Ultrasonic response ZnS-coated Lf piezoelectric nanoparticles and preparation method and application thereof

Abstract

The invention discloses ultrasonic response ZnS (at) Lf piezoelectric nanoparticles as well as a preparation method and application thereof, and relates to the technical field of piezoelectric materials. The ultrasonic response ZnS (at) Lf piezoelectric nano-particle comprises a ZnS nano-particle and lactoferrin loaded on the surface of the ZnS nano-particle. According to the invention, lactoferrin is used as a potent brain-targeting ligand, and passes through a blood-brain barrier to be accumulated in brain parenchyma under the cell transfer action of brain capillary endothelial cells. ZnS nano particles release charges under the ultrasonic action, an electric signal activates a voltage-gated calcium ion channel and extracellular calcium ion influx, calcium ion / calmodulin kinase induces tyrosine hydroxylase (TH) phosphorylation, the activity of tyrosine hydroxylase is enhanced, tyrosine hydroxylase is a key enzyme for generating dopamine, and the activity of the tyrosine hydroxylase is enhanced. Therefore, more tyrosine in vivo is converted into neurotransmitter dopamine under the action of the enzyme. Finally, the regeneration of dopaminergic neurons is realized, so that the Parkinson's disease is effectively improved.

Description

Technical Field

[0001] This invention relates to the field of piezoelectric materials technology, and more specifically, to an ultrasonically responsive ZnS@Lf piezoelectric nanoparticle, its preparation method, and its application. Background Technology

[0002] Population aging is inevitable, leading to an increased prevalence of neurodegenerative diseases, including Parkinson's disease. Generally, Parkinson's disease commonly affects people aged 50-60, with a very small percentage developing it before age 40; the prevalence is projected to rise by 50% by 2030. In 1817, James Parkinson first described Parkinson's disease as a condition characterized by motor disorders and various non-motor impairments, such as cognitive and autonomic dysfunction. From a pathogenesis perspective, Parkinson's disease is typically characterized by the loss of dopaminergic neurons and the abnormal accumulation of α-synuclein in the substantia nigra and striatum. The development of Parkinson's disease is influenced by multiple factors, such as genetic susceptibility, neuroinflammation, oxidative stress, and calcium homeostasis.

[0003] The occurrence of Parkinson's disease is closely related to the degeneration and death of dopaminergic neurons in the substantia nigra of the midbrain. The significant loss of these neurons directly leads to a substantial decrease in dopamine levels in their projection target area—the striatum. The production and release of dopamine requires a series of ordered processes. First, neurons directly or indirectly obtain tyrosine from food. This tyrosine is catalyzed by tyrosine hydroxylase within the neuron to generate L-DOPA. L-DOPA is then decarboxylated by dopa decarboxylase to convert into dopamine. The newly generated dopamine is then transported to synaptic vesicles at the axonal terminals of dopaminergic neurons for storage. When release is needed, this process depends on calcium... 2+ Regulation: When the cell membrane depolarizes, it triggers the opening of voltage-gated calcium ion channels on the membrane, leading to the release of calcium ions. 2+ Influx of calcium into the cell down its concentration gradient, when intracellular calcium... 2+ When the concentration rises to a certain threshold, Ca2+ will be activated. 2+ The dependent rapid regulation of vesicle exocytosis promotes the fusion of synaptic vesicles with the cell membrane and releases the dopamine stored therein.

[0004] Piezoelectric materials are a subset of inorganic and organic dielectric compounds that become electrically polarized when mechanically stimulated and vice versa, deforming when subjected to an electric field. Nanoscale piezoelectric materials have numerous applications in biomedicine; their small size allows them to travel to different regions of the organism and cross physiological barriers such as cell membranes and blood vessels. Different types of mechanical energy can be used to activate piezoelectric nanomaterials, with ultrasonic activation being the most widely used. This generates numerous cavitation bubbles and provides periodic mechanical pressure to dispersed nanoparticles. Therefore, ultrasonically activated piezoelectric nanomaterials have found widespread application in various wireless therapies, encompassing applications such as biosensing, bacterial disinfection, neuronal cell stimulation, anti-cancer effects, combating neurodegenerative diseases, regenerative drugs, and tissue engineering.

