ALGINIC ACID-BASED INJECTABLE HYDROGEL SYSTEM

Abstract

The present invention provides an alginic acid-based injectable hydrogel system for labeling an accurate position of a disease lesion and effectively delivering a drug to a target region. The formulation of the present invention can make it easy to locally inject a contrast agent and a drug into a target region while controlling the release rate of the contrast agent or the drug, with the formation of the hydrogel in the injected region. Through the advantages, the labeled position can be accurately determined from images, thereby enhancing the precision of surgical operation, with a minimal incision formed therefor. In addition, when used, the alginic acid-based injectable hydrogel system allows the effective local delivery of a drug to a target region while increasing the long-acting effect of the drug.

Claims

1. A composition for preparing an injectable hydrogel, comprising a first agent comprising alginic acid and an ionic crosslinking agent; and a second agent comprising alginic acid and a crosslinking rate controller.

2. The composition of claim 1, wherein the ionic crosslinking agent is selected from the group consisting of calcium carbonate, strontium carbonate, calcium disodium edetate (calcium EDTA), calcium citrate, calcium sulfate, calcium alginate, calcium gluconate, calcium phosphate dibasic (CaHPO.sub.4), barium carbonate, zinc carbonate, calcium chloride, calcium lactate, calcium aspartate, calcium saccharate, calcium oxovalerate, calcium lactobionate, calcium lactogluoconate, and a mixture thereof.

3. The composition of claim 1, wherein the crosslinking rate controller comprises any of one or more selected from the group consisting of an organic acid or a salt thereof, or a mixture thereof, and photoacid generator (PAG).

4. The composition of claim 3, wherein the organic acid is D-gluconic acid.

5. The composition of claim 1, wherein the first agent or the second agent comprises an image contrast agent or a drug.

6. The composition of claim 1, wherein the alginic acid is mixed with hyaluronic acid.

7. The composition of claim 5, wherein the image contrast agent is a magnetic resonance image contrast agent, a CT contrast agent, or a fluorescent dye.

8. The composition of claim 7, wherein the fluorescent dye is the complex of human serum albumin and a fluorescent dye connected through an ionic bond, a hydrophobic bond or a covalent bond.

9. The composition of claim 5, wherein the drug is an anticancer agent, an anti-inflammatory agent, an anesthetic agent, or an immunotherapeutic agent.

10. The composition of claim 9, wherein the anti-inflammatory agent is biodegradable microspheres, nanoparticles, liposome or a form mounted on micelle.

11. The composition of claim 10, wherein the anti-inflammatory agent reduces the removal rate of the fluorescent dye by suppressing the inflammatory reaction at the injection site.

12. A composition for labeling a lesion, comprising a first agent comprising alginic acid and an ionic crosslinking agent; and a second agent comprising alginic acid and a crosslinking rate controller.

13. A method for labeling a lesion, comprising injecting the composition of claim 12 into a subject.

Description

DESCRIPTION OF DRAWINGS

[0034] FIG. 1 is a configuration diagram of an alginic acid-based injectable hydrogel system.

[0035] FIG. 2A is a fluorescence graph according to the concentration of indocyanine green.

[0036] FIG. 2B is a graph showing the maximum fluorescence intensity according to the concentration of indocyanine green.

[0037] FIG. 2C is an absorbance graph according to the concentration of indocyanine green.

[0038] FIG. 3A is a fluorescence graph according to the concentration of indocyanin green-human serum albumin bound at 1:1.

[0039] FIG. 3B is a graph showing the maximum fluorescence intensity according to the concentration of indocyanine green-human serum albumin bound at 1:1.

[0040] FIG. 3C is an absorbance graph according to the concentration of indocyanin green-human serum albumin bound at 1:1.

[0041] FIG. 4A is a fluorescence graph according to the ratio of human serum albumin when the concentration of indocyanine green is fixed at 30 μM.

[0042] FIG. 4B is a graph showing the maximum fluorescence intensity according to the ratio of human serum albumin when the concentration of indocyanine green is fixed at 30 μM.

[0043] FIG. 4C is an absorbance graph according to the ratio of human serum albumin when the concentration of indocyanine green is fixed at 6 μM.

[0044] FIG. 5 is a photograph and graph showing the gelation time according to the concentration of calcium ions and D-gluconic acid in the alginic acid hydrogel.

[0045] FIG. 6A is a graph observing change in fluorescence intensity until 3 days after injecting the three conditions of alginic acid hydrogel containing 30 μM of indocyanine green solution, indocyanine green-human serum albumin solution, and 30 μM of indocyanine green-human serum albumin into 96 wells.

[0046] FIG. 6B is a fluorescence image taken immediately after injecting the three conditions of alginic acid hydrogel containing 30 μM of indocyanine green solution, indocyanine green-human serum albumin solution, and 30 μM of indocyanine green-human serum albumin into 96 wells.

[0047] FIG. 7 is a schematic diagram of subcutaneous injection of a nude mouse by putting an alginic acid solution as the first agent wherein indocyanine green-human serum albumin and calcium carbonate are mixed, in one side of a dual syringe, and putting an alginic acid solution mixed with D-gluconic acid as the second agent in the other side.

[0048] FIG. 8 is a fluorescence image obtained over time for three conditions of an indocyanine green solution, an indocyanine green-human serum albumin solution, and an alginic acid hydrogel containing an indocyanine green-human serum albumin injected subcutaneously of a nude mouse.

[0049] FIG. 9 is a photograph observing an appearance of the injected site over time for three conditions of an indocyanine green solution, an indocyanine green-human serum albumin solution, and an alginic acid hydrogel containing an indocyanine green-human serum albumin injected subcutaneously to a nude mouse.

[0050] FIG. 10A is a graph representing a region of interest (ROI) value over time for three conditions of an indocyanine green solution, an indocyanine green-human serum albumin solution, and an alginic acid hydrogel containing an indocyanine green-human serum albumin injected subcutaneously to a nude mouse.

