Sodium alginate gel with added biologically active substances, preparation method and application thereof

Abstract

The present invention relates to a sodium alginate gel with added bioactive substances, a preparation method and an application thereof. The gel comprises polydeoxyribonucleotides, sodium alginate hydrogel and metal halides, wherein PDRN and metal halides are coated in the sodium alginate hydrogel, the sodium alginate hydrogel is composed of sodium alginate and water, and the sodium alginate in the sodium alginate hydrogel is acetyl-modified sodium alginate. Compared with ordinary sodium alginate and ordinary sodium alginate combined with PDRN, the gel has a better wound healing effect. The gel has good strength and elasticity, and has a better effect of promoting wound healing than gauze.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a sodium alginate gel with added bioactive substances, a preparation method and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] As a dressing, hydrogel can overcome the shortcomings of poor air permeability and easy adhesion to wounds compared to traditional gauze. Gel products made from sodium alginate generally have safety risks and single functions. Although they have good hydrophilic and moisturizing properties, they do not have other functions such as anti-inflammatory and skin growth promotion. Their ability to promote wound healing is limited, and wounds are prone to inflammation and scarring after use. Although sodium alginate gel with added functional ingredients such as bioactive substances can help improve the medicinal effects of sodium alginate gel, the preparation process of the functional ingredients is relatively complicated. Summary of the invention

[0004] In view of the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide a sodium alginate gel with added bioactive substances, a preparation method and an application thereof.

[0005] In order to solve the above technical problems, the technical solution of the present invention is:

[0006] In a first aspect, a sodium alginate gel with added bioactive substances includes polydeoxyribonucleotide (PDRN), sodium alginate hydrogel, and metal halide. The metal halide serves as an initiator and cross-links with sodium alginate to form a hydrogel. The PDRN is coated in the sodium alginate hydrogel. The sodium alginate in the sodium alginate hydrogel is acetyl-modified sodium alginate.

[0007] The present invention relates to a sodium alginate hydrogel, which is used as a carrier, wherein the bioactive substance PDRN is coated, and the metal halide is used to promote cross-linking. The obtained hydrogel can be used as a dressing, has good strength and elasticity, and has a better effect of promoting wound healing than gauze.

[0008] The basic component of sodium alginate hydrogel is acetyl-modified sodium alginate. Compared with ordinary sodium alginate, acetyl-modified sodium alginate has enhanced hydrophilicity, which improves the moisturizing effect of the hydrogel dressing and is more conducive to wound healing.

[0009] PDRN has nearly 95% similarity with human DNA, is more easily absorbed by the human body, and has significant effects in promoting wound healing and scar repair. It also reduces inflammatory factors and has anti-inflammatory effects, thereby enhancing the repair and anti-inflammatory effects of hydrogel dressings.

[0010] The gel formed by the combination of sodium alginate hydrogel and PDRN can be used as a dressing to solve the problem that sodium alginate does not have other functions such as anti-inflammatory and skin growth promotion, has limited ability to promote wound healing, and is prone to inflammation and scarring on the wound surface after use.

[0011] Ordinary sodium alginate is composed of β-(1-4)-D-mannuronic acid and α-(1-4)-L-guluronic acid and has certain hydrophilic adsorption properties, but as a dressing for promoting wound healing, its anti-inflammatory and healing abilities are limited.

[0012] Compared with ordinary sodium alginate gel, the gel product formed by combining acetyl-modified sodium alginate gel and PDRN has better anti-inflammatory and wound healing effects. Compared with ordinary sodium alginate gel, the gel product formed by combining acetyl-modified sodium alginate gel and PDRN has better anti-inflammatory and wound healing effects.

[0013] Although the combination of existing sodium alginate (ordinary sodium alginate) and functional ingredients can enhance the medicinal effects of sodium alginate gel, the process of preparing gel from functional ingredients and sodium alginate is complicated, and some organic matter is added as solubilizing and preservative ingredients, which may pose a risk of allergy during patient use.

[0014] In some embodiments of the present invention, the PDRN is a bioactive substance extracted from the testes of salmon or salmon; in some embodiments, the molecular weight of the PDRN is 30-1000 kDa. The PDRN bioactive substance with the molecular weight has a better effect of being absorbed by the human body and promotes wound healing and scar repair.

