CN112480435B

CN112480435B - Injectable antibacterial hydrogel material and preparation method thereof

Info

Publication number: CN112480435B
Application number: CN202011389763.1A
Authority: CN
Inventors: 冯茜; 杨雅燕; 肖秀峰; 于永生
Original assignee: Fujian Normal University
Current assignee: Fujian Normal University
Priority date: 2020-12-02
Filing date: 2020-12-02
Publication date: 2022-07-05
Anticipated expiration: 2040-12-02
Also published as: CN112480435A

Abstract

The invention discloses an injectable antibacterial hydrogel material and a preparation method thereof, and relates to an antibacterial hydrogel which is composed of modified hyaluronic acid and oxidized chitosan and has injectability, good biocompatibility and tissue adhesion performance. In addition, the common bactericide hypochlorous acid is generated in the hydrogel forming process, the instant sterilization effect can be achieved, and the continuous antibiosis can be realized by the positive charge on the oxidized chitosan. The antibacterial hydrogel disclosed by the invention is convenient and quick to prepare, does not need expensive reagents, and is convenient to use.

Description

Injectable antibacterial hydrogel material and preparation method thereof

Technical Field

The invention belongs to the technical field of medical biomaterials, and particularly designs an antibacterial hydrogel and a preparation method and application thereof.

Background

Bacteria have extremely strong adaptability and are ubiquitous in the natural environment, and wound bacterial infection is very common in daily life. Such as mechanical damage, improper temperature and chemical-induced skin damage. If the potential infectious microorganisms and necrotic tissue cannot be removed in a timely manner, they are easily infected with bacteria. The selection of an appropriate wound dressing is important for the treatment of infection, and factors such as the good or poor hemostatic effect, the good or poor air permeability, and the excellent or poor moisture retention, comfort and flexibility are important factors for the wound dressing.

At present, the following antibacterial strategies include photodynamic antibacterial therapy, hydrophilic antifouling coatings, metal nano materials and the like. Photodynamic antibacterial therapy is a technique for diagnosing and treating diseases by utilizing the photodynamic effect generated by light and a photosensitizer. The disadvantage is that the photodynamic effect generated by using different photosensitizers has different curative effects on different types of bacterial infections, and the antibacterial effect is also related to various factors such as the action mechanism of the photosensitizers, the types of bacteria, the growth states of the bacteria and the like. The hydrophilic antifouling coating is widely applied to the fields of self-cleaning, antifogging, antifouling, oil-water separation and the like, but the process for preparing the hydrophilic antifouling coating mostly involves expensive instruments or complex process flows. In addition, the super-hydrophilic surface prepared by the prior art is easy to be damaged by external factors such as force, light, temperature and the like, and cannot meet the requirement of long-term use. The metal nano material is widely applied in the early development stage, but the application range of the organic antibacterial agent is limited due to poor heat resistance.

The above strategies have certain limitations, such as complex operation, harmful ion release and the like. The hydrogel has a three-dimensional polymer network structure, is similar to extracellular matrix (ECM), has good biocompatibility and degradability, and is suitable for the wound antibacterial dressing material.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide an antibacterial wound dressing which can be gelatinized in situ, has good antibacterial performance and good biocompatibility and a preparation method thereof. Meanwhile, the preparation method of the antibacterial hydrogel disclosed by the invention is simple and feasible.

The invention provides a preparation method of an antibacterial hydrogel wound dressing, which comprises the following steps:

the method comprises the following steps: firstly, dissolving chitosan powder in dilute glacial acetic acid aqueous solution, and then adding sodium periodate (NaIO)₄) Stirring for 24 h in the dark.Subsequently, dialyzed against deionized water to remove unreacted small molecule NaIO₄. The obtained oxidized chitosan sample is stored in a refrigerator at the temperature of-20 ℃ after being frozen and dried.

Step two: hyaluronic acid powder is first dissolved in a buffer solution. EDC and HOBT were added, and after stirring for 1 hour, adipic acid dihydrazide was added and the reaction was carried out for 24 hours. Subsequently, the HA-ADH sample obtained by dialysis with deionized water was freeze-dried and stored in a refrigerator at-20 ℃.

Step three: dissolving the obtained oxidized chitosan in salicylic acid solution to obtain solution 1, dissolving HA-ADH in calcium hypochlorite solution to obtain solution 2, and physically mixing the solution 1 and the solution 2 according to a certain proportion to obtain the novel injectable antibacterial hydrogel material.

In the scheme, the concentration of the glacial acetic acid solution in the first step is 0.5 wt%.