[0051] FIG. 10B is a graph representing TBR (target-to-background ratio=fluorescence ROI value at the labeling site fluorescence ROI value of the surrounding tissue) of the labeling site and the surrounding tissue over time for three conditions of an indocyanine green solution, an indocyanine green-human serum albumin solution, and an alginic acid hydrogel containing an indocyanine green-human serum albumin injected subcutaneously to a nude mouse.

[0052] FIG. 10C is a graph representing area of the spreading fluorescence at the labeling site over time for three conditions of an indocyanine green solution, an indocyanine green-human serum albumin solution, and an alginic acid hydrogel containing an indocyanine green-human serum albumin injected subcutaneously to a nude mouse.

[0053] FIG. 11A a structure of ZW800-1C NHS ester.

[0054] FIG. 11B is a graph of absorbance and fluorescence of covalently bound human serum albumin-ZW 800-1C conjugate.

[0055] FIG. 11C is a fluorescence image obtained over time for an alginic acid hydrogel containing a fluorescent dye in the form of a covalent bond of human serum albumin-ZW 800-1C injected subcutaneously to a nude mouse.

[0056] FIG. 12 is a view showing that doxorubicin, which is a drug, added to alginic acid hydrogel is injected into an ep-tube.

[0057] FIG. 13 is a fluorescence image obtained for distilled water, an alginic acid hydrogel, and alginic acid hydrogel containing doxorubicin.

[0058] FIG. 14 is a view showing that manganese chloride and ferridex, which is an MRI contrast agent, added to an alginic acid hydrogel are injected into an ep-tube.

[0059] FIG. 15 is an MRI T1 highlighted image obtained for distilled water, an alginic acid hydrogel, and an alginic acid hydrogel containing manganese chloride (0.05, 0.1, 0.2 mM).

[0060] FIG. 16 is an MRI T2 highlighted image obtained for distilled water, an alginic acid hydrogel, and an alginic acid hydrogel containing ferridex (25, 50, 100 μM).

[0061] FIG. 17 is a view showing that Rose Bengal, which is a CT contrast agent, added to an alginic acid hydrogel, is injected into an ep-tube.

[0062] FIG. 18 is a CT image obtained for distilled water, an alginic acid hydrogel, and an alginic acid hydrogel containing Rose Bengal.

[0063] FIG. 19 is a CT image obtained for distilled water, an alginic acid hydrogel, and an alginic acid hydrogel containing Rose Bengal.

[0064] FIG. 20 is a view showing that a hydrogel made by mixing alginic acid and hyaluronic acid is injected into an ep-tube.

[0065] FIG. 21 is a fluorescence detection image confirming the site where the hydrogel was injected through a fluorescent laparoscope 3 days after injecting an alginic acid hydrogel containing an indocyanine green-human serum albumin into the submucosal layer in the stomach of test group 1 (pig #1). The fluorescent signal shown in the dotted circle is a fluorescent signal generated at the site where the hydrogel was injected.

[0066] FIG. 22 is a fluorescence detection image confirming the site where the hydrogel was injected through a fluorescent laparoscope 30 minutes after injecting an alginic acid hydrogel containing an indocyanine green-human serum albumin into the submucosal layer in the stomach of test group 2 (pig #2). The fluorescent signal shown in the dotted circle is a fluorescent signal generated at the site where the hydration gel was injected.

[0067] FIG. 23 is a fluorescence image obtained using a fluorescent laparoscope after incision of the stomach of a pig (top: pig #1, bottom: pig #2).

[0068] FIG. 24 is a microscopic observation photograph of PLGA microspheres loaded with celecoxib.

[0069] FIG. 25A is an absorption spectrum obtained by dissolving celecoxib in an acetonitrile solution at various concentrations.

[0070] FIG. 25B is a standard curve obtained by measuring the absorbance of a celecoxib (acetonitrile) solution in each of concentration at a wavelength of 251 nm.

[0071] FIG. 25C is an absorption spectrum measured by dissolving a celecoxib-loaded PLGA microsphere solution at a concentration of 0.04 mg/mL in an acetonitrile solution.

[0072] FIG. 26A is a fluorescence image obtained 24 hours, hours, and 72 hours after injecting an alginic acid hydrogel containing PLGA microspheres loaded with celecoxib subcutaneously to a mouse.

[0073] FIG. 26B is a result for quantitatively analyzing the generated fluorescent signal over time at the injected site (region of interest, ROI) to which an alginic acid hydrogel is injected without celecoxib (Alg Gel) and an alginic acid hydrogel (microsphere/Alg Gel) containing celecoxib-loaded PLGA microspheres were injected.

BEST MODE

[0074] Hereinafter, preferred embodiments of the present disclosure will be described in more detail with reference to the accompanying drawings in order to describe the present disclosure in more detail. However, the present disclosure is not limited to the embodiments described herein and may be embodied in other forms. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by an expert skilled in the art to which the present disclosure belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.

Example 1: Experiment of Incorporating Surgical Marker to Alginic Acid Hydrogel

[0075] 1-1: Measurement of Fluorescence and Absorbance of Indocyanine Green and Indocyanine Green-Human Serum Albumin Complex

[0076] Before developing a surgical marker using indocyanine green and human serum albumin conjugate, in order to determine the appropriate concentration of indocyanine green and human serum albumin conjugate, the two substances were mixed by each concentration to observe change in fluorescence (λ.sub.ex=720 nm) and absorbance.

Preparation Example 1

[0077] 3 mL of distilled water was added to 1.5 mg of indocyanine green (ICG, purchased from Sigma-Aldrich) and dissolved by stirring to prepare a high concentration of indocyanine green solution. Distilled water was added to the high concentration of indocyanine green solution and mixed through stirring to prepared a 2 to 100 μM solution.

Preparation Example 2

[0078] 1 mL of the high concentration of indocyanine green solution was taken, added to 42.89 mg of human serum albumin (HSA, purchased from Sigma-Aldrich), and mixed with each other thoroughly by pipetting to obtain a high concentration of indocyanine green-human serum albumin complex. The high concentration of the indocyanine green-human serum albumin solution was added to distilled water and stirred to prepared a 2 to 100 μM of solution.

