A PROTAC-loaded biomimetic neuro-targeting plant exosome and its preparation and application

Abstract

This invention discloses a biomimetic neuro-targeting plant exosome loaded with PROTAC, its preparation, and its application, belonging to the field of biomedical technology. This nanomedicine uses plant-derived exosomes as the core carrier, loading PROTAC molecules and naturally carrying endogenous active molecules. Based on this, an engineered exosome delivery system is constructed through biomimetic cell membrane coating and modification with pH-responsive lysosomal escape polymers. After intranasal administration, this system can overcome the nasal mucosal barrier sequentially, achieving enrichment in brain lesion areas and efficiently releasing the therapeutic payload within cells. This invention utilizes the selective degradation of pathology-related proteins by PROTAC, combined with the regulatory effects of endogenous active molecules from plant exosomes on cellular metabolism and mitochondrial function-related pathways, to achieve synergistic regulation of neuronal protein homeostasis and metabolic homeostasis, thereby providing multi-target, multi-level intervention in the pathological processes of neurodegenerative diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical nanotechnology, specifically relating to a biomimetic neural-targeting plant exosome loaded with PROTAC and its preparation and application. Background Technology

[0002] Neurodegenerative diseases are a class of diseases characterized by the progressive, selective loss of neuronal structure or function (including neuronal death), leading to functional impairment. These include Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and spinal muscular atrophy (SMA). Currently, the specific causes of these diseases are unclear, and existing treatments have not significantly altered their progression. They not only seriously threaten global public health but also impose a heavy socioeconomic burden due to long-term care and treatment needs. The development of treatment strategies for neurodegenerative diseases faces multiple challenges: on the one hand, it requires overcoming the bottleneck of efficient, precise, and safe brain-targeted delivery; on the other hand, it needs to address the complex network of pathological mechanisms, such as neuronal exhaustion caused by neuronal mitochondrial dysfunction and kinase overactivation. Therefore, there is an urgent need for a treatment strategy that can achieve efficient brain-targeted delivery while simultaneously blocking kinases and remodeling mitochondrial metabolic support within the same spatiotemporal range.

[0003] Protein degradation-targeting chimeras (PROTACs) technology can catalyze ubiquitin-dependent protein degradation, eliminating the enzymatic activity and scaffold function of overactive pathological kinases at the source, providing an ideal solution for breaking the aforementioned pathological mechanism network. However, currently reported PROTAC molecules suffer from typical drug delivery defects such as poor blood-brain barrier (BBB) ​​penetration and low oral bioavailability, severely limiting their therapeutic potential.

[0004] To circumvent the blood-brain barrier, the nasobrain pathway, where drugs are administered directly to the brain, has garnered significant attention. However, this route still faces three delivery barriers. First, the nasal mucosa acts as a barrier; the rapid ciliary clearance mechanism and mucosal layer of the nasal cavity capture and remove most drug carriers, preventing their penetration into the epithelium. Second, there is insufficient targeting within the brain; unmodified carriers, once inside the brain, often exhibit non-specific distribution, failing to accumulate in the core lesions of Parkinson's disease—the substantia nigra pars compacta (SNpc). Finally, there is the challenge of lysosomal escape; even when drug carriers are taken up by cells, they are often isolated and degraded within lysosomes, preventing the therapeutic payload from being released into the cytoplasm to exert its effect.

[0005] Plant-derived exosomes are considered promising natural drug delivery carriers due to their excellent biocompatibility, low immunogenicity, and endogenous active ingredients (such as functional miRNAs and lipids). Some traditional Chinese medicinal plants possess blood-tonifying, blood-activating, meridian-clearing, and pain-relieving effects, and their extracts have been shown to have metabolic regulatory and neuroprotective activities. However, unmodified natural plant exosomes also struggle to overcome the multiple physiological barriers of naso-brain delivery, lacking the ability to target diseased brain regions with precise delivery and efficient cytoplasmic release. Therefore, developing a biomimetic intelligent nanomedicine system that integrates the degradation advantages of PROTAC with the metabolic regulatory functions of plant exosomes and synergistically overcomes multiple delivery barriers has significant scientific value and clinical potential for achieving precise and multidimensional treatment of neurodegenerative diseases. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a PROTAC-loaded biomimetic neuro-targeting plant exosome, its preparation, and its application. The PROTAC-loaded biomimetic neuro-targeting plant exosome provided by this invention can overcome the nasal mucosa and lysosomal barriers, precisely target brain lesions, and synergistically treat neurodegenerative diseases through a dual mechanism of kinase degradation and metabolic remodeling. The PROTAC-loaded biomimetic neuro-targeting plant exosome of this invention has a core derived from natural exosomes of traditional Chinese medicinal plants, which is loaded with a kinase-targeting protein degradation chimeric molecule; an outer shell is a cell membrane encapsulating the surface of the core; and a surface modification layer is a pH-responsive polymer intercalated into the lipid bilayer of the outer shell.

