HYDROGEL BASED ON γ-POLYGLUTAMIC ACID AND ε-POLYLYSINE CROSSLINKED POLYMER, AND PREPARATION METHOD THEREFOR

Abstract

A hydrogel based on a cross-linked -polyglutamic acid and -polylysine polymer is obtained by cross-linking of -polyglutamic acid with -polylysine, and it is a polymer having the following constitutional unit, wherein, m is a natural number of 15 to 45, n is a natural number of 3900 to 17000, and x is a natural number of 5 to 40. It also discloses a preparation method of as-described hydrogel and its application in preparation as a medical wound dressing.

Claims

1. A hydrogel based on a cross-linked polymer of -polyglutamic acid and -polylysine, characterized in that, it is obtained by cross-linking of the -polyglutamic acid with the -polylysine, and it is a polymer having the following constitutional unit: ##STR00002## wherein, m is a natural number of 15 to 45, n is a natural number of 3900 to 17000, and x is a natural number of 5 to 40.

2. The hydrogel according to claim 1, characterized in that, the -polyglutamic acid and -polylysine are obtained by microbial fermentation, respectively.

3. The hydrogel according to claim 1 or 2, characterized in that, molecular weight of the -polyglutamic acid is 500 thousand to 2.2 million Daltons, molecular weight of the -polylysine is 2000 to 5500 Daltons.

4. A process for preparing a hydrogel with a -polyglutamic acid and a -polylysine, characterized in that, it comprises the following steps: (1) adding dropwisely a 2-(N-morpholino)ethanesulfonic acid buffer containing the -polylysine into a 2-(N-morpholino)ethanesulfonic acid buffer containing the -polyglutamic acid, and stirring and mixing homogeneously; (2) adding a cross-linking agent into the mixture obtained in step (1), reacting in an ice bath for 10 to 120 min, then reacting for 2 to 24 hours at room temperature to form said hydrogel; (3) placing the hydrogel formed in step (2) into a dialysis bag, and dialyzing in deionized water until swelling equilibrium, then adopting freeze drying or vacuum drying, to obtain a sponge-like dressing.

5. The process according to claim 4, characterized in that, in step (1), the -polyglutamic acid and -polylysine are obtained by microbial fermentation, respectively.

6. The process according to claim 4, characterized in that, in step (1), molecular weight of the -polyglutamic acid is 500 thousand to 2.2 million Daltons, molecular weight of the -polylysine is 2000 to 5500 Daltons.

7. The process according to claim 4, characterized in that, in step (1), the MES buffer is of 0.1 mol/L and pH 5.0.

8. The process according to claim 4, characterized in that, in step (1), the MES buffer containing the -polylysine is a homogeneous solution, wherein concentration of the -polylysine is 20 g/L to 160 g/L; the MES buffer containing -polyglutamic acid is a homogeneous solution, wherein mass percentage content of the -polyglutamic acid is 40 g/L to 200 g/L.

9. The process according to claim 4, characterized in that, in step (2), the cross-linking agent is selected from a group consisting of a combination of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, a combination of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysulfosuccinimide, 1-cyclohexyl-2-morpholinoethylcarbodiimide-p-toluenesulfonate and Woodward's Reagent K.

10. The process according to claim 9, characterized in that, in steps (2), the cross-linking agent is a combination of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, wherein the mole ratio of carboxyl groups included in the -polyglutamic acid:amino groups included in the -polylysine:1-(3-dimethylaminopropyl)-3-ethylcarbodiimide:N-hydroxysuccinimide is 1:0.25 to 0.5:0.25 to 1:0.25 to 1.

11. The process according to claim 9, characterized in that, in step (2), the cross-linking agent is a combination of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysulfosuccinimide, wherein the mole ratio of carboxyl groups included in the -polyglutamic acid:amino groups included in the -polylysine:1-(3-dimethylaminopropyl)-3-ethylcarbodiimide:N-hydroxysulfosuccinimide is 1:0.25 to 0.5:0.25 to 1:0.25 to 1.

