Biocompatible composition for labeling target lesions and method for producing same

Abstract

The present invention relates to a target lesion labeling composition, which is in the form of microparticles containing a biocompatible polymer and a dye for staining biological tissue, and is administered directly to a target lesion during surgery at a target site to confirm the size and location of the target lesion in real time, and a method for manufacturing the same. The target lesion labeling composition of the present invention remains in the target lesion for up to several months after injection into the target lesion, and allows the location, size, etc. of the target lesion to be detected in real time during surgery, thereby improving the success rate of surgery on the target lesion as well as preventing excessive loss of normal tissue, and therefore can be widely used as an effective surgical marker.

Description

[Technical field]

[0001] The present invention relates to a biocompatible target lesion labeling composition and a method for producing the same. More specifically, the present invention relates to a target lesion labeling composition comprising a biocompatible polymer containing a dye for staining biological tissue and a biological protein, a method for visualizing information labeled at the site of a lesion using the target lesion labeling composition, and a target lesion surgical labeling agent comprising the target lesion labeling composition mixed with a biopolymer to improve stability and imaging ability in the body. [Background technology]

[0002] In the case of anti-cancer treatment, methods using various anti-cancer drugs have been developed, but to date, the most commonly used method is to remove cancer cells through surgical methods. However, when using surgical methods, it is essential to have the technology to minimize the surgical area during surgery for the health and welfare of patients after surgery. In particular, in the case of breast cancer, if the number of lesions to be removed during surgery in domestic women with small breasts is reduced compared to other countries, the goal of breast-conserving treatment cannot be achieved. The surgical area is determined by the lesion and the excess area around the lesion. If the surgeon does not know the exact location and area of ​​the lesion, he or she will have to leave a large excess area around the lesion. This is because if the surgical area is reduced carelessly, there is a risk that the tumor will remain in the resection margin. However, in actual clinical surgery, there are not many ways for the surgeon to accurately check the lesion in real time during surgery. Despite the development of very precise diagnostic methods, such diagnostic methods cannot be used during surgery, and during actual surgery, the surgeon mainly relies on the surgeon's touch and vision, in which case it is rare for the lesion to be clearly distinguished. Especially when the lesion is small, it is even less distinguishable. In order to achieve the objectives of minimally invasive surgery and surgery, techniques are required to inform the surgeon of the lesion intraoperatively.

[0003] In conventional tumor removal surgery, especially breast cancer surgery, the location of the patient's micro-lesion is confirmed before surgery using ultrasound, mammography, or magnetic resonance imaging, and the location of the confirmed lesion is displayed, and then the tissue at the indicated area is removed. Methods for displaying the location of the confirmed lesion include marking the surface of the skin, using a wire, and injecting a black pigment such as charcoal. However, the method of marking the skin with a pen to display the location of the lesion is the easiest to use but has the disadvantage of being the least accurate, because the shape of the breast changes significantly between the time of diagnosis and the operating room due to the characteristics of very soft breast tissue, and marking only on the surface of the skin is not sufficient for lesions deep in the breast. Next, the method of piercing the breast lesion with a wire has the disadvantage that the wire, which should be inserted perpendicular to the skin surface, must be inserted at an angle due to the possibility of affecting the ultrasound probe, and the position of the wire may move due to the movement of the breast, resulting in lower accuracy than expected, and the inserted wire gets in the way of the surgery, requiring further surgery to remove the wire insertion site. Finally, the method of injecting a dye such as charcoal has the advantage that the injected dye binds to the lesion and can accurately mark the location of the lesion, but has the disadvantage that if the lesion is deep in the breast, the black dye cannot be seen from the outside and the dye may contaminate the surgical site. The above-mentioned disadvantages can also be applied to surgical procedures to remove cancerous tissue other than breast cancer.

[0004] As such, with the technology developed to date, it is difficult to precisely determine the surgical area for surgically removing cancerous lesions. Therefore, when surgically removing cancerous lesions, the area of ​​resection must be expanded more than necessary, and post-surgery tests must be performed to confirm that the lesion was removed successfully.

[0005] Under these circumstances, the present inventors have confirmed that a marker (KR-10-2012-0153793) using a complex in which macroaggregated albumin is bound to a dye for staining biological tissue as a marker when surgically removing cancer lesions can effectively adsorb to cancer lesions to accurately label the location of the lesion, and that the dye can be tracked in real time during surgery to accurately confirm the extent of the lesion to be removed.

[0006] With general dyes and the like, the dye or fluorescent dye cannot remain at the injection site for a long time, but spreads around within a few hours, and is eventually excreted from the body and loses its ability to mark lesions. In the case of the conventional invention (KR-10-2012-0153793), although it remains at the injection site for a longer time than conventional general (fluorescent) dyes, it is difficult to do so beyond a few days. This is especially true when the injection site is a tissue that is constantly moving, such as the stomach and intestines, has a high blood flow, or is injected into a place where the tissue is relatively loose rather than hard. In other words, conventional markers have the problem that the dye cannot remain at the injection site for a long time, but as it spreads around within a few hours, it loses its ability to mark lesions in a short period of time.

[0007] In cases where cancer patients have large lesions and surgery cannot be performed immediately, there has been an increase in the number of preemptive anti-cancer treatments, where anti-cancer treatment is performed first, followed by surgery to remove the cancer. In this case, the lesion is displayed in advance before anti-cancer treatment, which takes several months, and the area is removed after anti-cancer treatment. In this case, if the tumor is well treated by anti-cancer treatment and reduced to a tiny size, it is often difficult to find the cancer area during surgery. The cancer area is identified by placing a metal clip during anti-cancer treatment, but in actual surgery, it is not easy to find the metal clip, and it is not uncommon for it to move during the long anti-cancer treatment period, so improvements are needed. It would be very useful if fluorescent dyes could remain at the injected area for several months, and the fluorescence and color could be confirmed even after several months.

[0008] In order for a (fluorescent) dye to remain at the injection site for a long time, it must be in the form of particles of an appropriate size (diameter: several to several hundred micrometers) so as not to spread to the surrounding area, and it must not be degraded or denatured by biological reactions and lose its color or fluorescence so that the dye is stable for a long time.

[0009] Microspheres, which are typical micrometer-sized particles, have been developed from a variety of materials. Various porous microspheres that can carry (fluorescent) dyes have been developed, but these are primarily used to expel the carried substance, in this case the (fluorescent) dye, at a slow, controlled rate. The main purpose is not to not expel drugs, but to expel them appropriately, which is completely different from the required purpose. In other words, the (fluorescent) dye must be in a form that is not expelled from the microsphere into the body, and the microspheres must be made of materials that can be used in the body.

[0010] Under these circumstances, the easiest method that can be devised is to mix a (fluorescent) dye with a biocompatible polymer to create microspheres, but the polymerization process, which is generally used to synthesize monomers, generates heat and radicals, which denature the (fluorescent) dye and cause it to lose its color and fluorescence, making it impossible to achieve the intended purpose.

[0011] The present invention devisees a method for completely embedding and encapsulating (fluorescent) dyes by dissolving a biocompatible polymer in a solvent and utilizing the double emulsion method. Since the dyes encapsulated in properly manufactured biocompatible polymer microspheres do not flow out, the above-mentioned drawbacks can be eliminated.

[0012] We also devised a method to stabilize the encapsulated (fluorescent) dye within the polymer microspheres for a long period of time, and confirmed that the dye was able to retain color and fluorescence for a longer period of time when bound to a protein rather than by itself.

[0013] In addition, when the surface is modified with hyaluronic acid, after injection, fibroblasts are activated in the surrounding tissue, collagen tissue is produced, and the microspheres can adhere well. However, hyaluronic acid does not dissolve in organic solvents, and it is difficult to coat polymer microspheres with hyaluronic acid using conventional methods. We devised a method to dissolve hyaluronic acid and collagen in a water-soluble solvent during the microsphere manufacturing process, and developed a manufacturing method that allows coating with hyaluronic acid and collagen during the process of making biocompatible polymer microspheres. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] KR10-2012-0153793 Summary of the Invention [Problem to be solved by the invention]

[0015] The present invention has been devised to solve the above-mentioned problems, and an object of the present invention is to provide a composition for labeling internal lesions, which comprises a dye or fluorescent dye and a biocompatible polymeric synthetic substance including a biological protein.

