A transnasal brain-targeting nanosphere loaded with miR-195, its preparation method and application

Abstract

This invention relates to a transnasal brain-targeting nanosphere loaded with miR-195, its preparation method, and its applications, belonging to the field of biomedical technology. To address the limitations of existing drug delivery carriers in simultaneously achieving efficient nasal delivery to the brain, precise targeting of Aβ lesions, and stable loading of nucleic acid drugs such as miR-195, this invention provides a transnasal brain-targeting nanosphere loaded with miR-195. From the inside out, it comprises a drug-loaded core, a biomimetic cell membrane shell, and a dual-ligand functionalized surface canopy. The miR-195 loading in the nanosphere is 0.2–0.8% w / w. This invention's nanospheres deliver directly to the lesion via the nose. The cationic GO-Cl4 and neutral polymer synergistically protect miR-195 from degradation. Dual-ligand modification enables naso-brain barrier penetration and Aβ targeting, improving treatment precision and providing an effective treatment option for Alzheimer's disease, with broad clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a transnasal brain-targeting nanosphere loaded with miR-195, its preparation method and application. Background Technology

[0002] Abnormal deposition of amyloid-β (Aβ) is a core pathological feature of Alzheimer's disease and many other central nervous system degenerative diseases. Its accumulation in the brain induces neuroinflammation and neuronal apoptosis, ultimately leading to cognitive impairment and memory decline, severely threatening patients' quality of life and becoming a major medical challenge urgently needing to be addressed in the global aging society. MicroRNAs (miRNAs) have emerged as a highly promising therapeutic strategy due to their ability to specifically regulate the expression of target genes related to Aβ production and clearance. Among them, miR-195 has been shown to significantly reduce Aβ deposition in the brain and alleviate neurological damage by inhibiting the expression of Aβ precursor protein (APP) and β-secretase (BACE1), making it a key active molecule for treating Aβ-related central nervous system diseases. However, as a nucleic acid drug, miR-195 is easily degraded by nucleases and rapidly cleared by the kidneys, requiring stable delivery via drug delivery carriers.

[0003] However, traditional intravenous injection and oral administration methods have low brain penetration efficiency and significant toxic side effects. Nasal-brain targeted delivery, due to its non-invasive nature, ability to directly bypass the blood-brain barrier, and reduced systemic toxicity, has become a research hotspot in this field of drug delivery systems, providing an ideal route for the delivery of nucleic acid drugs such as miR-195.

[0004] Some studies have used Aβ-binding peptides such as KLVFF to modify the surface of liposomes to construct nasal drug delivery systems, attempting to achieve drug delivery to the brain and direct targeting of Aβ plaques via the nasal route (Chemical Engineering Journal, 2024, 494, 153210). This system achieves targeting through hydrogen bonding between KLVFF and Aβ, which improves lesion affinity to some extent. However, its carrier structure design is only suitable for the physical encapsulation and stable delivery of hydrophobic small molecules or neutral nanozymes. For hydrophilic nucleic acid macromolecules with strong negative charges, such as miR-195, electrostatic repulsion and steric hindrance make it difficult to stably encapsulate nucleic acid drugs within liposomes, resulting in extremely low encapsulation efficiency and a high risk of drug leakage. Furthermore, this system lacks protective designs against ribonucleases, and miR-195 is easily and rapidly degraded in the nasal mucus and epithelial microenvironment, losing its biological activity. Therefore, this system cannot achieve efficient, stable, and functional delivery of miR-195.

[0005] The mucus penetration, enzyme stability, and long retention characteristics required by the nasal mucosal barrier are fundamentally contradictory to the high-density, high-activity targeting ligands required for Aβ targeting in the brain, which necessitates modifications to the carrier surface. Existing drug delivery carriers, when administered nasally, cannot simultaneously achieve efficient nasal transport to the brain, precise targeting of Aβ lesions, and stable loading of nucleic acid drugs such as miR-195, which greatly limits the precision treatment of Alzheimer's disease. Summary of the Invention

[0006] To address the limitations of existing drug delivery carriers in achieving efficient nasal delivery to the brain, precise targeting of Aβ lesions, and stable loading of nucleic acid drugs such as miR-195, this invention provides a transnasal brain-targeting nanosphere loaded with miR-195, its preparation method, and its applications.

[0007] The technical solution of this invention:

[0008] A transnasal brain-targeting nanosphere loaded with miR-195, the nanosphere having a core-shell-corona three-layer hierarchical structure, consisting of, from the inside out: a drug-loaded core, a biomimetic cell membrane shell, and a dual-ligand functionalized surface corona; the drug-loaded core is composed of the neutral polymer PLGA, the cationic organic compound GO-C14, and miR-195; the biomimetic cell membrane shell is composed of a bone marrow mesenchymal stem cell membrane and tightly encapsulates the surface of the drug-loaded core; covalently coupled... The polyethylene glycol-modified phospholipids of lecithin and the covalently coupled Aβ-targeting peptide KLVFF are spontaneously inserted into the lipid bilayer of the biomimetic cell membrane shell through hydrophobic fragments, while the hydrophilic polyethylene glycol chains extend to the outer side of the membrane, anchoring lecithin and Aβ-targeting peptide KLVFF to the surface of the biomimetic cell membrane shell, forming a dual-ligand functionalized surface canopy. The drug loading of miR-195 in the nanospheres is 0.2~0.8% w / w.

[0009] Furthermore, the nanospheres have a particle size of 140~180nm and a surface potential of -7.0~-9.0mV.

[0010] A method for preparing transnasal brain-targeting nanospheres loaded with miR-195 includes the following steps: Step 1: Dissolve the polyethylene glycol-modified phospholipid activator DSPE-PEG-NHS in organic solvent I, add leek lectin and buffer solution, mix and stir to react, then dialyze and dry to obtain leek lectin-modified polyethylene glycol phospholipid. The maleimide derivative of polyethylene glycol phospholipid, DSPE-PEG-Mal, was dissolved in organic solvent II. Aβ-targeting peptide KLVFF and a catalyst were added, and after mixing and stirring, the mixture was dialyzed and dried to obtain polyethylene glycol phospholipid modified with Aβ-targeting peptide KLVFF. Step 2: Control the dosage according to the mass ratio of cationic organic compound G0-C14, miR-195, neutral polymer PLGA, bone marrow mesenchymal stem cell membrane, pecan lectin-modified polyethylene glycol phospholipid and Aβ-targeting peptide KLVFF-modified polyethylene glycol phospholipid. Neutral polymer PLGA was dissolved in an organic solvent and mixed with cationic organic compound G0-C14 as the organic phase. miR-195 solution and the first part of Tris-HCl solution were added as the aqueous phase. After ultrasonic dispersion, an initial emulsion was formed. The second part of Tris-HCl solution and dichloromethane solution were added to the initial emulsion, and ultrasonic emulsification was continued to obtain a shaped emulsion. The third part of Tris-HCl solution was added to the shaped emulsion, and the mixture was stirred, centrifuged, and washed to obtain a drug-loaded core composed of PLGA / G0-C14 / miR-195 complex. The obtained drug-loaded core was suspended in a bone marrow mesenchymal stem cell membrane solution and treated with water bath so that the bone marrow mesenchymal stem cell membrane tightly coated the surface of the drug-loaded core. After centrifugation and washing, a drug-loaded core coated with a biomimetic cell membrane shell was obtained. The drug-loaded core coated with the obtained biomimetic cell membrane shell, the polyethylene glycol phospholipid modified with lectin obtained in step one, and the polyethylene glycol phospholipid modified with Aβ-targeting peptide KLVFF were co-incubated. The lectin and Aβ-targeting peptide KLVFF were anchored and modified on the outer side of the biomimetic cell membrane shell, and assembled to form a dual-ligand functionalized surface crown, resulting in a transnasal brain-targeting nanosphere loaded with miR-195 with a core-shell-crown three-layer hierarchical structure.

