An electrospun antibacterial piezoelectric nanofiber membrane and its preparation method

Abstract

This invention discloses an electrospun antibacterial piezoelectric nanofiber membrane and its preparation method. Polycaprolactone and polyvinylidene fluoride are mixed, zinc oxide nanoparticles and succinylnaringin are added, and the membrane is prepared via electrospinning. The invention utilizes electrospinning to blend PVDF with the biodegradable material PCL to improve the properties of the individual polymers. The addition of zinc oxide nanoparticles and succinylnaringin to the nanofiber membrane further enhances the material's piezoelectric, osteogenic, and antibacterial properties.

Description

Technical Field

[0001] The present invention belongs to the technical field of orthopedic material preparation, and specifically relates to an electrospun antibacterial piezoelectric nanofiber membrane and a preparation method thereof. Background Art

[0002] With the world's aging population and the increasing number of sports injuries in many countries, the treatment of orthopedic problems has become a major medical challenge. The field of orthopedics involves not only the healing of muscles, ligaments, or bones, but also the reduction of bacteria caused by orthopedic implants. Currently, it is difficult to find orthopedic materials that can eliminate bacterial colonization and promote osteoblast function in the same material without the use of antibiotics or pharmaceutical agents.

[0003] PVDF (polyvinylidene fluoride) is considered a potential bone tissue engineering material due to its piezoelectric properties and biocompatibility, but its in vivo application is limited by its non-degradability, making it unsuitable for long-term implantation. PCL (polycaprolactone) has been extensively studied in the biomedical and pharmaceutical fields due to its excellent biocompatibility, biodegradability, and suitable mechanical properties. PCL can be slowly degraded into non-toxic products through simple hydrolysis in physiological environments, making it highly attractive for long-term implant applications.

[0004] Although the piezoelectric and mechanical properties of the electrospun nanofiber membrane composited with polyvinylidene fluoride and polycaprolactone were improved, its antibacterial and osteogenesis-promoting properties were reduced. Summary of the Invention

[0005] The object of the present invention is to provide an electrospun antibacterial piezoelectric nanofiber membrane and a preparation method thereof.

[0006] An electrospun antibacterial piezoelectric nanofiber membrane is prepared by mixing polycaprolactone and polyvinylidene fluoride, adding zinc oxide nanoparticles and succinyl naringin, and using electrospinning technology.

[0007] Zinc oxide nanoparticles are ideal for reducing bacterial function and infection because they possess excellent antimicrobial activity. Under ultrasonic mechanical forces, zinc oxide can generate reactive oxygen species to kill bacteria, and zinc nanoparticles promote surface exposure of zinc, further reducing bacterial function. Furthermore, the ZnO structure, through sp3 hybridization, forms four equivalent atomic orbitals between Zn and O, creating a tetrahedral coordination configuration. This imparts piezoelectric properties similar to those of bone, further enhancing the piezoelectric strength of the nanofiber membrane. Succinylnaringin, a flavonoid compound, exhibits antibacterial, anti-inflammatory, and osteoblast-enhancing properties, and is clinically used to treat bacterial infections and prevent and treat osteoporosis.

[0008] The preparation method of the electrospun antibacterial piezoelectric nanofiber membrane is carried out according to the following steps:

[0009] (1) dissolving polycaprolactone (PCL) in a mixed liquid of dichloromethane and dimethylformamide to prepare a polycaprolactone solution;

[0010] (2) dissolving polyvinylidene fluoride (PVDF) in a mixture of dimethylformamide and acetone, adding zinc oxide nanoparticles (ZnONPs) and succinylnaringin to prepare a polyvinylidene fluoride solution;

[0011] (3) The polycaprolactone solution prepared in step (1) and the polyvinylidene fluoride solution prepared in step (2) are mixed and stirred uniformly, and after electrospinning, the mixture is placed in a vacuum drying oven to remove the residual solvent, thereby preparing an electrospun antibacterial piezoelectric nanofiber membrane.

[0012] The volume ratio of dichloromethane to dimethylformamide in step (1) is 2:3.

[0013] The mass volume ratio concentration of the polycaprolactone in step (1) is 20-30%.

[0014] The volume ratio of dimethylformamide to acetone in step (2) is 3:2.

[0015] In step (2), the mass volume ratio concentration of the polyvinylidene fluoride is 20-30%; the mass volume ratio concentration of the zinc oxide nanoparticles is 0.5-2%; and the mass volume ratio concentration of the succinyl naringin is 0.5-2%.