12. The process according to claim 9, characterized in that, in step (2), the cross-linking agent is 1-cyclohexyl-2-morpholinoethylcarbodiimide-p-toluenesulfonate, wherein the mole ratio of carboxyl groups included in the -polyglutamic acid:amino groups included in the -polylysine:1-cyclohexyl-2-morpholinoethylcarbodiimide-p-toluenesulfonatebeis is 1:0.25 to 0.5:0.25 to 1.

13. The process according to claim 9, characterized in that, in step (2), the cross-linking agent is Woodward's Reagent K, wherein the mole ratio of carboxyl groups included in the -polyglutamic acid:amino groups included in the -polylysine:Woodward's Reagent K is 1:0.25 to 0.5:0.25 to 1.

14. The process according to claim 4, characterized in that, grinding and crushing the sponge-like dressing, and split charging with an aluminum composite film, a xerogel powder is yielded.

15. The process according to claim 4, characterized in that, adding 1 to 10 fold weight of water to the sponge-like dressing to make a soft material, split charging in a polyethylene tube, sealing and packing, the hydrogel is yielded.

16. The process according to claim 4, characterized in that, adding 1 to 5 fold weight of water to the sponge-like dressing to prepare a soft material, pressing into a film-coated tablet and placing onto a polyethylene film, drying by an airflow of 70 to 90.0 making its water content being 20 to 60 wt %, and laminating a polyethylene breathable film, after cutting sealing with an aluminum composite film, thereby a gel film is made.

17. A hydrogel is prepared by the process of claim 4.

18. A process for preparing a medical wound dressing uses the hydrogel of claim 1 or claim 17.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 is a reaction principle diagram of the present invention.

[0037] FIG. 2 is a nuclear magnetic resonance spectroscopy (.sup.1H-NMR) of a hydrogel based on -polyglutamic acid and -polylysine cross-linked polymer.

[0038] FIG. 3 is an infrared spectrum (FTIR) of the hydrogel based on -polyglutamic acid and -polylysine cross-linked polymer in Example 1. (a) is -polylysine, (b) is -polyglutamic acid, (c) is -polylysine--polyglutamic acid hydrogel.

[0039] FIG. 4 is an electron microscope image (SEM) of the hydrogel based on -polyglutamic acid and -polylysine cross-linked polymer (Example 1).

[0040] FIG. 5 is a situation of detecting cytocompatibility by a confocal laser scanning microscope (CLSM), the cells grow on the hydrogel scaffold, the living cells are green, and the dead cells are red. a) growth of fibroblasts on the polyglutamic acid hydrogel scaffold; b) growth of fibroblasts on the -polyglutamic acid and -polylysine cross-linked polymer hydrogel scaffold.

[0041] FIG. 6 is back wound healing experiment of domestic rabbit, a) in a control group the back is covered only with a gauze then bandaged; b) a group treated using polyglutamic acid hydrogel dressing; c) a group treated using a -polyglutamic acid and -polylysine cross-linked polymer hydrogel dressing.

DESCRIPTION OF THE EMBODIMENTS

[0042] According to the following examples, the present invention can be better understood. However, a person skilled in the art will easily understood that, the contents described in the examples are only used to illustrate the present invention, and should not and will not restrain the present invention described in detail in the claims.

[0043] The resources of reagents used in the following examples are as follows:

-polyglutamic acid and -polylysine: purchased from Nanjing Shineking Biological Technology Co., Ltd.;
MES (2-(N-morpholino)ethanesulfonic acid), EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide), NHS (N-hydroxysuccinimide) and Sulfo-NHS (N-hydroxysulfosuccinimide): purchased from Sinopharm Chemical Reagent Co., Ltd;
CMC (1-cyclohexyl-2-morpholinoethylcarbodiimide p-toluenesulfonate) and (2-ethyl-5-phenylisoxazolium-3-sulfonate): purchased from Sigma-Aldrich Company.

