Self-assembled nanogel encapsulated smart plant exosome vesicles and preparation and application thereof

Abstract

The application discloses a self-assembled nanogel-encapsulated intelligent plant exosome vesicle and a preparation and application thereof, and belongs to the technical field of biological medicine. The application takes plant-derived exosome vesicles as core carriers, encapsulates the exosome vesicles based on self-assembled nanogel formed by siRNA and tannic acid, and constructs an intelligent exosome vesicle delivery system through multifunctional ligand modification of nerve cell-specific adhesion peptides and lysosome escape peptides. The system can carry specific siRNA, and after local administration, can realize the enrichment of nerve lesion areas, the responsive release of siRNA-loaded plant exosome vesicles, the nerve cell-specific recognition and internalization, the efficient endosome escape in cells and the efficient release of treatment loads in cells in sequence in local pathological environments such as diabetic peripheral neuropathy. The application realizes the synergistic regulation of mitochondrial dysfunction and neuroinflammation of nerve cells, thereby realizing multi-target and multi-level intervention on the pathological process of neurodegenerative diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical nanotechnology, specifically relating to a self-assembled nanogel-encapsulated intelligent plant exosome vesicle and its preparation and application. Background Technology

[0002] Peripheral neuropathy is a group of diseases characterized by the progressive, selective loss of neuronal structure or function (including neuronal death), leading to functional impairment and manifesting as typical focal lesions. It includes diabetic peripheral neuropathy (DPN) and amyotrophic lateral sclerosis (ALS). Currently, the specific etiology of these diseases is unclear, and existing treatments have not significantly altered their progression, posing a serious threat to global public health and imposing a heavy socioeconomic burden due to long-term care and treatment needs. The development of treatment strategies for peripheral neuropathy faces multiple challenges: on the one hand, it requires overcoming the neuronal delivery bottlenecks caused by insufficient stability of small nucleic acid drugs, insufficient neuronal specificity recognition, and insufficient intracellular endosome escape ability in the local pathological environment; on the other hand, it requires addressing complex pathological mechanisms such as neuronal mitochondrial dysfunction and blocked pathways of brain-derived neurotrophic factor (BDNF). Therefore, there is an urgent need for a treatment strategy that can simultaneously achieve efficient, precise, and safe neuronal delivery and mitochondrial metabolic support within a spatiotemporal context.

[0003] Plant-derived exosomes and vesicles, due to their excellent biocompatibility, low immunogenicity, and endogenous active components (such as functional miRNAs and lipids), are widely involved in intercellular communication and material exchange, and are considered promising natural drug delivery carriers. Some traditional Chinese medicinal plants possess blood-tonifying, blood-activating, meridian-clearing, and pain-relieving effects; their extracts have been shown to have metabolic regulatory and neuroprotective activities. Therefore, medicinal plant exosomes and vesicles may serve as therapeutic nanoplatforms to treat peripheral neuropathy. Simultaneously, using medicinal plant exosomes and vesicles to carry siRNA to specifically downregulate the expression of key pathogenic genes of PTEN (Phosphatase and Tensin Homolog) may be a precise and efficient supplementary strategy, further promoting neuronal survival, accelerating electrophysiological recovery, myelin regeneration, and nerve innervation.

[0004] Compared to oral administration, local drug delivery offers advantages such as direct action on the lesion site, rapid increase in drug concentration at the lesion site, and reduced systemic toxicity. However, the local microenvironment of peripheral neuropathy contains various enzymes, inflammatory factors, and secretions, often leading to non-specific uptake loss of plant exosomes and siRNA, as well as the shedding and degradation of their carried small nucleic acid drugs. Furthermore, unmodified carriers, once inside the lesion site, often exhibit non-specific distribution, making it difficult to identify and accumulate in peripheral nerves, thus hindering the targeted internalization of plant exosomes and their carried siRNA. Finally, there is the challenge of lysosomal escape; even if 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. Therefore, further intelligent engineering modifications to plant exosome vesicles are needed to develop an intelligent nanomedicine system that integrates protective and responsive self-assembled nanogels with miRNA / siRNA mitochondrial function-enhancing effects carried by plant exosome vesicles. This system would possess the ability to stabilize the local microenvironment, responsively release substances, recognize nerve cells, accelerate internalization, and promote endosome escape. Such a system would have significant scientific value and clinical potential for achieving precise and multidimensional treatment of peripheral neuropathy. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a self-assembled nanogel-encapsulated intelligent plant exosome vesicle, its preparation, and its application. The self-assembled nanogel-encapsulated intelligent plant exosome vesicles provided by this invention can overcome the degradation of drugs by the local microenvironment of peripheral neuropathy, accurately identify and adhere to nerve cells, and synergistically treat peripheral neuropathy through a dual mechanism of improving mitochondrial dysfunction and clearing pathogenic gene blockade. The core of the self-assembled nanogel-encapsulated intelligent plant exosome vesicles of this invention is a natural exosome vesicle derived from a traditional Chinese medicine plant (taking Angelica sinensis as an example) (using Angelica sinensis exosome vesicle AEV as an example), and the surface of the core is loaded with siRNA (siRNA targeting the PTEN gene). Pten The outer shell is a SiR that wraps around the surface of the core. Pten Nanogels formed by self-assembly with tannic acid; the surface modification layer is a TH multifunctional ligand inserted into the core lipid bilayer.