[0079] <Evaluation 1-Evaluation of Fluorescence Properties>

[0080] When only indocyanine green is in 2 to 100 μM, the maximum fluorescence intensity increased to about 20 μM, and then due to an aggregation of the fluorescent dye, the fluorescence decreased from the concentration or higher (FIGS. 2A and 2B). On the other hand, it was confirmed that the indocyanine green-human serum albumin complex bound in 1:1 had a higher fluorescence than the case of using only indocyanine green in the concentration range of 2 to 100 μM, and the maximum fluorescence intensity continued to increase until it became about 30 μM, and remained almost unchanged from the concentration or higher (FIGS. 3A and 3B).

[0081] <Evaluation 2-Evaluation of Absorbance Properties>

[0082] As a result of observing the absorbance of the indocyanine green and the indocyanine green-human serum albumin complex bound in 1:1 in concentration range of 2 to 10 μM, it was confirmed that both showed a tendency to increase the absorbance as the concentration increased, and the absorbance of the indocyanine green-human serum albumin complex appeared stronger (FIGS. 2C and 3C).

[0083] 1-2: Measurement of Fluorescence and Absorbance According to Mixing Ratio of Indocyanine Green-Human Serum Albumin Complex

[0084] Before developing a surgical marker using indocyanine green-human serum albumin conjugate, in order to determine the appropriate mixed ratio of indocyanine green-human serum albumin conjugate, the two substances were mixed by each concentration to observe change in fluorescence (λ.sub.ex=720 nm) and absorbance.

Preparation Example 3

[0085] In the same manner as in Preparation Example 1, a high concentration of indocyanine green solution was prepared. After adding 1 mL of distilled water to 42.89 mg of human serum albumin, pipetting was performed to thoroughly mix to prepare a high concentration of human serum albumin solution. The high concentration of indocyanine green solution was fixed to a final concentration of 30 μM by taking 0.125 mL, and only the human serum albumin solution was added in an amount corresponding to the molar ratio of indocyanine green of 0 to 2 to make a total of 1 mL of a solution with distilled water and stir to mix thoroughly.

[0086] <Evaluation 3-Evaluation of Fluorescence and Absorbance Properties>

[0087] Changes in fluorescence and absorbance were observed using the solution of Preparation Example 3 (FIGS. 4A to 4C). In the indocyanine green-human serum albumin conjugate, the fluorescence tended to increase as the amount of human serum albumin increased. In particular, fluorescence increased until the two substances were bound at a ratio of 1:1, and there was little change after the ratio of human serum albumin increased at or above that ratio. When looking at the absorbance according to the molar ratio of indocyanine green-human serum albumin conjugate, the absorbance tended to increase as the number of moles of human serum albumin increased.

[0088] 1-3: Observation of Gelation of Alginic Acid Solution Over Time after Addition of Calcium

[0089] In order to make an alginic acid hydrogel with an appropriate gelation time that may be used as a surgical marker, calcium ions of different concentrations as shown in Table 1 were added to the alginic acid solution through a dual syringe, and then the change in the alginic acid solution over time was observed (FIG. 5).

[0090] Calcium ions form a hydrogel through cross-linking with alginic acid. Since calcium carbonate (purchased from Sigma-Aldrich) has low solubility, it was made to allow rapid cross-linking with alginic acid by regulating the dissolution rate of calcium carbonate with a mix of D-gluconic acid (purchased from Sigma-Aldrich), which is an acidic condition through a dual syringe. Through this process, alginic acid may be gelled evenly throughout. The pH of the total solution was adjusted to neutral by adding two folds of the number of moles of D-gluconic acid with respect to calcium carbonate.

TABLE-US-00001 TABLE 1 Mixing condition 1 2 3 The first Alginic acid Alginic acid Alginic acid agent 1.0 w/v % 1.0 w/v % 1.0 w/v % calcium calcium calcium carbonate 0.1X carbonate 0.2X carbonate 0.3X The second Alginic acid Alginic acid Alginic acid agent 1.0 w/v % D- 1.0 w/v % D- 1.0 w/v % D- gluconic acid gluconic acid gluconic acid 0.2X 0.4X 0.6X

[0091] (The mixing condition of the alginic acid hydrogel through a dual syringe; X means the number of moles of the carboxy group contained in 1.0 w/v % of alginic acid.)

Preparation Example 4

[0092] 0.036 g of sodium alginate (purchased from Kimika) was dissolved in 3 mL of distilled water to prepare a 1.2 wt % solution of alginic acid. 0.25 mL of distilled water was added to 1.25 mL of a 1.2 wt % alginic acid solution to prepare a 1.0 wt % alginic acid solution. 1 mL of a 1.0 wt % alginic acid solution was added to 1.14 mg of calcium carbonate and mixed by pipetting or vortexing to prepare a first agent solution. 0.007 mL of a D-gluconic acid solution was added to 0.833 mL of 1.2 wt % of alginic acid solution, and made to a total of 1 mL with distilled water, followed by mixing completely through vortexing and stirring to prepare a second agent solution. Calcium carbonate and D-gluconic acid were used 0.1 and 0.2 times, respectively, based on the number of moles of carboxyl groups contained in 1.0 w/v % of alginic acid.

Preparation Example 5

[0093] A solution was prepared in the same manner as in Preparation Example 4, except that the calcium carbonate of the first agent was 2.29 mg and the D-gluconic acid solution of the second agent was 0.015 mL. Calcium carbonate and D-gluconic acid were used 0.2 and 0.4 times, respectively, based on the number of moles of carboxyl groups contained in 1.0 w/v % of alginic acid.

Preparation Example 6

[0094] A solution was prepared in the same manner as in Preparation Example 4, except that the calcium carbonate of the first agent was 3.43 mg and the D-gluconic acid solution of the second agent was 0.022 mL. Calcium carbonate and D-gluconic acid were used 0.3 and 0.6 times, respectively, based on the number of moles of carboxyl groups contained in 1.0 w/v % of alginic acid.

