Preparation of brain-neuron-mitochondria step-by-step targeting nano drug delivery system as well as product and application thereof

Abstract

The invention discloses preparation of a brain-neuron-mitochondria step-by-step targeting nano drug delivery system as well as a product and application thereof. The RVG is successfully expressed on the cell exosome in a lentivirus infection mode, the engineered exosome is used as a delivery carrier, drug molecules can penetrate through a blood brain barrier and target neurons, meanwhile, the engineered exosome is precisely targeted to neuron mitochondria by modifying mitochondrial targeting polypeptide, and therefore the effect of treating the neuronal mitochondria is achieved. The engineered exosome loaded with the drug molecules releases curcumin, and the therapeutic effects of inhibiting alpha-Syn aggregation, improving neuroinflammation, relieving mitochondrial dysfunction and relieving oxidative stress are achieved. The brain-neuron-mitochondrion step-by-step targeting nano drug delivery system prepared by the invention can load drugs and is applied to treatment of neurodegenerative diseases, drug delivery is performed through caudal veins, the preparation process is simple, and the brain-neuron-mitochondrion step-by-step targeting nano drug delivery system has good biological safety and potential clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the preparation of a brain-neuron-mitochondrial stepwise targeted nanodrug delivery system, its products, and applications. Background Technology

[0002] With the aging population, neurodegenerative diseases have become a major social problem. These diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple sclerosis. As the disease progresses, neurons gradually lose structure or function, eventually dying. This progressive deformation and death of neurons affects cognitive and motor functions. Mitochondria are central to neuronal energy metabolism and play a crucial role in neurotransmitter and signal transmission. Abnormalities in their function (such as increased oxidative stress, decreased adenosine triphosphate (ATP) production, and autophagy-apoptosis imbalance) can induce cellular senescence. Research has found that when mitochondria fail to transmit signals normally along neuronal axons, symptoms of neurodegenerative diseases appear, indicating that mitochondrial dysfunction plays a central role in the development of these diseases.

[0003] Currently, neurodegenerative diseases are primarily treated with medication, but long-term use can cause side effects such as gastrointestinal reactions, behavioral fluctuations, and motor disorders. Furthermore, in the treatment of brain diseases, the blood-brain barrier (BBB) ​​is a major obstacle, blocking approximately 98% of small molecule drugs and all large molecule drugs from entering the brain, preventing drugs from specifically reaching the lesion area. In recent years, exosomes have received widespread attention in the field of drug delivery. Exosomes are sac-like structures formed by a lipid bilayer membrane secreted by cells, possessing low immunogenicity, good biocompatibility, and the natural ability to cross biological barriers (such as the BBB), making them a highly promising drug delivery vehicle for the treatment of brain diseases. However, only 0.5% of naturally occurring exosomes reach the brain, and their brain-targeting performance needs further improvement.

[0004] Furthermore, the pathological microenvironment, including oxidative stress, neuroinflammation, and characteristic protein aggregation, further contributes to neuronal loss and the deterioration of neurodegenerative diseases. Natural polyphenols (such as curcumin and gallate esters) have the effects of inhibiting neuroinflammation and improving oxidative stress, showing significant advantages in the treatment of neurodegenerative diseases. Curcumin, in particular, can bind to α-Syn proteins to form soluble, low-molecular-weight oligomers, thereby effectively inhibiting α-Syn aggregation; it also improves neuroinflammation, reduces mitochondrial dysfunction, and alleviates oxidative stress. However, curcumin suffers from poor water solubility, a short in vivo half-life (only 1-2 hours), low bioavailability, and insufficient brain targeting, which further limits its clinical application.

[0005] In conclusion, current drug delivery systems still require further improvement. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a brain-neuron-mitochondrial stepwise targeted nanodrug delivery system, its products, and applications.

[0007] In a first aspect, the present invention provides a method for preparing a brain-neuron-mitochondrial stepwise targeted nanomedicine delivery system, comprising the preparation of brain-neuron-mitochondrial engineered exosomes loaded with drugs, including the following steps:

[0008] Step (1): Construct a lentiviral overexpression vector for the EGFP-RVG-Lamp2b (Lamp2b, lysosome-associated membrane protein 2b) fusion protein and transfect it into cells;

[0009] Step (2): After lentivirus infection, change the culture medium to complete culture medium, continue culturing, add puromycin to screen cells until there are no apoptotic cells, and obtain stable cells expressing RVG.