[0005] Currently, the common treatment for Parkinson's disease (PD) involves supplementing patients with dopamine or administering dopamine receptor agonists in an attempt to restore dopamine levels in the brain to normal. The current standard treatment is oral levodopa. As a precursor to dopamine, levodopa can cross the blood-brain barrier and enter the brain's remaining dopamine neurons, where it is catalyzed by dopamine decarboxylase to synthesize dopamine, which is then released from the nerve cells. Levodopa is effective because PD patients still have residual dopamine neurons in their brains. However, as the disease progresses, the number of these neurons decreases, and the drug's effectiveness gradually diminishes. Increasing the dosage can produce significant side effects. Therefore, levodopa only improves symptoms and alleviates suffering; it does not protect or replenish damaged dopamine neurons in the brain. Furthermore, long-term use can cause numerous serious side effects. Thus, while medication and neurotrophic factor injections can relieve symptoms, they cannot stop the progression of the disease, nor can they regenerate damaged neurons, and therefore cannot fundamentally treat PD.

[0006] In recent years, deep brain stimulation (DBS), an emerging therapy with advantages such as simple operation, safety, non-invasiveness, and no adverse reactions, has significantly alleviated pain in patients with Parkinson's disease (PD). It improves PD symptoms by implanting electrodes into the subthalamic nucleus and delivering electrical signals to stimulate it. Currently, PD patients who have received DBS treatment have experienced significant symptom relief, and their medication dosage has also decreased. While DBS can alleviate symptoms, it cannot treat damaged cells and carries the risks of invasive surgery. Long-term use can lead to tolerance, side effects, and other problems, and the medical costs are also relatively high.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The purpose of this invention is to provide an ultrasound-responsive ZnS@Lf piezoelectric nanoparticle, its preparation method, and its application. By activating voltage-gated calcium ion channels under ultrasound, the production of the neurotransmitter dopamine is promoted, thereby effectively treating Parkinson's disease. The combination of ultrasound and piezoelectric nanoparticles enables wireless and non-invasive neuronal electrical stimulation.

[0009] This invention is implemented as follows: In a first aspect, the present invention provides an ultrasonically responsive ZnS@Lf piezoelectric nanoparticle, comprising ZnS nanoparticles and lactoferrin loaded on the surface of the ZnS nanoparticles.

[0010] In an optional embodiment, the lactoferrin loading is 0.1-1 mg of lactoferrin per 1 mg of ZnS nanoparticles.

[0011] In an optional embodiment, the ZnS@Lf piezoelectric nanoparticles have a particle size of 180-200 nm; And / or, the ultrasonically responsive ZnS@Lf piezoelectric nanoparticles are suitable for ultrasonic power of 1.4-1.6 W / cm². 2 Under certain conditions, voltage-gated calcium ion channels are activated.

[0012] Secondly, the present invention provides a method for preparing ZnS@Lf piezoelectric nanoparticles as described in the foregoing embodiments, comprising: S1. Zinc chloride and glutathione are reacted under alkaline conditions to obtain ZnS nanoparticles; S2. The ZnS nanoparticles are mixed and reacted with polyethylene glycol dicarboxylic acid to obtain ZnS-COOH; S3. Add EDC and NHS to the ZnS-COOH dispersion, stir, then add lactoferrin, and continue stirring to obtain ZnS@Lf piezoelectric nanoparticles.

[0013] In an optional embodiment, the mass ratio of zinc chloride to glutathione is 1:3-4; And / or, before mixing the zinc chloride with the glutathione, the zinc chloride is first dissolved in a first deionized water and stirred for 20-40 minutes to dissolve it, wherein the solid-liquid ratio of the zinc chloride to the first deionized water is 1 mg: 0.2-0.3 mL; And / or, the alkaline conditions include adjusting the pH of the system to 11-13 using ammonia; And / or, the reaction temperature of the zinc chloride and the glutathione is 140-200℃, and the reaction time is 8-12h.

[0014] In an optional embodiment, the mass ratio of the ZnS nanoparticles to the polyethylene glycol dicarboxylic acid is 10-50 mg: 80-120 mg; And / or, before mixing the ZnS nanoparticles with the polyethylene glycol dicarboxylic acid, the ZnS nanoparticles are first dispersed in a second deionized water, and ammonia is added dropwise while stirring. The solid-liquid ratio of the ZnS nanoparticles, the second deionized water and the ammonia is 5-20 mg: 20-30 mL: 4-6 mL. And / or, the reaction temperature of the ZnS nanoparticles with the polyethylene glycol dicarboxylic acid is 60-70°C, and the mixing time is 2-4 hours; And / or, after the reaction between the ZnS nanoparticles and the polyethylene glycol dicarboxylic acid is completed, the ZnS nanoparticles are washed with water until neutral, and the ZnS-COOH is collected by centrifugation.

[0015] In an optional embodiment, the concentration of ZnS-COOH in the ZnS-COOH dispersion is 0.8-1.2 mg / mL; And / or, the solid-liquid ratio of the dispersion of the EDC, the NHS, the lactoferrin and the ZnS-COOH is 16-24 mg: 16-24 mg: 8-12 mg: 5-10 mL.

