Polymer microparticles loaded with near-infrared fluorescent dyes for lesion labeling and their formulations

Abstract

The present invention relates to microparticles loaded with near-infrared fluorescent dyes and their preparations, and more specifically, to microparticles loaded with near-infrared fluorescent dyes that can be injected into a lesion such as cancer to accurately identify the location of the lesion from a fluorescent image during surgery, thereby enabling precision surgery. The near-infrared fluorescent dye-loaded microparticles according to the present invention, when injected into a lesion such as cancer, can accurately determine the location of the lesion from a fluorescent image in real time, dramatically improving the accuracy of surgery for resecting the lesion and significantly shortening the time required for surgery. By forming a complex between a near-infrared fluorescent dye and human serum albumin or a near-infrared fluorescent dye and cyclodextrin and loading it into the microparticles, or by mixing the near-infrared fluorescent dye into a hydrogel polymer and loading it, the intensity of the fluorescence generated by the microparticles is stronger than when the microparticles are manufactured using only the near-infrared fluorescent dye, and the stability of the fluorescent dye in the microparticles is improved, making it possible to confirm the labeled site with a fluorescent image for a long period of time.

Description

[Technical Field]

[0001] The present invention relates to microparticles equipped with near-infrared fluorescent dyes and their formulations, and more specifically, to microparticles equipped with near-infrared fluorescent dyes that, when injected into a lesion site such as cancer, allow for precise identification of the location of the lesion site from a fluorescent image during surgery, thereby enabling precise surgery. [Background technology]

[0002] Fluorescence imaging technology has a significant advantage over magnetic resonance imaging, nuclear medicine imaging, and ultrasound imaging: it offers high sensitivity while enabling real-time image acquisition. Therefore, it is considered the most powerful tool for real-time confirmation and removal of lesions such as cancer during surgery. Furthermore, with the gradual commercialization of systems that incorporate near-infrared fluorescence imaging capabilities into endoscopes and laparoscopes, fluorescence imaging-based surgery is expected to be widely used in patient surgery in the future.

[0003] Laparoscopic surgery is a minimally invasive surgical method that reduces the damage and impact on the patient's body. Instead of making incisions in the abdomen or chest, it involves making small holes of 0.5 to 1.5 cm and inserting an endoscope (laparoscope) equipped with a special camera to visualize the abdominal cavity while performing surgery with surgical instruments (forceps, electrosurgical units, hemostatic sutures, etc.). Compared to open surgery, laparoscopic surgery has developed over the past few decades due to its advantages such as less postoperative pain, faster recovery due to fewer incisions, and shorter hospital stays. In recent years, with the introduction of robotic surgery that applies laparoscopic techniques, laparoscopic surgery has been accepted as a fundamental surgical technique. For the surgical treatment of colorectal cancer, gastric cancer, etc., it is necessary to insert an endoscope into the gastrointestinal tract or large intestine and visually confirm the treatment site, such as the cancer site, before surgically removing the cancerous area inside the body through laparoscopic surgery. However, in conventional surgical methods, the field of view and range obtained via endoscopy are completely different from those obtained via laparoscopy, making it difficult to accurately locate the target incision site. Consequently, the precision and extent of the incision vary depending on the surgeon's skill level, and the incision area inevitably becomes larger. Therefore, to mark the area to be removed during surgery, a method is used in which a drug such as Indian ink is injected into the lesion identified using an endoscope with an endoscopic injection needle to indicate the lesion site, and then the surgery is performed while visually confirming the area marked with Indian ink through a laparoscope. However, such marking methods have the disadvantage that complications can occur if the ink is accidentally injected into the muscle layer, and the area to be removed during surgery may expand due to the ink or dye spreading to the surrounding area at the injection site. Therefore, in order to minimize the area of ​​normal tissue removed during laparoscopic surgery, a contrast agent for "marking" or "marker" that indicates the appropriate location of the diseased lesion is necessary.

[0004] In recent years, attempts have been made to use near-infrared fluorescent dyes for labeling lesions. Fluorescence imaging techniques using near-infrared fluorescent dyes have the advantage of being able to detect labeled areas located in deep tissues from fluorescent images, and also being able to sensitively detect fluorescent signals even with small amounts of fluorescent dye. Fluorescence imaging techniques have the significant advantage of being able to acquire images in real time while maintaining high sensitivity compared to magnetic resonance imaging, nuclear medicine imaging, and ultrasound imaging. Therefore, they are considered the most powerful tool for confirming and removing lesions such as cancer in real time during surgery. Among the fluorescent imaging contrast agents that have been tried conventionally, indocyanine green, a near-infrared fluorescent dye approved for clinical use by the U.S. Food and Drug Administration (FDA), has been used in vascular imaging, but it has various limitations for clinical application. Indocyanine green exhibits a concentration-dependent aggregation phenomenon, resulting in decreased fluorescence intensity, and is chemically unstable in aqueous solutions, making it easily decomposed. Furthermore, because it rapidly spreads and is absorbed into surrounding tissues from the injection site, it generates fluorescent signals over a wider area than the injection site, and the fluorescent signal weakens quickly after injection. Therefore, there is a need to develop methods that improve the labeling efficiency of lesions and maintain a high, long-lasting fluorescence signal from near-infrared fluorescent dyes without blurring at the labeled site.

[0005] Wang, Long et al. manufactured microbubbles loaded with indocyanine green dye for the detection using ultrasonic images and fluorescence images. The microbubbles manufactured with poly(D,L-lactide-co-glycolide) (PLGA), a biodegradable polymer, showed an ultrasonic image contrast effect. By loading indocyanine green into the microbubbles, when the microbubbles moved from the place where they were injected and accumulated in the sentinel lymph nodes, the position of the sentinel lymph nodes could be confirmed from the ultrasonic images and near-infrared fluorescence images. However, as mentioned above, since indocyanine green tends to aggregate with each other on the aqueous solution and is unstable, when indocyanine green is used alone and loaded into microbubbles, the intensity of the fluorescence signal is greatly weakened due to the aggregation phenomenon of indocyanine green molecules, and further decrease in the fluorescence intensity due to the decomposition of indocyanine green over time becomes a problem. Also, in the case of microbubbles, when holes are formed in the shell layer during the manufacturing process or by hydrolysis in the living body, the loaded indocyanine green is rapidly released to the outside, resulting in a decrease in the fluorescence signal and a phenomenon of fluorescence signal leakage in the surrounding tissues. Detailed Description of the Invention

[0006] The present invention was devised to solve the problems of the prior art as described above, and completed the present invention by developing polymer microparticles loaded with a near-infrared fluorescent dye complex in which a near-infrared fluorescent dye and human serum albumin, or a near-infrared fluorescent dye and cyclodextrin form a complex. The microparticles loaded with a near-infrared fluorescent dye enable the position of the lesion into which the microparticles are injected to be accurately and for a long time discriminated through fluorescence images, in order to keep the fluorescent dye loaded in the microparticles from generating a strong fluorescence signal and staying in the microparticles for a long time.

[0007] When labeling the location of a lesion using the microparticles loaded with the near-infrared fluorescent dye developed in the present invention and its preparation, it becomes possible to accurately excise the lesion surgically while confirming the labeled location through a fluorescence image, and the operation time can also be significantly shortened. In addition, since the near-infrared fluorescent dye loaded in the microparticles can be tracked through images for a long time, it can also be used to track changes occurring at the labeled location for a long time.

Summary of the Invention

[0008] An exemplary object of the present invention is to provide polymer microparticles loaded with a near-infrared fluorescent dye for lesion labeling, wherein the near-infrared fluorescent dye forms a complex with human serum albumin or cyclodextrin, for lesion labeling.

[0009] Another exemplary object of the present invention is to provide a method for producing the polymer microparticles.

[0010] Another exemplary object of the present invention is to provide a composition for lesion labeling, comprising the polymer microparticles.

[0011] Another exemplary object of the present invention is to provide a method for labeling a lesion, comprising the step of injecting the composition into an individual.

[0012] Another exemplary object of the present invention is to provide the use of the composition for lesion labeling.

[0013] The technical problems to be achieved by the technical idea of the invention disclosed in this specification are not limited to the problems mentioned above for solving the problems, and other problems not mentioned will be clearly understood by those of ordinary skill from the following description.

Means for Solving the Problems

[0014] This can be explained in more detail as follows: On the other hand, each description and embodiment disclosed in this application can be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0015] As one embodiment for achieving the above objective, the present invention provides polymer microparticles for lesion labeling, wherein the near-infrared fluorescent dye forms a complex with human serum albumin or cyclodextrin.

[0016] The aforementioned lesion labeling involves marking the location of the lesion to be removed during surgery in advance, and then using fluorescence imaging to confirm the marked location in real time during surgery, thereby enabling accurate surgery and a reduction in surgical time.

[0017] In the present invention, the near-infrared fluorescent dyes are indocyanine green, IRDye 800CW carboxylate (IRDye 800CW), Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 780, Flamma 749, Flamma 774, Flamma 800, and FSD Fluor TM 647, FSD Fluor TM 680, FSD Fluor TM 750, FSD Fluor TM 800 may be selected from, but is not limited to, sulfo-cyanine 5 carboxylic acid (Cy5), sulfo-cyanine 5.5 carboxylic acid (Cy5.5), sulfo-cyanine 7 carboxylic acid (Cy7), or sulfo-cyanine 7.5 carboxylic acid (Cy7.5).

[0018] The near-infrared fluorescent dye may be a dye that generates a fluorescent signal at a wavelength of 650 nm or higher.

[0019] Indocyanine green, as mentioned above, has been used in vascular imaging and other applications as a fluorescent contrast agent approved for clinical use by the U.S. Food and Drug Administration (FDA). However, it has several limitations for clinical application. Indocyanine green exhibits a concentration-dependent aggregation phenomenon that reduces its fluorescence intensity, and it is easily degraded due to its chemical instability in aqueous solutions. Furthermore, because it rapidly spreads and is absorbed into surrounding tissues from the injection site, it either generates a fluorescence signal over a wider area than the injection site, or the fluorescence signal weakens quickly after injection. Therefore, there is a need to develop a method that improves the labeling efficiency of lesions and maintains the fluorescence signal of near-infrared fluorescent dyes at the labeled site for a long period without blurring.

[0020] Indocyanine green molecules tend to aggregate with each other in aqueous solutions and are unstable. Therefore, when using indocyanine green alone, the aggregation of indocyanine green molecules significantly weakens the fluorescence signal intensity, and over time, the decomposition of indocyanine green leads to a further decrease in fluorescence intensity, which becomes a problem.

[0021] The microparticles containing a near-infrared fluorescent dye and human serum albumin, or a complex of a near-infrared fluorescent dye and cyclodextrin, according to the present invention, allow the fluorescent dye contained within the microparticles to remain within the microparticles for a long period of time while generating a strong fluorescence signal, thereby enabling accurate and long-term identification of the target site using fluorescence imaging.

[0022] In the present invention, a water-in-oil-in-water (W1 / O / W2) emulsion method can be used as a manufacturing method for incorporating the composite into polymer microparticles. When a near-infrared fluorescent dye with low solubility in water is incorporated into microparticles alone, aggregation of the near-infrared fluorescent dyes occurs within the microparticles, resulting in a very weak fluorescence signal intensity in the manufactured microparticles and low chemical / physical stability of the near-infrared fluorescent dye within the microparticles. When a composite of a near-infrared fluorescent dye and human serum albumin, or a near-infrared fluorescent dye and cyclodextrin is formed and incorporated into microparticles, the aggregation phenomenon that occurs when the fluorescent dye is incorporated alone can be prevented, the fluorescence signal generated in the microparticles is much stronger, and the chemical / physical stability of the fluorescent dye is also improved.

[0023] In the present invention, the polymer may be, but is not limited to, poly(lactide-co-glycolide) (PLGA), poly(DL-lactide-co-glycolide) (PDLGA), poly(glycolic acid) (PGA), poly(lactide) (PLA), poly(hydroxybutyrate), polycaprolactone (PCL), polydioxanone (PDO), poly(amino acid), polyanhydride, polyorthoester, or polyphosphazene as a biodegradable polymer.

[0024] In the present invention, the polymer can form a block copolymer with poly(ethylene oxide)(PEG).

[0025] As an example, the polymer may also form a block copolymer with poly(D,L-lactide)-block-poly(ethylene glycol, PDLLA-PEG).

[0026] In the present invention, the polymer may be a non-biodegradable polymer such as poly(methyl methacrylate) (PMMA) or polycarbonate (PC).

[0027] In the present invention, the fine particles may further contain alginic acid or hyaluronic acid together with a complex of a near-infrared fluorescent dye and human serum albumin, or a near-infrared fluorescent dye and cyclodextrin. When the complex is dispersed and mounted in an alginic acid or hyaluronic acid hydrogel, aggregation of the fluorescent dyes can be prevented more effectively than when the complex is mounted in the fine particles alone, the fluorescence signal generated by the fine particles becomes significantly stronger, and the chemical and physical stability of the fluorescent dye is also improved.

[0028] Furthermore, mixing the near-infrared fluorescent dye present in the W1 phase with human serum albumin, or the near-infrared fluorescent dye with cyclodextrin, using a hydrogel made of a polymer such as alginic acid or hyaluronic acid, can further slow down the rate at which the fluorescent dye is released from the microparticles.

[0029] In the present invention, the fine particles contain one or more cavities (isolated spaces) within them, and the near-infrared fluorescent dye and human serum albumin, or the near-infrared fluorescent dye and cyclodextrin, may be mounted in the cavities.

[0030] In particular, if multiple isolated spaces are created within the microparticles and near-infrared fluorescent dyes are placed in these spaces, even if the outer shell of the microparticles decomposes and holes are created, the rapid release of the fluorescent dyes placed in these isolated spaces will not occur. By creating multiple isolated spaces within the microparticles in this way and placing the fluorescent dyes in them, the problem of unwanted release of fluorescent dyes that occurred with microbubbles can be solved.

[0031] In the present invention, the fine particles may further contain a surfactant.

[0032] The surfactant may be selected from polyvinyl alcohol, polyethylene glycol, Labrafil, Labrasol, medium chain triglyceride, lecithin, N-methyl pyrrolidone, polyvinyl pyrrolidone, hydropropyl methylcellulose, poloxamer (e.g., poloxamer 188 (P-188), poloxamer 407 (P-407)), or tween (e.g., tween-80, tween-20)).

[0033] The fine particles of the present invention can be dispersed in an aqueous solution containing the surfactant or hydrophilic polymer for injection into the labeled area. Examples of the hydrophilic polymer include carboxymethylcellulose, gelatin, collagen, alginic acid, and hyaluronic acid.

[0034] When the aforementioned microparticles are manufactured in pellet form together with a hydrogel polymer and injected into the site to be labeled using a syringe, it is possible to prevent the microparticles from moving away from the injection site and further suppress the release of the fluorescent dye into the surrounding tissue. The hydrogel polymer may be a temperature-sensitive polymer such as poloxamer, or alginic acid, hyaluronic acid, or derivatives thereof that trigger ionic crosslinking reactions.

[0035] In the present invention, the fine particles may be used for coating on the surface of a metal or nonmetallic material.

[0036] The aforementioned coating may be applied using adhesives or rubber agents.

[0037] In one embodiment of the present invention, a lesion marker using a metal and / or nonmetallic material can be produced by coating the fine particles together with an adhesive or rubber agent on the surface of a metal and / or nonmetallic material.

[0038] The adhesive may be, for example, urea-based, melamine-based, phenol-based, unsaturated polyester-based, epoxy-based, resorcinol-based, vinyl acetate-based, polyvinyl alcohol-based, vinyl chloride-based, polyvinyl acetal-based, acrylic-based, saturated polyester-based, polyamide-based, or polyethylene-based, but is not limited to these.

[0039] The rubber agent may, but is not limited to, but may be a butadiene rubber-based, nitrile rubber-based, butyl rubber-based, silicone rubber-based, chloroprene rubber-based, latex-free rubber (NFR), neoprene, natural latex, or synthetic latex.

[0040] As another embodiment for achieving the above objective, the present invention provides a method for producing polymer microparticles for labeling lesions, comprising the steps of: forming a complex of a near-infrared fluorescent dye with human serum albumin, or a near-infrared fluorescent dye with cyclodextrin; and integrating the complex within polymer microparticles.

[0041] In the present invention, a water-in-oil-in-water (W1 / O / W2) emulsion method can be used as a method for incorporating the composite into polymer microparticles.

[0042] The present invention may further include the step of mixing the near-infrared fluorescent dye with a hydrogel polymer in the above manufacturing method and incorporating it into polymer microparticles.

[0043] In the present invention, for the purpose of further increasing the fluorescence signal of the near-infrared fluorescent dye embedded in the microparticles, microparticles can be manufactured by mixing a polymer and a surfactant in an oil phase.

[0044] In yet another embodiment for achieving the above objective, the present invention provides a composition for labeling lesions, comprising the polymer microparticles.

[0045] The fluorescent dye, polymer, fine particles, and surfactant are as described above.

[0046] In the present invention, the fine particles may be a mixture of methylcellulose, alginic acid, hyaluronic acid, polyvinyl alcohol, collagen, gelatin, Tween, Poloxamer, or polyvinylpyrrolidone. Specifically, the fine particles may be dispersed in an aqueous solution containing methylcellulose, alginic acid, hyaluronic acid, polyvinyl alcohol, collagen, gelatin, Tween, Poloxamer, or polyvinylpyrrolidone.

[0047] In the present invention, the composition for labeling the lesion may be prepared so as to be injected via a syringe.

[0048] In the present invention, the composition for labeling the lesion may be in the form of a liquid or solid pellet, and more specifically, it may be in the form of a solid pellet.

[0049] In yet another embodiment for achieving the aforementioned objective, the present invention provides a method for labeling lesions, comprising the step of injecting the composition into an organism.

[0050] In one embodiment of the present invention, the individual is not particularly limited, but may include, for example, humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits, or guinea pigs, and more specifically may be mammals, and more specifically humans.