[0015] In some embodiments of the present invention, the metal halide is one of zinc chloride, calcium chloride and copper chloride.

[0016] In some embodiments of the present invention, the acetyl substitution degree of the acetyl-modified sodium alginate is 10% to 25%; further 12% to 20%. The acetyl-modified sodium alginate can be called acetylated sodium alginate. The acetyl-substituted sodium alginate has a better one-step gelling effect with metal halides and has better hydrophilicity.

[0017] In a second aspect, a method for preparing the above-mentioned sodium alginate gel with added bioactive substances is as follows: PDRN is added to an acetylated sodium alginate solution to obtain a mixed solution, and then the mixed solution is mixed with a metal halide solution to obtain the sodium alginate gel with added bioactive substances.

[0018] After acetylated sodium alginate and PDRN are mixed, they are molded in the presence of metal halides to obtain a gel.

[0019] In some embodiments of the present invention, the concentration of the acetylated sodium alginate solution is 1 to 10 wt %; further 1 to 8 wt %.

[0020] In some embodiments of the present invention, the concentration of PDRN in the solution after addition is 50 to 250 μg / mL. The dosage ratio of PDRN to acetylated sodium alginate affects the mechanical properties such as strength and elasticity of the obtained gel, thereby affecting the use experience and wound healing effect of the obtained gel.

[0021] In some embodiments of the present invention, the concentration of the metal halide solution is 0.1-5 wt %. The concentration of the metal halide solution affects the gelation of acetylated sodium alginate and the degree of cross-linking of acetylated sodium alginate. Hydrogel can only be obtained at a certain concentration.

[0022] In some embodiments of the present invention, the reaction time after adding the metal halide is 20-30 minutes, and then the reaction is allowed to stand for 15-35 hours.

[0023] In some embodiments of the present invention, the preparation method of acetylated sodium alginate is: mixing sodium alginate and acetic anhydride solution, performing acetylation reaction, and then mixing the reactant with water to obtain a precipitate, and the precipitate is acetylated sodium alginate. Further, the volume ratio of sodium alginate and acetic anhydride solution is 1:5-30; further 1:10-24; for example 1:12, 1:10, 1:16, 1:20, 1:24; further, the temperature of the acetylation reaction is 30-90°C, and the reaction time is 5-16h. The product finally obtained is washed, dissolved in water, and freeze-dried to obtain powdered acetylated sodium alginate.

[0024] In a third aspect, the sodium alginate gel with added bioactive substances is used in external dressing medicines; further, the external dressing is an anti-inflammatory, skin growth promoting, and wound healing promoting medicine.

[0025] One or more technical solutions of the present invention have the following beneficial effects:

[0026] The hydrogel obtained by the present invention uses natural polymer material sodium alginate as raw material, bioactive substance PDRN as additive, and metal halide as ion crosslinking agent, and has good anti-inflammatory, wound healing and scar repair effects. Compared with ordinary sodium alginate, ordinary sodium alginate combined with PDRN has better wound healing effect. It has good strength and elasticity, and has better wound healing effect than gauze.

[0027] The hydrogel dressing can be prepared by a conventional physical stirring method, the materials used are non-toxic and harmless, biodegradable, have no side effects on the human body, and the preparation process is simple. DETAILED DESCRIPTION

[0028] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0029] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0030] The present invention is further described below in conjunction with embodiments

[0031] Example 1

[0032] (a) placing sodium alginate in an acetic anhydride solution in a mass volume ratio of 1:12, stirring and reacting at 50° C. for 10 hours. After the reaction is completed, pouring the reaction solution into deionized water for precipitation, washing the precipitate 2 to 3 times, dissolving it in water and then freeze-drying it into powder to obtain acetylated sodium alginate with an acetyl substitution degree of 15%;

[0033] (b) dissolving acetylated sodium alginate in deionized water to prepare a solution with a concentration of 2 wt %;

[0034] (c) adding PDRN to the acetylated sodium alginate solution obtained in step (b) under mechanical stirring, with the addition amount being 150 μg / mL;

[0035] (d) Under mechanical stirring conditions, the mixed solution obtained in step (c) was added to a calcium chloride solution with a concentration of 0.2 wt%, stirred for 20 min, and then allowed to stand for 24 h to obtain a PDRN-sodium alginate gel.

