0.07–0.7 mg / mL. Internalization of these aggregates was observed, followed by disintegration into single AuNPs within the cytoplasm and / or intracellular organelles. Figure 2 shows the internalization of LiG-AuNPs in human neuroblastoma SH-SY5Y cells incubated with such particles at a concentration of 1 mg / mL for 24 hours. Given the similarities in morphology and composition between NaG-AuNPs and LiG-AuNPs, similar behavior can be expected during the internalization process of the latter, as demonstrated in the following paragraphs of this document. As a non-exhaustive example, LiG-AuNPs were used in Dulbecco's modified Eagle's medium (DMEM; Dulbecco et al., 1959), a culture medium with high salt concentrations, particularly sodium-rich, that is suitable for mimicking the extracellular environment. + The release of LiG-AuNPs dispersed in DMEM, which had been previously purified by centrifugal ultrafiltration, was mainly + / Na + By exchange, Li + Lithium tends to release ions, making these cations available for their extracellular pharmacological action. The nanoparticles were dispersed in the medium by sonication for 5 min, then recovered after 1 and 24 h by filtration through a small column (cutoff 5 kDa), and subsequently analyzed by inductively coupled plasma optical emission spectroscopy (ICP-OES). The lithium contents in pure MEM and MEM after 1 and 24 h of exposure to LiG-AuNPs were found to be 0.0017 ± 0.0001 mg / L, 16.71 ± 0.36 mg / L, and 18.28 ± 0.08 mg / L, respectively. Thus, the monodispersed nanoparticles released 83.5% and 91.4% of the lithium in MEM after 1 and 24 h. The remaining Li + The cations (10-15%) remain electrostatically attached to the internalized LiG-AuNPs and allow for significant accumulation of lithium within the cells.

[0101] Determining the optimum concentration to use The limiting concentration of LiG-AuNPs was preliminarily evaluated by measuring cell viability of 90% or more using trypan blue exclusion test in monkey kidney epithelial cells (VERO cells) and SH-SY5Y human neuroblastoma cells treated with increasing concentrations of LiG-AuNPs (ranging from 0.1 to 10.0 mg / mL) for 24 hours. The limiting concentration of LiG-AuNPs was 2.0 mg / mL (extracellular Li + corresponds to 6 mEq / L), within which cell viability is maintained at over 90% and the cytotoxic concentration (CC 50 was 4.3 to 5.0 mg / mL (Figure 3).

[0102] Determination of the efficiency of lithium internalization (uptake) into cells by LiG-AuNPs Li after extracellular treatment with LiG-AuNPs + The cellular uptake of was determined by ICP-OES spectroscopy of acid-digested cell lysates and compared with an equivalent treatment using lithium chloride (LiCl). SH-SY5Y human neuroblastoma cells in culture were treated with 3 mM LiCl (to remove extracellular Li + equivalent to 3 mEq / L) and 1 mg / mL LiG-AuNPs (extracellular Li + The cells were exposed to LiG-AuNPs (equivalent to 3 mEq / L) for 24 hours. At the end of the treatment, the intracellular Li levels in the cells treated with LiG-AuNPs were + The concentrations are approximately 26-fold (ranging from 9-fold to 44-fold) higher than those in cells treated with LiCl, with values ​​ranging from 0.154 to 0.3375 pg / cell in treatment with LiG-AuNPs and 0.007 to 0.0375 pg / cell with LiCl.

[0103] In vitro, GSK-3 inhibition was observed at extracellular LiCl concentrations ranging from 5 to 20 mM (IC 50 Considering that the LiG-AuNPs are typically obtained at 1 mM, the LiG-AuNPs are equally effective in intracellular Li +This allows concentrations to be obtained at significantly lower extracellular concentration levels.

[0104] Determining the efficacy of LiG-AuNPs on inhibitory phosphorylation of GSK-3β The efficacy of LiG-AuNPs' action on intracellular molecular targets was verified by assessing the inhibitory phosphorylation of GSK-3β (at serine 9) in various experimental paradigms suitable for determining both the effective concentration and transversality of the effect of LiG-AuNPs. In all of the experiments described below, LiG-AuNPs were used suspended in double-distilled water at a storage concentration of 100 mg / mL for approximately 7 days.