[0011] Furthermore, the organic solvent I mentioned in step one is dimethylformamide, and the buffer solution includes phosphate buffer and carbonate buffer; the mass-to-volume ratio of DSPE-PEG-NHS, dimethylformamide, lecithin, phosphate buffer and carbonate buffer is 400~600mg:4~6mL:1~3mg:1~3mL:0.5~2mL, the mixing and stirring reaction time is 0.5~1.5h, the dialysis is carried out sequentially with phosphate buffer and pure water, the total dialysis time is 7.5~8.5h, and the drying method is freeze drying.

[0012] Furthermore, in step one, the organic solvent II is chloroform, the Aβ-targeting peptide KLVFF is dissolved in methanol, and the catalyst is triethylamine; the mass-volume ratio of DSPE-PEG-Mal, chloroform, Aβ-targeting peptide KLVFF, methanol, and triethylamine is 400~600mg:5~15mL:3~5mg:1~3mL:0.5~2mL, the mixing and stirring reaction is carried out under argon protection, the reaction time is 19~25h, the dialysis time is 2.5~4.5h, and the drying method is freeze drying.

[0013] Furthermore, in step two, the mass ratio of the cationic organic compound G0-C14, miR-195, neutral polymer PLGA, bone marrow mesenchymal stem cell membrane, pecan lectin-modified polyethylene glycol phospholipid, and Aβ-targeting peptide KLVFF-modified polyethylene glycol phospholipid is 1:0.2:50:25.5:5.31:10.62, 1.5:0.5:40:20.5:4:8, or 2:1:60:29.5:8:12.

[0014] Furthermore, in step two, the organic solvent is dichloromethane, and the mass-to-volume ratio of the neutral polymer PLGA to dichloromethane is 40-60 mg: 0.5-2 mL; the concentration of the miR-195 solution is 1 mg / mL; the first portion of Tris-HCl solution is 1 M concentration, and the amount added is 1-4% v / v of the volume of the organic phase; the second portion of Tris-HCl solution is 10 mM concentration, and the amount added is 50-100% v / v of the volume of the initial emulsion; the third portion of Tris-HCl solution is 10 mM concentration, and the amount added is 100-200% v / v of the volume of the formed emulsion.

[0015] Furthermore, in step two, the ultrasonic power for both ultrasonic dispersion and ultrasonic emulsification is 700W, and the processing method is alternating on / off for 2 seconds; the stirring speed is 700rpm and the stirring time is 6h; the centrifugation speed is 10000g and the centrifugation time is 5min, and ultrapure water is used for washing.

[0016] Furthermore, in step two, the ultrasonic frequency of the water bath ultrasonic treatment is 42kHz, the ultrasonic power is 100W, and the ultrasonic time is 2min; the co-incubation time is 25~35min.

[0017] Application of a transnasal brain-targeting nanosphere loaded with miR-195 in the preparation of a drug for treating Alzheimer's disease.

[0018] The beneficial effects of this invention are:

[0019] The drug-loaded nanospheres with a core-shell-corona three-layer hierarchical structure provided by this invention achieve highly efficient and stable loading of miR-195, effectively resisting degradation and clearance, and solving the problem of nucleic acid drug delivery. In the drug-loaded core, the cationic organic compound G0-C14 and the strongly negatively charged miR-195 form a stable complex through electrostatic interaction. Combined with the encapsulation and protection effect of the neutral polymer PLGA, this effectively prevents miR-195 from being degraded by nasal mucus and nucleases in vivo, while reducing rapid renal clearance, significantly improving the circulatory stability and bioavailability of miR-195 in vivo. Moreover, the drug loading is controlled at 0.2~0.8% w / w, which can ensure the effective dose required for treatment and avoid the toxic side effects caused by drug overdose, achieving long-term stable delivery of miR-195.

[0020] Meanwhile, the lectin in the canopy of the dual-ligand functionalized surface of the nanospheres can specifically recognize glycoprotein receptors on the surface of nasal mucosal epithelial cells, mediating the efficient penetration of the nanospheres through the nasal mucosal barrier and directly bypassing the blood-brain barrier, thus achieving rapid and efficient transport of miR-195 from the nasal cavity to the brain. Compared with traditional intravenous injection and oral administration, this nasal delivery route is non-invasive, has no first-pass effect in the liver, significantly reduces systemic toxicity, and significantly improves the efficiency of drug entry into the brain, solving the key problems of difficult brain entry and low bioavailability of nucleic acid drugs. The Aβ-targeting peptide KLVFF in the dual-ligand-functionalized surface canopy can specifically bind to abnormally deposited Aβ plaques in the brain via hydrogen bonds. Combined with the brain-targeting effect of gentian lectin, it forms a dual targeting mechanism of "nasal mucosal penetration - brain targeting - precise binding to Aβ lesions". This allows the nanospheres to be efficiently enriched in the Aβ deposit area in the brain after nasal administration, specifically acting on neurons and microglia around the Aβ plaques. This avoids the indiscriminate distribution of the drug in normal brain tissue, significantly improves the precision of treatment, and reduces damage to normal brain tissue.

[0021] The biomimetic cell membrane shell is composed of bone marrow mesenchymal stem cell membrane, which not only has good biocompatibility and biodegradability, reducing the immunogenicity of the carrier itself, but also further protects miR-195 in the drug-carrying core and assists the nanospheres in escaping the body's clearance system. The core-shell-corona trilayer structure works together and synergistically enhances the effect, solving the problem of stable miR-195 loading and achieving the dual goals of efficient transnasal delivery to the brain and precise targeting of Aβ lesions. Ultimately, miR-195 can play an efficient role in the Aβ deposition area by inhibiting the expression of Aβ precursor protein (APP) and promoting the activity of Aβ degrading enzymes, significantly reducing Aβ deposition in the brain, alleviating neuroinflammation and neuronal apoptosis, improving cognitive dysfunction and memory decline, and showing significant therapeutic effects on Alzheimer's disease.