[0006] According to a first aspect of the present invention, the present invention first provides a method for preparing intelligent plant exosome vesicles (THAEV@siR-TAgel) encapsulated by self-assembled nanogels, which includes the following steps: 1) Modify the surface of plant exosome vesicles with TH multifunctional ligands, wherein the TH multifunctional ligands are polymerized from nerve cell-specific adhesion peptides, lysosomal escape peptides and palmitic acid groups; 2) Add siRNA targeting the pathogenic gene to the system of step 1) and load it onto the surface of the plant exosome vesicles modified in step 1); 3) Add an excess of tannic acid solution to the system in step 2) to form a nanogel layer on the surface of plant exosome vesicles through self-assembly. The nanogel layer is formed by the self-assembly of siRNA and tannic acid through hydrogen bonds, thereby obtaining the intelligent plant exosome vesicles encapsulated by the self-assembled nanogel.

[0007] According to a preferred embodiment of the present invention, step 1) includes: co-incubating the TH multifunctional ligand with a plant exosome vesicle solution at 37°C, so that the TH multifunctional ligand is modified on the surface of the plant exosome vesicle; the mass ratio of the plant exosome vesicle protein to the TH multifunctional ligand is 2:1 to 4:1.

[0008] According to a preferred embodiment of the present invention, the TH multifunctional ligand is composed of a nerve cell-specific adhesion peptide, a lysosomal escape peptide, and a palmitic acid group. The amino acid sequence of the nerve cell-specific adhesion peptide is HLNILSTLWKYR (SEQ ID NO. 1). This sequence can bind to the trisialic acid ganglioside receptor GT1b specific to the nerve cell membrane surface, thereby promoting the specific recognition, uptake, and internalization of exosome vesicles in nerve cells. Preferably, the palmitic acid group is a hexadecanoic acid fatty acid residue, which is linked to a lysine residue of the TH multifunctional ligand via an amide bond.

[0009] Furthermore, the lysosomal escape peptide is an oligohistidine sequence Hn, wherein the number n of histidine H is any number from 2, 4, 6, 8, 10, to 12. The lysosomal escape peptide triggers the proton sponge effect through the protonation of the contained imidazole ring, causing lysosomal swelling and membrane disruption.

[0010] The structure of the TH multifunctional ligand is represented as: HLNILSTLWKYR+Hn-Lys(Pal).

[0011] According to a preferred embodiment of the present invention, the plant is one or more selected from Schisandra chinensis, ginseng, Ziziphus jujuba var. spinosa, Ligusticum chuanxiong, Panax notoginseng, Angelica sinensis, Rabdosia rubescens, Acorus tatarinowii, Pueraria lobata, Gastrodia elata, and Astragalus membranaceus. Preferably, the plant is Angelica sinensis.

[0012] According to a preferred embodiment of the present invention, the plant exosome vesicles are extracted from fresh tissue of the main medicinal part of the plant using differential ultracentrifugation. Typically, but not limited to, the preparation method of the plant exosome vesicles is as follows: Add an appropriate amount of buffer salt solution such as PBS, thoroughly disrupt the fresh plant tissue to obtain a crude extract. Centrifuge the crude extract at 1000-150000 G, and then remove the supernatant. Resuspend the precipitate using buffer salt solution such as PBS and filter to obtain the plant exosome vesicles.

[0013] According to a preferred embodiment of the present invention, step 2) includes: adding siRNA modified with 3' cholesterol to the system obtained in step 1), co-incubating under ice-water bath conditions, so that the siRNA is loaded on the surface of the plant-derived exosome vesicles; measuring the mass of the plant exosome vesicles by the total amount of protein contained therein, and the ratio of the mass of the plant exosome vesicles to the absorbance value of the siRNA is 100~200 μg: 0.2~1 OD.

[0014] According to a preferred embodiment of the present invention, the siRNA is an siRNA that targets the pathogenic PTEN gene in diabetic peripheral neuropathy. Pten The siR Pten The sequences include, but are not limited to, Pten-Rat-1373, Pten-Rat-1511, Pten-Rat-1248, and Pten-Rat-1142. Their sense and antisense sequences are shown in Table 1. The sequences in Table 1 correspond to SEQ ID NO.2 to SEQ ID NO.9 in the sequence list from front to back.

