An anisotropic piezoelectric thin film, a preparation method and applications thereof

Abstract

The application discloses an anisotropic piezoelectric film, a preparation method and application thereof, and belongs to the technical field of biomedical materials. The anisotropic piezoelectric film has a wavy wrinkle structure which is parallel arranged and periodically repeated on the surface; and the surface of the anisotropic piezoelectric film also has a porous structure to form a through pore network. The anisotropic piezoelectric film has a regular structure, high order and anisotropy, simulates the parallel arrangement of nerve fiber bundles in vivo, has good bionics, and can guide nerve directional regeneration well. The anisotropic piezoelectric film is used for drug loading and repairing nerves, has the functions of physical guidance, biochemical factor release and electrical stimulation, and can effectively promote the directional growth of nerve axons and the regeneration and repair of nerve tissues.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and more specifically, relates to an anisotropic piezoelectric thin film, its preparation method, and its application. Background Technology

[0002] Spinal cord injury is one of the most serious central nervous system injuries in clinical practice. Due to the inhibitory microenvironment formed in the injured area and the limited regenerative capacity of central neurons, conventional treatments such as decompression surgery, internal fixation, drug therapy and rehabilitation training are difficult to achieve effective reconstruction of nerve function. Repairing spinal cord injury remains a major challenge for medicine today. Tissue engineering technology, through a comprehensive strategy of "material scaffold-seed cells-bioactive molecules," aims to reconstruct a microenvironment conducive to axonal regeneration and neural circuit remodeling, providing new insights for spinal cord injury repair. Biomaterial scaffolds play a crucial role in bridging injury cavities, carrying cells and growth factors, and regulating the local mechanical and biochemical environment. Currently, various forms have been developed, including hydrogel scaffolds, porous scaffolds, tubular catheters, and 3D-printed scaffolds. In recent years, the application of technologies such as 3D bioprinting and multi-material composites has enabled more refined scaffold design, component distribution, and cell spatial arrangement, helping to improve multiple aspects such as scar formation, inflammatory response, and axonal regeneration; however, there is still room for improvement in overall therapeutic efficacy.

[0003] Neural tissue exhibits significant anisotropic structural features in its natural state. For example, nerve bundles in the spinal cord white matter are highly oriented along the longitudinal axis. This ordered physical microenvironment plays an important regulatory role in the adhesion, migration, and axonal directional growth of neurons and glial cells. To simulate this structural feature, researchers have proposed a variety of biomimetic scaffolds with guiding functions, such as oriented nano / micro fiber scaffolds based on electrospinning and patterned scaffolds with regular microgrooves or microchannels. Wu S et al. prepared oriented PVDF-TrFE piezoelectric fiber scaffolds using electrospinning. The results showed that the oriented fiber topology and piezoelectric polymer material are beneficial for supporting neurite extension and myelination (Wu S, Chen MS, Maurel P, Lee Y, Bunge MB, Arinzeh T L. Aligned fibrous PVDF-TrFE scaffoldswith Schwann cells support neurite extension and myelination in vitro. Journal of Neural Engineering, 2018, 15(5): 056010.). However, such scaffolds are mostly formed by random stacking of one-dimensional oriented fibers. Their geometric structure has limited regularity on a macroscopic scale, and the pores between fibers are randomly distributed. It is difficult to form a highly ordered surface structure with parallel arrangement and periodic repetition on a large scale. Therefore, their biomimicry of the "regular and periodic orientation" of natural nerve bundles is still insufficient. Besides geometric guidance, inflammation suppression and growth factor supply are also crucial for promoting nerve regeneration. Studies have shown that timely relief of local inflammation and reduction of glial scar formation during the acute phase of injury, along with the continuous supply of neurotrophic factors during the subacute and chronic phases, helps improve the injury microenvironment and promote axonal regeneration and functional recovery. Therefore, many tissue-engineered scaffolds incorporate drug-release systems, such as loading anti-inflammatory drugs, neurotrophic factors, or chemokines into hydrogels, tubular scaffolds, or porous materials, aiming to achieve slow drug release and efficient local accumulation. However, the pore structures formed by traditional porous scaffolds through particle leaching, gas foaming, etc., are often irregular, with discrete pore size distribution and insufficient connectivity, leading to limited three-dimensional cell ingrowth and drug delivery. In recent years, the role of electrical stimulation in nerve repair has received increasing attention. Traditional exogenous electrical stimulation often requires the implantation of electrodes and connection to an external power source, which is not only complex and invasive but also presents challenges in long-term stability and patient compliance. Piezoelectric materials can generate charge or potential under mechanical force, providing electrical signals to local cells without the need for an external power source, and are therefore considered promising materials for constructing "self-generating" nerve repair scaffolds. Inorganic piezoelectric materials such as barium titanate, potassium sodium niobate, and lead-containing lead zirconate titanate (PZT) possess high piezoelectric properties, but they suffer from high stiffness, potential ion dissolution in body fluids, and the potential toxicity of some materials, limiting their biosafety and long-term implantability. In contrast, organic piezoelectric polymers such as polyvinylidene fluoride (PVDF) and its copolymer P (VDF-TrFE) have the advantages of light weight, good flexibility, strong processability and good biocompatibility. Their β-phase crystal structure endows the materials with good ferroelectricity and piezoelectricity. In recent years, they have been used to prepare flexible piezoelectric sensors and oriented fiber scaffolds for nerve repair. However, most of them exist in the form of flat membranes or simple electrospun fibers, lacking complex microstructure designs that are highly matched with the morphology of nerve tissue, making it difficult to achieve both morphology guidance and piezoelectric stimulation functions in a single scaffold. In summary, existing spinal cord nerve repair scaffolds still have significant shortcomings in the following aspects: First, although technologies such as 3D printing and electrospinning can construct oriented structures or regular channels to a certain extent, existing guiding structures still lag behind in vivo nerve bundles in terms of regularity, periodicity, and anisotropy simulation, resulting in insufficient biomimicry. Second, the pore structure regularity and connectivity of drug-loaded scaffolds are limited, making it difficult to precisely control drug loading and release processes, and failing to meet the comprehensive needs of long-term anti-inflammatory effects and continuous regeneration. Third, there is a lack of a system design that effectively integrates anisotropic microstructures, long-range ordered porous drug-loaded channels, and built-in piezoelectric stimulation functions into a single flexible scaffold, which is still insufficient in simulating the morphology and bioelectric microenvironment of in vivo nerve tissue and achieving multimodal synergistic therapy. Therefore, it is necessary to develop a novel multifunctional nerve repair membrane that integrates morphology guidance, controlled drug release, and spontaneous electric stimulation to more comprehensively simulate the in vivo nerve microenvironment and improve the repair effect of spinal cord injury. Summary of the Invention

