Nanometer delivery system and application thereof

Abstract

The invention belongs to the technical field of cerebral apoplexy treatment medicine research, and particularly relates to a nano delivery system and application thereof. The nano delivery system is prepared according to the following steps: modifying cationic starch spheres with mitochondrial targeting peptide, loading PDK4-IN-1, and carrying out self-assembly to obtain an inner core; the preparation method comprises the following steps: modifying aminated anionic starch with homing peptide, and loading carfilzomib to form a starch spiral shell; and mixing the inner core with the starch spiral shell, and self-assembling to form the nano drug delivery system. According to the invention, a three-targeting, double-controlled-release and double-drug nano drug delivery system is constructed and is accurately delivered to a matrix action site in cytoplasm or mitochondria, so that double-channel synergistic enhancement of IS mitochondrial autophagy and toxicity attenuation and synergistic treatment of IS are realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of stroke treatment drugs, and particularly relates to a nano delivery system and application thereof. BACKGROUND

[0002] Stroke is a major refractory disease with high mortality and high disability rate. It is caused by the rupture of blood vessels supplying blood to the brain or occlusion, resulting in cerebral ischemia and hypoxia, and can be divided into hemorrhagic and ischemic (Ischemic stroke, IS). Globally, there are about 50 million new strokes each year, of which 5.5 million people die and 5 million people are disabled, of which IS accounts for 87%. The IS treatment guidelines require rapid recanalization to restore blood flow to the ischemic brain region. Intravenous thrombolysis using tissue plasminogen activator within 4.5 h after the onset of symptoms, mechanical thrombectomy for large vessel occlusion treatment methods can extend the effective treatment window to 24 h. Due to the short treatment time window and many contraindications, although the success rate of IS recanalization treatment is as high as 80%, only 33.4% of IS survivors have good functional prognosis, and as high as 50% of recanalization patients die or are disabled. The reason for the poor prognosis of IS is that existing treatments focus on recanalization and do not consider neuroprotection, especially neuroprotection before recanalization can significantly extend the effective treatment time window, reduce neural damage and promote neural function recovery. In addition, as high as 25% of elderly patients have clinically unrecognized occult IS, which significantly increases cognitive decline, overt IS, and all-cause mortality. Therefore, based on the new mechanism of IS, exploring new strategies for neuroprotective IS treatment is an important clinical problem that needs to be solved and has broad application prospects.

[0003] Mitochondrial mass of neurons is an important factor determining the fate of neurons after IS and neural function. Mitophagy is a process by which cells clear redundant or damaged mitochondria through the autophagy-lysosome pathway, and is also the main way for cells to control mitochondrial mass at the terminal stage of neuronal differentiation. There is controversy about the damage and protection of mitochondrial autophagy after IS, but most reports support the protective effect of mitochondrial autophagy. In 2023 Autophagy It was reported that mitochondrial autophagy was weakened in IS animal models, and the Pink1 / Parkin pathway of microglial cell mitochondrial autophagy was activated, which reduced the inflammatory response and restored the open field motor function and reduced the cerebral infarction area. Upregulating the expression level of Pink1 / Parkin in IS models triggers mitochondrial autophagy, which can clear damaged mitochondria, reduce ROS production and pro-apoptotic factor release, inhibit secondary neural damage, maintain cellular energy homeostasis, and promote neuronal recovery. In 2023 Sci RepTranscriptome sequencing of IS tissue and normal brain tissue found that BNIP3L was significantly down-regulated in IS tissue. Down-regulation of mitochondrial autophagy is a key mechanism for the exacerbation of IS neuronal damage, and restoring neuronal mitochondrial autophagy is expected to treat IS. The main pathways of IS mitochondrial autophagy are Pink1 / Parkin and BNIP3L / NIX. The combination of drugs in different pathways is one of the concepts of precision medicine, which has been widely used in the clinical diagnosis and treatment of complex diseases such as cardiovascular disease, diabetes, cancer, and nervous system disease. However, due to toxicity and drug resistance, the clinical effect is not satisfactory. Drug delivery systems can promote the administration of synergistic drug combinations by adjusting drug pharmacokinetics, stability, absorption, and targeting. For IS mitochondrial autophagy Pink1 / Parkin and BNIP3L / NIX key pathways, it is urgent to solve the dilemma of reducing toxicity and improving efficiency in treating IS: ①Screening of double-drug combination to synergistically enhance the two main pathways of mitochondrial autophagy; ②Due to drug hydrophobicity, different action sites and brain structure barriers, intravenous administration of double drugs is difficult to achieve synergistic treatment of IS, and drug delivery systems are needed to improve hydrophilicity and precisely target brain / neuron / mitochondria action sites.

[0004] Pink1 / Parkin pathway is the most classic pathway of mitochondrial autophagy. When the mitochondria are damaged, Pink1 migrates from the mitochondrial matrix to the outer membrane of the mitochondria. Pink1 gradually accumulates and phosphorylates to activate Parkin phosphorylation. Parkin mediates mitochondrial membrane protein ubiquitination, which is recognized and combined with LC3 by P62 and other receptor proteins, recruits lysosomes, and triggers mitochondrial autophagy to remove damaged mitochondria. BNIP3L is a member of the Bcl-2 family, located on the outer membrane of mitochondria. BNIP3L / NIX dimers can recognize and combine with LC3, recruit lysosomes, and activate mitochondrial autophagy; this process is regulated by proteasomes, ubiquitinated BNIP3L is degraded by proteasomes, inhibiting mitochondrial autophagy function and leading to accumulation of damaged mitochondria. Currently, experimental mitochondrial autophagy activators are too toxic to be used as screening drugs. For example, carbon cyanide chloride CCCP and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone FCCP can cause all mitochondria in cells to depolarize rapidly and mitochondrial autophagy to occur, damage the cytoskeleton, and inhibit lysosome acidification. Valinomycin treatment induces Pink1 / Parkin-dependent apoptosis. Antimycin A is also a mitochondrial respiration inhibitor that binds to the mitochondrial electron transport system, severely hindering electron transfer from CoQ to cytochrome C. Chem Biol Interact It was reported that metformin induced Pink1 / Parkin mitochondrial autophagy activation, improved mitochondrial morphology and function, reduced brain infarction volume and neuronal apoptosis in high glucose ischemia-reperfusion models, and improved neurological function. However, metformin is more suitable for high glucose reperfusion injury and is not suitable for IS treatment. NPJ Regen MedThe report will activate IS rat neuron mitochondrial autophagy by intranasal administration of Mn / Co3O4 nano therapeutic agent, effectively remove ROS and damaged mitochondria, improve cognitive function and reduce cerebral infarction area. However, due to the abundance of oxidants in the blood, metal reductase is not suitable for intravenous preparation. Ligustilide is a natural compound extracted from Chuanxiong and Angelica sinensis. It promotes mitochondrial autophagy through Pink1 / Parkin, thereby improving IS neuron damage. Traditional Chinese medicine extract intervention pathway is complex and is not suitable as a single pathway example drug.

