Temperature sensitive hydrogel composition including nucleic acid and chitosan

Abstract

The present invention provides a temperature sensitive hydrogel composition including a nucleic acid and chitosan. Since the hydrogel has excellent biocompatibility and biostability, and simultaneously has sol-gel phase transition properties depending on temperature changes, the hydrogel is present in a sol state at room temperature and becomes a gel when the hydrogel is injected into the human body or applied on the surface of epithelial skin and the temperature increases. Thus, the temperature-sensitive hydrogel of the present invention can be directly injected into and applied on certain parts requiring treatment and the retention and attaching time of a drug is increased through gelation depending on the temperature so that drug efficacy is sufficiently exhibited. Therefore, it is expected that the temperature-sensitive hydrogel of the present invention can be utilized for various treatments.

Claims

1. A temperature-sensitive hydrogel composition comprising a nucleic acid and chitosan, wherein the content of the nucleic acid is 1 wt % to 3 wt % relative to the total weight of the composition, wherein the content of the chitosan is 0.001 wt % to 0.03 wt % relative to the total weight of the composition, wherein the weight ratio of the nucleic acid and the chitosan is 33.333:1 to 3000:1, and wherein the temperature-sensitive hydrogel composition has a pH value of 6-8.

2. The temperature-sensitive hydrogel composition of claim 1, wherein the weight ratio of the nucleic acid and the chitosan is 50:1 to 2000:1.

3. The temperature-sensitive hydrogel composition of claim 2, wherein the weight ratio of the nucleic acid and the chitosan is 100:1 to 1000:1.

4. The temperature-sensitive hydrogel composition of claim 1, wherein the content of the nucleic acid is 1-2 wt % relative to the total weight of the composition.

5. The temperature-sensitive hydrogel composition of claim 1, wherein the nucleic acid is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a mixture thereof.

6. The temperature-sensitive hydrogel composition of claim 1, wherein the content of the chitosan is 0.003 wt % to 0.02 wt % relative to the total weight of the composition.

7. The temperature-sensitive hydrogel composition of claim 6, wherein the molecular weight of the chitosan is 3 kDa to 1,000 kDa.

8. The temperature-sensitive hydrogel composition of claim 1, wherein the temperature-sensitive hydrogel composition contains a polymer material as an additional ingredient.

9. The temperature-sensitive hydrogel composition of claim 8, wherein the polymer material is at least one selected from the group consisting of hyaluronic acid, poly-γ-glutamic acid, cellulose, polyacrylic acid, polyamino acids, alginate, and a combination thereof.

10. A method for producing the temperature-sensitive hydrogel composition of claim 1, the method comprising: i) dissolving the nucleic acid in a buffer solution for 1-2 hours with stirring at 60-70° C., to prepare a nucleic acid stock solution; ii) dissolving chitosan in an acidic buffer solution to prepare a chitosan stock solution; iii) mixing together the nucleic acid stock solution in step i) and the chitosan stock solution in step ii) such that the weight ratio of the nucleic acid and the chitosan is 33.333:1 to 3000:1, followed by stirring at 55-65° C. for 1-2 hours; and iv) lowering the nucleic acid-chitosan mixture liquid in step iii) to room temperature with stirring, thereby obtaining the temperature-sensitive hydrogel composition comprising a nucleic acid and chitosan.

11. The method of claim 10, wherein the temperature-sensitive hydrogel composition has an osmotic pressure of 100-500 mOsm.

12. The method of claim 10, wherein the temperature-sensitive hydrogel composition has a pH value of 6.5-7.5.

13. The method of claim 10, wherein the weight ratio of the nucleic acid and the chitosan is 50:1 to 2000:1.

14. The method of claim 13, wherein the weight ratio of the nucleic acid and the chitosan is 100:1 to 1000:1.

15. The method of claim 10, wherein the content of the nucleic acid is 1-3 wt % relative to the total weight of the composition.

16. The method of claim 10, wherein the nucleic acid is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a mixture thereof.

17. The method of claim 10, wherein the content of the chitosan is 0.001 wt % to 0.03 wt % relative to the total weight of the composition.

18. The method of claim 10, wherein the molecular weight of the chitosan is 3 kDa to 1,000 kDa.

19. The method of claim 10, wherein the temperature-sensitive hydrogel composition contains a polymer material as an additional ingredient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the results of confirming physical property states of temperature-sensitive hydrogels according to the mixing ratio of a nucleic acid and chitosan. Each composition was subjected to mixing, and then was investigated for transparency and viscoelasticity (A) and precipitate generation and layer separation (B) for 3 days.

(2) FIG. 2 shows the results of confirming whether gels are dissolved.

(3) FIG. 3 shows the results of confirming the sol-gel transition of a temperature-sensitive hydrogel composition.

