Biomimetic piezoelectric nanoparticles, a preparation method thereof and application thereof in preparing a medicine for treating depression

Abstract

This invention relates to the field of biomedicine, specifically to a biomimetic piezoelectric nanoparticle, its preparation method, and its application in the preparation of drugs for treating depression. The biomimetic piezoelectric nanoparticle (referred to as NKM@ZnO-PDA) consists of a core of piezoelectric zinc oxide nanoparticles, encapsulated with polydopamine (PDA) and a natural killer cell membrane (NK cells). The preparation method includes: dispersing piezoelectric zinc oxide nanoparticles in an alkaline aqueous solution of dopamine for auto-oxidation to prepare the core of the biomimetic piezoelectric nanoparticle (referred to as ZnO-PDA); and encapsulating the ZnO-PDA with an NK cell membrane under ultrasonic incubation and co-extrusion to prepare the biomimetic piezoelectric nanoparticles. The biomimetic piezoelectric nanoparticles exhibit excellent blood-brain barrier permeability and biocompatibility, and can improve mitochondrial function after being phagocytosed by astrocytes. After being phagocytosed by cells, the biomimetic piezoelectric nanoparticles can polarize under low-power ultrasonic conditions, stimulating cellular calcium influx, promoting the release of extracellular vesicles containing mitochondria, and transferring healthy mitochondria to neurons, thereby restoring neuronal function.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a biomimetic piezoelectric nanoparticle, its preparation method, and its application in the preparation of drugs for treating depression. Background Technology

[0002] Major Depressive Disorder (MDD) is a severe subtype of depression, characterized by persistent mood disturbances and functional impairment. As a chronic and complex mental illness, the core features of MDD include persistent low mood, loss of interest or pleasure, and decreased energy. In the early stages, patients often experience sleep disturbances, changes in appetite, difficulty concentrating, fatigue, and excessive feelings of self-blame or worthlessness. As the disease progresses, patients may develop severe cognitive impairment and even suicidal ideation or behavior, which is the most dangerous and critical clinical manifestation of MDD. Furthermore, MDD patients often have co-existing anxiety disorders, substance abuse, and other chronic physical illnesses, further complicating the condition.

[0003] The pathological changes in MDD involve multiple brain regions, particularly the prefrontal cortex (PFC), hippocampus, and amygdala. Its pathogenesis is complex and multidimensional, involving the neurotransmitter system, neuroplasticity, neuroinflammation, the endocrine system, and the interaction of genetic and environmental factors. Current hypotheses include oxidative stress and mitochondrial dysfunction, dysregulation of the monoamine neurotransmitter system, decreased neuroplasticity and reduced neurotrophic factors (such as Brain-Derived Neurotrophic Factor, BDNF), and dysfunction of the hypothalamic-pituitary-adrenal axis. Antidepressant medication and cognitive behavioral therapy are currently the core clinical treatments for MDD; however, these medications have a slow onset of action and potential side effects with long-term use, while cognitive behavioral therapy requires significant time and financial investment. In addition, some novel therapies, such as transcranial magnetic stimulation (TMS), transcranial direct current stimulation (tDCS), and deep brain stimulation (DBS), are still under investigation.

[0004] Mitochondrial dysfunction plays a crucial role in the pathology of depression. As the cell's energy factories, mitochondria are central to the pathogenesis of oxidative stress and malignant depression (MDD). MDD patients exhibit mitochondrial dysfunction, including reduced activity of the mitochondrial respiratory chain complex, decreased ATP production, and a decreased mitochondrial membrane potential. These dysfunctions not only lead to energy metabolism disorders but also exacerbate oxidative stress. This redox imbalance results in oxidative damage to lipids, proteins, and DNA, thereby affecting normal neuronal function.

[0005] Strategies targeting mitochondria for the treatment of depression include mitochondrial-targeting drugs, modulation of mitochondrial quality control pathways, regulation of mitochondrial dynamics, and mitochondrial genetic engineering. Examples include mitochondrial-targeting antioxidants, enhancement of mitophagy, and regulation of mitochondrial calcium homeostasis. However, reversing cellular mitochondrial damage is difficult, as damaged cells struggle to generate healthy mitochondria. Mitochondrial transplantation has become an effective approach to address these challenges. Healthy mitochondria are isolated from mitochondrial-rich tissues (e.g., muscle tissue) and transplanted into damaged cells as a drug. Although mitochondrial transplantation has shown promising therapeutic effects in mouse models of depression, clinical application still faces challenges related to mitochondrial sourcing, isolation, preservation, and delivery. Directly utilizing cells as mitochondrial carriers for mitochondrial transfer offers a solution. This strategy provides a stable source of mitochondria and directionality for transplantation, avoiding repeated administration and immunogenicity. Huang T et al. used mesenchymal stem cells with high mitochondrial activity as mitochondrial carriers, using pioglitazone to promote mitoogenesis and ferric ions to promote contact-based mitochondrial transfer, thereby restoring alveolar cell function and treating pulmonary fibrosis. However, the ability of transplanted exogenous cells to spontaneously cross the blood-brain barrier is limited, and long-term application in the brain may pose risks.

