EMBOLIZATION HYDROGEL HAVING CONTROLLABLE DEGRADATION TIME, AND PREPARATION METHOD THEREFOR

Abstract

The present invention relates to: an amorphous of spherical embolisation hydrogel having a degradation time that can be precisely controlled in blood vessels; and a preparation method therefor.

Claims

1. An embolic composition comprising hydrogel microparticles prepared without using a cross-linking agent.

2. The embolic composition according to claim 1, wherein the hydrogel microparticles are configured such that particles can be produced by thermal denaturation.

3. The embolic composition according to claim 1, wherein the hydrogel microparticles comprise at least one selected from a biocompatible polymer group consisting of gelatin, collagen, gum, rosin, hyaluronic acid, heparin, dextran, alginic acid, albumin, chitosan, polyglycolide, polylactide, polyhydroxyvalerate, and silk fibroin.

4. The embolic composition according to claim 1, wherein degradation time in vivo of the hydrogel microparticles is adjusted by heat treatment and/or washing.

5. The embolic composition according to claim 4, wherein the washing is performed using a solvent with a 8P polar value of 14 or higher.

6. The embolic composition according to claim 1, wherein the hydrogel microparticles are emulsion type microparticles comprising an organic solvent.

7. The embolic composition according to claim 6, wherein the organic solvent is at least one selected from a group consisting of methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, hexyl acetate, ethyl formate, dimethyl carbonate, diethyl carbonate, 1,3-dioxolidin-2-one, cellulose acetate butyrate, medium chain triglyceride (MCT) oil, vegetable oil, wax, and infused oil.

8. The embolic composition according to claim 6, wherein the emulsion comprises no separate emulsifier.

9. The embolic composition according to claim 1, wherein at least one of a local anesthetic, an antibiotic, and a contrast agent is added.

10. A method of preparing hydrogel microparticles prepared through step (a) or step (b): (a) 1) preparing an aqueous solution of a biocompatible polymer; 2) adding an organic solvent to the aqueous solution of the biocompatible polymer of step 1) so as to be emulsified in order to form micro-sized particles; 3) washing and drying the micro-sized particles prepared in step 2) to obtain micro-sized microparticles; 4) thermally curing the micro-sized microparticles of step 3); and 5) washing, dehydrating, and drying the micro-sized microparticles obtained in step 4); or (b) 1) preparing an aqueous solution of a biocompatible polymer; 2) stirring and/or low-temperature curing the aqueous solution of the biocompatible polymer of step 1) to form a foam and freeze-drying the foam; 3) thermally curing the foamy material of step 2) to obtain micro-sized microparticles; 4) washing and freeze-drying the micro-sized microparticles obtained in step 3); and 5) crushing the foamy material obtained in step 4) to obtain micro-sized microparticles.

11. The method according to claim 10, wherein the biocompatible polymer is at least one selected from a group consisting of gelatin, collagen, gum, rosin, hyaluronic acid, heparin, dextran, alginic acid, albumin, chitosan, polyglycolide, polylactide, polyhydroxyvalerate, and silk fibroin.

12. The method according to claim 10, wherein the organic solvent is at least one selected from a group consisting of methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, hexyl acetate, ethyl formate, dimethyl carbonate, diethyl carbonate, 1,3-dioxolidin-2-one, cellulose acetate butyrate, medium chain triglyceride (MCT) oil, vegetable oil, wax, and infused oil.

13. The method according to claim 10, wherein cold-curing the micro-sized particles prepared in step 2) at room temperature or lower is added between step 2) of (a) and step 3) of (a).

14. The method according to claim 10, wherein crushing the micro-sized microparticles and sieving the crushed microparticles so as to be divided by particle size is further added between step 4) of (a) and step 5) of (a) or after step 5) of (a), or sieving the micro-sized microparticles so as to be divided by particle size is further added after step 5) of (b).

15. The method according to claim 10, wherein the thermal curing is performed at 100? C. to 200? C. for 10 minutes to 24 hours.

16. The method according to claim 10, wherein the washing is performed at a temperature of above 0? C. to 40? C. or lower.

17. The method according to claim 10, wherein the washing is performed using a solvent with a ?P polar value of 14 or higher.