[0016] The volume ratio of the polycaprolactone solution to the polyvinylidene fluoride solution is 1:1.

[0017] The electrospinning operation steps of step (3) are as follows: the mixed and stirred solution in step (3) is transferred into two syringes equipped with 22G metal needles, and placed on the injection pump, the distance between the injection pump and the receiver is adjusted to 15 cm, the injection speed is 0.2 mm / min, the voltage of the external electric field is (-1 kV, +18 kV), the speed of the receiving drum is 5000 rpm / min, and the spinning is performed for 5-6 hours.

[0018] Beneficial effects of the present invention: The present invention uses electrospinning technology to mix PVDF with the biodegradable material PCL to improve the performance of a single polymer. PVDF is considered to be a potential bone tissue engineering material due to its piezoelectric properties and biocompatibility, but its application in the body is limited because it is non-degradable and not conducive to long-term implantation. PCL has been widely studied in the biomedical and pharmaceutical fields due to its good biocompatibility, biodegradability and suitable mechanical properties. PCL can be slowly degraded into non-toxic products by simple hydrolysis in a physiological environment, which makes it very attractive for long-term implantation applications. The addition of zinc oxide nanoparticles and succinyl naringin to the nanofiber membrane further enhances the piezoelectric properties, osteogenic activity and antibacterial properties of the material. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the SEM scanning electron microscope image of the sample prepared in Example 1.

[0020] Figure 2 The piezoelectric force microscope surface morphology, phase image, and amplitude image of the sample prepared in Example 1.

[0021] Figure 3 This is the piezoelectric phase curve of the sample prepared in Example 1.

[0022] Figure 4 This is the piezoelectric amplitude curve of the sample prepared in Example 1.

[0023] Figure 5 This is the Fourier infrared spectrum.

[0024] Figure 6 Live-dead staining of cells co-cultured for 3 days (cytocompatibility).

[0025] Figure 7 Cytoskeleton staining of cell co-cultured for 3 days.

[0026] Figure 8 The graphs are quantitative graphs of cell proliferation after 3d and 5d of co-culture.

[0027] Figure 9 These are gross images of early osteogenesis (ALP alkaline phosphatase) and late osteogenesis (ARS alizarin red staining) in in vitro experiments.

[0028] Figure 10 These are microscopic images of early osteogenesis (ALP alkaline phosphatase) and late osteogenesis (ARS alizarin red staining) in vitro experiments.

[0029] Figure 11 For antibacterial test (spread plate method).

[0030] Figure 12 Antibacterial assay (live-dead staining). DETAILED DESCRIPTION

[0031] To facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0032] Example 1

[0033] The preparation method of the electrospun antibacterial piezoelectric nanofiber membrane is carried out according to the following steps:

[0034] (1) 5 g of PCL was dissolved in 20 ml of a mixture of dichloromethane (DCM) and dimethylformamide (DMF) (2:3) to prepare a 25% (w / v) PCL solution.

[0035] (2) 5 g of PVDF was dissolved in 20 ml of a mixture of dimethylformamide (DMF) and acetone (3:2), and 20 mg of ZnONPs and 20 mg of succinylnaringin (Suc) were added to prepare a PVDF solution with a PVDF concentration (w / v) of 25%, a ZnONPs concentration (w / v) of 1%, and a succinylnaringin concentration (w / v) of 1%;

[0036] (3) The PCL solution prepared in step (1) and the PVDF solution prepared in step (2) were stirred evenly until a transparent solution was obtained; then, the mixed solution was transferred to two syringes equipped with 22G metal needles and placed on a syringe pump. The distance between the syringe pump and the receiver was adjusted to 15 cm, the injection speed was 0.2 mm / min, the voltage of the external electric field was (-1 kV, +18 kV), and the speed of the receiving roller was 5000 rpm / min. After 5 h of spinning, the sample was removed and placed in a vacuum drying oven to remove the residual solvent.

[0037] Example 2

[0038] The preparation method of the electrospun antibacterial piezoelectric nanofiber membrane is carried out according to the following steps:

[0039] (1) 5 g of PCL was dissolved in 20 ml of a mixture of dichloromethane (DCM) and dimethylformamide (DMF) (2:3) to prepare a 25% (w / v) PCL solution.

