Preparation method and application of amphiphilic prodrug and nanoparticles thereof for mitochondrial targeted therapy

Abstract

This invention relates to the field of biomedicine, specifically disclosing an amphiphilic prodrug for mitochondrial targeted therapy, its preparation method, and applications. The target-containing amphiphilic prodrug of this invention can self-assemble in water to form nanomicelles with an outer layer of hydrophilic short peptides and an inner layer of hydrophobic mitochondrial protective drugs. Because the hydrophilic short peptide SS-31 on the surface of the nanomicelles is positively charged, it can act as a cell-penetrating peptide to rapidly cross the cell membrane and even the blood-brain barrier in a non-toxic manner, and can also specifically and selectively enter the mitochondrial matrix, exerting an antioxidant effect by inhibiting cardiolipin oxidation. Therefore, these nanomicelles can rapidly target and accumulate in the mitochondrial region. Under the action of ROS, the small molecule linkers that specifically respond to reactive oxygen species break down and consume ROS. Through electron transfer, the mitochondrial protective drugs are released, synergistically acting on the mitochondria of damaged cells with SS-31, jointly exerting anti-inflammatory and anti-apoptotic effects.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an amphiphilic prodrug and its nanoparticle preparation method and application. Background Technology

[0002] Alzheimer's disease is a progressive cognitive impairment that severely impacts daily life and is a leading cause of dependence, disability, and death. Currently, approximately 55 million people worldwide suffer from dementia. With an aging population, the number of dementia patients globally is projected to reach 150 million by 2050, with about 60-70% of them having Alzheimer's disease (AD). The FDA-approved drugs for treating AD include three acetylcholinesterase inhibitors and one N-methyl-D-aspartate receptor antagonist. Unfortunately, these drugs are designed to increase neurotransmitter transmission and have no effect on disease progression. Typical pathophysiological features of Alzheimer's disease include β-amyloid aggregation and neurofibrillary tangles caused by Tau protein hyperphosphorylation. In recent years, drug development targeting these classic pathological features has failed, while clinical autopsies have revealed pathological features in the brains of AD patients such as increased oxidative stress, microglia-mediated neuroinflammation, and mitochondrial dysfunction. In fact, with increasingly in-depth research into the neuroimaging of Alzheimer's disease, AD is considered to be a disease caused by multiple factors, and multi-target therapy for AD is more likely to produce the expected therapeutic effect than single-target targeted therapy.

[0003] Cyclosporin A (CsA) is a cyclic peptide composed of 11 amino acids. It can inhibit the abnormal and persistent opening of the mitochondrial permeability transition pore, reduce the levels of intracellular ROS and pro-inflammatory factors, and decrease the release of apoptotic factors such as cytochrome c, thus exerting anti-inflammatory and anti-apoptotic effects. CsA can also act as a calcineurin inhibitor, inhibiting the dephosphorylation of nuclear factors in activated T cells and significantly reducing the expression of soluble apolipoprotein E4 (APOE4). Reduced APOE4 expression can improve the deposition of β-amyloid protein in the brain. Animal experiments have shown that in mice treated with CsA, the reduction in APOE4 protein expression occurs simultaneously with the reduction in β-amyloid protein. The central nervous system is protected by the blood-brain barrier, which prevents most drugs from crossing it. Therefore, it is necessary to design drugs to target and deliver CsA to damaged nerve cells and microglia, enabling it to target the mitochondria of damaged cells to exert its effects. Studies have shown that positively charged cell-penetrating peptides can cross the blood-brain barrier in a non-toxic manner. SS-31, a cationic peptide, is one such example, capable of facilitating the passage of CsA across the blood-brain barrier. SS-31 can selectively enter and accumulate in the mitochondrial matrix, exerting antioxidant effects by inhibiting cardiolipin oxidation. It can also synergistically protect mitochondria with CsA, exerting an anti-apoptotic effect.

[0004] Clinical studies have shown that ROS levels in the inflammatory environment of the brain of AD patients are significantly higher than in other parts of the brain. ROS-responsive nanomedicine carriers can fully utilize ROS in the microenvironment to regulate drug release. Ketothiols (TK) have a specific response to mitochondrial reactive oxygen species (mainly O2-) and can be rapidly degraded in 1 mM O2- aqueous solution.

