Zinc-based nano-organic framework coating material loaded with microRNA and its application

Abstract

The present invention discloses a zinc-based nano-organic framework coating material loaded with microRNA and its application, belonging to the technical field of biomedicine. The zinc-based nano-organic framework coating material loaded with microRNA includes a zinc-based nano-organic framework material; microRNA-34 is loaded on the zinc-based nano-organic framework material; the surface of the zinc-based nano-organic framework material is coated with the cell membrane of neural stem cells. In the present invention, neural stem cells endocytose miR-34@ZIF-8-SCM to induce the differentiation of neural stem cells into neurons; the cell membrane of neural stem cells not only has homology with neural stem cells, but also increases the release rate of microRNA-34 by miR-34@ZIF-8-SCM, further increasing the differentiation rate of neural stem cells into neurons.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a zinc-based nano-organic framework coating material loaded with microRNA and its applications. Background Art

[0002] Parkinson's disease has become one of the most common diseases threatening global human health and causing 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 defect of dopamine neurons. Therefore, it is necessary to develop an effective strategy to combine the treatment of neural stem cells (NSCs) to guide the directed differentiation of NSCs into functional neurons for the treatment of Parkinson's disease.

[0003] Zn 2＋ is one of the trace elements with the highest content in the mammalian brain and has been proven to have a positive effect on the differentiation of neural stem cells. For example, "The Effect 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 patent Application No. CN202111528077.2, Application of a Metal-organic Ligand Framework ZIF-67 Modified Hollow Vanadium Dioxide Core-membrane Composite Structure Drug Carrier, etc. have all reported different forms of Zn 2＋ in the induction of cell differentiation, but single Zn 2＋ has an insignificant effect on inducing cell differentiation and needs to be compounded with other materials. miR-34 is one of the most abundant miRNAs in the human brain nervous system and is currently mainly used for the treatment of cancer or tumors. However, miR-34 has a polyanionic charge, is difficult to internalize through cell membranes, has a short half-life in the systemic circulation, and is sensitive to nuclease degradation. These characteristics have hindered the widespread clinical application of miR-34-based therapeutic methods. Metal-organic frameworks (MOFs) are assembled from metal nodes and organic linkers and are a class of typical crystalline porous materials with a high specific surface area, large porosity, customizable size and structure, chemical stability, and biodegradability. Although materials such as ZIF in the metal-organic framework class can be used to load miR-34, ZIF is a porous polymer, and it is difficult to release miR-34 quickly after physically adsorbing miR-34. Currently, there are few reports on the use of miR-34 to induce the differentiation of neural stem cells into neurons, nor are there any reports on the use of miR-34 for the treatment of Parkinson's disease. Therefore, a zinc-based nano-organic framework material loaded with microRNA is needed, which can quickly pass through cell membranes and quickly release miR-34, and utilize Zn 2＋Acting together with miR-34 to promote the differentiation of neural stem cells, with the expectation of being used for the treatment of Parkinson's disease in the future. Summary of the Invention

[0004] In view of the above-mentioned prior art, the object of the present invention is to provide a zinc-based nano-organic framework 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 coated outside to obtain miR-34@ZIF-8-SCM; neural stem cells endocytose miR-34@ZIF-8-SCM to induce the differentiation of neural stem cells into neurons; the cell membrane of neural stem cells not only has homology with neural stem cells, but also improves the release rate of microRNA-34 from miR-34@ZIF-8-SCM, further improving the differentiation rate of neural stem cells into neurons.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In the first aspect of the present invention, there is provided a zinc-based nano-organic framework coating material loaded with microRNA, and the zinc-based nano-organic framework coating material loaded with microRNA includes a zinc-based nano-organic framework material; microRNA is loaded on the zinc-based nano-organic framework material, 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 is from neural stem cells.

[0007] Preferably, the zinc-based nano-organic framework coating material loaded with microRNA is prepared by the following method:

[0008] (1) Dispersing ZIF-8 nanoparticles in a PBS solution, adding microRNA-34 and mixing evenly, incubating overnight at low temperature, and then centrifuging and drying to obtain miR-34@ZIF-8;

[0009] (2) At low temperature, neural stem cells are lysed with a hypotonic lysis solution. The lysed solution is centrifuged once at low temperature, and the supernatant is taken and centrifuged again at low temperature to collect the precipitate to obtain the cell membrane of neural stem cells. The cell membrane is added to the PBS solution of miR-34@ZIF-8, and ultrasonic treatment is carried out. After co-extrusion, miR-34@ZIF-8-SCM is obtained, which is the zinc-based nano-organic framework coating material loaded with microRNA.