[0006] Utilizing autologous cells to achieve in situ supply of healthy mitochondria is an effective strategy for addressing the aforementioned problems. However, current research on in situ mitochondrial transfer mainly focuses on pathological mechanisms. For example, in stroke-affected brain regions, astrocytes spontaneously provide mitochondria to damaged neurons to alleviate neuronal damage and repair; in some brain tumors, tumor cells steal mitochondria from surrounding cells to promote their own malignant proliferation. Regulating mitochondrial transfer remains a challenge. Furthermore, autologous cells may not be able to provide healthy mitochondria under pathological conditions.

[0007] Mitochondrial transfer is conserved, with common pathways including tunnel nanotubes, tight junctions, extracellular vesicles containing mitochondria, migratory bodies, and fusion of directly exposed extracellular mitochondria with recipient cells, transferring mitochondria from donor cells to recipient cells. Calcium influx has been shown to be involved in multiple mitochondrial transfer pathways. Therefore, it is hypothesized that controllable induction of cellular calcium influx is a potential mechanism for regulating mitochondrial transfer.

[0008] Piezoelectric materials can be classified into three main categories based on their composition and properties: inorganic piezoelectric materials, organic piezoelectric materials, and composite piezoelectric materials. Inorganic piezoelectric materials typically have a crystalline structure, and their piezoelectric effect originates from the asymmetric arrangement of ions within the crystal. When a piezoelectric material is subjected to external stress, the positive and negative charge centers within the material undergo relative displacement, resulting in the generation of charges on the material surface and the formation of an electric field; this process is called the positive piezoelectric effect. In recent years, the application of piezoelectric materials in neuroscience has attracted widespread attention, especially in neural modulation and regeneration. The combination of piezoelectric materials and ultrasound can achieve local, wireless, and minimally invasive modulation of neural activity by converting mechanical energy into electrical energy. For example, barium titanate (BaTiO3) nanoparticles, after being co-incubated with nerve cells for 24 hours, primarily localize on the cell membrane and activate nerve cells under ultrasound stimulation, exhibiting discharge activity. In a zebrafish model, BaTiO3 nanoparticles with a carbon shell on their surface responded to ultrasound stimulation, inducing the upregulation of tyrosine hydroxylase and synaptophysin, indicating that the combined application of piezoelectric materials and ultrasound can modulate neural plasticity and may be used to treat neurodegenerative diseases. Furthermore, boron nitride nanotubes (BNNTs) showed significantly enhanced differentiation of neuron-like cells in response to ultrasound stimulation in PC12 pheochromocytoma cells, a process dependent on calcium ion influx. Despite the enormous application potential of piezoelectric materials, challenges remain regarding biocompatibility, targeting, and clinical translation.

[0009] In summary, although mitochondrial transplantation has shown promising therapeutic effects in various diseases, including depression, its clinical application still faces significant challenges, including: the source of mitochondria, isolation techniques, preservation techniques, targeted delivery across various physiological barriers, and the uptake efficiency of target cells. While directly using cells as mitochondrial carriers for mitochondrial transfer offers a solution to these problems, this strategy provides a stable source of mitochondria and directionality for transplantation, avoiding repeated administration and immunogenicity. However, the ability of transplanted exogenous cells to spontaneously cross the blood-brain barrier is limited, and long-term application in the brain may pose risks. Even using autologous cells to achieve in situ supply of healthy mitochondria is an effective strategy to address these issues. However, current research on in situ mitochondrial transfer mainly focuses on pathological mechanisms, and regulating mitochondrial transfer remains a challenge. Furthermore, autologous cells may not be able to provide healthy mitochondria under pathological conditions. In recent years, intelligent nanomedicines have shown great promise in complex diagnostic and therapeutic scenarios.

[0010] Therefore, developing a nanomedicine that can overcome physiological barriers and has good targeting, selecting suitable donor cells, and regulating effective mitochondrial transfer in donor cells has become an urgent problem to be solved. Summary of the Invention

[0011] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a biomimetic piezoelectric nanoparticle, its preparation method, and its application in the preparation of drugs for treating depression. This invention designs and constructs piezoelectric nanoparticles with good brain targeting and biocompatibility. Under ultrasound, the piezoelectric nanoparticles polarize to generate an electric field, inducing calcium influx into cells, thereby wirelessly regulating mitochondrial transfer at the site of depression, thus developing a novel mitochondrial therapy for the treatment of depression.

[0012] The inventive concept of this invention is as follows: To improve the biocompatibility and blood-brain barrier permeability of nanoparticles, and to target the inflammatory tendency of pathological brain regions, this invention utilizes NK cell membrane biomimetic technology to achieve good brain targeting with no significant side effects in cells and animals. To improve the function of damaged cells, especially mitochondrial function, this invention utilizes the antioxidant properties of polydopamine. After being taken up by astrocytes, it can reduce the reactive oxygen species level of damaged astrocytes and restore mitochondrial membrane potential. To achieve effective transfer of healthy mitochondria, this invention utilizes the property of piezoelectric materials to convert mechanical signals into electrical signals. After astrocytes take up piezoelectric nanoparticles, they are subjected to ultrasonic treatment. The intracellular biomimetic piezoelectric nanoparticles can induce cellular calcium influx, increasing the quantity and quality of extracellular mitochondrial vesicle release, providing neurons with healthy mitochondria, and achieving in situ autologous mitochondrial transfer. This invention provides a new strategy for mitochondrial therapy in the treatment of depression.