[0040] (2) 5 g of PVDF was dissolved in 20 ml of a mixture of dimethylformamide (DMF) and acetone (3:2), and 40 mg of ZnONPs was added to prepare a PVDF solution with a PVDF concentration (w / v) of 25% and a ZnONPs concentration (w / v) of 2%.

[0041] (3) The PCL solution prepared in step (1) and the PVDF solution prepared in step (2) were stirred evenly until a transparent solution was obtained; then, the mixed solution was transferred to two syringes equipped with 22G metal needles and placed on a syringe pump. The distance between the syringe pump and the receiver was adjusted to 15 cm, the injection speed was 0.2 mm / min, the voltage of the external electric field was (-1 kV, +18 kV), and the speed of the receiving roller was 5000 rpm / min. After 5 h of spinning, the sample was removed and placed in a vacuum drying oven to remove the residual solvent.

[0042] Example 3

[0043] The preparation method of the electrospun antibacterial piezoelectric nanofiber membrane is carried out according to the following steps:

[0044] (1) 5 g of PCL was dissolved in 20 ml of a mixture of dichloromethane (DCM) and dimethylformamide (DMF) (2:3) to prepare a 25% (w / v) PCL solution.

[0045] (2) 5 g of PVDF was dissolved in 20 ml of a mixture of dimethylformamide (DMF) and acetone (3:2), and 40 mg of succinylnaringin (Suc) was added to prepare a PVDF solution with a PVDF concentration (w / v) of 25% and a succinylnaringin concentration (w / v) of 1%;

[0046] (3) The PCL solution prepared in step (1) and the PVDF solution prepared in step (2) were stirred evenly until a transparent solution was obtained; then, the mixed solution was transferred to two syringes equipped with 22G metal needles and placed on a syringe pump. The distance between the syringe pump and the receiver was adjusted to 15 cm, the injection speed was 0.2 mm / min, the voltage of the external electric field was (-1 kV, +18 kV), and the speed of the receiving roller was 5000 rpm / min. After 5 h of spinning, the sample was removed and placed in a vacuum drying oven to remove the residual solvent.

[0047] Experimental Example 1:

[0048] Morphology and composition characterization: Scanning electron microscopy (SEM) was used to characterize the diameter, thickness and porosity of the sample prepared in Example 1. Figure 1 As shown, the sample thickness is between 0.2-0.5 mm, and the spinning diameter is Figure 1 shown.

[0049] The surface morphology, phase and amplitude of the sample prepared in Example 1 were analyzed by piezoelectric force microscopy. Figure 2-4 As shown, the amplitude curve and phase curve are relatively symmetrical, the piezoelectric performance is good, and the d33 piezoelectric coefficient is about 15.9pm / v.

[0050] The β phase of the polymer film (prepared in Example 2) and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) were analyzed by Bruker Tensor FTIR ( Figure 5 ). Meanwhile, the ZnONPs were removed from the preparation process of Example 2 before the measurement was performed, and the PVDF or PCL was further removed before the measurement was performed.

[0051] Comparison of the β-phase content of each group of piezoelectric materials by FTIR and XRD showed that the piezoelectric film had a higher β-phase content with the addition of PVDF and ZnO.

[0052] Experimental Example 2: Effect of antibacterial piezoelectric nanofiber membrane on in vitro osteogenic activity

[0053] ALP activity detection: The antibacterial piezoelectric nanofiber membrane prepared in Example 1-3 was used to inoculate MC3T3-E1 cells at a rate of 2×10 4 / ml was inoculated onto the antibacterial piezoelectric nanofiber membrane placed in a 24-well plate. The piezoelectric group was vibrated for 20 minutes every day (10 minutes in the morning and 10 minutes in the afternoon). After 3 days of cell culture, live-dead staining and cytoskeleton staining were performed for 3 days of cell co-culture. After 7 days of continuous culture, the ALP activity of the cells was detected using an ALP detection kit to evaluate the in vitro osteogenic activity of the PPFZ nanofiber membrane.

[0054] Live-dead staining of cell co-culture for 3 days Figure 6 As shown, cytoskeleton staining of cells co-cultured for 3 days is shown Figure 7 As shown, cell proliferation was quantified after 3 and 5 days. Figure 8 As shown in the figure, the results showed that: ①Suc has a certain effect on promoting cell proliferation. ②ZnO has a certain effect on promoting cell proliferation. ③Suc+ZnO has a significant effect on promoting cell proliferation. The live-dead cell staining results are consistent with the cell proliferation results.