[0051] In yet another embodiment for achieving the aforementioned objective, the present invention provides the use of the composition for labeling lesions. [Effects of the Invention]

[0052] The near-infrared fluorescent dye-laden microparticles according to the present invention enable real-time and accurate identification of lesion locations, such as cancer, through fluorescence imaging when injected into these lesions. This dramatically improves the accuracy of surgical resections and significantly reduces surgical time. By forming a complex of near-infrared fluorescent dye with human serum albumin, or near-infrared fluorescent dye with cyclodextrin, and embedding this complex within the microparticles, or by mixing the near-infrared fluorescent dye with a hydrogel polymer and embedding it, the fluorescence intensity generated by the microparticles is stronger compared to when the near-infrared fluorescent dye is used alone. This improves the stability of the fluorescent dye within the microparticles and allows for long-term visualization of the labeled area using fluorescence imaging. [Brief explanation of the drawing]

[0053] [Figure 1] Figure 1 shows the morphology and composition for formulations of polymer microparticles containing near-infrared fluorescent dye complexes. [Figure 2] Figure 2 shows fluorescence spectral data obtained by dissolving indocyanine green (ICG) and the indocyanine green-human serum albumin (ICG-HSA) complex at the same concentration in aqueous solution. It can be seen that ICG shows a higher fluorescence signal when it forms a complex with HSA. [Figure 3] Figure 3 shows scanning electron microscope images of (a) ICG-PLGA (without P-188), (b) ICG-HSA-PLGA (without P-188), and (c) ICG-HSA-PLGA (with 0.1% P-188) fine particles. [Figure 4]Figure 4 shows fluorescence spectra for comparison of fluorescence signals of ICG-HSA solution and ICG-PLGA (without P-188), ICG-HSA-PLGA (without P-188), and ICG-HSA-PLGA (P-188 0.1%) particulate dispersions. All samples were concentrated to an ICG-based 30 μM equivalent. In the case of ICG-PLGA (without P-188) particulate dispersion with ICG alone, the fluorescence signal was 6.8 times lower than that of ICG-HSA, indicating that when the ICG-HSA complex is incorporated, it is possible to produce particulates that generate a higher fluorescence signal. [Figure 5] Figure 5 shows scanning electron microscope images of (a) the surface of ICG-HSA-PLGA (P-188 0.5%) microparticles, (b) a cross-section of ICG-HSA-PLGA (P-188 0.5%) microparticles, (c) the surface of ICG-HSA-PLGA (P-188 1.0%) microparticles, and (d) a cross-section of ICG-HSA-PLGA (P-188 1.0%) microparticles. [Figure 6] Figure 6 shows fluorescence spectra for comparison of fluorescence signals of ICG-HSA solution and ICG-PLGA (without P-188), ICG-HSA-PLGA (0.5% P-188), and ICG-HSA-PLGA (1.0% P-188) fine particle dispersions. [Figure 7] Figure 7 shows fluorescence images (λex=780 / 20 nm, λem=845 / 40 nm) of (a) ICG-HSA, (b) ICG-HSA-PLGA (P-188 0.5%), and (c) ICG-HSA-PLGA (P-188 1.0%) particulate dispersions. [Figure 8] Figure 8 shows fluorescence images (λex=780 / 20 nm, λem=845 / 40 nm) taken over time after subcutaneous injection of (a) ICG-HSA, (b) ICG-HSA-PLGA (P-188 0.5%), and (c) ICG-HSA-PLGA (P-188 1.0%) microparticle dispersions into SKH-1 hairless mice. [Figure 9]Figure 9 is a graph showing the region of interest (ROI) values ​​of the fluorescence signal at the injection site over time, after subcutaneous injection of ICG-HSA, ICG-HSA-PLGA (P-188 0.5%), and ICG-HSA-PLGA (P-188 1.0%) microparticle dispersions into SKH-1 hairless mice. [Figure 10] Figure 10 shows scanning electron microscope images of (a) the surface and (b) the cross-section of ICG-HSA-Alginate-PLGA, PDLLA-PEG(P-188 0.1%) microparticles. [Figure 11] Figure 11 shows fluorescence spectra for comparison of fluorescence signals of ICG-HSA solution and ICG-PLGA (without P-188), ICG-HSA-Alginate-PLGA, and PDLLA-PEG (P-188 0.1%) particle dispersions. [Figure 12] Figure 12 shows fluorescence images (λex=780 / 20 nm, λem=845 / 40 nm) of (a) ICG-HSA and (b) ICG-HSA-Alginate-PLGA, PDLLA-PEG (P-188 0.1%) fine particle dispersions. [Figure 13] Figure 13 shows fluorescence images (λex=780 / 20 nm, λem=845 / 40 nm) taken over time after subcutaneous injection of (a) ICG-HSA and (b) ICG-HSA-Alginate-PLGA, PDLLA-PEG (P-188 0.1%) microparticle dispersions into SKH-1 hairless mice. [Figure 14] Figure 14 is a graph showing the region of interest (ROI) values ​​of the fluorescence signal at the injection site over time, after subcutaneous injection of ICG-HSA, ICG-HSA-Alginate-PLGA, and PDLLA-PEG (P-188 0.1%) microparticle dispersions into SKH-1 hairless mice. [Figure 15] Figure 15 shows scanning electron microscope images of (a) the surface and (b) the cross-section of ICG-HSA-PMMA(P-188 1.0%) microparticles. [Figure 16]Figure 16 shows fluorescence spectra for comparison of fluorescence signals of ICG-HSA solution, ICG-PLGA (without P-188), and ICG-HSA-PMMA (P-188 1.0%) fine particle dispersions. [Figure 17] Figure 17 shows fluorescence images (λex=780 / 20 nm, λem=845 / 40 nm) of (a) ICG-HSA and (b) ICG-HSA-PMMA (P-188 1.0%) fine particle dispersions. [Figure 18] Figure 18 shows fluorescence images (λex=780 / 20 nm, λem=845 / 40 nm) taken over time after subcutaneous injection of (a) ICG-HSA and (b) ICG-HSA-PMMA (P-188 1.0%) microparticle dispersions into SKH-1 hairless mice. [Figure 19] Figure 19 is a graph showing the region of interest (ROI) values ​​of the fluorescence signal at the injection site over time, after subcutaneous injection of ICG-HSA and ICG-HSA-PMMA (P-188 1.0%) microparticle dispersions into SKH-1 hairless mice. [Figure 20] Figure 20 shows scanning electron microscope images of (a) ICG-Alginate-PLGA (without P-188) and (b) ICG-Alginate-PLGA (with 0.1% P-188) fine particles. [Figure 21] Figure 21 shows fluorescence spectra for comparison of fluorescence signals of ICG-HSA solution and ICG-PLGA (without P-188), ICG-Alginate-PLGA (without P-188), and ICG-Alginate-PLGA (P-188 1.0%) fine particle dispersions. [Figure 22] Figure 22 shows scanning electron microscope images of (a) the surface of ICG-HSA-Alginate-PLGA (P-188 0.1%) microparticles, (b) the surface of ICG-HSA-Alginate-PLGA (P-188 0.5%) microparticles, and (c) a cross-section of ICG-HSA-Alginate-PLGA (P-188 0.5%) microparticles. [Figure 23]Figure 23 shows fluorescence spectra for comparison of fluorescence signals of ICG-HSA solution and ICG-PLGA (without P-188), ICG-HSA-Alginate-PLGA (P-188 0.1%), and ICG-HSA-Alginate-PLGA (P-188 0.5%) fine particle dispersions. [Figure 24] Figure 24 shows fluorescence images (λex=780 / 20 nm, λem=845 / 40 nm) of (a) ICG-HSA, (b) ICG-HSA-Alginate-PLGA(P-188 0.1%), and (c) ICG-HSA-Alginate-PLGA(P-188 0.5%) particulate dispersions. [Figure 25] Figure 25 shows fluorescence images (λex=780 / 20 nm, λem=845 / 40 nm) taken over time after subcutaneous injection of (a) ICG-HSA and (b) ICG-HSA-Alginate-PLGA(P-188 0.5%) microparticle dispersions into SKH-1 hairless mice. [Figure 26] Figure 26 is a graph showing the region of interest (ROI) values ​​of the fluorescence signal at the injection site over time, after subcutaneous injection of ICG-HSA and ICG-HSA-Alginate-PLGA (P-188 0.5%) microparticle dispersions into SKH-1 hairless mice. [Figure 27] Figure 27 shows scanning electron microscope images of (a) the surface of an ICG-HSA-Alginate-PLGA (Tween 0.5%) microparticle sphere and (b) a cross-section of an ICG-HSA-Alginate-PLGA (Tween 0.5%) microparticle. [Figure 28] Figure 28 shows the fluorescence spectra for comparison of the fluorescence signals of ICG-HSA solution, ICG-PLGA (without P-188), and ICG-HSA-Alginate-PLGA (Tween 0.5%) fine particle dispersion. [Figure 29] Figure 29 shows fluorescence images (λex=780 / 20 nm, λem=845 / 40 nm) of (a) ICG-HSA and (b) ICG-HSA-Alginate-PLGA (Tween 0.5%) dispersions of fine particles. [Figure 30] Figure 30 shows fluorescence images (λex=780 / 20 nm, λem=845 / 40 nm) taken over time after subcutaneous injection of (a) ICG-HSA and (b) ICG-HSA-Alginate-PLGA (Tween 0.5%) microparticle dispersions into SKH-1 hairless mice. [Figure 31] Figure 31 is a graph showing the region of interest (ROI) values ​​of the fluorescence signal at the injection site over time, after subcutaneous injection of ICG-HSA and ICG-HSA-Alginate-PLGA (Tween 0.5%) microparticle dispersions into SKH-1 hairless mice. [Figure 32] Figure 32 shows scanning electron microscope images of (a) the surface of ICG-mCD-Alginate-PLGA(P-188 0.1%) microparticles and (b) a cross-section of ICG-mCD-Alginate-PLGA(P-188 0.1%) microparticles. [Figure 33] Figure 33 shows fluorescence spectra for comparison of fluorescence signals of (a) ICG solution and ICG-mCD, (b) ICG-HSA solution and ICG-PLGA (without P-188), and ICG-mCD-Alginate-PLGA (P-188 0.1%) fine particle dispersion. [Figure 34] Figure 34 shows fluorescence images (λex=780 / 20 nm, λem=845 / 40 nm) of (a) ICG-HSA and (b) ICG-mCD-Alginate-PLGA (P-188 0.1%) fine particle dispersions. [Figure 35] Figure 35 shows fluorescence images (λex=780 / 20 nm, λem=845 / 40 nm) taken over time after subcutaneous injection of (a) ICG-HSA and (b) ICG-mCD-Alginate-PLGA (P-188 0.1%) microparticle dispersions into SKH-1 hairless mice. [Figure 36]Figure 36 is a graph showing the region of interest (ROI) values ​​of the fluorescence signal at the injection site over time, after subcutaneous injection of ICG-HSA and ICG-mCD-Alginate-PLGA (P-188 0.5%) microparticle dispersions into SKH-1 hairless mice. [Figure 37] Figure 37 is a graph comparing the fluorescence signal intensity at different concentrations of Cy7 and Cy7-HSA solutions (λex=730 nm, λem=780 nm (Cy7), λem=786 nm (Cy7-HSA)). [Figure 38] Figure 38 shows scanning electron microscope images of (a) the surface of Cy7-PMMA (without P-188) microparticles, (b) a cross-section of Cy7-PMMA (without P-188) microparticles, (c) the surface of Cy7-HSA-PMMA (without P-188) microparticles, (d) a cross-section of Cy7-HSA-PMMA (without P-188) microparticles, (e) the surface of Cy7-HSA-PMMA (P-188 1.0%) microparticles, and (f) a cross-section of Cy7-HSA-PMMA (P-188 1.0%) microparticles. [Figure 39] Figure 39 is a graph comparing the fluorescence spectra of Cy7 aqueous solution, Cy7-HSA aqueous solution, Cy7-PMMA (without P-188) fine particle dispersion, Cy7-HSA-PMMA (without P-188) fine particle dispersion, and Cy7-HSA-PMMA (P-188 1.0%) fine particle dispersion, all prepared at the same Cy7 concentration (30 μM) (λex = 730 nm). [Figure 40] Figure 40 is a graph comparing the fluorescence signal intensity of Cy7.5 and Cy7.5-HSA solutions at different concentrations (λex=730 nm, λem=810 nm (Cy7.5), λem=824 nm (Cy7.5-HSA)). [Figure 41]Figure 41 shows scanning electron microscope images of (a) the surface of Cy7.5-PMMA (without P-188) microparticles, (b) a cross-section of Cy7.5-PMMA (without P-188) microparticles, (c) the surface of Cy7.5-HSA-PMMA (without P-188) microparticles, (d) a cross-section of Cy7.5-HSA-PMMA (without P-188) microparticles, (e) the surface of Cy7.5-HSA-PMMA (P-188 1.0%) microparticles, and (f) a cross-section of Cy7.5-HSA-PMMA (P-188 1.0%) microparticles. [Figure 42] Figure 42 is a graph comparing the fluorescence spectra of aqueous solutions of Cy7.5, Cy7.5-HSA, Cy7.5-PMMA (without P-188) fine particle dispersion, Cy7.5-HSA-PMMA (without P-188) fine particle dispersion, and Cy7.5-HSA-PMMA (P-188 1.0%) fine particle dispersion, all prepared at the same Cy7.5 concentration (30 μM) (λex = 730 nm). [Figure 43] Figure 43 shows scanning electron microscope images of (a) the surface of Cy7.5-PC (without P-188) microparticles, (b) a cross-section of Cy7.5-PC (without P-188) microparticles, (c) the surface of Cy7.5-HSA-PC (without P-188) microparticles, and (d) a cross-section of Cy7.5-HSA-PC (without P-188) microparticles. [Figure 44] Figure 44 is a graph comparing the fluorescence spectra of Cy7.5-PC (without P-188) and Cy7.5-HSA-PC (without P-188) fine particle dispersions prepared at the same Cy7.5 concentration (λex = 730 nm). [Figure 45] Figure 45 is a graph comparing the fluorescence signal intensity at different concentrations of Flamma774 and Flamma774-HSA solutions (λex=730 nm, λem=812 nm (Flamma774), λem=818 nm (Flamma774-HSA)). [Figure 46]Figure 46 shows scanning electron microscope images of (a) the surface of Flamma774-PMMA (without P-188) microparticles, (b) a cross-section of Flamma774-PMMA (without P-188) microparticles, (c) the surface of Flamma774-HSA-PMMA (without P-188) microparticles, (d) a cross-section of Flamma774-HSA-PMMA (without P-188) microparticles, (e) the surface of Flamma774-HSA-PMMA (P-188 1.0%) microparticles, and (f) a cross-section of Flamma774-HSA-PMMA (P-188 1.0%) microparticles. [Figure 47] Figure 47 shows the fluorescence spectra for comparing the fluorescence signals of Flamma774 solution, Flamma774-HSA solution, Flamma774-PMMA (without P-188) particulate dispersion, Flamma774-HSA-PMMA (without P-188) particulate dispersion, and Flamma774-HSA-PMMA (P-188 1.0%) particulate dispersion. [Figure 48] Figure 48 is a graph comparing the fluorescence signal intensity of Alexa750 and Alexa750-HSA solutions at different concentrations (λex=730 nm, λem=776 nm). [Figure 49] Figure 49 shows scanning electron microscope images of cross-sections of (a) Alexa750-PMMA (without P-188) microparticles, (b) Alexa750-HSA-PMMA (without P-188) microparticles, and (c) Alexa750-HSA-PMMA (P-188 1.0%) microparticles. [Figure 50] Figure 50 shows the fluorescence spectra for comparing the fluorescence signals of Alexa750 solution, Alexa750-HSA solution, Alexa750-PMMA (without P-188) particulate dispersion, Alexa750-HSA-PMMA (without P-188) particulate dispersion, and Alexa750-HSA-PMMA (P-188 1.0%) particulate dispersion. [Figure 51] Figure 51 is a graph comparing the fluorescence signal intensity at different concentrations of IRDye 800CW and IRDye 800CW-HSA solutions (λex=730 nm, λem=806 nm (IRDye 800CW), λem=810 nm (IRDye 800CW-HSA)). [Figure 52] Figure 52 shows scanning electron microscope images of (a) the surface of IRDye 800CW-PMMA (without P-188) microparticles, (b) a cross-section of IRDye 800CW-PMMA (without P-188) microparticles, (c) the surface of IRDye 800CW-HSA-PMMA (without P-188) microparticles, (d) a cross-section of IRDye 800CW-HSA-PMMA (without P-188) microparticles, (e) the surface of IRDye 800CW-HSA-PMMA (P-188 1.0%) microparticles, and (f) a cross-section of IRDye 800CW-HSA-PMMA (P-188 1.0%) microparticles. [Figure 53] Figure 53 shows fluorescence spectra for comparison of fluorescence signals of IRDye 800CW solution, IRDye 800CW-HSA solution, IRDye 800CW-PMMA (without P-188) particulate dispersion, IRDye 800CW-HSA-PMMA (without P-188) particulate dispersion, and IRDye 800CW-HSA-PMMA (P-188 1.0%) particulate dispersion. [Figure 54] Figure 54 is a photograph taken when ICG-HSA-PLGA(P-188 0.5%) fine particles were dispersed in an aqueous solution of 0.5% sodium methylcellulose / 0.1% Tween 80, 1% sodium alginate, 1% sodium hyaluronate, 2% sodium methylcellulose, 2% poly(vinyl alcohol), 0.1% collagen, 5% gelatin, 10% Tween 80, 40% poloxamer 188, and 5% polyvinyl pyrrolidone to produce a composition in which fine particles are dispersed in a surfactant or polymer solution. [Figure 55] Figure 55 is a photograph showing cylindrical pellets containing ICG-HSA-PLGA(P-188 0.5%) fine particles, which were dispersed in 1% sodium hyaluronate and 5% gelatin aqueous solutions, respectively, and then freeze-dried. [Figure 56]Figure 56 is a photograph taken when ICG-HSA-PMMA(P-188 1.0%) fine particles were dispersed in an aqueous solution of 0.5% sodium methylcellulose / 0.1% Tween 80, 1% sodium alginate, 1% sodium hyaluronate, 2% sodium methylcellulose, 2% poly(vinylalcohol), 0.1% collagen, 5% gelatin, 10% Tween 80, 40% poloxamer-188 (P-188), and 5% polyvinyl pyrrolidone to produce a composition in which fine particles are dispersed in a surfactant or polymer solution. [Figure 57] Figure 57 is a photograph showing cylindrical pellets containing fine particles produced by dispersing ICG-HSA-PMMA(P-188 1.0%) fine particles in an aqueous solution of 1% sodium alginate, 1% sodium hyaluronate, 2% sodium methylcellulose, 2% poly(vinylalcohol), 5% gelatin, 40% poloxamer-188, and 5% polyvinylpyrrolidone, and then freeze-drying the mixture. [Figure 58] Figure 58 is a photograph of a metal clip coated with fluorescent microparticles, which was manufactured by dispersing IRDye 800CW-HSA-PMMA (without P-188) microparticles in a latex solution, coating the surface of the metal clip with the solution, and then drying it. [Figure 59] Figure 59 shows near-infrared fluorescence images (λex=780 / 20 nm, λem=845 / 40 nm) of a metal clip coated with IRDye 800CW-HSA-PMMA (without P-188) nanoparticles. [Modes for carrying out the invention]

[0054] The present invention will be described in more detail below through the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

[0055] Example 1: Comparison of fluorescence signals of indocyanine green solution and indocyanine green / human serum albumin complex

[0056] A 30 μM indocyanine green solution was prepared by dissolving 0.5 mg of indocyanine green (Diagnogreen strain, ICG, Daiichi Pharmaceutical Co., Ltd.), a near-infrared fluorescent dye, in 1 mL of distilled water, and then diluting it with distilled water. To compare the fluorescence signal with the indocyanine green solution, an indocyanine green / human serum albumin (HSA, SK Plasma) complex was prepared by dissolving 0.5 mg of indocyanine green powder in a mixture of 0.214 mL of human serum albumin solution (20% concentration) and 0.786 mL of distilled water, and then diluting it with distilled water. For fluorescence spectrum comparison, 200 μL of each prepared solution was taken and placed in a 96-well plate, and the fluorescence spectra (λ) were measured using a multifunctional microplate reader (SPARK, Tecan Trading AG, Zurich, Switzerland). ex The wavelength (=730 nm) was measured.

[0057] Fluorescence spectral measurements of indocyanine green solution and indocyanine green / human serum albumin complex solution at the same concentration confirmed, as in Figure 2, that the indocyanine green / human serum albumin complex produced a fluorescence signal 1.5 times higher than that of indocyanine green alone.

[0058] Example 2: Manufacturing and analysis of PLGA microparticles containing ICG or ICG-HSA 2-1. PLGA microparticle manufacturing with ICG ICG-PLGA (without P-188), which is a PLGA microparticle containing indocyanine green, a near-infrared fluorescent dye, was manufactured using a water-in-oil-in-water (W1 / O / W2) emulsion method.

[0059] Specifically, 0.75 mg of indocyanine green (ICG, Selleck chem) was dissolved in 1.5 mL of distilled water to prepare solution W1. 3.6 g of polyvinyl alcohol (PVA, Sigma-Aldrich) was added to 180 mL of distilled water, dissolved at 85 °C with stirring at 600 rpm, and cooled to room temperature to prepare solution W2. 1.08 g of poly-D,L-lactide-co-glycolid copolymer (PLGA, MW 192,000-240,000 Da, Evonik) was dissolved in 15 mL of methylene chloride (DCM, Sigma-Aldrich) to prepare the oil solution. After adding the prepared W1 solution to the oil solution, ultrasonic treatment was performed for 40 seconds using a water bath type ultrasonic device to produce a primary emulsion (water-in-oil). The prepared primary emulsion was then mixed with the prepared W2 solution while stirring at 1000 rpm using a mechanical stirrer to produce a secondary emulsion (water-in-oil-in-water). The prepared emulsion was stirred at 1000 rpm for 10 minutes, and then stirred at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C to remove the methylene chloride solvent. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PLGA polymer microparticles containing indocyanine green. The supernatant was removed, and the microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed microparticles were rapidly cooled with liquid nitrogen and freeze-dried to obtain microparticle powder.

[0060] 2-2. Manufacturing of PLGA microparticles equipped with ICG-HSA To produce PLGA microparticles (ICG-HSA-PLGA (without P-188)) containing an indocyanine green / human serum albumin complex, solution W1 was prepared by dissolving 0.75 mg of indocyanine green (Diagnogreen strain, Daiichi Yakuhin) in a mixed solution of 0.322 mL (20% concentration, 64.335 mg) of human serum albumin (HSA, SK plasma) and 1.178 mL of distilled water. Solution W2 was prepared by adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, dissolving it at 85 °C while stirring at 600 rpm, and then cooling it to room temperature. Solution W2 was prepared by dissolving 1.08 g of PLGA in 15 mL of methylene chloride. After adding the prepared W1 solution to the oil solution, sonication was performed for 40 seconds using a water bath type ultrasonic device to produce a primary emulsion (water-in-oil). The prepared W2 solution was stirred at 1000 rpm using a mechanical stirrer while a primary W / O emulsion was added to produce a secondary emulsion (water-in-oil-in-water). The prepared emulsion was stirred at 1000 rpm for 10 minutes, and then stirred at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C to remove the methylene chloride solvent. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PLGA polymer microparticles containing indocyanine green. The supernatant was removed, and the microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed microparticles were rapidly cooled with liquid nitrogen and freeze-dried to obtain microparticle powder.