[0015] Table 1 According to a preferred embodiment of the present invention, step 3) specifically involves: adding a tannic acid solution to the system obtained in step 2), and co-incubating under ice-water bath conditions, so that the siRNA and the tannic acid are linked by hydrogen bonds to self-assemble on the surface of the plant-derived exosome vesicles to form a nanogel layer. Preferably, the tannic acid is in excess, and the specific amount added is not limited.

[0016] According to a second aspect of the present invention, the present invention also provides a self-assembled nanogel-encapsulated intelligent plant exosome vesicle, which is prepared by the aforementioned method.

[0017] According to a third aspect of the present invention, the present invention also provides an application of the self-assembled nanogel-encapsulated intelligent plant exosome vesicles, specifically for the preparation of a drug for treating peripheral neuropathy; wherein the peripheral neuropathy may be a disease caused by diabetic peripheral neuropathy, or amyotrophic lateral sclerosis, etc.

[0018] Compared with the prior art, the present invention has the following significant advantages: (1) Synergistic Dual Therapeutic Mechanism: This invention organically integrates exogenous active siRNA targeting pathogenic genes with endogenous bioactive components from plant exosome vesicles. Pten Targeting the pathogenic PTEN gene in diabetic peripheral neuropathy reduces PTEN protein expression, which causes BDNF dysfunction, at its source, restoring the neuronal repair process supported by BDNF. Simultaneously, endogenous active molecules carried by plant exosomes activate related signaling axes, promoting the improvement of mitochondrial dysfunction. This synergistic effect can more effectively break the vicious cycle of peripheral neuropathy.

[0019] (2) Precise lesion targeting: The surface-modified Tet1 peptide enhances the specific recognition, adhesion and internalization of plant exosome vesicles in nerve cells. The introduced Hn 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 the drug and avoiding degradation by lysosomes.

[0020] (3) Local microenvironment responsive release: By utilizing the property that the hydrogen bonds in the self-assembled nanogel dissociate under oxidative stress or low pH conditions at the lesion site, the responsive release of plant exosome vesicles and siRNA was achieved, avoiding their degradation in the lesion environment and supporting subsequent neuronal targeting.

[0021] (4) High safety: The main carrier material of this invention is derived from siRNA contained in natural plants and organisms, which has good biocompatibility and low immunogenicity. In vivo experiments have confirmed that administration did not cause significant toxicity to major organs such as the heart, liver, and kidneys, and it has good potential for clinical translation. Attached Figure Description

[0022] Figure 1 Morphological characterization of THAEV@siR-TAgel. (a1-a2) Atomic force microscopy (AFM) characterization of AEV (scale bar 50 nm); (a3) ​​Transmission electron microscopy (TEM) characterization of AEV (scale bar 200 nm); (b1-b2) AFM characterization of THAEV@siR-TAgel (scale bar 50 nm); (b3) TEM characterization of THAEV@siR-TAgel (scale bar 200 nm); (c) Appearance of THAEV@siR-TAgel and AEV solution.

[0023] Figure 2Physical characterization of THAEV@siR-TAgel. (a) Hydrated particle size of THAEV@siR-TAgel and AEV; (b) Zeta potential of THAEV@siR-TAgel and AEV; (c) Dissociation curves of TA in PBS at different pH; (d) Dissociation curves of TA in H2O2 at different concentrations.

[0024] Figure 3 Physical characterization of THAEV@siR-TAgel. (a) Liquid chromatography-mass spectrometry (LC-MS) chromatogram of TH peptide; (b) Grafting rate of TH peptide on AEV surface at different mass ratios; (c) Encapsulation efficiency of siRPten in THAEV@siR-TAgel.

[0025] Figure 4 Fourier transform infrared (FTIR) spectrum verification of the self-assembled nanogel formed by TA and siRNA.

[0026] Figure 5 Uptake of THAEV@siR-TAgel in Schwann cells. (a) Relative fluorescence intensities of CM-DiD labeled THAEV@siR-TAgel and AEV in different cells; (b) Uptake curves of THAEV@siR-TAgel and AEV in RSC96 cells.

[0027] Figure 6 Analysis of the uptake pathways of THAEV@siR-TAgel and AEV in RSC96 cells.

[0028] Figure 7 Evaluation of the lysosomal escape ability of THAEV@siR-TAgel in Schwann cells. (a) Confocal images of THAEV@siR-TAgel and AEV colocalizing with lysosomes (scale bar 40 μm); (b) Quantitative analysis of THAEV@siR-TAgel and AEV colocalizing with lysosomes (PCCs: 0 indicates 100% lysosomal escape, while 1 indicates 0% lysosomal escape).

[0029] Figure 8 :THAEV@siR-TAgel in siR Pten Uptake in Schwann cells. (a) FITC-labeled siR in THAEV@siR-TAgel and AEV Pten (a) Laser confocal images taken up in RSC96 cells (scale bar 20 μm); (b) FITC-labeled siR in THAEV@siR-TAgel and AEV. Pten Relative uptake rate in RSC96 cells.

