Dye tattoo

Abstract

A method of durably marking skin comprising: introducing a dye to the dermis of the skin by applying the dye to the skin so as to cause the dye to migrate to the dermis along a concentration gradient; and causing the dye to fix in the dermis. A method of tattooing skin comprising introducing a reactive dye to the dermis of the skin, wherein the reactive dye binds to a microenvironment in the dermis. There is provided a tattoo ink comprising a reactive dye, wherein the reactive dye is capable of binding to tissue in the dermis of skin.

Description

[0001] 1 KSF01 -134374PC

[0002] DYE TATTOO

[0003] This invention relates to methods of tattooing and a tattoo ink comprising dye.

[0004] BACKGROUND TO THE INVENTION

[0005] Tattooing is the practice of marking skin. Conventional tattooing methods use a needle, often in combination with an electric motor, to pierce the skin repeatedly. Conventional tattoo ink comprises pigments, especially inorganic pigments. Many inorganic pigments can cause inflammatory reactions when present in the skin. Such pigments are especially challenging to remove in a tattoo removal process, often requiring many sessions of laser tattoo removal. There is a need to produce tattoos that can be removed more simply and effectively than conventional tattoos. There is also a need to create tattoos by alternative methods that do not rely on standard tattoo machines.

[0006] The use of dyes in tattooing has previously been viewed as unsuitable for producing permanent or long-lasting tattoos. Soluble dyes in particular are broken down in the body by natural processes such as an immune response. It would be desirable to utilise dyes as tattoo ink in such a way that lasting tattoos may be made.

[0007] The methods of tattooing described herein may avoid the use of conventional pigment inks and instead use dye to produce the tattoo. The methods are particularly advantageous as the dye is made to fix in the dermis, thus a long-lasting tattoo is made.

[0008] SUMMARY OF INVENTION

[0009] According to a first aspect there is provided a method of durably marking skin comprising: introducing a dye to the dermis of the skin by applying the dye to the skin so as to cause the dye to migrate to the dermis along a concentration gradient; and causing the dye to fix in the dermis. 2 KSF01 -134374PC

[0010] Preferably the dye comprises a reactive species capable of forming a bond with a component of the dermis. Preferably the reactive species comprises one or more of a halo-heterocyclic group, an activated double bond, an epoxide-type system, a carbonyl-based electrophile, a sulfonyl group, a phosphoryl group, an aziridine, a photoreactive group, a click-chemistry group, a metal-coordination group, or a combination of two or more thereof.

[0011] The dye may comprise a reactive molecule. The dye may be covalently bonded to a reactive molecule. Preferably the reactive molecule contains one or more reactive functional groups capable of forming bonds with components of the dermis.

[0012] The dye may be a reactive dye. The dye may comprise a vinyl sulfone and / or a triazine functional group. The dye may comprise Reactive Black 5 and / or Reactive Orange 16.

[0013] Preferably the dye is caused to fix in the dermis by reaction with a dermal component, wherein the dermal component comprises collagen, elastin, fibronectin, laminin, proteoglycans, glycosaminoglycans, or a combination of two or more thereof. Preferably the dye does not bind to keratin.

[0014] The dye may be capable of binding to tissue in the dermis. Preferably the dye is capable of reacting with collagen, elastin, fibronectin, laminin, proteoglycans, glycosaminoglycans, or two or more thereof. Preferably the dye reacts with one or more components of the dermis, wherein the components comprise collagen, elastin, fibronectin, laminin, proteoglycans, glycosaminoglycans, or a combination of two or more thereof.

[0015] The method may further comprise, after introducing the dye, introducing a fixing solution to the dermis so as to promote fixation of the dye. The fixing solution may promote the better binding of the dye with tissue in the dermis. Preferably the fixing solution comprises an alkali.

[0016] The method may further comprise, after introducing the dye, introducing an alkali to the dermis so as to promote fixation of the dye. 3 KSF01 -134374PC

[0017] The method may further comprise, after introducing the dye, introducing a solution containing sodium chloride to the dermis so as to promote fixation of the dye in the dermis.

[0018] The method may further comprise, prior to introducing the dye, preparing the dye by dissolving a dry dye in a solvent.

[0019] The dye may be introduced to the dermis by a mechanical process of penetrating the skin.

[0020] The method may further comprise at least partially removing an area of epidermis to expose an area of the dermis. The method may comprise applying the dye to the exposed area of the dermis. The area of epidermis may be removed by laser ablation.

[0021] The method may further comprise creating voids extending from a surface of the epidermis into the dermis. Preferably the method comprises introducing a dye through the voids into the dermis.

[0022] Creating voids may comprise at least partially removing a basement membrane, the basement membrane being between the epidermis and the dermis. Creating voids may comprise removing a basement membrane.

[0023] The dye may be caused to fix in the dermis by binding of the dye to proteins.

[0024] The dye may be caused to fix in the dermis by the dye agglomerating.

[0025] The dye may be soluble in dermal exudate.

[0026] The method may further comprise applying a mixture for promoting healing and / or improving the cosmetic appearance of the skin marking. The mixture for promoting healing may comprise a steroid. The mixture for promoting healing may comprise an antibiotic. The mixture for promoting healing may comprise an antimycotic. 4 KSF01 -134374PC

[0027] The method may further comprise applying a mixture comprising at least one of a steroid, an antibiotic and an antimycotic. Preferably the mixture improves the cosmetic appearance of the skin marking.

[0028] The method may further comprise sonicating the area of skin to which dye has been applied.

[0029] According to a second aspect there is provided a tattoo ink comprising a reactive dye, wherein the reactive dye is capable of binding to tissue in the dermis of skin.

[0030] The reactive dye may be capable of binding to hydroxyl groups and / or amino groups. The reactive dye may be capable of binding to collagen.

