Lectin combined with targeting peptide modified nanomicrospheres, and preparation method and application thereof

Abstract

The present application relates to a kind of agglutinin joint targeting peptide modified nanosphere and its preparation method and application, belong to the biomedicine technical field.To solve the problem that the existing drug delivery system is difficult to have the integration of nasal brain barrier breakthrough, nasal long-term retention, brain lesion targeting, broad-spectrum drug loading and high biological stability, the present application provides a kind of agglutinin joint targeting peptide modified nanosphere, including polymer matrix core, biomimetic cell membrane shell and double-ligand functionalization surface crown layer in turn from inside to outside.The nanosphere of the present application can prolong the nasal retention time and efficiently penetrate the nasal mucosa barrier by relying on the synergistic effect of core-shell-crown three-layer structure, realize transnasal brain and lesion precise targeting, improve drug delivery efficiency and efficacy, and reduce toxic side effects.The nanosphere of the present application has a wide range of drug loading spectrum, suitable for the drug demand of various central nervous system diseases, and has excellent clinical transformation value and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a lectin-targeted peptide-modified nanosphere, its preparation method and application. Background Technology

[0002] Central nervous system diseases, such as Alzheimer's disease, Parkinson's disease, glioma, and major depressive disorder, have become a major medical challenge for aging societies worldwide due to their high disability rates and severe harm. The core bottleneck in the treatment of these diseases lies in the natural barrier of the blood-brain barrier (BBB). The BBB prevents approximately 98% of small-molecule drugs and almost all large-molecule drugs from entering the brain parenchyma via conventional routes of administration, such as intravenous or oral administration, resulting in drug concentrations at the brain lesion site far below the therapeutic threshold. Simultaneously, the metabolic burden on the liver and kidneys, peripheral systemic toxicity, and poor long-term medication adherence associated with systemic administration further restrict the clinical efficacy in treating central nervous system diseases.

[0003] To overcome the BBB limitation, brain-targeted drug delivery systems have become a research hotspot. Existing technologies mostly employ receptor-mediated strategies, such as the glycosyl-TAT dual-modified nanoliposomes in patents CN111249234A and CN111249235A. These nanoliposomes utilize glucose transporters to penetrate the BBB and enhance cell entry efficiency, achieving brain enrichment after intravenous administration. However, such carriers rely on intravenous administration, resulting in limited brain entry efficiency. They can only accumulate non-specifically in the brain, with a high risk of off-target effects. Furthermore, the structural stability of traditional liposomes is insufficient, making it difficult to meet the treatment needs of complex central nervous system diseases.

[0004] Direct nasal-to-brain drug delivery has become an important alternative due to its non-invasive nature and ability to bypass the brain barrier (BBB). Patent CN119074944A discloses a lectin-conjugated LNP nasal drug delivery system, which prolongs retention time by binding lectins to the nasal mucosa, thereby improving mRNA delivery efficiency to the brain. However, this system only overcomes the local nasal barrier and lacks the ability to target brain lesions. Furthermore, the carrier is mRNA-specific, resulting in a narrow drug loading spectrum. Additionally, the problems of nasal mucociliary clearance and nasal epithelial enzyme degradation remain unresolved, leading to a short drug absorption window and limiting delivery efficiency.

[0005] In summary, the core challenge of existing central nervous system drug delivery technologies lies in balancing "efficient brain delivery" with "precise lesion targeting." Nasal delivery to the brain requires adaptation to the nasal mucosal barrier, while targeting brain lesions requires specific ligands. Differences in ligand type and density requirements for carrier surface modification can lead to issues such as strong nasal penetration resulting in weak targeting, or strong targeting resulting in poor nasal retention. Existing carriers often target only a single barrier, failing to simultaneously overcome the triple obstacles of nasal mucociliary clearance, nasobrain barrier penetration, and brain lesion recognition. Therefore, developing novel precision drug delivery carriers is crucial for achieving precise treatment of central nervous system diseases. Summary of the Invention

[0006] To address the challenge of existing drug delivery systems that cannot simultaneously achieve nasobrain barrier penetration, long-lasting nasal retention, intracranial lesion targeting, broad-spectrum drug loading, and high biostability, this invention provides a lectin-modified nanosphere with targeting peptides, its preparation method, and its applications.

[0007] The technical solution of this invention: A nanosphere modified with lectin and targeting peptide is disclosed. The nanosphere has a three-layered structure of core-shell-crown, comprising, from the inside out, a polymer matrix core, a biomimetic cell membrane shell, and a dual-ligand functionalized surface crown. The polymer matrix core is composed of a biodegradable neutral polymer and serves as the main body for drug loading and nanoforming. The biomimetic cell membrane shell tightly coats the surface of the polymer matrix core, forming a biocompatible and immune-evading shell. Polyethylene glycol-modified phospholipids covalently coupled with lectin and polyethylene glycol phospholipids covalently coupled with targeting peptides are spontaneously inserted into the lipid bilayer of the biomimetic cell membrane shell through hydrophobic fragments, with hydrophilic polyethylene glycol chains extending to the outer side of the membrane, anchoring the lectin and targeting peptide to the surface of the biomimetic cell membrane shell, forming a dual-ligand functionalized surface crown. In the surface canopy, lectin ligands endow the nasal mucosa with the ability to penetrate and resist ciliary clearance, while targeting peptide ligands endow the brain with the ability to specifically recognize lesions. The three-layer structure works together to achieve precise delivery of the product to the brain and lesions via the nose.

[0008] Furthermore, the neutral polymer is one of polylactic acid-glycolic acid copolymer, polylactic acid, polyethylene glycol-polylactic acid, polyhydroxyalkanoate, polymethyl methacrylate, polystyrene, polycaprolactone, or poly-N-isopropylacrylamide; The biomimetic cell membrane shell is one of the following: autologous bone marrow mesenchymal stem cell membrane, allogeneic bone marrow mesenchymal stem cell membrane, adipose mesenchymal stem cell membrane, umbilical cord mesenchymal stem cell membrane, erythrocyte membrane, leukocyte membrane, platelet membrane, or neutrophil membrane. The lectin is one of the following: Vitex agglutinin (UEA-I) or AAL (Alania spp.) which recognizes fucose; wheat germ agglutinin (WGA) or DBA (Dysplasia spp.) which recognizes N-acetylglucosamine; soybean agglutinin (SBA) or peanut agglutinin (PNA) which recognizes galactose; and concanavalin A (ConA) or LCA (Lentinula spp.) which recognizes mannose. The targeting peptide is one of Aβ targeting peptide, α-synuclein targeting peptide, epidermal growth factor receptor targeting peptide, or integrin αvβ3 targeting peptide. The polyethylene glycol in both the lectin-functionalized PEGylated phospholipids and the targeted peptide-functionalized PEGylated phospholipids has a molecular weight of 600~5000 Da.

[0009] Furthermore, the Aβ targeting peptide is one of KLVFF, LPFFD, LVFFARK, LVFFARKHH, c-LVFFARK, car-LVFFARK, L-RTHLVFFARK, or D-RTHLVFFARK; the α-synuclein targeting peptide is one of SYN17 or VFFK; the epidermal growth factor receptor targeting peptide is one of GE11 or EGFR10R; and the integrin αvβ3 targeting peptide is one of RGD or c(RGDfK).

