Novel antimicrobial carbon dots

Abstract

The present invention relates to novel antiviral carbon dots, compositions comprising said carbon dots, materials and devices coated with said carbon dots, said nanoparticles for medical use, methods for the preparation of said carbon dots, compositions, materials and devices.

Description

[Technical field]

[0001] The present invention relates to novel antiviral carbon dots, compositions comprising said carbon dots, materials and devices coated with said carbon dots, said nanoparticles for medical use, methods for the preparation of said carbon dots, compositions, materials and devices. [Background technology]

[0002] Infectious diseases account for 20% of global mortality, and viruses are responsible for about one third of deaths from infectious diseases. The most common causes of death are lower respiratory tract infections and the human immunodeficiency virus (HIV), which represent a major cost for national health systems. In addition to these well-known viruses, new ones emerge every year (e.g., more recently, SARS-CoV-2). The best approach to combat viral infections concerns, first of all, the prevention of the spread of infections by respecting a number of hygienic and behavioral rules, as well as treatments represented by vaccines, although the number of effective vaccines is limited and often not available in all regions of the world.

[0003] Antiviral drugs and / or antiviral compounds play a further important role in the success of this combat response. However, said drugs are often limited by virus specificity and there are numerous cases where they are ineffective against the emergence of new unknown virus strains. The prevention of the spread of diseases (such as SARS-CoV-2) is promoted by a number of actions. Wearing suitable personal protective equipment and keeping surfaces and the environment clean and disinfected are significant parts of the fight against the spread of infectious diseases. In the case of a pandemic, the need for highly active antiviral compounds that allow rapid and effective inactivation of viruses in the air and on surfaces (animated and non-animated) is strongly felt.

[0004] Prevention (including but not limited to prevention via vaccination) has proven to be the only effective weapon against the spread of infectious diseases, especially for diseases for which effective treatments are not available or are not available globally and for diseases for which, despite the effectiveness of treatments to combat the infectious pathogen, extensive damage to the body coexists.

[0005] There is a continuing need for effective, non-toxic antimicrobial (especially virucidal, antifungal, and / or bactericidal) compounds, and in particular compounds that act via mechanisms that are effective against a class of pathogens rather than pathogen-specific are preferred in light of the constant evolution of new pathogens and their potentially devastating effects, as the global spread of SARS-CoV-2 has demonstrated.

[0006] In recent years, active research into carbon-based nanomaterials has revealed that some systems have interesting properties for nanomedicine purposes, among which are carbon dots (C-dots).

[0007] Carbon dots (CDs) are a large family of carbon-based materials that encompass amorphous carbon nanoparticles, partially graphitized core-shell carbon nanoparticles, amorphous fluorescent polymer nanoparticles, and graphene quantum dots. The synthesis of CDs is achieved either by a top-down approach via laser ablation of a graphite target or electrochemical oxidation of a graphite electrode, or by a via bottom-up approach by carbonization of organic precursors. The latter route allows for facile tailoring of CDs, especially via the appropriate selection of chemical reagents. In the last few years, much attention has been paid to the application of CDs as antimicrobial agents. So far, the antimicrobial effect has been attributed to functional groups on the surface, which in turn are responsible for their extraordinary optical properties as fluorophores. CDs have indeed shown an active role against infection, which seemed to be further increased when CDs were exposed to a UV light source (Innocenzi Chem Sci 2020). This evidence led the scientific community to hypothesize that reactive oxygen species (ROS) are produced when CDs are photoexcited. Since that hypothesis, therefore, the majority of CDs have been recognized as photosensitizer nanomaterials, which are efficient and biocompatible and capable of producing reactive oxygen species under excitation by UV or in a few cases visible light. In particular, CDs with high phosphorescence yield have proven to be efficient singlet oxygen (SO) photosensitizers and show promising characteristics as UV-promoted antimicrobial agents (ACS Biomater. Sci. Eng. 2018, 4, 12, 3983-3993). Indeed, among the different ROS, SO represents a powerful antibacterial and antimicrobial agent that causes alterations in the membrane organization (Muller-Breitkreutz J Photochem PhotoBiol B 1995, Ogilby Chem Soc Rev 2010). SO is also an effective broad-spectrum antiviral agent that acts directly on the envelope.

[0008] It has already been demonstrated that electrochemically produced graphene quantum dots (GQDs) generate ROS when photoexcited with visible light (470 nm, 1 W). The photogenerated ROS was able to kill, in this case, methicillin-resistant Staphylococcus aureus and Escherichia coli. In contrast to the response of graphene or graphene oxide, which can kill tumor cells by the release of heat, irradiated GQDs do not cause a significant temperature increase in bacterial suspensions (BZRistic et al. / Biomaterials 35(2014)4428-4435), while they produce a meaningful increase in ROS concentration. The majority of studies published so far attribute the pro-oxidant activity of CDs to SO formation. It has been proposed that, specifically for GQDs, SO is generated via photoexcitation by the pathways of energy transfer and electron transfer. This means that both hydroxyl radicals and superoxide anions result from the generation of electron-hole pairs and charge separation, reacting with the surrounding molecular oxygen or H2O, respectively (ACS Nano 2016,10,9,8690-8699). At present, there is no comprehensive explanation of ROS production induced by photoexcitation of CDs, but some results indicate that ROS generation may be related to oxygen functional groups in the structure of CDS. It has been demonstrated that the removal of oxygen functional groups (especially for ketonic carbonyl groups on the surface of GQDs) inhibits ROS formation by GQDs in cells and reduces light-induced cytotoxicity (Chem. Commun.,2017,53,10588-10591).

[0009] Shanaz et al 2013 (Shanaz et al “Oxidative Synthesis of Highly Fluorescent Boron / Nitrogen Co-Doped Carbon Nanodots Enabling Detection of Photosensitizer and Carcinogenic Dye” Analytical Chemistry 85(21)2013) discloses the synthesis of carbon dots under hydrothermal conditions starting from boric acid and N-(4-hydroxyphenyl)glycine. The document discloses various characterization parameters of said carbon dots.

[0010] Das et al 2021 (Das et al “Carbon Dots: An Emerging Smart Material for Analytical Application” Micromachines 2021 2(1):84) reports a number of different carbon dot syntheses resulting from hydrothermal, solvothermal, microwave, and purely thermal synthesis, inducing decomposition reactions (such as pyrolysis or combustion). Among the different protocols, a method of synthesis is disclosed that requires only glycine and citric acid to form carbon dots.

[0011] Kovacova et al 2018 (Kovacova et al “Carbon Quantum Dots Modified Polyurethane Nanocomposite as Effective Photocatalytic and Antibacterial Agents” ACS Biomater. Sci. Eng. 2018, 4, 12, 3983-3993) disclose the synthesis of carbon dot-based nanocomposites obtained by embedding nanoparticles in a polyurethane polymer matrix. Carbon dots are obtained by hydrothermal synthesis of tri-block copolymers in the presence of phosphoric acid.

[0012] Xiaolin Nie et al 2020 (Xiaolin Nie et al “Carbon quantum dots: A bright future as photosensitizers for in vitro antibacterial photodynamic inactivation” Photochem Photobiol B. 2020 Mar 23;206:111864) report a modified version of a previously published carbon dot synthesis. The protocol involves a one-pot solvothermal procedure using a 1:2 mass ratio of citric acid to 1,5-diaminonaphthalene. The document highlights the antibacterial activity of the carbon dots.

