WO2007115291A2

WO2007115291A2 - Tissue markings with discrete absorption particles

Info

Publication number: WO2007115291A2
Application number: PCT/US2007/065928
Authority: WO
Inventors: Ljiljana Kundakovic
Original assignee: Freedom-2, Inc.
Priority date: 2006-04-04
Filing date: 2007-04-04
Publication date: 2007-10-11
Also published as: WO2007115291A3

Abstract

The invention provides a tissue marking ink that includes at least one discrete absorption particle.

Description

TITLE

TISSUE MARKINGS WITH DISCRETE ABSORPTION PARTICLES

BACKGROUND OF THE INVENTION

[0001] The present invention relates to tissue markings with discrete absorption particles. Also, it relates to methods for preparing and removing such tissue markings.

[0002] Tissue markings, e.g., tattoos, have been used in almost every culture throughout history. They have been found on a five thousand year old human mummy, and decorated figurines suggest their use at least fifteen thousand years ago. Tattoos have been used for many purposes including identity, beauty, artistic and spiritual expression, medicine, and magic.

[0003] In the United States, official statistics are not kept on tattooing, but the practice has apparently been growing in popularity for the past few decades. The majority of tattoos are apparently obtained by people under forty years of age, including a significant proportion of teenagers. An estimated 2 million people are tattooed every year. A recent Harris Poll reported that 34 million Americans (16% of the population) have a tattoo. A recent national survey shows that the overall prevalence of tattoos among all U.S. adults is now 24%, peaking at 40% in the 25- 30 year cohort. Approximately one-third had considered getting the tattoo removed, but none had opted to do so (probably because of perceived high cost, hassle, lack of efficacy, or side effects). This means that approximately 8% of the entire population has thought about getting a tattoo removed and decided against it for some reason.

[0004] In the United States today, tattoo uses include not only the familiar artistic tattoo, but also permanent makeup (for example, permanent eyebrows, eyeliner, lip liner, and lip color); corrective or reconstructive pigmentation (for example, re- pigmentation of scar tissue or areola reconstruction on mastectomy patients); medical markings (for example, marking gastrointestinal surgery sites for future monitoring or marking locations for radiation treatment); and identification markings on animals (for example, pedigree "tags" on purebred pets).

[0005] The tissue marking procedure traditionally consists of piercing the skin with needles or similar instruments to introduce ink that typically includes inert and insoluble pigment particles having a wide distribution of sizes, which are suspended in a liquid carrier. Examples of machines typically used to apply a tattoo include an electromagnetic coil tattooing machine (such as that disclosed in U.S. Patent No. 4,159,659 to Nightingale); a rotary permanent cosmetics application machine (such as that disclosed in U.S. Patent No. 5,472,449 to Chou); or any manual tattooing device (such as the sterile single-use device marketed by Softap Inc., San Leandro, CA).

[0006] During the healing process, after tissue marking pigment has been applied, pigment particles can be affected in a variety of ways, many of which are detrimental to the appearance of the tissue marking. In particular, some small particles may readily diffuse and make the tissue marking blur. Other small particles may be taken up by the macrophages and phagocytes. Large particles may be removed from the implantation area directly through, for example, transdermal elimination, or sequestered in the extracellular matrix. Also, particles may be moved away from the implantation area to the lymphatic system.

[0007] Ultimately, what one sees as the tissue marking are the remaining particles of pigment located where they typically are engulfed by phagocytic skin cells (such as macrophages, phagocytes and fibroblasts) or are sequestered in the extracellular matrix. Transdermal elimination, diffusion and removal via the immune system tend to reduce the intensity and clarity of the tissue marking.

[0008] To create a permanent tattoo one must implant pigments that are not dissolved or digested by living tissue. Primitive pigments probably consisted of graphite and other carbon substances. Modern pigments also include inorganic metal salts and brightly colored organometallic complexes.

[0009] Tattoo ink ingredients have never yet been regulated or fully disclosed to the public. Ink composition and pigment sources remain trade secrets. Allergic reactions to these unknown and/or undisclosed substances, rare but in some cases severe, have been reported at the time of tattooing, well after the time of tattooing, and after exposure to sunlight or laser treatments.

[0010] The long-term health effects, including potential toxicity and/or carcinogenicity of tattoo pigments, have not been studied and are not known. Unfortunately, these pigments, chosen for their permanence, are believed to remain in the body for life, whether within the skin or in the lymph nodes. Even if the visible tattoo is "removed" or lightened from the marked area, for example, by laser treatment, the pigment may not be eliminated from the body.

[0011] A widely recognized problem with tattoos is that they cannot be easily removed. The above-mentioned recent Harris Pole estimates that about half of all Americans with tattoos would at some point wish they could remove them. Dissatisfaction can stem from undesired social disapproval; from the appearance of a tattoo that may be poorly executed, out-of- style, or inaccurate (commonly in the case of name-containing vow tattoos); or from changes in the wearer's self- perception or lifestyle. There is evidence that tattoo removal was an issue as early as the first century A.D. in Rome, when soldiers returned from barbaric regions with tattoos that were unacceptable to society.

[0012] Tattoo "removal" methods include overtattooing without ink, dermabrasion, and surgical excision, all of which may leave an unacceptable appearance and/or scarring. There currently does not publicly available efficacious, cost-effective method for tattoo removal [0013] One current tattoo removal method is treatment with Q-switched laser pulses, perhaps in the nanosecond domain, which method has been shown to remove and lighten tattoos with a low risk of scarring and skin pigment changes. A series of typically six to ten Q-switched laser treatments, which are expensive and usually cause discomfort, are administered at approximately one-month intervals. However, this method is generally inefficient and ineffective - only about 50% of tattoos are successfully removed in less than ten Q-switched laser treatments, and various wavelength lasers may be necessary to remove all of the types of ink used in a tattoo.

[0014] Q-switched laser treatment of tattoos is based on the concept of selective photo thermolysis, in which a selectively-absorbed pulse of light is used to locally heat and destroy dermal cells containing the tattoo ink. After a laser treatment, some ink particles are naturally eliminated, for example, by the lymphatic system. Other ink particles, however, are re-phagocytosed by dermal cells, or otherwise remain in the skin as a residual tattoo, requiring re-treatment.

[0015] Typically, fixed wavelength lasers are used for Q-switched laser treatment of tattoos. Because some tattoo inks absorb only weakly or, like yellow, orange, sky blue and some green colored inks, do not absorb laser energy at the fixed wavelengths used for the laser treatment, several treatments employing different lasers may be necessary in order to remove multi-colored tattoos. These multiple treatments may also have to be combined with other types of treatment, such as dermabrasion, in order to remove the resistant inks. On average, multi-colored tattoos require the use of at least three different fixed wavelength lasers. Since the number of fixed wavelength lasers available for treatment of tattoos is limited and since such lasers may still be ineffective to remove a variety of different colored tissue markings, a new approach to tattoo removal is clearly needed.

