Preparation method of high-stability antibacterial composite nanofiber membrane

Abstract

The application relates to the field of medical materials and discloses a preparation method of a high-stability antibacterial composite nanofiber membrane. Two polymers with large melting point differences are selected, coaxial nanofiber membranes with a core-shell structure are prepared through coaxial electrospinning technology, nanometers with antibacterial effects are uniformly sprayed on the surfaces of the coaxial nanofiber membranes, and then the low-melting-point polymers of the shell layers are melted through heating treatment to generate a physical bonding effect on the nanometers, so that the nanometers are stably compounded on the surfaces of the nanofiber membranes, and the activity of the nanometers is maximally exerted.

Description

Technical Field

[0001] This invention relates to the field of medical materials, and in particular to a method for preparing a highly stable antibacterial composite nanofiber membrane. Background Technology

[0002] Wounds are highly susceptible to bacterial infection during the healing process, and antibiotic therapy is the most common treatment for bacterial infections. However, with the widespread use of antibiotics, due to the short reproductive cycle and easy mutation of bacteria, more and more bacteria are exhibiting antibiotic resistance. To address the problem of bacterial resistance, there is an urgent need to develop novel antibacterial materials. Nanozymes, which exhibit catalytic properties similar to natural enzymes at the nanoscale, can regulate the level of reactive oxygen species (ROS). By destroying the active components of bacteria (such as proteins, DNA, and RNA) through ROS, bacteria die, thus avoiding the problem of bacterial resistance. Among them, peroxide-like (POD) nanozymes and oxide-like (OXD) nanozymes can rapidly catalyze the generation of ROS from hydrogen peroxide and oxygen for antibacterial purposes. Nanozymes have attracted widespread attention and research due to their high stability, ease of production, and low cost. However, in practical applications, the high surface energy of nanozymes can limit their activity due to aggregation or lack of a carrier, and direct application to the wound site may cause inflammation and other complications. In previous research, our team discovered that nanofibers of one-dimensional nanomaterials can provide support and increase the surface area of ​​nanoenzymes, thereby maximizing their function. Therefore, the two can be combined to construct composite nanofiber membrane dressings.

[0003] Electrospinning is a simple and effective method for preparing nanofibers. Nanozymes can be easily incorporated into nanofibers, typically by mixing nanozymes with polymer solutions and then directly electrospinning to obtain composite nanofiber membranes. However, nanozymes are usually embedded in the polymer matrix, which may reduce their functional activity, especially for nanozymes that need to react with substrates to generate ROS. Therefore, to ensure maximum nanozyme activity, they need to be composited onto the surface of the fibers. Our team initially used electrostatic adsorption to adsorb nanozymes onto the surface of the fiber membrane, but nanozymes tend to aggregate on the nanofiber surface, affecting their functional activity; furthermore, the weak adsorption force results in poor particle binding stability, making them prone to detachment and potentially posing a toxicity risk. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for preparing a highly stable antibacterial composite nanofiber membrane. This invention selects two polymers with significantly different melting points and prepares a coaxial nanofiber membrane with a core-shell structure using coaxial electrospinning technology. Then, an antibacterial nanoenzyme is uniformly sprayed onto the surface of the coaxial nanofiber membrane. Following this, a heat treatment is performed, causing the low-melting-point polymer in the shell to melt and physically bond the nanoenzyme, thereby stably bonding the nanoenzyme to the surface of the nanofiber membrane and maximizing its activity.

[0005] The specific technical solution of this invention is as follows: a method for preparing a highly stable antibacterial composite nanofiber membrane, comprising the following steps:

[0006] (1) Preparation of coaxial nanofiber membrane: Low melting point polymer is formulated into a shell spinning solution that can be used for electrospinning, and high melting point polymer is formulated into a core spinning solution that can be used for electrospinning. Coaxial nanofiber membrane is prepared by coaxial electrospinning.

[0007] The coaxial electrospinning parameters are as follows: needle type: inner needle 20-24G, outer needle 15-19G; electrospinning core-shell flow rate ratio: 1:1-1:4; core layer flow rate: 0.0006-0.0015mm / s; receiving distance: 8-17cm; receiving roller speed: 100-300r / min; voltage: 6-20kV; temperature: 25-35℃; humidity: 40-60%.

[0008] (2) Preparation of nanozyme dispersion: Nanozyme was added to anhydrous ethanol for dispersion, polyethylene glycol was added and ultrasonic treatment was performed to obtain a nanozyme dispersion that can be continuously and uniformly dispersed.

[0009] (3) Nanoenzyme composite: Nanoenzymes are sprayed onto a coaxial nanofiber membrane using a spraying technique, and the low-melting-point polymer in the shell is heated to melt, resulting in a highly stable antibacterial composite nanofiber membrane.

[0010] This invention selects two polymers with large differences in melting point and prepares a coaxial nanofiber membrane with a core-shell structure through coaxial electrospinning technology; then, an antibacterial nanoenzyme is uniformly sprayed onto the surface of the coaxial nanofiber membrane, and then heat treatment is performed to melt the low-melting-point polymer in the shell layer and physically bond it to the nanoenzyme, thereby stably compositing the nanoenzyme on the surface of the nanofiber membrane and maximizing the activity of the nanoenzyme.

[0011] In addition, the core-shell flow rate ratio in coaxial electrospinning in step (1) also needs to be considered. If the shell flow rate is too high, the shell polymer will melt too much during heat treatment, and the fibers will fuse and cross-link with each other, affecting the porosity and permeability of the fiber membrane. It will also cause the nanozyme to be completely embedded in the polymer matrix, affecting the enzyme activity. If the shell flow rate is too low, the core-shell structure will not be able to form, which will also affect the activity of the adhesive nanozyme.

