Medical adhesive composition and preparation method for same

Abstract

The present invention provides: a medical adhesive composition comprising azide group-containing albumin, gelatin, and 4-arm PEG-DBCO, and formed in a powder form; and a preparation method for same. The medical adhesive composition according to the present invention has advantages such as fast, strong, and lasting adhesion in vivo, biocompatibility, biodegradability, long-term storability, ease of application, and minimization of side effects. In addition, according to the present invention, the medical adhesive composition can be effectively prepared and effectively attached.

Description

Medical adhesive composition and method for manufacturing the same

[0001] The present invention relates to a medical adhesive composition and a method for manufacturing the same, and more specifically, to a medical adhesive composition having improved characteristics and a method for manufacturing the same.

[0002] The primary clinical method for treating damaged tissue is surgical suturing. However, treatment using sutures requires a high level of skill, is time-consuming, and carries a high risk of side effects. Surgical staplers can be used for convenience, but they also have disadvantages, such as device malfunction or tissue rupture. Since methods using sutures or staples are mechanical, they can cause additional tissue damage, increasing the likelihood of infection or bleeding.

[0003] Medical adhesives can be a promising alternative to these traditional surgical methods. They can easily form strong bonds with tissues, promoting healing and reducing the risk of infection and bleeding. Furthermore, they can be used in various medical applications, such as tissue adhesion, bleeding control, drug delivery, and the integration of bioelectronic devices.

[0004] Research is underway to develop medical adhesives using various biocompatible and biodegradable materials (fibrin, albumin, collagen, gelatin, hyaluronic acid, etc.). However, existing commercially available medical adhesives do not possess the characteristics of an ideal adhesive and have several drawbacks.

[0005] For example, cyanoacrylate (Dermabond) is a powerful tissue adhesive with a rapid curing time and strong adhesive properties; however, it is unsuitable for internal use due to cytotoxicity and inflammation, and its application is limited to external tissues. Additionally, its high rigidity after polymerization can cause problems when applied to flexible biological tissues, and the heat generated during the polymerization process can damage surrounding tissues.

[0006] Fibrin adhesive (Tisseel) is suitable for various medical applications due to its fast setting time and high biocompatibility, but it has limitations in cases requiring strong and long-term adhesion due to its low adhesive strength.

[0007] Albumin-based adhesives (BioGlue) have fluidity and strong adhesion and are suitable for medical applications such as tissue suturing and wound closure, but glutaraldehyde, a crosslinking agent, can cause severe toxicity, inflammatory reactions, tissue necrosis, and long-term health risks.

[0008] To address these issues, an ideal adhesive must possess the following characteristics: (1) biocompatibility and biodegradability to ensure safety within the body, (2) rapid and strong adhesion to biological tissues, (3) the ability to maintain adhesion even in moist biological tissues, (4) flexibility, elasticity, and fatigue resistance to ensure long-term adhesion in dynamic environments such as the heart, lungs, and stomach, (5) long-term storage capability, and (6) ease of application with various devices such as endoscopes. Additionally, it must minimize adverse effects by preventing adhesion to unintended tissues.

[0009] [Prior Art Literature]

[0010] [Patent Literature]

[0011] (Patent Document 1) Korean Registered Patent No. 10-2683263, July 10, 2024, Specification

[0012] One problem that the present invention aims to solve is to provide a medical adhesive having improved characteristics.

[0013] In addition, another problem that the present invention aims to solve is to provide a method for manufacturing the medical adhesive of the present invention.

[0014] The problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0015] The present invention provides a medical adhesive composition comprising azide-containing albumin, gelatin, and 4-arm PEG-DBCO, which is in powder form.

[0016] In addition, the present invention provides a method for preparing a medical adhesive composition, comprising: (a) a step of preparing an azide-containing albumin comprising mixing albumin and 2-Morpholinoethanesulphonic acid (MES) with a compound containing both an azide group and an amide group; and (b) a step of mixing the azide-containing albumin with 4-arm PEG-DBCO and gelatin.

[0017] The medical adhesive composition of the present invention has the following effects: biocompatibility and biodegradability that can ensure safety within the body; rapid and strong adhesion to biological tissues; the ability to maintain adhesion even in moist biological tissues; flexibility, elasticity, and fatigue resistance that can ensure long-term adhesion in dynamic environments such as the heart, lungs, and stomach; long-term storage capability; ease of application with various devices such as endoscopes; and the ability to minimize side effects by preventing adhesion to unintended tissues. Furthermore, the medical adhesive composition of the present invention can be effectively manufactured by the method of the present invention.

[0018] Figure 1 is a figure showing the results of a characteristic verification experiment of a composition, which is an embodiment of the present invention.

[0019] Figure 2 is an SEM image taken during a characteristic verification experiment of a composition, which is an embodiment of the present invention.

[0020] Figure 3 shows the results of a long-term storage test of a composition that is an embodiment of the present invention.

[0021] Figure 4 is a figure showing the results of an experiment confirming the adhesive properties of a composition, which is an embodiment of the present invention.

[0022] FIGS. 5a to 5e are figures showing the experimental results of confirming the mechanical properties of a composition which is an embodiment of the present invention.

[0023] Figure 6 is a figure showing the experimental results confirming the adhesive effect on an organ of one embodiment of the present invention.

[0024] Figure 7 is a figure showing the experimental results confirming the sealing effect on an organ of one embodiment of the present invention.

[0025] Figure 8 shows the results of an in vitro hemostatic effect test of one embodiment of the present invention.

[0026] Figure 9 shows the results of an experiment on red blood cell adhesion in one embodiment of the present invention.

[0027] FIG. 10 shows the results of a biocompatibility confirmation experiment of one embodiment of the present invention.

[0028] Figure 11 shows the experimental results confirming the in vivo hemostatic effect of one embodiment of the present invention.

[0029] FIGS. 12a to 12d show the results of an experiment confirming drug distribution in vivo according to one embodiment of the present invention.

[0030] FIGS. 13a to 13d show the results of an experiment confirming drug distribution by organ in vitro according to one embodiment of the present invention.

[0031] Figure 14 is a figure showing the experimental results confirming the photothermal therapy auxiliary effect in tumor cells according to one embodiment of the present invention.

[0032] FIG. 15 is a figure showing the experimental results confirming the auxiliary effect of photothermal therapy in the liver according to one embodiment of the present invention.

[0033] FIGS. 16a to 16c show the experimental results confirming the solid retention effect at the in vivo administration site according to one embodiment of the present invention.

[0034] FIG. 17 is a figure showing the experimental results of the application of an in vivo electronic device according to one embodiment of the present invention.

[0035] FIGS. 18a to 18d show the experimental results confirming the auxiliary effect of inhibiting tumor recurrence according to one embodiment of the present invention.

[0036] FIGS. 19a to 19d show the results of an in vitro safety confirmation experiment according to one embodiment of the present invention.

[0037] FIG. 20 shows the results of an in vivo safety confirmation experiment according to one embodiment of the present invention.

[0038] Figure 21 is a photograph showing the results of a biodegradability confirmation test in vivo of one embodiment of the present invention.

[0039] Figure 22 is a photograph showing the results of a test confirming the hemostatic effect on gastric bleeding of one embodiment of the present invention.

[0040] The advantages and features of the present invention and the methods for achieving them will become clear from the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0041] Throughout this specification, "and / or" includes each of the mentioned components and all combinations of one or more. The terms used herein are for describing the embodiments and are not intended to limit the invention. In this specification, the singular form includes the plural form unless specifically stated otherwise in the text. As used herein, "comprises" and / or "comprising" do not exclude the presence or addition of one or more other components in addition to the mentioned components.

[0042] A medical adhesive composition, which is one embodiment of the present invention, comprises azide-containing albumin, gelatin, and 4-arm PEG-DBCO, and is in powder form.

