CN114272206A - Injectable antibacterial polymer hydrogel and preparation method and application thereof - Google Patents

Abstract

The invention provides an injectable antibacterial polymer hydrogel and a preparation method and application thereof, belonging to the technical field of biomedical materials. The hydrogel is prepared from alginate, hyaluronate, a compound containing iron ions and disodium ethylene diamine tetraacetate. The ALG-HA hydrogel disclosed by the invention is uniform and stable, HAs excellent self-repairing performance, shear thinning performance and injectability, and also HAs excellent antibacterial performance. It can be used as injectable carrier, and can be adapted to local injection treatment of tissue defects with different sizes and shapes. In addition, the hydrogel is simple in raw materials, fast in forming, simple and convenient to prepare, green and environment-friendly, and has a good application prospect.

Description

Injectable antibacterial polymer hydrogel and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to an injectable antibacterial polymer hydrogel and a preparation method and application thereof.

Background

The injectable antibacterial hydrogel system has become a research hotspot of local injection antibacterial treatment due to the advantages of simple operation, minimal invasion/no wound, small toxic and side effect, long maintenance time of effective antibacterial drug concentration and the like.

In recent years, natural polymer organic compounds such as Alginate (ALG) and Hyaluronic Acid (HA) are popular candidates for synthesis of hydrogels due to their advantages such as good biocompatibility, degradability, and low cost, and the characteristic of rapid formation of supramolecular hydrogels by reaction with specific metal ions through metal-ligand interaction. However, such supramolecular hydrogels based on macromolecular compounds often lack shear thinning properties and clinical syringeability is not easily achieved.

In addition, the hydrogel does not have antibacterial performance, antibacterial property needs to be realized by encapsulating antibacterial drugs, and the problems of complex and time-consuming preparation steps, low drug encapsulation efficiency, inactivation of the encapsulated drugs, drug leakage, toxic and side effects on local cell tissues and the like exist.

Therefore, the supramolecular hydrogel with shear thinning characteristic and antibacterial performance constructed based on the natural macromolecular compound has potential research value and clinical application prospect.

Disclosure of Invention

The invention aims to provide an injectable antibacterial polymer hydrogel and a preparation method and application thereof.

The invention provides an injectable antibacterial polymer hydrogel which is prepared from alginate, hyaluronate, a compound containing iron ions and disodium ethylene diamine tetraacetate.

Further, the hydrogel is obtained by mixing a mixed solution of alginate and hyaluronate with a mixed solution of a compound containing iron ions and disodium ethylenediaminetetraacetate.

Further, the volume ratio of the mixed solution of the alginate and the hyaluronate to the mixed solution of the compound containing iron ions and the disodium ethylene diamine tetraacetate is (1-50): 1.

further, the volume ratio of the mixed solution of the alginate and the hyaluronate to the mixed solution of the compound containing iron ions and the disodium ethylene diamine tetraacetate is (1-10): 1;

preferably, the volume ratio of the mixed solution of the alginate and the hyaluronate to the mixed solution of the compound containing the iron ions and the disodium ethylene diamine tetraacetate is 5: 1.

further, the concentration of the alginate in the alginate and hyaluronate mixed solution is 1-5% (w/v), and the concentration of the hyaluronate is 0.1-1% (w/v);

preferably, the concentration of the alginate salt and the hyaluronic acid salt in the mixed solution of the alginate salt and the hyaluronic acid salt is 3% (w/v) and the concentration of the hyaluronic acid salt is 0.75% (w/v).

Further, the concentration of the iron ions in the mixed solution of the compound containing the iron ions and the disodium ethylene diamine tetraacetate is 0.1-1 mol/L, and the concentration of the disodium ethylene diamine tetraacetate is 0.01-0.1 mol/L;

preferably, the concentration of the iron ions in the mixed solution of the iron ion-containing compound and the disodium ethylene diamine tetraacetate is 0.1mol/L, and the concentration of the disodium ethylene diamine tetraacetate is 0.05 mol/L.

Further, the alginate is sodium alginate; and/or the hyaluronate is sodium hyaluronate; and/or the compound containing iron ions is Fe³⁺A compound of (1);

preferably, the Fe-containing³⁺The compound of (a) is ferric sulfate.