[0105] In vitro studies Human neuroblastoma cells (SH-SY5Y) were incubated for 1 and 24 hours with: i) vehicle (culture medium DMEM / Ham's F12); ii) LiG-AuNP aggregates (approximately 250 μm) at a concentration of 1 mg / mL (equivalent to 3 mEq / L of extracellular lithium); or iii) 6 mM LiCl (equivalent to 6 mEq / L of extracellular lithium), a concentration commonly used for in vitro studies of GSK-3 inhibition (Zhang et al., 2003). Western blot (WB) experiments on intracellular lysates of treated cells revealed phosphorylation of GSK-3β at serine 9 (pGSK-3β). S9 ) was evaluated relative to the total expression of the protein. S9 The ratio of the expression of pGSK-3β to the expression of the total amount of GSK-3β is set to "1" (pGSK-3β S9 / GSK-3β = 1), the following values ​​were obtained under the remaining experimental conditions (Figure 4): i) 1 hour treatment: 1 mg / mL LiG-AuNP: 1.60±0.20 (n=4 independent experiments; p=0.048 vs. vehicle); 6 mM LiCl: 1.78±0.25 (n=4; p=0.019 vs. vehicle; not significant vs. 1 mg / mL LiG-AuNP) ii) 24-hour treatment: 1 mg / mL LiG-AuNP: 2.23±0.26 (n=10 independent experiments; p<1×10 vs. vehicle) -3 ); 6 mM LiCl: 2.45±0.29 (n=10; p<1×10 vs. vehicle) -3 ; No significant difference compared to 1 mg / mL LiG-AuNPs (p=0.982)

[0106] Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test.

[0107] To demonstrate the effectiveness of treatment with LiG-AuNPs in inducing inhibitory phosphorylation (serine at position 9) of GSK-3β compared with lithium salts, SH-SY5Y cells were treated with a low concentration of LiG-AuNPs (0.05 mg / mL, extracellular Li) for 1 and 24 hours. + The LiG-AuNPs were also treated with a similar concentration of LiCl (equivalent to 0.15 mEq / L) under these conditions. Ser9 A significant increase in the ratio of GSK-3β [1.47±0.13 (n=4) after 1 hour and 1.54±0.12 (n=8) after 24 hours; p=0.023 and p<1×10 vs. vehicle, respectively] -3 ) can be determined. On the other hand, 0.15 mM LiCl (equivalent to 0.15 mEq / L) did not induce any significant changes after 24 hours (pGSK-3β Ser9 / GSK-3β = 1.14 ± 0.14; n = 4; not significantly different from vehicle - p = 0.495). A low dose (0.15 mM) LiCl condition for 1 hour was not tested given that it had no significant effect 24 hours after treatment.

[0108] For the phosphorylation of serine at position 9 of GSK-3β, NaG-AuNPs (1 mg / mL for 24 h) were used as a control for LiG-AuNPs based on their ability to be internalized into cells (Figure 4B). As expected, NaG-AuNPs inhibited the phosphorylation of pGSK-3β. S9No significant modification of α-glucan was determined (1.32±0.29, n=4; p=0.794 vs. vehicle=1; FIG. 4B; right black column).

[0109] Finally, the cross-sectional effect of LiG-AuNPs in other cell models was examined in mouse cortical astrocytes or human lung cancer cells (A549) treated with 1 mg / mL LiG-AuNPs for 24 hours, resulting in the expression of pGSK-3β. S9 was evaluated and confirmed by obtaining duplicate results.

[0110] These data demonstrate that LiG-AuNPs are more effective than LiCl in inducing inhibitory phosphorylation of GSK-3β under all test conditions, even at very low doses of extracellular lithium (0.15 mEq / L) where LiCl fails to exert any significant effect.