[0030] Figure 9 Local retention capacity of THAEV@siR-TAgel and AEV in vivo. (a) Fluorescence distribution imaging of THAEV@siR-TAgel and AEV in the sciatic nerve region of mice; (b) Quantitative analysis of fluorescence distribution of THAEV@siR-TAgel and AEV in the sciatic nerve region of mice.

[0031] Figure 10 Evaluation of the effects of THAEV@siR-TAgel and AEV on mitochondrial function and inflammatory pathways in Schwann cells. (a1-a2) Western blotting images and quantitative analysis of Tomm40, Tomm20, COX4i2, and COX7A1 expression in RSC96 cells; (b1-b2) Western blotting images and quantitative analysis of IL-17, AP-1, p-NFκB, and NFκB expression in RSC96 cells.

[0032] Figure 11 :THAEV@siR-TAgel in siR Pten The effectiveness of. (a) siR Pten Transfection rate in RSC96 cells by siRNA-mate transfection reagent; (b) siRNA Pten Knockdown efficacy of PTEN gene expression.

[0033] Figure 12 :THAEV@siR-TAgel in siR Pten Evaluation of inhibition of the pathogenic gene PTEN. (a1-a2) Western blot analysis of THAEV@siR-TAgel regulation of Pten and Akt; (b) Immunofluorescence staining images of total Pten and total Akt in RSC96 cells; (c) Relative expression level of BDNF in RSC96 cells.

[0034] Figure 13 Effects of THAEV@siR-TAgel and AEV on behavioral performance in diabetic mice with sciatic nerve pinching. (a) Schematic diagram of Vonfery test; (b) Schematic diagram of hot plate test; (c) Schematic diagram of SFI calculation of footprint test; (d) Pinprick withdrawal threshold of mice; (e) Thermal withdrawal latency of mice; (f) Sciatic nerve function index (SFI) of mice.

[0035] Figure 14 Effects of THAEV@siR-TAgel and AEV on sciatic nerve myelin sheath repair. (a) TEM characterization of myelin sheath in transverse sections of the sciatic nerve; (b) Quantitative analysis of myelin sheath thickness in transverse sections of the sciatic nerve; (c) Immunofluorescence staining of MBP, a myelin sheath marker, in longitudinal sections of the sciatic nerve (scale bar 500 μm); (d) Overall fluorescence density of MBP in longitudinal sections of the sciatic nerve.

[0036] Figure 15 Effects of THAEV@siR-TAgel and AEV on sciatic nerve neuronal repair. (a) Immunofluorescence staining of NF200, a neurofilament marker, in longitudinal sections of the sciatic nerve (scale bar 500 μm); (b) Overall fluorescence density of NF200 in longitudinal sections of the sciatic nerve.

[0037] Figure 16 HE staining results of major organs (scale bar 200 μm). Detailed Implementation

[0038] 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.

[0039] The preparation method of the present invention will be specifically implemented in the following specific embodiments, and the technical effects will be verified.

[0040] Schwann cells (RSC96), rat adrenal pheochromocytoma cells (PC12, highly differentiated), human fibroblasts (HSF), and human vascular endothelial cells (HUVEC) used in this embodiment were cultured in 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 high-glucose-induced Schwann cell model was constructed by inducing RSC96 cell damage in vitro with a glucose concentration of 200 mM. Experimental data are expressed as mean ± SD, and statistical significance was defined as P < 0.05. The plant exosome vesicles used were selected from Angelica sinensis exosome vesicles (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 traditional Chinese medicine producing area.

[0041] Example 1: Preparation and physicochemical characterization of THAEV@siR-TAgel This embodiment provides an intelligent preparation process for plant exosome vesicles and its physical characterization data. First, fresh Angelica sinensis roots are taken, washed, chopped, and subjected to tissue disruption by adding PBS buffer pre-cooled to 4°C. Natural exosome vesicles (AEVs) derived from Angelica sinensis are extracted using differential ultracentrifugation: impurities are removed by sequential centrifugation at 1000×g (10 min), 4000×g (20 min), and 10000×g (30 min). The supernatant is collected and ultracentrifuged at 150000×g for 1.5 h, and the precipitate is resuspended to obtain purified AEVs.

[0042] THAEV@siR-TAgel was designed and fabricated step by step based on AEV.

[0043] THAEV: AEV and TH multifunctional ligand (amino acid sequence: HLNILSTLWKYR+H10-Lys(Pal)) were incubated at 37℃ for 1.5 h at a mass ratio of 2:1 to 4:1. Ultimately, the grafting rate of the TH multifunctional ligand on the AEV surface reached 100%. Figure 3 (b) The LC-MS spectrum of the TH multifunctional ligand is shown in Figure 1. Figure 3 'a' in 'a'.