[0012] In step (2), in order to keep the nanozyme dispersion in a dispersed state for a long time, an appropriate amount of polyethylene glycol is added to the dispersion. Its molecular chains will unfold in the solution and easily form strong hydrogen bonds on the surface of the nanozyme particles. This hydrogen bond effect causes it to be adsorbed on the particles. The electrostatic repulsion and the steric hindrance effect of polyethylene glycol make the nanozyme dispersed stably.

[0013] Preferably, in step (1), the difference in melting points between the low-melting-point polymer and the high-melting-point polymer is at least greater than 20-40°C.

[0014] It is important to note that the two polymers selected should have a certain difference in melting points. This is to ensure that the high-melting-point polymer in the core layer will not be affected by heat and melt during heat treatment, thus preserving the fiber structure. If the difference in melting points is too small, both the core and shell polymers will melt when heated to temperatures close to their melting points, making it difficult to maintain the nanofiber structure.

[0015] Preferably, in step (1), the low-melting-point polymer is polycaprolactone with a Tm of 58-60℃, and the high-melting-point polymer is any one of polyglycolic acid with a Tm of 224-225℃, cellulose acetate with a Tm of 230-300℃, and polylactic acid with a Tm of 175-178℃.

[0016] Preferably, in step (1), the concentration of polycaprolactone in the shell spinning solution is 0.1-0.18 g / mL, and the solvent is at least one of trifluoroethanol and hexafluoroisopropanol, which is stirred and dissolved at room temperature.

[0017] Preferably, in step (1), the concentration of polyglycolic acid in the core spinning solution is 0.06-0.2 g / mL, the solvent is hexafluoroisopropanol, and it is dissolved by stirring under heating conditions at a temperature of 40-80°C; or, the concentration of cellulose acetate in the core spinning solution is 0.15-0.25 g / mL, the solvent is a mixture of acetone, N,N'-dimethylacetamide, and hexafluoroisopropanol or glacial acetic acid in a volume ratio of 3:2-2.5:1, and it is dissolved by stirring at room temperature; or, the concentration of polylactic acid spinning solution in the core spinning solution is 0.08-0.14 g / mL, the solvent is a mixture of dichloromethane and N,N'-dimethylformamide in a volume ratio of 4:1-1:1, and it is dissolved by stirring at room temperature.

[0018] Preferably, in step (2), the nanozyme is at least one of cerium dioxide nanoparticles, cobalt tetroxide nanoparticles and manganese oxide nanoparticles, and the concentration of the nanozyme dispersion is 0.1-3 mg / mL.

[0019] The antibacterial function of composite nanofiber membranes is mainly achieved through the action of nanozymes. As mentioned in the background section, nanozymes are prone to aggregation, which limits their functional activity. Therefore, to achieve uniform distribution on the fibers, it is first necessary to ensure uniform dispersion of the nanozymes in the solvent, requiring the preparation of a dispersion of a certain concentration. If the nanozyme concentration is too high, it is easy for them to aggregate and settle.

[0020] Preferably, in step (2), the molecular weight of the polyethylene glycol is 6000-8000.

[0021] Preferably, in step (2), the concentration of polyethylene glycol in the nanoenzyme dispersion is 0.001-0.01 g / mL.

[0022] The molecular weight of polyethylene glycol (PEG) is crucial. The dispersion of nanozymes is primarily determined by the steric hindrance of the adsorption layer on the particle surface, and the length of the molecular chain directly determines the magnitude of this steric hindrance. If the molecular weight is too small, the resulting steric hindrance cannot effectively disperse the particles. The concentration of PEG also affects the dispersion performance. If the concentration is too low, the particles tend to settle, which is detrimental to the subsequent spraying process; if the concentration is too high, the viscosity increases, resulting in excessive coating of the particles, which also affects the spraying process and the particle's functionality. To ensure uniform dispersion, the dispersion was also subjected to ultrasonic treatment.

[0023] Preferably, in step (2), the power of the ultrasonic treatment is 80-100W and the time is 10-30min.

[0024] Preferably, in step (3), the spraying technology is either air gun spraying or electrostatic spraying; wherein, the air gun spraying parameters are: pressure of 0.4-0.6 MPa, receiving distance of 10-15 cm, and moving speed of 10-30 cm / s; the electrostatic spraying parameters are: flow rate of 0.001-0.005 mm / s, receiving distance of 8-12 cm, receiving roller speed of 100-300 r / min, voltage of 18-30 kV, temperature of 25-35℃, and humidity of 40-60%.

[0025] Electrostatic spraying and air gun spraying are used to distribute nanozymes relatively evenly on the surface of fibers. To ensure the stability of the composite, the difference in melting points between the two polymers is used to carry out an appropriate heating process. If the temperature is too low or the time is too short, the shell polymer will not melt and bind the nanozymes. If the temperature is too high or the time is too long, the shell will melt excessively, resulting in the loss of fiber structure and the formation of a large area of ​​non-porous membrane.

[0026] Preferably, in step (3), the heating and melting temperature is 70-80°C and the time is 3-6 minutes.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention selects two polymers with large differences in melting point to prepare a coaxial nanofiber membrane with a core-shell structure through coaxial electrospinning technology; then, nanoenzymes with antibacterial effects are uniformly sprayed on the surface of the coaxial nanofiber membrane, and then heat treatment is carried out to melt the low-melting-point polymer in the shell layer and generate a physical bonding effect on the nanoenzymes, thereby stably compositing the nanoenzymes on the surface of the nanofiber membrane and maximizing the activity of the nanoenzymes. Attached Figure Description

[0028] Figure 1 The image shown is a scanning electron microscope (SEM) image of the material prepared in Example 1.

