Drug-loaded polyglutamic acid-based hydrogel and preparation method thereof

Abstract

The present invention provides a drug-loaded polyglutamic acid-based hydrogel and a preparation method thereof. The preparation method of the drug-loaded polyglutamic acid-based hydrogel provided by the present invention comprises the following steps: a) dissolving polyglutamic acid in water to obtain a polyglutamic acid solution; b) dissolving colcemid powder in an organic solvent to obtain a colcemid solution; c) dissolving polylysine in water to obtain a polylysine solution; d) mixing the polyglutamic acid solution, the colcemid solution and the polylysine solution, and standing to obtain a drug-loaded polyglutamic acid-based hydrogel; wherein, step a), step b) and step c) have no order restrictions. The drug-loaded polyglutamic acid-based hydrogel provided by the present invention can effectively improve the compressive strength and water retention rate of the material, and produce good antibacterial and anticancer abilities.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a drug-loaded polyglutamic acid-based hydrogel and a preparation method thereof. Background Art

[0002] Polyglutamic acid (γ-PGA) is a polyamino acid material obtained by microbial fermentation in nature. It is composed of glutamic acid units connected by peptide bonds via α-amino and γ-carboxyl groups. However, due to its high water absorption and water solubility, polyglutamic acid will dissolve immediately when it comes into contact with water. Even if it is placed in the air, it will quickly absorb moisture in the air and dissolve. Therefore, the scope of use of untreated polyglutamic acid is greatly limited.

[0003] The patent with publication number CN104606680 discloses a preparation method of a gamma-polyglutamic acid hydrogel loaded with drugs. It mainly uses polyglutamic acid as raw material, and one of erythromycin, penicillin, amoxicillin or azithromycin is used as the loaded drug, and the hydrogel is obtained by crosslinking with glutaraldehyde steam. The advantage of this invention is that the hydrogel that can be used for medical dressings with anti-inflammatory and biocompatibility is achieved by loading drugs such as erythromycin, and the mechanical strength of the hydrogel is improved by crosslinking with glutaraldehyde steam. However, the drugs such as erythromycin in the above method all have defects such as narrow antimicrobial spectrum, easy to produce drug resistance and adverse reactions, and the hydrogel after crosslinking with glutaraldehyde steam has certain toxicity, which not only increases the risk of the safety of the hydrogel, but also has cumbersome operation and too single functionalization, and in addition, the improvement effect of its mechanical strength and water retention is not good enough. Summary of the invention

[0004] In view of this, the present invention provides a drug-loaded polyglutamic acid-based hydrogel and a preparation method thereof. The drug-loaded polyglutamic acid-based hydrogel provided by the present invention can effectively improve the compression strength and water retention rate of the material, and produce good antibacterial and anticancer properties.

[0005] The present invention provides a method for preparing a drug-loaded polyglutamic acid-based hydrogel, comprising the following steps:

[0006] a) dissolving polyglutamic acid in water to obtain a polyglutamic acid solution;

[0007] b) dissolving colcemid powder in an organic solvent to obtain a colcemid solution;

[0008] c) dissolving polylysine in water to obtain a polylysine solution;

[0009] d) mixing the polyglutamic acid solution, the colcemid solution and the polylysine solution, and allowing the mixture to stand to obtain a drug-loaded polyglutamic acid-based hydrogel;

[0010] There is no order restriction for step a), step b) and step c).

[0011] Preferably, in step a), the weight average molecular weight of the polyglutamic acid is 700 to 2000 kDa.

[0012] Preferably, in step c), the weight average molecular weight of the polylysine is 2 to 5 kDa.

[0013] Preferably, the mass percentage concentration of polyglutamic acid in the drug-loaded polyglutamic acid-based hydrogel is 2% to 3%;

[0014] The mass percentage concentration of colchicine in the drug-loaded polyglutamic acid-based hydrogel is 1% to 3%;

[0015] The mass percentage concentration of polylysine in the drug-loaded polyglutamic acid-based hydrogel is 4% to 8%.

