MicroRNA-loaded zinc-based nano organic framework coating material and application thereof

Abstract

The invention discloses a microRNA-loaded zinc-based nano organic framework coating material and application thereof, and belongs to the technical field of biological medicines. The zinc-based nano organic framework coating material loaded with the microRNA comprises a zinc-based nano organic framework material, the microRNA-34 is loaded on the zinc-based nano organic framework material, and the microRNA-34 is loaded on the zinc- The surface of the zinc-based nano organic framework material is coated with cell membranes of neural stem cells. According to the invention, the neural stem cells endocytosis miR-34 (at) ZIF-8-SCM so as to induce the neural stem cells to differentiate into neurons; the cell membrane of the neural stem cell not only has homology with the neural stem cell, but also improves the speed of releasing the microRNA-34 by the miR-34 ZIF-8-SCM, and further improves the speed of differentiating the neural stem cell into neurons.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a zinc-based nano-organic framework coating material loaded with microRNA and an application thereof. Background Art

[0002] Parkinson's disease has become the most common disease that threatens human health worldwide and causes huge economic losses to society. The incidence of Parkinson's disease is increasing year by year, and the main cause of the disease is the degeneration and death of dopaminergic neurons. Currently, a series of stem cells from various sources are used to produce dopamine in in vitro experiments. However, the translation of these efforts in vivo currently lacks effectiveness and sustainability. Parkinson's disease is caused by the loss of dopamine neurons, so it is necessary to develop an effective strategy to combine neural stem cell (NSCs) therapy to guide the directional differentiation of NSCs into functional neurons to treat Parkinson's disease.

[0003] Zn 2＋ It is one of the trace elements with the highest content in the mammalian brain and has been shown to play a positive role in the differentiation of neural stem cells. For example, "Effects of zinc on the proliferation and differentiation of rat bone marrow mesenchymal stem cells into neural-like cells" (Yan Jun et al., 2016, DOI: CNKI: SUN: ZSZD.0.2016-07-001), the patented metal-organic ligand framework ZIF-67 modified hollow vanadium dioxide core-membrane composite structure drug carrier application number CN202111528077.2, etc. have reported different forms of Zn 2＋ Inducing cell differentiation, but single Zn 2＋ The effect of inducing cell differentiation is not significant and needs to be compounded with other materials. miR-34 is one of the most abundant miRNAs in the human brain nervous system. It is currently mainly used for the treatment of cancer or tumors. However, miR-34 has a polyanionic charge and is difficult to internalize through the cell membrane. It has a short half-life in the systemic circulation and is sensitive to nuclease degradation. These characteristics hinder the widespread clinical application of miR-34-based treatments. Metal-organic frameworks (MOFs) are assembled from metal nodes and organic connectors. They are a typical class of crystalline porous materials with high specific surface area, large porosity, customizable size and structure, chemical stability and biodegradability. Although metal-organic framework materials such as ZIF can be used to load miR-34, ZIF is a porous polymer. After physically adsorbing miR-34, it is difficult to release it quickly. At present, there are few reports on the use of miR-34 to induce neural stem cells to differentiate into neurons, and there are no reports on the use of miR-34 in the treatment of Parkinson's disease. Therefore, a zinc-based nano-organic frame material loaded with microRNA is needed, which can quickly pass through the cell membrane and quickly release miR-34. 2＋It works together with miR-34 to promote the differentiation of neural stem cells, in the hope of being used in the treatment of Parkinson's disease in the future. Summary of the invention

[0004] In view of the above-mentioned prior art, the purpose of the present invention is to provide a zinc-based nano-organic frame coating material loaded with microRNA and its application. In the present invention, microRNA-34 is loaded into ZIF-8, and then the cell membrane of neural stem cells is encapsulated to obtain miR-34@ZIF-8-SCM; neural stem cells internalize miR-34@ZIF-8-SCM to induce neural stem cells to differentiate into neurons; the cell membrane of neural stem cells not only has homology with neural stem cells, but also increases the speed of miR-34@ZIF-8-SCM releasing microRNA-34, further increasing the speed of neural stem cells to differentiate into neurons.

[0005] To achieve the above object, the present invention adopts the following technical solution: The first aspect of the present invention provides a zinc-based nano-organic framework coating material loaded with microRNA, wherein the zinc-based nano-organic framework coating material loaded with microRNA comprises a zinc-based nano-organic framework material; the zinc-based nano-organic framework material is loaded with microRNA, wherein the microRNA is microRNA-34; and the surface of the zinc-based nano-organic framework material is coated with a cell membrane, wherein the cell membrane is derived from a neural stem cell.

[0006] Preferably, the microRNA-loaded zinc-based nano-organic framework coating material is prepared by the following method: (1) Disperse ZIF-8 nanoparticles in PBS solution, add microRNA-34 and mix well, keep at low temperature overnight, then centrifuge and dry to obtain miR-34@ZIF-8; (2) Neural stem cells were lysed with hypotonic lysis buffer at low temperature, the lysed solution was centrifuged once at low temperature, the supernatant was centrifuged twice at low temperature, the precipitate was collected to obtain the cell membrane of the neural stem cell membrane, the cell membrane was added to the PBS solution of miR-34@ZIF-8, ultrasonically treated, and co-extruded to obtain miR-34@ZIF-8-SCM, which is a zinc-based nano-organic framework coating material loaded with microRNA.