[0111] The effectiveness of lithiated nanoparticles was further verified in a model of increased phosphorylation, for example, tau protein phosphorylation in HSV-1 infection (De Chiara G et al., Plos Pathogens, 2019). As shown in Figure 7, LiG-AuNPs (0.05 mg / L - Li + Application of 0.15 mEq / L of tau for 24 hours significantly reduces cellular immunoreactivity to phosphorylation of tau protein at threonine 205 under both infected and control conditions.

[0112] In vivo studies The effectiveness of LiG-AuNPs in determining the regulation of GSK-3β at the brain level was verified in a mouse animal model by intranasal administration as an alternative method to systemic lithium administration. In general, intranasal administration is the best option for intracerebral drug delivery, considering that it is a non-invasive method that bypasses the blood-brain barrier (Hanson and Frey, 2008). LiG-AuNPs were administered to C57B1 / 6 mice at 3 μL per nasal cavity at concentrations of 1 mg / mL (3 mEq / L of lithium (I)), 10, and 100 mg / mL (300 mEq / L of lithium (I)) in double distilled water for five consecutive days, and the animals were dissected for brain extraction 6 hours after the last administration.

[0113] The concentrations for mice were selected based on information from in vitro experiments and the usual lithium doses typically used in human patients. Indeed, commercially available lithium carbonate (Li2CO3) tablets containing 300 mg of lithium salt correspond to approximately 0.018 mg of lithium per gram of body weight (approximately 0.018 mg lithium / g body weight). Considering that LiG-AuNPs contain approximately 2% lithium, the maximum concentration tested (6 μL at 100 mg / mL for 5 days) corresponds to approximately 0.012 mg of lithium per gram of body weight (for mice), thus complying with the human dose.

[0114] For each extracted brain, one hemisphere was analyzed en bloc by ICP-OES after freeze-drying to determine the gold concentration, while the other hemisphere was used by WB measurements to determine the concentration of pGSK-3β in various brain regions, e.g., hippocampus, cortex, and olfactory bulb. Ser9 The analysis performed demonstrated that intranasal administration of LiG-AuNPs can regulate GSK-3β in the brain (Figure 9), particularly at the hippocampal level. In this region, the amount of GSK-3β relative to the total amount of protein (GSK-3β) was significantly increased. Ser9 Increased levels of pGSK-3β were found in mice treated with LiG-AuNPs compared to mice treated with vehicle.Ser9 Assuming the ratio of GSK-3β to GSK-3β was 1.00 ± 0.06, the other values ​​were 1.67 ± 0.27 (1 mg / mL, n = 7; p = 0.083 vs. vehicle), 2.02 ± 0.45 (10 mg / mL, n = 9; p = 0.016 vs. vehicle), and 2.37 ± 0.62 (100 mg / mL, n = 9; p = 0.006 vs. vehicle) (Figure 9A, B). No significant modifications were observed at the level of the cortex or olfactory bulb (Figure 9C, D).

[0115] ICP-OES spectroscopy detected the presence of gold in all analyzed hemispheres, with mean values ​​of 72.8 ± 18.6 and 84.2 ± 6.8 ng / hemisphere for 10 and 100 mg / mL LiG-AuNPs, respectively, indicating that the AuNPs reached the brain. At a concentration of 1 mg / mL, ICP-OES spectroscopy was unable to detect the presence of gold, likely because it was below the detection threshold of the instrument used.

[0116] These studies also show that 10 mg / mL of LiG-AuNPs is the optimal dose for intranasal administration to mice.

[0117] Determining the effectiveness of LiG-AuNPs in reducing the production of superoxide In LiG-AuNPs, lithium ions are covalently bound to gold via sulfur molecules present in glutathione (GSH). It is also known that GSH exerts important antioxidant effects through its sulfur (Mukwevho et al., Molecules. 2014). Here, we hypothesize that LiG-AuNPs may exert antioxidant effects to support the beneficial effects of lithium. The antioxidant effects of LiG-AuNPs (1 mg / mL) were examined in SH-SY5Y human neuroblastoma cells treated for 24 hours with lipopolysaccharide (LPS, 5 μg / mL), which is known to induce oxidative stress in cells. At the end of LPS treatment, the cells were incubated with a fluorescent probe for superoxide, called "dihydroethidium" (DHE). Our results show that treatment of cells with LPS induces a 28% increase in fluorescence intensity (p = 1.4 × 10 vs. vehicle). -33 Treatment with LiG-AuNPs returned the fluorescence to control values ​​(p = 7.6 × 10 vs. cells treated with LPS). -33 8A-D), demonstrating the antioxidant activity of LiG-AuNPs. As additional confirmation, we quantified NADPH oxidase 4 (NOX4) enzyme, which is known to be involved in the production of reactive oxygen species (ROS), and pro-oxidant interleukin-1β (both immature / inactive (IL-1β precursor, 31 KDa) and mature / active (IL-1β, 17 KDa) forms) in HSV-1-infected cells.