[0055] After the cells were grown on the materials for 7 days, the ALP activity of the cells was evaluated ( Figure 9-10 ), the results showed that: ①Suc has a certain effect in promoting early osteogenic differentiation of cells. ②ZnO has a certain effect in promoting cell differentiation. ③Suc+ZnO has a significant effect in promoting cell differentiation.

[0056] Experimental Example 3: Effect of antibacterial piezoelectric nanofiber membrane on bacterial activity in vitro

[0057] The antibacterial piezoelectric nanofiber membrane was disinfected with 75% alcohol and then sterilized under ultraviolet light for 60 minutes. The samples were placed in a 24-well culture plate. 6 Escherichia coli (E. coli) and Staphylococcus aureus (SA) were inoculated in 24-well culture plates at a concentration of 100 cells / ml in 30 g / L Tryptic Soy Broth (TSB) in a 5% / 95% CO2 / air atmosphere for 6 hours at 37°C. After inoculation, the 24-well plates were placed in an ultrasonic cleaner (100 W, 40 kHz) for piezoelectric stimulation. The control group received no treatment. After 6 hours of incubation, the bacterial solution was diluted 10 5 The diluted solution was dropped onto an agar plate to form a colony group. The agar plate was incubated in an incubator for 12 hours, and the colonies were counted (n1 / 43).

[0058] The results of the plate spread assay were as follows Figure 11 As shown: ①Suc can inhibit the growth of Staphylococcus aureus and Escherichia coli. ②ZnO can inhibit the growth of Staphylococcus aureus and Escherichia coli. ③Suc+ZnO has a significant inhibitory effect on Staphylococcus aureus and Escherichia coli.

[0059] Live-dead staining assay results Figure 12 As shown: ①Suc can inhibit the growth of Staphylococcus aureus and Escherichia coli. ②ZnO can inhibit the growth of Staphylococcus aureus and Escherichia coli. ③The effect of ZnO on the inhibition of Staphylococcus aureus and Escherichia coli is obvious.

[0060] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for preparing an electrospun antibacterial piezoelectric nanofiber membrane, characterized in that: Follow these steps: (1) dissolving polycaprolactone in a mixed liquid of dichloromethane and dimethylformamide to prepare a polycaprolactone solution; (2) dissolving polyvinylidene fluoride in a mixture of dimethylformamide and acetone, adding zinc oxide nanoparticles and succinyl naringin to prepare a polyvinylidene fluoride solution; the mass volume ratio concentration of the polyvinylidene fluoride is 20-30%; the mass volume ratio concentration of the zinc oxide nanoparticles is 0.5-2%; the mass volume ratio concentration of the succinyl naringin is 0.5-2%; (3) The polycaprolactone solution prepared in step (1) and the polyvinylidene fluoride solution prepared in step (2) are mixed and stirred evenly, and after electrospinning, the mixture is placed in a vacuum drying oven to remove the residual solvent, thereby preparing an electrospun antibacterial piezoelectric nanofiber membrane.

2. The method for preparing the electrospun antibacterial piezoelectric nanofiber membrane according to claim 1, characterized in that: The volume ratio of dichloromethane to dimethylformamide in step (1) is 2:

3.

3. The method for preparing the electrospun antibacterial piezoelectric nanofiber membrane according to claim 1, characterized in that: The mass volume ratio concentration of the polycaprolactone in step (1) is 20-30%.

4. The method for preparing the electrospun antibacterial piezoelectric nanofiber membrane according to claim 1, characterized in that: The volume ratio of dimethylformamide to acetone in step (2) is 3:

2.

5. The method for preparing the electrospun antibacterial piezoelectric nanofiber membrane according to claim 1, characterized in that: The volume ratio of the polycaprolactone solution to the polyvinylidene fluoride solution is 1:

1.

6. The method for preparing the electrospun antibacterial piezoelectric nanofiber membrane according to claim 1, characterized in that: The electrospinning operation steps of step (3) are as follows: the mixed and stirred solution in step (3) is transferred into a syringe equipped with a 21G metal needle and placed on a syringe pump, the distance between the syringe pump and the receiver is adjusted to 15 cm, the injection speed is 0.2 mm / min, the voltage of the external electric field is -1 kV, +18 kV, the speed of the receiving drum is 5000 rpm / min, and the spinning is carried out for 5-6 hours.

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