[0013] Innocenzi and Stagi 2020 (Innocenzi and Stagi “Carbon-based antiviral nanomaterials: graphene, C-dots, and fullerenes. A perspective” Chem Sci. 2020 Jul 14;11(26):6606-6622) is a review of carbon-based antiviral nanomaterials (carbon dots, graphene, fullerenes, and nanodiamons, etc.) as alternative innovative antiviral systems.

[0014] C-dots have been extensively investigated for their excellent properties in photonics, optoelectronics, and photocatalysis. In addition, C-dots seem to play a crucial role both as luminescent markers and as active systems for viral agents. One of the important features of C-dots is their ease of implementation and low cost, which allows the realization of systems with controlled chemical composition and size (typically less than 10 nm). In a typical synthesis, a carbon-based precursor undergoes controlled pyrolysis under wet or dry conditions. The products can be engineered by reacting a nitrogen source with the carbon precursor or by performing post-synthetic functionalization. Studies performed in Vero cells, MOLT-4 A549, MARC-145, PK-15, and many others confirm the excellent biocompatibility of C-dots, being non-cytotoxic even at high concentrations of several hundred μg / mL. C-dots with controlled morphology derived from various monomers have been shown to be effective against HSV-1, HIV-1, PRRSV, PRV, HCoV-229E, and flavivirus (JEV). Although there are numerous promising results, the mechanism of viral inhibition by C-dots is controversial and still debated. In particular, it is believed that C-dots with specific functional groups on the surface may interact with cell membranes and prevent virions from attacking the system. More recently, on the other hand, benzoxazine-derived C-dots have been shown to be effective in disrupting viral activity by directly interacting with virions, constituting the first true dot system capable of actively combating viral particles and exhibiting broad-spectrum antiviral activity.

[0015] Based on the state of the art, it is therefore clear that CDs can have highly variable antimicrobial effects depending on the exact CD type, and that the effectiveness of CDs as antimicrobial (e.g. antibacterial and / or antiviral and / or antifungal) effects is not a priori predictable.

[0016] Considering the increasing desire to improve the spread of infectious diseases in a more environmentally and safety friendly manner, the future development of new compounds that meet the above requirements and are not likely to induce resistance in pathogens is of great importance for global health, encompassing both the plant and animal kingdoms. Summary of the Invention

[0017] We investigated the potential of three different types of CDs, obtained by carbonization of similar precursors under hydrothermal conditions, as antiviral materials against SARS-COV 2. Among the multiple CD precursors used in the literature so far, we selected citric acid (probably the most used precursor for the bottom-up synthesis of CDs), 1,5-diaminonaphthalene, and glycine (an amino acid that can be thermally polymerized at low temperatures) (Scheme 1).

[0018] The glycine according to the present invention is a non-functionalized glycine. [ka]

[0019] The inventors also compared two CDs obtained from the same precursors using two different acids (hydrochloric acid or boric acid) with CDs obtained by substituting one of the two precursors in the preparation process, and surprisingly found that the use of specific combinations of precursors together with one or the other of the selected acids provided CDs with strong virucidal effect upon irradiation with either UV or visible light.

[0020] Notably, depending on the acid chosen, CDs prepared with boric acid showed strong differences in virucidal efficacy upon irradiation with visible light compared to the same precursors prepared with hydrochloric acid.

[0021] In particular, the virucidal activity of different CDs under visible or UV light exposure was evaluated against SARS-CoV-2 (human 2019-nCoV strain INMI1, from Istituto “L. Spallanzani”, Rome, Italy) according to the guidelines reported in EN14476 for the testing of chemical disinfectants.

[0022] The object of the present invention is therefore a. Dissolving glycine, 1,5-diaminonaphthalene, and boric acid or hydrochloric acid in distilled water; sonicating the mixture obtained in step b until the solution becomes cloudy grey; c. subjecting the cloudy grey solution thus obtained to a heat treatment, thereby solubilizing the product obtained in distilled water; d. filtering the mixture obtained in c. through a 10-30 μm pore size filter and collecting the eluate; e. drying the eluate thus obtained; A process for preparing carbon dots comprising: The process wherein glycine, 1,5-diaminonaphthalene, and boric acid or hydrochloric acid, and water are the only compounds used in the process; Carbon dots obtainable by the process for the preparation of carbon dots in any of the embodiments disclosed and claimed herein; A composition comprising the carbon dots disclosed and claimed herein and at least one carrier and / or excipient; an article of manufacture coated or loaded with said carbon dots or compositions disclosed and claimed herein; A medical device comprising said carbon dots or compositions disclosed and claimed herein. said carbon dots or compositions disclosed and claimed herein for use in medical treatment; Use of carbon dots or compositions disclosed and claimed herein for disinfecting, sanitizing, and / or sterilizing surfaces, objects, air, water, areas; 1. A method for disinfecting, sanitizing, and / or sterilizing a surface, object, air, water, or area, comprising: said method comprising placing an object to be disinfected, sanitized, and / or sterilized in contact with a suitable amount of said carbon dots or compositions disclosed and claimed herein and subjecting it to irradiation with visible and / or ultraviolet light for a period of at least 5 minutes; A medical treatment for the treatment of an infectious disease, wherein the carbon dots or pharmaceutical compositions disclosed and claimed herein are administered to a patient in need thereof, and the carbon dots or compositions disclosed and claimed herein are irradiated or exposed to ultraviolet and / or visible light; A medical treatment of a plant, wherein the plant or part thereof is treated with the carbon dots or the composition disclosed and claimed herein and the plant or part thereof is exposed to visible light; It is.

[0023] In accordance with the present invention, the only compounds used in the processes disclosed and claimed herein are glycine, 1,5-diaminonaphthalene, distilled water, and one of boric acid or hydrochloric acid.

[0024] Glossary As used herein, UV or ultraviolet light has the meaning commonly given in the art, ie, a form of electromagnetic radiation with wavelengths between 10 nm (with corresponding frequencies around 30 PHz) and 400 nm.

[0025] As used herein, visible light has the meaning commonly intended in the art, i.e., the visible light spectrum is the segment of the electromagnetic spectrum that the human eye can see. More simply, this range of wavelengths is referred to as visible light. Typically, the human eye can detect wavelengths from about 400 to 700 nanometers.

[0026] Carbon quantum dots (CQDs, C-dots, or CDs) are small carbon nanoparticles (<10 nm in size) that were discovered in 2004 and are a new class of fluorescent carbon nanomaterials. CQDs possess the attractive properties of high stability, good electrical conductivity, low toxicity, environmental friendliness, simple synthetic route, and optical properties comparable to those of quantum dots.

[0027] In this specification, the term irradiated (e.g., UV and / or visible light) may be replaced in any part by the term exposed (e.g., UV and / or visible light). The term glycine in the present invention refers to the unfunctionalized amino acid glycine.

[0028] Gly-DiaN in this specification refers to carbon dots prepared from glycine, 1,5-diaminonaphthalene, HCl dissolved in distilled water, as described in the Example "Synthesis of Gly-DiaN, DiaN-CA, and Gly-DiaN-BA" in the Examples section below.

[0029] DiaN-CA in this specification refers to carbon dots prepared from citric acid, 1,5-diaminonaphthalene, and HCl dissolved in distilled water, as described in the Example "Synthesis of Gly-DiaN, DiaN-CA, and Gly-DiaN-BA" in the Examples in the following section.

