CN115337448B

CN115337448B - Tannic acid-coupled polyphosphazene-based hydrogel wound dressing with anti-inflammatory, antibacterial and ROS responsive properties and preparation method thereof

Info

Publication number: CN115337448B
Application number: CN202211013349.XA
Authority: CN
Inventors: 俞豪杰; 倪志鹏; 王立
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-08-23
Filing date: 2022-08-23
Publication date: 2023-06-13
Anticipated expiration: 2042-08-23
Also published as: CN115337448A

Abstract

The invention discloses tannic acid coupled polyphosphazene hydrogel with anti-inflammatory, antibacterial and ROS response performances and a preparation method thereof. Polyphosphazene-based hydrogel wound dressings are obtained by sequentially reacting polyphosphazene with tannic acid and a polyvinyl alcohol solution. The obtained PPBA-TA-PVA hydrogel can effectively inhibit the growth of escherichia coli within 4 hours, and can effectively remove 1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radical and OH free radical and simultaneously show degradation behavior. The invention covalently bonds the ROS-responsive boric acid ester bond with the tannic acid anti-inflammatory small molecule, and has the characteristics of safe and low-toxicity ROS response release. The invention has good anti-inflammatory, antibacterial and ROS response performances, can effectively shorten the inflammation stage of the diabetes wound and accelerate the wound healing by reducing proinflammatory cytokines (IL-6 and IL-1 beta), and has wide application prospect in the field of biomedical materials.

Description

Tannic acid coupled polyphosphazene-based hydrogel wound dressing with anti-inflammatory, antibacterial and ROS response performances and preparation method thereof

Technical Field

The invention belongs to the technical field of high polymer materials, and particularly relates to a tannic acid coupled polyphosphazene hydrogel wound dressing with anti-inflammatory, antibacterial and ROS response performances and a preparation method thereof.

Technical Field

Diabetic wounds are a serious and common complication of diabetes, and current treatment regimens remain nonspecific. By 2035, patients with diabetic wounds are expected to reach 9000 tens of thousands to 1.5 million people, of whom about 20% of moderately severe diabetic wounds face amputation. There is a great deal of evidence that in diabetic wounds, long-term low-level inflammation accumulates excessive Reactive Oxygen Species (ROS) in the wound, disrupting vital processes of wound repair, such as angiogenesis, ECM remodeling and re-epithelialization, thereby preventing the wound from developing from the inflammatory phase to the healing, increasing phase. In addition, the hyperglycemic microenvironment of the diabetic wound is more susceptible to microbial infection than other wound surfaces, bacterial infection often causes additional accumulation of ROS, causes insufficient oxygenation, further slows down the healing of the diabetic wound surface, and also causes abnormal lesions (neuropathy, vascular lesions and other complex systemic effects) to cause difficult healing. Therefore, reducing ROS levels of the wound and effectively eliminating microorganisms are critical to diabetic wound healing.

An ideal diabetic wound dressing should have the following characteristics: 1) Has good ROS scavenging performance, reduces diabetes wound inflammation, and shortens inflammation stage; 2) Has good anti-infective property; 3) Good compatibility to human tissue, no tissue injury or infection risk; 4) The use is simple and convenient, and the use is convenient for wounded; 5) Can be stored for a long time effectively, and has stable property in a wider temperature range.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide a tannic acid coupled polyphosphazene-based hydrogel wound dressing with anti-inflammatory, antibacterial and ROS response performances and a preparation method thereof.

The invention is realized by the following technical scheme:

1. tannic acid coupled polyphosphazene-based hydrogel wound dressing with anti-inflammatory, antibacterial and ROS response performance

Adopting PPBA-TA nano conjugate containing phenylboronic acid group and quaternary ammonium salt group functionalized polyphosphazene PPBA and tannic acid TA as anti-inflammatory and antibacterial components; the PPBA-TA nano conjugate reacts with polyvinyl alcohol through forming ROS-responsive boric acid ester bond to obtain polyphosphazene-based hydrogel wound dressing (PPBA-TA-PVA) with ROS-responsive degradation performance.