[0016] Preferably, in step d), the mass ratio of polyglutamic acid in the polyglutamic acid solution: colcemid in the colcemid solution: polylysine in the polylysine solution is (1-2):1:4.

[0017] Preferably, in step d), the mass ratio of polyglutamic acid in the polyglutamic acid solution: colcemid in the colcemid solution: polylysine in the polylysine solution is 2:1:4.

[0018] Preferably, in step c), the standing temperature is 20-25° C. and the standing time is 6-8 hours.

[0019] Preferably, in step a), the mass ratio of polyglutamic acid to water is 1:(10-15);

[0020] In step b), the mass ratio of colchicine powder to organic solvent is 1:(5-8);

[0021] In step c), the mass ratio of polylysine to water is 2:(10-15).

[0022] Preferably, in step b), the organic solvent is at least one of ethanol, ether and chloroform;

[0023] Step d) specifically comprises:

[0024] d1) mixing the polyglutamic acid solution with the colcemid solution to obtain a mixed solution;

[0025] d2) mixing the mixed solution with a polylysine solution and allowing the mixture to stand to obtain a drug-loaded polyglutamic acid-based hydrogel.

[0026] The present invention also provides a drug-loaded polyglutamic acid-based hydrogel, which is prepared by the preparation method described in the above technical solution.

[0027] Experimental results show that the drug-loaded polyglutamic acid-based hydrogel provided by the present invention has a compression strength of more than 80 MPa and a water retention rate of more than 94%, showing excellent compression strength and water retention; the antibacterial rate against Escherichia coli reaches more than 84%, and the antibacterial rate against Staphylococcus epidermidis reaches more than 80%, showing excellent antibacterial properties; the 1-day anti-cancer rate reaches more than 79%, and the 3-day anti-cancer rate reaches more than 85%, showing excellent anti-skin cancer ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0029] Figure 1 This is a graph showing the relationship between the storage modulus and loss modulus of the hydrogel product obtained in Example 1 and the frequency. DETAILED DESCRIPTION

[0030] The present invention provides a method for preparing a drug-loaded polyglutamic acid-based hydrogel, comprising the following steps:

[0031] a) dissolving polyglutamic acid in water to obtain a polyglutamic acid solution;

[0032] b) dissolving colcemid powder in an organic solvent to obtain a colcemid solution;

[0033] c) dissolving polylysine in water to obtain a polylysine solution;

[0034] d) mixing the polyglutamic acid solution, the colcemid solution and the polylysine solution, and allowing the mixture to stand to obtain a drug-loaded polyglutamic acid-based hydrogel;

[0035] There is no order restriction for step a), step b) and step c).

[0036] In the present invention, the term "kDa" refers to 1000 Da; the term "Da" refers to the unit of weight average molecular weight.

[0037] Regarding step a):

[0038] In the present invention, the polyglutamic acid is γ-polyglutamic acid (i.e., γ-PGA), and its weight average molecular weight is preferably 700-2000kDa, specifically 700kDa, 800kDa, 900kDa, 1000kDa, 1100kDa, 1200kDa, 1300kDa, 1400kDa, 1500kDa, 1600kDa, 1700kDa, 1800kDa, 1900kDa, 2000kDa; if the molecular weight is too low, the biodegradability of the hydrogel product is reduced, and if the molecular weight is too high, the solubility of polyglutamic acid deteriorates, affecting the preparation and product performance. The present invention is controlled at the above molecular weight to ensure the compressive strength and water retention rate of the product, and to ensure the antibacterial and anti-skin cancer ability of the product by better matching with other components. In the present invention, the source of the polyglutamic acid is a commercial product available on the market, specifically a commercial product available on the market obtained by microbial fermentation, such as commercial products available on the market from Nanjing Xuankai Biotechnology Co., Ltd.

[0039] In the present invention, the water is preferably purified water.

[0040] In the present invention, the mass ratio of polyglutamic acid: water is preferably 1:(10-15), specifically 1:10, 1:11, 1:12, 1:13, 1:14, 1:15.