[0036] Example 2

[0037] (a) placing sodium alginate in an acetic anhydride solution at a mass volume ratio of 1:16, stirring and reacting at 70° C. for 16 hours. After the reaction is completed, pouring the reaction solution into deionized water for precipitation, washing the precipitate 2 to 3 times, dissolving it in water and then freeze-drying it into powder to obtain acetylated sodium alginate with an acetyl substitution degree of 18%;

[0038] (b) dissolving acetylated sodium alginate in deionized water to prepare a solution with a concentration of 5 wt %;

[0039] (c) adding PDRN to the acetylated sodium alginate solution obtained in step (b) under mechanical stirring, with the addition amount being 200 μg / mL;

[0040] (d) Under mechanical stirring conditions, the mixed solution obtained in step (c) was added to a zinc chloride solution with a concentration of 1 wt%, stirred for 25 min, and then allowed to stand for 24 h to obtain a PDRN-sodium alginate gel.

[0041] Example 3

[0042] (a) placing sodium alginate in an acetic anhydride solution at a mass volume ratio of 1:24, stirring and reacting at 50° C. for 8 hours. After the reaction is completed, pouring the reaction solution into deionized water for precipitation, washing the precipitate 2 to 3 times, dissolving it in water and then freeze-drying it into powder to obtain acetylated sodium alginate with an acetyl substitution degree of 20%;

[0043] (b) dissolving acetylated sodium alginate in deionized water to prepare a solution with a concentration of 8 wt %;

[0044] (c) adding PDRN to the acetylated sodium alginate solution obtained in step (b) under mechanical stirring, with the addition amount being 180 μg / mL;

[0045] (d) Under mechanical stirring conditions, the mixed solution obtained in step (c) was added to a 3 wt % calcium chloride solution, stirred for 30 min, and then allowed to stand for 24 h to obtain a PDRN-sodium alginate gel.

[0046] Example 4

[0047] (a) placing sodium alginate in an acetic anhydride solution at a mass volume ratio of 1:10, stirring and reacting at 30° C. for 5 hours. After the reaction is completed, pouring the reaction solution into deionized water for precipitation, washing the precipitate 2 to 3 times, dissolving it in water and then freeze-drying it into powder to obtain acetylated sodium alginate with an acetyl substitution degree of 12%;

[0048] (b) dissolving acetylated sodium alginate in deionized water to prepare a solution with a concentration of 1 wt %;

[0049] (c) adding PDRN to the acetylated sodium alginate solution obtained in step (b) under mechanical stirring, with the addition amount being 150 μg / mL;

[0050] (d) Under mechanical stirring conditions, the mixed solution obtained in step (c) was added to a 1 wt % copper chloride solution, stirred for 30 min, and then allowed to stand for 24 h to obtain a PDRN-sodium alginate gel.

[0051] Example 5

[0052] (a) placing sodium alginate in an acetic anhydride solution at a mass volume ratio of 1:20, stirring and reacting at 65° C. for 12 hours. After the reaction is completed, pouring the reaction solution into deionized water for precipitation, washing the precipitate 2 to 3 times, dissolving it in water and then freeze-drying it into powder to obtain acetylated sodium alginate with an acetyl substitution degree of 15%;

[0053] (b) dissolving acetylated sodium alginate in deionized water to prepare a solution with a concentration of 6 wt %;

[0054] (c) adding PDRN to the acetylated sodium alginate solution obtained in step (b) under mechanical stirring, with the addition amount being 150 μg / mL;

[0055] (d) Under mechanical stirring conditions, the mixed solution obtained in step (c) was added to a 2 wt % copper chloride solution, stirred for 30 min, and then allowed to stand for 24 h to obtain a PDRN-sodium alginate gel.

[0056] Example 6

[0057] Compared with Example 3, the concentration of the calcium chloride solution used is 1.5wt%, and the reagents, dosages and preparation methods in other steps are the same.

