Branched polyethylene glycol epoxy derivative crosslinked sodium hyaluronate gel, preparation and application thereof

Abstract

The present invention discloses a branched polyethylene glycol epoxy derivative crosslinked sodium hyaluronate gel, preparation and application thereof. The crosslinking agent used in the crosslinked sodium hyaluronate gel prepared by the present invention is a polyethylene glycol epoxy derivative, due to the existence of multiple ether bonds in the molecule of the crosslinking agent, there are more hydrogen bonds in the gel system; meanwhile, due to the particularity of the space structure of the branched polyethylene glycol epoxy derivative, the gel prepared has a more complex winding structure in its space, thus achieving better stability. Moreover, the branched polyethylene glycol epoxy derivative involved in the present invention is a compound with single molecular weight, therefore, the gel prepared thereby has better batch stability.

Claims

1. A branched polyethylene glycol epoxy derivative, having the following structure: ##STR00029## wherein, A is a core structure, which has the following structure: ##STR00030## wherein, B has the following structure: ##STR00031## r is an integer of 1-5, a, b, c and d are integers, independently selected from 0 and 1, s is an integer of 1-5, e, f and g are integers, independently selected from 0 and 1; X.sub.1 and X.sub.2 are linking groups, wherein X.sub.1 is —CH.sub.2CH.sub.2CONH—, and X.sub.2 is selected from any one or a combination of two or more of the group consisting of: —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CONH—, —CH.sub.2CH.sub.2CONH—, —CH.sub.2CONHCH.sub.2—, —CH.sub.2CONHCH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2NHCOCH.sub.2— and —CH.sub.2CH.sub.2NHCOCH.sub.2CH.sub.2—; PEG has the following structure: —(CH.sub.2CH.sub.2O).sub.m—, m is an integer of 4-200, n is an integer of 3-24, and the branched polyethylene glycol epoxy derivative is a compound with single molecular weight.

2. The derivative according to claim 1, wherein r is an integer of 1, 2 or 3, s is an integer of 1, 2 or 3.

3. The derivative according to claim 1, wherein the A is selected from the following structures: ##STR00032##

4. The derivative according to claim 1, wherein the m is an integer of 4-100.

5. The derivative according to claim 1, wherein the X.sub.2 is —CH.sub.2—.

6. The derivative according to claim 5, wherein the branched polyethylene glycol epoxy derivative has the following structure: ##STR00033## ##STR00034## the m is 4, 12 or 24.

7. A preparation method of the branched polyethylene glycol epoxy derivative according to claim 1, wherein the method comprises a step of catalyzing ##STR00035## and branched polyethylene glycol AX.sub.1-PEG-H).sub.n for reaction via a catalyst in solvent, wherein, A is a core structure, which has the following structure: ##STR00036## wherein, B has the following structure: ##STR00037## r is an integer of 1-5, a, b, c and d are integers, independently selected from 0 and 1, s is an integer of 1-5, e, f and g are integers, independently selected from 0 and 1, X.sub.1 and X.sub.2 are linking groups, wherein X.sub.1 is —CH.sub.2CH.sub.2CONH—, and X.sub.2 is selected from any one or a combination of two or more of the group consisting of: —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CONH—, —CH.sub.2CH.sub.2CONH—, —CH.sub.2CONHCH.sub.2—, —CH.sub.2CONHCH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2NHCOCH.sub.2— and —CH.sub.2CH.sub.2NHCOCH.sub.2CH.sub.2—; —X is a leaving group, PEG has the following structure: —(CH.sub.2CH.sub.2O).sub.m—, m is an integer of 4-200, n is an integer of 3-24, the catalyst is a base catalyst.

8. A crosslinking agent, wherein the crosslinking agent comprises the branched polyethylene glycol epoxy derivative according to claim 1.

9. A high-molecular polymer crosslinked by the branched polyethylene glycol epoxy derivative according to claim 1, wherein the molecular weight of the high-molecular polymer is 50,000-3000,000 Dalton.

10. The crosslinked high-molecular polymer according to claim 9, wherein the high-molecular polymer is a natural polymer and/or synthetic polymer; the polymer is selected from: one or more of chitin and chitin derivatives, chitosan and chitosan derivatives, carrageenan and carboxymethyl carrageenan, cellulose derivatives, starch and starch derivatives, sodium alginate, guar gum and carboxymethyl guar gum, collagen, hyaluronic acid and hyalurate; and/or, the synthetic polymer is selected from: one or more of polyvinylpyrrolidone, polyethylene glycol, polyethylene oxide, polyvinyl alcohol, polyvinyl acetate, polylactic acid, polyglycolic acid, polyacrylic acid, polyacrylamide, polytetrahydrofuran, polyepoxybutane, polyoxetane, polymaleic anhydride, poly (2-hydroxyethylmethacrylate), polypropylene glycol, polycaprolactone and derivatives thereof.