[0030] Gly-DiaN-BA in this specification refers to carbon dots prepared from glycine, 1,5-diaminonaphthalene, and boric acid dissolved in distilled water, as described in the Example "Synthesis of Gly-DiaN, DiaN-CA, and Gly-DiaN-BA" in the Examples in the following section.

[0031] The term antimicrobial in this specification may be intended as a compound or mixture thereof having virucidal and / or fungicidal and / or bactericidal activity, or as said activity per se.Antimicrobial substances according to the present invention may also refer to compounds having phytoplasmicidal activity or as such activity. [Brief description of the drawings]

[0032] [Figure 1] Experimental setup. Drops of virus suspension and nanomaterials were placed on the lids of 96-well plates for different treatments (exposed to visible light, UV light, or left in the dark). [Diagram 2] A, B, and C show the UV-Vis spectra of three samples characterized by their main absorption bands. The absorption band peaking at 320 nm is attributed to the n⇒π* transition of carbonyl and amide bonds (C=O and C=N) [1]. [Diagram 3] Excitation-emission intensity spectra of Gly-DiaN, DiaN-CA, and Gly-Dian-BA in aqueous solution (a, b, and c, respectively). Photoluminescence intensity is reported in a pseudocolor scale from blue to red. The figure shows 3D excitation-emission intensity maps of carbon dots of samples A) Gly-DiaN, B) DiaN-CA, and C) Gly-Dian-BA, respectively. Panels A and B exhibit maximum photoluminescence intensity with emission at 454 nm and excitation at 360 nm. In contrast, sample C shows a maximum photoluminescence emission peaked at 404 nm when excited at 350 nm. With regard to CD photoluminescence, the three systems show a narrower excitation range. So far, the excitation-dependent emission of CDS has been attributed to various emission centers in the nanoparticles formed by uncontrolled reactions of the carbonization process. According to this interpretation, a narrow excitation range may indicate a specific type of CD with a more controlled chemical composition and structure. [Figure 4]A, B, and C are FTIR spectra of Gly-DiaN, DiaN-CA, and Gly-DiaN-BA in the range of 4000-400 cm-1, respectively. 4D is the spectrum of the Gly-DiaN-BA sample in the range of 3700-2500 cm-1 compared to that of boric acid used as a reference. The bands at 3005 and 2823 cm-1 are attributed to C-H bond stretching, while the band at 1413 cm-1 is due to CO-NH amide groups. The significant difference between Gly-DiaN-BA (4C) and the other two samples (4A and 4B) is the presence of boric acid. This explains the band at 3215 cm-1 assigned to the OH stretching of the acid (Figure 4D). [Diagram 5] FTIR spectra of Gly-DiaN (5A and 5B) and Gly-DiaN-BA (5C and 5D) after heat treatment at 150° C. and 250° C. for 2 hours. As previously observed, the presence of boric acid catalyzes the polymerization of the reagents at low temperatures. The FTIR spectra in FIG. 5 show the difference between Gly-DiaN-BA and Gly-DiaN when treated at 150° C. and 250° C. for 2 hours, respectively. [Figure 6] The EPR spectra of Gly-DiaN, DiaN-CA, and Gly-DiaN-BA were collected under dark conditions (a, c, and e) and under UV irradiation (b, d, and f). The EPR spectra obtained from powder samples (Figure 6) show that all three systems produce radical species both in the dark and in the presence of UV irradiation. [Figure 7] TGA (black line) and DTA (dotted line) analysis of Gly-DiaN, DiaN-CA, and Gly-DiaN-BA samples (a, b, and c, respectively) from 25 °C to 500 °C. The TG-DTA analysis in Figure 7 shows the thermal decomposition of a) Gly-DiaN, b) DiaN-CA, and c) Gly-DiaN-BA. [Figure 8]Virucidal effect of carbon dots Gly-DiaN, DiaN-CA, and Gly-DiaN-BA exposed to UV light (365 nm for 5 min) and visible light (450 nm for 5 min). #1=Gly-DiaN #2=DiaN-CA #3=Gly-DiaN-BA [Figure 9] Fluorescence analysis in the range of 300-700 nm. The emission of aqueous solutions of dots (with a concentration of 0.1 mg / mL) was evaluated in the wavelength range of 300-700 nm. The emission maxima of Gly-DiaN are centered at 450 nm, DiaN-CA at 445 nm, and Gly-DiaN-BA at 404 nm. The emission of each dot was measured at three different excitation wavelengths: 320, 340, and 360 nm. Figure 9a is Gly-DiaN, Figure 9b is DiaN-CA, and Figure 9c is Gly-DiaN-Ba. [Figure 10] UV-Vis analysis in the range of 200-600 nm. The absorption of aqueous solutions of the dots (with a concentration of 0.1 mg / mL) was measured in the wavelength range of 200-600 nm. The maximum peak of absorption is between 310 nm and 340 nm, with the maximum absorption centered at 330 nm. The figure shows an absorption of more than 0.5 at the maximum peak for the test conditions used. [Figure 11] Stability over time. The stability of Gly-Dian-BA over time was evaluated over a period of 40 days. The absorbance at 330 nm of an aqueous solution of dots with a concentration of 0.1 mg / mL was measured at time 0, after 7 days, after 31 days, and after 40 days. As can be observed, the absorbance was consistent over the period. [Figure 12]Antiviral effect of surfaces functionalized with Gly-DiaN-BA (Wuhan strain of SARS-CoV-2 virus). A certain volume of virus suspension (5 μL) of SARS-CoV-2 virus (Wuhan strain) was poured onto the lid of a 96-well plate (12×8 cm). The virus suspension was then added to the coated plate. The substrate was then left in the dark or exposed to ultraviolet light (UV=365 nm) or visible light (VL=450 nm) for 5 minutes. After an additional 5 minutes of incubation, the virus suspension was collected and the virus titer was determined according to the methodology described below. When the surface functionalized with Gly-DiaN-BA CD was irradiated with UV, the virus titer dropped to 0, and also clearly decreased when the surface functionalized with Gly-DiaN-BA CD was exposed to visible light. [Figure 14] Untreated (14a) and treated (14b) slices from the same fruit (strawberry) were kept at room temperature under different conditions and the changes in the slices were monitored over time. The figure shows an untreated slice (14a) with extensive mold growth and decay after 3 days, while a treated slice (14b) shows signs of drying but no mold or decay after 196 days. [Figure 15] TEM analysis of Gly-DiaN and DiaN-CA is shown. Transmission electron microscopy images were obtained using a FEI TECNAI 200 microscope with a field emission gun operating at 200 kV. Sample preparation was performed by dispersing the nanoparticles in ethanol by sonication, then dropping them onto a carbon-coated copper grid and drying them for observation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] The present invention relates to a. Dissolving glycine, 1,5-diaminonaphthalene, and boric acid or hydrochloric acid in distilled water; sonicating the mixture obtained in step b until the solution becomes cloudy grey; c. subjecting the cloudy grey solution thus obtained to a heat treatment, thereby solubilizing the product obtained in distilled water; d. filtering the mixture obtained in c. through a 10-30 μm pore size filter and collecting the eluate; e. drying the eluate thus obtained; A process for the preparation of carbon dots comprising: Glycine, 1,5-diaminonaphthalene, water, and either boric acid or hydrochloric acid are the only compounds used in the process.

[0034] Thus, in one embodiment of the present invention, the process is carried out with the compounds glycine, 1,5-diaminonaphthalene, water, and boric acid, and in a second embodiment, the process is carried out with the compounds glycine, 1,5-diaminonaphthalene, water, and hydrochloric acid.