[0016] The majority of inks on the market today are, at best, of questionable safety. Tattoo inks are composed of ingredients that often include toxic heavy metals and organic dyes hazardous to human health. The above-mentioned laser "removal" of these materials may, in fact, create carcinogenic compounds, which are stored in the lymph nodes for the lifetime of the patient. [0017] Recently, there has been proposed a new approach to provide both safe and more easily removable permanent tissue markings. Specifically, this approach involves encapsulating, complexing or aggregating tissue marking pigments or dyes in or as a vehicle prior for application to tissue, as described, for example, in U.S. Patent No. 6,013,122 to Klitzman et al. and U.S. Patent No. 6,814,760 to Anderson et al. The encapsulated pigment or dye may be selected such that it safely interacts with living tissue, and allows the use of pigments or dyes that otherwise could not be used in a traditional tissue marking. Such encapsulated, complexed or aggregated pigments or dyes may include, in addition to those conventionally used in the art, pigments or dyes that are dispersible or biodegradable in living tissue, or pigments that could cause an adverse reaction if placed directly into a living organism.

[0018] Such improved tissue markings can be designed in advance to be susceptible to a specific type and amount of energy, which, when applied, ruptures or breaks apart the vehicle associated with the pigment or dye. Such a design may, but not necessarily, include the use of a wavelength-specific discrete absorption particles to assist in the rupturing or breaking apart of the vehicle.

[0019] Because the design characteristics of the vehicle, pigment or dye, and/or the discrete absorption particles can govern the specific type and amount of energy necessary to eliminate the tissue marking, a single fixed wavelength laser can be used to remove inks of different colors, permitting accurate and efficient removal. If the pigment or dye carried by the vehicle is readily dissolvable, digestible or dispersible in living tissue, rupturing or breaking apart the vehicle results in the substantial or entire removal of an otherwise permanent tissue marking.

[0020] Permanent, removable tissue markings prepared in accordance with U.S. Patent Nos. 6,013,122 and 6,814,760 mitigate some of the above-mentioned drawbacks of current removal methods, laser-based or otherwise. However, such tissue markings require the pigment or dye to be encapsulated, aggregated or otherwise complexed in order to be effectively and efficiently removed. A method is needed for making more easily removable conventional, non-encapsulated, non- aggregated or non-complexed tissue markings, which may be less time-consuming and expensive to produce.

SUMMARY OF THE INVENTION

[0021] To overcome the above-described deficiencies of the prior art, the present invention provides an improved tissue marking ink, and methods for implanting and removing tissue markings made from this ink.

[0022] In accordance with the present invention, the markings can be applied to tissue (whether human or animal) such as, but not limited to, skin, iris, sclera, muscles, tendons, organs, brain, small and large intestines, uterus, tumors and other cellular masses, legions, tissue beneath fingernails, tissue beneath toenails, tissue inside the mouth including the tongue, or tissue lining internal body passages. Most likely, the tissue is skin.

[0023] According to the present invention, the improved tissue marking ink includes at least one discrete absorption particle, which is designed to absorb a specific, exogenous energy. When implanted into the tissue, the tissue marking ink, and in particular the pigment or dye and the discrete absorption particles, are phagocytosed or otherwise internalized by cells and/or their cytoplasm or organelles, forming a tissue marking. The tissue marking will be permanent until it is desired to remove it. To remove the tissue marking, exogenous energy is applied to the tissue. Once a sufficient amount of such exogenous energy has been applied, the discrete absorption particles can act like an "egg tooth" that rupture, or otherwise disrupt, the cells and/or their cytoplasm or organelles, releasing the pigment or dye into the extracellular space. The pigment or dye may then be removed from the original tissue marking location by natural biological processes, such as via the lymphatic system or through expulsion through the skin.

[0024] The discrete absorption particles are, preferably, in particulate form, and are, preferably, of a size and shape and have physical and chemical properties that allow them to be used with tissue marking inks without affecting the desired color of the ink or the tissue. The discrete absorption particles are added to the tissue marking ink at a desired concentration, so that they can be efficiently and effectively used to release the pigment or dye from the tissue cells when exogenous energy is applied to the tissue marking.

[0025] Depending on the desired method of removal, the discrete absorption particles may be designed to absorb electromagnetic radiation or heat. Examples of electromagnetic radiation include visible radiation, near- IR, IR, near UV, high intensity visible radiation and visible radiation. Alternatively, or in addition, the discrete absorption particles may be designed from materials that exhibit magnetic properties, such as iron oxides. The discrete absorption particles preferably have a size, shape and physical and chemical properties to be eliminated by natural biological processes as discussed above.

[0026] The present invention provides an improved tissue marking ink, which includes discrete absorption particles, which are mixed, or otherwise combined, with the pigment or dye particles prior to implantation in tissue. The discrete absorption particles are preferably suspended or dispersed in the tissue marking ink, which, in addition to the pigment or dye, includes a carrier. The discrete absorption particles can be used with conventional tissue marking inks, as well as with a variety of modified tissue marking inks as discussed herein.

[0027] When and if it is desired to remove the tissue marking or tattoo prepared using discrete absorption particles, the cells may be ruptured, or otherwise made to release pigment or dye particles. This may be accomplished by applying a specific form of exogenous energy, which targets the discrete absorption particles, thereby generating heat and rupturing, or otherwise disrupting, the cell to release the pigment or dye and the discrete absorption particles. If, for example, a discrete absorption particle that absorbs visible, high intensity visible, near- IR, IR, near UV, or UV radiation, exposing it to such radiation should lead to heat generation that is sufficient to rupture, or otherwise disrupt, the cell to release the pigment or dye internalized by the cell. To target the magnetic discrete absorption particles phagocytosed by tissue cells, an external magnetic field generator producing focused alternating magnetic energy can be applied to force the magnetic poles of the discrete absorption particles to reverse with each alternation, resulting in a small discharge of heat. [0028] The cumulative heat generated in the cells that phagocytosed the pigment or dye along with the discrete absorption particles should be sufficient to rupture, or otherwise disrupt, the cell and release the pigment or dye into the extracellular space. The heating effect is very local and does not harm nearby tissue cells.

[0029] In one aspect of the present invention, a permanent, but removable on demand, tissue marking ink can be made by combining the tissue marking pigment or dye with at least one discrete absorption particle in a carrier. This carrier is preferably, but not limited to, a liquid, such as water or ethanol. The carrier may also be, for example, in a form of a gel.