[0031] The reactive dye may comprise Reactive Black 5 and Reactive Orange 16.

[0032] According to a third aspect there is provided a method of tattooing skin comprising: introducing a reactive dye to a dermis of the skin; applying a solution comprising an alkali and sodium chloride to the dermis; and washing the solution from the skin; wherein the reactive dye binds to a microenvironment in the dermis.

[0033] The method may further comprise, prior to washing the solution from the skin, observing a waiting period of 5 to 15 minutes.

[0034] According to a further aspect there is provided a method of durably marking skin comprising introducing a dye to a dermal layer of skin and causing the dye to fix in the dermal layer by reaction of the dye with components of the dermal layer.

[0035] Preferably the dye is caused to fix in the dermal layer by reaction with collagen, elastin, fibronectin, laminin, proteoglycans, glycosaminoglycans, or two or more thereof.

[0036] BRIEF DESCRIPTION OF THE FIGURES

[0037] The present invention will now be described by way of example with reference to the accompanying drawings. In the drawings: 5 KSF01 -134374PC

[0038] Figure 1 shows a schematic cross section of skin.

[0039] Figure 2 shows a scheme of making a dye tattoo.

[0040] Figure 3 shows a schematic concentration gradient of dye in skin.

[0041] Figure 4 shows a representation of a dye tattoo in a cross section of skin.

[0042] Figure 5 shows an SEM image of tattooed skin.

[0043] DETAILED DESCRIPTION

[0044] The following description is presented to enable any person skilled in the art to make and use the invention, and is provided in the context of a particular application. Various modifications to the disclosed embodiments will be readily apparent to those skilled in the art.

[0045] The general principles defined herein may be applied to other embodiments and applications without departing from the present invention. Thus, the present invention is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

[0046] There is provided a method of durably marking skin comprising introducing a dye to the dermis of the skin by applying the dye to the skin so as to cause the dye to migrate to the dermis along a concentration gradient and causing the dye to fix in the dermis. The dye may be fixed in the dermis by chemical means. Advantageously, there is no need to retain the dye in a polymer shell as the dye is caused to migrate to the dermis. The methods of durably marking skin described herein enable a dye which would otherwise dissolve or dissipate in the skin to be fixed in the skin. Without being bound by theory, it is thought that reaction of the dye with the extracellular matrix causes the dye to be durably fixed in the skin. 6 KSF01 -134374PC

[0047] A dye is a substance used to add colour, for example to cells or tissue. A dye molecule may comprise a chromophore, which is a moiety which absorbs light at a particular wavelength and reflects light at a wavelength in the visible spectrum, thereby appearing coloured.

[0048] As used herein ‘dye’ preferably means any substance or combination of substances capable of producing a visible colour in the dermis when applied according to the disclosed methods. Preferably the term ‘dye’ includes materials which are colourless or weakly coloured prior to fixation in the skin but which develop or reveal colour when situated in the skin. Such dyes may develop colour as a result of a chemical and / or physical reaction, for example by a change in pH, by oxidation or reduction, a photochemical activation, covalent bonding to constituents of the skin, or a combination of two or more thereof.

[0049] Examples of suitable dyes include pro-dyes, leuco-dyes, photochromic dyes, thermochromic dyes, solvatochromic dyes, coordination systems, soluble metal salts, or a combination of two or more thereof. The term ‘dye’ as used herein may encompass a colouring agent together with any solvent, carrier, or auxiliary substances forming a solution, dispersion, or mixture.

[0050] The dye may be provided in particulate form, for example as an organic or inorganic pigment, optionally a carbon-based pigment, optionally dispersed or suspended in a carrier. The dye may comprise particles. Such particulate colourants may be delivered via the voids according to the methods described herein. Particulate colourants are preferably retained or fixed within the dermis.

[0051] The dye is preferably not provided in particulate form, for example as an organic or inorganic pigment, such as a carbon-based pigment, such as dispersed or suspended in a carrier. Preferably the dye does not comprise particles.

[0052] The use of dye to produce a tattoo may be advantageous as the dye can be removed simply and effectively by chemical reaction with the dye. For example, a reactive dye can be decolourised by reaction with a reducing agent or oxidising agent. 7 KSF01 -134374PC

[0053] As used herein, ‘fix’ or ‘fixation’ in the dermis may describe binding of the dye to one or more components of the extracellular matrix. Such components include fibrous proteins (e.g. collagen, elastin), cell-adhesive glycoproteins (e.g. fibronectin, laminin), proteoglycans and glycosaminoglycans (e.g. hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate), constituents of the basement membrane, and constituents of the papillary dermis.

[0054] Figure 1 shows a schematic cross section of skin 100. The skin 100 has an upper layer, the epidermis 101 , a dermis layer 102 and a hypodermis layer 103. In undamaged skin, the epidermis is the outer layer of the skin, and the dermis is a layer between the epidermis and the hypodermis. The epidermis 101 has an upper surface exposed, e.g. to air, at the outer surface of the skin. On the opposing side of the epidermis, the epidermis has an inner boundary at its interface 104 with the dermis 102. The interface 104 comprises a basement membrane, also called a basal membrane. The dermis 102 comprises connective tissue, such as collagen, reticular and elastic fibres. The dermis 102 comprises a papillary layer, proximal to the epidermis, and a reticular layer, proximal to the hypodermis. The papillary layer is composed of looser connective tissue; that is to say connective tissue that is looser than the general density of the reticular layer. The reticular layer is composed of denser connective tissue; that is to say connective tissue that is denser than the general density of the papillary layer. The papillary and reticular layers may not be clearly distinguishable from one another, there is a gradient of transition from papillary to reticular as the depth from the surface of the skin increases.