[0061] 2-3. Manufacturing of PLGA microparticles equipped with ICG-HSA containing a surfactant (poloxamer) To observe the effect of adding a surfactant to the organic solvent phase along with polymers such as PLGA, microparticles with a surfactant added to PLGA were manufactured. To manufacture PLGA microparticles equipped with ICG-HSA containing 0.1% (oil phase) of the surfactant poloxamer-188 (P-188, BASF), solution W1 was prepared by dissolving 0.75 mg of indocyanine green (Diagnogreen strain, Daiichi Yakuhin) in a mixed solution of 0.322 mL (20% concentration, 64.335 mg) of human serum albumin (HSA, SK Plasma) and 1.178 mL of distilled water. Solution W2 was prepared by adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, dissolving it while stirring at 600 rpm at 85°C, and then cooling it to room temperature. An oil solution was prepared by dissolving 1.08 g of PLGA and 16.5 mg of Poloxamer-188 (P-188, BASF) in 15 mL of methylene chloride. After adding the prepared W1 solution to the oil solution, sonication was performed for 40 seconds using a water bath ultrasonic device to produce a primary emulsion (water-in-oil). A secondary emulsion (water-in-oil-in-water) was prepared by adding the prepared W2 solution to the primary emulsion while stirring at 1000 rpm using a mechanical stirrer. After stirring the prepared emulsion at 1000 rpm for 10 minutes, the methylene chloride solvent was removed by stirring at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PLGA polymer microparticles containing indocyanine green. The supernatant was removed, and the fine particles were washed by adding distilled water, redispersing, and centrifuging three times. The washed fine particles were rapidly cooled with liquid nitrogen and then freeze-dried to obtain fine particle powder.

[0062] 2-4. Analysis of indocyanine green content and fluorescence intensity in microparticles To evaluate the content and loading rate of indocyanine green in the manufactured microparticles, the concentration of indocyanine green in the microparticle dissolution solution was quantified using an ultraviolet / visible light absorbance spectrometer. A high-concentration indocyanine green / human serum albumin complex solution was prepared by dissolving 0.5 mg of indocyanine green in a mixed solution of 0.214 mL (20% concentration, 42.89 mg) of human serum albumin and 0.786 mL of distilled water. Thirteen standard solutions with concentrations between 0.065 and 6.54 μM were prepared by distilling the high-concentration complex solution with distilled water. The absorbance of these standard solutions was measured using an ultraviolet / visible light absorbance spectrometer to create a calibration curve. Then, 10 mg of PLGA microparticles were dissolved in 1 mL of dimethyl sulfoxide (DMSO, Daejung Chemicals & Metals Co. Ltd.) solvent. The absorbance of this solution was analyzed using an ultraviolet / visible light absorbance spectrometer, and the concentration was measured by substituting it into the calibration curve. The measured concentration of the composite was substituted into the following formula to evaluate the indocyanine green content and loading efficiency within the microparticles.

[0063] Indocyanine green content within microparticles = Weight of indocyanine green in 10 mg of microparticles / Weight of 10 mg of microparticles

[0064] Indocyanine green content within microparticles = (Weight of indocyanine green contained in the entire microparticle / Weight of indocyanine green used to contain the microparticles) × 100%

[0065] To analyze the particle morphology and size of the synthesized microparticles, scanning electron microscopy (JSM-7800F Prime, JEOL Ltd, Japan) analysis was performed. After attaching the microparticle powder to carbon tape mounted on a scanning electron microscope sample holder, Pt / Pd was coated onto the surface via sputtering for 60 seconds, and the scanning electron microscope images were analyzed with a 5 kV acceleration voltage. The particle size was determined by measuring the size of 100 randomly selected particles from the obtained images and averaging the result.

[0066] To compare the fluorescence signals of the aforementioned manufactured microparticles and the indocyanine green / human serum albumin complex, a complex solution and a microparticle dispersion of the same concentration of indocyanine green were prepared. 0.5 mg of indocyanine green powder was dissolved in a mixture of 0.214 mL of human serum albumin solution (20% concentration) and 0.786 mL of distilled water, and then diluted with distilled water to prepare a 30 μM indocyanine green / human serum albumin complex (ICG-HSA). Using the indocyanine green content in the PLGA microparticles evaluated by the aforementioned quantitative analysis, a dispersion was prepared by adding 30 μM indocyanine green to 0.3 mL of distilled water. For fluorescence spectrum comparison, 200 μL each of the prepared solution and dispersion were taken and placed in a 96-well plate, and the fluorescence spectra (λ) were measured using a multifunctional microplate reader (SPARK, Tecan Trading AG, Zurich, Switzerland). ex The wavelength (=730 nm) was measured.

[0067] Quantitative results of indocyanine green in microparticles: For ICG-PLGA (without P-188), the indocyanine green loading rate in microparticles was 3.99%, and it was confirmed that 43.6 ng was loaded in 1 mg of microparticles. For ICG-HSA-PLGA (without P-188) microparticles, the loading rate was 8.39%, and it was confirmed that the indocyanine green content in 1 mg of microparticles was 93.6 ng. For ICG-HSA-PLGA (P-188 0.1%) manufactured under conditions of containing 0.1% poloxamer-188 based on the primary emulsion phase during ICG-HSA-PLGA synthesis, the indocyanine green loading rate was 5.26% compared to ICG-HSA-PLGA (without P-188), and it was confirmed that the indocyanine green content in 1 mg of microparticles decreased to 59.7 ng. This is because poloxamer-188, which has both hydrophilic and hydrophobic functional groups, is evenly distributed on the PLGA, and the hydrophilic indocyanine green / human serum complex is evenly distributed on the PLGA, while at the same time a channel for water to pass through is formed, allowing some of the fluorescent dye to leach out during the synthesis process.

[0068] Scanning electron microscope images confirmed that the particle morphology of the three microparticles was spherical (Figure 3, (a) ICG-PLGA, (b) ICG-HSA-PLGA, (c) ICG-HSA-PLGA (P-188 0.1%)). The average particle sizes were 67.5±24.6 μm, 85.4±28.7 μm, and 77.5±20.2 μm for ICG-PLGA (without P-188), ICG-HSA-PLGA (without P-188), and ICG-HSA-PLGA (P-188 0.1%), respectively, confirming that all particles were 20 μm or larger, capable of evading macrophage action.

[0069] As can be seen from the fluorescence spectrum (Figure 4), in the case of ICG-PLGA (without P-188) microparticle dispersion with ICG alone, the fluorescence signal was confirmed to be 6.8 times lower than that of ICG-HSA. This is judged to be because when indocyanine green is mounted on PLGA, aggregation of the fluorescent dye cannot be prevented, resulting in a signal reduction effect. In the case of ICG-HSA-PLGA (without P-188) and ICG-HSA-PLGA (P-188 0.1%) microparticle dispersions, the fluorescence signal decreased by 2.8 times or less compared to ICG-HSA, but it was confirmed that the fluorescence signal was 2.4 times higher than that of ICG-PLGA (without P-188). This confirms that surgical marker microparticles with a higher fluorescence signal can be obtained when mounted on PLGA in the ICG-HSA form compared to mounting ICG on PLGA. This is because HSA can prevent the aggregation of ICG and partially prevent the reduction in fluorescence signal. Microparticles manufactured to contain 0.1% poloxamer-188 showed similar fluorescence intensity to PLGA microparticles with ICG-HSA but without poloxamer-188, and exhibited a higher fluorescence signal compared to ICG-PLGA microparticles.

[0070] Example 3: Production of ICG-HSA-PLGA microparticles at different concentrations of poloxamer-188 and fluorescence intensity analysis 3-1. Methods for Manufacturing and Experimenting with Microparticles In this example, the concentration of poloxamer-188 was increased to produce microparticles, and the effect on the fluorescence intensity of the microparticles was observed. To produce ICG-HSA-mounted PLGA microparticles (ICG-HSA-PLGA(P-188 0.5%)) containing 0.5% (oil phase) of primary emulsion-based poloxamer-188 (P-188, BASF), solution W1 was prepared by dissolving 0.75 mg of indocyanine green in a mixed solution of 0.322 mL of human serum albumin (20% concentration, 64.335 mg) and 1.178 mL of distilled water. After adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, it was dissolved while stirring at 600 rpm at 85 °C, and then cooled to room temperature to produce solution W2. An oil solution was prepared by dissolving 1.08 g of PLGA and 82.5 mg of poloxamer-188 in 15 mL of methylene chloride. After adding the prepared W1 solution to the oil solution, sonication was performed for 40 seconds using a water bath type ultrasonic device to produce a primary emulsion (water-in-oil). A secondary emulsion (water-in-oil-in-water) was prepared by adding the prepared W2 solution to the prepared primary emulsion while stirring at 1000 rpm using a mechanical stirrer. After stirring the prepared emulsion at 1000 rpm for 10 minutes, the methylene chloride solvent was removed by stirring at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40 °C. Then, the PLGA polymer microparticles carrying indocyanine green were obtained by centrifugation at 14,000 rpm for 5 minutes. The supernatant was removed, and the microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed fine particles were rapidly cooled using liquid nitrogen and then freeze-dried to obtain fine particle powder.

[0071] To produce ICG-HSA-equipped PLGA microparticles (ICG-HSA-PLGA(P-188 1.0%)) containing 1.0% (oil phase) of poloxamer-188 as a primary emulsion phase standard, solution W1 was prepared by dissolving 0.75 mg of indocyanine green in a mixed solution of 0.322 mL of human serum albumin (20% concentration, 64.335 mg) and 1.178 mL of distilled water. After adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, the mixture was dissolved at 85 °C with stirring at 600 rpm, and then cooled to room temperature to produce solution W2. Dissolve 1.08 g of PLGA and 165 mg of poloxamer-188 in 15 mL of methylene chloride to prepare an oil solution. After adding the prepared W1 solution to the oil solution, sonication was performed for 40 seconds using a water bath type ultrasonic device to produce a primary emulsion (water-in-oil). The prepared primary emulsion was used to produce a secondary emulsion (water-in-oil-in-water) by adding the prepared W2 solution while stirring at 1000 rpm using a mechanical stirrer. The prepared emulsion was stirred at 1000 rpm for 10 minutes, and then stirred at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C to remove the methylene chloride solvent. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PLGA polymer microparticles containing indocyanine green. The supernatant was removed, and the microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed microparticles were rapidly cooled with liquid nitrogen and freeze-dried to obtain microparticle powder.

[0072] To evaluate the indocyanine green content and loading efficiency in the manufactured microparticles, the concentration of indocyanine green in the microparticle dissolution solution was quantified using an ultraviolet / visible light absorbance spectrometer. A high-concentration indocyanine green / human serum albumin complex solution was prepared by dissolving 0.5 mg of indocyanine green in a mixed solution of 0.214 mL (20% concentration, 42.89 mg) of human serum albumin and 0.786 mL of distilled water. This high-concentration complex solution was diluted with distilled water to prepare 13 standard solutions with concentrations ranging from 0.065 to 6.54 μM. The absorbance of these standard solutions was measured using an ultraviolet / visible light absorbance spectrometer to create a calibration curve. Then, 10 mg of PLGA microparticles were dissolved in 1 mL of dimethyl sulfoxide solvent, and the absorbance of this solution was analyzed using an ultraviolet / visible light absorbance spectrometer. The concentration was then measured by substituting the results into the calibration curve. The measured complex concentrations were substituted into the following formula to evaluate the indocyanine green content and loading efficiency in the microparticles.

[0073] Scanning electron microscopy analysis was performed to analyze the particle morphology and size of the synthesized microparticles. For cross-sectional analysis of the manufactured microparticles, the microparticle powder was pulverized with a razor blade. After attaching the microparticle surface and cross-sectional microparticle powder to carbon tape attached to a scanning electron microscope sample holder, Pt / Pd was coated onto the surface by sputtering for 60 seconds, and the scanning electron microscope images were analyzed with a 5kV acceleration voltage. The particle size was determined by measuring the size of 100 randomly selected particles from the obtained images and averaging the result.

[0074] For the comparison of the fluorescence signals between the prepared microparticles and the indocyanine green / human serum albumin complex, complex solutions and microparticle dispersions of indocyanine green at the same concentration were prepared. After dissolving 0.5 mg of indocyanine green powder in a mixed solution of 0.214 mL of human serum albumin solution (20% concentration) and 0.786 mL of distilled water, it was diluted with distilled water to prepare 0.3 mL of indocyanine green / human serum albumin complex (ICG-HSA) at a concentration of 30 μM. The content of indocyanine green in the PLGA microparticles evaluated by the above quantification was added to 0.3 mL of distilled water using the information so that the concentration of indocyanine green became 30 μM to prepare a dispersion. For the comparison of fluorescence spectra, 200 μL each of the prepared solution and dispersion were aliquoted and placed in a 96-well plate, and the fluorescence spectrum (λ ex = 730 nm) was measured using a multifunctional microplate reader. Fluorescence images were obtained by putting 300 μL of the same solution into a microtube and then analyzing it using a fluorescence imaging device (IVIS Lumina XR, Xenogen Corporation-Caliper, CA, USA) (λ ex = 780 / 20 nm, λ em = 845 / 40 nm).

[0075] For the evaluation of the fluorescence persistence of the surgical marker microparticles in an animal model, 100 μL of an ICG-HSA solution and a microparticle dispersion with an ICG concentration of 30 μM prepared using physiological saline for injection containing 0.5% carboxymethyl cellulose (Sodium carboxymethyl cellulose, Sigma-Aldrich) and 0.1% Tween (Tween 80, Sigma-Aldrich) were injected subcutaneously into SKH-1 hairless mice (Orient Bio Inc.) using a 1 mL syringe (21G needle). Then, fluorescence signals over time were photographed using a fluorescence imaging device (IVIS Lumina XR) (λ ex = 780 / 20 nm, λ em = 845 / 40 nm).

[0076] 3-2. Comparative Analysis of Indocyanine Green Loading Rate and Fluorescence Intensity When the concentration of poloxamer-188 in the primary emulsion phase was increased to 0.5% during the production of ICG-HSA-PLGA microparticles, the ICG loading rate in the ICG-HSA-PLGA(P-188 0.5%) microparticles was 13.8%, and the indocyanine green content in 1 mg of microparticles was 124.2 ng. This confirmed an increase in both the loading rate and content of indocyanine green compared to ICG-HSA-PLGA(P-188 0.1%). When the concentration of poloxamer-188 in the primary emulsion phase was increased to 1.0% during the production of ICG-HSA-PLGA microparticles, the ICG loading rate in the ICG-HSA-PLGA(P-188 1.0%) microparticles was 8.71%, and the indocyanine green content in 1 mg of microparticles was 72.7 ng, confirming that both the loading rate and content of indocyanine green increased compared to ICG-HSA-PLGA(P-188 0.1%).

[0077] Scanning electron microscope (SEM) images of ICG-HSA-PLGA (P-188 0.5%) and ICG-HSA-PLGA (P-188 1.0%) microparticles show that the two microparticles are spherical in shape with average particle diameters of 59.8±19.6 μm and 68.0±27.6 μm, respectively (Figure 5, (a, b: ICG-HSA-PLGA (P-188 0.5%), c, d: ICG-HSA-PLGA (P-188) (1.0%), and after evaluating the cross-sectional area of ​​both microparticles using a scanning electron microscope, it was confirmed that the PLGA microparticles have multiple small spherical spaces inside. In the case of PLGA microbubbles, the surface thickness is thin, and if the surface decomposes and holes are created, the dye contained within the microbubbles rapidly dissolves. In contrast, in the case of the microparticles manufactured in this invention, where multiple spherical spaces containing the dye are isolated from each other, it is possible to prevent the dissolution of the fluorescent dye even if the surface of the microparticle partially decomposes and holes are created, and there is an advantage that the fluorescent dye can be maintained at a high concentration within the microparticle for a long period of time at the labeled site.

[0078] Figure 6 shows the fluorescence spectra of ICG-HSA-PLGA (P-188 0.5%) and ICG-HSA-PLGA (P-188 1.0%) dispersions. Higher fluorescence signals were observed compared to ICG-HSA (2.1 times and 3.6 times higher, respectively) and ICG-PLGA (without P-188) (14 times and 25 times higher, respectively). This result indicates that very high fluorescence signals can be obtained when manufacturing fine particles containing 0.5% or more poloxamer-188 relative to the primary emulsion phase.

[0079] Even when the particulate dispersion was imaged with a fluorescence imaging device (Figure 7, a: ICG-HSA, b: ICG-HSA-PLGA (P-188 0.5%), c: ICG-HSA-PLGA (P-188 1.0%)), it was confirmed that strong fluorescence signals were generated for ICG-HSA-PLGA (P-188 0.5%) and ICG-HSA-PLGA (P-188 1.0%) particulates.

[0080] Figure 8 shows the results of subcutaneous injection of ICG-HSA solution, ICG-HSA-PLGA (P-188 0.5%), and ICG-HSA-PLGA (P-188 1.0%) microparticle dispersions into SKH-1 hairless mice, with fluorescence images (λ) observed over time. ex =780 / 20 nm, λ em This shows the fluorescence distribution (=845 / 40 nm). When ICG-HSA was injected, there was significant fluorescence diffusion at the injection site, and it was confirmed that the fluorescence signal was hardly detectable from day 1 after injection (Figure 8-a). On the other hand, in the case of ICG-HSA-PLGA (P-188 0.5%) (Figure 8-b) and ICG-HSA-PLGA (P-188 1.0%) (Figure 8-c) microparticle dispersions, the fluorescence signal generated in the labeled region decreased slightly until day 1, but by day 30, there was no fluorescence diffusion and a strong fluorescence signal was maintained, and it was found that the labeled area could be clearly identified from the fluorescence image.

[0081] Figure 9 shows the ROI values ​​for the fluorescence signal at the labeled site, analyzed from the aforementioned fluorescence images. In the case of ICG-HSA, the fluorescence signal decreased rapidly after injection, dropping to a low level of 6.4% after one day, and almost completely disappearing after two days.

[0082] In the case of ICG-HSA-PLGA(P-188 0.5%) and ICG-HSA-PLGA(P-188 1.0%) microparticle dispersions, the highest fluorescence intensity was observed at 5 hours, with 46% and 65% of fluorescence remaining after 1 day, and 26% and 48% of fluorescence still remaining after 30 days. From these results, it was confirmed that in the case of ICG-HSA-PLGA(P-188 0.5%) and ICG-HSA-PLGA(P-188 1.0%) microparticles, there was no bleeding of the fluorescent dye at the labeled site, and a strong fluorescence signal could be maintained for more than 30 days.

[0083] Example 4: Production and analysis of fine particles using polymers other than PLGA 4-1: Production and analysis of microparticles using PLGA and PDLLA-PEG block copolymers For the production of microparticles, poly(D,L-lactide)-block-poly(ethylene glycol) (PDLLA-PEG, Sigma-Aldrich) was used, with PDLLA and PEG blocks having molecular weights of 20,000 and 5,000 Da, respectively. For the production of ICG-HSA-Alginate-PLGA and PDLLA-PEG (P-188 0.1%) microparticles, solution W1 was prepared by first dissolving 0.75 mg of indocyanine green (ICG) in 0.322 mL (20% concentration, 64.335 mg) of human serum albumin (HSA) and 0.278 mL of distilled water, and then mixing a solution of 6 mg of sodium alginate (ALG, Kimica) dissolved in 0.6 mL of distilled water. 3.6 g of polyvinyl alcohol was added to 180 mL of distilled water, dissolved at 85°C with stirring at 600 rpm, and cooled to room temperature to produce the W2 solution. 1.08 g of PLGA, 0.12 mg of PDLLA-PEG, and 16.5 mg of poloxamer-188 (P-188) were added to 15 mL of methylene chloride and dissolved to produce the oil solution. The prepared W1 solution was added to the oil solution and subjected to sonication for 40 seconds using a water bath type ultrasonic device. 0.3 mL of calcium chloride (CaCl2, Sigma-Aldrich) at a concentration of 4.304 mg / mL was further added and subjected to sonication for another 40 seconds. As a result, the alginic acid dissolved in the W1 phase underwent ionic crosslinking and gelled, producing a primary emulsion (water-in-oil). A secondary emulsion (water-in-oil-in-water) was prepared by adding the prepared primary emulsion to the W2 solution while stirring at 1000 rpm using a mechanical stirrer. The prepared emulsion was stirred at 1000 rpm for 10 minutes, and then stirred at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C to remove the methylene chloride solvent. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PLGA polymer microparticles containing indocyanine green. The supernatant was removed, and the microparticles were washed by adding distilled water for redispersion and repeating the centrifugation process three times.The washed microparticles were freeze-dried to obtain ICG-HSA-Alginate-PLGA, PDLLA-PEG (P-188 0.1%) microparticle powder.

[0084] To evaluate the indocyanine green content and loading rate in the manufactured microparticles, the microparticles were dissolved, and the concentration of indocyanine green in the solution was quantified using an ultraviolet / visible light absorption analyzer.

[0085] Scanning electron microscopy analysis was performed to analyze the particle morphology and size of the synthesized microparticles. For cross-sectional analysis of the manufactured microparticles, the particles were cut with a razor blade. The microparticle powder was attached to carbon tape mounted on a scanning electron microscope sample holder, and then coated with Pt / Pd via sputtering for 60 seconds. Scanning electron microscope images were then analyzed with a 5kV acceleration voltage. The particle size was determined by measuring the size of 100 randomly selected particles from the obtained images and averaging the result.