[0044] The resources of equipments used in the following examples are as follows:

Magnetic stirrer: Type 85-2C, Shanghai Niuhang Instrument and Equipment Co., Ltd.
Freeze dryer: Type FD-1C-50, Beijing Boyikang Experimental Instrument Co., Ltd.
Vacuum drying box: Type YZG-600, Nanjing Yantai Electrical Heating Equipment Co., Ltd.
Infrared spectrometer: Type Nicolet 380, Thermo Company, USA.
NMR spectrometer: Type AVANCE AV-500, Bruker Daltonics Company, USA.

Example 1

[0045] At room temperature 4.0 g -polyglutamic acid (1 million to 1.2 million Daltons, including 0.031 mole of carboxyl groups) was dissolved in 50 ml of 0.1 mol/L MES buffer (pH 5.0), and stirred until a clear solution was formed. At room temperature 1.78 g -polylysine (3000 to 4500 Daltons, including 0.014 mole of amino groups) was dissolved in 50 ml of 0.1 mol/L MES buffer (pH 5.0), and a -polylysine solution was added dropwisely into the polyglutamic acid solution, stirred and making the solution mixed homogenously. 4.17 g (0.0217 mol) EDC and 2.50 g (0.0217 mol) NHS were added into the above-described mixed solution of -polyglutamic acid and -polylysine, and the feeding mole ratio of carboxyl groups included in the -polyglutamic acid:amino group included in the -polylysine:EDC:NHS was controlled to 1:0.45:0.7:0.7, and reacted in an ice bath for 30 minutes, then reacted at room temperature for 2 hours to form a hydrogel. The hydrogel formed was placed into a dialysis bag, and dialyzed in deionized water until swelling equilibrium, then freeze drying or vacuum drying was adopted to obtain a sponge-like dressing, swelling rate of the hydrogel formed was 96.6 g/g. Its structure identification is seen in FIG. 2, in the HNMR spectra of -polyglutamic acid and -polylysine cross-linked polymer hydrogel, it can be seen that chemical binding of -polyglutamic acid with -polylysine d forms a cross-linked polymer. The infrared spectrum in FIG. 3 also demonstrate forming of -polyglutamic acid and -polylysine cross-linked polymer hydrogel. Because the -polylysine has many free amino groups, characteristic peaks at 1546 cm.sup.1 IA 1113 cm.sup.1 are obvious, after formation of the hydrogel by cross-linking, amino groups of the -polylysine reacted with carboxyl groups in the -polyglutamic acid to form amide bond, the number of free amino groups during formation of the polymer was greatly reduced, thus the characteristic peaks at these two sites are no longer obvious. Furthermore, one broad peak appearing at site of 3500 to 3300 cm.sup.1 is also a characteristic absorption peak of the hydrogel, it is mainly induced by stretching vibration of the hydroxyl group and NH stretching vibration on the amide bond. SEM in FIG. 4 shows a surface morphology of the hydrogel prepared, indicating that the hydrogel prepared by the present invention is a three-dimensional porous structure, and the pore size is between 100 and 200 m, it is suitable to be used as a wound dressing.

Example 2

[0046] At room temperature 5.0 g -polyglutamic acid (1 million to 1.2 million Daltons, including 0.039 mol of carboxyl groups) was dissolved in 50 mL of 0.1 mol/L MES buffer (pH=5.0), and stirred until a clear solution was formed. At room temperature 1.99 g -polylysine (3000 to 4500 Daltons, including 0.0156 mole of amino groups) was dissolved in 50 mL 0.1 mol/L MES buffer (pH 5.0), a -polylysine solution was added dropwisely into the polyglutamic acid solution, stirred and made the solution mixed homogenously. 4.49 g (0.023 mol) EDC and 5.08 g (0.023 mol) sulfo-NHS were added into the above-described mixed solution of -polyglutamic acid and -polylysine, the feeding mole ratio of carboxyl groups included in the -polyglutamic acid:amino groups included in the -polylysine:EDC:Sulfo-NHS was controlled to 1:0.4:0.6:0.6, and reacted in an ice bath for 30 minutes, then reacted at room temperature for 2 hours to form a hydrogel. The formed hydrogel was placed in a dialysis bag, and dialyzed in deionized water until swelling equilibrium, then freeze drying or vacuum drying was adopted to obtain a sponge-like dressing, the expansion rate of the hydrogel obtained was 73.8 g/g.