[0118] Through Western blot experiments, we confirmed that LiG-AuNP treatment performed 24 h postinfection significantly reduced the levels of NOX4 (−50%; Figure 8E ) and IL-1β ( Figure 8F, G ).

[0119] Determining the efficacy of LiG-AuNPs against downstream targets of GSK-3β (Herpes simplex virus type 1 infection and tau protein phosphorylation) GSK-3β is known to play a key role in viral infection, both RNA and DNA viruses. Among the various viruses that use GSK-3β to infect cells is herpes simplex virus type 1 (HSV-1). One of the present inventors' research groups previously demonstrated that pharmacological inhibition of GSK-3β with SB216763 significantly restricted HSV-1 infection (Li Puma et al., Glia, 69: 201–215, 202; doi: 10.1002 / glia.23895). Several studies have also demonstrated the antiviral effects of lithium, albeit at concentrations far above the toxic limit of this cation (>10 mM) at systemic levels. Regarding its antiviral effects, it has been demonstrated that glutathione exerts an important antiviral effect by counteracting the oxidative stress generated after viral invasion.

[0120] Subsequently, based on these studies, we evaluated the efficacy of LiG-AuNPs in exerting antiviral effects in an experimental model of HSV-1 infection in vitro.

[0121] In vitro HSV-1 infection involves a "contact" period between cells and virus, called the "adsorption phase," which typically lasts 1 hour and is performed at 37°C in a serum-free (e.g., fetal bovine serum - FBS) culture medium. During this time period, the virus can bind to the cell membrane and enter host cells. At the end of the adsorption period, unadsorbed virus was removed by washing with phosphate-buffered saline (PBS), and the cells were incubated in culture medium supplemented with 2% FBS for up to 24 hours [postinfection period (pi)] to allow virus replication. At the end of the 24 hours, the efficacy of infection was determined both by quantifying the virus titer in the extracellular medium via a plaque standard assay and by measuring viral protein expression (via WB and immunofluorescence).

[0122] Subsequently, the efficacy of LiG-AuNPs in inhibiting HSV-1 infection was investigated by adjusting the experimental conditions to evaluate both the concentration and the effective administration time used.

[0123] Confluent monolayers of primary human neuroblastoma cells (SH-SY5Y), monkey kidney epithelial cells (VERO), and mouse cortical astrocytes were infected with HSV-1 (multiplicity of infection [MOI] = 1) and subsequently analyzed at 24 h p.i. under the experimental conditions detailed in Figures 5 and 6. Specifically, treatment with vehicle or various concentrations (0.05 mg / mL, 1 mg / mL, and 2 mg / mL) of LiG-AuNPs during only the viral adsorption phase, only the postinfection phase, or during the entire infection phase.

[0124] Depending on the cell type, these experimental results demonstrate that LiG-AuNPs are effective in inhibiting / reducing HSV-1 infection in vitro at concentrations of 0.05 mg / mL (extracellular Li + This is demonstrated by the excellent efficacy of IFN-γ at concentrations lower than 0.15 mEq (Figure 6E) when applied throughout the entire infection period.

[0125] In some experiments (i.e., immunofluorescence), NaG-AuNPs were used as a control for LiG-AuNPs (Figure 5). It is important to keep in mind that although NaG-AuNPs do not contain lithium, they are protected by glutathione, which may exert a weak antiviral effect by antagonizing the oxidative stress produced by viral invasion.