[0007] According to a first aspect of the present invention, the present invention first provides a method for preparing biomimetic neural-targeting plant exosomes loaded with PROTAC, which includes the following steps: 1) Load PROTAC into plant exosomes, using the active molecules contained in the exosomes themselves and the loaded PROTAC as complementary therapeutic molecules; 2) Hybridize the cell membrane with the plant exosomes loaded with PROTAC from step 1); 3) By co-incubating or click chemistry, the surface of the membrane-hybridized PROTAC-loaded plant exosomes obtained in step 2) is modified with a pH-responsive multifunctional polymer to obtain the PROTAC-loaded biomimetic neural-targeting plant exosomes.

[0008] According to a preferred embodiment of the present invention, in step 1), the method of loading PROTAC into plant exosomes is co-incubation, sonication, electroporation, or freeze-thaw cycling; the mass ratio of exosome protein to PROTAC is 0.05~20:1.

[0009] According to a preferred embodiment of the present invention, the plant is one or more of the following: Schisandra chinensis, ginseng, Ziziphus jujuba var. spinosa, Ligusticum chuanxiong, Panax notoginseng, Angelica sinensis, Rabdosia rubescens, Acorus tatarinowii, Pueraria lobata, Gastrodia elata, and Astragalus membranaceus.

[0010] According to a preferred embodiment of the present invention, the PROTAC is one or more of ARV-110, ARV-471, XL01126, NX-2127, NX-5948, and KT-474.

[0011] According to a preferred embodiment of the present invention, the method for hybridizing the cell membrane with the PROTAC-loaded plant exosomes in step 1) is co-extrusion, PEG-mediated fusion, or co-incubation; wherein the mass ratio of plant exosome membrane surface proteins to cell membrane surface proteins is 0.05~20:1.

[0012] According to a preferred embodiment of the present invention, the cell membrane is one or more of embryonic stem cell membrane, adult stem cell membrane, neural stem cell membrane, induced pluripotent stem cell membrane, tumor cell membrane, T cell membrane, NK cell membrane, and macrophage membrane; and the cell membrane surface expresses one or more of chemokine receptors CXCR4, CXCR7, CXCR9, and CXCR10.

[0013] According to a preferred embodiment of the present invention, the pH-responsive polymer DSPE-His-PEG is composed of distearate phosphatidylethanolamine (DSPE) and histidine polypeptide His. 6-15 It is coupled with polyethylene glycol; wherein, the DSPE is a lipid molecule used to link to the membrane surface, and the histidine polypeptide has a proton sponge effect to promote the lysosomal escape of the PROTAC-loaded biomimetic neural-targeting plant exosomes; the polyethylene glycol is used to assist the PROTAC-loaded biomimetic neural-targeting plant exosomes in penetrating the mucus barrier and prolonging blood circulation time.

[0014] According to a second aspect of the present invention, the present invention also provides a biomimetic neural-targeting plant exosome loaded with PROTAC, which is prepared by the aforementioned method.

[0015] According to a third aspect of the present invention, the present invention also provides an application of the aforementioned PROTAC-loaded biomimetic neuro-targeting plant exosomes, specifically for the preparation of a drug for treating central nervous system degenerative diseases; wherein the central nervous system degenerative diseases may be Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, or spinal muscular atrophy.

[0016] Compared with the prior art, the present invention has the following significant advantages: (1) Synergistic dual therapeutic mechanism: This invention organically integrates exogenous chemical drugs (PROTAC) with endogenous bioactive components of plant exosomes. The proteolytic targeted chimera catalyzes the degradation of pathological proteins through the ubiquitin-proteasome pathway, blocking inflammatory signals at the source; at the same time, the endogenous active molecules carried by plant exosomes activate related metabolic signaling axes, promoting mitochondrial biogenesis and antioxidant defense. This synergistic effect of dekinase and metabolic remodeling can more effectively break the vicious cycle of neurodegenerative diseases.

[0017] (2) Overcoming multiple physiological barriers: The surface-modified PEG hydrophilic chain forms a hydration layer, reducing mucin adsorption, prolonging the residence time of nanomedicines in the nasal cavity and promoting penetration. The introduced His polypeptide fragment has a proton sponge effect, protonating in the acidic environment of endosomes / lysosomes, leading to membrane rupture, thereby achieving efficient cytoplasmic release of drugs and avoiding degradation by lysosomes.