[0030] In another aspect of the present invention, at least one discrete absorption particle is included in the sphere, capsule or aggregate as disclosed in Provisional Application Nos. 60/709,619 and 60/710,614. The discrete absorption particle(s), once the sphere, capsule or aggregate is phagocytosed by the tissue cell, can be used to efficiently rupture, or otherwise disrupt, the cell more to release the pigment or dye particles from the cell into the extracellular space.

[0031] As used herein, a "tissue marking" is any mark created by the introduction of the pigment into tissue, typically living tissue, with the intention of permanent or long-term endurance. Markings may be invisible or any visible color, and should be detectable, for example, by the naked eye or by using a detection device.

[0032] As used herein, a tissue marking "pigment" or "dye" is broadly defined as a substance, which, upon implantation into tissue, can provide a tissue marking having diverse colors or appearance properties. The pigment or dye can be comprised of graphite and other carbon substances, as well as any other conventional pigment or dye material. The pigment can include inorganic metal salts and brightly colored organometallic complexes, etc. The pigment or dye may be that disclosed in, for example, U.S. Patent Nos. 6,013,122; 6,800,122 (chromophores); 6,814,760; and 6,881,249; Provisional U.S. Patent Application Nos. 60/709,619 and 60/710,614; or U.S. Patent Application Publication No. 2005/0172852 Al.

[0033] As used herein, a "tattoo" is a type of tissue marking wherein the tissue is usually, but not limited to, skin. [0034] As used herein, "dispersed" or "suspended" discrete absorption particles are present in the tissue marking ink in a form, which is different from the encapsulation or complex of the discrete absorption particles in U.S. Patent Nos. 6,800,122; 6,814,760; and 6,881,249. For example, the discrete absorption particle(s) in accordance with the present invention may be dispersed or suspended in a carrier as individual particles, a group of particles, as a sphere, capsule or aggregate, as disclosed in Provisional U.S. Patent Application Nos. 60/709,619 and 60/710,614, or in addition to the pigment or dye particles in the sphere, capsule or aggregate disclosed in these provisional patent applications.

[0035] As used herein, "diameter" refers to a diameter of a spherical body and the largest linear dimension of a non- spherical body.

[0036] As used herein, "nanoparticle" with a diameter is a particle or a structure in the nanometer (nm) range, typically from about 1 to about 100 nm in diameter. Examples of a nanometer- sized structure in accordance with the present invention include, but are not limited to, nanoshells and nanometer- sized encapsulations (nanocapsules) of nanometer-sized materials.

[0037] As used herein, a material is "invisible" when essentially no color can be detected apart from the normal coloration of the its surroundings by the naked eye under normal lighting conditions, for example, diffuse sunlight or standard artificial lighting.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. [0039] The invention has numerous advantages over known tissue markings. For example, the present invention provides a tissue marking that is more easily removable than those conventionally known or used in the art.

[0040] Other features and advantages of the invention are apparent from the following detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1 is a schematic representation of a tissue marking ink containing conventional tissue marking pigment or dye particles and discrete absorption particles dispersed in a carrier.

[0042] FIG. 2 is a schematic representation of a tissue marking ink containing discrete absorption particles in a sphere, capsule or aggregate with pigment or dye particles.

[0043] FIG. 3 is a schematic representation of a tissue marking containing discrete absorption particles, both as individual particles and a sphere, capsule or aggregate of discrete absorption particles, dispersed in a carrier with spheres, capsules or aggregates of pigment or dye particles.

DETAILED DESCRIPTION

[0044] The concept of a removable on demand tissue markings, such as a tattoo, according to the present invention includes a pigment or dye and at least one discrete absorption particle, which may be combined in a carrier to form tissue marking ink. Once the pigment or dye and the discrete absorption particle(s) are phagocytosed by a tissue cell, the discrete absorption particle(s) may be used to lyse, or otherwise disrupt, the cell to release the pigment or dye from the cell. Since the energy necessary for the disruption would depend on the specific properties of the discrete absorption particles, such as absorption and/or polarization, and not on the type or color of the pigment, it is possible to use one type of exogenous energy, such as one frequency of laser light or an alternating magnetic field, to remove tissue markings of different colors if the same discrete absorption particles are used with the pigments or dyes of these colors.

[0045] As shown in FIG. 1, for use with conventional tattoo pigments or dyes 2, the discrete absorption particle(s) 1 having a desired size, structure and chemical, physical and energy absorption properties, are mixed with pigment or dye particles 2 at a suitable or desired concentration in a carrier 3 to form tissue marking ink. The ink is then implanted in tissue, for example, by conventional tattoo application methods.

[0046] The size of the discrete absorption particles can be adjusted to be in the same size range as that of conventional pigment or dye particles used to mark tissue. While the pigment or dye particle size distribution in conventional tissue marking or tattoo inks varies greatly, the pigment or dye ink particles are generally smaller than about 10 microns. The most commonly used tissue marking inks, such as India Ink or a suspension of amorphous carbon, contain particles smaller than 1 micron and, in some cases, smaller than 0.2 micron. Therefore, the size of the discrete absorption particles may be adjusted to be generally smaller than about 10 microns, and preferably smaller than about 1 micron. For some pigment or dye particles, it may be necessary to introduce the discrete absorption particles with the particle size smaller than about 0.2 micron.

[0047] For incorporation with certain types of pigments or dyes, it is desirable to prepare discrete absorption particles on a nanometer scale. It is preferable that these particles are smaller than about 100 nm or that they have a specific nanostructure, so that the particle properties, such as absorption of specific energy, are better controlled. Also, it may be desirable to introduce nanosized discrete absorption particles to facilitate particle elimination from the tissue by natural biological processes after the pigment or dye-containing cells are ruptured, or otherwise disrupted, during a removal treatment.

[0048] To control particle properties and their elimination from the tissue, the discrete absorption particles may, preferably, be nanoparticles that are smaller than about 100 nm, and more preferably smaller than about 20 nm. The discrete absorption particles of such a size can be introduced into the tissue marking ink either as individual particles or as aggregates, capsules or spheres to prevent their rapid elimination during introduction into tissue and to facilitate phagocytosis, as discussed in Provisional Application Nos. 60/709,619 and 60/710,614.

[0049] If the discrete absorption particles absorb in the visible, near IR and/or IR region, they are preferably introduced at a concentration that does not affect the desired color of the tissue marking. Generally, this concentration should be not more than about 10% (v/v), and is preferably about 2% (v/v) or less, to ensure efficient removal.

