Ultrasonic excited piezoelectric delivery exosome electrostatic spinning yarn and preparation method thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to ultrasonic-excited piezoelectric delivery exosome electrostatic spinning and a preparation method thereof.The piezoelectric delivery exosome electrostatic spinning comprises piezoelectric electrostatic spinning prepared by embedding barium titanate nanoparticles into polylactic acid, the surface of the piezoelectric electrostatic spinning is modified by chitosan and then loaded with exosome, and the surface of the piezoelectric electrostatic spinning is loaded with the exosome. The preparation method comprises the following steps: preparation of piezoelectric electrostatic spinning: dissolving polylactic acid in hexafluoroisopropanol, adding barium titanate nanoparticles, and carrying out ultrasonic dispersion to form a uniform precursor solution; piezoelectric electrostatic spinning containing barium titanate is prepared through high-voltage electrostatic spinning; piezoelectric electrostatic spinning surface modification: immersing the piezoelectric electrostatic spinning into a chitosan solution for incubation, and modifying the piezoelectric electrostatic spinning to form piezoelectric electrostatic spinning with positive electricity on the surface; the modified piezoelectric electrostatic spinning is soaked in an exosome suspension to be incubated, and piezoelectric exosome delivery electrostatic spinning is prepared and has the self-energy-supply function, the space-time response function and the biological delivery function, the defects of traditional biological medicine materials are hopefully overcome, and the piezoelectric exosome delivery electrostatic spinning can be applied to the field of biological medicine materials. The clinical transformation application of the intelligent biological material in the chronic wound repair of diabetes mellitus is promoted.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to an ultrasonically excited piezoelectric delivery exosome electrospinning method and its preparation method. Background Technology

[0002] Diabetic chronic wounds are a persistent challenge in clinical treatment, with complex mechanisms of occurrence and development involving multiple factors such as metabolic disorders, immune imbalances, persistent inflammatory responses, and impaired tissue regeneration. A high-glucose environment can continuously induce oxidative stress, leading to elevated levels of reactive oxygen species (ROS), inhibiting cell proliferation and migration, and disrupting normal processes of angiogenesis and collagen deposition. Simultaneously, chronic hyperglycemia can inhibit the polarization transformation of macrophages from pro-inflammatory M1 to reparative M2, resulting in persistent inflammation and further exacerbating tissue damage. The combined effect of impaired local angiogenesis and tissue ischemia and hypoxia leaves the wound in a state of chronic energy deficiency and microcirculatory disturbance. Abnormal biological functions of fibroblasts and keratinocytes lead to reduced extracellular matrix (ECM) synthesis and impaired epithelial remodeling, significantly delaying wound closure. According to a report by the International Diabetes Federation (IDF), the number of people with diabetes worldwide reached 537 million in 2021 and is projected to increase to 783 million by 2045. Among them, the lifetime risk of developing diabetic foot ulcers (DFU) is approximately 19–34%, and about 80% of diabetes-related lower limb amputations are due to ulcer formation. The five-year mortality rate is significantly higher than in non-diabetic individuals. These data clearly demonstrate that chronic diabetic wounds not only severely threaten patients' quality of life but also impose a heavy social and economic burden.

[0003] In clinical treatment, the management of diabetic wounds currently mainly includes basic measures such as decompression, debridement, infection control, revascularization, moist dressings, and metabolic regulation, supplemented by negative pressure wound therapy (NPWT), recombinant human platelet-derived growth factor (rhPDGF-BB), and bioengineered skin substitutes (such as...). Adjunctive therapies include hyperbaric oxygen therapy (HBOT). However, these therapies still face multiple limitations: long treatment cycles, high costs, significant individual differences in efficacy, and high recurrence rates. Furthermore, while some physical therapies, such as phototherapy, electrical stimulation, or ultrasound intervention, have shown positive effects in improving local microcirculation and cell activity, their clinical application remains in the exploratory stage due to limitations in energy penetration, stimulation intensity, and the non-specificity of biological responses. Especially in chronic hyperglycemic environments, single drug or energy stimulation often fails to achieve multidimensional synergistic regulation of inflammation, angiogenesis, and tissue remodeling. Summary of the Invention

[0004] To address the problems in existing technologies, this invention provides an ultrasonically excited piezoelectrically delivered exosome electrospinning method and its preparation. It innovatively integrates the "piezoelectric effect" with "exosome therapy" into a smart, adjustable platform. Furthermore, it utilizes low-frequency ultrasound to achieve a non-invasive and highly controllable activation method, providing a new strategy for precise intervention in chronic wounds. This invention's piezoelectrically delivered exosome electrospinning combines self-powered, spatiotemporally responsive, and bio-delivery functions, potentially overcoming the shortcomings of traditional biomaterials and promoting the clinical translational application of smart biomaterials in the repair of diabetic chronic wounds.