18. Hydrogel microparticles prepared using the method according to claim 10.

19. An embolic composition comprising the hydrogel microparticles according to claim 18.

20. Hydrogel microparticles prepared using the method according to claim 11.

Description

DESCRIPTION OF DRAWINGS

[0048] FIG. 1 is a micrograph of spherical hydrogel microparticles according to Example 1 of the present invention. A scale is shown under the micrograph, wherein the total length of gradations is 500 ?m.

[0049] FIG. 2 is a 160? micrograph of the spherical hydrogel microparticles according to Example 1 of the present invention swollen in water. A scale is shown under the right side of the micrograph, wherein the total length of a white bar is 250 ?m.

[0050] FIG. 3 shows the result of measurement of particle size distribution of the spherical hydrogel microparticles according to Example 1 of the present invention.

[0051] FIG. 4 shows the elution test results of Example 1 and Comparative Example 1.

[0052] FIG. 5 shows the cytotoxicity test results of Example 1 and Comparative Example 1.

[0053] FIG. 6 shows the results of adjusting degradation time using Examples 2 to 5.

[0054] FIG. 7 is a micrograph of amorphous hydrogel microparticles according to Example 13 of the present invention. A scale is shown under the micrograph, wherein the total length of gradations is 300 ?m.

[0055] FIG. 8 is a 160? micrograph of the amorphous hydrogel microparticles according to Example 13 of the present invention swollen in water. A scale is shown under the right side of the micrograph, wherein the total length of a white bar is 250 ?m.

BEST MODE

[0056] In the present application, it should be understood that the terms comprises, has, includes, etc., when used in this specification, specify the presence of stated features, numbers, steps, operations, elements, components, or combinations thereof, but do not preclude the presence or addition of one or more other features, numbers, steps, operations, elements, components, or combinations thereof.

[0057] It will be understood that, when a component is referred to as being connected to or coupled to another component, it may be directly connected to or coupled to the other component, or intervening components may be present. In contrast, when a component is referred to as being directly connected to or directly coupled to another component, there are no intervening components present. Other terms that describe the relationship between components, such as between and directly between or adjacent to and directly adjacent to, must be interpreted in the same manner.

[0058] In addition, unless otherwise defined, all terms, including technical and scientific terms, used in this specification have the same meanings as those commonly understood by a person having ordinary skill in the art to which the present invention pertains. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having meanings consistent with their meanings in the context of the relevant art and the present disclosure, and are not to be interpreted in an idealized or overly formal sense unless expressly so defined herein.

Example 1: Preparation of Spherical Hydrogel Microparticles

[0059] 1) 20 g of gelatin is completely dissolved in 100 ml of 50? ? C. distilled water. [0060] 2) The gelatin solution obtained in step 1) is slowly injected into 400 ml of medium-chain triglyceride (MCT) oil under a stirring condition of 450 rpm to prepare an emulsion. [0061] 3) The emulsion prepared in step 2) is cured at 4?C for 30 minutes, supernatant is removed, the emulsion is washed with acetone, and the emulsion is dried under vacuum. [0062] 4) The spherical microparticles obtained in step 3) are thermally treated at 150? C. for 4 hours. [0063] 5) The microparticles thermally treated in step 4) are washed with distilled water, are washed with acetone, and are dried under vacuum to obtain final spherical hydrogel microparticles.

Washing Method of Example 1

[0064] The washing method is influenced by the ratio of microspheres to a washing solution, washing intensity, and washing time.

[0065] 50 g of the microspheres according to Example 1 were mixed with 1.5 L of 15? C. distilled water, and stirring was performed at 200 rpm for 30 minutes to wash the microspheres. After 30 minutes, the swollen microspheres were completely settled, supernatant was removed, 1.5 L of 15? C. distilled water was added, and stirring was performed again at 200 rpm for 30 minutes to wash the microspheres. The washing process was performed until the supernatant was completely transparent. In the case of Example 1, washing was performed a total of 3 times.

[0066] In the following other examples or comparative example, washing was performed in the same manner as described above.

[0067] The washing solution was checked, and it was found that microspheres swelled in solvents with a OP polar value of 14 or higher.