[0010] Step (3): Culture stable cells with exosome-free serum, collect cell culture supernatant, collect exosomes by differential centrifugation, resuspend exosomes in PBS to obtain engineered exosomes;

[0011] Step (4): DSPE-PEG 2000 -NHS (distearate phosphatidylethanolamine-polyethylene glycol 2000-N-hydroxysuccinimide) and mitochondrial-targeting peptide SS31 were reacted with stirring at room temperature in the dark. Unreacted SS31 was removed by dialysis to obtain DSPE-PEG. 2000 -SS31;

[0012] Step (5): Combine the above-mentioned engineered exosomes with DSPE-PEG 2000 -SS31 target chain was incubated with ultrasound to obtain engineered exosomes targeting the brain-neuron-mitochondria;

[0013] Step (6): Dissolve the drug and mPEG (polyethylene glycol) separately in tetrahydrofuran, mix and sonicate, and quickly drop into ultrapure water while stirring at room temperature to obtain the drug self-assembly.

[0014] Step (7): The above-mentioned brain-neuron-mitochondrial targeted engineered exosomes and drug self-assemblies are ultrasonically incubated to obtain the brain-neuron-mitochondrial stepwise targeted nano-drug delivery system.

[0015] Preferably, in step (1), the cells transfected by the lentiviral overexpression vector are human embryonic kidney cells (HEK-293T), human neuroblastoma cells (SH-SY5Y), or microglia (BV2).

[0016] Preferably, in step (1), the multiplicity of infection (MOI) is 1:5-100.

[0017] Preferably, the lentivirus infection time in step (2) is 16 h.

[0018] Preferably, in step (2), the complete culture medium is DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0019] Preferably, the differential centrifugation method in step (3) is as follows: centrifuge at 500 g for 15 min to remove suspended cells and dead cells, centrifuge at 2000 g for 15 min to remove cell debris, centrifuge at 10000 g for 30 min to remove large microbubbles, collect the supernatant, filter it through a 0.22 μm filter, and then centrifuge at 100,000 g for 2 h.

[0020] Preferably, in step (4) DSPE-PEG 2000 The molar ratio of NHS to SS31 is 0.5-2:1.

[0021] Preferably, in step (5), the exosomes and DSPE-PEG 2000 The mass ratio of SS31 is 0.05-0.2:1.

[0022] Preferably, the volume ratio of the drug to mPEG in step (6) is 0.5-2:1.

[0023] Preferably, in step (7), the mass ratio of engineered exosomes to drug self-assemblies is 0.05-2:1.

[0024] Preferably, the drug molecule is curcumin.

[0025] Secondly, the present invention provides a brain-neuron-mitochondrial stepwise targeted nanodrug delivery system, which is prepared by the above method and includes brain-neuron-mitochondrial engineered exosomes loaded with drugs; wherein the brain-neuron-mitochondrial engineered exosomes serve as carriers to encapsulate and load drug nanoparticles, and their membrane surfaces overexpress rabies virus glycoprotein RVG targeting peptides and are modified with mitochondrial targeting peptide SS31.

[0026] Thirdly, the present invention provides the application of the above-mentioned brain-neuron-mitochondrial stepwise targeted nanomedicine delivery system in the preparation of products for treating neurodegenerative diseases.

[0027] Preferably, the neurodegenerative disease is Parkinson's disease.

[0028] Preferably, the administration method is intravenous administration.