[0005] Pyruvate dehydrogenase kinase (PDK) inhibits pyruvate dehydrogenase complex (PDH), thereby preventing mitochondrial oxidative metabolism of pyruvate. Normal mitochondria maintain mitochondrial autophagy balance through PDK2-mediated PARL activation and Pink1 rapid degradation. In IS, PDK4 mediates the Pink / Parkin pathway of mitochondrial autophagy. Although pyruvate levels can stimulate the mitochondrial autophagy pathway, the activity of PDK4 is affected by various factors such as ATP, NADH, and acetyl coenzyme A. PDK4 senses mitochondrial dysfunction according to the cell state and regulates mitochondrial autophagy. In 2022 Aging Cell It is also demonstrated that PDK4 is significantly higher in ischemic cardiomyocytes and myocardial cells of old mice than in normal mice, and PDK4-IN-1 treatment increases glucose oxidation dependence in young myocardial cells but not in old myocardial cells. Young hearts are more effective in dealing with ischemic injury stress than old hearts. In 2023 Nat Metab It is reported that PDK4-IN-1 intraperitoneal injection of 10 mg / kg for 3 consecutive days has good safety, reduces the severity of DNA damage in senescent cells, and limits the development of senescence-associated secretory phenotype. The combination of PDK4-IN-1 with anti-tumor drugs reduces the tumor volume by 75.1% on the whole.

[0006] In 2017 Autophagy It is reported that cerebral ischemia-reperfusion induces mitochondrial autophagy through BNIP3L / NIX, and BNIP3L knockout inhibits mitochondrial autophagy and aggravates cerebral ischemia-reperfusion injury. Overexpression of BNIP3L can promote mitochondrial autophagy through Park2 to treat nerve damage. In 2020 Autophagy It is reported that long-term ischemia in IS causes degradation of BNIP3L by proteasome, resulting in loss of mitochondrial autophagy. The proteasome inhibitor CFZ blocks BNIP3L degradation, restores mitochondrial autophagy, significantly reduces cerebral infarction volume and neurological deficit scores, and effectively extends the therapeutic time window to 3 h after IS. CFZ alone has many side effects, and in 2021Exp Physiol It is reported that CFZ combined with MAPK inhibitors for tumor treatment can achieve attenuation and synergistic effect.

[0007] Therefore, PDK4-IN-1 and CFZ are expected to be screened as example drugs for enhancing mitochondrial autophagy main pathway in combination, but intracerebral injection is an invasive operation, which can cause complications such as intracranial infection and pain. Due to hydrophobicity, different action sites and brain structure obstacles, it is difficult for intravenous injection of the two drugs to simultaneously and efficiently pass through the blood brain barrier (BBB) to reach the action site.

[0008] The PDK4-IN-1 drug delivery system needs to have brain targeting / neuron / mitochondria targeting function, deliver PDK4-IN-1 into the mitochondrial matrix, inhibit PDK4, enhance the Pink1 / Parkin pathway of mitochondrial autophagy, and protect the brain from ischemic injury, but its pharmaceutical properties make it difficult to efficiently reach the neuron mitochondrial matrix: ① Poor water solubility: PDK4-IN-1 is highly hydrophobic and difficult to dissolve in water, and dimethyl sulfoxide (DMSO) is often used as a solvent for dissolution. The solubility of DMSO is 125 mg / mL, and it is often administered orally or intraperitoneally. Intravenous injection embolization is risky, suggesting that PDK4-IN-1 needs to improve water solubility through a carrier. ② Low brain drug ratio: oral PDK4-IN-1 10 mg / kg reaches a peak blood drug concentration of 0.565 ± 0.208 μg / mL at 6 h, showing good bioavailability (64%), long half-life (>7 h), and moderate clearance rate (0.69) in rats. PDK4-IN-1 has anti-diabetic, anti-cancer and anti-allergic activity, and is commonly enriched in the liver and kidney, but not in the brain, suggesting that PDK4-IN-1 needs to be brain-targeted to improve brain drug ratio. ③ Action site in mitochondrial matrix: PDK4 is located in the mitochondrial matrix, where it inhibits the PDH complex by phosphorylating the E1α subunit of PDH, thereby regulating glucose metabolism. The action site of PDK4-IN-1, a specific inhibitor of PDK4, is in the mitochondrial matrix, suggesting that PDK4-IN-1 needs a carrier for brain, neuron, and mitochondrial targeting. ④ Large side effects: As a new drug developed in 2019, there are few reports on side effects. The side effects related to excessive inhibition of PDK4 can be used as a reference to avoid high-dose use. ⑤ Existing improved preparation: PDK4-IN-1 has the same biological activity as free PDK4-IN-1 at the same molar concentration, but salted PDK4-IN-1 usually has better water solubility and stability. There is currently no report on IS-targeted therapeutic carriers.