[0007] Preferably, in step (1), the weight ratio of microRNA-34 to ZIF-8 is 1:40.

[0008] Preferably, in step (1), the temperature of the low temperature overnight is 4° C.; and the speed of the centrifugation is 3000 rpm.

[0009] Preferably, in step (2), the low temperature is 4° C. and the lysis time is 4 h.

[0010] Preferably, in step (2), the first low-temperature centrifugation is performed at 3000 rpm at 4°C for 5 min; and the second low-temperature centrifugation is performed at 15000 rpm at 4°C for 30 min.

[0011] Preferably, in step (2), the power of the ultrasonic treatment is 40 W, the temperature is 4° C., and the time is 8 min; and the co-extrusion is performed by using a micro co-extruder to sequentially pass through polycarbonate membranes with pore sizes of 400 nm and 200 nm.

[0012] The second aspect of the present invention provides the use of a zinc-based nano-organic framework coating material loaded with microRNA in the preparation of a drug for inducing neural stem cells to develop into neurons.

[0013] Preferably, the concentration of the zinc-based nano-organic framework coating material loaded with microRNA is 25 µg / mL.

[0014] The third aspect of the present invention provides the use of a cell membrane of a neural stem cell in increasing the rate of release of microRNA-34 from a zinc-based nano-organic framework coating material loaded with microRNA.

[0015] Beneficial effects of the present invention: (1) The present invention loads microRNA-34 into ZIF-8, and then encapsulates the cell membrane of neural stem cells to obtain miR-34@ZIF-8-SCM; the neural stem cells internalize miR-34@ZIF-8-SCM to induce the neural stem cells to differentiate into neurons; the cell membrane of the neural stem cells not only has homology with the neural stem cells, but also increases the speed of miR-34@ZIF-8-SCM releasing microRNA-34, thereby further increasing the speed of neural stem cells differentiating into neurons.

[0016] (2) The miR-34@ZIF-8-SCM prepared by the present invention has high biocompatibility and strong targeting ability, and can regulate the differentiation of neural stem cells in a targeted manner. The problem of easy degradation of miR-34 in clinical application is solved, and ZIF-8 is used as a transport carrier of miR-34 to protect miR-34 from degradation by nucleases. At the same time, the surface of miR-34@ZIF-8 is coated with a biofilm to solve the cell's rejection reaction to miR-34@ZIF-8, a foreign substance, and simulate natural molecules to improve the biocompatibility and targeting ability of NSCs. The miR-34@ZIF-8-SCM nanoparticles improve the delivery and internalization of miR-34, enabling miR-34 to promote the differentiation of NSCs into mature neurons with electrophysiological functions within 5 days in vitro.

[0017] (3) The miR-34@ZIF-8-SCM nanoparticles of the present invention are expected to enhance the therapeutic effect of NSCs therapy on Parkinson's disease and improve the application of miR-34 and NSCs therapy in the treatment of traumatic nerve injury and neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :(a)Scanning electron microscopy of ZIF-8,(b)Transmission electron microscopy of ZIF-8; Figure 2 : X-ray diffraction pattern of ZIF-8; Figure 3 :(a) Agarose gel electrophoresis to explore the loading capacity of ZIF-8 on miR-34;(b) Analysis of agarose gel electrophoresis results of the loading capacity of ZIF-8 on miR-34; Figure 4 : (a) Transmission electron microscopy image of cell membrane, (b) transmission electron microscopy image of miR-34@ZIF-8-SCM; Figure 5 : (a) Nitrogen adsorption isotherms of ZIF-8 and miR-34@ZIF-8 and (b) pore size distribution of ZIF-8 and miR-34@ZIF-8; Figure 6 :(a) Zeta potential of ZIF-8, miR-34@ZIF-8, and miR-34@ZIF-8-SCM;(b) Fourier transform spectra of ZIF-8, miR-34@ZIF-8, and miR-34@ZIF-8-SCM; Figure 7 : Protection of miR-34 by miR-34@ZIF-8-SCM; Figure 8 :Effects of different concentrations of miR-34@ZIF-8-SCM on the activity of neural stem cells; Fig. 9 :(a) Confocal images of miR-34@ZIF-8 nanoparticles incubated in neural stem cells for 30 minutes and 5 hours, (b) Confocal images of miR-34@ZIF-8-SCM nanoparticles incubated in neural stem cells for 30 minutes and 5 hours, respectively; miR-34 was labeled with Cy5 (red), cell nuclei were stained with DAPI (blue), and neural stem cell membranes were stained with Dio (green); Fig.10 : (a) Transmission electron microscopy image of the distribution of miR-34@ZIF-8 in neural stem cells after 6 hours of incubation; (b) Transmission electron microscopy image of the distribution of miR-34@ZIF-8-SCM in neural stem cells after 6 hours of incubation; Fig.11 : (a) Intracellular uptake, confocal image of miR-34@ZIF-8-SCM incubated in neural stem cells for 30 minutes; (b) Intracellular uptake, confocal image of miR-34@ZIF-8-SCM incubated in neural stem cells for 5 hours; Fig.12 :Release of miR-34 from miR-34@ZIF-8 and miR-34@ZIF-8-SCM in a simulated lysosomal environment (pH=5.0); Fig.13 : After co-culture of neural stem cells with different nanoparticles for 5 days, RT-qPCR was used to analyze the secreted neural-related genes; Fig.14 :After co-culture of neural stem cells with different nanoparticles for 10 days, RT-qPCR was used to analyze the secreted neural-related genes; Fig.15 :(a) Immunofluorescence staining of neural-related proteins secreted by neural stem cells after co-culture with different nanoparticles for 5 days;(b) Fluorescence intensity analysis of Tuj1;(c) Fluorescence intensity analysis of MAP2;(d) Fluorescence intensity analysis of GFAP; Fig.16 :After co-culture of neural stem cells with different nanoparticles for 10 days, protein imprint analysis of neural-related genes secreted by them was performed; Fig.17 :(a)Analysis of calcium sparks at the beginning of differentiated neurons in the miR-34@ZIF-8-SCM nanoparticle group, the arrow indicates the location of calcium sparks after adding dopamine;(b)Analysis of calcium sparks at 48 seconds in differentiated neurons in the miR-34@ZIF-8-SCM nanoparticle group;(c)Fluorescence results were analyzed for the corresponding fluorescence intensity; Fig.18: Delivery and internalization of miR-34@ZIF-8-SCM in cells. DETAILED DESCRIPTION