[0004] 1. The problem to be solved To address at least one of the technical problems in existing technologies, such as poor orderliness and insufficient biomimicry of scaffolds for neural repair, difficulty in simultaneously possessing anisotropic and piezoelectric stimulation functions, and limited drug loading capacity, this invention provides anisotropic piezoelectric thin films, their preparation methods, and their applications.

[0005] 2. Technical Solution To solve the above problems, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides an anisotropic piezoelectric thin film, wherein the surface of the anisotropic piezoelectric thin film has a parallel, periodically repeating wavy fold structure; The wavelength of the wavy folds is 30~50μm, and the height difference between the crests and troughs is 10~30μm; The anisotropic piezoelectric film is an organic piezoelectric polymer film with a thickness of 5~500μm; The anisotropic piezoelectric film surface also has a porous structure, forming a network of interconnected pores; the porosity of the anisotropic piezoelectric film surface is 55-75%; the average pore diameter is 200-300 nm.

[0006] It should be noted that the thickness of the anisotropic piezoelectric film refers to the average thickness including the wavy wrinkled structure. The thickness of the films with wavy wrinkled surfaces involved in this invention refers to the average thickness.

[0007] The anisotropic piezoelectric film has a good qualitative and ordered structure, which can guide the regenerated nerve to grow precisely in a fixed direction, effectively avoiding disordered axon entanglement and neuroma formation, and significantly improving nerve regeneration efficiency and functional recovery level. In particular, the wavy folds provided by this invention have rounded edges, undulating contours distributed in a sinusoidal curve, and wavelengths of 30~50μm. The height difference between the peaks and troughs is 10~30μm, which is closer to the size of nerve tissue and has good biomimetic performance.

[0008] Meanwhile, under external ultrasonic stimulation, the piezoelectric film will spontaneously respond to generate weak electrical signals, simulating the physiological electric field in the body, directly activating nerve regeneration-related pathways, thereby greatly accelerating nerve repair. It also has the advantages of good flexibility and high biosafety.

[0009] As a preferred embodiment of any technical solution of the first aspect of the present invention, the wavelength of the wavy folds can be any of the following ranges: 30~50μm, 30~40μm, 35~50μm, 35~45μm, 45~50μm.

[0010] As a preferred embodiment of any technical solution of the first aspect of the present invention, the height difference between the crests and troughs of the wavy folds can be any of the following ranges: 10~30μm, 10~25μm, 10~20μm, 15~30μm, 15~25μm, 15~20μm.

[0011] As a preferred embodiment of any technical solution of the first aspect of the present invention, the thickness of the anisotropic piezoelectric film can be any of the following ranges: 5~500μm, 5~100μm, 5~90μm, 5~80μm, 5~70μm, 5~60μm, 5~50μm, 5~30μm, 10~500μm, 10~100μm, 10~90μm, 10~80μm, 10~70μm, 10~60μm, 10~50μm, 10~30μm.

[0012] As a preferred embodiment of any technical solution in the first aspect of the present invention, the organic piezoelectric polymer includes one or two of polyvinylidene fluoride-trifluoroethylene (P(VDF-TrFE)) and polyvinylidene fluoride (PVDF).

[0013] It should be noted that P(VDF-TrFE) and PVDF have excellent piezoelectric and ferroelectric properties. When stimulated by ultrasound, their piezoelectric effect will cause charge accumulation on the film surface, forming a local electric field, thereby providing electrical stimulation signals to the nerve cells attached to it and simulating the bioelectric microenvironment in the extracellular matrix of nerve cells. The electrical signal can activate the neuronal membrane ion channels and promote axonal sprouting, thereby accelerating the recovery of nerve function.

[0014] As a preferred embodiment of any technical solution in the first aspect of the present invention, the organic piezoelectric polymer is polyvinylidene fluoride-trifluoroethylene (P(VDF-TrFE)), wherein the molar ratio of vinylidene fluoride (VDF) to trifluoroethylene (TrFE) is (65~80):(22~35).

[0015] As a preferred embodiment of any technical solution in the first aspect of the present invention, the porous structure on the surface of the anisotropic piezoelectric film is an inverse opal porous structure.

[0016] It should be noted that during the preparation process, when the silica microspheres self-assemble into a colloidal crystal template, the microspheres in the colloidal crystal template have overlapping areas that are in contact with each other. After the template is removed, these overlapping areas form the interconnected channels in the inverse opal structure.

[0017] The interconnected pores form a three-dimensional, pervasive pore network, providing ample accessible space for drug molecule transport and loading. Compared to dense structures, this ordered, interconnected network significantly increases the material's effective specific surface area and internal usable volume, allowing the drug solution to penetrate more fully into the entire framework, rather than remaining only in the surface region.

[0018] The average pore size of the porous structure depends on the particle size of the silica microspheres, and the pore size on the surface of the anisotropic piezoelectric film can be adjusted by adjusting the particle size of the silica microspheres.