[0010] Preferably, in step (1), the weight ratio of microRNA-34 to ZIF-8 is 1:40.

[0011] Preferably, in step (1), the temperature of incubating overnight at low temperature is 4°C; the rotation speed of centrifugation is 3000 rpm.

[0012] Preferably, in step (2), the temperature of the low temperature is 4°C; the lysis time is 4 h.

[0013] Preferably, in step (2), the first low-temperature centrifugation is centrifugation at 3000 rpm for 5 min at 4°C; the second low-temperature centrifugation is centrifugation at 15000 rpm for 30 min at 4°C.

[0014] Preferably, 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 to sequentially pass through polycarbonate membranes with pore sizes of 400 nm and 200 nm using a micro co-extruder.

[0015] In the second aspect of the present invention, there is provided 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 differentiate into neurons.

[0016] Preferably, the concentration of the zinc-based nano-organic framework coating material loaded with microRNA is 25 µg / mL.

[0017] In the third aspect of the present invention, there is provided the use of the cell membrane of neural stem cells in increasing the release rate of microRNA-34 from the zinc-based nano-organic framework coating material loaded with microRNA.

[0018] Advantages of the present invention:

[0019] (1) In the present invention, microRNA-34 is loaded into ZIF-8 and then coated with the cell membrane of neural stem cells to obtain miR-34@ZIF-8-SCM; neural stem cells endocytose miR-34@ZIF-8-SCM to induce the differentiation of neural stem cells into neurons; the cell membrane of neural stem cells not only has homology with neural stem cells but also increases the release rate of microRNA-34 from miR-34@ZIF-8-SCM, further increasing the differentiation rate of neural stem cells into neurons.

[0020] (2) The miR-34@ZIF-8-SCM prepared by the present invention has high biocompatibility and strong targeting ability, and can directionally regulate the differentiation of neural stem cells. It solves the problem that miR-34 is easily degraded in clinical applications. ZIF-8 is used as a transport carrier for miR-34 to protect miR-34 from being degraded by nucleases. At the same time, a biological membrane is coated on the surface of miR-34@ZIF-8 to solve the rejection reaction of cells to this foreign substance miR-34@ZIF-8, simulate natural molecules, and improve the biocompatibility and targeting ability to 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.

[0021] (3) The miR-34@ZIF-8-SCM nanoparticles of the present invention are expected to enhance the therapeutic effect of NSCs treatment on Parkinson's disease, and are expected to improve the application of miR-34 and NSCs treatment in the treatment of traumatic nerve injury and neurodegenerative diseases. Brief Description of the Drawings

[0022] Figure 1 : (a) Scanning electron microscope of ZIF-8, (b) Transmission electron microscope image of ZIF-8;

[0023] Figure 2 : X-ray diffraction pattern of ZIF-8;

[0024] Figure 3 : (a) Agarose gel electrophoresis to explore the loading capacity of ZIF-8 for miR-34; (b) Analysis of the agarose gel electrophoresis results of the loading capacity of ZIF-8 for miR-34;

[0025] Figure 4 : (a) Transmission electron microscope image of the cell membrane, (b) Transmission electron microscope image of miR-34@ZIF-8-SCM;

[0026] Figure 5 : (a) Nitrogen adsorption isotherms of ZIF-8 and miR-34@ZIF-8 and (b) Pore size distributions of ZIF-8 and miR-34@ZIF-8;

[0027] Figure 6 : (a) Zeta potentials of ZIF-8, miR-34@ZIF-8, miR-34@ZIF-8-SCM; (b) Fourier transform spectra of ZIF-8, miR-34@ZIF-8, miR-34@ZIF-8-SCM;

[0028] Figure 7: Protection of miR-34 by miR-34@ZIF-8-SCM;

[0029] Figure 8 : Effects of different concentrations of miR-34@ZIF-8-SCM on the activity of neural stem cells;

[0030] Figure 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 the neural stem cell membrane was stained with Dio (green);

[0031] Figure 10 : (a) Transmission electron microscopy images of the distribution of miR-34@ZIF-8 in neural stem cells after 6-hour incubation; (b) Transmission electron microscopy images of the distribution of miR-34@ZIF-8-SCM in neural stem cells after 6-hour incubation;