[0016] In an optional embodiment, after the reaction in steps S1 and S3 is completed, solid-liquid separation is further included, and the precipitate is washed. Preferably, the solid-liquid separation includes centrifugation at 8000-12000 rpm for 8-12 min; The washing process includes washing with anhydrous ethanol and pure water 2-4 times in sequence; Thirdly, the present invention provides the use of ZnS@Lf piezoelectric nanoparticles as described in any of the foregoing embodiments, or ZnS@Lf piezoelectric nanoparticles prepared by the preparation method of ZnS@Lf piezoelectric nanoparticles as described in any of the foregoing embodiments, in the preparation of medicaments for treating or improving diseases caused by low expression of voltage-gated calcium ion channels.

[0017] In an alternative implementation, the disease includes Parkinson's disease.

[0018] The present invention has the following beneficial effects: The ultrasound-responsive ZnS@Lf piezoelectric nanoparticles provided by this invention utilize ZnS nanoparticles as active particles to activate voltage-gated calcium ion channels, and load lactoferrin (Lf) with targeting activity onto their surface. Lf, as a potent brain-targeting ligand, crosses the blood-brain barrier and accumulates in the brain parenchyma via transcellularity of brain capillary endothelial cells. Under ultrasound, the ZnS nanoparticles release charge, and the electrical signal activates the voltage-gated calcium ion channels, leading to an influx of extracellular calcium ions. Calcium ion / calmodulin kinase induces phosphorylation of tyrosine hydroxylase (TH), enhancing its activity. Tyrosine hydroxylase is a key enzyme in the production of dopamine; therefore, more tyrosine in the body is converted into the neurotransmitter dopamine under the action of this enzyme. Ultimately, this leads to the regeneration of dopaminergic neurons, effectively improving Parkinson's disease. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 A schematic diagram illustrating the preparation method of ultrasonically responsive ZnS@Lf piezoelectric nanoparticles provided by this invention; Figure 2 This is a transmission electron microscope image of the ZnS nanoparticles prepared in Example 1 of the present invention; Figure 3 This is a particle size distribution diagram of the ZnS nanoparticles prepared in Example 1 of the present invention; Figure 4 This is a transmission electron microscope image of the ultrasonically responsive ZnS@Lf piezoelectric nanoparticles prepared in Example 1 of this invention. Figure 5 This is a particle size distribution diagram of the ultrasonically responsive ZnS@Lf piezoelectric nanoparticles prepared in Example 1 of the present invention; Figure 6 The elemental distribution diagram of the ultrasonically responsive ZnS@Lf piezoelectric nanoparticles prepared in Example 1 of this invention is shown. Figure 7 The XPS spectrum of the ultrasonically responsive ZnS@Lf piezoelectric nanoparticles prepared in Example 1 of this invention is shown below. Figure 8 The image shows the FTIR spectrum of the ultrasonically responsive ZnS@Lf piezoelectric nanoparticles prepared in Example 1 of this invention. Figure 9This is a schematic diagram of the piezoelectric response force microscope of the ultrasonically responsive ZnS@Lf piezoelectric nanoparticles in Experimental Example 2 of the present invention, where a is the amplitude image, b is the phase image, c is the butterfly amplitude curve, and d is the phase hysteresis loop. Figure 10 The Ca2+ cells of SH-SY5Y cells in different treatment groups in Experimental Example 3 of this invention 2+ Inflow fluorescence images; Figure 11 This is a diagram showing the open field trajectory of mice in different treatment groups in Experiment Example 4 of this invention; Figure 12 These are immunofluorescence images of the substantia nigra region of mice in different treatment groups in Experimental Example 4 of this invention; Figure 13 These are in vivo fluorescence images of different treatment groups in Experimental Example 5 of this invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0022] This invention provides an ultrasonically responsive ZnS@Lf piezoelectric nanoparticle, which comprises ZnS nanoparticles and lactoferrin loaded on the surface of the ZnS nanoparticles.

[0023] The lactoferrin loading is 0.1-1 mg lactoferrin per 1 mg ZnS nanoparticles.

[0024] The particle size of ZnS@Lf piezoelectric nanoparticles is 180-200 nm; And / or, the ultrasonically responsive ZnS@Lf piezoelectric nanoparticles are available at ultrasonic power of 1.4-1.6 W / cm². 2 Under certain conditions, voltage-gated calcium ion channels are activated.