[0095] <Evaluation 4-Evaluation of Gelation of Aqueous Alginic Acid Solution According to Amount of Calcium Ions>

[0096] After injecting the solution of the first agent and the second agent of Preparation Examples 4 to 6 to be a total volume of 0.5 mL in a glass bottle through a dual syringe, the formation of gel over time was observed by turning the glass bottle over. As shown in Table 1 and FIG. 5, an alginic acid solution was injected into a glass bottle with a dual syringe for a calcium condition of 0.1× to 0.3×, and the change of the solution over time was observed.

[0097] In the 0.1× calcium condition, the solution was in a liquid state (sol) flowing immediately after injection, and then changed to a solid state (gel) after about 46 minutes. In the 0.2× calcium condition, the solution was in a liquid state (sol) slightly flowing immediately after injection, and then changed to a solid state (gel) after about 2 minutes. Finally, in the 0.3× calcium condition, the solution exists in a state that hardly flow immediately after injection, and then changed to a solid state (gel) after about 1 minute. This gelation time is plotted on a graph according to the concentration of calcium ions used in FIG. 5.

[0098] It was observed that the gelation time of alginic acid decreased as the concentration of calcium ions added to alginic acid increased through 0.1 to 0.3× calcium conditions. Especially, among them, in the case of a calcium condition of 0.3×, it existed in a solid state (gel) that hardly flows immediately after injection. In this experiment, in order to effectively prevent the diffusion of the indocyanine green-human serum albumin complex, a subsequent experiment was conducted by selecting a calcium condition of 0.3×, which becomes a gel immediately after injection.

[0099] 1-4: Fluorescence Measurement of Alginic Acid Hydrogel Containing Indocyanine Green-Human Serum Albumin Complex

[0100] In order to prevent the easy diffusion of indocyanine green in the living body, the indocyanine green-human serum albumin complex was incorporated into alginic acid, and then cross-linked with calcium ions through a dual syringe to form an injectable hydrogel, and fluorescence (λ.sub.ex=720 nm) and fluorescence images (λ.sub.ex=780 nm, λ.sub.em=845 nm) were confirmed under in vitro conditions.

Preparation Example 7

[0101] A high concentration of indocyanine green solution was prepared in the same manner as in Preparation Example 1. Distilled water was added to 0.139 mL of the high concentration of indocyanine green solution to make a total of 1.5 mL, followed by a mix with stirring to prepare the first agent solution. Distilled water was used as the second agent solution. The final solution of the first agent and the second agent was prepared so that the concentration of the indocyanine green was 30 μM.

Preparation Example 8

[0102] A high concentration of indocyanine green-human serum albumin solution was prepared in the same manner as in Preparation Example 2. Distilled water was added to 0.139 mL of the high concentration of indocyanine green-human serum albumin to make a total of 1.5 mL, followed by a mix with stirring to prepare the first agent solution. Distilled water was used as the second agent solution. The final solution mixed with the first agent and the second agent was prepared so that the concentration of indocyanine green-human serum albumin complex was 30 μM.

Preparation Example 9

[0103] A high concentration of indocyanine green-human serum albumin solution was prepared in the same manner as in Preparation Example 2. The first agent and the second agent solutions were prepared in the same method as in Preparation Example 6, except that 0.139 mL of the high concentration of indocyanine green-human serum albumin solution was added to 1.25 mL of a 1.2 wt % of alginic acid solution, and then the total volume was made to 1.5 mL with distilled water.

[0104] <Evaluation 5-Fluorescence Evaluation>

[0105] The solutions of Preparation Examples 7 to 9 were injected into 96 wells, respectively so that the total volume was 200 μL using a dual syringe. The fluorescence graph and fluorescence image of the injected gel were confirmed over time.

[0106] As shown in FIG. 6A, immediately after injection of each solution, the conditions of the indocyanine green-human serum albumin solution had the highest fluorescence, and the fluorescence was lowered in the order of the indocyanine green solution, alginic acid hydrogel containing indocyanine green-human serum albumin condition. 3 hours after injection, the fluorescence of the alginic acid hydrogel containing indocyanine green-human serum albumin was relatively high, and the fluorescence was lowered in the order of the indocyanine green-human serum albumin solution, alginic acid hydrogel containing indocyanine green-human serum albumin condition, and indocyanine green. The same results were also shown when these conditions were observed up to 3 days.

[0107] It was confirmed through the fluorescence image of FIG. 6B that the solution was well mixed and entered when the alginic acid hydrogel was injected with a dual syringe.

[0108] 1-5: Fluorescence Measurement of Alginic Acid Hydrogel Containing Indocyanine Green-Human Serum Albumin in Animal Model

[0109] In order to see the fluorescence persistence of the alginic acid hydrogel containing indocyanine green-human serum albumin in a nude mouse, it was injected in the form of a gel subcutaneously to the nude mouse through a dual syringe, and then the labeling performance over time was photographed with a fluorescent image device (IVIS Lumina XR, Xenogen Corporation-Caliper, CA, USA). Three solutions to be injected into the animal model were prepared in the same manner as in Preparation Examples 7 to 9. FIG. 7 is a schematic diagram of injecting an alginic acid hydrogel containing indocyanine green-human serum albumin subcutaneously to a nude mouse using a dual syringe.

[0110] <Evaluation 6-Evaluation of the Persistence of Fluorescence of Gel in Animal Model>

[0111] The solutions of Preparation Examples 7 to 9 were injected subcutaneously to a nude mouse, respectively so that the total volume was 100 μL using a dual syringe.

[0112] FIG. 8 is a fluorescence image (λ.sub.ex=780 nm, λ.sub.em=845 nm) obtained over time for three conditions of an indocyanine green solution, an indocyanine green-human serum albumin solution, and an alginic acid hydrogel containing an indocyanine green-human serum albumin injected subcutaneously to a nude mouse. At this time, it was confirmed that the fluorescence in the indocyanine green and indocyanine green-human serum albumin solution was maintained for only about one day, whereas in the case of the alginic acid hydrogel, fluorescence was continuously emitted for about 5 days. In addition, in the case of the alginic acid hydrogel, it was confirmed that the degree of the spreading of fluorescence dye was less than that of the other two conditions because the gel prevented the spreading of the fluorescence dye.

