Electroactive polymer-based microspheres and preparation method thereof

Abstract

The invention discloses an electroactive polymer-based microsphere and a preparation method thereof. The electroactive polymer-based microspheres are PHBV / inorganic piezoelectric material composite microspheres, self-polarization from inside to outside is achieved in the preparation process of the microspheres, and non-zero surface potential can be achieved without additional external field induced polarization. The electroactive polymer-based microsphere disclosed by the invention also has good biocompatibility and ultrasonic response characteristic. The electroactive polymer-based microspheres provided by the invention can be used in targeted therapy tissue repair, especially bone tissue repair; alternatively, the method may be used in smart drug delivery systems and implantable medical devices. The preparation method of the electroactive polymer-based composite microspheres is simple and easy to implement.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, specifically to an electroactive polymer-based microsphere and its preparation method. Background Technology

[0002] Clinical repair of bone defects remains a major challenge for orthopedics, oral and maxillofacial surgery, and neurosurgery. While autologous bone grafting remains the "gold standard," its availability is limited and it involves secondary surgical trauma. Therefore, developing high-performance artificial bone repair materials is crucial. Ideal materials not only need good osteoconductivity and biocompatibility but should also be able to actively regulate and accelerate the endogenous bone regeneration process. In recent years, the concept of "bioelectronic medicine," which uses physical stimulation (such as electrical and magnetic stimulation) to regulate cell behavior and promote tissue regeneration, has provided revolutionary insights for the design of bone repair materials.

[0003] Electrical stimulation has been widely proven to directly promote osteoblast adhesion, proliferation, differentiation, and extracellular matrix mineralization. Based on this, piezoelectric materials have attracted considerable attention because they can convert the body's everyday weak mechanical stresses (such as muscle contraction and walking) into beneficial electrical signals, achieving passive, endogenous electrical stimulation. However, existing piezoelectric materials face significant challenges in bone repair, particularly in terms of microstructure and practical manufacturing processes.

[0004] First, in terms of material morphology and clinical application, existing research mainly focuses on bulk ceramics, thin films, or coatings. These forms are difficult to adapt to complex and irregular non-weight-bearing bone defects, and cannot be implanted through minimally invasive injection methods, thus limiting their clinical application scenarios.

[0005] Secondly, the most critical technical bottleneck lies in the "polarization process." Traditional piezoelectric ceramics or piezoelectric polymers must undergo high-voltage electric field polarization treatment to ensure that their internal electric domains are arranged in an orderly manner, thereby exhibiting piezoelectricity. This post-processing is extremely unfriendly to micron-scale spherical materials: the strong electric field easily leads to charge accumulation, breakdown, or structural damage in the microspheres; the process is complex, making it difficult to achieve uniform and stable mass production; and the long-term stability of the piezoelectric properties of the polarized microspheres after implantation in the body is questionable.

[0006] Furthermore, traditional electroactive materials (such as conductive polymer scaffolds and piezoelectric films) primarily generate or conduct electrical signals through their inherent properties (such as conductivity and piezoelectricity). However, these static electroactive systems have significant limitations: their electrical output is fixed once implanted, making real-time, dynamic adjustment impossible according to the needs of different treatment stages, and they lack the ability to respond to the dynamic biological demands of the repair process. This limits their application in complex and dynamic regenerative medicine scenarios. For example, cells require different types, intensities, and durations of electrical stimulation at different stages of tissue repair (inflammatory phase, proliferative phase, and remodeling phase); for deep tissues, how to non-invasively activate and regulate these microparticles is also a major challenge. Moreover, traditional electrical stimulation modulation methods usually require the implantation of electrodes, which is an invasive procedure, increasing the risk of infection and surgical complexity, and making it difficult to perform long-term, dynamic regulation of deep tissues in vivo. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide an electroactive polymer-based microsphere and a method for preparing the same. The microsphere achieves self-polarization from the inside out during the preparation process, and can have a non-zero surface potential without external field-induced polarization. At the same time, it also has good biocompatibility and ultrasonic response characteristics.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect of the present invention, an electroactive polymer-based composite microsphere is provided, which is a PHBV / inorganic piezoelectric material composite microsphere.

[0010] In some specific embodiments of the present invention, the inorganic piezoelectric material is one of ZnO, BTO, or KNN.

[0011] In some specific embodiments of the present invention, the PHBV / inorganic piezoelectric material composite microspheres are self-polarized microspheres with non-zero surface potential.

[0012] In some specific embodiments of the present invention, the particle size of the microspheres is between 500 nm and 10 μm.

[0013] In some specific embodiments of the present invention, the surface potential of the microspheres is 400~700 mV.

[0014] In a second aspect of the present invention, a method for preparing the above-mentioned electroactive polymer-based composite microspheres is provided, comprising the following steps:

[0015] (1) PHBV powder is added to a first reaction vessel containing chloroform. After sealing the first reaction vessel, it is magnetically stirred at 30~50 °C for 15~25 h to obtain a PHBV-chloroform solution with a concentration of 0.01~0.1 g / mL. Inorganic piezoelectric nanoparticles are added to the first reaction vessel. The mass of the inorganic piezoelectric nanoparticles is 10~50% of the mass of the PHBV powder. The first reaction vessel is then resealed and magnetically stirred at room temperature for 10~20 h to mix evenly, thus obtaining a PHBV / inorganic piezoelectric material mixture.