11. The crosslinked high-molecular polymer according to claim 9, wherein the crosslinked high-molecular polymer is crosslinked sodium hyaluronate.

12. A preparation method of the crosslinked high-molecular polymer according to claim 9, comprising a step of crosslinking the high-molecular polymer with the branched polyethylene glycol epoxy derivative according to claim 1 in alkaline condition.

13. The preparation method according to claim 12, wherein the crosslinked high-molecular polymer is crosslinked sodium hyaluronate.

14. A gel, wherein the gel comprises the crosslinked high-molecular polymer according to claim 9.

15. A method for preparing a product for medicine, cosmetic plastic surgery, cosmetics or health-care food, the method comprising the step of preparing the products using the crosslinked high-molecular polymer of claim 9.

16. A soft tissue filler, comprising the crosslinked high-molecular polymer of claim 9.

17. A soft tissue filler, comprising the gel of claim 14.

18. The preparation method according to claim 13, wherein molar ratio of the branched polyethylene glycol epoxy derivative according to any one of claims 1-6 to polymer units in hyaluronic acid is 0.01-1:1; and/or, the reaction temperature is 35-45° C.; and/or, molecular weight of the sodium hyaluronate is 50,000-3000,000 Dalton.

19. A gel according to claim 14, wherein the crosslinked high-molecular polymer is crosslinked sodium hyaluronate.

20. A method according to claim 15, wherein the product for cosmetic plastic surgery is a soft tissue filler; and/or, the product for medicine comprises a postoperative anti-blocking agent, drug carrier and other drugs preventing and/or treating diseases.

Description

DETAILED DESCRIPTION OF THE EMBODIMENTS

(1) The technical solution of the present invention will be described clearly and completely hereafter with reference to embodiments of the present invention, apparently, embodiments described herein are only a part of embodiments of the present invention, and are not all of embodiments thereof. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without any creative efforts are within the protection scope of the present invention.

Embodiment 1: Synthesis of 4-Armed Dodecaethylene Glycol Tetraglycidyl Ether (Ia)

(2) Synthesis of 4-armed dodecaethylene glycol tetraglycidyl ether with the following structure:

(3) ##STR00023##

(4) 4-armed dodecaethylene glycol (CCH.sub.2OCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2O.sub.12H|.sub.4, 0.1 mol), tetrahydrofuran (THF, 100 mL) and potassium hydroxide (0.8 mol) were added to a three-port bottle for agitation in water bath, and then epoxy chloropropane (ECH, 1.2 mol) was dropwise added to the reaction system for reaction at room temperature over night, where the reaction temperature was controlled within 35° C. At the end of the reaction, the reaction liquid was filtered, filter residue was washed by dichloromethane, then filtrate was collected to remove dichloromethane by rotary evaporation, thus obtaining a crude product. The crude product was separated by a silicagel column to obtain purified 4-armed dodecaethylene glycol tetraglycidyl ether.

(5) .sup.1H-NMR (DMSO-d.sub.6): 2.26-2.30 (m, 8H), 2.54-2.55 (m, 4H), 2.72-2.73 (m, 4H), 3.09-3.10 (m, 4H), 3.17-3.28 (m, 20H), 3.35-3.64 (m, 192H), 3.70-3.71 (m, 4H), 7.88-7.92 (t, 4H);

(6) MALDI-TOF (2780.3, M+Na).

Embodiment 2: Synthesis of 4-Armed PEG24 Tetraglycidyl Ether (Ib)

(7) Synthesis of 4-armed PEG24 tetraglycidyl ether with the following structure:

(8) ##STR00024##

(9) 4-armed PEG24 (CCH.sub.2OCH.sub.2CH.sub.2CONHCH.sub.2CH.sub.2O.sub.12H|.sub.4, 0.1 mol), tetrahydrofuran (THF, 100 mL) and potassium hydroxide (0.8 mol) were added to a three-port bottle for stirring in water bath, and then epoxy chloropropane (ECH, 1.2 mol) was dropwise added to the reaction system for reaction at room temperature over night, where the reaction temperature was controlled within 35° C. At the end of the reaction, the reaction liquid was filtered, filter residue was washed by dichloromethane, then filtrate was collected to remove dichloromethane by rotary evaporation, thus obtaining a crude product. The crude product was separated by a silicagel column to obtain purified 4-armed PEG24 tetraglycidyl ether.