[0113] FIG. 9 is a result observing the injected site over time of an indocyanine green solution, an indocyanine green-human serum albumin solution, and an alginic acid hydrogel containing an indocyanine green-human serum albumin injected subcutaneously to a nude mouse. Specifically, it was confirmed that under the conditions of indocyanine green and indocyanine green-human serum albumin solution, the injection site appeared slightly convex immediately after injection, and after about 24 hours, the solution spreads and disappeared. On the other hand, in the case of alginic acid hydrogel, it was confirmed that the convex protruding region was clearly observed with the eye immediately after injection, and in the case of the gel, the decomposition was partly but maintained for about a month.

[0114] In FIG. 10A, the ROI value of the labeled region was compared to how long fluorescence of each condition persists in a nude mouse. When checking the ROI value, indocyanine green and indocyanine green-human serum albumin solutions showed almost similar patterns. It was confirmed that as time passed, the injected solution spreads and the fluorescence was greatest at 6 hours, and 16% and 12% of fluorescence remained, respectively, compared to immediately after injection when one day passed. On the other hand, in the case of the alginic acid hydrogel, it was confirmed that the fluorescence was the greatest at 6 hours as the solution spreads slightly after injection, but about 61% of the fluorescence signal still appeared compared to immediately after injection even after 2 days.

[0115] In FIG. 10B, a target-to-background ratio (TBR) was observed by dividing the fluorescence ROI value of the target site by the fluorescence ROI value of the surrounding tissue (background). As a result, it was confirmed that indocyanine green, indocyanine green-human serum albumin solution condition takes about 4 to 5 days for TBR to increase 5 times, whereas alginic acid hydrogel takes about 8 days for TBR to increase 5 times.

[0116] In FIG. 10C, the area where fluorescence appears in the nude mouse was confirmed. When comparing the area where the fluorescence spreads to the maximum in the conditions of indocyanine green and indocyanine green-human serum albumin solution and the area where the fluorescence spreads to the maximum in the alginic acid hydrogel, it can be seen that the fluorescence appears less diffuse about twice compared to that of the other conditions in the alginic acid hydrogel. In addition, the two conditions with different fluorescence signals almost disappeared in 48 hours, whereas the alginic acid hydrogel condition showed fluorescence signals up to 5 days.

[0117] Taking these results together, it was confirmed that the alginic acid hydrogel covered the fluorescent dye and prevented it from spreading at the target site under in vivo conditions, thereby effectively increasing the fluorescence duration.

[0118] 1-6: Human Serum Albumin-ZW800-1C Incorporation Test of Alginic Acid Hydrogel

[0119] Human serum albumin-ZW800-1C fluorescent dye, a covalently bound complex, was added to the alginic acid hydrogel to confirm the persistence of fluorescence under in vivo conditions.

Preparation Example 10

[0120] Human serum albumin (10 mg/mL) was dissolved in a phosphate buffer, and then reacted with ZW800-1C NHS ester (FIG. 11A) in 1 mL of phosphate buffer at a molar ratio of 1:2 for 1 hour at room temperature. After completion of the reaction, unbound fluorescent dyes and reaction by-products were removed using a PD-10 column (GE Healthcare). The human serum albumin-ZW-800-1C conjugate obtained after purification was concentrated using an Amicon Ultra-0.5 mL (cut off: 30 kDa) centrifugal filter and stored in a refrigerator at 4° C.

[0121] In order to analyze the ratio of fluorescent dye bound to human serum albumin, human serum albumin-ZW800-1C conjugate was dissolved in a denatured phosphate buffer (including 1% SDS/1 mM NaOH) and absorbance was measured. The concentration of human serum albumin protein was calculated using the molar extinction coefficient (34,445 M.sup.−1cm.sup.−1) of human serum albumin at 280 nm, and the fluorescent dye was used with a known molar extinction coefficient (125,000 M.sup.−1cm.sup.−1 at 779 nm in DMSO). As a result of the analysis, it was calculated that ZW800-1C was attached with 0.26 per one human serum albumin.

[0122] The degree of absorption and fluorescence of the human serum albumin-fluorescent dye conjugate was measured by diluting in a phosphate buffer at a concentration of 5 μM (based on the fluorescent dye concentration) (FIG. 11B).

Preparation Example 11

[0123] The first agent and the second agent were prepared in the same manner as in Preparation Example 9, except that the fluorescent dye covalently bound to 30 μM of human serum albumin-ZW800-1C was used in place of the indocyanine green-human serum albumin complex in the first agent.

[0124] <Evaluation 7-Evaluation of Persistence of Fluorescence of Gel in Animal Model>

[0125] The solutions of Preparation Example 11 were injected subcutaneously to a nude mouse, so that the total volume was 100 μL using a dual syringe. In the in vivo condition, it was confirmed that even in the case of the fluorescent dye covalently bound to human serum albumin-ZW800-1C, the fluorescence duration was effectively increased by preventing diffusion at the target site (FIG. 11C).

Example 2: Drug Incorporation Experiment of Alginic Acid Hydrogel

[0126] Doxorubicin (purchased from Sigma-Aldrich) as an anticancer agent, was prepared as a drug to be incorporated into the alginic acid hydrogel.

Preparation Example 12

[0127] 1.5 mg of doxorubicin was dissolved in 0.5 mL of distilled water to prepare a high concentration of doxorubicin solution. The first agent and the second agent solutions were prepared in the same method as in Preparation Example 6, except that 0.125 mL of the high concentration of doxorubicin was added to 1.25 mL of a 1.2 wt % of alginic acid solution, and then the total volume was made to 1.5 mL with distilled water.

[0128] <Evaluation 8-Doxorubicin Incorporation in Alginic Acid Hydrogel and Fluorescence Image Confirmation>

[0129] The solution of Preparation Example 12 was injected into the ep-tube so that the total volume was 0.5 mL using a dual syringe. Thereafter, it was confirmed that the alginic acid hydrogel formed by turning the ep-tube over as shown in FIG. 12 effectively contained doxorubicin.