[0016] (2) PVA is added to a second reaction vessel containing deionized water. After sealing the second reaction vessel, it is magnetically stirred at room temperature for 15-25 h to obtain a PVA solution with a concentration of 2-6 mg / mL.

[0017] (3) Add the PHBV / inorganic piezoelectric material mixture to the second reaction vessel containing the PVA solution, open the second reaction vessel, and magnetically stir at room temperature until it evaporates to dryness to obtain a solid precipitate; the volume ratio of the PHBV / inorganic piezoelectric material mixture to the PVA solution is 1: (8~10).

[0018] (4) Wash the solid precipitate obtained in step (3) with deionized water, and then centrifuge at 5000~8000 rpm for 2~6 min to separate the precipitate; wash the separated precipitate with deionized water, centrifuge at 5000~8000 rpm for 2~6 min to separate the precipitate again; repeat the steps of washing with deionized water, centrifugation and separation several times, and ultrasonically mix the finally separated precipitate with deionized water to obtain a mixture. The mixture is passed through a 1~10 μm sieve to obtain a second mixture. The second mixture is then filtered through a 100~300 nm filter paper, and the solid on the filter paper is collected to obtain the target product.

[0019] In some specific embodiments of the present invention, the inorganic piezoelectric material is one of ZnO, BTO, or KNN.

[0020] In some specific examples of the present invention, in step (4), the centrifugation speed is 8000 rpm and the time is 3 min.

[0021] In some specific examples of the present invention, in step (4), the mixture is passed through a 5-10 μm sieve to obtain a second mixture.

[0022] In a third aspect of the present invention, another method for preparing the above-mentioned electroactive polymer-based composite microspheres is also provided, comprising the following steps:

[0023] (1) PHBV powder is added to a first reaction vessel containing chloroform. After sealing the first reaction vessel, it is magnetically stirred at 30-50 °C for 15-25 h to obtain a PHBV-chloroform solution with a concentration of 0.01-0.1 g / mL. Inorganic piezoelectric nanoparticles are ultrasonically dispersed in deionized water at room temperature to obtain an inorganic piezoelectric aqueous dispersion with a concentration of 0.005-0.02 g / mL. The inorganic piezoelectric aqueous dispersion is added to a first reaction vessel containing the PHBV-chloroform solution. After sealing the first reaction vessel, it is magnetically stirred at room temperature for 10-20 h to mix evenly to obtain a PHBV / inorganic piezoelectric material mixture. The mass of the inorganic piezoelectric nanoparticles is 10-50% of the mass of the PHBV powder.

[0024] (2) PVA is added to a second reaction vessel containing deionized water. After sealing the second reaction vessel, it is magnetically stirred at 50-70 °C for 15-25 h to obtain a PVA solution with a concentration of 2-6 mg / mL. The PVA solution is added to a first reaction vessel containing the PHBV / inorganic piezoelectric material mixture obtained in step (1). After sealing the first reaction vessel, it is magnetically stirred for 2-6 h to obtain a mixture. The volume ratio of the PHBV / inorganic piezoelectric material mixture to the PVA solution is 1:(1.2-2).

[0025] (3) PVA is added to a third reaction vessel containing deionized water. After sealing the third reaction vessel, it is magnetically stirred at 50-70 °C for 15-25 h to obtain a PVA solution with a concentration of 0.2-0.6 mg / mL.

[0026] (4) Add the mixture obtained in step (2) to the third reaction vessel containing the PVA solution obtained in step (3), open the third reaction vessel, and stir magnetically at 25~45 °C until it evaporates to dryness to obtain a solid precipitate; wherein, the volume ratio of the mixture to the PVA solution is 1:(2.5~4).

[0027] (5) Wash the solid precipitate obtained in step (4) with deionized water, and then centrifuge at 5000~8000 rpm for 2~6 min to separate the precipitate; wash the separated precipitate with deionized water, centrifuge at 5000~8000 rpm for 2~6 min to separate the precipitate again; repeat the steps of washing with deionized water, centrifuging and separation several times, and the precipitate obtained in the end is the target product.

[0028] In some specific embodiments of the present invention, the inorganic piezoelectric material is one of ZnO, BTO, or KNN.

[0029] In some specific examples of the present invention, in step (5), the centrifugation speed is 8000 rpm and the time is 3 min.

[0030] In this invention, the room temperature is 15~25℃.

[0031] The electroactive polymer-based composite microspheres prepared by this invention achieve self-polarization from the inside out during the preparation process, possessing a non-zero surface potential without the need for additional external field-induced polarization, and are expected to regulate cell behavior through surface potential. Simultaneously, the composite microspheres of this invention exhibit good biocompatibility, and experiments have verified that they are beneficial to cell proliferation. Moreover, the size of the microspheres allows them to be injected directly into the lesion, potentially enabling precise and minimally invasive delivery. Furthermore, the composite microspheres of this invention can rapidly respond to externally applied ultrasound, generating stable electrical signals, and the strength of these signals can be controlled by adjusting ultrasound parameters to provide a targeted electrical microenvironment suitable for tissue repair. Therefore, in a fourth aspect, this invention also provides applications of the electroactive polymer-based microspheres, such as their use in targeted tissue repair therapy (especially bone tissue repair), and further, for example, in intelligent drug delivery systems and implantable medical devices.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The electroactive polymer-based composite microspheres of the present invention are self-polarized microspheres. They do not require additional complex post-processing polarization technology and have good piezoelectric properties. This means that the piezoelectric properties of the microspheres from the inside out are native and uniform. There is no performance gradient caused by incomplete polarization. Its piezoelectric response is completely determined by the intrinsic properties of the material and the crystal structure. This means that the performance of the self-polarized microspheres of the present invention is more stable. At the same time, the absence of destructive post-processing polarization technology can maintain the original spherical structure, surface morphology and internal structure of the microspheres, which promotes good compatibility with biological tissues and is conducive to the subsequent realization of the function of drug carrier and cell scaffold, thus providing a safer and more efficient solution for clinical application.