[0029] The beneficial effects of this invention are as follows:

[0030] This invention constructs a brain-neuron-mitochondrial hierarchical targeted nanodelivery system. RVG is successfully expressed on exosomes via lentiviral infection. These engineered exosomes serve as delivery carriers, allowing drug molecules to penetrate the blood-brain barrier and target neurons. Simultaneously, by modifying mitochondrial-targeting peptides, the engineered exosomes are precisely targeted to neuronal mitochondria. The engineered exosomes carrying drug molecules release curcumin, exerting therapeutic effects such as inhibiting α-Syn aggregation, improving neuroinflammation, reducing mitochondrial dysfunction, and alleviating oxidative stress. The brain-neuron-mitochondrial hierarchical targeted nanodelivery system prepared in this invention can load curcumin and be applied to the treatment of neurodegenerative diseases. Administered via tail vein, the preparation process is simple and exhibits good biosafety, demonstrating potential clinical application value. Attached Figure Description

[0031] Figure 1 This is a graph showing the transfection efficiency of the EGFP-RVG-Lamp2b and EGFP-blank-Lamp2b lentiviral systems in different cell types. In the graph, Control: untransfected cells; EGFP-blank-Lamp2b: cells transfected with EGFP-blank-Lamp2b virus; EGFP-RVG-Lamp2b: cells transfected with EGFP-RVG-Lamp2b virus.

[0032] Figure 2 This is the result of Western blot analysis verifying the expression of characteristic proteins of engineered exosomes; (A) transmission electron microscopy image of engineered exosomes; (B) particle size distribution map; (C) particle size and potential map; (D) marker protein expression status. The figures are labeled as follows: 293T-blank-Exo: engineered exosomes of RVG from HEK-293T cells that do not express RVG; 293T-RVG-Exo: engineered exosomes of RVG from HEK-293T cells that express RVG; SH-SY5Y-blank-Exo: engineered exosomes of RVG from SH-SY5Y cells that do not express RVG; SH-SY5Y-RVG-Exo: engineered exosomes of RVG from SH-SY5Y cells that express RVG; BV2-blank-Exo: engineered exosomes of RVG from BV2 cells that do not express RVG; BV2-RVG-Exo: engineered exosomes of RVG from BV2 cells that express RVG.

[0033] Figure 3 These are graphs showing the cellular uptake and quantitative analysis of engineered exosomes, where (A) shows the cellular uptake and (B) shows the quantitative analysis.

[0034] Figure 4This is an in vivo fluorescence distribution map of mice after tail vein injection of engineered exosomes at different time points.

[0035] Figure 5 The results of morphological characterization of the brain-neuron-mitochondrial stepwise targeted drug delivery system SS31-Exo-RVG / CurNP prepared in Example 2 using transmission electron microscopy, particle size analyzer, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are shown. (A) Transmission electron microscopy images of Exo-RVG, CurNP, and Exo-RVG / CurNP; (B) Fluorescence colocalization map of curcumin and engineered exosomes; (C) SDS-PAGE images of different drug delivery systems; (D) Particle size, potential, and polydispersity index of the drug delivery system. In the figure, the labels are as follows: CurNP: curcumin self-assembly; Exo-RVG: RVG-expressing engineered exosomes; Exo-RVG / CurNP: RVG-expressing engineered exosomes carrying curcumin self-assembly; Exo-blank: RVG-unexpressed engineered exosomes; Exo-blank / CurNP: RVG-unexpressed engineered exosomes carrying curcumin self-assembly; SS31-Exo-blank / CurNP: mitochondrial-targeted modified RVG-unexpressed engineered exosomes carrying curcumin self-assembly; SS31-Exo-RVG / CurNP: mitochondrial-targeted modified RVG-expressing engineered exosomes carrying curcumin self-assembly.

[0036] Figure 6 This describes the uptake of the nanomedicine delivery system in bEnd.3 cells and its co-localization with mitochondria.

[0037] Figure 7 This describes the uptake and co-localization of the nanomedicine delivery system with mitochondria in SH-SY5Y cells.

[0038] Figure 8 The in vivo fluorescence imaging system was used to observe the fluorescence distribution in small animals in real time after injecting an equal amount of DiR-labeled stepwise targeted drug delivery system; (A) In vivo fluorescence distribution map of mice after tail vein injection of the drug delivery system at different times; (B) Ex vivo fluorescence distribution map of different drug delivery systems in different tissues of mice; (C) Average fluorescence intensity of different drug delivery systems in the brain of mice.

