Sodium alginate-based hydrogel with immunomodulatory effect as well as preparation method and application of sodium alginate-based hydrogel

Abstract

The invention relates to the technical field of biological materials, and provides sodium alginate-based hydrogel with a natural immune regulation effect, a preparation method and application, and the sodium alginate-based hydrogel is used for skin injury repair. The preparation process comprises the following steps: weighing magnesium salt, quercetin and sodium alginate powder, adding deionized water, fully stirring and dissolving, and then adding a solution containing divalent cations for crosslinking to prepare the sodium alginate-based hydrogel containing magnesium and quercetin. The hydrogel has excellent degradation characteristics and mechanical properties, good biocompatibility, and anti-oxidation characteristics and anti-inflammatory characteristics to natural immune cells, and can be used for skin injury repair.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a sodium alginate-based hydrogel with immunomodulatory effect, a preparation method and application thereof. Background Art

[0002] Skin injury is one of the most common diseases, and skin wound healing is a complex and highly coordinated physiological process. Autologous tissue transplantation is currently the main means of treatment. However, this treatment method faces the problems of slow tissue expansion, skin retraction and graft skin necrosis. In recent years, stem cell therapy has been widely studied in the field of skin defect repair, but it also has problems such as poor stability and tumorigenicity. Mechanistically, acute inflammation of the skin wound is an important factor leading to abnormal wound healing. Innate immune cells are the main executors of acute inflammation, and neutrophils are the most important participants. In the early stage of injury, various internal and external factors can promote the recruitment of neutrophils in the blood to the injury site to play an anti-infection role, and then excessive infiltration will lead to the release of a large number of inflammatory mediators and recruit other immune cells to aggravate the inflammatory response, thereby inhibiting the normal growth of epithelial cells, resulting in prolonged injury healing time or abnormal healing. Therefore, the development of a new type of skin defect repair material with immune regulation, which can effectively regulate the inflammatory response of the wound while promoting skin regeneration, has become an important direction for studying skin regeneration and healing.

[0003] Sodium alginate is a natural polymer that is non-toxic, edible, abundant and inexpensive. When sodium alginate is dissolved in an aqueous solution, the Na on its G unit + Can react with some divalent cations such as Ca 2+ , Sr 2+ Ion exchange occurs, forming a network gel structure, which is conducive to drug loading and cell adhesion. In addition, alginate hydrogel has the advantages of high porosity, high water content, and easy preparation. Therefore, sodium alginate has been widely used in the biomedical field. However, sodium alginate hydrogel is a biologically inert hydrogel, and its degradation rate is slow, which does not match the regeneration rate of the skin, thus hindering its large-scale application.

[0004] In view of the above problems and the potential of sodium alginate hydrogel in the field of skin defect repair, the present invention discloses a sodium alginate-based hydrogel with natural immunomodulatory effect, a preparation method and application. Summary of the invention

[0005] The purpose of the present invention is to provide a sodium alginate-based hydrogel with natural immunomodulatory effect, a preparation method and application, so as to solve the problems existing in the prior art such as slow degradation rate, mismatch with skin regeneration rate, aggravated inflammation, prolonged injury healing time or abnormal healing, etc.

[0006] The technical solution of the present invention is: a sodium alginate-based hydrogel with natural immunomodulatory effect, wherein the sodium alginate-based hydrogel contains magnesium salt, quercetin and sodium alginate at the same time.

[0007] Preferably, the content of sodium alginate is 25-150 mmol / L.

[0008] Preferably, the content of the magnesium salt is 10-300 mmol / L.

[0009] Preferably, the content of quercetin is 0.05-0.5 mmol / L.

[0010] Preferably, the sodium alginate concentration is 75-125 mmol / L; the magnesium salt content is 25-100 mmol / L; and the quercetin content is 0.01-0.5 mmol / L.

[0011] The present invention also provides a method for preparing a sodium alginate-based hydrogel having a natural immunomodulatory effect, the preparation steps of which include:

[0012] S1. Weigh magnesium salt, quercetin and sodium alginate powder, add deionized water, stir and fully dissolve to prepare sodium alginate-based pregel;

[0013] S2. Adding a solution containing divalent cations into the sodium alginate-based pregel for cross-linking to prepare a sodium alginate-based hydrogel.

