Phosphorus-containing dendrimer nano-composite wrapped by exosome as well as preparation method and application of phosphorus-containing dendrimer nano-composite

Abstract

The invention relates to an exosome-coated phosphorus-containing dendrimer nano-composite as well as a preparation method and application thereof. The nano-composite is prepared from exosome derived from mesenchymal stem cells, a hydroxyl-terminal phosphorus-containing dendrimer and quercetin. The exosome derived from the mesenchymal stem cells is used as a carrier, so that not only are good biocompatibility and anti-inflammatory activity achieved, but also the bioavailability of the phosphorus-containing dendrimer and quercetin can be improved. The phosphorus-containing dendrimer and quercetin exert anti-inflammatory and anti-oxidation effects by promoting microglial cells to be polarized to M2 type and removing active oxygen, so that neuronal cells are protected from apoptosis. The preparation method has the advantages of being simple, easy to operate, good in biocompatibility and the like, and the provided strategy has good development prospects and application values in treatment of Parkinson's disease or other neurodegenerative diseases.

Description

Technical Field

[0001] The present invention belongs to the field of functional nanocarriers, and particularly relates to an exosome-encapsulated phosphorus-containing dendrimer nanocomposite, a preparation method thereof, and an application thereof. Background Art

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disorder in the world, second only to Alzheimer's disease. Characterized by high morbidity, high disability rates, and a slow progression, PD imposes a heavy economic burden on society. The primary pathological change in PD is the degeneration and death of dopamine (DA) neurons in the substantia nigra of the midbrain (Am. J. Med. 2019, 132, 802-807), resulting in a significant decrease in DA content (>70%). Furthermore, abnormal accumulation of α-synuclein (ɑ-syn) within dopamine neurons in the substantia nigra of the midbrain activates resting microglia and causes them to secrete proinflammatory cytokines (Proc. Natl. Acad. Sci. USA 2014, 111, 2402-2403). The neurotoxicity of ɑ-syn aggregates and the resulting neuroinflammation further trigger neuronal degeneration and death. Currently, drug therapy is the mainstay of PD treatment. However, free drugs used clinically for PD are limited by their inability to cross the blood-brain barrier (BBB) ​​to repair damaged neurons. Therefore, there is an urgent need to develop new, safe, and effective anti-PD drug delivery systems that can penetrate the BBB and overcome the inherent limitations of free drugs.

[0003] Exosomes are membrane vesicles released into the extracellular matrix following fusion of intracellular multivesicular bodies with the cell membrane. They are nanoscale lipid-encapsulated structures with a diameter of 30-150 nm, containing substances such as proteins, mRNA, and microRNA. Exosomes are nanometer-sized and disc-shaped, with low immunogenicity. As drug delivery vehicles, they possess natural material transport properties, long-term circulation in the body, and excellent biocompatibility. Exogenous substances such as chemicals, proteins, and nucleic acids can be loaded into exosomes through methods such as ultrasound, electroporation, and incubation, creating targeted, multifunctional biopharmaceutical delivery systems for delivering drugs to specific areas to enhance therapeutic efficacy. For example, Li et al. designed a nanovaccine platform that uses dendritic cell-derived exosomes as carriers for patient-specific neoantigens for personalized immunotherapy. This nanovaccine delivers antigens to lymph nodes, exhibits strong antigen specificity, and broadly stimulates T- and B-cell-mediated immune responses, significantly inhibiting tumor growth, prolonging survival, delaying tumorigenesis, and maintaining long-term memory. It can also eliminate lung metastases (J. Controlled Release 2023, 353, 423–433). Furthermore, as a vehicle for intercellular substance transfer, exosomes carry biological substances from parent cells and share similar biological functions with them, thereby exerting their biological activity. Exosomes derived from mesenchymal stem cells (MSCs) have anti-inflammatory, antioxidant, cell proliferation-promoting, and damaged tissue repair effects, and can also improve the immune microenvironment of lesions to a certain extent. Researchers have found that miR-106b in MSC-derived exosomes can alleviate neuronal apoptosis and enhance neuronal autophagy in PD patients (Neurosci. Lett. 2021, 760, 136094). MiR-133b, which is abundantly expressed in exosomes derived from mesenchymal stem cells, is specifically expressed in dopaminergic neurons in the substantia nigra of the midbrain, but is downregulated in the brain tissue of PD patients (Int. J. Mol. Sci. 2014, 15(3), 4142-4157). Therefore, an increase in miR-133b content can play a neuroprotective role, promote the growth of nerve axons, and reduce the extent of neurodegenerative diseases (Stem Cells 2013, 31(12), 2737-2746). In addition, studies have found that nasal administration of exosomes can bypass the blood-brain barrier at the tissue level and show better brain accumulation of exosomes than intravenous administration (ACSNano 2017, 11, 10883-10893). In general, MSC-derived exosomes are a promising therapeutic vector for Parkinson's disease.

[0004] Quercetin is a flavonoid substance widely found in plants, with good anti-inflammatory, antioxidant and anti-apoptotic biological activities. Quercetin can exert efficient anti-inflammatory / antioxidant effects by scavenging reactive oxygen species and inhibiting the production of NLRP3 inflammasomes (Nat.Biomed.Eng.2022,6,76-93). Studies have found that quercetin can protect neuronal cells by affecting apoptosis, autophagy and invadosomes, against cell damage induced by various toxic substances such as MPP+ (1-methyl-4-phenylpyridinium ion) (NeuralRegenerationRes.2015,10,1113-1119). Studies have shown that dendrimers with hydroxylated surfaces have anti-inflammatory effects, can penetrate the damaged blood-brain barrier, and can target activated microglia in damaged areas (Sci.Adv.2020,6,eaay8514). A series of studies by Majoral et al. found that phosphorus-containing dendrimers of different generations and terminal groups were able to inhibit the formation of α-syn fibrils. They proposed that phosphorus-containing dendrimers may inhibit aggregation by blocking the hydrophobic interactions between α-syn monomers (MolPharmaceutics 2013, 10, 1131-1137). Previous studies have shown that phosphorus-containing dendrimers with a high density of hydroxyl groups on their surfaces can not only penetrate the damaged BBB but also have anti-inflammatory activity and can be used for efficient protein delivery (Bioact. Mater. 2024, 38, 45–54).

[0005] The currently developed nanoplatforms still do not combine the efficacy of exosomes and phosphorus-containing dendrimers to penetrate the nasal mucosal barrier, bypass the BBB, and achieve the goal of effectively treating Parkinson's disease through synergistic anti-inflammatory / anti-oxidative / anti-apoptotic effects. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide an exosome-encapsulated phosphorus-containing dendrimer nanocomposite, its preparation method, and application. Using mesenchymal stem cell-derived exosomes as a carrier, the nanocomposite not only exhibits excellent biocompatibility and anti-inflammatory activity but also improves the bioavailability of the phosphorus-containing dendrimer and quercetin. The phosphorus-containing dendrimer and quercetin exert anti-inflammatory and antioxidant effects by promoting microglial polarization toward the M2 phenotype and scavenging reactive oxygen species, thereby protecting neurons from apoptosis.