[0025] The ultrasound-responsive ZnS@Lf piezoelectric nanoparticles provided in this invention can promote dopamine production by activating voltage-gated calcium ion channels, thereby restoring degenerated dopaminergic neurons. Lactoferrin (Lf) acts as a potent brain-targeting ligand, accumulating in the brain parenchyma via transcellularity of brain capillary endothelial cells across the blood-brain barrier. Under ultrasound, the nanoparticles release charge, and the electrical signal activates the voltage-gated calcium ion channels, causing extracellular calcium ion influx. Calcium ion / calmodulin kinase induces phosphorylation of tyrosine hydroxylase (TH), enhancing its activity. Tyrosine hydroxylase is a key enzyme in dopamine production; therefore, more tyrosine in the body is converted into the neurotransmitter dopamine under the action of this enzyme. Ultimately, this leads to the regeneration of dopaminergic neurons, effectively improving Parkinson's disease.

[0026] This invention also provides a method for preparing the above-mentioned ZnS@Lf piezoelectric nanoparticles, which includes the following steps: S1. Zinc chloride and glutathione are reacted under alkaline conditions to obtain ZnS nanoparticles; Before mixing zinc chloride with glutathione, zinc chloride is first dissolved in deionized water and stirred for 20-40 minutes until dissolved. The solid-liquid ratio of zinc chloride to deionized water is 1 mg: 0.2-0.3 mL; the mass ratio of zinc chloride to glutathione is 1:3-4; the reaction temperature of alkaline zinc chloride and glutathione is 140-200℃, and the reaction time is 8-12 h. Conditions include adjusting the pH of the system to 11-13 using ammonia.

[0027] Glutathione (GSH) is a tripeptide containing a thiol (-SH), an amino (-NH2), and a carboxyl (-COOH) group, with the structural formula γ-glutamyl-cysteyl-glycine. The sulfur atom in the thiol group may be released under alkaline conditions, serving as a sulfur source. In an alkaline environment with pH = 11-13, the thiol group of glutathione may deprotonate, forming a sulfide anion (S). 2- ), and then with Zn 2+ The reaction produces ZnS precipitate. By mixing zinc chloride and glutathione in a specific mass ratio and reacting under alkaline conditions, and by heating during the reaction, the reaction efficiency of zinc chloride and glutathione can be improved, thus promoting the formation of ZnS precipitate from zinc chloride. 2+ It reacts with the thiol groups in glutathione to generate ZnS nanoparticles.

[0028] After the reaction, solid-liquid separation is performed, followed by washing of the precipitate. Various methods of solid-liquid separation are available, including but not limited to centrifugation, pressure filtration, and filtration. In this invention, centrifugation is selected for solid-liquid separation. The centrifugation speed can be, for example, 8000-12000 rpm, and the centrifugation time can be, for example, 8-12 min. Centrifugation effectively achieves solid-liquid separation, thereby separating the reaction product, ZnS nanoparticles. Subsequent washing removes residual solvent from the surface of the ZnS nanoparticles. Washing includes sequential washing with anhydrous ethanol and pure water 2-4 times.

[0029] S2. Mix ZnS nanoparticles with polyethylene glycol dicarboxylic acid and react to obtain ZnS-COOH; Before mixing ZnS nanoparticles with polyethylene glycol dicarboxylic acid, ZnS nanoparticles are first dispersed in a second deionized water, and ammonia is added dropwise while stirring. The solid-liquid ratio of ZnS nanoparticles, second deionized water, and ammonia is 5-20 mg: 20-30 mL: 4-6 mL. The addition of ammonia helps maintain an alkaline environment in the system, which is beneficial for maintaining the stability of ZnS nanoparticles.

[0030] By mixing ZnS nanoparticles with polyethylene glycol dicarboxylic acid (PEG-COOH), which provides the -COOH group, a foundation is laid for subsequent lactoferrin loading. In this invention, the mass ratio of ZnS nanoparticles to PEG-COOH is 10-50 mg: 80-120 mg; the reaction temperature is 60-70°C, and the mixing time is 2-4 h. After the reaction is complete, the ZnS nanoparticles are washed with water until neutral, and the ZnS-COOH group is collected by centrifugation.

[0031] S3. Add EDC and NHS to the dispersion of ZnS-COOH, stir, then add lactoferrin, and continue stirring to obtain ZnS@Lf piezoelectric nanoparticles.

[0032] The concentration of ZnS-COOH in the ZnS-COOH dispersion was 0.8-1.2 mg / mL; the solid-liquid ratio of the dispersions of EDC, NHS, lactoferrin, and ZnS-COOH was 16-24 mg: 16-24 mg: 8-12 mg: 5-10 mL. EDC and NHS can activate -COOH, promoting the reaction between ZnS-COOH and lactoferrin and loading lactoferrin onto the surface of ZnS.