[0014] Preferably, the magnesium salt described in S1 includes one or more combinations of soluble magnesium salts such as magnesium chloride, magnesium nitrate, and magnesium sulfate.

[0015] Preferably, the magnesium salt described in S1 is magnesium chloride.

[0016] Preferably, the divalent cation used for cross-linking described in S2 is Ca 2+ , Sr 2+ 、Zn 2+ and Cu 2+ One or more combinations of these.

[0017] Preferably, the divalent cation-containing solution described in S2 is Ca-containing 2+ , Sr 2+ 、Zn 2+ and Cu 2+ A salt solution of nitrate, chloride, sulfate, carbonate, etc., which is a combination of one or more of them.

[0018] Preferably, the concentration of divalent cations in the divalent cation-containing solution is 70 to 110 mmol / L.

[0019] Preferably, the divalent cation is Ca 2+ .

[0020] Preferably, the Ca 2+ The concentration is 90mmo l / L.

[0021] Preferably, the solution of divalent cations is a calcium chloride solution.

[0022] The present invention also proposes the use of the sodium alginate-based hydrogel with natural immunomodulatory effect prepared according to the method in skin damage repair.

[0023] Compared with the prior art, the advantages of the present invention are:

[0024] (1) The present invention adds magnesium salt and quercetin into sodium alginate hydrogel to prepare a novel sodium alginate-based hydrogel. The sodium alginate-based hydrogel has a porous structure and can release Mg and Mg simultaneously. 2+ and quercetin, with excellent mechanical properties and good biocompatibility.

[0025] (2) The sodium alginate-based hydrogel prepared by the present invention can release magnesium ions. Since the atomic radius of divalent magnesium ions is small, they can react with the Na on the G unit of sodium alginate. + Compete for binding with other divalent ions (Ca 2+ , Sr 2+ 、Zn 2+ , Cu 2+ ), so that it has good swelling behavior, improves its degradation rate, and solves the problem of mismatch between its degradation rate and skin regeneration rate; at the same time, magnesium ions downregulate the NF-kB pathway in the immune response, so that it can play an anti-inflammatory role; in addition, the divalent cation is selected as Ca 2+ When added with water, the mechanical properties and safety of sodium alginate-based hydrogels can be improved.

[0026] (3) The sodium alginate-based hydrogel prepared by the present invention is added with quercetin, which belongs to the flavonoids and has extremely strong antioxidant properties. The released quercetin can upregulate the expression of Nrf2 and exert an antioxidant effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described below in conjunction with the accompanying drawings and embodiments:

[0028] Figure 1 The digital photos and SEM photos after freeze-drying of the gel A prepared in Comparative Example 1 of the present invention and the gel B prepared in Example 1;

[0029] Figure 2a Evaluation of magnesium ion release in the A gel prepared in Comparative Example 1 of the present invention and the B gel prepared in Example 1;

[0030] Figure 2b Evaluation of the release of quercetin in the gelatin A prepared in Comparative Example 1 of the present invention and the gelatin B prepared in Example 1;

[0031] Figure 2c The degradation of the glue A prepared in the comparative example 1 and the glue B prepared in the example 1 of the present invention is evaluated;

[0032] Figure 3 The mechanical properties of the glue A prepared in Comparative Example 1 and the glue B prepared in Example 1 of the present invention are evaluated;

[0033] Figure 4 The swelling performance of the A glue prepared in the comparative example 1 and the B glue prepared in the example 1 of the present invention is evaluated;

[0034] Figure 5a The A glue and Mg prepared in Comparative Examples 1-3 of the present invention 2+ Evaluation of compatibility of solution, Que solution and B gel prepared in Example 1 with neutrophils;

[0035] Figure 5b The A glue and Mg prepared in Comparative Examples 1-3 of the present invention 2+ Solution, Que solution and B gel prepared in Example 1 were co-cultured with neutrophils to evaluate cell activity;

[0036] Figure 6a The A glue and Mg prepared in Comparative Examples 1-3 of the present invention 2+ Compatibility evaluation of the solution, Que solution and the B glue prepared in Example 1 with skin epithelial cells;

[0037] Figure 6b The A glue and Mg prepared in Comparative Examples 1-3 of the present invention 2+ Evaluation of cell activity after co-culturing skin epithelial cells with solution, Que solution and B glue prepared in Example 1;