[0016] Specific objects of the present invention are (a) to provide a method for labeling internal lesions with color or fluorescent dyes that remain at the injection site for several months or more, (b) to provide methods for producing such compounds for labeling internal lesions and compositions for injection, and (c) to provide methods for using the same to provide information on the location of lesions. [Means for solving the problem]

[0017] As a means for achieving the above-mentioned object, the present invention provides a composition for marking a target lesion, which is in the form of microspheres containing a biocompatible polymer and a dye for staining a biological tissue, and is administered directly to a target lesion during a marking site operation to confirm the size and location of the target lesion in real time.

[0018] Here, the microspheres may further include a surface modifier coated on the surface.

[0019] Here, the biocompatible polymer may be polymethyl methacrylate (PMMA), polyglycolic acid (PGA), polylactic acid (PLA), polylactic acid-polyglycolic acid copolymer (PLGA), poly-ε-caprolactone (PCL), lactic acid-ε-caprolactone copolymer (PLCL), polydioxanone (PDO), polytrimethylene carbonate (PTMC), polyamino acid (polyamino methyl methacrylate (PMMA)), polyglycolic acid (PGA), polylactic acid-polyglycolic acid copolymer (PLGA), poly-ε-caprolactone (PC ...lactic acid-ε-caprolactone copolymer (PLCL), polydioxanone (PDO), polytrimethylene carbonate (PTMC), polyamino acid (polyamino methyl methacrylate (PDO)), polyglycolic acid (polyglycolic acid) copolymer (PLGA), polyglycolic acid-polyglycolic acid copolymer (PLGA), polyglycolic acid-polyglycolic acid copolymer (PLGA), polyglycolic acid-polyglycolic acid copolymer (PLGA), polyglycolic acid-polyglycolic acid copolymer (PLGA), polyglycolic acid-polyglycolic acid copolymer (PLCL), polyglycolic acid-polyglycolic acid copolymer (PLCL), polyglycolic acid-polyglycolic acid copolymer (PLCL), polyglycolic acid-polyglycolic acid copolymer (PLCL), polyglycolic acid-polyglycolic acid copolymer (PLCL), polyglycolic acid-polyglycolic acid copolymer (PLCL), polyglycolic acid-polyglycolic acid copolymer (PLCL), The polyiminocarbonate may be any one of a polyanhydride, a polyorthoester, a polyphosphazene, a polyiminocarbonate, a polyphosphoester, a polyhydroxyvalate, a copolymer thereof, and a mixture thereof.

[0020] Here, the dye for staining biological tissue may be a dye that can be visually confirmed or a fluorescent dye.

[0021] Here, the visually visible dye may be any one selected from the group consisting of neutral red, Nile blue, Bismarck brown, lithium carmine, trypan blue, Janus green, methyl violet, auramine, malagaite green, sapranin, eosin, Congo red, erythrosine, nigrosine, Alcian blue hematoxylin, aniline blue, light green, and combinations thereof.

[0022] Here, the fluorescent dye may be a near-infrared fluorescent dye.

[0023] Here, the fluorescent dye may be indocyanine green (ICG).

[0024] Here, the fluorescent dye may be used alone or conjugated to a biological protein.

[0025] Here, the surface modifier may be any one of hyaluronic acid, collagen, and a mixture thereof.

[0026] Here, the microspheres may further contain a biological protein.

[0027] Here, the biological protein may be albumin.

[0028] Here, the microspheres may have a diameter of 10 to 100 μm.

[0029] Here, the composition may have an average density of 1 to 3 g / ml.

[0030] In addition, as a means for achieving the above-mentioned object, the present invention provides a method for producing a composition for labeling a target lesion, comprising the steps of: mixing an organic solvent containing a biocompatible polymer with distilled water containing a dye for staining biological tissue; and pouring the mixed solution into an aqueous solvent and stirring to form the microspheres.

[0031] Here, the method for preparing a composition for labeling a target lesion may further include adding a surface modifier to the water-soluble solvent.

[0032] Here, the method for preparing a composition for labeling a target lesion may further include a step of washing and filtering the stirred solution to select microspheres of a specific size.

[0033] Here, the distilled water may further contain a biological tissue staining dye-biological protein conjugate.

[0034] Here, the organic solvent may be any one selected from the group consisting of dichloromethane (DCM), o-xylene, m-xylene, p-xylene, and combinations thereof.

[0035] Here, the concentration of the dye for staining biological tissue may be 1 to 100 μM.

[0036] Here, the water-soluble solvent may be any one selected from the group consisting of polyvinylpyrrolidonem (PVP), polyacrylic acid (PAA), polyacryl amide (PAAm), polyhydroxyethyl methacrylate (PHEMA), polyvinyl alcohol (PVA), and combinations thereof.

[0037] Furthermore, as a means for achieving the above-mentioned object, the present invention provides a target lesion labeling composition in the form of microspheres containing a biocompatible polymer and a dye for staining biological tissue; and an injectable composition for target lesion labeling containing hyaluronic acid (HA) having an average molecular weight of 0.5 to 3.0 MDa.

[0038] Here, hyaluronic acid (HA) may be contained in an amount of 0.3 to 1% by weight relative to 100% by weight of the injectable composition.

[0039] Here, the hyaluronic acid (HA) may have a viscosity of 24 to 1500 cps at room temperature.

[0040] Here, the target lesion into which the injectable composition is injected can retain the label for 4 to 12 months.

[0041] The present invention also provides a method for providing information on the location of a target lesion, comprising the steps of: directly administering a target lesion labeling composition in the form of microspheres containing a biocompatible polymer and a dye for staining biological tissue to a target lesion; and recognizing a location where the color of the tissue to which the target lesion labeling composition is administered changes as the target lesion, and confirming the location in real time during surgery at the labeled site. Effect of the Invention

[0042] The composition for labeling a target lesion of the present invention remains in the target lesion for up to several months after injection, and the location, size, etc. of the labeled lesion can be detected in real time during surgery. This not only improves the success rate of surgical procedures for the target lesion, but also prevents excessive loss of normal tissue, and therefore can be widely used as an effective surgical marker. [Brief description of the drawings]

[0043] [Figure 1] FIG. 1 is a schematic diagram showing a method for producing a biocompatible target lesion labeling composition of the present invention. [Diagram 2] FIG. 1 is a schematic diagram specifically illustrating a method for producing a biocompatible target lesion labeling composition of the present invention. [Diagram 3] These are actual images of PMMA containing ICG and HSA, images taken with a fluorescent camera, and fluorescent microscope images of PMMA with and without ICG-HSA binding. [Figure 4] 1 is a graph showing density as a function of the concentration of microspheres of the present invention relative to distilled water. [Diagram 5] 1 is a graph showing changes in near-infrared fluorescence signal intensity of microspheres due to changes in ICG and albumin concentrations. [Figure 6] 1 is an image showing the degree of dispersion of microspheres depending on the hyaluronic acid concentration in the biocompatible target lesion labeling injectable composition of the present invention. [Figure 7] 1 is a graph showing a comparison of changes in near-infrared fluorescence signal intensity over time for ICG, ICG-HSA, and ICG-HSA-PMMA particles under in vitro conditions, measured with IVIS (Perkin Elmer, Hopkinton, Mass.). [Figure 8] These images compare the stability of ICG-HSA-PMMA particles over time when the particles are placed in HSA, distilled water, and agarose gel. [Figure 9] The results are a comparison of the change in near-infrared fluorescence signal intensity over time measured by IVIS when ICG-HSA-PMMA particles were injected into ICR mice under in vivo conditions together with hyaluronic acid or distilled water. [Figure 10] The changes in near-infrared fluorescence signal intensity over time when ICG-HSA-PMMA particles were injected into ICR mice under in vivo conditions together with hyaluronic acid or distilled water were photographed with a fluorescent camera and analyzed using the Image J program. The graph shows the results of photographing mice with a fluorescent camera. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] The present invention can be modified in various ways and can have various embodiments, and specific embodiments are illustrated in the drawings and will be described in detail in the detailed description. However, this is not intended to limit the present invention to the specific embodiments, and it should be understood that the present invention includes all modifications, equivalents, and alternatives within the spirit and technical scope of the present invention.

