NO slow-release dressing and preparation method thereof
By combining metal organic frame materials with amino-polyethylene glycol 3400-polyarginine and inducible nitric oxide synthase recombinant proteins, the problem that existing dressings cannot produce NO in situ is solved. , achieved significant antibacterial properties and wound healing promotion effect.
Patent Information
- Application Number
- CN202411402011.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-09
- Publication Date
- 2025-01-07
AI Technical Summary
Existing dressings cannot produce NO molecules in situ by enzymatic methods, resulting in delayed wound healing or difficulty in healing, and repeated dressing changes and frequent use of antibiotics can lead to infection and damage.
By combining metal organic framework material, amino-polyethylene glycol 3400-polyarginine and inducible nitric oxide synthase recombinant protein with sodium alginate, carrier particles are formed and embedded using a three-dimensional network structure of calcium alginate. The enzyme reaction system achieves the immobilization and sustained release of NO, and combines Tween 80 and glycerol to form a NO sustained release dressing.
The continuous production of dressing and release of NO is achieved, significant antibacterial properties and promote wound healing effect, increase fibroblast proliferation and collagen production, and reduce the healing time of infected wounds.
Smart Images
Figure CN119258265A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of biomedical materials, and in particular to an NO sustained-release dressing and a preparation method thereof. Background Art
[0002] Skin wound healing requires a complex and lengthy self-healing process. Open wounds and wounds caused by certain skin diseases can last for months or even years. Repeated dressing changes and frequent use of disinfectants and antibiotics can lead to infections and wound damage caused by multidrug-resistant bacteria, which can be life-threatening in severe cases. Therefore, it is necessary to develop a new strategy to combat pathogens, promote wound healing, and avoid frequent dressing changes and overuse of antibiotics.
[0003] NO is one of the key factors in wound healing. It can promote the chemotaxis of inflammatory cells, stimulate cell proliferation, enhance the synthesis and deposition of collagen, and promote the formation of new blood vessels. At the same time, NO molecules and their byproduct peroxynitrite (ONOO) generated by the reaction with oxidants in the surrounding environment - ) and nitrogen trioxide (N 2 O 3 ) can easily lead to lipid peroxidation of bacterial membranes, thus showing significant antibacterial properties.
[0004] Currently, there is no dressing that can enzymatically generate NO molecules in situ to supplement the lack of endogenous NO in the wound, which may cause delayed wound healing or make it difficult for the wound to heal. Summary of the invention
[0005] In view of this, the object of the present invention is to provide a NO sustained-release dressing and a preparation method thereof, which can not only achieve antibacterial effect by producing peroxynitrite, but also produce an appropriate amount of NO to stimulate cell proliferation, enhance collagen synthesis and deposition, promote angiogenesis, and accelerate wound healing.
[0006] In order to solve the above technical problems, the present invention provides a method for preparing a NO sustained-release dressing, comprising the following steps:
[0007] S1. Add metal organic framework material, amino-polyethylene glycol 3400-polyarginine and polyvinyl pyrrolidone into water, adjust the pH value to 10.0-11.0, raise the temperature to 80-95°C, incubate for 20-24h, cool down to 4-6°C, add inducible nitric oxide synthase recombinant protein and genipin, stir well, centrifuge to remove the supernatant, and freeze-dry to obtain NOS2-loaded microparticles;
[0008] S2. Add the NOS2-loaded microparticles, Tween 80 and glycerol prepared in step S1 into the sodium alginate solution, fully disperse them and pour them into the holes of the polytetrafluoroethylene hydrogel mold, then immerse them in a calcium chloride solution, let them stand for 12-18 hours, take them out and wash them to obtain a NO sustained-release dressing.
[0009] Furthermore, the metal organic framework material is MIL-100 (Fe).
[0010] Furthermore, in step S1, in the amino-polyethylene glycol 3400-polyarginine, the degree of polymerization of polyarginine is 8-15, and the weight average molecular weight of polyvinyl pyrrolidone is 16000-30000.