[0044] THAEV@siR-TAgel: First, a siRNA solution containing four sequences at 0.2–1 OD was incubated with 100–200 μg of THAEV at 100 rpm / min and 4°C for 30 min. Then, the THAEV / siRNA solution was placed in an ice-water bath and stirred at 100 rpm / min using a magnetic stirrer. Over 3 min, 1 mL of tannic acid (TA) solution was added dropwise to the system, and after thorough mixing, the reaction was allowed to proceed for 30 min. The hydrogen bonding cross-linking effect was verified. Figure 4 Finally, the obtained THAEV@siR-TAgel nanogel was centrifuged and filtered (Amicon Ultra-4 ultra-100k, Millipore) to remove unreacted TA and siRPten at a molecular weight cutoff of 100 kDa. The THAEV@siR-TAgel was resuspended in RNase-free PBS and stored at 4°C or -80°C. The encapsulation efficiency of siRNA in the THAEV@siR-TAgel was greater than 80%. Figure 3 (c) In addition, an AEV / siR mixture was prepared as a negative control.

[0045] like Figure 1 As shown in a1-a3, AEV has a typical teacup-shaped vesicle structure, while THAEV@siR-TAgel exhibits a vesicle structure and a nanogel layer of approximately 10-20 nm thickness is visible. Figure 1 (b1-b3 in the original text). For example... Figure 1 As shown in c, both THAEV@siR-TAgel and AEV can be uniformly dispersed in PBS solution. A significant Tyndall effect was observed in the semi-transparent solutions they formed, exhibiting their characteristics as nanoparticles. Subsequently, a Malvern nanoparticle size analyzer revealed the particle size and potential changes during the preparation of THAEV@siR-TAgel. Figure 2 As shown in a, the average hydrated particle size of AEV is approximately 83.44 ± 16.09 nm, while the average hydrated particle size of THAEV@siR-TAgel increases to approximately 262.70 ± 26.99 nm. Figure 2As shown in b, the average Zeta potential of AEV is approximately -13.77 ± 0.83 mV, while the average Zeta potential of THAEV@siR-TAgel is approximately -21.17 ± 1.03 mV.

[0046] The reversible hydrogen bonds formed in the self-assembled nanogel (siR-TAgel) break under oxidative stress and acidic environments such as lysosomes (pH 4.5–5.5). pH 4.5–5 and H₂O₂ simulate the acid-base imbalance and oxidative stress state around nerve cells under DPN conditions, and the responsive release capacity in this environment directly affects the AEV, miRNAs, and siR in THAEV@siR-TAgel. Pten Delivery efficiency and bioavailability in nerve cells. Results of TA release ( Figure 2 Figures c and d show that the spontaneous release rate of TA at 8 h in PBS at pH 7.4 was only 44.15%, while it increased to 85.78% at PBS at pH 4.5, indicating that an acidic environment can accelerate the dissociation of the TAgel nanogel layer. Meanwhile, the spontaneous release rate of TA at 8 h in H2O2-free conditions (PBS) was 44.15%, while the release rates at 50 μM and 100 μM conditions increased to 68.47% and 93.25%, respectively, indicating that oxidative stress promoted the dissociation of the TAgel nanogel layer in a concentration-dependent manner. These results further provide evidence for the responsive dissociation of siR-TAgel based on reversible hydrogen bonds, and support the development of siR-TAgel for THAEV loading. Pten The responsive release within the DPN microenvironment provides the material basis.

[0047] Example 2: Evaluation of the in vitro delivery performance of THAEV@siR-TAgel To evaluate the cellular uptake characteristics of THAEV@siR-TAgel, AEVs were labeled with DiD. The labeled nanomedicine was co-incubated with RSC96 cells for different time periods. Flow cytometry analysis showed that, compared to AEVs (100% relative CM-DiD fluorescence intensity), the relative CM-DiD fluorescence intensities of THAEV@siR-TAgel in RSC96, PC12, HSF, and HUVEC cells at 2 h were 211.43%, 146.98%, 103.16%, and 71.73%, respectively. This indicates that the uptake efficiency of THAEV@siR-TAgel in the two neuronal cell types was significantly increased compared to AEVs, and significantly higher than that in non-neural cells (HSF and HUVEC). Simultaneously, it was found that the relative fluorescence intensity of THAEV@siR-TAgel at RSC96 increased by 137.11% at 4 h compared to 2 h, while the relative fluorescence intensity of THAEV@siR-TAgel at PC12 at 4 h showed no significant difference compared to 2 h. These results indicate that THAEV@siR-TAgel can specifically adhere to nerve cells such as SCs through the recognition and binding of TH multifunctional ligands and GT1b, thereby promoting their efficient uptake and internalization. Figure 5 (a) Meanwhile, the cellular uptake of THAEV@siR-TAgel in RSC96 neural cells significantly increased with increasing incubation time, demonstrating good biointerface affinity. Figure 5 (b) in the middle.