[0045] Throughout this specification, when a part is said to "comprise" certain elements, this means that it can further include other elements, rather than excluding other elements, unless specifically stated to the contrary.

[0046] As used herein, terms of degree such as "about," "substantially," and the like, when the tolerances of manufacturing and materials inherent in the referred meaning are given, are used to mean the numerical value or a approximation thereof, and are used to prevent unscrupulous infringers from taking unfair advantage of the disclosure in which precise or absolute numerical values ​​are referred to to aid in the understanding of the present application. In addition, throughout the present specification, "the step of" or "the step of" does not mean "the step for".

[0047] Since a person skilled in the art of the present invention can carry out various applications through the gist of the present invention, the scope of the present invention is not limited to the following examples. The scope of the present invention extends to the parts that are obvious to a person skilled in the art of the present invention to easily replace or modify using conventional techniques based on the matters described in the claims.

[0048] The present invention will now be described in more detail with reference to the accompanying drawings where necessary.

[0049] <Biocompatible composition for labeling lesions> As a means for achieving the above-mentioned object, the present invention provides a composition for marking a target lesion, which is in the form of microspheres containing a biocompatible polymer and a dye for staining a biological tissue, and is administered directly to a target lesion during a targeting site operation to confirm the size and location of the target lesion in real time.

[0050] The tissue to be labeled with the composition is preferably a solid tissue such as a solid cancer that can be penetrated and fixed by the composition, and more preferably prostate cancer, breast cancer, uterine cancer, skin cancer, cervical cancer, lung cancer, brain tumor, gastrointestinal tumor, liver cancer, soft tissue sarcoma, lymphoma, etc., but is not limited thereto as long as it is a cancer or other benign lesion that contains tissue that can be penetrated and fixed by the synthetic substance.

[0051] The term "biocompatible polymer" as used herein means a polymer that is non-toxic or has little toxicity in the body and can be used for medical purposes, such as poly(methyl methacrylate, PMMA). The biocompatible polymer of the present invention remains in the lesion for a long time after being injected into and labeled with a lesioned tissue in the body, and can be used to confirm the size and location of the lesioned tissue.

[0052] Here, the biocompatible polymer may be polymethyl methacrylate (PMMA), polyglycolic acid (PGA), polylactic acid (PLA), polylactic acid-polyglycolic acid copolymer (PLGA), poly-ε-caprolactone (PCL), lactic acid-ε-caprolactone copolymer (PLCL), polydioxanone (PО), polytrimethylene carbonate (PTMC), polyamino acid (polyamino α-amino β ... The biocompatible polymer may be any of polyanhydride, polyorthoester, polyphosphazene, polyiminocarbonate, polyphosphoester, polyhydroxyvalate, copolymers thereof, and mixtures thereof. In addition, the biocompatible polymer may have a transparent property and a high light transmittance property in order to embed a dye or fluorescent dye in the present invention.

[0053] The term "dye for staining biological tissue" as used herein means a substance that binds to biological tissue and labels the binding position, thereby enabling the labeled position to be confirmed visually or using a detection tool. Here, the dye for staining biological tissue may be a dye that can be confirmed visually or a fluorescent dye. For the purposes of the present invention, the dye for staining biological tissue may be used to label a lesion site by being contained in a biocompatible polymer in a form bound to a biological protein, and preferably may be a dye that can be confirmed visually, a fluorescent dye that generates fluorescence at the binding site and can be detected using equipment such as a fluorescent camera, etc., used alone or in combination, but is not particularly limited thereto.

[0054] The term "visibly visible dye" as used herein means a type of dye in which a labeling substance bound to biological tissue exhibits a color in the visible light region, allowing the labeled site to be visually identified. For the purposes of the present invention, the visually visible dye can be injected into a lesion site in the body to allow the lesion to be clearly visually identified in the surgical field, thereby improving the success rate of surgery. The visually visible dye may be neutral red, Nile blue, Bismarck brown, lithium carmine, trypan blue, Janus green, methyl violet, auramine, malagaite green, sapranin, eosin, Congo red, erythrosine, nigrosine, Alcian blue hematoxylin, aniline blue, light green, etc., which may be used alone or in combination, but is not limited thereto, as long as the target lesion tissue can be identified.

[0055] The term "fluorescent dye" as used herein means an organic compound that absorbs light of a certain wavelength to form an excited state, and then generates fluorescence that maximizes the penetration distance of light and minimizes error signals due to moisture. The near-infrared fluorescent dye may be an organic compound that generates fluorescence of a near-infrared wavelength of preferably 700 nm to 3000 nm, more preferably 750 nm to 900 nm. The near-infrared fluorescence generated from the near-infrared fluorescent dye may be captured in the form of an image or monitored in real time using equipment such as a fluorescent camera, a fluorescent sensing probe (PCT / KR2011 / 009271), or a surgical fluorescent camera. The near-infrared fluorescence of the present invention is absorbed less in vivo than other wavelength bands, and the near-infrared generated at a relatively deep site in the organism can be detected outside the organism. For the purposes of the present invention, the fluorescent dye in the near-infrared region can be injected into a lesion site in the body to accurately confirm the lesion using a fluorescent imaging device in the surgical field, thereby improving the success rate of the surgery. In particular, unlike the dyes that can be visually confirmed, the location of the lesion can be detected outside the body before making an incision to directly confirm the lesion, thereby enabling rapid and accurate surgery to be performed. As the near-infrared fluorescent dye, indocyanine green (ICG) or the like may be preferably used, and any near-infrared fluorescent dye that can be used on the human body may be included in the scope of the present invention.

[0056] The complex in which the near-infrared fluorescent dye is bound to a biocompatible polymer labels the target lesion and has the advantage of being more stable and accurate in the detected fluorescent signal than complexes in which the near-infrared fluorescent dye is bound to other substances, thereby increasing the rate at which minute lesions can be detected and improving the accuracy of lesion resection.

[0057] The term "indocyanine green (ICG)" in the present invention means a fluorescent imaging dye in the near infrared region that is widely used in the fields of biology and medicine, and has the property of being decomposed and disappearing within about one hour after being injected into the human body, or being excreted in feces and urine, making it advantageous for clinical application as a fluorescent dye that can be used in the human body. In fact, cases of using indocyanine green for application to the human body have been reported in several papers, and as an example, it has been reported that it was safely used clinically in 18 breast cancer patients (T. Kitai, et al., Breast Cancer, 12:211-215, 2005). Currently, it is already widely used clinically.

[0058] Fluorescent dyes such as indocyanine green are taken up by the liver from tissues, broken down, and excreted through the biliary system. In order to prevent dyes such as indocyanine green from migrating to the veins and lymphatic vessels of surrounding tissues, it is advantageous to bind them to relatively large particles of a certain size (10 to 100 μm). The composition may be produced in such a form that the fluorescent dye is completely encapsulated in the microspheres so that the microspheres of the composition remain at the injection site for several months while continuing to emit fluorescence. The water-soluble fluorescent dye encapsulated in the microspheres in this way is trapped in the walls of the hydrophobic polymeric microspheres, so that it is expected to remain at the injection site for a long time.

[0059] Even if the fluorescent dye is well enclosed in the microspheres and does not flow into the surroundings, the dye must not lose its properties due to quenching caused by continuous excitation light or decomposition caused by biological reactions. Quenching here means that when the fluorescent dye is exposed to excitation light, it emits fluorescence in the excited state and falls to a stable state, but is decomposed by the oxygen generated at this time, or forms dimers or tetramers and no longer emits fluorescence, losing its ability to emit fluorescence. If quenching occurs, the dye will not emit fluorescence even if it is at the injection site, and will not be able to function as a marker. Therefore, in order for the fluorescent dye to be retained for several months, it must be well bound, such as embedded in microspheres of an appropriate size so that the fluorescent dye will remain at the injection site, and quenching must be minimal.