[0007] The present invention provides an exosome-encapsulated phosphorus-containing dendrimer nanocomposite, comprising exosomes derived from mesenchymal stem cells (MSCs), a hydroxyl-terminated phosphorus-containing dendrimer, and quercetin. The nanocomposite uses MSC-derived exosomes as a carrier and internally loads the hydroxyl-terminated phosphorus-containing dendrimer and quercetin, an anti-inflammatory and antioxidant drug.

[0008] The hydroxyl-terminated phosphorus-containing dendrimer is AK76, and its structural formula is

[0009] The present invention also provides a method for preparing an exosome-encapsulated phosphorus-containing dendrimer nanocomposite, comprising the following steps:

[0010] (1) adding a hydroxyl-terminated phosphorus-containing dendrimer dissolved in methanol to an exosome solution dissolved in PBS, mixing, sonicating, incubating, and then centrifuging for purification, collecting the supernatant, and obtaining an exosome-encapsulated hydroxyl-terminated phosphorus-containing dendrimer complex EXO-AK76 (i.e., AE);

[0011] (2) The quercetin solution dissolved in methanol was added to the exosome-encapsulated hydroxyl-terminated phosphorus-containing dendrimer complex solution, mixed, sonicated, incubated, and then centrifuged for purification, and the supernatant was collected to obtain the exosome-encapsulated phosphorus-containing dendrimer nanocomplex EXO-AK76-Que (i.e., QAE).

[0012] Preferably, the mass ratio of the exosomes to the hydroxyl-terminated phosphorus-containing dendrimers in step (1) is 1:1.

[0013] Preferably, the mass ratio of quercetin and the exosome-encapsulated hydroxyl-terminated phosphorus-containing dendrimer complex in step (2) is 1:1.

[0014] Preferably, the ultrasound in steps (1) and (2) is specifically as follows: ultrasound is applied under 20% pulse conditions for 3 minutes, and the process is repeated 6 times, with a 2-minute pause between each cycle.

[0015] Preferably, the incubation in steps (1) and (2) is specifically: incubation at 37° C. for 30 minutes.

[0016] Preferably, the centrifugal purification in steps (1) and (2) is specifically: centrifugal purification at 3000-5000 rpm for 8-10 min.

[0017] The present invention also provides an application of an exosome-encapsulated phosphorus-containing dendrimer nanocomposite in the preparation of anti-inflammatory, antioxidant, and anti-apoptotic drugs.

[0018] The present invention also provides an application of an exosome-encapsulated phosphorus-containing dendrimer nanocomposite in the preparation of a drug for treating Parkinson's disease.

[0019] The present invention uses MSC-derived exosomes as a carrier, and after ultrasonic disruption, incubates at 37°C to restore the membrane, thereby combining with hydroxyl-terminated phosphorus-containing dendrimers to form a nanocomplex, which is further ultrasonically loaded with quercetin. The nanocomplex provided by the present invention has the advantages of simple preparation method, easy operation, good biocompatibility, and low immunogenicity. On the one hand, the prepared nanocomplex is administered through nasal drops, bypassing the BBB at the tissue level, effectively increasing the accumulation level of the drug in the brain; on the other hand, the nanomedicine relieves neuroinflammation, scavenges reactive oxygen species, thereby reducing oxidative stress and repairing damaged neurons, thereby achieving a synergistic treatment effect for Parkinson's disease, such as Figure 1 The strategy provided by the present invention has good development prospects and application value in the treatment of Parkinson's disease or other neurodegenerative diseases.

[0020] The nanocomplex is a method in which exosomes are ultrasonically disrupted and incubated at 37°C to restore the membrane, thereby loading hydroxyl-terminated phosphorus-containing dendrimers with quercetin to form nanocomposite particles QAE for synergistic anti-inflammatory / anti-oxidative / anti-apoptotic treatment of Parkinson's disease.

[0021] The present invention uses Zeta potential and dynamic light scattering analysis (DLS), fluorescence spectrophotometer, transmission electron microscopy (TEM), protein immunoblotting (Western Blot, WB), nanoparticle tracking analysis technology (NTA), resistive pulse sensing (RPS) and other means to characterize the physical and chemical properties of the extracted MSC-derived exosomes and nanocomposite particles QAE. Then, the CCK-8 method is used to analyze and evaluate the cytotoxicity of QAE and related control materials; the phagocytosis of the materials by microglia is detected by laser confocal microscopy; the effect of the materials on the intracellular ROS level is evaluated by laser confocal microscopy and flow cytometry; the effect of the nanomaterials on microglia typing is evaluated by flow cytometry; the inflammatory-related factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and interleukin-1β are detected by enzyme-linked immunosorbent assay (ELISA) kit. -10 (IL-10) expression level; the effect of the material on the intracellular mitochondrial membrane potential level and cell apoptosis was evaluated by flow cytometry; the expression of cellular p-p65, NLRP3 and Caspase-3 proteins was detected by Western blot; a PD mouse model was established, and the improvement of motor recovery was evaluated using a series of behavioral tests; H&E staining was used to analyze the damage of brain tissue after treatment; and the anti-inflammatory / antioxidant effect and dopamine recovery effect of the material in the brain were evaluated by immunofluorescence staining of brain sections.

[0022] Beneficial effects

[0023] (1) The present invention uses mesenchymal stem cell-derived exosomes as carriers, which not only have good biocompatibility and anti-inflammatory activity and can actively target the brain, but also improve the bioavailability of phosphorus-containing dendrimers and quercetin, enhancing their intracellular delivery efficiency. Phosphorus-containing dendrimers and quercetin exert anti-inflammatory and antioxidant effects by promoting microglial polarization to M2 type and scavenging reactive oxygen species, thereby protecting neuronal cells from apoptosis. The present invention has the advantages of simple preparation method, easy operation, and good biocompatibility. The strategy provided has good development prospects and application value in the treatment of Parkinson's disease or other neurodegenerative diseases.

[0024] (2) On the one hand, the present invention can bypass the BBB at the tissue level through nasal drop administration, significantly increasing the accumulation level of the drug in the brain; on the other hand, it can alleviate neuroinflammation by promoting the polarization of microglia to M2 type, eliminate reactive oxygen species, reduce cell apoptosis and necrosis rates, and thus protect neuronal cells, achieving a synergistic therapeutic effect in treating Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the synthesis of the nanocomplex QAE of the present invention (A) and schematic diagram of its delivery and potential therapeutic mechanism (B).

[0026] Figure 2 Phase contrast microscopy image of the mesenchymal stem cells extracted in Example 1 (A) and flow cytometric analysis images of the cell surface antigens CD45 and CD90.1 (B, C).