[0010] A method for preparing lectin-modified targeted peptide nanospheres includes the following steps: Step 1: Dissolve the polyethylene glycol-modified phospholipid activator DSPE-PEG-NHS in organic solvent I, add lectin and buffer solution, mix and stir to react, then dialyze and dry to obtain lectin-functionalized polyethylene glycol-modified phospholipid. The maleimide derivative of polyethylene glycol phospholipid, DSPE-PEG-Mal, was dissolved in organic solvent II. The target peptide and catalyst were added, and after mixing and stirring, the mixture was dialyzed and dried to obtain the target peptide-functionalized polyethylene glycol phospholipid. Step 2: Control the dosage by using neutral polymer, cell membrane, lectin-functionalized polyethylene glycol phospholipid and target peptide-functionalized polyethylene glycol phospholipid in a mass ratio of 40~60:20~30:4~8:8~12; Neutral polymers were dissolved in an organic solvent and vortexed to form the organic phase. An aqueous phase was then added and ultrasonically dispersed to form an emulsion. After stirring, centrifugation, and washing, neutral polymer nanospheres were obtained. The obtained neutral polymer nanospheres were suspended in a cell membrane solution and ultrasonically treated to tightly coat the surface of the neutral polymer nanospheres with cell membranes. After centrifugation and washing, biomimetic cell membrane shell-coated nanospheres were obtained. The obtained biomimetic cell membrane shell-coated nanospheres, lectin-functionalized polyethylene glycol phospholipids obtained in step one, and target peptide-functionalized polyethylene glycol phospholipids were co-incubated to anchor and modify the lectin and target peptides on the outer side of the biomimetic cell membrane shell, assembling them to form a dual-ligand functionalized surface crown layer, resulting in lectin-modified nanospheres 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, lectin, phosphate buffer and carbonate buffer is 400~600mg:4~6mL:1~3mg:1~3mL:0.5~2mL, the mixing and stirring reaction time is 0.5~2h, the dialysis is carried out sequentially with phosphate buffer and pure water, the total dialysis time is 7~9h, and the drying method is freeze drying.

[0012] Furthermore, in step one, the organic solvent II is chloroform, the targeting peptide is dissolved in methanol, and the catalyst is triethylamine; the mass-to-volume ratio of DSPE-PEG-Mal, chloroform, targeting peptide, 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 20~30h, the dialysis time is 3~5h, and the drying method is freeze drying.

[0013] Furthermore, in step two, the mass ratio of the neutral polymer, cell membrane, lectin-functionalized polyethylene glycol phospholipid, and target peptide-functionalized polyethylene glycol phospholipid is 50:25.5:5.31:10.62, 40:20.5:4:8, or 60:29.5:8:12. The organic solvent is dichloromethane, and the mass-to-volume ratio of the neutral polymer to dichloromethane is 40-60 mg: 0.5-2 mL; the ultrasonic dispersion is performed with an ultrasonic power of 700 W, and the treatment method is to alternate between on and off for 2 seconds, repeated 2-3 times; the stirring speed is 700 rpm and the stirring time is 6 hours; the centrifugation speed is 10000 g and the centrifugation time is 5 minutes; and ultrapure water is used for washing.

[0014] Furthermore, the ultrasonic treatment in step two is water bath ultrasonic treatment with an ultrasonic frequency of 42kHz, an ultrasonic power of 100W, and an ultrasonic time of 2min; the co-incubation time is 20~40min, and the final obtained nanospheres have a particle size of 140~180nm and a negatively charged surface.

[0015] Application of a lectin-modified nanosphere combined with a targeting peptide in the preparation of a nasal drug delivery carrier targeting the central nervous system.

[0016] Furthermore, the drug delivery carrier may carry one or more of the following: nucleic acid drugs, insoluble small molecule drugs, protein drugs, peptide drugs, or vaccine antigens.

[0017] The beneficial effects of this invention are: This invention constructs a nano-drug delivery system that integrates naso-brain barrier breakthrough, long-term nasal retention, intracranial lesion targeting, broad-spectrum drug delivery, and high biostability. This system achieves the following effects through the synergistic effect of its core-shell-corona layered hierarchical structure: (1) The biomimetic cell membrane shell of the nanospheres of this invention has excellent biocompatibility, which avoids the body's immune recognition and rapid clearance from the source. At the same time, it can resist the degradation of various hydrolytic enzymes in the nasal epithelium, protecting the overall structure of the carrier and the encapsulated drug from damage. The lectin modified on the surface can specifically bind to the glycogroups on the surface of the nasal mucosal epithelial cells. With the help of specific adhesion, it can effectively resist the active clearance effect of nasal mucocili, and significantly prolong the retention time of the carrier at the nasal drug delivery site. The two work together to prolong the local action time of the drug, so that the carrier can smoothly penetrate the nasal mucosal barrier, open up the delivery channel from the nasal cavity to the central nervous system, and lay the foundation for the subsequent drug delivery to the brain.

[0018] (2) The polymer matrix core of the nanospheres of this invention is made of a biodegradable neutral polymer with excellent granulation properties. It has a loose and porous internal structure, which can efficiently load poorly soluble small molecule drugs through various methods such as physical encapsulation and hydrophobic interaction, thus meeting the stable encapsulation requirements of hydrophobic drugs. The biomimetic cell membrane shell has good membrane encapsulation and structural stability, and can completely encapsulate easily degradable bioactive substances such as nucleic acid drugs, protein drugs, polypeptide drugs and vaccine antigens, thus avoiding the inactivation of these drugs during delivery. The polyethylene glycol-modified phospholipid component of the surface can precisely optimize the overall hydrophilicity-hydrophobicity balance of the carrier, adapting to drug molecules with different polarities and different physicochemical properties, further broadening the drug loading range. The three-layer hierarchical structure complements each other to achieve efficient encapsulation of multiple types of drugs, with a wide drug loading spectrum, adapting to the diverse medication needs of central nervous system diseases.

[0019] (3) The biomimetic cell membrane shell of the nanospheres of this invention has excellent membrane fusion and barrier-crossing capabilities, which can assist the carrier in further penetrating the nasobrain barrier; the specific targeting peptides modified on the surface can accurately identify specific targets corresponding to lesions in the brain, achieve targeted enrichment for different central nervous system diseases, ensure accurate drug delivery to the core area of ​​the lesion, increase drug concentration at the lesion site, and reduce drug exposure and potential side effects in normal brain tissue. The two work together to complete the targeted delivery link of "nasobrain penetration-lesion enrichment", realizing targeted treatment of central nervous system diseases.

[0020] The nanospheres of this invention can directly enter the brain via the nose through the olfactory nerve and trigeminal nerve pathways, without passing through systemic circulation. This avoids the first-pass effect of intravenous administration and completely bypasses the strict barrier of the blood-brain barrier. In vitro cell experiments and in vivo tissue distribution experiments have verified that this system can efficiently cross the nasal barrier, smoothly enter the central nervous system, and specifically accumulate in the lesion area of ​​the brain, significantly improving the efficiency of intracranial drug delivery and ensuring that the drug accurately reaches the lesion site and exerts its effect.

[0021] With its integrated drug delivery design, the nanospheres of this invention can stably reside in brain cells surrounding lesions, maintaining a consistently high local drug concentration. This not only significantly prolongs the duration of drug action and enhances therapeutic efficacy but also substantially reduces the dosage. Simultaneously, the precise lesion-targeted delivery design minimizes unnecessary drug exposure in the systemic bloodstream, reducing systemic toxicity and immune-related adverse reactions at their source, further enhancing carrier safety. This design possesses extremely high clinical translational value and broad application prospects. Attached Figure Description

[0022] Figure 1 Transmission electron microscopy image of the lectin-modified Aβ-targeting peptide nanospheres prepared in Example 1; Figure 2 The particle size distribution diagram of the lectin-modified Aβ-targeting peptide nanospheres prepared in Example 1 is shown. Figure 3 Zeta potential diagram of the lectin-modified Aβ-targeting peptide nanospheres prepared in Example 1; Figure 4 This is a dynamic fluorescence imaging image of the four nanospheres crossing the nasal mucosal barrier model in Experiment Example 2; Figure 5 This is a comparison of the cumulative fluorescence intensity of the four nanospheres crossing the nasal mucosal barrier model in Experiment Example 2; Figure 6 This is an in vivo fluorescence imaging image showing the dynamic brain distribution of the four types of nanospheres in Aβ hippocampal deposited mice in Experiment Example 3. Figure 7 This is a comparison of the cumulative fluorescence intensity of the four types of nanospheres in Aβ hippocampal deposited mouse brain tissue 6 hours after drug administration in Experiment Example 3; Figure 8 This is an in vivo fluorescence imaging image showing the dynamic distribution of the four types of nanospheres in the brain of APP / PS1 transgenic mice in Experiment Example 4. Figure 9 This is a comparison of the cumulative fluorescence intensity of the four types of nanospheres in the isolated brain tissue of APP / PS1 transgenic mice 6 hours after drug administration in Experiment Example 4; Figure 10 Transmission electron microscopy image of the lectin-modified Aβ-targeting peptide nanospheres prepared in Example 4; Figure 11 The particle size distribution diagram of the lectin-modified Aβ-targeting peptide nanospheres prepared in Example 4 is shown. Figure 12 Zeta potential diagram of the lectin-modified Aβ-targeting peptide nanospheres prepared in Example 4; Figure 13This is an in vivo fluorescence imaging image showing the dynamic distribution of the four types of nanospheres in the brain of APP / PS1 transgenic mice in Experiment Example 6. Figure 14 This is a comparison of the cumulative fluorescence intensity of the four types of nanospheres in the isolated brain tissue of APP / PS1 transgenic mice 6 hours after drug administration, as shown in Experiment Example 6. Detailed Implementation

[0023] 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.