[0034] (2) The electroactive polymer-based composite microspheres of the present invention have good biocompatibility and can significantly promote cell proliferation.

[0035] (3) The electroactive polymer-based composite microspheres of this invention possess ultrasonic response characteristics and exhibit superior piezoelectric effects when excited by ultrasound, generating an electric potential response on the surface of the microspheres. Therefore, through non-invasive external field modulation of ultrasonic stimulation, remote, real-time, and dynamic "switching" and "programming" of electroactivity can be achieved. Based on treatment needs, specific modes (intensity, frequency, waveform) of electrical stimulation can be applied to specific sites at specific times, thereby achieving personalized, adaptive, and efficient tissue regeneration and repair. This provides a novel non-invasive modulation scheme, broadening the application prospects of external field-modulated electroactive materials in the biomedical field.

[0036] (4) The preparation method of the electroactive polymer-based composite microspheres of the present invention is simple and easy to implement. Attached Figure Description

[0037] Figure 1 This is the Fourier transform infrared (FTIR) spectrum of the target product obtained in Example 1.

[0038] Figure 2 The X-ray diffraction (XRD) spectra of the target products obtained in Example 1 and Comparative Example 1 are compared.

[0039] Figure 3 This is a scanning electron microscope (SEM) image of the target product obtained in Example 1.

[0040] Figure 4 This is the EDS result of the target product obtained in Example 1.

[0041] Figure 5 This is a scanning electron microscope (SEM) image of the target product obtained in Comparative Example 1.

[0042] Figure 6 This is the EDS result of the target product obtained in Comparative Example 1.

[0043] Figures 7a-7c The figures show the height distribution, amplitude, and phase of the PHBV / ZnO microspheres obtained in Example 1, measured using a piezoelectric microscope.

[0044] Figures 8a-8c The figures show the height distribution, amplitude, and phase of the PHBV microspheres obtained in Comparative Example 1, measured using a piezoelectric microscope.

[0045] Figure 9a and Figure 9b The figures show the height distribution and potential distribution of the PHBV / ZnO microspheres obtained in Example 1, measured using a Kelvin probe force microscope.

[0046] Figure 10a and Figure 10bThe figures show the height and potential distribution of the PHBV microspheres obtained in Comparative Example 1, measured using a Kelvin probe force microscope.

[0047] Figure 11 The results are from ultrasonic performance tests on the target product PHBV / ZnO microspheres obtained in Example 1.

[0048] Figure 12 The proliferation of MC3T3-E1 cells cultured with blank control group, PHBV microspheres or PHBV / ZnO microspheres was determined by CCK-8 assay.

[0049] Figure 13 This is a scanning electron microscope (SEM) image of the target product obtained in Example 2.

[0050] Figure 14 This is the EDS result of the target product obtained in Example 2.

[0051] Figure 15a and Figure 15b The figures show the height distribution and potential distribution of the PHBV / ZnO microspheres obtained in Example 2, as measured using a Kelvin probe force microscope.

[0052] Figure 16 The results are from ultrasonic performance tests on the PHBV / ZnO microspheres obtained in Example 2. Detailed Implementation

[0053] To better illustrate the present invention and facilitate understanding of its technical solutions, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and do not represent or limit the scope of protection of the present invention; the scope of protection of the present invention is defined by the claims.

[0054] In the following examples and comparative examples, reagents or instruments whose manufacturers are not specified are all commercially available products. For example,

[0055] PHBV, full name poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid), was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., with a purity of ≥98%, item number: P909981-100g; nano zinc oxide (ZnO) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with a purity of ≥99.9%; nano barium titanate (BaTiO3, BTO) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., with a purity of 99.9%; nano potassium sodium niobate (K... 0.5 Na 0.5NbO3 (KNN) was purchased from Jiangxi Changyanke New Materials Co., Ltd., with a purity of 99.9%; cryopreserved mouse embryonic osteoblast precursor cells (MC3T3-E1) were purchased from Shanghai Guandao Biotechnology Co., Ltd.; α-MEM culture medium was purchased from Hangzhou Gino Biotechnology Co., Ltd.

[0056] The room temperature is 15~25℃.

[0057] Example 1: Preparation of PHBV / ZnO microspheres

[0058] (1) Add 0.15 g of PHBV powder to a culture bottle containing 5 mL of chloroform, then seal the bottle opening and stir magnetically at 40 °C for 24 h to obtain a PHBV-chloroform solution with a concentration of 0.03 g / mL (the solution volume is about 5 mL); open the bottle opening and add 0.03 g of ZnO powder, reseal the bottle opening, and stir magnetically at room temperature for 10 h to mix evenly to obtain a PHBV / ZnO mixture.