2. Preparation method of tannic acid coupled polyphosphazene-based hydrogel wound dressing with anti-inflammatory, antibacterial and ROS response performances

The functionalized polyphosphazene PPBA containing phenylboronic acid groups and quaternary ammonium salt groups is specifically poly [ N, N-dimethyl-aminoethyl-p-methylbenzeneammonium bromide ] (N, N-dimethyl ethylenediamine) ] phosphazene; the grafting rate of boric acid groups of polyphosphazene is 15-30%.

Firstly, respectively and fully dissolving phenylboronic acid group-containing and quaternary ammonium salt group-containing functionalized polyphosphazene PPBA, tannic acid TA and polyvinyl alcohol PVA in deionized water to respectively obtain polyphosphazene PPBA solution, tannic acid TA solution and polyvinyl alcohol PVA solution; and adding the polyphosphazene PPBA solution into the tannic acid TA solution, mixing and stirring to obtain a PPBA-TA nano conjugate, adding the PPBA-TA nano conjugate into the polyvinyl alcohol PVA solution, mixing and stirring to obtain the tannic acid coupled polyphosphazene hydrogel wound dressing with anti-inflammatory, antibacterial and ROS response properties.

The modified poly phosphazene PPBA comprises 10-30 parts by mass of phenylboronic acid groups and quaternary ammonium salt groups, 1-3 parts by mass of tannic acid TA, 10-30 parts by mass of polyvinyl alcohol PVA, 1000-2000 parts by mass of the sum of deionized water of the three solutions, and the proportion of the deionized water in the three solutions is equal to the mass part ratio of the three solutions.

The mixing and stirring time is 10-30 minutes.

The rotational speed of the mixing and stirring is 500-1000 rpm.

The particle size of the PPBA-TA nano conjugate is 50-200 nm.

The hydrogel wound dressing has good contact antibacterial performance, and the gram-negative bacterium escherichia coli suspension is coated on the surface of the hydrogel obtained in the step 3) for culture at 37 ℃, so that the 4h growth inhibition rate of the hydrogel on escherichia coli reaches 93.1+/-1.1%, and the 8h growth inhibition rate reaches 99.4+/-0.6%.

The hydrogel wound dressing can effectively remove 1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radical and OH free radical. Each hydrogel was able to scavenge 100 parts DPPH free radical (0.1 mM) with 100 parts OH free radical (1 mM) in 20 hours, in parts by mass.

The hydrogel wound dressing has an ROS response function, can be degraded in an OH free radical solution (1 mM), and has a positive correlation with the coupling concentration of TA, and the hydrogel wound dressing is immersed in the OH free radical solution, so that the hydrogel degradation rate is 40-98% in 96 hours.

The hydrogel wound dressing is capable of reducing proinflammatory cytokines (IL-6, IL-1. Beta.).

The invention has the following beneficial effects:

1. the tannic acid coupled polyphosphazene-based hydrogel wound dressing with anti-inflammatory, antibacterial and ROS response performances has good antibacterial performances, rich quaternary ammonium salt groups and protonatable tertiary amine groups provided by poly [ N, N-dimethyl-aminoethyl-p-methylbenzeneammonium bromide) (N, N-dimethyl ethylenediamine) ] phosphazene (PPBA) in a hydrogel skeleton can be adsorbed to the surface of a thallus after being contacted with the thallus, penetrate through the cell wall, disturb the composition of cell membranes through the change of osmotic pressure and the decomposition of organic matters, promote the leakage of intracellular substances (DNA and RNA), further lead the thallus to die, avoid the use of antibiotics, slow down the appearance of multi-drug resistant bacteria, and have long-term and effective antibacterial performances.

2. The tannic acid coupled polyphosphazene-based hydrogel wound dressing with anti-inflammatory, antibacterial and ROS response performances has good anti-inflammatory performances. The hydrogel is coupled with natural polyphenol tannic acid with anti-inflammatory effect, and phenolic hydroxyl groups in the tannic acid can be combined with free radicals to form a o-benzoquinone structure, so that the effect of free radical scavenging is exerted.