MODE FOR CARRYING OUT THE INVENTION

(4) Hereinafter, preferable embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

Example 1

Production of Nucleic Acid-Chitosan Hydrogels

(5) For the production of nucleic acid and chitosan hydrogels, nucleic acid and chitosan stock solutions were prepared with concentrations corresponding examples shown in table 1 below. Here, nucleic acid was put in a buffer solution of 200 mM sodium phosphate dibasic dodecahydrate, and then dissolved therein using a heat stirrer at 65° C. for 1 hour.

(6) In addition, chitosan was dissolved using 100 mM acetic acid.

(7) The nucleic acid and chitosan stock solutions prepared with the concentrations in table 1 below were mixed at a weight ratio of 1:1, and stirred in a heat stirrer at 60° C. for 1 hour. Thereafter, the temperature was lowered to room temperature, followed by stirring for 1 hour, to produce nucleic acid-chitosan hydrogels.

Comparative Example 1

Production of Comparative Nucleic Acid-Chitosan Hydrogel

(8) Nucleic acid and chitosan stock solutions were prepared with concentrations corresponding to comparative examples shown in table 1 below. Comparative nucleic acid-chitosan hydrogels were produced by the same method as in example 1.

(9) TABLE-US-00001 TABLE 1 Stock solution concentration (wt %) Final concentration (wt %) Constitution Nucleic acid Chitosan Nucleic acid Chitosan Example 1-1 2 0.1 1 0.05 Example 1-2 2 0.04 1 0.02 Example 1-3 2 0.02 1 0.01 Example 1-4 2 0.002 1 0.001 Example 1-5 2 0.001 1 0.0005 Example 1-6 2 0.0004 1 0.0002 Example 1-7 2 0.0002 1 0.0001 Example 1-8 0.02 0.0002 0.01 0.0001 Example 1-9 0.02 0.00002 0.01 0.00001 Example 1-10 6 0.06 3 0.03 Example 1-11 6 0.006 3 0.003 Comparative 2 0 1 0 example 1-1 Comparative 0 0.02 0 0.01 example 1-2 Comparative 2 0.2 1 0.1 example 1-3 Comparative 2 0.0001 1 0.00005 example 1-4

Example 2

Production of Nucleic Acid-Chitosan-Hyaluronic Acid Hydrogel

(10) A nucleic acid-chitosan-hyaluronic acid hydrogel was produced through the following procedure.

(11) A nucleic acid was dissolved in a buffer solution of 200 mM sodium phosphate dibasic dodecahydrate to have a concentration of 2.2 wt %. Here, the nucleic acid were dissolved using a heat stirrer at 65° C. for 1 hour.

(12) In addition, chitosan was dissolved in 100 mM acetic acid to have a concentration of 0.4 wt %.

(13) Sodium hyaluronic acid was dissolved in a buffer solution of 200 mM sodium phosphate dibasic dodecahydrate to have a concentration of 2 wt %. Here, the sodium hyaluronic acid was dissolved using a heat stirrer at 40° C. for 30 minutes, followed by cooling to room temperature with stirring using a stirrer.

(14) Then, 2.2 wt % of the prepared nucleic acid solution and 0.4 wt % of the prepared chitosan solution were mixed at a weight ratio of 9:1, followed by stirring in a heat stirrer at 65° C. for 10 minutes. 2 wt % of sodium hyaluronic acid was added to the nucleic acid-chitosan mixture solution at a weight ratio of 1:1, followed by stirring in a heat stirrer at 65° C. for 1 hour, and then the resulting solution was cooled to room temperature while the stirring maintained, thereby producing nucleic acid-chitosan-hyaluronic acid hydrogels of example 2.

Comparative Example 2

Production of Comparative Chitosan-Hyaluronic Acid Hydrogel

(15) Chitosan and hyaluronic acid stock solutions were prepared with concentrations in table 2 below.

(16) Chitosan was dissolved using 100 mM acetic acid.

(17) Sodium hyaluronic acid was dissolved in a buffer solution of 200 mM sodium phosphate dibasic dodecahydrate. Here, sodium hyaluronic acid was dissolved using a heat stirrer at 40° C. for 30 minutes, and then the resulting solution was cooled to room temperature while the stirring was maintained. The prepared chitosan and hyaluronic acid solutions were mixed at a weight ratio of 1:1, followed by stirring in a heat stirrer at 65° C. for 1 hour. Thereafter, the temperature was lowered to room temperature, followed by stirring for 1 hour, to produce chitosan-hyaluronic acid hydrogels.