[0022] The nanospheres of this invention exhibit excellent biocompatibility, a simple and controllable preparation process, and can be mass-produced. They combine safety, effectiveness, and economy, meeting the needs of clinical treatment and possessing broad prospects for clinical promotion and practical application value. They provide new technical solutions and product support for the precision treatment of Alzheimer's disease. Attached Figure Description

[0023] Figure 1 This is a comparison chart of the in vitro release rates of the four drug-loaded nanospheres in Experiment Example 1;

[0024] Figure 2 This is a confocal image showing the uptake of four drug-loaded nanospheres by SH-SY5Y cells in Experiment Example 1.

[0025] Figure 3 This is a comparison of the dynamic changes in the relative expression levels of miR-195 in the hippocampus and cortex of mice in the drug-treated group and the model control group in Experiment Example 2.

[0026] Figure 4 This is a comparison of the escape latency changes of APP / PS1 transgenic model mice in each group during the training period in the Barnes maze experiment in Experiment Example 3;

[0027] Figure 5 This is a comparison of the escape latency of APP / PS1 transgenic mouse models in different groups on the test day of the Barnes maze experiment in Example 3;

[0028] Figure 6 This is a comparison of the escape latency changes of 5×FAD transgenic model mice in each group during the training period in the Barnes maze experiment in Experiment Example 3;

[0029] Figure 7 This is a comparison of the escape latency of 5×FAD transgenic mice in each group on the test day of the Barnes maze experiment in Example 3. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0031] Example 1

[0032] This embodiment provides a method for preparing transnasal brain-targeting nanospheres loaded with miR-195.

[0033] In this embodiment, the PEGylated phospholipid activator was DSPE-PEG2000-NHS, with a polyethylene glycol molecular weight of 2000; the PEGylated phospholipid maleimide derivative was DSPE-PEG2000-Mal, with a polyethylene glycol molecular weight of 2000; and the targeting peptide was Aβ-targeting peptide KLVFF. All three materials were purchased from Xi'an Qiyue Biotechnology Co., Ltd.; the lectin was Vitex lecithin (UEA-I), purchased from Xinbosheng Biotechnology Co., Ltd.; miR-195 mimics (miR-195 mimics: chemically synthesized double-stranded RNA with a structure identical to endogenous mature miRNA, which causes overexpression of miRNA in cells after transfection) were purchased from Jiangsu Saisofe Biotechnology Co., Ltd. (Jiangsu, China); the cationic organic compound was G0-C14, purchased from MedChemExpress (Shanghai) Co., Ltd.; and the neutral polymer was polylactic-co-glycolic acid copolymer (PLGA), purchased from Jinan Daigang Bioengineering Co., Ltd.

[0034] In this embodiment, the cell membrane material was a mouse bone marrow mesenchymal stem cell membrane, which was prepared using cell membrane extraction reagent A (P0033-1 kit) purchased from Shanghai Beyotime Biotechnology Co., Ltd. The preparation process is as follows:

[0035] C57BL / 6 mice were euthanized by cervical dislocation after isoflurane anesthesia, disinfected by immersion in 75% alcohol for 5 minutes, and transferred to a laminar flow hood for aseptic processing. The femur and tibia were separated, and soft tissues were removed. The cells were placed in DMEM / F12 (1:1) medium containing 1% penicillin-dextrose antibody. The epiphyses at both ends of the bones were cut off. Using a 1 mL sterile syringe, the culture medium was drawn up and the bone marrow cavity was flushed bidirectionally three times each. Cell suspension was prepared by pipetting and collected in 15 mL sterile centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, 3 mL of erythrocyte lysis buffer was added, and the cells were incubated at room temperature for 5 minutes. The cells were centrifuged again at 1000 rpm for 5 minutes, and the supernatant was discarded. 10 mL of DMEM / F12 medium containing 15% fetal bovine serum was added to prepare a suspension, and the suspension was prepared by centrifugation at 1×10⁻⁶. 4 / cm 2 Cells were seeded in T75 culture flasks and cultured at 37°C with 5% CO2. The medium was changed at 48h and 96h. Cells were passaged at a 1:3 ratio on day 6 and collected after 3 passages for later use.

[0036] Third-generation bone marrow mesenchymal stem cells were harvested and their concentration adjusted to 3 × 10⁻⁶. 7Add 1 mL of cell membrane extraction reagent A to each sample, resuspend thoroughly, and incubate on ice for 15 min. Quickly freeze in liquid nitrogen for 3 s, thaw at room temperature for 30 s, repeating this freeze-thaw cycle 3 times. Centrifuge at 700 g for 10 min and collect the supernatant containing cell membrane fragments. Centrifuge at 14000 g for 30 min to precipitate the cell membrane fragments, discard the supernatant, and repeat the process 3 × 10⁻⁶ times. 7 Approximately 240 μg of membrane proteins can be extracted from each cell. The cell membrane fragments were resuspended in sterile PBS buffer (pH 7.4) and the concentration was adjusted to 25.5 mg / mL to prepare a solution containing bone marrow mesenchymal stem cell membranes. The solution was stored at 4°C.

[0037] Although this invention uses mouse bone marrow mesenchymal stem cell membranes extracted from mouse bone marrow as an example for verification, the technical solution has cross-species applicability and is equally applicable to human bone marrow mesenchymal stem cell membranes. According to the International Society for Cell Therapy (ISCT) standards, mesenchymal stem cells highly express key surface markers such as CD73, CD90, and CD105. These membrane proteins determine the high similarity of their interfacial properties during physical encapsulation. The immune escape function and homing effect of mesenchymal stem cell membranes, unique to them, are highly conserved in mammalian evolution, and their cross-species biological functions are predictable. The extraction-encapsulation process used in this invention does not depend on the cell biological activity of a specific species; therefore, the encapsulation ratio and physicochemical properties verified on mouse bone marrow mesenchymal stem cell membranes can be equivalently derived to human membrane carriers.

[0038] The preparation method of transnasal brain-targeting nanospheres loaded with miR-195 in this embodiment is as follows:

[0039] Step 1: Preparation of polyethylene glycol-modified phospholipids modified with leek lectin and polyethylene glycol-modified phospholipids modified with Aβ-targeting peptide KLVFF:

[0040] 500 mg of DSPE-PEG2000-NHS was dissolved in 5 mL of dimethylformamide, and 2 mg of lecithin, 2 mL of phosphate buffer and 1 mL of carbonate buffer were added to adjust the pH to 9. The mixture was stirred at room temperature for 1 h. The resulting reaction solution was dialyzed against phosphate buffer for 4 h and then against pure water for 4 h. The solution was then freeze-dried to obtain lecithin-modified polyethylene glycol phospholipid DSPE-PEG2000-UEA-I.