[0029] Figure 2 The image shows a scanning electron microscope (SEM) image of the material prepared in Comparative Example 5. Detailed Implementation

[0030] The present invention will be further described below with reference to embodiments.

[0031] Example 1

[0032] (1) Preparation of coaxial nanofiber membranes: Two polymers were prepared: polycaprolactone (PCL, Tm = 58-60℃) and polyglycolic acid (PGA, Tm = 224-225℃), which were respectively prepared into polymer solutions suitable for electrospinning. The concentration of the polycaprolactone spinning solution was 0.1 g / mL, the solvent was trifluoroethanol, and it was dissolved by stirring at room temperature; the concentration of the polyglycolic acid spinning solution was 0.08 g / mL, the solvent was hexafluoroisopropanol, and it was dissolved by stirring under heating conditions at a temperature of 40℃. Polyglycolic acid spinning solution was used as the core layer and polycaprolactone spinning solution was used as the shell layer. The needles used for coaxial electrospinning were 22G inner needles and 17G outer needles. The core-shell flow rate ratio was 1:3, the core layer flow rate was 0.0008 mm / s, the receiving distance was 15 cm, the receiving roller speed was 200 r / min, the voltage was 12 kV, the temperature was 30 ℃, the humidity was 50%, and the spinning time was 4 h, resulting in a coaxial nanofiber membrane of polycaprolactone / polyglycolic acid.

[0033] (2) Preparation of nanoenzyme dispersion: Cerium dioxide nanoparticles (CeO2) were added to anhydrous ethanol for dispersion, and the concentration of the ethanol dispersion was 0.5 mg / mL. Polyethylene glycol 6000 was added at a concentration of 0.01 g / mL, and then ultrasonic dispersion was performed at a power of 100 W for 20 min to obtain CeO2NPs dispersion.

[0034] (3) Nanoenzyme composite: CeO2 was sprayed onto a coaxial nanofiber membrane using electrostatic spraying. The electrostatic spraying parameters were: flow rate of 0.003 mm / s, receiving distance of 10 cm, receiving drum speed of 200 r / min, voltage of 20 kV, temperature of 30 °C, and humidity of 40%. The membrane was then heat-treated at 80 °C for 5 min to obtain a heat-treated cerium dioxide nanoparticle (CeO2) composite polycaprolactone / polyglycolic acid nanofiber membrane.

[0035] Example 2

[0036] (1) Preparation of coaxial nanofiber membranes: Two polymers were prepared: polycaprolactone (PCL, Tm = 58-60℃) and polylactic acid (PLA, Tm = 175-178℃), which were respectively prepared into polymer solutions suitable for electrospinning. The concentration of the polycaprolactone spinning solution was 0.1 g / mL, the solvent was trifluoroethanol, and it was dissolved by stirring at room temperature; the concentration of the polylactic acid spinning solution was 0.12 g / mL, the solvent was a mixed solvent of dichloromethane and N,N'-dimethylformamide (volume ratio of 1:1), and it was dissolved by stirring at room temperature. Polylactic acid spinning solution was used as the core layer and polycaprolactone spinning solution was used as the shell layer. The needles used for coaxial electrospinning were 22G inner needles and 17G outer needles. The core-shell flow rate ratio was 1:4, the core layer flow rate was 0.0006 mm / s, the receiving distance was 13 cm, the receiving roller speed was 200 r / min, the voltage was 15 kV, the temperature was 28 ℃, the humidity was 60%, and the spinning time was 5 h, resulting in a polycaprolactone / polylactic acid coaxial nanofiber membrane.

[0037] (2) Preparation of nanoenzyme dispersion: Cobalt tetroxide nanoparticles (Co3O4NPs) were added to anhydrous ethanol for dispersion, and the concentration of the ethanol dispersion was 0.8 mg / mL. Polyethylene glycol 6000 was added at a concentration of 0.005 g / mL, and then ultrasonic dispersion was performed at a power of 80 W for 30 min to obtain PB NPs dispersion.

[0038] (3) Nanoenzyme composite: Co3O4NPs were sprayed onto a coaxial nanofiber membrane using electrostatic spraying. The electrostatic spraying parameters were: flow rate of 0.002 mm / s, receiving distance of 12 cm, receiving drum speed of 200 r / min, voltage of 18 kV, temperature of 35 ℃, and humidity of 40%. The membrane was then heat-treated at 75 ℃ for 6 min to obtain a heat-treated cobalt tetroxide nanoparticle (Co3O4NPs) composite polycaprolactone / polylactic acid nanofiber membrane.

[0039] Example 3

[0040] (1) Preparation of coaxial nanofiber membranes: Two polymers were prepared: polycaprolactone (PCL, Tm = 58-60℃) and cellulose acetate (CA, Tm = 230-300℃), which were respectively prepared into polymer solutions suitable for electrospinning. The concentration of the polycaprolactone spinning solution was 0.1 g / mL, the solvent was trifluoroethanol, and it was stirred and dissolved at room temperature; the concentration of the cellulose acetate spinning solution was 0.18 g / mL, the solvent was a mixed solvent of hexafluoroisopropanol and glacial acetic acid (volume ratio of 2:1), and it was stirred and dissolved at room temperature. Polycaprolactone spinning solution was used as the core layer, and cellulose acetate spinning solution was used as the shell layer. The needles used for coaxial electrospinning were 22G inner needles and 17G outer needles. The core-shell flow rate ratio was 1:2, the core layer flow rate was 0.0012 mm / s, the receiving distance was 12 cm, the receiving roller speed was 180 r / min, the voltage was 18 kV, the temperature was 28 ℃, the humidity was 50%, and the spinning time was 4 h, resulting in a polycaprolactone / cellulose acetate coaxial nanofiber membrane.

