CN113150362A

CN113150362A - Preparation method and application of porous hydrogel integrating capturing and killing of bacteria

Info

Publication number: CN113150362A
Application number: CN202110361959.8A
Authority: CN
Inventors: 吴小泉; 孙静; 叶玮; 孙阿勇
Original assignee: Huaiyin Institute of Technology
Current assignee: Huaiyin Institute of Technology
Priority date: 2021-04-02
Filing date: 2021-04-02
Publication date: 2021-07-23

Abstract

The invention relates to the technical field of biomedical materials, and discloses a preparation method and application of a porous hydrogel integrating bacteria capturing and killing, wherein a diketone compound is loaded on a zeolite imidazole framework ZIF-8 by using a one-step synthesis method to obtain diketone @ ZIF-8, and then QC and the diketone @ ZIF-8 are mixed to prepare the diketone @ ZIF-8/QC. Based on the pH responsiveness of ZIF-8, the invention promotes the antibacterial drug to be slowly released in the weak acid environment for the survival of bacteria, improves the utilization efficiency of the drug, enhances the long-term effect of the antibacterial effect and realizes the safe and effective antibacterial curative effect; meanwhile, the quaternized chitosan has the capability of actively capturing and killing bacteria, achieves the aim of synergistic antibacterial, improves the antibacterial curative effect, and has important application value in the aspects of treating wound infection, promoting wound healing and the like.

Description

Preparation method and application of porous hydrogel integrating capturing and killing of bacteria

Technical Field

The invention relates to the technical field of biomedical materials, in particular to a preparation method and application of porous hydrogel integrating capturing and killing of bacteria.

Background

In recent years, bacterial infections have become a major health-threatening problem. Bacteria are adhered to the surface of food or various articles, so that people can contact the bacteria inadvertently and enter the mouth, nose or body epidermis to threaten the health of human beings directly. The number of deaths caused by bacterial infection is increasing dramatically every year, and especially in the areas with low income and undeveloped areas, the diseases caused by bacterial infection are very painful. The use of traditional antibiotics in large quantities has long cured diseases related to bacterial infections to a great extent and selectively inhibited or killed bacteria. However, the excessive use of antibiotics leads to the formation and evolution of resistant bacterial strains, seriously jeopardizing the physical health of the patient.

The skin plays an important role in the functions of human body defense and the like, can protect the body from mechanical damage, microorganisms and external environmental factors, and can ensure the loss of electrolytes in the human body and prevent dehydration. However, once the skin is damaged, wound repair is required. Under good environmental conditions, the wound surface can heal quickly. But under some microenvironment conditions which can not ensure the rapid healing of the wound surface, the bacteria invasion can be caused, so that the recovery of the wound surface is delayed, and the wound surface can be worsened in severe cases. An ideal wound healing microenvironment needs to ensure the wettability of the wound environment, absorb the excess exudate from the wound, have good air permeability, and prevent the invasion of bacteria or kill bacteria. The hydrogel wound dressing is characterized by excellent biocompatibility, biodegradability, no toxicity, no harm and the like, and is very colorful in the field of wound dressings. The hydrogel material can be well attached to the wound surface due to the porous structure and the proper swelling ratio of the hydrogel material, so that redundant wound surface exudates are absorbed, the moist environment of the wound surface is kept, and the dissolution of fibrin and necrotic tissues is facilitated. In addition, the hydrogel material can resist bacterial infection by loading different medicaments, reduce the frequency of dressing change and promote wound healing.

As an antibacterial drug which is not easy to cause bacterial drug resistance, the diketone compound has pharmacological actions of antibiosis, anti-inflammation, anticancer, antioxidation and the like, has a protective effect on partial tissues and organs, is a natural product in nature, is low in price and small in toxic and side effects, and therefore has a very wide application prospect in the fields of clinical medicine and the like.

As a novel porous material, the Metal Organic Frameworks (MOFs) have high porosity and high specific surface area, can be used as drug carriers to release drugs at targeted sites, and have good application prospects in the field of medical treatment. The zeolite imidazole framework (ZIF-8) has good biocompatibility and pH sensitivity, and can be used as an ideal nano-drug carrier.

Aiming at the requirements of the existing wound antibacterial dressing, the novel wound dressing integrating capturing and killing of bacteria is designed by combining the good antibacterial performance of diketone compounds and the excellent characteristics of chitosan hydrogel, the weak acidic environment for the existence of bacteria can be intelligently identified, the bacteria are actively captured, meanwhile, the slow release of antibacterial drugs is realized, the healing efficacy of wound infection is enhanced, the toxic and side effects are reduced, and the wound dressing has important values in the aspects of treating wound infection and promoting the healing of wounds.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a preparation method and application of a porous hydrogel integrating capturing and killing bacteria, and the method is based on the pH responsiveness of a zeolite imidazole framework ZIF-8, so that the antibacterial drug is promoted to be slowly released in a weakly acidic environment where bacteria exist, the utilization efficiency of the drug is improved, the long-acting property of the antibacterial effect is enhanced, and the safe and effective antibacterial curative effect is realized.