[0032] Figure 11 : (a) Intracellular uptake, confocal images of miR-34@ZIF-8-SCM incubated in neural stem cells for 30 minutes; (b) Intracellular uptake, confocal images of miR-34@ZIF-8-SCM incubated in neural stem cells for 5 hours;

[0033] Figure 12 : Release of miR-34 by miR-34@ZIF-8 and miR-34@ZIF-8-SCM under simulated lysosomal environment (pH = 5.0);

[0034] Figure 13 : After co-culturing neural stem cells with different nanoparticles for 5 days, RT-qPCR analysis of neural-related genes secreted by them;

[0035] Figure 14 : After co-culturing neural stem cells with different nanoparticles for 10 days, RT-qPCR analysis of neural-related genes secreted by them;

[0036] Figure 15 : (a) After co-culturing neural stem cells with different nanoparticles for 5 days, immunofluorescence staining of neural-related proteins secreted by them; (b) Fluorescence intensity analysis of Tuj1; (c) Fluorescence intensity analysis of MAP2; (d) Fluorescence intensity analysis of GFAP;

[0037] Figure 16: After co-culturing neural stem cells with different nanoparticles for 10 days, Western blot analysis was performed on the neural-related genes secreted by them;

[0038] Figure 17 : (a) Analysis of the onset of calcium sparks in differentiated neurons in the miR-34@ZIF-8-SCM nanoparticle group. The arrow indicates the location where calcium sparks occurred after adding dopamine; (b) Analysis of calcium sparks in differentiated neurons in the miR-34@ZIF-8-SCM nanoparticle group at 48 seconds; (c) Corresponding fluorescence intensity analysis of the fluorescence results;

[0039] Figure 18 : Delivery and internalization of miR-34@ZIF-8-SCM in cells. Detailed implementation manners

[0040] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0041] As introduced in the background art section, 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. There are few reports on the use of miR-34 to induce the differentiation of neural stem cells into neurons, and there are no related reports on the use of miR-34 for the treatment of Parkinson's disease.

[0042] Based on this, the object of the present invention is to provide a zinc-based nano-organic framework coating material loaded with microRNA and its application. The present invention loads microRNA-34 into ZIF-8 and then coats the cell membrane of neural stem cells on the outside to obtain miR-34@ZIF-8-SCM. ZIF-8 can load nucleic acids through electrostatic and coordination interactions to form miR-34@ZIF-8. Then, the NSCs membrane and miR-34@ZIF-8 are coated on by mechanical co-extrusion to finally form miR-34@ZIF-8-SCM. miR-34@ZIF-8-SCM 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 neuronal differentiation of NSCs. This combination strategy is crucial for the directional regulation of neural stem cell differentiation, the promotion of the functional recovery of damaged nerve tissues, and the treatment of Parkinson's disease.

[0043] 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 an extremely high centrifugation speed. Although miR-34@ZIF-8 nanoparticles have ideal acid sensitivity and exhibit a pH- and time-dependent miR-34 release pattern, the release rate of miR-34@ZIF-8 to release microRNA-34 is not high. However, after miR-34@ZIF-8 coats the cell membrane, it will instead promote the release of miR-34. miR-34@ZIF-8-SCM nanoparticles are encapsulated in membrane-bound vesicles through endocytosis of cells. Then, the vesicles encapsulating miR-34@ZIF-8-SCM nanoparticles are transported to lysosomes (see Figure 18 ). Therefore, miR-34@ZIF-8-SCM nanoparticles are successfully internalized into NSCs and accumulate in lysosomes. Subsequently, the acidic environment of lysosomes triggers the dissociation of hydrogen bonds between miR-34 and ZIF-8, and miR-34 is released from the endosome-lysosome pathway. Most importantly, the cell membrane not only does not block the release of miR-34 from ZIF-8, but also increases the release rate of the zinc-based nano-organic framework coating material loaded with microRNA to release microRNA-34. miR-34@ZIF-8 coated with the cell membrane shows a faster release of miR-34 under the condition of pH 5.0. Therefore, even if miR-34@ZIF-8 is coated with the cell membrane, it can instead increase the release rate of miR-34 from ZIF-8 in neural stem cells, enabling ZIF-8 and miR-34 to act together to increase the differentiation rate of neural stem cells.

[0044] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in conjunction with specific embodiments.

[0045] It is illustrated that microRNA-34 in the present invention is purchased from Shanghai GenePharma Co., Ltd.