[0041] Under argon protection, 500 mg of DSPE-PEG2000-Mal was dissolved in 10 mL of chloroform to obtain solution I, and 4 mg of Aβ-targeting peptide KLVFF was dissolved in 2 mL of methanol to obtain solution II. Solutions I and II were mixed and 1 mL of triethylamine was added. The mixture was stirred at room temperature for 24 h, and the solvent was evaporated. The resulting product was dissolved in chloroform, dialyzed for 4 h, and freeze-dried to obtain PEGylated phospholipid DSPE-PEG2000-KLVFF modified with Aβ-targeting peptide KLVFF.

[0042] Step 2: Preparation of transnasal brain-targeting nanospheres loaded with miR-195:

[0043] The dosage was controlled according to the mass ratio of G0-C14, miR-195, PLGA, bone marrow mesenchymal stem cell membrane, DSPE-PEG2000-UEA-I and DSPE-PEG2000-KLVFF as 1:0.2:50:25.5:5.31:10.62.

[0044] The miR-195 mimics lyophilized powder was dissolved in RNase-free water to prepare a miR-195 mimics solution with a concentration of 1 mg / mL.

[0045] Weigh 50 mg PLGA and dissolve it in 1 mL of dichloromethane solution. Vortex for 2 min to completely dissolve it to obtain a PLGA dichloromethane solution with a concentration of 50 mg / mL. Take 200 μL of PLGA dichloromethane solution and mix it with 200 μg GO-C14 as the organic phase. Then add 41.25 μL of miR-195 mimics solution and 10 μL of 1M Tris-HCl (pH 7.4) solution to the organic phase as the aqueous phase. Use an ultrasonic cell disruptor to disperse the mixture at 700 W ultrasonic power, alternating between on and off for 2 s, for a total duration of 1 min to form an initial emulsion.

[0046] 1M Tris-HCl (pH 7.4) was added to the system as the first Tris-HCl solution to construct a stable weakly alkaline buffer environment, preventing miR-195 from degrading and unwinding under acidic conditions, thus protecting the structural integrity of the miRNA. On the other hand, the neutral to slightly alkaline environment (pH 7.4) allows G0-C14 to maintain a moderate positive charge, which can efficiently adsorb and recombine with the negatively charged miR-195 through electrostatic interactions, forming a stable G0-C14 / miRNA complex.

[0047] 2.5 mL of 10 nM Tris-HCl (pH 7.4) solution and 100 μL of dichloromethane solution were added to the initial emulsion. Ultrasonic emulsification was performed at 700 W, alternating between on and off cycles for a total duration of 2 min. This process was repeated twice to obtain a shaped emulsion. A second portion of Tris-HCl solution was added to the initial emulsion as an aqueous buffer system to further stabilize the pH environment and prevent sudden changes in local pH caused by continuous evaporation of the organic phase and vigorous mixing of the two phases, thus maintaining the structural stability of miR-195. Simultaneously, by adjusting the ratio of the aqueous to organic phases, the interfacial tension was optimized for secondary ultrasonic emulsification, which is more conducive to the formation of smaller and more uniformly distributed nanodroplets. This allows for preliminary morphological shaping before complete solidification of the nanoparticles, improving the uniformity and structural integrity of the final nanoparticles.

[0048] 5 mL of 10 mM Tris-HCl (pH 7.4) solution was added to the resulting emulsion, and the mixture was magnetically stirred at 700 rpm for 6 h. Finally, the mixture was centrifuged at 10000 g for 8 min and washed twice with ultrapure water to obtain a drug-loaded core composed of nanospheres of the PLGA / G0-C14 / miR-195 complex. A third portion of Tris-HCl solution was added to the emulsion to further dilute the system. Lowering the organic solvent concentration facilitated the volatilization of dichloromethane, driving the PLGA / G0-C14 / miR-195 complex to gradually precipitate from the organic phase and solidify into dense, solid nanoparticles. The large volume of aqueous phase, under gentle stirring, effectively inhibited nanoparticle aggregation, ensuring particle size uniformity and dispersion stability. Throughout the preparation process, the Tris-HCl solution maintained a near-neutral physiological pH environment in the system, protecting the structural integrity and drug loading efficiency of the miRNA.

[0049] The obtained drug-loaded core was suspended in 1 mL of a solution containing a bone marrow mesenchymal stem cell membrane. The mixture was subjected to water bath sonication at a frequency of 42 kHz and a power of 100 W for 2 min to allow the bone marrow mesenchymal stem cell membrane to tightly coat the surface of the drug-loaded core, forming a biomimetic cell membrane shell. The drug-loaded core coated with the biomimetic cell membrane shell was collected by centrifugation at 10000 g for 5 min, washed twice with ultrapure water, and set aside for later use.

[0050] The obtained biomimetic cell membrane shell-coated drug-loaded core, DSPE-PEG2000-UEA-I and DSPE-PEG2000-KLVFF obtained in step one were co-incubated for 30 min. During co-incubation, DSPE-PEG2000-UEA-I and DSPE-PEG2000-KLVFF spontaneously inserted into the lipid bilayer of the biomimetic cell membrane shell through hydrophobic DSPE fragments, while the hydrophilic PEG chains extended to the outer side of the membrane as spatial arms, anchoring lectins and targeting peptides to the outer side of the biomimetic cell membrane shell, assembling to form a dual-ligand functionalized surface crown, resulting in transnasal brain-targeting nanospheres BMUK@PL / G0-195 with a core-shell-crown three-layer hierarchical structure and loaded with miR-195. The obtained transnasal brain-targeting nanospheres loaded with miR-195 were suspended in 10% sucrose at a drug-loaded core concentration of 10 mg / mL and stored at 4℃ for later use.

[0051] Example 2

[0052] The only difference between this embodiment and Example 1 is that step two, preparing transnasal brain-targeting nanospheres loaded with miR-195:

[0053] The dosage was controlled according to the mass ratio of G0-C14, miR-195, PLGA, bone marrow mesenchymal stem cell membrane, DSPE-PEG2000-UEA-I and DSPE-PEG2000-KLVFF as 1.5:0.5:40:20.5:4:8.

[0054] Example 3

[0055] The only difference between this embodiment and Example 1 is that step two, preparing transnasal brain-targeting nanospheres loaded with miR-195:

[0056] The dosage was controlled according to the mass ratio of G0-C14, miR-195, PLGA, bone marrow mesenchymal stem cell membrane, DSPE-PEG2000-UEA-I and DSPE-PEG2000-KLVFF as 2:1:60:29.5:8:12.

[0057] Experimental Example 1

[0058] To detect the encapsulation efficiency, in vitro release, and cellular uptake of transnasal brain-targeting nanospheres loaded with miR-195, this study prepared drug-loaded nanospheres with different modifications using Cy3-miR-195 as the fluorescently labeled nucleic acid.

[0059] Cy3-miR-195 was purchased from Jiangsu Cysobio Biotechnology Co., Ltd. (Jiangsu, China). When preparing fluorescently labeled nanospheres, the amount of Cy3-miR-195 added was calculated based on the required mass of miR-195 for the experiment to ensure that the actual amount of miR-195 added was consistent with the design value.