[0005] The technical problem solved by this invention is achieved by the following technical solution:

[0006] The present invention aims to provide an ultrasonically excited piezoelectric delivery exosome electrospinning method, comprising a piezoelectric electrospinning fiber made of polylactic acid embedded barium titanate nanoparticles, wherein the surface of the piezoelectric electrospinning fiber is modified with chitosan and loaded with exosomes.

[0007] Piezoelectric materials, due to their ability to spontaneously generate electric charges and local electric fields under mechanical stress or acoustic stimulation, are ideal candidates for simulating bioelectrical signals in vivo. Studies have shown that piezoelectric signals can modulate cell membrane potential and promote calcium ion influx, thereby enhancing key repair processes such as fibroblast proliferation, angiogenesis, and collagen deposition. Unlike traditional external electrical stimulation systems, piezoelectric materials possess self-powered properties, generating continuous and stable electric field signals under non-invasive activation conditions (such as activity or low-frequency ultrasound) in vivo, achieving safe and controllable electrical stimulation. In particular, barium titanate (BaTiO3) possesses a high dielectric constant, excellent piezoelectric properties, and biocompatibility. Using polylactic acid (PLA) as a matrix, BaTiO3 is synthesized through electrospinning technology. … By embedding nanoparticles, a composite membrane with flexibility, biodegradability, and stable piezoelectric output can be obtained, which can generate controllable electrical signals under ultrasonic stimulation, providing a continuous electrical microenvironment for cell growth and wound regeneration.

[0008] Meanwhile, exosomes, as important mediators of intercellular communication, exhibit significant multi-target regulatory capabilities in tissue repair due to their carrying of various bioactive molecules such as miRNAs, mRNAs, and proteins. Studies have confirmed that exosomes derived from platelets or mesenchymal stem cells (PRP-Exos, MSC-Exos) can promote endothelial cell tube formation, stimulate fibroblast migration and collagen synthesis, and improve inflammation and angiogenesis disorders in diabetic wounds by regulating signaling pathways such as YAP / TAZ, NF-κB, and PI3K–AKT / VEGF. In particular, PRP-Exos have the advantages of safe source, stable composition, and strong autologous adaptability, and can significantly accelerate the closure of diabetic wounds and promote angiogenesis and epithelial regeneration. However, exosome therapy still faces challenges such as short local retention time, unstable release, and low delivery efficiency, which limit its long-term efficacy.

[0009] To address this, this invention proposes an innovative "physical-biological" dual-mode synergistic strategy: constructing a PLA / BaTiO3@PRP-Exos piezoelectric electrospun composite membrane that can be triggered by low-frequency ultrasound. This composite dressing uses PLA as the supporting matrix, with BaTiO3 nanoparticles endowing it with piezoelectric responsiveness. The surface is modified and loaded with bioactive platelet-derived exosomes (PRP-Exos). Under ultrasound stimulation, the composite membrane can simultaneously generate a local piezoelectric field and trigger the on-demand release of exosomes, achieving a spatial-temporal coupling effect of inflammation regulation, electrophysiological signal modulation, and biological factor delivery. Exosome delivery further enhances fibroblast migration and ECM remodeling. The synergistic effect of both allows for dynamic regulation of the wound microenvironment in three stages: inflammation resolution, angiogenesis, and matrix remodeling, ultimately achieving efficient healing of diabetic chronic wounds.

[0010] A method for preparing ultrasonically excited piezoelectrically delivered exosomes via electrospinning, comprising:

[0011] Preparation of piezoelectric electrospinning: Polylactic acid was dissolved in hexafluoroisopropanol, and barium titanate nanoparticles were added and ultrasonically dispersed to form a uniform precursor solution; piezoelectric electrospinning containing barium titanate was obtained by high voltage electrospinning.

[0012] Surface modification of piezoelectric electrospinning: piezoelectric electrospinning is incubated in chitosan solution to modify the piezoelectric electrospinning to form piezoelectric electrospinning with a positively charged surface;

[0013] Piezoelectric electrospinning loaded with exosomes: The modified piezoelectric electrospinning fiber is immersed in an exosome suspension and incubated to prepare a piezoelectric electrospinning fiber for delivering exosomes.

[0014] Furthermore, the added barium titanate nanoparticles have a mass of 2-3 wt% of the polylactic acid mass.

[0015] Furthermore, the ultrasound was dispersed at 100-300W for 10–20 minutes.

[0016] Furthermore, the high-voltage electrospinning flow rate is 1 mL / h, and the spinning voltage is set to 15–20 kV.

[0017] Furthermore, in the surface modification of piezoelectric electrospinning, piezoelectric electrospinning fibers are immersed in chitosan solution and incubated at room temperature with shaking for 20-40 minutes.

[0018] Furthermore, the modified piezoelectric electrospinned fibers were immersed in an exosome suspension and incubated with shaking at 3-5°C for 0.5-1.5 hours.

[0019] Furthermore, the exosomes used were extracted from platelet-enriched plasma using differential centrifugation. After stepwise centrifugation at 300×g for 5-15 min, 1500×g for 10-20 min, and 10000×g for 25-35 min, the exosomes were purified by centrifugation at 120000×g for 60-80 min, and the resulting exosomes had a particle size of 100–200 nm.