[0013] The technical solution of this invention to solve the technical problem is as follows: In a first aspect of the present invention, a biomimetic piezoelectric nanoparticle is provided, which is composed of nanoparticle ZnO-PDA and an NK cell membrane wrapped on the surface of ZnO-PDA; wherein the ZnO-PDA is formed by wrapping polydopamine with nano zinc oxide having a piezoelectric effect as the core; The ZnO-PDA has a particle size of 238.31±14 nm; The biomimetic piezoelectric nanoparticles, referred to as NKM@ZnO-PDA, have a particle size of 263.46±17 nm.

[0014] In a second aspect of the invention, a method for preparing biomimetic piezoelectric nanoparticles as described in the first aspect is provided.

[0015] The preparation method involves dispersing nano-zinc oxide in an alkaline solution of dopamine, causing dopamine to self-polymerize on the surface of nano-piezoelectric zinc oxide. Appropriately sized nanoparticles of ZnO-PDA are collected by centrifugation. NK cell membranes are then coated onto the surface of the ZnO-PDA through ultrasonic incubation and co-extrusion to prepare NKM@ZnO-PDA.

[0016] Furthermore, the specific steps of the preparation method are as follows: 1) Dissolve dopamine hydrochloride in water, add ZnO aqueous dispersion, stir vigorously and slowly add alkaline buffer under 50 ℃ water bath heating, and continue the reaction for 5 hours; after the reaction is completed, first centrifuge the whole solution at 10000 rpm for 10 minutes, discard the precipitate and keep the supernatant, then centrifuge the supernatant at 4000 rpm for 10 minutes, discard the supernatant and keep the precipitate, wash the obtained precipitate twice with pure water until the supernatant is clear and resuspend it to finally obtain ZnO-PDA; 2) The NK cell membrane vesicles prepared by ultrasound were mixed with ZnO-PDA and sonicated in an ice bath for 5 min to ensure that the NK cell membrane vesicles and ZnO-PDA nanoparticles were fully mixed. Then, the nanoparticles were repeatedly extruded 10 times through a 200 nm polycarbonate membrane using a microporous extruder to obtain NKM@ZnO-PDA with uniform particle size.

[0017] Further, in step 1), the mass ratio of dopamine to zinc oxide is between 20:1 and 5:1 (preferably 15:1). The alkaline buffer solution includes sodium hydroxide solution or Tris or Tris-HCl buffer, adjusted to pH 8-9.5.

[0018] Further, in step 2), the method for preparing NK cell membrane vesicles by ultrasound is as follows: the NK cell membrane is homogenized before use, and ultrasound is performed using a probe at a power of 100 W for 5 minutes, with a 1-second pause during ultrasound. The NK cell membrane fragments are then broken up to form NK cell membrane vesicles.

[0019] Further, in step 2), the NK cell membrane preparation method is as follows: NK cells are cultured, centrifuged at 1000 rpm for 3 minutes to collect the cells, and resuspended in a hypotonic sucrose solution. The cells are then sonicated using a probe under ice bath conditions to lyse the NK cells for 5 minutes, with a 2-second pause and 5-second break during sonication, followed by an ice bath. Subsequently, the cells are centrifuged at 3000 g to remove unlyseeded cells and organelles, and the supernatant is collected. The NK cell membrane is then collected by centrifugation at 14000 g and resuspended in PBS. The NK cell membrane needs to maintain its physiological activity during use.

[0020] Further, in step 2), the ultrasonic preparation method for ZnO-PDA is as follows: before use, ultrasonic dispersion is performed, ultrasonication is performed using a probe at a power of 100 W for 5 minutes, with a 1-second interval between working and 1-second pauses during ultrasonication, followed by an ice bath.

[0021] In a third aspect of the invention, the use of biomimetic piezoelectric nanoparticles as described in the first aspect in the preparation of medicaments for treating depression is provided.

[0022] Furthermore, the drug is used in combination with brain ultrasound therapy.

[0023] The drug can be taken up by astrocytes, improving oxidative stress in astrocytes and restoring mitochondrial membrane potential. Under ultrasound conditions, intracellular piezoelectric nanoparticles generate a micro-electric field, stimulating the opening of cellular calcium ion channels, causing calcium influx, increasing the number and quality of extracellular mitochondrial vesicles, which are then taken up by neurons, improving the function of damaged neurons.

[0024] In a fourth aspect of the invention, a pharmaceutical composition for treating depression is provided, comprising a therapeutically effective amount of the biomimetic piezoelectric nanoparticles as described in the first aspect and at least one pharmaceutically acceptable carrier.