[0060] I. Drug-loaded nanospheres BMUK@PL / G0-195-Cy3 modified with UEA-I and KLVFF

[0061] Preparation method of drug-loaded nanospheres modified with lectin and Aβ-targeting peptide:

[0062] The dosage of G0-C14, miR-195, PLGA, bone marrow mesenchymal stem cell membrane, DSPE-PEG2000-UEA-I, and DSPE-PEG2000-KLVFF was controlled at a mass ratio of 1:0.2:50:25.5:5.31:10.62. The only difference from Example 1 was that Cy3-miR-195 was used instead of miR-195 mimics as the loaded drug.

[0063] II. Drug-loaded nanospheres BMU@PL / G0-195-Cy3 modified only with UEA-I

[0064] Preparation method of drug-loaded nanospheres modified with only UEA-I:

[0065] The dosage of G0-C14, miR-195, PLGA, bone marrow mesenchymal stem cell membrane and DSPE-PEG2000-UEA-I was controlled at a mass ratio of 1:0.2:50:25.5:5.31. The difference from Example 1 is that Cy3-miR-195 was used instead of miR-195 mimics as the drug loading core and the drug loading core coated with the biomimetic cell membrane shell.

[0066] The obtained biomimetic cell membrane shell-coated drug-loaded core was co-incubated with DSPE-PEG2000-UEA-I for 30 min to obtain drug-loaded nanospheres modified only with UEA-I. The obtained drug-loaded nanospheres modified only with UEA-I were suspended in 10% sucrose at a drug core concentration of 10 mg / mL and stored at 4℃ for later use.

[0067] III. Drug-loaded nanospheres modified with KLVFF only: BMK@PL / G0-195-Cy3

[0068] Preparation method of drug-loaded nanospheres modified with KLVFF only:

[0069] The dosage of G0-C14, miR-195, PLGA, bone marrow mesenchymal stem cell membrane and DSPE-PEG2000-KLVFF was controlled at a mass ratio of 1:0.2:50:25.5:10.62. The difference from Example 1 is that Cy3-miR-195 was used instead of miR-195 mimics as the drug loading core and the drug loading core coated with the biomimetic cell membrane shell.

[0070] The obtained biomimetic cell membrane shell-coated drug-loaded core was co-incubated with DSPE-PEG2000-KLVFF for 30 min to obtain KLVFF-modified drug-loaded nanospheres. The obtained KLVFF-modified drug-loaded nanospheres were suspended in 10% sucrose at a drug core concentration of 10 mg / mL and stored at 4℃ for later use.

[0071] IV. Unmodified drug-loaded nanospheres BM@PL / G0-195-Cy3

[0072] Preparation method of unmodified drug-loaded nanospheres:

[0073] The dosage of G0-C14, miR-195, PLGA, and bone marrow mesenchymal stem cell membrane was controlled at a mass ratio of 1:0.2:50:25.5. The difference from Example 1 was that Cy3-miR-195 was used instead of miR-195 mimics as the drug loading material to prepare the drug-loaded core and the biomimetic cell membrane shell-coated drug-loaded core, thus obtaining unmodified drug-loaded nanospheres. The obtained unmodified drug-loaded nanospheres were suspended in 10% sucrose at a drug loading core concentration of 10 mg / mL and stored at 4°C for later use.

[0074] V. Drug-carrying core—PLGA / G0-C14 / miR-195-Cy3

[0075] Preparation method of drug-loaded core:

[0076] The dosage of G0-C14, miR-195, and PLGA was controlled at a mass ratio of 1:0.2:50. The difference from Example 1 was that Cy3-miR-195 was used instead of miR-195 mimics as the drug loading material to prepare the PLGA / G0-C14 / miR-195 complex. The resulting unmodified drug-loaded nanospheres were suspended in 10% sucrose at a drug core concentration of 10 mg / mL and stored at 4°C for later use.

[0077] The particle size, zeta potential, encapsulation efficiency, in vitro release rate, and SH-SY5Y cell uptake of the five drug-loaded nanospheres with different modification types were characterized.

[0078] (1) Characterization of nanospheres:

[0079] At room temperature, 100 μL of each group of drug-loaded nanosphere solutions prepared above were taken, diluted with 900 μL of ultrapure water, mixed evenly, and added to a special cuvette of a dynamic light scattering particle size analyzer to test the average particle size and zeta potential of each group of nanospheres.

[0080] (2) Determination of the encapsulation efficiency of nanospheres:

[0081] Dissolve 0.132 mg miR-195-Cy3 in 1 mL of ultrapure water to obtain a miR-195-Cy3 solution with a concentration of 0.132 mg / mL. Add 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 μL of the solution to cuvettes, respectively, and add ultrapure water to a final volume of 3 mL. Measure the fluorescence value at excitation and emission using a fluorescence spectrophotometer at 550 / 570 nm and plot a standard curve.

[0082] Different modified nanospheres PLGA / G0-C14 / miR-195-Cy3, BM@PL / G0-195-Cy3, BMK@PL / G0-195-Cy3, BMU@PL / G0-195-Cy3 and BMUK@PL / G0-195-Cy3 were placed into dialysis bags with a molecular cutoff of 30 KD and dialyzed with 10 ml of DEPC saline as the dialysis fluid to remove free miR-195-Cy3. 10 μL of dialysis-modified nanospheres were added to a special cuvette, and ultrapure water was added to 3 ml. The fluorescence value after dialysis was measured by a fluorescence spectrophotometer at excitation and emission at 550 / 570 nm. The encapsulation efficiency (%) of cy3-miR-195 in the drug-loaded nanospheres was calculated according to the formula: Encapsulation efficiency (%) = (Total amount of cy3-miR-195 added - Amount of cy3-miR-195 in the supernatant) / Total amount of cy3-miR-195 added × 100%.

[0083] The particle size, zeta potential, PDI dispersion coefficient, and encapsulation efficiency of each nanosphere are shown in Table 1.

[0084] Table 1

[0085]

[0086] As can be seen from the data in Table 1, the average particle size of the drug-loaded nanospheres with different modifications is 140-180 nm, they are negatively charged, and the encapsulation efficiency is all above 70%. The comparison of PDI and polydispersity index shows that the PDI values ​​of the drug-loaded nanospheres are all less than 0.3, indicating that the particle size distribution of the four types of nanospheres is good.

[0087] (3) In vitro release test

[0088] 1 mL of each of the following dialysis agents (BM@PLGA / G0-195-Cy3, BMU@PL / G0-195-Cy3, BMK@PL / G0-195-Cy3, and BMUK@PL / G0-195-Cy3) was added to a dialysis bag with a molecular weight cutoff of 30 KD. The dialysate consisted of 10 mL of PBS + 10% artificial cerebrospinal fluid. Dialysis was performed at 4°C with magnetic stirring under light-protected conditions. The fluorescence value of the dialysate was measured every 4, 8, 12, 24, 36, and 48 hours, and the dialysis bag was removed and placed into freshly prepared dialysate each time. Release curves were plotted after 48 hours. According to the formula: Release rate (%) = (T1 + T2 + T3 + T4 + T5 + T6) / Ttotal × 100% (Ttotal: total drug amount (10μg); T = C × V; C: release concentration (μg / L); V: dialysate volume (10mL), the release rate at each time point was obtained, and the results are as follows. Figure 1 As shown.