[0086] To compare the fluorescence signals of the aforementioned manufactured microparticles and the indocyanine green / human serum albumin complex, a complex solution and a microparticle dispersion of the same concentration of indocyanine green were prepared. 0.5 mg of indocyanine green powder was dissolved in a mixture of 0.214 mL of human serum albumin solution (20% concentration) and 0.786 mL of distilled water, and then diluted with distilled water to prepare 0.3 mL of a 30 μM indocyanine green / human serum albumin complex (ICG-HSA). Using the information obtained from the quantitative analysis of indocyanine green content in PLGA and PDLLA-PEG microparticles, a microparticle dispersion was prepared by adding it to 0.3 mL of distilled water to achieve a concentration of 30 μM. For fluorescence spectrum comparison, 200 μL each of the prepared solution and microparticle dispersion were taken and placed in a 96-well plate, and the fluorescence spectra (λ) were analyzed using a multifunctional microplate reader. ex The fluorescence image was measured at λ = 730 nm. 300 μL of the same solution was placed in a microtube and analyzed using a fluorescence imaging device (IVIS Lumina XR) (λ). ex=780 / 20 nm, λ em (=845 / 40 nm)

[0087] To evaluate the changes in fluorescence intensity of microparticles injected from an animal model, a microparticle dispersion was prepared using injectable saline containing 0.5% sodium carboxymethyl cellulose (Sigma-Aldrich) and 0.1% Tween (Sigma-Aldrich). 100 μL of this dispersion was injected subcutaneously into SKH-1 hairless mice. The fluorescence signals over time were captured using a fluorescence imaging system (IVIS Lumina XR) (λ ex =780 / 20 nm, λ em (=845 / 40 nm)

[0088] The indocyanine green content of ICG-HSA-Alginate-PLGA and PDLLA-PEG (P-188 0.1%) microparticles was 15.8%, and the indocyanine green content per 1 mg of microparticle was 234.6 ng, confirming an increase in indocyanine green content compared to conventional conditions.

[0089] The ICG-HSA-Alginate-PLGA and PDLLA-PEG (P-188 0.1%) microparticles were confirmed to be spherical particles with an average particle diameter of 74.3 ± 18.2 μm, possessing an appropriate size to avoid phagocytic activity by macrophages (Figure 10(a)). Furthermore, scanning electron micrographs of the particle cross-sections (Figure 10(b)) showed that the interior of the particles contained multiple isolated small spherical spaces, similar to ICG-HSA-PLGA (P-188 0.5%) and ICG-HSA-PLGA (P-188 1.0%), which prevented the elution of the carried-on fluorescent dye, confirming that this morphology is highly suitable for long-term fluorescent dye labeling.

[0090] Figure 11 shows that the fluorescence spectra of ICG-HSA-Alginate-PLGA and PDLLA-PEG (P-188 0.1%) particle dispersions exhibited fluorescence signals 2.3 times higher than ICG-HSA and 15 times higher than ICG-PLGA (without P-188).

[0091] The fluorescence image comparison results of the particulate dispersions (Figure 12) reconfirmed that the fluorescence signals of ICG-HSA-Alginate-PLGA and PDLLA-PEG (P-188 0.1%) particulate dispersion (Figure 12-b) were higher than those of the ICG-HSA solution (Figure 12-a).

[0092] Figure 13 shows the fluorescence images (λ) obtained by subcutaneous injection of ICG-HSA solution, ICG-HSA-Alginate-PLGA, and PDLLA-PEG (P-188 0.1%) microparticle dispersion into SKH-1 hairless mice. ex = 780 / 20 nm, λ em The image was taken at 845 / 40 nm. In the case of ICG-HSA, fluorescence was observed up to 5 hours, and it was confirmed that fluorescence was not observed after 1 day (Figure 13-a). On the other hand, in the case of ICG-HSA-Alginate-PLGA and PDLLA-PEG (P-188 0.1%) microparticle dispersion (Figure 13-b), the fluorescently labeled area gradually decreased after 7 days, but it was confirmed that the fluorescence signal remained high until 30 days. The ROI values ​​analyzed from the fluorescence images are shown in a graph (Figure 14). Unlike ICG-HSA, it was confirmed that when ICG-HSA-Alginate-PLGA and PDLLA-PEG (P-188 0.1%) microparticles were administered, 79% of the fluorescence signal was maintained after 1 day and 32% after 30 days. This confirms that fluorescence labeling of lesion sites is possible for more than 30 days when using ICG-HSA-Alginate-PLGA and PDLLA-PEG (P-188 0.1%) microparticles.

[0093] Example 4-2: Production and analysis of fine particles using PMMA polymer To produce PMMA microparticles, polymethyl methacrylate (PMMA; MW 120,000 Da, Sigma-Aldrich) polymer was used to produce ICG-HSA-PMMA (P-188 1.0%) microparticles. Solution W1 was prepared by dissolving 0.75 mg of indocyanine green (ICG) in a mixed solution of 0.322 mL (20% concentration, 64.335 mg) of human serum albumin (HSA) and 1.178 mL of distilled water. Solution W2 was prepared by adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, dissolving it at 85°C with stirring at 600 rpm, and then cooling it to room temperature. An oil solution was prepared by dissolving 1.08 g of PMMA and 165 mg of poloxamer-188 (P-188) in 15 mL of methylene chloride. After adding the prepared W1 solution to the oil solution, the mixture was ultrasonically treated with a probe-type ultrasonic device for 3 minutes (1 minute ultrasonic treatment + 30 second rest, repeated) to produce a primary emulsion (water-in-oil). The prepared W2 solution was stirred at 1000 rpm using a mechanical stirrer, and the prepared primary emulsion solution was added at a rate of 30 mL / min using a syringe pump to produce a secondary emulsion (water-in-oil-in-water). The prepared emulsion was stirred at 1000 rpm for 10 minutes, and then stirred at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C to remove the methylene chloride solvent. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PMMA polymer microparticles containing indocyanine green. The supernatant was removed, and the PMMA microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed microparticles were freeze-dried to obtain microparticle powder.

[0094] To evaluate the indocyanine green content and loading rate in the manufactured microparticles, the microparticles were dissolved, and the concentration of indocyanine green in the solution was quantified using an ultraviolet / visible light absorption analyzer.

[0095] Scanning electron microscopy analysis was performed to analyze the particle morphology and size of the synthesized microparticles. For cross-sectional analysis of the manufactured microparticles, the particles were cut with a razor blade. The microparticle powder was attached to carbon tape mounted on a scanning electron microscope sample holder, and then coated with Pt / Pd via sputtering for 60 seconds. Scanning electron microscope images were then analyzed with a 5kV acceleration voltage. The particle size was determined by measuring the size of 100 randomly selected particles from the obtained images and averaging the result.

[0096] To compare the fluorescence signals of the aforementioned manufactured microparticles and the indocyanine green / human serum albumin complex, a complex solution and a microparticle dispersion of the same concentration of indocyanine green were prepared. 0.5 mg of indocyanine green powder was dissolved in a mixture of 0.214 mL of human serum albumin solution (20% concentration) and 0.786 mL of distilled water, and then diluted with distilled water to prepare 0.3 mL of a 30 μM indocyanine green / human serum albumin complex (ICG-HSA). Using the information obtained from the quantitative analysis of indocyanine green content in PMMA microparticles, the indocyanine green was added to 0.3 mL of distilled water to prepare a microparticle dispersion with a concentration of 30 μM. For fluorescence spectrum comparison, 200 μL each of the prepared solution and microparticle dispersion were taken and placed in a 96-well plate, and the fluorescence spectra (λ) were analyzed using a multifunctional microplate reader. ex The fluorescence image was measured at λ = 730 nm. 300 μL of the same solution was placed in a microtube and analyzed using a fluorescence imaging device (IVIS Lumina XR) (λ). ex =780 / 20 nm, λ em (=845 / 40 nm)

[0097] To evaluate the changes in fluorescence intensity of microparticles injected from an animal model, a microparticle dispersion was prepared using injectable saline containing 0.5% sodium carboxymethyl cellulose (Sigma-Aldrich) and 0.1% Tween (Sigma-Aldrich). 100 μL of this dispersion was injected subcutaneously into SKH-1 hairless mice. The fluorescence signals over time were captured using a fluorescence imaging system (IVIS Lumina XR) (λ ex =780 / 20 nm, λ em (=845 / 40 nm)

[0098] The indocyanine green content of the ICG-HSA-PMMA (P-188 1.0%) microparticles was 44.0%, and the indocyanine green content was high at 370.3 ng per 1 mg of microparticles.

[0099] The ICG-HSA-PMMA(P-188 1.0%) microparticles were confirmed to be spherical particles with an average particle diameter of 49.0 ± 18.7 μm, possessing an appropriate size to avoid phagocytic activity by macrophages (Figure 15(a)). Furthermore, scanning electron microscopy images of the particle cross-section (Figure 15(b)) showed that the inside of the particles contained multiple isolated small spherical spaces, which prevented the elution of fluorescent dyes carried within these spaces, confirming that this morphology is highly suitable for long-term fluorescent dye labeling.

[0100] Figure 16 shows that the fluorescence spectrum of the ICG-HSA-PMMA (P-188 1.0%) fine particle dispersion exhibits a fluorescence signal at a similar level to that of ICG-HSA, and a fluorescence signal seven times higher than that of ICG-PLGA (without P-188).

[0101] The fluorescence image comparison results of the ICG-HSA-PMMA (P-188 1.0%) fine particle dispersion (Figure 17) confirmed that it generated a high fluorescence signal similar to that of the ICG-HSA solution.

[0102] Figure 18 shows the results of subcutaneous injection of ICG-HSA solution and ICG-HSA-PMMA (P-188 1.0%) microparticle dispersion into SKH-1 hairless mice, with fluorescence images (λ) obtained over time. ex =780 / 20 nm, λ em This is the result of imaging at 845 / 40 nm. Unlike ICG-HSA (Figure 18-a), when ICG-HSA-PMMA (P-188 1.0%) microparticles (Figure 18-b) were administered, there was no bleeding of the fluorescent dye, and a high fluorescence signal was maintained for up to 30 days.

[0103] Figure 19 shows a graph of ROI values ​​analyzed from fluorescence images. Unlike ICG-HSA, the ICG-HSA-PMMA (P-188 1.0%) microparticle dispersion retained 55% fluorescence after 1 day, and maintained a fluorescence intensity of 36% even after 30 days. This result confirms that ICG-HSA-PMMA (P-188 1.0%) microparticles are useful as a fluorescent marker that can maintain a fluorescence signal for more than 30 days in labeled lesions.

[0104] From the above examples, it was confirmed that when microparticles are manufactured using ICG alone, microparticles that generate a very weak fluorescence signal are obtained, while microparticles that generate a high fluorescence signal can be manufactured by manufacturing microparticles using an ICG-HSA composite, or by mixing ICG-HSA with an alginate polymer and then gelling it via ion crosslinking.

[0105] Example 5: Production and analysis of ICG-equipped PLGA microparticles using alginate hydrated gel In this example, we analyzed whether the fluorescence signal of ICG-containing microparticles could be increased even when ICG was mixed with alginic acid to make solution W1 and the alginic acid was ionically crosslinked using calcium. To produce PLGA microparticles containing ICG in an alginic acid hydrate gel (ICG-Alginate-PLGA (without P-188)), solution W1 was prepared by mixing a solution of 0.75 mg of indocyanine green (ICG) dissolved in 0.6 mL of distilled water with a solution of 6 mg of sodium alginate dissolved in 0.6 mL of distilled water. After adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, it was dissolved while stirring at 600 rpm at 85 °C and cooled to room temperature to produce solution W2. 1.08 g of PLGA was added to 15 mL of methylene chloride and dissolved to produce an oil solution. After adding the prepared W1 solution to the oil solution, sonication was performed for 40 seconds using a water bath type ultrasonic device. To induce ionic crosslinking of alginate, 0.3 mL of calcium chloride (CaCl2, Sigma-Aldrich) at a concentration of 4.304 mg / mL was further added, and sonication was performed for another 40 seconds to produce a primary emulsion (water-in-oil) in which alginate had gelled. Using a mechanical stirrer, the W2 solution was stirred at 1000 rpm while the prepared primary emulsion was added to produce a secondary emulsion (water-in-oil-in-water). The prepared emulsion was stirred at 1000 rpm for 10 minutes, and then stirred at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C to remove the methylene chloride solvent. Subsequently, centrifugation was performed at 14,000 rpm for 5 minutes to obtain ICG-Alginate-PLGA (without P-188) particles, which are PLGA polymer microparticles carrying indocyanine green. The supernatant was removed, distilled water was added for redispersion, and the particles were washed by repeating this process three times. The washed particles were then freeze-dried to obtain a fine particle powder.

[0106] Next, ICG-Alginate-PLGA (P-188 0.1%) fine particles were prepared by further mixing 0.1% (oil phase) of poloxamer-188 (P-188) based on the primary emulsion phase. Solution W1 was prepared by mixing a solution of 0.75 mg of indocyanine green (ICG) dissolved in 0.6 mL of distilled water with a solution of 6 mg of sodium alginate dissolved in 0.6 mL of distilled water. Solution W2 was prepared by adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, dissolving it at 85°C while stirring at 600 rpm, and then cooling it to room temperature. An oil solution was prepared by dissolving 1.08 g of PLGA and 16.5 mg of poloxamer-188 (P-188) in 15 mL of methylene chloride. After adding the prepared W1 solution to the oil solution, sonication was performed for 40 seconds using a water bath type ultrasonic device. To induce the formation of ionic crosslinks of the alginate polymer, 0.3 mL of calcium chloride (CaCl2, Sigma-Aldrich) at a concentration of 4.304 mg / mL was further added, and sonication was performed for another 40 seconds to produce a primary emulsion in which the alginate had gelled. The W2 solution was added to the prepared primary emulsion while stirring with a mechanical stirrer at 1000 rpm to produce a secondary emulsion (water-in-oil-in-water). The prepared emulsion was stirred at 1000 rpm for 10 minutes, and then stirred with a magnetic stirrer at 280 rpm for 21 hours at a temperature of 40°C to remove the methylene chloride solvent. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PLGA polymer microparticles containing indocyanine green. The supernatant was removed, and the microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed fine particles were freeze-dried to obtain a fine particle powder.

[0107] To evaluate the content and loading rate of indocyanine green in the manufactured microparticles, the concentration of indocyanine green in the microparticle dissolution solution was quantified using an ultraviolet / visible light absorption spectrometer. 10 mg of PLGA microparticles were placed in 1 mL of dimethyl sulfoxide (DMSO) solvent, and the absorbance of the dissolved solution was analyzed using an ultraviolet / visible light absorption spectrometer. The concentration of indocyanine green was measured by comparing it with a standard calibration curve.

[0108] To analyze the particle morphology and size of the synthesized microparticles, scanning electron microscopy (JSM-7800F Prime) analysis was performed. The microparticle powder was attached to carbon tape mounted on a scanning electron microscope sample holder, and then coated with Pt / Pd via sputtering for 60 seconds. The scanning electron microscope images were then analyzed with a 5kV acceleration voltage. The particle size was determined by measuring the size of 100 randomly selected particles from the obtained images and averaging the result.

[0109] To compare the fluorescence signals of the aforementioned manufactured microparticles and the indocyanine green / human serum albumin complex, a complex solution and a microparticle dispersion of the same concentration of indocyanine green were prepared. 0.5 mg of indocyanine green powder was dissolved in 0.214 mL of a 20% human serum albumin solution and 0.786 mL of a distilled water mixture, and then diluted with distilled water to prepare 0.3 mL of a 30 μM indocyanine green / human serum albumin complex (ICG-HSA). Using the information obtained from the quantitative analysis of indocyanine green content in the PLGA microparticles, a microparticle dispersion was prepared by adding indocyanine green to 0.3 mL of distilled water to achieve a concentration of 30 μM. For fluorescence spectrum comparison, 200 μL each of the prepared solution and microparticle dispersion were taken and placed in a 96-well plate, and the fluorescence spectra (λ) were analyzed using a multifunctional microplate reader. ex The wavelength (=730 nm) was measured.

[0110] The indocyanine green loading rate in ICG-Algiante-PLGA (without P-188) microparticles was 5.79%, and the indocyanine green content in 1 mg of microparticles was 69.0 ng. In the case of ICG-Algiante-PLGA (P-188 0.1%) microparticles with 0.1% poloxamer-188 added based on the primary emulsion phase, the indocyanine green loading rate increased to 9.21%, and the indocyanine green content in 1 mg of microparticles also increased to 84.3 ng.

[0111] In the scanning electron microscope image in Figure 20, it was confirmed that the two microparticles, ICG-Algiante-PLGA (without P-188) and ICG-Algiante-PLGA (with 0.1% P-188), were spherical particles with average particle sizes of 84.5 ± 33.2 μm and 70.2 ± 21.1 μm, respectively, confirming that they could effectively evade phagocytic activity by macrophages.

[0112] Figure 21 shows the fluorescence spectra of ICG-Algiante-PLGA (without P-188) and ICG-Algiante-PLGA (with P-188 0.1%) microparticle dispersions. Compared to ICG-PLGA (without P-188) microparticles, the ICG-Algiante-PLGA (without P-188) microparticles showed a slight increase in fluorescence, while the ICG-Algiante-PLGA (with P-188 0.1%) microparticles showed a five-fold higher fluorescence signal. From these results, it was found that by adding poloxamer-188 to the alginate gelation conditions, it is possible to produce microparticles that exhibit a high fluorescence signal.

[0113] Example 6: Preparation and analysis of PLGA microparticles by mixing ICG-HSA with alginate hydrate gel The ICG-HSA complex was mixed with an alginate polymer, and polymer microparticles were produced under conditions where the alginate polymer was ionically crosslinked with calcium. The fluorescence signals were then analyzed.

[0114] To produce ICG-HSA-Alginate-PLGA(P-188 0.1%) microparticles, which are ICG-HSA-mounted PLGA microparticles obtained by mixing alginic acid with ICG-HSA and adding poloxamer-188 (P-188) to the oil phase, a solution was prepared by mixing 75 mg of indocyanine green (ICG) with 0.322 mL of human serum albumin (HSA) (20% concentration, 64.335 mg) and 0.278 mL of distilled water. To this solution, a solution of 6 mg of sodium alginate (Soidum Alginate, ALG, Kimica) dissolved in 0.6 mL of distilled water was mixed to produce solution W1. After adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, it was dissolved while stirring at 600 rpm at 85°C and cooled to room temperature to produce solution W2. An oil solution was prepared by dissolving 1.08 g of PLGA and 16.5 mg of poloxamer-188 (P-188) in 15 mL of methylene chloride. After adding the prepared W1 solution to the oil solution, it was sonicated for 40 seconds using a water bath type ultrasonic device, and then 0.3 mL of calcium chloride (CaCl2, Sigma-Aldrich) at a concentration of 4.304 mg / mL was added and sonicated for another 40 seconds to prepare a primary emulsion (water-in-oil) in which alginic acid had gelled. A secondary emulsion (water-in-oil-in-water) was prepared by adding the primary emulsion to the W2 solution while stirring at 1000 rpm using a mechanical stirrer. After stirring the prepared emulsion at 1000 rpm for 10 minutes, the methylene chloride solvent was removed by stirring at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C. Subsequently, the PLGA polymer microparticles containing indocyanine green were obtained by centrifugation at 14,000 rpm for 5 minutes. The supernatant was removed, and the microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed microparticles were freeze-dried to obtain a microparticle powder.

[0115] To produce ICG-HSA-Alginate-PLGA(P-188 0.5%) microparticles, which are ICG-HSA-mounted PLGA microparticles obtained by mixing alginic acid with ICG-HSA and adding poloxamer-188 (P-188) to the oil phase, a solution was prepared by mixing 75 mg of indocyanine green (ICG) with 0.322 mL of human serum albumin (HSA) (20% concentration, 64.335 mg) and 0.278 mL of distilled water. To this solution, a solution of 6 mg of sodium alginate (Soidum Alginate, ALG, Kimica) dissolved in 0.6 mL of distilled water was added to prepare solution W1. After adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, it was dissolved at 85°C while stirring at 600 rpm, and then cooled to room temperature to produce solution W2. An oil solution was prepared by dissolving 1.08 g of PLGA and 82.5 mg of poloxamer-188 (P-188) in 15 mL of methylene chloride. After adding the prepared W1 solution to the oil solution, it was sonicated for 40 seconds using a water bath type ultrasonic device, and 0.3 mL of calcium chloride (CaCl2, Sigma-Aldrich) at a concentration of 4.304 mg / mL was added and sonicated for another 40 seconds to prepare a primary emulsion (water-in-oil) in which alginic acid had gelled. A secondary emulsion (water-in-oil-in-water) was prepared by adding the primary emulsion to the W2 solution while stirring at 1000 rpm using a mechanical stirrer. After stirring the prepared emulsion at 1000 rpm for 10 minutes, the methylene chloride solvent was removed by stirring at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C. Subsequently, the PLGA polymer microparticles containing indocyanine green were obtained by centrifugation at 14,000 rpm for 5 minutes. The supernatant was removed, and the microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed microparticles were freeze-dried to obtain a microparticle powder.