[0050] When and if it is desired to remove the tissue marking or tattoo that includes at least one discrete absorption particle that is susceptible to laser light, light from only a single fixed wavelength laser need be applied. Since each phagocytic tissue cell that engulfed the tissue marking ink contains a certain number of discrete absorption particles, along with what is typically, but not necessarily, a larger number of pigment or dye particles, a laser is selected to specifically target the absorption of the discrete absorption particles, not the absorption of the pigment or dye particles as it is currently done in conventional tattoo removal. The discrete absorption particles absorb the light and generate heat to rupture, or otherwise disrupt, the cells and release the pigment or dye particles, as well as the discrete absorption particles, into the extracellular space, where the released particles are partly or completely eliminated by the lymphatic system.

[0051] The released discrete absorption particles that were not removed by the lymphatic system may potentially be re-phagocytosed by other, undamaged tissue cells. To avoid such re-phagocytosis, the discrete absorption particles are preferably dispersible in tissue. Alternatively, or in addition, it may be desirable to use a near-infrared absorbing material to form discrete absorption particles that are visibly transparent, or nearly transparent, at the concentrations and sizes used, so that the perceived color of the tissue marking or of the tissue after the removal procedure is not affected even if the material is not dispersible. The discrete absorption particles are thus substantially or completely invisible when in tissue.

[0052] In another embodiment of the present invention, discrete absorption particles may be introduced into tissue marking inks disclosed in Provisional U.S. Patent Application Nos. 60/709,619 and 60/710,614, as shown in FIGs. 2 and 3. In this case, the discrete absorption particles particle(s) 1 may be included in (e.g., FIG. 2) or with (e.g., FIG. 3) the sphere, capsule or aggregate at a concentration, which would be sufficient to rupture, or otherwise disrupt, the cell after phagocytosis, releasing the pigment or dye particles into the extracellular space. In addition, as shown in FIG. 3, the discrete absorption particles may themselves form a sphere, capsule or aggregate 5 akin to that formed by pigment or dye particles. As with conventional tissue marking inks, the discrete absorption particles can be designed to absorb near- IR, IR, UV, near UV and/or visible radiation. The discrete absorption particles exhibiting magnetic properties may also be used.

[0053] When and if it is desired to remove the tissue marking, even the one formed using conventional tissue marking pigment or dye, the cells may be disrupted by using only one fixed wavelength laser selected based on the absorption properties of the discrete absorption particles and/or by alternating an external magnetic field, releasing the pigment or dye particles, as well as the discrete absorption particles, into the extracellular space.

[0054] Non-limiting examples of materials that can be used to form discrete absorption particles include metals, such as gold, silver and platinum. Particles formed from such metals, particularly those on a nanometer scale and their derived nanostructures, exhibit specific absorption features associated with the plasmon resonance of conduction electrons confined in the nanoparticle. The absorption frequency, such as absorption maxima or color, and other absorption features, such as the shape and the width of the absorption peaks, of these nanoparticles and other metallic nanostructures, such as nanoshells, are determined by the type of material, size and shape of nanoparticles and nanostructures, size distribution and the environment that surrounds these particles (Flutter and Fendler, Advanced Materials, 2004, 16(19), p. 1685). For example, gold nanoparticles have a strong plasmon resonance absorption at 520 nm. Silver nanoparticles have a plasmon resonance absorption at 390 nm. The absorption cross-section of metallic nanoparticles and nanostructures, such as nanoshells, is a million times larger than that of typical molecular pigments or dyes, resulting in an efficient light to heat conversion (Brongersma, Nature Materials, 2002, 2, p. 296), which is sufficient to efficiently rupture tissue, particularly dermal tissue, cells that contain pigment or dye particles.

[0055] Metallic and composite metallic discrete absorption nanoparticles and nano structures that exhibit plasmon resonance can be prepared as nanospheres, nanoshells, rods, rings, disks and cubes. Several different preparation techniques, such as colloidal metallic preparation methods, microemulsions, surfactant stabilized micelles, reverse micelles, surfactant vesicles, as well as laser ablation methods, vacuum deposition and electron beam lithography may be used to form these structures (Advanced Materials, 2004, 16(19), p. 1685 and its references).

[0056] Particularly useful materials are metallic nanoshells (Halas et al. Journal of Optical Society of America B, 1999, 16(10), pl824; Halas et al. Science, 2003, 302, p. 419). Nanoshells are optically tunable nanoparticles composed of a dielectric (for example, silica) core coated with an ultra-thin metallic layer. Gold nanoshells, for example, those developed by Nanospectra Biosciences, Inc., Houston, TX, have physical properties, particularly a strong plasmon resonance, similar to a gold colloid. The maximum absorption (plasmon resonance) of nanoshells can be varied over a wide range by varying the ratio of inner to outer diameter of the shell, yielding plasmon resonance tunable from about 600 nm to greater than about 1000 nm (Halas, Nano Letters, 2003, 3(10), p. 1411). For example, gold silica nanoshells with a 100 nm core and a 5 nm shell thickness show a maximum absorption at about 1000 nm, while those having the shell thickness of 20 nm have a maximum absorption at about 700 nm.

[0057] Single and multiple layer nanoshells ranging in size from a few nanometers to a few hundred nanometers may be prepared and used as discrete absorption particles in tissue markings. Such nanoshells may be, for example, about 800 nm in diameter (Halas et al. Science, 2003, 302, p. 419).

[0058] The metallic nanoparticles and nanostructures can be stabilized by covalently bound thiol and disulfide functionalized monolayers. If desired, the surface of metallic nanoparticles can be functionalized to induce a specifically desired immune response or to target specific surface receptors of antigen- presenting tissue cells to induce surface receptor mediated endocytosis, as discussed in Provisional Application No. 60/587,864 and International Application No. PCT/US2005/024865, which are incorporated herein by reference.

[0059 j Other materials that may be used as discrete absorption particles with IR and near IR absorption include those disclosed in U.S. Patents No. 6,800,122 to Anderson. In particular, these materials include, but are not limited to, graphite and other forms of carbon and metal oxides, such as iron oxide (red/brown or black), glasses (BG-7 and KG-3 filter glass made by Schott, Inc.), cyanine dyes (including indocyanine green and other colors), phthalocyanine dyes (green-blue), and pyrylium dyes (multiple colors).

[0060] Visible-colored materials that can be targeted by visible radiation include, but are limited to, dispersible colorants approved by Food and Drug Administration (FDA) for use in foods, pharmaceutical preparations, medical devices, or cosmetics, such as the non-soluble salts and lakes of FD&C and D&C dyes, as disclosed in U.S. Patents No. 6,800,122. Additional FDA approved dyes and colored drugs that can be used to form discrete absorption particles are listed in the Code of Federal Regulations (CFR) for Food and Drugs (see Title 21 of CFR chapter 1, parts 1-99).