TABLE-US-00001 Solvent ?P polar Washable Acetone 10.4 X Ethanol 8.8 X Dimethylformamide 13.7 X Dimethyl sulfoxide 16.4 ? Water 15.1 ?

Comparative Example 1

[0068] Identical to Example 1 except that the last step 5), among the preparation steps of Example 1, was not performed.

Examples 2 to 5: Preparation of Spherical Hydrogel Microparticles

[0069] The heat treatment temperature and time in step 4) of Example 1 were changed to 120? C. and 3 hours, respectively. After heat treatment, the microparticles are divided into four parts and washed in distilled water at 4? C. (Example 2), 10? C. (Example 3), 20? C. (Example 4), and 27? C. (Example 5), respectively. After distilled water washing, the microparticles are washed with acetone and dried under vacuum to obtain final spherical hydrogel microparticles.

Examples 6 to 12: Preparation of Spherical Hydrogel Microparticles

[0070] The heat treatment temperature and time in step 4) of Example 1 were changed as shown in Table 4 below.

Example 13: Preparation of Amorphous Hydrogel Microparticles

[0071] 1) 10 g of gelatin is completely dissolved in 100 ml of 50? C. distilled water. [0072] 2) The gelatin solution obtained in step 1) is stirred at 14000 rpm for 30 minutes to form enough foam, is cured for 2 hours in a ?50? C. environment, and is freeze-dried. [0073] 3) The sponge-like gelatin obtained after freeze-drying in step 2) is thermally treated at 150? C. for 5 hours. [0074] 4) The sponge-like gelatin thermally treated in step 3) is washed with distilled water, is cured for 2 hours in a ?50? C. environment, and is freeze-dried again. [0075] 5) The sponge obtained after freeze-drying in step 4) is crushed to obtain final amorphous sponge particles.

Experiment 1: Microscope Observation

[0076] The spherical hydrogel microparticles prepared according to Example 1 and the amorphous hydrogel microparticles prepared according to Example 3 were observed using a microscope (see FIGS. 1 and 7, respectively).

[0077] In addition, the spherical hydrogel microparticles prepared according to Example 1 and the amorphous hydrogel microparticles prepared according to Example 13 were immersed in distilled water for 20 minutes to swell, and were observed using the microscope (see FIGS. 2 and 8, respectively).

[0078] As can be seen from FIGS. 1 and 2, the hydrogel microparticles prepared according to Example 1 have a spherical shape both before and after swelling.

[0079] As can be seen from FIGS. 7 and 8, the amorphous hydrogel microparticles prepared according to Example 13 are amorphous both before and after swelling.

Experiment 2: Measurement of Particle Size Distribution

[0080] The particle size distribution of the spherical hydrogel microparticles prepared according to Example 1 was measured using a particle size analyzer.

[0081] FIG. 3 shows the result of measurement of particle size distribution of the spherical hydrogel microparticles according to Example 1 of the present invention. The x-axis is the particle size in ?m, and the y-axis is the volume (%) occupied by the particle size.

[0082] It can be seen that the spherical hydrogel microparticles according to Example 1 of the present invention have a very narrow degree of particle distribution with an average size of about 300 ?m. In addition, the particle size distribution is a desirable size for embolization.

Experiment 3: Elution Test

[0083] Eluates were observed for Example 1 and Comparative Example 1.

[0084] The elution test was performed by adding 30 ml of a 1?PBS solution to 2 g of microparticles to sufficiently swell the microparticles and eluting the same in a 37? C. shaking water bath for 24 hours.

[0085] FIG. 4 shows the elution test results of Example 1 and Comparative Example 1. In FIG. 4, the left side is the result of Comparative Example 1 and the right side is the result of Example 1. It can be seen that Comparative Example 1 has more eluate than Example 1.

Experiment 4: Toxicity Test

[0086] A cytotoxicity test (ISO10993-5), an endotoxin test (ISO10993-11), and a pyrogenic test (ISO10993-11) were performed for each of Example 1 and Comparative Example 1.

[0087] Table 1 and FIG. 5 show the cytotoxicity test results, Table 2 shows the endotoxin test results, and Table 3 shows the pyrogenic test results. Negative Control, Positive Control, and Blank are all based on the above ISO standards.