[0046] Dio brand: MCE, product number: HY-D0969;

[0047] Lysosome tracer brand: Thermo Fisher, product number: L7525;

[0048] 5×RNA nucleic acid buffer brand: Solarbio, product number: R1050;

[0049] Tuj1 primary antibody brand: Abcam, product number: ab131034;

[0050] Nestin primary antibody, brand: proteintech, catalog number: 19483-1-AP;

[0051] GFAP primary antibody, brand: proteintech, catalog number: 16825-1-AP;

[0052] MAP2 primary antibody, brand: Abcam, catalog number: ab254264;

[0053] TH primary antibody, brand: proteintech, catalog number: 25859-1-AP;

[0054] Alexa Fluor 488 secondary antibody, brand: Cisco Jie, catalog number: EF0007;

[0055] Alexa Fluor 594 secondary antibody, brand: Cisco Jie, catalog number: EF0011;

[0056] Goat anti-rabbit secondary antibody, brand: abcam, model: ab150113;

[0057] Goat anti-mouse secondary antibody, brand: abcam, model: ab7148;

[0058] Neural basal medium, brand: Gibco, catalog number: 2994579;

[0059] BCA protein quantification kit, brand: Cisco Jie, catalog number: EC0001-A;

[0060] Unless otherwise specified, the pH of the PBS solution used in the present invention is 7.4.

[0061] The test materials used in the examples of the present invention are all conventional test materials in the art and can be obtained through commercial channels.

[0062] Example 1: Preparation method of miR-34@ZIF-8-SCM

[0063] (1) At room temperature, 4 ml of an aqueous solution of zinc nitrate hexahydrate (0.585 g) was added dropwise to 40 ml of an aqueous solution of 2-methylimidazole (16.2 g) with continuous stirring, and the mixed solution gradually became milky white. After reacting for 15 minutes, it was centrifuged at 10000 rpm for 15 minutes and washed with deionized water multiple times. Finally, it was freeze-dried and dried for 24 hours to obtain ZIF-8.

[0064] (2)Take 1 mg of ZIF-8 and dilute it with 1 ml of PBS, and disperse it evenly by ultrasonic treatment to prepare a PBS-ZIF-8 solution with a concentration of 1 mg / mL. After centrifuging 0.025 mg of miR-34 powder, add 125 μL of DEPC-treated nuclease-free water to obtain a miR-34 solution with a concentration of 20 μM. Take 100 μL of miR-34 and add it to the 1 mg / mL ZIF-8 solution, and incubate overnight at 4 °C to obtain a 1 mg / mL miR-34@ZIF-8 PBS solution.

[0065] (3)When the diameter of the neurospheres reaches 150 μm, collect the neurospheres, with a quantity of approximately 2×10 8 cells. Centrifuge at 1000 rpm for 5 minutes to obtain neural stem cells. At 4 °C, lyse the neural stem cells with a hypotonic lysis solution (1 mM NaHCO3, 0.2 mM EDTA, 1 mM PMSF) for 4 hours. After lysis, centrifuge the solution at 3000 rpm for 5 minutes at 4 °C. Take the supernatant and centrifuge at 15000 rpm for 30 minutes at 4 °C to collect the precipitate to obtain the cell membrane of neural stem cells. Add the cell membrane to the miR-34@ZIF-8 PBS solution, perform ultrasonic treatment at a power of 40 W, a temperature of 4 °C, and a time of 8 min. Then, pass the resulting 1 ml solution through polycarbonate membranes with pore sizes of 400 nm and 200 nm in turn, and extrude back and forth 15 times. After extrusion, miR-34@ZIF-8-SCM is obtained, which is a zinc-based nano-organic framework coating material loaded with microRNA.

[0066] Example 2: Characterization of miR-34@ZIF-8-SCM

[0067] (1)The morphology of ZIF-8 nanoparticles was determined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). As Figure 1 shown, the ZIF-8 nanoparticles prepared from Zn(NO3)2·6H2O and 2-methylimidazole at room temperature are monodisperse, with a polyhedral morphology, and an average size of ≈100 nm. There is evidence that this size is the optimal size for cell uptake. Figure 2 Furthermore, X-ray diffraction (XRD) analysis was used to characterize the structure of ZIF-8 nanoparticles. The main peak of ZIF-8 was observed at 7.3° in the XRD.