[0126] Example 1: Preparation of lithiated reduced glutathione-coated gold nanoparticles (LiG-AuNPs) As an example, a generally effective, scalable, and open-ended procedure is described for the synthesis of lithiated reduced glutathione-coated gold nanoparticles (LiG-AuNPs). The types and amounts of reagents and solvents, equipment, and experimental conditions described below are not intended to be limitations of the present patent proposal.

[0127] The preparation method of LiG-AuNPs includes the following detailed steps: I. A 100 mL round-bottom glass reaction flask equipped with a magnetic stirrer was charged at room temperature and atmospheric pressure with the following compounds: tetrachloroauric acid trihydrate (HAuCl4·3H2O, CAS No. 16961-25-4, 0.333 g equivalent to 0.846 mmol), methanol (CAS No. 67-56-1, 27.8 mL), water (22.2 mL), reduced glutathione (GSH, CAS No. 70-18-8, 0.581 g equivalent to 1.89 mmol), and lithium hydroxide monohydrate (LiOH·H2O, CAS No. 1310-66-3, 0.388 g equivalent to 9.25 mmol). The addition of reduced glutathione caused the reaction mixture to become cloudy, but the cloudiness quickly disappeared within a few seconds after the addition of lithium hydroxide, yielding a clear, colorless solution. II. The solution obtained in step (I) is transferred at room temperature and atmospheric pressure into a round-bottom glass reaction flask having a volume of 3 L equipped with a magnetic anchor for stirring. III. The solution obtained in step (II) is diluted with 260 mL of methanol and 760 mL of water at room temperature and atmospheric pressure. IV. The solution obtained in step (III) is rapidly treated with a freshly prepared aqueous solution of sodium borohydride (NaBH4, CAS number: 16940-66-2; 0.145 g, corresponding to 3.83 mmol, dissolved in 15 mL of deionized water) at room temperature and atmospheric pressure, with vigorous stirring at room temperature. The addition of the reducing agent causes the formation of a dark brown colloidal suspension (see Figure 1). V. The intermediate obtained in step (IV) is kept under magnetic stirring at room temperature and atmospheric pressure for 48 hours, after which lithium chloride (LiCl, CAS number: 7447-41-8, amount 15.38 g corresponding to 0.363 mol) and methanol (700 mL) are added. VI. The colloidal suspension obtained in step (V) is transferred into an Imhoff-type glass cone, where the LiG-AuNP particles are allowed to settle for approximately 48-72 hours. Subsequently, the supernatant is removed, and the particles are transferred to a 50 mL centrifuge tube and centrifuged (6500 rpm for 10 minutes). The supernatant is carefully removed from the centrifuge tube, and the particles are dried in vacuum. Approximately 0.25 g of LiG-AuNP can be obtained from this procedure.

Claims

1. The method comprises the steps of: + Preparation of gold nanoparticles coated with: i) a gold precursor at a concentration of 0.0001 M to 10 M, preferably 0.001 to 0.1 M, more preferably 0.010 M to 0.020 M, even more preferably 0.017 M; at least one polar solvent; glutathione in a molar ratio to said gold precursor of 0.1:1 to 100:1, preferably 1:1 to 1:0.1; a basic lithium compound in a molar ratio relative to said gold precursor of 10:0.01 to 0.01:10, preferably 1:1 to 1:0.1, more preferably 1:0.05 to 1:0.15, even more preferably 1:0.09; at a temperature ranging from -20° to 120°C, preferably at room temperature, in an environment equipped with a stirring and shaking system to obtain a clear, colorless solution; ii) diluting the solution obtained in step i) with at least one polar solvent in the range of 1:0 to 1:1000, preferably 1:5 to 1:50, more preferably 1:5 to 1:30, even more preferably 1:20; iii) adding a reducing agent for the gold precursor to the diluted solution of step ii) in a molar ratio relative to the gold precursor of 1:1 to 100:0.01, preferably 1:1 to 1:0.1, more preferably 1:0.1 to 1:0.3, even more preferably 1:0.22, at a temperature in the range of -20° to 120°C, preferably at room temperature, in an environment equipped with a stirring and shaking system, to obtain a colloidal suspension of gold nanoparticles; iv) adding a lithium salt to the gold precursor in a molar ratio of 0.1:1 to 100:0.001, preferably 1:0.001 to 1:1, more preferably 1:0.001 to 1:0.005, even more preferably 1:0.002; v) diluting the solution obtained in step iv) with at least one polar solvent in the range of 1:0 to 1:1000, preferably 1:5 to 1:50, more preferably 1:5 to 1:30, even more preferably 1:20; vi) purifying the nanoparticles obtained in steps iv)-v).