In the scheme, the molecular mass of the chitosan in the first step is 30000Da, the concentration of the chitosan is 1g/100mL, and the mass ratio of the added chitosan to the sodium periodate is 1: 0.66.

In the scheme, the dialysis in the step one is to dialyze the mixture for 5 days by using deionized water, and the deionized water is replaced 4 times every day.

In the above scheme, the buffer solution in step two is morpholine ethanesulfonic acid buffer solution, and the pH value thereof is 6.5.

In the scheme, the molecular weight of the hyaluronic acid in the step two is 920000 Da, the concentration of the hyaluronic acid is 1g/100mL, the mass ratio of the added hyaluronic acid to EDC is 1:1.25, the mass ratio of the added hyaluronic acid to HOBT is 1:0.9, and the mass ratio of the added hyaluronic acid to adipic dihydrazide is 1: 4.5.

In the scheme, 8 mg, 10 mg, 12 mg and 14 mg of chitosan oxide are respectively dissolved in every 100 uL of salicylic acid solution in the step three, and 12 mg of HA-ADH is dissolved in every 100 uL of calcium hypochlorite solution; and step three, physically mixing the solution 1 and the solution 2 according to the volume ratio of 1:1.

The invention provides an antibacterial hydrogel wound dressing prepared according to the preparation method.

The invention has the beneficial effects that: in the preparation method of the antibacterial hydrogel wound dressing disclosed by the invention, the hyaluronic acid containing the modified natural polymer oxidized chitosan and the grafted hydrazide can be chemically crosslinked under a mild condition to form a three-dimensional reticular hydrogel structure, and the antibacterial hydrogel wound dressing has the advantages of good hydrophilicity, proper swelling property, good biocompatibility, self-healing property and tissue adhesion property. In addition, the slow release of the common bactericide in the hydrogel forming process can achieve instant disinfection; and the oxidized chitosan has positive charges in the hydrogel, so that sustainable antibiosis can be realized. These properties make the hydrogel have wide application prospects in clinical treatment.

Drawings

FIG. 1 is a schematic diagram of the preparation and antibacterial mechanism of the hydrogel of the present invention;

FIG. 2 shows the preparation of OCS and HA-ADH and its IR spectrum according to the present invention;

FIG. 3 is a graph of the swelling behavior of an OCS/HA hydrogel;

FIG. 4 is a graph of OCS/HA hydrogel degradation behavior;

FIG. 5a is a graph of time-scanning rheological behavior of an OCS/HA hydrogel;

FIG. 5b is a graph of time-scanning rheological behavior of an OCS/HA-HClO hydrogel;

FIG. 5c is a graph of frequency-sweep rheological behavior of an OCS/HA-HClO hydrogel;

FIG. 5d is a graph of strain-swept rheological behavior of an OCS/HA-HClO hydrogel;

FIG. 6a is a diagram of a hydrogel self-healing process;

FIG. 6b is a representation of the injectability of a hydrogel;

FIG. 6c is an electron photograph of hydrogel adhering to pigskin;

FIG. 7a is a hydrogel in vitro antibacterial performance test;

figure 7b is a hydrogel biocompatibility test.

Detailed Description

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention, which is to be given the full breadth of the appended claims and any and all equivalents thereof which may be modified by one skilled in the art after reading this disclosure.

OCS = oxidized chitosan; HA-ADH = hydrazide modified hyaluronic acid; SA = salicylic acid; ca (ClO)₂= sodium hypochlorite

FIG. 1 is a schematic diagram of the preparation and antibacterial mechanism of the hydrogel of the present invention.

Examples

The invention provides a preparation method of an injectable antibacterial hydrogel, which comprises the following steps:

(a) and (3) synthesis of OCS:

firstly, 2 g of chitosan with the molecular weight of 30000Da is dissolved in 200 ml of 0.5 wt% dilute glacial acetic acid aqueous solution, and then 1.325 g of sodium periodate (NaIO) is added₄). Stirred at room temperature for 24 h in the dark. Subsequently, dialyzing with deionized water for 4 days to remove unreacted small molecule NaIO₄. And freeze-drying to obtain Oxidized Chitosan (OCS), and storing in a refrigerator at-20 ℃.

The samples were analyzed using fourier transform infrared spectroscopy (FT-IR). Tabletting with KBr at 25 deg.C at 500-4000 cm⁻¹Is measured within the range of (1). The obtained infrared spectrum results are shown in FIG. 2, and the comparison shows that the modified oxidized chitosan is at 2900-^-1The characteristic peak of aldehyde group appears, which indicates that the oxidized chitosan is successfully prepared.