[0009] The CFZ delivery system needs to have brain targeting / neuron targeting function to deliver CFZ into the cytoplasm of neurons, inhibit proteasome degradation of BNIP3L, enhance BNIP3L / NIX pathway of mitochondrial autophagy to protect the brain from ischemic injury. However, its pharmaceutical properties make it difficult to efficiently reach the cytoplasm of neurons: ① Poor water solubility: CFZ is highly hydrophobic and difficult to dissolve in water, but it can be dissolved in DMSO at 80 mg / mL and ethanol at 25 mg / mL. The clinical name of the commercially available product is Kelos. The instructions indicate that CFZ injection uses sulfobutyl betadex sodium, citric acid, and sodium hydroxide to help solubility. Only 20 mg of CFZ requires 50-100 mL of 5% glucose injection for intravenous administration, with a continuous infusion duration of more than 30 minutes. To reduce kidney toxicity, 250-500 mL / kg of oral and intravenous fluids are required before administration, indicating that CFZ needs to use a carrier to improve water solubility. ② Low brain drug occupancy: The blood concentration of CFZ rapidly decreases after intravenous injection, with a 30-minute blood concentration of 1 μmol / L. After 24 hours, it is enriched in the heart, lungs, adrenal glands, and liver, but not in the brain. CFZ is a proteasome inhibitor mainly used to treat peripheral disease multiple myeloma, indicating that CFZ needs to be brain-targeted to increase brain drug occupancy. ③ Short action time: After intravenous administration, the plasma concentration of CFZ rapidly decreases in a biphasic manner. CFZ exhibits a high plasma clearance rate of 195-319 mL / min / kg, a short terminal half-life of 5-20 min, and a rapid and extensive tissue distribution. The high clearance rate is mainly mediated by extrahepatic metabolism through peptidase cleavage and epoxide hydrolysis. The main metabolites in urine and bile account for about 26% and 31% of the total dose, respectively, and a total of 57% within 24 hours after administration, indicating that CFZ needs to use a carrier to prolong the action time. ④ Lysosomal escape: CFZ structure contains multiple amine groups, which can be protonated in the acidic environment of lysosomes and escape lysosomal degradation through the proton sponge effect, indicating that CFZ can achieve lysosomal pH-responsive controlled release and escape. ⑤ Action site in cytoplasm: CFZ acts on the proteasome in the cytoplasm, blocks BNIP3L degradation, restores mitochondrial autophagy, significantly reduces brain infarction volume and neurological deficit scores, and effectively extends the therapeutic time window to 3 hours after IS. ⑥ Large side effects: During CFZ treatment, severe adverse reactions may occur, including but not limited to cardiac toxicity, pulmonary toxicity, thrombocytopenia, venous thrombosis, peripheral neuropathy, etc. Therefore, brain targeting needs to be improved to reduce peripheral blood drug concentration and reduce peripheral toxicity. Some patients developed reversible encephalopathy syndrome after receiving larger doses of CFZ, presenting with convulsions, headaches, drowsiness, confusion, blindness, changes in consciousness, and hypertension, and even progressive multifocal white matter disease, leading to patient death, indicating that CFZ needs to be attenuated and potentiated by a carrier to avoid high-dose central toxicity.⑦ Existing improved formulations lack brain-targeting and controlled-release functions: In 2022, a CFZ nanoemulsion suspension lyophilized formulation was reported. This formulation effectively solved the problems of poor water solubility and low drug content of CFZ, but it lacks brain-targeting and controlled-release functions. Summary of the Invention

[0010] In view of the technical problems existing in the prior art, the present invention constructs a three-target, dual-controlled-release, dual-drug nano-delivery system to precisely deliver drugs to the matrix action sites in the cytoplasm or mitochondria, thereby achieving dual-pathway synergistic enhancement of IS mitochondrial autophagy and reducing toxicity and increasing efficacy in the treatment of IS.

[0011] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a nano-drug delivery system, which is prepared according to the following steps: The core was obtained by modifying cationic starch globules with mitochondrial-targeting peptides and loading them with PDK4-IN-1; Aminated anionic starch was modified with homing peptides and then loaded with carfilzomib to form a starch helical shell. The core is mixed with the starch helical shell and self-assembled to form the nano-drug delivery system.

[0012] Preferably, the specific steps for modifying cationic starch globules with mitochondrial-targeting peptides are as follows: Using cationic starch spheres and chloroacetic acid as raw materials, a halogenation reaction was carried out to obtain an intermediate product; The intermediate product was mixed with the mitochondrial-targeting peptide and subjected to an amidation reaction to obtain mitochondrial-targeting peptide-modified cationic starch spheres.

[0013] Preferably, the halogenation reaction is carried out at 50℃~70℃ for 2h~6h, and the amidation reaction is carried out at 30℃~40℃ for 6h~8h.

[0014] Preferably, the mass ratio of the cationic starch spheres to chloroacetic acid is 1:0.2~1; The mitochondrial targeting peptide is mitochondrial targeting peptide SS-31, and the mass ratio of the intermediate product to the mitochondrial targeting peptide is 1:0.1~0.2.

[0015] More preferably, the mass ratio of the mitochondrial-targeting peptide-modified cationic starch to the PDK4-IN-1 is 1:0.1~0.2.

[0016] Preferably, the amination anionic starch is prepared according to the following steps: Amination of anionic starch was obtained by halogenation reaction using starch, chloroacetic acid and 3-chloro-1-(N,N-dimethyl)propylamine as raw materials.

[0017] Preferably, the halogenation reaction is carried out at 50-70℃ for 2-4h.

[0018] Preferably, the mass ratio of the starch, halogenated acetic acid and 3-chloro-1-(N,N-dimethyl)propylamine is 1:1:0.1-1.

[0019] Preferably, the specific operation process of the homing peptide modified aminated anionic starch is as follows: The amide reaction is carried out with the aminated anionic starch and the homing peptide as raw materials, and the product is obtained.

[0020] Preferably, the amide reaction of the aminated anionic starch and the homing peptide is stirred at room temperature for 48h.

[0021] Preferably, the mass ratio of the aminated anionic starch and the homing peptide is 1:0.2-0.5.

[0022] Preferably, the mass ratio of the product of the homing peptide modified aminated anionic starch and Carfilzomib (CFZ) is 1:0.004-0.1.

[0023] Further preferably, when the drug is CFZ, the mass ratio of the inner core and the starch helix shell is 1:0.1-1.

[0024] Preferably, the self-assembly is that the starch helix shell is self-assembled and gathered around the inner core PDK4-IN-1 under the action of stirring, electrostatic adsorption, van der Waals force and hydrogen bond.

[0025] Preferably, the nano drug delivery system is a core-shell structure, the particle size is 40nm, and the surface can see a shell composed of multiple 10nm nanospheres.

[0026] In the second aspect of the present application, the use of the nano delivery system in the preparation of a brain targeting product is provided.

[0027] In the third aspect of the present application, the use of the nano delivery system in the preparation of a neuron targeting product is provided.

[0028] In the fourth aspect of the present application, the use of the nano delivery system in the preparation of a mitochondrion targeting product is provided.

[0029] In the fifth aspect of the present application, the use of the nano delivery system in the preparation of a product for enhancing brain neuron mitochondrion autophagy is provided.

[0030] Preferably, the nano delivery system is used for preparing a product for treating ischemic cerebral stroke.

[0031] Compared with the prior art, the present application has the following beneficial effects: The present application proposes a new strategy for treating IS by enhancing brain neuron mitochondrial autophagy through a double-channel, and constructing a three-targeting, double-controlled-release, double-drug nano drug delivery system to achieve the new strategy. Through brain / neuron / mitochondria targeting, pH-responsive shell structure precise control release of cafitozomib and PDK4-IN-1 to the cytoplasm and mitochondrial matrix respectively, precise enhancement of Pink1 / Parkin and BNIP3L / NIX pathways, and synergistic enhancement of mitochondrial autophagy, the effective treatment time window of IS is significantly prolonged, and the recovery of neurological function is promoted.