[0024] Example 1 This embodiment provides a method for preparing BMUK@PLGA nanospheres modified with lectin and Aβ-targeting peptide.

[0025] In this embodiment, the PEGylated phospholipid activator was DSPE-PEG2000-NHS, with a polyethylene glycol molecular weight of 2000, purchased from Xi'an Qiyue Biotechnology Co., Ltd.; 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, purchased from Xi'an Ruixi Biotechnology Co., Ltd.; the lectin was Vitex lectin (UEA-I), purchased from Xinbosheng Biotechnology Co., Ltd.; and the neutral polymer was polylactic-co-glycolic acid copolymer (PLGA), purchased from Jinan Daigang Bioengineering Co., Ltd.

[0026] 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: 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.

[0027] Third-generation bone marrow mesenchymal stem cells were harvested and their concentration adjusted to 3 × 10⁻⁶. 7 Add 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.

[0028] 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.

[0029] The preparation method of lectin-modified Aβ-targeting peptide-modified nanospheres in this embodiment is as follows: Step 1: Preparation of lectin-functionalized polyethylene glycol phospholipids and target peptide-functionalized polyethylene glycol phospholipids: 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 lectin-functionalized polyethylene glycol phospholipid DSPE-PEG2000-UEA-I. 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 the targeted peptide-functionalized polyethylene glycol phospholipid DSPE-PEG2000-KLVFF.

[0030] Step 2: Preparation of BMUK@PLGA nanospheres modified with lectin and Aβ-targeting peptide: The dosage was controlled according to the mass ratio of neutral polymer PLGA, bone marrow mesenchymal stem cell membrane, DSPE-PEG2000-UEA-I, and DSPE-PEG2000-KLVFF of 50:25.5:5.31:10.62.

[0031] Weigh 50 mg of PLGA and dissolve it in 1 mL of dichloromethane solution. Vortex for 2 min to ensure complete dissolution, forming the organic phase. Add 5 mL of 10 mM Tris-HCl solution as the aqueous phase. Use an ultrasonic cell disruptor at 700 W, alternating between on and off cycles for a total of 1 min to form an emulsion. Repeat the ultrasonic treatment three times to ensure a stable and homogeneous emulsion. Place the ultrasonically treated emulsion on a magnetic stirrer and stir at 700 rpm for 6 h to allow the dichloromethane to evaporate and the PLGA to precipitate into nanospheres. Finally, centrifuge at 10000 g for 5 min to collect the PLGA nanospheres, wash twice with ultrapure water, and set aside for later use.

[0032] The obtained PLGA nanospheres were suspended in 1 mL of a solution containing bone marrow mesenchymal stem cell membranes. The mixture was subjected to water bath sonication at a frequency of 42 kHz and a power of 100 W for 2 min, so that the bone marrow mesenchymal stem cell membranes tightly coated the surface of the PLGA nanospheres, which served as the polymer matrix core, forming a biomimetic cell membrane shell. The nanospheres coated with the biomimetic cell membrane shell were collected by centrifugation at 10000 g for 5 min, washed twice with ultrapure water, and set aside for later use.

[0033] The obtained biomimetic cell membrane shell-coated nanospheres, DSPE-PEG2000-UEA-I, and DSPE-PEG2000-KLVFF were co-incubated for 30 min. During co-incubation, DSPE-PEG2000-UEA-I and DSPE-PEG2000-KLVFF spontaneously inserted and anchored into the lipid bilayer of the biomimetic cell membrane shell through hydrophobic DSPE fragments. The hydrophilic PEG chain extended to the outer side of the membrane as a spatial arm, anchoring and modifying lectin and targeting peptide on the outer side of the biomimetic cell membrane shell, assembling to form a dual-ligand functionalized surface crown, resulting in nanospheres with a core-shell-crown three-layer hierarchical structure modified by lectin and Aβ targeting peptide.

[0034] Take 100 μL of the prepared nanospheres, dilute with 900 μL of ultrapure water, mix thoroughly, and add to a dedicated cuvette of the Zetasizer Nano ZS 90 dynamic light scattering particle size analyzer to test the average particle size and Zeta potential of the nanospheres; the morphology of the nanospheres is photographed using a transmission electron microscope, and the results are as follows. Figures 1-3 As shown, the nanospheres are mostly spherical in shape, with a particle size of 140~180nm, and are negatively charged.

[0035] Example 2 The only difference between this embodiment and Example 1 is that the preparation method of step two, the preparation of lectin-modified Aβ-targeting peptide-modified nanospheres, is as follows: The dosage was controlled according to the mass ratio of neutral polymer PLGA, bone marrow mesenchymal stem cell membrane, DSPE-PEG2000-UEA-I, and DSPE-PEG2000-KLVFF of 40:20.5:4:8.

[0036] Weigh 40 mg of PLGA and dissolve it in 1 mL of dichloromethane solution. Vortex for 2 min to ensure complete dissolution, forming the organic phase. Add 5 mL of 10 mM Tris-HCl solution as the aqueous phase. Use an ultrasonic cell disruptor at 700 W, alternating between on and off cycles for a total of 1 min to form an emulsion. Repeat the ultrasonic treatment three times to ensure a stable and homogeneous emulsion. Place the ultrasonically treated emulsion on a magnetic stirrer and stir at 700 rpm for 6 h to allow the dichloromethane to evaporate and the PLGA to precipitate into nanospheres. Finally, centrifuge at 10000 g for 5 min to collect the PLGA nanospheres, wash twice with ultrapure water, and set aside for later use.

[0037] The obtained PLGA nanospheres were suspended in 1 mL of a solution containing bone marrow mesenchymal stem cell membranes. The mixture was subjected to water bath sonication at a frequency of 42 kHz and a power of 100 W for 2 min, so that the bone marrow mesenchymal stem cell membranes tightly coated the surface of the PLGA nanospheres, which served as the polymer matrix core, forming a biomimetic cell membrane shell. The nanospheres coated with the biomimetic cell membrane shell were collected by centrifugation at 10000 g for 5 min, washed twice with ultrapure water, and set aside for later use.

[0038] The obtained biomimetic cell membrane shell-coated nanospheres, DSPE-PEG2000-UEA-I, and DSPE-PEG2000-KLVFF were co-incubated for 30 min. During co-incubation, DSPE-PEG2000-UEA-I and DSPE-PEG2000-KLVFF spontaneously inserted and anchored into the lipid bilayer of the biomimetic cell membrane shell through hydrophobic DSPE fragments. The hydrophilic PEG chain extended to the outer side of the membrane as a spatial arm, anchoring and modifying lectin and targeting peptide on the outer side of the biomimetic cell membrane shell, assembling to form a dual-ligand functionalized surface crown, resulting in nanospheres with a core-shell-crown three-layer hierarchical structure modified by lectin and Aβ targeting peptide.

[0039] Example 3 The only difference between this embodiment and Example 1 is that the preparation method of step two, the preparation of lectin-modified Aβ-targeting peptide-modified nanospheres, is as follows: The dosage was controlled according to the mass ratio of neutral polymer PLGA, bone marrow mesenchymal stem cell membrane, DSPE-PEG2000-UEA-I, and DSPE-PEG2000-KLVFF of 60:29.5:8:12.