[0033] After the reaction, solid-liquid separation is performed, followed by washing of the precipitate. Various methods of solid-liquid separation are available, including but not limited to centrifugation, pressure filtration, and filtration. In this invention, centrifugation is selected for solid-liquid separation. The centrifugation speed can be, for example, 8000-12000 rpm, and the centrifugation time can be, for example, 8-12 min. Centrifugation effectively achieves solid-liquid separation, thereby separating the reaction product, ZnS@Lf piezoelectric nanoparticles. Subsequent washing removes residual solvent from the surface of the ZnS@Lf piezoelectric nanoparticles. Washing includes sequential washing with anhydrous ethanol and pure water 2-4 times.

[0034] Furthermore, this invention also provides the use of the aforementioned ZnS@Lf piezoelectric nanoparticles in the preparation of medicaments for treating or improving diseases caused by low expression of voltage-gated calcium ion channels. These diseases include Parkinson's disease.

[0035] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0036] Example 1 This embodiment provides an ultrasonically responsive ZnS@Lf piezoelectric nanoparticle; please refer to the preparation method for details. Figure 1 Specifically, it includes the following steps: 148 mg of ZnCl2 was dissolved in 35 mL of deionized water and stirred for 30 minutes. Then, 500 mg of glutathione was added while stirring. 25% ammonia solution was added dropwise to the reaction mixture until the pH reached 12. Finally, the resulting solution was heated to 160 °C in a 100 mL PTFE-lined autoclave. After 10 h, the solution was cooled to room temperature and centrifuged at 10,000 rpm for 10 minutes. The precipitate was collected and washed three times each with anhydrous ethanol and ultrapure water to prepare ZnS nanoparticles.

[0037] 15 mg of the above solid product was dispersed in 25 mL of water, followed by the addition of 5 mL of NH3·H2O. After stirring for 10 min, 100 mg of polyethylene glycol dicarboxylic acid was added, and the mixture was then stirred at 65 °C for 3 h. After the reaction, the mixture was thoroughly washed with water until neutral pH was reached, and ZnS-COOH was collected by centrifugation.

[0038] 20 mg EDC and 20 mg NHS were added to 10 mL of ZnS-COOH dispersion (ZnS-COOH concentration in the dispersion was 1 mg / mL), and stirred at room temperature for 2 h. Then 10 mg lactoferrin was added, and the mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 10 minutes, the precipitate was collected, and washed three times to prepare ultrasonically responsive ZnS@Lf piezoelectric nanoparticles.

[0039] Example 2 This embodiment provides an ultrasonically responsive ZnS@Lf piezoelectric nanoparticle; please refer to the preparation method for details. Figure 1 Specifically, it includes the following steps: 148 mg of ZnCl2 was dissolved in 30 mL of deionized water and stirred for 40 minutes. Then, 445 mg of glutathione was added while stirring. 25% ammonia solution was added dropwise to the reaction mixture until the pH reached 11. Finally, the resulting solution was heated to 140 °C in a 100 mL PTFE-lined autoclave. After 12 h, the solution was cooled to room temperature and centrifuged at 8000 rpm for 12 minutes. The precipitate was collected and washed three times each with anhydrous ethanol and ultrapure water to prepare ZnS nanoparticles.

[0040] 10 mg of the above solid product was dispersed in 20 mL of water, followed by the addition of 4 mL of NH3·H2O. After stirring for 10 min, 80 mg of polyethylene glycol dicarboxylic acid was added, and the mixture was then stirred at 60 °C for 4 h. After the mixture was stirred, it was thoroughly washed with water until neutral pH was reached, and ZnS-COOH was collected by centrifugation.

[0041] 16 mg EDC and 16 mg NHS were added to 5 mL of ZnS-COOH dispersion (ZnS-COOH concentration in the dispersion was 0.8 mg / mL), and the mixture was stirred at room temperature for 2 h. Then, 8 mg lactoferrin was added, and the mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was centrifuged at 8000 rpm for 12 minutes, the precipitate was collected, and washed three times to prepare ultrasonically responsive ZnS@Lf piezoelectric nanoparticles.

[0042] Example 3 This embodiment provides an ultrasonically responsive ZnS@Lf piezoelectric nanoparticle; please refer to the preparation method for details. Figure 1 Specifically, it includes the following steps: 148 mg of ZnCl₂ was dissolved in 44 mL of deionized water and stirred for 20 minutes. Then, 592 mg of glutathione was added while stirring. 25% ammonia solution was added dropwise to the reaction mixture until the pH reached 13. Finally, the resulting solution was heated to 200 °C in a 100 mL PTFE-lined autoclave. After 8 h, the solution was cooled to room temperature and centrifuged at 12000 rpm for 8 minutes. The precipitate was collected and washed three times each with anhydrous ethanol and ultrapure water to prepare ZnS nanoparticles.