(b) Synthesis of HA-ADH:

first 2 g HA of molecular weight 920000 Da was dissolved in 200 ml morpholine ethanesulfonic acid buffer solution (pH = 6.5). 2.5 g EDC and 1.78 g HOBT were added and stirring was continued at room temperature for 1 h. 9 g of adipic Acid Dihydrazide (ADH) was added thereto, and stirring was continued at room temperature for 24 hours to obtain a crude product, HA-ADH. Dialyzing with deionized water for 4 days, freeze-drying, and storing in a refrigerator at-20 ℃.

The samples were analyzed using fourier transform infrared spectroscopy (FT-IR). Tabletting with KBr at 25 deg.C at 500-4000 cm⁻¹Is measured within the range of (1). The obtained infrared spectrogram result is shown in figure 2, and the comparison can find that the modified HA-ADH HAs a characteristic peak of hydrazide at 1130 cm-1, which indicates that the preparation is successfulHA-ADH。

(c) The preparation method of the antibacterial hydrogel comprises the following steps:

in the first step, PBS is used as a solvent to prepare HA-ADH solution and OCS solution with different concentrations respectively. The two solutions were then mixed in a volume ratio of 1:1 using a double syringe. The hydrogels with different component concentrations were labeled as OCSx/HA hydrogels, i.e., the HA-ADH concentration was constantly 3% and the OCS concentration was labeled as y% (4%, 5%, 6%, 7%).

And secondly, preparing the OCS/HA-HClO hydrogel on the basis of preparing the OCS/HA hydrogel. HA-ADH dissolved in 125 mg/L Ca (ClO)₂In the solution, OCS was dissolved in 2.5 mM SA solution, respectively. The two precursor solutions were then added to the two separate chambers of the dual barrel syringe. The prepared two solutions were mixed and injected into a model or a skin surface in a volume ratio of 1:1, and a series of OCS/HA-HClO hydrogels were prepared by an in situ gel method.

Gel formation time test of hydrogels:

gelation time was measured by vial pouring. HA-ADH and OCS were dissolved in PBS solution, respectively. And mixing and injecting the two solutions by using a double-barrel injector to obtain the HA/OCS hydrogel, wherein the gelling time is determined when the hydrogel HAs no flowing capacity. The OCS/HA-HClO hydrogel gel time was determined in a similar manner and is shown in Table 1. We found that gel time is inversely related to OCS concentration. The OCS/HA hydrogels and OCS/HA-HClO hydrogels showed almost the same gel time, indicating that Ca (ClO)₂The addition of the solution and the SA solution had no effect on the preparation of the hydrogel.

TABLE 1 measurement of gel formation time of hydrogels by vial pouring

Swelling performance test of hydrogel:

all samples (100 uL, n =5) were prepared in teflon molds (diameter 10 mm, height 1 mm), respectively. All samples were soaked in 500uL of PBS buffer for 24 hours at room temperature. The hydrogel was then removed from the PBS buffer at a specified time point of 0Weighing is carried out for 5 h, 1h, 3 h, 7 h, 12 h and 24 h. This weight mark is W_s. The hydrogel was then lyophilized to obtain a dry weight (W)_d). The calculation formula is as follows: swelling ratio (%) =

As shown in FIG. 3, we found that the swelling ratio was 1.5 times the weight of the hydraulic collagen when the OCS concentration was more than 6%. Each group of OCS/HA hydrogel achieves swelling balance after being soaked in PBS for 10 h, which shows that the OCS/HA hydrogel HAs good water absorption performance.

Testing of the degradation properties of the hydrogels:

the OCS₄/HA-HClO、OCS₅/HA-HClO、OCS₆/HA-HClO、OCS₇the/HA-HClO (100 uL, n =5) was soaked in 1 mL PBS buffer at 37 ℃. At specified time intervals (1 h, 4 h, 10 h, 1 d, 2 d, 4 d and 7 d) 100 uL of solution was collected and supplemented with the same volume of PBS solution. The concentration of the collected solution after the background of PBS is subtracted is measured by a microplate reader. As shown in fig. 4, the degradation rate of the hydrogel decreased with increasing OCS concentration, with the degradation rate being minimal when OCS concentration exceeded 6%.