[0040] Weigh 60 mg of PLGA and dissolve it in 1 mL of dichloromethane solution. Vortex for 2 min to ensure complete dissolution, forming the organic phase. Add 5 mL of 10 mM Tris-HCl solution as the aqueous phase. Use an ultrasonic cell disruptor at 700 W, alternating between on and off cycles for a total of 1 min to form an emulsion. Repeat the ultrasonic treatment three times to ensure a stable and homogeneous emulsion. Place the ultrasonically treated emulsion on a magnetic stirrer and stir at 700 rpm for 6 h to allow the dichloromethane to evaporate and the PLGA to precipitate into nanospheres. Finally, centrifuge at 10000 g for 5 min to collect the PLGA nanospheres, wash twice with ultrapure water, and set aside for later use.

[0041] The obtained PLGA nanospheres were suspended in 1 mL of a solution containing bone marrow mesenchymal stem cell membranes. The mixture was subjected to water bath sonication at a frequency of 42 kHz and a power of 100 W for 2 min, so that the bone marrow mesenchymal stem cell membranes tightly coated the surface of the PLGA nanospheres, which served as the polymer matrix core, forming a biomimetic cell membrane shell. The nanospheres coated with the biomimetic cell membrane shell were collected by centrifugation at 10000 g for 5 min, washed twice with ultrapure water, and set aside for later use.

[0042] The obtained biomimetic cell membrane shell-coated nanospheres, DSPE-PEG2000-UEA-I, and DSPE-PEG2000-KLVFF were co-incubated for 30 min. During co-incubation, DSPE-PEG2000-UEA-I and DSPE-PEG2000-KLVFF spontaneously inserted and anchored into the lipid bilayer of the biomimetic cell membrane shell through hydrophobic DSPE fragments. The hydrophilic PEG chain extended to the outer side of the membrane as a spatial arm, anchoring and modifying lectin and targeting peptide on the outer side of the biomimetic cell membrane shell, assembling to form a dual-ligand functionalized surface crown, resulting in nanospheres with a core-shell-crown three-layer hierarchical structure modified by lectin and Aβ targeting peptide.

[0043] Example 4 This embodiment provides a method for preparing lectin-modified Aβ-targeting peptide-modified nanospheres hBMWL@PLGA.

[0044] In this embodiment, the PEGylated phospholipid activator was DSPE-PEG2000-NHS, with a polyethylene glycol molecular weight of 2000, purchased from Xi'an Qiyue Biotechnology Co., Ltd.; 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 LPFFD, purchased from Xi'an Ruixi Biotechnology Co., Ltd.; the lectin was wheat germ lectin (WGA), purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; and the neutral polymer was polylactic-co-glycolic acid copolymer (PLGA), purchased from Jinan Daigang Bioengineering Co., Ltd.

[0045] In this embodiment, the cell membrane material is a human bone marrow mesenchymal stem cell membrane. The human bone marrow mesenchymal stem cells were purchased from Suzhou Cyagen Biotech Co., Ltd. The cell membrane was prepared using cell membrane extraction reagent A (P0033-1 kit) purchased from Shanghai Beyotime Biotechnology Co., Ltd. The preparation process is as follows: 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.

[0046] The preparation method of lectin-modified Aβ-targeting peptide-modified nanospheres in this embodiment is as follows: Step 1: Preparation of lectin-functionalized polyethylene glycol phospholipids and target peptide-functionalized polyethylene glycol phospholipids: 500 mg of DSPE-PEG2000-NHS was dissolved in 5 mL of dimethylformamide, and 2 mg of wheat germ lectin, 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 lectin-functionalized polyethylene glycol phospholipid DSPE-PEG2000-WGA. 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 LPFFD 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 the targeted peptide-functionalized polyethylene glycol phospholipid DSPE-PEG2000-LPFFD.

[0047] Step 2: Preparation of lectin-modified Aβ-targeting peptide-modified nanospheres hBMWL@PLGA: The dosage was controlled according to the mass ratio of neutral polymer PLGA, bone marrow mesenchymal stem cell membrane, DSPE-PEG2000-WGA and DSPE-PEG2000-LPFFD of 50:25.5:5.31:10.62.

[0048] Weigh 50 mg of PLGA and dissolve it in 1 mL of dichloromethane solution. Vortex for 2 min to ensure complete dissolution, forming the organic phase. Add 5 mL of 10 mM Tris-HCl solution as the aqueous phase. Use an ultrasonic cell disruptor at 700 W, alternating between on and off cycles for a total of 1 min to form an emulsion. Repeat the ultrasonic treatment three times to ensure a stable and homogeneous emulsion. Place the ultrasonically treated emulsion on a magnetic stirrer and stir at 700 rpm for 6 h to allow the dichloromethane to evaporate and the PLGA to precipitate into nanospheres. Finally, centrifuge at 10000 g for 5 min to collect the PLGA nanospheres, wash twice with ultrapure water, and set aside for later use.

[0049] The obtained PLGA nanospheres were suspended in 1 mL of a solution containing bone marrow mesenchymal stem cell membranes. The mixture was subjected to water bath sonication at a frequency of 42 kHz and a power of 100 W for 2 min, so that the bone marrow mesenchymal stem cell membranes tightly coated the surface of the PLGA nanospheres, which served as the polymer matrix core, forming a biomimetic cell membrane shell. The nanospheres coated with the biomimetic cell membrane shell were 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 nanospheres, DSPE-PEG2000-WGA, and DSPE-PEG2000-LPFFD were co-incubated for 30 min. During co-incubation, DSPE-PEG2000-WGA and DSPE-PEG2000-LPFFD spontaneously inserted and anchored into the lipid bilayer of the biomimetic cell membrane shell through hydrophobic DSPE fragments. The hydrophilic PEG chains extended to the outer side of the membrane as spatial arms, anchoring and modifying lectins and targeting peptides on the outer side of the biomimetic cell membrane shell, assembling to form a dual-ligand functionalized surface crown, resulting in nanospheres modified with lectins and Aβ targeting peptides with a core-shell-crown three-layer hierarchical structure.

[0051] Take 100 μL of the prepared nanospheres, dilute with 900 μL of ultrapure water, mix thoroughly, and add to a dedicated cuvette of the Zetasizer Nano ZS 90 dynamic light scattering particle size analyzer to test the average particle size and Zeta potential of the nanospheres; the morphology of the nanospheres is photographed using a transmission electron microscope, and the results are as follows. Figures 10-12 As shown, the nanospheres are mostly spherical in shape, with a particle size of 140~180nm, and are negatively charged.

[0052] Experimental Example 1 To investigate the drug delivery effect of the lectin-modified Aβ-targeting peptide nanospheres prepared in Example 1, this experiment prepared PLGA nanospheres encapsulated in bone marrow mesenchymal stem cell membranes with different fluorescent labels and different modification types. Cy7 fluorescent labeling was used to detect the in vitro cellular uptake of the nanospheres, and DiR fluorescent labeling was used to detect the in vivo tissue distribution and in vivo uptake of the nanospheres.

[0053] In this embodiment, the cyanine dye Cy7 and 1,1'-bis(octadecyl)-3,3,3'3'-tetramethyl-diiodocyanine cyanide (DiR) were purchased from HEDE Biotechnology Co., Ltd. (Beijing, China); DSPE-PEG2000-UEA-I, DSPE-PEG2000-KLVFF and the solution containing bone marrow mesenchymal stem cell membrane were all prepared according to the method provided in Example 1.

[0054] I. Nanospheres modified with different fluorescently labeled lectins and Aβ-targeting peptides The dosage was controlled according to the mass ratio of neutral polymer PLGA, bone marrow mesenchymal stem cell membrane, DSPE-PEG2000-UEA-I, and DSPE-PEG2000-KLVFF of 50:25.5:5.31:10.62.