[0032] To achieve brain / neuron / mitochondria three targeting, the present application respectively selects homing peptide and mitochondrial targeting peptide: ①In the focal IS model, the present application obtains a stroke homing peptide SHp based on phage display technology screening, and the amino acid sequence of the homing peptide SHp is CLEVSRKNC. The homing peptide SHp can not only bind to the vascular endothelial transferrin receptor to mediate blood-brain barrier penetration, but also bind to the IS neuron glutamate receptor to mediate the drug delivery system to enter the neurons of the ischemic brain area.

[0033] ②The homing peptide is selected as the neuron target to realize the drug delivery system into the neuron: brain tissue cells include neurons and neuroglial cells, and neurons are the most important cell type in IS injury. Neurons are a kind of cells with high structural polarity, and extracellular substances can enter neurons through multiple pathways such as clathrin, rapid endocytosis, glycosyl phosphatidylinositol-anchored protein, macropinocytosis, phagocytosis, and caveolin. The neuron target is a kind of polypeptide obtained by in vitro phage display technology, which can bind to the GT1B ganglioside highly expressed on the neuron cell, and enter the neuron cell through clathrin and macropinocytosis. The combination of endosome and lysosome can realize the pH-responsive controlled release of lysosome. As the neuron target, the homing peptide combined with the brain target group makes 72% of the drug in the brain enriched in neurons, and the drug enrichment amount in neurons is 3.6 times that of the brain target group alone.

[0034] ③The mitochondrial polypeptide SS-31 is selected as the mitochondrial target to realize the entry of PDK4-IN-1 into the mitochondrial matrix: the mitochondrial inner membrane has high negative charge, and the mitochondrial targeting strategy mediated by lipophilic cation triphenylphosphonium (TPP) has been widely used, but TPP is difficult to label damaged mitochondria, a large number of insertions can directly damage mitochondria, and it can only transport drugs to the surface of mitochondrial membrane. The SS-31 polypeptide is a small molecule polypeptide of four amino acids, which can realize multiple functions such as cardiolipin peroxidase inhibition, mitochondrial targeting, and cell membrane penetration. With the specificity of mitochondrial inner membrane cardiolipin as the target, the present application reduces the Ca 2+Overload, oxidative damage. PDK4-IN-1 acts on mitochondrial matrix PDK4, and SS-31 is needed to target PDK4-IN-1 to the inner matrix of mitochondria, and CFZ acts on cytoplasmic proteasomes, reduces BNIP3L degradation on the mitochondrial membrane, and does not need to be targeted to the mitochondria, but needs to be released in the cytoplasm of neurons.

[0035] To release PDK4-IN-1 in the mitochondrial matrix and CFZ in the cytoplasm of neurons, the present application selects nanometer starch as the skeleton, and adopts pH-shell core double control to release CFZ and PDK4-IN-1: ① Selecting nanometer starch as the skeleton, three target heads, pH-responsive amine bonds can be connected, and a shell-core double-layer drug loading structure can be presented: starch has the advantages of non-toxicity, biodegradability, and good biocompatibility, etc., and the maximum daily dosage of intravenous injection of hydroxyethyl starch can reach 3.0 g / kg. Compared with inorganic nanometer drug delivery system skeletons, nanometer starch has high rigidity, large specific surface area, diverse morphology, high specific strength, and is rich in modifiable hydroxyl groups (1.505 x 10 21 / g), which can be cross-linked with different chemical bonds to prepare various modified starches.

[0036] ② Selecting lysosomal pH-responsive shell-core to release CFZ and inner core: the drug delivery system is easily digested by lysosomes when entering neurons, and the amine bond can be protonated in the lysosome to realize the function of pH-responsive drug release, and also can realize lysosome escape through the proton sponge effect. A shell-core structure starch drug delivery system can be prepared, the outer shell has amine bonds to load CFZ, which is released under pH response, and CFZ itself contains amine bonds that can escape from lysosomes and reach the cytoplasm to act on proteasomes; the inner core is a cationic starch cross-linked ball, which is stable in structure and not easy to release under pH response, and can slowly release PDK4-IN-1 over time, has a positive charge that can escape from lysosomes, and under the mediation of SS-31, enters the mitochondrial matrix to slowly release PDK4-IN-1 to act on PDK4; CFZ and PDK4-IN-1 synergistically enhance the treatment of IS by mitochondrial autophagy. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is (Lf-TET-AAS@CFZ):(SS31-CS@PDK4-IN-1) characterization; a, particle size comparison of the inner core and the whole material; b, transmission electron micrograph of the shell-core drug delivery system; c, zeta potential values of the inner shell, outer core and whole; d, change of nanoparticles in 10% fetal bovine serum; e, pH-responsive drug release curve; Figure 2PDK4 is a key enzyme of IS, PI1 enhances the level of mitophagy through Pink1 / Parkin; a, screening of differentially expressed genes in stroke; b, intersection of differentially expressed genes in stroke and mitochondrial genes; c, PPI network between diagnostic genes and their top 20 interaction genes; d, model effect in training set; e, nomogram model of diagnostic genes; f, KEGG pathway analysis of intersection genes; g~k, Western Blot analysis and statistics of mitochondrial autophagy-related proteins in different groups.

[0038] Figure 3 PI1 and CFZ synergistically enhance mitochondrial autophagy; a~b, SuperPred database predicts CFZ, PI1 drug target results; c, electron microscope images of mitochondrial autophagy levels in different groups; d, Western Blot analysis and statistics of autophagy-related proteins in different groups.

[0039] Figure 4 Molecular simulation results confirm the feasibility of the stable structure formed by the two drugs and yin and yang amyloid; a~b, structural changes during the simulation of cationic amyloid and PDK4-IN-1; c~d, structural changes during the simulation of anionic amyloid and CFZ; e, the RMSD value of atoms in each molecule in the system changes with simulation time; f, the solvent accessible surface area of each molecule in the system changes with simulation time; g~i, the interaction between each molecule in the system; Figure 5 a is the fluorescence imaging of the mouse brain in vivo at each time point, b is the quantitative analysis of the fluorescence of the mouse brain at each time point, c is the fluorescence imaging of the mouse brain, d is the fluorescence imaging of the mouse main organs; e is the quantitative analysis of the fluorescence intensity of the mouse main organs, f is the fluorescence imaging of the brain section; Figure 6 (Lf-TET-AAS@Cy3):(SS31-CS@PDK4-IN-1) fluorescence 4h and 1h distribution in each organ; Figure 7 (Lf-TET-AAS@CFZ):(SS31-CS@C6) and mitochondrial co-localization in neurons; Figure 8 a, b are the performance statistics of different groups in the Y maze experiment; c, d are representative TTC staining images of brain sections and infarct area statistics, respectively; e is the HE staining image of brain paraffin sections. DETAILED DESCRIPTION

[0040] The application will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the application are not limited thereto.