The invention also provides a preparation method for preparing the hydrogel, which comprises the following steps:

(1) dissolving alginate and hyaluronate in water to obtain a mixed solution A;

(2) dissolving a compound containing iron ions and disodium ethylene diamine tetraacetate in water to obtain a mixed solution B;

(3) adding the mixed solution B into the mixed solution A to obtain a mixed solution A;

preferably, the mixed solution B is stirred when being added into the mixed solution A.

The invention also provides application of the hydrogel in preparation of an injectable antibacterial drug carrier.

The invention also provides application of the hydrogel in preparation of a tissue repair material.

In the present invention, w/v represents g/mL.

The ALG-HA hydrogel disclosed by the invention is uniform and stable, HAs excellent self-repairing performance, shear thinning performance and injectability, and also HAs excellent antibacterial performance. It can be used as injectable carrier, and can be adapted to local injection treatment of tissue defects with different sizes and shapes. In addition, the hydrogel is simple in raw materials, fast in forming, simple and convenient to prepare, green and environment-friendly, and has a good application prospect.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 shows the results of the formation of the ALG-HA hydrogels of the present invention: a is the result of determining the formation of each group of gels in the vial inversion experiment; b is the result of confirming the formation of the hydrogel prepared in example 1 by the rheological test time sweep.

FIG. 2 is the rheological profile of the hydrogel prepared in example 1: a is the amplitude scanning result of the ALG-HA hydrogel; and B is the frequency scanning result of the ALG-HA hydrogel.

FIG. 3 is a graph depicting the results of self-healing performance of various groups of hydrogels by rheological cyclic strain time scanning: a is the ALG-HA hydrogel prepared in example 1; b is the HA hydrogel prepared in comparative example 2; c is ALG hydrogel prepared in comparative example 1.

FIG. 4 is a graph of shear-thinning and injectability results for ALG-HA hydrogels of the present invention: a is the shear rate test result for the hydrogel prepared in example 1; b is the injection inversion experimental result of the hydrogel prepared in example 1; c is the injection experimental result of the hydrogel prepared in example 1, wherein the specification of the syringe of a is 10ml, the specification of the syringe of b is 5ml, and the specification of the syringe of C is 1 ml; d is the writing ability of the hydrogel prepared in example 1.

FIG. 5 shows the antibacterial results of the ALG-HA hydrogel of the present invention: a is a colony culture macro graph of S.aureus and E.coli; b is the count results of colony cultures of s.aureus and e.coli; in the figure, Control represents the blank Control group, and Gel represents the ALG-HA hydrogel group.

Detailed Description

Unless otherwise indicated, the starting materials and equipment used in the embodiments of the present invention are known products and obtained by purchasing commercially available products.

Example 1 preparation of injectable antibacterial Polymer hydrogel of the present invention

Sodium Alginate (ALG) and sodium Hyaluronate (HA) were accurately weighed and dissolved in ultrapure water at room temperature to prepare a sodium alginate-sodium hyaluronate mixed solution (ALG-HA solution) in which the ALG concentration was 3% (w/v) and the HA concentration was 0.75% (w/v).

Fe was prepared by accurately weighing ferric sulfate and disodium Ethylenediaminetetraacetate (EDTA) respectively, dissolving them in ultrapure water at room temperature³⁺EDTA complex solution, Fe³⁺Has a concentration of 0.1mol/L and EDTA concentration of 0.05mol/L, i.e. Fe³⁺The molar ratio to EDTA was 2: 1.

2mL of ALG-HA solution was placed in a vial, and Fe in a total volume of 400. mu.L was added dropwise with mechanical stirring³⁺-EDTA complex solution. And (3) after full reaction (reaction at room temperature for 0.5-1 min), obtaining the injectable antibacterial polymer hydrogel (ALG-HA hydrogel).

Comparative example 1 preparation of sodium alginate hydrogel

Sodium Alginate (ALG) was precisely weighed and dissolved in ultrapure water at room temperature to prepare a sodium alginate solution (ALG solution) in which the ALG concentration was 3% (w/v).

Fe was prepared by accurately weighing ferric sulfate and disodium Ethylenediaminetetraacetate (EDTA) respectively, dissolving them in ultrapure water at room temperature³⁺EDTA complex solution, Fe³⁺Has a concentration of 0.1mol/L and EDTA concentration of 0.05mol/L, i.e. Fe³⁺The molar ratio to EDTA was 2: 1.

2mL of ALG solution was placed in a vial, and Fe in a total volume of 400. mu.L was added dropwise with mechanical stirring³⁺-EDTA complex solution. And (3) after full reaction (reaction at room temperature for 0.5-1 min), obtaining the sodium alginate hydrogel (ALG hydrogel).