Example 3

[0047] At room temperature 6.0 g -polyglutamic acid (1 million to 1.2 million Daltons, including 0.047 mol of carboxyl groups) was dissolved in 50 mL of 0.1 mol/L MES buffer (pH=5.0), and stirred until a clear solution was formed. At room temperature 3.0 g -polylysine (3000 to 4500 Daltons, including 0.0235 mol of amino groups) was dissolved in 50 mL 0.1 mol/L MES buffer (pH=5.0), a -polylysine solution was added dropwisely into the polyglutamic acid solution, and stirred and made the solution mixed homogenously. 7.95 g (0.0188 mol) CMC was added into the above-described mixed solution of -polyglutamic acid and -polylysine, and feeding mole ratio of carboxyl groups included in the -polyglutamic acid:amino groups included in the -polylysine:CMC was controlled to 1:0.5:0.4, and reacted in an ice bath for 30 minutes, then reacted at room temperature for 2 hours to form a hydrogel. The hydrogel formed was placed in a dialysis bag, dialyzed in a deionized water until swelling equilibrium, then freeze drying or vacuum drying was adopted to obtain a sponge-like dressing, the expansion rate of the hydrogel obtained was 48.4 g/g.

Example 4

[0048] At room temperature 4.5 g -polyglutamic acid (1 million to 1.2 million Daltons, including 0.035 mol of carboxyl groups) dissolved in a 50 ml of 0.1 mol/L MES buffer (pH 5.0), and stirred to form a clear solution. At room temperature 2.24 g -polylysine (3000 to 4500 Daltons, including 0.0175 mol of amino groups) was dissolved in a 50 mL 0.1 mol/L MES buffer (pH 5.0), a -polylysine solution was added dropwisely into the polyglutamic acid solution, stirred and made the solution mixed homogenously. 7.08 g (0.028 mol) Woodward's Reagent K was added into the above-described mixed solution of -polyglutamic acid and -polylysine, and the feeding mole ratio the carboxyl groups included in the -polyglutamic acid:amino groups included in the -polylysine:Woodward's Reagent K was 1:0.5:0.8, and reacted in an ice bath for 30 minutes, then reacted at room temperature for 2 hours to form a hydrogel. The hydrogel formed was placed into a dialysis bag, and dialyzed in a deionized water until swelling equilibrium, then freeze drying or vacuum drying was adopted to obtain a sponge-like dressing, the expansion rate of the hydrogelo obtained was 52.7 g/g.

Example 5

[0049] The method is the same as Example 1, the difference is controlling the feeding mole ratio of carboxyl groups included in the -polyglutamic acid:amino groups included in the -polylysine:EDC:NHS to 1:0.25:0.25:0.25, the expansion rate of the hydrogel obtained was 12.5 g/g.

Example 6

[0050] The method is the same as Example 1, the difference is controlling the feeding mole ratio of carboxyl groups included in the -polyglutamic acid:amino groups included in the -polylysine:EDC:NHS to 1:0.5:1:1, the expansion rate of the hydrogel obtained was 38.6 g/g.

Example 7

[0051] The method is the same as Example 2, the difference is controlling the feeding mole ratio of carboxyl groups in the -polyglutamic acid:amino groups included in the -polylysine:EDC:Sulfo-NHS to 1:0.25:0.25:0.25, the expansion rate of the hydrogel obtained was 30.2 g/g.