[0019] A second aspect of the present invention provides a method for preparing anisotropic piezoelectric thin films, comprising the following steps: (1) Prepare a polymer elastic film with a thickness of 0.8~1.2 mm, stretch it along the length direction to 2~3 times the original length, keep it stretched, treat it with ultraviolet light for 30~60 min, and then release the tensile stress at a speed of 0.01~0.1 mm / min, so that the polymer elastic film shrinks back to the original length and forms a first template with wavy wrinkles on the surface. (2) Prepare a light-curing prepolymer and pour the light-curing prepolymer onto the wavy folds of the first template, allowing it to flow naturally until it covers the entire surface of the first template; then, cure the prepolymer, demold it, and obtain a second template that is complementary to the structure of the first template.

[0020] (3) Coat the surface of the second template with wavy folds with silica microsphere suspension, let it stand and dry at 20~35℃, and form a silica microsphere colloidal crystal layer with opal structure on the surface of the second template. (4) Prepare a solution containing organic piezoelectric polymer and coat it on the surface of the silica microsphere colloidal crystal layer described in step (3). Dry it at 45~75℃ for 2~8 h and cool it naturally to room temperature to form an organic piezoelectric polymer film embedded with silica microspheres on the second template. Then, peel it off from the second template and immerse it in hydrofluoric acid solution until the silica microspheres are completely dissolved. Clean and dry the product to obtain a film with an inverse opal porous structure on the surface. (5) After annealing the film obtained in step (4) at 120~140℃ for 1~3h, it is naturally cooled to room temperature. Then, the annealed film is placed in a corona polarization device and polarized at 11000~14000 V and 65~85℃ for 0.5~1.5h to obtain an anisotropic piezoelectric film.

[0021] It should be noted that step (1) involves aging the polymer elastic film using UV light and the generated ozone to form wrinkles on its surface. The surface of the polymer elastic film treated with UV light forms a wavy wrinkle structure perpendicular to the stretching direction. The wavy wrinkles have a consistent height and change periodically, similar to a sine curve, and have good directional order.

[0022] Among them, the stretching length, ultraviolet light irradiation time and conditions in step (1) have a crucial impact on the final morphology of the wrinkles. If the stretching length is insufficient or the ultraviolet light irradiation time is too short, it will be difficult to form regular and orderly wrinkles; if the stretching length is too large or the ultraviolet light irradiation time is too long, the wrinkles formed will break or have cracks.

[0023] As a preferred embodiment of any technical solution in the second aspect of the present invention, in step (1), the polymer elastic membrane includes one or two of polydimethylsiloxane (PDMS) membrane and polybutylene terephthalate (PBAT) membrane.

[0024] As a preferred embodiment of any technical solution in the second aspect of the present invention, in step (1), the wavelength of the ultraviolet light in the ultraviolet irradiation treatment includes 185 nm and 254 nm; the distance between the ultraviolet light source and the surface of the polymer elastic film is 4~8 cm.

[0025] As a preferred embodiment of any technical solution in the second aspect of the present invention, in step (2), the photocurable prepolymer liquid includes a photocurable monomer and a photoinitiator, and the mass ratio of the photocurable monomer to the photoinitiator is (75~125):1.

[0026] As a preferred embodiment of any technical solution in the second aspect of the present invention, the photocurable monomer includes one or more of ethoxylated trimethylolpropane triacrylate (ETPTA), trifunctional acrylate (TMPTA), and polyurethane acrylate (PUA).

[0027] Further preferably, the photocurable monomer is ethoxylated trimethylolpropane triacrylate (ETPTA).

[0028] As a preferred embodiment of any technical solution in the second aspect of the present invention, the photoinitiator includes one or two of 2-hydroxy-2-methyl-1-phenyl-1-propanone (photoinitiator 1173) and 1-hydroxycyclohexylphenyl methyl ketone (photoinitiator 184).

[0029] It should be noted that the photoinitiator can also be selected to match the photoinitiator used in the photocuring monomer and the light.

[0030] As a preferred embodiment of any technical solution in the second aspect of the present invention, in step (2), the curing treatment is a UV irradiation treatment, and the UV irradiation treatment satisfies one or more of the following conditions (i) to (iv): (i) The wavelength of ultraviolet light is 365 nm; (ii) Ultraviolet light intensity is 1000~1500 mw / cm 2 ; (iii) The distance between the ultraviolet light source and the surface of the photocurable prepolymer liquid is 5~15cm; (iv) Irradiation time is 45~90s.

[0031] As a preferred embodiment of any technical solution of the second aspect of the present invention, in step (2), the thickness of the second template film layer is 0.5~1mm.

[0032] As a preferred embodiment of any technical solution of the second aspect of the present invention, in step (2), before pouring the photocurable prepolymer onto the surface of the wavy folds of the first template, the surface of the wavy folds of the first template is further modified to be hydrophilic.

[0033] The hydrophilic modification is performed by oxygen plasma treatment for 15-90 seconds under conditions of oxygen flow rate of 10-20 sccm and power of 30-80 W.

[0034] The hydrophilic modification allows the photocurable prepolymer to spread more evenly on the first template.

[0035] As a preferred embodiment of any technical solution in the second aspect of the present invention, in step (3), the mass fraction of silica in the silica microsphere suspension is 1~5%, and the coating amount of the silica microsphere suspension is 18~30 g / m 2 ; The diameter of the silica microspheres in the silica microsphere suspension can be any of the following ranges: 100~1000nm, 100~500nm, 400~1000nm, 200~300nm.

[0036] The particle size deviation (CV) of the silica microspheres in the silica microsphere suspension is ≤3%.

[0037] It should be noted that in a colloidal system of slow evaporation / settling, silica microspheres physically self-assemble into a face-centered cubic close-packed structure through gravity and capillary forces, forming a long-range ordered opal structure layer on the surface of the second template.

[0038] As a preferred embodiment of any technical solution in the second aspect of the present invention, in step (4), the organic piezoelectric polymer includes one or two of polyvinylidene fluoride-trifluoroethylene (P(VDF-TrFE)) and polyvinylidene fluoride (PVDF).