[0025] The pharmaceutical composition can be formulated into a variety of dosage forms suitable for administration. In one or more embodiments, the pharmaceutical composition is an oral dosage form, including but not limited to: tablets, capsules, granules, powders, oral solutions, oral suspensions, or oral gels. In other embodiments, the pharmaceutical composition is an injectable dosage form, which can be administered via intravenous, intramuscular, or subcutaneous routes. The pharmaceutical composition can also be formulated into other dosage forms, such as transdermal patches, sublingual tablets, or nasal sprays.

[0026] The biomimetic piezoelectric nanoparticles of this invention are based on nano-zinc oxide, with polydopamine and natural killer cell (NK cell) membranes coated on their surfaces to enhance biocompatibility and achieve good brain region targeting function. Simultaneously, these nanoparticles can be taken up by astrocytes, generating an electric field under ultrasound, thereby inducing calcium influx into astrocytes at safe ultrasound power. This promotes the transfer of mitochondria from astrocytes to neurons via vesicles, thereby restoring the normal function of damaged neurons and treating depression.

[0027] The polydopamine involved in this invention increases the hydrophilicity of zinc oxide surface, thereby improving the monodispersity of zinc oxide in aqueous solution, and is called ZnO-PDA with a particle size of 238.31±14 nm; the NK cell membrane is wrapped on the surface of ZnO-PDA to form biomimetic piezoelectric nanoparticles, called NKM@ZnO-PDA, with a particle size of 263.46±17 nm.

[0028] This invention involves dispersing nano-zinc oxide in an alkaline solution of dopamine, causing dopamine to self-polymerize on the surface of nano-piezoelectric zinc oxide. Appropriately sized nanoparticles of ZnO-PDA are collected by centrifugation. NK cell membranes are then coated onto the ZnO-PDA surface through ultrasonic incubation and co-extrusion to prepare NKM@ZnO-PDA. The NK cell membranes are quantified using the BCA method before use.

[0029] This invention involves direct co-incubation with cells, allowing for uptake by astrocytes, improving oxidative stress in astrocytes, and restoring mitochondrial membrane potential. Under ultrasound conditions, intracellular piezoelectric nanoparticles generate a micro-electric field, stimulating the opening of cellular calcium ion channels, resulting in calcium influx and increasing the quantity and quality of extracellular mitochondrial vesicles. These therapeutic vesicles are uptaken by neurons, improving the function of damaged neurons. This invention achieves wireless regulation of the transfer of healthy mitochondria from astrocytes to neurons via vesicles. In a mouse model of depression, regular tail vein administration combined with brain ultrasound therapy demonstrated good antidepressant effects.

[0030] The core innovation of this invention compared to existing technologies lies in: utilizing piezoelectric nanoparticles to achieve wireless control of mitochondrial transfer, as detailed below: 1. The piezoelectric nanoparticles of the present invention use nano zinc oxide with piezoelectric effect and polydopamine as raw materials, and are surface-modified with NK cell membranes, exhibiting good biocompatibility and brain targeting.

[0031] 2. The piezoelectric nanoparticles of this invention have the function of improving in-situ damaged cells. After treating cells with oxidative stress damage using the piezoelectric nanoparticles, the intracellular reactive oxygen species level decreased, the mitochondrial membrane potential was restored, and the quality of mitochondria, which serve as mitochondrial donor cells, was improved.

[0032] 3. The piezoelectric nanoparticles of this invention promote mitochondrial transfer via vesicles by activating cellular calcium influx. The piezoelectric nanoparticles are directly co-incubated with cells and can be taken up by the cells. Under ultrasound conditions, the intracellular piezoelectric nanoparticles generate a micro-electric field, stimulating cellular calcium influx and increasing the quantity and quality of extracellular mitochondrial vesicles. These therapeutic vesicles are taken up by neurons, enabling wireless regulation of the transfer of healthy mitochondria from astrocytes to neurons via vesicles.

[0033] The present invention has the following technical effects: 1. This invention has good brain targeting, inflammation targeting and biosafety.

[0034] 2. This invention has a good piezoelectric effect, which generates a micro-electric field under ultrasonic conditions, which can induce calcium influx into cells, promote the release of extracellular vesicles containing mitochondria, and promote mitochondrial transfer.

[0035] 3. This invention can alleviate cellular oxidative stress to a certain extent and promote mitochondrial repair.

[0036] 4. Based on compliance with animal ethics regulations, this invention has undergone pharmacodynamic testing at the animal level, achieving a relatively ideal antidepressant effect. Attached Figure Description

[0037] Figure 1This is a schematic diagram showing the preparation of biomimetic piezoelectric nanoparticles ZnO-PDA and NKM@ZnO-PDA.

[0038] Figure 2 The particle size (A) and potential (B) of the biomimetic piezoelectric nanoparticles ZnO-PDA and NKM@ZnO-PDA are shown.

[0039] Figure 3 For the quantitative analysis of intracellular reactive oxygen species (A) and mitochondrial membrane potential (B) in astrocytes by biomimetic piezoelectric nanoparticles ZnO-PDA and NKM@ZnO-PDA (US represents ultrasound).

[0040] Figure 4 This study aims to quantitatively analyze the changes in intracellular calcium flow in astrocytes induced by biomimetic piezoelectric nanoparticles NKM@ZnO-PDA.