[0061] As discussed above, materials with magnetic properties can also be used to form discrete absorption particles. Specifically, black iron oxide particles, such as superparamagnetic iron oxide (SPIO, particle size greater than about 50 nm), ultrasmall superparamagnetic iron oxide (USPIO, particle size smaller than about 50 nm) and monodisperse iron oxide nanoparticles (MION, particle size smaller than about 20 nm) may be used. Superparamagnetic iron oxide consists of nonstoichiometric microcrystalline magnetite cores, which are coated with dextrans (in ferumoxides) or siloxanes (in ferumoxsils). The most common form of iron oxide that may be used is magnetite, which is a mixture of Fe₂O₃ and FeO. Fe₃O₄ may be used in lieu of FeO. These materials are available as tissue specific MRI contrast agents (Feridex®, Endorem™, GastroMARK®, Lumirem®, Sinerem®, Resovist®). USPIO particles are also available as MRI contrast agents (Sinerem®, Combidex®, Clariscan™ ). [0062] If desired, magnetic iron oxide nanoparticles can be functionalized to induce a specifically desired immune response or to target specific surface receptors of antigen presenting cells in the tissue to induce surface receptor mediated endocytosis, as disclosed in Provisional Application No. 60/587,864 and International Application No. PCT/US2005/024865.

[0063] The iron oxide nanoparticles described above are particularly advantageous, because they may be successfully targeted using different methods. For example, the iron-oxide nanoparticles may be targeted by a conventional laser, such as Q-switched Nd: YAG (1064 nm), frequency doubled Nd: YAG (532 nm), Alexandrite (755 nm) or Ruby (694 nm) laser. Alternatively, iron oxide nanoparticles phagocytosed by tissue cells can be targeted by an external magnetic field generator, such as the one being developed by Triton Biosciences for treatment of cancer, which generator produces focused, alternating magnetic energy.

[0064] A magnetic field may be applied to the tissue to force the magnetic poles of each nanoparticle or aggregate of nanoparticles to reverse with each alternation, which results in a small discharge of heat. Because the magnetic field alternates thousands of times per second, the cumulative heat generation in the pigment or dye-containing cells is sufficient to rupture the cell and release the pigment or dye into the extracellular space. The nanoparticles or their aggregates are so small that the heating effect is very local and does not harm nearby tissue cells.

[0065] Tissue markings in accordance with the present invention may be implanted using conventional tattooing methods. However, any method that would deliver the ink so that a tissue marking is formed can be used.

[0066] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims

What is claimed is:

1. A tissue marking ink comprising: pigment or dye; a carrier for the pigment or dye; and at least one discrete absorption particle, which is dispersed in the carrier and which is capable of disrupting or rupturing a tissue cell and/or its components, upon being internalized thereby along with the pigment or dye, to release the pigment or dye from the tissue cell when exposed to exogenous energy.

2. The ink according to claim 1, wherein the discrete absorption particle is metallic or a composite of metallic nanoparticles that exhibit plasmon resonance.

3. The ink according to claim 1, wherein the discrete absorption particle is in a form of a nanosphere, nanoshell, rod, ring, disk or cube.

4. The ink according to claim 3, wherein the discrete absorption particle is in the form of the nanoshell.

5. The ink according to claim 4, wherein the nanoshell is an optically tunable nanoparticle.

6. The ink according to claim 4, wherein the nanoshell is composed of a dielectric core coated with a metallic layer.

7. The ink according to claim 6, wherein the dielectric core comprises silica.

8. The ink according to claim 7, wherein the metallic layer comprises gold.

9. The ink according to claim 6, wherein the dielectric core comprises silica and the metallic layer comprises gold.

10. The ink according to claim 9, wherein the metallic layer is from about 5 nm to about 20 nm thick.

11. The ink according to claim 2, wherein a maximum absorption of the discrete absorption particle is from about 390 nm to about 1000 nm.

12. The ink according to claim 1, wherein the discrete absorption particle is selected from the group consisting of graphite, a carbon or metal oxide, glass, a cyanine dye, a phthalocyanine dye, and pyrylium dye.

13. The ink according to claim 12, wherein the discrete absorption particle is back, brown or red iron oxide or indocyanine green.

14. The ink according to claim 1, wherein a concentration of the discrete absorption particles in the carrier is about 10% (v/v) or less.

15. The ink according to claim 1, wherein a concentration of the discrete absorption particles in the carrier is about 2% (v/v) or less.

16. The ink according to claim 1, wherein a diameter of the discrete absorption particle is from about 1 nm to about 10 microns.

17. The ink according to claim 16, wherein the diameter is from about 1 to about 200 nm.

18. The ink according to claim 16, wherein the diameter is from about 1 to about 100 nm.

19. The ink according to claim 16, wherein the diameter is from about 1 to about 20 nm.

20. The ink according to claim 1, wherein the discrete absorption particle is dispersible in tissue.

21. The ink according to claim 1, wherein the discrete absorption particle absorbs visible, near- IR, IR, near UV or UV radiation.

22. The ink according to claim 1, wherein the discrete absorption particle has magnetic properties.

23. The ink according to claim 22, wherein the discrete absorption particle is selected from the group consisting of superparamagnetic iron oxide and monodisperse iron oxide particles.

24. The ink according to claim 23, wherein the superparamagnetic iron oxide particle is at least about 50 nm in diameter.

25. The ink according to claim 23, wherein the superparamagnetic iron oxide particle is smaller than about 50 nm in diameter.

26. The ink according to claim 1, wherein the discrete absorption particles are monodisperse iron oxide particles.

27. The ink according to claim 26, wherein the monodisperse iron oxide particles are smaller than about 20 nm in diameter.

28. The ink according to claim 1, wherein the discrete absorption particle is substantially invisible when in tissue.

29. The ink according to claim 1, wherein the discrete absorption particle is transparent.

30. The ink according to claim 1, wherein the discrete absorption particles form a sphere, capsule or aggregate.

31. The ink according to claim 30, wherein the discrete absorption particles are bound by a biodegradable or bioabsorbable material to form the sphere, capsule or aggregate.

32. The ink according to claim 31, wherein the biodegradable or bioabsorbable material degrades to release the discrete absorption particles into a tissue cell.

33. The ink according to claim 31, wherein the biodegradable or bioabsorbable material is at least one material selected from the group consisting of zinc alginate poly(lactic acid), poly(vinyl alcohol), a polyanhydride, a polylactide, poly(glycolic acid), poly-L-lactic acid, poly D-lactic acid, poly-lactic-glycolic acid, and a triblock copolymer of poly caprolactone-polyethylene glycol-poly caprolactone.