[0130] As a result of examining the fluorescence image (λ.sub.ex=420 nm, λ.sub.em=570 nm) of the hydrogel containing doxorubicin using a fluorescence image device (IVIS) as shown in FIG. 13, it was confirmed that doxorubicin was well spread throughout the gel.

Example 3: Magnetic Resonance Image (MRI) Contrast Agent Incorporation Experiment of Alginic Acid Hydrogel

[0131] A manganese chloride solution (manganese chloride, purchased from Sigma-Aldrich, 1M) and Ferdex I.V. (Feridex, purchased from Taejun Pharmaceutical co., Ltd., 11.2 mg Fe/mL) as the magnetic resonance image contrast agent were used as they are, as commercial products themselves are dispersed in aqueous solution.

Preparation Example 13

[0132] A manganese chloride solution was diluted to 1/100 by adding 990 μL of distilled water to 10 μL of a manganese chloride solution. After adding 10, 20, and 40 μL of the diluted manganese chloride solution to 1.25 mL of a 1.2 wt % of alginic acid solution, respectively, the first agent and the second agent solutions were prepared in the same manner as in Preparation Example 6, except for using solutions of three concentrations (final concentrations 0.05, 0.1, 0.2 mM) made to 1.5 mL with distilled water.

Preparation Example 14

[0133] Feridex solution was diluted to 1/20 by adding 950 μL of distilled water to 50 μL of Feridex solution. After adding 5, 10, and 20 μL of the diluted Feridex solution to 1.25 mL of a 1.2 wt % of alginic acid solution, respectively, the first agent and the second agent solutions were prepared in the same manner as in Preparation Example 6, except for using solutions of three concentrations (final concentrations 25, 50, 100 μM) made to 1.5 mL with distilled water.

[0134] <Evaluation 9-Magnetic Resonance Image Contrast Agent Incorporation in Alginic Acid Hydrogel and Magnetic Resonance Image Confirmation>

[0135] In the solutions of Preparation Examples 13 and 14, 0.2 mM of manganese chloride and 100 μM of Feridex solutions were injected into ep-tubes, respectively so that the total volume was 0.5 mL using a dual syringe. Thereafter, it was confirmed that the alginic acid hydrogel formed by turning the ep-tube over as shown in FIG. 14 effectively contained manganese chloride and Feridex which are magnetic resonance image contrast agent.

[0136] Three concentrations of the manganese chloride solutions of Preparation Example 13 were injected into the micro-tube so that the total volume was 0.25 mL using a dual syringe. Thereafter, as shown in FIG. 15, distilled water, alginic acid hydrogel, and manganese chloride (0.05, 0.1, 0.2 mM) were checked for MRI T1 highlighted images. It was confirmed that the MRI signal appeared brighter as the amount of manganese chloride added to the alginic acid hydrogel increased.

[0137] Three concentrations of the Feridex solutions of Preparation Example 14 were injected into the micro-tube so that the total volume was 0.25 mL using a dual syringe. Thereafter, as shown in FIG. 16, distilled water, alginic acid hydrogel, and Feridex (25, 50, 100 μM) were checked for MRI T2 highlighted images. It was confirmed that the MRI signal appeared brighter as the amount of Feridex added to the alginic acid hydrogel increased.

Example 4: Computed Tomography (CT) Contrast Agent Incorporation Experiment of Alginic Acid Hydrogel

[0138] It was confirmed whether the CT contrast agent may be incorporated into the alginic acid hydrogel using Rose bengal (purchased from Sigma-Aldrich) as a CT contrast agent.

Preparation Example 15

[0139] 100 mg of Rose bengal was dissolved in 1 mL of distilled water to prepare a high concentration of Rose bengal solution. The first agent and the second agent solutions were prepared in the same manner as in Preparation Example 6, except for using a solution dissolved with stirring by adding 0.729 mL of a high concentration of Rose bengal and 0.771 mL of distilled water to 0.015 g of sodium alginate.

[0140] <Evaluation 10-Rose Bengal Incorporation in Alginic Acid Hydrogel and CT Image Conformation>

[0141] The solution of Preparation Example 15 was injected into the ep-tube so that the total volume was 0.5 mL using a dual syringe. Thereafter, it was confirmed that the alginic acid hydrogel formed by turning the ep-tube over as shown in FIG. 17 effectively contained Rose bengal.

[0142] As shown in FIGS. 18 and 19, the CT images of the hydrogel containing distilled water, alginic acid hydrogel, and Rose bengal were confirmed. Through this, it was confirmed that Rose bengal was well spread throughout the alginic acid hydrogel.

Example 5: Hydrogel Experiment According to Mixing Ratio of Alginic Acid and Hyaluronic Acid

[0143] It was confirmed whether a hydrogel was formed under the conditions wherein alginic acid and hyaluronic acid were mixed at 50:50 and 1:99.

Preparation Example 16

[0144] 0.06 g of sodium hyaluronate (purchased from Lifecore Biomedical, Inc.) was dissolved in 2 mL of distilled water to prepare a 3 wt % of hyaluronic acid solution.

[0145] 0.036 g of sodium alginate was dissolved in 3 mL of distilled water to prepare a 1.2 wt % of alginic acid solution. In the manner as in Preparation Example 1, a high concentration of indocyanine green solution was prepared.

[0146] 0.334 mL of a 3 wt % of hyaluronic acid solution and 0.093 mL of a high concentration of indocyanine green solution were added to 0.417 mL of a 1.2 wt % of alginic acid solution, and the total volume was made 1 mL with distilled water.

[0147] 1 mL of the mixed alginic acid and hyaluronic acid solution was added to 3.43 mg of calcium carbonate and vortexed to prepare the first agent solution. After adding 0.334 mL of a 3 wt % of hyaluronic acid solution and 0.022 mL of D-gluconic acid to 0.417 mL of a 1.2 wt % of alginic acid solution, the total volume was made 1 mL with distilled water, and then the solution of the second agent was prepared by mixing with vortexing and stirring.

Preparation Example 17

[0148] In the same manner as in Preparation Example 16, a 3 wt % of hyaluronic acid solution, a 1.2 wt % of alginic acid solution, and a high concentration of indocyanine green solution were prepared.