[0041] (2) Preparation of nanoenzyme dispersion: Manganese oxide nanoparticles (MnO NPs) were added to anhydrous ethanol for dispersion, and the concentration of the ethanol dispersion was 1 mg / mL. Polyethylene glycol 8000 was added at a concentration of 0.005 g / mL, and then ultrasonic dispersion was performed at a power of 100 W for 10 min to obtain the MnO NPs dispersion.

[0042] (3) Nanoenzyme composite: MnONPs were sprayed onto a coaxial nanofiber membrane using an air gun spraying technique. The air gun spraying parameters were: pressure 0.6 MPa, receiving distance 10 cm, and moving speed 20 cm / s. The membrane was then heat-treated at 78 °C for 5 min to obtain a heat-treated manganese oxide nanoparticle (MnONPs) composite polycaprolactone / cellulose acetate nanofiber membrane.

[0043] Example 4

[0044] (1) Preparation of coaxial nanofiber membranes: Two polymers were prepared: polycaprolactone (PCL, Tm = 58-60℃) and cellulose acetate (CA, Tm = 230-300℃), which were respectively prepared into polymer solutions suitable for electrospinning. The concentration of the polycaprolactone spinning solution was 0.14 g / mL, the solvent was trifluoroethanol, and it was dissolved by stirring at room temperature; the concentration of the cellulose acetate spinning solution was 0.18 g / mL, the solvent was a mixed solvent of hexafluoroisopropanol and glacial acetic acid (volume ratio of 2:1), and it was dissolved by stirring at room temperature. Polycaprolactone spinning solution was placed in the core layer, and cellulose acetate spinning solution was placed in the shell layer. The needles used for coaxial electrospinning were 22G inner needles and 17G outer needles. The core-shell flow rate ratio was 1:1, the core layer flow rate was 0.002 mm / s, the receiving distance was 15 cm, the receiving roller speed was 200 r / min, the voltage was 13 kV, the temperature was 28 ℃, the humidity was 60%, and the spinning time was 3 h, thus obtaining a coaxial nanofiber membrane of polycaprolactone / cellulose acetate.

[0045] (2) Preparation of nanoenzyme dispersion: Cobalt tetroxide nanoparticles (Co3O4NPs) were added to anhydrous ethanol for dispersion, and the concentration of the ethanol dispersion was 0.5 mg / mL. Polyethylene glycol 8000 was added at a concentration of 0.004 g / mL, and then ultrasonic dispersion was performed at a power of 100 W for 30 min to obtain Co3O4NPs dispersion.

[0046] (3) Nanoenzyme composite: Co3O4NPs were sprayed onto a coaxial nanofiber membrane using an air gun spraying technique. The air gun spraying parameters were: pressure 0.5 MPa, receiving distance 10 cm, and moving speed 15 cm / s. The membrane was then heat-treated at 80 °C for 5 min to obtain a heat-treated cobalt tetroxide nanoparticle (Co3O4NPs) composite polycaprolactone / cellulose acetate nanofiber membrane.

[0047] Comparative Example 1 (Shell flow velocity too low)

[0048] (1) Preparation of coaxial nanofiber membranes: Two polymers were prepared: polycaprolactone (PCL, Tm = 58-60℃) and polyglycolic acid (PGA, Tm = 224-225℃), which were respectively prepared into polymer solutions suitable for electrospinning. The concentration of the polycaprolactone spinning solution was 0.1 g / mL, the solvent was trifluoroethanol, and it was dissolved by stirring at room temperature; the concentration of the polyglycolic acid spinning solution was 0.08 g / mL, the solvent was hexafluoroisopropanol, and it was dissolved by stirring under heating conditions at a temperature of 40℃. Polyglycolic acid spinning solution was used as the core layer and polycaprolactone spinning solution was used as the shell layer. The needles used for coaxial electrospinning were 22G inner needles and 17G outer needles. The core-shell flow rate ratio was 1:0.5, the core layer flow rate was 0.0008 mm / s, the receiving distance was 15 cm, the receiving roller speed was 200 r / min, the voltage was 8 kV, the temperature was 30 ℃, the humidity was 50%, and the spinning time was 4 h, resulting in a coaxial nanofiber membrane of polycaprolactone / polyglycolic acid.

[0049] (2) Preparation of nanoenzyme dispersion: Cerium dioxide nanoparticles (CeO2NPs) were added to anhydrous ethanol for dispersion, and the concentration of the ethanol dispersion was 0.5 mg / mL. Polyethylene glycol 6000 was added at a concentration of 0.01 g / mL, and then ultrasonic dispersion was performed at a power of 100 W for 20 min to obtain CeO2NPs dispersion.

[0050] (3) Nanoenzyme composite: CeO2NPs were sprayed onto a coaxial nanofiber membrane using electrostatic spraying. The electrostatic spraying parameters were: flow rate of 0.003 mm / s, receiving distance of 10 cm, receiving drum speed of 200 r / min, voltage of 20 kV, temperature of 30 °C, and humidity of 40%. The membrane was then heat-treated at 80 °C for 5 min to obtain a heat-treated cerium dioxide nanoparticle (CeO2NPs) composite polycaprolactone / polyglycolic acid nanofiber membrane.