[0019] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0020] As described in the background technology section, although metal organic framework materials such as ZIF can be used to load miR-34, ZIF is a porous polymer and it is difficult to quickly release miR-34 after physical adsorption. Currently, there are few reports on the use of miR-34 to induce neural stem cells to differentiate into neurons, and there are no reports on the use of miR-34 in the treatment of Parkinson's disease.

[0021] Based on this, the purpose of the present invention is to provide a zinc-based nano-organic frame coating material loaded with microRNA and its application. The present invention loads microRNA-34 in ZIF-8, and then outsources the cell membrane of neural stem cells to obtain miR-34@ZIF-8-SCM. ZIF-8 can load nucleic acids to form miR-34@ZIF-8 through electrostatic and coordination interactions. Then the NSCs membrane and miR-34@ZIF-8 are mechanically co-extruded and coated to finally form miR-34@ZIF-8-SCM, which can effectively prevent miR-34 from being degraded, promote the absorption of miR-34 by NSCs, and can accurately target NSCs. Cell experiments show that miR-34@ZIF-8-SCM nanoparticles have high cell compatibility and can significantly promote the directional differentiation of neurons in NSCs. This combination strategy is crucial for directional regulation of neural stem cell differentiation, promoting functional recovery of damaged neural tissue, and treating Parkinson's disease.

[0022] However, although ZIF-8 can load microRNA-34, it can be seen from the preparation process of the present invention that microRNA-34 can still be firmly loaded in ZIF-8 at extremely high centrifugal speeds. Although miR-34@ZIF-8 nanoparticles have ideal acid sensitivity and show a pH- and time-dependent miR-34 release pattern, the rate at which miR-34@ZIF-8 releases microRNA-34 is not high. After miR-34@ZIF-8 is coated with the cell membrane, it will promote the release of miR-34. The miR-34@ZIF-8-SCM nanoparticles are encapsulated in membrane-bound vesicles through endocytosis of the cell, and then the vesicles encapsulating the miR-34@ZIF-8-SCM nanoparticles are transported to the lysosomes (see Fig.18). Therefore, miR-34@ZIF-8-SCM nanoparticles were successfully internalized into NSCs and accumulated in lysosomes. Afterwards, the acidic environment of the lysosome triggered the dissociation of the hydrogen bond between miR-34 and ZIF-8, and miR-34 was released from the endosome-lysosome pathway. Most importantly, the cell membrane not only did not block the release of miR-34 from ZIF-8, but also increased the rate of release of microRNA-34 from the zinc-based nano-organic framework coating material loaded with microRNA. The miR-34@ZIF-8 wrapped in the cell membrane showed a faster release of miR-34 under pH 5.0. Therefore, even if miR-34@ZIF-8 is coated with the cell membrane, it can increase the release rate of miR-34 from ZIF-8 in neural stem cells, so that ZIF-8 and miR-34 work together to increase the differentiation rate of neural stem cells.

[0023] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0024] It is noted that the microRNA-34 in the present invention was purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd.

[0025] Dio Brand: MCE, Item No.: HY-D0969; Lysosomal tracer brand: Thermo Fisher, catalog number: L7525; 5× RNA Nucleic Acid Buffer Brand: Solarbio, Product Number: R1050; Tuj1 primary antibody brand: Abcam, catalog number: ab131034; Nestin primary antibody brand: proteintech, catalog number: 19483-1-AP; GFAP primary antibody brand: proteintech, catalog number: 16825-1-AP; MAP2 primary antibody brand: Abcam, catalog number: ab254264; TH primary antibody brand: proteintech, catalog number: 25859-1-AP; Alexa Fluor 488 secondary antibody brand: Sikorje, catalog number: EF0007; Alexa Fluor 594 secondary antibody brand: Sikogene, catalog number: EF0011; Goat anti-rabbit secondary antibody brand: abcam, model: ab150113; Goat anti-mouse secondary antibody brand: abcam, model: ab7148; Neurobasal medium brand: Gibco, catalog number: 2994579; BCA protein quantification kit brand: Sikojet, catalog number: EC0001-A; Unless otherwise specified, the pH of the PBS solution used in the present invention is 7.4.