[0055] Mix 1000 μL of 50 mg / mL PLGA dichloromethane solution with 1000 μL of 20 μg / mL Cy7 solution or 20 μg / mL DiR solution. Treat the mixture with an ultrasonic cell disruptor at 700 W for 1 min, alternating between on and off cycles for 2 seconds each, for a total duration of 1 min. Then add 10 mL of 10 mM Tris-HCl solution and 200 μL of dichloromethane solution, and continue ultrasonic treatment at 700 W for 2 seconds each, alternating between on and off cycles for a total duration of 2 min, to form a stable emulsion. Finally, add 20 mL of 10 mM Tris-HCl solution to the emulsion and magnetically stir at 700 rpm for 2 h. Centrifuge and wash to obtain Cy7 or DiR labeled PLGA nanospheres.

[0056] 1 mL of Cy7 or DiR-labeled PLGA nanospheres were suspended in a solution containing bone marrow mesenchymal stem cell membranes (25.5 mg / mL). The mixture was subjected to water bath sonication at 42 kHz and 100 W for 2 min to coat the surface of the Cy7 or DiR-labeled PLGA nanospheres, which served as the polymer matrix core, with the bone marrow mesenchymal stem cell membrane, forming a biomimetic cell membrane shell. The nanospheres coated with the biomimetic cell membrane shell were collected by centrifugation at 10000 g for 5 min, washed twice with ultrapure water, and set aside for later use.

[0057] The obtained biomimetic cell membrane shell-coated Cy7 or DiR-labeled nanospheres, DSPE-PEG2000-UEA-I, and DSPE-PEG2000-KLVFF were co-incubated for 30 min to obtain Cy7 or DiR-labeled lectins combined with Aβ-targeting peptide-modified nanospheres with a core-shell-crown three-layer hierarchical structure, denoted as BMUK@PLGA.

[0058] II. Nanospheres modified with lectins only by different fluorescent labels The dosage was controlled according to the mass ratio of neutral polymer PLGA, bone marrow mesenchymal stem cell membrane and DSPE-PEG2000-UEA-I of 50:25.5:5.31.

[0059] Mix 1000 μL of 50 mg / mL PLGA dichloromethane solution with 1000 μL of 20 μg / mL Cy7 solution or 20 μg / mL DiR solution. Treat the mixture with an ultrasonic cell disruptor at 700 W for 1 min, alternating between on and off cycles for 2 seconds each, for a total duration of 1 min. Then add 10 mL of 10 mM Tris-HCl solution and 200 μL of dichloromethane solution, and continue ultrasonic treatment at 700 W for 2 seconds each, alternating between on and off cycles for a total duration of 2 min, to form a stable emulsion. Finally, add 20 mL of 10 mM Tris-HCl solution to the emulsion and magnetically stir at 700 rpm for 2 h. Centrifuge and wash to obtain Cy7 or DiR labeled PLGA nanospheres.

[0060] Cy7 or DiR-labeled PLGA nanospheres were suspended in 1 mL of a solution containing bone marrow mesenchymal stem cell membranes (25.5 mg / mL). The mixture was subjected to water bath sonication at 42 kHz and 100 W for 2 min to allow the bone marrow mesenchymal stem cell membranes to tightly coat the surface of the Cy7 or DiR-labeled PLGA nanospheres, which served as the polymer matrix core, forming a biomimetic cell membrane shell. The nanospheres coated with the biomimetic cell membrane shell were collected by centrifugation at 10000 g for 5 min, washed twice with ultrapure water, and set aside for later use.

[0061] The obtained biomimetic cell membrane shell-coated Cy7 or DiR-labeled nanospheres were co-incubated with DSPE-PEG2000-UEA-I for 30 min to obtain Cy7 or DiR-labeled lectin-modified nanospheres with a core-shell-crown three-layer hierarchical structure, denoted as BMU@PLGA.

[0062] III. Nanospheres modified with Aβ-targeting peptides only by different fluorescent labels The dosage was controlled according to the mass ratio of neutral polymer PLGA, bone marrow mesenchymal stem cell membrane and DSPE-PEG2000-KLVFF of 50:25.5:10.62.

[0063] Mix 1000 μL of 50 mg / mL PLGA dichloromethane solution with 1000 μL of 20 μg / mL Cy7 solution or 20 μg / mL DiR solution. Treat the mixture with an ultrasonic cell disruptor at 700 W for 1 min, alternating between on and off cycles for 2 seconds each, for a total duration of 1 min. Then add 10 mL of 10 mM Tris-HCl solution and 200 μL of dichloromethane solution, and continue ultrasonic treatment at 700 W for 2 seconds each, alternating between on and off cycles for a total duration of 2 min, to form a stable emulsion. Finally, add 20 mL of 10 mM Tris-HCl solution to the emulsion and magnetically stir at 700 rpm for 2 h. Centrifuge and wash to obtain Cy7 or DiR labeled PLGA nanospheres.

[0064] Cy7 or DiR-labeled PLGA nanospheres were suspended in 1 mL of a solution containing bone marrow mesenchymal stem cell membranes (25.5 mg / mL). The mixture was subjected to water bath sonication at 42 kHz and 100 W for 2 min to allow the bone marrow mesenchymal stem cell membranes to tightly coat the surface of the Cy7 or DiR-labeled PLGA nanospheres, which served as the polymer matrix core, forming a biomimetic cell membrane shell. The nanospheres coated with the biomimetic cell membrane shell were collected by centrifugation at 10000 g for 5 min, washed twice with ultrapure water, and set aside for later use.

[0065] The obtained biomimetic cell membrane shell-coated Cy7 or DiR-labeled nanospheres were co-incubated with DSPE-PEG2000-KLVFF for 30 min to obtain Cy7 or DiR-labeled nanospheres modified only with Aβ-targeting peptides and having a core-shell-crown three-layer hierarchical structure, denoted as BMK@PLGA.

[0066] IV. Unmodified biomimetic cell membrane shell-coated nanospheres with different fluorescent labels The dosage was controlled according to the mass ratio of neutral polymer PLGA to bone marrow mesenchymal stem cell membrane of 50:25.5.

[0067] Mix 1000 μL of 50 mg / mL PLGA dichloromethane solution with 1000 μL of 20 μg / mL Cy7 solution or 20 μg / mL DiR solution. Treat the mixture with an ultrasonic cell disruptor at 700 W for 1 min, alternating between on and off cycles for 2 seconds each, for a total duration of 1 min. Then add 10 mL of 10 mM Tris-HCl solution and 200 μL of dichloromethane solution, and continue ultrasonic treatment at 700 W for 2 seconds each, alternating between on and off cycles for a total duration of 2 min, to form a stable emulsion. Finally, add 20 mL of 10 mM Tris-HCl solution to the emulsion and magnetically stir at 700 rpm for 2 h. Centrifuge and wash to obtain Cy7 or DiR labeled PLGA nanospheres.

[0068] Cy7 or DiR-labeled PLGA nanospheres were suspended in 1 mL of a solution containing bone marrow mesenchymal stem cell membranes (25.5 mg / mL). The mixture was subjected to water bath sonication at 42 kHz and 100 W for 2 min to allow the bone marrow mesenchymal stem cell membranes to tightly coat the surface of the Cy7 or DiR-labeled PLGA nanospheres, which served as the polymer matrix core, forming a biomimetic cell membrane shell. The unmodified Cy7 or DiR-labeled nanospheres coated with the biomimetic cell membrane shell were collected after centrifugation at 10000 g for 5 min and denoted as BM@PLGA.

[0069] Experimental Example 2 This experiment investigated the ability of the four nanospheres prepared in Experiment 1 to cross the nasal mucosal barrier using the Transwell in vitro nasal mucosal barrier model.

[0070] (1) Establishing a Transwell in vitro nasal mucosal barrier model Human nasal epithelial cells (HNEpC) were selected as the model cells and cultured in complete culture medium, namely MEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). The cells were placed in a cell culture incubator at 37°C and 5% CO2 and passaged using the adherent culture method.