[0039] Figure 9The study evaluated the efficacy of a drug delivery system in mice after injection of different drugs using rotarod, water maze, and pole climbing tests. The results included: (A) a schematic diagram of the construction, treatment, and behavioral observation of a Parkinson's disease mouse model; (B) the descent latency of mice in the rotarod test; (C) the fall speed of mice in the rotarod test; (D) the turning time of mice in the pole climbing test; (E) the total time of mice in the pole climbing test; (F) a typical path diagram of mice in the water maze test; (G) the time mice spent in the target quadrant in the water maze test; (H) the number of times mice traversed the water maze test; and (I) the escape latency of mice in the water maze test. Detailed Implementation

[0040] To make the technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0041] As previously stated, this invention provides a brain-neuron-mitochondrial stepwise targeted nanodelivery system, comprising brain-neuron-mitochondrial engineered exosomes loaded with a drug, wherein the engineered exosome membrane surface overexpresses an RVG-targeting peptide and is modified with a mitochondrial-targeting peptide SS31, and the drug is curcumin; the brain-neuron-mitochondrial stepwise targeted nanodelivery system is prepared using the following method:

[0042] Step (1): Construct a lentiviral overexpression vector for the EGFP-RVG-Lamp2b fusion protein, infect multiple MOIs with a virus ratio of 1:5-100, and transfect them into human embryonic kidney cells (HEK-293T), human neuroblastoma cells (SH-SY5Y), and microglia (BV2), respectively.

[0043] Step (2): After 16 hours of lentivirus infection, replace the culture medium with complete medium and continue culturing. Observe the infection efficiency of the EGFP (enhanced green fluorescent protein) tag using an inverted fluorescence microscope. Add puromycin to select cells for 1 week until no apoptotic cells are found.

[0044] Step (3): Culture stable cells with exosome-free serum, collect cell culture supernatant, and collect engineered exosomes by differential centrifugation;

[0045] Step (4): Dissolve curcumin and mPEG (polyethylene glycol) in tetrahydrofuran respectively, mix them at a volume ratio of 0.5-2:1 and sonicate. Then quickly drop them into ultrapure water and stir at room temperature to obtain curcumin self-assembly (CurNP).

[0046] Step (5): The engineered exosomes and curcumin self-assemblies are ultrasonically incubated at a mass ratio of 0.05-2:1 to obtain the engineered exosome nanodelivery system.

[0047] Step (6): DSPE-PEG 2000 -NHS and mitochondrial targeting peptide SS31 were reacted at a molar ratio of 0.5-2:1 at room temperature in the dark with stirring, and unreacted SS31 was removed by dialysis.

[0048] Step (7): The above-mentioned engineered exosome nanodelivery system and the DSPE-PEG-SS31 target chain are ultrasonically incubated at a mass ratio of 0.05-0.2:1 to obtain a brain-neuron-mitochondrial stepwise targeted nanodelivery system.

[0049] In the preparation method provided by this invention, the proportions of each raw material can be selected according to actual needs. For example, in some embodiments, the multiplicity of infection (MOI) can be selected from 1:5, 1:20, 1:50, 1:100, or other values ​​within the range, which are not limited here; the volume ratio of curcumin and mPEG can be selected from 0.5:1, 1:1, 1.5:1, 2:1, or other values ​​within the range, which are not limited here; DSPE-PEG 2000 The molar ratio of NHS to SS31 can be selected from 0.5:1, 1:1, 2:1, or other values ​​within the range, and is not limited here; engineered exosomes and DSPE-PEG 2000 The mass ratio of -SS31 can be selected from 0.05:1, 0.1:1, 0.2:1, or other values ​​within the range. It can be selected according to actual needs and is not limited here.

[0050] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0051] Example 1:

[0052] This embodiment prepared an engineered exosome, and the specific process is as follows:

[0053] S1: Construction of stable transgenic lines expressing RVG: Human embryonic kidney cells (HEK-293T), human neuroblastoma cells (SH-SY5Y), and microglia (BV2) were constructed. Cells were seeded 24 h before transfection into 6-well plates. When the cells reached approximately 60% confluency, transfection was performed. The EGFP-RVG-Lamp2b lentiviral overexpression vector, synthesized by a biotechnology company, was transfected into the three cell types (HEK-293T, SH-SY5Y, and BV2) at MOI ratios of 1:5, 1:50, and 1:100, respectively. DMEM medium containing 1% penicillin-streptomycin was added to the culture volume, and the cells were incubated at 37 °C. Sixteen hours after infection, the virus-containing culture medium was discarded and replaced with fresh DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The medium was then incubated at 37 °C for another 48 hours. EGFP fluorescence expression was observed using an inverted fluorescence microscope. The EGFP-blank-Lamp2b lentivirus system was used as a control.