Example 8

[0052] The method is the same as Example 2, the difference is controlling the feeding mole ratio of carboxyl groups included in the -polyglutamic acid:amino groups included in the -polylysine:EDC:Sulfo-NHS to 1:0.5:1:1, the expansion rate of the hydrogel obtained was 42.3 g/g.

Example 9

[0053] The method is the same as Example 3, the difference is controlling the feeding mole ratio of carboxyl groups in the -polyglutamic acid:amino groups included in the -polylysine:CMC to 1:0.25:0.25, the expansion rate of the hydrogel obtained was 33.7 g/g.

Example 10

[0054] The method is the same as Example 3, the difference is controlling the feeding mole ratio of carboxyl groups included in the -polyglutamic acid:amino groups included in the -polylysine:CMC to 1:0.5:1, the expansion rate of the hydrogel obtained was 39.4 g/g.

Example 11

[0055] The method is the same as Example 4, the difference is controlling the feeding mole ratio of carboxyl groups included in the -polyglutamic acid:amino groups included in the -polylysine:Woodward's Reagent K to 1:0.25:0.25, the expansion rate of the hydrogel obtained was 36.6 g/g.

Example 12

[0056] The method is the same as Example 4, the difference is controlling the feeding mole ratio of carboxyl groups included in the -polyglutamic acid:amino groups included in the -polylysine:Woodward's Reagent K to 1:0.5:1, the expansion rate of the hydrogel obtained was 43.4 g/g.

Example 13

[0057] The method is the same as Example 4, the difference is reacting in an ice bath for 10 minutes, and reacted at room temperature for 5 hours to form a hydrogel, the expansion rate of the hydrogel obtained was 35.8 g/g.

Example 14

[0058] The method is the same as Example 4, the difference is reacting in an ice bath for 120 minutes, and reacted at room temperature for 24 hours to form a hydrogel, the expansion rate of the hydrogel obtained was 26.9 g/g.

Comparative Example 1

[0059] At room temperature 4.0 g -polyglutamic acid (1 million to 1.2 million Daltons, including 0.031 mole of carboxyl groups) were dissolved in 50 mL of 0.1 mol/L MES buffer (pH=5.0), and stirred until a clear solution was formed. 4.17 g (0.0217 mol) EDC and 2.50 g (0.0217 mol) NHS were added into the -polyglutamic acid solution, and the feeding mole ratio of carboxyl groups included in the -polyglutamic acid:EDC:NHS was controlled to 1:0.7:0.7, and reacted in an ice bath for 30 minutes, then reacted at room temperature for 6 hours to form a hydrogel. The formed hydrogel was placed into a dialysis bag, and dialyzed in deionized water until swelling equilibrium, then freeze drying or vacuum drying was adoped to obtain a sponge-like dressing, the expansion rate of the hydrogel obtained was 3.4 g/g.

Comparative Example 2

[0060] At room temperature, 1.78 g -polylysine (3000 to 4500 Daltons, including 0.014 mol of amino groups) was dissolved in 50 mL of 0.1 mol/L MES buffer (pH 5.0), stirred until a clear solution was formed. 4.16 g EDC (0.0217 mmol) and 2.50 g NHS (0.0217 mmol) were added into the -polylysine solution, the feeding mole ratio of amino groups included in the -polylysine:EDC:NHS was controlled to 0.45:0.7:0.7. Reacted in an ice bath for 30 minutes, then reacted at room temperature for 9 hours to form a hydrogel. The hydrogel formed was placed into a dialysis bag, and dialyzed in a deionized water until swelling equilibrium, then freeze drying or vacuum drying was adopted to obtain a sponge-like dressing, the expansion rate of the hydrogel obtained was 2.8 g/g.