[0058] Example 7

[0059] Compared with Example 2, in step (a), sodium alginate is placed in acetic anhydride solution, the reaction time is 5 hours, other preparation methods are the same, and the acetyl substitution degree is 10 wt%.

[0060] The preparation methods of steps (b), (c) and (d) are the same.

[0061] Example 8: PDRN-sodium alginate gel strength test

[0062] The gel strength and elasticity of Examples 1 to 6 were measured using a QTS-25 texture analyzer, using a TA41 cylindrical probe, measuring mode TPA, a trigger value of 5 g, and a pressing speed of 30 mm / min. The instrument's own analysis program displayed the gel strength and elasticity of the tested samples in real time.

[0063] The gel strength and elasticity of Examples 1-5 and Comparative Example 1 are shown in Table 1. The gel used in Comparative Example 1 is the gel prepared in Example 6.

[0064] Table 1 PDRN-sodium alginate gel strength test results

[0065] name Gel strength / g Elasticity / mm Example 1 404.56±3.04 1.55 Example 2 425.73±2.56 1.60 Example 3 489.69±3.46 1.71 Example 4 400.61±2.67 1.55 Example 5 456.43±2.89 1.62 Comparative Example 1 320.65±1.39 1.48

[0066] It can be seen from Table 1 that the gels prepared in Examples 1-6 have good appearance characteristics, high gel strength and good elasticity. Compared with Example 3, the gel strength and elasticity of Comparative Example 1 are reduced, indicating that when the concentration of the acetylated sodium alginate solution is high, the concentration of the metal halide used should also be increased accordingly to achieve better crosslinking and obtain a gel with better strength and elasticity.

[0067] Example 9: In vitro cytotoxicity assay

[0068] According to GB T16886.5-2017 / ISO10993-5:2009 "Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test", in vitro cytotoxicity tests were performed on the acetylated sodium alginate, PDRN and the PDRN-sodium alginate gel prepared in the present invention to verify biocompatibility.

[0069] The MTT cytotoxicity test evaluation method was selected, L929 cells were selected as the cell line, 10% DMSO solution was used as the positive control group, 3% fetal calf serum culture medium was used as the negative control group, and the experimental group was added with 3% fetal calf serum culture medium extract of the test sample, and cultured at 37°C with shaking for 24 hours. The added amount of the test sample was 0.2g / mL.

[0070] The specific experimental steps are as follows:

[0071] (1) L929 cells that had been subcultured for 48 h were prepared into 1×10 5 The cell suspension of 100 cells / mL is ready for use;

[0072] (2) 1×10 5 The cells / mL cell suspension was inoculated into a 96-well cell culture plate (100 μL / well) and placed in a 37°C, 5% CO 2 Culture in an incubator for 24 h to form a semiconfluent monolayer;

[0073] (3) After 24 h, the original culture medium was discarded, and then 100 μL of sample extract, positive control solution, and negative control solution were added, 5 wells per group, and placed at 37°C and 5% CO. 2 Culture in an incubator for 24 h.

[0074] (4) Add 50 μL of MTT solution to each well and continue to incubate at 37°C and 5% CO 2 Continue incubating in the incubator for 2 h;

[0075] The MTT solution was discarded, and 100 μL of DMSO solution was added to each well. The plate was shaken and placed on a microtiter plate photometer to measure the absorbance at 570 nm. The cell proliferation rate (RGR, %) was calculated and the cytotoxicity was evaluated according to the standards in the table below.

[0076] Table 2 In vitro cytotoxicity grading table

[0077] level Relative proliferation rate / % 0 ≥100 1 75-99 2 50-74 3 25-49 4 1-24 5 0

[0078] The experimental results are shown in Table 3. The in vitro cytotoxicity of the materials used in the present invention and the prepared gel are both level 1, indicating that they have good biocompatibility and will not produce toxicity or cause toxic residue when applied to the wound surface.

[0079] Table 3 In vitro cytotoxicity test results

[0080] sample Toxicity level Acetylated Sodium Alginate 1 PDRN 1 Example 1 Gel 1 Example 2 Gel 1 Example 3 Gel 1 Example 4 Gel 1 Example 5 Gel 1 Example 6 Gel 1 Example 7 Gel 1 Positive control group 5

[0081] Example 10: Wound healing experiment

[0082] The experiment was an animal experiment, with a total of 6 groups of experiments, of which 1 group was a blank control group and 5 groups were test sample groups.