The technical scheme is as follows: the invention provides a preparation method of porous hydrogel integrating capturing and killing bacteria, which comprises the following steps: 1) adding zinc nitrate hexahydrate, 2-methylimidazole and diketone compounds into an organic solvent, uniformly mixing, centrifuging, removing supernate, washing with methanol, and drying in vacuum to obtain a diketone compound-loaded zeolite imidazole framework; 2) adding glacial acetic acid, chitosan and quaternary ammonium compounds into a solvent, uniformly stirring and mixing, performing centrifugal separation, taking supernate, adding an organic solvent, standing, centrifuging again, collecting sediments, dialyzing, freeze-drying and drying to obtain quaternized chitosan; 3) dissolving the quaternized chitosan in water, adding the diketone compound-loaded zeolite imidazole framework, and stirring until the mixture is uniformly mixed to obtain a hydrogel compound; and storing the hydrogel composite at 4 ℃, and freeze-drying to obtain the porous hydrogel composite.

Further, the diketone compound in the step 1) is 2, 4-pyrrolidine diketone or curcumin.

Further, the organic solvent in step 1) is acetone, methanol or ethanol.

Further, in the step 1), uniformly mixing the materials in a magnetic stirring mode for 0.5-1 h and then in an ultrasonic mode, wherein the ultrasonic time is 3-5 min and the ultrasonic temperature is 10-70 ℃; in the step 1), the centrifugal speed is 6000-8000 rpm, the centrifugal time is 0.5-1 h, and the drying time is 10-24 h.

Further, in the step 2), the molecular weight of the chitosan is 100000-300000 Da; stirring at 40-60 ℃ for 0.5-1 h; the centrifugal speed is 6000-8000 rpm, and the centrifugal time is 10-18 h.

Further, in the step 2), the quaternary ammonium compound is acryloyloxyethyltrimethyl ammonium chloride or 2, 3-epoxypropyltrimethylammonium chloride.

Further, in step 2), the organic solvent is acetone or methanol.

Further, in the step 2), adding an organic solvent into the supernatant, standing for 5-10 min, centrifuging again, and collecting precipitates; the centrifugal speed is 6000-8000 rpm, the centrifugal time is 0.5-1 h, the deionized water dialysis is carried out, the dialysis time is 2-3 days, and the freeze-drying time is 2-3 days.

Further, in the step 1), the mass ratio of the zinc nitrate hexahydrate to the 2-methylimidazole is 1: 1-2; the mass ratio of zinc nitrate hexahydrate to diketone compounds is 20-50: 1; in the step 2), the mass ratio of the glacial acetic acid to the chitosan is 0.15-0.2: 1; the mass ratio of the chitosan to the quaternary ammonium compound is 1-2: 1; in the step 3), the mass ratio of the diketone compound-loaded zeolite imidazole framework to the quaternized chitosan is 2% -10%.

The invention provides application of porous hydrogel prepared by a preparation method of the porous hydrogel integrating capturing and killing of bacteria in wound dressing.

Has the advantages that: the invention synthesizes porous hydrogel integrating trapping and killing bacteria by utilizing a one-step synthesis method, loads a diketone compound to a zeolite imidazole framework ZIF-8 to obtain the zeolite imidazole framework (diketone @ ZIF-8) loading the diketone compound, and then mixes Quaternized Chitosan (QC) and the diketone @ ZIF-8 to prepare the porous hydrogel composite material (diketone @ ZIF-8/QC).

Compared with the prior art, the diketone @ ZIF-8/QC porous hydrogel composite material disclosed by the invention can intelligently identify a weakly acidic environment in which bacteria exist based on the pH responsiveness of ZIF-8, and meanwhile, the antibacterial drug is slowly released in the weakly acidic environment in which the bacteria exist, the drug utilization efficiency is improved, and the long-acting property of the antibacterial effect is enhanced; the porous hydrogel composite material is used in wound dressing, so that the healing effect of wound infection can be enhanced, the toxic and side effects are reduced, and the safe and effective antibacterial purpose is achieved. Meanwhile, the ZIF-8 slowly releases the drug in a weak acid environment to kill bacteria, so that the purpose of synergistic antibacterial is achieved, the antibacterial curative effect is improved, and the chitosan has important application values in the aspects of treating wound infection, promoting wound healing and the like.