[0055] Dye introduced only to the epidermis 101 will be removed by the natural exfoliation of epidermal cells over time. It is not believed to be possible to produce a lasting tattoo by superficial application of a dye. Dye introduced to the dermis 102 may remain in the dermis such that a durable tattoo is made. Dye may be introduced to the dermis by various methods. Dye can be introduced into the dermis in various ways, for example by creating channels (voids) in the skin, without the use of standard tattoo machines. Preferably, voids may be formed penetrating into the dermis. The voids may be formed by a laser, preferably by laser ablation. A region of skin below the epidermis may be exposed prior to introducing the dye, for example by chemical or mechanical peeling of the epidermis, or by laser ablation. 8 KSF01 -134374PC

[0056] A durable tattoo may substantially resist removal from the skin under exposure to biocompatible agents. A durable tattoo may substantially resist degradation under typical exposure to sunlight. A durable tattoo may be capable of enduring visibly in the skin of a living subject for a period of greater than one year, greater than five years or greater than ten years.

[0057] The process of making a dye tattoo will be discussed with reference to Figure 2. The process steps are represented in sequence, viewing the figure from left to right. Figure 2 shows a region of skin 200 having an external surface 209, epidermal layer 201 , dermal layer 202, and hypodermal layer 203. These layers may be referred to as the epidermis, dermis, and hypodermis, respectively. A channel through the epidermis 201 and into the dermis 202 is indicated at 204. A plurality of channels may be formed in the skin to produce a tattoo design. The channel 204 may be made by any suitable means, such as a needle, or by a laser. A dye 205 is introduced to the dermis 202 by applying the dye to the skin. The dye 205 may be applied at the skin surface 209, the external surface of the epidermis. The dye may be applied by a needle puncturing the epidermis. The dye may be injected into the dermis. The dye may enter the dermis via channel 204. Preferably the dye is introduced to a depth in the skin greater than the basement membrane between the epidermal layer and dermal layer. Preferably the dye is not introduced into the hypodermal layer 203 as dye in the hypodermis may appear indistinct in the skin and produce an undesirable tattoo.

[0058] The dye is introduced to the dermis by applying the dye to the skin so as to cause the dye to migrate to the dermis along a concentration gradient. The dye may be applied in a dry form, for example as a powder, or as a solution wherein the dye is dissolved in a solvent. In an example, a tattoo ink is prepared by dissolving dry powdered dye in water. The dye may dissolve in dermal exudate. Dermal exudate may comprise extracellular fluid (which may include interstitial fluid, plasma, and transcellular fluid) and / or blood and / or sweat. The dye may flow from a region of relatively higher concentration at the surface of the skin to a region of relatively lower concentration inside the dermis. The concentration gradient may be a gradient of concentration of the dye and / or of a preparation comprising the dye in which the dye is applied to the skin. 9 KSF01 -134374PC

[0059] Molecules of the dye are schematically represented as dots 207. Figure 2 shows dye molecules 207 which have migrated into the dermis 202 from a channel in the skin.

[0060] The dye may comprise a reactive molecule. The dye may be covalently bonded to a reactive molecule. Preferably the reactive molecule contains one or more reactive functional groups capable of forming bonds with nucleophilic or electrophilic sites in dermal tissue, for example amino, hydroxyl, thiol, or carboxyl groups. Examples of reactive functional groups include: halo-heterocyclic groups, activated double bond systems (Michael acceptors), epoxide-type systems (ring-opening alkylation), carbonyl-based electrophiles, sulfonyl or phosphoryl groups, leaving-group-activated systems, aziridines, photoreactive groups, click-chemistry groups (for in-situ activation), and metal-coordination groups. Examples of suitable dyes containing these functional groups is provided below.

[0061] A reactive dye may be used. A reactive dye is a dye of the class known as reactive dyes. The reactive dye can for bind to tissue in the dermis. A reactive dye comprises a chromophore which is capable of reacting with another substance. The reactive dye or dyes selected for use are preferably reactive with hydroxyl groups present in tissue. The reactive dye or dyes selected for use are preferably reactive with free amino groups present in tissue. The application of an alkali may promote binding by supplying an excess of hydroxyl groups and / or amino groups. The dye may be selected for its ability to remain in the dermis. The dye may be selected for its ability to react with components of the dermis or extracellular matrix. Preferably the dye is capable of binding to tissue in the dermis. The dye may bind to collagen. The dye may agglomerate. The dye may bind to the basement membrane of the skin. The dye may polymerize with the help of auxiliary components (for example quaternary amines). It is to be appreciated that the dye polymerizing itself or polymerizing due to reaction with components of the dermis is distinct from introducing the dye with a polymer, or introducing the dye encapsulated in a polymer.

[0062] The reactive dye may be a vinyl sulfone and / or triazine dye. The triazine dye may be a chlorotriazine dye. In an example, the dye comprises Reactive Black 5 (C26H2iN5Na4Oi9Se), shown as formula 1 , and Reactive Orange 16 10 KSF01 -134374PC

[0063] (C2oHi?N3Na20nS3), shown as formula 2. Reactive Black 5 and Reactive Orange 16 both comprise vinyl sulfone functional groups. Each molecule of the dye may comprise one or more vinyl sulfone functional groups. The combination of Reactive Black 5 and Reactive Orange 16 provides a combined colour that appears black. The addition of orange improves the appearance of the dye as a black tattoo, as Reactive Black 5 used alone appears dark blue in the skin.