[0011] Furthermore, in step S1, the feed ratio of the metal organic framework material, amino-polyethylene glycol 3400-polyarginine, water, polyvinyl pyrrolidone, inducible nitric oxide synthase recombinant protein and genipin is (10-18) mg: (8-16) mg: 10 ml: (1.0-2.0) mg: (50-100) μg: (8-16) μg.
[0012] Furthermore, in step S1, the concentration of the sodium alginate solution is 2.0-4.0 w / v%.
[0013] Furthermore, in step S2, the feed ratio of the NOS2-loaded microparticles, sodium alginate solution, Tween 80 and glycerol is (2-3) mg: (10-20) ml: (0.2-0.4) g: (0.05-0.10) g.
[0014] Furthermore, in step S2, the concentration of the calcium chloride solution is 1.5-2.0 w / v%.
[0015] Furthermore, in step S2, the washing is performed by immersion washing with a PBS solution for 3 times.
[0016] The present invention also provides a NO sustained-release dressing prepared by the above-mentioned preparation method.
[0017] Furthermore, the thickness of the NO sustained-release dressing is 5-8 mm.
[0018] Compared with the prior art, the present invention has the following beneficial effects:
[0019] 1) The present invention forms a carrier by shuttle entanglement with block copolymer amino-polyethylene glycol 3400-polyarginine through organic metal framework (MOFs) material, which is used to adsorb inducible nitric oxide synthase (NOS2) recombinant protein. The three-dimensional network structure of calcium alginate is used for embedding to finally obtain an immobilized enzyme reaction system that generates NO in situ. The system can convert the arginine residues in the polyarginine segment in the block copolymer into L-citrulline and release nitric oxide (NO). In particular, this process requires the recombinant protein to first combine with oxygen and produce superoxide free radicals through the heme iron intermediate in MOFs. The latter forms hydrogen peroxide and oxygen through superoxide dismutase. Hydrogen peroxide then reacts with the amino group on arginine to release NO. Therefore, the present invention constructs an immobilized enzyme system that continuously produces and releases NO.
[0020] 2) NO molecules and their byproduct peroxynitrite (ONOO) generated by the reaction with oxygen and other oxidants in the surrounding environment - ) and nitrogen trioxide (N 2 O 3 ) can easily lead to lipid peroxidation of bacterial membranes, thus showing significant antibacterial properties. At the same time, NO can also promote wound healing, increase fibroblast proliferation and collagen production. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 This is a scanning electron microscope photograph of the freeze-dried NO sustained-release dressing prepared in Example 1;
[0022] Figure 2 The antibacterial test results obtained in Experimental Example 1;
[0023] Figure 3 This is the mouse infection wound healing experiment obtained in Experimental Example 2. DETAILED DESCRIPTION
[0024] In order to further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, rather than limiting the claims of the present invention.
[0025] MIL-100(Fe) was purchased from Xi'an Qiyue.
[0026] Amino-polyethylene glycol 3400-polyarginine was purchased from Xi'an Qiyue.
[0027] Example 1
[0028] A NO sustained-release dressing and a preparation method thereof, comprising the following steps:
[0029] 1. Add 15 mg of MIL-100 (Fe) and 10 mg of amino-polyethylene glycol 3400-polyarginine with a polymerization degree of 10 to 10 ml of water, add 1.4 mg of polyvinyl pyrrolidone with a weight average molecular weight of 25,000, adjust the pH to 10.8, raise the temperature to 85°C, incubate for 21 hours, cool to 5°C, add 80 μg of inducible nitric oxide synthase (NOS2) recombinant protein and 10 μg of genipin, stir well, centrifuge to remove the supernatant, and lyophilize to obtain NOS2-loaded microparticles;
[0030] 2. Add 2.8 mg of NOS2-loaded microparticles prepared in step 1 into 15 ml of 2.8 w / v% sodium alginate solution, add 0.32 g of Tween 80 and 0.07 g of glycerol, and after fully dispersing, pour into the holes of a polytetrafluoroethylene hydrogel mold and immerse in a 1.8 w / v% calcium chloride solution. After standing for 16 h, take out and wash three times with a PBS solution to obtain a NO sustained-release dressing with a thickness of 7 mm. Figure 1 This is a scanning electron microscope photo of the NO sustained-release dressing after freeze-drying. It can be observed that the dressing has a three-dimensional network of channels, and the channels contain particles with a particle size of <2μm, which are NOS2 particles.