[0027] Figure 3 Figure 1 shows the particle concentration of mesenchymal stem cell-derived exosomes extracted in Example 1 analyzed by NTA (A) and by RPS (B).

[0028] Figure 4 The hydrated particle size and PDI (A) and surface potential diagram (B) of the exosomes (EXO), AE and QAE prepared in Example 1 and Example 2.

[0029] Figure 5 TEM images of EXO (A), AE (B), and QAE (C) prepared in Example 1 and Example 2.

[0030] Figure 6 The WB results of protein expression on EXO and QAE prepared in Example 1 and Example 2 are shown.

[0031] Figure 7 Graph showing cell viability of EXO and QAE prepared in Example 1 and Example 2 after incubation with HNEpC cells, BV2 cells, and SH-SY5Y cells for 24 h.

[0032] Figure 8 (A) Analysis of intracellular PKH26 fluorescence intensity and (B) fluorescence quantitative analysis of QAE and BV2 cells after co-incubation for 2h, 4h, and 8h, respectively; all cell experiments involving EXO were labeled with PKH26.

[0033] Figure 9 Figure 2 shows the intracellular PKH26 fluorescence imaging (A) and QAE penetration efficiency (B) of HNEpC cells and SH-SY5Y cells after they were co-incubated with EXO and QAE in transwell chambers for 24 hours.

[0034] Figure 10 Figure 3 (A) shows the laser confocal microscopy analysis of intracellular ROS levels in BV2 cells after they were treated with MPP+ and co-incubated with AK76, AE, Que, and QAE for 24 hours; flow cytometric analysis of ROS (B) and fluorescence quantification (C); PBS and MPP+ were the negative and positive control groups, respectively.

[0035] Figure 11 Figure 2: Flow cytometric analysis of CD86 and CD206 expression levels after BV2 cells were treated with MPP+ and co-incubated with AK76, AE, Que, and QAE for 24 hours, and quantitative results of the M2 microglia / M1 microglia ratio (B); PBS and MPP+ were negative and positive control groups, respectively.

[0036] Figure 12 Figure 2 shows the WB detection results (A) and corresponding quantitative graphs (B, C) of intracellular p-p65 and NLRP3 protein expression in BV2 cells after being treated with MPP+ and co-incubated with AK76, AE, Que, and QAE for 24 hours; PBS and MPP+ were the negative and positive control groups, respectively.

[0037] Figure 13 Figure 2 is a quantitative analysis of the expression levels of pro-inflammatory cytokines TNF-α (A), IL-1β (B), IL-6 (C) and anti-inflammatory cytokine IL-10 (D) in the cell supernatant after BV2 cells were co-incubated with AK76, AE, Que, and QAE for 24 hours after treatment with MPP+; PBS and MPP+ were the negative and positive control groups, respectively.

[0038] Figure 14 Figure 2: Flow cytometric analysis (A) and quantitative analysis (B) of mitochondrial membrane potential in SH-SY5Y cells after they were treated with MPP+ and then co-incubated with AK76, AE, Que, and QAE for 24 hours; PBS and MPP+ were the negative and positive control groups, respectively.

[0039] Figure 15 Figure 2: Flow cytometric analysis (A) and quantitative analysis (B) of apoptosis levels in SH-SY5Y cells after they were treated with MPP+ and then co-incubated with AK76, AE, Que, and QAE for 24 hours; PBS and MPP+ were the negative and positive control groups, respectively.

[0040] Figure 16 Figure 2 shows the WB detection results (A) and corresponding quantitative graph (B) of intracellular caspase-3 protein expression in SH-SY5Y cells treated with MPP+ and co-incubated with BV2 cells with AK76, AE, Que, and QAE for 24 hours; PBS and MPP+ were the negative and positive control groups, respectively.

[0041] Figure 17 Figure 3. In vivo fluorescence imaging of Cy5.5-NHS fluorescence images (A) and corresponding quantitative images (B) of MPTP-induced Parkinson's mice treated with EXO and QAE nasal drops, or in vitro fluorescence imaging (C) and corresponding quantitative images (D) of the main organs (heart, liver, spleen, lung, kidney and brain) of mice after nasal drops administration. All animal experiments involving EXO used Cy5.5-NHS-labeled EXO.

[0042] Figure 18 Behavioral test results of MPTP-induced Parkinson's mice after 7 days of treatment with PBS, AK76, AE, and QAE nasal drops: (A) peak grip strength test; (B) time required for climbing pole test; (C) immobility time in tail suspension test; (D) time required to fall in fatigue rotarod test; and (E) floating time in forced swimming.

[0043] Figure 19 Results of the open field test in MPTP-induced Parkinson's mice after 7 days of treatment with PBS, AK76, AE, and QAE nasal drops: (A) Representative trajectories of the open field test and time heat map of representative trajectories; (B) Total distance moved by the mice; (C) Average movement speed; (D) Percentage of time spent in the center area; (E) Percentage of time spent immobile.

[0044] Figure 20 These are the results of Nissl staining of the hippocampal CA1 region in MPTP-induced Parkinson's mice after 7 days of treatment with PBS, AK76, AE, and QAE nasal drops.

[0045] Figure 21 Immunofluorescence staining images of GFAP and α-syn in the striatum of MPTP-induced Parkinson's mice after 7 days of treatment with PBS, AK76, AE, and QAE nasal drops (A) and the corresponding α-syn quantitative images (B).

[0046] Figure 22Immunofluorescence staining of reactive oxygen species in brain sections of MPTP-induced Parkinson's mice after 7 days of treatment with PBS, AK76, AE, and QAE nasal drops (A) and the corresponding quantitative graph (B).

[0047] Figure 23 Figure 3 (A) Immunofluorescence staining of CD206 and CD86 in microglia of brain sections of MPTP-induced Parkinson's mice after 7 days of treatment with PBS, AK76, AE, and QAE nasal drops, and quantitative results of the CD206 / CD86 ratio (B).