[0051] Comparative Example 2 (Excessive Shell Flow Velocity)

[0052] 1) Preparation of coaxial nanofiber membranes: Two polymers were prepared: polycaprolactone (PCL, Tm = 58-60℃) and polyglycolic acid (PGA, Tm = 224-225℃), which were respectively formulated into polymer solutions suitable for electrospinning. The concentration of the polycaprolactone spinning solution was 0.1 g / mL, the solvent was trifluoroethanol, and it was dissolved by stirring at room temperature; the concentration of the polyglycolic acid spinning solution was 0.08 g / mL, the solvent was hexafluoroisopropanol, and it was dissolved by stirring under heating conditions at a temperature of 40℃. Polyglycolic acid spinning solution was used as the core layer and polycaprolactone spinning solution was used as the shell layer. The needles used for coaxial electrospinning were 22G inner needles and 17G outer needles. The core-shell flow rate ratio was 1:6, the core layer flow rate was 0.0008 mm / s, the receiving distance was 15 cm, the receiving roller speed was 200 r / min, the voltage was 18 kV, the temperature was 30 ℃, the humidity was 50%, and the spinning time was 4 h, resulting in a coaxial nanofiber membrane of polycaprolactone / polyglycolic acid.

[0053] (2) Preparation of nanozymes: Cerium dioxide nanoparticles (CeO2NPs) were dispersed in anhydrous ethanol at a concentration of 0.5 mg / mL. Polyethylene glycol 6000 was added at a concentration of 0.01 g / mL, and then ultrasonic dispersion was performed at a power of 100 W for 20 min to obtain a CeO2NPs dispersion.

[0054] (3) Nanoenzyme composite: CeO2NPs were sprayed onto a coaxial nanofiber membrane using electrostatic spraying. The electrostatic spraying parameters were: flow rate of 0.003 mm / s, receiving distance of 10 cm, receiving drum speed of 200 r / min, voltage of 20 kV, temperature of 30 °C, and humidity of 40%. The membrane was then heat-treated at 80 °C for 5 min to obtain a heat-treated cerium dioxide nanoparticle (CeO2NPs) composite polycaprolactone / polyglycolic acid nanofiber membrane.

[0055] Comparative Example 3 (Too little polyethylene glycol)

[0056] (1) Preparation of coaxial nanofiber membranes: Two polymers were prepared: polycaprolactone (PCL, Tm = 58-60℃) and polyglycolic acid (PGA, Tm = 224-225℃), which were respectively prepared into polymer solutions suitable for electrospinning. The concentration of the polycaprolactone spinning solution was 0.1 g / mL, the solvent was trifluoroethanol, and it was dissolved by stirring at room temperature; the concentration of the polyglycolic acid spinning solution was 0.08 g / mL, the solvent was hexafluoroisopropanol, and it was dissolved by stirring under heating conditions at a temperature of 40℃. Polyglycolic acid spinning solution was used as the core layer and polycaprolactone spinning solution was used as the shell layer. The needles used for coaxial electrospinning were 22G inner needles and 17G outer needles. The core-shell flow rate ratio was 1:3, the core layer flow rate was 0.0008 mm / s, the receiving distance was 15 cm, the receiving roller speed was 200 r / min, the voltage was 12 kV, the temperature was 30 ℃, the humidity was 50%, and the spinning time was 4 h, resulting in a coaxial nanofiber membrane of polycaprolactone / polyglycolic acid.

[0057] (2) Preparation of nanoenzyme dispersion: Cerium dioxide nanoparticles (CeO2NPs) were added to anhydrous ethanol for dispersion, and the concentration of the ethanol dispersion was 0.5 mg / mL. Polyethylene glycol 6000 was added at a concentration of 0.0001 g / mL, and then ultrasonic dispersion was performed at a power of 100 W for 20 min to obtain CeO2NPs dispersion.

[0058] (3) Nanoenzyme composite: CeO2NPs were sprayed onto a coaxial nanofiber membrane using electrostatic spraying. The electrostatic spraying parameters were: flow rate of 0.003 mm / s, receiving distance of 10 cm, receiving drum speed of 200 r / min, voltage of 20 kV, temperature of 30 °C, and humidity of 40%. The membrane was then heat-treated at 80 °C for 5 min to obtain a heat-treated cerium dioxide nanoparticle (CeO2NPs) composite polycaprolactone / polyglycolic acid nanofiber membrane.

[0059] Comparative Example 4 (Excessive Polyethylene Glycol)

[0060] (1) Preparation of coaxial nanofiber membranes: Two polymers were prepared: polycaprolactone (PCL, Tm = 58-60℃) and polyglycolic acid (PGA, Tm = 224-225℃), which were respectively prepared into polymer solutions suitable for electrospinning. The concentration of the polycaprolactone spinning solution was 0.1 g / mL, the solvent was trifluoroethanol, and it was dissolved by stirring at room temperature; the concentration of the polyglycolic acid spinning solution was 0.08 g / mL, the solvent was hexafluoroisopropanol, and it was dissolved by stirring under heating conditions at a temperature of 40℃. Polyglycolic acid spinning solution was used as the core layer and polycaprolactone spinning solution was used as the shell layer. The needles used for coaxial electrospinning were 22G inner needles and 17G outer needles. The core-shell flow rate ratio was 1:3, the core layer flow rate was 0.0008 mm / s, the receiving distance was 15 cm, the receiving roller speed was 200 r / min, the voltage was 12 kV, the temperature was 30 ℃, the humidity was 50%, and the spinning time was 4 h, resulting in a coaxial nanofiber membrane of polycaprolactone / polyglycolic acid.

[0061] (2) Preparation of nanoenzyme dispersion: Cerium dioxide nanoparticles (CeO2NPs) were added to anhydrous ethanol for dispersion, and the concentration of the ethanol dispersion was 0.5 mg / mL. Polyethylene glycol 6000 was added at a concentration of 0.1 g / mL, and then ultrasonic dispersion was performed at a power of 100 W for 20 min to obtain CeO2NPs dispersion.