[0048] To investigate the cellular entry pathway of THAEV@siR-TAgel, RSC96 cells were pretreated with various endocytosis inhibitors and then co-incubated with THAEV@siR-TAgel. Flow cytometry results showed that ( Figure 6 When the clathrin-mediated endocytosis pathway was inhibited, the uptake inhibition rates of THAEV@siR-TAgel and AEV reached 56.91% and 75.80%, respectively. Meanwhile, inhibition of caveolin had the least impact on the uptake inhibition rates of THAEV@siR-TAgel and AEV, at 4.07% and 16.58%, respectively. These results indicate that the uptake and internalization of THAEV@siR-TAgel and AEV mainly depend on clathrin-mediated endocytosis.

[0049] Subsequently, the Hn sequence promoted the lysosomal escape of THAEV@siR-TAgel and THAEV. AEVs and lysosomes were first labeled using DiO and LysoTracker, respectively. Under fluorescence confocal microscopy, the colocalization of AEVs and lysosome fluorescence was used to determine whether EVs were retained in lysosomes. Fluorescence confocal images showed significant colocalization (yellow fluorescence) of AEVs (green fluorescence) and lysosomes (red fluorescence) at 2 h, 4 h, and 6 h. Figure 7 (a) and PCCs continuously increased, reaching 0.55, 0.64 and 0.67 respectively. Figure 4 (3b) indicates that AEV does not possess rapid lysosomal escape capability. THAEV@siR-TAgel showed less colocalization with lysosomes at 2 h, 4 h, and 6 h, with PCCs of 0.43, 0.37, and 0.32, respectively, indicating that it possesses rapid lysosomal escape capability. Meanwhile, AEV and siR... Pten The lysosomal escape ability of the products of simple mixing shows the same trend as that of AEV.

[0050] Given the aforementioned advantages, THAEV@siR-TAgel possesses the ability to enable siR Pten The potential for efficient internalization in nerve cells. First, siRNA was transfected using the transfection reagent siRNA-mate. Pten Transfection was used as a positive control to evaluate the delivery of siR via THAEV@siR-TAgel. Pten The uptake efficiency was up to RSC96. Laser confocal microscopy images show ( Figure 8 (a) FITC-labeled siR was observed in both the siRNA-mate group and the THAEV@siR-TAgel group. Pten This indicates that they are capable of carrying SiR. Pten It was taken up by RSC96 cells. However, FITC-labeled siR was not observed in the AEV / siR simple mixture group images. Pten Based on the fluorescence intensity results provided by flow cytometry, the effects of RSC96 cells on siR in different groups were analyzed. Pten Quantitative analysis of relative uptake rate ( Figure 8 (b) shows that RSC96 cells in the siRNA-mate group and the THAEV@siR-TAgel group showed resistance to siRNA. Pten All showed a time-dependent uptake pattern, with relative uptake rates of 57.98%, 95.97%, and 100% at 1 h, 3 h, and 5 h, and 47.61%, 81.37%, and 100%, respectively. However, in the AEV / siR simple mixture group, siR... Pten The highest relative uptake rate was 7.19%, occurring at 1 h, indicating that AEV cannot directly deliver siR.Pten .

[0051] Furthermore, the affinity of THAEV@siR-TAgel and other agents for nerve tissue was assessed using in vivo imaging techniques in small animals. These agents were directly injected into the sciatic nerve region, and changes in the fluorescent region were observed. Imaging results showed ( Figure 9 (a) THAEV@siR-TAgel showed more uniform distribution in the sciatic nerve region within 4–24 h post-injection and a longer retention time in the nerve area, indicating its retention capacity in nerve tissue. However, the fluorescence intensity of AEV in the sciatic nerve region significantly decreased 8 h after injection, representing a decrease in retention. Quantitative analysis of the radioactivity of fluorescence imaging also confirmed these results. Figure 9 (b) After 12 h, the radiation efficiency of THAEV@siR-TAgel was significantly higher than that of AEV, and the value did not change significantly, while the radiation efficiency of AEV decreased continuously from 4 to 24 h.

[0052] Example 3: Study on the in vitro neuroprotective activity and mechanism of THAEV@siR-TAgel Western blotting analysis validated the molecular mechanism by which THAEV@siR-TAgel reverses mitochondrial dysfunction by enhancing mitochondrial oxidative phosphorylation pathways and improving neuroinflammation. THAEV@siR-TAgel improves mitochondrial function by enhancing substrate utilization by extramitochondrial membrane transport enzymes (TOM) and mitochondrial respiratory chain complex IV (MRC-IV). Western blotting results showed that THAEV@siR-TAgel intervention upregulated the protein expression levels of Tomm40, Tomm20, COX4i2, and COX7A1 by 3.35-fold, 2.26-fold, 2.39-fold, and 2.01-fold, respectively, compared to the HG environment (Model group). Figure 10 (a1-a2 in the text). Simultaneously, THAEV@siR-TAgel inhibited the IL-17 neuroinflammatory pathway, which helped improve mitochondrial dysfunction and promote SCs repair. The expression of IL-17, AP-1, and p-NFκB signaling was downregulated by 0.77-fold, 0.74-fold, and 0.55-fold, respectively, compared to the HG environment. Figure 10 (b1-b2 in the middle).