3. The tannic acid coupled polyphosphazene-based hydrogel wound dressing with anti-inflammatory, antibacterial and ROS response performance has good ROS response performance, ROS can attack a single bond between boron and benzene ring to form an unstable peroxide intermediate, and then hydrolysis and boric acid group shedding are carried out, so that the separation of the boric acid group and the benzene ring is caused. The hydrogel is capable of releasing linked TAs as needed at high ROS levels, thereby maintaining the ROS at reasonable concentrations in the wound.

4. The tannin-coupled polyphosphazene-based hydrogel wound dressing with anti-inflammatory, antibacterial and ROS response performances can greatly shorten the inflammatory stage in a rat diabetes wound model by reducing proinflammatory cytokines (IL-6 and IL-1 beta).

Drawings

FIG. 1 shows the contact inhibition of E.coli by the hydrogel prepared in example 1.

FIG. 2 shows the DPPH radical scavenging rate of the hydrogel prepared in example 1.

FIG. 3 shows the OH radical scavenging rate of the hydrogels prepared in example 1.

FIG. 4 shows the degradation rate of the hydrogel prepared in example 1 in OH radical solution.

FIG. 5 is a graph showing the healing rate of the hydrogel prepared in example 1 on diabetic wounds of rats.

FIG. 6 shows the contact inhibition of E.coli by the hydrogel prepared in example 2.

FIG. 7 shows the DPPH radical scavenging rate of the hydrogel prepared in example 2.

FIG. 8 shows the OH radical scavenging rate of the hydrogels prepared in example 2.

FIG. 9 shows the degradation rate of the hydrogel prepared in example 2 in OH radical solution.

Fig. 10 is a graph showing the healing rate of the hydrogel prepared in example 2 to diabetic wounds of rats.

FIG. 11 shows the contact inhibition ratio of E.coli by the hydrogel prepared in example 3.

FIG. 12 shows the DPPH radical scavenging rate of the hydrogel prepared in example 3.

FIG. 13 shows the OH radical scavenging rate of the hydrogels prepared in example 3.

FIG. 14 shows the degradation rate of the hydrogel prepared in example 3 in OH radical solution.

FIG. 15 is a graph showing the healing rate of diabetic wounds in rats by the hydrogel prepared in example 3.

Detailed Description

The present invention will be described in more detail with reference to the following specific examples, but the embodiments of the present invention are not limited thereto.

Embodiments of the invention are as follows:

the functionalized polyphosphazene PPBA containing phenylboronic acid groups and quaternary ammonium salt groups is specifically poly [ N, N-dimethyl-aminoethyl-p-methylbenzeneammonium bromide ] (N, N-dimethyl ethylenediamine) ] phosphazene.

Example 1

30mg of polyphosphazene (PPBA), 1mg of Tannic Acid (TA) and 30mg of polyvinyl alcohol (PVA) are dissolved in 900 mu l, 100 mu l and 1000 mu l of deionized water respectively, and the mixture is completely dissolved.

Firstly, respectively and fully dissolving phenylboronic acid group-containing and quaternary ammonium salt group-containing functionalized polyphosphazene PPBA, tannic acid TA and polyvinyl alcohol PVA in deionized water to respectively obtain polyphosphazene PPBA solution, tannic acid TA solution and polyvinyl alcohol PVA solution; and then adding the polyphosphazene PPBA solution into the tannic acid TA solution, mixing and stirring to obtain the PPBA-TA nano-conjugate, wherein the particle size of the PPBA-TA nano-conjugate is 50-200 nm. And finally, adding the PPBA-TA nano conjugate into a polyvinyl alcohol PVA solution, mixing and stirring to obtain the tannic acid coupled polyphosphazene-based hydrogel wound dressing with anti-inflammatory, antibacterial and ROS response performances. Wherein the mixing and stirring time is 10 minutes, and the rotating speed is 1000rpm.

