Removable tatoo ink

Abstract

It is an object of the present invention to provide a removable tattoo ink for tattoos that is functionalized carbon black, where said functionalized carbon black is oxidized carbon black in which at least a fraction of the alcohol (-OH) groups are esterified with azide-substituted acyl halides, a method for obtaining it and methods for removing it from the skin.

Description

[0001] REMOVABLE TATTOO INK

[0002] TECHNICAL FIELD

[0003] The tattoo industry is continuously growing globally. At the same time, the demand for tattoo removal is also increasing. In Europe and the United States alone, more than 24 million individuals, growing at a rate of about 12% each year, wish to remove at least one tattoo. In Italy, out of 1.2 million people who say they want to remove a tattoo, only 4% have effectively removed it1. The size of the global tattoo removal market was estimated at $4.34 billion in 2021. It is estimated to reach $12.15 billion by 2030, with a CAGR (compound annual growth rate) of 12.1% over the forecast period 2021-20302.

[0004] The chemical inertness of the inks used for tattoos constitutes a significant barrier to complete and effective tattoo removal, generating dissatisfaction and limiting the options available to tattoo removal professionals.

[0005] To date, no effective solutions are available, where the chemical inertness characterizing inks has required the development of aggressive physical removal methods. The current possibilities are laser, a long removal process that can take years, is very painful and can leave scars, and "cover up", a process whereby the tattoo is covered by a new, larger and darker one, thus without it being removed.

[0006] Both alternatives are ineffective and have several contraindications, which is why, in a market with millions of potential customers, only a very small fraction decides to carry out removal.

[0007] New technologies are being developed, including "rapid acoustic" technology that uses sound to make laser tattoo removal faster (in three sessions) and each treatment session more effective.

[0008] Alternatives to laser are, by way of example, based on the use of cold plasma, i.e. a highly reactive low-temperature gas that, by exerting an energy field, leads to the detachment and degradation of tattoo ink particles from their biological anchors, as described in US10716611.

[0009] EP2696922 describes an apparatus for the removal of a pigmented section of skin comprising a) a device for puncturing the skin; b) an array of one or more needles connected to said device for puncturing the skin at said pigmented section; and c) an intermediate member connected to said device and to the suction means for enabling said suction means to collect the pigment mixture with cellular fluids at said punctured section of skin.

[0010] US20130123746A1 describes the on-site injection of a liquid containing e.g. a-hydroxy acids that can dissolve tattoo pigments. However, the need for a chemical, non-physical, effective and non-invasive method of tattoo removal remains strongly perceived.

[0011] Bio-orthogonal chemistry comprises chemical reactions that can take place in a living organism without interfering with biochemical processes. A bio- orthogonal reaction involves the incorporation of a chemical marker into the target biomolecule, followed by a chemical reaction leading to the formation of a covalent bond with an exogenous probe3.

[0012] To increase the reaction speed and lower cytotoxicity, one of the most widely used click chemistry reactions is Strain Promoted Azide-Alkyne [3+2] Cycloaddition SPA AC. To overcome the cytotoxicity of the copper catalyst, azide-alkyne [3+2] cycloaddition with strained reagents excludes the use of copper. In this reaction, the alkyne partner is bound within a ring of eight members4. This geometry produces about 18 kcal / mol strain, resulting in a dramatic increase in reaction speed compared to linear alkynes. However, SPAAC reactions with simple cyclooctins are still slow reactions: their second- order rate constants have values comparable to Staudinger ligation, leading to the formation of an amide (-lO^M^S'1). Therefore, much effort has been devoted to the development of new, more reactive cyclooctin families, achieving up to 400-fold faster kinetics with the biarylazacyclooctinone derivative BARAC (k = 0.96 M^s-1)5. Typical solutions to improve the reactivity of cyclooctins include the introduction of electron-attracting groups such as fluorine or additional strain elements, e.g. a cyclopropane ring opposite the triple bond, leading to a bicyclo[6.1.0]non-4-yne (BCN) structure and azadibenzocyclooctin (ADIBO or DBCO) fused structures. However, it is important to note that increased reactivity often results in lower stability, increased hydrophobicity and complex, low-yield synthetic procedures.