[0068] (2) The loading capacity of miRNA carriers strongly affects the delivery efficiency. miR-34 and ZIF-8 in each group were co-incubated overnight in deionized water. miR-34 was bound to the surface of ZIF-8 through electrostatic and coordination interactions to obtain miR-34@ZIF-8 with different weight ratios. The maximum mass of miR-34 that could be loaded on ZIF-8 nanoparticles was explored by agarose gel electrophoresis. First, miR-34@ZIF-8 nanoparticles were formed by mixing according to the weight ratios of miR-34:ZIF-8 of 0, 1:20, 1:40, 1:60, 1:80, 1:100, or 1:140 (w / w) and incubated overnight in deionized water at 4°C. Then, the unloaded miR-34 was removed by centrifugation at 1500 rpm. All miR-34@ZIF-8 nanoparticles synthesized with different concentrations of miR-34 were diluted with PBS (pH 7.4). After that, 10 μL of miR-34@ZIF-8 nanoparticles with the weight ratios of miR-34:ZIF-8 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 carried out at 110 V for 30 minutes on a 5% agarose gel containing 10 μL of nucleic acid dye (Solarbio). Naked miR-34 (100 nM) was used as a reference.

[0069] 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 completely loaded onto ZIF-8, and the effect of ZIF-8 loading miR-34 is the best.

[0070] (3) Figure 4 In (a), the cell membrane was collected and purified with cell lysate, and the structure of the membrane was observed by transmission electron microscopy (TEM). The morphology of the cell membrane was uniform. Figure 4 In (b), miR-34@ZIF-8-SCM nanoparticles were measured by transmission electron microscopy (TEM). An obvious core-shell structure could be seen, and the membrane layer could be clearly identified on the surface of the miR-34@ZIF-8 polyhedron.

[0071] (4)The specific surface area was determined by nitrogen adsorption method, and the storage possibility of molecules in the pores and channels of ZIF-8 was analyzed by BET (brunauer - emmet - teller). As shown in Figure 5, the N2 adsorption isotherms of ZIF-8 and miR-34@ZIF-8 at 77 K (-196 °C) are of type I. At low relative pressures, the adsorption volume increases rapidly, indicating a microporous structure. It is proved that miR-34 can not only be adsorbed on the surface of ZIF-8 through electrostatic and coordination interactions, but also its own porous nature allows miR-34 to penetrate into the pores, playing a dual protection role for miR-34. However, at the same time, the type I adsorption isotherm also indicates that ZIF-8 has strong physical adsorption, which affects the release rate of miR-34.

[0072] (5)The self-assembly of cell membranes is driven by the thermodynamic driving force and electrostatic interaction between the negatively charged protein-lipid membrane and the surface of positively charged ZIF-8 nanoparticles. Figure 6 The zeta potential data in (a) show that due to the electronegativity of the coated cell membrane, the surface potential of miR-34@ZIF-8 changes from +11.6 mV to -28.0 mV of miR-34@ZIF-8-SCM. In addition, the increase in the absolute value confirms that the miR-34@ZIF-8-SCM structure is more stable.

[0073] (6)To further verify the structure of miR-34@ZIF-8-SCM, Figure 6 In (b) are 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, the peaks at 3135 cm -1 and 2928 cm -1 and 1580 cm -1 correspond to the C-H stretching, C-H stretching, and C-N stretching of imidazole, respectively. After treatment with miR-34, some peaks belonging to miR-34 appear; for example, a peak at 1170 cm -1 corresponds to the asymmetric stretching vibration of C-O-C. In addition, in the miR-34@ZIF-8-SCM spectrum, there is an inconspicuous peak at 1249 cm -1 which may correspond to the phosphate - asymmetric stretching of cell membrane phospholipids. The above data indicate that miR-34@ZIF-8-SCM with high stability has been successfully constructed.

[0074] (7) The key to enhancing the clinical application value of miR-34 therapy is to solve the problem that miR-34 is easily degraded by nucleases during the process of phagocytosis. miR-34 is anchored in ZIF-8 to enhance the stability of miR-34 and prevent the destruction of miR-34 by nucleases 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 the miR-34@ZIF-8-SCM nanoparticles prepared in Example 1 (20 μM) were respectively placed in a solution containing 10 μg / mL nuclease 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, the samples were thawed and run on a 2% agarose gel containing 10 μL nucleic acid dye (Solarbio), and electrophoresis was carried out at 110 V for 30 minutes.