[0059] (2) Add 0.25 g of polyvinyl alcohol (PVA) particles to a beaker containing 50 mL of deionized water, then seal the mouth of the beaker and stir magnetically for 24 h at room temperature to obtain a PVA solution with a concentration of 5 mg / mL (the solution volume is about 50 mL).

[0060] (3) Add all of the PHBV / ZnO mixture obtained in step (1) into a beaker containing all of the PVA solution obtained in step (2), open the mouth of the beaker, and stir magnetically at room temperature (1~2 days) until dry to obtain a solid precipitate;

[0061] (4) Wash the solid precipitate obtained in step (3) with deionized water, and then centrifuge at 8000 rpm for 3 min to separate the precipitate; wash the separated precipitate again with deionized water, centrifuge at 8000 rpm for 3 min to separate the precipitate, and repeat the above steps of washing with deionized water, centrifugation and separation 3 to 5 times. Then mix the finally separated precipitate with 30 mL of deionized water in a centrifuge tube and sonicate for 15 min to obtain a mixture. Pass the mixture through a 5 μm sieve to obtain a second mixture. Then filter the second mixture through a 100 nm filter paper and collect the solid on the filter paper to obtain the target product.

[0062] Characterization and identification:

[0063] The target product obtained in Example 1 was subjected to Fourier transform infrared spectroscopy analysis, and the results are as follows: Figure 1 As shown. Figure 1 Middle, 1724cm -1 The absorption peak at 1132 cm⁻¹ corresponds to the stretching vibration of C=O.-1 1182 cm -1 1280cm -1 The absorption peaks at these locations correspond to the stretching vibrations of CO, and these characteristic peaks are all infrared spectral characteristic peaks of PHBV. This indicates that PHBV is present in the obtained target product.

[0064] The target product obtained in Example 1 was subjected to X-ray diffraction analysis, and its XRD pattern is shown below. Figure 2 The peaks in the spectrum are shown in green. The peaks on this spectrum are aligned with the peaks on the standard card PDF#36-1451 of ZnO. For example, the peak at 31.8° corresponds to the (100) crystal plane in ZnO, the peak at 34.4° corresponds to the (002) crystal plane, the peak at 36.2° corresponds to the strongest peak in ZnO (101) crystal plane, the peak at 47.5° corresponds to the (102) crystal plane in ZnO, and the peak at 56.6° corresponds to the (110) crystal plane in ZnO.

[0065] The surface morphology of the target product obtained in Example 1 was tested using scanning electron microscopy. The SEM results are as follows: Figure 3 As shown. By Figure 3 As can be seen, the target product obtained in Example 1 is microspheres with a particle size of about 3~4 μm. Tiny particles are visible on the surface of the microspheres, and the particle size distribution is uniform.

[0066] The target product obtained in Example 1 was subjected to EDS analysis, and the EDS results are as follows: Figure 4 As shown. By Figure 4 It can be seen that C, O, and Zn elements are distributed at the positions corresponding to the microspheres in the SEM image, indicating that C, O, and Zn elements are present in the microspheres.

[0067] In summary, the target product obtained in Example 1 is PHBV / ZnO microspheres.

[0068] Comparative Example 1: Preparation of PHBV Microspheres

[0069] (1) Add 0.15 g of PHBV powder to a culture bottle containing 5 mL of chloroform, then seal the bottle opening and stir magnetically at 40 °C for 24 h to obtain a PHBV-chloroform solution with a concentration of 0.03 g / mL (the solution volume is about 5 mL).

[0070] (2) Add 0.25 g of PVA particles to a beaker containing 50 mL of deionized water, then seal the mouth of the beaker and stir magnetically for 24 h at room temperature to obtain a PVA solution with a concentration of 5 mg / mL (the solution volume is about 50 mL).

[0071] (3) Add all of the PHBV-chloroform solution obtained in step (1) into a beaker containing all of the PVA solution obtained in step (2), open the mouth of the beaker, and stir magnetically at room temperature (1~2 days) until it evaporates to dryness (the solution is completely evaporated) to obtain a solid precipitate;

[0072] (4) Wash the solid precipitate obtained in step (3) with deionized water, and then centrifuge at 8000 rpm for 3 min to separate the precipitate; wash the separated precipitate again with deionized water, centrifuge at 8000 rpm for 3 min to separate the precipitate, and repeat the above steps of washing with deionized water, centrifugation and separation 3 to 5 times. Finally, mix the separated precipitate with 30 mL of deionized water in a centrifuge tube by sonication for 15 min to obtain a mixture. Pass the mixture through a 5 μm sieve to obtain a second mixture. Centrifuge the second mixture at 8000 rpm for 3 min to separate the target product.

[0073] The product was characterized and identified using the same method as in Example 1. The results showed that the target product obtained in Comparative Example 1 was PHBV. Its XRD pattern is shown below. Figure 2 The SEM results of its surface morphology, as shown by the yellow curve in the middle, are as follows: Figure 5 As shown. By Figure 5 As can be seen, the target product obtained in Comparative Example 1 is microspheres with a particle size of approximately 6 μm, and the surface of the microspheres is free of tiny particles. EDS analysis results are as follows: Figure 6 As shown. By Figure 6 As can be seen, C and O elements are distributed at positions corresponding to the microspheres in the SEM image, indicating the presence of C and O elements in the microspheres. In conclusion, the target product obtained in Comparative Example 1 is PHBV microspheres.