[0020] Furthermore, after piezoelectric electrospinning and loading exosomes, the exosomes are dried and fixed in a vacuum drying oven at room temperature and a vacuum degree of -0.05-0.1 MPa for 10-15 hours.

[0021] Preparation method of exosomes by ultrasonically excited piezoelectric delivery electrospinning:

[0022] (1) Extraction of exosomes

[0023] Exosomes were extracted from platelet-enriched plasma (PRP) using differential centrifugation. After removing impurities and large vesicles by stepwise centrifugation at 300×g, 1500×g, and 10000×g, the exosomes were purified by ultracentrifugation at 120000×g. The resulting PRP-derived exosomes had a particle size of 100–200 nm and a typical bilateral vesicle structure, making them suitable for subsequent loading and bioactive delivery.

[0024] (2) Preparation of piezoelectric electrospinning

[0025] Preparation of piezoelectric electrospun membranes: Polylactic acid (PLA) was dissolved in a hexafluoroisopropanol mixed solvent, and barium titanate (BaTiO3) nanoparticles were added and ultrasonically dispersed to form a uniform precursor solution. BaTiO3-containing piezoelectric fiber membranes with fiber diameters of approximately 0.50–1.35 μm were prepared by high-voltage electrospinning (15–20 kV, spinning flow rate: 1 ml / L), exhibiting stable piezoelectric response characteristics.

[0026] (3) Exosome loading

[0027] By modifying the surface of a piezoelectric electrospun membrane with chitosan solution to form a positively charged interfacial layer, and then immersing it in an exosome suspension, the surface charge and porous structure jointly drive the adsorption and immobilization of exosomes. The resulting PLA / BaTiO3@PRP-Exos composite membrane exhibits high loading efficiency and enables subsequent ultrasonic-triggered release.

[0028] (4) Fixation after loading exosomes

[0029] The electrospun membrane with adsorbed exosomes was vacuum dried under room temperature and low pressure at 20–30℃ to accelerate moisture removal and achieve preliminary fixation under mild conditions, thereby enhancing the binding stability of exosomes on the fiber surface. This effectively reduces the shedding of exosomes during storage and application, maintaining their structural integrity and biological activity, making them suitable for subsequent ultrasonic-triggered release.

[0030] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0031] 1. This invention combines the electrical stimulation effect of piezoelectric materials with the bioregulatory function of exosomes to construct an intelligent composite dressing with ultrasonic response characteristics, thereby achieving multidimensional and precise regulation of the local microenvironment of diabetic chronic wounds.

[0032] 2. The composite dressing of the present invention uses polylactic acid (PLA) as the matrix and embeds barium titanate (BaTiO2). … Nanoparticles are used to form a piezoelectric electrospun membrane, which is then surface-modified to load platelet-derived exosomes (PRP-Exos) and utilizes low-frequency ultrasound as a non-invasive trigger signal. This invention enables the synchronous generation of local electrical signals under ultrasound stimulation and achieves on-demand release of exosomes, thereby producing synergistic effects in inflammation regulation, angiogenesis, and tissue remodeling. Specifically, the local electric field generated by the piezoelectric effect can modulate the cell membrane potential. In vitro experimental results show that PLA / BaTiO2... … The @PRP-Exos composite membrane can effectively inhibit inflammation, enhance the tubular formation ability of endothelial cells, and promote fibroblast migration and collagen deposition under ultrasound triggering. In vivo diabetic rat wound model further verified the promoting effect of the composite membrane on wound healing, which was manifested by reduced inflammatory cell infiltration, increased blood vessel density, and more complete and orderly epidermal regeneration and collagen arrangement.

[0033] 3. The ultrasonically responsive PLA / BaTiO3 constructed in this invention … @PRP-Exos piezoelectric composite dressing achieves precise regulation of diabetic chronic wounds through "physical-biological" dual-mode signal synergy. It combines self-powered, controllable release, and spatiotemporal response characteristics, providing a novel non-invasive intelligent strategy for chronic wound repair, demonstrating excellent biocompatibility and clinical translational potential.

[0034] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, it can be implemented according to the contents of the specification. Furthermore, in order to make the above contents, objectives, features and advantages of the present invention more obvious and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0035] Figure 1 This is a transmission electron microscope image of exosomes in an ultrasonically excited piezoelectric delivery electrospinning process according to the present invention.

[0036] Figure 2 This is a diagram showing the average particle size distribution of exosomes during electrospinning of exosomes via ultrasonically excited piezoelectric delivery according to the present invention.

[0037] Figure 3 This is a SEM image of an ultrasonically excited piezoelectric delivery exosome electrospinning process at multiple scales from 10 μm, 2 μm, 500 nm to 200 nm, as shown in Experimental Example 1 of this invention.