(10) .sup.1H-NMR (DMSO-d.sub.6): 2.26-2.30 (m, 8H), 2.54-2.55 (m, 4H), 2.72-2.73 (m, 4H), 3.09-3.10 (m, 4H), 3.17-3.28 (m, 20H), 3.35-3.64 (m, 384H), 3.70-3.71 (m, 4H), 7.88-7.92 (t, 4H);

(11) MALDI-TOF (4665.9, M+Na).

Embodiment 3: Synthesis of 8-Armed Tetraethylene Glycol Octaglycidyl Ether (Ic)

(12) Synthesis of 8-armed tetraethylene glycol octaglycidyl ether with the following structure:

(13) ##STR00025##

(14) 8-armed tetra ethylene glycol

(15) ##STR00026##
0.1 mol), tetrahydrofuran (THF, 100 mL) and potassium hydroxide (1.6 mol) were added to a three-port bottle for stirring in water bath, and then epoxy chloropropane (ECH, 2.4 mol) was dropwise added to the reaction system for reaction at room temperature over night, where the reaction temperature was controlled within 35° C. At the end of the reaction, the reaction liquid was filtered, filter residue was washed by dichloromethane, then filtrate was collected to remove dichloromethane by rotary evaporation, thus obtaining a crude product. The crude product was separated by a silicagel column to obtain purified 8-armed tetraethylene glycol octaglycidyl ether.

(16) .sup.1H-NMR (DMSO-d.sub.6): 2.27-2.33 (m, 16H), 2.54-2.55 (m, 8H), 2.72-2.73 (m, 8H), 3.09-3.10 (m, 8H), 3.16-3.26 (m, 24H), 3.28-3.44 (m, 24H), 3.48-3.50 (m, 116H), 3.55-3.60 (m, 8H), 3.66-3.71 (m, 16H), 7.87-7.90 (t, 8H);

(17) MALDI-TOF (2880.8, M+Na).

Embodiment 4: Synthesis of 8-Armed Dodecaethylene Glycol Octaglycidyl Ether (Id)

(18) Synthesis of 8-armed dodecaethylene glycol octaglycidyl ether with the following structure:

(19) ##STR00027##

(20) 8-armed dodecaethylene glycol

(21) ##STR00028##
0.1 mol), tetrahydrofuran (THF, 100 mL) and potassium hydroxide (1.6 mol) were added to a three-port bottle for stirring in water bath, and then epoxy chloropropane (ECH, 2.4 mol) was dropwise added to the reaction system for reaction at room temperature over night, where the reaction temperature was controlled within 35° C. At the end of the reaction, the reaction liquid was filtered, filter residue was washed by dichloromethane, then filtrate was collected to remove dichloromethane by rotary evaporation, thus obtaining a crude product. The crude product was separated by a silicagel column to obtain purified 8-armed dodecaethylene glycol octaglycidyl ether.

(22) .sup.1H-NMR (DMSO-d.sub.6): 2.28-2.33 (m, 16H), 2.54-2.55 (m, 8H), 2.72-2.73 (m, 8H), 3.09-3.10 (m, 8H), 3.16-3.27 (m, 24H), 3.29-3.44 (m, 24H), 3.47-3.50 (m, 372H), 3.55-3.60 (m, 8H), 3.66-3.71 (m, 16H), 7.87-7.90 (t, 8H);

(23) MALDI-TOF (5698.4, M+Na).

Embodiment 5: 4-Armed Dodecaethylene Glycol Tetraglycidyl Ether Crosslinked Sodium Hyaluronate Gel (IIa)

(24) The crosslinking agent, 4-armed dodecaethylene glycol tetraglycidyl ether (0.2 mol, prepared by Embodiment 1) was dissolved into NaOH solution and added powdered sodium hyaluronate (1 mol polymer unit) for reaction with agitation at 40° C. to form crosslinked sodium hyaluronate gel; secondly, an appropriate amount of hydrochloric acid was added to the gel to adjust pH=7.0, then an appropriate amount of PBS buffer solution to swell the gel; afterwards, the gel was sieved by a standard pharmacopeia sieve to collect gel particles, finally, the gel was repeatedly dialyzed, sieved by the standard pharmacopeia sieve, filled and sterilized by steam to obtain the modified sodium hyaluronate gel for injection.