The hydrogel wound dressing prepared by the invention has excellent antibacterial performance, and the preparation method is simple and feasible and has good practicability.

Detailed Description

The present invention will be described in detail with reference to specific examples.

Comparative example 1 (no quaternized chitosan):

1 g of chitosan (molecular weight 100000-300000 Da) was added to 36 ml of deionized water. Then, 180. mu.l of glacial acetic acid was added thereto, and the mixture was stirred at 55 ℃ for 30 min. The solution was then removed and centrifuged at 5000 rpm. After centrifugation, the supernatant was extracted and dialyzed against deionized water. After three days, the sediment was collected and lyophilized.

Comparative example 2 (quaternized chitosan):

1 g of chitosan (100000-300000 Da) was added to 36 ml of deionized water. Thereafter, 180. mu.l of glacial acetic acid were added. After stirring at 55 ℃ for 30 min, 2, 3-epoxypropyltrimethylammonium chloride was added and stirred at 55 ℃ for 12 h. Then, the solution was taken out and centrifuged at 7000 rpm for 15 h. After centrifugation, the supernatant was extracted and added to acetone. After standing for a few minutes, the solution was centrifuged again. The sediment was collected and dialyzed against deionized water. After three days, the sediment was collected and freeze dried to give Quaternized Chitosan (QC).

Comparative example 3 (zeolitic imidazole framework without supported diketones with quaternized chitosan):

preparation of ZIF-8 drug carriers: uniformly stirring zinc nitrate hexahydrate and deionized water at normal temperature, adding 2-methylimidazole into ethanol, stirring, mixing the two solutions, magnetically stirring, centrifuging, removing supernatant, washing with methanol, and vacuum drying to obtain the ZIF-8 drug carrier.

Preparation of quaternized chitosan QC: exactly the same as in comparative example 2, and will not be described herein.

Preparation of a composite material ZIF-8/QC: QC was dispersed in water at a concentration of 50 mg/ml and stirred until it became clear. Adding 5 mg of ZIF-8 drug carrier into 1 ml of QC solution, stirring the solution again to obtain a uniform solution, and freeze-drying to obtain ZIF-8/QC.

Example 1

Preparation of curcumin @ ZIF-8: uniformly stirring zinc nitrate hexahydrate and deionized water at normal temperature, adding 2-methylimidazole and curcumin into methanol, stirring, mixing the two solutions, magnetically stirring for 40min, performing ultrasonic treatment for 3min, centrifuging (the centrifugal speed is 7000 rpm, the centrifugal time is 40 min), removing supernatant, washing with methanol, and drying in vacuum for 15h to obtain curcumin @ ZIF-8.

Preparation of curcumin @ ZIF-8/QC: QC was dispersed in water at a concentration of 50 mg/ml and stirred until it became clear. Adding 1 mg of curcumin @ ZIF-8 into 1 ml of QC solution, stirring the solution again to obtain a uniform solution, and freeze-drying to obtain curcumin @ ZIF-8/QC-2%.

Example 2

The preparation of curcumin @ ZIF-8 and the preparation of quaternized chitosan QC in this example are exactly the same as in example 1 and are not described herein.

Preparation of curcumin @ ZIF-8/QC: QC was dispersed in water at a concentration of 50 mg/ml and stirred until clean. Adding 2 mg of curcumin @ ZIF-8 into 1 ml of QC solution, stirring the solution again to obtain a uniform solution, and freeze-drying to obtain curcumin @ ZIF-8/QC-4%.

Example 3

Preparation of curcumin @ ZIF-8/QC: QC was dispersed in water at a concentration of 50 mg/ml and stirred until clean. Adding 3 mg curcumin @ ZIF-8 to 1 ml QC solution, stirring the solution again to obtain a uniform solution, and freeze-drying to obtain curcumin @ ZIF-8/QC-6%.

Example 4

Preparation of curcumin @ ZIF-8/QC: QC was dispersed in water at a concentration of 50 mg/ml and stirred until clean. Adding 4 mg curcumin @ ZIF-8 to 1 ml QC solution, stirring the solution again to obtain a uniform solution, and freeze-drying to obtain curcumin @ ZIF-8/QC-8%.

Example 5

Preparation of curcumin @ ZIF-8/QC: QC was dispersed in water at a concentration of 50 mg/ml and stirred until clean. Adding 5 mg curcumin @ ZIF-8 to 1 ml QC solution, stirring the solution again to obtain a uniform solution, and freeze-drying to obtain curcumin @ ZIF-8/QC-10%.

The products obtained in the comparative examples 1 to 3 and the examples 1 to 5 are applied to wound dressings, and the bacteriostatic effects of the products are respectively tested, and the test results are shown in the following table 1.