[0005] Therefore, based on the pathological characteristics of increased oxidative stress, microglia-mediated neuroinflammation, and mitochondrial dysfunction in the brains of AD patients, TK was used to link lipophilic CsA and hydrophilic SS-31. CsA-TK-SS-31 can self-assemble in water to form reactive oxygen species-responsive and positively charged nanoparticles. The positively charged surface of the nanoparticles increases the blood-brain barrier penetration efficiency and cell entry efficiency, targeting ROS-producing neurons and microglia in the brain. Under the action of ROS, TK breaks and consumes ROS. Through electron transfer, the ester bond and amide bond of cyclosporine A glycine ester-ketothiol-SS-31 break, releasing CsA and SS-31, which further act on the mitochondria of damaged cells, exerting an "anti-inflammatory and anti-apoptotic" effect and reversing the chronic neuroinflammatory microenvironment in AD. Summary of the Invention

[0006] The first and second objectives of this invention are to provide an amphiphilic prodrug and its nanoparticles for mitochondrial targeted therapy. Through the mitochondrial targeting and positively charged transmembrane action of SS-31, the drug can quickly reach the damaged mitochondrial site. At the same time, the small molecule linker that specifically responds to reactive oxygen species breaks and consumes ROS, releasing the mitochondrial protective drug to further act on the mitochondria of damaged cells in conjunction with SS-31, and jointly exert anti-inflammatory and anti-apoptotic effects.

[0007] A third objective of this invention is to provide an amphiphilic prodrug for mitochondrial targeted therapy and a method for preparing the nanoparticle thereof.

[0008] A fourth objective of this invention is to provide an amphiphilic prodrug for mitochondrial targeted therapy and its nanoparticles for pharmaceutical use.

[0009] The first technical solution of the present invention is an amphiphilic prodrug for mitochondrial targeted therapy, characterized in that it is obtained by covalently bonding a hydrophilic short peptide for mitochondrial targeting with a hydrophobic mitochondrial protective drug through a small molecule linker that specifically responds to reactive oxygen species.

[0010] Furthermore, the mitochondrial-targeting hydrophilic short peptide is SS-31; the hydrophobic mitochondrial protective drug is selected from cyclosporine A; and the small molecule linker with specific reactive oxygen species response is selected from small molecule linkers containing ketithiolide structures.

[0011] The second technical solution of the present invention is an amphiphilic prodrug nanoparticle for mitochondrial targeted therapy, characterized in that the amphiphilic prodrug is obtained by self-assembly in water.

[0012] Furthermore, the outer layer of the nanoparticle is composed of the mitochondrial-targeting hydrophilic short peptide SS-31, and the inner layer is composed of the hydrophobic mitochondrial protective drug. The nanoparticle has a particle size of less than 200 nm and a potential of greater than 10 mV.

[0013] The third technical solution of the present invention is a method for preparing an amphiphilic prodrug for mitochondrial targeted therapy, characterized by comprising the following steps:

[0014] Step (1): Covalently bind the hydrophobic mitochondrial protective drug to a small molecule linker, and then covalently bind it to the mitochondrial-targeting hydrophilic short peptide SS-31 to obtain an amphiphilic prodrug with a targeting head group.

[0015] The amphiphilic prodrugs described in steps (2) and (1) are synthesized in an organic solvent. After the reaction is completed, the reaction solution is transferred to a dialysis bag and dialyzed in deionized water for 24 hours to remove the organic solvent, thereby obtaining an aqueous solution of targeted amphiphilic prodrug nanoparticles.

[0016] Furthermore, the organic solvent is selected from at least one of dimethyl sulfoxide, N,N'-dimethylformamide, pyridine, dichloromethane, and methanol.

[0017] The fourth technical solution of the present invention is an amphiphilic prodrug for mitochondrial targeted therapy and its nanoparticles, and their application in the preparation of drugs for treating Alzheimer's disease.

[0018] An amphiphilic prodrug for mitochondrial targeted therapy and its nanoparticles are used in the preparation of drugs for treating myocardial ischemia-reperfusion injury.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides a targeted amphiphilic prodrug with a target head group that can self-assemble in water to form nanoparticles with an outer layer of hydrophilic short peptide and an inner layer of hydrophobic mitochondrial protective drug. Because the hydrophilic short peptide SS-31 on the surface of the nanoparticles is positively charged, it can act as a cell-penetrating peptide to rapidly cross the cell membrane and even the blood-brain barrier in a non-toxic manner, and can also specifically and selectively enter the mitochondrial matrix to exert an antioxidant effect by inhibiting cardiolipin oxidation. Therefore, these nanoparticles can rapidly target and accumulate in the mitochondrial region. Under the action of ROS, the small molecule linkers that specifically respond to reactive oxygen species break down and consume ROS. Through electron transfer, the mitochondrial protective drug is released, synergistically acting on the mitochondria of damaged cells with SS-31 to jointly exert anti-inflammatory and anti-apoptotic effects.