[0043] 50 mg of the above solid product was dispersed in 30 mL of water, followed by the addition of 6 mL of NH3·H2O. After stirring for 10 min, 120 mg of polyethylene glycol dicarboxylic acid was added, and the mixture was then stirred at 70 °C for 2 h. After the mixture was stirred, it was thoroughly washed with water until neutral pH was reached, and ZnS-COOH was collected by centrifugation.

[0044] 24 mg EDC and 24 mg NHS were added to 10 mL of ZnS-COOH dispersion (ZnS-COOH concentration in the dispersion was 1.2 mg / mL), and stirred at room temperature for 2 h. Then 12 mg lactoferrin was added, and the mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was centrifuged at 12000 rpm for 8 minutes, the precipitate was collected, and washed 3 times to prepare ultrasonically responsive ZnS@Lf piezoelectric nanoparticles.

[0045] Comparative Example 1 This comparative example provides a ZnS@BSA nanoparticle, the preparation method of which is basically the same as that of Example 1, except that the lactoferrin in Example 1 is replaced with BSA.

[0046] Experimental Example 1 The ZnS nanoparticles and the final product, ultrasonically responsive ZnS@Lf piezoelectric nanoparticles, obtained during the preparation process in Example 1 were characterized.

[0047] Please refer to the transmission electron microscope image of the ZnS nanoparticles. Figure 2 ,from Figure 2 As can be seen, the ZnS nanoparticles are uniform spheres with consistent morphology and well-dispersed size. Please refer to the particle size distribution diagram of the ZnS nanoparticles. Figure 3 ,from Figure 3 It can be seen that the average particle size of ZnS nanoparticles is about 180 nm.

[0048] Please refer to the transmission electron microscope image of the ultrasonically responsive ZnS@Lf piezoelectric nanoparticles. Figure 4 ,from Figure 4 As can be seen, a distinct film forms on the surface of the ZnS nanoparticles, indicating effective binding of lactoferrin. Please refer to the particle size distribution diagram of the ultrasonically responsive ZnS@Lf piezoelectric nanoparticles. Figure 5 ,from Figure 5 It can be seen that the particle size of ZnS@Lf is about 190 nm.

[0049] For further details, please refer to the elemental distribution diagram of the ultrasonically responsive ZnS@Lf piezoelectric nanoparticles. Figure 6 ,from Figure 6 It can be seen that Zn and S elements are uniformly distributed in the ZnS nanoparticles. Please refer to the XPS spectrum of the ultrasonic response ZnS@Lf piezoelectric nanoparticles. Figure 7 ,from Figure 7 It can be seen that Zn and S elements are indeed present, as well as the bond energies of Zn 2p and S 2p. For the FTIR spectra of the ultrasonically responsive ZnS@Lf piezoelectric nanoparticles, please refer to [link to FTIR spectra]. Figure 8 ,from Figure 8 It can be seen that 1640.44 cm -1 and 1544.05 cm -1 The bands are clearly visible, which belong to amide I and amide II bands in lactoferrin, further confirming the successful binding of lactoferrin to ZnS nanoparticles.

[0050] The above characterization demonstrates that Example 1 of the present invention effectively synthesized ultrasonically responsive ZnS@Lf piezoelectric nanoparticles.

[0051] Experimental Example 2: Evaluation of the piezoelectric response performance of ultrasonically responsive ZnS@Lf piezoelectric nanoparticles The piezoelectric behavior of nanoparticles was verified using piezoelectric force microscopy (PFM). The specific operation included: first, fixing the conductive sample and cleaning the surface; then, installing the conductive probe and calibrating the optical path and resonant frequency; parameter settings covering scanning mode (conventional / contact resonance / DART-PFM), electric field parameters (AC / DC voltage), and scanning range; applying an AC electric field to the sample through probe contact to excite the inverse piezoelectric effect; demodulating the amplitude and phase signals using a lock-in amplifier; and finally generating an amplitude / phase map.

[0052] Please refer to the test results. Figure 9 ,from Figure 9 It can be seen that in the amplitude diagram ( Figure 9 a) and phase diagram ( Figure 9 In diagram b), the polarization state of ZnS can be clearly observed, proving the piezoelectric response of ZnS and demonstrating that ZnS has different polarization directions; simultaneously, ZnS exhibits a typical butterfly amplitude curve (…). Figure 9 c) and phase hysteresis loop ( Figure 9 (d). The phase hysteresis loop confirms excellent local hysteresis response, and the 180° phase angle change confirms the polarization switching process.

