HYALURONIC ACID-BASED HYDROGEL USING PEPTIDE CROSSLINKING AGENT, AND METHOD FOR PRODUCING SAME

Abstract

The present invention relates to a hyaluronic acid-based hydrogel which is a hyaluronic acid-peptide crosslinked body crosslinked using a peptide crosslinking agent. More specifically, the present invention relates to a hyaluronic acid-based hydrogel and a method for producing same, wherein a crosslinked body having novel physical properties is obtained using a relatively small amount of a crosslinking agent that forms peptide bonds, unlike conventional crosslinking agents, and the hyaluronic acid-based hydrogel has the advantages of: being safe and having few side effects; the physical properties of a filler being adjustable according to the amount of peptide crosslinking; and having a high elasticity ratio.

Claims

1. A hyaluronic acid-based hydrogel comprising a peptide cross-linking agent comprising 3 kinds to 10 kinds of amino acids and hyaluronic acid or a salt thereof, wherein the 3 kinds to 10 kinds of amino acids are selected from the group consisting of glutamic acid (Glu), aspartic acid (Asp), glutamine (Gln), asparagine (Asn), histidine (His), lysine (Lys), arginine (Arg), serine (Ser), cysteine (Cys), beta alanine (β-Ala), alanine (Ala), glycine (Gly), leucine (Leu), isoleucine (Ile), valine (Val), threonine (Thr), methionine (Met), proline (Pro), phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp).

2. The hyaluronic acid-based hydrogel according to claim 1, wherein the peptide cross-linking agent has any one structure of the following Chemical formulas 2 to 4,
X.sub.1—X.sup.2—X.sub.3   [Chemical formula 2]
Y.sub.1—Y.sub.2—Y.sub.3—Y.sub.4   [Chemical formula 3]
Z.sub.1—Z.sub.2—Z.sub.3—Z.sub.4—Z.sub.5   [Chemical formula 4] and in the formula, X.sub.3, Y.sub.4 and Z.sub.5 are each independently lysine (Lys), and X.sub.1, X.sub.2, Y.sub.1, Y.sub.2, Y.sub.3, Z.sub.1, Z.sub.2, Z.sub.3 and Z.sub.4 are each independently selected from the group consisting of glutamine (Gln), asparagine (Asn), serine (Ser), cysteine (Cys), beta alanine β-Ala), alanine (Ala), glycine (Gly), leucine (Leu), isoleucine (Ile), valine (Val), threonine (Thr), methionine (Met), proline (Pro), phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp), and in the Chemical formulas 2 to 4, lysine of X.sub.3, Y.sub.4 and Z.sub.5 comprises an amide group (—CONH.sub.2) instead of a carboxyl group (—COOH).

3. The hyaluronic acid-based hydrogel according to claim 1, having the structure of the following Chemical formula 1: ##STR00005## and in the formula, n is an integer of 500 to 1,400, and L comprises 3 kinds to 10 kinds of amino acids.

4. The hyaluronic acid-based hydrogel according to claim 1, wherein the hyaluronic acid has a molecular weight of 200,000˜1,200,000 Da.

5. (canceled)

6. A peptide cross-linking agent comprising a peptide having any one structure of the following Chemical formulas 2 to 4:
X.sub.1—X.sup.2—X.sub.3   [Chemical formula 2]
Y.sub.1—Y.sub.2—Y.sub.3—Y.sub.4   [Chemical formula 3]
Z.sub.1—Z.sub.2—Z.sub.3—Z.sub.4—Z.sub.5   [Chemical formula 4] and in the formula, X.sub.3, Y.sub.4 and Z.sub.5 are each independently lysine (Lys), and X.sub.1, X.sub.2, Y.sub.1, Y.sub.2, Y.sub.3, Z.sub.1, Z.sub.2, Z.sub.3 and Z.sub.4 are each independently selected from the group consisting of glutamine (Gln), asparagine (Asn), serine (Ser), cysteine (Cys), beta alanine (β-Ala), alanine (Ala), glycine (Gly), leucine (Leu), isoleucine (Ile), valine (Val), threonine (Thr), methionine (Met), proline (Pro), phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp), and in the Chemical formulas 2 to 4, lysine of X.sub.3, Y.sub.4 and Z.sub.5 is an amino acid comprising an amide group (—CONH.sub.2) instead of a carboxyl group (—COOH).