[0035] No other compounds are used in the process of the present invention, therefore the mixture resulting from step a. consists of glycine, 1,5-diaminonaphthalene, and boric acid or hydrochloric acid dissolved in distilled water. No other reagents, catalysts, solvents are used in the entire process.

[0036] As indicated in the Abstract and demonstrated in the Experimental Part and Figures, the presence of boric acid or hydrochloric acid in the process of the present invention provides carbon dots with remarkable virucidal activity. In particular, when boric acid is used instead of HCl in the same manner and with the same amount of glycine and 1,5-diaminonaphthalene, the virucidal activity is further enhanced. In particular, the carbon dots obtainable by the process disclosed and claimed herein exhibit unexpected and excellent virucidal properties upon irradiation with visible light.

[0037] In one embodiment of the present invention, when boric acid is used in step a., the ratio (by weight) of glycine:1,5-diaminonaphthalene:boric acid used in a. is about 1.0:2.0:0.8, preferably the ratio is 1.0:2.06:0.8.

[0038] In other words, in the process of the present invention, 100% by weight of the glycine, 1,5-diaminonaphthalene, and boric acid are therefore 25-26% glycine by weight, 53-54% by weight of 1,5-diaminonaphthalene; 20-21% boric acid by weight It is composed of:

[0039] In one embodiment, in step a., 0.15 g of glycine, 0.31 g of 1,5-diaminonaphthalene, and 0.12 g of boric acid are dissolved in 15-25 ml of distilled water, preferably 20 ml of distilled water. A mixture of glycine, 1,5-diaminonaphthalene, boric acid, and water is thereby obtained. More than one of the above may be used.

[0040] When hydrochloric acid is used in step a., the molar ratio of glycine:1,5-diaminonaphthalene:hydrochloric acid in step a. is about 1.0:1.0:0.25-0.75, preferably the ratio is 1.0:1.0:0.5.

[0041] In one embodiment, 0.15 g of glycine, 0.31 g of 1,5-diaminonaphthalene, and 0.0415-0.1245 mL (preferably 0.083 mL) of 1 M HCL are dissolved in 20 ml of distilled water.

[0042] As is clear from the specification and claims, boric acid and hydrochloric acid are mutually exclusive, and therefore, in step a., either one or the other acid will be used together with only glycine, 1,5 diaminonaphthalene, and water. More than one of the above may be used.

[0043] According to the process of the present invention, in b., the mixture obtained in a. is ultrasonicated until it becomes cloudy gray, then it is subjected to heat treatment in c., and the product obtained after heat treatment is suspended in distilled water.

[0044] The heat treatment in c. of any of the embodiments of the process for preparation of carbon dots disclosed herein can be carried out in any of the techniques commonly used by those skilled in the art, such as in a microwave with a suitable W-time balance, or in an oven at 200°C for a time range of 12 hours or more, or in an autoclave under hydrothermal conditions at 200°C for at least 8 hours, etc.

[0045] In a preferred embodiment, the heat treatment is carried out in a microwave oven at 800-1200 W for a period of 2-5 minutes, more preferably at 1000 W for a period of about 2 minutes.

[0046] Once the heat treatment was carried out, the product obtained thereby was suspended in distilled water.

[0047] Preferably, the amount of distilled water used in c. is similar or the same as the amount of distilled water used in a.

[0048] In d., the mixture obtained in c. is dried and the resulting carbon dots are in the form of a black-purple powder.

[0049] The drying procedure of the mixture can be carried out according to any drying procedure commonly used in the preparation of carbon dots in the protocol. As an example, the mixture can be dried by heating for a suitable period of time, or in air at room temperature for about 48 hours, or under vacuum for about 24 hours.

[0050] A typical drying procedure is to carry out the drying by subjecting the eluate to a temperature of 50-70° C. for a period of 12-18 hours.

[0051] The present invention therefore relates to carbon dots obtainable by the process disclosed and claimed above.

[0052] As shown in the experimental examples and figures below, the carbon dots of the present invention exhibit strong antiviral effects when irradiated with UV and / or visible light.

[0053] The carbon dots prepared according to this method (also referred to herein and in the figures as Gly-DiaN-BA of the present invention) were compared to carbon dots prepared according to the process disclosed above, in which in step a. the boric acid catalyst is replaced by an HCl catalyst to prepare the carbon dots (defined herein and in the figures as Gly-DiaN), and to carbon dots prepared according to the process disclosed above, in which in step a. no catalyst is used and citric acid is used instead of glycine in a w:w ratio of citric acid:1,5-diaminonaphthalene of 1:0.81 (defined herein and in the figures as DiaN-CA). CD Gly-DiaN-BA and Gly-DiaN prepared according to the process of the present invention show unexpected and unpredictable improvement in antiviral properties when compared to CD DiaN-CA, and in particular Gly-DiaN-BA shows further improvement in antiviral properties when compared to CD Gly-DiaN and DiaN-CA.

[0054] The CDs of the present invention and comparable CDs were characterized as disclosed in FIGS.

[0055] Ultraviolet-visible (UV-vis) spectra of the samples solubilized in water were measured in the absorption mode from 200 to 600 nm by using a “Nicolet Evolution 300” UV-vis spectrophotometer (Thermo Fisher) (Fig. 2).

[0056] Fourier transform infrared spectroscopy (FTIR) analysis of the samples was performed by a “Vertex 70” interferometer (Bruker) in the range of 4000–400 cm−1 with a resolution of 4 cm−1 (Figs. 4 and 5).

[0057] Fluorescence analysis of samples solubilized in water was performed with a "Horiba Jobin Yvon NanoLog" spectrofluorometer with excitation and emission wavelengths of 300-700 nm.

[0058] Transmission electron microscopy analysis was performed on a Fei Technai G2 F20 Twin TMP with a field emission electron gun operating at 200 kV.

[0059] Thermogravimetric analysis (TGA) was measured from room temperature to 500 °C with a ramp rate of 10 °C min-1 under a nitrogen flow of 20 mL min-1 by an SDT-Q600 apparatus (TA Instruments) (Fig. 7).

[0060] TEM characterization of the inventive and control CDs was performed, resulting in all CDs having an average size around 5-6 nm, with no evidence of crystalline structure observed.

[0061] Fluorescence analysis was performed in the range of 300 nm to 700 nm. The measurements were performed at a CD concentration of 0.1 mg / ml in water. The emission of aqueous solutions of CDs of Gly-DiaN, DiaN-CA, and Gly-DiaN-BA (each solution with a concentration of 0.1 mg / mL) was evaluated in the wavelength range of 300 to 700 nm as reported in Figure 9. The maximum emission peak observed for Gly-DiaN is at an excitation wavelength of 360 nm, with a peak between 425 and 475, and the maximum emission centered at 450 nm (Figure 9a). The maximum emission peak observed for DiaN-CA is at an excitation wavelength of 360 nm, with a peak between 425 and 475, and the maximum emission centered at 445 nm (Figure 9b). The maximum emission peak observed for Gly-Dian-BA is at an excitation wavelength of 320 nm, with peaks between 375 nm and 425 nm, and the maximum emission centered at 404 nm (Figure 9c). The emission of each dot was measured at three different excitation wavelengths: 320, 340, and 360 nm. The data obtained show that no shift of the peak is observed at the measured wavelengths.