[0064] Each molecule of the dye may comprise one or more groups selected from the following list: monochlorotriazine, monofluorochlorotriazine, dichlorotriazine, difluorochloropyrimidine, dichloroquinoxaline, vinyl sulfone, vinyl amide. The dye may be capable of covalently bonding to dermis tissue, as described in more detail below

[0065] As shown in figure 2, the dye 205 may be caused to better fix in the dermis by applying a solution 206 subsequently to the dye 205. The solution may comprise an alkali. For example, a solution of sodium carbonate in water may be applied. The alkali may promote better fixation of the dye in the dermis. The alkali may promote binding of the dye by supplying hydroxyl groups and / or amino groups. The alkali may promote polymerization of the dye. 11 KSF01 -134374PC

[0066] The solution may further comprise a salt, preferably sodium chloride. The salt may improve retention of the dye in the dermis by reducing the repulsion between dye species and tissue in the dermis. The salt may promote better fixation of the dye in the dermis. The salt may promote the binding of the dye to proteins in the dermis. For example, sodium chloride can reduce repulsion between collagen and dye, so that dye molecules can more easily associate with collagen.

[0067] Other additives may be incorporated in the formulation that is applied to the skin. Examples of additives may include those contained in known tattooing preparations, not excluding pigments.

[0068] Figure 2 shows a dye tattoo 208 in a healed region of skin. After application of the dye, the tattoo may be covered with a bandage to reduce the risk of infection. A mixture for promoting healing may be applied. The mixture is preferably able to improve the aesthetic appearance of the tattoo. The mixture may comprise a steroid, an antibiotic, an antimycotic, or a combination of two or more thereof. The steroid reduces inflammation and may promote retention of the dye in the dermis. Retention of the dye may be promoted by the accelerated and / or improved healing of tissue subjected to the tattooing process. Retention of the dye may be promoted by local inhibition of immune activity. An example of a suitable group of steroids is glucocorticoids.

[0069] Conventional tattoo ink comprises pigments, especially inorganic pigments. These pigments may be solid particles having a size between 200 nm - 200 pm. The presently described method of tattooing and tattoo ink preferably avoid the use of pigments and instead uses a dye to produce the colour of the tattoo. In some embodiments, the dye may be in particulate form.

[0070] The term “dye” may be used to refer to the combination of a colouring agent and solvent. The solvent may be an aqueous solvent or a non-aqueous solvent. It is preferred that the solvent is an aqueous solvent since an aqueous solvent may have better biocompatibility. 12 KSF01 -134374PC

[0071] The dye is preferably soluble. The solubility of the dye at 37°C and at a pH in the range 6.5 - 8.0 is preferably in the range from 1 msv / / ??d to 50 msv / / ??d, where msvis the mass of solvent required to dissolve one unit of mass ma of dye (as a solute). The dye may be selected so as to be suitable for at least partially dissolving in extracellular fluid. The dye may be selected so as to be suitable for at least partially dissolving in interstitial fluid and / or blood. Preferably the dye is water soluble. Preferably the dye is at least partly soluble in oil. The dye may be soluble in fatty tissue. Such solubility may improve the ability of the dye to penetrate and / or fix to tissue.

[0072] The dye may fix in the dermis through covalent interactions, non-covalent interactions, or a combination of the two. Non-covalent interactions include ionic or electrostatic forces, hydrogen bonding, hydrophobic and n - n stacking interactions. Fixation of the dye may occur by coordination or chelation with metal ions present in tissue. The binding or interaction of the dye may involve chemical and / or physicochemical bonding. Such bonding may include covalent bonding, for example formation of new ff or 7i bonds between reactive groups of the dye (e.g., vinyl sulfone, triazine, isocyanate, aldehyde, carboxyl, amino, hydroxyl groups) and functional groups of components of the dermis and / or extracellular matrix (e.g., -NH2, -OH, -COOH, -SH groups).

[0073] The dye may fix in the dermis by ionic bonding. For example, cationic dyes can ionically bond to anionic sulfate or carboxylate groups of glycosaminoglycans.

[0074] The dye may fix in the dermis by hydrogen bonding, such as by donor and acceptor atoms (e.g., N-H- 0 or O-H- O) linking dye molecules and polar moieties in the dermis. The dye may fix in the dermis by Van der Waals interactions, such as dipoledipole, dipole-induced dipole, and London dispersion forces. The dye may fix in the dermis by hydrophobic interactions, for example non-polar association between aromatic or aliphatic groups of the dye and hydrophobic domains of proteins or lipids. Ti - Ti stacking and Tr-cation interactions may occur between aromatic rings of dye chromophores and aromatic amino acids or cationic residues. 13 KSF01 -134374PC

[0075] The dye may fix in the dermis by coordinate bonding (chelation). The dye may form a complex with metal ions present in tissue (e.g. calcium, iron, copper, zinc ions. Dipole and induced-dipole interactions, halogen bonds, or other weak non-covalent forces may contribute to the stabilization of the dye in the extracellular matrix. Fixation of the dye may result from one or more interactions as described above. Reaction of the dye may form a network that immobilizes dye molecules within the dermis. Preferably, the network containing the dye molecules forms in the extracellular matrix of the dermis.

[0076] Preferably, the dye comprises a reactive molecule or a functional group which reacts with a moiety in the dermis. The dye may comprise an azo dye. The dye may comprise sulfur. The dye may be prepared prior to application by dissolving a powder dye in water. Preferably the dye is prepared immediately prior to application, for example not longer than 1 hour before application. Additionally, salt (NaCI) may be added to the dye. Alternatively, the dye may be formed by applying more than one solution sequentially to an area of skin.

[0077] The dye may comprise or be covalently bonded to a reactive molecule. The reactive molecule preferably contains one or more reactive functional groups capable of forming bonds with constituents of the dermis and / or extracellular matrix. Preferably the dye reacts with nucleophilic or electrophilic sites in dermal tissue, for example amino, hydroxyl, thiol, or carboxyl groups.