2. 2. The method of claim 1, wherein the gold precursor is selected from gold halides, gold chalcogens, gold pycnogens, gold crystallogen complexes, and gold clusters (I or III) or organogold compounds, or mixtures thereof.

3. 3. The method of claim 1, wherein the gold precursor is tetrachloroauric acid in the trihydrate form.

4. 4. The method of claim 1, wherein the polar solvent is at least one of aprotic polar solvents including acetone, acetonitrile, tetrahydrofuran, dioxane, dimethyl sulfoxide, dimethylformamide, peralkylated ureas, such as tetramethylurea and 1,3-dimethyl-2-imidazolidinone, and hexamethylphosphoramide, or protic polar solvents including water, alcohols, carboxylic acids, amines, aqueous alcoholic solutions, or sulfonated or nitrated compounds, or a mixture thereof.

5. 5. The method of claim 4, wherein the polar solvent is methanol, an aqueous alcoholic solution of methanol, or a mixture comprising water and methanol.

6. 6. The method according to claim 1, wherein the glutathione is a reduced form of glutathione (CAS number: 70-18-8).

7. 7. The method of any one of claims 1 to 6, wherein the basic lithium compound is selected from lithium hydroxide (anhydrous) or lithium hydroxide monohydrate, lithium oxide, lithium hydride, lithium alkoxide, lithium amide, lithium carbonate, lithium bicarbonate, lithiated zinc compound, metallic lithium, lithium / ammonia solution, lithium amalgam, lithiated anion resin, lithium phosphate, lithium sulfate, lithium carboxyl compound, lithium tetraborate, lithiated borate, lithium fluoride, lithium hypochlorite, lithium chlorite, lithium oxyanion, organic soluble compound, or mixtures thereof.

8. 8. The method according to any one of claims 1 to 7, wherein the reducing agent for the gold precursor is selected from sodium borohydride, lithium borohydride, lithium aluminum hydride.

9. 9. The method according to any one of claims 1 to 8, wherein the lithium salt is an inorganic or organic lithium compound, preferably selected from lithium chloride, lithium iodide, lithium fluoride, lithium bromide, lithium oxide, lithium hydroxide, lithium sulfide, or mixtures thereof.

10. 10. The method of any one of claims 1 to 9, wherein step (iii) is carried out with constant stirring for a period ranging from 1 to 120 hours, preferably 48 hours.

11. 11. The method of any one of claims 1 to 10, wherein step (iv) is carried out by removing the supernatant liquid by sedimentation, centrifugation, dialysis, filtration, centrifugal ultrafiltration, or a combination thereof, followed by drying under vacuum or air, or by heating, or by gas flow, or a combination thereof.

12. Glutathione and Li + 1. A gold nanoparticle coated with a hydroxybenzoate, the coated nanoparticle having a diameter of 0.1 to 100 nm.

13. 13. The nanoparticles of claim 12, wherein the gold is present in an amount of 40-60% w / w, the glutathione is present in an amount of 20-30% w / w, and the lithium is present in an amount of 0.1-10% w / w.

14. Nanoparticles as defined in any one of claims 12 to 13, obtainable by the method according to any one of claims 1 to 11.

15. 14. A method for preparing an aggregate of nanoparticles as defined in claims 12-13, comprising dispersing the particles in a solvent selected from deionized water, alcohol or hydroalcoholic solution, or a pharmaceutically acceptable solution or suspension at a temperature ranging from +4 to 56°C, preferably from 35 to 40°C, in an amount to give a concentration of the nanoparticles of 0.00001 to 1000 mg / mL, preferably from 1 to 100 mg / mL, for at least 1 minute.