TABLE 1 results of bacteriostatic tests on the products obtained in comparative examples 1 to 3 and examples 1 to 5

As can be seen from table 1 above, the bacteriostatic effect of the quaternized chitosan is significantly improved compared to the non-quaternized chitosan, the bacteriostatic effect of the composite material of the quaternized chitosan and the zeolite imidazole framework without the supported diketone compound is better than that of the quaternized chitosan, and the bacteriostatic effect of the composite material of the quaternized chitosan and the zeolite imidazole framework with the supported diketone compound is also significantly better than that of the composite material of the quaternized chitosan and the zeolite imidazole framework without the supported diketone compound. In the composite material of quaternized chitosan and zeolite imidazole framework loaded with diketone compounds, the antibacterial effect is gradually increased along with the increase of the proportion of the zeolite imidazole framework loaded with diketone compounds.

The above embodiments are merely illustrative of the technical concepts and features of the present invention, and the purpose of the embodiments is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. A preparation method of porous hydrogel integrating capturing and killing bacteria is characterized in that: the method comprises the following steps:

1) adding zinc nitrate hexahydrate, 2-methylimidazole and diketone compounds into an organic solvent, uniformly mixing, centrifuging, removing supernate, washing with methanol, and drying in vacuum to obtain a diketone compound-loaded zeolite imidazole framework;

2) adding glacial acetic acid, chitosan and quaternary ammonium compounds into a solvent, uniformly stirring and mixing, performing centrifugal separation, taking supernate, adding an organic solvent, standing, centrifuging again, collecting sediments, dialyzing, freeze-drying and drying to obtain quaternized chitosan;

3) dissolving the quaternized chitosan in water, adding the diketone compound-loaded zeolite imidazole framework, and stirring until the mixture is uniformly mixed to obtain a hydrogel compound; and storing the hydrogel composite at 4 ℃, and freeze-drying to obtain the porous hydrogel composite.

2. The method for preparing a porous hydrogel integrating capturing and killing of bacteria according to claim 1, wherein: the diketone compound in the step 1) is 2, 4-pyrrolidine diketone or curcumin.

3. The method for preparing a porous hydrogel integrating capturing and killing of bacteria according to claim 1, wherein: the organic solvent in the step 1) is acetone, methanol or ethanol.

4. The method for preparing a porous hydrogel integrating capturing and killing of bacteria according to claim 1, wherein: in the step 1), uniformly mixing the materials in a magnetic stirring mode for 0.5-1 h and then in an ultrasonic mode, wherein the ultrasonic time is 3-5 min and the ultrasonic temperature is 10-70 ℃;

in the step 1), the centrifugal speed is 6000-8000 rpm, the centrifugal time is 0.5-1 h, and the drying time is 10-24 h.

5. The method for preparing a porous hydrogel integrating capturing and killing of bacteria according to claim 1, wherein the method comprises the following steps: in the step 2), the molecular weight of the chitosan is 100000-300000 Da;

stirring at 40-60 ℃ for 0.5-1 h;

the centrifugal speed is 6000-8000 rpm, and the centrifugal time is 10-18 h.

6. The method for preparing a porous hydrogel integrating capturing and killing of bacteria according to claim 1, wherein: in the step 2), the quaternary ammonium compound is acryloyloxyethyl trimethyl ammonium chloride or 2, 3-epoxypropyl trimethyl ammonium chloride.

7. The method for preparing a porous hydrogel integrating capturing and killing of bacteria according to claim 1, wherein: in the step 2), the organic solvent is acetone or methanol.

8. The method for preparing a porous hydrogel integrating capturing and killing of bacteria according to claim 1, wherein: adding an organic solvent into the supernatant in the step 2), standing for 5-10 min, centrifuging again, and collecting precipitates;

the centrifugal speed is 6000-8000 rpm, the centrifugal time is 0.5-1 h, the deionized water dialysis is carried out, the dialysis time is 2-3 days, and the freeze-drying time is 2-3 days.

9. The method for preparing a porous hydrogel integrating capturing and killing of bacteria according to claim 1, wherein:

in the step 1), the mass ratio of the zinc nitrate hexahydrate to the 2-methylimidazole is 1: 1-2; the mass ratio of zinc nitrate hexahydrate to diketone compounds is 20-50: 1;

in the step 2), the mass ratio of the glacial acetic acid to the chitosan is 0.15-0.2: 1; the mass ratio of the chitosan to the quaternary ammonium compound is 1-2: 1;

in the step 3), the mass ratio of the diketone compound-loaded zeolite imidazole framework to the quaternized chitosan is 2% -10%.

10. Use of a porous hydrogel obtained by the method for preparing a porous hydrogel integrated with capturing and killing of bacteria according to any one of claims 1 to 9 in a wound dressing.