[0038] Figure 4 This is a particle size analysis diagram of an ultrasonically excited piezoelectric delivery exosome electrospinning process in Experiment Example 1 of the present invention.

[0039] Figure 5 This is an EDS energy spectrum of an ultrasonically excited piezoelectric delivery exosome electrospinning process in Experimental Example 1 of the present invention.

[0040] Figure 6 This is a time-domain output voltage response diagram of an ultrasonically excited piezoelectric delivery exosome electrospinning process in Experimental Example 2 of the present invention.

[0041] Figure 7 This is an FTIR analysis diagram of an ultrasonically excited piezoelectric delivery exosome electrospinning method in Experiment Example 3 of the present invention.

[0042] Figure 8 This is a graph showing the water contact angle measurement results of an ultrasonically excited piezoelectric delivery of exosomes electrospinning in Experiment Example 4 of this invention. Detailed Implementation

[0043] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0044] In addition, unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or prepared by existing methods.

[0045] Example 1

[0046] Extraction and characterization of exosomes: Fresh peripheral venous blood containing an anticoagulant (sodium citrate) was centrifuged at 1500×g for 10 min to remove red blood cells and most white blood cells. The supernatant was collected and transferred to a new centrifuge tube. The supernatant was then centrifuged at 2000×g for 15 min to further precipitate platelets. Approximately two-thirds of the plasma at the top was discarded, retaining the concentrated platelets at the bottom. After mixing, PRP was obtained. PRP was transferred to centrifuge tubes and centrifuged sequentially at 300×g for 10 min, 1500×g for 15 min, and 10000×g for 30 min to progressively remove residual cells, cell debris, and large vesicles, retaining the supernatant at each step. The supernatant was passed through a 0.22 μm filter, and the final supernatant was transferred to an ultracentrifuge tube and ultracentrifuged at 120000×g for 70 min. The supernatant was discarded, and the final precipitate was platelet-derived exosomes (PRP-Exos).

[0047] Resuspend PRP-Exos in an appropriate amount of PBS. Take 10 μL of the exosome sample, aspirate it, and drop it onto a 200-mesh carbon copper mesh. Let it stand for 1 min to allow for full adsorption of the particles. Then, gently blot off the excess liquid by touching the edge of the copper mesh with filter paper. Add 10 μL of uranium acetate staining solution to the copper mesh, continue precipitation for 1 min, and then blot off the excess staining solution again with filter paper. Afterward, allow it to air dry at room temperature for several minutes to completely fix the sample. Place the treated copper mesh in a transmission electron microscope and image it with an accelerating voltage of 80 kV. Figure 1 As shown, typical vesicle-like morphology and membrane structure characteristics of exosomes were obtained. The extracted exosome solution was diluted with sterile PBS at a ratio of 1:100–1:500 to bring the particle concentration into the suitable range for nanoparticle tracking analysis (NTA) instrument (approximately 1×10⁻⁶). 10 –1×10 11 (particles / mL).

[0048] The diluted sample was injected into the NanoSight NS300, the laser wavelength was set to 488nm, and the camera level was 12–15. Three sets of 60-second videos were continuously acquired. NTA software (automatically performing particle tracking, data extraction, and statistical analysis, such as…) was used. Figure 2 As shown, the average particle size of the exosomes obtained was approximately 150 nm.

[0049] 2. Preparation of piezoelectric electrospinning

[0050] A certain amount of polylactic acid (PLA) powder was weighed and dissolved in hexafluoroisopropanol (HFIP) to adjust the final mass concentration to 15% (w / v). The solution was then stirred thoroughly on a magnetic stirrer for 6 hours until completely dissolved, yielding a transparent and homogeneous PLA solution. Subsequently, 2.5 wt% of barium titanate (BaTiO2) was added. … Nanoparticles (approximately 200 nm in diameter) were ultrasonically treated (200 W) for 10–20 min to ensure thorough dispersion, followed by magnetic stirring for 6 h to obtain a uniform electrospinning precursor solution. This precursor solution was loaded into a 10 mL syringe and connected to a metal needle, which was then mounted on the injection pump of the electrospinning apparatus, with a flow rate adjusted to 1 mL / h. The spinning voltage was set to 16 kV, and the distance from the needle to the collecting roller was set to 15 cm. A high-speed rotating roller with a linear velocity of 1300 rpm was used as the collector. Spinning was performed at room temperature and a relative humidity of 25%–40%. The nanofiber membrane collected on the aluminum foil was the PLA / BaTiO2 membrane. … Piezoelectric electrospun film.