[0043] Albumin is one of the proteins that constitute the basic material of cells; it is present in very large quantities in the blood and refers to a protein produced in the liver. Albumin has the smallest molecular weight among simple proteins existing in nature. Serum albumin in the blood functions to maintain and restore plasma volume, so it is used to prevent shock caused by excessive bleeding and in the treatment of surgeries and burns. It is also known to possess oxygen-carrying capabilities similar to hemoglobin.

[0044] Albumin may include all albumin that can be formulated and may be derived from mammalian plasma, including human plasma. It may also be recombinant serum albumin produced by genetic engineering.

[0045] Genetic information regarding the albumin of the present invention can be obtained from known databases such as NCBI GenBank.

[0046] In the present invention, the amino groups (-NH2) exposed on the surface of albumin may be 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 40, 10 to 35, 10 to 30, 10 to 20, 15 to 40, 15 to 30, or 15 to 20.

[0047] Azide-containing albumin may be albumin in which the COOH functional group of albumin is modified to an azide (N3) functional group. For example, it may be azide-modified serum albumin and may be manufactured using known techniques.

[0048] The azide group is a reactive group composed of three nitrogen atoms that possesses high reactivity and is known to act as an electron donor in the 1,3-dipolar cycloaddition reaction, a type of Cu-Free click chemistry, to form a triaza-5-membered ring. The number of azide groups introduced onto the surface of albumin may be 1 to 20, 1 to 17, 1 to 14, 1 to 10, 1 to 5, 4 to 15, 4 to 10, 7 to 15, or 9 to 12. If the number of azide groups exceeds the above range, it may be immediately absorbed by the liver upon injection into the body, which may restrict absorption to other target disease sites.

[0049] Such azide-containing albumins exhibit very rapid adhesion to wet biological tissue surfaces in combination with gelatin and 4-arm PEG-DBCO. This can be attributed primarily to bioorthogonal click chemistry (e.g., DBCO-azide reaction).

[0050] 4-arm PEG-DBCO is a molecule composed of 4-arm PEG and DBCO, with DBCO functional groups attached to the ends of each of the four PEG arms. The DBCO functional groups can selectively bind to azide-containing albumin. PEG stands for polyethylene glycol, a polymer characterized by excellent biocompatibility and high water solubility. 4-arm PEG is a form of the basic PEG structure in which four arm-like branches are formed. This provides multiple junction sites, enabling linkage with various other molecules or functional groups.

[0051] Dibenzocyclooctyne (DBCO) is a functional group used in click chemistry reactions. It can act in Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC) reactions and reacts selectively with azide groups. Since DBCO can react efficiently even under physiological conditions, it is suitable for both in vivo and in vitro applications.

[0052] Gelatin, a degradation product of collagen, is used in various fields ranging from food to pharmaceuticals due to its biocompatibility, low immunogenicity, easily deformable functional groups, and low cost.

[0053] Such azide-containing albumin, gelatin, and 4-arm PEG-DBCO are combined and formed into a powder.

[0054] The term "powder" implies that it comprises a plurality of independent particles. The average particle size of the powder is not limited thereto, but may be, for example, 50 to 500 μm. Each of the azide-containing albumin, gelatin, and 4-arm PEG-DBCO may consist of separate, independent particles, or two or more of the azide-containing albumin, gelatin, and 4-arm PEG-DBCO may form a single independent particle through contact or bonding. Additionally, the powder may comprise one or more freeze-dried powders selected from the azide-containing albumin, gelatin, and 4-arm PEG-DBCO. For example, the azide-containing albumin may be in the form of a powder (e.g., freeze-dried powder or dry powder). Additionally, the 4-arm PEG-DBCO may be in a state mixed with the azide-containing albumin under conditions without moisture or without the addition of moisture. For example, 4-arm PEG-DBCO may be mixed with azide-containing albumin in a freeze-dried state while existing in an organic solvent, and then the organic solvent is removed, so that 4-arm PEG-DBCO is more effectively adsorbed or mixed with the porous azide-containing albumin. At this time, the organic solvent is not limited as long as it is capable of dissolving 4-arm PEG-DBCO without dissolving the azide-containing albumin, but for example, it may be acetonitrile. In this way, the azide-containing albumin may be in the form of a freeze-dried powder and may be mixed with 4-arm PEG-DBCO under moisture-free conditions. Gelatin may be a freeze-dried powder or a non-freeze-dried powder. Furthermore, it goes without saying that 4-arm PEG-DBCO may be in the form of a freeze-dried powder or a non-freeze-dried powder before being mixed with the azide-containing albumin.

[0055] One embodiment of the present invention, in which such azide-containing albumin, gelatin, and 4-arm PEG-DBCO are combined and formed into a powder, has effects confirmed by experimental results.

[0056] Specifically, since one embodiment of the present invention possesses strong adhesive properties, it can be utilized for hemostasis, wound closure, surgical site closure, and gastric perforation repair. Furthermore, one embodiment of the present invention can be easily applied even in humid environments, making it effective for application to living organisms containing moisture. Rather, since one embodiment of the present invention exhibits strong adhesion within a shorter time due to moisture and maintains that adhesion, it can be applied more effectively to living organisms that inevitably contain moisture. In this way, the powder form is more effective in terms of generating adhesive properties through moisture absorption. This is because the powder form is in a state capable of absorbing moisture more effectively. This powder-form embodiment of the present invention also has the advantage of being able to be stored for a long period. Additionally, one embodiment of the present invention can be applied more easily to body organs with curved shapes and can be easily applied with various devices such as endoscopes. Therefore, one embodiment of the present invention has no limitations on the application site and can be easily applied to living organisms in various ways. Moreover, since it decomposes after a certain period of time, it is possible to control the release of encapsulated drugs according to the decomposition, in addition to its simple adhesive use.

[0057] In addition, one embodiment of the present invention also has excellent properties such as flexibility, elasticity, and fatigue resistance, which can ensure long-term adhesion in dynamic environments such as the heart, lungs, and stomach.

[0058] Although not limited thereto, the gelatin may be 10 to 200 parts by weight, preferably 10 to 150 parts by weight, per 100 parts by weight of azide-containing albumin. Additionally, 4-arm PEG-DBCO may be 10 to 100 parts by weight, preferably 10 to 50 parts by weight, more preferably 10 to 30 parts by weight, per 100 parts by weight of azide-containing albumin. Such content may be based on azide-modified fetal bovine serum albumin. Within this range, it is more preferable in terms of biocompatibility, biodegradability, rapid and strong adhesion to biological tissues, maintenance of adhesion in moist biological tissues, flexibility, elasticity, fatigue resistance, storage stability, etc.

[0059] In addition, a method for preparing a medical adhesive composition, which is an embodiment of the present invention, comprises: (a) a step of preparing an azide-containing albumin comprising mixing albumin and 2-Morpholinoethanesulphonic acid (MES) with a compound containing both an azide group and an amide group; and (b) a step of mixing the azide-containing albumin with 4-arm PEG-DBCO and gelatin.

[0060] Compounds containing both azide and amide groups are not limited to those containing both azide and amide groups, but may be, for example, one or more selected from Azide-HN2, Azide-PEG-HN2, Azide-PEG2-HN2, Azide-PEG3-HN2, and Azide-PEG4-HN2.

[0061] The azide-containing albumin may be a freeze-dried material. Additionally, 4-arm PEG-DBCO and gelatin may each be in powder form. That is, by mixing the freeze-dried azide-containing albumin with the powdered 4-arm PEG-DBCO and gelatin, the freeze-dried material granulates during the process, making it easier to form a powder. Furthermore, since moisture absorption is prevented during the manufacturing process, long-term storage is possible. Additionally, the freeze-dried material makes it easier to generate adhesive strength through moisture absorption when applied to the body.