[0041] The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used are all purchased from conventional biochemical reagent manufacturers unless otherwise specified.

[0042] PDK4-IN-1 and Carfilzomib (CFZ) used in the present application are purchased from MCE, and the rest of the chemical reagents not mentioned are purchased from Aladdin. The sequence of SS-31 polypeptide is NH2-DArg-Dmt-Lys-Phe-NH2, and the amino acid sequence of stroke homing peptide SHp is C-L-E-V-S-R-K-N-C.

[0043] Example 1 Preparation and characterization of (SHp-AAS@CFZ):(SS31-CS@PDK4-IN-1) shell-core nanodrug delivery system (1) Preparation of cationic starch CS As shown in synthesis route 1, 1.0 g of starch was weighed into a 50 mL conical flask, and 19 mL of deionized water was added to dissolve it. At the same time, sodium hydroxide (0.5 g, 12 mmol) was weighed and dissolved in 1 mL of deionized water, and then added dropwise into the conical flask under stirring. Alkalization was carried out at room temperature for 30 minutes. Then 2,3-epoxypropyltrimethylammonium chloride (GTAC) (0.7 g, 5 mmol) was added, and the reaction was carried out in a 60°C water bath for 4 h. After the reaction was completed, the solution pH was adjusted to about 7.0 by glacial acetic acid. The obtained solution was added to a dialysis bag (Mw=8000) and dialyzed overnight. Finally, the dialyzed solution was concentrated and added dropwise into a large amount of anhydrous ethanol for sedimentation. The product was collected by centrifugation and dried to obtain cationic starch CS.

[0044] Synthesis route 1: preparation of cationic starch (2) Preparation of CS spheres: CS was prepared by reverse emulsion method (W / O). Water phase (W): 0.1 g of cationic starch CS prepared was weighed into a 50 mL conical flask, and 10 mL of deionized water was added to dissolve it. 0.5 mol / L sodium hydroxide standard solution was added dropwise to adjust the pH to 10, and alkalization was carried out for 30 min to obtain the water phase. Oil phase (O): 0.6 g of Span60 was taken in a 250 ml conical flask, and 200 ml of liquid paraffin was used to dissolve it at 60°C to obtain the oil phase. In a 50°C water bath, W phase was slowly added to O phase at a volume ratio (v / v) of 1-20, and 2 ml of epichlorohydrin was added as a crosslinking agent for reaction for 4 h. Finally, the product was removed by centrifugation at 4000 rpm x 10 min to remove the liquid paraffin, and chloroform was added to wash away span60. It was placed in a high-speed centrifuge (10000 rpm x 5 min) and repeated repeatedly. Finally, dialysis was carried out to obtain CS spheres.

[0045] (0) Preparation of SS31-CS spheres: As shown in synthesis route 2, 3, 1.0 g of CS spheres was weighed into a 50 mL conical flask and dissolved in 10 mL of deionized water. At the same time, 0.5 g of sodium hydroxide was dissolved in 1 mL of deionized water and added dropwise to the conical flask under stirring, and alkalized at room temperature for 30 min; then 0.2 g of chloroacetic acid was added and reacted in a 60°C water bath for 4 h. After the reaction was completed, the solution was cooled to room temperature, a small amount of solution was taken with a pipette, and the pH of the solution was monitored in real time with pH paper, and the solution was neutralized to pH 7 with appropriate amount of glacial acetic acid. The obtained solution was added to an 8000 Mw dialysis bag and dialyzed in deionized water overnight. Finally, the dialyzed solution was concentrated and added dropwise to a large amount of anhydrous ethanol to precipitate; the product was collected by centrifugation and dried to obtain the intermediate product CS-1. 1.0 g of CS-1 spheres was weighed into a 50 mL conical flask, 20 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 50 mg of N-hydroxysuccinimide (NHS) were added, dissolved in PBS buffer, and activated in a 37°C water bath for 2 h, then 20 mg of SS31 peptide was added and reacted in a 37°C water bath for 6 h. After the reaction was completed, the solution was dialyzed overnight, concentrated and freeze-dried to obtain SS31-CS spheres.

[0046] Synthesis of CCS Synthesis of SS31-CS (1) Preparation of core SS31-CS@PDK4-IN-1 The obtained SS31-CS spheres were added with 2 mg of PDK4-IN-1 and continuously stirred for 12 h to allow SS31-CS to fully encapsulate PDK4-IN-1, obtaining SS31-CS@PDK4-IN-1.

[0047] (2) Preparation of aminated anionic starch (AAS) As shown in synthetic route 4, 1.6 g of starch was weighed into a 50 mL conical flask and stirred with 30 mL of deionized water. At the same time, sodium hydroxide (0.8 g, 20 mmol) was weighed into the conical flask and allowed to alkalinize at room temperature for 30 min; then, the conical flask was placed in a 60°C water bath, and chloroacetic acid (0.6 g, 6 mmol) and 3-chloro-1-(N,N-dimethyl)propylamine (0.8 g, 6.6 mmol) were added to react for 2 h; after the reaction was completed, the solution pH was adjusted to about 7.0, and the resulting solution was added to a dialysis bag (Mw=3500) and dialyzed overnight; finally, the dialyzed solution was concentrated and added dropwise to a large amount of anhydrous ethanol to precipitate, centrifuged (4000 rpm x 10 min) to collect the product, washed repeatedly with anhydrous ethanol, and dried to obtain AAS.

[0048] Synthetic route 4: preparation of amine anionic starch (3) Preparation of SHp-AAS 0.1 g of AAS was weighed into a 25 mL conical flask, and EDC 100 mg and NHS 20 mg were added in sequence and stirred at room temperature for 2 h. Then, SHp 20 mg was added and stirred at room temperature for 48 h. After the reaction was completed, the resulting solution was added to a dialysis bag (Mw=3500) and dialyzed overnight; finally, the dialyzed solution was concentrated and vacuum freeze-dried to obtain the homing peptide-modified amine anionic starch, labeled as SHp-AAS.

[0049] (4) Preparation of shell SHp-AAS@CFZ: 0.1 g of SHp-AS was weighed into a 10 mL conical flask and dissolved in 2 mL of deionized water. 40 uL of CFZ (10 mg / mL, DMSO solution) was taken and added to the conical flask and continuously stirred for 12 h to allow the SHp-AS to fully encapsulate the CFZ, obtaining a starch helical shell, labeled as SHp-AAS@CFZ.