[0031] Example 2
[0032] A NO sustained-release dressing and a preparation method thereof, comprising the following steps:
[0033] 1. Add 10 mg of MIL-100 (Fe) and 8 mg of amino-polyethylene glycol 3400-polyarginine with a polymerization degree of 8 to 10 ml of water, add 1.0 mg of polyvinyl pyrrolidone with a weight average molecular weight of 16,000, adjust the pH to 10.0, raise the temperature to 80°C, incubate for 24 hours, cool to 4°C, add 50 μg of inducible nitric oxide synthase (NOS2) recombinant protein and 8 μg of genipin, stir well, centrifuge to remove the supernatant, and lyophilize to obtain NOS2-loaded microparticles;
[0034] 2. Add 2 mg of NOS2-loaded microparticles prepared in step 1 into 10 ml of 2.0 w / v% sodium alginate solution, add 0.2 g of Tween 80 and 0.05 g of glycerol, and pour into the holes of a polytetrafluoroethylene hydrogel mold after being fully dispersed. Immerse in a 1.5 w / v% calcium chloride solution, let stand for 12 h, take out and wash three times with a PBS solution to obtain a NO sustained-release dressing with a thickness of 5 mm.
[0035] Example 3
[0036] A NO sustained-release dressing and a preparation method thereof, comprising the following steps:
[0037] 1. Add 18 mg of MIL-100 (Fe) and 16 mg of amino-polyethylene glycol 3400-polyarginine with a polymerization degree of 15 to 10 ml of water, add 2.0 mg of polyvinyl pyrrolidone with a weight average molecular weight of 30,000, adjust the pH to 11.0, raise the temperature to 95°C, incubate for 20 hours, cool to 6°C, add 100 μg of inducible nitric oxide synthase (NOS2) recombinant protein and 16 μg of genipin, stir well, centrifuge to remove the supernatant, and lyophilize to obtain NOS2-loaded microparticles;
[0038] 2. Add 3 mg of NOS2-loaded microparticles prepared in step 1 into 20 ml of 4.0 w / v% sodium alginate solution, add 0.4 g of Tween 80 and 0.10 g of glycerol, and after fully dispersing, pour into the holes of a polytetrafluoroethylene hydrogel mold and immerse in a 2 w / v% calcium chloride solution. After standing for 18 h, take out and wash three times with a PBS solution to obtain a NO sustained-release dressing with a thickness of 8 mm.
[0039] Comparative Example 1 (without MOFs)
[0040] A dressing and a preparation method thereof, comprising the following steps:
[0041] 10 mg of amino-polyethylene glycol 3400-polyarginine with an arginine polymerization degree of 10, 1.4 mg of polyvinyl pyrrolidone with a weight average molecular weight of 25,000, 80 μg of inducible nitric oxide synthase (NOS2) recombinant protein and 10 μg of genipin were added to 15 ml of 2.8 w / v% sodium alginate solution, supplemented with 0.32 g of Tween 80 and 0.07 g of glycerol, and after being fully dispersed, poured into the holes of a polytetrafluoroethylene hydrogel mold, immersed in a 1.8 w / v% calcium chloride solution, and after standing for 16 hours, taken out and washed three times with a PBS solution to obtain a dressing with a thickness of 7 mm.