[0054] S2: Collection of exosomes from HEK293T, SH-SY5Y, and BV2 cells expressing RVG: HEK293T, SH-SY5Y, and BV2 cells transfected in step S1 were cultured in DMEM medium prepared with 10% exosome-free fetal bovine serum and 1% penicillin-streptomycin. After 48 h, the cell supernatant was collected and centrifuged differentially. Specifically, centrifugation was performed at 500 g for 15 min to remove suspended and dead cells, at 2000 g for 15 min to remove cell debris, and at 10000 g for 30 min to remove large microvesicles. The supernatant was collected, filtered through a 0.22 μm filter, and then centrifuged at 100,000 g for 2 h. The exosomes were resuspended in PBS and stored at -80 ℃ for later use.

[0055] Example 2:

[0056] This embodiment prepares a brain-neuron-mitochondrial hierarchical targeted nanodelivery system, the specific process of which is as follows:

[0057] S1: Preparation of curcumin self-assemblies: Weigh 1 mg of curcumin and 30 mg of mPEG, dissolve them separately in 1 mL of tetrahydrofuran, and mix them at a 1:1 volume ratio using sonication for 5 min. Quickly add the mixture dropwise to 3 mL of ultrapure water and stir at room temperature for 10 min. Then transfer the resulting reactants to a dialysis bag with a molecular weight of 3500 Da and dialyze them in ultrapure water for 24 h to remove organic solvents and free drug, thus obtaining the curcumin self-assemblies.

[0058] S2: Preparation of engineered exosome nanodelivery system: The engineered exosome solution prepared in Example 1 was mixed with curcumin self-assembly at a mass ratio of 1:10, and incubated at 4 ℃ for 10 min and 37 ℃ for 1 h, respectively, to obtain engineered exosomes loaded with curcumin self-assembly.

[0059] S3: Preparation of a brain-neuron-mitochondrial hierarchical targeted nanodelivery system: DSPE-PEG 2000 -NHS and SS31 were dissolved in N,N-dimethylformamide at a molar ratio of 0.5:1 and reacted at room temperature in the dark for 12 h. The solution was then transferred to a dialysis bag with a molecular weight of 2000 Da and dialyzed in ultrapure water for 48 h to obtain DSPE-PEG. 2000 -SS31. The resulting DSPE-PEG 2000 - SS31 was mixed with the engineered exosome nanodelivery system synthesized in step S2 above at a mass ratio of 0.2:1 and incubated at 37 °C for 1 h to obtain a brain-neuron-mitochondrial stepwise targeted drug delivery system (SS31--Exo-RVG / CurNP).

[0060] Test Example 1: Evaluation of Transfection Efficiency of Stable Transfectants

[0061] Following the method described in Example 1, the EGFP-RVG-Lamp2b and EGFP-blank-Lamp2b lentiviral systems were transfected into HEK293T, SH-SY5Y, and BV2 cells. The expression of EGFP fluorescence was observed using an inverted fluorescence microscope. The results are shown in [Figure 1]. Figure 1 (A) Figure 1 (B) From Figure 1 As can be seen in (B), obvious fluorescent expression can be observed in all three stable cell types, indicating the successful expression of EGFP-RVG-Lamp2b and EGFP-blank-Lamp2b, which can be used for the collection of engineered exosomes.

[0062] Test Example 2: Structural Characterization of Engineered Exosomes

[0063] 10 μL of the engineered exosome material from Example 1 was dropped onto a copper grid. After 10 min, it was blotted dry, negatively stained with phosphotungstic acid, blotted dry again, and air-dried. Its morphology was observed using a transmission electron microscope. The particle size and potential of the engineered exosomes were analyzed using a particle size analyzer. The expression of characteristic proteins of the exosomes was verified by Western blot experiments. The results are as follows: Figure 2 (A) Figure 2As shown in (D), the six exosomes (293T-blank-Exo, 293T-RVG-Exo, SH-SY5Y-blank-Exo, SH-SY5Y-RVG-Exo, BV2-blank-Exo, and BV2-RVG-Exo) all exhibited a distinct saucer-like structure, with particle sizes ranging from approximately 100 to 150 nm and potentials from -15 to -30 mV. Western blot experiments confirmed that the six exosomes successfully expressed exosome-related proteins, thus verifying the successful extraction of exosomes.