34. The ink according to claim 30, wherein the discrete absorption particles are polarized.

35. The ink according to claim 30, wherein the discrete absorption particles are modified to form the sphere, capsule or aggregate.

36. The ink according to claim 35, wherein the modification is physical or chemical.

37. The ink according to claim 30, wherein the sphere, capsule or aggregate comprises a compound on its outer surface, which said compound increases protein adsorption to the outer surface.

38. The ink according to claim 30, wherein the sphere, capsule or aggregate comprises on its outer surface molecules that interact with specific receptors on a surface of the tissue cell to heighten aggressiveness of an immune system reaction to the sphere, capsule or aggregate.

39. The ink according to claim 38, wherein the molecules are selected from the group consisting of integrins, endotoxin, lipopolysaccharide and any combination thereof.

40. The ink according to claim 37, wherein the compound is covalently or ionically bonded to the outer surface.

41. The ink according to claim 37, wherein the compound is a lipopolysaccharide.

42. The ink according to claim 41, wherein the lipopolysaccharide is obtained from E. CoIi or Porphyromonas gingivalis.

43. The ink according to claim 37, wherein the compound is a cytokine.

44. The ink according to claim 43, wherein the cytokine is TNF- α.

45. The ink according to claim 37, wherein the compound is a leukotriene.

46. The ink according to claim 45, wherein the leukotriene is LTB4.

47. The ink according to claim 30, wherein the sphere, capsule or aggregate according has an outer surface, which comprises at least one projection, at least one recess, at least one indentation and/or at least one pore.

48. The ink according to claim 47, wherein the outer surface of the sphere, capsule or aggregate has projections, which are spaced apart by and/or have a length of about 0.1 microns to about 100 microns.

49. The ink according to claim 47, wherein the outer surface of the sphere, capsule or aggregate has said at least one pore with a diameter or minor axis of about 0.1 micron to about 100 microns.

50. The ink according to claim 47, wherein the outer surface of the sphere, capsule or aggregate has indentations, which are spaced apart by and/or have a length of about 0.1 microns to about 100 microns.

51. The ink according to claim 30, wherein the sphere, capsule or aggregate has an edge with a curvature radius of about 0.1 to about 10 microns.

52. The ink according to claim 30, wherein the discrete absorption particles aggregate to form the sphere, capsule or aggregate without modification.

53. A tissue marking ink, comprising a sphere, capsule or aggregate comprising at least one pigment or dye particle and at least one discrete absorption particle, wherein the pigment or dye particle is from about 1 to about 100 nm in diameter, wherein the sphere, capsule or aggregate is capable of being internalized by a tissue cell, and releasing the pigment or dye particle into the tissue cell upon or after phagocytosis, and wherein the at least one discrete absorption particle is capable of disrupting or rupturing the tissue cell and/or its components to release the pigment or dye from the tissue cell when exposed to exogenous energy.

54. The ink according to claim 53, wherein the discrete absorption particle is selected from the group consisting of graphite, a carbon or metal oxide, glass, a cyanine dye, a phthalocyanine dyes, and pyrylium dye.

55. The ink according to claim 54, wherein the discrete absorption particle is back, brown or red iron oxide or indocyanine green.

56. The ink according to claim 53, wherein a concentration of the discrete absorption particles in the carrier is about 10% (v/v) or less.

57. The ink according to claim 53, wherein a concentration of the discrete absorption particles in the carrier is about 2% (v/v) or less.

58. The ink according to claim 53, wherein a diameter of the discrete absorption particle is from about 1 nm to about 10 microns.

59. The ink according to claim 58, wherein the diameter is from about 1 to about 200 nm.

60. The ink according to claim 58, wherein the diameter is from about 1 to about 100 nm.

61. The ink according to claim 58, wherein the diameter is from about 1 to about 20 nm.

62. The ink according to claim 53, wherein the discrete absorption particle is dispersible in tissue.

63. The ink according to claim 53, wherein the discrete absorption particle absorbs visible, near- IR, IR, near UV or UV radiation.

64. The ink according to claim 53, wherein the discrete absorption particle has magnetic properties.

65. The ink according to claim 64, wherein the discrete absorption particle is selected from the group consisting of superparamagnetic iron oxide and monodisperse iron oxide particles.

66. The ink according to claim 65, wherein the superparamagnetic iron oxide particle is at least about 50 nm in diameter.

67. The ink according to claim 65, wherein the superparamagnetic iron oxide particle is smaller than about 50 nm in diameter.

68. The ink according to claim 53, wherein the discrete absorption particles is monodisperse iron oxide particles.

69. The ink according to claim 68, wherein the monodisperse iron oxide particles are smaller than about 20 nm in diameter.

70. The ink according to claim 53, wherein the discrete absorption particle is substantially invisible when in tissue.

71. The ink according to claim 53, wherein the discrete absorption particle is transparent.

72. The ink according to claim 53, wherein the at least one discrete absorption particle and/or the at least one pigment or dye particles in the sphere, capsule or aggregate are bound by a biodegradable or bioabsorbable material.

73. The ink according to claim 72, wherein the biodegradable or bioabsorbable material degrades to release the discrete absorption particles and the one more pigment or dye particles into a tissue cell.

74. The ink according to claim 72, wherein the biodegradable or bioabsorbable material is at least one material selected from the group consisting of zinc alginate poly(lactic acid), poly(vinyl alcohol), a polyanhydride, a polylactide, poly(glycolic acid), poly-L-lactic acid, poly D-lactic acid, poly-lactic-glycolic acid, and a triblock copolymer of poly caprolactone-polyethylene glycol-poly caprolactone.

75. The ink according to claim 53, wherein the at least one discrete absorption particle and/or the at least one pigment or dye particle are polarized.

76. The ink according to claim 53, wherein the at least one discrete absorption particle and/or the at least one pigment or dye particle are modified to form the sphere, capsule or aggregate.

77. The ink according to claim 76, wherein the modification is physical or chemical.

78. The ink according to claim 53, wherein the sphere, capsule or aggregate comprises a compound on its outer surface, which said compound increases protein adsorption to the outer surface.

79. The ink according to claim 78, wherein the sphere, capsule or aggregate comprises on its outer surface molecules that interact with specific receptors on a surface of the tissue cell to heighten aggressiveness of an immune system reaction to the sphere, capsule or aggregate.

80. The ink according to claim 79, wherein the molecules are selected from the group consisting of integrins, endotoxin, lipopolysaccharide and any combination thereof.

