CN113876995B - Nanofiber membrane for skin wound and preparation method thereof - Google Patents
Nanofiber membrane for skin wound and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a nanofiber membrane for skin wounds and a preparation method thereof, wherein the nanofiber membrane is of a core-shell structure, and a shell layer material contains gelatin; the core layer material contains polycaprolactone and nano enzyme composite material. The invention utilizes the activities of PBAs nano enzyme, hydrogen peroxide and superoxide enzyme to inhibit the wound inflammation, and antisense oligonucleotide AOND selectively inhibits TGF-beta 1 expression, inhibits the formation of scar and promotes the healing of wound.
Description
Technical Field
The invention belongs to the field of functional nanofiber membranes and preparation thereof, and particularly relates to a nanofiber membrane for skin wound surfaces and a preparation method thereof.
Background
During wound healing, signaling pathways are activated that act together to restore the tissue microenvironment, consisting of cells and the extracellular matrix (ECM), including enzymes, cytokines, and growth factors. The process of wound healing comprises four stages: coagulation, inflammation, repair and remodeling stages. Reactive Oxygen Species (ROS), such as hydrogen peroxide, hydroxyl radicals, and superoxide radicals, are generated in excess at the site of injury. Although ROS have important biological functions in the body, excessive reactive oxygen species affect cell proliferation and cause apoptosis through oxidative damage to cell membranes, DNA and proteins, and unresolved inflammation is a major cause of impaired wound healing because they overwhelm the cellular antioxidant system and impede the healing process. Thus, continued oxidative stress at the wound site compromises the function of skin cells (fibroblasts, keratinocytes, etc.), promotes inflammation, necrosis and fibrotic scarring, ultimately delaying the tissue repair process. In mammals, enzymes having ROS-degrading ability such as Catalase (CAT), superoxide dismutase (SOD), glutathione Peroxidase (POD), etc. constitute an antioxidant defense system against intracellular increased ROS.
The nano enzyme is used as a nano material with a high-efficiency catalytic function of natural enzyme characteristics, and has the characteristics of low cost, good stability, high catalytic activity and the like. Nanoenzymes regulate skin wound healing by modulating reactive oxygen species and inhibiting inflammation, reportedly inhibiting inflammatory mediators of Lipopolysaccharide (LPS) -induced macrophages. The Prussian Blue (PB) nanoenzyme with a Metal Organic Framework (MOF) structure not only has the degradation activity of hydrogen peroxide, but also has strong superoxide and peroxide removing capability, and has application potential in wound treatment.
CN110772379A a preparation method of a compound nanometer fibrous membrane of load nanometer enzyme and its wound surface are applied, use nanometer enzyme to balance the active oxygen expression in vivo, achieve the goal of diminishing inflammation and promoting healing, but can only play a weak role to scar hyperplasia, increase and closely relate to fibrosis of the tissue in the scar plastic phase of wound healing, and transforming growth factor TGF-beta 1 expresses and is the main factor that fibrosis regulates and control, the invention can inhibit wound healing TGF-beta 1 over-expression in the scar plastic phase effectively, achieve the function of inhibiting the scar.
Disclosure of Invention
The invention aims to solve the technical problem of providing a nanofiber membrane for skin wounds and a preparation method thereof, and overcoming the defect that scars cannot be effectively inhibited in the prior art.
According to the nanofiber membrane, the nanofiber membrane is of a core-shell structure, wherein a shell layer material contains gelatin; the core layer material contains polycaprolactone and a nano-enzyme composite material.
The nano enzyme composite material is a PBAs/AOND nano enzyme composite material, and specifically comprises the following components: containing inorganic iron salt, inorganic zinc salt, surfactant and K 4 [Fe(CN) 6 ]The raw materials are subjected to solvothermal reaction to obtain a prussian blue analogue PBAs powdery product, and then the prussian blue analogue PBAs powdery product is mixed and stirred with antisense oligonucleotide AOND of TGF-beta 1 to obtain the PBAs/AOND nano enzyme composite material.
The TGF-beta 1 antisense oligonucleotide AOND sequence is specifically 5.