[0062] The Gly-DiaN-BA emission peak is 1.6×10 between 357 and 425 nm upon excitation at 320 nm. 7 is higher, up to about 1.8 × 10 7 Indicates the intensity / arbitrary unit.

[0063] UV.vis analysis was also performed in the range of 200 nm to 600 nm for Gly-DiaN, DiaN-CA, and Gly-DiaN-BA CD, as reported in Figure 10. The absorption of aqueous solutions of the dots (with a concentration of 0.1 mg / mL) was measured in the wavelength range of 200 to 600 nm. For all samples tested, the maximum absorption is centered at 330 nm.

[0064] All CDs exhibit a maximum peak of absorption between 310 and 340 nm, with the peak centered at approximately 330 nm.

[0065] FIG. 11 reports the stability of Gly-DiaN-BA over time.

[0066] The TEM analysis is shown in FIG.

[0067] Once characterized, the cytotoxicity of the CDs of the invention and the control CDs was assessed and their virucidal efficacy by UV and visible light (Figures 8, 12, and 13) was analyzed in various experiments. The virucidal activity of the CDs of the invention and the control CDs was assessed in Vero E6 cells (kidney epithelial cells from African green monkeys, ATCC CRL-1586) infected with SARS-Cov2 (various strains), and the results shown in Figures 8, 12, and 13, as well as in the tables in the experimental section below, show that the virucidal activity of CD Gly-DiaN-BA of the invention and CD Gly-DiaN is comparable after irradiation with UV light (0% virus titer), and only CD Gly-DiaN-BA of the invention shows equal virucidal activity after irradiation with visible light (0% virus titer), while CD Gly-DiaN shows lower virucidal activity (30% virus titer). CD DiaN-CA exhibits very low virucidal activity after irradiation with visible light (63% virus titer).

[0068] Thus, the CDs of the present invention exhibit unexpected advantageous properties, and in particular, Gly-DiaN-BA exhibits an additional unexpected advantage over Gly-DiaN, especially when the virucidal effect is tested in visible light.

[0069] A further object of the present invention is a composition comprising the carbon dots of the present invention and at least one carrier and / or excipient.

[0070] Depending on the final form of the composition, a person skilled in the art will readily select suitable carrier(s) and / or excipient(s).

[0071] The composition may be in the form of, by way of example only, a gel, a solution, an emulsion, a spray, an aerosol, or a powder.

[0072] According to an embodiment of the present invention, the composition can be a sanitizing or cleaning composition, paint, varnish, gloss, pigment. In one embodiment, the composition can be loaded onto an absorbent substrate to form a sanitizing wipe or sanitizing filter, or the like. The composition can also be loaded into a dispensing device, which includes a reservoir for its storage and a means for release, preferably in a dose-controlled manner, and even more preferably in a time-controlled manner, so that it is released into the air or liquid in a predefined amount in one or more time periods. In a further embodiment, the sanitizing or cleaning composition can be of food grade quality.

[0073] In a further embodiment, the carrier(s) and / or excipient(s) are suitable for administration of the composition onto a plant or selected parts of a plant, thereby allowing the treatment of a plant infectious disease (such as a disease caused by a plant virus, or a plant bacterial disease, or a plant fungal disease, or a plant disease caused by a phytoplasma).

[0074] When plants are treated, the infected parts of the plants can, by way of example, be dusted or sprayed onto the affected plant parts and then the plants are exposed to visible light if they are not already under visible light during and after treatment.

[0075] Thus, the present invention also relates to a medical treatment of a plant, wherein said plant or part thereof is treated with said carbon dots or the composition disclosed and claimed in the present specification, and said plant or part thereof is exposed to visible light.

[0076] In a preferred embodiment, the suitable carrier(s) and / or excipient(s) are of pharmaceutical grade (i.e. pharma-ceutically acceptable) and the composition is a pharmaceutical composition.

[0077] A pharmaceutical composition of the present invention may be a composition suitable for oral, parenteral, subcutaneous, intravenous, inhalation, topical, rectal, or vaginal administration.

[0078] Thus, the carbon dots or compositions defined and claimed herein may be administered orally, systemically, parenterally, by injection, by intravenous injection, by aerosol, by nebulization, topically, intranasally, nasopharynx, and / or oropharynx, rectally, or intravaginally.

[0079] The pharmaceutical composition may thus be in the form of a powder, granules, solution, emulsion, cream, gel, ointment, spray, aerosol, suspension, syrup, injectable liquid, or the like.

[0080] Those skilled in the art of pharmaceutical formulation will readily identify suitable carriers and / or excipients for formulating the compositions of the present invention.

[0081] The composition may be provided already subdivided into single doses or single dose fractions, or may be provided in a dispenser that is easy to release the single doses or single dose fractions (such as, for example, a dispenser that is easy to release a predefined amount of a solution in the form of a spray).

[0082] An object of the present invention is also an article coated with said carbon dots or a composition according to any of the embodiments disclosed herein or defined in the claims.

[0083] Without being limited thereto, the product may be a sanitization or cleaning product, a medical device, medical equipment, a personal protective device, a system for air filtration, a fabric, a medicated bandage, a medicated patch, a surface, or an object.

[0084] In a preferred embodiment, the medicated bandage or patch will be transparent to UV and / or visible light, thereby allowing the carbon dots of the present invention to exert their antimicrobial activity.

[0085] Exposure of each and every one of the above products (even to merely visible light) will result in activation of the carbon dots of the present invention, thereby triggering their antimicrobial activity.

[0086] The present invention also relates to a medical device comprising / loaded with carbon dots or compositions according to any of the embodiments disclosed herein or defined in the claims. In a preferred embodiment, said medical device is a drug delivery device and thus comprises a reservoir for storage of said carbon dots or compositions and a means for their release in a dose-controlled or dose-controllable manner. The drug delivery device can be a self-administration device or a surgical device (e.g. a laparoscopic device).

[0087] Preferably, the device will further comprise a means for emission of UV and / or visible light for a desired period of time.

[0088] As an example, the device may be a device for nasopharyngeal delivery and may be equipped with a light-emitting moiety designed to produce light when the drug is administered to activate the carbon dots of the present invention in situ and enable them to exert their antimicrobial activity.

[0089] In other embodiments, when the device is a surgical device for laparoscopy, the device may be equipped with a light-emitting portion, or light suitable for activating the carbon dots of the present invention may be emitted by an additional device such as those commonly used in laparoscopy. The light may be UV and / or visible light.

[0090] Considering the very short amount of time required for activation of the antimicrobial activity of the carbon dots of the present invention, both the carbon dots, or pharmaceutical compositions containing the carbon dots, are highly suitable for laparoscopic surgery for the prevention of infection resulting from the surgery itself, since the activation of the carbon dots kills microbial infectious pathogens that may be introduced by the laparoscope itself, and acts on local internal infections, thereby avoiding or strongly limiting the use of antibiotics and / or antiviral drugs.

[0091] The object of the present invention is therefore represented by said carbon dots or compositions according to any of the embodiments disclosed herein or defined in the claims, for use in medical treatment.

[0092] The medical treatment of the present invention includes administering the carbon dots or pharmaceutical compositions disclosed and claimed herein to a patient in need thereof, and irradiating or exposing the carbon dots or compositions disclosed and claimed herein to ultraviolet and / or visible light.