[0075] As Figure 7 shown, after miR-34@ZIF-8-SCM was incubated in nuclease for 90 min, the fluorescence intensity of miR-34 in the miR-34@ZIF-8-SCM group basically did not change, while naked miR-34 had been completely degraded after 30 min. This result confirmed that the miR-34@ZIF-8-SCM nanoparticles could significantly protect miR-34 from nuclease degradation. This protective effect may be related to the steric hindrance of the nanoparticles.

[0076] Example 3: Biocompatibility and targeting ability of miR-34@ZIF-8-SCM

[0077] (1) 10 5 cells / mL of NSCs (isolated from 13- to 15-day-old C57 mouse embryos) were seeded 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, 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 in a 96-well plate, and the light absorption at 450 nm was measured using a microplate reader (BioTek, USA). The parallel experiments were repeated three times.

[0078] As Figure 8As shown, the cells treated with ZIF-8-SCM NPs had good viability and proliferation ability. As the concentration of ZIF-8-SCM NPs increased to 50 µg / mL, the number of NSCs did not increase compared to the blank group on the first day, and was significantly lower than the blank group (0 μg / mL) and the low-concentration groups (5 µg / mL, 25 µg / mL). Therefore, a concentration of 25 µg / mL was selected.

[0079] (2) To demonstrate the good biocompatibility and high targeting ability of the miR-34@ZIF-8-SCM delivery system to NSCs. 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 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 times (30 minutes and 5 hours), and then the distribution of miR-34 nanoparticles in the cells was observed with a confocal microscope ( Figure 9 ). After incubating NSCs with miR-34@ZIF-8 nanoparticles for 30 minutes, red fluorescence (Cy5) was hardly observed in NSCs, and red fluorescence (Cy5) was only observed in NSCs after incubating for 5 hours. 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 indicates that miR-34@ZIF-8-SCM nanoparticles can be successfully absorbed by NSCs, and the encapsulation of the 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 coated with NSCs membrane is more easily internalized and aggregated by NSCs ( Figure 10 ).

[0080] (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 and neural stem cells were co-incubated for 30 minutes or 5 hours respectively. miR-34 was labeled with Cy5 dye, the neural stem cell membrane was labeled with Dio dye, and lysosomes were labeled with a lysosome tracer (Thermo Fisher). Under confocal microscopy, at 30 minutes, a small amount of miR-34 could be observed in lysosomes, and miR-34 and the neural stem cell membrane were co-localized in lysosomes. After 5 hours, miR-34 was released from lysosomes, and the neural stem cell membrane, miR-34, and lysosomes were all separated. The results showed that the miR-34@ZIF-8-SCM nanoparticles had ideal acid sensitivity and exhibited a pH- and time-dependent miR-34 release pattern. The miR-34@ZIF-8-SCM nanoparticles were encapsulated in membrane-bound vesicles through endocytosis of cells. Then, the vesicles containing miR-34@ZIF-8-SCM nanoparticles could be transported to lysosomes. Therefore, the miR-34@ZIF-8-SCM nanoparticles were successfully internalized into NSCs and accumulated in lysosomes. Subsequently, the acidic environment of lysosomes triggered the dissociation of hydrogen bonds between miR-34 and ZIF-8, resulting in the release of miR-34 from the endosome-lysosome pathway and entering the cellular mechanism, further accelerating neuronal differentiation ( Figure 11 ).

[0081] Experimental Example: Differentiation of Neural Stem Cells

[0082] (1) Observe the rate of miR-34 release of miR-34@ZIF-8-SCM prepared in step (3) of Example 1 and miR-34@ZIF-8 prepared in step (2) of Example 1 in neural stem cells. miR-34@ZIF-8 and miR-34@ZIF-8-SCM nanoparticles were incubated at 4 °C in Tris−HCl (pH 5.0). 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 obtained are shown in Figure 12 .

[0083] (2) q-PCR was used to evaluate the acceleration of NSCs differentiation into neurons by miR-34@ZIF-8-SCM

[0084] 10 7Individual / mL NSCs (isolated from 13-15-day-old C57 mouse embryos) were respectively placed 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 (25 μg / mL) prepared in step (1) of Example 1, miR-34@ZIF-8 (25 μg / mL) prepared in step (2) of Example 1, and miR-34@ZIF-8-SCM nanoparticles (25 μg / mL) prepared in step (3) of Example 1 for 5 days or 10 days, and then RNA was isolated from the NSCs using TRIzol reagent (Invitrogen). The concentration and purity of the 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 genes related to neurogenesis, including Nestin, Tuj1, MAP2, and GFAP. The relative transcriptional levels of the target gene expression were normalized with Actin and expressed as mean ± SD (n = 3).