[0051] 3. Surface charge modification of electrospun films

[0052] First, a 1% (v / v) dilute acetic acid solution was prepared, and chitosan was dissolved in this solution to prepare a 1% (w / v) chitosan solution. The solution was then magnetically stirred at room temperature for 2–4 hours until completely dissolved. Electrospun PLA / BaTiO3 fiber membranes were cut into standard 2cm × 2cm pieces and completely immersed in the chitosan solution. The membranes were then gently incubated at room temperature with slight shaking for 30 minutes to allow chitosan to uniformly adsorb onto the fiber surface, imparting a positive charge to the material. After incubation, the membranes were removed and rinsed 3–4 times (5–10 minutes each time) with ultrapure water to remove any loosely bound chitosan. The treated membranes were then dried at a temperature below 40°C for 30 minutes to obtain a positively charged chitosan-modified PLA / BaTiO3 nanofiber membrane, providing a suitable interface for subsequent electrostatic adsorption of exosomes.

[0053] 4. Exosome load:

[0054] Frozen PRP exosomes were slowly thawed on ice and diluted to 150 μg / mL with PBS. Chitosan-modified PLA / BaTiO3 fiber membranes were placed in 24-well plates, and exosome PBS suspension was added to each well to completely submerge the membrane. The plates were incubated with gentle shaking at 4°C for 1 h. Through electrostatic adsorption and the synergistic effect of the porous structure, the exosomes were fully bound to the fiber surface and pore network. After loading, the membranes were gently rinsed with ultrapure water (1–2 times) to remove unbound free exosomes and avoid interference with subsequent use. The treated membrane is the PLA / BaTiO3@PRP-Exos piezoelectric composite membrane.

[0055] 5. Dressing drying and fixing:

[0056] After washing, the PLA / BaTiO3@PRP-Exos composite membrane was placed on sterile filter paper to remove excess liquid, and then transferred to a vacuum drying oven. It was dried at room temperature (20–25℃) and a vacuum of approximately -0.08 MPa until the membrane mass remained constant (12 h). This yielded a structurally stable PLA / BaTiO3@Exo composite dressing that can be stored long-term. The dried membrane can be sealed and stored at 4℃ or -20℃ for later use. Before use, it can be lightly moistened with sterile PBS.

[0057] Experimental Example 1: SEM Characterization

[0058] Experimental Methods: Pure PLA membranes, PLA / BaTiO3 composite membranes, and exosome-loaded PLA / BaTiO3@PRP-Exos membranes were cut into approximately 5×5mm pieces and directly adhered to conductive adhesive. Gold was sputtered onto the substrate using an Oxford Quorum SC7620 sputtering system for 45 seconds at 10mA. Subsequently, the morphology of the samples was imaged and energy dispersive spectroscopy (EDS) mapping was performed using a ZEISS GeminiSEM 300 scanning electron microscope. The accelerating voltage for morphology imaging was 3kV, and for EDS mapping, it was 15kV. An SE2 secondary electron detector was used. Imaging analysis was performed to determine the fiber diameter, pore structure, and distribution of BaTiO3 nanoparticles within the fibers.

[0059] Test results

[0060] (1) Fiber particle size distribution and uniformity: such as Figure 4 As shown, the particle size distribution results indicate that the diameter of the obtained electrospun fibers is mainly concentrated in the range of 0.50–1.35 μm, with the highest frequency distribution located at approximately 0.78 μm, indicating that most fibers in the material have a consistent micron-scale size. With stable control of spinning voltage, flow rate, and BaTiO3 content, the fiber diameter distribution exhibits a typical positively skewed characteristic, with a very low proportion of high-diameter regions (>1.6 μm), indicating that the preparation process parameters are uniform and controllable, and the fiber structure is stable and reproducible. This reflects a good balance between solution viscosity, conductivity, and solvent evaporation rate in the spinning system, enabling the material to form a continuous and uniform fiber network structure, providing a structural basis for subsequent piezoelectric response and exosome loading.

[0061] (2) SEM morphology analysis: such as Figure 3As shown in the SEM images, the fibers exhibit a highly continuous, smooth, and bead-free typical electrospun morphology at multiple scales from 10 μm, 2 μm, 500 nm to 200 nm. Cross-linking between fibers forms a dense network, yet the spun filaments maintain a good porous structure, facilitating air permeation, cell migration, and material transport and exchange. This continuous and unbroken fiber structure not only ensures the mechanical stability of the material but also provides the necessary dielectric pathway for piezoelectric properties. In high-magnification areas, dispersed nanoscale bright spots or protrusions are visible. Some particles only form slight protrusions on the fiber surface without large-area particle aggregation, indicating good dispersion of nanoparticles within the fiber and a stable composite system structure. In high-magnification (500–200 nm) images, granular structures with diameters of approximately 100–200 nm are observed on the fiber surface, exhibiting discrete attachment or localized aggregation.

[0062] (3) Energy-dispersive X-ray spectroscopy analysis: such as Figure 5 As shown, the energy dispersive spectroscopy (EDS) results further validated the successful introduction of the inorganic phase BaTiO3 into the fiber. Characteristic peaks of Ba (L-line), Ti (K-line), and O (K-line) were clearly detected in the spectrum, and the peak intensity distribution ratio was consistent with the theoretical composition of BaTiO3. The wt% values ​​of Ba and Ti were approximately 1.61 wt% and 0.56 wt%, respectively, consistent with the typical content characteristics of low-added nanofillers in polymer fibers. Furthermore, the main peak was carbon, originating from the PLA polymer matrix, perfectly consistent with the expected structure. Elemental distribution analysis showed that Ba and Ti signals exhibited slight aggregation in localized areas of the fiber but were uniform overall, indicating that the BaTiO3 nanoparticles were well dispersed in the fiber without significant agglomeration. This uniform distribution is crucial for improving the consistency of the piezoelectric response.