Comparative Example 3

[0061] At room temperature 4.0 g -polyglutamic acid (1 milliontol 2 million Daltons, including 0.031 mol of carboxyl groups) was dissolved in 50 mL of 0.1 mol/L MES buffer (pH 5.0), and stirred until a clear solution was formed. At room temperature 1.78 g -polylysine (3000 to 4500 Daltons, including 0.014 mol of amino groups) was dissolved in 50 mL 0.1 mol/L MES buffer (pH 5.0), a -polylysine solution was added dropwisely into the polyglutamic acid solution, the feeding mole ratio of carboxyl groups included in the -polyglutamic acid:amino groups included in the -polylysine was controlled to 1:0.45, stirred and making the solution mixed homogenously, but the hydrogel cannot be formed.

Example 15

[0062] The sponge-like dressing in Example 1 to 4 was grinded and crushed, and split charged with an aluminum composite film, to prepare a xerogel powder.

Example 16

[0063] 5 g of the sponge-like dressing in Example 1 to 4 was weighed, and 30 g water was added to make a soft material, and split charged in a polyethylene tube, sealed to prepare a hydrogel.

Example 17

[0064] 5 g of the sponge-like dressing in Example 1 to 4 was weighed, 20 g of water was added to make a soft material, pressed into a film-coated tablet and placed onto a polyethylene film, and dried by a airflow of 80 C., making its water content being 40 wt %, and a polyethylene breathable film was laminated, sealed with an aluminum composite film after cutting, to prepare a gel film.

Experiment Example 18

Cytocompatibility Experiment

[0065] 3 fold weight of dressing of water was added to the sponge-like dressing in Example 1 to make a soft material hydrogel, and fibroblast was inoculate onto the surface of hydrogel at a concentration of 510.sup.4/cm.sup.2, and cultured at 37 C. for 6 hours. The cells were stained by a LIVE/DEAD fluorescent reagent, the living cells were stained with green fluorescent material (calcein-AM), and the dead cells were stained with red fluorescent material (EthD-I). Then, the cell survival was observed by a confocal laser scanning microscope (CLSM). See FIG. 5, red fluorescent material in a) are obviously higher than in b), indicating that a considerable amount of cells among the cells on the -polyglutamic acid hydrogels caffold are dead, while most of the cells on the -polyglutamic acid and -polylysine cross-linked polymer hydrogel scaffold are living, this indicated a good biocompatibility of the hydrogel of the present invention.

Experiment Example 19

Wound Healing Experiment

[0066] After the back of domestic rabbit was sheared, and unhaired with a sodium sulfide solution for 48 hours, each domestic rabbit was subcutaneously injected 0.5 mL of 0.5% lidocaine injection for local anesthesia, totally at 3 sites, at the local anesthesia site circular skin incisions of about 1.0 cm diameter were sheared at three sites with surgical scissors, disinfected with a 70 v/v % ethanol, The wound dressing in Example 1 was coated at the wound, and covered with a wax degreased cotton gauze, and bandaged, in the control groups one group was covered only with a gauze then bandaged, the another group was coated with a -polyglutamic acid hydrogel dressing. During experiment each group had no bacteria infection, and the wound healing condition were observed respectively after 0, 7, 14 days. See FIG. 6, with extension of treatment time, in all three groups the wound of the domestic rabbits were all healed, the hydrogel dressing treated groups were all superior to the group treated only with the gauze; in the group treated by using -polyglutamic acid and -polylysine cross-linked polymer hydrogel dressing, the wound area was only 40% of that of control group of -polyglutamic acid hydrogel, the wound was obviously decreased, and the surface was smooth and flat, showing a good biocompatibility and ability of promoting wound healing.

[0067] Comprehensive evaluation: the -polyglutamic acid/-polylysine hydrogel wound dressing of the present invention has a good biocompatibility, and contributes to cell adhesion and growth, it has a promotion effect to wound healing, and can effectively reduce leakage of tissue fluid, having an extensive application prospect in the medical wound dressing field.