[0149] 0.66 mL of a 3 wt % of hyaluronic acid solution and 0.093 mL of a high concentration of indocyanine green solution were added to 0.005 mL of a 1.2 wt % of alginic acid solution, and the total volume was made 1 mL with distilled water. 1 mL of the mixed alginic acid and hyaluronic acid solution was added to 3.43 mg of calcium carbonate and vortexed to prepare the first agent solution.

[0150] After adding 0.66 mL of a 3 wt % of hyaluronic acid solution and 0.022 mL of D-gluconic acid to 0.005 mL of a 1.2 wt % of alginic acid solution, the total volume was made 1 mL with distilled water, and then the solution of the second agent was prepared by mixing with vortexing and stirring.

TABLE-US-00002 TABLE 2 Preparative example Preparative example 16 17 Ratio of alginic 50:50 1:99 acid and hyaluronic acid

[0151] <Evaluation 11-Evaluation of Mixed Hydrogel of Alginic Acid and Hyaluronic Acid>

[0152] The solution of Preparation Examples 16 and 17 was injected into the ep-tube, respectively so that the total volume was 0.5 mL using a dual syringe. Thereafter, as shown in FIG. 20, it was confirmed that the hydrogel was formed even under the condition of mixing alginic acid and hyaluronic acid formed by turning the ep-tube over.

Example 6: Performance Evaluation of Alginic Acid Hydrogel Containing Indocyanine Green-Human Serum Albumin in Pig Model

[0153] Pigs (mini-pigs) have similar metabolic processes, organ structures, and sizes to those of the human body, making it easy to perform experimental treatment and follow-up using an endoscope. In order to confirm the fluorescent labeling performance of alginic acid hydrogel containing indocyanine green-human serum albumin, which is a fluorescent surgical marker in pigs, alginic acid hydrogel containing indocyanine green-human serum albumin was injected in the inner submucosal layer of the stomach of two pigs, and on the injected day and 3 days after the injection of the hydrogel, the fluorescent signal generated from the fluorescent dye injected into the stomach was detected as an image through a fluorescent laparoscope to evaluate whether the labeled location could be identified.

Preparation Example 18

[0154] A high concentration of indocyanine green-human serum albumin solution was prepared in the same manner as in Preparation Example 2. After adding 0.558 mL of the high concentration of indocyanine green-human serum albumin solution to 8.4 mL of a 1 wt % of alginic acid solution, distilled water was further added to make the total volume to 12 mL, thereby preparing a fluorescent surgical marker solution. In this test, an experiment was conducted without adding an ionic crosslinking agent.

[0155] <Evaluation 12-Confirmation of Fluorescent Labeling Performance Using Fluorescent Laparoscope on Pigs>

[0156] Two pigs (pig #1, pig #2) were fasted for 48 hours before injecting the prepared alginic acid hydrogel, and the experiment was conducted after systemic anesthesia.

[0157] On the first day of the test, an endoscope and a catheter (Clear-Jet injector, 23G, Finemedix Co., Ltd.) were inserted through the mouth of test group 1 (pig #1), and the indocyanine green-human serum albumin-containing alginic acid hydrogel of Preparation Example 18 was injected by 1 mL into each of the three lower mucous layers in the pig's stomach. In order to confirm the injected location, the hemostasis clip was clipped right next to the hydrogel injected location. Three days after injection, a fluorescent laparoscopy system was installed in the pig's abdominal cavity, and the fluorescent signal generated from a fluorescent surgical marker located inside the stomach was detected through a fluorescent laparoscope located outside the stomach, and thereby it was evaluated whether it can be confirmed with real-time detection of the location where the surgical marker was labeled.

[0158] On the day of the fluorescent laparoscopy evaluation of test group 1, fluorescent surgical markers were injected into the three submucosal layers in the stomach in the same manner as performed in test group 1 for test group 2 (pig #2). In order to confirm the injected location, the hemostasis clip was clipped right next to the injected location. After injection of the hydrogel, a fluorescent laparoscopy system was installed in the pig's abdominal cavity, and the fluorescent signal generated from a fluorescent surgical marker located inside the stomach was detected through a fluorescent laparoscope located outside the stomach, and thereby it was evaluated whether it can be confirmed with real-time detection of the location where the surgical marker was labeled.

[0159] As a result of observing the location where alginic acid hydrogel containing indocyanine green-human serum albumin was injected, from an image using a fluorescent laparoscopic system, the locations where the hydration gel was injected could be confirmed from the fluorescence image both in test group 1 where a fluorescent marker was injected prior to 3 days (FIG. 21) and observed test group 2 where it was injected on the same day (FIG. 22).

[0160] Subsequently, in order to confirm that the location identified from the fluorescence image was the location where the hydrogel was actually injected, the pig was euthanized and the pig's stomach was incised. It was confirmed that a fluorescent signal was generated next to the location where a haemostasis clip clipped right next to the location where the hydrogel was injected, and from this result, it was found that the fluorescence confirmed from the fluorescence laparoscopic image of the pig experiment was a fluorescence signal generated from the actual hydrogel (FIG. 23).

[0161] Taking these results together, it can be seen that the alginic acid gel containing indocyanine green-human serum albumin had excellent performance as an image marker for the area to be removed by surgery.

Example 7: Image Signal Enhancement Test in Alginic Acid Hydrogel Containing Anti-Inflammatory Agent

[0162] When an alginic acid hydrogel is injected into the lower mucosa for image labeling, the patient's immune system recognizes that a foreign substance has invaded into the living body and may cause an inflammatory reaction in removing the foreign substance. At this time, proteins including various enzymes, complement systems, antibodies, and immune cells are introduced into the injection site, and actions to remove alginic acid and fluorescent dyes present at the injection site occur. Therefore, there is a possibility that the rate at which the fluorescent dye contained in the hydrogel is removed due to the inflammatory reaction occurring at the injection site would be fast.