[0048] Figure 24 Immunofluorescence staining of IBA-1 and TH in the striatum of MPTP-induced Parkinson's mice after 7 days of treatment with PBS, AK76, AE, and QAE nasal drops (A), quantitative images of IBA-1 (B), and quantitative images of TH (C). DETAILED DESCRIPTION

[0049] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0050] Unless otherwise specified, all chemical reagents were used directly without further purification. SD rats and C57BL / 6 mice were purchased from Shanghai Regen Biotechnology Co., Ltd. The hydroxyl-terminated phosphorus dendrimer AK76 was obtained from the group of Professor J.P. Majoral at the Laboratory of Coordination Chemistry, National Center for Scientific Research, France. Quercetin was obtained from Shanghai Bailingwei Biotechnology Co., Ltd. Mouse microglia (BV2) were obtained from Shanghai EK Biotechnology Co., Ltd. Human nasal epithelial cells (HNEpC) and SH-SY5Y cells were obtained from Shanghai Meiwan Biotechnology Co., Ltd. MEM, DMEM, fetal bovine serum, penicillin-streptomycin, and trypsin were obtained from Hangzhou Jinuo Biomedical Technology Co., Ltd. DME / F-12 was obtained from Shanghai Anjin Biotechnology Co., Ltd. Dimethyl sulfoxide (DMSO) and other chemicals and solvents were obtained from Sinopharm Chemical Reagent Co., Ltd. The exosome isolation and purification kit and the exosome fluorescent labeling dye PKH26 were obtained from Shanghai Yumeibo Biotechnology Co., Ltd. Cy5.5-NHS was obtained from Sigma-Aldrich (St. Louis, MO). Cell viability test kit-8 (CCK-8), BCA assay kit, ROS assay kit, and JC-1 assay kit were all purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Apoptosis detection kit was purchased from Jiangsu Keygene Biotechnology Co., Ltd. Antibodies such as anti-CD86-PE, anti-CD206-FITC, anti-CD45-FITC, and anti-CD90.1-FITC were purchased from Thermo Fisher Scientific (Waltham, MA). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-1β, IL-6, and IL-10 were obtained from Wuhan Sewell Technology Co., Ltd. MPP+ was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. MPTP was purchased from MedChemExpress (MCE). Water with a resistivity greater than 18.2 MΩ·cm used in all experiments was purified using a PURIST UV ultrapure water system (Shanghai Ruifulai Biotechnology Co., Ltd.).

[0051] Example 1

[0052] (1) Bone marrow mesenchymal stem cells (BMSCs) were isolated and purified from 2-week-old male SD rats using the whole bone marrow attachment method. First, the SD rats were disinfected with 75% alcohol and euthanized. Then, the skin and muscle tissue of the limbs were separated to obtain complete femurs and tibias. The bone marrow cavity was exposed, and DMEM / F-12 culture medium was aspirated with a sterile syringe. The cells in the bone marrow cavity were repeatedly rinsed into a 6-well plate. Subsequently, the cell suspension was centrifuged at 1000 rpm for 5 minutes, the cell pellet was collected, and the cells were resuspended with DMEM / F-12 culture medium containing 10% fetal bovine serum by volume. After mixing, the cells were inoculated into a culture dish and placed in a 5% CO2, 37°C incubator for static culture. After primary inoculation, the culture was statically cultured. The medium was changed for the first time after 24 hours, and the medium was changed every 3 days thereafter. When the cells grew to 80%-90% confluence, they were digested with trypsin and subcultured at a ratio of 1:3. BMSCs cells in the logarithmic growth phase were collected and plated at a ratio of 2×10 5 The cells were seeded into 6-well plates at a density of 10 cells per well and cultured at 5% CO2 and 37°C for 24 hours. Figure 2 As shown in A. Flow cytometry was used to analyze the expression of typical markers CD45 and CD90.1 in BMSCs. The culture medium was discarded, the cells were washed twice with PBS, and the cells were collected by trypsin digestion. The cell pellet was resuspended with 200 μL PBS, and Anti-CD45-FITC and Anti-CD90.1-FITC antibodies were added to the cells and incubated on ice in the dark for 30 minutes. After washing three times with PBS to remove the probes that were not bound to the cells, the cell pellet was resuspended with 300 μL PBS and transferred to a flow tube. The expression of CD45 and CD90.1 in the primary extracted BMSCs cells was evaluated by flow cytometry. The results are shown in Figure 2 As shown in BC, the extracted BMSCs expressed CD45 negatively but CD90.1 positively, which was consistent with the characteristics of BMSCs.

[0053] (2) Select 3rd-6th generation mesenchymal stem cells with good growth conditions to extract exosomes (EXO). When the cells grow to 70-80% confluence, replace the mesenchymal stem cell complete culture medium with serum-free mesenchymal stem cell culture medium (for exosome culture). After 48 hours of culture, the cell supernatant is centrifuged at 3000g and 4°C for 10 minutes to remove cell debris, and the supernatant from the previous step is centrifuged at 10000g and 4°C for 10 minutes to remove impurities. Add exosome concentrate (ECS) to the supernatant after centrifugation to remove impurities, mix with a vortex oscillator for 1 minute, and let it stand at 4°C for more than 8 hours. Centrifuge at 10000g for 60 minutes at 4°C and discard the supernatant. Take an appropriate amount of 1×PBS to evenly disperse the centrifugal precipitate and transfer it to a 1.5mL centrifuge tube. Centrifuge at 4°C and 12000g for 2 minutes, and retain the supernatant. The supernatant was transferred to the upper chamber of the exosome purification filter (EPF column) and centrifuged at 4°C and 3000g for 10 minutes. After centrifugation, the liquid at the bottom of the EPF column was collected to purify the exosome particles. Resuspended in PBS. In addition, the exosome concentration was quantitatively determined by the BCA kit and was 0.25 mg / mL. Figure 3 As shown in AB, the concentration of EXO measured by NTA was 2.1×10 9 particles / mL, and the RPS results showed that the concentration of EXO at 60-200 nm was 1.03×10 10 particles / mL.

[0054] Example 2

[0055] (1) 25 μg of hydroxyl-terminated phosphorus dendrimer AK76 was dissolved in 10 μL of methanol and then added dropwise to a 25 μg / mL exosome PBS solution (1 mL). The mixed solution was sonicated under the following conditions: amplitude 20%, temperature 4°C, 6 cycles of 30 s each, 3 min each, and 2 min of cooling between each cycle. After sonication, the mixed solution was incubated at 37°C for 30 min to restore the exosome membrane. The above solution was then ultrafiltered and centrifuged at 5000 rpm at 4°C for 5 min to remove free AK76, and the supernatant was collected as EXO-AK76 (AE).

[0056] (2) 25 μg of quercetin (Que) was dissolved in 10 μL of methanol and then added dropwise to the above AE solution. The mixed solution was sonicated under the following conditions: amplitude 20%, temperature 4°C, 6 cycles, 30 s each, 3 min each, and two minutes of cooling between each cycle. After sonication, the QAE solution was incubated at 37°C for 30 min. The above solution was ultrafiltered and centrifuged at 1000 rpm for 10 min at 4°C to remove free quercetin, and the supernatant was collected as the nanocomplex QAE. At the same time, the quercetin content in the precipitate was measured by ultraviolet spectrophotometry, and the amount of unloaded quercetin was calculated, thereby calculating the quercetin loading rate to be 48.1% and the encapsulation efficiency to be 96.2%.