[0038] Figure 7a The A glue, B glue, Mg 2+ Evaluation of the antioxidant properties of solution and Que solution on neutrophils;

[0039] Figure 7b The A glue and Mg prepared in Comparative Examples 1-3 of the present invention 2+ Evaluation of the antioxidant properties of solution, Que solution and gelatin B prepared in Example 1 on neutrophils;

[0040] Figure 8The RNA-Seq test results of the Que solution prepared in Comparative Example 3 of the present invention on the antioxidant properties of neutrophils;

[0041] Fig. 9 The A gel and Mg prepared in Comparative Examples 1-3 of the present invention 2+ Evaluation of the anti-inflammatory properties of solution, Que solution and B gel prepared in Example 1 on neutrophils;

[0042] Fig.10 The Mg prepared in Comparative Example 2 of the present invention 2+ RNA-Seq test results of the solution’s anti-inflammatory properties on neutrophils;

[0043] Fig.11 RNA-Seq test results of the anti-inflammatory properties of B gel prepared in Example 1 of the present invention on neutrophils;

[0044] Fig.12 The A glue and Mg prepared in Comparative Examples 1-3 of the present invention 2+ Western blot test results of solution, Que solution and gel B prepared in Example 1. DETAILED DESCRIPTION

[0045] The present invention is further described in detail below in conjunction with specific embodiments:

[0046] Example 1

[0047] Weigh 610 mg of magnesium chloride, 3.5 mg of quercetin and 2000 mg of sodium alginate in 100 mL of sterilized deionized water and stir thoroughly to dissolve to prepare SAB+Mg 2+ +Que sodium alginate-based pregel, gel B; add calcium chloride solution to the sodium alginate-based pregel, remove the crosslinking solution after crosslinking for 1 minute, and prepare the sodium alginate-based hydrogel after washing with deionized water.

[0048] Comparative Example 1

[0049] Weigh 2000 mg of sodium alginate in 100 mL of sterilized deionized water to prepare sodium alginate-based pregel SAB, gel A; add calcium chloride solution to the pregel, crosslink for 1 min, and then remove the crosslinking solution. Wash with deionized water to prepare sodium alginate hydrogel.

[0050] Comparative Example 2

[0051] Preparation of a single component Mg with the same magnesium ion concentration as in Example 1 2+ Solution.

[0052] Comparative Example 3

[0053] A single component Que solution having the same quercetin concentration as in Example 1 was prepared.

[0054] Compared with Comparative Example 1, Example 1 Figure 1 As shown, the B glue (SAB+Mg 2+ +Que) and the A glue (SAB) prepared in Comparative Example 1 both have a porous structure; Figure 2a , Figure 2b As shown, based on the magnesium ion concentration and the quercetin concentration, it can be seen that the B glue prepared in Example 1 can release magnesium ions and quercetin; Figure 2c As shown, the B gel prepared in Example 1 is rich in magnesium ions and has a significantly faster degradation rate, which proves that the addition of magnesium ions accelerates the degradation of sodium alginate hydrogel; Figure 3 The storage modulus and loss modulus of the B glue prepared in Example 1 are lower than those of the A glue prepared in Comparative Example 1, and the loss factor of the B glue is greater than that of the A glue. Compared with the A glue, the B glue has more excellent mechanical properties; and combined with Figure 4 As shown, glue B has better swelling behavior.

[0055] The B glue, A glue, Mg prepared in Example 1 and Comparative Examples 1-3 were 2+ Solution and Que solution were co-cultured with neutrophils, such as Figure 5a , Figure 5b As shown, compared with Comparative Examples 1-3, Example 1 has better cell morphology and cell activity; the B glue, A glue, Mg 2+ Solution and Que solution were co-cultured with skin epithelial cells, such as Figure 6a , Figure 6b As shown, compared with Comparative Examples 1-3, Example 1 also has better cell morphology and cell activity; the results show that the B glue prepared in Example 1 and the Mg prepared in Comparative Examples 2-3 2+ Both the solution and Que solution have no obvious cytotoxicity and have good biocompatibility.