[0041] Figure 5 This study aimed to quantitatively analyze the number (A) and quality (B) of extracellular mitochondrial vesicles in astrocytes using biomimetic piezoelectric nanoparticles NKM@ZnO-PDA (mdACM represents mitochondrial-cleared astrocyte conditioned medium, and Calcium chelator is a calcium ion antagonist).

[0042] Figure 6 Representative images of mitochondria transferred via mitochondrial vesicles in astrocytes under different treatments (scale bar = 20 μm) (md indicates astrocyte conditioned medium with mitochondrial clearance).

[0043] Figure 7 These are in vivo imaging images showing the whole-body distribution of biomimetic piezoelectric nanoparticles. A, a representative image showing the change in local fluorescence intensity in the mouse brain over time. B, representative images of organs such as the heart, liver, spleen, lungs, and kidneys after 24 hours of dissection of mice in each group. (Heart: H, Liver: Li, Spleen: S, Lung: Lu, Kidney: K).

[0044] Figure 8 This is a representative image of the open field trajectory in mice after treatment with biomimetic piezoelectric nanoparticles (MDD stands for Major Depression). Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0046] The experimental materials and their sources involved in the embodiments of this invention are as follows:

[0047] Example 1: Preparation and Characterization of Key Parameters of Biomimetic Piezoelectric Nanoparticles Preparation process as follows Figure 1 Specifically, as shown, 180 mg of dopamine hydrochloride was dissolved in 90 ml of pure water, and 34 μl of ZnO aqueous dispersion (containing approximately 12 mg of ZnO) was added. The mixture was stirred vigorously in a 50°C water bath while 760 μl of 1M NaOH solution was slowly added, and the reaction was continued for 5 hours. After the reaction was completed, the entire solution was centrifuged at 10,000 rpm for 10 minutes, the precipitate was discarded and the supernatant was retained. The supernatant was then centrifuged at 4,000 rpm for 10 minutes, the supernatant was discarded and the precipitate was retained. The precipitate was washed twice with pure water until the supernatant was clear and then resuspended to finally obtain ZnO-PDA.

[0048] NK cells were cultured in T75 culture medium, centrifuged at 1000 rpm for 3 minutes to collect the cells, and resuspended in hypotonic sucrose solution. The cells were then sonicated using a probe under ice bath conditions for 5 minutes (2 seconds on, 5 seconds off, ice bath). Subsequently, the cells were centrifuged at 3000 g to remove unlysaturated cells and organelles. The supernatant was collected, and the NK cell membranes were collected by centrifugation at 14000 g. The cells were resuspended in 1 ml of PBS and stored at -80°C for use within one week.

[0049] NK cell membranes needed to be homogenized before use. This was achieved using a probe-based sonication method at 100 W for 5 minutes (1 second on, 1 second off, ice bath) to break up NK cell membrane fragments into NK cell membrane vesicles. ZnO-PDA needed to be dispersed by sonication before use. This was also done using a probe-based sonication method at 100 W for 5 minutes (1 second on, 1 second off, ice bath). The sonicated NK cell membrane vesicles were then mixed with ZnO-PDA and sonicated on ice for 5 minutes to ensure thorough mixing. Subsequently, a microporous extruder was used to repeatedly extrude the mixture through a 200 nm polycarbonate membrane 10 times to obtain NKM@ZnO-PDA with uniform particle size. NK cell membranes were quantified using the BCA method before use.

[0050] Figure 1 This is a schematic diagram showing the preparation of biomimetic piezoelectric nanoparticles ZnO-PDA and NKM@ZnO-PDA.

[0051] Figure 2 The particle size (A) and potential (B) of the biomimetic piezoelectric nanoparticles ZnO-PDA and NKM@ZnO-PDA in Example 1 are shown. Figure 2 (A) It can be seen that: ZnO-PDA has a uniform particle size of 238.31±14 nm; NKM@ZnO-PDA after coating has a uniform particle size of 263.46±17 nm. Figure 2(B) indicates that the nanoparticles all have a negative potential and good biocompatibility.

[0052] Example 2: Biomimetic piezoelectric nanoparticles promote the recovery of mitochondrial function in astrocytes When donor cells are damaged, they cannot provide healthy mitochondria to recipient cells. Therefore, this study investigated the effects of biomimetic piezoelectric nanoparticles on astrocytes, particularly mitochondrial function. Logarithmic-phase astrocytes were collected and a cell suspension was prepared, using 10 nanoparticles per well. 4 Cells were seeded at a density of 100 μM into 12-well plates and allowed to adhere overnight. Astrocytes were incubated with 600 μM hydrogen peroxide for 12 h to serve as a model of cellular oxidative stress and mitochondrial damage. The culture medium was then discarded and replaced with fresh medium containing the drugs for another 12 h. The concentrations of ZnO-PDA and NKM@ZnO-PDA were 10 μg / ml. Untreated healthy astrocytes were used as a control. Healthy cells were treated with piezoelectric nanoparticles, and the safety of the nanoparticles was observed. For the sonication group, sonication (0.5 W / cm²) was administered 4 h after the medium was replaced with the drug-containing medium. 2 Stimulate with 1 MHz for 3 minutes, then continue culturing for 12 h.