[0041] In the present invention, there is no particular restriction on the method of dissolving polyglutamic acid in water, and the material can be mixed, such as stirring. In the present invention, there is no particular restriction on the operating temperature of dissolving polyglutamic acid in water, and it can be carried out at room temperature. After the above treatment, a polyglutamic acid solution is obtained.

[0042] Regarding step b):

[0043] In the present invention, the source of the colcemid powder is not particularly limited and can be a commercial product.

[0044] In the present invention, the organic solvent is preferably at least one of ethanol, ether and chloroform, and more preferably ethanol. When ethanol is used, toxicity can be reduced and anesthesia is not easily caused.

[0045] In the present invention, the mass ratio of colcemid powder: organic solvent is preferably 1:(5-8), specifically 1:5, 1:6, 1:7, 1:8.

[0046] In the present invention, there is no particular restriction on the method of dissolving colcemid powder in an organic solvent, and the materials can be mixed, such as stirring. In the present invention, there is no particular restriction on the operating temperature of dissolving colcemid powder in an organic solvent, and it can be carried out at room temperature. After the above treatment, a colcemid solution is obtained.

[0047] Regarding step c):

[0048] In the present invention, the weight average molecular weight of the polylysine is preferably 2 to 5 kDa, specifically 2 kDa, 3 kDa, 4 kDa, and 5 kDa; if the molecular weight is too low, the antibacterial activity is lost. The present invention has no particular limitation on the source of the polylysine, which can be a commercial product or prepared by conventional methods known to those skilled in the art.

[0049] In the present invention, the water is preferably purified water.

[0050] In the present invention, the mass ratio of polylysine: water is preferably 2: (10-15), specifically 2:10, 2:11, 2:12, 2:13, 2:14, 2:15.

[0051] In the present invention, there is no particular restriction on the method of dissolving polylysine in water, and the material can be mixed, such as stirring. In the present invention, there is no particular restriction on the operating temperature of dissolving polylysine in water, and it can be carried out at room temperature. After the above treatment, a polylysine solution is obtained.

[0052] There is no order restriction for the above step a), step b) and step c) in the present invention.

[0053] Regarding step d):

[0054] In the present invention, the step of mixing in step d) preferably specifically comprises:

[0055] d1) mixing the polyglutamic acid solution with the colcemid solution to obtain a mixed solution;

[0056] d2) mixing the mixed solution with a polylysine solution and allowing the mixture to stand to obtain a drug-loaded polyglutamic acid-based hydrogel.

[0057] Wherein, in step d1), there is no special restriction on the way of mixing the polyglutamic acid solution and the colcemid solution, and the materials can be mixed evenly, such as stirring evenly. In step d2), the way of mixing the mixed solution with the polylysine solution is preferably: slowly adding the mixed solution to the polylysine solution and stirring evenly. In step d2), after all the solutions are mixed evenly, they are allowed to stand. The standing temperature is room temperature, which can be 20 to 25°C, specifically 20°C, 21°C, 22°C, 23°C, 24°C, and 25°C. The standing time is preferably 6 to 8h, specifically 6h, 7h, and 8h. After blending and standing, polyglutamic acid and polylysine are physically cross-linked to form a three-dimensional network structure, and colcemid is evenly distributed in the three-dimensional network structure, and finally a drug-loaded hydrogel with a three-dimensional network structure is obtained to form a gel-state drug delivery.

[0058] In the present invention, the mass percentage concentration of polyglutamic acid in the drug-loaded polyglutamic acid-based hydrogel is preferably 2% to 3%, specifically 2% and 3%. The mass percentage concentration of colchicine in the drug-loaded polyglutamic acid-based hydrogel is preferably 1% to 3%, specifically 1%, 2%, and 3%. The mass percentage concentration of polylysine in the drug-loaded polyglutamic acid-based hydrogel is 4% to 8%, specifically 4%, 5%, 6%, 7%, and 8%. In the present invention, more preferably, in step d), when the polyglutamic acid solution, the colcemic acid solution and the polylysine solution are mixed, the mass ratio of polyglutamic acid:colcemic acid:polylysine is preferably (1-2):1:4, specifically 1:1:4, 2:1:4, more preferably 2:1:4; specifically, this can be achieved by controlling the mass ratio of polyglutamic acid in step a):colcemic acid in step b):polylysine in step c) to the above ratio; under the above ratio, the components can form an optimal combination, so that the hydrogel product has good biodegradability, antibacterial and anti-skin cancer capabilities.