[0039] Further preferred, in step (4), the organic piezoelectric polymer is polyvinylidene fluoride-trifluoroethylene (P(VDF-TrFE)), wherein the molar ratio of vinylidene fluoride (VDF) to trifluoroethylene (TrFE) is (65~80):(22~35).

[0040] As a preferred embodiment of any technical solution in the second aspect of the present invention, in step (4), the concentration of the solution containing the organic piezoelectric polymer is 10~15wt%, and the coating amount is 450~750 g / m. 2 .

[0041] The anisotropic piezoelectric thin film provided by the first aspect of the present invention can be prepared by using the method provided by the second aspect of the present invention.

[0042] A third aspect of the present invention provides a composite dressing or scaffold comprising an anisotropic piezoelectric film and a drug loaded on the anisotropic piezoelectric film; The anisotropic piezoelectric thin film is an anisotropic piezoelectric thin film provided by any technical solution of the first aspect of the present invention or an anisotropic piezoelectric thin film prepared using any technical solution of the second aspect of the present invention. The fourth aspect of the present invention provides an anisotropic piezoelectric film provided by any technical solution of the first aspect of the present invention or an anisotropic piezoelectric film prepared using any technical solution of the second aspect of the present invention, or the third aspect of the present invention provides the application of a composite dressing or scaffold in the field of nerve repair.

[0043] 3. Beneficial effects Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The anisotropic piezoelectric film provided by this invention has a parallel, periodically repeating wavy fold structure. This structure is regular and highly ordered, providing clear guidance for cell growth and enabling oriented growth of cells or nerve axons. Furthermore, this highly ordered structure exhibits anisotropy, mimicking the parallel arrangement of nerve fiber bundles in vivo, exhibiting good biomimicry. This facilitates the bridging of newly formed axons along the injured vertebral segment, avoiding disordered growth. This significantly improves the directionality and effectiveness of axon regeneration at both ends of the injured spinal cord, outperforming the effect of traditional disordered structures in guiding nerve regeneration.

[0044] (2) The anisotropic piezoelectric film provided by the present invention is made of organic piezoelectric polymer material, whose good piezoelectric properties can promote nerve repair. In addition, the organic piezoelectric polymer used also has the advantages of good flexibility, high biosafety, and no toxic side effects, and can be safely implanted into the body and integrate well with the surrounding tissues.

[0045] (3) The method for preparing anisotropic piezoelectric thin films provided by the present invention uses silica microspheres to self-assemble into an opal structure, and uses this as a template to prepare a porous thin film with an inverse opal structure. The anisotropic piezoelectric thin film prepared by this method has long-range ordered pores on the surface with good connectivity, which can ensure the uniformity and predictability of drug release after drug loading, and effectively avoid the disadvantages of uneven drug loading and excessively fast initial release of traditional stents.

[0046] Meanwhile, by adjusting the amount and particle size of silica microspheres, the porosity and pore size of the final film can be flexibly controlled to suit different drug loading requirements.

[0047] (4) The anisotropic piezoelectric thin film preparation method provided by the present invention can form a specific structure simply by stretching and ultraviolet light irradiation. The preparation method is simple, low cost, reusable, and fast. It has a short response time for customized sizes and has important clinical application value.

[0048] (5) The composite dressing or scaffold provided by the present invention can simultaneously provide three functions: physical guidance, release of biochemical factors and electrical stimulation by loading the drug onto the anisotropic piezoelectric film provided by the present invention, effectively promoting the directional growth of nerve axons and the regeneration and repair of nerve tissue. Attached Figure Description

[0049] Figure 1 This is a schematic diagram illustrating the preparation process and application of PVDF-O in Example 1; Figure 2 This is a schematic diagram of the structure of the first template prepared in Example 1; Figure 3 This is a SEM image of the surface morphology of the P(VDF-TrFE) thin film in Example 1; Figure 4 The images shown are magnified SEM images of the P(VDF-TrFE) thin film in Example 1, where: a is an SEM image from one viewpoint, b is a magnified image of image a, and c is an SEM image from another viewpoint. Figure 5 An optical microscope image of the first template prepared in Comparative Example 1; Figure 6 An optical microscope image of the first template prepared in Comparative Example 2; Figure 7 An optical microscope image of the second template prepared in Comparative Example 6; Figure 8 These are fluorescent staining images of AM / PI live and dead cells from different experimental groups in Experiment Example 1; Figure 9 The cell viability results for different experimental groups in Experiment Example 1; Figure 10 These are fluorescent staining images of phalloidin / DAPI cells from different experimental groups in Experiment Example 2. Detailed Implementation

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0051] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0052] Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as "less than about 4.5," which should be interpreted to include all the values ​​and ranges described above. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0053] The present invention will be further described below with reference to specific embodiments.

[0054] Information on raw materials and equipment used in the embodiments Polydimethylsiloxane (PDMS) elastic silicone film: Dow Corning SYLGARD™ 184 silicone rubber (SYLGARD™ 184 Silicone Elastomer Kit); Polyvinylidene fluoride-trifluoroethylene (P(VDF-TrFE)): The molar ratio of vinylidene fluoride (VDF) to trifluoroethylene (TrFE) is 75:25; Ethoxylated trimethylolpropane triacrylate (ETPTA) monomer: average number-average molecular weight approximately 428 g / mol (Mn≈428), Sigma-Aldrich brand, product number 409073; Rat adrenal pheochromocytoma cells (PC12 cells): BOSTER brand; Calcein-AM / PI Live / Dead Cell Double Staining Kit: Solarbio, China, Catalog No. CA1630; CCK-8 Cell Proliferation and Toxicity Assay Kit: Solarbio, China, Catalog No. CA1210; Phalloidin staining kit: Solarbio, China, catalog number CA1610; 4',6-Diamidinyl-2-phenylindole Staining Kit (DAPI Staining Kit): Solarbio, China, Catalog No. C0065; UV ozone cleaning equipment: Novascan PSD PRO series UV ozone cleaner.