[0042] Comparative Example 2 (no block copolymer)
[0043] A dressing and a preparation method thereof, comprising the following steps:
[0044] 1. Add 15 mg of MIL-100 (Fe) to 10 ml of water, add 1.4 mg of polyvinyl pyrrolidone with a weight average molecular weight of 25,000, adjust the pH to 10.8, raise the temperature to 85°C, incubate for 21 hours, cool to 5°C, add 80 μg of inducible nitric oxide synthase (NOS2) recombinant protein and 10 μg of genipin, stir well, centrifuge to remove the supernatant, and lyophilize to obtain NOS2-loaded microparticles;
[0045] 2. Add 2.8 mg of NOS2-loaded microparticles prepared in step 1 into 15 ml of 2.8 w / v% sodium alginate solution, add 0.32 g of Tween 80 and 0.07 g of glycerol, and after fully dispersing, pour into the holes of a polytetrafluoroethylene hydrogel mold and immerse in a 1.8 w / v% calcium chloride solution. After standing for 16 h, take out and wash three times with a PBS solution to obtain a dressing with a thickness of 7 mm.
[0046] Comparative Example 3 (without genipin)
[0047] A dressing and a preparation method thereof, comprising the following steps:
[0048] 1. Add 15 mg of MIL-100 (Fe) and 10 mg of amino-polyethylene glycol 3400-polyarginine with a polymerization degree of 10 to 10 ml of water, add 1.4 mg of polyvinyl pyrrolidone with a weight average molecular weight of 25,000, adjust the pH to 10.8, raise the temperature to 85°C, incubate for 21 hours, cool to 5°C, add 80 μg of inducible nitric oxide synthase (NOS2) recombinant protein, stir well, centrifuge to remove the supernatant, and lyophilize to obtain NOS2-loaded microparticles;
[0049] 2. Add 2.8 mg of NOS2-loaded microparticles prepared in step 1 into 15 ml of 2.8 w / v% sodium alginate solution, add 0.32 g of Tween 80 and 0.07 g of glycerol, and after fully dispersing, pour into the holes of a polytetrafluoroethylene hydrogel mold and immerse in a 1.8 w / v% calcium chloride solution. After standing for 16 h, take out and wash three times with a PBS solution to obtain a dressing with a thickness of 7 mm.
[0050] Experimental Example 1: Antibacterial Experiment
[0051] The flask oscillation method was used to test the antibacterial properties of the dressings of Examples 1-3 and Comparative Examples 1-3 and two commercially available hydrogel dressings (A: Hi-Shine disposable hydrocolloid dressing; B: Hartmann hydrogel wound dressing) against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The results are shown in Figure 2 .
[0052] Depend on Figure 2It can be seen that the dressing prepared by the embodiment of the present invention has excellent antibacterial ability. In comparison, the antibacterial ability of the dressing of comparative example 3 is relatively poor, followed by the dressing of comparative example 1, and the worst antibacterial ability is the dressing of comparative example 2. The two commercially available hydrogel dressings also did not show obvious antibacterial properties. The reason is that MOFs were not added during the preparation of the dressing of comparative example 1. In the process of catalyzing the oxidation of arginine to synthesize NO, the recombinant protein of inducible nitric oxide synthase (NOS2) needs to synthesize superoxide radicals through the heme iron intermediates in MOFs. The lack of MIL-100 (Fe) leads to the obstruction of superoxide radical formation and the greatly reduced NO generation efficiency, which directly affects the antibacterial properties of the dressing. Comparative Example 2 does not add the block copolymer amino-polyethylene glycol 3400-polyarginine, which contains arginine, a catalytic substrate of the inducible nitric oxide synthase (NOS2) recombinant protein, which directly leads to insufficient NO production and affects the three-dimensional structure of the NOS2-carrying microparticles, resulting in an adsorption barrier to the inducible nitric oxide synthase (NOS2) recombinant protein. Therefore, the lack of NO causes the dressing to almost lose its antibacterial ability. Comparative Example 3 does not add genipin, which can increase the cross-linking of the inducible nitric oxide synthase (NOS2) recombinant protein with the block copolymer and increase the protein adsorption amount. Its absence will affect the generation efficiency of NO, and then affect the antibacterial performance. In addition, the two commercially available hydrogel dressings do not have a NO generation system, and no other antibacterial measures are added, so the antibacterial effect is poor.