7. A method for manufacturing of the peptide cross-linking agent of claim 6 comprising steps of i) binding a side chain or C-terminus of the first amino acid to a polymer supporter resin; ii) removing a protective group at the N-terminus of the first amino acid; iii) binding amino acids in order using a coupling agent to synthesize a peptide; and iv) removing the resin and protective group from the peptide synthesized in the iii).

8. (canceled)

9. The method for manufacturing according to claim 7, wherein the amino acid in the step i) and step iii) is selected from the group consisting of glutamic acid (Glu), aspartic acid (Asp), glutamine (Gln), asparagine (Asn), histidine (His), lysine (Lys), arginine (Arg), serine (Ser), cysteine (Cys), beta alanine (β-Ala), alanine (Ala), glycine (Gly), leucine (Leu), isoleucine (Ile), valine (Val), threonine (Thr), methionine (Met), proline (Pro), phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp).

10. The method for manufacturing according to claim 7, wherein in the amino acid in the step i) and step iii), the amine terminus and side chain are protected by a protective group selected from the group consisting of Boc (tert-butoxycarbonyl), Fmoc (9-fluorenylmethoxycarbonyl), Cbz (benzyloxycarbonyl), tBu (tert-butyl), StBu (tert-butylthio), Trt (triphenylmethyl; trityl), Acm (acetamidomethyl) and Tacm (trimethylacetamido-methyl).

11. (canceled)

12. (canceled)

13. (canceled)

14. (canceled)

15. A method for manufacturing the hyaluronic acid-based hydrogel according to claim 1, comprising i) manufacturing a peptide using 1 kind to 10 kinds of amino acids selected from the group consisting of glutamic acid (Glu), aspartic acid (Asp), glutamine (Gln), asparagine (Asn), histidine (His), lysine (Lys), arginine (Arg), serine (Ser), cysteine (Cys), beta alanine (β-Ala), alanine (Ala), glycine (Gly), leucine (Leu), isoleucine (Ile), valine (Val), threonine (Thr), methionine (Met), proline (Pro), phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp); and ii) manufacturing a hyaluronic acid cross-linked body comprising a peptide cross-linking agent by adding the peptide cross-linking agent synthesized in the step i), a coupling agent, an auxiliary coupling agent, and a base after adding distilled water to hyaluronic acid.

16. The method for manufacturing according to claim 15, wherein the step i) is performed by the synthesis method of claim 7.

17. The method for manufacturing according to claim 15, wherein the peptide cross-linking agent used in the step ii) is 0.01-0.1 equivalent compared to a disaccharide (D-glucuronic acid and N-acetylglucosamine) which is a component of hyaluronic acid.

18. (canceled)

19. (canceled)

20. (canceled)

21. The method for manufacturing according to claim 15, wherein the step ii) is performed at a temperature of 20° C. to 40° C. for 2 hours, 4 hours, 8 hours, 16 hours or 24 hours.

22. The method for manufacturing according to claim 15, further comprising iii) washing the cross-linked body manufactured in the step ii) with phosphate-buffered saline (PBS) solution to remove unreacted substances; iv) pulverizing the cross-linked body washed in the step iii); and v) sterilizing the cross-linked body pulverized in the step iv), after the step ii).

23. The method for manufacturing according to claim 22, wherein in the step iv) the cross-linked body is pulverized in a particle size of about 100˜500 μm.

24. The method for manufacturing according to claim 22, wherein in the step v), sterilizing is performed at a temperature of 121° C. to 135° C. for 8 minutes and 20 minutes.

25. A composition for injection into a body, comprising the hyaluronic acid-based hydrogel according to claim 1.

26. An implant for tissue repair comprising the composition for injection into a body according to claim 25.

27. A filler for repair of wrinkles, fine wrinkles, wound or skin depression comprising the composition for injection into a body according to claim 25.

28. A method for improvement of wrinkles, comprising injecting the hyaluronic acid-based hydrogel according to claim 1 into a body.

29. A method for tissue repair or volume increase, comprising injecting the hyaluronic acid-based hydrogel according to claim 1 into a body.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0067] FIG. 1 is a drawing of schematizing one embodiment of the process of manufacturing the hyaluronic acid-based hydrogel using a peptide cross-linking agent according to the present invention.

MODE FOR INVENTION

[0068] Hereinafter, the present invention will be described in detail by examples. However, the following examples illustrates the present invention only, but the present invention is not limited by the following examples.