81. The ink according to claim 78, wherein the compound is covalently or ionically bonded to the outer surface.

82. The ink according to claim 78, wherein the compound is a lipopolysaccharide.

83. The ink according to claim 82, wherein the lipopolysaccharide is obtained from E. CoIi or Porphyromonas gingivalis.

84. The ink according to claim 78, wherein the compound is a cytokine.

85. The ink according to claim 84, wherein the cytokine is TNF- α.

86. The ink according to claim 78, wherein the compound is a leukotriene.

87. The ink according to claim 86, wherein the leukotriene is LTB4.

88 The ink according to claim 53, wherein the sphere, capsule or aggregate according has an outer surface, which comprises at least one projection, at least one recess, at least one indentation and/or at least one pore.

89. The ink according to claim 88, wherein the outer surface of the sphere, capsule or aggregate has projections, which are spaced apart by and/or have a length of about 0.1 microns to about 100 microns.

90. The ink according to claim 88, wherein the outer surface of the sphere, capsule or aggregate has said at least one pore with a diameter or minor axis of about 0.1 micron to about 100 microns.

91. The ink according to claim 88, wherein the outer surface of the sphere, capsule or aggregate has indentations, which are spaced apart by and/or have a length of about 0.1 microns to about 100 microns.

92. The ink according to claim 72, wherein the sphere, capsule or aggregate having an edge with a curvature radius of about 0.1 to about 10 microns.

93. A method for preparing tissue marking ink comprising the steps of: providing pigment or dye particles, at least one discrete absorption particle and a carrier; combining the pigment or dye particles, the at least one discrete absorption particle and the carrier such that the at least one discrete absorption particle is dispersed in the carrier, wherein the at least one discrete absorption particle is capable of disrupting or rupturing a tissue cell and/or its components, upon being internalized thereby along with the pigment or dye, to release the pigment or dye from the tissue cell when exposed to an exogenous energy.

94. The method according to claim 93, wherein the discrete absorption particle is selected from the group consisting of graphite, a carbon or metal oxide, glass, a cyanine dye, a phthalocyanine dyes, and pyrylium dye.

95. The ink according to claim 94, wherein the discrete absorption particle is back, brown or red iron oxide or indocyanine green.

96. The method according to claim 93, wherein a concentration of the discrete absorption particles in the carrier is about 10% (v/v) or less.

97. The method according to claim 93, wherein a concentration of the discrete absorption particles in the carrier is about 2% (v/v) or less.

98. The method according to claim 93, wherein a diameter of the discrete absorption particle is from about 1 nm to about 10 microns.

99. The method according to claim 98, wherein the diameter is from about 1 to about 200 nm.

100. The method according to claim 98, wherein the diameter is from about 1 to about 100 nm.

101. The method according to claim 98, wherein the diameter is from about 1 to about 20 nm.

102. The method according to claim 93, wherein the discrete absorption particle is dispersible in tissue.

103. The method according to claim 93, wherein the discrete absorption particle absorbs visible, near- IR, IR, near UV or UV radiation.

104. The method according to claim 93, wherein the discrete absorption particle has magnetic properties.

105. The method according to claim 104, wherein the discrete absorption particle is selected from the group consisting of superparamagnetic iron oxide and monodisperse iron oxide particles.

106. The method according to claim 105, wherein the superparamagnetic iron oxide particle is at least about 50 nm in diameter.

107. The method according to claim 105, wherein the superparamagnetic iron oxide particle is smaller than about 50 nm in diameter.

108. The method according to claim 93, wherein the discrete absorption particles is monodisperse iron oxide particles.

109. The method according to claim 108, wherein the monodisperse iron oxide particles are smaller than about 20 nm in diameter.

110. The method according to claim 93, wherein the discrete absorption particle is substantially invisible when in tissue.

111. The method according to claim 93, wherein the discrete absorption particle is transparent.

112. The method according to claim 93, wherein the discrete absorption particles form a sphere, capsule or aggregate.

113. The method according to claim 112, wherein the discrete absorption particles are bound by a biodegradable or bioabsorbable material to form the sphere, capsule or aggregate.

114. The method according to claim 113, wherein the biodegradable or bioabsorbable material degrades to release the discrete absorption particles into a tissue cell.

115. The method according to claim 113, wherein the biodegradable or bioabsorbable material is at least one material selected from the group consisting of zinc alginate poly(lactic acid), poly(vinyl alcohol), a polyanhydride, a polylactide, poly(glycolic acid), poly-L-lactic acid, poly D-lactic acid, poly-lactic - glycolic acid, and a triblock copolymer of poly caprolactone-polyethylene glycol- poly caprolactone.

116. The method according to claim 112, wherein the discrete absorption particles are polarized.

117. The method according to claim 112, wherein the discrete absorption particles are modified to form the sphere, capsule or aggregate.

118. The method according to claim 117, wherein the modification is physical or chemical.

119. The method according to claim 112, wherein the sphere, capsule or aggregate comprises a compound on its outer surface, which said compound increases protein adsorption to the outer surface.

120. The method according to claim 112, wherein the sphere, capsule or aggregate comprises on its outer surface molecules that interact with specific receptors on a surface of the tissue cell to heighten aggressiveness of an immune system reaction to the sphere, capsule or aggregate.

121. The method according to claim 21, wherein the molecules are selected from the group consisting of integrins, endotoxin, lipopolysaccharide and any combination thereof.

122. The method according to claim 119, wherein the compound is covalently or ionically bonded to the outer surface.

123. The method according to claim 119, wherein the compound is a lipopolysaccharide.

124. The method according to claim 123, wherein the lipopolysaccharide is obtained from E. CoIi or Porphyromonas gingivalis.

125. The method according to claim 119, wherein the compound is a cytokine.

126. The method according to claim 125, wherein the cytokine is TNF- α.

127. The method according to claim 119, wherein the compound is a leukotriene.

128. The method according to claim 127, wherein the leukotriene is LTB4.

129. The method according to claim 112, wherein the sphere, capsule or aggregate according has an outer surface, which comprises at least one projection, at least one recess, at least one indentation and/or at least one pore.

130. The method according to claim 129, wherein the outer surface has projections, which are spaced apart by and/or have a length of about 0.1 microns to about 100 microns.

131. The method according to claim 129, wherein the outer surface has said at least one pore with a diameter or minor axis of about 0.1 micron to about 100 microns.

132. The method according to claim 129, wherein the outer surface has indentations, which are spaced apart by and/or have a length of about 0.1 microns to about 100 microns.

133. The method according to claim 112, wherein the sphere, capsule or aggregate has an edge with a curvature radius of about 0.1 to about 10 microns.

134. The method according to claim 112, wherein the discrete absorption particles aggregate to form the sphere, capsule or aggregate without modification.