[0063] Combining particle morphology characteristics and EDS results, it is clear that these particles originate from two different components: 1) BaTiO3 nanoparticles (approximately 200 nm) with clear edges, high electron density, and relatively rigid morphology, some exhibiting a semi-embedded distribution. Their distribution matches the characteristic element peaks of Ba, Ti, and O detected by EDS in the corresponding region, confirming that BaTiO3 nanoparticles are successfully embedded or fixed on the fiber surface and interior, providing a structural basis for piezoelectric properties. 2) Exosomes (approximately 100–150 nm) with rounded contours and soft edges, exhibiting a typical spherical vesicle morphology, mostly attached to the outer layer of the fiber through surface adsorption or localized accumulation. This characteristic is consistent with their biological vesicle structure, indicating that the positively charged chitosan layer on the fiber surface can effectively capture exosomes. The exosomes maintain their intact morphology during loading and exhibit stable binding with the surface-modified PLA fibers.

[0064] Overall, the particle size statistics, SEM multi-scale morphology and EDS elemental analysis are in good agreement, proving that the electrospinning process is stable and reliable. BaTiO3 nanoparticles were successfully embedded in the PLA fiber structure, forming a piezoelectric composite material with excellent structural integrity and uniformity.

[0065] Experimental Example 2: Measurement of Piezoelectric Voltage Output:

[0066] Experimental Method: The prepared PLA / BaTiO3 fiber membrane was cut into rectangular samples of 3cm × 5cm. Conductive copper sheets of the same size were cut and attached to the upper and lower surfaces of the fiber membrane as upper and lower electrodes. Insulating tape was used to wrap and fix the electrodes and copper wire connections on both sides to avoid leakage and environmental interference. Before testing, a layer of ultrasonic coupling agent was uniformly coated on the upper surface of the membrane to ensure full acoustic coupling with the ultrasonic probe. Subsequently, a low-frequency ultrasonic probe (frequency 1MHz, sound intensity 0.1W / cm²) was used. 2 An ultrasonic pulse (with a duty cycle of 50%) was applied perpendicularly to the membrane surface, and the transient piezoelectric voltage signal generated by the fiber membrane under ultrasonic stimulation was recorded in real time using a mixed-signal oscilloscope (RIGOL MSO5354 series). The oscilloscope sampling frequency was set to 10–100 kHz, and the peak-to-peak output voltage (Vpp) and complete waveform at different time periods were recorded to evaluate the piezoelectric output capability of the PLA / BaTiO3 fiber membrane and its response characteristics to ultrasonic stimulation.

[0067] Experimental results: at 1MHz, 0.1W / cm 2 Under ultrasonic stimulation with a 50% duty cycle, the PLA / BaTiO3 piezoelectric fiber membrane generated a stable and repeatable voltage oscillation signal. Figure 6 As shown, the voltage-time curve reveals that during the ultrasound "off" phase, the voltage waveform amplitude is small, exhibiting only low-amplitude fluctuations around the baseline (approximately ±60mV), primarily reflecting environmental mechanical disturbances and system background noise. Upon entering the ultrasound "on" phase, the waveform amplitude significantly increases, with the peak-to-peak voltage rising from approximately 120mV to approximately 0.24mV, exhibiting regular periodic oscillations. Furthermore, the positive and negative peaks are essentially symmetrical, indicating that the piezoelectric film can effectively convert periodic ultrasonic mechanical stress into an alternating electrical signal. The significant change in voltage amplitude before and after ultrasound not only demonstrates the material's sensitive piezoelectric response to low-intensity ultrasound but also suggests that its output voltage can be amplified and controlled on demand by adjusting the ultrasound duty cycle, laying the foundation for subsequent electrical stimulation applications.

[0068] Experimental Example 3: Fourier Transform Infrared Spectroscopy (FTIR) Analysis

[0069] Experimental methods: Chemical functional groups of the samples were detected using a Thermo Fisher IS10 infrared spectrometer (Nicolet iS10). PLA / BaTiO3@PRP-Exos membranes were freeze-dried and then pressed into tablets or tested directly in ATR mode, with a wavenumber range of 4000–400 cm⁻¹. -1 32 scans, 4cm resolution -1 The focus of the analysis is on the C=O stretching vibration peak of PLA (approximately 1750 cm⁻¹). -1 The Ti–O stretching peak of BaTiO3 (approximately 500–600 cm⁻¹) -1 ) and characteristic peaks of exosome proteins (approximately 1650 / 1550 cm⁻¹) -1 ) Changes.