[0062] In addition, to avoid the influence of moisture during the manufacturing process and to further improve the applicability of the composition, 4-arm PEG-DBCO can be mixed with azide-containing albumin while present in an organic solvent. In this way, the azide-containing albumin can be prevented from being affected by moisture. Also, in this way, the gelatin can be prevented from being affected by moisture. By removing such organic solvent by means such as evaporation, 4-arm PEG-DBCO can be more effectively immobilized on the porous structure of the azide-containing albumin freeze-dried material. Furthermore, when applied to the body, the 4-arm PEG-DBCO immobilized on the porous structure of the azide-containing albumin freeze-dried material absorbs moisture along with the azide-containing albumin and gels within a shorter time, making it possible to generate adhesive strength quickly.

[0063] Accordingly, one embodiment of the present invention may further include a step of removing an organic solvent.

[0064] As such, in step (b), it is more preferable to perform the mixing in a state where no moisture is present or added.

[0065] In addition, one embodiment of the present invention may further include a step of grinding the mixture mixed in step (b). Such a grinding step may be included after the step of removing the organic solvent. Through such a grinding process, the composition can be made to have a desired particle size distribution.

[0066] Unless otherwise noted, the details mentioned in the medical adhesive composition and manufacturing method, which is an embodiment of the present invention, are applied identically within the scope of identity, provided they do not contradict each other.

[0067] The present invention will be explained in more detail below through examples, reference examples, and experimental examples; however, the following examples, reference examples, and experimental examples are merely for illustrating the present invention and the content of the present invention is not limited by the following examples.

[0068] The materials and experimental animals used in the examples, reference examples, and experimental examples were prepared as follows.

[0069] Examples

[0070] <Preparation of Ingredients>

[0071] 4T1 cell lines were purchased from the American Type Culture Collection. Dulbecco's modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Dublin, Ireland). 4-arm PEG-DBCO (molecular weight (MW): 20 kDa) and azide-PEG4-amine were purchased from Creative PEGWorks (Chapel Hill, North Carolina, USA) and LumiProbe (Hunt Valley, Maryland, USA), respectively. Indocyanine Green was purchased from TCI (Chuo-ku, Tokyo, Japan). Unless otherwise specified, all remaining chemicals and reagents were purchased from Sigma-Aldrich. Porcine skin, blood vessels, spleen, and heart tissues were purchased from local markets.

[0072] <Preparation of Experimental Animals>

[0073] ICR mice (male, 6 weeks old) and BALB / c mice (male, 6 weeks old) were purchased from Orient Bio (South Korea) and used as a 4T1 tumor xenograft model. All mice were housed in a pathogen-free environment. All in vivo experiments were performed in accordance with the guidelines and approval of the Kyungpook National University Animal Care and Treatment Committee (IACUC-2024-0239).

[0074] <Manufacture of Medical Adhesive Composition>

[0075] Example 1

[0076] First, azide-modified bovine serum albumin (azide-BSA) was prepared. First, 1 g of BSA was dissolved in 100 mL of 100 mM MES buffer (pH 4.75), and 3 g of azide-PEG4-NH₂ was added. Then, the pH was adjusted back to 4.75 using 1 M HCl. Subsequently, 2 g of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) was slowly added while stirring at 500 rpm. The reaction was carried out at 20°C for 6 hours. The reaction was stopped by adding 4 mL of 4 M acetic acid buffer at pH 4.75. After the reaction, unreacted material was removed by dialyzing five times with distilled water containing a molecular weight barrier (MW barrier: 10 kDa). After dialysis, the obtained azide-BSA was freeze-dried.

[0077] To obtain high-density azide-BSA containing indocyanine green (ICG-azide-BSA), 1 g of freeze-dried azide-BSA was dissolved in 10 mL of distilled water (DW) containing 2 mg of indocyanine green (ICG) and thoroughly mixed. This mixture was freeze-dried to prepare high-density ICG-azide-BSA. Subsequently, 1 g of ICG-azide-BSA powder, 1 g of gelatin, and 250 mg of 4-arm PEG-DBCO were thoroughly mixed to prepare ICG-AGP powder. At this time, gelatin and 4-arm PEG-DBCO were used in powder form.

[0078] Example 2

[0079] A powder formulation (AGP powder) not containing indocyanine was prepared. Specifically, 1 g of freeze-dried azide-BSA prepared in the same manner as in Example 1 was thoroughly mixed with 1 g of gelatin and 250 mg of 4-arm PEG-DBCO to prepare AGP powder.

[0080] Example 3

[0081] A powder formulation of the same type as in Example 1 was prepared by a different method from Example 1. Specifically, first, 1 g of freeze-dried high-density ICG-azide-BSA prepared in the same manner as in Example 1 was prepared. Then, 250 mg of 4-arm PEG-DBCO was dissolved in 2 mL of acetonitrile and mixed with 1 g of freeze-dried high-density ICG-azide-BSA. To remove acetonitrile from the mixture, it was dried at 37°C for 2 hours. After removing acetonitrile, 1 g of gelatin was added and mixed.

[0082] Example 4

[0083] A formulation of the same type as in Example 2 {a powder formulation not containing indocyanine (AGP powder)} was prepared using a method different from that of Example 2. Specifically, 1 g of freeze-dried azide-BSA prepared in the same manner as in Example 1 was prepared. Then, 250 mg of 4-arm PEG-DBCO was dissolved in 2 mL of acetonitrile and mixed with 1 g of freeze-dried high-density ICG-azide-BSA. To remove acetonitrile from the mixture, it was dried at 37°C for 2 hours. After removing acetonitrile, 1 g of gelatin was added and mixed.

[0084] Example 5

[0085] The mixed powder of Example 5 was prepared in the same manner as in Example 3, except that 100 mg of 4-arm PEG-DBCO was used instead of 250 mg.

[0086] Example 6

[0087] The mixed powder of Example 6 was prepared in the same manner as in Example 1, except that 100 mg of 4-arm PEG-DBCO was used instead of 250 mg.

[0088] Reference Example 1

[0089] A formulation (ICG-AP powder) that is identical to Example 1 but does not contain gelatin was prepared as Reference Example 1. Specifically, 1 g of freeze-dried azide-BSA prepared in the same manner as in Example 1 was thoroughly mixed with 250 mg of 4-arm PEG-DBCO to prepare a mixed powder (ICG-AP powder).

[0090] Experimental Example

[0091] The effects of the present invention could be confirmed through the following experimental examples.

[0092] In the experimental example, all statistical analyses were performed using Microsoft Excel. Data were analyzed using a two-sample Student's t-test. Differences were considered statistically significant when the p-value was less than 0.05.

[0093] <Experimental Example 1> Characterization Experiment

[0094] For Example 1 and Reference Example 1, an experiment was conducted to verify the adhesive properties.

[0095] 20 mg each of Example 1 (ICG-AGP powder) and Reference Example 1 (ICG-azide-BSA) were placed in respective vials, and 100 μl of PBS was added. After 30 seconds, 2 mL of PBS was added. Subsequently, after 1 hour, if the substance adhered to the vial when the vial was inverted, it was determined that gelation had occurred. The results are shown in Fig. 1. Fig. 1 is a figure showing the results of a characteristic confirmation experiment of a composition that is an embodiment of the present invention.

[0096] The top of FIG. 1 shows the results for Example 1, and the bottom of FIG. 1 shows the results for Reference Example 1. As shown in FIG. 1, Example 1, in powder form, gelled within 30 seconds upon contact with moisture and maintained a stable state for more than one hour even after water was added. Subsequently, even when the vial was inverted, Example 1 transformed into a gel state and remained strongly attached to the inside of the vial. However, Reference Example 1 could be seen as having dissolved rather than adhering to the inside of the vial. From these results, it can be seen that according to the present invention, strong gel formation is possible in the presence of moisture, allowing for strong adhesion when applied to bleeding sites or wounds where moisture is present in the form of blood or body fluids. However, in the case of Reference Example, since gel formation is not possible in the presence of moisture, it can be seen that it is difficult to apply in the presence of moisture. In other words, according to the present invention, azide-containing albumin, gelatin, and 4-arm PEG-DBCO are organically combined, so it can be applied more effectively not only to bleeding sites or wounds where water is present in the form of blood or body fluids, but also in vivo.