16. 12. The method according to claim 1, further comprising the steps i) to vi) of the method according to claim 1, further comprising the step of dispersing glutathione and Li + Method for preparing aggregates of gold nanoparticles coated with PEG-coated gold nanoparticles.

17. 17. The method of claim 15, wherein the solvent is deionized water at a pH of 5.5 to 6.

5.

18. Glutathione and Li according to any one of claims 12 to 14, wherein the aggregates have a diameter of 0.1 to 5000 nm. + Aggregates of gold nanoparticles coated with .

19. Glutathione and Li according to claim 18, obtainable by the method according to any one of claims 16 or 17. + Aggregates of nanoparticles coated with

20. Glutathione and Li according to claim 18 or 19 + and a solvent selected from deionized water, alcohol, an aqueous alcoholic solution, a pharmaceutically acceptable solvent, or a pharmaceutically acceptable suspending agent, and at least one pharmaceutically acceptable excipient and / or carrier.

21. 21. The pharmaceutical composition of claim 20, in a form suitable for oral, systemic, parenteral, injectable, intravenous, aerosol, spray, topical, intranasal, nasopharyngeal and / or oropharyngeal, rectal, or vaginal administration.

22. 22. A pharmaceutical composition according to any one of claims 20 and 21 in the form of a cream, ointment, salve, aerosol, solution, suspension, gel, hydrogel, emulsion, soft or hard gelatin capsule, sprayable solution or suspension.

23. Glutathione and Li according to any one of claims 12 to 14 + and one or more aliquots of a solvent selected from deionized water, alcohol, a pharmaceutically acceptable solvent, a pharmaceutically acceptable suspending agent, or an aqueous alcoholic solution.

24. Glutathione and Li according to any one of claims 12 to 14 for use in therapeutic treatment. + or the glutathione and Li nanoparticles according to claim 18 or 19. + or a pharmaceutical composition according to any one of claims 20 to 22.

25. Glutathione and Li for use according to claim 24 for therapy as an adjuvant in the treatment of infectious diseases, infections caused by DNA or RNA viruses, neurodegenerative diseases, or mood disorders associated with the activation or positive regulation of glycogen synthase kinase-3 (GSK-3). + Gold nanoparticles coated with glutathione and Li + 2. A pharmaceutical composition or kit of gold nanoparticles coated with a hydroxybenzoate.

26. the infectious, neurodegenerative, and mood disorder associated with activation or positive regulation of glycogen synthase kinase-3 (GSK-3) is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, or a tauopathy; The infectious disease caused by a DNA or RNA virus is selected from the group consisting of infectious diseases caused by coronaviruses, orthomyxoviruses, filoviruses, flaviviruses, hepadnaviruses, hepeviruses, herpesviruses, papillomaviruses, pneumoviruses, poxviruses, rhinoviruses, reoviruses, togaviruses, and influenza viruses. Glutathione and Li for use according to claims 24 and 25 + Gold nanoparticles coated with glutathione and Li + 2. A pharmaceutical composition or kit of gold nanoparticles coated with a hydroxybenzoate.

27. 27. The method of claim 26, wherein the herpes virus is herpes simplex virus type 1 (HSV-1), and the coronavirus is SARS-CoV-1 and SARS-CoV-2. + Gold nanoparticles coated with glutathione and Li + 2. A pharmaceutical composition or kit of gold nanoparticles coated with a hydroxybenzoate.

28. Glutathione and Li for use according to any one of claims 24 to 27, wherein the treatment or the adjuvant of the treatment occurs by oral, systemic, parenteral, injection, intravenous, aerosol, spray, topical, intranasal, nasopharyngeal and / or oropharyngeal, rectal, intravaginal administration of a therapeutically effective dose of the gold nanoparticles, or nanoparticle aggregates, or composition. + Gold nanoparticles coated with glutathione and Li + 2. A pharmaceutical composition or kit of gold nanoparticles coated with a hydroxybenzoate.

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