(18) TABLE-US-00002 TABLE 2 Final concentration Stock solution concentration (wt %) (wt %) Hyaluronic Constitution Chitosan Hyaluronic acid Chitosan acid Comparative 0 2 0 1 example2-1 Comparative 0.02 2 0.01 1 example 2-2

Experimental Example 1

Confirmation of Physical Properties of Nucleic Acid-Chitosan Hydrogels

(19) The hydrogel compositions of example 1, example 2, comparative example 1, and comparative example 2 were used to investigate gelation, gel stability, and solubility thereof.

(20) Each composition was subjected to mixing, and then the transparency and gelation state thereof were observed to the naked eye for 3 days. The gelation was examined by viscoelasticity and the gel stability was examined by precipitate generation and layer separation.

(21) For the solubility of gel, the hydrogel composition of each of example 1, example 2, comparative example 1, and comparative example 2 was dropped in an aqueous solution at 37.5° C., followed by gelation, and then the solubility of gel was examined while the stirring was conducted at 400 rpm for 5 minutes with the temperature maintained at 37.5° C. The results were shown in table 3 and FIGS. 1 and 2.

(22) TABLE-US-00003 TABLE 3 Results of stirring Results after three days at 37.5° C. Precipitate Layer for 5 minutes Constitution Viscoelasticity generation separation Gel solubility Example 1-1 ◯ X X X Example 1-2 ◯ X X X Example 1-3 ◯ X X X Example 1-4 ◯ X X X Example 1-5 ◯ X X X Example 1-6 ◯ X X X Example 1-7 ◯ X X X Example 1-8 ◯ X X X Example 1-9 ◯ X X X Example 1-10 ◯ X X X Example 1-11 ◯ X X X Example 2 ◯ X X X Comparative ◯ X X ◯ example 1-1 Comparative X X X ◯ example 1-2 Comparative ◯ ◯ ◯ ◯ example 1-3 Comparative ◯ ◯ X ◯ example 1-4 Comparative X X X ◯ example 2-1 Comparative ◯ X X ◯ example 2-2

(23) It can be seen from table 3 and FIG. 1 that, in the temperature-sensitive hydrogel compositions of examples 1-1 to 1-11, the precipitate isolation and the layer separation did not occur with viscoelasticity maintained, even three days after the nucleic acid and chitosan were mixed. However, as for comparative examples 1-1 to 1-4, which got out of the weight ratio range of nucleic acid and chitosan of the present invention, the precipitate generation, the layer separation, or the viscoelasticity was not observed.

(24) In addition, it can be seen from the solubility of gel in table 3 and FIG. 2 that the temperature-sensitive hydrogel composition of each of examples 1-1 to 1-11 formed a gel at 37.5° C. and the formed gel was continuously maintained, whereas comparative examples 1-1 to 1-4 were not gelated at 37.5° C. or were completely dissolved within 20 seconds even if the gelation occurred.

(25) It was confirmed that, even in example 2 in which hyaluronic acid as an additional ingredient was added in the nucleic acid and chitosan, the viscoelasticity was maintained even while the precipitate isolation and the layer separation were not shown and the gel was continuously maintained after the gelation, whereas in comparative example 2-2 in which the nucleic acid was not contained, the precipitate isolation and the layer separation were not shown with viscoelasticity maintained, but the gel was dissolved within 20 seconds after the gel was formed at 37.5° C. (see table 3 and FIG. 1).

(26) Through these results, it can be seen that the hydrogel composition of the present invention shows temperature sensitivity, high stability, and a gel form continuously maintained after gelation, and here, it was confirmed that the weight ratio of nucleic acid and chitosan plays a key role.

Experimental Example 2

Confirmation of Sol-Gel Transition with Temperature

(27) The temperature-sensitive hydrogel composition produced in each of example 1, example 2, comparative example 1, and comparative example 2 was examined for sol-gel transition.

(28) For the confirmation of sol-gel transition, a rheometer was used. The measurement conditions used here were PU20, gap of 0.5 mm, 0.1 Hz, and 1% stress-strain, and the changes of G′ (elasticity) and G″ (viscosity) were measured while the temperature was raised from 24° C. to 40° C. by 1° C. and then was maintained for 1 minute. In addition, the sol-gel transition after and before 36° C. was observed to the naked eye while the temperature of each composition was raised, and the results were shown in FIG. 3.

(29) As shown in FIG. 3, it was observed that the temperature-sensitive hydrogel of example 1-3 exhibited a gentle decrease width of G′ (elasticity) and an increase of G″ (viscosity) with an increasing temperature (FIG. 3A), which correspond to the feature of a temperature-sensitive hydrogel. In addition, it was observed that the temperature-sensitive hydrogel of example 1-3 exhibited a sol state at a temperature lower than 36° C. but was gelated at a temperature exceeding 36° C. (FIG. 3B). It was confirmed that these results were identical in all of the temperature-sensitive hydrogel compositions in examples 1 and 2. It can be seen from these results that the hydrogel compositions of the present invention show temperature sensitivity.