[0071] When HNEpC cells proliferated to 90% confluence density, they were digested with 0.25% trypsin-EDTA digestion solution. After digestion was terminated, the cells were collected by centrifugation, resuspended in the complete culture medium described above, and the cell density was adjusted to 1×10⁶ cells / year. 6 / mL. Take 200μL of HNEpC cell suspension with adjusted density and seed it evenly in the upper chamber of Transwell; add 600μL of the above complete culture medium to the lower chamber and place it in a 37℃, 5% CO2 cell culture incubator for 5-7 days. Change the culture medium every 2 days until the HNEpC cells in the upper chamber form a dense and complete nasal mucosal epithelial monolayer, thus completing the establishment of the Transwell in vitro nasal mucosal barrier model.

[0072] (2) Detection of the ability of nanospheres to cross the Transwell nasal mucosal barrier in vitro The complete culture medium in the Transwell was replaced with DMEM medium, and after culturing for another 4 hours, four types of Cy7 fluorescently labeled nanospheres (BM@PLGA, BMU@PLGA, BMK@PLGA, and BMUK@PLGA) prepared in Experiment 1 were added, and the cells were cultured at 37°C and 5% CO2. After a total incubation of 24 hours, HNEpC cells in the lower chamber of the Transwell were collected in 1.5 mL EP tubes at 0, 4, 8, 12, and 24 hours. Fluorescence images were acquired at 740 nm using a fluorescence imaging system, and the cumulative fluorescence intensity was counted. The results are as follows: Figure 4 and Figure 5 As shown.

[0073] Figure 4 The results showed that the double-modified group (BMUK@PLGA) with lectin and Aβ-targeting peptide was the first to show obvious fluorescence at 4h, and its brightness was significantly higher than that of the other three groups; the single-modified group (BMU@PLGA, BMK@PLGA) had weaker fluorescence than the double-modified group, and the unmodified group (BM@PLGA) had the weakest fluorescence; at 24h, the fluorescence brightness and range of the double-modified group reached the peak, which was much higher than that of the single-modified group and the unmodified group.

[0074] Figure 5 The results showed that the highest cumulative amount of nanospheres penetrating the in vitro nasal mucosal barrier and reaching the lower chamber of Transwell within 24 hours was observed in the dual-modification group. This indicates that the nanospheres modified with lectin and Aβ-targeting peptide have a stronger ability to penetrate the nasal mucosal barrier. Compared with single-ligand modified and unmodified nanospheres, the dual-ligand modification of lectin UEA-I and targeting peptide KLVFF can play a synergistic role, significantly improving the efficiency of nanospheres penetrating the nasal mucosal barrier, and providing experimental evidence for drug delivery targeting the central nervous system via the nose.

[0075] Experimental Example 3 This experiment investigated the ability of four types of nanospheres prepared in Experiment 1 to be introduced into the brain via the nose and target the Aβ lesion area using an Aβ hippocampal deposition mouse model.

[0076] (1) Constructing an Aβ hippocampal deposition mouse model 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). After a 7-day recovery period following surgery, stable Aβ deposition was ensured to form a pathological model for subsequent targeted experiments.

[0077] (2) Small animal in vivo imaging to detect the brain targeting of nanospheres.

[0078] Aβ hippocampal deposited mice were randomly divided into five groups. They were given intranasal administration of pure PLGA nanospheres with DiR fluorescent labeling prepared in Experiment 1, as well as four different modified nanospheres BM@PLGA, BMU@PLGA, BMK@PLGA and BMUK@PLGA. The concentration of the nanospheres was 50 mg / mL and the dosage was 20 μL per mouse.

[0079] Four groups of mice were placed in a small animal in vivo imaging chamber. In vivo imaging was performed at 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after drug administration, at an excitation wavelength of 720 nm and an emission wavelength of 790 nm, to capture the fluorescence distribution in the mouse brain. Subsequently, isolated brain tissue from the mice was harvested at each time point and subjected to fluorescence imaging again to quantitatively assess the accumulation of nanospheres in the brain. The results are as follows: Figure 6 and Figure 7 As shown.

[0080] Figure 6 The results showed that the fluorescence intensity of the nanospheres modified with lectin and Aβ-targeting peptide was significantly higher than that of other groups at all time points. The fluorescence intensity of the single-modification group (BMU@PLGA, BMK@PLGA) was significantly weaker than that of the double-modification group, while the fluorescence intensity of the unmodified group (BM@PLGA) and the pure PLGA nanosphere group was the weakest. This indicates that the nanospheres modified with lectin and Aβ-targeting peptide have the highest efficiency of entering the brain via the nose, the strongest enrichment ability in the Aβ-lesioned brain region marked by circles in the figure, and the targeting effect remains stable over time.

[0081] Figure 7 The results showed that the highest drug accumulation in the isolated brain was achieved by nanospheres modified with lectin and Aβ-targeting peptide. This indicates that nanospheres modified with lectin and Aβ-targeting peptide have a stronger targeting ability to the brain Aβ region. Compared with the single modification group, the dual modification of lectin UEA-I and targeting peptide KLVFF can play a synergistic role, significantly improving the efficiency of drug penetration through the nasal mucosal barrier and targeting the Aβ region in the brain.

[0082] Test Example 4 This experiment investigated the ability of four types of nanospheres prepared in Experiment 1 to be introduced into the brain via the nose and target the Aβ lesion area using an APP / PS1 transgenic mouse model.

[0083] The APP / PS1 transgenic mouse model in this embodiment was purchased from Jiangsu Airingfei Biotechnology Co., Ltd. This model mouse spontaneously forms Aβ plaques in brain regions such as the hippocampus and cortex, simulating the core pathological features of Alzheimer's disease patients.

[0084] Aβ hippocampal deposited mice were randomly divided into five groups. After being anesthetized with isoflurane, they were given intranasal administration of pure PLGA nanospheres with DiR fluorescent labeling prepared in Experiment 1, as well as four different modified nanospheres BM@PLGA, BMU@PLGA, BMK@PLGA and BMUK@PLGA. The concentration of the nanospheres was 50 mg / mL and the dosage was 20 μL per mouse.

[0085] Four groups of mice were placed in a small animal in vivo imaging chamber. In vivo imaging was performed at 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after drug administration, at an excitation wavelength of 720 nm and an emission wavelength of 790 nm, to capture the fluorescence distribution in the mouse brain. Subsequently, isolated brain tissue from the mice was harvested at each time point and subjected to fluorescence imaging again to quantitatively assess the accumulation of nanospheres in the brain. The results are as follows: Figure 8 and Figure 9 As shown.

[0086] Figure 8 The results showed that the brain fluorescence intensity of the nanospheres modified with lectin and Aβ-targeting peptide was significantly higher than that of other groups at all time points, indicating that the nanospheres modified with lectin and Aβ-targeting peptide had the highest efficiency of entering the brain via the nose and demonstrated the ability to enrich the brain region with Aβ lesions in the AD model.

[0087] Figure 9 The results showed that the highest drug accumulation in the isolated brain was achieved by nanospheres modified with lectin and Aβ-targeting peptide. This indicates that nanospheres modified with lectin and Aβ-targeting peptide have a stronger targeting ability to the brain Aβ region. Compared with the single modification group, the dual modification of lectin UEA-I and targeting peptide KLVFF can play a synergistic role, significantly improving the efficiency of drug penetration through the nasal mucosal barrier and targeting the Aβ region in the brain.

[0088] Experimental Example 5 To investigate the in vivo drug delivery effect of the lectin-modified Aβ-targeting peptide nanospheres prepared in Example 4, this experiment prepared PLGA nanospheres encapsulated in a DiR fluorescently labeled human bone marrow mesenchymal stem cell membrane to detect the uptake of the nanospheres in the brain.

[0089] In this example, 1,1'-bis(octadecyl)-3,3,3'3'-tetramethyl-diiodocyanine (DiR) was purchased from HEDE Biotechnology Co., Ltd. (Beijing, China); DSPE-PEG2000-WGA, DSPE-PEG2000-LPFFD, and the solution containing bone marrow mesenchymal stem cell membrane were all prepared according to the method provided in Example 4.