[0018] (3) Precise lesion targeting: By utilizing the chemotactic interaction between relevant receptors on the cell membrane surface and ligands highly expressed at the lesion site, the active enrichment of nanomedicines was achieved, which significantly increased the drug concentration at the lesion site in the brain and reduced the distribution of non-target sites.

[0019] (4) High safety: The main carrier materials of this invention are derived from natural plants and biological membranes, which have good biocompatibility and low immunogenicity. In vivo experiments have confirmed that long-term nasal administration did not cause significant toxicity to major organs such as the heart, liver, and kidneys, and has good potential for clinical translation. Attached Figure Description

[0020] Figure 1 :Structural confirmation of XL01126 and loading characterization of AEV-X. (a) Hydrated particle size distribution and Zeta potential detection of AEV-X; (b) Transmission electron microscopy morphology characterization of AEV-X (scale bar: 200 nm).

[0021] Figure 2 Construction and efficiency evaluation of AEV-XN membrane fusion. (a) Statistical histogram of fusion efficiency; (b) Quantitative statistical bar chart of membrane fusion efficiency; (c) Transmission electron micrograph of AEV-XN (scale bar: 200 nm); (d) Histogram of particle size distribution of AEV-XN.

[0022] Figure 3 Construction and physicochemical characterization of AEV-XNH. (a) Quantitative statistics of PEG grafting rate and XL01126 encapsulation rate; (b) Particle size distribution detection results of AEV-XNH; (c) TEM morphology characterization of AEV-XNH; (d) Zeta potential histogram of the system; (e) In vitro drug release kinetic curve of AEV-XNH.

[0023] Figure 4 Characterization of cell uptake kinetics of AEV-XNH and analysis of endocytosis pathway based on inhibitor screening. (a) Cell uptake at different time points observed by CLSM; (b) Quantitative statistical analysis of the proportion of DiO positive cells; (c) Quantitative statistical graph of positive cell uptake rate under various inhibitory conditions.

[0024] Figure 5 Evaluation of the lysosomal escape ability of AEV-XNH. (a) Confocal intracellular colocalization images at different time points; (b) Quantitative statistical analysis of Pearson colocalization coefficient (PCC).

[0025] Figure 6 Evaluation of the transnasal transport efficiency of AEV-XNH across the nasal mucosal epithelial barrier. (a) Statistical graph of fluorescence intensity of nanoparticles penetrating the culture medium in the lower chamber; (b) Statistical graph of fluorescence intensity related to target cells in the lower chamber.

[0026] Figure 7 MPP + Modeling concentration screening and in vitro neuroprotective activity of AEV-XNH. (a) Different concentrations of MPP + (a) Survival rate of SH-SY5Y cells after treatment; (b) Survival rate of SH-SY5Y cells after systemic intervention Figure 8 The regulatory effect of AEV-XNH on glial cell viability. Cell viability of BV2 cells after 12h (a) and 24h (b) intervention; cell viability of U87 cells after 12h (c) and 24h (d) intervention.

[0027] Figure 9 Degradation effect of AEV-XNH on LRRK2 protein. (a) Representative Western blot bands of LRRK2 and internal reference β-actin; (b) Quantitative analysis of relative expression of LRRK2 by grayscale.

[0028] Figure 10 The inhibitory effect of AEV-XNH on neuronal oxidative stress. (a) Statistical graph of relative ROS levels in each group; (b) Statistical graph of MDA content in each group.

[0029] Figure 11 The restorative effect of AEV-XNH on intraneuronal GSH levels.

[0030] Figure 12 AEV-XNH on neuronal NADP + / Regulatory role of NADPH in redox balance. (a) Statistical graph of NADPH content in each group; (b) NADP content in each group + / NADPH ratio statistics chart.

[0031] Figure 13 The repair effect of AEV-XNH on neuronal mitochondrial function. (a) Quantitative detection of intracellular ATP content in each group; (b) Quantitative statistical analysis of TMRE fluorescence intensity.

[0032] Figure 14 Quantitative statistical analysis of the intensity of fluorescence signals in brain regions after intranasal administration of AEV-XNH.

[0033] Figure 15 Evaluation of the behavioral improvement of AEV-XNH in PD mouse models. (a) Quantitative analysis of the resting time and (b) average velocity of mice in the open field test; (c) Statistical analysis of the fall latency of mice in the rotarod test; (d) Statistical analysis of the immobility time of mice in the tail suspension test.

[0034] Figure 16 : Protective effect of AEV-XNH on the function of dopaminergic neurons in the substantia nigra of mice. (a) Immunofluorescence double-labeled confocal images of dopamine transporter (DAT) and tyrosine hydroxylase (TH) (scale bar: 200 μm); (b) Quantitative statistics of the positive mean signal intensity of dopamine transporter; (c) Quantitative statistics of the positive mean signal intensity of tyrosine hydroxylase.