[0026] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0027] Example 1: Preparation method of miR-34@ZIF-8-SCM (1) At room temperature, 4 ml of zinc nitrate hexahydrate (0.585 g) aqueous solution was added dropwise to 40 ml of 2-methylimidazole (16.2 g) aqueous solution and stirred continuously. The mixed solution gradually turned milky white. After 15 minutes of reaction, the mixture was centrifuged at 10,000 rpm for 15 minutes and washed with deionized water several times. Finally, the mixture was freeze-dried and dried for 24 hours to obtain ZIF-8.

[0028] (2) Take 1 mg ZIF-8 and dilute it with 1 ml PBS, disperse it evenly by ultrasonication, and prepare a PBSZIF-8 solution with a concentration of 1 mg / mL. After centrifugation of 0.025 mg miR-34 powder, add 125 μL of nuclease-free water DEPC to obtain a miR-34 solution with a concentration of 20 μM. Take 100 μL miR-34 and add it to the 1 mg / mL ZIF-8 solution, incubate it at 4°C overnight, and obtain a 1 mg / mL miR-34@ZIF-8 PBS solution.

[0029] (3) When the diameter of neurospheres reaches 150 μm, the neurospheres are collected, and the number is about 2×10 8 , 1000rpm, centrifuged for 5 minutes to obtain neural stem cells. At 4°C, neural stem cells were lysed with hypotonic lysis solution (1 mM NaHCO3, 0.2 mM EDTA, 1 mMPMSF) for 4 hours, and the lysed solution was centrifuged at 4°C, 3000rpm for 5 minutes. After taking the supernatant, it was centrifuged at 4°C, 15000 rpm for 30 minutes, and the precipitate was collected to obtain the cell membrane of the neural stem cell membrane. The cell membrane was added to the PBS solution of miR-34@ZIF-8, and ultrasonic treatment was performed at a power of 40W, a temperature of 4°C, and a time of 8min. Then, the obtained 1ml solution was sequentially passed through nanoscale polycarbonate membranes with pore sizes of 400nm and 200nm, and extruded back and forth 15 times. After co-extrusion, miR-34@ZIF-8-SCM was obtained, which is a zinc-based nano-organic framework coating material loaded with microRNA.

[0030] Example 2: Characterization of miR-34@ZIF-8-SCM (1) The morphology of ZIF-8 nanoparticles was determined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Figure 1 As shown, ZIF-8 nanoparticles prepared from Zn(NO3)2·6H2O and 2-methylimidazole at room temperature are monodisperse, have a polyhedral morphology, and have an average size of ≈100 nm, which there is evidence to be the optimal size for cellular uptake. Figure 2 X-ray diffraction (XRD) analysis was further used to characterize the structure of ZIF-8 nanoparticles, and a main peak of ZIF-8 at 7.3° was observed in XRD.

[0031] (2) The loading capacity of the miRNA carrier strongly affects the delivery efficiency. The miR-34 of each group was co-incubated with ZIF-8 in deionized water overnight. MiR-34 was bound to the ZIF-8 surface through electrostatic and coordination interactions to obtain miR-34@ZIF-8 with different weight ratios. The maximum mass of miR-34 that can be loaded on ZIF-8 nanoparticles was investigated by agarose gel electrophoresis. First, miR-34@ZIF-8 nanoparticles were mixed at a miR-34:ZIF-8 weight ratio of 0, 1:20, 1:40, 1:60, 1:80, 1:100 or 1:140 (w / w) and incubated in deionized water at 4°C overnight. The unloaded miR-34 was then centrifuged at 1500 rpm to remove it. All miR-34@ZIF-8 nanoparticles synthesized with different concentrations of miR-34 were diluted with PBS (pH 7.4). Afterwards, 10 μL of miR-34@ZIF-8 nanoparticles with a miR-34:ZIF-8 weight ratio of 0, 1:20, 1:40, 1:60, 1:80, 1:100, or 1:140 (w / w) were diluted with 5×TAE buffer (Merck). Then, electrophoresis was performed on a 5% agarose gel containing 10 μL of nucleic acid dye (Solarbio) at 110 V for 30 min. Naked miR-34 (100 nM) was used as a reference.

[0032] according to Figure 3 It can be seen that as the amount of miR-34 loaded onto ZIF-8 nanoparticles increases, the free miR-34 gradually decreases. When the weight ratio of miR-34:ZIF-8 is 1:40, miR-34 is fully loaded into ZIF-8, and the effect of ZIF-8 loading miR-34 is best.

[0033] (3) Figure 4 (a) The cell membrane was collected and purified using cell lysis buffer, and the membrane structure was observed using a transmission electron microscope (TEM). The cell membrane morphology was uniform. Figure 4(b) Transmission electron microscopy (TEM) was used to measure the miR-34@ZIF-8-SCM nanoparticles, and an obvious core-shell structure can be seen. The membrane layer can be clearly identified on the surface of the miR-34@ZIF-8 polyhedron.