The preparation method of the nanofiber membrane comprises the following steps:
dissolving gelatin in a solvent to obtain a shell solution; mixing polycaprolactone, a PBAs/AOND nano enzyme composite material and a solvent to obtain a core layer solution; and carrying out coaxial electrostatic spinning to obtain the nanofiber membrane.
The preferred mode of the above preparation method is as follows:
the molecular weight of the polycaprolactone is 6-8 ten thousand;
the solvent is hexafluoroisopropanol; the mass concentration of gelatin in the shell layer solution is 5-15%; the mass concentration of polycaprolactone in the core layer solution is 6-15%; the mass concentration of the PBAs/AOND nano enzyme composite material is 0.001-0.1%;
the PBAs/AOND nano enzyme composite material is specifically as follows:
mixing inorganic iron salt solution, inorganic zinc salt solution and surfactant, and adding K 4 [Fe(CN) 6 ]Reacting the solution for 1-60 min at 40-80 ℃, centrifugally washing at 10000-25000 r/min, and freeze-drying to obtain Prussian blue analog PBAs;
mixing the Prussian blue analogue PBAs with AOND, stirring, centrifugally washing at 10000-25000 r/min, and freeze-drying to obtain the PBAs/AOND nano-enzyme composite material.
The inorganic ferric salt is one of ferric chloride, ferric nitrate, ferric sulfate and ferric acetate; the inorganic zinc salt is one of zinc chloride, zinc nitrate, zinc sulfate and zinc acetate; the concentrations of the inorganic ferric salt solution and the inorganic zinc salt solution are both 1-10 mM; the surfactant is polyvinylpyrrolidone PVP and/or ethylene diamine tetraacetic acid EDTA.
The mole ratio of the Prussian blue analogues PBAs to the AOND is 1:10 3 -10 4 (ii) a The stirring is carried out for more than 8 hours at 25 ℃.
The specific technological parameters of the coaxial electrostatic spinning are as follows: the spinning voltage is as follows: 6-20kv, the receiving distance is 6-20cm, the flow rate of the core solution is 0.1-1 mL/min, and the flow rate of the shell solution is 0.1-2 mL/min.
The invention relates to application of the nanofiber membrane in skin wound materials.
The invention can fully utilize the programmability of nucleic acid molecules, and successfully fuse the nucleic acid molecules with Prussian blue analogue metal organic framework Structures (PBAs) through phosphoric acid coordination to prepare the functionalized nano enzyme. The material has good anti-inflammatory performance, and can inhibit tissue inflammation and promote tissue regeneration when used as a wound dressing.
Advantageous effects
The invention coordinates and compounds a phosphorylation TGF-beta 1 antisense nucleotide sequence 5 'GTAGCCTTGGCTCGTG-3' with metal ions in PBAs to obtain a functional nano-enzyme composite material PBAs/AOND, and finally prepares the functional nano-enzyme-loaded fiber membrane with a core-shell structure through coaxial electrostatic spinning. The PBAs nanoenzyme activity of the technology can inhibit wound inflammation, AOND selectively inhibits TGF-beta 1 expression, inhibits scar formation, and promotes wound healing. The preparation method is simple, and the obtained product has good stability and can meet the requirements of clinical application.
The preparation method is simple and can be used for mass preparation. The prepared nano-fiber has good air permeability, good drug-loading performance and stable performance, and the addition of the functional nano-enzyme can promote the rapid healing of wound tissues.
Drawings
FIG. 1 is a scanning electron microscope SEM image of PBAs/AOND in example 1.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims. The polyvinylpyrrolidone has a molecular weight of 3-5 ten thousand
The molecular weight of polycaprolactone is 6-8 ten thousand
Example 1
(1) The preparation method by the solvothermal method specifically comprises the following steps: respectively preparing ferric chloride solution and zinc acetate solution with concentration of 5mM, and mixing 1mL and 2mL of polyvinylpyrrolidone with concentration of 28mg/mLHomogeneous, 2mL of K at 5mM concentration 4 [Fe(CN) 6 ]Slowly adding the solution into the mixed solution, and reacting at 50 ℃ for 10min to obtain Prussian Blue Analogues (PBAs). Centrifugal washing is carried out at a rotation speed of 20000r/min, and then freeze drying is carried out to obtain the PBAs powdery product.