[0097] <Experimental Example 2> Experiment to verify state upon moisture absorption

[0098] When Example 1 became a gel state due to water absorption, its morphological characteristics were analyzed using a field emission scanning electron microscope (FE-SEM). The results are shown in Fig. 2. Fig. 2 is an SEM image taken during a characteristic verification experiment of a composition that is an embodiment of the present invention. As shown in Fig. 2, it can be seen that Example 1 becomes a hydrogel state with a porous structure due to water absorption. It appears that this porous structure exhibits suitability in a biological environment.

[0099] <Experimental Example 3> Long-term storage test

[0100] Example 1: 20g was stored in a vial at 37°C (relative humidity 40%). After 8 weeks, the powder state was checked by inverting the vial. The results are shown in Fig. 3. Fig. 3 shows the results of a long-term storage test of a composition that is an embodiment of the present invention. As shown in Fig. 3, it can be seen that an embodiment of the present invention maintains a powder state even during long-term storage.

[0101] From these results, it can be seen that the composition of the present invention maintains stability and non-adhesion in a dry state, but rapidly transforms into a state of strong adhesion in the presence of moisture, and can maintain that state for a long time. Therefore, it can be seen that the present invention enables long-term storage and can exhibit strong adhesion when necessary.

[0102] <Experimental Example 4> Test to Verify Adhesion Properties

[0103] To evaluate the tissue adhesion properties of Example 1, 30 mg of Example 1 was applied to porcine skin (5 cm x 2 cm) soaked in PBS. The skin, formed into a gel, was bent, stretched, returned to its original state, twisted, and washed with water to observe the adhesion properties.

[0104] In addition, rapid adhesion was confirmed through an experiment in which 20 mg of Example 1 was applied to a glass plate soaked in PBS and pig skin (2 cm x 1.5 cm) was pressed for up to 5 seconds.

[0105] The results are shown in Fig. 4. Fig. 4 is a figure showing the results of an experiment confirming the adhesive properties of a composition, which is an embodiment of the present invention. Fig. 4A is the result of confirming the adhesive properties, and Fig. 4B is the result of confirming the adhesion speed.

[0106] As shown in FIG. 4A, the adhesive area of ​​the present invention remained firmly in place even under various movements such as bending, stretching, recovery, and twisting. In addition, it did not wash away easily and maintained strong adhesive strength even in an environment where it was washed with water.

[0107] In addition, as shown in Fig. 4B, it can be seen that when Example 1 is applied to a glass plate soaked in PBS, stable adhesion can be achieved within 2.61 seconds (16.51 seconds - 13.90 seconds). This is a relatively very fast adhesion speed compared to conventional adhesives, which take several seconds to tens of seconds to achieve complete adhesion.

[0108] In addition, to quantitatively evaluate the adhesive strength, 40 mg of the mixed powder of Example 1, Example 5, or Example 6 was applied to pig skin (4 cm x 2 cm) soaked in PBS. Then, another piece of pig skin (4 cm x 2 cm) was placed on top (overlapping area 2 cm x 1 cm) and pressed for a certain period of time, after which the adhesion was checked using a 1 kg weight. The measurement time to check the adhesion was 30 seconds. The results are shown in Table 1.

[0109] Example Adhesion Retention 1 Adhesion Retention 5 Adhesion Retention 6 Adhesion Retention

[0110] As described in Table 1, when one embodiment was adhered to pig skin and a load of 1 kg was applied in the vertical direction to the adhesion site, the adhesive force was maintained, demonstrating strong physical properties. At this time, the overlapping adhesion area was approximately 2 cm², indicating that it is effective even when applied to a small area within the body. From these results, it can be seen that the present invention exhibited rapid adhesion in a humid environment and demonstrated stable adhesion even under mechanical deformation and washing conditions. Therefore, it can be seen that the present invention is widely applicable to hemostasis, tissue suture support, local drug delivery patches, etc.

[0111] <Experimental Example 5> Experiment to Verify Mechanical Properties

[0112] To be used as an adhesive in vivo, it is necessary to have strong adhesive strength, appropriate flexibility, elasticity, and fatigue resistance. Therefore, the mechanical properties of Example 1 were measured and evaluated.

[0113] First, the lap shear adhesion strength was evaluated using a moist pig skin sample (2 cm x 0.5 cm). A total of 40 mg of the powder from Example 1 was applied to an adhesion area of ​​1 cm x 0.5 cm and pressure was applied for 30 seconds. The shear strength was measured using a universal testing machine (5900R Instron, USA) at a crosshead speed of 50 mm / min.

[0114] In addition, tensile adhesion strength was evaluated using two moist porcine skin samples measuring 1.5 cm x 1.5 cm. A total of 80 mg of the powder from Example 1 was applied, and the two skin pieces were pressed together for 30 seconds. The tensile test was performed using a universal testing machine (5900R Instron, USA) at a crosshead speed of 50 mm / min. Wrap shear adhesion strength and tensile adhesion strength were calculated by dividing the maximum force by the adhesion area.

[0115] In addition, the compressibility of Example 1 (length, width, and thickness: 10 mm × 15 mm × 5 mm, respectively), which was swollen in PBS for 1 hour after gelation, was tested using a universal testing machine (5565 Instron, USA). For the compression test, the crosshead speed was set to 10 mm / min and the strain limit to 70%. Additionally, the repeated compression test was performed 30 times with the strain limit set to 50%.

[0116] In addition, tensile tests were performed using a universal testing machine (5900R Instron, USA), and the Young's modulus and tensile strength of Reference Example 1 and Example 1, which were swollen in PBS for 1 hour after gelation, were measured. Specimens were prepared with dimensions of 6 mm × 6 mm × 1.2 mm in length, width, and thickness, respectively, and measurements were taken at a crosshead speed of 50 mm / min. Maximum strength refers to the stress measured at the fracture point, and strain was also evaluated at the same location. Young's modulus was derived from the slope of the stress-strain curve with a strain range between 1 / 6 and 1 / 5.

[0117] The results are shown in FIGS. 5a to 5e. FIGS. 5a to 5e are figures showing the experimental results confirming the mechanical properties of a composition that is an embodiment of the present invention. FIG. 5a is a graph showing the experimental results of lap shear adhesion strength for Example 1, where the x-axis represents displacement and the y-axis represents shear stress. FIG. 5b shows the experimental results of tensile adhesion strength for Example 1. FIG. 5c shows the experimental results of compressive strength for Example 1, and FIG. 5d shows the experimental results of cyclic compressive strength for Example 1. FIG. 5e shows the experimental results of tensile strength for Example 1 and Reference Example 1.

[0118] As shown in Fig. 5, the shear stress in the wrap shear adhesive strength test results was measured to be 60.30 ± 4.67 kPa, which represents the maximum stress the adhesive can withstand when a shear load is applied between the two surfaces {Fig. 5a}. In the tensile adhesive strength test results, the tensile stress was measured to be 68.99 ± 3.17 kPa, which represents the maximum stress the adhesive can withstand when a tensile load is applied {Fig. 5b}. These results demonstrate that Example 1 has sufficient adhesive strength to be used as a bioadhesive and can function effectively in a biological environment.

[0119] In addition, the durability of Example 1 can be evaluated from the results of the compressive strength test. Example 1 maintained its physical shape and exhibited high fatigue resistance during the compressive strength test performed at a maximum strain of 70% and the 30-cycle compressive strength test at a strain of 50%. These results indicate that Example 1 can maintain structural stability even under repeated compression conditions {Figs. 5c, 5d}.