[0090] I. DiR fluorescently labeled lectin combined with Aβ-targeting peptide-modified nanospheres The dosage was controlled according to the mass ratio of neutral polymer PLGA, bone marrow mesenchymal stem cell membrane, DSPE-PEG2000-WGA and DSPE-PEG2000-LPFFD of 50:25.5:5.31:10.62.

[0091] Mix 1000 μL of 50 mg / mL PLGA dichloromethane solution with 1000 μL of 20 μg / mL DiR solution, and treat with an ultrasonic cell disruptor at 700 W power for 1 min, alternating between 2 s on and 2 s off, for a total duration of 1 min. Then add 10 mL of 10 mM Tris-HCl solution and 200 μL of dichloromethane solution, and continue ultrasonic treatment at 700 W power, alternating between 2 s on and 2 s off, for a total duration of 2 min, to form a stable emulsion. Finally, add 20 mL of 10 mM Tris-HCl solution to the emulsion, and magnetically stir at 700 rpm for 2 h. After centrifugation and washing, obtain DiR-labeled PLGA nanospheres.

[0092] DiR-labeled PLGA nanospheres were suspended in 1 mL of a solution containing bone marrow mesenchymal stem cell membranes (25.5 mg / mL). The mixture was subjected to water bath sonication at 42 kHz and 100 W for 2 min to tightly coat the surface of the DiR-labeled PLGA nanospheres, which served as the polymer matrix core, forming a biomimetic cell membrane shell. The nanospheres coated with the biomimetic cell membrane shell were collected by centrifugation at 10000 g for 5 min, washed twice with ultrapure water, and set aside for later use.

[0093] The obtained biomimetic cell membrane shell-coated DiR-labeled nanospheres, DSPE-PEG2000-WGA, and DSPE-PEG2000-LPFFD were co-incubated for 30 min to obtain DiR-labeled lectin-modified Aβ-targeting peptide nanospheres with a core-shell-crown three-layer hierarchical structure, denoted as hBMWL@PLGA.

[0094] II. DiR fluorescently labeled lectin-modified nanospheres The dosage was controlled according to the mass ratio of neutral polymer PLGA, bone marrow mesenchymal stem cell membrane and DSPE-PEG2000-WGA of 50:25.5:5.31.

[0095] Mix 1000 μL of 50 mg / mL PLGA dichloromethane solution with 1000 μL of 20 μg / mL DiR solution, and treat with an ultrasonic cell disruptor at 700 W power for 1 min, alternating between 2 s on and 2 s off, for a total duration of 1 min. Then add 10 mL of 10 mM Tris-HCl solution and 200 μL of dichloromethane solution, and continue ultrasonic treatment at 700 W power, alternating between 2 s on and 2 s off, for a total duration of 2 min, to form a stable emulsion. Finally, add 20 mL of 10 mM Tris-HCl solution to the emulsion, and magnetically stir at 700 rpm for 2 h. After centrifugation and washing, obtain DiR-labeled PLGA nanospheres.

[0096] DiR-labeled PLGA nanospheres were suspended in 1 mL of a solution containing bone marrow mesenchymal stem cell membranes (25.5 mg / mL). The mixture was subjected to water bath sonication at 42 kHz and 100 W for 2 min to tightly coat the surface of the DiR-labeled PLGA nanospheres, which served as the polymer matrix core, forming a biomimetic cell membrane shell. The nanospheres coated with the biomimetic cell membrane shell were collected by centrifugation at 10000 g for 5 min, washed twice with ultrapure water, and set aside for later use.

[0097] The obtained biomimetic cell membrane shell-coated DiR-labeled nanospheres were co-incubated with DSPE-PEG2000-WGA for 30 min to obtain DiR-labeled lectin-modified nanospheres with a core-shell-crown three-layer hierarchical structure, denoted as hBMW@PLGA.

[0098] III. DiR fluorescently labeled nanospheres modified with only Aβ-targeting peptide The dosage was controlled according to the mass ratio of neutral polymer PLGA, bone marrow mesenchymal stem cell membrane and DSPE-PEG2000-LPFFD of 50:25.5:10.62.

[0099] Mix 1000 μL of 50 mg / mL PLGA dichloromethane solution with 1000 μL of 20 μg / mL DiR solution, and treat with an ultrasonic cell disruptor at 700 W power for 1 min, alternating between 2 s on and 2 s off, for a total duration of 1 min. Then add 10 mL of 10 mM Tris-HCl solution and 200 μL of dichloromethane solution, and continue ultrasonic treatment at 700 W power, alternating between 2 s on and 2 s off, for a total duration of 2 min, to form a stable emulsion. Finally, add 20 mL of 10 mM Tris-HCl solution to the emulsion, and magnetically stir at 700 rpm for 2 h. After centrifugation and washing, obtain DiR-labeled PLGA nanospheres.

[0100] DiR-labeled PLGA nanospheres were suspended in 1 mL of a solution containing bone marrow mesenchymal stem cell membranes (25.5 mg / mL). The mixture was subjected to water bath sonication at 42 kHz and 100 W for 2 min to tightly coat the surface of the DiR-labeled PLGA nanospheres, which served as the polymer matrix core, forming a biomimetic cell membrane shell. The nanospheres coated with the biomimetic cell membrane shell were collected by centrifugation at 10000 g for 5 min, washed twice with ultrapure water, and set aside for later use.

[0101] The obtained biomimetic cell membrane shell-coated DiR-labeled nanospheres were co-incubated with DSPE-PEG2000-LPFFD for 30 min to obtain DiR-labeled nanospheres modified only with Aβ-targeting peptides and having a core-shell-crown three-layer hierarchical structure, denoted as hBML@PLGA.

[0102] IV. DiR fluorescently labeled unmodified biomimetic cell membrane shell-coated nanospheres The dosage was controlled according to the mass ratio of neutral polymer PLGA to bone marrow mesenchymal stem cell membrane of 50:25.5.

[0103] Mix 1000 μL of 50 mg / mL PLGA dichloromethane solution with 1000 μL of 20 μg / mL DiR solution, and treat with an ultrasonic cell disruptor at 700 W power for 1 min, alternating between 2 s on and 2 s off, for a total duration of 1 min. Then add 10 mL of 10 mM Tris-HCl solution and 200 μL of dichloromethane solution, and continue ultrasonic treatment at 700 W power, alternating between 2 s on and 2 s off, for a total duration of 2 min, to form a stable emulsion. Finally, add 20 mL of 10 mM Tris-HCl solution to the emulsion, and magnetically stir at 700 rpm for 2 h. After centrifugation and washing, obtain DiR-labeled PLGA nanospheres.

[0104] DiR-labeled PLGA nanospheres were suspended in 1 mL of a solution containing bone marrow mesenchymal stem cell membranes (25.5 mg / mL). The mixture was subjected to water bath sonication at 42 kHz and 100 W for 2 min to tightly coat the surface of the DiR-labeled PLGA nanospheres, which served as the polymer matrix core, forming a biomimetic cell membrane shell. The nanospheres coated with the unmodified DiR-labeled biomimetic cell membrane shell were collected after centrifugation at 10000 g for 5 min and denoted as hBM@PLGA.

[0105] Experimental Example 6 This experiment investigated the ability of four types of nanospheres prepared in Experiment 5 to be introduced into the brain via the nose and target the Aβ lesion area using an APP / PS1 transgenic mouse model.

[0106] The APP / PS1 transgenic mouse model in this embodiment was purchased from Jiangsu Airingfei Biotechnology Co., Ltd. This model mouse spontaneously forms Aβ plaques in brain regions such as the hippocampus and cortex, simulating the core pathological features of Alzheimer's disease patients.

[0107] Aβ hippocampal deposited mice were randomly divided into five groups. After being anesthetized with isoflurane, they were given intranasal administration of pure PLGA nanospheres with DiR fluorescent labeling prepared in Experiment 5, as well as four different modified nanospheres hBM@PLGA, hBMW@PLGA, hBML@PLGA and hBMWL@PLGA. The concentration of the nanospheres was 50 mg / mL and the dosage was 20 μL per mouse.