[0116] The content and density of indocyanine green in the manufactured microparticles were analyzed.

[0117] Scanning electron microscopy analysis was performed to observe the particle morphology, size, and cross-sectional shape of the synthesized fine particles. The particle diameter was determined by measuring the diameter of 100 randomly selected particles in the obtained photographs and averaging the result.

[0118] To compare the fluorescence signals of the aforementioned manufactured microparticles with those of the indocyanine green / human serum albumin complex, a complex solution and a microparticle dispersion of the same concentration of indocyanine green were prepared. For fluorescence spectrum comparison, 200 μL each of the prepared solution and microparticle dispersion were taken and placed in a 96-well plate, and the fluorescence spectra (λ) were measured using a multifunctional microplate reader. ex The fluorescence image was measured at λ = 730 nm. 300 μL of the same solution was placed in a microtube and analyzed using a fluorescence imaging device (IVIS Lumina XR) (λ). ex =780 / 20 nm, λ em (=845 / 40 nm)

[0119] To evaluate the fluorescent labeling efficacy of fluorescent marker microparticles in an animal model, a 30 μM ICG concentration ICG-HSA solution and microparticles were dispersed in injectable saline containing 0.5% carboxymethyl cellulose (sodium carboxymethyl cellulose, Sigma-Aldrich) and 0.1% Tween (Tween 80, Sigma-Aldrich). 100 μL of the microparticle dispersion was injected subcutaneously into SKH-1 hairless mice using a syringe, and the fluorescence signal over time was recorded using a fluorescence imaging device (IVIS Lumina XR) (λ ex =780 / 20 nm, λ em (=845 / 40 nm)

[0120] The indocyanine green content in ICG-HSA-Alginate-PLGA(P-188 0.1%) and ICG-HSA-Alginate-PLGA(P-188 0.5%) microparticles was 10.5% and 7.62%, respectively, and the indocyanine green content per 1 mg microparticle was analyzed to be 108.7 ng and 61.6 ng.

[0121] The ICG-HSA-Alginate-PLGA(P-188 0.1%) and ICG-HSA-Alginate-PLGA(P-188 0.5%) microparticles were confirmed to have a suitable size for avoiding phagocytic activity by macrophages, as spherical particles with average particle diameters of 77.8±26.5 μm and 67.9±24.1 μm (Figure 22). Furthermore, as can be seen from the cross-sectional image of the microparticles in Figure 22(c), the inside of the microparticles is composed of multiple isolated spherical spaces, which shows that the release of fluorescent dyes can be suppressed more effectively than with microbubble-type structures.

[0122] Figure 23 shows a comparison of fluorescence signals based on fluorescence spectra. ICG-HSA-Alginate-PLGA(P-188 0.1%) showed a fluorescence signal similar to ICG-HSA, and a fluorescence signal 6.4 times higher than ICG-PLGA(without P-188). In the case of ICG-HSA-Alginate-PLGA(P-188 0.5%) nanoparticles, it was found that the fluorescence signal was 2.5 times higher than ICG-HSA and 17 times higher than ICG-PLGA(without P-188).

[0123] Fluorescence images (Figure 24) also confirmed that the manufactured ICG-HSA-Alginate-PLGA (P-188 0.1%) (Figure 8C-b) and ICG-HSA-Alginate-PLGA (P-188 0.5%) (Figure 8C-c) fine particle dispersions generated strong fluorescence signals.

[0124] Figure 25 shows fluorescence images taken over time after subcutaneous injection of ICG-HSA solution and ICG-HSA-Alginate-PLGA(P-188 0.5%) microparticle dispersion into SKH-1 hairless mice (λ). ex =780 / 20 nm, λ em This is a photograph (845 / 40 nm). Unlike the case of ICG-HSA (Figure 25-a), where no fluorescence signal was observed after 1 day, it was confirmed that a high fluorescence signal was maintained until 30 days after administration of ICG-HSA-Alginate-PLGA (P-188 0.5%) microparticle dispersion (Figure 25-b).

[0125] The ROI values ​​analyzed using the aforementioned fluorescence images are shown in a graph (Figure 26). In the case of the ICG-HSA-Alginate-PLGA (P-188 0.5%) microparticle dispersion, 80% fluorescence remained after 1 day of administration, and a high fluorescence signal of 46% was observed even after 30 days.

[0126] These results confirmed that ICG-HSA-Alginate-PLGA(P-188) nanoparticles are highly useful as a fluorescent marker.

[0127] Example 7: Production and analysis of microparticles using Tween as a surfactant In this example, to demonstrate that other types of surfactants can be used instead of the surfactant P-188, microparticles were prepared and analyzed using Tween. Solution W1 was prepared by mixing 0.75 mg of indocyanine green (ICG) with 0.322 mL (20% concentration, 64.335 mg) of human serum albumin (HSA) and 0.278 mL of distilled water, and then mixing this solution with 6 mg of sodium alginate dissolved in 0.6 mL of distilled water. Solution W2 was prepared by adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, dissolving it at 85°C with stirring at 600 rpm, and then cooling it to room temperature. An oil solution was prepared by dissolving 1.08 g of PLGA and 82.5 mg of Tween (Sigma-Aldrich) in 15 mL of methylene chloride. After adding the prepared W1 solution to the oil solution, sonication was performed for 40 seconds using a water bath type ultrasonic device. Then, 0.3 mL of calcium chloride (CaCl2, Sigma-Aldrich) at a concentration of 4.304 mg / mL was added, and sonication was performed for another 40 seconds to produce a primary emulsion (water-in-oil) in which alginic acid had gelled. The W2 solution was added to the primary emulsion while stirring with a mechanical stirrer at 1000 rpm to produce a secondary emulsion (water-in-oil-in-water). The prepared emulsion was stirred at 1000 rpm for 10 minutes, and then stirred at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C to remove the methylene chloride solvent. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PLGA polymer microparticles containing indocyanine green. The supernatant was removed, and the microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed microparticles were freeze-dried to obtain a microparticle powder.

[0128] To evaluate the indocyanine green content and loading efficiency in the manufactured microparticles, the concentration of indocyanine green in the microparticle dissolution solution was quantified using an ultraviolet / visible light absorption analyzer. The measured composite concentration was used to evaluate the indocyanine green content and loading efficiency in the microparticles.

[0129] Scanning electron microscopy analysis was performed to analyze the particle morphology, size, and cross-section of the synthesized microparticles. After attaching the microparticle powder for surface and cross-sectional analysis to carbon tape attached to a scanning electron microscope sample holder, Pt / Pd was coated onto the surface via sputtering for 60 seconds, and the scanning electron microscope images were analyzed with a 5kV acceleration voltage. The particle size was determined by measuring the size of 100 randomly selected particles from the obtained images and averaging the result.

[0130] To compare the fluorescence signals of the manufactured microparticles with those of the indocyanine green / human serum albumin complex, a complex solution and a microparticle dispersion of the same concentration of indocyanine green were prepared. For fluorescence spectrum comparison, 200 μL each of the prepared solution and microparticle dispersion were taken and placed in a 96-well plate, and the fluorescence spectra (λ) were measured using a multifunctional microplate reader. ex The fluorescence image was measured at λ = 730 nm. 300 μL of the same solution was placed in a microtube and analyzed using a fluorescence imaging device (IVIS Lumina XR) (λ). ex =780 / 20 nm, λ em (=845 / 40 nm)

[0131] To evaluate the fluorescent labeling efficacy of fluorescent marker microparticles in an animal model, a 30 μM ICG concentration ICG-HSA solution and microparticles were dispersed in injectable saline containing 0.5% carboxymethyl cellulose (sodium carboxymethyl cellulose, Sigma-Aldrich) and 0.1% Tween (Tween 80, Sigma-Aldrich). 100 μL of the microparticle dispersion was injected subcutaneously into SKH-1 hairless mice using a syringe, and the fluorescence signal over time was recorded using a fluorescence imaging device (IVIS Lumina XR) (λ ex =780 / 20 nm, λ em (=845 / 40 nm)

[0132] In the case of ICG-HSA-Algiante-PLGA (Tween 0.5%) microparticles, in which poloxamer-188, a surfactant that dissolves in the oil phase under the manufacturing conditions of ICG-HSA-Algiante-PLGA (P-188 0.5%), was replaced with tween, the indocyanine green loading rate within the microparticles was 9.68%, and the indocyanine green content in 1 mg of microparticles was confirmed to be 81.0 ng.

[0133] As can be seen in the scanning electron microscope image in Figure 27, the ICG-HSA-Algiante-PLGA(Tween 0.5%) microparticles have a spherical particle morphology (Figure 27(a)), and the average particle diameter is 66.1 ± 26.0 μm, which is a size suitable for avoiding phagocytic activity by macrophages. Furthermore, as can be confirmed in the cross-sectional scanning electron microscope image of the particles (Figure 27(b)), the inside of the microparticles is fractionated into multiple isolated small spheres, confirming that they have a structure that suppresses the release of the onboard fluorescent dye, similar to the results of previous examples.

[0134] The ICG-HSA-Algiante-PLGA (Tween 0.5%) microparticle dispersion showed a fluorescence signal twice as high as ICG-HSA and 13 times higher than ICG-PLGA (without P-188) (Figure 28).

[0135] From the fluorescence image in Figure 29, it was confirmed that the ICG-HSA-Algiante-PLGA(P-188 0.5%) (Figure 29-b) fine particle dispersion generates a strong fluorescence signal.

[0136] Figure 30 shows the results of subcutaneous injection of ICG-HSA solution (Figure 30-a) and ICG-HSA-Alginate-PLGA (Tween 0.5%) microparticle dispersion (Figure 30-b) into SKH-1 hairless mice, with fluorescence images (λ) obtained over time. ex =780 / 20 nm, λ emThis is a photograph taken at 845 / 40 nm. Unlike when ICG-HSA is injected, with the ICG-HSA-Alginate-PLGA(P-188 0.5%) microparticle dispersion, there was no fluorescence leakage into the surrounding tissue, and it was confirmed that the fluorescence signal was maintained at a high level at the administration site for 30 days.

[0137] The ROI values ​​analyzed from the aforementioned fluorescence images are shown in a graph (Figure 31). In the case of the ICG-HSA-Alginate-PLGA (Tween 0.5%) microparticle dispersion, 70% of the fluorescence remained after 1 day, and 52% of the fluorescence still remained after 30 days. From these results, it was found that even when the surfactant poloxamer-188 is replaced with another surfactant such as Tween to manufacture the microparticles, it is possible to manufacture microparticles that can fluorescently label lesion sites for a long period of time while generating a high fluorescence signal.

[0138] Example 8: Production and analysis of microparticles containing an indocyanine green / cyclodextrin complex Indocyanine green was complexed with cyclodextrin (mCD), and microparticles containing this complex were manufactured. The fluorescence intensity and efficacy as a fluorescent marker were then analyzed. To produce PLGA microparticles containing an indocyanine green / cyclodextrin complex ((ICG-mCD-Alginate-PLGA(P-188 0.1%)), a solution of 0.375 mg of indocyanine green (ICG) dissolved in 0.3 mL of ethanol and a solution of 1.790 mg of methylcyclodextrin (mCD, Sigma-Aldrich) dissolved in 0.6 mL of distilled water were mixed and stirred for 6 hours. This was mixed with a solution of 3.6 mg of sodium alginate dissolved in 0.648 mL of distilled water to produce solution W1. After adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, it was dissolved at 85°C with stirring at 600 rpm and cooled to room temperature to produce solution W2. 1.08 g of PLGA and 16.5 g of poloxamer-188 (P-188) were added. A solution was prepared by dissolving mg of alginate in 15 mL of methylene chloride. After adding the prepared W1 solution to the oil solution, it was ultrasonically treated for 40 seconds using a water bath type ultrasonic device, and 0.072 mL of calcium chloride (CaCl2, Sigma-Aldrich) at a concentration of 10.76 mg / mL was added and ultrasonically treated for another 40 seconds to prepare a primary emulsion (water-in-oil) in which alginate had gelled. A secondary emulsion (water-in-oil-in-water) was prepared by adding the primary emulsion to the W2 solution while stirring at 1000 rpm using a mechanical stirrer. After stirring the prepared emulsion at 1000 rpm for 10 minutes, the methylene chloride solvent was removed by stirring at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C. The PLGA polymer microparticles containing indocyanine green were obtained by centrifugation at rpm for 5 minutes. The supernatant was removed, and the microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed microparticles were freeze-dried to obtain a microparticle powder.

[0139] The content and density of indocyanine green in the manufactured microparticles were analyzed.

[0140] Scanning electron microscopy analysis was performed to observe the particle morphology and size analysis, as well as the cross-sectional shape of the synthesized fine particles. The particle diameter was determined by measuring the diameter of 100 randomly selected particles in the obtained photographs and averaging the result.

[0141] To compare the fluorescence signals of the aforementioned manufactured microparticles and the indocyanine green / human serum albumin complex, a complex solution and a microparticle dispersion of the same concentration of indocyanine green were prepared. 0.5 mg of indocyanine green powder was dissolved in a mixture of 0.214 mL of human serum albumin solution (20% concentration) and 0.786 mL of distilled water, and then diluted with distilled water to prepare 0.3 mL of a 30 μM indocyanine green / human serum albumin complex (ICG-HSA). Using the information obtained from the quantitative analysis of indocyanine green content in the PLGA microparticles, the indocyanine green concentration was adjusted to 30 μM by adding it to 0.3 mL of distilled water to prepare a microparticle dispersion. For fluorescence spectrum comparison, 200 μL each of the prepared solution and dispersion were taken and placed in a 96-well plate, and the fluorescence spectra (λ) were analyzed using a multifunctional microplate reader. ex The fluorescence image was measured at λ = 730 nm. 300 μL of the same solution was placed in a microtube and analyzed using a fluorescence imaging device (IVIS Lumina XR) (λ). ex =780 / 20 nm, λ em (=845 / 40 nm)

[0142] To evaluate the fluorescent labeling efficacy of fluorescent marker microparticles in an animal model, a 30 μM ICG concentration ICG-HSA solution and microparticles were dispersed in injectable saline containing 0.5% carboxymethyl cellulose (sodium carboxymethyl cellulose, Sigma-Aldrich) and 0.1% Tween (Tween 80, Sigma-Aldrich). 100 μL of the microparticle dispersion was injected subcutaneously into SKH-1 hairless mice using a syringe, and the fluorescence signal over time was captured using a fluorescence imaging device (IVIS Lumina XR) (λ ex =780 / 20 nm, λ em (=845 / 40 nm)

[0143] The indocyanine green content within the ICG-mCD-Alginate-PLGA (P-188 0.1%) microparticles containing indocyanine green / cyclodextrin was 11.4%, and the indocyanine green content per 1 mg microparticle was confirmed to be 76.3 ng.

[0144] The ICG-mCD-Alginate-PLGA(P-188 0.1%) microparticles, as spherical particles (Figure 32(a)), had an average size of 65.0 ± 26.0 μm, which was suitable for avoiding phagocytic activity by macrophages. Furthermore, as can be seen from the cross-sectional image of the microparticles in Figure 32(b), the inside of the microparticles is composed of multiple isolated spherical spaces, which indicates that the release of fluorescent dyes can be suppressed more effectively than with microbubble-shaped structures.

[0145] As can be seen from the fluorescence spectrum in Figure 33(a), ICG-mCD exhibits a fluorescence signal 1.5 times higher than that of ICG. This means that forming a complex with mCD can prevent the aggregation of ICG molecules in aqueous solution and also increase the chemical and physical stability of ICG.

[0146] We confirmed that an ICG-mCD-Algiante-PLGA (P-188 0.1%) microparticle dispersion prepared using an ICG-mCD complex exhibited a fluorescence signal 4.2 times higher than that of an ICG-PLGA (P-188-free) microparticle dispersion.

[0147] Furthermore, the fluorescence image in Figure 34 confirmed that the ICG-mCD-Algiante-PLGA microparticle dispersion (right) generates a high fluorescence signal.

[0148] In Figure 35, it was confirmed that the ICG-mCD-Algiante-PLGA (P-188 0.1%) microparticle dispersion (Figure 35-b) showed no fluorescence blurring at the administration site and the fluorescence signal was maintained for up to 30 days. In the case of ICG-HSA (Figure 35-a), the same configuration as in the previous example was shown.

[0149] The ROI values ​​analyzed from the aforementioned fluorescence images are shown in a graph (Figure 36). In the case of the ICG-mCD-Algiante-PLGA(P-188 0.1%) microparticle dispersion, 32% fluorescence remained after 1 day from injection, and 29% of fluorescence remained high even after 30 days. As a result, it was confirmed that microparticles (ICG-mCD-Algiante-PLGA(P-188 0.1%)) equipped with the indocyanine green / cyclodextrin complex could maintain their fluorescence signal for more than 30 days, demonstrating their efficacy as a fluorescent marker.

[0150] Example 9: Production and analysis of microparticles containing Cy7-human serum albumin complex In addition to indocyanine green, we also conducted experiments to demonstrate that microparticles containing other near-infrared fluorescent dyes can be manufactured and emit bright fluorescence. For this purpose, we used sulfo-cyanine 7 carboxylic acid (Cy7), sulfo-cyanine 7.5 carboxylic acid (Cy7.5), Flamma 774 carboxylic acid (Flamma774), Alexa fluor 750 carboxylic acid (Alexa 750), and IRDye 800CW carboxylate (IRDye 800CW) as fluorescent dyes to be incorporated into the microparticles. Microparticles were manufactured using polymethyl methacrylate (Poly(methyl methacrylate)) and polycarbonate (PC) polymers, and their efficacy was evaluated.

[0151] 9-1: Comparison of fluorescence signals at different concentrations of Cy7 solution and Cy7-HSA complex 0.48 mg of sulfo-cyanine7 carboxylic acid (Cy7, Lumiprobe), a near-infrared fluorescent dye, was dissolved in 1 mL of distilled water, and then diluted with distilled water to prepare Cy7 solutions at concentrations of 100, 70, 50, 40, 30, 20, 10, 5, 4, 3, 2, and 1 μM. Cy7-human serum albumin (HSA, SK plasma) complexes were prepared to compare the fluorescence signals with the Cy7 solutions. 0.48 mg of Cy7 powder was dissolved in a mixture of 0.214 mL of human serum albumin solution (20% concentration) and 0.786 mL of distilled water, and then diluted with distilled water to prepare Cy7-human serum albumin complexes (Cy7-HSA) at concentrations of 100, 70, 50, 40, 30, 20, 10, 5, 4, 3, 2, and 1 μM. For fluorescence spectrum comparison, 200 μL of each prepared solution was taken and placed in a 96-well plate, and the fluorescence intensity (λ) was measured using a multifunctional microplate reader (SPARK, Tecan Trading AG, Zurich, Switzerland). ex =730 nm, λ em =780 nm (Cy7), λ emWe measured the emission peak at 786 nm (Cy7-HSA). The Cy7 fluorescent dye shows an emission peak at 780 nm, but after forming a complex with HSA, it showed an emission peak at 786 nm.

[0152] Similar to Figure 37, which compares the fluorescence intensity of Cy7 solutions and Cy7-HSA complex solutions at concentrations of 1-100 μM, it was confirmed that Cy7-HSA solutions generate higher fluorescence signals than Cy7 solutions at concentrations of 10 μM or higher. Furthermore, it was confirmed that Cy7.5-HSA solutions generate 1.2 times higher fluorescence signals than Cy7.5 solutions at a concentration of 30 μM and 1.3 times higher at the maximum measured concentration of 100 μM.

[0153] 9-2: Manufacturing and analysis of PMMA microparticles containing Cy7 and Cy7-HSA composites To produce PMMA microparticles containing the near-infrared fluorescent dye Cy7, Cy7-PMMA (without P-188) and 0.81 mg of Cy7 were dissolved in 1.691 mL of distilled water to prepare solution W1. 3.6 g of polyvinyl alcohol was added to 180 mL of distilled water, dissolved at 85°C with stirring at 600 rpm, and cooled to room temperature to prepare solution W2. 1.217 g of PMMA was dissolved in 16.909 mL of methylene chloride to prepare an oil solution. After adding the prepared W1 solution to the oil solution, sonication was performed using a probe-type ultrasonic device for 3 minutes (1 minute sonication + 30 second rest repeated) to produce a primary emulsion (water-in-oil). The prepared W2 solution was stirred at 1000 rpm using a mechanical stirrer, while the manufactured primary emulsion solution was added at a rate of 10 mL / min using a syringe pump to produce a secondary emulsion (water-in-oil-in-water). The manufactured emulsion was stirred at 1000 rpm for 10 minutes, and then stirred at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C to remove the methylene chloride solvent. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PMMA polymer microparticles containing Cy7. The supernatant was removed, and the PMMA microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed microparticles were freeze-dried to obtain microparticle powder.