[0089] Figure 1 The results showed that the in vitro release rate of drug-loaded nanospheres modified with UEA-I and KLVFF after 48 hours was less than 40%, which was significantly lower than that of the single-modification group and the blank group. This indicates that the dual modification of UEA-I and KLVFF can effectively enhance the structural stability of nanoparticles, delay the release of miR-195, and has a good sustained-release effect, providing a basis for long-term in vivo delivery of miR-195 and improving the therapeutic effect.

[0090] (4) SH-SY5Y cell uptake

[0091] The uptake of drug-loaded nanospheres with different modifications by human neuroblastoma SH-SY5Y cells was investigated. The specific steps are as follows:

[0092] SH-SY5Y cells were cultured in DMEM / F12 medium containing 1% penicillin-streptomycin and 15% fetal bovine serum. The cells were then incubated in a cell culture incubator at 37°C and 5% CO2.

[0093] Cultured cells were seeded into 24-well plates with slides at the bottom and cultured overnight. The medium was then changed with DMEM / F12, and the cells were cultured for another 12 hours. Four different drug-loaded nanospheres were then added, and the cells were incubated at 37°C and 5% CO2 for 8 hours. Afterward, the old culture medium was discarded, the cells were washed twice with PBS, stained with DAPI for 5 minutes, washed twice with PBS, and the slides were mounted with anti-quenching mounting medium. The intracellular Cy3 fluorescence intensity was detected using confocal microscopy. The results are shown below. Figure 2 As shown.

[0094] Figure 2The results showed that drug-loaded nanospheres modified with UEA-I and KLVFF were more easily taken up by SH-SY5Y cells than unmodified and single-modified drug-loaded nanospheres, indicating that the dual-modified nanospheres were more likely to enter the cells.

[0095] Experimental Example 2

[0096] This experiment used qRT-PCR technology to detect the expression level of miRNA-195 in the hippocampus and cortex of mice after intranasal administration of the brain-targeting nanospheres BMUK@PL / G0-195 prepared in Example 1 to mice with Aβ hippocampal brain injection.

[0097] I. Constructing an Aβ hippocampal deposition mouse model

[0098] C57BL / 6 mice were anesthetized with isoflurane and placed on a stereotaxic apparatus. 500 pmol of pre-incubated Aβ protein was slowly injected into a unilateral hippocampal site (posterior fontanelle 2.2 mm, 1.2 mm to the right, depth 1.8 mm). Seven days of postoperative recovery were allowed to ensure stable Aβ deposition and the formation of a pathological model for subsequent targeted experiments.

[0099] II. Group Dosing

[0100] Aβ hippocampal deposited mice were randomly divided into a treatment group and a model control group (CON group). Mice in the treatment group were administered the intranasal brain-targeting nanospheres BMUK@PL / G0-195 prepared in Example 1 at a dose of 0.1 mg / kg. The model control group received an equal volume of physiological saline intranasally.

[0101] III. Extraction of RNA from Brain Tissue

[0102] Samples were collected from mice in the drug administration group and the model control group on days 1, 3, 5, 7 and 14 after intranasal administration to detect the expression level of miR-195 in the hippocampus and cortex of the two groups of mice.

[0103] Each 50-100 mg sample of mouse brain tissue was thoroughly ground under liquid nitrogen, followed by homogenization with 1 mL of TRIzol reagent. The homogenized sample was then incubated at room temperature (15-30°C) for 5 minutes to ensure complete separation of nucleic acids and proteins. Next, 0.2 mL of chloroform was added, the mixture was shaken vigorously for 15 seconds, and incubated at room temperature for another 3 minutes. The sample was then centrifuged at 13500 rpm for 15 minutes at 2-8°C. After centrifugation, the sample separated into three layers: a lower yellow organic phase, a middle protein interface, and an upper colorless aqueous phase, in which RNA was mainly distributed. The upper aqueous phase was carefully transferred to a new centrifuge tube, and 0.5 mL of isopropanol was added to precipitate RNA. After incubation at room temperature for 10 minutes, the tube was centrifuged again at 13500 rpm for 10 minutes at 2-8°C. After centrifugation, the RNA precipitate adhered to the bottom and walls of the tube; the supernatant was discarded. Wash the RNA precipitate with 1 mL of 75% ethanol prepared with DEPC-treated water, and centrifuge at 10600 rpm for 5 minutes at 2–8 °C. Discard the ethanol, air-dry the RNA precipitate on ice for 5–10 minutes, and finally add an appropriate amount of DEPC-treated water to dissolve the RNA. Measure the RNA concentration using a UV spectrophotometer and record the A260 / A280 ratio, with an optimal range of 1.8–2.0. Store the extracted RNA solution at −80 °C for later use.

[0104] Fourth, the extracted RNA was reverse transcribed into cDNA.

[0105] The reverse transcription reaction was performed according to the operating procedures of the Applied Biosystems (ABI) High-Capacity cDNA Reverse Transcription Kit. The reverse transcription primer Mmu-miR-195-5p-RT is shown in SEQ ID NO.1, and the internal control primer U6-RT is shown in SEQ ID NO.2.

[0106] SEQ ID NO.1:

[0107] 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGCCAATAT-3';

[0108] SEQ ID NO. 2: 5'-CGCTTCACGAATTTGGCGTGTCAT-3'.

[0109] Place the required reagents on ice until they are completely melted, then gently tap to mix. The reaction system is shown in Table 2.

[0110] Table 2

[0111]

[0112] The reaction conditions were: 25℃ for 10 min, 37℃ for 120 min, 85℃ for 5 min, and finally the temperature was lowered to 4℃. The product was stored at -20℃.

[0113] V. Real-time quantitative PCR

[0114] This experiment was performed using an Applied Biosystems 7500 FAST Real-time PCR instrument.

[0115] The upstream specific primer Mmu-miR-195-5p-F for miR-195 is shown in SEQ ID NO.3, and the downstream specific primer Mmu-miR-195-5p-R is shown in SEQ ID NO.4; the upstream primer U6-F for the U6 internal reference is shown in SEQ ID NO.5, and the downstream primer U6-R is shown in SEQ ID NO.6.

[0116] SEQ ID NO.3: 5'-ACACTCCAGCTGGGTAGCAGCACAGAAATATTG-3';

[0117] SEQ ID NO.4: 5'-CTCAACTGGTGTCGTGGA-3';

[0118] SEQ ID NO.5: 5'-CGCTTCACGAATTTGGCGTGTCAT-3';

[0119] SEQ ID NO. 6: 5'-GCTTCGGCAGCACATATACTAAAAT-3'.