(2) Dissolving the obtained 10mL of 5nM PBAs and 10mL of 5uM AOND in water, stirring at 25 ℃ for 8 hours, centrifuging and washing with deionized water at the rotating speed of 20000r/min, and freeze-drying to obtain the PBAs/AOND nano-enzyme composite material. (3) Dissolving gelatin in hexafluoroisopropanol to obtain gelatin solution with mass fraction of 8% as shell solution; dissolving polycaprolactone in hexafluoroisopropanol to obtain a polycaprolactone solution with the mass fraction of 10%, and then adding 0.1mg of AOND-PBANPs to obtain a core solution; preparing the nanofiber membrane under the condition of regulating and controlling a spinning process under the conditions that the temperature is 20 ℃ and the humidity is 40%. Co-spinning to obtain PCL/AOND-PBANPs @ GE.
The specific spinning conditions are as follows: voltage 15kv, receiving distance 12cm, flow rate of core 0.1mL/min and flow rate of shell solution 0.2mL/min.
Example 2
(1) The preparation method by the solvothermal method specifically comprises the following steps: the molar ratio is 2:1, 2mL and 1mL of ferric chloride and zinc acetate with the concentration of 8mM respectively, and uniformly mixing with 3mL of 28mg/mL polyvinylpyrrolidone, and 3mL of K with the concentration of 5mM 4 [Fe(CN) 6 ]Slowly adding the solution into the mixed solution, and reacting at 70 deg.C for 30min to obtain Prussian Blue Analogues (PBAs). Centrifugal washing at 15000r/min, and freeze drying to obtain PBAs powder.
(2) And d, dissolving 10mL of 5nM PBAs obtained in the step a and 10mL of 5uM AOND in water, stirring for 10 hours at 25 ℃, centrifuging and washing with deionized water at the rotating speed of 15000r/min, and freeze-drying to obtain the PBAs/AOND nano enzyme composite material.
(3) Dissolving gelatin in hexafluoroisopropanol to obtain a gelatin solution with the mass fraction of 12 percent as a shell solution; dissolving polycaprolactone into hexafluoroisopropanol to obtain a polycaprolactone solution with the mass fraction of 12%, and then adding 0.2mg of AOND-PBANPs to obtain a core solution; preparing the nanofiber membrane under the condition of regulating and controlling a spinning process under the conditions that the temperature is 20 ℃ and the humidity is 40%. Co-spinning to obtain PCL/AOND-PBANPs @ GE.
The specific spinning conditions are as follows: the voltage was 10kv, the receiving distance was 15cm, the flow rate of the core was 0.2mL/min, and the flow rate of the shell solution was 0.2mL/min.
Example 3
(1) The preparation method by the solvothermal method specifically comprises the following steps: ferric chloride and zinc acetate, each with a concentration of 5mM, 1mL each, were mixed with 3mL of 28mg/mL polyvinylpyrrolidone, and 2mL of 5mM K was added 4 [Fe(CN) 6 ]Slowly adding the solution into the mixed solution, and reacting at 70 deg.C for 30min to obtain Prussian Blue Analogues (PBAs). Centrifugal washing at 15000r/min, followed by freeze-drying gave the product as a powder of PBAs.
(2) Dissolving gelatin in hexafluoroisopropanol to obtain gelatin solution with mass fraction of 8% as shell solution; dissolving polycaprolactone in hexafluoroisopropanol to obtain a polycaprolactone solution with the mass fraction of 10%, and then dissolving 0.1mg of PBANPs to obtain a nuclear solution; the nanofiber membrane is prepared under the condition that the temperature is 20 ℃ and the humidity is 40% by regulating and controlling the spinning process. Co-spinning to obtain PCL/AOND-PBANPs @ GE.