[0059] The present invention also provides a drug-loaded polyglutamic acid-based hydrogel, which is prepared by the preparation method described in the above technical solution.

[0060] The drug-carrying polyglutamic acid-based hydrogel provided by the present invention is formed into a gel-state drug delivery by a blending method of polyglutamic acid, polylysine and colcemid, and a hydrogel with a three-dimensional network structure is obtained by physical crosslinking of polyglutamic acid and polylysine and standing at room temperature. The present invention uses polylysine as a crosslinking agent, and reduces the size of the phase separation structure inside the gel by introducing electrostatic interaction inside the gel, promotes the formation of a dense polymer crosslinking network, improves the water retention rate of the overall material, and improves the compressive strength and viscoelasticity of the material. Moreover, colcemid is evenly distributed on the above-mentioned polymer three-dimensional network, so that each component is combined in a specific structure and distribution form, which is conducive to improving the antibacterial and anti-skin cancer capabilities of the product, and ensuring the compressive strength and water retention of the material. In addition, the above-mentioned hydrogel product is biodegradable, biocompatible, non-toxic and harmless, and has long-lasting and stable performance, and can be widely used in the fields of medical dressings and tissue engineering materials.

[0061] Experimental results show that the drug-loaded polyglutamic acid-based hydrogel provided by the present invention has a compression strength of more than 80 MPa and a water retention rate of more than 94%, showing excellent compression strength and water retention; the antibacterial rate against Escherichia coli reaches more than 84%, and the antibacterial rate against Staphylococcus epidermidis reaches more than 80%, showing excellent antibacterial properties; the 1-day anti-cancer rate reaches more than 79%, and the 3-day anti-cancer rate reaches more than 85%, showing excellent anti-skin cancer ability.

[0062] In order to further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.

[0063] In the following examples, unless otherwise specified, the raw materials are commercially available, wherein γ-polyglutamic acid was purchased from Nanjing Xuankai Biotechnology Co., Ltd.

[0064] Example 1

[0065] a) Weigh 1.0 g of γ-polyglutamic acid (M W =700 kDa) in a 50 mL beaker, add 10 g of purified water, stir evenly to obtain a polyglutamic acid solution;

[0066] b) weighing 0.5 g of colcemid powder, adding 5 g of ethanol, and stirring evenly to obtain a colcemid solution;

[0067] c) Weigh 2.0 g polylysine (M W =3kDa) powder, add 10g purified water, stir evenly to obtain a polylysine solution;

[0068] d) After the polyglutamic acid solution and the colcemid solution are mixed, they are slowly added into the polylysine solution, stirred evenly, and allowed to stand at room temperature 25° C. for 8 hours to obtain a drug-loaded polyglutamic acid-based hydrogel.

[0069] The product obtained in Example 1 was tested, specifically the relationship between the storage modulus and loss modulus of the product and the frequency. The results are as follows: Figure 1 As shown, it can be seen that its storage modulus is greater than the loss modulus, proving that the obtained product is a hydrogel product.

[0070] Example 2

[0071] a) Weigh 1.0 g of γ-polyglutamic acid (M W =1100 kDa) in a 50 mL beaker, add 10 g of purified water, stir evenly to obtain a polyglutamic acid solution;

[0072] b) weighing 0.5 g of colcemid powder, adding 5 g of ethanol, and stirring evenly to obtain a colcemid solution;

[0073] c) Weigh 2.0 g polylysine (M W =3kDa) powder, add 10g purified water, stir evenly to obtain a polylysine solution;

[0074] d) After the polyglutamic acid solution and the colcemid solution are mixed, they are slowly added into the polylysine solution, stirred evenly, and allowed to stand at room temperature 25° C. for 8 hours to obtain a drug-loaded polyglutamic acid-based hydrogel.