[0064] Test Example 3: Evaluation of the Targeting Performance of Engineered Exosomes

[0065] In this embodiment, engineered exosomes are labeled using lipophilic membrane dyes DiI or DiR.

[0066] Mouse brain microvascular endothelial cells (bEnd.3) and SH-SY5Y cells were used at a concentration of 1×10⁻⁶. 5 Seeds were placed in confocal dishes at 1 / 2 well and incubated overnight. 1-Methyl-4-phenylpyridine (MPP) was then used as the inoculum. + SH-SY5Y cells were treated as a model of neurodegenerative diseases using SH-SY5Y-MPP. + An equal volume of DiI-labeled engineered exosomes was added and incubated at 37 °C for 4 h. After removing the liquid, the cells were washed three times with PBS, fixed with paraformaldehyde, and then stained with 4',6-diamidinyl-2-phenylindole (DAPI) to stain the nuclei. The fluorescence uptake of each group of exosomes was observed and imaged using a confocal microscope. The results are as follows: Figure 3 (A) Figure 3 As shown in (B). RVG-Exo engineered exosomes (SH-SY5Y-RVG-Exo) derived from SH-SY5Y cells were used in three cell lines (bEnd.3, SH-SY5Y, SH-SY5Y-MPP). + Both exhibit strong fluorescence.

[0067] An equal volume of DiR-labeled engineered exosomes was injected into C57BL / 6 mice via the tail vein. The fluorescence distribution within the mice was observed in real-time using a small animal in vivo fluorescence imaging system. The results are as follows: Figure 4 As shown, SH-SY5Y-RVG-Exo exhibits strong fluorescence in the brain.

[0068] The above results indicate that SH-SY5Y-RVG-Exo can be well taken up by brain microvascular endothelial cells and neurons, and can serve as a brain-neuron targeted delivery carrier.

[0069] Test Example 4: Characterization of the Brain-Neuron-Mitochondria Stepwise Targeted Drug Delivery System

[0070] A brain-neuron-mitochondrial stepwise targeted drug delivery system (SS31-Exo-RVG / CurNP) was prepared according to the method described in Example 2. The morphology of the drug delivery system was characterized by transmission electron microscopy, particle size analyzer, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results are as follows: Figure 5 (A) Figure 5 As shown in (D), the prepared curcumin self-assemblies are spherical structures with uniform morphology and a particle size of approximately 120 nm. DiI-labeled exosomes showed a clear overlap in fluorescence with the curcumin self-assemblies (CurNP). SDS-PAGE electrophoresis results showed that after coating with engineered exosomes, SS31-Exo-RVG / CurNP exhibited a distinct protein band compared to the CurNP group, confirming the successful coating of exosomes.

[0071] Test Example 5: Evaluation of the stepwise targeting performance of the brain-neuron-mitochondrial stepwise targeted drug delivery system

[0072] bEnd.3 and SH-SY5Y cells were fed at a rate of 1×10⁻⁶. 5 Cells were seeded per well in confocal microscopy dishes and cultured overnight. An equal volume of DiI-labeled nanoparticle drug delivery system was added, and the mixture was incubated at 37 °C for 4 h. After removing the liquid, the cells were washed three times with PBS, fixed with paraformaldehyde, and stained with DAPI on the nuclei. The fluorescence uptake of each drug delivery system was observed and imaged using a confocal microscope. The results are shown below. Figures 6-7 As shown, the SS31-Exo-RVG / CurNP group exhibited the most significant cellular uptake and mitochondrial co-localization, indicating that the targeting modification of RVG and SS31 can significantly improve the brain-neuron-mitochondrial targeting efficiency of the nanodelivery system.

[0073] An equal volume of DiR-labeled stepwise targeted drug delivery system was injected into C57BL / 6 mice via the tail vein. The fluorescence distribution within the mice was observed in real-time using a small animal in vivo fluorescence imaging system. The results are as follows: Figure 8 (A) Figure 8 As shown in (C), the fluorescence intensity of SS31-Exo-RVG / CurNP in the brain significantly increased over time, demonstrating good brain targeting effects.