[0053] After treatment, astrocytes were digested with trypsin and a single-cell suspension was prepared. Subsequently, the cells were stained with DCFH-AD using the Beyotime reactive oxygen species (ROS) detection kit according to the manufacturer's instructions, and intracellular ROS levels were quantitatively analyzed using flow cytometry.

[0054] Further investigation was conducted into changes in mitochondrial membrane potential. The plating, modeling, and drug administration of astrocytes were performed as described above. The treated astrocytes were prepared into single-cell suspensions, and the cell membrane potential was stained with JC-1 dye. JC-1 was prepared in serum-free DMEM medium at a working concentration of 10 μg / ml. Cells in each well were resuspended in 300 μl of dye, stained at 37°C for 20 min, centrifuged to remove free dye, and resuspended in PBS. The mitochondrial membrane potential was analyzed using flow cytometry. The key indicator for evaluating mitochondrial membrane potential was the dye, specifically the red / green fluorescence intensity ratio = JC-1 red / JC-1 green. The relative membrane potential was calculated using healthy controls as a relative value.

[0055] Figure 3 Quantitative analysis of intracellular reactive oxygen species (ROS) levels (A) and mitochondrial membrane potential (B) in astrocytes using biomimetic piezoelectric nanoparticles ZnO-PDA and NKM@ZnO-PDA (US represents ultrasound). Experimental results ( Figure 3A) shows that when astrocytes are damaged, intracellular ROS levels increase significantly. ZnO-PDA treatment can reduce ROS levels in damaged astrocytes, restoring them to normal levels. After treatment with ZnO-PDA or NKM@ZnO-PDA and ultrasound (US), intracellular ROS levels further decrease, even falling below normal levels. Further analysis of the treatment's effect on the mitochondrial membrane potential of damaged astrocytes... Figure 3 B). Ultrasound therapy in all groups had the function of increasing mitochondrial membrane potential, and this function was amplified by biomimetic piezoelectric nanoparticles ZnO-PDA and NKM@ZnO-PDA. At the same time, the biomimetic piezoelectric nanoparticles themselves also had a good effect on improving mitochondrial membrane potential.

[0056] Example 3: Biomimetic piezoelectric nanoparticles regulate calcium flow in astrocytes Astrocytes in the logarithmic growth phase were collected and a cell suspension was prepared, with 10 cells per well. 5 Cells were seeded at a density of 10 μg / ml into 6-well plates and allowed to adhere overnight. The medium was then replaced with fresh medium containing NKM@ZnO-PDA (10 μg / ml) and incubated for 4 h. Cells were then digested to prepare single-cell suspensions and incubated with Fluo-4 AM for 30–60 min. The working solution for Fluo-4 AM was a 5 μM Hanks solution. After staining, cells were washed with Hanks solution, resuspended in incomplete medium, and incubated for 20 min to allow for complete hydrolysis of Fluo-4 AM by intracellular esterases.

[0057] Before using flow cytometry, discard the incomplete culture medium and replace it with Hanks buffer. Collect cells that have not been sonicated for one minute, then transfer the cells to wells and sonicate (0.5 W / cm²). 2 The sample was stimulated with 1 MHz for 3 minutes, and then immediately transferred to a flow cytometer for detection. The change in the average fluorescence intensity of Fluo-4 AM with the application of 1 MHz was quantitatively analyzed.

[0058] Figure 4 This study presents a quantitative analysis of the effects of biomimetic piezoelectric nanoparticles NKM@ZnO-PDA on calcium flow changes in astrocytes, as described in Example 3. The results show that NKM@ZnO-PDA can induce significant calcium influx in cells under low-power ultrasound. After ultrasounding, the intracellular fluorescence intensity generated by calcium influx was twice the baseline, and the calcium flow slowly decreased after ultrasounding was stopped.

[0059] Example 4: Biomimetic piezoelectric nanoparticles promote the secretion of healthy mitochondrial vesicles by astrocytes Astrocytes were cultured using T75 culture medium. During the logarithmic growth phase, a portion of the astrocytes were used to induce a model for 12 h using 600 μM hydrogen peroxide. The modeling medium was then discarded and replaced with fresh medium containing NKM@ZnO-PDA (10 μg / ml) for another 12 h. The medium containing nanoparticles was then discarded and replaced with 8 ml of new incomplete medium. Groups requiring sonication were then sonicated again (0.5 W / cm²). 2 Stimulate with 1 MHz for 6 minutes, then incubate for 24 h.

[0060] Collect the supernatant from each group, centrifuge at 500 g for 3 min to remove suspended cells, and centrifuge at 1000 g for 5 min to remove cell debris. Transfer the centrifuged supernatant to an ultrafiltration tube (MWCO 100K) and centrifuge at 3500 g for 10 min. Collect the supernatant from the ultrafiltration tube and centrifuge at 10000 g for 10 min. Collect the pellet and resuspend it using JC-1 dye. JC-1 was prepared in serum-free DMEM medium at a working concentration of 10 μg / ml, stained at 37°C for 20 min, washed with PBS, and resuspended. After adjusting the flow cytometry parameters using 1 μm silica microspheres, the stained vesicles were detected.