[0070] Test results: such as Figure 7 As shown, at 4000–400cm -1 Within this range, several representative characteristic absorption peaks appeared in the sample: among them, 2995 cm⁻¹ -1 With 2944cm -1 Corresponding to typical C–H stretching vibration, 1750 cm -1 The significant C=O stretching absorption peak clearly indicates the ester structure of polylactic acid (PLA); simultaneously, at 1452 cm⁻¹... -1 With 1360cm -1 C–H bending vibration, 1182 cm -1 C–O stretching vibrations and 1128, 1085, and 1044 cm -1 A series of C–O–C stretching vibration peaks together constitute the complete characteristic spectrum of the PLA molecule, indicating that PLA maintains good chemical structural stability in the composite system. In addition to the PLA characteristics, the 3381 cm⁻¹ peak in the sample... -1 The appearance of broad and distinct O–H stretching vibration peaks, as well as 1128, 1085, and 1044 cm⁻¹. -1 The C–O–C absorption peaks at 1632 cm⁻¹ further corroborate the successful introduction of chitosan. The presence of these peaks indicates that the hydroxyl groups and sugar ring structure of chitosan are fully preserved, suggesting that it can provide a stable hydrogen bond network in the system, contributing to enhanced interfacial interactions. -1 The distinct amide I absorption peak observed at 533 cm⁻¹ is a typical characteristic of the protein backbone structure in exosomes, usually originating from the C=O stretching vibration in proteins. Its presence indicates successful loading of the exosome during complexation, and that its main protein structure remained intact. The appearance of this absorption peak not only demonstrates the effective introduction of exosomes but also suggests the potential formation of hydrogen bonds or intermolecular interactions between the exosomes and the chitosan / PLA matrix, providing a foundation for subsequent biological activity. Furthermore, the sample showed an absorption peak at 533 cm⁻¹.-1 The characteristic absorption peak corresponds to the Ti–O vibration in BaTiO3, which is an important infrared signature signal for ferroelectric ceramic materials. This absorption peak clearly characterizes the presence of BaTiO3, indicating that its crystal structure was effectively maintained during the preparation process. The successful introduction of BaTiO3 means that the material possesses a piezoelectric response basis, providing possibilities for subsequent mechanoelectric coupling or stimulus-response functions.

[0071] Experimental Example 4: Contact Angle Measurement

[0072] Experimental Method: Samples were cut into 1cm × 1cm pieces and placed on a contact angle measuring instrument platform (Shanghai Xuanzhun SZ-CAMC32). 3μL of deionized water was added to the membrane surface using a micro-syringe. The water droplet morphology was automatically recorded within 1 second after imaging, and the static contact angle was calculated. The test temperature was 25℃. The contact angle changes of PLA membrane and chitosan-modified PLA / BaTiO3 membrane were measured respectively to evaluate surface wettability and modification effect.

[0073] Test results: such as Figure 8 As shown, without exosome loading, the pure PLA fiber membrane exhibits a high contact angle of 132–135°, indicating that the material is highly hydrophobic and has low surface energy. After loading exosomes, the contact angle of the PLA / BaTiO3@PRP-Exos piezoelectric composite membrane decreased to approximately 130°, with a slight increase in hydrophilicity. This change indicates that the exosomes form localized hydrophilic protein / lipid regions on the fiber surface, resulting in a slight improvement in overall wettability. However, the contact angle remains within the hydrophobic range, indicating that the macroscopic surface properties of the material are still dominated by PLA nanofibers, and exosome loading did not change the overall hydrophobic structure. In summary, exosomes impart slight hydrophilicity without compromising material stability, enabling the material to possess both mechanical stability and biocompatibility.

[0074] Based on the above experimental results, a comprehensive analysis yields the following conclusions:

[0075] 1) Network Structure Changes: The PLA / BaTiO3 piezoelectric fiber membrane loaded with exosomes maintained a continuous and uniform overall fiber network structure with a stable fiber diameter distribution, indicating that the main network of the material was not destroyed during loading. Exosomes adhered to the fiber surface to form a granular bioactive layer. This biofilm layer altered the arrangement of local chain segments and the distribution of interfacial energy on the fiber surface, transforming the network structure from a "pure inorganic-polymer interface" to a "biological-inorganic-polymer" ternary interface system, providing new interfacial functional regions for cell-cell interactions.

[0076] 2) Electrical Properties and Ultrasonic Stimulation Response: The PLA / BaTiO3 piezoelectric fiber membrane loaded with exosomes outputs a clear, periodic piezoelectric voltage signal under ultrasonic stimulation. The voltage-time curve exhibits regular oscillations, with a peak-to-peak value of approximately 200-250 mV, remaining stable throughout the entire ultrasonic stimulation phase. The waveform regularity and stable oscillation amplitude indicate that the piezoelectric units within the membrane can effectively respond to the mechanical stimulation provided by ultrasound, generating a continuous and identifiable electrical signal. No interruptions in the electrical signal, waveform disorder, or rapid amplitude decay were observed during the experiment, indicating that the material maintains good piezoelectric conversion capability and can provide continuous and stable electrical stimulation output.