[0074] Test Example 6: Evaluation of the therapeutic effect of the brain-neuron-mitochondrial stepwise targeted drug delivery system in mice with neurodegenerative diseases

[0075] Parkinson's disease mice were used as a model of neurodegenerative disease. Mice were intraperitoneally injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 10 consecutive days. After modeling, saline, levodopa (L-DOPA), CurNP, Exo-RVG / CurNP, and SS31-Exo-RVG / CurNP were administered via tail vein injection, once daily for 7 days. The efficacy of the drug delivery system was evaluated using the rotarod test, water maze test, and pole climbing test. The results are as follows: Figure 9 (A) Figure 9 As shown in (G). Compared with the MPTP model group, the SS31-Exo-RVG / CurNP drug administration group showed significantly improved motor ability, indicating that it has a good anti-Parkinson's disease effect.

[0076] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. A method for preparing a brain-neuron-mitochondrial stepwise targeted nanomedicine delivery system, characterized in that, This includes the preparation of brain-neuron-mitochondrial engineered exosome-loaded drugs; The preparation of the brain-neuron-mitochondrial engineered exosome-loaded drug includes: A lentiviral overexpression vector for the EGFP-RVG-Lamp2b fusion protein was constructed and transfected into cells. After lentiviral infection, the culture medium was replaced with complete culture medium and cultured for a longer period. Puromycin was added to select cells until no apoptotic cells were obtained, thus obtaining stable transfected cells. Stable cells were cultured with exosome-free serum, cell culture supernatant was collected, exosomes were collected by differential centrifugation, and exosomes were resuspended in PBS to obtain engineered exosomes; DSPE-PEG 2000 -NHS and mitochondrial targeting peptide SS31 were reacted at room temperature with stirring, and unreacted mitochondrial targeting peptide SS31 was removed by dialysis to obtain DSPE-PEG. 2000 -SS31; The above-mentioned engineered exosomes were combined with DSPE-PEG 2000 -SS31 target chain was incubated with ultrasound to obtain engineered exosomes targeting the brain-neuron-mitochondria; The drug and polyethylene glycol were dissolved separately in tetrahydrofuran, mixed and sonicated, and then rapidly dropped into ultrapure water and stirred at room temperature to obtain the drug self-assembly. The engineered exosomes targeting the brain-neuron-mitochondria were incubated with the drug self-assembly by ultrasound to obtain a brain-neuron-mitochondria stepwise targeted nanodelivery system.

2. The method according to claim 1, characterized in that, Cells transfected with lentiviral overexpression vectors are human embryonic kidney cells, human neuroblastoma cells, or microglia.

3. The method according to claim 1, characterized in that, The DSPE-PEG 2000 The molar ratio of -NHS to mitochondrial targeting peptide SS31 is 0.5-2:

1.

4. The method according to claim 1, characterized in that, The exosomes and DSPE-PEG 2000 The mass ratio of the SS31 target chain is 0.05-0.2:

1.

5. The method according to claim 1, characterized in that, The volume ratio of curcumin to polyethylene glycol is 0.5-2:

1.

6. The method according to claim 1, characterized in that, The mass ratio of the engineered exosomes to the curcumin self-assembly is 0.05-2:

1.

7. The method according to claim 1, characterized in that, The drug molecule is curcumin.

8. A brain-neuron-mitochondrial stepwise targeted nanomedicine delivery system, prepared by the method according to any one of claims 1-7, characterized in that, The invention includes brain-neuron-mitochondrial engineered exosomes loaded with drugs; wherein the brain-neuron-mitochondrial engineered exosomes serve as carriers to encapsulate and load drug nanoparticles, and their membrane surfaces overexpress RVG-targeting peptides and are modified with mitochondrial-targeting peptide SS31.

9. The application of the brain-neuron-mitochondrial stepwise targeted nanomedicine delivery system of claim 8 in the preparation of products for neurodegenerative diseases.

10. The application according to claim 9, characterized in that, The neurodegenerative disease mentioned is Parkinson's disease.

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