[0055] Example 1 According to such Figure 1 The process shown is used to prepare anisotropic piezoelectric thin films. The specific steps are as follows: (1) Preparation of the first template 1-1) Prepare a PDMS elastic silicone film (hereinafter referred to as PDMS film) with a length of 40mm, a width of 10mm and a thickness of 1mm. Fix it on a self-made stretching fixture and stretch it along the length direction to 2.5 times the original length. Keep it in this stretched state and use an ultraviolet ozone cleaning device (ultraviolet wavelengths include 185 nm and 254 nm) to irradiate the PDMS elastic silicone film at a distance of 5cm for 60 minutes to perform surface treatment on the PDMS elastic silicone film.

[0056] 1-2) Subsequently, a stepper motor is used to slowly release the clamp at a speed of 0.03 mm / min, allowing the PDMS film length to gradually return to its initial length, thus obtaining the first template, the structure of which is as follows: Figure 2 As shown.

[0057] After retraction, the PDMS film (first template) forms a parallel, wave-like fold structure perpendicular to the stretching direction on its UV-treated surface. The wave-like folds exhibit periodic variations, with an undulating profile resembling a sine curve. The wavelength of the wave-like folds is approximately 50 µm, and the height difference between the peaks and troughs is 20 µm. The entire fold surface is uniform and continuous, without any breaks.

[0058] (2) Preparation of the second template 2-1) Preparation of UV-curable prepolymer solution 5g of ethoxylated trimethylolpropane triacrylate (ETPTA) monomer was weighed into a brown, light-proof sample bottle, and photoinitiator 1173 was added. The mixture was stirred at 500 rpm for 30 minutes in the dark using a magnetic stirrer. Subsequently, the bottle was placed in a vacuum drying oven and evacuated until all air bubbles generated during stirring were completely removed, forming a photocurable prepolymer solution (hereinafter referred to as EPTA prepolymer solution). The mass ratio of EPTA to photoinitiator in the EPTA prepolymer solution is approximately 100:1.

[0059] 2-2) Cleaning and pretreatment of the first template: Gently wipe the wavy, wrinkled surface of the first template with anhydrous ethanol and isopropanol in sequence, and dry it with nitrogen. Place the cleaned first template into a plasma cleaner (60W power) and treat it with oxygen plasma (oxygen flow rate 15sccm) for about 1 minute to improve its surface hydrophilicity.

[0060] 2-3) Fix the first template, after treatment in 2-2), onto a flat substrate (such as a glass slide) with the pleated side facing up. Use a pipette to draw an appropriate amount of ETPTA prepolymer solution and carefully drip it onto one end of the wavy, pleated surface of the first template. Slowly tilt the first template, allowing the ETPTA prepolymer solution to flow naturally across the entire surface due to its own gravity and low viscosity. Then place it in a UV curing machine and cure it under UV light at a wavelength of 365nm and a light intensity of 1300mw / cm². 2 Under the condition that the distance between the ultraviolet light source and the surface of the ETPTA prepolymer liquid is 10cm, the ETPTA prepolymer liquid is irradiated for 1 minute to solidify into an ETPTA film. Then, the solidified ETPTA film is gently peeled off from the first template with tweezers or a scalpel blade to obtain an ETPTA mold with a structure complementary to the first template, which is the second template. The thickness of the second template film layer is 8mm.

[0061] (3) Preparation of inverse opal template 3-1) Monodisperse silica microspheres were prepared using the modified Stöber method. 8 mL of tetraethoxysilane (TEOS) was dissolved in 200 mL of anhydrous ethanol. 20 mL of deionized water and 5 mL of ammonia were added as catalysts, and the mixture was magnetically stirred at room temperature for 12 hours to generate a silica sol. The product was centrifuged, washed several times with ethanol, and vacuum dried at 60 °C to obtain loose white SiO2 microsphere powder. Transmission electron microscopy (TEM) confirmed that the SiO2 microspheres were solid spheres with a diameter of approximately 300 nm, uniform particle size distribution, and a particle size deviation ≤3% (silica microspheres of the same specifications can also be purchased commercially).

[0062] 3-2) The SiO2 microspheres from 3-1) were dispersed in anhydrous ethanol to prepare a 5% (w / w) SiO2 microsphere suspension. After cleaning the surface of the second template, it was placed horizontally in a clean environment, and approximately 15 μL of the SiO2 microsphere suspension was slowly added dropwise to the wavy, wrinkled surface of the second template. The coating amount of the SiO2 microsphere suspension was 22.15 g / m². 2 Subsequently, a clean glass slide was used to gently and evenly spread the SiO2 microsphere suspension across the entire surface of the second template, allowing it to expand and fill the entire surface. The template was then left to dry naturally at room temperature (25°C) for approximately 30 minutes under vibration-free conditions. At this point, a layer of shimmering, iridescent microspheres was observed to have formed on the surface of the second template, indicating that an opal-structured colloidal crystal layer of SiO2 microspheres had formed on the ETPTA casting surface (i.e.,...). Figure 1 (Photonic crystal template in the image). Simultaneously, the uniform color of the microsphere colloidal crystal layer was observed, indicating that its thickness was essentially consistent.

[0063] (4) P(VDF-TrFE) film forming 4-1) Dissolve P(VDF-TrFE) powder (VDF to TrFE molar ratio 75:25) in N,N-dimethylformamide (DMF) and stir gently at 50°C for 2 hours until a clear and homogeneous P(VDF-TrFE) solution is formed. The concentration of P(VDF-TrFE) in the P(VDF-TrFE) solution is 12 wt%.

[0064] 4-2) The P(VDF-TrFE) solution is drop-coated onto the surface of the SiO2 microsphere colloidal crystal layer in step (3) (the coating amount of P(VDF-TrFE) solution is 472 g / m²). 2 The film was dried at 60°C for 6 hours and then naturally cooled to room temperature, forming a thin film on the second template. It was then carefully peeled off the second template using tweezers. The resulting film was translucent, with white SiO2 microspheres embedded on the side in contact with the second template. The peeled film had a wavy, wrinkled structure complementary to the structure of the second template, and a thickness of 10 μm.