[0078] Examples of reactive functional groups include: halo-heterocyclic groups, activated double bond systems (Michael acceptors), epoxide-type systems (ring-opening alkylation), carbonyl-based electrophiles, sulfonyl or phosphoryl groups, leaving- group-activated systems, aziridines, photoreactive groups, click-chemistry groups (for in-situ activation), and metal-coordination groups.

[0079] Halo-heterocyclic groups bond by nucleophilic substitution. Examples of dyes having a halo-heterocyclic group include: monochlorotriazine, monofluorochlorotriazine, dichlorotriazine, difluorochloropyrimidine, trichloropyrimidine, dichloroquinoxaline, monofluorotriazine and mixed halotriazines. 14 KSF01 -134374PC

[0080] Activated double-bond groups (known as Michael acceptors) form bonds by donating an electron pair. Examples of activated double-bond systems include vinyl sulfone (- SO2-CH=CH2), vinyl amide (-CH=CH-CONH-), acrylamide, acryloyl, crotonyl, maleimide derivatives, and haloacrylamido groups (such as a-bromo- or a-chloro- acrylamides).

[0081] Epoxide-type systems react by ring-opening alkylation. Examples of epoxide-type groups include glycidyl, epoxy groups, epichlorohydrin, 3-chloro-2-hydroxypropyl, and oxirane derivatives.

[0082] Examples of carbonyl-based electrophiles include acyl halides (-COCI, -COBr), acid anhydrides (-CO-O-CO-), isocyanates (-N=C=O), isothiocyanates (-N=C=S), aldehydes and activated ketones (which may be used for Schiff base formation)

[0083] Examples of sulfonyl and phosphoryl groups include sulfonyl halides (-SO2CI), sulfonic anhydrides, sulfates, phosphoryl chlorides and phosphoram idates.

[0084] Examples of leaving-group-activated systems include tosylates, mesylates, triflates, sulfonate esters, activated esters (e.g., N-hydroxysuccinimide esters).

[0085] The reactive functional group aziridines react by nucleophilic ring-opening. Other strained ring structures similar to aziridines which are capable of nucleophilic ringopening may also provide the reactive molecule.

[0086] Photoreactive and click-chemistry groups may provide the reactive molecule for in-situ activation. Examples of photoreactive and / or click-chemistry groups include aryl azides (-N3), diazirines, alkynes and azides for 1 ,3-dipolar cycloaddition (“click” reaction), tetrazoles (photo-activated nitrile imine sources).

[0087] Examples of metal-coordination groups include carboxylate, catechol, and imidazole moieties. Without being bound by theory, such moieties are thought to form coordinate bonds with metal ions naturally present in skin tissue. 15 KSF01 -134374PC

[0088] An optional additional step of the method of tattooing will now be described. After introducing the dye into the dermis, a treatment solution may be applied. The treatment solution comprises one or more components which promote fixation of the dye to components of the dermis or the extracellular matrix. Preferably the treatment solution comprises soluble components in aqueous solution. The treatment solution may contain activators which react with reactive groups to enhance covalent or coordination bonding between dye functional groups and tissue constituents. In this way, the treatment solution is applied to promote fixation of the dye. The treatment solution may enhance the reaction of reactive groups of the dye with components of the extracellular matrix.

[0089] Examples of treatment solutions include alkaline solutions, acidic solutions, solutions containing metal salts, organic or inorganic catalysts, buffers, high-pH activators (pH 8-11 ), and low-pH activators (pH 3-5). Examples of each type of treatment solution will now be given.

[0090] Alkaline solutions may include sodium carbonate, sodium hydroxide, potassium carbonate, or two or more thereof. Alkaline solutions may provide hydroxide ions which promote activation of vinyl sulfone, triazine, or epoxide reactive groups.

[0091] Acidic solutions may include citric acid, acetic acid, lactic acid, or two or more thereof. Acidic solutions may provide protonic activation for carbonyl-based or amide-based reactive systems.

[0092] Metal salts may including chlorides, sulfates, or acetates of Na+, K+, Ca2+, Mg2+, Zn2+, Cu2+, Fe2+ / Fe3+, Al3+, capable of forming coordination complexes with dye molecules and tissue ligands, thereby improving fixation.

[0093] Organic or inorganic catalysts and buffers may include ammonium salts, phosphate buffers, borates, imidazoles, amines (e.g., triethanolamine, morpholine), or two or more thereof. Such substances in the treatment solution may be used to adjust reaction kinetics and / or pH of the solution. 16 KSF01 -134374PC

[0094] High-pH activators (pH 8-11 ) are suitable for nucleophilic substitution, for example with triazine, vinyl sulfone, and epoxide groups.

[0095] Low-pH activators (pH 3-5) are suitable for condensation or Schiff-base reactions of aldehyde and carbonyl groups with amino-containing biomolecules.

[0096] The solution may also contain ionic salts (NaCI, KCI, CaCl2, MgCl2, ZnCk) to modulate electrostatic interactions between the dye and tissue and to minimize charge-based repulsion.

[0097] Figure 3 shows a cross section of skin 300 having an epidermal layer 301 , dermal layer 302, and hypodermal layer 303. Dye 305 is applied to the skin. The dye is shown as having entered the epidermal layer 301 and partially entered the dermal layer 302. The dye 305 is shown in a puncture 304 into the skin. The puncture 304 may be described as a void. The puncture 304 may have been formed by a needle. The dye 305 enters the dermis in the direction of the arrow. The dye 305 is shown in the void as having a representative concentration gradient. The relatively more concentrated region of dye is shown as the darker region proximal to the hypodermal layer 303. The relatively less concentrated region of dye is shown as the lighter region proximal to the epidermal layer 301 . Preferably the dye is caused to migrate along a concentration gradient as the dye dissolves in dermal exudate. The dermal layer may exude fluid in response to being penetrated and / or the epidermal layer being removed to expose the dermis.