Embodiment 6: 4-Armed PEG24 Tetraglycidyl Ether Crosslinked Sodium Hyaluronate Gel (IIb)

(25) The crosslinking agent, 4-armed PEG24 tetraglycidyl ether (0.2 mol, prepared by Embodiment 2) was dissolved into NaOH solution and added powdered sodium hyaluronate (1 mol polymer unit) for reaction with agitation at 40° C. to form crosslinked sodium hyaluronate gel; secondly, an appropriate amount of hydrochloric acid was added to the gel to adjust pH=7.0, then an appropriate amount of PBS buffer solution to swell the gel; afterwards, the gel was sieved by a standard pharmacopeia sieve to collect gel particles, finally, the gel was repeatedly dialyzed, sieved by the standard pharmacopeia sieve, loaded and sterilized by steam to obtain the modified sodium hyaluronate gel for injection.

Embodiment 7: 8-Armed Tetraethylene Glycol Octaglycidyl Ether Crosslinked Sodium Hyaluronate Gel (IIc)

(26) The crosslinking agent, 8-armed tetraethylene glycol octaglycidyl ether (0.2 mol, prepared by Embodiment 3) was dissolved into NaOH solution and added powdered sodium hyaluronate (1 mol polymer unit) for reaction with agitation at 40° C. to form crosslinked sodium hyaluronate gel; secondly, an appropriate amount of hydrochloric acid was added to the gel to adjust pH=7.0, then an appropriate amount of PBS buffer solution to swell the gel; afterwards, the gel was sieved by a standard pharmacopeia sieve to collect gel particles, finally, the gel was repeatedly dialyzed, sieved by the standard pharmacopeia sieve, loaded and sterilized by steam to obtain the modified sodium hyaluronate gel for injection.

Embodiment 8: 8-Armed Dodecaethylene Glycol Octaglycidyl Ether Crosslinked Sodium Hyaluronate Gel (IId)

(27) The crosslinking agent, 8-armed dodecaethylene glycol octaglycidyl ether (0.2 mol, prepared by Embodiment 4) was dissolved into NaOH solution and added powdered sodium hyaluronate (1 mol polymer unit) for reaction with agitation at 40° C. to form crosslinked sodium hyaluronate gel; secondly, an appropriate amount of hydrochloric acid was added to the gel to adjust pH=7.0, then an appropriate amount of PBS buffer solution to swell the gel; afterwards, the gel was sieved by a standard pharmacopeia sieve to collect gel particles, finally, the gel was repeatedly dialyzed, sieved by the standard pharmacopeia sieve, loaded and sterilized by steam to obtain the modified sodium hyaluronate gel for injection.

Embodiment 9: In-Vitro Stability Test

(28) The crosslinked sodium hyaluronate gel was degraded by hyaluronidase solution to test the in-vitro stability of the gel.

(29) Test Method:

(30) 0.5 g crosslinked sodium hyaluronate gel was taken and added to 2 mL 300 U/ml hyaluronidase solution for degradation at 37° C. for 40 h, PBS was added to 5 mL, 1 ml solution was taken, added 4 mL absolute ethyl alcohol and centrifuged for 15 min at 10000 r/min, 2 mL supernatant was taken and adjusted to the volume of 5 mL with PBS, to produce solution A; another 0.5 g crosslinked sodium hyaluronate gel was taken and added 10 mL 0.5 mol/L sulfuric acid solution, hydrolyzed for 15 min in boiling water bath, the solution was added water to 100 mL, to produce solution B. 1 mL solution A and 1 mL B was respectively taken to test the content of glucuronic acid by modified carbazole developing process. In-vitro enzymatic degradation resistance of the gel is denoted by coefficient R, R=1-0.625A/B, in which, A is the content of glucuronic acid in solution A, and B is the content of glucuronic acid in solution B. The higher the R, the better the in-vitro enzymatic degradation resistance is, and the more stable is the crosslinked gel. Test results were as shown in table 1.

(31) TABLE-US-00001 TABLE 1 Test results of in-vitro enzymolysis stability of the crosslinked sodium hyaluronate gel Test group IIa IIb IIc IId BDDE-HA Coefficient of 91% 88% 81% 89% 71% enzymatic degradation resistance

(32) Test results show that compared with conventional crosslinking agents, e.g., BDDE crosslinked sodium hyaluronate gel, the branched polyethylene glycol epoxy derivative crosslinked sodium hyaluronate gel has better in-vitro stability.

(33) What is described above are merely preferred embodiments of the present invention, and are not to limit the present invention; any modification and equivalent replacement, etc. within the spirit and principle of the present invention shall be covered in the protection scope of the present invention.