[0075] Example 3

[0076] a) Weigh 1.0 g of γ-polyglutamic acid (M W =2000 kDa) in a 50 mL beaker, add 10 g of purified water, stir evenly to obtain a polyglutamic acid solution;

[0077] b) weighing 0.5 g of colcemid powder, adding 5 g of ethanol, and stirring evenly to obtain a colcemid solution;

[0078] c) Weigh 2.0 g polylysine (M W =3kDa) powder, add 10g purified water, stir evenly to obtain a polylysine solution;

[0079] d) After the polyglutamic acid solution and the colcemid solution are mixed, they are slowly added into the polylysine solution, stirred evenly, and allowed to stand at room temperature 25° C. for 8 hours to obtain a drug-loaded polyglutamic acid-based hydrogel.

[0080] Example 4

[0081] The method was carried out in accordance with Example 3, except that in step a), the amount of γ-polyglutamic acid was reduced to 0.5 g.

[0082] Comparative Example 1

[0083] The method was carried out in accordance with Example 3, except that in step c), the polylysine powder was replaced by chitosan powder.

[0084] Comparative Example 2

[0085] The method was carried out in accordance with Example 3, except that in step c), the polylysine powder was replaced with γ-(2,3-epoxypropoxy)propyltrimethoxysilane.

[0086] Example 5: Product Testing

[0087] The products obtained in Examples 1-4 and Comparative Examples 1-2 were tested for various properties, as follows:

[0088] 1. Compression strength and water retention test

[0089] The compressive strength was tested using a universal testing machine. The water retention rate was measured using an electronic moisture meter. The test process was as follows: the hydrogel samples were weighed separately at room temperature, and then the samples were placed in an oven at 25°C, and weighed after 48 hours, thereby obtaining the water retention rate of the samples within 48 hours. The test results are shown in Table 1.

[0090] Table 1: Compression strength and water retention

[0091]

[0092] It can be seen from the test results in Table 1 that the compressive strength of the products obtained in Examples 1-4 of the present invention is above 80 MPa, and the water retention rate is above 94%, showing excellent compressive strength and water retention. Compared with the examples, the compressive strength and water retention of the products of Comparative Examples 1-2 are significantly reduced, proving that if other crosslinking agents are used, the mechanical properties and water retention of the material cannot be effectively improved. The present invention uses specific polylysine in combination with other components of the present invention to effectively improve the compressive strength and water retention of the material.

[0093] 2. Antibacterial test

[0094] Escherichia coli and Staphylococcus epidermidis were cultured to the logarithmic phase, the bacteria were washed with sterile saline solution, and then the hydrogel sample was added to 2 mL of the bacterial suspension with a good concentration. After irradiation with visible light for 10 minutes, a certain amount of bacterial solution was diluted and spread on LB solid culture medium, and then placed in a 37°C biochemical incubator for 16 hours. Finally, the number of bacterial colonies in the bacterial solution after different illumination times was counted by counting method. The test results are shown in Table 2.

[0095] Table 2: Antibacterial test results

[0096]

[0097] It can be seen from the test results in Table 2 that the antibacterial rate of the products of Examples 1-4 of the present invention against Escherichia coli is more than 84%, and the antibacterial rate against Staphylococcus epidermidis is more than 80%, showing excellent antibacterial properties. Among them, compared with Example 4, the mass ratio of polyglutamic acid: colcemid: polylysine in Example 3 is at the most preferred ratio of 2:1:4, and the result further significantly improves the antibacterial property of the material, which is significantly better than Comparative Examples 1-2.

[0098] 3. Anti-cancer test

[0099] The sterilized hydrogel was placed in a 48-well plate, with 1×10 4 Squamous cells were inoculated at a density of 10 cells / well, and the hydrogel samples were co-cultured with squamous cells for 1 day and 3 days, and then the cell viability was detected by CCK8 method. The test results are shown in Table 3.