Will be in logarithmic phase at a concentration of 10. Mu.l10 ⁶ The CFU bacterial suspension of escherichia coli ATCC 25922 was dispersed on the surface of the hydrogel and cultured for 1h, 2h, 4h and 8h in a constant temperature incubator at 37 ℃. After co-incubation, 1ml of PBS solution was added to the hydrogel surface and sonicated for 30 minutes. 100. Mu.l of the solution was plated with LB solid medium and incubated overnight in a 37℃incubator, and the number of colonies on the medium was counted.

0.4ml of the hydrogel provided by the invention was incubated with 30ml of DPPH radical solution (0.1 mM) at 37 ℃ in the absence of light, and the DPPH radical scavenging ability of the PPBA-TA-PVA hydrogel was measured at different times by means of 517nm ultraviolet absorption intensity. 0.4ml of the hydrogel provided by the invention was incubated with 25ml of OH free radical solution (1 mM) at 37℃in the absence of light, and the amount of solution was taken at various times and mixed with an equal volume of TMB (10 mM, DMSO) for 10min. The ability of PPBA-TA-PVA hydrogels to scavenge hydroxyl radicals at various times was measured by UV absorbance at 904 nm.

1ml of the hydrogel provided by the invention was incubated with 50ml of OH free radical solution (1 mM) at 37℃in the absence of light, and the ROS-responsive degradation capacity of the hydrogel wound dressing was determined by observing the consumption of the hydrogel at different times.

Taking a streptomycin-induced rat diabetes wound model as an object, filling the hydrogel provided by the invention in a wound, periodically changing dressing, and observing the healing condition of the wound to determine the property of the hydrogel wound dressing in promoting the healing of the diabetes wound.

In the healing process of the diabetes wound, the tissues around the wound are taken on the 10 th day respectively, genes related to inflammatory factors IL-6 and IL-1 beta are subjected to fluorescence quantification pcr, and the performance of the hydrogel wound dressing in the aspect of reducing the inflammation level of the wound is measured.

The results of this example are shown in fig. 1 to 5, and fig. 1 shows the contact antibacterial performance of the obtained hydrogel against escherichia coli, and it can be seen from the graph that the gel material has excellent contact antibacterial performance. The bacteriostasis rate of 1h can reach 46.7+/-5.2%, and the bacteriostasis rate of 8h can reach 98.3+/-1.1%, so that bacteria can be basically killed. Figures 2 and 3 show the scavenging properties of the resulting hydrogels for DPPH radicals and OH radicals, and it can be seen from the figures that the gel materials have good ROS scavenging properties. It can scavenge 94.7+ -1.9% DPPH free radical in 70h and 93.6+ -0.3% OH free radical in 8h. FIG. 4 is a graph showing the ROS response degradation of the resulting hydrogel, as can be seen from the graph, the gel material degrades about 27.8.+ -. 2.0% of the hydrogel in an OH free radical solution for 96 hours. Fig. 5 shows the healing of a diabetic wound treated with a gel material, and shows that the hydrogel group has a wound healing rate of 65.0±3.2% on the 6 th day of wound healing, and the present invention can promote the healing of a diabetic wound more than the control group (44.0±4.4%). In the healing process of the diabetes wound, the expression amount of the inflammatory factors IL-6 and IL-1 beta in the group of the invention is 0.53 times and 0.59 times of that of the blank control group on the 10 th day.

Example 2

30mg of polyphosphazene (PPBA), 2mg of Tannic Acid (TA) and 30mg of polyvinyl alcohol (PVA) are dissolved in 800 μl, 200 μl and 1000 μl deionized water respectively, and the solution is completely dissolved.

Firstly, respectively and fully dissolving phenylboronic acid group-containing and quaternary ammonium salt group-containing functionalized polyphosphazene PPBA, tannic acid TA and polyvinyl alcohol PVA in deionized water to respectively obtain polyphosphazene PPBA solution, tannic acid TA solution and polyvinyl alcohol PVA solution; and then adding the polyphosphazene PPBA solution into the tannic acid TA solution, mixing and stirring to obtain the PPBA-TA nano-conjugate, wherein the particle size of the PPBA-TA nano-conjugate is 50-200 nm. And finally, adding the PPBA-TA nano conjugate into a polyvinyl alcohol PVA solution, mixing and stirring to obtain the tannic acid coupled polyphosphazene-based hydrogel wound dressing with anti-inflammatory, antibacterial and ROS response performances. Wherein the mixing and stirring time is 30 minutes, and the rotating speed is 500rpm.