EXAMPLE 1

Synthesis of Ala-Leu-Lys-NH2, which is the Peptide Cross-Linking Agent According to the Present Invention

[0069] For manufacturing the peptide cross-linking agent according to the present invention, the process of [Reaction formula 3] was conducted.

[0070] [Reaction Formula 3]

[0071] Synthesis of tri-peptide, which is the peptide cross-linking agent of the present invention

##STR00004##

[0072] 1) Rink amide MBHA resin (0.502 mmol/g) 996 mg (500 umol) purchased from Novabiochem was weighed and placed in a reaction container. The resin was swelled for 5 minutes using DMF (N,N-Dimethylformamide) and the solvent was removed under reduced pressure. After removing Fmoc (9-Fluorenylmethoxycarbonyl) using DMF solution diluted with 20% piperidine in the reaction container, amino acids (3 equivalents) and HOBt (1-hydroxybenzotriazole) (3 equivalents), HBTU

[0073] (N,N,N,N-Tetramethyl-O-(1H-benzotriazol-1-yl)uranium hexafluorophosphate) (3 equivalents), DIPEA (N,N-Diisopropylethylamine) (6 equivalents) were added, and the reaction was carried out for 4 hours.

[0074] 2) After reaction, solvent removal and washing of the resin were performed, and then Fmoc (9-Fluorenylmethoxycarbonyl) removal reaction was carried out using DMF solution diluted with 20% piperidine (5 min×2 times).

[0075] 3) The coupling reaction of the amino acid and resin was performed using amino acids (3 equivalents)/HOBt (3 equivalents)/HBTU (3 equivalents)/DIPEA (6 equivalents) reagents for 4 hours.

[0076] 4) The coupling reaction was confirmed using a qualitative method called kaiser test (E. Kaiser et al., Anal. Biochem. 1970, 34, 595), and then, the Kaiser solution consists of three kinds. [a. ninhydrin (5 g)+ethanol (100 ml); b. phenol (80 g)+ethanol (20 ml); c. pyridine (98 ml)+0.002 M aq. KCN (2 ml)] After completing the coupling reaction, the resin was washed, and then 2-3 drops of the three kinds of the Kaiser test solution were added, and then heat was applied at 120° C. for 3 minutes. When the unreacted part remained, the color of the resin showed a blue light, and when it was completely reacted, there was no change in the color of the resin.

[0077] 5) After synthesizing a desired peptide derivative by repeating 2)-4) processes, the resin and protective group removal was carried out using cleavage cocktail [TFA:H2O=95:5 (v/v)] solution.

EXAMPLE 2

Manufacturing of Cross-Linked Body of Hyaluronic Acid-Peptide According to the Present Invention

[0078] For manufacturing of the hyaluronic acid-based hydrogel according to the present invention, the following process was conducted.

[0079] The reaction was carried out at a room temperature for 24 hours by adding 10 ml distilled water to 1 g hyaluronic acid, making the tri-peptide synthesized in Example 1 to be 0.012 equivalents (Example 1a), 0.014 equivalents (Example 1b), 0.016 equivalents (Example 1c), and 0.018 equivalents (Example 1d), and adding EDC [1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide; 0.01˜0.1 equivalents], HOBt [1-Hydroxybenzotriazole; 0.01˜0.1 equivalents], DIPEA [N,N-Diisopropylethylamine; 0.02˜0.2 equivalents) reagents. The manufactured cross-linked body was washed with phosphate-buffered saline (PBS) solution several times to remove unreacted substances, and the washed cross-linked body was pulverized to adjust to a particle size of about 100˜500 μm, and then it was sterilized at 121° C. for 15 minutes.

EXAMPLE 3

Analysis of Physical Properties of Cross-Linked Body of Hyaluronic Acid-Peptide Manufactured by the Present Invention

[0080] For analysis of physical properties of manufactured Example 2, the concentration of the cross-linked body was manufactured as 20 mg/PBS 1 ml, and then it was analyzed using a rotational rheometer. Then, the rheometer analysis was measured within a frequency range of 0.02 Hz, and after adding the sample to Plate-Plate, a normal force value was measured when it was rotated at a shear rate of 10.sup.−1 for 0.1 second and the upper plate was dropped at a rate of 0.1 mm/s.