[0021] This invention is based on the fact that the concentration of reactive oxygen species (ROS) in cells at the site of inflammation is much higher than that in normal cells. It introduces a small molecule linker containing a ketithiotillane structure that specifically responds to ROS, linking the hydrophilic SS-31 and the hydrophobic cyclosporine A to form an amphiphilic prodrug. By self-assembling in water to form uniformly sized nanoparticles, it not only has excellent mitochondrial targeting ability and positively charged transmembrane ability, but also significantly reduces its toxic side effects on normal tissues and cells because the ketithiotillane bond can only be broken inside the cell at the site of inflammation to release cyclosporine A. It also synergistically exerts anti-inflammatory and anti-apoptotic effects on mitochondria. Attached Figure Description

[0022] Figure 1 The MALDI-TOF mass spectrum of the amphiphilic prodrug A with a targeted head group in Example 1 is shown below.

[0023] Figure 2 The particle size and potential diagram of the nanoparticles with targeted prodrug A in Example 1;

[0024] Figure 3 Example 1: Nanoparticles with targeted prodrug A for use with Aβ 1-42 The therapeutic effect of damaged SH-SY5Y cells.

[0025] Figure 4 Example 1 illustrates the therapeutic effect of nanoparticles containing targeted prodrug A on hypoxia / reoxygenation-damaged H9C2 cardiomyocytes. Detailed Implementation

[0026] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Improvements and adjustments made by those skilled in the art based on the present invention in practical applications still fall within the scope of protection of the present invention.

[0027] Example 1

[0028]

[0029] 1.1 Synthesis of intermediate A-1

[0030] Accurately measure 15.0 mL of anhydrous pyridine into a 100 mL round-bottom flask. Accurately weigh CsA (3610.2 mg, 3.0 mmol) and add it to the flask. Sonicate for 30 seconds until completely dissolved. Purge with nitrogen and stir in an ice-water bath at 0°C for 10 minutes. Accurately weigh 1540.2 mg, 9.0 mmol of chloroacetic anhydride into the same flask. Stir in an ice-water bath for 10 minutes until completely dissolved. Remove the ice-water bath and stir overnight at room temperature. After the reaction is complete, transfer the reaction mixture to a separatory funnel. Accurately measure 45.0 mL of diethyl ether into the separatory funnel. Shake thoroughly to mix and allow to separate into layers. Discard the lower layer. Wash the ether layer with Grade I water (3 × 15.0 mL) until the ether is clear. After adding anhydrous sodium sulfate (5.0 g) and drying for 1 h, the solution was filtered and concentrated under reduced pressure to a pale yellow solid. Purification was achieved by silica gel column chromatography (50.0 g) using dichloromethane:ethyl acetate (V / V = 1:1) as the eluent. Thin-layer chromatography identified the target product. The eluent containing the target product was concentrated under reduced pressure to a white solid and crystallized in diethyl ether (20.0 mL). The crystals were placed in a vacuum drying oven and dried under vacuum at 37.0 °C for 24 h to obtain cyclosporine A chloroacetate (1921.5 mg, 1.5 mmol), with a yield of 50.0%.