[0108] Four groups of mice were placed in a small animal in vivo imaging chamber. In vivo imaging was performed at 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after drug administration, at an excitation wavelength of 720 nm and an emission wavelength of 790 nm, to capture the fluorescence distribution in the mouse brain. Subsequently, isolated brain tissue from the mice was harvested at each time point and subjected to fluorescence imaging again to quantitatively assess the accumulation of nanospheres in the brain. The results are as follows: Figure 13 and Figure 14 As shown.

[0109] Figure 13 The results showed that the brain fluorescence intensity of the nanospheres modified with lectin and Aβ-targeting peptide was significantly higher than that of other groups at all time points, indicating that the nanospheres modified with lectin and Aβ-targeting peptide had the highest efficiency of entering the brain via the nose and demonstrated the ability to enrich the brain region with Aβ lesions in the AD model.

[0110] Figure 14The results showed that the highest drug accumulation in the isolated brain was achieved by nanospheres modified with lectin and Aβ-targeting peptide. This indicates that nanospheres modified with lectin and Aβ-targeting peptide have a stronger targeting ability to the brain Aβ region. Compared with the single modification group, the dual-ligand modification of lectin WGA and targeting peptide LPFFD can play a synergistic role, significantly improving the efficiency of drug penetration through the nasal mucosal barrier and targeting the Aβ region in the brain.

Claims

1. A lectin-targeted peptide-modified nanosphere, characterized in that, The nanospheres have a core-shell-crown three-layer hierarchical structure, comprising, from the inside out, a polymer matrix core, a biomimetic cell membrane shell, and a dual-ligand functionalized surface crown. The polymer matrix core is composed of a biodegradable neutral polymer. The biomimetic cell membrane shell tightly coats the surface of the polymer matrix core. Polyethylene glycol-modified phospholipids covalently coupled to lectins and polyethylene glycol phospholipids covalently coupled to targeting peptides are spontaneously inserted into the lipid bilayer of the biomimetic cell membrane shell through hydrophobic fragments. The hydrophilic polyethylene glycol chains extend to the outer side of the membrane, anchoring the lectins and targeting peptides to the surface of the biomimetic cell membrane shell, forming a dual-ligand functionalized surface crown.

2. The nanospheres according to claim 1, characterized in that, The neutral polymer is one of polylactic acid-glycolic acid copolymer, polylactic acid, polyethylene glycol-polylactic acid, polyhydroxyalkanoate, polymethyl methacrylate, polystyrene, polycaprolactone, or poly-N-isopropylacrylamide; The biomimetic cell membrane shell is one of the following: autologous bone marrow mesenchymal stem cell membrane, allogeneic bone marrow mesenchymal stem cell membrane, adipose mesenchymal stem cell membrane, umbilical cord mesenchymal stem cell membrane, erythrocyte membrane, leukocyte membrane, platelet membrane, or neutrophil membrane. The lectin is one of the following: *Vitex negundo* lectin or *Cyclocarya paliurus* lectin that recognizes fucose; wheat germ lectin or *Hymenopus hyacinthus* lectin that recognizes N-acetylglucosamine; soybean lectin or peanut lectin that recognizes galactose; and concanavalin A lectin or lentil lectin that recognizes mannose. The targeting peptide is one of Aβ targeting peptide, α-synuclein targeting peptide, epidermal growth factor receptor targeting peptide, or integrin αvβ3 targeting peptide. The polyethylene glycol in both the lectin-functionalized PEGylated phospholipids and the targeted peptide-functionalized PEGylated phospholipids has a molecular weight of 600~5000 Da.

3. The nanospheres according to claim 2, characterized in that, The Aβ targeting peptide is one of KLVFF, LPFFD, LVFFARK, LVFFARKHH, c-LVFFARK, car-LVFFARK, L-RTHLVFFARK, or D-RTHLVFFARK; the α-synuclein targeting peptide is one of SYN17 or VFFK; the epidermal growth factor receptor targeting peptide is one of GE11 or EGFR10R; and the integrin αvβ3 targeting peptide is one of RGD or c(RGDfK).

4. A method for preparing nanospheres as described in any one of claims 1-3, characterized in that, Includes the following steps: Step 1: Dissolve the polyethylene glycol-modified phospholipid activator DSPE-PEG-NHS in organic solvent I, add lectin and buffer solution, mix and stir to react, then dialyze and dry to obtain lectin-functionalized polyethylene glycol-modified phospholipid. The maleimide derivative of polyethylene glycol phospholipid, DSPE-PEG-Mal, was dissolved in organic solvent II. The target peptide and catalyst were added, and after mixing and stirring, the mixture was dialyzed and dried to obtain the target peptide-functionalized polyethylene glycol phospholipid. Step 2: Control the dosage by using neutral polymer, cell membrane, lectin-functionalized polyethylene glycol phospholipid and target peptide-functionalized polyethylene glycol phospholipid in a mass ratio of 40~60:20~30:4~8:8~12; Neutral polymers were dissolved in an organic solvent and vortexed to form the organic phase. An aqueous phase was then added and ultrasonically dispersed to form an emulsion. After stirring, centrifugation, and washing, neutral polymer nanospheres were obtained. The obtained neutral polymer nanospheres were suspended in a cell membrane solution and ultrasonically treated to tightly coat the surface of the neutral polymer nanospheres with cell membranes. After centrifugation and washing, biomimetic cell membrane shell-coated nanospheres were obtained. The obtained biomimetic cell membrane shell-coated nanospheres, lectin-functionalized polyethylene glycol phospholipids obtained in step one, and target peptide-functionalized polyethylene glycol phospholipids were co-incubated to anchor and modify the lectin and target peptides on the outer side of the biomimetic cell membrane shell, assembling them to form a dual-ligand functionalized surface crown layer, resulting in lectin-modified nanospheres with a core-shell-crown three-layer hierarchical structure.

5. The method for preparing nanospheres according to claim 4, 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-volume ratio of DSPE-PEG-NHS, dimethylformamide, lectin, phosphate buffer and carbonate buffer is 400~600mg:4~6mL:1~3mg:1~3mL:0.5~2mL. The mixing and stirring reaction time is 0.5~2h. Dialysis is performed sequentially using phosphate buffer and pure water. The total dialysis time is 7~9h. The drying method is freeze drying.

6. The method for preparing nanospheres according to claim 4 or 5, characterized in that, The organic solvent II mentioned in step one is chloroform, the targeted peptide is dissolved in methanol, and the catalyst is triethylamine; the mass-volume ratio of DSPE-PEG-Mal, chloroform, targeted peptide, 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 20~30h, the dialysis time is 3~5h, and the drying method is freeze drying.

7. The method for preparing nanospheres according to claim 6, characterized in that, The mass ratio of the neutral polymer, cell membrane, lectin-functionalized polyethylene glycol phospholipid, and target peptide-functionalized polyethylene glycol phospholipid described in step two is 50:25.5:5.31:10.62, 40:20.5:4:8, or 60:29.5:8:

12. The organic solvent is dichloromethane, and the mass-to-volume ratio of the neutral polymer to dichloromethane is 40-60 mg: 0.5-2 mL; the ultrasonic dispersion is performed with an ultrasonic power of 700 W, and the treatment method is to alternate between on and off for 2 seconds, repeated 2-3 times; the stirring speed is 700 rpm and the stirring time is 6 hours; the centrifugation speed is 10000 g and the centrifugation time is 5 minutes; and ultrapure water is used for washing.

8. The method for preparing nanospheres according to claim 7, characterized in that, The ultrasonic treatment in step two is water bath ultrasonic treatment, with an ultrasonic frequency of 42kHz, an ultrasonic power of 100W, and an ultrasonic time of 2min; the co-incubation time is 20~40min, and the final obtained nanospheres have a particle size of 140~180nm and a negatively charged surface.

9. The use of the nanospheres as described in any one of claims 1-3 in the preparation of a nasal drug delivery carrier targeting the central nervous system.

10. The application according to claim 9, characterized in that, The drug delivery carrier carries one or more of the following: nucleic acid drugs, insoluble small molecule drugs, protein drugs, peptide drugs, or vaccine antigens.

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