[0053] Additionally, siR delivered by THAEV@siR-TAgel Pten Inhibition of PTEN expression led to the restoration of BDNF-activated p-Akt expression levels in RSC96 cells. First, four rat siR... PtenSequences: Pten-Rat-1373, Pten-Rat-1511, Pten-Rat-1248, and Pten-Rat-1142 after transfection with siRNA-mate ( Figure 11 (a) significantly knocked down the expression level of the Pten gene (relative value of RT-qPCR) to 0.49, 0.43, 0.21 and 0.10, respectively, demonstrating their effectiveness. Figure 11 (b) . WB analysis further confirmed the effectiveness of THAEV@siR-TAgel in modulating the BDNF-PI3K-Akt pathway by inhibiting Pten ( Figure 12 (a1-a2 in the model group). The results showed that THAEV@siR-TAgel reduced the relative expression level of p-Pten in RSC96 cells to 30.25% of the model group and increased the relative expression level of p-Akt to 248.88% of the model group, while AEV itself did not have the effect of regulating Pten and Akt. Subsequently, laser confocal imaging showed ( Figure 12 (b) In RSC96 cells cultured in an HG environment, significant Pten total protein fluorescence was observed, indicating that HG infiltration can lead to high Pten expression in SCs. The Pten total protein fluorescence intensity in the THAEV@siR-TAgel group was significantly decreased, producing an inhibitory effect similar to that of the positive group siRNA-mate transfection. Finally, ELISA analysis showed that under HG conditions, the BDNF level in RSC96 cells decreased to 45.93% compared to normal cells, while the relative level of THAEV@siR-TAgel recovered to 88.39%.

[0054] Example 4: In vivo pharmacodynamic evaluation of THAEV@siR-TAgel This embodiment establishes a sciatic nerve compression injury (DPNI) model in diabetic mice: 6-week-old C57 db / db mice (n=5). Experimental groups include a sham-operated group (Sham), a model group, a positive drug group (acetyl-L-carnitine hydrochloride ALCAR), a THAEV@siR-TAgel treatment group, and an AEV / siR mixed group. The drugs were injected in situ into the sciatic nerve region once every other day.

[0055] Behavioral evaluation results showed that THAEV@siR-TAgel treatment significantly restored sensory innervation of the sciatic nerve. Following a sciatic nerve injury model, diabetic mice exhibited no response to mechanical or thermal stimuli in the hind paw soles, with a significant increase in paw withdrawal threshold (PWT) and thermal withdrawal latency (TWL). The PWT and TWL in the THAEV@siR-TAgel group were significantly lower than those in the model group on day 7, indicating that THAEV@siR-TAgel treatment promoted the recovery of mechanical and thermal pain sensation in mice. Figure 13 (ab in the text). The sciatic nerve function index (SFI) was calculated based on mouse footprint images to assess the motor function of the damaged sciatic nerve. The SFI in the THAEV@siR-TAgel group was significantly higher than that in the model group on day 7, indicating that THAEV@siR-TAgel treatment promoted the innervation of the sciatic nerve motor function in mice. Figure 13 (c in the text)

[0056] The recovery of myelin sheath was assessed using TEM characterization of cross-sections of the sciatic nerve tissue. The myelin sheath of a normal sciatic nerve was relatively uniform in thickness, with an orderly and dense layered structure within the ring. However, significant myelin sheath fragmentation and degeneration were observed in the model group. Treatment with THAEV@siR-TAgel maintained the structural integrity of the myelin sheath, with uniform thickness and a dense layered structure. Figure 14 (a) Quantitative analysis of myelin thickness also showed that THAEV@siR-TAgel treatment improved myelin repair ( Figure 14 (b) in the middle.

[0057] To further observe the effect of THAEV@siR-TAgel on myelin formation in a sciatic nerve compression injury model, immunofluorescence staining of myelin basic protein (MBP) was performed on sciatic nerve tissue 14 days postoperatively. Figure 6 .2d) to assess myelin sheath repair and distribution. To better characterize the differences in nerve regeneration, we specifically selected the proximal, injured, and distal ends of the sciatic nerve for side-by-side comparison. Immunofluorescence images showed that Schwann cells in the Sham, ALCAR, and THAEV@siR-TAgel groups were arranged in an orderly manner, indicating that the myelin sheath of the sciatic nerve was effectively repaired. Simultaneously, the area of ​​MBP fluorescence was quantified to reflect the structural integrity and degree of myelin sheath regeneration. Figure 14(c) The results showed that the overall fluorescence density of MBP in the DPNI group and the AEV / siR mixed group decreased to 57.06% and 69.55% of that of the normal sciatic nerve, respectively, which is consistent with the TEM results of myelin sheath. Meanwhile, the overall fluorescence density of MBP in the ALCAR and THAEV@siR-TAgel groups was not significantly different from that in the Sham group, at 92.14% and 94.63% of that of the normal sciatic nerve, respectively, indicating that their myelination levels were restored. In conclusion, THAEV@siR-TAgel treatment has myelin sheath repair capabilities in the treatment of the DPNI model.