[0053] Experiment Example 3 To verify whether ZnS@Lf nanoparticles can activate voltage-gated calcium ion channels under ultrasound, this invention divided SH-SY5Y cells (commercially purchased from Servicebio) into four groups (control group, US group, ZnS@Lf group, and ZnS@Lf+US group). In the control group, SH-SY5Y cells were cultured normally in DMEM medium, while in the US group, SH-SY5Y cells were subjected to ultrasound treatment at 1.5 W / cm². 2Ultrasound, ZnS@Lf group applied ZnS@Lf nanoparticles to SH-SY5Y cells, ZnS@Lf+US group applied ZnS@Lf nanoparticles to SH-SY5Y cells and added 1.5W / cm 2 Ultrasound. After 4 hours of treatment, staining was performed. Live cells were stained with a calcium ion fluorescent probe (Fluo-4 AM) to label intracellular free calcium. 2+ The cells initially exhibited green fluorescence; subsequently, Hoechst staining was used to label the cell nucleus, resulting in blue fluorescence. Single-channel images of the Fluo-4 channel (green) and the Hoechst channel (blue) were acquired separately. The two single-channel images were merged to obtain an overlay image of "green calcium signal + blue cell nucleus," allowing for clear observation of "the distribution of calcium signal within the cell (e.g., whether it is close to the cell nucleus)" and "the correlation between changes in calcium concentration and cell structure."

[0054] Please see Figure 10 The results showed that intracellular Ca2+ only decreased when cells were treated with nanoparticles under ultrasound. 2+ Only when the concentration increases will ultrasound or nanoparticles alone not change the intracellular Ca2+ concentration. 2+ concentration.

[0055] Experiment Example 4 This experimental case study investigated the feasibility of using ultrasound-stimulated nanoparticles to treat a mouse model of Parkinson's disease.

[0056] The specific experimental method included: 8-week-old male C57BL / 6 mice were intraperitoneally injected with MPTP to establish a Parkinson's disease model, with injections lasting for 5 consecutive days. All mice were randomly divided into (1) Control group; (2) MPTP group; (3) MPTP + US group; (4) MPTP + ZnS@Lf group; and (5) MPTP + ZnS@Lf + US group. Treatment conditions are detailed in the table below.

[0057] Please see Figure 11 Behavioral results from the open field experiment showed that mice in the MPTP+ZnS@Lf+US group were more active and covered a greater total distance. Please refer to [link / reference needed]. Figure 12 In terms of pathological features, this experimental case mainly focused on two indicators: α-synuclein (α-syn) and tyrosine hydroxylase (TH). Immunofluorescence results in the substantia nigra region showed that the expression of TH in the MPTP+ZnS@Lf+US group was increased compared with other groups, while the expression of α-syn was decreased.

[0058] Therefore, the ultrasound-responsive ZnS@Lf piezoelectric nanoparticles constructed in this application can regenerate dopaminergic neurons and have good therapeutic application value for Parkinson's disease.

[0059] Experimental Example 5 To investigate the brain-targeting capabilities of ZnS@Lf provided in Example 1 and ZnS@BSA provided in Comparative Example 1, Cy5-labeled ZnS@Lf and ZnS@BSA were injected into mice via the tail vein. Fluorescence imaging was performed at 0, 3, 6, 9, 12, and 24 hours.

[0060] from Figure 13 The provided in vivo fluorescence images showed that ZnS@Lf-Cy5 reached its peak brain fluorescence at 6 hours and still accumulated significantly in the brain at 24 hours. This is attributed to Lf's brain-targeting ability and blood-brain barrier (BBB) ​​permeability. However, since BSA lacks this ability, ZnS@BSA lacks brain-targeting ability and blood-brain barrier (BBB) ​​permeability, resulting in weak fluorescence signals for ZnS@BSA-Cy5, which cannot achieve effective accumulation in the brain parenchyma and is not conducive to subsequent treatment.