[0035] Figure 17 Representative bands of LRRK2 / PPAR signaling pathway-related proteins in the substantia nigra of the brain, as shown by Western Blot.

[0036] Figure 18 AEV-XNH affects (a) NADPH content and (b) NADP content in the substantia nigra of the brain. + Quantitative analysis of NADPH ratio, (c) MDA content and (d) GSH content.

[0037] Figure 19 : Curves showing the changes in body weight of mice in each group during the experiment.

[0038] Figure 20 Representative pathological images of major organs (heart, liver, spleen, lung, kidney) stained with hematoxylin and eosin (scale bar: 200 μm).

[0039] Figure 21 Quantitative statistical graphs of serum biochemical indicators (a) AST; (b) ALT; (c) CREA; (d) UREA. Detailed Implementation

[0040] Unless otherwise specified, all raw materials, reagents, and equipment involved in this invention can be purchased commercially. Unless otherwise specified, the experimental methods used in the following examples are conventional biochemical experimental methods.

[0041] The PROTAC-loaded biomimetic neural-targeting plant exosomes of the present invention are prepared using the following three steps: 1) Loading PROTAC into plant exosomes In an alternative embodiment of the present invention, PROTAC can be loaded into plant exosomes by means of co-incubation, sonication, electroporation, or freeze-thaw cycles, with the active molecules contained in the exosomes themselves and the loaded PROTAC serving as complementary therapeutic molecules.

[0042] The plant exosomes can be derived from one or more of the following: Schisandra chinensis, ginseng, Ziziphus jujuba var. spinosa, Ligusticum chuanxiong, Panax notoginseng, Angelica sinensis, Rabdosia rubescens, Acorus tatarinowii, Pueraria lobata, Gastrodia elata, and Astragalus membranaceus. The PROTAC is one or more of the following: ARV-110, ARV-471, PROTAC, NX-2127, NX-5948, and KT-474. The plant exosomes can activate mitochondrial metabolic reprogramming-related signaling pathways in target cells; the PROTAC is used to catalyze the degradation of pathological target proteins.

[0043] Typical, but not limited, co-incubation can be performed as follows: plant exosomes are mixed with PROTAC, wherein the mass ratio of exosome protein to PROTAC is 20:1 to 1:20, incubated at 37°C for 0.5 to 2 h, centrifuged at 100,000 × g to 150,000 × g for 0.5 to 1 h, the free PROTAC in the supernatant is removed, and the precipitate is collected to obtain the PROTAC-loaded plant exosomes.

[0044] Ultrasonic treatment can be performed as follows: Mix plant exosomes with PROTAC, wherein the mass ratio of exosome protein to PROTAC molecules is 20:1-1:20, sonicate, centrifuge at 100,000×g-150,000×g for 0.5-1h, remove free PROTAC from the supernatant, and collect the precipitate to obtain PROTAC-loaded plant exosomes.

[0045] Electroporation can be performed as follows: Plant exosomes are mixed with PROTAC, wherein the mass ratio of exosome protein to PROTAC is 20:1-1:20. Electroporation is performed under the following conditions: voltage (100-1000 V), electric field strength (5-20 V / cm), single pulse of 0.1-0.5 seconds, 2-10 pulses per group, pulse interval of 0.1-1 seconds, and buffer (low conductivity, such as sucrose solution or HEPES buffer). Centrifuge at 100,000×g-150,000×g for 0.5-1 h, remove the free PROTAC from the supernatant, and collect the precipitate to obtain the PROTAC-loaded plant exosomes.

[0046] The specific operation of a freeze-thaw cycle is as follows: Plant exosomes were mixed with PROTAC, with the mass ratio of exosome protein to PROTAC being 20:1 to 1:20. The mixture was subjected to repeated freeze-thaw cycles at 37°C and liquid nitrogen for 3 to 10 times. After centrifugation at 100,000×g to 150,000×g for 0.5 to 1 h, the free PROTAC in the supernatant was removed, and the precipitate was collected to obtain the PROTAC-loaded plant exosomes.

[0047] 2) Hybridize the cell membrane with the PROTAC-loaded plant exosomes from step 1). Specifically, the cell membrane can be hybridized with the plant exosomes loaded with PROTAC in step 1) by physical fusion (co-extrusion), chemical induction (PEG-mediated fusion), or spontaneous recombination of the biomembrane (co-incubation).