135. A method for preparing tissue marking ink comprising the steps of: providing pigment or dye particles and at least one discrete absorption particle; combining the pigment or dye particles and the at least one discrete absorption particle into a sphere, capsule or aggregate, which is capable of being phagocytosed by a tissue cell, and is capable of releasing the pigment or dye particle into the tissue cell upon or after phagocytosis, wherein the at least one discrete absorption particle is capable of disrupting or rupturing the tissue cell and/or its components to release the pigment or dye from the tissue cell when exposed to exogenous energy.

136. The method according to claim 135, wherein the discrete absorption particle is selected from the group consisting of graphite, a carbon or metal oxide, glass, a cyanine dye, a phthalocyanine dyes, and pyrylium dye.

137. The method according to claim 136, wherein the discrete absorption particle is back, brown or red iron oxide or indocyanine green.

138. The method according to claim 135, wherein a concentration of the discrete absorption particles in the carrier is about 10% (v/v) or less.

139. The method according to claim 135, wherein a concentration of the discrete absorption particles in the carrier is about 2% (v/v) or less.

140. The method according to claim 135, wherein a diameter of the discrete absorption particle is from about 1 nm to about 10 microns.

141. The method according to claim 140, wherein the diameter is from about 1 to about 200 nm.

142. The method according to claim 140, wherein the diameter is from about 1 to about 100 nm.

143. The method according to claim 140, wherein the diameter is from about 1 to about 20 nm.

144. The method according to claim 135, wherein the discrete absorption particle is dispersible in tissue.

145. The method according to claim 135, wherein the discrete absorption particle absorbs visible, near- IR, IR, near UV or UV radiation.

146. The method according to claim 135, wherein the discrete absorption particle has magnetic properties.

147. The method according to claim 146, wherein the discrete absorption particle is selected from the group consisting of superparamagnetic iron oxide and monodisperse iron oxide particles.

148. The method according to claim 147, wherein the superparamagnetic iron oxide particle is at least about 50 nm in diameter.

149. The method according to claim 147, wherein the superparamagnetic iron oxide particle is smaller than about 50 nm in diameter.

150. The method according to claim 135, wherein the discrete absorption particles is monodisperse iron oxide particles.

151. The method according to claim 150, wherein the monodisperse iron oxide particles are smaller than about 20 nm in diameter.

152. The ink according to claim 135, wherein the discrete absorption particle is substantially invisible when in tissue.

153. The method according to claim 135, wherein the discrete absorption particle is transparent.

154. The method according to claim 135, wherein the at least one discrete absorption particles and/or one or more of the pigment or dye particles in the sphere, capsule or aggregate are bound by a biodegradable or bioabsorbable material.

155. The method according to claim 154, wherein the biodegradable or bioabsorbable material degrades to release the at least one discrete absorption particle and one or more of the pigment or dye particles into a tissue cell.

156. The method according to claim 154, wherein the biodegradable or bioabsorbable material is at least one material selected from the group consisting of zinc alginate poly(lactic acid), poly(vinyl alcohol), a polyanhydride, a polylactide, poly(glycolic acid), poly-L-lactic acid, poly D-lactic acid, poly-lactic - glycolic acid, and a triblock copolymer of poly caprolactone-polyethylene glycol- poly caprolactone.

157. The method according to claim 135, wherein the at least one discrete absorption particle and/or one or more of the pigment or dye particles are polarized.

158. The method according to claim 135, wherein the at least one discrete absorption particle and/or one or more of the pigment or dye particles are modified to form the sphere, capsule or aggregate.

159. The method according to claim 158, wherein the modification is physical or chemical.

160. The method according to claim 135, wherein the sphere, capsule or aggregate comprises a compound on its outer surface, which said compound increases protein adsorption to the outer surface.

161. The method according to claim 160, wherein the sphere, capsule or aggregate comprises on its outer surface molecules that interact with specific receptors on a surface of the tissue cell to heighten aggressiveness of an immune system reaction to the sphere, capsule or aggregate.

162. The method according to claim 161, wherein the molecules are selected from the group consisting of integrins, endotoxin, lipopolysaccharide and any combination thereof.

163. The method according to claim 160, wherein the compound is covalently or ionically bonded to the outer surface.

164. The method according to claim 160, wherein the compound is a lipopolysaccharide.

165. The method according to claim 164, wherein the lipopolysaccharide is obtained from E. CoIi or Porphyromonas gingivalis.

166. The method according to claim 160, wherein the compound is a cytokine.

167. The method according to claim 166, wherein the cytokine is TNF- α.

168. The method according to claim 160, wherein the compound is a leukotriene.

169. The method according to claim 168, wherein the leukotriene is LTB4.

170 The method according to claim 135, wherein the sphere, capsule or aggregate according has an outer surface, which comprises at least one projection, at least one recess, at least one indentation and/or at least one pore.

171. The method according to claim 170, wherein the outer surface of the sphere, capsule or aggregate has projections, which are spaced apart by and/or have a length of about 0.1 microns to about 100 microns.

172. The method according to claim 170, wherein the outer surface of the sphere, capsule or aggregate has said at least one pore with a diameter or minor axis of about 0.1 micron to about 100 microns.

173. The method according to claim 170, wherein the outer surface of the sphere, capsule or aggregate has indentations, which are spaced apart by and/or have a length of about 0.1 microns to about 100 microns.

174. The method according to claim 154, wherein the sphere, capsule or aggregate has an edge with a curvature radius of about 0.1 to about 10 microns.

175. A method of preparing a tissue marking comprising: preparing a tissue marking ink by a method as in claim 93; and implanting the tissue marking ink in tissue.

176. A method of preparing a tissue marking comprising: preparing a tissue marking ink by a method as in claim 135; and implanting the tissue marking ink in tissue.

177. A method of removing a tissue marking prepared according to claim 175, comprising applying the exogenous energy to the tissue to release the pigment or dye from the tissue cell.

178. The method according to claim 177, wherein the exogenous energy is electromagnetic radiation or heat.

179. The method according to claim 177, wherein the exogenous energy is near-infrared, infrared, near-ultra violet, ultraviolet, high intensity visible radiation or visible radiation.

180. The method according to claim 177, wherein the exogenous energy is magnetic energy.

181. The method according to claim 180, comprising applying focused alternating magnetic energy using a magnetic field generator.

182. A method of removing a tissue marking prepared according to claim 176, comprising applying the exogenous energy to the tissue to release the pigment or dye from the tissue cell.

183. The method according to claim 182, wherein the exogenous energy is electromagnetic radiation or heat.

184. The method according to claim 182, wherein the exogenous energy is near- infrared, infrared, near-ultra violet, ultraviolet, visible or high intensity visible radiation.

185. The method according to claim 182, wherein the exogenous energy is magnetic energy.

186. The method according to claim 185, comprising applying focused alternating magnetic energy using a magnetic field generator.