[0013] The functionalization of nanoparticles (NPs) with strained alkynes in vitro and in vivo represents a particularly demanding challenge. The chemistry used to attach the strained alkyne to the nanoparticle must not alter the stability and functionality / reactivity of either the NP or the alkyne. More reactive cyclooctypes are often not the best choice due to their hydrophobicity, which can affect the colloidal stability of nanoparticles, especially in a high-density modification of the NP surface.

[0014] Furthermore, attention must be paid to the presentation and possible steric hindrance of the cyclooctin group on the surface of the nanomaterial in order to ensure the success of the SPAAC reaction with the azide-modified biomolecule.

[0015] Functionalizing nanomaterials with azides as partners of alkynes in the bio-orthogonal reaction has several advantages. Azides offer superior biocompatibility, ensuring minimal interference with biological functions, and exhibit greater stability, contributing to longer life and ease of handling. These properties make azide-functionalized nanomaterials particularly advantageous for various biomedical applications, including drug delivery, bioimaging and therapies. Despite the advantages, the commercial supply of azides for bio-ortho chemistry is distinctly limited compared to that of commercially available alkynes.

[0016] The great potential of bio-orthogonal chemistry, coupled with the limited alternatives on the functionalization of nanomaterials with azides, underlines the need for the development of a new synthetic route for azide functionalization on carbon black by a non-physical, but chemical method that is effective and non- invasive aimed at tattoo removal.

[0017] DESCRIPTION

[0018] A chemically modified tattoo ink, wherein said ink is removable, forms the subject matter of the present invention. Said ink was advantageously obtained by the authors of the present invention via a new carbon black functionalization route by strain-promoted [3+2] azide-alkyne cycloaddition.

[0019] Subject matter of the present invention is thus to provide a chemically modified ink that facilitates removal by overcoming the chemical inertness associated with carbon black, thus making it targetable with a functional group suitable for bio-orthogonal chemistry. This modification aims to simplify the process of tattoo removal by allowing the ink to participate in bio-orthogonal reactions that enable removal through, but not limited to, extraction by non- invasive methods or increased laser effectiveness. DESCRIPTION OF THE FIGURES

[0020] Figure 1: reaction product analysis after oxidation of carbon black (A) UV analysis, comparison between carbon black (black line) and reacted carbon black (grey line); (B)1HNMR spectrum of oxidized carbon black.

[0021] Figure 2: (A, B) FT-IR spectra of the product after esterification.

[0022] Figure 3: FT-IR spectrum of the product after addition of azide.

[0023] DETAILED DESCRIPTION

[0024] The subject matter of the present invention to provide an ink which is carbon black functionalized, wherein functionalized carbon black means an oxidized carbon black in which at least a fraction of the alcohol (-OH) groups are esterified by means of suitable acyl halides.

[0025] In an embodiment, said fraction of esterified alcohol groups is greater than or equal to 50% of the OH groups, or is greater than or equal to 60% of the OH groups. In a preferred embodiment, it is greater than or equal to 80% of the OH groups. In an even more preferred embodiment, almost all the hydroxyl groups are esterified.

[0026] In an embodiment, said acyl halides have a linear or branched organic chain, comprised between C2 and CIO, or between C3 and C8, containing a halide selected from F, Cl, and Br, preferably bromine, and a leaving group selected from C-halogen, mesyl or tosyl.

[0027] The presence of said C-halogen groups on the ester functionalizations allows the introduction of an azide group (C-N3) by nucleophilic substitution with the sodium azide salt (NaNs). This allows the carbon black to be functionalized with an azide group, making it compatible for click chemistry reactions with, but not limited to, a strained alkyne group.

[0028] It is further subject matter of the present invention to provide a method for obtaining ink that is carbon black functionalized, wherein said method comprises: a) Oxidation of carbon black, where said oxidation increases the OH groups on the surface of the carbon black; b) Functionalization of the oxidized carbon black by esterification between said OH groups and an acyl halide containing a C-halogen group useful for the subsequent reaction with an azide.

[0029] In an embodiment, said oxidation is achieved by the Hummers6method in water.

[0030] In an embodiment, said acyl halide is selected as a functionalized C- halogen, preferably identified as 2-bromo-2- methylpropanoylbromide.