Comparative example 2 preparation of sodium hyaluronate hydrogel

Sodium Hyaluronate (HA) was accurately weighed and dissolved in ultrapure water at room temperature to prepare a sodium hyaluronate solution (HA solution) in which the concentration of HA was 0.75% (w/v).

Fe was prepared by accurately weighing ferric sulfate and disodium Ethylenediaminetetraacetate (EDTA) respectively, dissolving them in ultrapure water at room temperature³⁺EDTA complex solution, Fe³⁺Has a concentration of 0.1mol/L and EDTA concentration of 0.05mol/L, i.e. Fe³⁺The molar ratio to EDTA was 2: 1.

2mL of HA solution was placed in a vial, and Fe in a total volume of 400. mu.L was added dropwise with mechanical stirring³⁺-EDTA complex solution. And (3) after full reaction (reaction at room temperature for 0.5-1 min), obtaining the sodium hyaluronate hydrogel (HA hydrogel).

The advantageous effects of the present invention are demonstrated by specific test examples below.

Test example 1 basic rheological characterization of the hydrogels according to the invention

1. Test method

Hydrogels were prepared in vials according to the methods described in example 1, comparative example 1 and comparative example 2, the formation of the hydrogels was confirmed by inverting the vials, and the observation and photographic recording were performed.

The rheological testing was performed by an Anton Paar modular intelligent rotary rheometer (MCR 302). And (3) loading the hydrogel on a test board preset at 25 ℃, and adding a layer of silicone oil around the test board after loading the hydrogel so as to prevent water evaporation in the test process. The rheological properties of each hydrogel were analyzed by frequency, time, and amplitude sweeps at 25 ℃.

2. Test results

As can be seen from fig. 1A: mixing ALG-HA solution with Fe³⁺After mixing the EDTA complex solution, formation of a tan ALG-HA hydrogel (ALG-HA-Fe-EDTA in fig. 1A) was observed in the vial, and after inverting the vial, the hydrogel remained in a stable state and did not flow or collapse, indicating successful preparation of the ALG-HA hydrogel; respectively mixing HA solution or ALG solution with Fe at the same concentration and ratio³⁺After mixing the EDTA complex solution, the HA solution was observed with F^e3+EDTA complex solution forming a clear yellow liquid with fluidity (HA-Fe-EDTA in FIG. 1A), ALG solution with Fe³⁺The EDTA complex solution formed a heterogeneous yellow-like solid/liquid mixture (ALG-Fe-EDTA in FIG. 1A). Description of the drawings: at the same concentration and ratio, only the mixed solution of ALG and HA is mixed with F^e3+-EDTA complex solutionThe hydrogel can be successfully prepared after mixing reaction.

In ALG-HA solution with Fe³⁺The formation of the hydrogel was further confirmed by rheological characterization immediately after the reaction of the EDTA complex solution. The time sweep (FIG. 1B) shows that after the autoreaction HAs taken place, the storage modulus G 'is greater than the loss modulus G', demonstrating ALG-HA vs Fe³⁺-EDTA, forming a hydrogel with solid-like physical properties; within 30 minutes of the test, there was little change in the values of G 'and G', indicating ALG-HA and Fe³⁺The reaction between EDTA is immediate and the hydrogel formed has a certain homogeneity and stability.

The results of the amplitude sweep experiments are shown in fig. 2A, where the strain (γ) is less than 10%, G '> G ", the sample has solid-like physical properties, being in a gel state, and where the strain is greater than 10%, G' < G", the sample has liquid-like physical properties, i.e. a transition from a gel state to a sol state. The results show that the ALG-HA hydrogel maintains the gel state within 0.01% -10% of strain, and G' have almost no change within 0.01% -1% of strain, which indicates that the hydrogel maintains stable mechanical properties within the range.

The hydrogel was subjected to a frequency sweep in the range of 1-100rad/s with a strain of 1%, and the results (FIG. 2B) showed that G 'and G "were slightly larger with increasing frequency (. omega.) and G' was always larger than G", indicating that the ALG-HA hydrogel maintained the gel state in this range.

The above test results demonstrate mixing of ALG and HA mixed solution with Fe³⁺After the EDTA complex solution is mixed, the ALG-HA hydrogel with uniform and stable performance can be quickly formed.