[0093] According to embodiments of the present invention, the carbon dots or pharmaceutical compositions disclosed and claimed herein may be used in the treatment of microbial infections, or as adjuvants in the treatment of microbial infections, or in the treatment for the prevention of microbial infections or infectious diseases.

[0094] Due to the observed antiviral activity, the carbon dots or compositions of the invention in any of the embodiments provided herein can be used in the treatment or prevention, or as an adjuvant in the treatment or prevention of bacterial, fungal, or viral infections.

[0095] The antiviral activity induced by simple visible light, as demonstrated in the examples below, supports the prophylactic and therapeutic effects of the carbon dots and compositions of the present invention.

[0096] Thus, the present invention also encompasses a method for the treatment or prevention, or for the adjuvant effect of the treatment or prevention of bacterial, fungal, or viral infection, or bacterial, fungal, or viral disease, in which a therapeutically effective dose of said carbon dots or the composition described above is administered in a therapeutically effective dosage to a subject in need thereof and irradiated (exposed) with UV or visible light.

[0097] Administration can be multiple times per day which can be repeated and the carbon dots or composition can be supplied already in single dosage form or by a single dose dispenser.

[0098] A therapeutically effective amount means a therapeutically effective amount or an amount sufficient to provide a desired therapeutic effect. A therapeutically effective amount is an amount that produces one or more desired biological activities during the treatment or prevention of a disease.

[0099] As described above in the section relating to medical devices / surgical devices, the carbon dots or compositions of the present invention can be administered via laparoscopic surgery, a procedure that is commonly performed in the presence of a light-emitting tool.

[0100] In a preferred embodiment, the medical treatment is the treatment or prevention of a viral infection as defined herein.

[0101] According to one embodiment, the virus is an animal DNA virus or an animal RNA virus.

[0102] The viral disease may be an animal disease, wherein the animal is a mammal, a fish, a reptile, or a bird.

[0103] In particular, the animal may be a human, horse, pig, bird, small ruminant (such as goats, sheep, and other wild small ruminants), large ruminant (such as cows, horses, or other wild large ruminants), rodent, dog, cat, primate, feline, small or large bird, chicken, fish, insect.

[0104] As an example, the insect may be a bee, and the carbon dots of the present invention may be used to treat a viral infection in the bee.

[0105] According to a preferred embodiment, the animal is a human.

[0106] By way of example, diseases include African horse sickness (ASH), African swine fever (ASF), Avian influenza (AIV), Bluetongue (BT), Crimean-Congo hemorrhagic fever (CCHF), Foot and mouth disease (FMD), Newcastle disease (ND), Petit ruminant disease (PPPR), Porcine epidemic diarrhea (PED), Rift Valley fever (RFV), Rinderpest (RPV), Schmallenberg virus (SBV), and Schmallenberg swine fever (SBF). Infectious diseases, West Nile Virus (WNV), Canine Distemper, Canine Influenza, Canine Parvovirus, Rabies, Canine Coronavirus Gastroenteritis, Feline Immunodeficiency Virus (FIV), Feline Leukemia Virus (FelV), Feline Coronavirus, Feline Infectious Peritonitis (FIP), Feline Parvovirus, Hepatitis, Hemorrhagic Fever (Ebola), Encephalitis, Mononucleosis, Covid-19, MERS, Herpes Virus Foci, HIV, Chicken Pox, Gastroenteritis, and others.

[0107] Non-limiting examples of suitable animal viruses are provided in the table below. [Table 1] From Table 201.2 in “Classification of Human Viruses” R,D,Siegel;Principles and Practice of Pediatric Infectious Diseases.2018:1044-1048.e1.

[0108] In further embodiments, the virus is an animal or human coronavirus, orthomyxovirus, filovirus, flavivirus, hepadnavirus, hepevirus, herpesvirus, papillomavirus, pneumovirus, poxivirus, rhinovirus, reovirus, and / or togavirus.

[0109] In a particularly preferred embodiment, the coronavirus is one of SARS, MERS or SARS-CoV-2, FCoV.

[0110] According to the present invention, the carbon dots or compositions may be administered orally, systemically, parenterally, by injection, by aerosol, by nebulization, locally intranasally, nasopharynx, and / or oropharynx, rectally, or intravaginally.

[0111] An object of the present invention is also the use of said carbon dots or compositions as defined and claimed herein for the disinfection, sanitization and / or sterilization of surfaces, objects, air, water, areas.

[0112] As an example, they can be used to treat water (such as drinking water and water for animal breeding) by mixing the water with the carbon dots or the composition, or treating the water with the carbon dots or the composition, and exposing the water to visible and / or ultraviolet light.

[0113] Therefore, the present invention also relates to a method for disinfection, sanitization and / or sterilization of surfaces, objects, air, water, areas, said method comprising placing the object to be disinfected, sanitized and / or sterilized in contact with a suitable amount of the defined carbon dots or compositions defined in the present specification and claims, and subjecting or exposing it to irradiation with visible and / or ultraviolet light for a period of at least 5 minutes.

[0114] All experiments in the following examples were carried out with the commercially available cell line Vero E6.

[0115] In any part of the specification and claims, the word comprising may be replaced by the word "consisting of."

[0116] All examples were carried out with nanoparticles comprising L-lysine (hyper)branched nanopolymers prepared according to Example 7, also referred to as "Lysine-B nanomaterial".

[0117] It is stated herein that all experiments involving cells were performed with commercially available African green monkey Vero E6 cells. EXAMPLES

[0118] Synthesis of Gly-DiaN, DiaN-CA, and Gly-DiaN-BA To obtain CDs, three different solutions containing different precursors were prepared: glycine and 1,5-diaminonaphthalene (Gly-DiaN), 1,5-diaminonaphthalene and citric acid (DiaN-CA), and glycine, 1,5-diaminonaphthalene, and boric acid (Gly-DiaN-BA). Gly-DiaN: 0.15 g of glycine and 0.31 g of 1,5-diaminonaphthalene and 0.83 mL of 1 M HCl were dissolved in distilled water (20 mL). DiaN-CA: 0.38 g of citric acid and 0.31 g of 1,5-diaminonaphthalene and 0.83 mL of 1 M HCl were dissolved in distilled water (20 mL). Gly-DiaN-BA: 0.15 g of glycine, 0.31 g of 1,5-diaminonaphthalene and 0.12 g of boric acid were dissolved in distilled water (20 mL).

[0119] The solution was sonicated until it turned cloudy grey, then treated in a microwave oven for 2 min at 1000 W. The resulting product was solubilized in 20 mL of distilled water, filtered through a 0.22 μm pore size filter, and dried in an oven at 60° C. for 15 h to obtain a black-purple powder.

[0120] Carbon dots obtained from 0.15 g of glycine and 0.31 g of 1,5-diaminonaphthalene are designated as Gly-DiaN in the present specification and figures.

[0121] Carbon dots obtained from 0.38 g of citric acid and 0.31 g of 1,5-diaminonaphthalene are designated as DiaN-CA in the present specification and figures.

[0122] Carbon dots obtained from 0.15 g of glycine, 0.31 g of 1,5-diaminonaphthalene, and 0.12 g of boric acid are designated as Gly-DiaN-BA in the present specification and figures.

[0123] Material characterization The ultraviolet-visible (UV-vis) spectra of each of the carbon dot samples Gly-DiaN, DiaN-CA, and Gly-DiaN-BA solubilized in water were measured in the absorption mode from 200 to 600 nm by using a “Nicolet Evolution 300” UV-vis spectrophotometer (Thermo Fisher).