[0031] The functionalization of the carbon black makes it possible to act specifically on the ink according to the present invention, solving the main limitation for tattoo removal: chemical inertia. The presence of the appropriate functional group makes the pigment targetable through click-chemistry reactions. The click-chemistry reaction allows functionalization with a second compound, molecule and / or particle that makes removal possible by, but not limited to, extraction of the ink with microneedles suitable for biofluid extraction, or increased effectiveness of laser removal by conjugation with elements that enhance the absorption and excitation capabilities of the inks.

[0032] It is therefore further subject matter of the present invention to provide a method for removing tattoos, wherein said method comprises:

[0033] - Applying a tattoo using an ink that is carbon black functionalized according to the present invention;

[0034] - Conducting a SPAAC reaction between said ink functionalized with azide groups and nanomaterials comprising cycloalkynes, e.g. gold nanoparticles containing cycloalkynes.

[0035] In an embodiment, said method also comprises an additional step of ink extraction with microneedles.

[0036] In an embodiment, said method also comprises an additional laser removal step.

[0037] The following examples are only meant to show some embodiments of the invention and are not meant to be limiting. The scope of the invention is defined by the claims.

[0038] Example 1: Insertion of an azide group on carbon black nanoparticles extracted from tattoo ink.

[0039] The method comprises a first oxidation step and a second functionalization step. Oxidation, based on the Hummers method6, is required to increase the surface OH groups.

[0040] Oxidation procedure: to 1 g of carbon black, 2 g of NaNOa is added; it is then solubilized in 23 mL of concentrated sulfuric acid (96%) by immersing the reaction flask in an ice bath until the temperature is brought to and maintained at 0°C.

[0041] At the above temperature, 3 g KMnO4 is added.

[0042] The mixture is diluted with 46 mL of H2O and heated to 98°C while stirring, using a condenser to prevent evaporation of the solvent. After 30 minutes, the heat is removed and the mixture takes on a brown tinge. The mixture is cooled to room temperature and diluted with an additional 140 mL of H2O and 10 mL of 30% H2O2.

[0043] The reaction mixture is separated through a PTFE filter, then several washes are performed with distilled water and the anhydrous precipitate is recovered by treatment in a vacuum dryer in the presence of P2O5. The highly acidic filtrate is appropriately disposed of. UV and1H-NMR analyses of the solid product (Figures 1A, IB) support oxidation. Figure 1A shows the comparison of the UV spectrum of pure and oxidized carbon black by the Hummers method6. Figure IB shows the1HNMR (400MHz) spectrum in DMSO-d6 (at 2.50 ppm) of an oxidized carbon black sample, which shows signals for unsaturations at 7.2 and 6.7 ppm and for HC-O at 4.2 ppm.

[0044] The subsequent functionalization process of the oxidized carbon black (CBO) involves the esterification of the OH groups present on the surface with 2- bromo-2-methylpropanoylbromide in order to insert a bromine atom that will then be replaced with an azide group (Diagram 1).

[0045] Diagram 1: Diagram of the reaction between CBO and 2-bromo-2- methylpropanoylbromide, followed by subsequent nucleophilic substitution with sodium azide to obtain the insertion of the azide group.

[0046] Functionalization process: 1 g CBO is sonicated in 100 mL of dimethylformamide (DMF) for 30 minutes or until a homogeneous suspension is obtained. To the suspension 30 mL of 2-bromo-2- methylpropanoylbromide is added and 20 mL of triethylamine or another organic base necessary to neutralize the hydrobromic acid generated during the reaction. The reaction is then left under magnetic stirring at room temperature for 48 hours.

[0047] The product is filtered through a PTFE filter and washed with dichloromethane. The formation of the esterification product is verified by recording the FT-IR spectrum (Figure 2A, 2B). FT-IR spectrum of the CBO esterification product, showing in the region at 1730 cm-1 absorption of the C=O group, in the range 1300-1000 cm absorption of C-O, as well as bands in the region 1667-1653 for C=C absorption of the carbon black structure.