[0154] To produce PMMA microparticles containing the near-infrared fluorescent dye Cy7-HSA complex (Cy7-HSA-PMMA (without P-188)), 0.81 mg of Cy7 was dissolved in 0.363 mL of human serum albumin (HSA) (20% concentration, 72.553 mg) and 1.328 mL of distilled water to prepare solution W1. 3.6 g of polyvinyl alcohol was added to 180 mL of distilled water, dissolved at 85°C with stirring at 600 rpm, and cooled to room temperature to prepare solution W2. 1.217 g of PMMA was dissolved in 16.909 mL of methylene chloride to prepare an oil solution. After adding the prepared W1 solution to the oil solution, sonication was performed using a probe-type ultrasonic device for 3 minutes (1 minute sonication + 30 second rest repeated) to produce a primary emulsion (water-in-oil). The prepared W2 solution was stirred at 1000 rpm using a mechanical stirrer, and the manufactured primary emulsion solution was added at a rate of 10 mL / min using a syringe pump to produce a secondary emulsion (water-in-oil-in-water). The manufactured emulsion was stirred at 1000 rpm for 10 minutes, and then stirred at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C to remove the methylene chloride solvent. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PMMA polymer microparticles carrying Cy7-HSA. The supernatant was removed, and the PMMA microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed microparticles were freeze-dried to obtain microparticle powder.

[0155] To produce PMMA microparticles containing a 1.0% primary emulsion phase of poloxamer-188 and equipped with a near-infrared fluorescent dye Cy7-HSA complex (Cy7-HSA-PMMA(P-188 1.0%)), solution W1 was prepared by dissolving 0.81 mg of Cy7 in 0.363 mL of human serum albumin (HSA) (20% concentration, 72.553 mg) and 1.328 mL of distilled water. Solution W2 was prepared by adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, dissolving it at 85°C with stirring at 600 rpm, and then cooling to room temperature. An oil solution was prepared by dissolving 1.217 g of PMMA and 186 mg of poloxamer-188 (P-188) in 16.909 mL of methylene chloride. After adding the prepared W1 solution to the oil solution, sonication was performed using a probe-type ultrasonic device for 3 minutes (1 minute sonication + 30 second rest repeated) to produce a primary emulsion (water-in-oil). While stirring the prepared W2 solution at 1000 rpm using a mechanical stirrer, the prepared primary emulsion solution was added at a rate of 10 mL / min using a syringe pump to produce a secondary emulsion (water-in-oil-in-water). After stirring the prepared emulsion at 1000 rpm for 10 minutes, the methylene chloride solvent was removed by stirring at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C. Subsequently, the PMMA polymer microparticles carrying Cy7-HSA were obtained by centrifugation at 14,000 rpm for 5 minutes. The supernatant was removed, and the PMMA microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed fine particles were freeze-dried to obtain a fine particle powder.

[0156] To evaluate the Cy7 content and loading rate in the manufactured microparticles, the microparticles were dissolved, and the concentration of Cy7 in the solution was quantified using an ultraviolet / visible light absorption analyzer.

[0157] Scanning electron microscopy analysis was performed to analyze the particle morphology and size of the synthesized microparticles. For cross-sectional analysis of the manufactured microparticles, the particles were cut with a razor blade. The microparticle powder was attached to carbon tape mounted on a scanning electron microscope sample holder, and then coated with Pt / Pd via sputtering for 60 seconds. Scanning electron microscope images were then analyzed with a 5kV acceleration voltage. The particle size was determined by measuring the size of 100 randomly selected particles from the obtained images and averaging the result.

[0158] To compare the fluorescence signals of the aforementioned manufactured microparticles and the Cy7-human serum albumin complex, a Cy7 complex solution and a microparticle dispersion of the same concentration were prepared. 0.48 mg of Cy7 powder was dissolved in a mixture of 0.214 mL of human serum albumin solution (20% concentration) and 0.786 mL of distilled water, and then diluted with distilled water to prepare 0.3 mL of a 30 μM Cy7-human serum albumin complex (Cy7-HSA). Using the information obtained from the quantitative analysis of Cy7 content in the PMMA microparticles, a microparticle dispersion was prepared by adding Cy7 to 0.3 mL of distilled water to achieve a Cy7 concentration of 30 μM. For fluorescence spectrum comparison, 200 μL each of the prepared solution and microparticle dispersion were taken and placed in a 96-well plate, and the fluorescence spectra (λ) were measured using a multifunctional microplate reader. ex The wavelength (=730 nm) was measured.

[0159] The Cy7 content in Cy7-PMMA (without P-188) microparticles, manufactured by incorporating only Cy7 during PMMA microparticle production, was 6.98%, and the Cy7 content per 1 mg microparticle was confirmed to be 46.5 ng. The Cy7 content in Cy7-HSA-PMMA (without P-188), manufactured by incorporating Cy7-HSA, and Cy7-HSA-PMMA (1.0% P-188), manufactured by adding 1.0% poloxamer-188 as a primary emulsion phase, was 25.8% and 19.1%, respectively, and the Cy7 content per 1 mg microparticle was 162.0 ng and 104.8 ng, confirming that these microparticles incorporated a higher Cy7 content than Cy7-PMMA (without P-188) microparticles incorporating only Cy7.

[0160] Analysis of scanning electron microscope images of Cy7-PMMA (without P-188), Cy7-HSA-PMMA (without P-188), and Cy7-HSA-PMMA (1.0% P-188) microparticles revealed that each microparticle was a spherical particle with average particle sizes of 56.7±21.3 μm, 53.7±16.1 μm, and 59.0±20.0 μm, respectively, confirming that it possessed an appropriate size to avoid phagocytic activity by macrophages (Figure 38(a, c, d)). Furthermore, observation of scanning electron microscope cross-sections of each microparticle (Figure 38(b, d, f)) showed that the PMMA microparticles contained multiple isolated small spherical spaces within them, which prevented the elution of the attached fluorescent dye, confirming that this morphology is highly suitable for long-term fluorescent dye labeling.

[0161] Figure 39 shows that the fluorescence spectrum of the Cy7-PMMA (without P-188) microparticle dispersion showed a fluorescence signal 4.2 times lower than that of Cy7-HSA. The fluorescence spectra of the Cy7-HSA-PMMA (without P-188) and Cy7-HSA-PMMA (1.0% P-188) microparticle dispersions showed fluorescence signals 2.5 times and 2.8 times higher than that of Cy7-HSA, respectively, and 10 times and 12 times higher than that of Cy7-PMMA (without P-188), respectively. From these results, it can be seen that a much higher fluorescence signal can be obtained when Cy7 is incorporated in a composite form with Cy7-HSA than when Cy7, a near-infrared fluorescent dye, is incorporated alone.

[0162] Example 10: Production and analysis of microparticles containing Cy7.5-human serum albumin complex Comparison of fluorescence signals at different concentrations of 10-1:Cy7.5 solution and Cy7.5-HSA complex. 0.70 mg of the near-infrared fluorescent dye sulfo-cyanine 7.5 carboxylic acid (Cy7.5, Lumiprobe) was dissolved in 1 mL of distilled water, and then diluted with distilled water to prepare Cy7.5 solutions at concentrations of 100, 70, 50, 40, 30, 20, 10, 5, 4, 3, 2, and 1 μM. To compare the fluorescence signals with the Cy7.5 solutions, Cy7.5-human serum albumin (HSA, SK plasma) complexes were prepared by dissolving 0.70 mg of Cy7.5 powder in a mixture of 0.214 mL of human serum albumin solution (20% concentration) and 0.786 mL of distilled water, and then diluting with distilled water to prepare Cy7.5-human serum albumin complexes (Cy7.5-HSA) at concentrations of 100, 70, 50, 40, 30, 20, 10, 5, 4, 3, 2, and 1 μM. For fluorescence spectrum comparison, 200 μL of each prepared solution was taken and placed in a 96-well plate, and the fluorescence spectra (λ) were measured using a multifunctional microplate reader (SPARK, Tecan Trading AG, Zurich, Switzerland). ex =730 nm, λ em =810 nm (Cy7.5), λ em We measured the emission peak at 824 nm (Cy7.5-HSA). The Cy7.5 fluorescent dye shows an emission peak at 810 nm, but after forming a complex with HSA, it showed an emission peak at 824 nm.

[0163] Similar to Figure 40, which compares the fluorescence intensity of Cy7.5 solutions and Cy7.5-HSA complex solutions at concentrations of 1-100 μM, it was confirmed that Cy7.5-HSA solutions generate higher fluorescence signals than Cy7.5 solutions at concentrations of 10 μM or higher. It was also confirmed that Cy7.5-HSA solutions generate 1.1 times higher fluorescence signals than Cy7.5 solutions at a concentration of 30 μM and 1.2 times higher at the maximum measured concentration of 100 μM.

[0164] 10-2: Manufacturing and analysis of PMMA microparticles containing Cy7.5 and Cy7.5-HSA composites To produce PMMA (Cy7.5-PMMA (without P-188)) microparticles containing only the near-infrared fluorescent dye Cy7.5, solution W1 was prepared by dissolving 1.18 mg of Cy7.5 in 1.691 mL of distilled water. After adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, it was dissolved at 85°C while stirring at 600 rpm, and then cooled to room temperature to produce solution W2. 1.217 g of PMMA was dissolved in 16.909 mL of methylene chloride to prepare an oil solution. After adding the prepared W1 solution to the oil solution, sonication was performed using a probe-type ultrasonic device for 3 minutes (1 minute sonication + 30 second rest repeated) to produce a primary emulsion (water-in-oil). The prepared W2 solution was stirred at 1000 rpm using a mechanical stirrer, and the manufactured primary emulsion solution was added at a rate of 10 mL / min using a syringe pump to produce a secondary emulsion (water-in-oil-in-water). The manufactured emulsion was stirred at 1000 rpm for 10 minutes, and then stirred at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C to remove the methylene chloride solvent. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PMMA polymer microparticles containing Cy7.5. The supernatant was removed, and the PMMA microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed microparticles were freeze-dried to obtain microparticle powder.

[0165] Cy7.5-HSA-PMMA (without P-188), which is a PMMA microparticle containing a near-infrared fluorescent dye Cy7.5-HSA complex, was prepared. Solution W1 was prepared by dissolving 1.18 mg of Cy7.5 in 0.363 mL of human serum albumin (HSA) (20% concentration, 72.553 mg) and 1.328 mL of distilled water. Solution W2 was prepared by adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, dissolving it at 85°C with stirring at 600 rpm, and cooling to room temperature. An oil solution was prepared by dissolving 1.217 g of PMMA in 16.909 mL of methylene chloride. After adding the prepared W1 solution to the oil solution, sonication was performed for 3 minutes (1 minute sonication + 30 second rest repeated) using a probe-type ultrasonic device to prepare a primary emulsion (water-in-oil). The prepared W2 solution was stirred at 1000 rpm using a mechanical stirrer, and the manufactured primary emulsion solution was added at a rate of 10 mL / min using a syringe pump to produce a secondary emulsion (water-in-oil-in-water). The manufactured emulsion was stirred at 1000 rpm for 10 minutes, and then stirred at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C to remove the methylene chloride solvent. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PMMA polymer microparticles carrying Cy7.5-HSA. The supernatant was removed, and the PMMA microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed microparticles were freeze-dried to obtain microparticle powder.

[0166] Cy7.5-HSA-PMMA (P-188 1.0%), which is a PMMA microparticle containing a 1.0% primary emulsion phase of poloxamer-188 and equipped with a near-infrared fluorescent dye Cy7.5-HSA complex, was prepared. Solution W1 was prepared by dissolving 1.18 mg of Cy7.5 in 0.363 mL of human serum albumin (HSA) (20% concentration, 72.553 mg) and 1.328 mL of distilled water. Solution W2 was prepared by adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, dissolving it at 85°C with stirring at 600 rpm, and cooling to room temperature. An oil solution was prepared by dissolving 1.217 g of PMMA and 186 mg of poloxamer-188 (P-188) in 16.909 mL of methylene chloride. After adding the prepared W1 solution to the oil solution, ultrasonic treatment was performed using a probe-type ultrasonic device for 3 minutes (1 minute ultrasonic treatment + 30 second rest repeated) to produce a primary emulsion (water-in-oil). While stirring the prepared W2 solution at 1000 rpm using a mechanical stirrer, the prepared primary emulsion solution was added at a rate of 10 mL / min using a syringe pump to produce a secondary emulsion (water-in-oil-in-water). After stirring the prepared emulsion at 1000 rpm for 10 minutes, the methylene chloride solvent was removed by stirring at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C. Then, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PMMA polymer microparticles carrying Cy7.5-HSA. The supernatant was removed, and the PMMA microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed fine particles were freeze-dried to obtain a fine particle powder.

[0167] To evaluate the Cy7.5 content and loading rate in the manufactured microparticles, the microparticles were dissolved, and the concentration of Cy7.5 in the solution was quantified using an ultraviolet / visible light absorption analyzer.

[0168] Scanning electron microscopy analysis was performed to analyze the particle morphology and size of the synthesized microparticles. For cross-sectional analysis of the manufactured microparticles, the particles were cut with a razor blade. The microparticle powder was attached to carbon tape mounted on a scanning electron microscope sample holder, and then coated with Pt / Pd via sputtering for 60 seconds. Scanning electron microscope images were then analyzed with a 5kV acceleration voltage. The particle size was determined by measuring the size of 100 randomly selected particles from the obtained images and averaging the result.

[0169] To compare the fluorescence signals of the aforementioned manufactured microparticles and the Cy7.5-human serum albumin complex, a Cy7.5 complex solution and a microparticle dispersion of the same concentration were prepared. 0.70 mg of Cy7.5 powder was dissolved in a mixture of 0.214 mL of human serum albumin solution (20% concentration) and 0.786 mL of distilled water, and then diluted with distilled water to prepare 0.3 mL of a 30 μM Cy7.5-human serum albumin complex (Cy7-HSA). Using the information obtained from the quantitative analysis of Cy7.5 content in the PMMA microparticles, a microparticle dispersion was prepared by adding Cy7.5 to 0.3 mL of distilled water to achieve a concentration of 30 μM. For fluorescence spectrum comparison, 200 μL each of the prepared solution and microparticle dispersion were taken and placed in a 96-well plate, and the fluorescence spectra (λ) were measured using a multifunctional microplate reader. ex The wavelength (=730 nm) was measured.

[0170] The Cy7.5 loading rate in Cy7.5-PMMA (without P-188) microparticles, which were manufactured by loading Cy7.5 during the production of PMMA microparticles, was 4.10%, and the Cy7.5 content in 1 mg of microparticles was confirmed to be 39.8 ng. The Cy7.5 loading rates in Cy7.5-HSA-PMMA (without P-188), manufactured by loading Cy7.5-HSA, and Cy7.5-HSA-PMMA (1.0% P-188), manufactured by adding 1.0% poloxamer-188 based on the primary emulsion phase, were 14.0% and 15.6%, respectively, and the Cy7.5 content in 1 mg of microparticles was 128.7 ng and 124.7 ng, respectively, confirming that these microparticles contained a higher content of Cy7.5 than Cy7.5-PMMA (without P-188) microparticles loaded with Cy7.5 alone.

[0171] Analysis of scanning electron microscope images of Cy7.5-PMMA (without P-188), Cy7.5-HSA-PMMA (without P-188), and Cy7.5-HSA-PMMA (1.0% P-188) microparticles revealed that each microparticle was a spherical particle with average particle sizes of 57.9±18.4 μm, 58.0±16.0 μm, and 65.9±24.4 μm, respectively, confirming that it possessed an appropriate size to avoid phagocytic activity by macrophages (Figure 41(a, c, e)). Furthermore, evaluation of scanning electron microscope cross-sections of each microparticle (Figure 41(b, d, f)) showed that the inside of the PMMA microparticles contained multiple isolated small spherical spaces, which prevented the elution of the attached fluorescent dye, confirming that it is a morphology very suitable for long-term fluorescent dye labeling.

[0172] Figure 42 shows that the fluorescence spectrum of the Cy7.5-PMMA (without P-188) microparticle dispersion showed a fluorescence signal 5.0 times lower than that of Cy7.5-HSA. The fluorescence spectra of the Cy7.5-HSA-PMMA (without P-188) and Cy7.5-HSA-PMMA (1.0% P-188) microparticle dispersions showed fluorescence signals 1.3 times and 1.6 times higher than Cy7.5-HAS, respectively, and 6.4 times and 8.2 times higher than Cy7.5-PMMA (without P-188). From these results, it can be seen that when Cy7.5, a near-infrared fluorescent dye, is incorporated in the form of Cy7.5-HSA, a much higher fluorescence signal can be obtained than when it is incorporated alone.

[0173] 10-3: Manufacturing and analysis of polycarbonate microparticles containing Cy7.5 and Cy7.5-HSA Cy7.5-PC (without P-188), which is polycarbonate (PC) microparticles equipped with the near-infrared fluorescent dye Cy7.5, was manufactured. Solution W1 was prepared by dissolving 1.18 mg of Cy7.5 in 1.691 mL of distilled water. After adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, it was dissolved at 85°C while stirring at 600 rpm, and then cooled to room temperature to prepare solution W2. Solution oil was prepared by adding 1.217 g of PC to 16.909 mL of methylene chloride and dissolving it. After adding the prepared W1 solution to the oil solution, sonication was performed for 3 minutes (1 minute sonication + 30 second rest repeated) using a probe-type ultrasonic device to prepare a primary emulsion (water-in-oil). The prepared W2 solution was stirred at 1000 rpm using a mechanical stirrer, and the manufactured primary emulsion solution was added at a rate of 10 mL / min using a syringe pump to produce a secondary emulsion (water-in-oil-in-water). The manufactured emulsion was stirred at 1000 rpm for 10 minutes, and then stirred at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C to remove the methylene chloride solvent. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PC polymer microparticles containing Cy7.5. The supernatant was removed, and the PC microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed microparticles were freeze-dried to obtain microparticle powder.

[0174] Cy7.5-HSA-PC (without P-188), which is a PC microparticle containing a Cy7.5-HSA complex, was manufactured. Solution W1 was prepared by dissolving 1.18 mg of Cy7.5 in 0.363 mL of human serum albumin (HSA) (20% concentration, 72.553 mg) and 1.328 mL of distilled water. Solution W2 was prepared by adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, dissolving it at 85°C with stirring at 600 rpm, and cooling to room temperature. Solution W2 was prepared by adding 1.217 g of PC to 16.909 mL of methylene chloride and dissolving it. An oil solution was prepared. After adding the prepared W1 solution to the oil solution, sonication was performed for 3 minutes (1 minute sonication + 30 second rest repeated) using a probe-type ultrasonic device to prepare a primary emulsion (water-in-oil). The prepared W2 solution was stirred at 1000 rpm using a mechanical stirrer, and the manufactured primary emulsion solution was added at a rate of 10 mL / min using a syringe pump to produce a secondary emulsion (water-in-oil-in-water). The manufactured emulsion was stirred at 1000 rpm for 10 minutes, and then stirred at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C to remove the methylene chloride solvent. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PC polymer microparticles containing Cy7.5-HSA. The supernatant was removed, and the PC microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed microparticles were freeze-dried to obtain microparticle powder.

[0175] To evaluate the Cy7.5 content and loading rate in the manufactured microparticles, the microparticles were dissolved, and the concentration of Cy7.5 in the solution was quantified using an ultraviolet / visible light absorption analyzer.

[0176] Scanning electron microscopy analysis was performed to analyze the particle morphology and size of the synthesized microparticles. For cross-sectional analysis of the manufactured microparticles, the particles were cut with a razor blade. The microparticle powder was attached to carbon tape mounted on a scanning electron microscope sample holder, and then coated with Pt / Pd via sputtering for 60 seconds. Scanning electron microscope images were then analyzed with a 5kV acceleration voltage. The particle size was determined by measuring the size of 100 randomly selected particles from the obtained images and averaging the result.

[0177] To compare the fluorescence signals of the manufactured microparticles and the Cy7.5-human serum albumin complex, a Cy7.5 complex solution and a microparticle dispersion of the same concentration were prepared. 0.70 mg of Cy7.5 powder was dissolved in a mixture of 0.214 mL of human serum albumin solution (20% concentration) and 0.786 mL of distilled water, and then diluted with distilled water to prepare 0.3 mL of a 30 μM Cy7.5-human serum albumin complex (Cy7-HSA). Using the Cy7.5 content in the PC microparticles evaluated by the quantitative analysis, a microparticle dispersion was prepared by adding Cy7.5 to 0.3 mL of distilled water to achieve a concentration of 30 μM. For fluorescence spectrum comparison, 200 μL each of the prepared solution and microparticle dispersion were taken and placed in a 96-well plate, and the fluorescence spectra (λ) were measured using a multifunctional microplate reader. ex The wavelength (=730 nm) was measured.