[0031] 1.2 Synthesis of intermediate A-2

[0032] Accurately measure 10.0 mL of dimethyl sulfoxide into a 100 mL round-bottom flask, and accurately weigh 1921.5 mg (1.5 mmol) of sporein A chloroacetate into it. Dissolve the flask completely by heating and sonication. Accurately measure 12 mL of 0.5 mol / L sodium azide solution into the flask, purge with nitrogen, and stir at room temperature in the dark for 6 hours. After the reaction is complete, transfer the reaction mixture to a separatory funnel. Accurately measure 45.0 mL of diethyl ether into the separatory funnel, mix thoroughly by shaking, and allow to stand for separation. Discard the lower phase. Wash the ether layer with grade I water (3 × 15.0 mL) until the ether is clear. After adding anhydrous sodium sulfate (5.0 g) and drying for 1 h, the solution was filtered and concentrated under reduced pressure to a white solid. It was then placed in a vacuum drying oven and dried at 37.0 °C for 24 h to obtain cyclosporine A azidoacetate (1573.1 mg, 1.2 mmol). The obtained cyclosporine A azidoacetate (1573.1 mg, 1.2 mmol) was dissolved in methanol (10.0 mL), and grade I water (0.5 mL) was slowly added. A precise volume of 1.0 mol / L anhydrous stannous chloride solution (6 mL) was added to a round-bottom flask, and the mixture was stirred at room temperature for 6 h. After 6 h, ammonia water was slowly added to the round-bottom flask until the formation of a white precipitate ceased. The precipitate was removed by filtration, and the remaining reaction solution was transferred to a separatory funnel. A precise volume of diethyl ether (40.0 mL) was added to the separatory funnel, and the mixture was thoroughly shaken and allowed to stand for separation. The lower phase was discarded. The ether layer was washed with saturated sodium chloride solution (3 × 15.0 mL) until the ether was clear. After adding anhydrous sodium sulfate (5.0 g) and drying for 1 h, the solution was filtered and concentrated under reduced pressure to a white solid. The solid was then placed in a vacuum drying oven and dried under vacuum at 37.0 °C for 24 h to obtain sporein A glycine ester (985.3 mg, 0.78 mmol), with a yield of 52.0%.

[0033] 1.3 Synthesis of intermediate A-3

[0034] Accurately measure 10.0 mL of dichloromethane into a 100 mL round-bottom flask. Accurately weigh cyclosporine A glycine ester (985.3 mg, 0.78 mmol) and add it to the flask. Sonicate for 30 seconds until completely dissolved. Accurately weigh 5,5-dimethyl-4,6-dithio-azelanoic acid (ketothiols, TK) (300.4 mg, 1.2 mmol), 4-dimethylaminopyridine (24.4 mg, 0.2 mmol), and dicyclohexylcarbodiimide (206.9 mg, 1.0 mmol) and add them sequentially to the round-bottom flask. Stir the mixture at room temperature for 12 hours. After the reaction is complete, filter to remove the white precipitate and concentrate under reduced pressure to a white solid. Purify by silica gel column chromatography (50.0 g) using dichloromethane:methanol (V / V = 30:1) as the eluent. Thin-layer chromatography was used to identify the target product. The eluent containing the target product was concentrated under reduced pressure to a white solid and then placed in a vacuum drying oven. After vacuum drying at 37.0 °C for 24 h, cyclosporine A glycine ester-ketithiolide intermediate (789.6 mg, 0.53 mmol) was obtained, with a yield of 67.9%.

[0035] 1.4 Synthesis of Prodrug A

[0036] Accurately measure 5.0 mL of dimethyl sulfoxide into a 100 mL round-bottom flask. Accurately weigh 150.2 mg (0.1 mmol) of cyclosporine A glycine ester-ketothiol and add it to the flask. Sonicate for 30 seconds until completely dissolved and then purge with nitrogen for protection. Accurately weigh 38.3 mg (0.2 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 34.5 mg (0.3 mmol) of N-hydroxysuccinimide and add them to the flask. Activate at room temperature in the dark for 4 hours. Then accurately weigh 57.6 mg (0.1 mmol) of SS-31 and add it to the reaction solution. Continue the reaction for 24 hours. After 24 hours, the reaction solution was transferred to a dialysis bag (MWCO = 300D), dialyzed with deionized water for 24 hours, and the dialysate was freeze-dried to obtain cyclosporine A glycine ester-ketithiolide-SS-31 compound (42.5 mg, 0.02 mmol), with a yield of 20.1%.

[0037] Adding the lyophilized powder of the amphiphilic prodrug A prepared above to Grade I water yields an aqueous solution of the amphiphilic prodrug nanoparticles, which is the nanoparticle prepared in this embodiment based on prodrug A for mitochondrial targeted therapy, exhibiting reactive oxygen species response and synergistic anti-inflammatory and anti-apoptotic effects.

[0038] This embodiment uses the particle size and potential of the mitochondrial-protective nanoparticles based on prodrug A as follows: Figure 2 As shown, the average size of the nanoparticles is around 100 nm, and the potential is around 20 mV.

[0039] Good uniformity of nanoparticles can effectively reduce the differences between different batches during nanoparticle preparation, ensuring reliable quality stability. Secondly, good uniformity can ensure that drug nanoparticles have consistent pharmacokinetic, pharmacodynamic, and metabolic pathways during in vivo circulation, which helps them pass clinical evaluation.