Test example 2 shear-thinning injectability study of the hydrogel of the present invention

1. Test method

(1) Cyclic strain time sweep

The self-healing performance was performed by an Anton Paar modular intelligent rotational rheometer (MCR302), and the sample application method was the same as in test example 1. A cyclic strain time scan was performed at a setting of ω ═ 1rad/s, with a strain setting of 0.1% to place the sample in a stable gel-like state, followed by a rapid increase in strain from 0.1% to 100%, after a certain time the sample had broken to a sol-like state, reducing the strain from 100% to 0.1%, for observation of the self-healing of the ALG-HA hydrogel (hydrogel prepared in example 1) and the control samples (hydrogel prepared in comparative examples 1 and 2). All tests were repeated at least three times.

(2) Shear rate scanning

Shear thinning was performed by an Anton Paar modular intelligent rotary rheometer (MCR302) using the same sample loading method as in test example 1. Setting the shear rate from 0 to 100s^-1The viscosity of the sample (hydrogel prepared in example 1) was measured and the test was repeated at least three times.

(3) In vitro injection experiment

The ALG-HA hydrogel prepared in example 1 was loaded into a 10mL syringe and manually injected into a glass vial at room temperature (25 ℃), and the vial was inverted immediately after injection and the phenomenon was observed and recorded. The ALG-HA hydrogel prepared in example 1 was stained with methyl blue, loaded with the methyl blue-stained ALG-HA hydrogel using 10mL, 5mL, and 1 mL-sized syringes, and manually injected at room temperature (25 ℃) into the dH-containing chamber₂O glass vials, observed and recorded. The ALG-HA hydrogel prepared in example 1, which was stained with methyl blue, was loaded into a 10mL syringe, manually injected onto the surface of a glass plate at room temperature (25 ℃), and the writability of the hydrogel was visually observed.

2. Test results

Self-healing results of hydrogels as shown in fig. 3, ALG-HA hydrogel (fig. 3A) appeared gel-like at 0.1% strain, with G 'and G "dropping rapidly and G' < G" as the strain increased rapidly from 0.1% to 100%, indicating that the gel network was broken, transitioning from gel to sol. When the strain is reduced to 0.1% again, the hydrogel completes the sol-to-gel transition in a short time (about 0.8min), the gel state is restored, and G' and G ″ are completely restored to be equal to the initial values; after 4 cycles of strain, G' and G "are still equal to the initial values. The result shows that the ALG-HA hydrogel HAs rapid self-repairing capability, almost no loss of mechanical strength and 100 percent of self-repairing capability.

In contrast, HA hydrogels that are liquid in the macroscopic state (HA-Fe-EDTA, fig. 3B) always behave G "> G', i.e. fluid properties; while the ALG hydrogel (ALG-Fe-EDTA, fig. 3C) is G ' > G ", it is considered that G ' and G" are significantly decreased and G "> G" is significantly decreased after being damaged by high stress in the cyclic strain time scanning, and G ' and G "are increased to some extent and show G ' > G" after high stress removal, but the difference from the initial modulus value is large and the values of G ' and G "are very close to each other, and thus the self-repairing performance is not achieved.

Shear rate sweep experiments (fig. 4A) show that as the shear rate increases, the viscosity of the ALG-HA hydrogel decreases, indicating that the hydrogel HAs shear-thinning properties. In-vitro injection experiments further define the injectability of the ALG-HA hydrogel. As shown in fig. 4B, ALG-HA hydrogel was injected into the glass vial at room temperature using a syringe, and the vial was inverted immediately after injection, the hydrogel remained in a solid nature without falling off, i.e., the sol-gel transition of ALG-HA hydrogel occurred rapidly within seconds. Fig. 4C shows that the ALG-HA hydrogel can be injected into water by different sized syringes and remains continuous without breaking, without spreading out during this process, illustrating that it can be adapted to local injection treatment of tissue defects of different sizes and shapes. Finally, the writability of the ALG-HA hydrogel was tested, and at room temperature, the ALG-HA hydrogel was injected on the glass surface, and had self-repairing ability without any external force interference, and could be formed into any three-dimensional shape (fig. 4D). These findings indicate that the ALG-HA hydrogels of the present invention have excellent self-healing, shear thinning, and syringeability, further confirming the potential for the application of the hydrogels as injectable carriers.