[0065] 4-3) The film from step 4-2) was immersed in a 2wt% hydrofluoric acid dilution solution (prepared using a 40wt% hydrofluoric acid aqueous solution) at room temperature (25℃) for 24 hours until the white color on the film surface disappeared, indicating that the silica microspheres were completely dissolved. Subsequently, the film was removed using polytetrafluoroethylene tweezers and rinsed with deionized water to thoroughly remove the acid and reaction byproducts. Finally, the film was laid flat on a clean glass slide and air-dried to obtain a P(VDF-TrFE) film with anisotropic wrinkles and an inverse opal structure. The P(VDF-TrFE) film is a semi-transparent flexible film (approximately 10µm thick) with a uniformly arranged pore structure on its surface. Due to the photonic crystal effect generated by the nanopore array, it exhibits structural color under illumination. The porosity of the P(VDF-TrFE) film was measured to be 70%.

[0066] The P(VDF-TrFE) thin film was characterized by scanning electron microscopy (SEM), and the results are as follows: Figure 3 , Figure 4 As shown: Figure 3 Here are SEM images of the surface morphology of the P(VDF-TrFE) thin film, such as... Figure 3 As shown, the surface of the P(VDF-TrFE) thin film has a parallel, periodically repeating wavy fold structure. The undulating contour of the wavy folds is distributed in a sinusoidal curve, and the wavelength of the wavy folds is about 50µm, which is consistent with the first template.

[0067] Figure 4This is a magnified SEM image of a P(VDF-TrFE) thin film. The results show that the surface of the P(VDF-TrFE) thin film is covered with pores, which are arranged in an orderly manner and have a similar pore size of approximately 300 nm. Figure 4 a in Figure 4 (b) confirms the successful construction of an inverse opal porous structure on the surface of the P(VDF-TrFE) film; simultaneously, tilting the film at a certain angle reveals clear interconnections between the pores, exhibiting a three-dimensional interconnected pore network. Figure 4 (c in the text)

[0068] (5) Annealing and polarization treatment 5-1) Place the P(VDF-TrFE) film obtained in step (4) in an oven at 135°C for annealing for 2 hours. Turn off the oven heating function and allow the P(VDF-TrFE) film to cool slowly to room temperature with the oven. Annealing can improve the crystallinity of P(VDF-TrFE) and stabilize the wavy wrinkled structure of the P(VDF-TrFE) film.

[0069] 5-2) The annealed P(VDF-TrFE) film is placed in a corona polarization device and polarized for 1 hour at 13000V and 80℃ to obtain the P(VDF-TrFE) piezoelectric film, which is an anisotropic piezoelectric film, denoted as PVDF-O.

[0070] The morphology of PVDF-O is consistent with that of the P(VDF-TrFE) film in step 4-3). Its wavy folds have a wavelength of 50 μm, a height difference of 20 μm between the crests and troughs, a film thickness of 10 μm, and a surface porosity of 70%. The average pore diameter is approximately 300 nm. Simultaneously, its piezoelectric coefficient (d33) was measured to be 10 pC / N using a quasi-static method.

[0071] Example 2 The only difference between this embodiment and embodiment 1 is step (1). Step (1) in this embodiment is as follows: (1) Preparation of the first template 1-1) Prepare a PDMS elastic silicone film (hereinafter referred to as PDMS film) with a length of 40mm, a width of 10mm and a thickness of 1mm. Fix it on a self-made stretching fixture and stretch it along the length direction to 2.5 times the original length. Keep it in this stretched state and use an ultraviolet ozone cleaning device (ultraviolet wavelengths include 185 nm and 254 nm) to irradiate the PDMS elastic silicone film at a distance of 8cm from the surface for 60 minutes to perform surface treatment on the PDMS elastic silicone film.

[0072] 1-2) Subsequently, the clamp is slowly released at a speed of 0.05 mm / min using a stepper motor control, so that the length of the PDMS film gradually returns to the initial length, thus obtaining the first template, whose wavy folds have a wavelength of about 45 µm and a height difference of 15 µm between the peaks and troughs.

[0073] The anisotropic piezoelectric thin film prepared in this embodiment has a wavy fold structure with parallel arrangement and periodic repetition. The wavelength of the wavy folds is 45 μm, the height difference between the peaks and troughs is 15 μm, the film thickness is 10 μm, the porosity of the film surface is 70%, and the average pore diameter is about 300 nm.

[0074] Comparative Example 1 The only difference between this comparative example and Example 1 is the UV irradiation time in step 1-1), where the UV irradiation time in this comparative example is 15 min.

[0075] The difference in Young's modulus between the upper and lower surfaces of the first template prepared in step (1) of this comparative example is insufficient, and only some areas can form wavy folds, and the folds are discontinuous. Figure 5 ).

[0076] Comparative Example 2 The only difference between this comparative example and Example 1 is the ultraviolet light irradiation time in step 1-1), which is 120 min.

[0077] In this comparative example, the hardened layer on the upper surface of the first template prepared in step (1) is too thick, and the wavy folds are prone to breakage or branching. Figure 6 ).

[0078] Comparative Example 3 The only difference between this comparative example and Example 1 is the stretching length of the PDMS film in step 1-1). In this comparative example, the PDMS film is stretched to 1.5 times its original length.

[0079] In this comparative example, wrinkles can only be formed in the stretching edge region during the preparation of the first template in step (1).

[0080] Comparative Example 4 The only difference between this comparative example and Example 1 is the stretching length of the PDMS film in step 1-1). In this comparative example, the PDMS film is stretched to 3.5 times its original length.

[0081] In this comparative example, during the preparation of the first template in step (1), the PDMS film will break when exposed to ultraviolet light, or the surface of the prepared first template will have cracks, making it impossible to obtain folds with a sinusoidal shape.