[0050] (5) Preparation of (SHp-AAS@CFZ):(SS31-CS@PDK4-IN-1) shell-core nanomedicine delivery system: 2 mL of the inner core SS31-CS@PDK4-IN-1 was added, and 2 mL of the shell SHp-AAS@CFZ was taken, and the shell was self-assembled and aggregated around the inner core SS31-CS@PDK4-IN-1 by electrostatic adsorption, van der Waals force, and hydrogen bonding, and stirred for 12 h to form a double-shell core structure starch ball.

[0051] Example 2 Preparation of (SHp-AAS@CFZ):(SS31-CS@PDK4-IN-1) shell-core nanomedicine delivery system The difference from Example 1 is only in the preparation process of amine anionic starch (AAS), specifically: 1.6 g of starch was weighed into a 50 mL conical flask and dissolved in 29 mL of deionized water. At the same time, 0.8 g of sodium hydroxide was dissolved in 1 mL of deionized water, and was added dropwise into the conical flask under stirring, and alkalized at room temperature for 30 min; then, the conical flask was placed in a 70°C water bath, and 1.6 g of chloroacetic acid and 1.6 g of 3-chloro-1-(N,N-dimethyl)propylamine were reacted for 2 h; after the reaction was completed, appropriate glacial acetic acid was added to adjust the solution pH to 7; and the solution was left to settle in a large amount of anhydrous ethanol overnight, the next day the supernatant was discarded, and the settled material was dried to obtain AAS.

[0052] Example 3 Preparation of (SHp-AAS@CFZ):(SS31-CS@PDK4-IN-1) shell-core nanodrug delivery system The difference from Example 1 is only in the preparation process of amine anionic starch (AAS), specifically: 1.6 g of starch was weighed into a 50 mL conical flask and dissolved in 29 mL of deionized water. At the same time, 0.8 g of sodium hydroxide was dissolved in 1 mL of deionized water, and was added dropwise into the conical flask under stirring, and alkalized at room temperature for 30 min; then, the conical flask was placed in a 70°C water bath, and 1.6 g of chloroacetic acid and 1.6 g of 3-chloro-1-(N,N-dimethyl)propylamine were reacted for 2 h; after the reaction was completed, appropriate glacial acetic acid was added to adjust the solution pH to 7; and the solution was left to settle in a large amount of anhydrous ethanol overnight, the next day the supernatant was discarded, and the settled material was dried to obtain AAS.

[0053] Example 4 Preparation of (SHp-AAS@CFZ):(SS31-CS@PDK4-IN-1) shell-core nanodrug delivery system The difference from Example 1 is only in the preparation process of amine anionic starch (AAS), specifically: 1.6 g of AAS was weighed into a 50 mL conical flask, and EDC 20 mg and NHS 50 mg were added, dissolved in PBS buffer, and activated in a 37°C water bath for 2 h, then SHp 800 mg was added, and stirred at room temperature for 48 h. After the reaction was completed, the obtained solution was added to a dialysis bag (Mw=3500) and dialyzed overnight; finally, the dialyzed solution was concentrated and freeze-dried under vacuum to obtain homing peptide modified amine anionic starch, labeled as SHp-AAS.

[0054] Example 5 Preparation of (SHp-AAS@CFZ):(SS31-CS@PDK4-IN-1) shell-core nanodrug delivery system The difference between Example 1 is only in the preparation process of the shell SHp-AAS@CFZ, specifically: Take 200 mg SHp-AAS dissolved in 100 mL deionized water, fully stirred to dissolve, then add CFZ 20 mg and continue to stir for 12 h, so that SHp-AAS fully wraps CFZ, to obtain SHp-AAS@CFZ.

[0055] Example 6 (SHp-AAS@CFZ):(SS31-CS@PDK4-IN-1) shell-core nanomedicine system preparation The difference between Example 1 is only in the preparation process of the shell SHp-AAS@CFZ, specifically: Take 1.0 g CS ball and place it in a 50 mL conical flask, add 10 mL deionized water to dissolve. At the same time, take sodium hydroxide 0.5 g, dissolve with 1 mL deionized water, and add dropwise to the conical flask under stirring, alkali at room temperature for 30 min; then add chloroacetic acid 0.2 g, and place it in a 70°C water bath for 3 h. After the reaction is completed, cool to room temperature, dip a small amount of solution with a pipette, and monitor the pH value of the solution with pH paper in real time, and add appropriate amount of glacial acetic acid to neutralize the solution to pH 7. The obtained solution is added to an 8000 Mw dialysis bag and dialyzed in deionized water overnight. Finally, the dialyzed solution is concentrated and added dropwise to a large amount of anhydrous ethanol to settle; centrifugal collection of the product and drying to obtain the intermediate product CS-1. Take 1.0 g CS-1 ball in a 50 mL conical flask, and at the same time add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) 20 mg and N-hydroxysuccinimide (NHS) 50 mg, dissolve with PBS buffer, and activate in a 37°C water bath for 2 h, then add SS31 peptide 20 mg, and react in a 30°C water bath for 8 h. After the reaction is completed, the solution is dialyzed overnight, concentrated and freeze-dried to obtain the SS31-CS ball.

[0056] Example 7 (SHp-AAS@CFZ):(SS31-CS@PDK4-IN-1) shell-core nanomedicine system preparation The difference between Example 1 is only in the preparation process of the shell SHp-AAS@CFZ, specifically: Weigh 1.0 g of CS beads and place them in a 50 mL Erlenmeyer flask, then add 10 mL of deionized water to dissolve them. Simultaneously, weigh 0.5 g of sodium hydroxide, dissolve it in 1 mL of deionized water, and add it dropwise to the Erlenmeyer flask with stirring. Alkalize at room temperature for 30 min. Then add 0.2 g of chloroacetic acid and react in a 50 °C water bath for 6 h. After the reaction is complete, cool to room temperature. Use a pipette to take a small amount of the solution and monitor the pH value in real time with pH paper. Add an appropriate amount of glacial acetic acid to neutralize the solution to pH 7. Add the resulting solution to an 8000 Mw dialysis bag and dialyze overnight in deionized water. Finally, concentrate the dialyzed solution and add it dropwise to a large amount of anhydrous ethanol for precipitation. Centrifuge to collect the product and dry it to obtain the intermediate product CS-1. Weigh 1.0 g of CS-1 beads into a 50 mL Erlenmeyer flask, and simultaneously add 20 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 50 mg of N-hydroxysuccinimide (NHS). Dissolve in PBS buffer and activate in a 37°C water bath for 2 h. Then add 20 mg of SS31 peptide and react in a 40°C water bath for 6 h. After the reaction is complete, dialyze the solution overnight, concentrate, and lyophilize to obtain SS31-CS beads.