[0077] 3) Changes in physical morphology and interfacial properties after exosome loading: Exosome loading causes protein characteristic peaks to appear on the fiber surface, and its biomembrane structure covers the outer surface of the fiber, forming a new interfacial chemical composition. The water contact angle slightly decreases from the original strong hydrophobicity, exhibiting an interfacial characteristic of "overall hydrophobicity, local hydrophilicity". This change indicates that the protein lipid layer of the exosome forms a hydrophilic region at the microscale, improving interfacial wettability without destroying the original hydrophobic main structure of the fiber. The introduction of exosomes changes the surface chemical composition and interfacial properties at the microscale, enabling the material to obtain a certain degree of interfacial biocompatibility while maintaining hydrophobicity.

[0078] 4) Functional Changes and Overall Structure – Performance Correlation: The piezoelectric fiber membrane loaded with exosomes, while maintaining the stability of the fiber network structure and continuous piezoelectric voltage output, acquires a biofunctional layer endowed by exosomes. The presence of exosomes on the fiber surface endows the material with potential biological signal delivery and cell regulation capabilities, while the stable piezoelectric signal generated by the membrane under ultrasound can serve as an additional physical stimulus to participate in cell regulation. The combination of these two aspects means that the material can simultaneously achieve electrical stimulation output and exosome delivery under ultrasound triggering, possessing the potential to synergistically regulate cell behavior and promote tissue repair.

[0079] Overall, PLA / BaTiO3 piezoelectric fibers loaded with exosomes possess structural integrity, electrical activity, interfacial biological functions, and exosome delivery capabilities, laying the foundation for a multi-dimensional and controllable approach to chronic wounds.

[0080] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0081] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. An ultrasonically excited piezoelectric delivery electrospinning method for exosomes, characterized in that, This includes piezoelectric electrospinning made of polylactic acid embedded with barium titanate nanoparticles, with the surface of the piezoelectric electrospinning fiber modified with chitosan and loaded with exosomes.

2. The method for preparing ultrasonically excited piezoelectrically delivered exosomes via electrospinning as described in claim 1, characterized in that: include: Preparation of piezoelectric electrospinning: Polylactic acid was dissolved in hexafluoroisopropanol, and barium titanate nanoparticles were added and ultrasonically dispersed to form a uniform precursor solution; Barium titanate-containing piezoelectric electrospun fibers were prepared by high-voltage electrospinning. Surface modification of piezoelectric electrospinning: piezoelectric electrospinning is incubated in chitosan solution to modify the piezoelectric electrospinning to form piezoelectric electrospinning with a positively charged surface; Piezoelectric electrospinning loaded with exosomes: The modified piezoelectric electrospinning fiber is immersed in an exosome suspension and incubated to prepare a piezoelectric electrospinning fiber for delivering exosomes.

3. The method for preparing ultrasonically excited piezoelectrically delivered exosomes via electrospinning as described in claim 2, characterized in that: The added barium titanate nanoparticles account for 2-3 wt% of the mass of polylactic acid.

4. The method for preparing ultrasonically excited piezoelectrically delivered exosomes via electrospinning as described in claim 3, characterized in that: Ultrasonic dispersion is 100-300W for 10-20 minutes.

5. The method for preparing ultrasonically excited piezoelectrically delivered exosomes via electrospinning as described in claim 4, characterized in that: The high-voltage electrospinning flow rate was 1 mL / h, and the spinning voltage was set to 15–20 kV.

6. The method for preparing ultrasonically excited piezoelectrically delivered exosomes via electrospinning as described in claim 2, characterized in that: In the surface modification of piezoelectric electrospinning, piezoelectric electrospinning fibers are immersed in chitosan solution and incubated with shaking at room temperature for 20-40 minutes.

7. The method for preparing ultrasonically excited piezoelectrically delivered exosomes via electrospinning as described in claim 2, characterized in that: The modified piezoelectric electrospinned fibers were immersed in an exosome suspension and incubated with shaking at 3-5℃ for 0.5-1.5h.

8. The method for preparing ultrasonically excited piezoelectrically delivered exosomes via electrospinning as described in claim 2, characterized in that: The exosomes used were extracted from platelet-enriched plasma by differential centrifugation. They were centrifuged in stages at 300×g for 5-15 min, 1500×g for 10-20 min, and 10000×g for 25-35 min, and then purified by centrifugation at 120000×g for 60-80 min. The resulting exosomes had a particle size of 100–200 nm.

9. The method for preparing ultrasonically excited piezoelectrically delivered exosomes via electrospinning as described in claim 2, characterized in that: After piezoelectric electrospinning to load exosomes, the fibers are dried and fixed in a vacuum drying oven at room temperature and a vacuum degree of -0.05-0.1 MPa for 10-15 hours.

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