[0048] The cell membrane of this invention is one or more of the following: embryonic stem cell membrane, adult stem cell membrane, neural stem cell membrane, induced pluripotent stem cell membrane, tumor cell membrane, T cell membrane, NK cell membrane, and macrophage membrane. The cell membrane surface expresses one or more of the following chemokine receptors: CXCR4, CXCR7, CXCR9, and CXCR10. These receptors can specifically recognize and bind to one or more of the following chemokine receptors expressed at the lesion site: CXCL12, CXCL19, CXCL21, CXCL25, and CXCL27. The expression of chemokine receptors on the cell membrane surface endows nanomedicines with the ability to actively hom to the lesion region.

[0049] Typical, but not limited, physical fusion (co-extrusion) can be performed as follows: The obtained PROTAC-loaded plant exosomes and cell membranes are mixed, wherein the mass ratio of exosomes to cell membrane proteins is 20:1-1:20. The mixture is sonicated for 2-10 min, and then repeatedly extruded through polycarbonate membranes with pore sizes of 400 nm and 200 nm for 10-200 times. The resulting product is centrifuged at 100,000×g-150,000×g for 30-120 min, and the bottom precipitate is collected. This bottom precipitate is the membrane-hybridized PROTAC-loaded plant exosomes.

[0050] The chemical induction method (PEG-mediated fusion) can be carried out as follows: Mix the obtained PROTAC-loaded plant exosomes with the cell membrane, wherein the mass ratio of exosomes to cell membrane proteins is 20:1-1:20, add PEG-8000 (final concentration 1-20%) and incubate for 10-60 min, add PBS to dilute and terminate the reaction, and remove free PEG by ultrafiltration to obtain membrane-hybridized PROTAC-loaded plant exosomes.

[0051] The spontaneous recombination method of biomembranes (co-incubation) can be carried out as follows: Plant exosomes loaded with PROTAC are mixed with cell membranes, wherein the mass ratio of exosome protein to cell membrane protein is 20:1-1:20. This mixture is then placed in a Ca-containing environment. 2+ Incubate in (1-10 mM) buffer for 30-120 min, centrifuge at 100000×g-150000×g for 30-120 min, and collect the bottom precipitate. This bottom precipitate is the membrane-hybridized PROTAC-loaded plant exosome.

[0052] 3) By co-incubating or click chemistry, the surface of the membrane-hybridized PROTAC-loaded plant exosomes obtained in step 2) is modified with a pH-responsive multifunctional polymer to obtain the PROTAC-loaded biomimetic neural-targeting plant exosomes.

[0053] Typically, but not limited to, the co-incubation method can be carried out as follows: membrane-hybridized PROTAC-loaded plant exosomes are mixed with a pH-responsive polymer DSPE-His-PEG, with the polymer concentration controlled at 50 to 500 μg / mL, and incubated in a buffer solution at pH 7.0 to 7.4 at 37°C for 0.5 to 2 hours to obtain the PROTAC-loaded biomimetic neural-targeting plant exosomes; The click chemistry method can be carried out as follows: an azide or alkynyl group is introduced onto the surface of a membrane-hybridized PROTAC-loaded plant exosome, and it undergoes an addition reaction with a pH-responsive polymer carrying the corresponding click chemistry functional group; after the reaction is complete, the mixture is centrifuged at 100,000 × g to 150,000 × g for 0.5 to 1 hour, the unreacted polymer is removed, and the precipitate is collected to obtain the PROTAC-loaded biomimetic neural-targeting plant exosome.

[0054] The preparation method of the present invention will be specifically implemented in the following specific embodiments, and the technical effects will be verified.

[0055] The neural stem cells (NE-4C), human neuroblastoma cells (SH-SY5Y), and human nasal mucosal epithelial cells (HNEPC) used in this embodiment were cultured in a complete medium containing 10% fetal bovine serum, penicillin (50 U / mL), and streptomycin (50 U / mL) at 37°C in a 5% CO2 environment. The Parkinson's disease (PD) model was constructed as follows: SH-SY5Y cell damage was induced in vitro with MPP+ (2 mM), and C57BL / 6 mice were induced in vivo with MPTP (30 mg / kg / day for 7 consecutive days). Experimental data are expressed as mean ± SD, and statistical significance was defined as P < 0.05. The plant exosomes used were Angelica sinensis exosomes (extracted using differential ultracentrifugation). The active Angelica sinensis part suitable for this invention was fresh Angelica sinensis root purchased from Minxian County, Gansu Province, a renowned medicinal herb producing area.