[0178] The Cy7.5 loading rate in Cy7.5-PMMA (without P-188) microparticles, manufactured by loading Cy7.5 during PMMA microparticle production, was 2.08%, and the Cy7.5 content per 1 mg microparticle was confirmed to be 20.2 ng. The Cy7.5 loading rate in Cy7.5-HSA-PMMA (1.0% P-188) microparticles, manufactured by loading Cy7.5-HSA and adding 1.0% poloxamer-188 based on the primary emulsion phase, was 11.8%, and the Cy7.5 content per 1 mg microparticle was 108.6 ng, confirming that it loaded a higher content of Cy7.5 than Cy7.5-PMMA (without P-188) microparticles.

[0179] Analysis of scanning electron microscope images of Cy7.5-PMMA (without P-188) and Cy7.5-HSA-PMMA (without P-188) microparticles revealed that each microparticle was a spherical particle with average particle diameters of 58.3±19.0 μm and 55.2±19.5 μm, respectively, confirming that it possessed an appropriate size to avoid phagocytic activity by macrophages (Figure 43(a, c)). Furthermore, observation of scanning electron microscope cross-sections of each microparticle (Figure 43(b, d)) showed that the inside of the PMMA microparticles contained multiple isolated small spherical spaces, which prevented the elution of the attached fluorescent dye, confirming that it is a morphology very suitable for long-term fluorescent dye labeling.

[0180] Figure 44 shows the fluorescence spectrum of the Cy7.5-HSA-PC (without P-188) microparticle dispersion, which exhibited a fluorescence signal 2.2 times higher than that of Cy7.5-PC (without P-188). This result indicates that a significantly higher fluorescence signal can be obtained when Cy7.5-HSA is incorporated into a composite form of PC polymer-based microparticles compared to when Cy7.5 is incorporated alone.

[0181] Example 11: Production and analysis of microparticles containing Flamm774-human serum albumin complex 11-1: Comparison of fluorescence signals at different concentrations of Flamma774 solution and Flamma774-HSA complex. 0.60 mg of the near-infrared fluorescent dye Flamma774 carboxylic acid (Flamma774, Bioacts) was dissolved in 1 mL of distilled water, and then diluted with distilled water to prepare Flamma774 solutions at concentrations of 100, 70, 50, 40, 30, 20, 10, 5, 4, 3, 2, and 1 μM. To compare the fluorescence signals with the Flamma774 solutions, Flamma774-human serum albumin (HSA, SK plasma) complexes were prepared by dissolving 0.60 mg of Flamma774 powder in a mixture of 0.214 mL of human serum albumin solution (20% concentration) and 0.786 mL of distilled water, and then diluting with distilled water to prepare Flamma774-human serum albumin complexes (Flamma774-HSA) at concentrations of 100, 70, 50, 40, 30, 20, 10, 5, 4, 3, 2, and 1 μM. For fluorescence spectrum comparison, 200 μL of each prepared solution was taken and placed in a 96-well plate, and the fluorescence spectra (λ) were measured using a multifunctional microplate reader (SPARK, Tecan Trading AG, Zurich, Switzerland). ex =730 nm, λ em =812 nm (Flamma774), λ em We measured the emission peak at 818 nm (Flamma774-HSA). The Flamma774 fluorescent dye shows an emission peak at 812 nm, but after forming a complex with HSA, it showed an emission peak at 818 nm.

[0182] Similar to Figure 45, which compares the fluorescence intensity of Flamma774 solutions and Flamma774-HSA complex solutions at concentrations of 1 to 100 μM, it was confirmed that Flamma774-HSA solutions generated higher fluorescence signals than Flamma774 solutions across the entire concentration range. Specifically, Flamma774-HSA solutions generated 1.3 times higher fluorescence signals than Flamma774 solutions at a concentration of 30 μM and 1.3 times higher at the maximum measured concentration of 100 μM.

[0183] 11-2: Manufacturing and analysis of PMMA microparticles containing Flamma774 and Flamma774-HSA composite. Flamma774-PMMA (without P-188), which is PMMA microparticles equipped with the near-infrared fluorescent dye Flamma774, was manufactured. Solution W1 was prepared by dissolving 1.01 mg of Flamma774 in 1.691 mL of distilled water. After adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, it was dissolved at 85°C while stirring at 600 rpm, and then cooled to room temperature to prepare solution W2. An oil solution was prepared by dissolving 1.217 g of PMMA in 16.909 mL of methylene chloride. After adding the prepared W1 solution to the oil solution, sonication was performed for 3 minutes (1 minute sonication + 30 second rest repeated) using a probe-type ultrasonic device to produce a primary emulsion (water-in-oil). The prepared W2 solution was stirred at 1000 rpm using a mechanical stirrer, and the manufactured primary emulsion solution was added at a rate of 10 mL / min using a syringe pump to produce a secondary emulsion (water-in-oil-in-water). The manufactured emulsion was stirred at 1000 rpm for 10 minutes, and then stirred at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C to remove the methylene chloride solvent. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PMMA polymer microparticles carrying Flamma774. The supernatant was removed, and the PMMA microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed microparticles were freeze-dried to obtain microparticle powder.

[0184] Flamma774-HSA-PMMA (without P-188), which is PMMA microparticles containing the Flamma774-HSA complex, was manufactured. Solution W1 was prepared by dissolving 1.01 mg of Flamma774 in 0.363 mL of human serum albumin (HSA) (20% concentration, 72.553 mg) and 1.328 mL of distilled water. After adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, it was dissolved at 85°C with stirring at 600 rpm, and cooled to room temperature to prepare solution W2. An oil solution was prepared by dissolving 1.217 g of PMMA in 16.909 mL of methylene chloride. After adding the prepared W1 solution to the oil solution, sonication was performed for 3 minutes (1 minute sonication + 30 second rest repeated) using a probe-type ultrasonic device to prepare a primary emulsion (water-in-oil). The prepared W2 solution was stirred at 1000 rpm using a mechanical stirrer, and the manufactured primary emulsion solution was added at a rate of 10 mL / min using a syringe pump to produce a secondary emulsion (water-in-oil-in-water). The manufactured emulsion was stirred at 1000 rpm for 10 minutes, and then stirred at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C to remove the methylene chloride solvent. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PMMA polymer microparticles carrying Flamma774-HSA. The supernatant was removed, and the PMMA microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed microparticles were freeze-dried to obtain microparticle powder.

[0185] Flamma774-HSA-PMMA (P-188 1.0%), which is a PMMA microparticle containing a 1.0% primary emulsion phase of poloxamer-188 and equipped with a near-infrared fluorescent dye Flamma774-HSA complex, was prepared. Solution W1 was prepared by dissolving 1.01 mg of Flamma774 in 0.363 mL of human serum albumin (HSA) (20% concentration, 72.553 mg) and 1.328 mL of distilled water. Solution W2 was prepared by adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, dissolving it at 85°C with stirring at 600 rpm, and cooling to room temperature. An oil solution was prepared by dissolving 1.217 g of PMMA and 186 mg of poloxamer-188 (P-188) in 16.909 mL of methylene chloride. After adding the prepared W1 solution to the oil solution, sonication was performed using a probe-type ultrasonic device for 3 minutes (1 minute sonication + 30 second rest repeated) to produce a primary emulsion (water-in-oil). While stirring the prepared W2 solution at 1000 rpm using a mechanical stirrer, the prepared primary emulsion solution was added at a rate of 10 mL / min using a syringe pump to produce a secondary emulsion (water-in-oil-in-water). After stirring the prepared emulsion at 1000 rpm for 10 minutes, the methylene chloride solvent was removed by stirring at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PMMA polymer microparticles carrying Flamma774-HSA. The supernatant was removed, and the PMMA microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed fine particles were freeze-dried to obtain a fine particle powder.

[0186] To evaluate the content and loading rate of Flmma774 in the manufactured microparticles, the microparticles were dissolved, and the concentration of Flmma774 in the solution was quantified using an ultraviolet / visible light absorption analyzer.

[0187] Scanning electron microscopy analysis was performed to analyze the particle morphology and size of the synthesized microparticles. For cross-sectional analysis of the manufactured microparticles, the particles were cut with a razor blade. The microparticle powder was attached to carbon tape mounted on a scanning electron microscope sample holder, and then coated with Pt / Pd via sputtering for 60 seconds. Scanning electron microscope images were then analyzed with a 5kV acceleration voltage. The particle size was determined by measuring the size of 100 randomly selected particles from the obtained images and averaging the result.

[0188] To compare the fluorescence signals of the aforementioned manufactured microparticles and the Flmma774-human serum albumin complex, a complex solution and a microparticle dispersion of the same concentration of Flmma774 were prepared. 0.60 mg of Flmma774 powder was dissolved in a mixture of 0.214 mL of human serum albumin solution (20% concentration) and 0.786 mL of distilled water, and then diluted with distilled water to prepare 0.3 mL of Flmma774-human serum albumin complex (Flmma774-HSA) at a concentration of 30 μM. Using the information obtained from the quantitative analysis of Flmma774 content in the PMMA microparticles, a microparticle dispersion was prepared by adding Flmma774 to 0.3 mL of distilled water to achieve a concentration of 30 μM. For fluorescence spectrum comparison, 200 μL each of the prepared solution and microparticle dispersion were taken and placed in a 96-well plate, and the fluorescence spectra (λ) were measured using a multifunctional microplate reader. ex The wavelength (=730 nm) was measured.

[0189] The Flamma774 content in Flamma774-PMMA (without P-188) microparticles, which were manufactured by incorporating Flamma774 during the production of PMMA microparticles, was 5.08%, and the Flamma774 content per 1 mg microparticle was confirmed to be 42.3 ng. The Flamma774 content in Flamma774-HSA-PMMA (without P-188), manufactured by incorporating Flamma774-HSA, and Flamma774-HSA-PMMA (1.0% P-188), manufactured by adding 1.0% poloxamer-188 as a primary emulsion phase, was 23.3% and 21.6%, respectively, and the Flamma774 content per 1 mg microparticle was 183.0 ng and 148.6 ng, respectively, confirming that these microparticles contained a higher content of Flamma774 than Flamma774-PMMA (without P-188) microparticles that incorporated only Flamma774.

[0190] Analysis of scanning electron microscope images of Flamma774-PMMA (without P-188), Flamma774-HSA-PMMA (without P-188), and Flamma774-HSA-PMMA (1.0% P-188) microparticles revealed that each microparticle was a spherical particle with average particle sizes of 56.8±19.2 μm, 62.5±19.3 μm, and 55.8±19.3 μm, respectively, confirming that it possessed an appropriate size to avoid phagocytic activity by macrophages (Figure 46(a, c, e)). Furthermore, analysis of scanning electron microscope cross-sections of each microparticle (Figure 46(b, d, f)) showed that the inside of the PMMA microparticles contained multiple isolated small spherical spaces, which prevented the elution of the attached fluorescent dye, confirming that it is a morphology very suitable for long-term fluorescent dye labeling.

[0191] Figure 47 shows the fluorescence spectrum of the Flamma774-PMMA (without P-188) microparticle dispersion, which showed a fluorescence signal 4.5 times lower than that of Flamma774-HSA. The fluorescence spectra of the Flamma774-HSA-PMMA (without P-188) and Flamma774-HSA-PMMA (1.0% P-188) microparticle dispersions showed fluorescence signals 1.4 times and 2.3 times higher than Flamma774-HSA, respectively, and 6.2 times and 10.4 times higher than Flamma774-PMMA (without P-188), respectively. From these results, it can be seen that when Flamma774, a near-infrared fluorescent dye, is incorporated in the Flamma774-HSA composite form, a much higher fluorescence signal is obtained than when it is incorporated alone.

[0192] Example 12: Production and analysis of microparticles containing Alexa750-human serum albumin complex 12-1: Comparison of fluorescence signals at different concentrations of Alexa750 solution and Alexa750-HSA complex. Alexa fluor, a near-infrared fluorescent dye TM0.77 mg of 750 carboxylic acid, tris(triethylammonium)salt (Alexa750, Thermo Fisher Scientific) was dissolved in 1 mL of distilled water, and then diluted with distilled water to prepare Alexa750 solutions at concentrations of 100, 70, 50, 40, 30, 20, 10, 5, 4, 3, 2, and 1 μM. To compare the fluorescence signals with the Alexa750 solutions, Alexa750-human serum albumin (HSA, SK plasma) conjugates were prepared by dissolving 0.77 mg of Alexa750 powder in a mixture of 0.214 mL of human serum albumin solution (20% concentration) and 0.786 mL of distilled water, and then diluting with distilled water to prepare Alexa750-human serum albumin conjugates (Alexa750-HSA) at concentrations of 100, 70, 50, 40, 30, 20, 10, 5, 4, 3, 2, and 1 μM. For fluorescence spectrum comparison, 200 μL of each prepared solution was taken and placed in a 96-well plate, and the fluorescence spectra (λ) were measured using a multifunctional microplate reader (SPARK, Tecan Trading AG, Zurich, Switzerland). ex =730 nm, λ em We measured (=776 nm).

[0193] Similar to Figure 48, which compares the fluorescence intensity of Alexa750 solutions and Alexa750-HSA complex solutions at concentrations of 1-100 μM, it was confirmed that the Alexa750-HSA solution produced a higher fluorescence signal than the Alexa750 solution across the entire concentration range. It was also confirmed that the Alexa750-HSA solution produced a fluorescence signal 1.1 times higher than the Alexa750 solution at a concentration of 30 μM and 1.1 times higher at the maximum measured concentration of 100 μM.

[0194] 12-2: Manufacturing and analysis of PMMA microparticles equipped with Alexa750 and Alexa750-HSA composites Alexa750-PMMA (without P-188), which is PMMA microparticles equipped with the near-infrared fluorescent dye Alexa750, was manufactured. Solution W1 was prepared by dissolving 1.31 mg of Alexa750 in 1.691 mL of distilled water. After adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, it was dissolved at 85°C while stirring at 600 rpm, and then cooled to room temperature to prepare solution W2. Solution oil was prepared by adding 1.217 g of PMMA to 16.909 mL of methylene chloride and dissolving it. After adding the prepared W1 solution to the oil solution, ultrasonic treatment was performed for 3 minutes (1 minute ultrasonic treatment + 30 second rest repeated) using a probe-type ultrasonic device to prepare a primary emulsion (water-in-oil). The prepared W2 solution was stirred at 1000 rpm using a mechanical stirrer, and the manufactured primary emulsion solution was added at a rate of 10 mL / min using a syringe pump to produce a secondary emulsion (water-in-oil-in-water). The manufactured emulsion was stirred at 1000 rpm for 10 minutes, and then stirred at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C to remove the methylene chloride solvent. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PMMA polymer microparticles equipped with Alexa750. The supernatant was removed, and the PMMA microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed microparticles were freeze-dried to obtain microparticle powder.

[0195] Alexa750-HSA-PMMA (without P-188), which is a PMMA microparticle containing the near-infrared fluorescent dye Alexa750-HSA complex, was manufactured. Solution W1 was prepared by dissolving 1.31 mg of Alexa750 in 0.363 mL of human serum albumin (HSA) (20% concentration, 72.553 mg) and 1.328 mL of distilled water. Solution W2 was prepared by adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, dissolving it at 85°C with stirring at 600 rpm, and cooling to room temperature. Solution W2 was prepared by dissolving 1.217 g of PMMA in 16.909 mL of methylene chloride. After adding the prepared W1 solution to the oil solution, sonication was performed for 3 minutes (1 minute sonication + 30 second rest repeated) using a probe-type ultrasonic device to prepare a primary emulsion (water-in-oil). The prepared W2 solution was stirred at 1000 rpm using a mechanical stirrer, and the manufactured primary emulsion solution was added at a rate of 10 mL / min using a syringe pump to produce a secondary emulsion (water-in-oil-in-water). The manufactured emulsion was stirred at 1000 rpm for 10 minutes, and then stirred at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C to remove the methylene chloride solvent. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PMMA polymer microparticles equipped with Alexa750-HSA. The supernatant was removed, and the PMMA microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed microparticles were freeze-dried to obtain microparticle powder.

[0196] Alexa750-HSA-PMMA (P-188 1.0%), which is a PMMA microparticle containing a 1.0% primary emulsion phase of poloxamer-188 and equipped with a near-infrared fluorescent dye Alexa750-HSA complex, was prepared. Solution W1 was prepared by dissolving 1.31 mg of Alexa750 in 0.363 mL of human serum albumin (HSA) (20% concentration, 72.553 mg) in 1.328 mL of distilled water. Solution W2 was prepared by adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, dissolving it at 85°C with stirring at 600 rpm, and cooling to room temperature. An oil solution was prepared by dissolving 1.217 g of PMMA and 186 mg of poloxamer-188 (P-188) in 16.909 mL of methylene chloride. After adding the prepared W1 solution to the oil solution, sonication was performed using a probe-type ultrasonic device for 3 minutes (1 minute sonication + 30 second rest repeated) to produce a primary emulsion (water-in-oil). While stirring the prepared W2 solution at 1000 rpm using a mechanical stirrer, the prepared primary emulsion solution was added at a rate of 10 mL / min using a syringe pump to produce a secondary emulsion (water-in-oil-in-water). After stirring the prepared emulsion at 1000 rpm for 10 minutes, the methylene chloride solvent was removed by stirring at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PMMA polymer microparticles equipped with Alexa750-HSA. The supernatant was removed, and the PMMA microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed fine particles were freeze-dried to obtain a fine particle powder.

[0197] To evaluate the content and encapsulation rate of Alexa750 in the manufactured microparticles, the microparticles were dissolved, and the concentration of Alexa750 in the solution was quantified using an ultraviolet / visible light absorption analyzer.

[0198] Scanning electron microscopy analysis was performed to analyze the particle morphology and size of the synthesized microparticles. For cross-sectional analysis of the manufactured microparticles, the particles were cut with a razor blade. The microparticle powder was attached to carbon tape mounted on a scanning electron microscope sample holder, and then coated with Pt / Pd via sputtering for 60 seconds. Scanning electron microscope images were then analyzed with a 5kV acceleration voltage. The particle size was determined by measuring the size of 100 randomly selected particles from the obtained images and averaging the result.

[0199] To compare the fluorescence signals of the aforementioned manufactured microparticles and the Alexa750-human serum albumin complex, a complex solution and a microparticle dispersion of Alexa750 at the same concentration were prepared. 0.77 mg of Alexa750 powder was dissolved in a mixture of 0.214 mL of human serum albumin solution (20% concentration) and 0.786 mL of distilled water, and then diluted with distilled water to prepare 0.3 mL of Alexa750-human serum albumin complex (Alexa750-HSA) at a concentration of 30 μM. The Alexa750 content in the PMMA microparticles, as evaluated by the quantitative analysis, was used to prepare a microparticle dispersion by adding Alexa750 to 0.3 mL of distilled water so that the concentration of Alexa750 was 30 μM. For fluorescence spectrum comparison, 200 μL each of the prepared solution and microparticle dispersion were taken and placed in a 96-well plate, and the fluorescence spectra (λ) were measured using a multifunctional microplate reader. ex The wavelength (=730 nm) was measured.

[0200] In Alexa750-PMMA (without P-188), which was manufactured by incorporating Alexa750 during the production of PMMA microparticles, the Alexa750 content within the microparticles was 14.1%, and it was confirmed that the Alexa750 content per 1 mg of microparticle was 151.8 ng. In Alexa750-HSA-PMMA (without P-188), which was manufactured by incorporating Alexa750-HSA, the Alexa750 content within the microparticles was 9.61%, and it was confirmed that the Alexa750 content per 1 mg of microparticle was 97.7 ng. In Alexa750-HSA-PMMA (1.0% P-188), which was manufactured by adding 1.0% poloxamer-188 based on the primary emulsion phase, the Alexa750 content within the microparticles was 21.2%, and it was confirmed that the Alexa750 content per 1 mg of microparticle was 188.1 ng, indicating that it contained a higher content of Alexa750 than the other two microparticle types.

[0201] The Alexa750-PMMA (without P-188), Alexa750-HSA-PMMA (without P-188), and Alexa750-HSA-PMMA (1.0% P-188) microparticles were confirmed to be spherical particles with average particle sizes of 53.0±15.8 μm, 54.6 ±20.3 μm, and 57.7±19.6 μm, respectively, indicating an appropriate size to avoid phagocytic activity by macrophages. Evaluation of scanning electron microscope cross-sections of each microparticle (Figure 49) revealed that the PMMA microparticles contain multiple isolated small spherical spaces, which prevents the elution of the attached fluorescent dye, confirming that this morphology is highly suitable for long-term fluorescent dye labeling.