[0057] Since the performance and application effects of the (SHp-AAS@CFZ):(SS31-CS@PDK4-IN-1) shell-core nanoparticle drug delivery systems prepared in Examples 1 to 7 are basically the same, the following description will only take the (SHp-AAS@CFZ):(SS31-CS@PDK4-IN-1) shell-core nanoparticle drug delivery system prepared in Example 1 as an example to illustrate its performance and application effects.

[0058] The morphology and particle size of the (SHp-AAS@CFZ):(SS31-CS@PDK4-IN-1) core-shell nanoparticle drug delivery system of Example 1 were observed using TEM. Figure 1 As shown in Figure a, the core particle size of (SHp-AAS@CFZ):(SS31-CS@PDK4-IN-1) is approximately 50 nm, and the overall particle size is approximately 105 nm. TEM images show that (SHp-AAS@CFZ):(SS31-CS@PDK4-IN-1) exhibits a uniform and regular core-shell structure, and the overall particle size is consistent with the particle size analyzer measurement results. No obvious aggregation was observed between the drug delivery system particles, indicating good dispersibility. Figure 1 Figure b. The Zeta potential is approximately -0.68 ± 0.18 mV, verifying the attraction between the negatively charged outer shell and the positively charged inner core. The surface micro-negativity effectively prevents particle aggregation (see figure c). The drug delivery system exhibits minimal particle size variation over one week in a 10% fetal bovine serum environment, consistently remaining below 200 nm, indicating good stability (see figure c). Figure 1Middle d. Cy2-NH2, Nile Red fluorescence was loaded on the drug delivery system to facilitate the detection of carrier release curve. At physiological pH, the release amount of the inner core and the shell structure did not exceed 10% within 24 hours; while in the acidic environment of pH=5.6, the inner core structure released about 45%, and the shell structure released about 85%, and the release rate of the shell structure was significantly higher than that of the inner core structure, indicating that the shell was pH-responsive release, and the inner core was pH-insensitive slow release, see Figure 1 Middle e.

[0059] Experimental example 1 GSE16561, GSE37587 two sets of data as the training set of this analysis, GSE58294 as the validation set of this analysis, bioinformatics analysis training set IS and control samples, P Value<0.05, |logFC|>0.263 (i.e. difference ratio 1.2 times), identify differential genes and use heat map to display, Venn analysis of differential expression genes and mitochondrial genes, get 7 intersection genes as differential mitochondrial genes; The feature genes screened by three machine learning algorithms (LASSO regression model, SVM-RFE model, random forest model) are intersected, and 2 diagnostic genes PDK4, GMF1 , AUC is 0.792. Using GeneMANIA database to analyze the PPI regulatory relationship network of diagnostic genes and their 20 interaction genes, it is found that PDK4, GMF1 is the core gene in these differential genes.

[0060] Using the regression coefficient of lasso analysis, the diagnostic score is constructed by the following formula: Riskscore = ∑βgene×Expgene, Where βgene represents the LASSO regression coefficient of the gene, and Expgene represents the expression level of the gene in each sample.

[0061] Draw the distribution box plot of RiskScore in different grouping samples, compared with GMF1, PDK4 the difference is more significant, the training set and the validation set are significantly different, and the two constitute a joint diagnostic nomogram, PDK4 Basically all constitute the diagnostic nomogram; through KEGG analysis of differential gene related signal pathway, the result shows that the mitochondrial autophagy pathway has changed significantly. Using OGD cell model verification, PDK4 inhibitor PDK4-IN-1, namely PI1, increases the expression levels of Pink1, Parkin and LC3B-II proteins, and reduces the mitochondrial autophagy substrate SQSTM1. The specific results are shown in Figure 2 .

[0062] The CFZ and PI1 drug target points are predicted by the SuperPred database, and the intersection of the IS mitochondrial autophagy difference genes is obtained. The CFZ and PI1 can regulate mitochondrial autophagy through 5 target genes. The double-drug combination increases the target genes, and can regulate mitochondrial autophagy through 9 target genes. The OGD cell model is used for verification. Compared with single drug, TEM observation shows that the number of mitochondrial autophagosomes in the CFZ and PI1 double-drug combination group is significantly increased; the expression level of LC3B-II protein is increased, and the expression of mitochondrial autophagy substrate SQSTM1 is significantly reduced. The results are shown in Figure 3 .

[0063] Experimental Example 2 Molecular simulation predicts self-assembly of double-drug and anion-starch In order to study the conformational changes of the two systems in the simulation process and the tightness of the complex structure finally formed, the Gromacs 2019.6 program is used to construct the System1 and System2 complex systems in turn. 10 cationic starch CS molecules and 20 PDK4-IN-1 molecules are randomly filled into System1, as shown in a~b in Figure 4 ; System2 is randomly filled with 35 AS molecules and 50 CFZ molecules into the system after the simulation of System1 is completed, as shown in c~d in Figure 4 . The steepest descent method is used for energy minimization to eliminate the close contact between atoms, and 100 ps of NVT equilibrium simulation is carried out at 298.15 K. The simulation visualization is completed by using the Gromacs embedded program and VMD.

[0064] The root mean square deviation RMSD is an important basis for measuring the stability of the system: the RMSD values of the two systems quickly stabilize after the initial fluctuation, indicating that all molecules in the two systems can quickly form stable nanoclusters, confirming that the force field parameters used in the application are real and reliable, and subsequent analysis can be based on this, as shown in e in Figure 4 .

[0065] The solvent accessible surface area SASA can be used to evaluate the tightness of molecular aggregation in the system: the SASA of the two systems decreases to a stable state after different degrees of decrease in the simulation process, further indicating that all molecules in the two systems can finally aggregate and self-assemble to form stable nanocluster structures, as shown in f in Figure 4 .

[0066] Intermolecular interaction force: PDK4-IN-1 and CA mainly self-assemble through electrostatic force, van der Waals force and hydrogen bond. Although a relatively large amount of AS and CFZ molecules were added in System 2 to weaken the energy, it did not change the result of PDK4-IN-1 binding with CA; AS tightly binds with CS to form a nanoshell core cluster structure by encapsulating CFZ with van der Waals force, electrostatic force and hydrogen bond, as shown in Figure 4 g~i.