[0098] Optionally, a solution may be applied which contains a substance that liquifies the exudate, helping the dye penetrate into the dermis through the basement membrane. The dye may comprise a substance to liquify the dermal exudate.

[0099] The dye may be comprised in a tattoo ink. A tattoo ink may be prepared by combining a colouring agent with a solvent. Preferably, the method of tattooing uses a tattoo ink comprising a reactive dye, wherein the reactive dye is capable of binding to tissue in the dermis of skin. 17 KSF01 -134374PC

[0100] Figure 4 shows a cross section of tattooed skin 400 having an epidermal layer 401 , dermal layer 402, and hypodermal layer 403. The skin has a basement membrane 404 at the interface between the epidermal layer 401 and dermal layer 402. The skin 400 contains area of dye, indicated generally at 405 and 406. Without being bound by theory, it is thought that the dye is fixed in the dermis by binding to proteins such as collagen. The dye may bind to the basement membrane 404 and / or to tissue in the dermis proximal to the basement membrane, as shown at 405. The dye may bind to a microenvironment in the dermis, as shown at 406. A plurality of dye molecules may aggregate in the microenvironment. The dye may react with constituents of the extracellular matrix and become fixed. The area of dye 406 may comprise an agglomeration of dye molecules and tissue. In an example, a reactive dye binds to collagen fibres in the dermis and is thereby durably fixed in the dermis.

[0101] Figure 5 shows a scanning electron microscope (SEM) image of a cross section of skin having a dye tattoo. Figure 5 shows the epidermis 501 and dermis 502. At the interface between the epidermis 501 and dermis 502, a region having dye present 505 is shown. Without being bound by theory, it is thought that areas of aggregated dye are most concentrated in the dermis at the interface of a dermal layer with an epidermal layer. The region having dye present 505 may be the basement membrane.

[0102] An exemplary method of tattooing skin will be described. The method comprises introducing a reactive dye to a dermis of the skin. The reactive dye may comprise a vinyl sulfone and / or triazine functional group. The dye may be introduced to the dermis by exposing the dermis, such as by laser ablation of the epidermis. The dye may be introduced to the dermis by dermal penetration, such as by a tattoo needle. Preferably the method comprises creating voids extending from a surface of the epidermis into the dermis and introducing a dye through the voids into the dermis. Creating voids may comprise at least partially removing a basement membrane to expose the dermis.

[0103] The dye may migrate into the dermis along a concentration gradient. The method further comprises applying a solution containing an alkali and a salt to the dermis. Preferably the salt is sodium chloride. Preferably the alkali is sodium carbonate or sodium hydroxide. The method further comprises a step of washing the solution from 18 KSF01 -134374PC the skin. Excess dye may be removed by washing. Between the step of applying the dye and washing the solution from the skin, a waiting period of between 2 and 20 minutes may be observed, preferably a waiting period of 5 to 15 minutes. The observation of the waiting period may permit the dye to react with components of the dermis and / or extracellular matrix.

[0104] In an example where a laser is used to ablate the epidermis, a region of skin to be tattooed is positioned proximal to the laser. The laser may be focused by a lens. The lens may be adjusted to focus the laser about the surface of the region of skin. Preferably, the laser beam is focused at a depth just below the surface of the skin, i.e. below or at the epidermis. The laser may be configured to pattern the region of skin with a selected design. The laser may create voids in the skin. The epidermis may be removed by laser ablation. Part of the dermis may be removed by laser ablation. The laser may be pulsed. Once voids have been formed and / or the dermis has been exposed, a dye is applied to the region of skin. The method then proceeds as described above with reference to figure 2.

[0105] Reaction of the dye with tissue and retention of the dye in the dermis may be promoted by sonicating the area of skin to which dye has been applied. An ultrasonic probe may be used to sonicate the area. Sonication may be performed after applying the dye to distribute the dye in an area of skin.

[0106] Fixing the dye in the dermis may comprise forming water-insoluble precipitates, polymers, or coordination complexes of the dye within the dermis. Following formation of voids into the dermis and at least partial removal of the basement membrane, one or more reagent solutions may be applied to the skin. The reagent solutions may be applied sequentially. The reagents may diffuse into the dermis. The reagents may diffuse into the dermis along a concentration gradient. The reagents preferably react with each other and / or with components of the extracellular matrix (e.g., collagen, elastin, fibronectin, glycoproteins, proteoglycans, hyaluronic acid, integrins), to produce water-insoluble species. In this way, the dye is durably fixed in the dermis. Advantageously, a water-soluble dye can be delivered to the dermis via the voids and fixed in the dermis such that a substantially water-insoluble species remains. The insoluble species may form in the papillary dermis and / or in the reticular dermis. 19 KSF01 -134374PC

[0107] Examples of combinations of reagents suitable for use as a dye and an overview of the reactions will be provided. In each example, voids are formed in a region of skin using a CO2 laser prior to application of a reagent solution. Optionally, after application of a first reagent, the region of skin may be rinsed with deionised water. Optionally, after application of the reagents, a neutralising solution is applied to the region of skin to dilute and / or remove residual reactants. The neutralising solution may contain one or more buffers to restore the pH of the region of skin to around 5.5 (typical skin pH).

[0108] In a first example, a first solution of aqueous p-phenylenediamine (PPD) was applied to the skin. A waiting period of between about 1 to about 10 minutes was observed. A second solution comprising hydrogen peroxide (H2O2) was applied to the skin. Optionally, tannic acid and / or a solution containing iron (III) ions may be applied to catalyse the reaction between PPD and hydrogen peroxide. The formation of dark (brown-black) oligomers was observed. It is theorised that dye species are retained by covalent bonding within the dermis. It is understood that oxidative polymerisation of the PPD occurs.