[0085] Quantitative analysis of the expression of NSCs marker gene Nestin, neuron marker class III β-tubulin (Tuj1, an early indicator of neuronal differentiation), neuron marker microtubule-associated protein 2 (MAP2, an indicator of mature neuron differentiation), and astrocyte marker glial fibrillary acidic protein (GFAP) was performed by real-time fluorescence PCR. As shown in the figure, on day 5, 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-fold. When comparing the expression of Tuj1, which represents early neuronal differentiation, on day 5, the culture of NSCs with miR-34@ZIF-8-SCM nanoparticles, miR-34@ZIF-8 nanoparticles, and ZIF-8 nanoparticles was approximately 2.0-fold, 1.2-fold, and 0.9-fold that of the blank group, respectively. The expression of mature neuron MAP2 in the culture of NSCs with miR-34@ZIF-8-SCM nanoparticles, miR-34@ZIF-8 nanoparticles, and ZIF-8 nanoparticles was approximately 2.3-fold, 1.2-fold, and 1.1-fold that of the blank group, respectively. The expression of astrocyte representative gene GFAP in the culture of NSCs with miR-34@ZIF-8-SCM nanoparticles, miR-34@ZIF-8 nanoparticles, and ZIF-8 nanoparticles was approximately 0.7-fold, 0.8-fold, and 1.3-fold that of the blank group, respectively. After 10 days of culture, the gene expression of Tuj1 and MAP2 was significantly up-regulated in the co-culture group of miR-34@ZIF-8-SCM nanoparticles and NSCs, while the gene expression of GFAP decreased instead. The results showed that miR-34@ZIF-8-SCM nanoparticles could effectively accelerate the neuronal differentiation of NSCs and inhibit the glial differentiation of NSCs. The obtained results are shown in Figure 13 , Figure 14 .

[0086] (3) Evaluation of the acceleration of NSCs differentiation into neurons by miR-34@ZIF-8-SCM through immunofluorescence staining

[0087] To more intuitively show the differentiation of NSCs, immunofluorescence staining was also performed on cells co-cultured for 5 days. After NSCs were co-cultured with blank (containing neurobasal 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 on glass slides for 5 days, the glass slides were fixed with 4% paraformaldehyde for 15 minutes and then washed three times with PBS. Then, they 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 glass slides of the blank group, ZIF-8 nanoparticle group, miR-34@ZIF-8 group, and miR-34@ZIF-8-SCM nanoparticle group were incubated overnight at 4°C with primary antibodies against MAP2 (mouse polyclonal anti-MAP2), Tuj1 (mouse monoclonal anti-Tuj1), or GFAP (rabbit polyclonal anti-GFAP). The samples were incubated with Alexa Fluor 488 secondary antibody and Alexa Fluor 594 secondary antibody at 37°C for 1 hour. Then, the cells were washed three times with PBS and then treated with DAPI for 10 min to stain the cell nuclei. Images of the stained samples were obtained by a laser confocal microscope.

[0088] As Figure 15 shown, NSCs cultured with the miR-34@ZIF-8-SCM nanoparticle group showed stronger expression of the neuronal-specific markers Tuj1 and MAP2 and had multiple neurites, significantly more than those in other groups (the blank group, ZIF-8 nanoparticles, and miR-34@ZIF-8 nanoparticle group). This figure also demonstrated that miR-34@ZIF-8-SCM nanoparticles effectively promoted the direct differentiation of NSCs into neurons rather than glial cells, which was consistent with the results of RT-qPCR.

[0089] (4) Western Blot assessment of the acceleration of NSCs differentiation into neurons by miR-34@ZIF-8-SCM

[0090] To further verify the promoting effect of miR-34@ZIF-8-SCM nanoparticles on the differentiation of neural stem cells into neurons, 10 7NSCs per mL were placed in 6-well plates containing the blank group (containing neural basal 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) and cultured for 5 days. Then, total proteins were isolated from the NSCs using cell lysis buffer containing protease and phosphatase inhibitors. Proteins were also isolated from untreated NSCs cultured on the blank group as a control. The protein concentration was determined using a BCA protein assay kit (Cusabio). Then, 5× protein buffer (ComWin Biotech) was added to each sample, and the proteins were separated by SDS−PAGE. Then, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and blocked with blocking 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, soaked with chemiluminescent HPR substrate (brand Thermo Fisher), and scanned using a fluorescence chemistry HD2 system (ProteinSimple, USA).