[0034] (4) The specific surface area was determined by nitrogen adsorption, and the storage possibility of molecules in the pores and channels of ZIF-8 was analyzed by BET (Brunauer-Emmett-Teller). As shown in Figure 5, the N2 adsorption isotherms of ZIF-8 and miR-34@ZIF-8 at 77 K (-196°C) are type I. Under low relative pressure, the adsorption volume increases rapidly, and it is a microporous structure. It proves that miR-34 can not only be adsorbed on the surface of ZIF-8 through electrostatic and coordination effects, but also its own porous nature allows miR-34 to penetrate into the pores, which plays a dual protective role for miR-34. But at the same time, the type I adsorption isotherm also shows that ZIF-8 has strong physical adsorption, which affects the release rate of miR-34.

[0035] (5) The self-assembly of the cell membrane is driven by the thermodynamic driving force and electrostatic interaction between the negatively charged protein-lipid membrane and the positively charged surface of ZIF-8 nanoparticles. Figure 6 The zeta potential data in (a) show that the surface potential of miR-34@ZIF-8 changes from +11.6 mV to −28.0 mV of miR-34@ZIF-8-SCM due to the electronegativity of the coated cell membrane. In addition, the increase in the absolute value confirms that the miR-34@ZIF-8-SCM structure is more stable.

[0036] (6) To further verify the structure of miR-34@ZIF-8-SCM, Figure 6 (b) is the Fourier transform infrared (FTIR) spectra of ZIF-8 prepared in step (1) of Example 1, miR-34@ZIF-8 prepared in step (2) of Example 1, and miR-34@ZIF-8-SCM prepared in step (3) of Example 1. In the ZIF-8 spectrum, at 3135 cm -1 and 2928cm -1 and 1580cm -1 The peaks at 1170 cm-1 correspond to the CH stretching, CH stretching, and CN stretching of imidazole, respectively. After miR-34 treatment, some peaks belonging to miR-34 appeared; for example, -1 A peak at 1249 cm-1 corresponds to the antisymmetric stretching vibration of COC. -1There is an inconspicuous peak at , which may correspond to the phosphate-antisymmetric stretching of cell membrane phospholipids. The above data show that miR-34@ZIF-8-SCM with high stability has been successfully constructed.

[0037] (7) The key to improving the clinical application value of miR-34-based therapy is to solve the problem that miR-34 is easily degraded by nucleases during cell phagocytosis. miR-34 was anchored in ZIF-8 to enhance the stability of miR-34 and prevent nucleases from destroying miR-34 during cell uptake. Therefore, the stability of miR-34 on ZIF-8 was verified by agarose gel electrophoresis, and the process of nuclease degradation was simulated in vitro. Naked miR-34 (20 μM) and miR-34@ZIF-8-SCM nanoparticles prepared in Example 1 (20 μM) were placed in a solution containing 10 μg / mL nuclease degrading enzyme and incubated at 37°C for 0, 30, 60 or 90 minutes. Samples were collected at each experimental time point, diluted with 5× RNA nucleic acid buffer (Solarbio), denatured at 100°C, and then frozen at 4°C. After sample collection was completed, samples were thawed and run on a 2% agarose gel containing 10 μL of nucleic acid stain (Solarbio) at 110 V for 30 min.

[0038] like Figure 7 As shown in the figure, after incubation of miR-34@ZIF-8-SCM in nuclease for 90 minutes, the fluorescence intensity of miR-34 in the miR-34@ZIF-8-SCM group remained basically unchanged, while the naked miR-34 was completely degraded after 30 minutes. This result confirms that miR-34@ZIF-8-SCM nanoparticles can significantly protect miR-34 from degradation by nucleases. This protective effect may be related to the steric hindrance of the nanoparticles.

[0039] Example 3: Biocompatibility and targeting ability of miR-34@ZIF-8-SCM (1) 10 5 NSCs (isolated from 13-15 day C57 mouse embryos) were inoculated into 48-well plates containing different concentrations of miR-34@ZIF-8-SCM nanoparticles (0, 5, 25, 50 μg / mL) and cultured for 1, 2, and 3 days. Subsequently, 500 μL of CCK-8 solution was added to each well and incubated at 37°C for 2 hours. 100 μL of the incubation solution was added to each well of a 96-well plate, and the light absorption at 450 nm was measured using a microplate reader (BioTek, USA). Parallel experiments were repeated three times.

[0040] like Figure 8As shown in the figure, the cells treated with ZIF-8-SCM NPs had good viability and proliferation ability. When the concentration of ZIF-8-SCM NPs increased to 50 µg / mL, the number of NSCs did not increase compared with the blank group on the first day, and was significantly lower than that of the blank group (0 μg / mL) and the low concentration group (5 µg / mL, 25 µg / mL), so the concentration of 25 µg / mL was selected.