[0109] In a second example, an iron-gall type dye was fixed in the dermis. A first solution of tannic acid was applied to the skin. A second solution of ferric chloride (FeCh) was applied to the skin. A dark bluish-black precipitate was observed. Similarly, a first solution of gallic acid may be reacted with ferric chloride to produce a dark precipitate in the dermis. Similarly, either gallic or tannic acid may be reacted with ferric sulfate (Fe2(SO4)s) to produce a dark precipitate in the dermis.

[0110] In a third example, a first solution containing ferric nitrate (Fe(NOs)3) and ferrous sulfate (FeSC ) in a molar ratio of about 2:1 Fe3+:Fe2+was applied to the skin. A second solution containing ammonium hydroxide was applied to the skin. Preferably the ammonium hydroxide is dilute in solution, having a pH of about 8 to about 9. A black species was observed. The reaction is understood to produce iron oxide, known as magnetite (FesC ).

[0111] In a fourth example, a Prussian blue dye was made to fix in the dermis. A first solution of potassium ferrocyanide (K4[Fe(CN)e] ■ 3H2O) was applied to the skin. A second 20 KSF01 -134374PC solution comprising iron (III) ions was applied. The second solution may contain one or more of ferrous chloride and ferrous nitrate. The resulting dye contained the insoluble complex Fe4[Fe(CN)e]3 which appeared dark blue. The Prussian blue dye may be produced by reacting sodium ferrocyanide with Fe3+ions.

[0112] In a fifth example, a copper (II) sulfate (CuSC -SFbO) solution was applied to the skin. A second solution containing a basic sodium compound such as sodium carbonate or sodium bicarbonate solution was applied to the skin. The first and second solutions react to produce basic copper carbonate (CuCO3'Cu(OH)2), an insoluble green solid. The precipitate was observed to develop from a pale green colour to a vivid green. Optionally, a third solution comprising an acid may be added to reduce the copper carbonate and turn the precipitate blue.

[0113] As described herein, in addition to agglomeration, dye fixation in the dermis may occur by polymerization and / or polycondensation of the dye in the dermis. The dye may be fixed by other mechanisms of immobilization, including ionic complexation, coordination / chelation, supramolecular assembly, photo-activated or enzyme-mediated covalent deposition, and bio-orthogonal reactions.

[0114] The dye may be fixed in the dermis by polymerization of the dye or dye-bearing moieties. The dye may be fixed in the dermis by polycondensation of the dye or dye-bearing moieties. The dye may be fixed in the dermis by reaction to form an ionic complex, coordination, chelation, supramolecular assembly, photoactivated or enzyme-mediated covalent deposition, bio-orthogonal reactions, or a combination of two or more thereof.

[0115] In a further example, a dye marking is produced by oxidative polymerization of aniline. A first solution of aniline (phenylamine) was prepared. The first solution may be prepared under acidic or neutral conditions. The first solution was introduced to the dermis. A second solution of an oxidizing agent was introduced to the dermis. The oxidizing agents may be one or more of potassium dichromate, potassium chlorate (optionally with a metal salt catalyst such as vanadium), and ammonium persulfate. The oxidation of aniline monomers yielded an insoluble black emeraldine polymer 21 KSF01 -134374PC known as aniline black (polyaniline). It is understood that the process may involve formation of intermediate aniline radicals and p-aminodiphenylamine, which further oxidize and combine into a high molecular weight polymer having a black appearance. The reaction of the first and second solutions produces a dark polymer in the dermis.

[0116] In a further example, a dye of polymerized dopamine was produced by reacting dopamine (3,4-dihydroxyphenethylamine) with water, optionally with a mild alkali to adjust the pH to about 8, to produce a dopamine solution. The dopamine solution was exposed to air. Optionally, hydrogen peroxide solution may be reacted with the dopamine solution to accelerate the oxidation of dopamine. The dopamine undergoes autoxidation to dopamine-quinone and further reacts to form a black-brown polydopamine polymer. The polydopamine polymer may deposit in the dermis or become fixed by reaction with the extracellular matrix. Polydopamine contains indole and catechol units and is thought to strongly adhere to biological tissue. As such, a persistent dark substance can be formed in the dermis. The reaction may be considered analogous to melanin production.

[0117] A dye may be produced by the oxidation of one or more polyphenol, such as pyrogallol and catechol. In an example, a polyphenolic compound such as pyrogallol (1 ,2,3- trihydroxybenzene) or catechol (1 ,2-dihydroxybenzene) in aqueous solution is applied to the dermis. The polyphenolic compound may be allowed to oxidise in air or an oxidizing agent such as hydrogen peroxide may be applied. The phenolic compound oxidize to ortho-quinones which polymerize into complex polyphenolic pigments. Polyphenolic pigments may be considered as similar to natural melanin precursors. A brown-black dye is observed in the dermis. The polyphenolic pigments are generally insoluble in water and can bind to proteins or other components of the dermis to produce a lasting brown colour.

[0118] Skin may be durably marked according to methods described herein with an enzymatic reaction producing the dye, where coloured reaction products are the dye. An enzymatic reaction may be described as a biochemical reaction catalyzed by enzymes. Advantageously, since enzymes operate in physiological environments, enzymatic reactions can occur in the dermis without substantial damage to surrounding tissue. 22 KSF01 -134374PC

[0119] In a first example of an enzymatic dye, tyrosinase (a copper-containing oxidase present in skin melanocytes and many organisms) is reacted with L-tyrosine or L- DOPA (3,4-dihydroxyphenylalanine). The reaction is understood as tyrosinase hydroxylating tyrosine to DOPA, then oxidizing L-DOPA to dopaquinone, which undergoes a series of reactions (the Raper-Mason pathway) leading to polymerization into melanin pigments. A dark form of melanin, eumelanin is produced from L- DOPA / tyrosine oxidation. Advantageously, the dye is highly biocompatible.