Rheological properties of the hydrogel:

the rheological properties of the hydrogels were obtained by malvern Kinexus rheometer test, all samples were tested with 8 mm flat plates versus 8 mm plate-to-plate spacing. An 80 uL prepared hydrogel sample is placed on a rheometer, and a time scanning test is carried out for 120 s by adopting 1% strain and 1Hz frequency, so as to obtain the storage modulus and the loss modulus of the hydrogel material. Frequency scanning is carried out by adopting strain of 1% and frequency of 0.1-1000 Hz, so as to obtain the law that the storage modulus and the loss modulus of the hydrogel material change along with the frequency. In the shear thinning test, the hydrogel is scanned alternately for 120 s under the conditions of 1% strain and 500% strain, namely after 60 s of low shear under 1% strain, 60 s of high shear under 2000% strain are carried out for 4 cycles, and after the cycle is finished, the low shear under 1% strain of 60 s is measured again, and the frequency is kept unchanged at 1Hz in the whole process.

In a time-sweep experiment, comparing FIGS. 5a and 5b, the results demonstrate SA and Ca (ClO)₂The addition of (b) has no negative effect on the formation of the hydrogel. Combining swelling and degradation experiments simultaneously, we chose OCS₇the/HA-HClO hydrogel was subjected to all the following tests. As shown in FIG. 5c, stress rates above 200% can destroy OCS₇The majority of the crosslinks in the/HA-HClO hydrogel. The hydrogel was thus in a completely fragmented state under the condition of 500% strain rate. A continuous transition test with two strain rates of 1% and 500% was chosen and it was observed from the shear thinning experiment of fig. 5d that the hydrogel repeatedly broke at a strain rate of 500% and recovered at a strain rate of 1%, indicating that the hydrogel has self-healing capabilities.

Self-healing, injectability and tissue adhesion testing of hydrogels:

the self-healing, injectable and adhesive properties were determined by macroscopic experiments. To test the self-healing properties of the hydrogels, we dyed two triangular hydrogel pieces 10 mm across and 1 mm thick with methylene blue and methyl orange, respectively. These behaviors of the hydrogels were monitored by digital photographs at specific time intervals. In the aspect of injection behavior testing, the dyed hydrogel was sequentially injected directly into a pentagram PVC mold. The adhesion of the hydrogels was tested using purchased pigskin after methylene blue staining of the hydrogels.

In the self-healing photomicrograph of fig. 6a, two triangular hydrogels of blue and orange color were in contact with each other, forming an intact rhombohedral hydrogel after 15 minutes. And the color of the contact interface between the two hydrogels became bright green, indicating a good fusion of the two triangles, also indicating excellent self-healing properties of the hydrogels due to the reversible dynamic schiff base reaction occurring between the aldehyde groups on OCS and the hydrazide on HA-ADH. The methylene blue dyed HA-ADH solution and OCS solution were mixed and injected into the left half of a Polytetrafluoroethylene (PTFE) mold. The methylene orange dyed HA-ADH solution and OCS were then mixed and injected into the right half of the same mold. Finally, the OCS/HA-HClO hydrogel was removed from the PTFE mold and the perfect pentagram shape was maintained (FIG. 6 b). These results indicate that the OCS/HA-HClO hydrogel HAs the ability to perfectly match various wound shapes. Furthermore, the OCS/HA-HClO hydrogel had strong tissue adhesion to wet pig skin even with continuous folding and twisting (fig. 6 c). The OCS/HA-HClO hydrogel can be tightly attached to the wound surface after covering the wound surface, and provides comprehensive protection for the wound surface.

In vitro antimicrobial performance testing of hydrogels:

the hydrogels were evaluated for antibacterial activity using escherichia coli and staphylococcus aureus. The antibacterial activity of OCS/HA hydrogel and OCS/HA-HClO hydrogel was evaluated in vitro by agar plate diffusion assay. OCS/HA hydrogels and OCS/HA-HClO hydrogels were selected as the experimental group. Then the prepared solution is mixed with about 1 × 10⁶ CFU ml^-1The following groups were added to the bacterial suspension: 1. PBS buffer (990uL), 2. agarose hydrogel; OCS/HA hydrogel and 4 OCS/HA-HClO hydrogel; 5. penicillin-streptomycin (PS) solution (990 uL). All samples were incubated at 37 ℃ for 2 h and the bacteria were resuspended in 1 ml PBS buffer. Diluting the bacterial suspension (10)⁴ CFU mL⁻¹) Inoculated on the surface of Luria-Bertani (LB) agar and incubated at 37 ℃ for 24 h.