[0041] (2) To demonstrate the good biocompatibility and efficient targeting ability of the miR-34@ZIF-8-SCM delivery system for NSCs. The miR-34 in the nanoparticles was labeled with Cy5 dye (red), the NSCs membrane was labeled with Dio dye (green), and the cell nuclei in the NSCs were labeled with DAPI dye (blue). The labeled miR-34@ZIF-8 nanoparticles and the labeled miR-34@ZIF-8-SCM nanoparticles were cultured with NSCs for different time periods (30 minutes and 5 hours), and then the distribution of miR-34 nanoparticles in the cells was observed using confocal microscopy ( Fig. 9 ). After NSCs were incubated with miR-34@ZIF-8 nanoparticles for 30 minutes, basically no red fluorescence (Cy5) was observed in NSCs, and red fluorescence (Cy5) was not observed in NSCs until 5 hours after incubation. However, when NSCs were incubated with miR-34@ZIF-8-SCM nanoparticles for 30 minutes, obvious red fluorescence (Cy5) could be observed in NSCs, and the fluorescence intensity increased after 5 hours. This result shows that miR-34@ZIF-8-SCM nanoparticles can be successfully absorbed by NSCs, and the wrapping of NSCs membrane successfully disguises miR-34@ZIF-8-SCM nanoparticles as natural molecules, greatly enhancing the biocompatibility and targeting ability of miR-34@ZIF-8-SCM nanoparticles to NSCs. TEM images further confirmed that the number of miR-34@ZIF-8-SCM nanoparticles increased significantly, indicating that miR-34@ZIF-8 is more easily internalized and aggregated by NSCs after coating the NSCs membrane ( Fig.10 ).

[0042] (3) To verify that the miR-34@ZIF-8-SCM delivery system can successfully release miR-34 in NSCs, miR-34@ZIF-8-SCM nanoparticles were co-incubated with neural stem cells for 30 minutes or 5 hours, miR-34 was labeled with Cy5 dye, neural stem cell membranes were labeled with Dio dye, and lysosomes were labeled with lysosomal tracer (Thermo Fisher). Under confocal microscopy, it can be observed that at 30 minutes, a small amount of miR-34 can be observed in the lysosomes, and miR-34 and neural stem cell membranes are co-localized in the lysosomes. After 5 hours, miR-34 was released from the lysosomes, and the neural stem cell membranes were separated from miR-34 and lysosomes. The results show that miR-34@ZIF-8-SCM nanoparticles have ideal acid sensitivity and exhibit pH- and time-dependent miR-34 release patterns. miR-34@ZIF-8-SCM nanoparticles are encapsulated in membrane-bound vesicles through endocytosis of cells. The vesicles encapsulating miR-34@ZIF-8-SCM nanoparticles can then be transported to lysosomes. Therefore, miR-34@ZIF-8-SCM nanoparticles are successfully internalized into NSCs and aggregated in lysosomes. Afterwards, the acidic environment of the lysosome triggers the dissociation of the hydrogen bond between miR-34 and ZIF-8, resulting in the release of miR-34 from the endosome-lysosome pathway and entering the cellular machinery, further accelerating the directed differentiation of neurons ( Fig.11 ).

[0043] Experimental example: Differentiation of neural stem cells (1) Observe the release rate of miR-34 in neural stem cells by miR-34@ZIF-8-SCM prepared in step (3) of Example 1 and miR-34@ZIF-8 prepared in step (2) of Example 1. miR-34@ZIF-8 and miR-34@ZIF-8-SCM nanoparticles were incubated in Tris-HCl (pH 5.0) at 4°C. At specific time intervals (0, 12, 24, 36, 48, 60 hours), the supernatant was collected by centrifugation. The concentration of miR-34 released into the supernatant was measured at a wavelength of 260 nm using a spectrophotometer (Q-5000, Quawell). The results are shown in Table 1. Fig.12 .

[0044] (2) q-PCR evaluation of miR-34@ZIF-8-SCM to accelerate the differentiation of NSCs into neurons 10 7NSCs (isolated from 13-15 day old C57 mouse embryos) were cultured in 6-well plates containing blank (containing neurobasal medium, 2% B27 supplement, 1% glutaMAX-1, 2% fetal bovine serum and 1% P / S), ZIF-8 nanoparticles prepared in step (1) of Example 1 (25 μg / mL), miR-34@ZIF-8 prepared in step (2) of Example 1 (25 μg / mL), and miR-34@ZIF-8-SCM nanoparticles prepared in step (3) of Example 1 (25 μg / mL) for 5 or 10 days, and then RNA was isolated from NSCs using TRIzol reagent (Invitrogen). The concentration and purity of RNA were measured at 260 / 280 nm using a spectrophotometer (Q-5000, Quawell). After reverse transcription, q-PCR was performed using a real-time fluorescence quantitative PCR system (LightCycler96 Roche) to analyze the housekeeping gene actin and neurogenesis-related genes, including Nestin, Tuj1, MAP2, and GFAP. The relative transcription level of target gene expression was normalized with Actin and expressed as mean ± SD (n = 3).