[0061] In summary, the ultrasound-responsive ZnS@Lf piezoelectric nanoparticles provided by this invention utilize ZnS nanoparticles as active particles to activate voltage-gated calcium ion channels, and load their surface with lactoferrin (Lf), which has a targeting effect. Lf, as a potent brain-targeting ligand, crosses the blood-brain barrier and accumulates in the brain parenchyma through transcellularity of brain capillary endothelial cells. Under ultrasound, the ZnS nanoparticles release charge, and the electrical signal activates the voltage-gated calcium ion channels, causing extracellular calcium ion influx. Calcium ion / calmodulin kinase induces phosphorylation of tyrosine hydroxylase (TH), enhancing its activity. Tyrosine hydroxylase is a key enzyme in the production of dopamine; therefore, more tyrosine in the body is converted into the neurotransmitter dopamine under the action of this enzyme. Ultimately, this leads to the regeneration of dopaminergic neurons, effectively improving Parkinson's disease.

[0062] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An ultrasonically responsive ZnS@Lf piezoelectric nanoparticle, characterized in that, It includes ZnS nanoparticles and lactoferrin loaded on the surface of the ZnS nanoparticles.

2. The ZnS@Lf piezoelectric nanoparticles according to claim 1, characterized in that, The lactoferrin loading is 0.1-1 mg of lactoferrin per 1 mg of ZnS nanoparticles.

3. The ZnS@Lf piezoelectric nanoparticles according to claim 1, characterized in that, The ZnS@Lf piezoelectric nanoparticles have a particle size of 180-200 nm; And / or, the ultrasonically responsive ZnS@Lf piezoelectric nanoparticles are suitable for ultrasonic power of 1.4-1.6 W / cm². 2 Under certain conditions, voltage-gated calcium ion channels are activated.

4. A method for preparing ZnS@Lf piezoelectric nanoparticles as described in claim 1, characterized in that, It includes: S1. Zinc chloride and glutathione are reacted under alkaline conditions to obtain ZnS nanoparticles; S2. The ZnS nanoparticles are mixed and reacted with polyethylene glycol dicarboxylic acid to obtain ZnS-COOH; S3. Add EDC and NHS to the ZnS-COOH dispersion, stir, then add lactoferrin, and continue stirring to obtain ZnS@Lf piezoelectric nanoparticles.

5. The method for preparing ZnS@Lf piezoelectric nanoparticles according to claim 4, characterized in that, The mass ratio of zinc chloride to glutathione is 1:3-4; And / or, before mixing the zinc chloride with the glutathione, the zinc chloride is first dissolved in a first deionized water and stirred for 20-40 minutes to dissolve it, wherein the solid-liquid ratio of the zinc chloride to the first deionized water is 1 mg: 0.2-0.3 mL; And / or, the alkaline conditions include adjusting the pH of the system to 11-13 using ammonia; And / or, the reaction temperature of the zinc chloride and the glutathione is 140-200℃, and the reaction time is 8-12h.

6. The method for preparing ZnS@Lf piezoelectric nanoparticles according to claim 4, characterized in that, The mass ratio of the ZnS nanoparticles to the polyethylene glycol dicarboxylic acid is 10-50 mg: 80-120 mg; And / or, before mixing the ZnS nanoparticles with the polyethylene glycol dicarboxylic acid, the ZnS nanoparticles are first dispersed in a second deionized water, and ammonia is added dropwise while stirring. The solid-liquid ratio of the ZnS nanoparticles, the second deionized water and the ammonia is 5-20 mg: 20-30 mL: 4-6 mL. And / or, the reaction temperature of the ZnS nanoparticles with the polyethylene glycol dicarboxylic acid is 60-70°C, and the mixing time is 2-4 hours; And / or, after the reaction between the ZnS nanoparticles and the polyethylene glycol dicarboxylic acid is completed, the ZnS nanoparticles are washed with water until neutral, and the ZnS-COOH is collected by centrifugation.

7. The method for preparing ZnS@Lf piezoelectric nanoparticles according to claim 4, characterized in that, The concentration of ZnS-COOH in the ZnS-COOH dispersion is 0.8-1.2 mg / mL; And / or, the solid-liquid ratio of the dispersion of the EDC, the NHS, the lactoferrin and the ZnS-COOH is 16-24 mg: 16-24 mg: 8-12 mg: 5-10 mL.

8. The method for preparing ZnS@Lf piezoelectric nanoparticles according to claim 4, characterized in that, After the reactions in steps S1 and S3 are completed, solid-liquid separation is also performed, and the precipitate is washed. Preferably, the solid-liquid separation includes centrifugation at 8000-12000 rpm for 8-12 min; The washing process includes washing with anhydrous ethanol and pure water 2-4 times in sequence.

9. The use of ZnS@Lf piezoelectric nanoparticles prepared by any one of claims 1-3 or by any one of claims 4-8 in the preparation of medicaments for treating or improving diseases caused by low expression of voltage-gated calcium ion channels.

10. The application according to claim 9, characterized in that, The diseases mentioned include Parkinson's disease.

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