[0061] Figure 5 This study presents a quantitative analysis of the number (A) and quality (B) of extracellular mitochondrial vesicles in astrocytes using the biomimetic piezoelectric nanoparticle NKM@ZnO-PDA in Example 4 (mdACM represents conditioned medium for astrocytes with mitochondrial clearance, and Calcium chelator is a calcium ion antagonist). The results showed a significant decrease in the number of mitochondrial vesicles released from damaged astrocytes, reaching half the normal value, and extremely low mitochondrial membrane potential. Direct ultrasound could not alter the decreased quality and quantity of mitochondria. After treatment with NKM@ZnO-PDA, the number and quality of mitochondria improved to some extent. Simultaneously, ultrasound promoted the release of both the number and quality of mitochondrial vesicles, particularly significantly improving their quality. Filtering with a 0.22 μm filter membrane resulted in a sharp decrease in the number of vesicles (mdACM group). Blocking calcium influx using a calcium flow inhibitor (Calcium chelator group) reduced both the quality and number of released mitochondria.

[0062] Example 5: Biomimetic piezoelectric nanoparticles promote the transfer of mitochondria from astrocytes to neurons in vesicle form. Astrocytes were seeded into 6-well plates and allowed to adhere overnight. Mitochondrial markers of the astrocytes were labeled using Mito-Tracker Red CMXRos staining. A modeling process was then established using 600 μM hydrogen peroxide for 12 h. The modeling medium was then discarded and replaced with fresh medium containing NKM@ZnO-PDA (10 μg / ml) for 12 h. After discarding the medium containing nanoparticles, the cells were sonicated (0.5 W / cm²). 2 Stimulate with 1 MHz for 3 minutes. Continue culturing for 12 hours, and collect the supernatant of astrocytes as conditioned medium.

[0063] Neurons were seeded into confocal microscopy dishes and allowed to adhere overnight. The neurons were then labeled with CFSE. Subsequently, a model was established for 24 h using 400 μM corticosterone. The modeling medium was then discarded and replaced with astrocyte conditioned medium for 12 h. The conditioned medium was filtered through a 0.22 μm filter as a negative control. The amount of labeled mitochondria in the neurons was observed using confocal microscopy.

[0064] Figure 6 Representative images (scale bar = 20 μm) of mitochondria transferred via mitochondrial vesicles from astrocytes under different treatments in Example 5 (md represents the conditioned medium for astrocytes with mitochondrial clearance). The results showed that mitochondrial vesicle secretion was significantly downregulated after astrocyte damage. Combined treatment with biomimetic piezoelectric nanoparticles and ultrasound promoted the transfer of mitochondria from astrocytes to neurons via vesicles. When vesicles in the culture medium were removed using a filter membrane, almost no fluorescently labeled exogenous mitochondria were found in the neurons, indicating no false positives caused by leakage of free dye, and demonstrating that vesicles are an important form of mitochondrial transfer.

[0065] Example 6: Biomimetic piezoelectric nanoparticles targeting the brain of a mouse model of depression to treat depression. To evaluate the brain-targeting properties of biomimetic piezoelectric nanoparticles, the distribution of the nanoparticle formulation in vivo was detected using an in vivo imaging system. Mice were randomly divided into four groups of four each: ZnO-PDA, ZnO-PDA+US, NKM@ZnO-PDA, and NKM@ZnO-PDA+US. A formulation labeled with DiR (5 μM) was prepared and administered via tail vein injection (0.1 ml) at a dose of 10 mg / kg. One hour after injection, ultrasound was performed on the groups requiring ultrasound (1 W / cm²). 2 The cells were stimulated with 1 MHz for 5 min, and fluorescence in vivo imaging was performed at 3, 6, 12, 24, and 48 h, and the image results were recorded.

[0066] Twenty-four hours after administration, one mouse from each group was sacrificed, and normal organs (heart, liver, spleen, lung, kidney, and brain) were dissected and examined under an in vivo imaging system. The images were recorded to assess the extent to which the formulation reached the brain and its distribution in normal tissues.

[0067] A mouse model of chronic restraint stress was established: A specially designed adjustable restraint tube (3 cm in diameter, 12 cm in length) was used to restrict mouse movement without affecting respiration. Mice were restrained at fixed times each day (e.g., 12:00-18:00) for 28 consecutive days, for 6 hours each time. Behavioral changes were used as markers of successful model establishment. During the restraint period, ZnO-PDA or NKM@ZnO-PDA (10 mg / kg) was injected via the tail vein every two days, and ultrasound was performed 24 hours later using a handheld ultrasound device (1 W / cm²). 2 The mice were stimulated with 1 MHz for 5 min. After treatment, the open field test was used to evaluate the spontaneous activity and exploratory behavior of the mice.