[0202] Figure 50 shows the fluorescence spectrum of the Alexa750-PMMA (without P-188) particulate dispersion, which showed a fluorescence signal 6.5 times lower than that of Alexa750-HSA. The fluorescence spectra of the Alexa750-HSA-PMMA (without P-188) and Alexa750-HSA-PMMA (1.0% P-188) particulate dispersions showed fluorescence signals 2.0 times and 1.9 times higher than Alexa750-HAS, respectively, and 13 times and 12 times higher than Alexa750-PMMA (without P-188). These results indicate that when the near-infrared fluorescent dye Alexa750 is incorporated in the Alexa750-HSA composite form, a much higher fluorescence signal can be obtained compared to when it is incorporated alone.

[0203] Example 13: Production and analysis of microparticles containing IRDye 800CW-human serum albumin complex 13-1: Comparison of fluorescence signals at different concentrations of IRDye 800CW solution and IRDye 800CW-HSA complex. 0.70 mg of IRDye® 800CW carboxylate (IRDye 800CW, LI-COR, Inc.), a near-infrared fluorescent dye, was dissolved in 1 mL of distilled water, and then diluted with distilled water to prepare Alexa750 solutions at concentrations of 100, 70, 50, 40, 30, 20, 10, 5, 4, 3, 2, and 1 μM. To compare the fluorescence signals of IRDye 800CW solution and human serum albumin (HSA, SK plasma) conjugates, 0.70 mg of IRDye 800CW powder was dissolved in a mixture of 0.214 mL of human serum albumin solution (20% concentration) and 0.786 mL of distilled water, and then diluted with distilled water to prepare IRDye 800CW / human serum albumin conjugates (IRDye 800CW-HSA) at concentrations of 100, 70, 50, 40, 30, 20, 10, 5, 4, 3, 2, and 1 μM. For fluorescence spectral comparison, 200 μL of each prepared solution was taken and placed in a 96-well plate, and the fluorescence spectra (λ) were measured using a multifunctional microplate reader (SPARK, Tecan Trading AG, Zurich, Switzerland).ex =730 nm, λ em =806 nm (IRDye 800CW), λ em We measured emission at 810 nm (IRDye 800CW-HSA). The IRDye 800CW fluorescent dye shows an emission peak at 806 nm, but after forming a complex with HSA, it showed an emission peak at 810 nm.

[0204] Similar to Figure 51, which compares the fluorescence intensity of IRDye 800CW solution and IRDye 800CW-HSA complex solution at concentrations of 1-100 μM, it was confirmed that the IRDye 800CW-HSA solution generates a higher fluorescence signal than the IRDye 800CW solution at concentrations of 3 μM or higher. It was also confirmed that the IRDye 800CW-HSA solution generates a fluorescence signal 1.1 times higher than the IRDye 800CW solution at a concentration of 30 μM and 1.1 times higher at the maximum measured concentration of 100 μM.

[0205] 13-2: Manufacturing and analysis of PMMA microparticles containing IRDye 800CW and IRDye 800CW-HSA composite IRDye 800CW-PMMA (without P-188), which are PMMA microparticles equipped with the near-infrared fluorescent dye IRDye 800CW, were manufactured. Solution W1 was prepared by dissolving 1.19 mg of IRDye 800CW in 1.691 mL of distilled water. After adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, it was dissolved at 85°C while stirring at 600 rpm, and then cooled to room temperature to prepare solution W2. Solution oil was prepared by adding 1.217 g of PMMA to 16.909 mL of methylene chloride and dissolving it. After adding the prepared W1 solution to the oil solution, ultrasonic treatment was performed for 3 minutes (1 minute ultrasonic treatment + 30 second rest repeated) using a probe-type ultrasonic device to prepare a primary emulsion (water-in-oil). The prepared W2 solution was stirred at 1000 rpm using a mechanical stirrer, and the manufactured primary emulsion solution was added at a rate of 10 mL / min using a syringe pump to produce a secondary emulsion (water-in-oil-in-water). The manufactured emulsion was stirred at 1000 rpm for 10 minutes, and then stirred at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C to remove the methylene chloride solvent. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PMMA polymer microparticles loaded with IRDye 800CW. The supernatant was removed, and the PMMA microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed microparticles were freeze-dried to obtain microparticle powder.

[0206] IRDye 800CW-HSA-PMMA (without P-188), which is a PMMA microparticle containing the near-infrared fluorescent dye IRDye 800CW-HSA complex, was manufactured. Solution W1 was prepared by dissolving 1.19 mg of IRDye 800CW in 0.363 mL of human serum albumin (HSA) (20% concentration, 72.553 mg) and 1.328 mL of distilled water. Solution W2 was prepared by adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, dissolving it at 85°C with stirring at 600 rpm, and cooling to room temperature. Solution W2 was prepared by dissolving 1.217 g of PMMA in 16.909 mL of methylene chloride. After adding the prepared W1 solution to the oil solution, sonication was performed for 3 minutes (1 minute sonication + 30 second rest repeated) using a probe-type ultrasonic device to prepare a primary emulsion (water-in-oil). The prepared W2 solution was stirred at 1000 rpm using a mechanical stirrer, and the manufactured primary emulsion solution was added at a rate of 10 mL / min using a syringe pump to produce a secondary emulsion (water-in-oil-in-water). The manufactured emulsion was stirred at 1000 rpm for 10 minutes, and then stirred at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C to remove the methylene chloride solvent. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PMMA polymer microparticles loaded with IRDye 800CW-HSA. The supernatant was removed, and the PMMA microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed microparticles were freeze-dried to obtain microparticle powder.

[0207] IRDye 800CW-HSA-PMMA (P-188 1.0%), which are PMMA microparticles containing a 1.0% primary emulsion phase of poloxamer-188 and equipped with the near-infrared fluorescent dye IRDye 800CW-HSA complex, were prepared. Solution W1 was prepared by dissolving 1.19 mg of IRDye 800CW in 0.363 mL of human serum albumin (HSA) (20% concentration, 72.553 mg) and 1.328 mL of distilled water. Solution W2 was prepared by adding 3.6 g of polyvinyl alcohol to 180 mL of distilled water, dissolving it at 85°C with stirring at 600 rpm, and cooling to room temperature. An oil solution was prepared by dissolving 1.217 g of PMMA and 186 mg of poloxamer-188 (P-188) in 16.909 mL of methylene chloride. After adding the prepared W1 solution to the oil solution, ultrasonic treatment was performed using a probe-type ultrasonic device for 3 minutes (1 minute ultrasonic treatment + 30 second rest repeated) to produce a primary emulsion (water-in-oil). While stirring the prepared W2 solution at 1000 rpm using a mechanical stirrer, the prepared primary emulsion solution was added at a rate of 10 mL / min using a syringe pump to produce a secondary emulsion (water-in-oil-in-water). After stirring the prepared emulsion at 1000 rpm for 10 minutes, the methylene chloride solvent was removed by stirring at 280 rpm for 21 hours using a magnetic stirrer at a temperature of 40°C. Subsequently, the mixture was centrifuged at 14,000 rpm for 5 minutes to obtain PMMA polymer microparticles mounted on an IRDye 800CW-HSA. The supernatant was removed, and the PMMA microparticles were washed by repeating the process of redispersion with distilled water and centrifugation three times. The washed fine particles were freeze-dried to obtain a fine particle powder.

[0208] To evaluate the content and loading rate of IRDye 800CW in the manufactured microparticles, the microparticles were dissolved, and the concentration of IRDye 800CW in the solution was quantified using an ultraviolet / visible light absorption analyzer.

[0209] Scanning electron microscopy analysis was performed to analyze the particle morphology and size of the synthesized microparticles. For cross-sectional analysis of the manufactured microparticles, the particles were cut with a razor blade. The microparticle powder was attached to carbon tape mounted on a scanning electron microscope sample holder, and then coated with Pt / Pd via sputtering for 60 seconds. Scanning electron microscope images were then analyzed with a 5kV acceleration voltage. The particle size was determined by measuring the size of 100 randomly selected particles from the obtained images and averaging the result.

[0210] To compare the fluorescence signals of the aforementioned manufactured microparticles and the IRDye 800CW-human serum albumin complex, a complex solution and a microparticle dispersion of the same concentration of Alexa750 were prepared. 0.70 mg of IRDye 800CW powder was dissolved in 0.214 mL of a 20% human serum albumin solution and 0.786 mL of a distilled water mixture, and then diluted with distilled water to prepare 0.3 mL of a 30 μM IRDye 800CW-human serum albumin complex (IRDye 800CW-HSA). Using the information obtained from the quantitative analysis of IRDye 800CW content in the PMMA microparticles, a microparticle dispersion was prepared by adding IRDye 800CW to 0.3 mL of distilled water to achieve a concentration of 30 μM. For fluorescence spectrum comparison, 200 μL each of the prepared solution and microparticle dispersion were taken and placed in a 96-well plate, and the fluorescence spectra (λ) were analyzed using a multifunctional microplate reader. ex The wavelength (=730 nm) was measured.

[0211] In the manufacturing of PMMA microparticles, the IRDye 800CW-PMMA (without P-188) microparticles, which were manufactured using IRDye 800CW, was found to have an IRDye 800CW loading rate of 10.7%, and the IRDye 800CW content in 1 mg of microparticles was confirmed to be 104.4 ng. The loading rates of IRDye 800CW in IRDye 800CW-HSA-PMMA (without P-188) microparticles manufactured using IRDye 800CW-HSA and IRDye 800CW-HSA-PMMA (1.0% P-188) manufactured by adding 1.0% poloxamer-188 as a primary emulsion phase were 19.5% and 19.0%, respectively. The IRDye 800CW content per 1 mg of microparticle was 179.6 ng and 152.8 ng, confirming that the latter contained a higher content of IRDye 800CW than the IRDye 800CW-PMMA (without P-188) microparticles.

[0212] Analysis of scanning electron microscope images of IRDye 800CW-PMMA (without P-188), IRDye 800CW-HSA-PMMA (without P-188), and IRDye 800CW-HSA-PMMA (1.0% P-188) microparticles revealed that each microparticle was a spherical particle with average particle sizes of 55.8±19.7 μm, 66.6±13.8 μm, and 59.8±17.3 μm, respectively, confirming that it possessed an appropriate size to avoid phagocytic activity by macrophages (Figure 52(a, c, e)). Furthermore, analysis of scanning electron microscope cross-sections of each microparticle (Figure 52(b, d, f)) showed that the inside of the PMMA microparticles contained multiple isolated small spherical spaces, which prevented the elution of the attached fluorescent dye, confirming that it is a morphology very suitable for long-term fluorescent dye labeling.

[0213] Figure 53 shows that the fluorescence spectrum of the IRDye 800CW-PMMA (without P-188) particulate dispersion showed a fluorescence signal 6.2 times lower than that of IRDye 800CW-HSA. The fluorescence spectra of the IRDye 800CW-HSA-PMMA (without P-188) and IRDye 800CW-HSA-PMMA (1.0% P-188) particulate dispersions showed fluorescence signals 4.0 times and 3.9 times higher than IRDye 800CW-HSA, respectively, and 25 times and 24 times higher than IRDye 800CW-PMMA (without P-188), respectively. From these results, it can be seen that when the near-infrared fluorescent dye IRDye 800CW is incorporated in the form of IRDye 800CW-HSA, a much higher fluorescence signal is obtained than when it is incorporated alone.

[0214] Example 14: Preparation of a dispersion composition of PLGA microparticles and preparation and analysis of a pellet-type marker containing the microparticles. As a representative example of a biodegradable polymer, fine particles produced using PLGA polymer were dispersed in various other surfactants or biocompatible polymer substances to prepare a dispersed aqueous solution composition for injection administration. The fine particles used in this example were ICG-HSA-PLGA(P-188 0.5%). To disperse the fine particles, a solution of 0.5% sodium methylcellulose (Sigma-Aldrich) / 0.1% Tween 80 (Sigma-Aldrich) dissolved in physiological saline for injection, 1% Sodium Alginate (Kimica), 1% Sodium Hyaluronate (MW 1M) (Lifecore Biomedical), 2% sodium methylcellulose (Sigma-Aldrich), 2% poly(vinyl alcohol) (Sigma-Aldrich), 0.1% Collagen (Collagen from calf skin, Sigma-Aldrich), 5% gelatin (Gelatin from porcine, Sigma-Aldrich), 10% Tween 80 (Sigma-Aldrich), 40% poloxamer 188 (BASF), and 5% Polyvinyl pyrrolidone (Sigma-Aldrich) was prepared, followed by the preparation of ICG-HSA-PLGA (P-188) The microparticles were dispersed in each solution so that the concentration of indocyanine green within the 0.5% microparticles was 30 μM.

[0215] To produce a solid-form lesion labeling formulation, dried pellet-type markers were manufactured. For this purpose, fine particles dispersed in 1% sodium hyaluronate and 5% gelatin aqueous solutions were transferred to a cylindrical frame, rapidly cooled with liquid nitrogen, and freeze-dried to produce solid pellet-type fluorescent markers.

[0216] Figure 54 shows the results of preparing compositions in which the fine particles were dispersed in various other surfactants and biocompatible polymers. It was confirmed that the ICG-HSA-PLGA(P-188 0.5%) fine particles were uniformly dispersed in all the composition solutions in which they were used.

[0217] Furthermore, Figure 55 confirms that fluorescent markers in the form of solid pellets can be produced using fine particles dispersed in a 1% sodium hyaluronate and 5% gelatin solution.

[0218] Example 15: Preparation of a dispersion composition of PMMA microparticles and preparation and analysis of a pellet-type marker containing the microparticles. As a representative example of a non-biodegradable polymer, fine particles produced using PMMA polymer were dispersed in various other surfactants or biocompatible polymer substances to prepare a dispersed aqueous solution composition for injection administration. The fine particles used in this example are ICG-HSA-PMMA(P-188 1.0%) fine particles.

[0219] To disperse the microparticles, solutions were prepared using 0.5% sodium methylcellulose (Sigma-Aldrich) / 0.1% Tween 80 (Sigma-Aldrich) dissolved in physiological saline for injection, and a solution of 1% sodium alginate, 1% sodium hyaluronate, 2% sodium methylcellulose, 2% poly(vinyl alcohol), 0.1% collagen, 5% gelatin, 10% Tween 80, 40% poloxamer 188, and 5% polyvinyl pyrrolidone. The microparticles were then dispersed in each solution so that the concentration of indocyanine green within the ICG-HSA-PMMA (P-188 1.0%) microparticles was 30 μM.

[0220] To produce a solid-form lesion labeling formulation, dried pellet-type markers were manufactured. For this purpose, fine particles dispersed in an aqueous solution of 1% sodium alginate, 1% sodium hyaluronate, 2% sodium methylcellulose, 2% poly(vinyl alcohol), 5% gelatin, 40% poloxamer-188, and 5% polyvinylpyrrolidone were transferred to a cylindrical frame, rapidly cooled with liquid nitrogen, and freeze-dried to produce solid pellet-type fluorescent markers.

[0221] As a result of dispersion, as in Fig. 56, a composition in which ICG-HSA-PMMA (P-188 1.0%) microparticles were dispersed in various other surfactants and biocompatible polymeric substances was produced. As a result, it was confirmed that the ICG-HSA-PMMA (P-188 1.0%) microparticles were uniformly dispersed in all the composition solutions in which they were used.

[0222] Also, according to the results of Fig. 57, it was confirmed that fluorescent marker in the form of solid pellets could be produced using microparticles dispersed in 1% sodium alginate, 1% sodium hyaluronate, 2% sodium methylcellulose, 2% poly(vinyl alcohol), 5% gelatin, 40% poloxamer-188, and 5% polyvinylpyrrolidone solutions.

[0223] Example 16: Production and Analysis of Near-Infrared Fluorescent Marker in the Form of a Metal Clip Coated with Microparticles In this example, an attempt was made to show that it is possible to produce fluorescent markers using metal and non-metal materials by coating the surfaces of metal and non-metal materials with microparticles loaded with a fluorescent dye. For this purpose, IRDye 800CW-HSA-PMMA (without P-188) microparticles, which are microparticles produced using PMMA polymer, were dispersed in a latex solution, then coated on the clip surface and dried to produce a metal clip coated with fluorescent dye-loaded microparticles.

[0224] To prepare a coating solution containing dispersed IRDye 800CW-HSA-PMMA (without P-188) microparticles, the microparticles were dispersed in a latex solution (Acrylonitrile-Butadinene Copolymer 36%, water 64%) to a concentration of 135.03 mg / mL. Then, 2 μL of the coating solution was uniformly coated onto both surfaces of a metal clip. After coating the metal clip surfaces with the solution, it was dried at room temperature for 24 hours, followed by heat treatment at 100°C for 30 minutes to produce a metal clip with fluorescent marker functionality coated with IRDye 800CW-HSA-PMMA (without P-188) microparticles.

[0225] To evaluate the efficacy of IRDye 800CW-HSA-PMMA (without P-188) microparticle-coated metal clips as fluorescent markers, one side of a fluorescent clip coated with IRDye 800CW-HSA-PMMA (without P-188) microparticles was cut, and fluorescence images were taken with the coated side facing upwards (λ). ex =780 / 20 nm, λ em (=845 / 40 nm).

[0226] As shown in the photograph in Figure 58, it was confirmed that the microparticles were uniformly coated on the surface of the metal clip.

[0227] The ROI (Region of Interest) value analyzed from the fluorescence image of a metal clip coated with microparticles was 4.92 × 10⁻¹⁴. 5 It was confirmed that this generated a stronger fluorescence signal compared to ICG-HSA composites of the same concentration. From the results in Figures 58 and 59, it was confirmed that various forms of fluorescent markers can be manufactured by coating microparticles containing near-infrared fluorescent dyes onto metal clips or the like.

[0228] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, which are described later in the detailed description, and their equivalent concepts.

Claims

1. Polymer microparticles equipped with near-infrared fluorescent dyes for lesion labeling, The polymer microparticle contains one or more cavities, The particle size of the polymer microparticles is 20 μm or larger. The near-infrared fluorescent dye forms a complex with human serum albumin or cyclodextrin. The composite is a polymer microparticle for labeling lesions, which is placed in the cavity.

2. The aforementioned near-infrared fluorescent dyes are indocyanine green, IRDye 800CW carboxylate (IRDye 800CW), Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 780, Flamma 749, Flamma 774, Flamma 800, and FSD Fluor TM 647, FSD Fluor TM 680, FSD Fluor TM 750, FSD Fluor TM The polymer fine particles according to claim 1, which are selected from 800, sulfocyanine 5-carboxylate (Cy5), sulfocyanine 5.5-carboxylate (Cy5.5), sulfocyanine 7-carboxylate (Cy7), or sulfocyanine 7.5-carboxylate (Cy7.5).

3. The polymer fine particles according to claim 1, wherein the polymer is poly(lactide-co-glycolide) (PLGA), poly(DL-lactide-co-glycolide) (PDLGA), poly(glycolic acid) (PGA), poly(lactide) (PLA), poly(hydroxybutyrate), polycaprolactone (PCL), polydioxanone (PDO), poly(amino acid), polyanhydride, polyorthoester, or polyphosphazene.

4. The polymer fine particles according to claim 3, comprising a block copolymer of the polymer and poly(ethylene oxide) (PEG).

5. The polymer fine particles according to claim 1, wherein the polymer is poly(methyl methacrylate) (PMMA) or polycarbonate (PC).

6. The polymer microparticles according to claim 1, wherein the microparticles further comprise alginic acid or hyaluronic acid.

7. The polymer microparticles according to claim 1, wherein the microparticles further comprise a surfactant.

8. The polymer microparticles according to claim 7, wherein the surfactant is selected from polyvinyl alcohol, polyethylene glycol, Labrafil, Labrasol, medium chain triglyceride, lecithin, N-methyl pyrrolidone, polyvinyl pyrrolidone, hydropropyl methylcellulose, Poloxamer, or Tween.

9. The polymer fine particles according to claim 1, wherein the fine particles are for coating on the surface of a metal or nonmetallic material.

10. The polymer fine particles according to claim 9, wherein the coating is performed by adhesives or rubber agents.

11. A method for producing polymer microparticles for labeling lesions, comprising the steps of: forming a complex of a near-infrared fluorescent dye and human serum albumin, or a near-infrared fluorescent dye and cyclodextrin; and loading the complex into the cavity of a polymer microparticle having one or more cavities and a particle size of 20 μm or more, using a water-in-oil-in-water (W1 / O / W2) emulsion method.

12. A method for producing polymer microparticles for labeling lesions according to claim 11, further comprising the step of mixing the near-infrared fluorescent dye with a hydrogel polymer and incorporating it into polymer microparticles.

13. A composition for labeling lesions, comprising polymeric microparticles according to any one of claims 1 to 10.

14. The composition for labeling lesions according to claim 13, wherein the fine particles are mixed with methylcellulose, alginic acid, hyaluronic acid, polyvinyl alcohol, collagen, gelatin, Tween, Poloxamer, or polyvinylpyrrolidone.

15. The composition for labeling lesions according to claim 14, wherein the composition is injectable via a syringe.

16. The composition for labeling lesions according to claim 13, wherein the composition is in the form of solid pellets.

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