[0060] Here, the fluorescent dye may be used alone or conjugated with a biological protein. That is, in the biocompatible target lesion labeling composition of the present invention, the microspheres may further include a biological protein. When the fluorescent dye is conjugated with the biological protein, it may exhibit stronger fluorescence and exhibit a longer duration than when the fluorescent dye is used alone. Here, the biological protein may be, for example, albumin, but is not limited thereto, and may be included in the scope of the present invention as long as it forms a conjugated bond with the fluorescent dye.

[0061] In addition, the target lesion labeling composition of the present invention may have a stronger fluorescence intensity as the biological protein is contained at a higher concentration. More specifically, the biological protein may be contained at a concentration of 0.5 to 20% w / v in the target lesion labeling composition. Specific details regarding the biological protein will be described in more detail below.

[0062] In the composition for labeling a target lesion of the present invention, the microspheres may further include a surface modifier coated on the surface thereof.

[0063] The surface modifier may be any one of hyaluronic acid, collagen, and a mixture thereof. The microspheres containing the biocompatible polymer and the dye for biological tissue staining of the present invention may cause side effects such as foreign body reaction caused by macrophages and foreign body giant cells when they remain in the living body for a long time for follow-up observation of lesions. Therefore, in order to reduce the occurrence of such side effects, hyaluronic acid or collagen, which is a biopolymer material widely present in the living body, may be used as the surface modifier.

[0064] Here, the microspheres may have a diameter of 10 to 100 μm. More preferably, the diameter may be 30 to 70 μm, and may have an average diameter of 40 μm. Our body tissues are finely composed of collagen fibers, reticular fibers, and elastic fibers with a thickness of tens to hundreds of nm, and the pore size of the capillary walls and lymphatic walls passing through the tissues is also tens of nm. In order to prevent the microspheres (ICG-albumin-PMMA microspheres) of the present invention from flowing out of the tissue, a micrometer size larger than the pore size of the fibers and capillary / lymphatic walls in the tissue is appropriate, and it has been confirmed through animal experiments that the above particle size is effective. To obtain the above microsphere particle size, the microspheres can be obtained through filtering.

[0065] Here, the composition may have an average density of 1 to 3 g / ml. The density of such a composition is higher than the density of distilled water at room temperature (0.99821 g / ml), and when it is diluted with distilled water to be used as an injection, a problem of precipitation may occur. In an injection, the occurrence of such precipitation may cause a problem of clogging of the injection needle during injection, or may make it difficult to uniformly introduce the composition of the present invention into the target lesion. Therefore, in order to suppress the occurrence of precipitation, the injection composition containing the target lesion labeling composition of the present invention may be further mixed with a biocompatible polymer such as hyaluronic acid. The average density of the target lesion labeling composition of the present invention and the specific details of the injection composition are described in more detail in the following paragraphs <Target Lesion Labeling Injection Composition> and {Examples and Evaluation}.

[0066] <Method of producing a biocompatible composition for labeling a target lesion> The present invention provides a method for producing a composition for labeling a target lesion, which is a means for achieving the above-mentioned object. More specifically, the method for producing a composition for labeling a target lesion of the present invention includes the steps of mixing an organic solvent containing a biocompatible polymer and distilled water containing a dye for staining a biological tissue; and adding the mixed solution to an aqueous solvent and stirring to form microspheres.

[0067] In addition, the method of the present invention may further include a step of adding a surface modifier to the water-soluble solvent, so that the surface of the formed microspheres can be coated with the surface modifier.

[0068] Here, the organic solvent may be any one selected from the group consisting of dichloromethane (DCM), o-xylene, m-xylene, p-xylene, and combinations thereof, but is not limited thereto.

[0069] Here, the water-soluble solvent may be any one selected from the group consisting of polyvinylpyrrolidone (PVP), polyacrylic acid (PAA), polyacryl amide (PAAm), polyhydroxyethyl methacrylate (PHEMA), polyvinyl alcohol (PVA), and combinations thereof, but is not limited thereto.

[0070] Here, the distilled water may further contain a biological tissue staining dye-biological protein conjugate.

[0071] In the method for producing a biocompatible target lesion labeling composition of the present invention, the biocompatible polymer, the dye for staining biological tissue, the surface modifier, and the biological protein are as described above.

[0072] Fig. 1 is a schematic diagram showing a method for producing a biocompatible target lesion labeling composition of the present invention. More specifically, the process for producing a biocompatible target lesion labeling composition is shown using polymethyl methacrylate (PMMA) as the biocompatible polymer, indocyanine green (ICG) as the biological tissue staining dye, hyaluronic acid as the surface modifier, and human serum albumin (HSA) as the biological protein.

[0073] Referring to FIG. 1, it can be seen that the biocompatible target lesion labeling composition produced by the above-mentioned manufacturing method appears as spheres with a diameter size of 10 to 100 μm, and can be visually confirmed to have a white powder form.

[0074] Here, the concentration of the dye for staining biological tissue may be 1 to 100 μM. More preferably, the concentration may be 3 to 30 μM. In the present invention, the concentration of the dye for staining biological tissue falls within the above range, so that the intensity of the fluorescence can be more strongly expressed. Specific details regarding the concentration of the dye for staining biological tissue will be described in more detail in the following {Examples and Evaluation}.

[0075] Here, the concentration of the water-soluble solvent may be 0.1 to 10% w / w, and the concentration of the surface modifier may be 0.01 to 0.3% w / v.

[0076] In the present invention, in order to coat the microspheres with hyaluronic acid or collagen, there is a method of adding hyaluronic acid or collagen to the raw material of polymer microspheres, but hyaluronic acid is not melted by heating, and there is a high possibility that hyaluronic acid will be denatured during the heat melting process. In addition, there is a method of dissolving the hyaluronic acid in a solvent to prepare a hyaluronic acid solution, which is then coated on the microspheres, but this is impossible because the hyaluronic acid does not dissolve well in general organic solvents. In the present invention, a process of preparing a solution in which hyaluronic acid dissolves well at the stage of making microspheres is developed, and a coating of hyaluronic acid is provided. The hyaluronic acid is known to have the effect of stimulating fibroblasts to produce collagen. The collagen thus produced can be expected to have the effect of the target lesion labeling composition injected into tissue being well fixed at the injection site and not flowing away even after several months.

[0077] Here, the method for producing a composition for labeling a target lesion may further include a step of washing and filtering the stirred solution to select microparticles of a specific size. As described above, it is desirable for dyes such as indocyanine green to bind to relatively large particles of a certain size (10 to 100 μm) so as not to move to veins or lymphatic vessels in surrounding tissues, so the method for producing a composition for labeling a target lesion of the present invention may further include a step of washing and filtering the stirred solution to select microparticles of a size of 10 to 100 μm.

[0078] <Injection composition for labeling target lesion> Furthermore, the present invention provides a means for achieving the above-mentioned object, which provides an injectable composition for labeling a target lesion, comprising a composition for labeling a target lesion in the form of microspheres containing a biocompatible polymer and a dye for staining biological tissue; and hyaluronic acid (HA) having an average molecular weight of 0.5 to 3.0 MDa.

[0079] In order to maximize the utility of the biocompatible polymer containing the biological tissue staining dye of the present invention as an in vivo marker, the physical properties of the target lesion labeling composition of the present invention can be enhanced using a biopolymer. That is, the target lesion labeling composition of the present invention is injected into a lesion site in a living body and serves to clearly identify the lesion site during surgery by displaying the lesion, but in order for such a composition to smoothly perform the above-mentioned purpose as a marker, it must be possible to prevent diffusion at the injected lesion site as much as possible and to continuously emit a high fluorescent signal. As one means for achieving this purpose, hyaluronic acid may be used. When the target lesion labeling composition of the present invention is mixed with a hyaluronic acid solution, the hyaluronic acid solution can improve the in vivo retention of the composition.

[0080] In addition, when the hyaluronic acid is further mixed into the injection composition for target lesion labeling of the present invention, it can delay or prevent the precipitation of microspheres, thereby preventing the needle of the syringe from being clogged by the precipitated microspheres, which can prevent the injection from failing. This will be described in more detail in the following [Examples and Evaluation].