[0081] <Analysis Condition>

[0082] (1) Test equipment: MCR Rheometer (Anton Paar)

[0083] (2) Frequency: 1˜10 Hz

[0084] (3) Temperature: 25±0.01° C.

[0085] (4) Strain: 1%

[0086] (5) Minimum torque oscillation: 10 nN*m

[0087] (6) Maximum torque: 150 nN*m

[0088] (7) Torque resolution: 0.1 nN*m

[0089] (8) Measuring geometry: 25 mm plate

[0090] (9) Measuring Gap: 1.0 mm

[0091] In addition, for the syringe ability (injection force) of the manufactured cross-linked body, the injection force value was measured by analyzing it at a rate of 12 mm/min, after equipping a syringe comprising 1 ml of the cross-linked body in a syringe ability test machine.

[0092] Through Table 1, it could be seen that the complex viscosity varied depending on the amount of the peptide to be cross-linked. Since the complex viscosity increased as the amount of the peptide increased, it could be seen that the complex viscosity could be controlled by adjusting the peptide amount during cross-linking.

TABLE-US-00001 TABLE 1 Complex Cross-linked Elasticity Viscosity viscosity Injection sample (G′ ) (G″ ) Tan δ (mPa .Math. s) force (N) Example 1a 101 36 0.35 852,000 15 Example 1b 169 38 0.22 1,380,000 23 Example 1c 310 53 0.17 2,500,000 28 Example 1d 444 108 0.24 3,640,000 31

EXAMPLE 4

Evaluation of Decomposition Ability of Cross-Linked Body of Hyaluronic Acid-Peptide Manufactured by the Present Invention

[0093] In order to evaluate the decomposition ability of manufactured Example 2, the weight of 0.6 g of the hyaluronic acid-peptide cross-linked body in a micro 2 ml tube was measured, and then hyaluronidase solution (33 mg/mL) (MP biomedicals) of 6 μL was added in each tube. Samples of the mixture cross-linked body was collected at 37° C. by time, and the complex viscosity of the samples was confirmed with a rheometer equipment, thereby confirming the decomposition ability of the cross-linked body.

[0094] Through Table 2, the complex viscosity change over time was confirmed, thereby the decomposition ability of the cross-linked body. As a result of confirming the degree of decomposition in vitro, it could be confirmed that Example 1d had a viscosity of 3,640,000 at 0 hour, but had a viscosity of 1,740,000 after 4 hours, and therefore, it was decomposed about 52.2% after 4 hours.

TABLE-US-00002 TABLE 2 Cross-linked Complex viscosity of cross-linked body over time (mPa .Math. s) sample 0 hour 1 hours 2 hours 4 hours Example 1a 852,000 321,000 186,000 142,000 Example 1b 1,380,000 749,000 471,000 313,000 Example 1c 2,500,000 1,350,000 1190,000 678,000 Example 1d 3,640,000 2,680,000 2,330,000 1,740,000

EXAMPLE 5

Evaluation of Absorption Ability of Cross-Linked Body of Hyaluronic Acid-Peptide Manufactured by the Present Invention

[0095] In order to evaluate the absorption ability of manufactured Example 2, the hyaluronic acid-peptide cross-linked body (Example 1a-1d) 0.6 g and phosphate buffer solution 10 ml were added to a falcon 50 ml tube, and then stirred for 5 minutes. Such a mixture was left at 37° C. for 72 hours, and then Coomassie Brilliant Blue solution 100 μl was added to confirm the layer separation and color difference. Using the layer separation as a boundary, the volume of the hyaluronic acid-peptide complex hydrogel sinking in the lower layer was confirmed, and it was applied to Equation 1 below to confirm the absorption ability.

Absorption solvent amount=swelled volume÷weight of specimen (ml/g)   [Equation 1]

[0096] As can be confirmed in Table 3, general fillers had a swelled volume between 5˜8, but the swelled volume of the hyaluronic acid-peptide cross-linked body (cross-linked sample) according to the present invention showed a value between 2˜3. This means that the peptide filter has a characteristic that does not swell well.

TABLE-US-00003 TABLE 3 Cross-linked Swelled volume Test liquid Absorption solvent sample (ml) weight (g) amount (ml/g) Example 1a 3.0 0.6 5.00 Example 1b 3.0 0.6 5.00 Example 1c 2.5 0.6 4.17 Example 1d 2.1 0.6 3.50