[0124] Fourier transform infrared spectroscopy (FTIR) analysis of the samples was performed with a “Vertex 70” interferometer (Bruker) in the range of 4000–400 cm−1 with a resolution of 4 cm−1.

[0125] Fluorescence analysis of samples solubilized in water was performed with a "Horiba Jobin Yvon NanoLog" spectrofluorometer with excitation and emission wavelengths of 300-700 nm.

[0126] Transmission electron microscopy analysis was performed on a Fei Technai G2 F20 Twin TMP with a field emission gun operating at 200 kV.

[0127] Thermogravimetric analysis (TGA) was measured by an SDT-Q600 apparatus (TA Instruments) from room temperature to 500 °C with a ramp rate of 10 °C min-1 under a nitrogen flow of 20 mL min-1.

[0128] The results are provided in Figures 2-7.

[0129] Cytotoxicity analysis The cytotoxicity of each of the carbon dot samples Gly-DiaN, DiaN-CA, and Gly-DiaN-BA was studied in Vero E6 cells (Cercopithecus aethiops, kidney, ATCC CRL-1586). The cell line is routinely maintained in DMEM supplemented with 1% glutamine, 1% penicillin / streptomycin, and 10% fetal bovine serum.

[0130] Exponentially growing Vero E6 cells were seeded into 96-well plates at optimal density in complete medium and after 24 hours, cells were exposed to different concentrations of nanomaterials for 72 hours. Duplicates for each concentration point were examined. Cytotoxic effects were assessed via microscopic observation (determination of cell monolayer integrity). Drug dilutions were performed in culture medium. Cytotoxic concentration 50% (CC50, the concentration that results in 50% loss of cell viability compared to untreated control) was calculated.

[0131] The table below shows the effect of nanomaterials on cell viability in terms of cell monolayer integrity after 72 hours of treatment at the concentrations shown.

[0132] For carbon dots #1 and #3, the highest concentration that could be tested was 5 mg / mL due to water solubility constraints, and therefore the cytotoxic concentration 50% (CC50) was calculated via dose-response curve interpolation. For #2, it was possible to reach 25 mg / mL. [Table 2]

[0133] Determination of virucidal activity A quantitative suspension test of the virucidal activity for chemical disinfectants and antiseptics in the medical field was carried out according to the guidelines in EN 14476:2013+A2:2019 / UNI EN 14476:2019.

[0134] A fixed volume of SARS-CoV-2 (Wuhan strain) virus suspension (as indicated for each experiment below in the Results section) was placed on the lid of a 96-well plate (12 x 8 cm). A fixed volume of nanomaterial was added to the virus suspension (at the indicated volume and final concentration below in the Results section).

[0135] The suspensions were then either left in the dark or exposed to visible light (450 nm) or ultraviolet light (365 nm) for the times indicated in each experiment (ranging from 2 to 5 minutes).

[0136] Culture medium was used as a control and was added to the virus suspension and exposed to dark or visible / UV light in the same manner as the nanomaterials. The general experimental setup is shown in Figure 1.

[0137] After an additional 5 min of incubation, the virus suspension was collected and the virus titer was determined according to the methodology described below.

[0138] Viral titer determination An in vitro system was used to determine the viral titer of the SARS-CoV-2 suspension.

[0139] Vero E6 cells (kidney epithelial cells from African green monkeys, ATCC CRL-1586) were maintained at optimal density based on the ATCC data sheet. On day 1 of the experiment, cells were transferred into 96-well plates (20.000 cells per well). On day 2, serial virus dilutions (10 -2 , 10 -3 , 10 -4 ...) Importantly, the treatment concentration of each nanomaterial was determined to obtain a non-cytotoxic concentration in the first serial dilution added to the cells.

[0140] After an additional three days, infection in each test well was determined by observation of cytopathic effects. The data were then used to determine titers of treated and control viruses according to the method of Reed and Muench (Reed, LJ; Muench, H. (1938). "A simple method of estimating fifty percent endpoints." The American Journal of Hygiene. 27:493-49).

[0141] The infection data was used to create the following table: [Table 3]

[0142] Viral titer determination is as follows. Place the virus dilution of 6 / 6 affected wells in the first row and the virus dilution of 0 / 6 affected wells in the last row. Enter the number of affected wells in column B and the number of unaffected wells in column C. Column D represents the cumulative number of wells affected, calculated by adding the numbers starting at the bottom of column B and adding up. Column E represents the cumulative number of unaffected wells, calculated by adding the numbers starting at the top of column C and summing down. Column F represents the cumulative percentage of affected wells (%): the value from column D divided by the sum of the values ​​in column D+E and expressed as a percentage.

[0143] The values ​​from column F are used to calculate the proportionate distance between the two dilutions closest to 50%. [Table 4] Add the negative index of the next virus dilution that exceeds 50%.

[0144] The calculated value was used as an exponent to the base of 10 to obtain the virus titer within the infection volume (i.e., TCID50 / 100 μL).

[0145] Viricidal Efficacy of Nanomaterials by UV and Visible Light Treatment concentrations of each nanomaterial were selected to obtain a non-cytotoxic concentration at the first serial dilution added to cells during the virus titration experiment. The following table and associated figures show the virucidal effect of the nanomaterials in the presence or absence of either visible light (450 nm) or ultraviolet light (365 nm) in different experiments. Control = virus-infected cells #1 = Glycine + 1,5-diaminonaphthalene + HCl #2 = Citric acid + 1,5-diaminonaphthalene + HCl #3 = Glycine + 1,5-diaminonaphthalene + Boric acid

[0146] UV exposure Experimental parameters: 5 μL of virus suspension + 10 μL carbon dots (#1, #2, and #3 at 3.3 mg / mL). Conditions: UV light 365nm (or dark place) Exposure time: 5 minutes in light (or in darkness) + 5 minutes incubation [Table 5]

[0147] The results are illustrated in FIG.

[0148] visible light exposure Experimental parameters: 5 μL virus suspension + 10 μL carbon dots (#1, #2, and #3 at 3.3 mg / mL). Conditions: Visible light (VL) 450 nm (or dark place) Exposure time: 5 minutes in light (or in darkness) + 5 minutes incubation [Table 6]

[0149] The results are illustrated in FIG.

[0150] Antiviral effect of surfaces functionalized with Gly-DiaN-BA CDs (SARS-CoV-2 virus Wuhan strain) surface functionalization Silicon substrates were first coated with hybrid silica films bearing amino groups via a sol-gel process and then functionalized with Gly-Dian-BA via an EDC / NHS coupling reaction.

[0151] Experimental parameters: A fixed volume of SARS-CoV-2 virus (Wuhan strain) suspension (5 μL) was poured onto the lid of a 96-well plate (12 × 8 cm). The virus suspension was then added to the coated plate.

[0152] The substrates were then left in the dark or exposed to ultraviolet (UV=365 nm) or visible light (VL=450 nm) for 5 minutes. After an additional 5 minutes of incubation, the virus suspension was collected and the virus titer was determined according to the methodology described below.

[0153] Viral titer determination An in vitro system was used to determine the viral titer of the SARS-CoV-2 suspension.

[0154] Vero E6 cells (kidney epithelial cells from African green monkeys, ATCC CRL-1586) were maintained at optimal density based on the ATCC data sheet. On day 1 of the experiment, cells were transferred into 96-well plates (20.000 cells per well). On day 2, serial virus dilutions (10 -2 , 10 -3 , 10 -4 ...) Importantly, the treatment concentration of each nanomaterial was determined to obtain a non-cytotoxic concentration in the first serial dilution added to the cells.