[0056] Example 1: Preparation and Physicochemical Characterization of AEV-XNH This embodiment provides a preparation process and physical characterization data of PROTAC-loaded biomimetic neural-targeting plant exosomes. First, fresh Angelica sinensis roots were taken, washed, chopped, and subjected to tissue disruption with pre-cooled PBS buffer at 4°C. Natural exosomes (AEVs) derived from Angelica sinensis were extracted using differential ultracentrifugation: impurities were removed by centrifugation at 1000g (10 min), 4000g (20 min), and 10000g (30 min) sequentially. The supernatant was collected and ultracentrifuged at 150000g for 1.5 h, and the precipitate was resuspended to obtain purified AEVs. The LRRK2-targeting PROTAC molecule XL01126 was mixed with AEVs at a mass ratio of 1:10 and co-incubated at 37°C for 1 h. Free drug was removed by centrifugation at 150000g to obtain the drug-loaded core AEV-X. Figure 1 As shown in a and b, AEV-X has a typical vesicle structure with an average hydrated particle size of approximately 87.9 ± 4.4 nm.

[0057] Neural stem cells were collected, and neural stem cell membranes (NSCMs) were extracted through repeated freeze-thaw lysis and gradient centrifugation. AEV-X and NSCMs were mixed at a protein ratio of 6:1, and repeatedly extruded through a 200nm filter membrane 100 times at 37°C using a lipoextruder to construct a biomimetic core-shell structure, AEV-XN. Figure 2 As shown in a and b, nanofluid cytometry analysis confirmed that NSCM was successfully coated on the surface of AEV-X. TEM observation showed that AEV-XN still maintained its vesicle structure, with clear outer membrane boundaries. Figure 2 c). DLS analysis showed that the average particle size further increased to 120.4 ± 15.0 nm ( Figure 2 (d in the text)

[0058] Add DSPE-His7-PEG 2000AEV-XNH was prepared by incubation at 37°C for 1 hour using the post-insertion method. Figure 3 As shown in a, the PEG grafting rate and XL01126 encapsulation efficiency were quantitatively determined and characterized. The final particle size of AEV-XNH was 139.3 ± 2.1 nm. Figure 3 (b and c in the text). Stepwise construction of the product's Zeta potential characterization showed that loading, fusion, and modification led to a potential neutralization ( Figure 3 (d in the text). In vitro release kinetics curves confirmed the release characteristics of AEV-XNH in PBS, and its release curves conformed to a first-order kinetic model ( ). Figure 3 (e in the text).

[0059] Example 2: Evaluation of the in vitro delivery performance of AEV-XNH To evaluate the cellular uptake characteristics of AEV-XNH, DiO2 was used to label AEV-XNH. The labeled nanomedicine was co-incubated with SH-SY5Y for different time periods. CLSM and flow cytometry analysis showed that the cellular uptake of AEV-XNH significantly increased with increasing incubation time, exhibiting good biointerface affinity. Figure 4 (a and b in the text).

[0060] To investigate the cellular entry pathway of AEV-XNH, SH-SY5Y cells were pretreated with various endocytosis inhibitors and then co-incubated with AEV-XNH. Flow cytometry results showed that ( Figure 4 (c) Compared with the 37℃ control group, the 4℃ low-temperature treatment significantly inhibited the uptake of AEV-XNH by cells, indicating that this process is an energy-dependent active endocytosis. Both the clathrin inhibitor chlorpromazine and the lipid raft inhibitor methyl-β-cyclodextrin significantly reduced the uptake rate of AEV-XNH, indicating that its endocytosis depends on both clathrin-mediated and lipid raft-mediated pathways.

[0061] SH-SY5Y cells were co-incubated with DiO-labeled AEV-XNH for different times (4, 8, 12 h), and stained with LysoTrackerRed, a red fluorescent lysosomal probe, 30 min before the end of the incubation period. CLSM observation and co-localization analysis showed that ( Figure 5 (a and b in the original text) After AEV-XNH enters the cell, the colocalization coefficient (PCC) of green and red fluorescence is significantly reduced, proving that the His7 fragment mediates efficient lysosomal escape and protects AEV-XNH from degradation.

[0062] An in vitro Transwell co-culture model simulating the nose-brain barrier was constructed. Human nasal mucosal epithelial cells (HNEPC) were seeded in the upper chamber to form a dense monolayer, while SH-SY5Y cells were seeded in the lower chamber. DiO-labeled AEV or AEV-XNH was added to the upper chamber. Fluorescence intensity in the lower chamber culture medium and within the SH-SY5Y cells was measured at different time points. Results showed ( Figure 6 (a and b in the figure) At each time point, the lower chamber permeation fluorescence intensity and target cell uptake fluorescence intensity of the AEV-XNH group were significantly higher than those of the AEV group, indicating that its PEG-modified layer effectively enhanced the mucus penetration ability and promoted drug delivery across the barrier.