[0074] Performance testing:

[0075] The piezoelectric response of the PHBV / ZnO microspheres obtained in Example 1 and the PHBV microspheres prepared in Comparative Example 1 was analyzed using piezoelectric microscopy. The microsphere samples were dispersed in anhydrous ethanol, dropped onto a silicon wafer, and dried in a 37°C oven until the anhydrous ethanol evaporated. A 2 μm × 2 μm microarea on the silicon wafer surface was selected for surface scanning. The height distribution, amplitude, and phase diagrams of the PHBV / ZnO microspheres are shown below. Figures 7a-7c As shown, the height distribution diagram, amplitude diagram, and phase diagram of the PHBV microspheres are respectively as follows: Figures 8a-8c As shown.

[0076] Figures 7a-7cIn the amplitude diagram of PHBV / ZnO microspheres, there are multiple clearly defined and discrete microspherical regions, which correspond to the microspherical regions in the height distribution diagram of PHBV / ZnO microspheres. Similarly, in the phase diagram of PHBV / ZnO microspheres, there are multiple clearly defined and discrete microspherical regions, which correspond to the microspherical regions in the height distribution diagram of PHBV / ZnO microspheres.

[0077] Figures 8a-8c In the amplitude diagram of the PHBV microspheres, multiple discrete microspherical regions are faintly visible, corresponding to the microspherical regions in the height distribution diagram of the PHBV microspheres. Similarly, multiple discrete microspherical regions are faintly visible in the phase diagram of the PHBV microspheres, corresponding to the microspherical regions in the height distribution diagram of the PHBV microspheres.

[0078] The comparison reveals that the signal intensity in the amplitude diagram of PHBV / ZnO microspheres is higher than that of PHBV microspheres, and the contrast in the phase diagram of PHBV / ZnO microspheres is also more pronounced. This indicates that, compared to PHBV microspheres, PHBV / ZnO microspheres have stronger amplitude signals, clearer phase contrast, and a stronger piezoelectric response.

[0079] The surface potential of the PHBV / ZnO microspheres obtained in Example 1 and the PHBV microspheres prepared in Comparative Example 1 were analyzed using Kelvin probe force microscopy. The microsphere samples were dispersed in anhydrous ethanol, dropped onto a silicon wafer, and dried in a 37°C oven until the anhydrous ethanol evaporated. A 2 μm × 2 μm microarea on the silicon wafer surface was selected for surface scanning. The height and potential distribution maps of the PHBV / ZnO microspheres are shown below. Figure 9a and Figure 9b As shown, the height distribution and potential distribution diagrams of the PHBV microspheres are respectively as follows: Figure 10a and Figure 10b As shown.

[0080] Depend on Figure 9a and Figure 9b It is evident that PHBV / ZnO microspheres possess a non-zero surface potential even without polarization treatment. This indicates that self-polarization of the entire microsphere from the inside out was achieved during the microsphere fabrication process. Compared to other materials that require external field-induced polarization to obtain surface potential, the self-polarized PHBV / ZnO microspheres exhibit a more stable surface potential and require less complex fabrication.

[0081] Depend on Figure 10a and Figure 10b It is evident that PHBV microspheres also possess a non-zero surface potential.

[0082] Furthermore, the surface potential of PHBV / ZnO microspheres is approximately 483 mV, while that of PHBV microspheres is approximately 793 mV. The surface potential of the microspheres decreases after being combined with ZnO, which means that the PHBV / ZnO composite microspheres achieve mild regulation of surface potential, and may be used to regulate changes in the electrophysiological microenvironment to control cell behavior.

[0083] In an ultrasonic field, the ultrasonic properties of the target product PHBV / ZnO microspheres obtained in Example 1 were tested using an oscilloscope. Specifically, 5 mg of PHBV / ZnO microspheres were dispersed in 4 mL of deionized water and injected into one well of a six-well plate. An ultrasonic probe was placed under the plate to generate ultrasonic waves. The output parameters of the ultrasonic therapy device were set as follows: power 3 W / cm², frequency 1 MHz, duty cycle 20%, and pulse duration 3 minutes. The positive and negative electrodes were placed below the liquid surface, and the other end was connected to an oscilloscope to measure the electrical signal generated by the PHBV / ZnO microspheres.

[0084] The results are as follows Figure 11 As shown. According to Figure 11 Without the application of ultrasound, there was only noise from the oscilloscope itself, and no obvious voltage was generated. However, after the application of ultrasound, periodic and regular voltage changes occurred. The generation, duration, and disappearance of the voltage were synchronized with the on-off cycle of the ultrasound. This indicates that the target product PHBV / ZnO microspheres obtained in Example 1 have good ultrasonic piezoelectricity and can generate stable and measurable electrical signals under the action of ultrasound.

[0085] The biocompatibility of the material was assessed by measuring the proliferation of MC3T3-E1 cells cultured with blank control group, PHBV microspheres or PHBV / ZnO microspheres using the CCK-8 (Cell Counting Kit-8) method.