In vitro antibacterial experiments showed that the OCS/HA hydrogel had significant antibacterial properties due to the positive charge of the OCS backbone (FIG. 7 a). More importantly, the bacterial mortality rate of the OCS/HA-HClO hydrogel group reaches 100 percent and is at the same level as that of the antibiotic PS positive control group. These results demonstrate the antimicrobial behavior of the hydrogel, since the freshly released HClO during the formation of the OCS/HA-HClO hydrogel is responsible for the transient disinfection, while the positive charge in the OCS/HA-HClO hydrogel is responsible for the long-term antimicrobial activity.

Biocompatibility testing of hydrogels:

OCS/HA hydrogel and OCS/HA-HClO hydrogel were prepared in 24-well plates (200 uL/hydrogel, n = 4). To a 24-well plate was added 1 ml growth medium containing 20 ten thousand 3T3 cells. After 2 d or 5d incubation, the OCS/HA and OCS/HA-HClO hydrogels were removed from the 24-well plates and the cell viability of the hydrogel surface 3T3 cells was examined by live/dead cell staining.

The biocompatibility of OCS/HA and OCS/HA-HClO hydrogels was tested using 3T3 cells as model cells (FIG. 7 b). 3T3 cells on the surface of OCS/HA and OCS/HA-HClO hydrogels survived most of the time and were not significantly different from the positive control group, indicating that OCS/HA and OCS/HA-HClO hydrogels had good biocompatibility.

The embodiments of the present invention have been described in detail with reference to the above examples, but the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the scope of the present invention, which is defined in the appended claims.

Claims

1. The preparation method of the antibacterial hydrogel is characterized in that the hydrogel consists of oxidized chitosan, hydrazide grafted hyaluronic acid, salicylic acid and calcium hypochlorite solution according to the weight ratio, and the preparation method comprises the following steps:

the method comprises the following steps: firstly, dissolving chitosan powder in dilute glacial acetic acid aqueous solution, and then adding sodium periodate NaIO₄Stirring for 24 hours in a dark place; subsequently, dialyzed against deionized water to remove unreacted small molecule NaIO₄(ii) a Freeze-drying the obtained oxidized chitosan sample, and storing the sample in a refrigerator at the temperature of-20 ℃;

step two: firstly, dissolving hyaluronic acid HA powder in a buffer solution; adding EDC and HOBT, stirring for 1h, adding adipic acid dihydrazide ADH, and reacting for 24 h; then, dialyzing with deionized water to obtain an HA-ADH sample, and storing the HA-ADH sample in a refrigerator at the temperature of-20 ℃ after freeze drying;

step three: and (3) dissolving the oxidized chitosan obtained in the step one in a salicylic acid solution to obtain a solution 1, dissolving the HA-ADH obtained in the step two in a calcium hypochlorite solution to obtain a solution 2, and physically mixing the solution 1 and the solution 2 according to a certain proportion to obtain the novel injectable antibacterial hydrogel material.

2. The method for preparing an antibacterial hydrogel according to claim 1, wherein the concentration of the glacial acetic acid solution in the first step is 0.5 wt%.

3. The method for preparing an antibacterial hydrogel according to claim 1, wherein the molecular mass of the chitosan in the first step is 30000Da, the concentration of the chitosan is 1g/100mL, and the mass ratio of the added chitosan to the sodium periodate is 1: 0.66.

4. The method of claim 1, wherein the dialysis of step one is performed for 5 days with deionized water, and the deionized water is changed 4 times per day.

5. The method for preparing the antibacterial hydrogel according to claim 1, wherein the buffer solution in the second step is morpholine ethanesulfonic acid buffer solution, and the pH value thereof is 6.5.

6. The method of preparing an antibacterial hydrogel according to claim 1, wherein the molecular mass of hyaluronic acid in step two is 920000 Da, the concentration of hyaluronic acid is 1g/100mL, the mass ratio of added hyaluronic acid to EDC is 1:1.25, the mass ratio of added hyaluronic acid to HOBT is 1:0.9, and the mass ratio of added hyaluronic acid to adipic acid dihydrazide is 1: 4.5.

7. The method for preparing antibacterial hydrogel according to claim 1, wherein 8 mg, 10 mg, 12 mg, 14 mg of oxidized chitosan are dissolved in each 100 μ L of salicylic acid solution in the third step, and 12 mg of HA-ADH is dissolved in each 100 μ L of calcium hypochlorite solution; and step three, physically mixing the solution 1 and the solution 2 according to the volume ratio of 1:1.

8. An injectable antibacterial hydrogel obtained by the method for preparing an antibacterial hydrogel according to any one of claims 1 to 7.