[0120] The reaction conditions were: 95℃ for 10 min pre-denaturation, 95℃ for 15 sec, 60℃ for 30 sec, 72℃ for 30 sec, for a total of 40 cycles. The dissolution curves were: 95℃ for 30 sec, 60℃ for 1 min, 95℃ for 30 sec, 60℃ for 1 min.

[0121] VI. Results Analysis

[0122] In this experiment, the amplification efficiency of the target gene miR-195 and the housekeeping gene U6 snRNA was the same. Mathematical derivation yielded the formula: Target gene amount = 2. -△△Ct And △△Ct=(Ct 目的基因 -Ct 管家基因 ) 实验组 -(Ct 目的基因 -Ct 管家基因 ) 对照组The results were normalized with the expression level of CON in the control group as 1, and statistical analysis was performed using an independent samples t-test. The results are as follows: Figure 3 As shown.

[0123] Figure 3 The results showed that the relative levels of miR-195 in the hippocampus of mice in the treatment group were significantly higher than those in the model control group on days 1, 3, 5, and 7, showing a significant upregulation. However, the miR-195 levels in the cortex at each time point were similar to those in the control group, with no significant difference. This indicates that the nanospheres BMUK@PL / G0-195 prepared in Example 1 can achieve hippocampal targeting of the negative pole after nasal administration, and are more effective in targeting the lesion area of ​​Alzheimer's disease.

[0124] The relative level of miR-195 in the hippocampus of mice in the drug-treated group reached its peak on day 1, then gradually decreased, returning to the level of the model control group on day 14. This indicates that nanodelivery can rapidly increase the level of miR-195 in the brain in the early stages and maintain high expression for several days. The dual-targeting modification of UEA-I and KLVFF significantly enhanced the delivery and enrichment of miR-195 in the hippocampus through synergistic recognition and endocytosis mechanisms, demonstrating the advantages of synergistic targeted delivery.

[0125] Experimental Example 3

[0126] This study investigated the effect of the drug-loaded nanospheres BMUK@PL / G0-195 prepared in Example 1 on improving the learning and memory abilities of APP / PS1 double transgenic Alzheimer's disease (AD) model mice using the Barnes maze test.

[0127] The Barnes maze test is a classic method for measuring spatial memory and learning abilities in animals, and is particularly used to assess their ability to remember target locations.

[0128] The experimental animals were divided into three groups:

[0129] (1) WT group: 7-month-old wild-type mice were given an equal volume of physiological saline;

[0130] (2) APP / PS1 group: 7-month-old APP / PS1 transgenic mice were used as a model control and were given an equal amount of physiological saline;

[0131] (3) APP / PS1+BMUK@PL / G0-195 group: 7-month-old APP / PS1 transgenic model mice were given drug-loaded nanospheres BMUK@PL / G0-195 prepared in Example 1 via nasal administration at a dose of 0.1 mg / kg and a volume of 20 μL. The administration was repeated every three days for a total of 26 times.

[0132] During behavioral testing, mice were trained to find and enter a target escape hole under light and airflow stimulation from day 1 to day 6. After a 3-day rest period following the training, the escape hole was removed on day 10. The escape latency of the mice to reach the original target location was recorded. The results are as follows: Figure 4 and Figure 5 As shown.

[0133] Figure 4 and Figure 5 The results showed that the escape latency of wild-type mice remained consistently low (approximately 20-30 seconds) and exhibited a slow decreasing trend. This indicates that normal mice have good spatial learning and memory abilities, enabling them to quickly adapt to their environment and remember target locations. The escape latency of APP / PS1 transgenic mice was significantly higher than that of wild-type mice, peaking on day 2 of training (approximately 120 seconds). Although it subsequently decreased, it remained at a high level on day 5 (approximately 80 seconds). This suggests that AD model mice suffer from severe spatial learning and memory impairment, making it difficult for them to quickly locate target locations. After treatment with drug-loaded nanospheres BMUK@PL / G0-195, the escape latency of APP / PS1 transgenic mice was significantly lower than that of the model group, showing a rapid decreasing trend. This indicates that intranasally administered nanospheres significantly improved the spatial learning ability of APP / PS1 mice, helping them quickly learn to find target locations.

[0134] The Barnes maze experiment demonstrated that dual-targeted modified BMUK@PL / G0-195 nanospheres, delivered nasally, significantly reversed cognitive impairment in APP / PS1 mice. This therapeutic effect was attributed to the synergistic targeting effect of the nanosystem: the dual modification with UEA-I and KLVFF not only enhanced the nanoparticles' ability to penetrate the nasal mucosal barrier but also enabled them to precisely target cells surrounding Aβ plaques in the hippocampus, releasing miR-195 in situ, thereby restoring the mice's learning and memory abilities at the molecular level.

[0135] Test Example 4

[0136] This study investigated the effect of the drug-loaded nanospheres BMUK@PL / G0-195 prepared in Example 1 on improving the learning and memory abilities of 5XFAD transgenic Alzheimer's disease (AD) model mice using the Barnes maze test.

[0137] The experimental animals were divided into three groups:

[0138] (1) WT group: 3-month-old wild-type mice were given an equal volume of physiological saline;

[0139] (2) 5XFAD group: 3-month-old 5XFAD transgenic mice were used as model controls and given an equal amount of physiological saline;

[0140] (3) 5XFAD+BMUK@PL / G0-195 group: 3-month-old 5XFAD transgenic model mice were given drug-loaded nanospheres BMUK@PL / G0-195 prepared in Example 1 via nasal administration at a dose of 0.1 mg / kg and a volume of 20 μL. The administration was repeated every three days for a total of 26 times.

[0141] During behavioral testing, mice were trained to find and enter a target escape hole under light and airflow stimulation from day 1 to day 6. After a 3-day rest period following the training, the escape hole was removed on day 10. The escape latency of the mice to reach the original target location was recorded. The results are as follows: Figure 6 and Figure 7 As shown.

[0142] Figure 6 and Figure 7 The results showed that the escape latency of 5XFAD mice treated with drug-loaded nanospheres BMUK@PL / G0-195 was significantly lower than that of the model group, with the curve rapidly decreasing and approaching the level of wild-type mice. This indicates that intranasally administered nanospheres helped 5XFAD mice regain their learning ability, enabling them to quickly locate targets after training and reversing cognitive decline. This demonstrates that intranasally delivered BMUK@PL / G0-195 can target the mouse brain via the nose, cross the nasal mucosal barrier, enter the cells surrounding Aβ plaques in the mouse brain tissue, accumulate, and release miR-195 to exert its therapeutic effect.