[0088] In Table 1 below, a cell viability of 80% or more is required to pass; in Table 2, an endotoxin value of 20 EU/device or more is required to pass; and in Table 3, if fever of 0.5? C. or more is generated, this is not a pass.

[0089] In all three toxicity tests, Comparative Example 1 was a Fail and Example 1 was a Pass.

TABLE-US-00002 TABLE 1 Cell viability (%) Morphology Negative control 100.00 0 Positive control 18.07 4 Blank 118.61 0 Comparative Example 1 10.33 1 Example 1 81.95 2 * To pass, cell viability should be more than 80%.

TABLE-US-00003 TABLE 2 Sample Endotoxin Range Re- No. Endotoxin value (50%~200%) mark Comparative 1 10.3 EU/device 5.2~20.6 EU/device Fail Example 1 2 12.1 EU/device 6.0~24.2 EU/device Fail 3 10.2 EU/device 5.1~20.4 EU/device Fail Example 1 1 1.1 EU/device 0.6~2.3 EU/device Pass 2 1.5 EU/device 0.8~3.0 EU/device Pass 3 1.8 EU/device 0.9~3.6 EU/device Pass * To pass: 20 EU/device

TABLE-US-00004 TABLE 3 Comparative Example 1 Example 1 Rabbit- Rabbit- Rabbit- Rabbit- Rabbit- Rabbit- 1 2 3 4 5 6 Initial 39.3 39.0 39.2 39.0 39.4 39.1 0 min 39.3 39.3 38.9 39.1 39.4 39.0 30 min 39.8 39.3 39.0 39.2 39.5 39.1 60 min 40.5 39.9 39.9 39.3 39.6 39.1 90 min 40.8 40.4 40.6 39.3 39.6 39.2 120 min 40.9 40.5 40.7 39.3 39.6 39.3 150 min 41.4 40.6 40.9 39.2 39.6 39.3 180 min 41.4 37.8 41.0 39.2 39.6 39.2 Max 2.1 1.6 1.8 0.3 0.2 0.2 Remark Fail Fail Fail Pass Pass Pass * Fail: body temperature rises above 0.5? C.

Experiment 5: Observation of Degradation Time Based on Washing Temperature

[0090] The microparticles of Examples 2 to 5 were sieved to collect only microparticles of 100 to 300 ?m in size, were introduced into 1? phosphate-buffered saline (PBS), and were observed for degradation time in a constant temperature water bath at 37?C, which is similar to the human body temperature. The results of the degradation time are shown in FIG. 6. As can be seen from Examples 2 to 5, the degradation time can be adjusted at intervals of about 10 minutes by changing only the temperature of the last distilled water washing step in the preparation method according to the present invention. It can also be seen that all of the microparticles according to the present invention can be degraded within a very relatively short time of 1 hour.

Experiment 6: Observation of Degradation Time Based on Heat Treatment Temperature

[0091] The microparticles of Examples 6 to 12 were sieved to collect only microparticles of 100 to 300 ?m in size, were introduced into 1? phosphate-buffered saline (PBS), and were observed for degradation time in a constant temperature water bath at 37? C., which is similar to the human body temperature. The results of the degradation time are shown in Table 4. Experiment 5 shows that the degradation time can be finely adjusted by changing the washing temperature after the same heat treatment. Experiment 6 clearly shows that the degradation time of the microparticles in large units can be adjusted by changing the heat treatment temperature and the heat treatment time.

TABLE-US-00005 TABLE 4 Heat Heat treatment treatment Degradation temperature time time Example 6 110? C. 24 hours 5 minutes Example 7 120? C. 3 hours 1 hour Example 8 130? C. 3 hours 24 hours Example 9 140? C. 1 hour 4 hours Example 10 140? C. 2 hours 40 hours Example 11 150? C. 4 hours 40 days Example 12 200? C. 10 minutes 7 days

[0092] It can be seen from Experiments 5 and 6 that, when designing the degradation time of microparticles, it is possible to set the degradation time in large units (days or hours) by adjusting the heat treatment temperature and time first and then to adjust the time in minutes by adjusting the washing temperature.