[0057] Example 3

[0058] The EXO, AE and QAE prepared in Examples 1 and 2 were prepared into solutions for measuring the hydrated particle size and surface potential. Figure 4 As shown, the hydrated particle size of EXO was 145.3±15.75nm and the potential was -9.38±0.49mV. After loading with AK76, the hydrated particle size was 229.3±4.55nm and the potential changed to -9.07±0.57mV. The changes in hydrated particle size and potential confirmed the successful synthesis of AE. When AE was loaded with the drug Que, the hydrated particle size was 317.0±8.95nm and the potential was -11.3±0.4mV. The increase in particle size without significant change in potential indicates that the exosomes were successfully loaded with AK76 and Que through the ultrasound-incubation recovery drug loading method.

[0059] Example 4

[0060] The EXO, AE and QAE prepared in Examples 1 and 2 were characterized for size and morphology. The TEM results of the nanocomposites are shown in Figure 1. Figure 5 As shown, EXO, AE and QAE are uniform spherical. After incubation with ultrasonic loading of AK76 and Que, the membrane of the exosomes recovered well. TEM images show that after two ultrasounds, the size of QAE increased compared with EXO and AE, which is consistent with the hydration particle size results. In addition, the protein concentration of EXO and QAE was determined by BCA, followed by SDS-PAGE electrophoresis, membrane transfer, immunoreaction, and ECL chemical developer fixation experiments. The CD63 and TSG101 protein content of EXO and QAE were studied, with β-actin used as an internal reference. The results are shown in Figure 6 As shown, after EXO was loaded with AK76 and Que, the surface characteristic marker proteins still existed to ensure the integrity of exosome function.

[0061] Example 5

[0062] HNEpC, BV2 and SH-SY5Y cells were used as cell models to test the cytotoxicity of QAE and related materials prepared in Example 2. HNEpC, BV2 or SH-SY5Y cells in the logarithmic growth phase were collected and 1×10 4 The cells were seeded in a 96-well plate at a density of , and the culture medium used was a complete culture medium supplemented with 100U / mL penicillin, 100U / mL streptomycin and 10% FBS, and incubated at 5% CO2 and 37°C for 24 hours. The original culture medium was discarded, and culture medium containing different concentrations of Que or QAE (relative Que concentrations of 0, 12.5, 25, 50, 75, 100μg / mL) was added to each well plate and co-cultured with the cells at 5% CO2 and 37°C for 24 hours. After that, the well plate was taken out, the original culture medium was discarded, and the plate was washed three times with PBS. Fresh culture medium containing 10% (v / v) CCK-8 was added, and the plate was incubated in the incubator for another 3 hours. Finally, the absorbance of each well was tested at a wavelength of 450nm using a multifunctional microplate reader, and the cells treated with PBS were used as blank controls. The results are shown in the figure below. Figure 7 As shown in the figure, within the experimental concentration range, with the increase of Que concentration, each group showed no obvious cytotoxicity to HNEpC, BV2 and SH-SY5Y cells.

[0063] Example 6

[0064] BV2 cells were used as a cell model to evaluate the phagocytic effect of cells on QAE. 5 BV2 cells were seeded in a laser confocal microscopy-specific culture dish at a density of 100 cells per well. The culture medium used was DMEM complete culture medium supplemented with 100U / mL penicillin, 100U / mL streptomycin and 10% FBS. The cells were incubated under 5% CO2 and 37°C for 24 hours. The culture medium in each dish was replaced with fresh culture medium containing QAE ([Que] = 25μg / mL) and cultured continuously for 2h, 4h or 8h. The cells were washed three times with PBS, fixed with 1mL 2.5% glutaraldehyde at room temperature for 15min, washed three times with PBS, and incubated with 1mL DAPI for 5min. After washing three times with PBS, the changes in cell fluorescence intensity were observed under a laser confocal microscope. The phagocytosis results are shown in Figure 2. Figure 8 As shown in A, as the incubation time increases, more red fluorescence accumulates in the cells, indicating that BV2's uptake of the material is also enhanced. Figure 8 As shown in Figure B, it further demonstrates the efficient uptake of QAE by BV2.

[0065] Example 7

[0066] In order to explore the permeability of QAE nanoparticles in the nasal mucosa, an in vitro nasal mucosal barrier model was established. HNEpC cells were cultured at 2×10 5Cells were seeded at a density of 100 cells / well in the upper chamber of a 6-well transwell plate. The bottom of the chamber had a polycarbonate membrane with 0.4 μm pores. The upper chamber was placed in the lower chamber of the transwell plate. The culture medium used was complete MEM supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS. The cells were cultured at 37°C with 5% CO2 for 5-7 days until the transendothelial electrical resistance (TEER) reached 200-300 Ω·cm. 2 , indicating that the cells formed a tightly connected monolayer similar to the nasal mucosal barrier. Subsequently, SH-SY5Y cells were plated at 1.2×10 5 The cells were seeded at a density of 10 cells / well in the lower chamber. After the cells adhered, fresh culture medium containing PBS, EXO-PKH26, or QAE-PKH26 ([Que] = 25 μg / mL) was added to the upper chamber and co-cultured with the lower chamber for 6 hours. The fluorescence intensity of the HNEpC cells in the upper layer and the SHSY5Y cells in the lower layer were measured using a PerkinElmer IVIS Lumina imaging system (Waltham, MA). The results are shown in Figure 2. Figure 9 As shown in the figure, the results showed that exosomes alone could slightly penetrate the simulated nasal mucosa, while the penetration effect of the nanocomplex was improved after loading AK76 and quercetin compared with exosomes alone, which may be because the hydroxyl groups on the surface of AK76 increased the penetration level of nanoparticles in the nasal mucosa.

[0067] Example 8

[0068] BV2 cells were used as a cell model to evaluate the ability of QAE to alleviate oxidative stress. In order to verify the effect of QAE nanocomplex on ROS scavenging in BV2 cells, BV2 cells in the logarithmic growth phase were collected and the cells were cultured at a rate of 1×10 5 The cells were seeded at a density of 2×10 5Cells were seeded at a density of 100 cells per well in a 6-well plate. The culture medium used was DMEM complete medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS. The cells were cultured at 5% CO2 and 37°C for 24 h. The culture medium was then replaced with DMEM medium containing MPP+ (at a concentration of 1 mM) and incubated with the cells for 6 h. A control group was supplemented with PBS (served as the control group). Subsequently, the cells were incubated with fresh DMEM medium containing PBS, AK76, AE, Que, or QAE ([Que] = 25 μg / mL) for 24 h. For confocal laser scanning microscopy observation, the cells were treated as follows: the culture medium was discarded, the cells were washed three times with PBS, the ROS fluorescent probe DCFH-DA was added and incubated with the cells at 37°C for 20 min, washed three times with PBS, fixed with 1 mL of 2.5% glutaraldehyde at room temperature for 15 min, washed three times with PBS, and incubated with 1 mL of DAPI for 5 min. After washing three times with PBS, the cell fluorescence intensity changes were observed under a confocal laser scanning microscopy. Flow cytometric analysis After trypsin digestion and collection of cells, the cell pellet was resuspended in 1 mL of PBS, and the ROS fluorescent probe DCFH-DA was added and incubated with the cells at 37°C for 20 min. After washing three times with PBS to remove the probe that was not bound to the cells, the cell pellet was resuspended in 300 μL of PBS and transferred to a flow tube. The effect of each nanomaterial on ROS scavenging in BV2 cells was evaluated by flow cytometry. Figure 10 As shown in the confocal microscope results (A), the fluorescence signal of DCFH-DA was almost unobservable in the negative control group treated with PBS, but the positive control group treated with MPP+ showed strong green fluorescence. After treatment with AE, Que, and QAE, the fluorescence intensity was weakened to varying degrees. Quantitative results of flow cytometry ( Figure 10 BC) further showed that, except for the negative control group, the ROS level in the QAE group was the lowest among all groups, and the ROS fluorescence was significantly reduced, revealing that QAE has significant ROS scavenging ability and good antioxidant effect.