[0063] Example 3: Study on the in vitro neuroprotective activity and mechanism of AEV-XNH Using different concentrations of MPP + SH-SY5Y cells were treated for 24 hours, and cell viability was detected by CCK-8 assay. Results showed ( Figure 7 a) 2mMMPP + This concentration reduced cell viability to approximately 42%, hence it was chosen to establish an in vitro PD model. Cell viability was assessed using the CCK-8 assay, and the results showed that, compared to the model group, AEV-XNH treatment most significantly restored cell viability to approximately 70.58%. Figure 7 (b) Meanwhile, AEV-XNH also exhibited good biocompatibility and anti-MPP activity in BV2 microglia and U87 astrocytes. + Protective effect against damage ( Figure 8 (ad in the text).

[0064] Detection of MPP by Western Blot + The expression level of LRRK2 protein in damaged SH-SY5Y cells. The results showed ( Figure 9 MPP + LRRK2 expression was significantly upregulated in the model group. Compared with free XL01126 or AEV alone, AEV-XNH treatment most effectively reduced LRRK2 protein levels, confirming that PROTAC can efficiently perform catalytic degradation function when delivered by this biomimetic vector.

[0065] Further analysis was conducted on intracellular redox and metabolic indicators. Results showed that AEV-XNH treatment significantly reduced MPP. + Induced reactive oxygen species (ROS) bursts and accumulation of malondialdehyde (MDA), a lipid peroxidation product (MDA) Figure 10 (a and b in the text). Furthermore, AEV-XNH effectively reversed the depletion of intracellular glutathione (GSH). Figure 11 It significantly increased the reserves of reduced coenzyme II (NADPH) and improved NADP.+ / NADPH ratio ( Figure 12 (a and b in the text). Further evaluation of mitochondrial function showed that AEV-XNH treatment partially restored mitochondrial function lost due to MPP. + Damage reduces intracellular adenosine triphosphate (ATP) levels and significantly restores mitochondrial membrane potential. Figure 13 (a and b in the text).

[0066] Example 4: Evaluation of in vivo brain-targeted delivery of AEV-XNH This embodiment uses in vivo imaging technology to verify the brain distribution characteristics of AEV-XNH after intranasal administration. After intranasal instillation of the DiR-labeled nanodrug into C57BL / 6 mice, IVIS in vivo imaging system monitoring showed that the fluorescence signal in the brain of the AEV-XNH group significantly increased over time, reaching a peak at 12 hours post-administration. Furthermore, the brain accumulation at each time point was significantly higher than that of the unmodified AEV group, indicating its excellent naso-brain transport efficiency. Figure 14 ).

[0067] Example 5: In vivo pharmacodynamic evaluation of AEV-XNH This embodiment validated the overall therapeutic efficacy of AEV-XNH in an MPTP-induced Parkinson's disease mouse model. Behavioral test results showed that AEV-XNH treatment significantly improved motor dysfunction in PD mice. In the open field test, the average movement speed of the treated group mice recovered to near the level of the healthy control group, and the resting time was significantly shortened ( Figure 15 (a and b in the text). In the rotundus experiment, the time mice spent on the accelerated rotundus was significantly prolonged, indicating that their motor coordination and balance abilities were effectively restored. Figure 15 c). The tail suspension test showed that the depressive-like behavior in mice was also alleviated ( Figure 15 (d in the text)

[0068] Immunofluorescence staining of brain tissue sections showed a significant increase in the density of tyrosine hydroxylase (TH)-positive neurons and dopamine transporter (DAT)-positive nerve fibers in the substantia nigra pars compacta (SNpc) of the AEV-XNH group. Figure 16 (a) Quantitative image analysis showed that AEV-XNH treatment restored the area of ​​DAT-positive regions to 21.3% and the area of ​​TH-positive regions to 7.5%, with significantly better neuroprotective effects than other control groups ( Figure 16 (b and c in the text).

[0069] Mouse substantia nigra tissue was collected for Western blotting and biochemical analysis. Western blotting results showed that AEV-XNH treatment significantly upregulated the expression of PPARγ, PPARδ, and their downstream target proteins CD36 and ME1 in the brain, while effectively reducing the pathological LRRK2 protein level. Figure 17 Further biochemical analysis of brain tissue confirmed that AEV-XNH treatment significantly reduced MDA levels in response to oxidative stress in the brain and significantly restored the levels of key antioxidant molecules GSH and NADPH, confirming the restoration of neuronal metabolic homeostasis in vivo. Figure 18 (ad in the text).