[0081] Here, hyaluronic acid (HA) may be contained in an amount of 0.3 to 1% by weight based on 100% by weight of the injection composition. If the amount of hyaluronic acid is less than 0.3% by weight, the target lesion labeling composition may precipitate, and the precipitated composition may clog the needle of the syringe. On the other hand, if the amount of hyaluronic acid is more than 1% by weight, the injection composition may be excessively viscous, which may also cause inconvenience during injection. Therefore, the amount of hyaluronic acid is preferably 0.3 to 1% by weight based on 100% by weight of the injection composition of the present invention. The specific details regarding this will be described in more detail in the following {Examples and Evaluation}.

[0082] Here, the hyaluronic acid (HA) may have a viscosity of 24 to 1500 cps at room temperature. If the viscosity of the hyaluronic acid is less than 24 cps, the viscosity of the injectable composition of the present invention is too low, which may cause problems such as difficulty in controlling the injection amount during injection or the occurrence of precipitation. On the other hand, if the viscosity exceeds 1500 cps, the viscosity is too high and excessive force is required to be applied to the syringe, which may cause problems such as difficulty in injection. Therefore, the viscosity of the hyaluronic acid is preferably 24 to 1500 cps at room temperature.

[0083] Here, the target lesion into which the injectable composition is injected can retain the label for several months or more. More specifically, the labeling of the target lesion via the injectable composition is preferably retained for 4 to 12 months. After the composition is injected into the target lesion before the preceding anticancer treatment (anticancer treatment before surgery), the composition must continue to retain fluorescence even after the preceding anticancer treatment so that the target lesion can be monitored. The specific period of the preceding anticancer treatment varies depending on the type of cancer and the type of anticancer treatment, and can change according to the progress of the anticancer treatment, but can generally be performed for a period of about 4 to 8 months. Thus, the target lesion into which the injectable composition for labeling the target lesion is injected preferably retains the label (fluorescence) for 4 months or more, more preferably retains the label for 6 months or more, and even more preferably retains the label for 8 months or more. Specific details regarding the stability of the injectable composition in the target lesion will be described in more detail in the following {Examples and Evaluation}.

[0084] <Method of providing information on the location of the target lesion> The present invention also provides a method for providing information on the location of a target lesion using the above-mentioned target lesion labeling composition. More specifically, the present invention provides a method for providing information on the location of a target lesion, comprising the steps of: directly administering a target lesion labeling composition in the form of microspheres containing a biocompatible polymer and a dye for staining biological tissue to a target lesion or around the target lesion; and recognizing a location where the color of the tissue to which the target lesion labeling composition is administered changes as the target lesion, and confirming the location in real time during surgery at the target site.

[0085] When the composition for labeling a target lesion provided by the present invention is administered to a lesioned tissue in a living body, the location of the lesion can be labeled using color, near-infrared fluorescence, or a combination of these. By detecting the label, the location, size, etc. of the target lesion can be detected in real time during surgery, thereby improving the accuracy of surgical removal of the target lesion and preventing excessive loss of normal tissue.

[0086] Furthermore, since the target lesion labeling composition of the present invention contains a biocompatible polymer, it can remain in the body for a long time while showing a high fluorescent signal in the injected lesion compared to a complex in which a dye for staining biological tissue is bound to another substance, so that the surgical resection accuracy of the target lesion can be easily confirmed. For example, the composition of the present invention can be injected into the position of a microlesion before surgery, and the lesion site can be stably and accurately confirmed by visual inspection or a fluorescent camera, etc., even during surgery several days to several weeks later.

[0087] [Mode for carrying out the invention] The contents claimed in this specification will be described in more detail with reference to the accompanying drawings and examples. However, the drawings and examples presented in this specification can be modified in various ways by ordinary skilled in the art and can have various forms, and the description in the present invention should not be considered to limit the present invention to the specific disclosed form, but to include all equivalents or alternatives within the spirit and technical scope of the present invention. In addition, the accompanying drawings are presented to help ordinary skilled in the art understand the present invention more accurately, and may be exaggerated or reduced in size.

[0088] [Examples and Evaluation] <Example> Example 1 1) Preparation of a solution of indocyanine green (ICG) bound to human serum albumin (HSA) A 3.2mM solution is prepared by dissolving 25mg of indocyanine green (ICG) in 10ml of distilled water. 312.5μl of this 10ml solution is transferred to a 1.5ml Eppendorf tube and diluted with 687.5μl of distilled water to produce 1ml of 0.1mM solution containing 78μg of ICG. Meanwhile, 0.4ml of 20% human serum albumin (HSA) solution is prepared and diluted with 0.6ml of distilled water to produce an 8% solution with a volume of 1ml. 125μl each of the two solutions, 1ml of 0.1mM ICG and 1ml of 8% HSA, are transferred to one 1.5ml Eppendorf tube to produce 0.25ml of a solution combining 50μM ICG and 4% HSA.

[0089] 2) Preparation of polymethyl methacrylate (PMMA) solution The beads were prepared by completely dissolving 300 mg (15 pieces) of ACRYPET polymethyl methacrylate beads (PMMA beads, Lotte MRC) in 3 ml of dichloromethane (DCM) solution.

[0090] 3) Preparation of polyvinylpyrrolidone (PVP) suspension stabilizer solution 4 g of polyvinylpyrrolidone (PVP) was completely dissolved in 100 ml of distilled water to prepare a 4% suspension stabilizer solution.

[0091] 4) Preparation of polymethyl methacrylate (PMMA) microspheres conjugated with indocyanine green (ICG) and human serum albumin (HSA) 0.25ml of the ICG-HSA aqueous solution prepared in Preparation Example 1 and 3ml of the PMMA solution prepared in Preparation Example 2 are placed in a 15ml tube, and the aqueous solution layer and the organic solution layer are vigorously mixed with each other using a homogenizer at room temperature for 20 minutes at a speed of 7500 rpm. This mixture is placed in 40ml of suspension stabilizer solution that is being stirred at room temperature at 900 rpm. After stirring for 90 minutes to allow the organic solvent gas to evaporate, the mixture is divided into 2ml Eppendorf tubes and washed three times for 10 minutes at a speed of 2000 rpm using a centrifuge. The dispersed ICG-HSA-PMMA microspheres are all combined in one tube. This material is filtered through 70μm and 30μm size filters to obtain a product with a size of 30~60μm. After filtering, the powdered ICG-HSA-PMMA dried at room temperature is placed in a gamma ray irradiator and sterilized at a strength of 75Gy for 17 seconds.

[0092] Example 2 A composition for labeling a target lesion was prepared in the same manner as in Example 1, except that in the process of preparing the polyvinylpyrrolidone (PVP) suspension stabilizer solution in Example 1, 4 g of polyvinylpyrrolidone (PVP) was completely dissolved in 100 ml of distilled water to prepare a 4% polyvinylpyrrolidone (PVP) solution, and then 0.15 g of hyaluronic acid was added to prepare a suspension stabilizer solution (hereinafter referred to as "Example 2").

[0093] The process of the above embodiment is diagrammatically shown in Figure 2. Figure 2 is a schematic diagram specifically illustrating the method for producing a biocompatible target lesion labeling composition of the present invention. Referring to the above-mentioned examples and FIG. 2, a biocompatible target lesion labeling composition of the present invention was prepared.

[0094] <Evaluation> The target lesion labeling composition prepared in Example 2 was used to carry out various evaluations below.

[0095] 1. Physical property evaluation of ICG-HSA-PMMA 1-1. Physical property evaluation Figure 3 shows actual images of PMMA containing ICG and HAS, images taken with a fluorescent camera, and fluorescent microscope images of PMMA with and without ICG-HAS binding. More specifically, Figure 3a shows actual images of PMMA containing ICG and HSA, images taken with a fluorescent camera, Figure 3b shows an image of PMMA not bound to ICG-HSA, and Figure 3c shows an image of ICG-HSA-PMMA, both taken with a fluorescent microscope.

[0096] Referring to Figures 3a to 3c, it can be seen that the composition for labeling target lesions of the present invention exhibits fluorescence, and that the composition exhibits spherical microspheres because it contains a biocompatible polymer (PMMA).

[0097] 1-2. Density evaluation It is difficult to directly measure the density in an aqueous solution in the actual usage form, so the density of the microspheres was measured indirectly by referring to the change in density according to the concentration. The density was measured using the representative ICG 6.5μM 4% HSA microspheres prepared above, and the density was indirectly measured using microspheres with a concentration of about 20mg / ml to 200mg / ml (2 to 20% (w / v)) as follows.