[0045] The expression of NSCs marker gene Nestin, neuron marker class III tubulin (Tuj1, an early indicator of neuronal differentiation), neuron marker microtubule separation protein 2 (MAP2, an indicator of mature neuronal differentiation) and astrocyte marker glial fibrillary acidic protein (GFAP) were quantitatively analyzed by real-time fluorescence PCR. As shown in the figure, at 5 days, the expression of NSCs marker Nestin in the blank group, ZIF-8 nanoparticle group, miR-34@ZIF-8 group and miR-34@ZIF-8-SCM nanoparticle group was basically about 1 times. By comparing the expression of Tuj1, which represents an early neuronal differentiation marker, at 5 days, it was found that NSCs cultured with miR-34@ZIF-8-SCM nanoparticles, miR-34@ZIF-8 nanoparticles, and ZIF-8 nanoparticles were about 2.0 times, about 1.2 times, and about 0.9 times that of the blank group, respectively. The expression of mature neuron MAP2 in miR-34@ZIF-8-SCM nanoparticles, miR-34@ZIF-8 nanoparticles, and NSCs cultured with ZIF-8 nanoparticles was approximately 2.3 times, approximately 1.2 times, and approximately 1.1 times that of the blank group, respectively. The expression of the representative gene GFAP of astrocytes in miR-34@ZIF-8-SCM nanoparticles, miR-34@ZIF-8 nanoparticles, and NSCs cultured with ZIF-8 nanoparticles was approximately 0.7 times, approximately 0.8 times, and approximately 1.3 times that of the blank group, respectively. After 10 days of culture, the expression of Tuj1 and MAP2 genes in the group co-cultured with miR-34@ZIF-8-SCM nanoparticles and NSCs was significantly upregulated, while the expression of GFAP gene decreased. The results show that miR-34@ZIF-8-SCM nanoparticles can effectively accelerate the neuronal differentiation of NSCs and inhibit the glial differentiation of NSCs. The results are shown in Fig.13 , Fig.14 .

[0046] (3) Immunofluorescence staining to evaluate the effect of miR-34@ZIF-8-SCM on the acceleration of NSCs differentiation into neurons In order to more intuitively show the differentiation of NSCs, immunofluorescence staining was performed on cells co-cultured for 5 days. After NSCs were co-cultured on glass slides with blank (containing neural basal medium, 2% B27 supplement, 1% glutaMAX-1, 2% fetal bovine serum and 1% P / S), ZIF-8 nanoparticles, miR-34@ZIF-8 and miR-34@ZIF-8-SCM nanoparticles for 5 days, the slides were fixed with 4% paraformaldehyde for 15 minutes and then washed three times with PBS. Then, the slides were permeabilized in 0.1% Triton X-100 for 5 minutes, washed three times with PBS, and blocked with 1% bovine serum albumin solution for 1 hour. Subsequently, the slides of the blank group, ZIF-8 nanoparticle group, miR-34@ZIF-8 group, and miR-34@ZIF-8-SCM nanoparticle group were incubated with primary antibodies for MAP2 (mouse polyclonal anti-MAP2), Tuj1 (mouse monoclonal anti-Tuj1), or GFAP (rabbit polyclonal anti-GFAP) at 4°C overnight. The samples were incubated with Alexa Fluor 488 secondary antibody and Alexa Fluor 594 secondary antibody for 1 hour at 37°C. Then, the cells were washed three times with PBS and then treated with DAPI for 10 min to stain the nuclei. Images of the stained samples were obtained by laser confocal microscopy.

[0047] like Fig.15 As shown in the figure, NSCs cultured with miR-34@ZIF-8-SCM nanoparticles showed stronger expression of neuron-specific markers Tuj1 and MAP2, and had multiple neurites, significantly more than other groups (blank group, ZIF-8 nanoparticles and miR-34@ZIF-8 nanoparticles group). This figure also proves that miR-34@ZIF-8-SCM nanoparticles effectively promote NSCs to differentiate directly into neurons rather than glial cells, which is consistent with the results of RT-qPCR.

[0048] (4) Western Blot evaluation of miR-34@ZIF-8-SCM to accelerate the differentiation of NSCs into neurons In order to further verify the effect of miR-34@ZIF-8-SCM nanoparticles on the differentiation of neural stem cells into neurons, 10 7100 μg / mL NSCs were cultured in 6-well plates containing blank (containing neurobasal medium, 2% B27 supplement, 1% glutaMAX-1, 2% fetal bovine serum and 1% P / S), ZIF-8 (25 μg / mL), miR-34@ZIF-8 (25 μg / mL), and miR-34@ZIF-8-SCM (25 μg / mL) for 5 days, and then total protein was isolated from NSCs using cell lysis buffer containing protease and phosphatase inhibitors. Protein was also isolated from NSCs cultured on the blank without treatment as a control. Protein concentration was determined using a BCA protein assay kit (Scottje). Then, 5× protein buffer (Conway Century) was added to each sample, and proteins were separated by SDS-PAGE. The proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane and blocked with thawed milk powder (5%) at 37°C for 1 hour. Then, the PVDF membrane was incubated with TH primary antibody (mouse polyclonal anti-TH), Tuj1 primary antibody (mouse polyclonal anti-Tuj1), MAP2 primary antibody (mouse polyclonal anti-MAP2), and GFAP primary antibody (rabbit polyclonal anti-GFAP) at 4°C for 10 hours. After washing with TBST, the PVDF membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (brand abcam, model ab150113) and goat anti-mouse secondary antibody (brand abcam, model ab7148) at 37°C for 1 hour. The membrane was washed with TBST again, and the membrane was soaked with chemiluminescent HPR substrate (brand Thermo Fisher) and scanned using the fluorescent chemistry HD2 system (ProteinSimple, USA).