[0120] In a second example of an enzymatic dye, a [3-galactosidase enzyme is applied to the dermis with X-gal (5-bromo-4-chloro-3-indolyl-[3-D-galactopyranoside). It is understood that the enzyme cleaves the X-gal into galactose and 5-bromo-4-chloro- 3-hydroxyindole, an indole derivative. The indole derivative spontaneously dimerizes and oxidizes to 5,5'-dibromo-4,4'-dichloro-indigo, an insoluble blue dye. A bright blue colour is observed.

[0121] In a third example of an enzymatic dye, a peroxidase enzyme such as horseradish peroxidase (HRP) is reacted with 3,3'-diaminobenzidine (DAB) and hydrogen peroxide (H2O2). DAB is oxidized into a reactive radical form that spontaneously polymerizes. The DAB is converted into an insoluble brown polymer that precipitates in the presence of the enzyme, forming a dark brown deposit in the dermis. A peroxidase enzyme may be reacted with tetramethylbenzidine (3,3',5,5'-tetramethylbenzidine) and hydrogen peroxide to yield a diimine derivative. The oxidized TMB forms a charge-transfer complex that appears as an intense blue colour in solution.

[0122] The applicant hereby discloses in isolation each individual feature described herein and any combination of two or more such features, to the extent that such features or combinations are capable of being carried out based on the present specification as a whole in the light of the common general knowledge of a person skilled in the art, irrespective of whether such features or combinations of features solve any problems disclosed herein. The applicant indicates that aspects of the present invention may consist of any such individual feature or combination of features. In view of the foregoing description, it will be evident to a person skilled in the art that various modifications may be made within the scope of the invention. 23 KSF01-134374PC

[0123] The foregoing description of example embodiments has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the present disclosure to the precise forms disclosed. Within this specification embodiments have been described in a way which enables a clear and concise specification to be written, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the invention. Many modifications and variations are possible in light of this disclosure. It is intended that the scope of the present disclosure be limited not by this detailed description, but rather by the claims appended hereto.

Claims

24 KSF01 -134374PCCLAIMS1 . A method of durably marking skin comprising: introducing a dye to the dermis of the skin by applying the dye to the skin so as to cause the dye to migrate to the dermis along a concentration gradient; and causing the dye to fix in the dermis.

2. A method as claimed in claim 1 , wherein the dye comprises a reactive species capable of forming a bond with a component of the dermis.

3. A method as claimed in claim 2, wherein the reactive species comprises one or more of a halo-heterocyclic group, an activated double bond, an epoxide-type system, a carbonyl-based electrophile, a sulfonyl group, a phosphoryl group, an aziridine, a photoreactive group, a click-chemistry group, a metal-coordination group, or a combination of two or more thereof.

4. A method as claimed in any preceding claim, wherein the dye is a reactive dye.

5. A method as claimed in any preceding claim, wherein the dye comprises a vinyl sulfone and / or a triazine functional group.

6. A method as claimed in any preceding claim, wherein the dye comprises Reactive Black 5 and / or Reactive Orange 16.

7. A method as claimed in any preceding claim, wherein the dye is capable of binding to tissue in the dermis.

8. A method as claimed in any preceding claim, wherein the dye is caused to fix in the dermis by reaction with a dermal component, wherein the dermal component comprises collagen, elastin, fibronectin, laminin, proteoglycans, glycosaminoglycans, or a combination of two or more thereof.

9. A method as claimed in any preceding claim, further comprising, after introducing the dye, introducing an alkali to the dermis so as to promote fixation of the dye.25 KSF01 -134374PC10. A method as claimed in any preceding claim, further comprising, after introducing the dye, introducing a solution containing sodium chloride to the dermis so as to promote fixation of the dye in the dermis.

11. A method as claimed in any preceding claim, further comprising, prior to introducing the dye, preparing the dye by dissolving a dry dye in a solvent.

12. A method as claimed in any preceding claim, wherein the dye is introduced to the dermis by a mechanical process of penetrating the skin.

13. A method as claimed in any preceding claim, further comprising: at least partially removing an area of epidermis to expose an area of the dermis; and applying the dye to the exposed area of the dermis.

14. A method as claimed in claim 13, wherein the area of epidermis is removed by laser ablation.

15. A method as claimed in any preceding claim, wherein the dye is caused to fix in the dermis by binding of the dye to proteins.

16. A method as claimed in any preceding claim, wherein the dye is caused to fix in the dermis by the dye agglomerating.

17. A method as claimed in any preceding claim, wherein the dye is soluble in dermal exudate.

18. A method as claimed in any preceding claim, further comprising applying a mixture comprising a steroid, an antibiotic, and an antimycotic.

19. A method as claimed in any preceding claim, further comprising sonicating the area of skin to which dye has been applied.26 KSF01 -134374PC20. A tattoo ink comprising a reactive dye, wherein the reactive dye is capable of binding to tissue in the dermis of skin.21 .A tattoo ink as claimed in claim 20, wherein the reactive dye is capable of binding to collagen, elastin, fibronectin, laminin, proteoglycans, glycosaminoglycans, or a combination of two or more thereof22. A tattoo ink as claimed in claim 20 or 21 , wherein the reactive dye comprises Reactive Black 5 and Reactive Orange 16.

23. A method of tattooing skin comprising: introducing a reactive dye to a dermis of the skin; applying a solution comprising an alkali and sodium chloride to the dermis; and washing the solution from the skin; wherein the reactive dye binds to a microenvironment in the dermis.

24. A method of tattooing as claimed in claim 23, further comprising, prior to washing the solution from the skin, observing a waiting period of 5 to 15 minutes.

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