[0048] In the subsequent conversion reaction of alkyl bromide to azide, it is assumed for simplicity's sake that all the OH groups have been functionalized. Then, 1 g of the product obtained is again dispersed by sonication in 100 mL of DMF and 2.5 g of NaNs is added to the solution. The solution is left under magnetic stirring at room temperature for 24 hours. The product is then filtered through a PTFE filter and the precipitate is obtained, washed and resuspended in ethanol. The precipitate that separates is further filtered and the supernatant disposed of. This procedure is repeated three times to eliminate unreacted NaNs. The structure of the desired product was verified by comparison with similar literature data9and checked by IR spectroscopy (Figure 3). The analysis revealed the presence of absorption bands at 2130 cm4attributable to the C-N3 group of the desired product, in addition to the signal of the unreacted NaNs salt.

[0049] Bibliography:

[0050] 1. Renzoni, A. et al. 2018 The tattooed population in Italy: a national survey on demography, characteristics and perception of health risks. Ann. 1st. Super. Sanita 54, 126-136.

[0051] 2. Tattoo removal market size, industry growth report, 2030. https: / 1 www.strategicmarketresearch.com / market-report / tattoo-removal-market.

[0052] 3. Idiago-Lopez, J., et al. 2021. Nanoparticles and bioorthogonal chemistry joining forces for improved biomedical applications. Nanoscale Adv 3, 1261-1292.

[0053] 4. Agard, N. J., et al. 2004. A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems. J. Am. Chem. Soc. 126.

[0054] 5. Jewett, J. C., et al. 2010. Rapid Cu-free click chemistry with readily synthesized biarylazacyclooctynones. J. Am. Chem. Soc. 132, 3688-3690.

[0055] 6. Hummers, W. S., Jr & Offeman, R. E. 1958. Preparation of Graphitic Oxide. J. Am. Chem. Soc. 80.

[0056] 7. Gamez, S. et al. 2022 Carbon supports for the oxidative cleavage of oleic acid: Influence of textural properties. Mol. Catal. 533, 112797. 8. Kang, J. H. et al. 2016 Hidden Second Oxidation Step of Hummers Method.

[0057] Chem. Mater. 28, 756-764.

[0058] 9. Eigler, S. et al. 2013 Controlled functionalization of graphene oxide with sodium azide. Nanoscale 5, 12136-12139.

Claims

CLAIMS1. Removable tattoo ink that is functionalized carbon black, where said functionalized carbon black is oxidized carbon black in which at least a fraction of the alcohol (-OH) groups are esterified with azide-substituted acyl halides.

2. The ink according to claim 1, wherein said fraction of esterified -OH groups is greater than or equal to 50% of the hydroxyl groups, or it is greater than or equal to 60% of the hydroxyl groups, or it is greater than or equal to 80% of the hydroxyl groups on said carbon black.

3. The ink according to claim 1, wherein said acyl halides have a linear or branched organic chain, comprised between C2 and CIO, or between C3 and C8 and said halide is selected from F, Cl, and Br, with the presence of a C-leaving group selected from the group comprising halogen or mesyl, or tosyl.

4. The ink according to claim 1, wherein said acyl halide is 2-bromo-2- methylpropanoylbromide.

5. A method for obtaining a removable tattoo ink that is functionalized carbon black, wherein said method comprises: a) Carbon black oxidation; b) Oxidized carbon black functionalization, by esterification between the - OH groups that are formed in said oxidation and an acyl halide and subsequent reaction with sodium azide, where said functionalization introduces N3 groups.

6. The method according to claim 5, where said oxidation is obtained by the Hummers method in water.

7. The method according to claim 5, wherein said acyl halide is selected from the group comprising acyl halides having a C-leaving group, preferably it is 2-bromo-2-methylpropanoylbromide.

8. A tattoo removal method that comprises:- Applying a tattoo using an ink which is ink according to one of claims 1to 4;- Conducting a SPAAC reaction between said ink functionalized with azide groups and nanomaterials comprising cyclo-alkynes.

9. The method according to claim 8, where said nanomaterials are gold nanoparticles.

10. The method according to claim 8 or 9, wherein said SPAAC reaction occurs with the application of a microneedle patch.

11. The method according to one of claims 8-10, which also comprises a laser removal step.

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