[0053] Experimental Example 2: Mouse infection wound healing experiment
[0054] The wounds of mice infected with Pseudomonas aeruginosa were treated with the dressings of Examples 1-3 and Comparative Examples 1-3 and two commercially available hydrogel dressings (A: Hi-Shine disposable hydrocolloid dressing; B: Hartmann hydrogel wound dressing), and the untreated wounds were used as controls to measure the changes in wound size. The results are shown in Table 1. Figure 3 .
[0055] Depend on Figure 3 It can be seen that the dressing prepared in the embodiment of the present invention has excellent ability to promote the healing of infected wounds. In comparison, the dressings in the comparative example and the commercially available hydrogel dressings have poor ability to promote the healing of infected wounds. The dressings in comparative examples 1-3 all have obstacles in the NO generation mechanism, resulting in low NO production efficiency to varying degrees. The specific reasons are Figure 2 The NO molecule and its byproduct peroxynitrite (ONOO) react with oxidants such as oxygen in the surrounding environment. - ) and nitrogen trioxide (N 2 O 3) can easily lead to lipid peroxidation of bacterial membranes, thereby showing significant antibacterial properties. At the same time, NO can also promote wound healing, increase the proliferation of fibroblasts and the production of collagen. Therefore, the healing rate of infected wounds exhibited by the dressings of Comparative Examples 1-3 is not as good as that of the dressings of Examples 1-3. Similarly, the other two commercially available hydrogel dressings do not have a NO generating system, so their ability to promote the healing of infected wounds is relatively poor.
[0056] The present invention provides a NO sustained-release dressing and a method for preparing the same. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be considered as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.
Claims
1. A method for preparing a NO sustained-release dressing, characterized in that: The steps include: S1. Add metal organic framework material, amino-polyethylene glycol 3400-polyarginine and polyvinyl pyrrolidone into water, adjust the pH value to 10.0-11.0, raise the temperature to 80-95°C, incubate for 20-24h, cool down to 4-6°C, add inducible nitric oxide synthase recombinant protein and genipin, stir well, centrifuge to remove the supernatant, and freeze-dry to obtain NOS2-loaded microparticles; S2. Add the NOS2-loaded microparticles, Tween 80 and glycerol prepared in step S1 into the sodium alginate solution, fully disperse them and pour them into the holes of the polytetrafluoroethylene hydrogel mold, then immerse them in a calcium chloride solution, let them stand for 12-18 hours, take them out and wash them to obtain a NO sustained-release dressing.
2. The preparation method according to claim 1, characterized in that: The metal organic framework material is MIL-100 (Fe).
3. The preparation method according to claim 1, characterized in that: In step S1, in the amino-polyethylene glycol 3400-polyarginine, the degree of polymerization of polyarginine is 8-15, and the weight average molecular weight of polyvinyl pyrrolidone is 16000-30000.
4. The preparation method according to claim 2, characterized in that: In step S1, the feed ratio of the metal organic framework material, amino-polyethylene glycol 3400-polyarginine, water, polyvinyl pyrrolidone, inducible nitric oxide synthase recombinant protein and genipin is (10-18) mg: (8-16) mg: 10 ml: (1.0-2.0) mg: (50-100) μg: (8-16) μg.
5. The preparation method according to claim 1, characterized in that: In step S1, the concentration of the sodium alginate solution is 2.0-4.0 w / v%.
6. The preparation method according to claim 1, characterized in that: In step S2, the feed ratio of the NOS2-loaded microparticles, sodium alginate solution, Tween 80 and glycerol is (2-3) mg: (10-20) ml: (0.2-0.4) g: (0.05-0.10) g.
7. The preparation method according to claim 1, characterized in that: In step S2, the concentration of the calcium chloride solution is 1.5-2.0 w / v%.
8. The preparation method according to claim 1, characterized in that: In step S2, the washing is performed by immersion washing with a PBS solution for 3 times.
9. The NO sustained-release dressing prepared by the preparation method according to any one of claims 1 to 8.
10. The NO sustained-release dressing according to claim 9, characterized in that: The thickness of the NO sustained-release dressing is 5-8 mm.