[0120] In addition, the stiffness and flexibility of Example 1 and Reference Example 1 were evaluated through tensile strength tests. As a result of the tensile test, it was found that the stiffness was significantly improved, as the Young's modulus of Example 1 increased approximately 3.3 times from 15 kPa to 50 kPa compared to Reference Example 1. Furthermore, the maximum tensile strength of Example 1 was measured at 16.3 kPa, which is a 60% increase compared to Reference Example 1 (8.8 kPa) {Fig. 5e}. These results indicate that the resistance to external tensile loads in Example 1 was significantly improved compared to Reference Example 1. Additionally, the elastic modulus, measured by strain at fracture, doubled from 60% of Reference Example 1 to 120% of Example 1. These results indicate that, according to the present invention, azide-containing albumin, gelatin, and 4-arm PEG-DBCO are organically combined to significantly improve both flexibility and stiffness.

[0121] Therefore, from the experimental results examined above, it can be seen that the composition of the present invention is a highly effective bioadhesive, possessing strong adhesive strength, durability, and mechanical properties.

[0122] <Experimental Example 6> Experiment to Confirm Adhesion Effect on Organs

[0123] The adhesive effect of Example 1 was evaluated in various organs of pigs (intestines, stomach, kidneys, liver, spleen, lungs). After applying 50 mg of the powder of Example 1 to each organ, the adhesive retention was checked by washing it off with water.

[0124] The results are shown in Fig. 6. Fig. 6 is a figure showing the experimental results confirming the adhesion effect on organs of one embodiment of the present invention. As shown in Fig. 6, when the powder of Example 1 was applied to various pig organs (heart, spleen, kidney, lung, liver), the powder adhered effectively, and the adhesive strength was maintained even after washing. These results indicate that the adhesive forms a stable bond and maintains its adhesive strength even after washing.

[0125] Therefore, it can be seen that the composition of the present invention is effectively attached to and maintained in various organs.

[0126] <Experimental Example 7> Experiment to Confirm Sealing Effect on Organs

[0127] To evaluate the adhesion of Example 1 to damaged gastric tissue, a 1 cm defect was created in the stomach of a pig. Then, 300 mg of Example 1 was applied to the defect. The stomach was filled with simulated gastric fluid (SGF), shaken using a vertical shaker, and the sealing performance was observed for 24 hours. The SGF was prepared by dissolving 1 g of pepsin and 2 g of NaCl in 1000 mL of deionized water, and then adjusting the pH to 1.5 ± 0.1 using HCl. Since the stomach contains strong acid and digestive enzymes such as pepsin and moves constantly, a similar environment was simulated using SGF with added strong acid and 1 mg / mL pepsin, and continuous motion was applied using a vertical shaker to reproduce physiological conditions.

[0128] In addition, the sealing effect was evaluated in the presence of food by placing bread and monitoring for an additional 4 hours.

[0129] The results are shown in Fig. 7. Fig. 7 is a figure showing the experimental results confirming the sealing effect on an organ of one embodiment of the present invention.

[0130] As shown in Fig. 7, leakage was observed when SGF was administered to the abdomen prior to treatment in Example 1. However, after treatment in Example 1, SGF leakage was prevented, and the sealing effect was maintained for 24 hours. This indicates that Example 1, a protein-based bioadhesive such as gelatin and albumin, maintains a long-term sealing effect even under harsh conditions involving digestive enzymes and strong acids. The long-term sealing effect can be attributed to the fact that the components are strongly bound through the DBCO-azide reaction, thereby preventing the internal penetration of digestive enzymes. To further evaluate stability under physiological conditions, food (bread) was added; after 4 hours, the bread was digested by SGF and turned into porridge, but the sealing effect of Example 1 remained intact.

[0131] Therefore, it can be seen that the present invention maintains a sealing function even in a demanding biological environment containing food and gastric juice.

[0132] <Experimental Example 8> Experiment to Confirm In Vitro Hemostatic Effect

[0133] To evaluate the blood coagulation ability of Example 1, 360 μL of citric acid-treated whole blood and 40 μL of 0.1 M CaCl₂ were mixed to prepare a final amount of 400 μL of activated blood. Experiments were performed by adding 10 mg of the Example 1 powder and 40 μL of activated blood to each well, or by adding only 40 μL of activated blood without the Example 1 powder. The samples were incubated at 37°C for predetermined times (30, 60, 120, 240, and 480 seconds). Afterward, 2 mL of deionized water was added to each well and gently mixed. Subsequently, the samples were photographed, and the supernatant was collected and analyzed. Samples from each time interval were processed independently in separate wells. The absorbance of the collected supernatant was measured at 540 nm, and the negative control was prepared by adding 2 mL of deionized water (DI water) to 40 μL of activated blood. The blood coagulation index (BCI) was calculated using the following mathematical formula.

[0134] [Mathematical Formula 1]

[0135] BCI = (As / Ac) × 100

[0136] As: Absorbance of the sample supernatant

[0137] Ac: Absorbance of the supernatant of the negative control group

[0138] The results are shown in Fig. 8. Fig. 8 shows the results of an in vitro test confirming the hemostatic effect of an embodiment of the present invention. Fig. 8A is a photograph showing the state over time, and Fig. 8B is a graph showing the blood coagulation index over time. As shown in Fig. 8, blood coagulation occurred after about 7 minutes in the negative control (Blank). On the other hand, when Example 1 (ICG-AGP powder) was administered to the blood, blood coagulation was completed within 30 seconds. Example 1 appears to utilize a click chemical reaction to rapidly form covalent bonds between adhesive components, thereby inducing gelation and forming a barrier that promotes coagulation. Furthermore, the microporous structure of the powder in Example 1 appears to absorb blood components, further enhancing the hemostatic ability.

[0139] <Experimental Example 9> Red Blood Cell Attachment Experiment

[0140] To evaluate the erythrocyte adhesion ability, 100 μL of citric acid-treated whole blood was centrifuged at 400 g for 10 minutes to separate the erythrocyte pellet, and then the pellet was diluted 10-fold with PBS to prepare an erythrocyte suspension. 20 mg of the powder of Example 1 was added to each well of a 24-well plate, followed by the addition of 40 μL of the erythrocyte suspension. The plate was incubated at 37°C for a set time (1 min, 5 min, 10 min). Unattached erythrocytes were removed by washing with 2 mL of PBS. Samples from each time interval were processed independently in separate wells. Subsequently, 2 mL of deionized water was added and the mixture was incubated at 37°C for 1 hour to lyse the attached erythrocytes, and the absorbance of the supernatant was measured at 540 nm. The reference value was set as the absorbance measured by mixing 40 μL of the erythrocyte suspension with 2 mL of deionized water. The red blood cell adhesion rate (%) was calculated using the following mathematical formula.

[0141] [Mathematical Formula 2]

[0142] Red blood cell adhesion rate (%) = As / Ar × 100

[0143] As: Absorbance of the sample supernatant

[0144] Ar: Absorbance of the supernatant relative to the reference value

[0145] The results are shown in Fig. 9. Fig. 9 shows the results of an erythrocyte adhesion experiment of one embodiment of the present invention. As shown in Fig. 9, it can be seen that Example 1 (ICG-AGP powder) effectively promotes erythrocyte adhesion, with approximately 70% of erythrocytes adhering within 1 minute and approximately 90% within 10 minutes. Erythrocytes (RBCs) play an important role in hemostasis, and the aggregation of these cells helps to form denser blood clots and stabilize the newly formed fibrin network. Therefore, it can be seen that the rapid aggregation resulting from the present invention creates a denser cellular matrix, thereby enhancing the integrity of the blood clot and contributing to faster and more effective hemostasis.

[0146] <Experimental Example 10> Biocompatibility Confirmation Experiment

[0147] To evaluate biocompatibility, the hemolytic rate of red blood cells was measured after exposure to Example 1 (ICG-AGP powder), PBS, and Triton. Specifically, to evaluate hemolytic activity, 20 mg of the Example 1 powder and 500 μL of red blood cell suspension (5% PBS, v / v) were placed in a 1.5 mL tube and incubated at 37°C for 1 hour. 0.1% Triton X-100 was used as a positive control, and a red blood cell suspension with PBS added instead of the Example 1 powder was used as a negative control. After incubation, each sample was centrifuged at 2000 rpm for 10 minutes to separate the supernatant, and the hemolytic rate was evaluated by measuring the absorbance of the supernatant at 540 nm. The hemolytic rate was calculated using the following mathematical formula.