[0067] Experimental Example 3 The hydrophobic fluorescent dye IR780 was used to replace CFZ to load the shell of the drug delivery system to prepare (SHp-AAS@IR780):(SS31-CS). Normal mice were injected intravenously with an injection amount of 0.1 mL. Small animal imaging showed that the SHp nanodrug delivery system targeted the brain tissue 5 min after injection, reached the peak at 6 h, and after dissection of the organs, the brain tissue was significantly enriched, and the fluorescence accumulation in the liver, lung, kidney and other organs was significantly reduced, indicating that SHp can achieve brain targeting. The PT model of cerebral ischemia was induced by photochemical method, and (SHp-AAS@Ce6):(SS31-CS) was injected intravenously into PT mice. Red fluorescence was enriched in the ischemic brain area with DAPI cell nucleus blue light dyeing, indicating that SHp has ischemic brain area targeting, as shown in Figure 5 .

[0068] The fluorescent dye Cy2-NH2 (green) containing amine was used to replace CFZ to load the shell, and (SHp-AAS@Cy2-NH2):(SS31-CS) was co-incubated with nerve cells in an incubator at 37°C, 5% CO2 and 95% air culture environment. Lysotracker was used to label (red) lysosomes, and DAPI was used to label (blue) cell nuclei. At 1 h, the drug delivery system was co-localized with lysosomes in neurons, and at 4 h, lysosomes were significantly reduced, and the green fluorescence of the drug delivery system was located in the cytoplasm, with no co-localization with lysosomes, indicating that the drug in the shell can achieve pH-responsive controlled release and escape from lysosomes, as shown in Figure 6 .

[0069] The hydrophobic fluorescent dye Cy2-NH2 (green) was used to replace PI1, and nerve cells were co-incubated with (SHp-AAS):(SS31-CS@Cy2-NH2) in an incubator at 37°C, 5% CO2 and 95% air culture environment. MitoTracker was used to label (red) mitochondria in nerve cells, and DAPI was used to label (blue) cell nuclei. Co-localization of the core in the drug delivery system with mitochondria indicates that the core has mitochondrial targeting function, as shown in Figure 7 .

[0070] Experimental Example 4 In vivo and in vitro experiments verify that PDK4-IN-1 and CFZ synergistically enhance the treatment of IS by mitochondrial autophagy C57BL / 6 mice were randomly divided into model group and control group, after induction of anesthesia, fixed in the stereotaxic instrument, the hair on the top of the head was shaved, the scalp was cut to expose the skull, hydrogen peroxide was used to wipe the exposed foramen, the stereotaxic instrument was used to locate, and the skull was opened to expose the brain tissue at the position of 1.5-2 mm on the right side of the foramen, rose solution was injected intraperitoneally, 5 min later, the cold light source was opened, and it was placed close to the opening and fixed for 15 min. After the modeling was successful, the mice were randomly divided into control group, PT group, PT+CFZ group, PT+PI1 group, PT+Nano group. Control group: C57BL / 6 mice injected with normal saline, PT group: model mice injected with normal saline, PT+CFZ group: model mice injected with 2 mg / kg CFZ, PT+PI1 group: model mice injected with 1 mg / kg PI1, PT+Nano group: model mice injected with nano drug delivery system (2 mg CFZ+1 mg PI1) / kg.

[0071] 100 in each group, PT model was injected once by tail vein, TTC staining method was used to evaluate 24 h infarction volume, and the treatment was effective when the infarction volume was significantly reduced compared with the PT group, and the ED50 of each group was determined by Bliss method.

[0072] The results show that Figure 8 The correct alternation frequency and alternation rate of the PT+Nano group were significantly increased, the infarction area was significantly reduced by TTC staining, and the vacuolation and karyopyknosis of the neuronal cells were obviously reduced by HE staining, which preliminarily indicated that the nano drug delivery system could improve the cognitive function of IS and reduce the infarction area by synergistically improving IS with CFZ and PI1.

[0073] The above embodiments are used to illustrate the detailed features and detailed methods of the present application, but the present application is not limited to the above detailed features and detailed methods. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of selected components of the present application, addition of auxiliary components, selection of specific modes, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. A nanodelivery system, characterized in that, It is prepared according to the following steps: After the cationic starch ball is modified by a mitochondria-targeting peptide, PDK4-IN-1 is loaded, and the inner core is self-assembled; After the aminated anionic starch is modified by a homing peptide, carfilzomib is loaded, and a starch helix shell is formed; Mix the inner core and the starch helix shell to self-assemble the nano drug delivery system.

2. The nanodelivery system of claim 1, wherein, The specific operation steps of the mitochondria-targeting peptide modified cationic starch ball are: Take cationic starch ball and chloroacetic acid as raw materials to carry out halogenation reaction to obtain an intermediate product; Mix the intermediate product with the mitochondria-targeting peptide to carry out amidation reaction.

3. The nano delivery system according to claim 2, wherein, The mass ratio of the cationic starch ball to chloroacetic acid is 1:0.2-1; The mitochondria-targeting peptide is mitochondria-targeting peptide SS-31, and the mass ratio of the intermediate product to the mitochondria-targeting peptide is 1:0.1-0.

2.

4. The nanodelivery system of claim 3, wherein, The mass ratio of the mitochondria-targeting peptide modified cationic starch to the PDK4-IN-1 is 1:0.1-0.

2.

5. The nanodelivery system of claim 1, wherein, The aminated anionic starch is obtained by halogenation reaction with starch, chloroacetic acid and 3-chloro-1-(N,N-dimethyl) propylamine as raw materials.

6. The nanodelivery system of claim 5, wherein, The mass ratio of the starch, chloroacetic acid and 3-chloro-1-(N,N-dimethyl) propylamine is 1:1:0.1-1.

7. The nanodelivery system of claim 1, wherein, The specific operation process of the homing peptide modified aminated anionic starch is: take the aminated anionic starch and homing peptide as raw materials to carry out amidation reaction.

8. The nanodelivery system of claim 7, wherein, The mass ratio of the aminated anionic starch to the homing peptide is 1:0.2-0.

5.

9. The nano delivery system according to claim 8, wherein, The mass ratio of the product after the homing peptide modified aminated anionic starch to carfilzomib is 1:0.004-0.1; The mass ratio of the inner core to the starch helix shell is 1:0.1-1.

10. Use of the nanodelivery system according to any one of claims 1 to 9, characterized in that, The nano delivery system has the following uses: (1) preparation of neuron-targeting products; (2) preparation of mitochondria-targeting products; (3) preparation of products for enhancing brain neuron mitochondrial autophagy; (4) preparation of products for treating ischemic stroke.

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