[0082] Comparative Example 5 The only difference between this comparative example and Example 1 is the curing conditions in steps 2-3). The curing conditions for this comparative example are: ultraviolet light intensity of 800 mw / cm². 2 Under the condition that the distance between the ultraviolet light source and the surface of the ETPTA prepolymer liquid is 20 cm, irradiation is performed for 20 seconds. In this comparative example, during the preparation of the second template in step (2), the ETPTA resin was not completely cured and adhered to the first template, making it impossible to peel it off to obtain a complete second template.

[0083] Comparative Example 6 The only difference between this comparative example and Example 1 is the curing conditions in steps 2-3). The curing conditions for this comparative example are: ultraviolet light intensity of 2000 mw / cm². 2 Under the condition that the distance between the ultraviolet light source and the surface of the ETPTA prepolymer liquid is 2.5 cm, irradiation for 120 s, In the preparation of the second template in step (2) of this comparative example, burst polymerization occurred during the curing of ETPTA resin, which caused the wavy wrinkled structure to be destroyed. Furthermore, due to excessively high local temperatures, the wavy wrinkles deformed, making it impossible to obtain a second template that complements the structure of the first template. In other words, the second template obtained in this comparative example has poor orderliness. Figure 7 ).

[0084] Comparative Example 7 The only difference between this comparative example and Example 1 is the annealing temperature in step 5-1) and the polarization conditions in step 5-2). This comparative example: The annealing temperature corresponding to step 5-1) is 110 ℃; The polarization conditions in step 5-2) are 10000 V, 60 ℃, and polarization treatment for 20 min.

[0085] The anisotropic piezoelectric thin film prepared in this comparative example has a piezoelectric coefficient of 2.3 pC / N, which is unsatisfactory.

[0086] Comparative Example 8 The only difference between this comparative example and Example 1 is the annealing temperature in step 5-1) and the polarization conditions in step 5-2). This comparative example: The annealing temperature corresponding to step 5-1) is 150 ℃; The polarization conditions in step 5-2) are 15000V and 85℃.

[0087] The structure of the anisotropic piezoelectric thin film prepared in this comparative example was destroyed, and the film was broken down.

[0088] Comparative Example 9 In this comparative example, a planar P(VDF-TrFE) film with a thickness of 10 μm (in which the molar ratio of vinylidene fluoride (VDF) to trifluoroethylene (TrFE) is 75:25) was placed in a corona polarization device and polarized for 1 h at 13000 V and 80 °C to obtain a planar piezoelectric film, denoted as PVDF.

[0089] Experimental Example 1 This experiment used the AM / PI live / dead cell fluorescence staining method and the Cell Counting Kit-8 (CCK-8) method to test the biosafety of the PVDF-O prepared in Example 1. The specific experimental methods are as follows: (1) AM / PI live / dead cell fluorescence staining detection PC12 cells were seeded on sterile piezoelectric films to be tested. After 24 hours of static incubation to allow complete cell adhesion, the cells were cultured for 3 days, after which the culture medium was discarded, and the cells were washed three times with phosphate-buffered saline (PBS). Subsequently, the cells were stained with a calcein-AM / PI live / dead cell double staining kit, and the fluorescence images were observed using an inverted optical microscope (Olympus IMT-2, Tokyo, Japan). Calcein (AM) labeled live cells (green fluorescence), and propidium iodide (PI) labeled dead cells (red fluorescence).

[0090] (2) CCK-8 detection PC12 cells were seeded on sterilized piezoelectric films and incubated for 24 hours until complete cell adhesion. Cells were then tested on days 1 and 3. Serum-free medium containing 10% (v / v) CCK-8 cell proliferation and toxicity assay kit was added to each well, and the cells were incubated at 37°C for 1 hour. The optical density (OD) of each well was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, USA). The OD value was positively correlated with cell viability and could indirectly reflect cell proliferation and survival.

[0091] Based on the above "(1) AM / PI live / dead cell fluorescence staining detection" and "(2) CCK-8 detection", external stimulation can be added for detection. For example, after the cells have been placed in stillness for 24 hours and are fully adhered to the wall, PC12 cells can be subjected to ultrasound stimulation (ultrasound conditions are 1 MHz frequency and 250 mW / cm). 2 Ultrasonic stimulation with high power intensity, 10 min / session).

[0092] Different piezoelectric films of the same shape and thickness were cut and subjected to AM / PI live / dead cell fluorescence staining and CCK-8 assays according to the above testing methods. Two main groups were established based on different testing conditions: a group without ultrasound stimulation (Sham) and a group with ultrasound stimulation (US). Within each main group, two experimental groups were set up based on the different piezoelectric films being tested: PVDF (PVDF in Comparative Example 9) and PVDF-O (PVDF-O in Example 1), as well as a control group Con (no piezoelectric film). The test results are shown below: Figure 8 The results of AM / PI live / dead cell fluorescence staining showed that PC12 cells exhibited good survival status with almost no cell death when tested with different piezoelectric films, whether used alone (Sham) or under ultrasonic stimulation (US).

[0093] Figure 9 The results of CCK-8 assay showed that PC12 cell viability remained above 95% after 1 and 3 days of culture in all groups, and there was no statistically significant difference in cell proliferation among the groups.

[0094] Figure 8 , Figure 9 The results showed that PVDF-O in Example 1 had good biocompatibility.

[0095] Experimental Example 2 To investigate the effect of PVDF-O prepared in Example 1 on nerve repair, the following experiment was conducted in this experimental case: PC12 cells were seeded on sterilized piezoelectric films and allowed to adhere completely for 24 hours. After 3 days, the culture medium was discarded, and the cells were washed three times with PBS. The cells were then stained with phalloidin and DAPI staining kits, respectively, and fluorescence images were observed using an inverted optical microscope (Olympus IMT-2, Tokyo, Japan). The cytoskeleton produced a green fluorescent signal, while the cell nucleus produced a blue fluorescent signal.