Will be in logarithmic phase at a concentration of 10. Mu.l of 10 ⁶ The CFU bacterial suspension of escherichia coli ATCC 25922 was dispersed on the surface of the hydrogel and cultured for 1h, 2h, 4h and 8h in a constant temperature incubator at 37 ℃. After co-incubation, 1ml of PBS solution was added to the hydrogel surface and sonicated for 30 minutes. 100. Mu.l of the solution was plated with LB solid medium and incubated overnight in a 37℃incubator, and the number of colonies on the medium was counted.

The results of this example are shown in fig. 6 to 10, and fig. 6 shows the contact antibacterial performance of the obtained hydrogel against escherichia coli, and it can be seen from the graph that the gel material has excellent contact antibacterial performance. The bacteriostasis rate of 1h can reach 32.2+/-4.0%, and the bacteriostasis rate of 8h can reach 89.5+/-1.2%, so that bacteria can be basically killed. Fig. 7 and 8 show the scavenging properties of the resulting hydrogels for DPPH radicals and OH radicals, and it can be seen that the gel materials have good ROS scavenging properties. It can scavenge 99.6+ -1.4% DPPH free radical in 70h and scavenge 97.4+ -0.1% OH free radical in 8h. FIG. 9 is a graph showing the ROS response degradation of the resulting hydrogel, as can be seen from the graph, the gel material degrades about 60.5.+ -. 2.6% of the hydrogel in an OH free radical solution for 96 hours. Fig. 10 shows the healing of a diabetic wound treated with a gel material, and shows that the hydrogel group has a wound healing rate of 83.1±2.6% on the 6 th day of wound healing, and the present invention can promote the healing of a diabetic wound more than the control group (44.0±4.4%). In the healing process of the diabetes wound, the expression amount of the inflammatory factors IL-6 and IL-1 beta in the group of the invention is 0.53 times and 0.50 times of that of the blank control group on the 10 th day.

Example 3

30mg of polyphosphazene (PPBA), 3mg of Tannic Acid (TA) and 30mg of polyvinyl alcohol (PVA) are dissolved in 700 mu l, 300 mu l and 1000 mu l of deionized water respectively, and the solution is completely dissolved.

Firstly, respectively and fully dissolving phenylboronic acid group-containing and quaternary ammonium salt group-containing functionalized polyphosphazene PPBA, tannic acid TA and polyvinyl alcohol PVA in deionized water to respectively obtain polyphosphazene PPBA solution, tannic acid TA solution and polyvinyl alcohol PVA solution; and then adding the polyphosphazene PPBA solution into the tannic acid TA solution, mixing and stirring to obtain the PPBA-TA nano-conjugate, wherein the particle size of the PPBA-TA nano-conjugate is 50-200 nm. And finally, adding the PPBA-TA nano conjugate into a polyvinyl alcohol PVA solution, mixing and stirring to obtain the tannic acid coupled polyphosphazene-based hydrogel wound dressing with anti-inflammatory, antibacterial and ROS response performances. Wherein the mixing and stirring time is 30 minutes, and the rotating speed is 1000rpm.

In the healing process of the diabetes wound, the tissues around the wound are taken on

days

3, 6 and 10 respectively, genes related to inflammatory factors IL-6 and IL-1 beta are subjected to fluorescence quantification pcr, and the performance of the hydrogel wound dressing in the aspect of reducing the inflammation level of the wound is measured.