[0086] After thawing frozen pre-osteoblastic cells MC3T3-E1, they were cultured in α-MEM medium for 3 days, then digested with trypsin, resuspended, and prepared into a concentration of 6.0 × 10⁻⁶. 5 Cell suspension at 1 / mL was prepared. For each sample, 0.25 mL of cell suspension was mixed with 0.375 mL of sample dispersion (4 mg / mL) and 0.875 mL of α-MEM medium by pipetting. 0.5 mL of this mixture was added to each well of a 24-well plate, and the plates were then incubated in a constant temperature and humidity incubator at 37 °C and 5% CO2. After 24 h (1 day) or 72 h (3 days) of incubation, 500 μL of α-MEM medium and 50 μL of CCK-8 were added to each well under dark conditions, and the plates were incubated at 37 °C for 2 h. Finally, the absorbance (OD value) at 450 nm was measured using a microplate reader to analyze cell proliferation. The results are shown below. Figure 12 As shown. Figure 12 In the diagram, the vertical axis represents the absorbance of CCK-8 cells; higher absorbance indicates a greater number of cells.

[0087] The samples described above were either the PHBV microspheres obtained in Comparative Example 1 or the PHBV / ZnO microspheres obtained in Example 1. A blank control group was also set up, in which the sample dispersion was replaced with ultrapure water.

[0088] exist Figure 12 In this study, by comparing the data of the PHBV / ZnO microspheres obtained in Example 1 with the data of the PHBV microspheres obtained in Comparative Example 1 and the data of the blank control group, it can be found that:

[0089] After culturing with the PHBV / ZnO microspheres obtained in Example 1, the cell number continuously increased from day 1 to day 3, indicating that the PHBV / ZnO microspheres had no toxic effect on MC3T3-E1 cells, exhibited good biocompatibility, and supported cell proliferation. Furthermore, compared to the blank control group and the culture with the PHBV microspheres obtained in Comparative Example 1, the absorbance of the culture with the PHBV / ZnO microspheres obtained in Example 1 was the highest after both 1 and 3 days of culture, meaning that the PHBV / ZnO composite microspheres significantly promoted cell proliferation. In contrast, the absorbance of the culture with the PHBV microspheres obtained in Comparative Example 1 after 3 days was lower than that of the blank control group. This indicates that the PHBV / ZnO microspheres have better biocompatibility than the PHBV microspheres.

[0090] Example 2: Preparation of PHBV / ZnO microspheres

[0091] (1) Add 0.15 g of PHBV powder to a culture bottle containing 5 mL of chloroform, then seal the bottle and stir magnetically at 40 °C for 24 h to obtain a PHBV-chloroform solution with a concentration of 0.03 g / mL (approximately 5 mL). Add 0.03 g of ZnO to 2 mL of deionized water and disperse by sonication for 15 min to obtain a ZnO aqueous dispersion. Add all the ZnO aqueous dispersion to a culture bottle containing all the PHBV-chloroform solution, then reseal the bottle and stir magnetically at room temperature for 12 h to mix evenly to obtain a PHBV / ZnO mixture.

[0092] (2) Add 0.05 g of PVA particles to a culture bottle containing 10 mL of deionized water, then seal the bottle opening and stir magnetically at 60°C for 24 h to obtain a PVA solution with a concentration of 5 mg / mL (the solution volume is about 10 mL); add the PVA solution to a culture bottle containing all the PHBV / ZnO mixture obtained in step (1), seal the bottle opening, and stir magnetically for 2 h to obtain the mixture.

[0093] (3) Add 0.025 g of PVA particles to a beaker containing 50 mL of deionized water, then seal the mouth of the beaker and stir magnetically at 60 °C for 24 h to obtain a PVA solution with a concentration of 0.5 mg / mL (the solution volume is about 50 mL).

[0094] (4) Add the mixture obtained in step (2) into a beaker containing all the PVA solution obtained in step (3), open the mouth of the beaker, and stir magnetically at 35°C (1~2 days) until it evaporates to dryness to obtain a solid precipitate.

[0095] (5) Wash the solid precipitate obtained in step (4) with deionized water, and then centrifuge at 8000 rpm for 3 min to separate the precipitate; wash the separated precipitate with deionized water again, centrifuge at 8000 rpm for 3 min to separate the precipitate, and repeat the above steps of washing with deionized water, centrifugation and separation 3 to 5 times. The precipitate obtained in the end is the target product.

[0096] The product was characterized and identified using the same method as in Example 1, confirming that the target product obtained in Example 2 was PHBV / ZnO microspheres. Its surface morphology is as follows: Figure 13 As shown, the target product obtained in Example 2 is microspheres with a particle size of approximately 6-7 μm. Tiny particles are visible on the surface of the microspheres, and the microspheres are of uniform size. The EDS results are as follows: Figure 14 As shown, C, O, and Zn elements are distributed at positions corresponding to the microspheres in the SEM image, indicating the presence of C, O, and Zn elements in the microspheres.

[0097] The performance was tested using the same method as in Example 1, confirming that the target product obtained in Example 2 had a good piezoelectric response. A non-zero surface potential was measured using a Kelvin probe force microscope. A 500 nm × 500 nm micro-region on the silicon wafer surface was selected for surface scanning, and its height and potential distribution maps are shown below. Figure 15a and Figure 15b As shown, its surface potential is 659 mV. Its ultrasonic properties are as follows: Figure 16 As shown. By Figure 16 It is evident that without the application of ultrasound, there is only noise from the oscilloscope itself, and no significant voltage is generated. However, after the application of ultrasound, periodic and regular voltage changes occur, and the generation, duration, and disappearance of the voltage are synchronized with the on / off cycle of the ultrasound. This indicates that the PHBV / ZnO microspheres prepared in Example 2 have good ultrasonic piezoelectricity and can generate stable and measurable electrical signals under the action of ultrasound.