[0148] [Mathematical Formula 3]

[0149] Hemolysis Rate (%) = (As-An) / (Ap-An) × 100

[0150] As: Absorbance of the sample supernatant

[0151] An: Absorbance of the supernatant of the negative control group

[0152] Ap: Absorbance of the supernatant of the positive control (0.1% Triton X-100)

[0153] The results are shown in Fig. 10. Fig. 10 shows the results of a biocompatibility confirmation experiment of one embodiment of the present invention. Fig. 10A is a photograph of the experimental process, and Fig. 10B is a graph showing the hemolysis rate for each sample.

[0154] As shown in Fig. 10, the powder of Example 1 promoted noticeable red blood cell aggregation even before centrifugation, which is likely due to the positive charge of the powder inducing electrostatic interactions with the negatively charged red blood cell membrane. In contrast, no aggregation was observed in the PBS or Triton groups before centrifugation (Fig. 10 A). Analysis of hemolysis rates revealed minimal hemolysis in the Example 1 (ICG-AGP powder) (2.9%) and PBS (2.5%) groups, whereas Triton (positive control) induced almost complete hemolysis (100%) (Fig. 10 B). The hemolysis rate of Example 1 was significantly lower than the internationally accepted standard of 5%, indicating that the cytotoxic effect on blood cells was negligible. The strong red blood cell aggregation ability and low hemolysis rate demonstrate that the composition of the present invention can be applied as a safe and effective hemostatic agent.

[0155] <Experimental Example 11> Experiment to Confirm In Vivo Hemostatic Effect

[0156] The hemostatic ability of Example 1 was further verified using a mouse liver hemorrhage model. First, hemorrhage was induced by making an incision of approximately 10 mm in the liver of mice using surgical scissors. The control group received no treatment, while the treatment group was immediately administered 100 mg of the powder of Example 1 to stop the bleeding. Both groups were observed for 5 minutes. Filter paper was placed under the liver to observe the amount of bleeding.

[0157] The results are shown in Fig. 11. Fig. 11 shows the experimental results confirming the in vivo hemostatic effect of one embodiment of the present invention. Fig. 11A is a photograph of the in vivo hemostatic process, Fig. 11B is a photograph of blood collected on filter paper, and Fig. 11C is a graph showing the amount of blood loss by test group. As shown in the figure, bleeding stopped immediately as soon as the powder of Example 1 was applied to the damaged liver of mice (Fig. 11A). Meanwhile, the filter paper used to absorb the blood showed significant bleeding (267 ± 80 mg) in the control group. On the other hand, the group to which Example 1 was applied showed minimal bleeding (71 ± 16 mg) (Figs. 11B, C). These results confirm that the composition of the present invention demonstrates an effective hemostatic effect in in vivo experiments and support the evaluation that it is suitable for clinical application.

[0158] <Experimental Example 12> Drug Distribution Confirmation Experiment

[0159] To evaluate whether the powder of Example 1 attaches to organs and delivers drugs, experiments were conducted on the liver and stomach of model mice. After abdominal incision, the control group (ICG solution) was administered an indocyanine green solution to the liver or stomach, while the treatment group (ICG-AGP powder) was administered 20 mg of Example 1 (indocyanine green dose: 18 μg) to the liver or stomach. After suturing the incision site, the mice were observed at set times for 10 days using a fluorescence imaging device (FOBI). Additionally, experiments were performed in the same manner to confirm the organ distribution of indocyanine green after treatment with the powder of Example 1. On the first day after treatment, dissection was performed, and organs (liver, lungs, kidneys, spleen, heart, stomach, and intestines) were analyzed using a fluorescence imaging device.

[0160] The results are shown in FIGS. 12a to 12d and FIGS. 13a to 13d. FIGS. 12a to 12d show the results of an in vivo drug distribution confirmation experiment according to one embodiment of the present invention, and FIG. 13 shows the results of an in vitro drug distribution confirmation experiment by organ according to one embodiment of the present invention. FIGS. 12a to 12d are photographs ( FIGS. 12a, 12b) showing results for the liver and stomach, respectively, and graphs ( FIG. 12c, 12d) showing fluorescence intensity over time. In addition, FIGS. 13a to 13d are photographs ( FIG. 13a, 13b) showing the results of confirming the drug distribution by organ after administration to the liver and stomach, respectively, and graphs ( FIG. 13c, 13d) showing fluorescence intensity by organ. In the graphs, n=3, **p<0.01, and ***p<0.001.

[0161] As shown in FIGS. 12a to 12d, the treatment group of Example 1 maintained a strong fluorescence signal for up to 10 days in the liver and stomach at the injection site. In contrast, the control group showed rapid absorption, distribution, or excretion within about 1 day, and thereafter, almost no fluorescence was detected.

[0162] In addition, as shown in FIGS. 13a to 13d, the powder of Example 1 attached to the liver and stomach tissues and exhibited a strong fluorescent signal. Most of the fluorescence observed in the liver and stomach was due to the attached powder of Example 1, while limited fluorescence was observed in other areas. In contrast, the fluorescence in the indocyanine green solution injection group was very weak, including in the liver and stomach.

[0163] However, when the indocyanine green solution was administered into the stomach, a significant fluorescent signal was observed in the intestine. This appears to be because some of the indocyanine green solution entered the intestine due to the injection pressure, causing a noticeable fluorescent signal in the intestinal region. These results indicate that the indocyanine green solution may be unintentionally delivered to non-target sites after injection.

[0164] Indocyanine green solution is rapidly excreted through the renal or biliary excretion pathways, resulting in a significant decrease in the concentration of free indocyanine green in the body within a relatively short time after injection. This rapid excretion necessitates repeated administration, which once again highlights the fundamental limitations of using free indocyanine green. In contrast, the powder of Example 1 can effectively attach to the target site to deliver the drug. Furthermore, the powder of Example 1 contains albumin as its main component, a protein known to bind to various drugs through multiple binding sites. In particular, albumin has a strong affinity for indocyanine green, allowing it to encapsulate and gradually release the indocyanine green after administration of the powder of Example 1. Consequently, a single administration of the powder of Example 1 can maintain drug release for up to 10 days, indicating the potential for more effective and long-term treatment or diagnostic intervention.

[0165] In addition, the powder of Example 1 and the indocyanine green solution were sequentially administered to the skin of each mouse, and changes in drug delivery and tissue distribution were observed using a fluorescence imaging device for 14 days. In this experiment, the powder of Example 1 maintained a strong fluorescence signal for up to 14 days, whereas the indocyanine green solution was eliminated within 1 day. Therefore, it can be seen that the powder of Example 1 can be released for a long period in various tissues. These results indicate that the composition of the present invention can be directly applied to surgical sites, such as liver tumor resection or gastrointestinal lesion surgery, to increase long-term local drug utilization and enhance therapeutic efficacy.

[0166] <Experimental Example 13> Experiment to Confirm the Auxiliary Effect of Photothermal Therapy

[0167] Photothermal therapy (PTT) is a treatment method that selectively eliminates cancer cells by converting laser radiation into heat. Indocyanine green has excellent ability to absorb radiation emitted from near-infrared lasers and convert it into heat, and as an FDA-approved substance, it is suitable for application in photothermal therapy. Example 1 was designed to include such indocyanine green to verify whether it is effective as an adjuvant for photothermal therapy.

[0168] Example 1 To evaluate the intratumoral PTT effect of the powder, 4T1 cells (1 × 10⁶ 6 Cells (100 μL) were injected into the flanks of mice. When the tumor size reached 100–200 mm³, the mice were divided and treated with PBS, ICG solution (ICG: 18 μg), Example 2 powder (AGP powder), and Example 1 powder (ICG-AGP powder) (ICG: 18 μg). After 24 hours, a near-infrared laser (808 nm, 1 W / cm², 5 min) was irradiated, and temperature changes were recorded using an infrared thermal imaging camera.