[0155] After an additional three days, infection in each test well was determined by observation of cytopathic effects, and the data were then used to determine titers of treated and control viruses according to the method of Reed and Muench (see Reed, LJ; Muench, H. (1938). "A simple method of estimating fifty percent endpoints." The American Journal of Hygiene. 27:493-49).

[0156] The results are illustrated in FIG.

[0157] Antiviral effect of surfaces functionalized with Gly-DiaN-BA CDs (SARS-CoV-2 virus delta strain). surface functionalization Silicon substrates were first coated with hybrid silica films bearing amino groups via a sol-gel process and then functionalized with Gly-Dian-BA via an EDC / NHS coupling reaction.

[0158] Experimental parameters: A fixed volume of SARS-CoV-2 virus (strain delta) virus suspension (5 μL) was poured onto the lid of a 96-well plate (12 × 8 cm). The virus suspension was then added to the coated plate.

[0159] The substrates were then left in the dark or exposed to ultraviolet (UV=365 nm) or visible light (VL=450 nm) for 5 minutes. After an additional 5 minutes of incubation, the virus suspension was collected and the virus titer was determined according to the methodology described below.

[0160] Viral titer determination An in vitro system was used to determine the viral titer of the SARS-CoV-2 suspension.

[0161] Vero E6 cells (kidney epithelial cells from African green monkeys, ATCC CRL-1586) were maintained at optimal density based on the ATCC data sheet. On day 1 of the experiment, cells were transferred into 96-well plates (20.000 cells per well). On day 2, serial virus dilutions (10 -2 , 10 -3 , 10 -4 ...) Importantly, the treatment concentration of each nanomaterial was determined to obtain a non-cytotoxic concentration in the first serial dilution added to the cells.

[0162] After an additional three days, infection in each test well was determined by observation of cytopathic effects, and the data were then used to determine titers of treated and control viruses according to the method of Reed and Muench (see Reed, LJ; Muench, H. (1938). "A simple method of estimating fifty percent endpoints." The American Journal of Hygiene. 27:493-49).

[0163] The results are illustrated in FIG.

[0164] Bactericidal effect of Gly-DiaN-BA CD Fruit slices were treated with an aqueous solution of Gly-DiaN-BAC dots according to the invention (concentration 1 mg / mL). The aqueous solution was sprayed onto the strawberry slices at a distance of 20 cm into the air at room temperature.

[0165] Untreated and treated slices from the same fruit (strawberry) were kept at room temperature under different conditions and changes in the slices were monitored over time.

[0166] The results of the observations over time are reported in Figure 14. The figure shows an untreated slice (14a) with extensive mold growth and decay after 3 days, whereas a treated slice (14b) shows signs of drying after 196 days but no mold or decay.

[0167] The antifungal and general antimicrobial effects of the CDs of the present invention are evident from the experimental results.

Claims

1. a. Dissolve glycine, 1,5-diaminonaphthalene, and boric acid in distilled water; b. Sonicate the mixture obtained in a. until the solution becomes cloudy gray; c) heat treating the cloudy grey solution thus obtained and dissolving the product in distilled water; d. The mixture obtained in c. is filtered through a 10-30 μm pore size filter and the eluate is collected; e. Drying the eluate thus obtained; 1. A process for preparing carbon dots, comprising: The process wherein glycine, 1,5-diaminonaphthalene, boric acid, and water are the only compounds used in the process.

2. 2. The process of claim 1, wherein the ratio by weight of glycine:1,5-diaminonaphthalene:boric acid in step a. is about 1.0:2.0:0.8, preferably said ratio is 1.0:2.06:0.

8.

3. 3. The process according to claim 2, wherein 0.15 g of glycine, 0.31 g of 1,5-diaminonaphthalene, and 0.12 g of boric acid are dissolved in 15-25 ml of distilled water, preferably in 20 ml of distilled water.

4. 2. The process of claim 1, wherein the heat treatment is carried out in a microwave oven at 800-1200 W for a period of 2-5 minutes, or in an oven at 200°C for a time ranging from 12 hours or more, or in an autoclave under hydrothermal conditions at 200°C for at least 8 hours.

5. 2. The process of claim 1, wherein the drying is carried out by subjecting the eluate to a temperature of 50-70°C for a period of 12-18 hours.

6. A carbon dot having a maximum emission peak between 357 nm and 425 nm in the 300-700 nm wavelength range and a maximum absorption peak between 310 nm and 340 nm in the 200-600 nm wavelength range under excitation at 320 nm when measured at a carbon dot concentration of 0.1 mg / ml in water.

7. Carbon dots according to claim 6, obtainable by the process according to any one of claims 1 to 5.

8. A composition comprising the carbon dots described in claim 6 and at least one carrier and / or excipient, preferably wherein the composition is a pharmaceutical composition and the at least one carrier and / or excipient is pharmaceutically acceptable.

9. 10. An article of manufacture coated or loaded with the carbon dots of claim 6 or the composition of claim 8.

10. 10. The article of manufacture of claim 9, wherein the article is selected from the group consisting of a sanitizing product, a cleaning product, a medical device, medical equipment, a personal protective device, a system for air filtration, a surgical mask, a filtering facepiece respirator, a surgical instrument, a device comprising a reservoir for storage of the carbon dots or composition, and a means for release of the carbon dots or composition.

11. A medical device comprising the carbon dots of claim 6 or the composition of claim 8, preferably wherein the medical device is a drug delivery device.

12. 12. The medical device of claim 11, further comprising means for emitting visible or ultraviolet light or being a drug delivery device that is transparent to visible and / or ultraviolet light.

13. 10. The carbon dots of claim 6 or the composition of claim 8 for use in medical treatment.

14. A carbon dot or composition for use as described in claim 13 for use in the treatment of a microbial infection, or as an adjuvant in the treatment of a microbial infection, or in the treatment for the prevention of a microbial infection or infectious disease, preferably wherein the infection affects an animal or a plant.

15. The carbon dot or composition according to claim 14, wherein the microbial infection is a viral infection, a bacterial infection, a fungal infection, or a phytoplasma infection.

16. The carbon dot or composition of claim 14, wherein the microbial infection is a viral infection caused by an animal DNA virus or an animal RNA virus.

17. The carbon dot or composition of claim 14, wherein the animal is a mammal.

18. The carbon dot or composition of claim 16, wherein the microbial infection is caused by a coronavirus, an orthomyxovirus, a filovirus, a flavivirus, a hepadnavirus, a hepevirus, a herpesvirus, a papillomavirus, a pneumovirus, a poxivirus, a rhinovirus, a reovirus, a togavirus, or an influenza virus.

19. The carbon dot or composition for use according to claim 18, wherein the coronavirus is SARS, MERS, SARS-CoV-2, or FCoV.

20. 10. Use of the carbon dots of claim 6 or the composition of claim 8 for disinfecting, sanitizing and / or sterilizing surfaces, objects, air, water, areas.

21. 1. A method for disinfecting, sanitizing, and / or sterilizing a surface, object, air, water, or area, comprising: contacting the object to be disinfected, sanitized, and / or sterilized with a suitable amount of the carbon dots of claim 6 or the composition of claim 8; Irradiating with visible and / or ultraviolet light for a period of at least 5 minutes; The method comprising:

Images