[0098] Example 3 Preparation of PHBV / BTO microspheres

[0099] (1) Add 0.15 g of PHBV powder to a culture bottle containing 5 mL of chloroform, then seal the bottle opening and stir magnetically at 40 °C for 24 h to obtain a PHBV-chloroform solution with a concentration of 0.03 g / mL (the solution volume is about 5 mL); open the bottle opening and add 0.015 g of BTO powder, reseal the bottle opening, and stir magnetically at room temperature for 10 h to mix evenly to obtain a PHBV / BTO mixture.

[0100] (2) Add 0.25 g of PVA particles to a beaker containing 50 mL of deionized water, then seal the mouth of the beaker and stir magnetically for 24 h at room temperature to obtain a PVA solution with a concentration of 5 mg / mL (the solution volume is about 50 mL).

[0101] (3) Add all of the PHBV / BTO mixture obtained in step (1) into a beaker containing all of the PVA solution obtained in step (2), open the mouth of the beaker, and stir magnetically at room temperature (1~2 days) until it evaporates to dryness (the solution is completely evaporated) to obtain a solid precipitate;

[0102] (4) Wash the solid precipitate obtained in step (3) with deionized water, and then centrifuge at 8000 rpm for 3 min to separate the precipitate; wash the separated precipitate again with deionized water, centrifuge at 8000 rpm for 3 min to separate the precipitate, and repeat the above steps of washing with deionized water, centrifugation and separation 3 to 5 times. Then mix the finally separated precipitate with 30 mL of deionized water in a centrifuge tube and sonicate for 15 min to obtain a mixture. Pass the mixture through a 5 μm sieve to obtain a second mixture. Then filter the second mixture through a 100 nm filter paper and collect the solid on the filter paper to obtain the target product.

[0103] The same method as in Example 1 was used for characterization, and the target product obtained in Example 3 was identified as PHBV / BTO composite microspheres.

[0104] Example 4

[0105] (1) Add 0.15 g of PHBV powder to a culture bottle containing 5 mL of chloroform, then seal the bottle opening and stir magnetically at 40 °C for 24 h to obtain a PHBV-chloroform solution with a concentration of 0.03 g / mL (the solution volume is about 5 mL); open the bottle opening and add 0.015 g of KNN powder, reseal the bottle opening, and stir magnetically at room temperature for 10 h to mix evenly to obtain a PHBV / KNN mixture.

[0106] (2) Add 0.25 g of PVA particles to a beaker containing 50 mL of deionized water, then seal the mouth of the beaker and stir magnetically for 24 h at room temperature to obtain a PVA solution with a concentration of 5 mg / mL (the solution volume is about 50 mL).

[0107] (3) Add all of the PHBV / KNN mixture obtained in step (1) into a beaker containing all of the PVA solution obtained in step (2), open the mouth of the beaker, and stir magnetically at room temperature (1~2 days) until it evaporates to dryness (the solution is completely evaporated) to obtain a solid precipitate;

[0108] (4) Wash the solid precipitate obtained in step (3) with deionized water, and then centrifuge at 8000 rpm for 3 min to separate the precipitate; wash the separated precipitate again with deionized water, centrifuge at 8000 rpm for 3 min to separate the precipitate, and repeat the above steps of washing with deionized water, centrifugation and separation 3 to 5 times. Then mix the finally separated precipitate with 30 mL of deionized water in a centrifuge tube and sonicate for 15 min to obtain a mixture. Pass the mixture through a 5 μm sieve to obtain a second mixture. Then filter the second mixture through a 100 nm filter paper and collect the solid on the filter paper to obtain the target product.

[0109] The same method as in Example 1 was used for characterization, and the target product obtained in Example 4 was identified as PHBV / KNN composite microspheres.

[0110] In summary, the electroactive polymer-based composite microspheres prepared by this invention achieve self-polarization from the inside out during the preparation process, possessing a non-zero surface potential without the need for additional external field-induced polarization, and are expected to regulate cell behavior through surface potential. Simultaneously, the composite microspheres of this invention exhibit good biocompatibility, and experiments have verified that they are beneficial to cell proliferation. Moreover, the microsphere size allows them to be injected directly into the lesion, potentially enabling precise and minimally invasive delivery. Furthermore, the composite microspheres of this invention can rapidly respond to externally applied ultrasound, generating stable electrical signals, and the strength of these signals can be controlled by adjusting ultrasound parameters to provide a targeted electrical microenvironment suitable for tissue repair. The electroactive polymer-based microspheres of this invention can be used for targeted therapy of tissue repair, especially bone tissue repair; or, they can be used in intelligent drug delivery systems and implantable medical devices.

[0111] Therefore, it is evident that the objective of this invention has been fully and effectively achieved. The function and structural principles of this invention have been demonstrated and explained in the embodiments. Any modifications can be made to the implementation methods without departing from these principles. Therefore, this invention includes all modified embodiments based on the spirit and scope of the claims.