[0062] (3) Nanoenzyme composite: CeO2NPs were sprayed onto a coaxial nanofiber membrane using electrostatic spraying. The electrostatic spraying parameters were: flow rate of 0.003 mm / s, receiving distance of 10 cm, receiving drum speed of 200 r / min, voltage of 20 kV, temperature of 30 °C, and humidity of 40%. The membrane was then heat-treated at 80 °C for 5 min to obtain a heat-treated cerium dioxide nanoparticle (CeO2NPs) composite polycaprolactone / polyglycolic acid nanofiber membrane.

[0063] Comparative Example 5 (without heat treatment)

[0064] (1) Preparation of coaxial nanofiber membranes: Two polymers were prepared: polycaprolactone (PCL, Tm = 58-60℃) and polyglycolic acid (PGA, Tm = 224-225℃), which were respectively prepared into polymer solutions suitable for electrospinning. The concentration of the polycaprolactone spinning solution was 0.1 g / mL, the solvent was trifluoroethanol, and it was dissolved by stirring at room temperature; the concentration of the polyglycolic acid spinning solution was 0.08 g / mL, the solvent was hexafluoroisopropanol, and it was dissolved by stirring under heating conditions at a temperature of 40℃. Polyglycolic acid spinning solution was used as the core layer and polycaprolactone spinning solution was used as the shell layer. The needles used for coaxial electrospinning were 22G inner needles and 17G outer needles. The core-shell flow rate ratio was 1:3, the core layer flow rate was 0.0008 mm / s, the receiving distance was 15 cm, the receiving roller speed was 200 r / min, the voltage was 12 kV, the temperature was 30 ℃, the humidity was 50%, and the spinning time was 4 h, resulting in a coaxial nanofiber membrane of polycaprolactone / polyglycolic acid.

[0065] (2) Preparation of nanoenzyme dispersion: Cerium dioxide nanoparticles (CeO2NPs) were added to anhydrous ethanol for dispersion, and the concentration of the ethanol dispersion was 0.5 mg / mL. Polyethylene glycol 6000 was added at a concentration of 0.01 g / mL, and then ultrasonic dispersion was performed at a power of 100 W for 20 min to obtain CeO2NPs dispersion.

[0066] (3) Nanoenzyme composite: CeO2NPs were sprayed onto a coaxial nanofiber membrane using electrostatic spraying. The electrostatic spraying parameters were: flow rate of 0.003 mm / s, receiving distance of 10 cm, receiving drum speed of 200 r / min, voltage of 20 kV, temperature of 30 °C, and humidity of 40%. Without heat treatment, a nanofiber membrane of cerium dioxide nanoparticles (CeO2NPs) composite with polycaprolactone / polyglycolic acid was obtained.

[0067] Comparative Example 6 (Heat Treatment Temperature Too High)

[0068] (1) Preparation of coaxial nanofiber membranes: Two polymers were prepared: polycaprolactone (PCL, Tm = 58-60℃) and polyglycolic acid (PGA, Tm = 224-225℃), which were respectively prepared into polymer solutions suitable for electrospinning. The concentration of the polycaprolactone spinning solution was 0.1 g / mL, the solvent was trifluoroethanol, and it was dissolved by stirring at room temperature; the concentration of the polyglycolic acid spinning solution was 0.08 g / mL, the solvent was hexafluoroisopropanol, and it was dissolved by stirring under heating conditions at a temperature of 40℃. Polyglycolic acid spinning solution was used as the core layer and polycaprolactone spinning solution was used as the shell layer. The needles used for coaxial electrospinning were 22G inner needles and 17G outer needles. The core-shell flow rate ratio was 1:3, the core layer flow rate was 0.0008 mm / s, the receiving distance was 15 cm, the receiving roller speed was 200 r / min, the voltage was 12 kV, the temperature was 30 ℃, the humidity was 50%, and the spinning time was 4 h, resulting in a coaxial nanofiber membrane of polycaprolactone / polyglycolic acid.

[0069] (2) Preparation of nanoenzyme dispersion: Cerium dioxide nanoparticles (CeO2NPs) were added to anhydrous ethanol for dispersion, and the concentration of the ethanol dispersion was 0.5 mg / mL. Polyethylene glycol 6000 was added at a concentration of 0.01 g / mL, and then ultrasonic dispersion was performed at a power of 100 W for 20 min to obtain CeO2NPs dispersion.

[0070] (3) Nanoenzyme composite: CeO2NPs were sprayed onto a coaxial nanofiber membrane using electrostatic spraying. The electrostatic spraying parameters were: flow rate of 0.003 mm / s, receiving distance of 10 cm, receiving roller speed of 200 r / min, voltage of 20 kV, temperature of 30 °C, and humidity of 40%. The membrane was then heat-treated at 100 °C for 5 min to obtain a heat-treated cerium dioxide nanoparticle (CeO2NPs) composite polycaprolactone / polyglycolic acid nanofiber membrane.

[0071] Comparative Example 7 (Heat Treatment Time Too Short)

[0072] (1) Preparation of coaxial nanofiber membranes: Two polymers with large differences in melting points were prepared: polycaprolactone (PCL, Tm = 58-60℃) and polyglycolic acid (PGA, Tm = 224-225℃). These were prepared into polymer solutions suitable for electrospinning. The concentration of the PCL spinning solution was 0.1 g / mL, and the solvent was trifluoroethanol, dissolved by stirring at room temperature. The concentration of the PGA spinning solution was 0.08 g / mL, and the solvent was hexafluoroisopropanol, dissolved by stirring under heating conditions at 40℃. Polyglycolic acid spinning solution was used as the core layer and polycaprolactone spinning solution was used as the shell layer. The needles used for coaxial electrospinning were 22G inner needles and 17G outer needles. The core-shell flow rate ratio was 1:3, the core layer flow rate was 0.0008 mm / s, the receiving distance was 15 cm, the receiving roller speed was 200 r / min, the voltage was 12 kV, the temperature was 30 ℃, the humidity was 50%, and the spinning time was 4 h, resulting in a coaxial nanofiber membrane of polycaprolactone / polyglycolic acid.