[0096] The above experiments can also be performed with external stimulation, such as adding ultrasound stimulation to PC12 cells after 24 hours of static incubation to allow complete cell adhesion (ultrasound conditions: 1 MHz frequency, 250 mW / cm²). 2 Ultrasonic stimulation with high power intensity, 10 min / session).

[0097] Similar to Example 1, this experiment also included two main groups: Sham and US. Within each main group, based on the different piezoelectric films under test, there were two experimental groups: PVDF and PVDF-O, as well as a control group (Con). The experimental results for each group are as follows: Figure 10 As shown: Figure 10The results of fluorescence staining of phalloidin / DAPI cells show the effects of different piezoelectric films on cell adhesion and extension. Specifically, in the non-ultrasound stimulation group (Sham), cells in the PVDF group showed no directionality in growth, while cells in the PVDF-O group grew directionally along parallel folds and grooves (see macroscopic schematic diagram). Figure 1 This confirms that the highly ordered wavy folded structure and inverse opal structure prepared in Example 1 can enhance the spreading and directional alignment of the cytoskeleton. Comparison between the US group and the Sham group revealed that the US group exhibited better cell arrangement order, reflecting a more pronounced promoting effect on cytoskeleton spreading and directional alignment under ultrasound stimulation (US).

[0098] The above description provides an illustrative overview of the present invention and its embodiments. This description is not restrictive, and the embodiments shown are merely one example of the invention's implementation. Actual implementations are not limited to these examples. Therefore, if those skilled in the art are inspired by this description and design similar implementations and examples without departing from the spirit of the invention, such designs should fall within the scope of protection of the present invention.

Claims

1. A method for preparing an anisotropic piezoelectric thin film, characterized in that, Includes the following steps: (1) Prepare a polymer elastic film with a thickness of 0.8~1.2 mm, stretch it along the length direction to 2~3 times the original length, keep it stretched, treat it with ultraviolet light for 30~60 min, and then release the tensile stress at a speed of 0.01~0.1 mm / min, so that the polymer elastic film shrinks back to the original length and forms a first template with wavy wrinkles on the surface. (2) Prepare a light-curing prepolymer and pour the light-curing prepolymer onto the wavy folds of the first template, allowing it to flow naturally until it covers the entire surface of the first template. Subsequently, after curing and demolding, a second template complementary to the structure of the first template is obtained; (3) Coat the surface of the second template with wavy folds with silica microsphere suspension, let it stand and dry at 20~35℃, and form a silica microsphere colloidal crystal layer with opal structure on the surface of the second template. (4) Prepare a solution containing organic piezoelectric polymer and coat it on the surface of the silica microsphere colloidal crystal layer described in step (3). Dry it at 45~75℃ for 2~8 h and cool it naturally to room temperature to form an organic piezoelectric polymer film embedded with silica microspheres on the second template. Then, peel it off from the second template and immerse it in hydrofluoric acid solution until the silica microspheres are completely dissolved. Clean and dry the product to obtain a film with an inverse opal porous structure on the surface. (5) After annealing the film obtained in step (4) at 120~140℃ for 1~3h, it is naturally cooled to room temperature. Then, the annealed film is placed in a corona polarization device and polarized at 11000~14000 V and 65~85℃ for 0.5~1.5h to obtain an anisotropic piezoelectric film. In step (1), the polymer elastic membrane is a polydimethylsiloxane membrane.

2. The method according to claim 1, characterized in that, In step (1), The wavelengths of the ultraviolet light used in the ultraviolet irradiation treatment include 185 nm and 254 nm; the distance between the ultraviolet light source and the surface of the polymer elastic film is 4~8 cm.

3. The method according to claim 1 or 2, characterized in that, In step (2), the photocurable prepolymer liquid includes a photocurable monomer and a photoinitiator, and the mass ratio of the photocurable monomer to the photoinitiator is (75~125):

1.

4. The method according to claim 3, characterized in that, In step (2), the curing process is a UV irradiation treatment, which satisfies one or more of the following conditions (i) to (iv): (i) The wavelength of ultraviolet light is 365 nm; (ii) Ultraviolet light intensity is 1000~1500 mw / cm 2 ; (iii) The distance between the ultraviolet light source and the surface of the photocurable prepolymer liquid is 5~15cm; (iv) Irradiation time is 45~90s.

5. The method according to claim 1 or 4, characterized in that, In step (2), before the photocurable prepolymer is poured onto the surface of the wavy folds of the first template, the surface of the wavy folds of the first template is further modified to be hydrophilic.

6. The method according to claim 4, characterized in that, The silica microsphere suspension in step (3) has a silica mass fraction of 1-5%, and the coating amount of the silica microsphere suspension is 18-30 g / m³. 2 ; In step (4), the concentration of the solution containing the organic piezoelectric polymer is 10-15 wt%, and the coating amount is 450-750 g / m. 2 .

7. The anisotropic piezoelectric thin film prepared by the method according to any one of claims 1 to 6, characterized in that, The anisotropic piezoelectric film surface has a parallel, periodically repeating wavy fold structure; The wavelength of the wavy folds is 30~50μm, and the height difference between the crests and troughs is 10~30μm; The anisotropic piezoelectric film is an organic piezoelectric polymer film with a thickness of 5~500μm; The anisotropic piezoelectric film surface also has a porous structure, forming a network of interconnected pores; the porosity of the anisotropic piezoelectric film surface is 55-75%; the average pore diameter is 200-300 nm.

8. A composite dressing or scaffold, characterized in that, Includes an anisotropic piezoelectric thin film and a drug loaded on the anisotropic piezoelectric thin film; The anisotropic piezoelectric thin film is the anisotropic piezoelectric thin film of claim 7 or the anisotropic piezoelectric thin film prepared by the method of any one of claims 1 to 6.

9. The use of the anisotropic piezoelectric film of claim 7 or the anisotropic piezoelectric film prepared by the method of any one of claims 1 to 6 in the preparation of nerve repair dressings or scaffolds.

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