Claims

1. An electroactive polymer-based composite microsphere, characterized in that, It is a composite microsphere of PHBV / inorganic piezoelectric material.

2. The electroactive polymer-based composite microspheres as described in claim 1, characterized in that, The inorganic piezoelectric material is one of ZnO, BTO, or KNN.

3. The electroactive polymer-based composite microspheres as described in claim 1, characterized in that, The PHBV / inorganic piezoelectric composite microspheres are self-polarized microspheres with a non-zero surface potential.

4. The electroactive polymer-based composite microspheres as described in claim 1, characterized in that, The particle size of the microspheres is between 500 nm and 10 μm.

5. The electroactive polymer-based composite microspheres as described in claim 3, characterized in that, The surface potential of the microspheres is 400~700 mV.

6. A method for preparing electroactive polymer-based composite microspheres as described in any one of claims 1 to 5, comprising the following steps: (1) PHBV powder is added to a first reaction vessel containing chloroform. After sealing the first reaction vessel, it is magnetically stirred at 30~50 °C for 15~25 h to obtain a PHBV-chloroform solution with a concentration of 0.01~0.1 g / mL. Inorganic piezoelectric nanoparticles are added to the first reaction vessel. The mass of the inorganic piezoelectric nanoparticles is 10~50% of the mass of the PHBV powder. The first reaction vessel is then resealed and magnetically stirred at room temperature for 10~20 h to mix evenly, thus obtaining a PHBV / inorganic piezoelectric material mixture. (2) PVA is added to a second reaction vessel containing deionized water. After sealing the second reaction vessel, it is magnetically stirred at room temperature for 15-25 h to obtain a PVA solution with a concentration of 2-6 mg / mL. (3) Add the PHBV / inorganic piezoelectric material mixture to the second reaction vessel containing the PVA solution, open the second reaction vessel, and magnetically stir at room temperature until it evaporates to dryness to obtain a solid precipitate; the volume ratio of the PHBV / inorganic piezoelectric material mixture to the PVA solution is 1: (8~10). (4) Wash the solid precipitate obtained in step (3) with deionized water, and then centrifuge at 5000~8000 rpm for 2~6 min to separate the precipitate; wash the separated precipitate with deionized water, centrifuge at 5000~8000 rpm for 2~6 min to separate the precipitate again; repeat the steps of washing with deionized water, centrifugation and separation several times, and ultrasonically mix the finally separated precipitate with deionized water to obtain a mixture. The mixture is passed through a 1~10 μm sieve to obtain a second mixture. The second mixture is then filtered through a 100~300 nm filter paper, and the solid on the filter paper is collected to obtain the target product.

7. The method as described in claim 6, characterized in that, The inorganic piezoelectric material is one of ZnO, BTO, or KNN.

8. A method for preparing electroactive polymer-based composite microspheres as described in any one of claims 1 to 5, comprising the following steps: (1) PHBV powder is added to a first reaction vessel containing chloroform. After sealing the first reaction vessel, it is magnetically stirred at 30-50 °C for 15-25 h to obtain a PHBV-chloroform solution with a concentration of 0.01-0.1 g / mL. Inorganic piezoelectric nanoparticles are ultrasonically dispersed in deionized water at room temperature to obtain an inorganic piezoelectric aqueous dispersion with a concentration of 0.005-0.02 g / mL. The inorganic piezoelectric aqueous dispersion is added to a first reaction vessel containing the PHBV-chloroform solution. After sealing the first reaction vessel, it is magnetically stirred at room temperature for 10-20 h to mix evenly to obtain a PHBV / inorganic piezoelectric material mixture. The mass of the inorganic piezoelectric nanoparticles is 10-50% of the mass of the PHBV powder. (2) PVA is added to a second reaction vessel containing deionized water. After sealing the second reaction vessel, it is magnetically stirred at 50-70 °C for 15-25 h to obtain a PVA solution with a concentration of 2-6 mg / mL. The PVA solution is added to a first reaction vessel containing the PHBV / inorganic piezoelectric material mixture obtained in step (1). After sealing the first reaction vessel, it is magnetically stirred for 2-6 h to obtain a mixture. The volume ratio of the PHBV / inorganic piezoelectric material mixture to the PVA solution is 1:(1.2-2). (3) PVA is added to a third reaction vessel containing deionized water. After sealing the third reaction vessel, it is magnetically stirred at 50-70 °C for 15-25 h to obtain a PVA solution with a concentration of 0.2-0.6 mg / mL. (4) Add the mixture obtained in step (2) to the third reaction vessel containing the PVA solution obtained in step (3), open the third reaction vessel, and stir magnetically at 25~45 °C until it evaporates to dryness to obtain a solid precipitate; wherein, the volume ratio of the mixture to the PVA solution is 1:(2.5~4). (5) Wash the solid precipitate obtained in step (4) with deionized water, and then centrifuge at 5000~8000 rpm for 2~6 min to separate the precipitate; wash the separated precipitate with deionized water, centrifuge at 5000~8000 rpm for 2~6 min to separate the precipitate again; repeat the steps of washing with deionized water, centrifugation and separation several times, and the precipitate obtained in the end is the target product.

9. The method as described in claim 8, characterized in that, The inorganic piezoelectric material is one of ZnO, BTO, or KNN.

10. The application of the electroactive polymer-based composite microspheres as described in any one of claims 1 to 5.

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