[0056] Under the condition of 5% serum, the B gel, A gel, Mg gel prepared in Example 1 and Comparative Examples 1-3 were tested respectively. 2+ Solution and the antioxidant properties of Que solution on neutrophils, e.g. Figure 7a , Figure 7bAs shown, compared with Example 1, Example 1 can significantly inhibit the upregulation of reactive oxygen species (ROS) induced by lipopolysaccharide (LPS), indicating that it has significant antioxidant properties; compared with Example 1 and Comparative Examples 2-3, it can be seen that the main antioxidant in Example 1 is quercetin, not magnesium ions. In order to further clarify the antioxidant properties of quercetin, the exon high-throughput sequencing method was used to identify the effect of the Que solution prepared in Comparative Example 3 on the gene expression of neutrophils, as shown in FIG. Figure 8 As shown, quercetin can significantly change the expression levels of a small number of genes in neutrophils. Signaling pathway (KEGG) enrichment analysis showed that these changed genes were mainly enriched in signaling pathways involved in antioxidant reactions. The results further showed that the quercetin component released from B gelatin exerts an antioxidant effect on neutrophils.

[0057] The B glue, A glue, Mg glue prepared in Example 1 and Comparative Examples 1-3 were tested respectively. 2+ Solution and the anti-inflammatory properties of Que solution on neutrophils; e.g. Fig. 9 As shown, compared with Comparative Example 1, the prepared B glue in Example 1 can significantly inhibit the secretion of multiple pro-inflammatory factors (TNF-α, IL-8 and IL-1β) by neutrophils induced by lipopolysaccharide (LPS), indicating that it has significant anti-inflammatory properties; compared with Comparative Examples 2-3, it can be seen that the main anti-inflammatory effect in B glue is magnesium ions, not quercetin. In order to further clarify the anti-inflammatory properties of magnesium ions, the exon high-throughput sequencing method was used to identify the Mg prepared in Comparative Example 2. 2+ The effect of solution on gene expression of neutrophils, e.g. Fig.10 As shown, magnesium ions can significantly change the expression levels of some genes in neutrophils. Signal pathway enrichment analysis (KEGG) showed that these changed genes were mainly enriched in signal pathways involved in inflammatory responses, further indicating that the magnesium ion component in B gelatin exerts an anti-inflammatory effect on neutrophils.

[0058] Exon high-throughput sequencing was used to identify the effect of the B gel prepared in Example 1 on the gene expression of neutrophils. Fig.11 As shown, it was shown that B glue could significantly change the expression levels of some genes in neutrophils, and the signal pathway enrichment analysis (KEGG) showed that these changed genes were mainly enriched in the signal pathways involved in inflammatory response and antioxidant response; the results proved the above demonstration that the B glue prepared in Example 1 had anti-inflammatory and antioxidant effects, and the magnesium ions and quercetin released by it played anti-inflammatory and antioxidant effects, respectively.

[0059] In order to further explore the reaction mechanism of the anti-inflammatory and oxidative effects of the B glue prepared in Example 1, the B glue, A glue, Mg glue prepared in Example 1 and Comparative Examples 1-3 were stained with protein immunoblotting technology. 2+The mechanism of action of the solution and Que solution was tested, such as Fig.12 As shown, the results showed that B glue down-regulated the expression of various proteins in the pro-inflammatory signaling pathway NF-kB pathway through magnesium ions, thereby exerting an anti-inflammatory effect on neutrophils; and up-regulated the expression of various proteins in the antioxidant signaling pathway NRF2 through quercetin, thereby exerting an antioxidant effect on neutrophils.

[0060] Example 2

[0061] Weigh 950 mg of magnesium chloride, 8 mg of quercetin and 2000 mg of sodium alginate in 100 mL of sterilized deionized water, stir and dissolve them thoroughly to prepare a sodium alginate-based pregel. Add strontium chloride solution to the pregel, remove the crosslinking solution after crosslinking for 1 min, and wash with deionized water to prepare a sodium alginate-based hydrogel.

[0062] Example 3

[0063] 1800 mg of magnesium chloride, 2 mg of quercetin and 2500 mg of sodium alginate were weighed and dissolved in 100 mL of deionized water, and a sodium alginate-based pregel was prepared. Calcium chloride solution was added to the pregel, and the crosslinking solution was removed after crosslinking for 1 min. After washing with deionized water, a sodium alginate-based hydrogel was prepared.