[0169] In addition, to evaluate the PTT effect of the powder of Example 1 on the liver, the abdomen of mice was incised and the liver was treated with PBS, ICG solution (ICG 18 μg), or the powder of Example 1 (ICG 18 μg). After treatment, the incision site was sutured, and after 24 hours, the mice were irradiated with a near-infrared (NIR) laser under the same conditions, and temperature changes were observed using an infrared thermal imaging camera.

[0170] The results are shown in FIGS. 14 and FIGS. 15. FIGS. 14 is a figure showing the experimental results confirming the photothermal therapy auxiliary effect in tumor cells according to one embodiment of the present invention, and FIGS. 15 is a figure showing the experimental results confirming the photothermal therapy auxiliary effect in the liver according to one embodiment of the present invention. FIGS. 14 and FIGS. 15 are graphs showing temperature over time, respectively.

[0171] As described above, in the treatment group of Example 1, the temperature of the target site rose to 55°C, reaching a temperature sufficient to induce cancer cell necrosis. In contrast, the groups treated with only ICG solution, PBS, and AGP powder were limited to 36°C, 35°C, and 34°C, respectively, failing to achieve an effective PTT (Fig. 14). A similar pattern was observed when the powder of Example 1 was surgically administered to the liver prior to laser treatment. While the powder of Example 1 reached approximately 54°C, the ICG solution raised the temperature only to 42°C, and the group administered only AGP powder reached 44°C (Fig. 15).

[0172] Ultimately, the powder of Example 1 remained at the treatment site for a long time and exhibited a strong photothermal effect. In contrast, the ICG solution disappeared quickly, so the temperature-raising effect was insufficient, and no photothermal effect was observed when only the AGP powder was used. These results demonstrate that by applying indocyanine green to the present invention, the photothermal effect can be maximized by maintaining a high concentration of indocyanine green at the treatment site. Therefore, it can be seen that the present invention can assist in sustaining the drug's efficacy by facilitating the application of the drug to the target.

[0173] <Experimental Example 14> Experiment to Confirm Solid Retention Effect at In Vivo Administration Site

[0174] To evaluate the feasibility of solid delivery using Example 2 (AGP powder), 10 mg of gold powder was compressed into a sheet-shaped aggregate (gold leaf). This gold leaf was applied to the liver using the powder from Example 2, and the distribution of the gold leaf was observed via CT imaging. Each mouse was administered 10 mg of liver gold leaf and 40 mg of AGP powder after abdominal incision, and CT scans were performed at set intervals (4 hours, 2 days, and 4 days). As a control group, mice administered only 10 mg of gold leaf without AGP powder were used. Due to the limitations of the CT scanner, different mice were used at each experimental time point (4 hours, 2 days, and 4 days).

[0175] Prior to administration, CT images were taken of a tube mixed with gold leaf and AGP powder, and a tube mixed only with AGP powder, respectively, to check the CT signal of the gold leaf. As a result, a clear CT signal was observed only in the tube mixed with gold leaf and AGP powder. This demonstrates that the gold leaf induces a strong CT signal.

[0176] The experimental results are shown in FIGS. 16a to 16c. FIGS. 16a to 16c show the experimental results confirming the solid retention effect at the in vivo administration site according to one embodiment of the present invention. FIG. 16a shows an example in which gold leaf was applied to the liver as Example 2, FIG. 16b shows an example in which only PBS was administered, and FIG. 16c shows an example in which only gold leaf was administered.

[0177] As shown in the figure, by comparing the liver tissue excised after administration with CT scan results using different mice at each time point, it was confirmed that the gold leaf coated with AGP powder strongly adhered to the liver at the administration site and remained there for a long time. In particular, a clear CT signal was observed in the liver 4 days after administration, confirming that the gold leaf had attached to the liver via the AGP powder. On the other hand, no separate signal was detected in the negative control group (PBS) during CT image analysis. Meanwhile, when only the gold leaf was administered, it lacked adhesive force, so the gold leaf did not stay fixed in place in the liver but migrated to the abdominal cavity, and the CT signal was observed in the abdominal cavity rather than the liver.

[0178] In addition, the same results were reproduced when repeated experiments were conducted on three mice, including this experiment, and the gold foil combined with the powder of Example 2 was attached to the liver. These results indicate that the gold foil combined with the powder of Example 2 adheres strongly to the surface of the liver and can maintain its position at the desired site for a long time. This characteristic indicates that the composition of the present invention adheres well to the desired location and can locally deliver and maintain solid materials. Therefore, the present invention can be applied to therapeutic strategies for target organs by combining it with various biomaterials or therapeutic substances.

[0179] <Experimental Example 15> Experiment on Application of In vivo electronic devices

[0180] An experiment was conducted to confirm whether a small electronic chip (RFID chip) can be stably attached to an organ using Example 2 (AGP powder) and whether the chip operates normally.

[0181] It was verified in advance whether the RFID chip was recognized by an RFID measuring device and whether a unique ID was displayed. Subsequently, six mice were divided into two groups (three mice per group). One group was administered a micro RFID chip (7 mm × 1.25 mm) and Example 2 to the liver via abdominal surgery, while the other group was administered Example 2 without an attached RFID chip as a control group. One day later, measurements were taken randomly from the mice using an RFID reader. Additionally, the livers were dissected and observed through a dissection experiment. The experimental results are shown in Fig. 17. Fig. 17 is a figure showing the experimental results of the application of an in vivo electronic device according to an embodiment of the present invention. As shown in the figure, the unique ID was displayed normally in the group with the RFID chip attached to Example 2. However, in the control group without the chip, no signal was recognized, so the unique tag ID was not displayed. Furthermore, the dissection experiment confirmed that the RFID chip was firmly attached to the liver due to the adhesive force of Example 2.

[0182] These results indicate that the composition of the present invention can stably attach a small electronic chip to a desired site (an internal organ such as the liver). Furthermore, it was shown that the RFID chip transmits and receives signals while attached in this manner. Therefore, the present invention can also be used for the purpose of directly attaching small medical electronic devices (sensors, drug delivery devices) to an organ when performing local treatment and monitoring simultaneously.

[0183] <Experimental Example 16> Experiment to Confirm Adjuvant Effect of Tumor Recurrence Inhibition

[0184] An experiment was conducted to confirm whether one embodiment of the present invention has an adjuvant effect of inhibiting tumor recurrence. A total of 1×10 6 100 μL of 4T1 cells were subcutaneously injected into the flanks of BALB / c mice. When the tumor size reached 250–350 mm³, the tumor was surgically removed. Mice were randomly assigned to four treatment groups {Example 1 (ICG-AGP powder, ICG: 18 μg) + PTT, Example 1 (ICG-AGP powder) alone, Indocyanine Green solution (ICG solution, ICG: 18 μg) + PTT, PBS}. After tumor removal, the sample for each treatment group was applied to the remaining tumor site, and after 24 hours, the PTT treatment group was irradiated with a near-infrared laser (808 nm, 1 W / cm², 5 min). Tumor size was measured every two days, and 20 days after treatment, the tumor was resected and photographed.

[0185] The results are shown in FIGS. 18a to 18d. FIGS. 18a to 18d show the experimental results confirming the auxiliary effect of inhibiting tumor recurrence according to one embodiment of the present invention. FIG. 18a is a photograph taken after tumor resection surgery and during the sample processing process. FIG. 18b is a graph showing tumor size according to elapsed time (***p<0.001). FIG. 18c is a photograph of a surgically removed tumor, and FIG. 18d is a photograph of a recurrent tumor (n=4 per group).