The specific spinning conditions are as follows: voltage 15kv, receiving distance 12cm, flow rate of core 0.1mL/min and flow rate of shell solution 0.2mL/min.
Claims (8)
1. The nanofiber membrane is characterized by being of a core-shell structure, wherein a shell layer material contains gelatin; the core layer material contains polycaprolactone and nano enzyme composite material;
the nano enzyme composite material is a PBAs/AOND nano enzyme composite material, and specifically comprises the following components: containing inorganic iron salt, inorganic zinc salt, surfactant and K 4 [Fe(CN) 6 ]The raw materials of (1) are subjected to solvothermal reaction to obtain Prussian blue analogues PBAs, and then the Prussian blue analogues PBAs are mixed and stirred with antisense oligonucleotide AOND to obtain a PBAs/AOND nano enzyme composite material; wherein the antisense oligonucleotide AOND sequence is specifically 5 'GTAGCCTTGGCTCGTG-3'.
2. A method of making the nanofiber membrane of claim 1 comprising:
dissolving gelatin in a solvent to obtain a shell solution; mixing polycaprolactone, a PBAs/AOND nano enzyme composite material and a solvent to obtain a core layer solution; and carrying out coaxial electrostatic spinning to obtain the nanofiber membrane.
3. The production method according to claim 2, wherein the solvents are hexafluoroisopropanol; the mass concentration of gelatin in the shell solution is 5-15%; the mass concentration of polycaprolactone in the core layer solution is 6 to 15 percent; the mass concentration of the PBAs/AOND nano-enzyme composite material is 0.001-0.1%.
4. The preparation method according to claim 2, wherein the PBAs/AOND nanoenzyme composite material is specifically:
mixing inorganic iron salt solution, inorganic zinc salt solution and surfactant, and adding K 4 [Fe(CN) 6 ]Reacting the solution at 40 to 80 ℃ for 1 to 60min, centrifuging, washing, and freeze-drying to obtain Prussian blue analog PBAs;
mixing the Prussian blue analogue PBAs and AOND, stirring, centrifuging, washing, and freeze-drying to obtain the PBAs/AOND nano-enzyme composite material.
5. The preparation method of claim 4, wherein the inorganic ferric salt is one of ferric chloride, ferric nitrate, ferric sulfate and ferric acetate; the inorganic zinc salt is one of zinc chloride, zinc nitrate, zinc sulfate and zinc acetate; the concentrations of the inorganic ferric salt solution and the inorganic zinc salt solution are both 1 to 10mM; the surfactant is polyvinylpyrrolidone PVP and/or ethylene diamine tetraacetic acid EDTA.
6. The preparation method according to claim 2, wherein the mole ratio of the Prussian blue analogues PBAs to AOND is 1:10 3 -10 4 (ii) a The stirring is carried out for more than 8 hours at 25 ℃.
7. The preparation method according to claim 2, wherein the specific process parameters of the coaxial electrostatic spinning are as follows: the spinning voltage is as follows: 6-20kv, the receiving distance is 6-20cm, the flow rate of the core solution is 0.1-1mL/min, and the flow rate of the shell solution is 0.1-2mL/min.
8. Use of the nanofiber membrane of claim 1 in the preparation of a skin wound material.
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| EP0737071A1 (en) * | 1993-06-15 | 1996-10-16 | Il- Yang Pharm. Co., Ltd. | Anti-sense oligodeoxynucleotide to fibrogenic cytokines and use thereof |
| CN106075539B (en) * | 2016-06-20 | 2019-04-12 | 浙江大学城市学院 | Load nanofiber membrane preparation method and the wound dressing application of the core/shell structure of Chinese medicine asiaticosid |
| CN109381732A (en) * | 2017-08-03 | 2019-02-26 | 国家纳米科学中心 | Electrostatic spinning dressing, preparation method and the application of growth factor-loaded micromolecular inhibitor |
| CN110772379B (en) * | 2019-11-01 | 2020-11-13 | 山东汉方制药有限公司 | Preparation method of composite nanofiber membrane loaded with nanoenzyme and wound surface application thereof |
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