[0064] Example 4

[0065] 1800 mg of magnesium chloride, 10 mg of quercetin and 2500 mg of sodium alginate were weighed and dissolved in 100 mL of deionized water, and a sodium alginate-based pregel was prepared. Zinc chloride solution was added to the pregel, and the crosslinking solution was removed after crosslinking for 1 min. After washing with deionized water, a sodium alginate-based hydrogel was prepared.

[0066] Example 5

[0067] 1000 mg of magnesium chloride, 3.5 mg of quercetin and 1500 mg of sodium alginate were weighed and dissolved in 100 mL of deionized water, and a sodium alginate-based pregel was prepared. Strontium nitrate solution was added to the pregel, and the crosslinking solution was removed after crosslinking for 1 min. After washing with deionized water, a sodium alginate-based hydrogel was prepared.

[0068] The sodium alginate-based hydrogels prepared in Examples 1 to 5 all have excellent mechanical properties, excellent degradation and swelling behaviors, good cell compatibility, and antioxidant and anti-inflammatory effects on neutrophils.

[0069] Examples 1 to 5 use salts containing different divalent cations as crosslinking agents, and all of them can prepare sodium alginate-based hydrogels with the above-mentioned excellence. However, when calcium chloride aqueous solution is used as a crosslinking agent, the prepared sodium alginate-based hydrogel has more excellent mechanical properties and cell compatibility. The role of the calcium chloride crosslinking agent is to crosslink multiple molecules in a chemical substance into a whole, thereby enhancing the cohesion of the substance and changing the structure of the substance; the calcium chloride crosslinking agent is applied to the present invention to enhance the binding force of the sodium alginate-based hydrogel, increase water absorption, improve corrosion resistance, and improve the safety of the sodium alginate-based hydrogel.

[0070] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the scope of protection of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is limited by the attached claims rather than the above description, and it is intended to include all changes within the meaning and scope of the equivalent elements of the claims in the present invention.

Claims

1. A sodium alginate-based hydrogel with natural immunomodulatory effect, characterized in that: The sodium alginate-based hydrogel contains magnesium salt, quercetin and sodium alginate simultaneously.

2. The sodium alginate-based hydrogel with natural immunomodulatory effect according to claim 1, characterized in that: The concentration of the sodium alginate is 25-150 mmol / L; the concentration of the magnesium salt is 10-300 mmol / L; and the concentration of the quercetin is 0.05-0.5 mmol / L.

3. The method for preparing the sodium alginate-based hydrogel having natural immunomodulatory effect according to claim 2, characterized in that: The concentration of the sodium alginate is 75-125 mmol / L; the concentration of the magnesium salt is 25-100 mmol / L; and the concentration of the quercetin is 0.1-0.3 mmol / L.

4. A method for preparing a sodium alginate-based hydrogel having natural immunomodulatory effect as claimed in any one of claims 1 to 3, characterized in that: Prepared by the following steps: (1) weighing magnesium salt, quercetin and sodium alginate powder, adding deionized water, stirring and fully dissolving to prepare a sodium alginate-based pregel; (2) Adding a solution containing divalent cations to the sodium alginate-based pregel for cross-linking to prepare a sodium alginate-based hydrogel with natural immunomodulatory effect.

5. The method for preparing the sodium alginate-based hydrogel having natural immunomodulatory effect according to claim 4, characterized in that: The magnesium salt is selected from one or more combinations of magnesium chloride, magnesium nitrate and magnesium sulfate.

6. The method for preparing a sodium alginate-based hydrogel having natural immunomodulatory effect according to claim 5, characterized in that: The magnesium salt is magnesium chloride.

7. The method for preparing the sodium alginate-based hydrogel having natural immunomodulatory effect according to claim 4, characterized in that: The divalent cation is selected from Ca 2+ , Sr 2+ 、Zn 2+ , Cu 2+ A combination of multiple.

8. The method for preparing the sodium alginate-based hydrogel with natural immunomodulatory effect according to claim 4, characterized in that: The concentration of divalent cations in the divalent cation-containing solution is 70-110 mmol / L.

9. The method for preparing the sodium alginate-based hydrogel with natural immunomodulatory effect according to claim 7 or 8, characterized in that: The divalent cation is Ca 2+ ; the Ca 2+ The concentration is 90mmo l / L.

10. Use of the sodium alginate-based hydrogel with natural immunomodulatory effect as claimed in any one of claims 1 to 3 in repairing skin damage.

Images