[0069] Example 9

[0070] In order to verify the effect of QAE nanoparticles on microglial polarization, BV2 cells in the logarithmic growth phase were collected and cultured at a rate of 2×10 5The cells were seeded at a density of 10 cells per well in a 6-well plate and cultured at 5% CO2 and 37°C for 24 hours. The culture medium was replaced with DMEM medium containing MPP+ (at a concentration of 1 mM) and incubated with the cells for 6 hours. PBS was added to the control group (as the Control group). Subsequently, the culture medium was discarded and replaced with fresh DMEM medium containing PBS, AK76, AE, Que, and QAE (the corresponding Que concentration was 25 μg / mL) and incubated with the cells for 24 hours. Subsequently, the culture medium was discarded, washed twice with PBS, and the cells were collected by trypsin digestion. The cell pellet was resuspended with 200 μL PBS, and Anti-CD206-FITC and Anti-CD86-PE antibodies were added to the cells and incubated with the cells on ice in the dark for 30 minutes. After washing three times with PBS to remove the antibodies that were not bound to the cells, the cell pellet was resuspended with 300 μL PBS and transferred to a flow tube. The effect of nanoparticles on microglial polarization was evaluated by detecting changes in the expression levels of CD86 and CD206 in the cells. The results are shown in the figure. Figure 11 As shown in A, compared with the positive control MPP+ group, the expression level of CD86 decreased and the expression level of CD206 increased after cells were treated with QAE, indicating that QAE nanoparticles can induce microglia to polarize from the M1 pro-inflammatory phenotype to the M2 anti-inflammatory phenotype. Figure 11 B Quantitative results further showed that among all groups, the microglia treated with QAE had the highest M2 / M1 ratio. This was because QAE could be effectively taken up by BV2 cells, and AK76 and Que exerted a highly effective anti-inflammatory effect, thereby promoting the polarization of microglia to the M2 type.

[0071] Example 10

[0072] In order to explore the molecular mechanism by which QAE nanoparticles regulate BV2 cells to fight against inflammation, BV2 cells in the logarithmic growth phase were collected and cultured at a rate of 2×10 5 The cells were seeded at a density of 10 cells per well in a 6-well plate and cultured at 5% CO2 and 37°C for 24 hours. The culture medium was replaced with DMEM medium containing MPP+ (at a concentration of 1 mM) and incubated with the cells for 6 hours. PBS was added to the control group (as the Control group). Subsequently, the culture medium was discarded and replaced with fresh DMEM medium containing PBS, AK76, AE, Que, and QAE (the corresponding Que concentration was 25 μg / mL) and incubated with the cells for 24 hours. Subsequently, the culture medium was collected, the cells were washed twice with PBS, and after trypsin digestion and collection of the cells, the cells were washed twice with PBS to collect the cells. The protein concentration was determined, and then SDS-PAGE electrophoresis, membrane transfer, immunoreaction, and ECL chemical developer fixation experiments were performed in sequence. The content of p-p65 and NLRP3 proteins in the cells was studied, with β-actin used as an internal reference. The results are shown in Figure 2. Figure 12As shown in the figure, under the induction of MPP+, the expression of p-p65 and NLRP3 pro-inflammatory pathway-related proteins was significantly upregulated, indicating that microglia were in a state of oxidative stress and strong inflammatory response under the induction of MPP+. After treatment with QAE, the expression levels of p-p65 and NLRP3 proteins in microglia decreased. The corresponding ELISA kits were used to detect the content of pro-inflammatory factors (TNF-α, IL-1β, IL-6) and anti-inflammatory factors (IL-10) in the supernatants of each group in the collected culture medium to further evaluate the anti-inflammatory effect of QAE nanoparticles. The results are shown in the figure. Figure 13 As shown, among all groups except the negative control group, the QAE group had the lowest expression levels of TNF-α, IL-1β, and IL-6, while the highest expression level of the anti-inflammatory factor IL-10, indicating that QAE has the strongest anti-inflammatory properties among all the studied materials. This is because the exosome-encapsulated AK76 and Que can be massively taken up by microglia, synergistically enhancing the anti-inflammatory capacity of the carrier and drug, alleviating the inflammatory response by scavenging ROS and promoting M2 microglial polarization.

[0073] Example 11

[0074] In order to verify the effect of QAE nanoparticles on the mitochondrial membrane potential of SH-SY5Y cells, SH-SY5Y cells in the logarithmic growth phase were collected and cultured at a rate of 2×10 5 Cells were seeded at a density of 100 cells per well in a 6-well plate and cultured at 37°C with 5% CO2 for 24 hours. The cells were then incubated with fresh DMEM medium containing PBS, AK76, AE, Que, and QAE (Que concentration was 25 μg / mL) for 24 hours. Subsequently, the cells were incubated with DMEM medium containing MPP+ (at a concentration of 1 mM) for 6 hours. PBS was added to the control group (serving as the control group). After trypsinization, the cells were collected and 0.5 mL of JC-1 staining working solution was added to resuspend the cell pellet and incubated in a cell culture incubator at 37°C for 20 minutes. After incubation, the cells were centrifuged at 1000 rpm for 5 minutes to pellet the cells, and the supernatant was discarded. After washing twice with JC-1 staining buffer, the cell pellet was collected and resuspended in 500 μL of JC-1 staining buffer. Flow cytometry was used to analyze changes in mitochondrial membrane potential in the different treatment groups. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix, forming polymers (J-aggregates) that produce red fluorescence. When the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondrial matrix. At this time, JC-1 is a monomer (J-monomer) and produces green fluorescence. The change of mitochondrial membrane potential can be detected by the change of fluorescence color. Figure 14As shown, the red fluorescence of cells in the positive control group decreased significantly, while the green fluorescence increased significantly. This indicates that MPP+ can significantly induce a decrease in mitochondrial membrane potential, leading to an imbalance in mitochondrial functional homeostasis. With the exception of the negative control group, the JC-1 aggregate / monomer ratio in SH-SY5Y cells treated with QAE nanoparticles was higher than in all other groups, demonstrating its strongest ability to restore mitochondrial membrane potential. This also demonstrates that QAE significantly enhances the protective capacity of SH-SY5Y cells' mitochondrial membrane potential.