[0083] Eighteen clean-grade rabbits weighing 2.0±0.1kg were selected, three in each group, numbered 1-6, and anesthetized with 30g / L sodium pentobarbital (30mg / kg) injection. The rabbits were depilated on their backs, and a circular skin defect with a diameter of 2cm was made on the backs. The wound should be deep to the fascia layer.

[0084] From the second day, the tested sample was evenly applied to the wound surface of the back of the rabbit at 8 am and 8 pm every day. The first group of white rabbits were smeared with common sodium alginate gel (common sodium alginate gel and cross-linking agent dosage are the same as in Example 2), the second group of white rabbits were smeared with acetyl sodium alginate gel (acetyl sodium alginate and cross-linking agent dosage are the same as in Example 2), the third group of white rabbits were smeared with PDRN-common sodium alginate gel (PDRN, common sodium alginate and cross-linking agent dosage are the same as in Example 2), the fourth group of white rabbits were smeared with gel (PDRN-acetyl sodium alginate gel) prepared by Example 2, the fifth group of white rabbits were blank control group, smeared with normal saline, and the sixth group of white rabbits were gel (PDRN-acetyl sodium alginate gel) prepared by Example 7. Gauze and elastic bandage were used to bandage and fix the wound surface to prevent the gel from falling off, and the wound surface condition, average wound healing time and average scar area after healing were recorded, and the results are shown in Table 4.

[0085] Table 4 Wound healing experiment results

[0086]

[0087] When applying ordinary sodium alginate and acetylated sodium alginate gel, the wound is prone to inflammation and the healing cycle is slow, while applying the gel added with PDRN will not cause inflammation and the wound healing cycle is short; when applying ordinary sodium alginate, acetylated sodium alginate and PDRN-ordinary sodium alginate gel, scars appear after wound healing, while applying the gel prepared by the present invention does not produce scars; in addition, by comparing the 4th group of white rabbits with the 6th group of white rabbits, it can be seen that the acetylation degree of sodium alginate affects the recovery effect of the wound. Comprehensive experimental results show that the PDRN-acetylated sodium alginate gel prepared by the present invention has excellent performance, can significantly promote wound healing, and has certain anti-inflammatory and scar repair effects.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A sodium alginate gel with added biologically active substances, characterized in that: It includes polydeoxyribonucleotide PDRN, sodium alginate and metal halide, wherein the metal halide is used as an initiator to generate cross-linking with the sodium alginate to form a hydrogel, the PDRN is coated in the sodium alginate hydrogel, and the sodium alginate in the sodium alginate hydrogel is acetyl-modified sodium alginate; The metal halide is one of zinc chloride, calcium chloride and copper chloride; The acetyl substitution degree of acetyl-modified sodium alginate is 10%~25%; The concentration of the acetyl-modified sodium alginate solution is 1 to 10wt%; The concentration of PDRN in the solution after addition was 50 ~ 250 μg / mL; The concentration of the metal halide solution is 0.1 ~ 5 wt%.

2. The sodium alginate gel with added biologically active substances as claimed in claim 1, characterized in that: The molecular weight of the PDRN is 30-1000 kDa.

3. The sodium alginate gel with added biologically active substances as claimed in claim 1, characterized in that: The acetyl substitution degree of acetyl-modified sodium alginate is 12%~20%.

4. The method for preparing sodium alginate gel with added biologically active substances according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: adding PDRN to an acetyl-modified sodium alginate solution to obtain a mixed solution, and then mixing the mixed solution with a metal halide solution to obtain a sodium alginate gel with added bioactive substances.

5. The method for preparing sodium alginate gel with added biologically active substances as claimed in claim 4, characterized in that: After adding the metal halide, the reaction time is 20-30 minutes, and then the mixture is allowed to stand for 15-35 hours.

6. Use of the sodium alginate gel with added bioactive substances according to any one of claims 1 to 3 in the preparation of external dressing medicines.

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