[0040] This embodiment is based on the fact that the concentration of reactive oxygen species (ROS) produced by mitochondria in neurons and microglia in the brains of Alzheimer's disease patients is much higher than that in normal cells. A small molecule linker with a ketithiothiol bond that specifically responds to ROS was designed and introduced to link the hydrophilic mitochondrial-targeting peptide SS-31 and the hydrophobic mitochondrial protective drug cyclosporine A to form an amphiphilic conjugate. Through self-assembly in water, uniformly sized nanoparticles are formed. The positively charged surface of the nanoparticles increases the blood-brain barrier penetration efficiency and cell entry efficiency. Under the influence of large amounts of ROS produced by damaged neurons and microglia, the ketithiothiol bond breaks and consumes ROS. Through electron transport, the ester bond and amide bond of cyclosporine A glycine ester-ketithiothiol-SS-31 break, releasing cyclosporine A and SS-31. These further act on the mitochondria of damaged cells, synergistically exerting an "anti-inflammatory and anti-apoptotic" effect, reversing the chronic neuroinflammatory microenvironment in AD, and providing a new approach for multi-target therapy of Alzheimer's disease.

[0041] In this embodiment, the mitochondrial protective drug is cyclosporine A. A small molecule linker containing a ketithiothiolate bond first undergoes an acylation reaction with cyclosporine A esterified with glycine, and then undergoes an acylation reaction with SS-31 to obtain an amphiphilic prodrug containing a targeting head group. In other alternative embodiments, the mitochondrial protective drug may also be other existing mitochondrial protective drugs. In this case, the mitochondrial protective drug needs to introduce a group to achieve bonding with the small molecule linker containing the ketithiothiolate bond; these will not be elaborated further here.

[0042] Example 2

[0043] This embodiment evaluates the effect of amphiphilic prodrug nanoparticles with reactive oxygen species responsiveness and synergistic anti-inflammatory and anti-apoptotic effects on β-amyloid protein 1-42 (Aβ) for mitochondrial targeted therapy. 1-42 The study investigated the protective effect against damage to SH-SY5Y cells (human neuroblastoma cells). SH-SY5Y cells in logarithmic growth phase were trypsinized, resuspended in MEM / F12 medium, counted under a microscope, and diluted to 5×10⁻⁶. 4 Aβ cells / mL were seeded at 200 μL per well in a 96-well plate and cultured for 24 h. The normal control group was incubated with 200 μL of blank MEM / F12 medium for 24 h. 1-42 Model group: Add 200 μL of 10 μM Aβ 1-42Solution stimulation for 24 hours; CsA-TK-SS-31 prevention group: 2 hours beforehand, 200 μL of 5 μM and 15 μM CsA-TK-SS-31 were administered as pretreatment, followed by 22.2 μL of 100 μM Aβ. 1-42 Co-incubation in solution for 24 hours; CsA-TK-SS-31 treatment group: 200 μL of 10 μM Aβ added. 1-42 After 24 hours of solution stimulation, patients were treated with 200 μL of 5 μM and 15 μM sA-TK-SS-31 for 24 hours, respectively. The 96-well plates were then removed and, under aseptic conditions in a laminar flow hood, the supernatant was discarded. 200 μL of blank MEM / F12 medium containing 10% CCK-8 was added to each well, and the plates were returned to the cell culture incubator for another 3 hours. After 3 hours, the culture plates were removed, and the OD values ​​of each well were read at 450 nm using a microplate reader. A cell viability curve was plotted, and the cell viability was calculated using the formula: Cell viability (%) = (Experimental group OD value - Blank OD value) / (Normal control group OD value - Blank OD value) × 100. Results are as follows: Figure 3 As shown, SH-SY5Y cells were treated with 10 μM Aβ. 1-42 Following cell damage, CsA-TK-SS-31 at concentrations of 5 μM and 15 μM significantly enhanced cell viability. Furthermore, pretreatment of SH-SY5Y cells with 5 μM and 15 μM CsA-TK-SS-31 also reduced Aβ levels. 1-42 The results showed that CsA-TK-SS-31 has a strong cytoprotective effect against damage caused by 10 μM Aβ. In summary, CsA-TK-SS-31 exhibits protective effects against damage caused by 10 μM Aβ. 1-42 The protective effect of CsA-TK-SS-31 on damaged SH-SY5Y cells, and the significant reduction in Aβ levels when prevented in advance. 1-42 Damage to it suggests that CsA-TK-SS-31 affects Aβ-mediated... 1-42 Damaged SH-SY5Y cells have good preventive and therapeutic effects.