The results of this example are shown in fig. 11 to 15, and fig. 11 shows the contact antibacterial performance of the obtained hydrogel against escherichia coli, and it can be seen from the graph that the gel material has excellent contact antibacterial performance. The bacteriostasis rate of 1h can reach 73.1+/-4.6%, and the bacteriostasis rate of 8h can reach 99.4+/-0.6%, so that bacteria can be basically killed. Fig. 12 and 13 show the scavenging properties of the resulting hydrogels for DPPH radicals and OH radicals, and it can be seen that the gel materials have good ROS scavenging properties. It can scavenge 98.4+ -2.1% DPPH free radical in 70h and scavenge 81.1+ -4.9% OH free radical in 8h. FIG. 14 is a graph showing ROS response degradation of the hydrogel, as can be seen from the graph, the gel material degrades about 60.5.+ -. 2.6% of the hydrogel in an OH free radical solution for 96 hours. Fig. 15 shows the healing of a diabetic wound treated with a gel material, and shows that the hydrogel group has a wound healing rate of 93.1±2.6% on the 6 th day of wound healing, and the present invention can promote the healing of a diabetic wound more than the control group (44.0±4.4%). In the healing process of the diabetes wound, the expression amount of the inflammatory factors IL-6 and IL-1 beta of the tissue around the wound is taken on the 10 th day, and the inflammatory factors of the group of the invention are obviously less than 0.40 times and 0.76 times of that of a blank control group.

Claims

1. A polyphosphazene-based hydrogel wound dressing with anti-inflammatory, antibacterial and ROS response properties coupled with tannic acid, characterized in that, the functionalized polyphosphorus polyphosphate containing phenylboronic acid group and quaternary ammonium salt group is used The PPBA-TA nanoconjugates of nitrile PPBA and tannic acid TA are used as anti-inflammatory and antibacterial components; PPBA-TA nanoconjugates react with polyvinyl alcohol through the formation of ROS-responsive borate bonds to obtain polymers with ROS-responsive degradation properties. Phosphazene-based hydrogel wound dressings.

2. a kind of preparation method of the polyphosphazene-based hydrogel wound dressing that has anti-inflammatory, antibacterial and ROS response properties of tannic acid coupling according to claim 1, is characterized in that, comprises the following steps:

Firstly, functionalized polyphosphazene PPBA containing phenylboronic acid groups and quaternary ammonium salt groups, tannic acid TA and polyvinyl alcohol PVA were fully dissolved in deionized water to obtain polyphosphazene PPBA solution and tannic acid TA solution respectively. and polyvinyl alcohol PVA solution; then add polyphosphazene PPBA solution to tannic acid TA solution, mix and stir to obtain PPBA-TA nano-conjugates, and finally add PPBA-TA nano-conjugates to polyvinyl alcohol PVA solution In this process, a tannic acid-coupled polyphosphazene-based hydrogel wound dressing with anti-inflammatory, antibacterial and ROS responsive properties was obtained by mixing and stirring.

3. a kind of preparation method of the polyphosphazene-based hydrogel wound dressing that has anti-inflammatory, antibacterial and ROS responsive performance of tannic acid coupling according to claim 2, it is characterized in that: in parts by mass, The functionalized polyphosphazene PPBA containing phenylboronic acid groups and quaternary ammonium salt groups is 10 to 30 parts, 1 to 3 parts of tannic acid TA, and 10 to 30 parts of polyvinyl alcohol PVA. The sum of ionized water is 1000-2000 parts.

4. a kind of preparation method of the polyphosphazene-based hydrogel wound dressing that has anti-inflammatory, antibacterial and ROS response properties of tannic acid coupling according to claim 2, is characterized in that: the time of described mixing and stirring 10 to 30 minutes.

5. a kind of preparation method of the polyphosphazene-based hydrogel wound dressing that has anti-inflammatory, antibacterial and ROS response properties of tannic acid coupling according to claim 2, is characterized in that: the rotating speed of described mixing and stirring 500-1000rpm.

6. The preparation method of a polyphosphazene-based hydrogel wound dressing with anti-inflammatory, antibacterial and ROS responsive properties coupled with tannic acid according to claim 2, characterized in that: the PPBA-TA nano The particle size of the conjugate is 50-200nm.