[0098] More specifically, after filling a transparent container with 20 ml of distilled water at room temperature, the above-mentioned microspheres were added to measure the change in density of the aqueous solution containing the microspheres, and in particular, the change in density according to the concentration of the microspheres was measured. Incidentally, after the microspheres were added to the distilled water, they were not easily dissolved in the distilled water, and it was confirmed that they showed a white suspension when shaken and mixed, and if the measurement was not performed quickly, the particles began to sink to the bottom, and after a few minutes, all the particles had settled.

[0099] Table 1 shows the density of distilled water containing microspheres, and lists the density according to the concentration of the microspheres.

[0100] [Table 1]

[0101] A linear regression equation was obtained by linear regression analysis with reference to Table 1, and the density at 100% was measured by extrapolation.

[0102]

number

[0103] Fig. 4 is a graph showing the density according to the concentration of the microspheres of the present invention in distilled water. Referring to Fig. 4, the density of the microspheres was estimated to be 1.2574g / ml according to the above formula. In addition, when the distilled water in which the microspheres were dissolved was checked after 20 minutes, it was confirmed that a white suspension had sunk to the bottom of the container. As a result, it was determined that the density of the microspheres was 1.2574g / ml, which is higher than the density of distilled water at room temperature (0.99821g / ml).

[0104] 1-3.Fluorescence evaluation To determine the optimal ICG and albumin concentrations for the ICG-albumin-PMMA microsphere fluorescent marker that exhibits near-infrared fluorescence, the near-infrared fluorescence of microspheres prepared with various ICG and albumin concentrations was measured.

[0105] In order to determine the mixing ratio of ICG and albumin that would produce the strongest near-infrared fluorescence, 1.3 to 1032 μM ICG was reacted with 0 to 1% w / v (10 mg / ml) albumin in various ratios to produce ICG-albumin-PMMA microspheres, and the signal intensities of the near-infrared fluorescence appearing in the prepared microspheres were measured. The results are shown in Table 2.

[0106] [Table 2]

[0107] The results of Table 2 are shown in a graph in Figure 5. Figure 5 is a graph showing the change in near-infrared fluorescence signal intensity of microspheres depending on the change in ICG and albumin concentrations.

[0108] Referring to Table 2 and FIG. 5, when no albumin was added, it was confirmed that 25.8 μM ICG showed the highest near-infrared fluorescence signal intensity of the microspheres, and when 0.05% w / v (0.5 mg / ml) albumin was treated, 3.9 μM ICG showed the highest near-infrared fluorescence signal intensity, but overall it was confirmed that the fluorescence was significantly low. When treated with 0.25% w / w (2.5 mg / ml) albumin, 9 μM ICG showed the highest near-infrared fluorescence signal intensity, and when treated with higher albumin concentrations, 0.5% w / v (5 mg / ml), 0.7% w / v (7 mg / ml), 1% w / v (10 mg / ml), 10% w / v (100 mg / ml), and 20% w / v (200 mg / ml), 6.5 μM ICG showed the highest near-infrared fluorescence signal intensity.

[0109] From the above experimental results, the ICG concentration at which the fluorescence intensity was strongest at the same albumin concentration was 6.5 to 25.8 μM. Therefore, in the present invention, the ICG concentration range is preferably 1 to 100 μM, and more preferably 3 to 30 μM.

[0110] Moreover, the fluorescence intensity at the same ICG concentration continued to increase up to 20% w / v, which was the maximum albumin concentration used in the experiment. In reality, the maximum concentration of human serum albumin required for the production of microspheres is 20% w / v, and it was not possible to produce microspheres at a higher albumin concentration. Therefore, the albumin concentration at which the fluorescence intensity is maximized could not be determined in the experimental results, but the experimental results confirmed that the maximum fluorescence begins to be greater from microspheres with 0.5% albumin than from microspheres without albumin. Therefore, the albumin used for the production of microspheres should ideally be 0.5% w / v or more, and is suitable for the production of microspheres up to 20% w / v, which is the maximum albumin concentration experimentally possible.

[0111] 2. Evaluation of the usefulness of the injectable composition of ICG-HSA-PMMA 2-1.Evaluation of the usefulness of hyaluronic acid To determine the usefulness of hyaluronic acid as an injectable composition, the suspension retention time of ICG-albumin-PMMA / hyaluronic acid compositions was measured as a function of hyaluronic acid concentration. A concentration of 2mg / ml of ICG-albumin-PMMA was used, and the final injection of ICG-albumin-PMMA microspheres appeared as a very light green or white suspension when completely dissolved in the hyaluronic acid composition.

[0112] More specifically, the time to maintain the suspending property after the ICG-albumin-PMMA injection composition was measured, and the time to maintain the suspending property was measured according to the concentration of the added hyaluronic acid. Here, the suspending property was defined as the time when the criterion for distinguishing between the sinking part and the floating part was visible based on the transmittance of 20% of 500 nm light. The results are shown in Table 3.

[0113] Table 3 shows the time during which the suspension of ICG-albumin-PMMA microspheres was maintained after preparation according to the concentration of the hyaluronic acid solvent.

[0114] [Table 3]

[0115] Referring to Table 3, it was confirmed that the suspension retention time of the injectable composition without hyaluronic acid was shorter than that of the composition with hyaluronic acid, and the suspension retention time became longer as the proportion of hyaluronic acid added increased.

[0116] On the other hand, when added to a 0.1% hyaluronic acid injection composition, the suspension retention time increased within 60 minutes, but it was confirmed that the suspension retention time was 1 to 3 hours from a hyaluronic acid concentration of 0.2% (w / v), and the suspension retention time exceeded 3 hours from a hyaluronic acid concentration of 0.3% (w / v).When performing injections in actual clinical practice, the time from preparation of the injection to actual injection is within 1 hour, so it was determined that 0.2% (w / v) is effective as an actual injection preparation.

[0117] Fig. 6 is an image showing the dispersion degree of microparticles according to hyaluronic acid concentration in the biocompatible target lesion labeling injection composition of the present invention.More specifically, the images are taken immediately after preparing the ICG-HSA-PMMA / hyaluronic acid injection prepared above, and after 30, 60, and 120 minutes have passed, and the image on the left (a) is an injection prepared by mixing microparticles with water for injection (distilled water), and the image on the right (b) is an injection prepared with 0.3% hyaluronic acid.

[0118] Referring to Figure 6, it was found that the injection composition prepared in water for injection quickly sinks, whereas the injection composition to which 0.3% hyaluronic acid was added does not sink completely for a long time (up to 120 minutes). In other words, the results of Figure 3 indicate that the ICG-HSA-PMMA / hyaluronic acid injection composition, in which 0.3% hyaluronic acid is added to microspheres, is very useful as an injection composition because it does not sink quickly during preparation for injection.

[0119] 2-2. Evaluation of usefulness as an injectable composition The force exerted on the finger during injection (gliding force, N) was measured under various conditions depending on the concentration of hyaluronic acid, and the usefulness of ICG-albumin-PMMA / hyaluronic acid as an injectable composition was determined.

[0120] More specifically, when the final injection product was used as an injection, the ease of injection was evaluated using 21G, 22G, 23G, and 26G needles (Kovax, Korea Vaccine Co., Ltd.), a 20G spinal needle (puncture needle, Tae Chang Industrial Co., Ltd.), and a 3cc disposable syringe (3cc, BD, Luer-Lock Tip, Singapore) equipped with a 7Fr endoscopic syringe (20G (needle inner diameter: 0.603mm), 21G (needle inner diameter: 0.495mm), 22G (needle inner diameter: 0.394mm), 23G (needle inner diameter: 0.318mm), and 26G (needle inner diameter: 0.241mm)).

[0121] The force applied to the syringe handle (force exerted on the finger during injection, N) was measured for each injection needle. In addition, the resistance was measured using an endoscopic syringe (7 Fr, length 180 cm, MTW, Germany) with a 1.8 m connecting tube.