Claims

1. A transnasal brain-targeting nanosphere loaded with miR-195, characterized in that, The nanospheres have a core-shell-crown three-layer hierarchical structure, consisting of, from the inside out: a drug-loaded core, a biomimetic cell membrane shell, and a dual-ligand functionalized surface crown. The drug-loaded core is composed of the neutral polymer PLGA, the cationic organic compound GO-C14, and miR-195. The biomimetic cell membrane shell is composed of bone marrow mesenchymal stem cell membrane and tightly encapsulates the surface of the drug-loaded core. Polyethylene glycol-modified phospholipids covalently coupled with strychnine and Aβ-targeting peptide KLVFF are spontaneously inserted into the lipid bilayer of the biomimetic cell membrane shell through hydrophobic fragments, while hydrophilic polyethylene glycol chains extend to the outer side of the membrane, anchoring strychnine and Aβ-targeting peptide KLVFF to the surface of the biomimetic cell membrane shell, forming a dual-ligand functionalized surface crown. The miR-195 loading in the nanospheres is 0.2-0.8% w / w.

2. The transnasal brain-targeting nanospheres carrying miR-195 according to claim 1, characterized in that, The nanospheres have a particle size of 140~180nm and a surface potential of -7.0~-9.0mV.

3. A method for preparing transnasal brain-targeting nanospheres loaded with miR-195 as described in claim 1 or 2, characterized in that, Includes the following steps: Step 1: Dissolve the polyethylene glycol-modified phospholipid activator DSPE-PEG-NHS in organic solvent I, add leek lectin and buffer solution, mix and stir to react, then dialyze and dry to obtain leek lectin-modified polyethylene glycol phospholipid. The maleimide derivative of polyethylene glycol phospholipid, DSPE-PEG-Mal, was dissolved in organic solvent II. Aβ-targeting peptide KLVFF and a catalyst were added, and after mixing and stirring, the mixture was dialyzed and dried to obtain polyethylene glycol phospholipid modified with Aβ-targeting peptide KLVFF. Step 2: Control the dosage according to the mass ratio of cationic organic compound G0-C14, miR-195, neutral polymer PLGA, bone marrow mesenchymal stem cell membrane, pecan lectin-modified polyethylene glycol phospholipid and Aβ-targeting peptide KLVFF-modified polyethylene glycol phospholipid. Neutral polymer PLGA was dissolved in an organic solvent and mixed with cationic organic compound G0-C14 as the organic phase. miR-195 solution and the first part of Tris-HCl solution were added as the aqueous phase. After ultrasonic dispersion, an initial emulsion was formed. The second part of Tris-HCl solution and dichloromethane solution were added to the initial emulsion, and ultrasonic emulsification was continued to obtain a shaped emulsion. The third part of Tris-HCl solution was added to the shaped emulsion, and the mixture was stirred, centrifuged, and washed to obtain a drug-loaded core composed of PLGA / G0-C14 / miR-195 complex. The obtained drug-loaded core was suspended in a bone marrow mesenchymal stem cell membrane solution and treated with water bath so that the bone marrow mesenchymal stem cell membrane tightly coated the surface of the drug-loaded core. After centrifugation and washing, a drug-loaded core coated with a biomimetic cell membrane shell was obtained. The drug-loaded core coated with the obtained biomimetic cell membrane shell, the polyethylene glycol phospholipid modified with lectin obtained in step one, and the polyethylene glycol phospholipid modified with Aβ-targeting peptide KLVFF were co-incubated. The lectin and Aβ-targeting peptide KLVFF were anchored and modified on the outer side of the biomimetic cell membrane shell, and assembled to form a dual-ligand functionalized surface crown, resulting in a transnasal brain-targeting nanosphere loaded with miR-195 with a core-shell-crown three-layer hierarchical structure.

4. The method for preparing transnasal brain-targeting nanospheres loaded with miR-195 according to claim 3, characterized in that, The organic solvent I mentioned in step one is dimethylformamide, and the buffer solution includes phosphate buffer and carbonate buffer. The mass-to-volume ratio of DSPE-PEG-NHS, dimethylformamide, lecithin, phosphate buffer and carbonate buffer is 400~600mg:4~6mL:1~3mg:1~3mL:0.5~2mL. The mixing and stirring reaction time is 0.5~1.5h. Dialysis is performed sequentially using phosphate buffer and pure water. The total dialysis time is 7.5~8.5h. The drying method is freeze drying.

5. The method for preparing transnasal brain-targeting nanospheres carrying miR-195 according to claim 3 or 4, characterized in that, The organic solvent II mentioned in step one is chloroform, the Aβ-targeting peptide KLVFF is dissolved in methanol, and the catalyst is triethylamine; the mass-volume ratio of DSPE-PEG-Mal, chloroform, Aβ-targeting peptide KLVFF, methanol, and triethylamine is 400~600mg:5~15mL:3~5mg:1~3mL:0.5~2mL, the mixing and stirring reaction is carried out under argon protection, the reaction time is 19~25h, the dialysis time is 2.5~4.5h, and the drying method is freeze drying.

6. The method for preparing transnasal brain-targeting nanospheres loaded with miR-195 according to claim 5, characterized in that, The mass ratios of the cationic organic compound G0-C14, miR-195, neutral polymer PLGA, bone marrow mesenchymal stem cell membrane, pecan lectin-modified polyethylene glycol phospholipid, and Aβ-targeting peptide KLVFF-modified polyethylene glycol phospholipid in step two are 1:0.2:50:25.5:5.31:10.62, 1.5:0.5:40:20.5:4:8, or 2:1:60:29.5:8:

12.

7. The method for preparing transnasal brain-targeting nanospheres loaded with miR-195 according to claim 6, characterized in that, The organic solvent in step two is dichloromethane, and the mass-to-volume ratio of neutral polymer PLGA to dichloromethane is 40-60 mg: 0.5-2 mL; the concentration of the miR-195 solution is 1 mg / mL; the first part of the Tris-HCl solution is 1 M concentration and is added at 1-4% v / v of the volume of the organic phase; the second part of the Tris-HCl solution is 10 mM concentration and is added at 50-100% v / v of the volume of the initial emulsion; the third part of the Tris-HCl solution is 10 mM concentration and is added at 100-200% v / v of the volume of the formed emulsion.

8. The method for preparing transnasal brain-targeting nanospheres loaded with miR-195 according to claim 7, characterized in that, In step two, the ultrasonic power for both ultrasonic dispersion and ultrasonic emulsification is 700W, and the processing method is alternating on / off for 2 seconds; the stirring speed is 700rpm and the stirring time is 6h; the centrifugation speed is 10000g and the centrifugation time is 5min; and ultrapure water is used for washing.

9. The method for preparing transnasal brain-targeting nanospheres loaded with miR-195 according to claim 8, characterized in that, The ultrasonic frequency of the water bath ultrasonic treatment in step two is 42kHz, the ultrasonic power is 100W, and the ultrasonic time is 2min; the co-incubation time is 25~35min.

10. The use of a transnasal brain-targeting nanosphere carrying miR-195 as described in claim 1 or 2 in the preparation of a medicament for treating Alzheimer's disease.

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