[0073] (2) Preparation of nanoenzyme dispersion: Cerium dioxide nanoparticles (CeO2NPs) were added to anhydrous ethanol for dispersion, and the concentration of the ethanol dispersion was 0.5 mg / mL. Polyethylene glycol 6000 was added at a concentration of 0.01 g / mL, and then ultrasonic dispersion was performed at a power of 100 W for 20 min to obtain CeO2NPs dispersion.

[0074] (3) Nanoenzyme composite: CeO2NPs were sprayed onto a coaxial nanofiber membrane using electrostatic spraying. The electrostatic spraying parameters were: flow rate of 0.003 mm / s, receiving distance of 10 cm, receiving drum speed of 200 r / min, voltage of 20 kV, temperature of 30 ℃, and humidity of 40%. The membrane was then heat-treated at 80 ℃ for 1 min to obtain a heat-treated cerium dioxide nanoparticle (CeO2NPs) composite polycaprolactone / polyglycolic acid nanofiber membrane.

[0075] Comparative Example 8 (excessive heat treatment time)

[0076] (1) Preparation of coaxial nanofiber membranes: Two polymers were prepared: polycaprolactone (PCL, Tm = 58-60℃) and polyglycolic acid (PGA, Tm = 224-225℃), which were respectively prepared into polymer solutions suitable for electrospinning. The concentration of the polycaprolactone spinning solution was 0.1 g / mL, the solvent was trifluoroethanol, and it was dissolved by stirring at room temperature; the concentration of the polyglycolic acid spinning solution was 0.08 g / mL, the solvent was hexafluoroisopropanol, and it was dissolved by stirring under heating conditions at a temperature of 40℃. Polyglycolic acid spinning solution was used as the core layer and polycaprolactone spinning solution was used as the shell layer. The needles used for coaxial electrospinning were 22G inner needles and 17G outer needles. The core-shell flow rate ratio was 1:3, the core layer flow rate was 0.0008 mm / s, the receiving distance was 15 cm, the receiving roller speed was 200 r / min, the voltage was 12 kV, the temperature was 30 ℃, the humidity was 50%, and the spinning time was 4 h, resulting in a coaxial nanofiber membrane of polycaprolactone / polyglycolic acid.

[0077] (2) Preparation of nanoenzyme dispersion: Cerium dioxide nanoparticles (CeO2NPs) were dispersed in anhydrous ethanol at a concentration of 0.5 mg / mL. Polyethylene glycol 6000 was added at a concentration of 0.01 g / mL, and then ultrasonic dispersion was performed at a power of 100 W for 20 min to obtain CeO2NPs dispersion.

[0078] (3) Nanoenzyme composite: CeO2NPs were sprayed onto a coaxial nanofiber membrane using electrostatic spraying. The electrostatic spraying parameters were: flow rate of 0.003 mm / s, receiving distance of 10 cm, receiving roller speed of 200 r / min, voltage of 20 kV, temperature of 30 °C, and humidity of 40%. The membrane was then heat-treated at 80 °C for 20 min to obtain a heat-treated cerium dioxide nanoparticle (CeO2NPs) composite polycaprolactone / polyglycolic acid nanofiber membrane.

[0079] Performance testing

[0080] The morphology and structure of the prepared nanozyme composite nanofiber membrane were observed using scanning electron microscopy. Nanozyme binding stability testing: 1*1cm pieces were cut... 2 Nanoenzyme composite nanofiber membranes, before and after heat treatment, were immersed in centrifuge tubes containing 5 ml of ultrapure water and ultrasonically cleaned for 20 min at 100 W. Afterward, the membranes were gently removed, and the remaining aqueous solution was subjected to UV-Vis absorbance testing. The particle concentration in the solution was determined by the position of the particle absorption peaks and the magnitude of the absorbance, thus obtaining the particle binding stability of the nanoenzyme composite nanofiber membrane. The ROS release performance of the nanoenzyme was tested by cutting 1*1 cm nanofibers... 2The nanoenzyme composite nanofiber membrane was ultrasonically treated, and then removed and added to a 2 mL aqueous solution containing 100 μL (0.2 M, pH 4.0) sodium acetate buffer, 20 μL (40 mM) 3,3′,5,5′-tetramethylbenzidine (TMB) and 20 μL (100 mM) hydrogen peroxide. After reacting for 1 hour, the absorbance at 652 nm was measured using a UV spectrophotometer (with Example 1 as a control) to determine the ROS generation.

[0081] The results are as follows:

[0082]