[0122] The injection used in the experiment had a concentration of 2 mg / ml of ICG-albumin-PMMA microspheres, and the concentration of hyaluronic acid in the final injection was 0.1-3.0% (w / v). After the injection was poured into the syringe, the experiment was immediately performed to prevent the microspheres from sinking, and the strain was measured using a strain-gauge dynamometer (Digital Force Gauge DPU-500N, IMADA, Toyohashi, Japan) while the final injection was being injected at a rate of 0.1 cc / sec. The results are shown in Table 4 below.

[0123] [Table 4]

[0124] In general, when performing injections in clinical settings, a force of 0.6 to 1.4 N was applied to the fingers during the injection. The grip strength of an average adult is 400 N, and the grip strength that the fingers can withstand is weaker than this, so much less force is required to make delicate adjustments such as adjusting the injection volume. Ten men and ten women were fitted with a force gauge between the thumb and index finger / middle finger to measure the maximum force (MaxPower) when applying maximum force and the force when giving an easy injection (EasyPower). The maximum force (MaxPower) was 98.1 N (113.5-78.2, standard deviation 9.8) for adult men and 43.1 N (50.6-34.2, standard deviation 6.1) for adult women, and the easy force was 25.4 N (29.7-20.1, standard deviation 3.3) for adult men and 14.5 N (18.3-11.5, standard deviation 2.2) for adult women. The ideal resistance of the injection was determined based on the maximum pressure of 43N when injecting an adult female and the comfortable pressure of 14.5N. In other words, the maximum pressure possible for injection was determined to be 40N, and the ideal resistance for comfortable injection was determined to be 10N.

[0125] When using a general injection needle (21~26G), the resistance during injection was not large at 10N up to a concentration of 1% hyaluronic acid, making it easy to use. Even with a 20G needle, which is much longer than other needles, the resistance did not exceed 10N at 1%.

[0126] On the other hand, when using an endoscopic syringe needle (7Fr, 180cm) with a long duct and high resistance, a force of 1.9N was required for injection water, and a force of 39.5N was required for hyaluronic acid with a concentration of 1% (w / v). Unless an extremely high pressure was applied, injection was not possible, and it was impossible to adjust the injection volume or speed.

[0127] Although there are individual differences, when a force of 40 N is applied to the fingers, it is difficult to inject into tissues and it is difficult to inject precisely. On the other hand, when using an endoscopic syringe needle (7 Fr, 180 cm), resistance increases (>40 N) when the hyaluronic acid exceeds 1%, making it impossible to inject with bare hands.

[0128] Under conditions where injection was possible by hand with maximum force (resistance of 40 N or less), it was possible to inject hyaluronic acid at 3% (w / v) or more, and even with a long puncture needle (20G), it was possible to inject up to 3% (w / v). Even with an extremely long special syringe (endoscopic syringe) connected to the needle by a thin conduit, it was possible to inject up to 1.0% (w / v) without difficulty.

[0129] On the other hand, when using a general syringe (26G-20G) that directly contacts the tissue, it is preferable to inject gently into the tissue, ideally with a force of less than 10 N. In the case of hyaluronic acid, it is possible to inject up to 2% (w / v) with a general needle (21-26G) with a force of less than 10 N, and even with a puncture needle (20G), it was possible to inject up to 1.5% (w / v). With an endoscopic syringe, it was possible to inject up to 0.2-0.3% (w / v) without difficulty.

[0130] 2-3. Evaluation of syringe pressure change due to precipitation The pressure (A) when the injection composition containing hyaluronic acid was immediately injected, and the pressure (B) applied to the syringe at the start of injection after the injection composition was left for 120 minutes and the composition had settled, and the pressure (C) applied during the injection were measured and are shown in Table 5 below.

[0131] The injection used in this experiment had a concentration of 2 mg / ml of ICG-albumin-PMMA microspheres, and the concentration of hyaluronic acid in the final injection was 0.1-3.0% (w / v). A 26G syringe was used, and the pressure applied to the syringe was measured in N. Furthermore, the gliding force was measured in N (Digital Force Gauge DPU-500N, IMADA, Toyohashi, Japan).

[0132] [Table 5]

[0133] Referring to Table 5, it was confirmed that the pressure (A) when the injection composition is immediately injected and the pressure (B) applied to the syringe after the injection composition is left for 120 minutes and the composition is precipitated, the higher the concentration of hyaluronic acid, the higher the pressure (A, B). The difference between A and B was almost 0.3% or more (-0.2 to 0.2N). As the concentration of hyaluronic acid increases above 0.3%, the difference between the pressure (B) applied to the syringe after the injection composition is left for 120 minutes and the pressure (A) when the injection composition is immediately injected is almost the same. The average BA value from the concentration of hyaluronic acid 0.3% (w / v) to 3.0% (w / v) was 0.07N, and the standard deviation was 0.15N.

[0134] Looking at the data for hyaluronic acid concentrations of 0.1% and 0.2% in Table 5, the pressure after leaving it for 2 hours was 45.2 and 34.7N, respectively, which made injection difficult. This means that the needle was clogged with the injection. The pressure after the injection moved decreased rapidly and was not different from the pressure (A) when the injection was immediately injected.

[0135] In other words, by adding an appropriate amount of hyaluronic acid (0.3% or more), the precipitation rate can be slowed down, and the drawback of the precipitated composition clogging the injection needle, making it impossible to start the injection without applying excessive force, can be solved.

[0136] On the other hand, when the concentration of hyaluronic acid exceeds 1% (w / v), it is found that the pressure during injection increases due to the viscosity of hyaluronic acid. Such an increase in pressure also increases the resistance during injection, which may cause inconvenience when used as an injection.

[0137] 3. Stability evaluation of ICG-HSA-PMMA PMMA microspheres bound to ICG and HSA, which can generate near-infrared fluorescent signals, were expected to serve as a marker that would function more stably in vivo, so the microspheres were produced and their stability was confirmed under two conditions: in vitro and in vivo.

[0138] 3-1. Confirmation of stability of near-infrared fluorescence signal in vitro Figure 7 is a graph showing a comparison of changes in near-infrared fluorescence signal intensity over time for ICG, ICG-HSA, and ICG-HSA-PMMA particles under in vitro conditions, measured with IVIS (Perkin Elmer, Hopkinton, MA).

[0139] 7, according to an embodiment of the present invention, when preparing PMMA microspheres bound with ICG-HSA (ICGHSA-PMMA), it was confirmed that the suitable mixing ratio for producing microspheres exhibiting a high level of near-infrared fluorescence signal is to combine 50μM ICG with 4% HSA and mix it with 300mg of PMMA beads. In this way, when bound to a biopolymer such as albumin, it shows stronger and longer-lasting fluorescence than when ICG is embedded in the microspheres alone.

[0140] The near-infrared fluorescence signal intensity emitted by the ICG-HSA-PMMA particles prepared in Preparation Example 4 was measured by IVIS for 5 months under in vitro conditions, and the results are shown in FIG. 8. FIG. 8 shows images comparing the stability of ICG-HSA-PMMA particles over time when the particles are placed in HSA, distilled water, and agarose gel. More specifically, FIG. 8a shows images comparing the stability of ICG-HSA-PMMA particles over time when the particles are placed in HSA and distilled water. FIG. 8b shows images showing the stability of ICG-HSA-PMMA particles over time when the particles are placed in agarose gel.

[0141] As shown in Figure 8a, the particles containing PMMA showed a relatively small decrease in the intensity of the near-infrared fluorescence signal, indicating stability. Also, as shown in Figure 8b, the particles were placed in (a) 20% HSA, distilled water, and (b) agarose gel, respectively, and the near-infrared fluorescence signal emitted for 17 weeks was measured using a fluorescence camera, and it was confirmed that the particles showed stability by visual observation.

[0142] 3-2. Confirmation of stability of in vivo near-infrared fluorescence signal The ICG-HSA-PMMA microspheres prepared in Preparation Example 4 were placed in hyaluronic acid solution or distilled water, and 100 μl of each solution was injected into ICR mice, and the intensity of near-infrared fluorescent signals generated from each mouse was measured using IVIS and fluorescent camera equipment for 4 months.