[0068] Figure 7 This is an in vivo imaging image of the whole-body distribution of biomimetic piezoelectric nanoparticles in Example 6. A is a representative image showing the change in local fluorescence intensity in the mouse brain over time. B is a representative image of organs such as heart, liver, spleen, lung, and kidney after dissection of mice in each group 24 hours later (H: heart, Li: liver, S: spleen, Lu: lung, K: kidney, B: brain). Experimental results show that NK cell membrane modification can significantly improve the brain targeting of biomimetic piezoelectric nanoparticles, with the highest accumulation in the brain at 24 hours after injection. In addition, appropriate ultrasound of the brain can improve the brain penetration efficiency of biomimetic piezoelectric nanoparticles. Figure 7 A). Images from dissections of mice in each group showed that NKM@ZnO-PDA modified with NK cell membranes had the highest brain accumulation and the lowest accumulation in other organs. Figure 7 (B) This indicates that the NK cell membrane plays an important role in improving the brain targeting of biomimetic nanoparticles.

[0069] Figure 8 These are representative images of the open field trajectories of mice after treatment with biomimetic piezoelectric nanoparticles in Example 6. Experimental results showed that spontaneous activity was significantly reduced in depressed (MDD) mice, and the complexity of their movement trajectories decreased. Neither ultrasound alone nor the application of biomimetic piezoelectric nanoparticles could effectively improve spontaneous activity in the mice. Only the combination of biomimetic piezoelectric nanoparticles and ultrasound treatment restored spontaneous activity in the depressed mice, demonstrating a good antidepressant effect.

[0070] The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A biomimetic piezoelectric nanoparticle, characterized in that, It is composed of ZnO-PDA nanoparticles and NK cell membranes encapsulating the surface of ZnO-PDA; the ZnO-PDA is formed by encapsulating polydopamine with nano zinc oxide having a piezoelectric effect as the core; The ZnO-PDA has a particle size of 238.31±14 nm; The biomimetic piezoelectric nanoparticles, referred to as NKM@ZnO-PDA, have a particle size of 263.46±17 nm.

2. The method for preparing biomimetic piezoelectric nanoparticles as described in claim 1, characterized in that, The process involves dispersing nano-zinc oxide in an alkaline solution of dopamine, causing dopamine to self-polymerize on the surface of nano-piezoelectric zinc oxide. Appropriately sized nanoparticles of ZnO-PDA are collected by centrifugation, and NK cell membranes are coated onto the surface of the ZnO-PDA through ultrasonic incubation and co-extrusion to prepare NKM@ZnO-PDA.

3. The preparation method according to claim 2, characterized in that, The specific steps are as follows: 1) Dissolve dopamine hydrochloride in water, add ZnO aqueous dispersion, stir vigorously and slowly add alkaline buffer under 50 ℃ water bath heating, and continue the reaction for 5 hours; after the reaction is completed, first centrifuge the whole solution at 10000 rpm for 10 minutes, discard the precipitate and keep the supernatant, then centrifuge the supernatant at 4000 rpm for 10 minutes, discard the supernatant and keep the precipitate, wash the obtained precipitate twice with pure water until the supernatant is clear and resuspend it to finally obtain ZnO-PDA; 2) The NK cell membrane vesicles prepared by ultrasound were mixed with ZnO-PDA and sonicated in an ice bath for 5 min to ensure that the NK cell membrane vesicles and ZnO-PDA nanoparticles were fully mixed. Then, the nanoparticles were repeatedly extruded 10 times through a 200 nm polycarbonate membrane using a microporous extruder to obtain NKM@ZnO-PDA with uniform particle size.

4. The preparation method according to claim 3, characterized in that, In step 1), the mass ratio of dopamine to zinc oxide is between 20:1 and 5:1; the alkaline buffer solution includes sodium hydroxide solution or Tris or Tris-HCl buffer solution, and the pH is adjusted to 8 to 9.

5.

5. The preparation method according to claim 3, characterized in that, In step 2), the method for preparing NK cell membrane vesicles by ultrasound is as follows: the NK cell membrane is homogenized before use, and ultrasound is performed using a probe at a power of 100 W for 5 minutes, with a 1-second pause during ultrasound. The NK cell membrane fragments are broken down to form NK cell membrane vesicles.

6. The preparation method according to claim 5, characterized in that, In step 2), the NK cell membrane preparation method is as follows: culture NK cells, centrifuge at 1000 rpm for 3 minutes to collect the cells, resuspend them in a hypotonic sucrose solution, use a probe to sonicate, and break the NK cells under ice bath conditions for 5 minutes, working for 2 seconds and stopping for 5 seconds during sonication, and then ice bath; then centrifuge at 3000 g to remove unbroken cells and organelles, collect the supernatant, and then centrifuge at 14000 g to collect the NK cell membrane.

7. The preparation method according to claim 3, characterized in that, In step 2), the ultrasonic preparation method for ZnO-PDA is as follows: before use, ultrasonic dispersion is performed, ultrasonication is performed using a probe at a power of 100 W for 5 minutes, with a 1-second interval between working and 1-second pauses during ultrasonication, followed by an ice bath.

8. The use of the biomimetic piezoelectric nanoparticles as described in claim 1 in the preparation of a drug for treating depression.

9. The application according to claim 8, characterized in that, The drug is used in combination with brain ultrasound therapy.

10. A pharmaceutical composition for treating depression, characterized in that, It comprises a therapeutically effective amount of the biomimetic piezoelectric nanoparticles as described in claim 1, and at least one pharmaceutically acceptable carrier.

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