[0091] As Figure 16 shown, the protein expression levels of Tuj1 and MAP2 in the miR-34@ZIF-8-SCM nanoparticle group were significantly higher than those in other groups, and there was little difference in the protein expression level of GFAP between the miR-34@ZIF-8-SCM group and other groups (blank group, ZIF-8 nanoparticle group, and miR-34@ZIF-8 nanoparticle group). Meanwhile, to more precisely determine the types of functional neurons, the protein expression of functional neurons in NSCs of each group at 10 days was analyzed by Western blot. TH, as a specific marker of dopaminergic neurons, is widely used to identify and study dopaminergic neurons in the central nervous system. Therefore, the protein expression level of TH was also measured. Finally, as Figure 16 shown, the protein expression level of TH in the miR-34@ZIF-8-SCM nanoparticle group was significantly higher than that in other groups.

[0092] (5) Calcium spark assessment of the acceleration of NSC differentiation into functional neurons by miR-34@ZIF-8-SCM

[0093] An important indicator of the accelerated successful directed differentiation of NSCs into neurons mediated by miR-34@ZIF-8-SCM nanoparticles is the generation of functional neurons. Calcium spark detection was performed on NSCs incubated with miR-34@ZIF-8-SCM nanoparticles on the 5th day. After treating NSCs with miR-34@ZIF-8-SCM NPs for 5 days, the cells were stained with 200 μL of the working solution of the calcium fluorescent probe (Flu-4AM) (2 μM, diluted with PBS), and then incubated at 37 °C for 20 minutes. After treating the cells with dopamine neurotransmitter, the change in fluorescence intensity was recorded with a confocal microscope and analyzed with ImageJ software.

[0094] The fluorescence intensity of calcium in the cells was low in the resting state, as Figure 17 shown. When exposed to an appropriate amount of neurotransmitter, such as dopamine (DA), the intracellular Ca 2+ fluorescence increased rapidly within a short time and then returned to the resting state over time. These results indicate that the derived neurons generated by miR-34@ZIF-8-SCM nanoparticles stimulating NSCs are functional neurons with electrophysiological properties.

[0095] 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 can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. Zinc-based nano-organic framework coating material loaded with microRNA, characterized in that, The zinc-based nano-organic framework coating material loaded with microRNA includes a zinc-based nano-organic framework material; microRNA is loaded on the zinc-based nano-organic framework material, 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 is derived from neural stem cells. The zinc-based nano-organic framework coating material loaded with microRNA is prepared by the following method: (1) Disperse ZIF-8 nanoparticles in PBS solution, add microRNA-34 and mix evenly, incubate overnight at low temperature, then centrifuge and dry to obtain miR-34@ZIF-8; the weight ratio of microRNA-34 to ZIF-8 is 1:

40. (2) At low temperature, neural stem cells are lysed with a hypotonic lysis solution. The lysed solution is centrifuged once at low temperature, and the supernatant is taken and centrifuged twice at low temperature to collect the precipitate to obtain the cell membrane of neural stem cells. The cell membrane is added to the PBS solution of miR-34@ZIF-8 and subjected to ultrasonic treatment. After co-extrusion, miR-34@ZIF-8-SCM is obtained, which is the zinc-based nano-organic framework coating material loaded with microRNA.

2. The zinc-based nano-organic framework coating material loaded with microRNA according to claim 1, wherein, In step (1), the temperature for incubating overnight at low temperature is 4 °C; the rotation speed of the centrifuge is 3000 rpm.

3. The zinc-based nano-organic framework coating material loaded with microRNA according to claim 1, characterized in that, In step (2), the temperature of the low temperature is 4 °C; the lysis time is 4 h.

4. The zinc-based nano-organic framework coating material loaded with microRNA according to claim 1, characterized in that, In step (2), the first low-temperature centrifugation is centrifugation at 3000 rpm for 5 min at 4 °C; the second low-temperature centrifugation is centrifugation at 15000 rpm for 30 min at 4 °C.

5. The zinc-based nano-organic framework coating material loaded with microRNA according to claim 1, wherein, 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 to use a micro co-extruder to pass through polycarbonate membranes with pore sizes of 400 nm and 200 nm in sequence.

Images