[0163] Therefore, in the Example, biodegradable microspheres loaded with an anti-inflammatory agent was prepared and injected into a living body together with a hydrogel to suppress the inflammatory reaction at the injection site, and accordingly, a strength of the fluorescence image signal at the injected site was evaluated whether the strength could also be maintained higher. Celecoxib was used as an anti-inflammatory agent, and drug-loaded microspheres were prepared using a biodegradable polymer, poly(lactic-co-glycolic acid) (PLGA).

[0164] 7-1. Preparation and Analysis of Biodegradable Microspheres Loaded with Anti-Inflammatory Agents.

Preparation Example 19

[0165] 135 mg of PLGA (50:50, Mw 38,000-54,000, purchased from Sigma-Aldrich) and 13.5 mg of celecoxib (purchased from Sigma-Aldrich) were dissolved in 4.5 mL of dichloromethane. 0.5 g of polyvinyl alcohol (PVA, Mw 13,000-23,000, purchased from Sigma-Aldrich) was added to mL of distilled water, heated to 65° C. to dissolve completely, and then the solution was cooled at 25° C. A beaker containing an aqueous solution wherein polyvinyl alcohol as a surfactant is dissolved, was immersed in ice, and a dichloromethane solution wherein celecoxib and PLGA are dissolved, was added dropwise, while a homogenizer (Homogenizer PT 3100 Polytron, Kinematica AG, Littau-Lucerne, Switzerland) was stirred vigorously at 5,000 rpm for 5 minutes to prepare an oil-in-water emulsion solution. To remove the organic solvent, dichloromethane, the prepared emulsion solution was mixed with 100 mL of distilled water, and stirred at 300 rpm for 8 hours under a temperature condition of 25° C. Centrifugation and washing were repeated 3 times at 3,000 rpm for 5 minutes in order to remove PVA, which is a surfactant present on the surface of the prepared microspheres. Thereafter, the aqueous solution was freeze-dried, and the obtained microspheres were stored at 4° C.

[0166] <Evaluation 13-Analysis of Microspheres Loaded with Prepared Anti-Inflammatory Agent>

[0167] After dispersing the PLGA microspheres loaded with celecoxib in distilled water at a concentration of 0.5 mg/mL, a few drops ware added on a slide glass and the shape and size was observed by using an optical microscope (Axio Imager M2 microscope, Zeiss, Germany) (FIG. 24). It was confirmed that the prepared microspheres had a round shape, and when the size of 100 microspheres was measured, it was confirmed that microspheres having a size of 9.4±2.7 μm were prepared. The microspheres loaded with celecoxib were dispersed in distilled water at a concentration of 0.5 mg/mL, and the zeta potential measured using a Zetasizer (ZetasizerNanoZS, Malvern Instruments Worces-tershire, UK) was measured to −20.7 mV.

[0168] <Evaluation 14-Analysis of Amount of Drug Loaded in Microspheres>

[0169] After dissolving 1 mg of celecoxib in 10 mL of acetonitrile, a standard solution of concentrations of 0.625, 1.25, 2.5, 5, 10, 12.5, and 25 μg/mL was prepared while diluting with acetonitrile. Each solution was measured for absorbance at a wavelength of 251 nm to obtain a standard curve for celecoxib (FIGS. 25A and 25B).

[0170] 5 mg of PLGA microspheres loaded with celecoxib were completely dissolved in 2.5 mL of acetonitrile, diluted 50 times with acetonitrile solution, absorbance was measured at a wavelength of 251 nm, and the amount of drug contained in the microspheres was analyzed by comparison with the standard curve (FIG. 25C). As a result, it was confirmed that the amount of celecoxib contained in 2 mg of microspheres was 196.4 μg. That is, the drug was loaded with 9.8 wt/wt % in the microspheres.

[0171] 7-2. Animal Test Evaluation

[0172] When alginic acid hydrogel containing indocyanine green-human serum albumin was mixed with celecoxib-loaded microspheres to suppress the inflammatory reaction, it was evaluated through animal experiments whether the fluorescent dye was maintained at the injected site for a longer time and the fluorescence intensity.

Preparation Example 20

[0173] Indocyanine green-human serum albumin solution was prepared in the same manner as in Preparation Example 2. After adding 0.093 mL of an indocyanine green-human serum albumin solution to 1.167 mL of a 1.2 wt % of alginic acid solution, distilled water was further added to make the total volume of 2 mL, followed by stirring.

Preparation Example 21

[0174] In order to prepare an alginic acid hydrogel containing an anti-inflammatory agent, 0.136 mL of a distilled water solution wherein PLGA microspheres loaded with celecoxib were dispersed at a concentration of 9 mg/mL was prepared. 2 mL of an alginic acid aqueous solution containing indocyanine green-human serum albumin was prepared as described above, and 0.136 mL of an aqueous microsphere solution and 2 mL of an alginic acid solution were mixed and stirred. The final concentration of celecoxib drug contained in the alginic acid hydrogel was 600 μg/mL.

[0175] <Evaluation 15-Evaluation of Persistence of Fluorescence of Gel in Animal Model>

[0176] Using a syringe, each of 100 μL of the solutions of Preparation Examples 20 and 21 was subcutaneously injected to a white mouse. FIG. 26A is a fluorescence image (λ.sub.ex=780 nm, λ.sub.em=845 nm) obtained at 24 hours, 48 hours, and hours after injecting alginic acid hydrogel into a subcutaneous location of the mouse. When the fluorescence intensity generated at the region of interest (ROI) to which the hydrogel was injected was quantitatively analyzed for each time period, the fluorescence intensity which is 1.2 times higher at 24 hours, 1.4 times higher at 48 hours, and 1.6 times higher at 72 hours compared to the hydrogel without microspheres was detected (FIG. 26B). This indicates that the inflammatory reaction at the site where the hydrogel was injected was suppressed due to the action of celecoxib, an anti-inflammatory drug loaded in the microspheres, and thereby that the fluorescent dye contained in the hydrogel was maintained at a higher concentration at the injected site.

[0177] As described above, a specific part of the content of the present disclosure has been described in detail, and for one skilled in this art, it will be obvious that this specific technology is only a preferred embodiment, and the scope of the present disclosure is not limited thereby. Therefore, it will be said that the substantial scope of the present disclosure is defined by the appended claims and their equivalents.