[0075] Example 12

[0076] In order to verify the protective effect of QAE nanoparticles on SH-SY5Y cells, SH-SY5Y cells in the logarithmic growth phase were collected and cultured at a rate of 2×10 5 The cells were seeded at a density of 10 cells per well in a 6-well plate and cultured at 5% CO2 and 37°C for 24 hours. The culture medium was replaced with fresh DMEM medium containing PBS, AK76, AE, Que, and QAE (the corresponding Que concentration was 25 μg / mL) and incubated with the cells for 24 hours. Subsequently, the culture medium was replaced with DMEM medium containing MPP+ (at a concentration of 1 mM) and incubated with the cells for 6 hours. PBS was added to the control group (as the Control group). The cells in the 6-well plate were then digested, collected by centrifugation, and washed. The instructions of the Annexin V-FITC / PI Cell Apoptosis Detection Kit (KeyGen Biotech, China) were used to detect the inhibition of cell apoptosis and necrosis by different materials after the cells were treated with MPP+. The results are shown in the figure. Figure 15 As shown in the results, after pretreatment with MPP+, the necrosis and apoptosis rates of SHSY5Y cells reached as high as 26.2%, indicating severe cell damage. Compared with the positive control group, the prepared AK76, AE, Que, and QAE all reduced the apoptosis and necrosis rates of SH-SY5Y cells. The QAE group had the lowest apoptosis rate of 7.6% for SH-SY5Y cells, demonstrating that QAE has a potent anti-apoptotic effect.

[0077] Example 13

[0078] To further elucidate the mechanism by which QAE nanoparticles regulate apoptosis in SH-SY5Y cells, the expression of the anti-apoptotic protein Caspase-3 in SH-SY5Y cells after different treatments was studied. SH-SY5Y cells in the logarithmic growth phase were collected and cultured at a concentration of 2×10 5The cells were seeded at a density of 10 cells per well in a 6-well plate and cultured at 5% CO2 and 37°C for 24 hours. The culture medium was replaced with fresh DMEM medium containing PBS, AK76, AE, Que, and QAE (the corresponding Que concentration was 25 μg / mL) and incubated with the cells for 24 hours. Subsequently, the culture medium was replaced with DMEM medium containing MPP+ (at a concentration of 1 mM) and incubated with the cells for 6 hours. PBS was added to the control group (as the Control group). Subsequently, the culture medium was collected, the cells were washed twice with PBS, and the cells were collected by trypsin digestion and then washed twice with PBS. The protein concentration was determined, and then SDS-PAGE electrophoresis, membrane transfer, immunoreaction, and ECL chemical developer fixation experiments were performed in sequence. The Caspase-3 protein content in the cells was studied, and β-actin was used as an internal reference. The results are shown in Figure 2. Figure 16 As shown in the results, under the induction of MPP+, the expression of Caspase-3 was upregulated, while after QAE treatment, the expression of Caspase-3 was significantly downregulated. This is due to the intrinsic anti-inflammatory properties of AK76 and the antioxidant and anti-apoptotic biological activities of Que. EXO loading of Que and AK76 improves the bioavailability of free drugs, effectively exerting their biological functions, and thus protecting neurons from MPP+-induced apoptosis.

[0079] Example 14

[0080] All animal experiments were conducted in strict accordance with the standards of the Animal Protection Association. Male SPF-grade 8-10-week-old C57BL / 6 mice used in the experiment were purchased from Shanghai Slake Experimental Animal Center. In order to induce a Parkinson's disease model, mice were continuously injected with MPTP intraperitoneally at a dose of 30 mg / kg body weight for 5-7 consecutive days. Parkinson's mice were selected and administered 20 μL EXO and QAE ([Que] = 2 mg / kg) via nasal drops (10 μL each in the left and right nose). The PerkinElmer IVIS Lumina imaging system (Waltham, MA) was used to perform in vivo imaging of the small animals, and AE and QAE were labeled with the dye Cy5.5. The results are shown in Figure 2. Figure 17 As shown in AB, the brains of mice in the QAE group showed obvious red fluorescence signals. As time went on, the fluorescence intensity gradually increased and reached a peak at 8 hours, then began to decline. However, the fluorescence signal in the brains of mice in the EXO group was always weak. Subsequently, heart, liver, spleen, lung, kidney and brain tissues were taken for in vitro fluorescence imaging. Figure 17 As shown in Figures CD, more pronounced fluorescence was observed in the brains of mice in the QAE group compared to the EXO group, consistent with the in vivo imaging results. These results indicate that compared to EXO alone, loading with AK76 and Que enhanced drug accumulation in the mouse brain. This is likely due to the hydroxyl groups on the surface of AK76, which enhance the penetration of the nanoparticles through the nasal mucosa and their slow metabolism through the RES over time.

[0081] Example 15

[0082] All animal experiments were conducted in strict accordance with the standards of the Animal Protection Association. Male SPF-grade 8-10-week-old C57BL / 6 mice used in the experiment were purchased from Shanghai Slake Experimental Animal Center. In order to induce Parkinson's disease, mice were continuously injected with MPTP intraperitoneally at a dose of 30 mg / kg body weight for 5-7 consecutive days. The healthy mice in the control group were injected with PBS (the negative control group was recorded as the PBS group). The Parkinson's mice were randomly divided into 4 groups (6 mice in each group) and administered 20 μL PBS (the positive control group was recorded as the MPTP group), AK76, AE, QAE (Que=2 mg / kg) (10 μL each in the left and right nose) through nasal drops every day for seven consecutive days. After the treatment, animal behavioral training was carried out, mainly including fatigue rotarod and climbing pole test training. After 5 days of animal behavioral training, a series of behavioral tests including fatigue rotarod, grip strength, tail suspension test, forced swimming, climbing pole and open field test training were carried out. The results are as follows. In the grip strength test, as Figure 18 As shown in A, except for the PBS negative control group, the grip strength of mice in the QAE group recovered to the best level in all treatment groups. Figure 18 As shown in B, in the pole climbing test, the MPTP mice treated with QAE nanoparticles took the shortest time among all treatment groups except the normal mice treated with PBS. Figure 18 As shown in C and E, in the forced swimming test and tail suspension test, the floating time and immobility time of mice treated with QAE nanoparticles were significantly shorter than those in the MPTP group, and close to the level of the normal group. Figure 18 As shown in Figure D, in the fatigue rotarod test, MPTP mice treated with QAE could stay on the rotarod for a long time, about 136 seconds, which is close to the 168 seconds of normal mice. The above results show that the treatment of QAE nanoparticles can significantly improve the behavioral ability of MPTP-induced PD mice, significantly restore the mice's anti-fatigue ability and grip strength level, and improve their depressive behavior, showing a strong desire to struggle and survive in a desperate environment. Figure 19 As shown in A, in the open field test, it can be noticed that MPTP mice tend to stay at the corners and edges of the open field. In contrast, after QAE treatment, Parkinson's mice were able to explore the center of the open field, similar to the normal group. After QAE treatment, the exploration time of the central area of ​​MPTP mice was significantly prolonged and returned to the level close to that of the normal group. In addition, as shown in the quantitative results Figure 19 As shown in Figures BE, the total distance traveled, average speed, percentage of time spent in the central zone, and immobility in MPTP mice treated with QAE approached those of the normal control group, indicating that Parkinson's symptoms were significantly alleviated and successfully returned to normal levels. The mice's activity levels increased, and bradykinesia and anxiety were alleviated, suggesting that QAE nanoparticles are an effective treatment strategy for Parkinson's disease.