[0044] Example 3

[0045] This embodiment evaluates the therapeutic effect of amphiphilic prodrug nanoparticles with reactive oxygen species response and synergistic anti-inflammatory and anti-apoptotic effects on hypoxia / reoxygenation-damaged H9C2 cells (rat cardiomyocytes) for mitochondrial targeted therapy.

[0046] H9C2 cells in logarithmic growth phase were harvested, digested with trypsin, resuspended in DMEM high-glucose medium, counted under a microscope, and diluted to 5 × 10⁻⁶. 4Cells / mL were seeded at 200 μL per well in 96-well plates and cultured for 24 h. The normal control group was incubated for 16 h with 200 μL of blank DMEM high-glucose medium. The hypoxia / reoxygenation injury group was incubated for 4 h with 200 μL of hypoxic solution in a hypoxic incubator, followed by 12 h with 200 μL of serum-free DMEM high-glucose medium. The CsA-TK-SS-31 treatment group was incubated for 4 h with 200 μL of hypoxic solution in a hypoxic incubator, followed by 12 h with serum-free DMEM high-glucose medium containing different concentrations of CsA-TK-SS-31. After incubation, the 96-well plates were removed and, under aseptic conditions in a laminar flow hood, the supernatant was discarded. 200 μL of blank DMEM high-glucose medium containing 10% CCK-8 was added to each well, and the plates were returned to the cell culture incubator for another 2 h. Two hours later, the culture plate was removed, and the OD values ​​of each well were read at 450 nm using a microplate reader. A cell viability curve was plotted, and the cell viability was calculated using the formula: Cell viability (%) = (Experimental group OD value - Blank OD value) / (Normal control group OD value - Blank OD value) × 100. The results are as follows: Figure 4 As shown, CsA-TK-SS-31 at concentrations of 5 μM and 15 μM significantly improved the activity of H9C2 cells under hypoxia / reoxygenation injury, indicating that CsA-TK-SS-31 has a strong protective effect against hypoxia / reoxygenation injury to H9C2 cells.

Claims

1. An amphiphilic prodrug nanoparticle for mitochondrial targeted therapy, characterized in that, A hydrophilic short peptide targeting mitochondria is covalently bonded to a hydrophobic mitochondrial protective drug via a small molecule linker that specifically responds to reactive oxygen species, resulting in an amphiphilic prodrug with a targeting head group. The mitochondrial-targeting hydrophilic short peptide is SS-31, the hydrophobic mitochondrial protective drug is cyclosporine A, and the small molecule linker that specifically responds to reactive oxygen species is ketthioglycol. Ketothioglycol is first acylated with glycine-esterified cyclosporine A, and then acylated with SS-31 to obtain an amphiphilic prodrug containing a targeting head group. An amphiphilic prodrug with a targeting head group self-assembles in water to form uniformly sized nanoparticles with an outer layer of hydrophilic short peptide SS-31 and an inner layer of cyclosporine A; the nanoparticles have a particle size of less than 200 nm and a potential of greater than 10 mV. Because the hydrophilic short peptide SS-31 on the surface of the nanoparticles is positively charged, it can act as a cell-penetrating peptide to rapidly cross the cell membrane and even the blood-brain barrier in a non-toxic manner, and quickly target mitochondria. It specifically and selectively enters the mitochondrial matrix, exerting an antioxidant effect by inhibiting cardiolipin oxidation. At the same time, under the action of ROS, the small molecule linker that specifically responds to reactive oxygen species breaks and consumes ROS. Through the electron transfer effect, it releases the mitochondrial protective drug cyclosporine A, which works synergistically with SS-31 to further act on the mitochondria of damaged cells, restore the dynamic balance of mitochondrial division and fusion, and jointly exert anti-inflammatory and anti-apoptotic effects. At the same time, because the ketithiothiolate bond can only be broken in the intracellular space of the inflamed area to release cyclosporine A, its toxic side effects on normal tissues and cells are significantly reduced.

2. The use of the amphiphilic prodrug nanoparticles for mitochondrial targeted therapy as described in claim 1 in the preparation of drugs for treating Alzheimer's disease.

3. The use of the amphiphilic prodrug nanoparticles for mitochondrial targeted therapy as described in claim 1 in the preparation of drugs for treating myocardial ischemia-reperfusion injury.

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