[0058] Subsequently, the damaged sciatic nerve recovered, and the regeneration of neurofilaments was verified by NF200 protein immunofluorescence staining. The proximal, injured, and distal ends of the sciatic nerve were also marked for side-by-side comparison. Immunofluorescence images showed that the NF200-positive axons in the Sham, ALCAR, and THAEV@siR-TAgel groups were arranged in an orderly manner, indicating that the nerve fibers of the sciatic nerve were effectively repaired. Figure 15 (a) Simultaneously, the area of ​​NF200 positive fluorescence was quantified to reflect the structural integrity and regeneration degree of axons. The overall density of NF200 fluorescence in the sciatic nerve of the THAEV@siR-TAgel group was not significantly different from that of the Sham group. Figure 15 (b) in the middle.

[0059] Example 5: Safety Evaluation of AEV-XNH Example 4: After treatment, the heart, liver, spleen, lungs, and kidneys of mice were pathologically evaluated. H&E staining results showed that the major organs in the THAEV@siR-TAgel treatment group had normal morphology, with no obvious tissue damage or inflammatory infiltration. Figure 16 This demonstrates that THAEV@siR-TAgel has excellent in vivo biocompatibility.

[0060] 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 intelligent plant exosome vesicles encapsulated in a self-assembled nanogel, characterized in that, Includes the following steps: 1) Modify the surface of plant exosome vesicles with a TH multifunctional ligand, wherein the TH multifunctional ligand is composed of a combination of a nerve cell-specific adhesion peptide, a lysosomal escape peptide, and a palmitic acid group; 2) Add siRNA targeting the pathogenic gene to the system of step 1) and load it onto the surface of the plant exosome vesicles modified in step 1); 3) Add an excess of tannic acid solution to the system in step 2) to form a self-assembled nanogel layer on the surface of plant exosome vesicles. The nanogel layer is formed by the self-assembly of the siRNA and tannic acid through hydrogen bonding, thus obtaining the intelligent plant exosome vesicles.

2. The method according to claim 1, characterized in that, 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; the plant exosome vesicles are extracted from fresh tissues of the main medicinal parts of the above-mentioned medicinal plants by differential ultracentrifugation.

3. The method according to claim 1, characterized in that, The amino acid sequence of the nerve cell-specific adhesion peptide is HLNILSTLWKYR.

4. The method according to claim 1, characterized in that, The lysosomal escape peptide is an oligohistidine sequence Hn, where the number of histidines n is any number from 2, 4, 6, 8, 10, to 12.

5. The method according to claim 1, characterized in that: Step 1) Specifically: The TH multifunctional ligand was co-incubated with plant exosome vesicle solution at 37°C to modify the surface of the plant exosome vesicles with the TH multifunctional ligand; the mass of the plant exosome vesicles was measured by the total amount of protein contained in the plant exosome vesicles, and the mass ratio of plant exosome vesicles to TH multifunctional ligand was 2:1 to 4:

1.

6. The method according to claim 1, characterized in that, In step 2), the siRNA is an siRNA that targets the pathogenic PTEN gene in diabetic peripheral neuropathy. Pten PTEN refers to homologs of phosphatase and tensin.

7. The method according to claim 1, characterized in that: Step 2) specifically involves adding siRNA modified with 3' cholesterol to the system obtained in step 1) and co-incubating it under ice-water bath conditions to load the siRNA onto the surface of the plant-derived exosome vesicles; the mass of the plant exosome vesicles is measured by the total protein content contained in the plant exosome vesicles, and the ratio of the mass of the plant exosome vesicles to the absorbance value of the siRNA is 100~200 μg: 0.2~1 OD.

8. The method according to claim 1, characterized in that: Step 3) Specifically involves adding a tannic acid solution to the system obtained in step 2) and co-incubating it under ice-water bath conditions, so that the siRNA and the tannic acid are linked by hydrogen bonds and encapsulated on the surface of the plant exosome vesicles to form a self-assembled nanogel layer.

9. A self-assembled nanogel-encapsulated intelligent plant exosome vesicle, characterized in that, It is prepared by the method described in any one of claims 1-8.

10. An application of the intelligent plant exosome vesicles encapsulated in the self-assembled nanogel as described in claim 9, characterized in that, This is used to prepare a drug for treating peripheral neurodegenerative diseases, including sciatic nerve injury caused by diabetes, sensory / motor dysfunction, and cataracts.

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