[0186] The average tumor volumes of the group treated with Example 1 and PTT, the group administered only Example 1, the group treated with indocyanine green solution and PTT, and the group administered PBS were 57.50 ± 114.99 mm³, 1113.49 ± 236.65 mm³, 1072.11 ± 231.87 mm³, and 947.25 ± 387.88 mm³, respectively. As a result, the group treated with Example 1 and PTT showed the greatest tumor size inhibition effect and effectively inhibited tumor recurrence. Conversely, the group administered only Example 1, the group treated with indocyanine green solution and PTT, and the PBS-administered group (control group) showed almost no tumor recurrence inhibition effect. The results of this experiment demonstrate that when indocyanine green is applied to the composition of the present invention, it effectively adheres to the residual tumor site after surgery, thereby removing the residual tumor through photothermal therapy and maximizing the tumor recurrence inhibition effect. These results indicate that the present invention can be applied as a treatment strategy to prevent tumor recurrence after surgery.

[0187] <Experimental Example 17> Safety Confirmation Experiment

[0188] The cytotoxicity of each component (azide-BSA, gelatin, 4-arm PEG-DBCO) was evaluated using the MTT assay. A total of 1 × 10⁻⁶ 5 4T1 cells at a concentration of 100 μL were seeded into 96-well plates and cultured for 24 hours. Subsequently, the 4T1 cells were divided into three groups and treated with azide-BSA, gelatin, or 4-arm PEG-DBCO at concentrations of 0.1, 1, 10, and 100 μg / mL for 24 hours. After treatment, cytotoxicity was evaluated using the MTT assay.

[0189] In addition, to confirm the effect of ICG-AGP powder, 4T1 cells (1 × 10⁶ 5cells / mL (1 mL) were cultured in a 24-well plate for 24 hours. Subsequently, 10 mg of gelled ICG-AGP powder and AGP powder alone were treated for 4 hours, followed by irradiation with a near-infrared laser (808 nm, 1 W / cm², 5 min). Cytotoxicity was evaluated using the MTT assay 24 hours after laser irradiation.

[0190] The results are shown in FIGS. 19a to 19d. FIGS. 19a to 19d show the results of an in vitro safety confirmation experiment according to an embodiment of the present invention. FIGS. 19a to 19d each show a graph of cell viability by concentration for 4-arm PEG-DBCO (Fig. 19a), a graph of cell viability by concentration for gelatin (Fig. 19b), a graph of cell viability by concentration for azide-BSA (Fig. 19c), and a graph of cell viability according to the presence or absence of laser irradiation upon treatment with Example 1 (ICG-AGP powder) and Example 2 (AGP powder) (Fig. 19d) (n=8, **p<0.01, ***<0.001). As shown in the figure, at the highest test concentration (100 μg / mL), the components of the ICG-AGP powder (azide-BSA, 4-arm PEG-DBCO, and gelatin) did not cause significant cytotoxicity, suggesting that the powder is non-toxic even at high concentrations. Similarly, gelled ICG-AGP did not exhibit toxicity but promoted PTT under laser irradiation to induce targeted cell death.

[0191] In addition, in vivo safety was confirmed by examining major organs (liver, lungs, kidneys, heart, and spleen) via H&E staining after administering Example 1 to mice. Specifically, 40 mg of Example 1 (ICG-AGP powder) was implanted into the flanks of mice, and major organs were resected and harvested after 14 days. Mice treated with PBS were used as a control group. H&E staining was performed according to the manufacturer's protocol, and the results were observed using a digital microscope. The results are shown in Fig. 20. Fig. 20 shows the results of an in vivo safety confirmation experiment according to an embodiment of the present invention. In Fig. 20, the results for the PBS-treated group and the Example 1-treated group can be seen for the heart, kidneys, liver, lungs, and spleen, respectively. As shown in the figure, no significant signs of toxicity were observed in any organ, indicating that the powder does not induce toxicity. These results indicate that the composition of the present invention possesses excellent biocompatibility and demonstrates suitability for clinical application.

[0192] <Experimental Example 18> In vivo biodegradability verification experiment

[0193] To investigate the biodegradability of Example 1 (ICG-AGP powder), the powder was implanted subcutaneously into the flanks of mice and monitored for 1 hour and 14 days. The results are shown in Fig. 21. Fig. 21 is a photograph showing the results of an in vivo biodegradability verification experiment of an embodiment of the present invention. Fig. 21 shows a photograph taken 1 hour after application of Example 1 (left) and a photograph taken 14 days after application (right). As shown, 1 hour after application of Example 1, Example 1 rapidly gelled in situ, indicating efficient cross-linking behavior in a physiological environment. Furthermore, by day 14 after application of Example 1, the gel had mostly degraded, and no evidence of long-term inflammation or tissue damage was observed. This indicates that since the composition of the present invention degrades effectively in the body, the risk of long-term foreign substance residue and the need for additional removal procedures can be minimized.

[0194] <Experimental Example 19> Experiment to Confirm Hemostatic Effect on Gastric Hemorrhage

[0195] The hemostatic effect of the embodiment of the present invention was evaluated in a large animal model using a minipig weighing 25–30 kg. Bleeding was induced in the stomach of the minipig using an endoscope equipped with a knife and forceps. After spraying Example 1 onto the bleeding site, the process of the powder gelling and depositing on the hemostatic site was observed via the endoscope for approximately 2 hours. When bleeding was induced in the stomach of the minipig, the powder rapidly adhered to the damaged tissue to form a stable hydrogel, thereby effectively achieving hemostasis. Through endoscopic observation for 2 hours thereafter, it was confirmed that the hydrogel remained undamaged, maintained strong adhesive strength, and formed a robust seal on the wound site. The results are shown in Fig. 22. Fig. 22 is a photograph showing the results of an experiment confirming the hemostatic effect of one embodiment of the present invention on gastric bleeding. These results indicate that the endoscopic spray administration system according to the present invention is feasible. Furthermore, from these results, it can be seen that strong and continuous bleeding control is possible through the present invention.

[0196] From the experimental results above, it can be seen that the medical adhesive composition of the present invention possesses the following effects: biocompatibility and biodegradability that ensure safety within the body; rapid and strong adhesion to biological tissues; the ability to maintain adhesion even in moist biological tissues; flexibility, elasticity, and fatigue resistance that ensure long-term adhesion in dynamic environments such as the heart, lungs, and stomach; long-term storage capability; ease of application with various devices such as endoscopes; and the ability to minimize side effects by preventing adhesion to unintended tissues. Furthermore, it can be seen that the medical adhesive composition of the present invention can be effectively manufactured by the method of the present invention.

[0197] According to the present invention, a medical adhesive composition having improved characteristics and a method for manufacturing the same can be provided. Therefore, the present invention is industrially applicable.

Claims

1. A medical adhesive composition comprising azide-containing albumin, gelatin, and 4-arm PEG-DBCO, in powder form.

2. In Paragraph 1, A medical adhesive composition comprising, for every 100 parts by weight of the azide-containing albumin, 10 to 200 parts by weight of the gelatin and 10 to 100 parts by weight of the 4-arm PEG-DBCO.

3. A medical adhesive composition according to claim 1, wherein the azide group-containing albumin is a freeze-dried product.

4. (a) a step of preparing azide-containing albumin comprising mixing albumin and 2-Morpholinoethanesulphonic acid (MES) with a compound containing both an azide group and an amide group; and (b) A method for preparing a medical adhesive composition comprising the step of mixing the azide group-containing albumin with 4-arm PEG-DBCO and gelatin.

5. A method for preparing a medical adhesive composition in which, in paragraph 4, the azide group-containing albumin is a freeze-dried product.

6. A method for preparing a medical adhesive composition in which the 4-arm PEG-DBCO is included in an organic solvent, as described in paragraph 4.

7. A method for preparing a medical adhesive composition according to claim 6, wherein the 4-arm PEG-DBCO is mixed with the azide-containing albumin in a state present in an organic solvent.

8. A method for manufacturing a medical adhesive composition according to claim 7, further comprising the step of removing the organic solvent.

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