[0070] Example 6: Safety Evaluation of AEV-XNH This study evaluated the biosafety of long-term intranasal administration of AEV-XNH. After 7 consecutive days of intranasal instillation of AEV-XNH in healthy C57BL / 6 mice, the weight gain curve was consistent with that of the saline control group, with no abnormal fluctuations observed. Figure 19 H&E staining of major organs (heart, liver, spleen, lungs, and kidneys) showed that all tissue structures were clear and intact, with no pathological changes such as congestion, edema, necrosis, or inflammatory cell infiltration. Figure 20 Serum biochemical test results showed that the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which reflect liver function, and creatinine (CREA) and urea (UREA), which reflect kidney function, were all within the normal range, with no statistically significant differences compared to the control group. Figure 21 The above results confirm that AEV-XNH has excellent biocompatibility and good systemic safety when administered via the nasal route.

[0071] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for preparing biomimetic neural-targeting plant exosomes loaded with PROTAC, characterized in that, Includes the following steps: 1) Load PROTAC into plant exosomes, using the active molecules contained in the exosomes themselves and the loaded PROTAC as complementary therapeutic molecules; 2) Hybridize the cell membrane with the plant exosomes loaded with PROTAC from step 1); 3) By co-incubating or click chemistry, the surface of the membrane-hybridized PROTAC-loaded plant exosomes obtained in step 2) is modified with a pH-responsive multifunctional polymer to obtain the PROTAC-loaded biomimetic neural-targeting plant exosomes.

2. The method according to claim 1, characterized in that: Step 1) describes loading PROTAC into plant exosomes using methods such as co-incubation, ultrasonic treatment, electroporation, or freeze-thaw cycles. The mass ratio of the exosome protein to PROTAC is 0.05~20:

1.

3. The method according to claim 1, characterized in that, The plants mentioned are one or more of the following: Schisandra chinensis, ginseng, Ziziphus jujuba var. spinosa, Ligusticum chuanxiong, Panax notoginseng, Angelica sinensis, Rabdosia rubescens, Acorus tatarinowii, Pueraria lobata, Gastrodia elata, and Astragalus membranaceus.

4. The method according to claim 1, characterized in that, The PROTAC is one or more of ARV-110, ARV-471, XL01126, NX-2127, NX-5948, and KT-474.

5. The method according to claim 1, characterized in that: The method for hybridizing the cell membrane with the PROTAC-loaded plant exosomes in step 1) is co-extrusion, PEG-mediated fusion, or co-incubation; wherein the mass ratio of plant exosome membrane surface proteins to cell membrane surface proteins is 0.05~20:

1.

6. The method according to claim 5, characterized in that, The cell membrane is one or more of the following: embryonic stem cell membrane, adult stem cell membrane, neural stem cell membrane, induced pluripotent stem cell membrane, tumor cell membrane, T cell membrane, NK cell membrane, and macrophage membrane; the cell membrane surface expresses one or more of the following chemokine receptors: CXCR4, CXCR7, CXCR9, and CXCR10.

7. The method according to claim 1, characterized in that, In step 3), the co-incubation method is as follows: the membrane-hybridized PROTAC-loaded plant exosomes are mixed with the pH-responsive polymer DSPE-His-PEG, the polymer concentration is controlled at 50 to 500 μg / mL, and incubated in a buffer solution at pH 7.0 to 7.4 at 37°C for 0.5 to 2 hours to obtain the PROTAC-loaded biomimetic neural-targeting plant exosomes; The click chemistry method involves introducing azide or alkynyl groups onto the surface of a membrane-hybridized PROTAC-loaded plant exosome, and then reacting it with a pH-responsive polymer carrying the corresponding click chemistry functional group. After the reaction is complete, the exosome is centrifuged at 100,000 × g to 150,000 × g for 0.5 to 1 hour, and the unreacted polymer is removed and the precipitate is collected to obtain the PROTAC-loaded biomimetic neural-targeting plant exosome.

8. The method according to claim 7, characterized in that, The pH-responsive polymer DSPE-His-PEG is composed of distearate phosphatidylethanolamine (DSPE) and histidine peptide His. 6-15 It is coupled with polyethylene glycol; wherein, the DSPE is a lipid molecule used to link to the membrane surface, and the histidine polypeptide has a proton sponge effect to promote the lysosomal escape of the PROTAC-loaded biomimetic neural-targeting plant exosomes; the polyethylene glycol is used to assist the PROTAC-loaded biomimetic neural-targeting plant exosomes in penetrating the mucus barrier and prolonging blood circulation time.

9. A biomimetic neural-targeting plant exosome loaded with PROTAC, characterized in that, It is prepared by the method described in any one of claims 1-8.

10. The application of the PROTAC-loaded biomimetic neural-targeting plant exosome as described in claim 9, characterized in that, This is used to prepare a drug for treating central nervous system degenerative diseases; the central nervous system degenerative diseases are Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, or spinal muscular atrophy.

Images