[0049] like Fig.16 As shown, the protein expression levels of Tuj1 and MAP2 in the miR-34@ZIF-8-SCM nanoparticle group were significantly higher than those in the other groups, and the protein expression level of GFAP in the miR-34@ZIF-8-SCM group was not much different from that in the other groups (blank group, ZIF-8 nanoparticle group and miR-34@ZIF-8 nanoparticle group). At the same time, in order to further determine the type of functional neurons, the functional neuronal protein expression of NSCs in each group at 10 days was analyzed by Western blot. TH, as a specific marker for dopaminergic neurons, is widely used to identify and study dopaminergic neurons in the central nervous system. Therefore, the expression level of TH protein was also measured. Finally, as shown in Fig.16 As shown, the protein expression level of TH in the miR-34@ZIF-8-SCM nanoparticle group was significantly higher than that in the other groups.

[0050] (5) Calcium spark evaluation of miR-34@ZIF-8-SCM to accelerate the differentiation of NSCs into functional neurons An important indicator of the successful acceleration of NSCs-to-neuron differentiation mediated by miR-34@ZIF-8-SCM nanoparticles is the generation of functional neurons. Calcium sparks were detected on NSCs incubated with miR-34@ZIF-8-SCM nanoparticles on day 5. After NSCs were treated with miR-34@ZIF-8-SCM NPs for 5 days, the cells were stained with 200 μL of calcium ion fluorescent probe (Flu-4AM) working solution (2 μM, diluted in PBS) and then incubated at 37°C for 20 minutes. After the cells were treated with dopamine neurotransmitters, the changes in fluorescence intensity were recorded by confocal microscopy and analyzed by ImageJ software.

[0051] The fluorescence intensity of calcium in cells at rest is low, e.g. Fig.17 When exposed to appropriate amounts of neurotransmitters, such as dopamine (DA), intracellular Ca 2+ The fluorescence increased rapidly in a short period of time and then returned to the resting state over time. These results indicate that the derived neurons generated by NSCs stimulated by miR-34@ZIF-8-SCM nanoparticles are functional neurons with electrophysiological properties.

[0052] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A zinc-based nano-organic framework coating material loaded with microRNA, characterized in that: The microRNA-loaded zinc-based nano-organic framework coating material comprises a zinc-based nano-organic framework material; the zinc-based nano-organic framework material is loaded with microRNA, and the microRNA is microRNA-34; the surface of the zinc-based nano-organic framework material is coated with a cell membrane, and the cell membrane comes from a neural stem cell.

2. The microRNA-loaded zinc-based nano-organic framework coating material according to claim 1, characterized in that: The microRNA-loaded zinc-based nano-organic framework coating material is prepared by the following method: (1) Disperse ZIF-8 nanoparticles in PBS solution, add microRNA-34 and mix well, keep at low temperature overnight, then centrifuge and dry to obtain miR-34@ZIF-8; (2) Neural stem cells were lysed with hypotonic lysis buffer at low temperature, the lysed solution was centrifuged once at low temperature, the supernatant was centrifuged again at low temperature, the precipitate was collected to obtain the cell membrane of the neural stem cell membrane, the cell membrane was added to the PBS solution of miR-34@ZIF-8, ultrasonically treated, and co-extruded to obtain miR-34@ZIF-8-SCM, which is the zinc-based nano-organic framework coating material loaded with microRNA.

3. The microRNA-loaded zinc-based nano-organic framework coating material according to claim 2, characterized in that: In step (1), the weight ratio of microRNA-34 to ZIF-8 is 1:

40.

4. The microRNA-loaded zinc-based nano-organic framework coating material according to claim 2, characterized in that: In step (1), the temperature of the low temperature overnight is 4°C; and the speed of the centrifugation is 3000 rpm.

5. The microRNA-loaded zinc-based nano-organic framework coating material according to claim 2, characterized in that: In step (2), the low temperature is 4°C; the lysis time is 4 hours.

6. The microRNA-loaded zinc-based nano-organic framework coating material according to claim 2, characterized in that: In step (2), the first low-temperature centrifugation is performed at 3000 rpm at 4°C for 5 min; the second low-temperature centrifugation is performed at 15000 rpm at 4°C for 30 min.

7. The microRNA-loaded zinc-based nano-organic framework coating material according to claim 2, characterized in that: In step (2), the power of the ultrasonic treatment is 40 W, the temperature is 4° C., and the time is 8 min; the co-extrusion is performed by using a micro co-extruder to sequentially pass through polycarbonate membranes with pore sizes of 400 nm and 200 nm.

8. Use of the microRNA-loaded zinc-based nano-organic framework coating material according to any one of claims 1 to 7 in the preparation of a drug for inducing neural stem cells to transform into neurons.

9. The use according to claim 8, characterized in that: The concentration of the microRNA-loaded zinc-based nano-organic framework coating material is 25 µg / mL.

10. Use of a cell membrane of a neural stem cell in increasing the rate of release of microRNA-34 from a microRNA-loaded zinc-based nano-organic framework coating material as claimed in any one of claims 1 to 7.

Images