[0143] Figure 9 shows the results of comparing the change in near-infrared fluorescence signal intensity over time when ICG-HSA-PMMA particles were injected into ICR mice under in vivo conditions together with hyaluronic acid or distilled water, as measured by IVIS. More specifically, Figure 9a is a graph quantitatively showing the change in near-infrared fluorescence signal intensity over time of ICG-HSA-PMMA particles injected into ICR mice, as measured by IVIS, and Figure 9b is an image taken by IVIS over time of ICG-HSA-PMMA particles injected into ICR mice.

[0144] Referring to FIG. 9, the IVIS observation results showed that the ICG-HSA-PMMA / hyaluronic acid mixture exhibited a high intensity near-infrared fluorescence signal and maintained stability, whereas the mixture mixed with distilled water exhibited a very low fluorescence signal.

[0145] Figure 10 shows a graph of the change in near-infrared fluorescence signal intensity over time when ICG-HSA-PMMA particles were injected into ICR mice together with hyaluronic acid or distilled water under in vivo conditions, photographed with a fluorescent camera and analyzed with the Image J program, and the results of photographing a mouse with a fluorescent camera. More specifically, Figure 10a is a graph quantitatively showing the change in near-infrared fluorescence intensity over time of ICG-HSA-PMMA particles injected into ICR mice, and Figure 10b is an image taken with a fluorescent camera over time of ICG-HSA-PMMA particles injected into ICR mice.

[0146] Referring to FIG. 10, in the case of a fluorescent camera, the fluorescence intensity was measured using the Image J program after shooting. Both mixtures showed high intensity near-infrared fluorescence signals. However, while the ICG-HSA-PMMA / hyaluronic acid mixture remained stable over time, the distilled water mixture showed a gradually decreasing fluorescence signal.

[0147] Also, referring to Figures 9 and 10, when ICG-HSA-PMMA microspheres are mixed with a hyaluronic acid solution, it can be seen that the solution improves the in vivo tissue retention of the microspheres. The hyaluronic acid plays a role in gathering the complex injected into the body and increasing the fluorescent signal, and can maximally prevent the complex injected into the body from diffusing, thereby allowing a high fluorescent signal to be obtained for up to several months. Therefore, the injection composition of the complex of the present invention may further contain hyaluronic acid.

[0148] The composition for labeling a target lesion of the present invention remains in the target lesion for several months after injection, and the position and size of the labeled lesion can be detected in real time during surgery, improving the success rate of surgical procedures for the target lesion and preventing excessive loss of normal tissue, so it can be widely used as an effective surgical marker. In the experiment, only the results of imaging up to 20 weeks were disclosed, but this was due to other reasons such as the termination of the experiment due to animal welfare-related experimental regulations, and it was confirmed that the fluorescence of ICG-HAS-PMMA microspheres was well maintained up to 25 weeks. Furthermore, although not shown in the graph, it was confirmed that the fluorescence of the microspheres lasted for more than one year in vitro.

[0149] The above description is merely an illustrative example of the technical concept of the present invention, and a person having ordinary knowledge in the technical field to which the present invention pertains may make various modifications and variations without departing from the essential characteristics of the present invention.

[0150] Therefore, the embodiments disclosed in the present invention are for illustration purposes only and are not intended to limit the technical idea of ​​the present invention, and the scope of the technical idea of ​​the present invention is not limited by such embodiments. The scope of protection of the present invention should be interpreted according to the following claims, and all technical ideas within the scope equivalent thereto should be interpreted as being included in the scope of the present invention. [Industrial Applicability]

[0151] The composition for labeling a target lesion of the present invention remains in the target lesion for up to several months after injection, and the location, size, etc. of the labeled lesion can be detected in real time during surgery. This not only improves the success rate of surgical procedures for the target lesion, but also prevents excessive loss of normal tissue, and therefore can be widely used as an effective surgical marker.

Claims

1. A biocompatible polymer; and a dye for staining a biological tissue; A target lesion marking composition that is administered directly to a target lesion during a targeting site surgery to confirm the size and location of the target lesion in real time.

2. The composition for labeling a target lesion according to claim 1 , wherein the microspheres further comprise a surface modifier coated on the surface thereof.

3. The biocompatible polymer includes polymethyl methacrylate (PMMA), polyglycolic acid (PGA), polylactic acid (PLA), polylactic acid-polyglycolic acid copolymer (PLGA), poly-ε-caprolactone (PCL), lactic acid-ε-caprolactone copolymer ( ... The target lesion labeling composition according to claim 1, characterized in that the compound is any one of polyamino acids, polyanhydrides, polyorthoesters, polyphosphazenes, polyiminocarbonates, polyphosphoesters, polyhydroxyvalates, copolymers thereof, and mixtures thereof.

4. The composition for labeling a target lesion according to claim 1 , wherein the dye for staining a biological tissue is a visually identifiable dye or a fluorescent dye.

5. The composition for labeling a target lesion according to claim 4, characterized in that the visually visible dye is any one selected from the group consisting of neutral red, Nile blue, Bismarck brown, lithium carmine, trypan blue, Janus green, methyl violet, auramine, malagaite green, sapranin, eosin, Congo red, erythrosine, nigrosine, Alcian blue hematoxylin, aniline blue, light green, and combinations thereof.

6. The composition for labeling a target lesion according to claim 4 , wherein the fluorescent dye is a near-infrared fluorescent dye.

7. The composition for labeling a target lesion according to claim 4, wherein the fluorescent dye is indocyanine green (ICG).

8. The composition for labeling a target lesion according to claim 4, wherein the fluorescent dye is used alone or conjugated to a biological protein.

9. The composition for labeling a target lesion according to claim 2, wherein the surface modifier is any one of hyaluronic acid, collagen, and a mixture thereof.

10. The composition for labeling a target lesion according to claim 1 , wherein the microspheres further comprise a biological protein.

11. The composition for labeling a target lesion according to claim 10, wherein the biological protein is albumin.

12. The composition for labeling a target lesion according to claim 1, wherein the microspheres have a diameter of 10 to 100 μm.

13. The composition for labeling a target lesion according to claim 1, characterized in that the composition has an average density of 1 to 3 g / ml.

14. Mixing an organic solvent containing a biocompatible polymer with distilled water containing a dye for staining biological tissue; and The mixed solution is added to an aqueous solvent, and then stirred to form microspheres.

15. The method for preparing a target variable labeling composition according to claim 14, further comprising the step of adding a surface modifier to the water-soluble solvent.

16. The method for producing a composition for labeling a target lesion according to claim 14, further comprising the step of washing and filtering the stirred solution to select microspheres of a particular size.

17. The method for preparing a composition for labeling a target lesion according to claim 14, wherein the distilled water further contains a dye-biological protein conjugate for staining biological tissue.

18. The method for producing a target lesion labeling composition according to claim 14, wherein the organic solvent is any one selected from the group consisting of dichloromethane (DCM), o-xylene, m-xylene, p-xylene, and combinations thereof.

19. The method for producing a target lesion labeling composition according to claim 14, wherein the concentration of the dye for staining biological tissue is 1 to 100 μM.

20. 15. The method for producing a composition for labeling a target lesion according to claim 14, wherein the water-soluble solvent is any one selected from the group consisting of polyvinylpyrrolidone (PVP), polyacrylic acid (PAA), polyacrylamide (PAAm), polyhydroxyethyl methacrylate (PHEMA), polyvinyl alcohol (PVA), and combinations thereof.

21. A composition for labeling a target lesion in the form of microspheres containing a biocompatible polymer and a dye for staining a vital tissue; and An injectable composition for labeling a target lesion, comprising hyaluronic acid (HA) having an average molecular weight of 0.5 to 3.0 MDa.

22. The injectable composition for labeling a target lesion according to claim 21, wherein the injectable composition contains 0.3 to 1% by weight of hyaluronic acid (HA) based on 100% by weight of the injectable composition.

23. The injectable composition for labeling a target lesion according to claim 21, wherein the target lesion into which the injectable composition is injected retains its label for 4 to 12 months.

24. Administering a target lesion labeling composition in the form of microspheres containing a biocompatible polymer and a dye for staining vital tissue directly to the target lesion or around the target lesion; and A method for providing information on the location of a target lesion, comprising: recognizing the location where the color changes in the tissue to which the target lesion labeling composition is administered as the target lesion, and confirming the location in real time during surgery at the target site.

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