[0083] The results above show that by first selecting a suitable polymer spinning solution and adjusting the flow rate ratio of the core and shell layers, a suitable core-shell nanofiber structure can be obtained. A nanoenzyme dispersion that can maintain uniform dispersion for a long time is obtained by adding polyethylene glycol. Then, the particles are sprayed onto the fiber surface by spraying, and after heat treatment, a stable composite nanoenzyme nanofiber membrane is obtained (Examples 1-4). Figure 1 The image shown is a scanning electron microscope (SEM) image of Example 1. It can be seen that CeO2 NPs are relatively uniformly distributed on the surface of the nanofibers. The shell layer, due to thermal melting, successfully bonds the particles to the surface, and the particle stability is improved by approximately 30%. When preparing the coaxial nanofiber membrane, if the core-shell flow rate ratio is too small (Comparative Example 1), a core-shell nanofiber structure cannot be formed, resulting in the inability to achieve physical bonding of the particles through thermal melting on the fiber surface after heat treatment. If the shell flow rate ratio is too large (Comparative Example 2), the proportion of the core-shell nanofiber shell layer is too high, leading to large-area film formation after thermal melting, making it difficult to maintain the fiber structure, and the particles cannot remain on the fiber surface. If the added polyethylene glycol content is too low (Comparative Example 3), the particle dispersion cannot be continuously and uniformly dispersed, which is detrimental to the spraying process. The particles sprayed on the fiber surface are unevenly distributed and agglomerated. If the polyethylene glycol content is too high (Comparative Example 4), the high viscosity affects the spraying process, and the particles will be coated. If no heat treatment is performed (Comparative Example 5), from... Figure 2 It can be seen that the particles simply adhere to the fiber surface, lacking binding force and unable to achieve stable composite bonding, making them prone to detachment. If the heat treatment temperature is too high (Comparative Example 6) or the time is too long (Comparative Example 8), the shell polymer will over-melt and flow and fuse, resulting in the loss of the fiber structure and the formation of a non-porous membrane, making it impossible for the particles to remain on the fiber surface. If the heat treatment time is too short (Comparative Example 7), the heat energy required to melt the shell polymer is insufficient, making effective thermal bonding impossible. The ROS release performance of the nanoenzyme composite nanofiber membrane after ultrasonic treatment was tested. The results showed that excessively high or low core-shell flow rates, excessively high or low polyethylene glycol content, excessively high or low heat treatment temperatures, and excessively long or short heat treatment times all reduced the ROS release of the composite nanofiber membrane (Comparative Examples 1-8).

[0084] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0085] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing a highly stable antibacterial composite nanofiber membrane, characterized in that... Includes the following steps: (1) Preparation of coaxial nanofiber membrane: Low melting point polymer is formulated into shell spinning solution that can be used for electrospinning, and high melting point polymer is formulated into core spinning solution that can be used for electrospinning. Coaxial nanofiber membrane is prepared by coaxial electrospinning. The coaxial electrospinning parameters are as follows: needle type: inner needle 20-24G, outer needle 15-19G; electrospinning core-shell flow rate ratio: 1:1-1:4; core layer flow rate: 0.0006-0.0015 mm / s; receiving distance: 8-17 cm; receiving roller speed: 100-300 r / min; voltage: 6-20 kV; temperature: 25-35℃; humidity: 40-60%. (2) Preparation of nanozyme dispersion: Nanozyme was added to anhydrous ethanol for dispersion, polyethylene glycol was added and ultrasonic treatment was performed to obtain a continuously uniformly dispersed nanozyme dispersion. (3) Composite of nanoenzymes: Nanoenzymes are sprayed onto coaxial nanofiber membranes using spraying technology. The low-melting-point polymer of the shell is heated and melted, which causes the low-melting-point polymer of the shell to melt and physically bond the nanoenzymes, thereby stably composited the nanoenzymes on the surface of the nanofiber membrane to obtain a highly stable antibacterial composite nanofiber membrane. In step (1), the low-melting-point polymer is polycaprolactone with a Tm of 58-60℃, and the high-melting-point polymer is any one of polyglycolic acid with a Tm of 224-225℃, cellulose acetate with a Tm of 230-300℃, and polylactic acid with a Tm of 175-178℃.

2. The preparation method according to claim 1, characterized in that: In step (1), The concentration of polycaprolactone in the shell spinning solution is 0.1-0.18 g / mL, and the solvent is at least one of trifluoroethanol and hexafluoroisopropanol, which is stirred and dissolved at room temperature. The concentration of polyglycolic acid in the core spinning solution is 0.06-0.2 g / mL, the solvent is hexafluoroisopropanol, and it is dissolved by stirring under heating conditions at a temperature of 40-80℃; or The concentration of cellulose acetate in the core spinning solution is 0.15-0.25 g / mL, and the solvent is a mixture of acetone, N,N'-dimethylacetamide, and hexafluoroisopropanol or glacial acetic acid in a volume ratio of 3:2-2.5:1, dissolved by stirring at room temperature; or The polylactic acid spinning solution in the core layer spinning solution has a concentration of 0.08-0.14 g / mL, and the solvent is a mixture of dichloromethane and N,N'-dimethylformamide with a volume ratio of 4:1-1:1, which is stirred and dissolved at room temperature.

3. The preparation method according to claim 1, characterized in that: In step (2), the nanozyme is at least one of cerium dioxide nanoparticles, cobalt tetroxide nanoparticles and manganese oxide nanoparticles, and the concentration of the nanozyme dispersion is 0.1-3 mg / mL.

4. The preparation method according to claim 1 or 3, characterized in that: The molecular weight of the polyethylene glycol is 6000-8000.

5. The preparation method according to claim 1 or 3, characterized in that: In step (2), the concentration of polyethylene glycol in the nanoenzyme dispersion is 0.01-0.1 g / mL.

6. The preparation method according to claim 1 or 3, characterized in that: In step (2), the power of the ultrasonic treatment is 80-100 W and the time is 10-30 min.

7. The preparation method according to claim 1, characterized in that: In step (3), the spraying technology is either air gun spraying or electrostatic spraying; wherein, the air gun spraying parameters are: pressure of 0.4-0.6 MPa, receiving distance of 10-15 cm, and moving speed of 10-30 cm / s; the electrostatic spraying parameters are: flow rate of 0.001-0.005 mm / s, receiving distance of 8-12 cm, receiving roller speed of 100-300 r / min, voltage of 18-30 kV, temperature of 25-35℃, and humidity of 40-60%.

8. The preparation method according to claim 1 or 7, characterized in that: In step (3), the heating and melting temperature is 70-80℃ and the time is 3-6 min.

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