[0100] Table 3: Anti-cancer test results

[0101]

[0102] It can be seen from the test results in Table 3 that the 1-day anticancer rate of the products of Examples 1-4 of the present invention reaches more than 79%, and the 3-day anticancer rate reaches more than 85%, which is significantly higher than Comparative Examples 1-2, showing excellent anticancer ability, proving that if other cross-linking agents are used, the anticancer ability of the material cannot be effectively improved. The present invention uses specific polylysine to match with other components of the present invention, and the components can interact well with each other, effectively improving the anticancer ability of the material. In each embodiment and the comparative example, the amount of colcemid with anticancer effect is the same, but the anticancer effect of the product is different, which also proves that the effect of each component in the system is not a simple addition, but will interact and influence each other. The present invention uses specific component matching to effectively improve the anti-skin cancer ability of the material.

[0103] It can be seen from the test results in Tables 1 to 3 that the present invention uses polyglutamic acid, polylysine and colcemid in a certain proportion to effectively improve the compressive strength and water retention of the hydrogel product, and can also effectively improve the antibacterial and anticancer capabilities of the material.

[0104] Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core ideas, including the best mode, and also enable any technician in the field to practice the present invention, including making and using any device or system, and implementing any combined method. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, the present invention can also be improved and modified, and these improvements and modifications also fall within the scope of protection of the claims of the present invention. The scope of patent protection of the present invention is defined by the claims and may include other embodiments that can be thought of by those skilled in the art. If these other embodiments have structural elements similar to the literal expression of the claims, or if they include equivalent structural elements that are not substantially different from the literal expression of the claims, then these other embodiments should also be included in the scope of the claims.

Claims

1. A method for preparing a drug-loaded polyglutamic acid-based hydrogel, characterized in that: The following steps are involved: a) dissolving polyglutamic acid in water to obtain a polyglutamic acid solution; the weight average molecular weight of the polyglutamic acid is 700-2000 kDa; b) dissolving colcemid powder in an organic solvent to obtain a colcemid solution; the organic solvent is ethanol; c) dissolving polylysine in water to obtain a polylysine solution; the weight average molecular weight of the polylysine is 2-5 kDa; d) mixing the polyglutamic acid solution, colcemid solution and polylysine solution, and allowing to stand to obtain a drug-loaded polyglutamic acid-based hydrogel; the mass percentage concentration of polyglutamic acid in the drug-loaded polyglutamic acid-based hydrogel is 2% to 3%; The mass percentage concentration of colchicine in the drug-loaded polyglutamic acid-based hydrogel is 1% to 3%; The mass percentage concentration of polylysine in the drug-loaded polyglutamic acid-based hydrogel is 4% to 8%; There is no order restriction for step a), step b) and step c).

2. The preparation method according to claim 1, characterized in that: In step d), the mass ratio of polyglutamic acid in the polyglutamic acid solution: colcemid in the colcemid solution: polylysine in the polylysine solution is (1-2):1:

4.

3. The preparation method according to claim 2, characterized in that: In step d), the mass ratio of polyglutamic acid in the polyglutamic acid solution: colcemid in the colcemid solution: polylysine in the polylysine solution is 2:1:

4.

4. The preparation method according to claim 1, characterized in that: In step d), the standing temperature is 20-25° C. and the standing time is 6-8 hours.

5. The preparation method according to claim 1, characterized in that: In step a), the mass ratio of polyglutamic acid to water is 1: (10-15); In step b), the mass ratio of colcemid powder to organic solvent is 1:(5-8); In step c), the mass ratio of polylysine to water is 2: (10-15).

6. The preparation method according to claim 1, characterized in that: Step d) specifically includes: d1) mixing the polyglutamic acid solution and the colcemid solution to obtain a mixed solution; d2) mixing the mixed solution with a polylysine solution and allowing the mixture to stand to obtain a drug-loaded polyglutamic acid-based hydrogel.

7. A drug-loaded polyglutamic acid-based hydrogel, characterized in that: Prepared by the preparation method according to any one of claims 1 to 6.

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