[0083] Example 16

[0084] To further verify the in vivo therapeutic effect of QAE on Parkinson's mice, the brain tissues of PD mice were observed using immunohistochemistry and immunofluorescence staining.

[0085] (1) Nissl staining was used to analyze the status of striatal neurons. Compared with the normal group, the MPTP group had significantly fewer Nissl bodies. After QAE treatment, striatal Nissl bodies increased significantly, approaching that of the normal group. This may be due to the enhanced intracellular delivery of QAE nanoparticles, which in turn exerted the better anti-inflammatory and antioxidant activities of Que and AK76, thereby enhancing the therapeutic effect on Parkinson's disease by protecting neurons from damage.

[0086] (2) The brains of each group of mice were sliced ​​and stained with glial fibrillary acidic protein (GFAP) / α-syn antibodies for immunofluorescence analysis. GFAP is a marker of astrocyte activation, and α-syn is closely related to the pathogenesis and related functional disorders of Parkinson's disease. Synuclein aggregation in pathological conditions can lead to neuronal cell death. Fluorescence results are as follows Figure 21 As shown in A, in contrast to the significant upregulation of GFAP and α-syn in the MPTP group, QAE treatment resulted in a significant downregulation of GFAP and α-syn, returning them to the normal group level. Quantitative analysis of α-syn expression showed that ( Figure 21 B), QAE can significantly inhibit the accumulation of α-syn in the brain and prevent further deterioration of Parkinson's disease.

[0087] (3) The brains of each group of mice were sliced ​​and stained with the ROS fluorescent probe DCFH-DA for immunofluorescence analysis. Figure 22 As shown in A, the MPTP group had the highest level of reactive oxygen species, while the QAE treatment group significantly reduced the level of reactive oxygen species, reducing it to the level of the normal group. This is because after nasal administration, QAE reaches the brain and releases Que, which greatly increases the accumulation of the drug in the brain and achieves an antioxidant effect. The quantitative results are shown in Figure 22 As shown in Figure 2B, the lowest ROS level was observed in the QAE group except for the negative control group, further demonstrating the antioxidant capacity of QAE.

[0088] (4) To examine the proportions of different types of microglia in the brain, immunofluorescence staining of CD206 and CD86 in the striatum was performed. Figure 23As shown in A, the expression of CD206 (green fluorescence), a marker of M2 microglia, decreased in the brains of the MPTP group, while the expression of CD86 (red fluorescence), a marker of M1, increased. After QAE treatment, a significant upregulation of CD206 expression and a downregulation of CD86 expression were observed in the mouse brains. Further quantification of the green / red fluorescence intensity ratio was performed as shown in Figure 23 As shown in B, the QAE-treated group had the highest CD206 / CD86 ratio among all groups, indicating that the proportion of M2 microglia was the highest, proving that QAE can promote the transformation of microglia in the brain of PD mice from the M1 pro-inflammatory phenotype to the M2 anti-inflammatory phenotype.

[0089] (5) The expression levels of ionized calcium binding adaptor molecule-1 (IBA-1, a marker of mouse brain microglial inflammation) and TH (a marker indicating dopamine synthesis capacity) in the mouse striatum were analyzed by immunofluorescence staining. Figure 24 As shown in A, QAE nanoparticle treatment downregulated the expression of IBA-1 and upregulated the expression of TH. The relative fluorescence intensity was quantitatively determined as Figure 24 As shown in Figures BC, MPTP-induced increases in IBA-1 expression and decreases in TH expression. Treatment with QAE reversed these changes, alleviating microglial inflammation and improving dopamine synthesis in the brain, thereby alleviating Parkinson's disease symptoms. These results suggest that MPTP protects brain neurons from inflammation and damage through its synergistic antioxidant and anti-inflammatory effects.

Claims

1. An exosome-encapsulated phosphorus-containing dendrimer nanocomposite, characterized by: The nanocomplex consists of exosomes derived from mesenchymal stem cells, hydroxyl-terminated phosphorus-containing dendrimers and quercetin.

2. A method for preparing an exosome-encapsulated phosphorus-containing dendrimer nanocomposite, comprising the following steps: (1) adding a hydroxyl-terminated phosphorus-containing dendrimer dissolved in methanol to an exosome solution dissolved in PBS, mixing, sonicating, incubating, and then centrifuging and purifying, collecting the supernatant to obtain an exosome-encapsulated hydroxyl-terminated phosphorus-containing dendrimer complex EXO-AK76; (2) The quercetin solution dissolved in methanol was added to the exosome-encapsulated hydroxyl-terminated phosphorus-containing dendrimer complex solution, mixed, sonicated, incubated, and then centrifuged for purification, and the supernatant was collected to obtain the exosome-encapsulated phosphorus-containing dendrimer nanocomplex EXO-AK76-Que.

3. The preparation method according to claim 2, wherein: The mass ratio of the exosomes to the hydroxyl-terminated phosphorus-containing dendrimers in step (1) is 1:

1.

4. The preparation method according to claim 2, wherein: The mass ratio of quercetin and the exosome-encapsulated hydroxyl-terminated phosphorus-containing dendrimer complex in step (2) is 1:

1.

5. The preparation method according to claim 2, wherein: The ultrasound in steps (1) and (2) is specifically as follows: ultrasound is performed for 3 minutes under 20% pulse conditions, and the cycle is repeated 6 times, with a 2-minute pause between each cycle.

6. The preparation method according to claim 2, wherein: The incubation in steps (1) and (2) is specifically: incubation at 37° C. for 30 minutes.

7. The preparation method according to claim 2, characterized in that: The centrifugal purification in steps (1) and (2) is specifically: centrifugal purification at 3000-5000 rpm for 8-10 minutes.

8. Use of the exosome-encapsulated phosphorus-containing dendrimer nanocomplex according to claim 1 in the preparation of anti-inflammatory, antioxidant, and anti-apoptotic drugs.

9. Use of the exosome-encapsulated phosphorus-containing dendrimer nanocomplex according to claim 1 in the preparation of a drug for treating Parkinson's disease.

Images