Test example 3 study of antibacterial Properties of hydrogel of the present invention

1. Test method

1.5mL of the ALG-HA hydrogel prepared in example 1 was added to a 15mL centrifuge tube, and 3mL of a bacterial solution (10 mL) was added after UV sterilization in a clean bench for 40min⁵CFU/mL) in equal volume of sterilized dH₂O as a blank control (n ═ 3). Co-culturing at 37 deg.C for 24 hr, taking bacteria liquid, diluting with gradient, and inoculating on agarSolid broth medium. The in vitro antimicrobial activity of the hydrogels was determined by bacterial colony counting. The bacteria were selected from Escherichia coli and Staphylococcus aureus.

2. Statistical analysis

The mean and standard deviation are used for each group of data. Using SPSS 16.0 software, the relevant experimental data were analyzed using the t-test, and differences were statistically significant when p < 0.05. Use, < 0.05; p < 0.01; p <0.01, representing significant differences.

3. Test results

Bacterial colony count statistics (fig. 5) show: compared with a control group, the bacterial colony number of the ALG-HA hydrogel group is obviously reduced, which shows that the ALG-HA hydrogel HAs obvious antibacterial effect on both E.coli and S.aureus.

In conclusion, the ALG-HA hydrogel disclosed by the invention is uniform and stable, HAs excellent self-repairing performance, shear thinning performance and injectability, and also HAs excellent antibacterial performance. It can be used as injectable carrier, and can be adapted to local injection treatment of tissue defects with different sizes and shapes. In addition, the hydrogel is simple in raw materials, fast in forming, simple and convenient to prepare, green and environment-friendly, and has a good application prospect.

Claims (10)

1. An injectable antibacterial polymer hydrogel, which is characterized in that: it is prepared from alginate, hyaluronate, compound containing iron ions and disodium ethylenediamine tetraacetate as raw materials.

2. The hydrogel of claim 1, wherein: the compound is prepared by mixing a mixed solution of alginate and hyaluronate with a mixed solution of a compound containing iron ions and disodium ethylenediamine tetraacetic acid.

3. The hydrogel of claim 2, wherein: the volume ratio of the mixed solution of the alginate and the hyaluronate to the mixed solution of the compound containing the iron ions and the disodium ethylene diamine tetraacetate is (1-50): 1.

4. the hydrogel of claim 3, wherein: the volume ratio of the mixed solution of the alginate and the hyaluronate to the mixed solution of the compound containing the iron ions and the disodium ethylene diamine tetraacetate is (1-10): 1;

preferably, the volume ratio of the mixed solution of the alginate and the hyaluronate to the mixed solution of the compound containing the iron ions and the disodium ethylene diamine tetraacetate is 5: 1.

5. the hydrogel of claim 2, wherein: the concentration of alginate in the alginate and hyaluronate mixed solution is 1-5% (w/v), and the concentration of hyaluronate is 0.1-1% (w/v);

preferably, the concentration of the alginate salt and the hyaluronic acid salt in the mixed solution of the alginate salt and the hyaluronic acid salt is 3% (w/v) and the concentration of the hyaluronic acid salt is 0.75% (w/v).

6. The hydrogel of claim 2, wherein: the concentration of the iron ions in the mixed solution of the compound containing the iron ions and the disodium ethylene diamine tetraacetate is 0.1-1 mol/L, and the concentration of the disodium ethylene diamine tetraacetate is 0.01-0.1 mol/L;

preferably, the concentration of the iron ions in the mixed solution of the iron ion-containing compound and the disodium ethylene diamine tetraacetate is 0.1mol/L, and the concentration of the disodium ethylene diamine tetraacetate is 0.05 mol/L.

7. The hydrogel according to any one of claims 1 to 6, wherein: the alginate is sodium alginate; and/or the hyaluronate is sodium hyaluronate; and/or the compound containing iron ions is Fe³⁺A compound of (1);

preferably, the Fe-containing³⁺The compound of (a) is ferric sulfate.

8. A method for producing the hydrogel according to any one of claims 1 to 7, characterized in that: it comprises the following steps:

(1) dissolving alginate and hyaluronate in water to obtain a mixed solution A;

(2) dissolving a compound containing iron ions and disodium ethylene diamine tetraacetate in water to obtain a mixed solution B;

(3) adding the mixed solution B into the mixed solution A to obtain a mixed solution A;

preferably, the mixed solution B is stirred when being added into the mixed solution A.

9. Use of the hydrogel of any one of claims 1 to 7 for the preparation of an injectable antimicrobial drug carrier.

10. Use of the hydrogel of any one of claims 1 to 7 in the preparation of a tissue repair material.

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