Prodrug nano-platform for activating ferroptosis and eliminating senescent cells as well as preparation method and application of prodrug nano-platform

Abstract

The invention discloses a prodrug nano-platform for activating ferroptosis and eliminating senescent cells as well as a preparation method and application of the prodrug nano-platform. According to the invention, an adamantylated beta-galactosidase (beta-gal) probe and a photosensitizer are assembled to a calix [4] arene macrocycle in a subject-object manner, and functionalization is carried out by using ferritin targeted polypeptide. The prepared targeted prodrug nano-platform (HK-PCGC) does not need exogenous radiation, can realize the effects of efficiently removing senescent cells in a targeted manner, reducing the inflammatory senescence degree and improving senescence-related pathological symptoms by virtue of a photodynamic cascade reaction triggered by senescent cells and a ferroptosis excitation technology, has no obvious influence on normal tissues, and is suitable for clinical application. Therefore, the polypeptide has the potential of being developed into drugs and technologies for resisting aging and preventing and treating aging-related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomaterials, and particularly relates to a prodrug nanoplatform for activating ferroptosis to eliminate senescent cells, its preparation method and application. The present invention relates to a prodrug nanoplatform capable of achieving on-demand activation of ferroptosis to eliminate senescent cells and its preparation method. This prodrug nanoplatform can be applied to the targeted delivery of senescent cell-eliminating drugs. Background Art

[0002] Aging is an irreversible process of declining tissue function and physical activity ability, which is closely related to the occurrence and development of degenerative diseases such as Alzheimer's disease, cardiovascular diseases, cancer, age-related macular degeneration, and arthritis under pathological conditions. Although its underlying mechanisms have not been fully elucidated, cellular senescence is considered to be the core event underlying progressive aging. Cellular senescence refers to a state mainly characterized by cell cycle arrest and the secretion of pro-inflammatory cytokines and extracellular matrix proteins, namely the senescence-associated secretory phenotype (SASP). Senescence is also accompanied by organelle dysfunction, such as mitochondrial distortion, endoplasmic reticulum stress, and lysosomal defects, which are mainly manifested as impaired degradation ability and high expression of senescence-associated β-galactosidase (SA-β-Gal). Although it has beneficial effects in some biological processes such as wound healing and embryogenesis, the excessive accumulation of senescent cells is associated with aging and age-related diseases. Therefore, selective elimination of senescent cells (SnCs) is regarded as a promising method for delaying aging and alleviating age-related pathologies. Some anti-aging drugs, such as dasatinib and quercetin (DQ), ABT-263 / 737, HSP90 inhibitors, cardiotonic glycosides, and procyanidin C1, have been continuously discovered and shown positive results in preclinical and clinical trials. However, these preparations still face a series of problems in truly entering the clinic, such as insufficient specificity, limited therapeutic effect, unsatisfactory biocompatibility, and side effects.

[0003] Ferroptosis is a newly discovered non-apoptotic form of cell death, which is mainly triggered by the accumulation of intracellular lipid peroxides (LPO) and iron overload. Lipid peroxidation is closely related to ferroptosis and is mainly triggered by non-enzymatic (Fenton reaction) and enzymatic (such as lipoxygenase) processes. The excessive deposition of lipid oxides on biomembranes can increase membrane permeability and promote cell death. Iron overload is another important hallmark of ferroptosis. Free ferrous ions can convert H2O2 into toxic hydroxyl radicals (OH-), which act on polyunsaturated fatty acids (PUFA) and related phospholipids, thereby promoting lipid peroxidation and ferroptosis. Under physiological conditions, intracellular iron balance can be regulated by transferrin receptors (uptake), ferroportin (export), and ferritin (storage), etc. The occurrence of ferroptosis requires the autophagic degradation of ferritin to release stored iron, thus becoming an important target for regulating iron ion balance and ferroptosis.

[0004] Recently, ferroptosis-based cell therapy has shown encouraging results in combating cancer, but its potential as a senescent cell clearance method has not been explored. Existing studies have shown that although senescent cells are characterized by iron accumulation, most of the intracellular iron ions are trapped in ferritin. How to release free iron by inducing ferritin degradation is the key to achieving selective cell ferroptosis. On the other hand, although senescent cells show increased levels of reactive oxygen species, cytotoxic free radicals such as HO- and RO- are not sufficiently produced, which hinders the generation of lipid peroxides and the autophagic degradation of ferritin. This suggests that by cleverly utilizing the iron accumulation characteristics of senescent cells and regulating ferritin autophagy and oxygen free radical levels, it is promising to develop an anti-aging therapy driven by endogenous ferroptosis. Summary of the Invention

[0005] Object of the Invention: The technical problem to be solved by the present invention is to provide a prodrug nanoplatform that can achieve on-demand activation of ferroptosis to eliminate senescent cells, and its preparation method and application, in view of the deficiencies of the prior art.

[0006] To achieve targeted removal of SnCs, reduce the level of senescence-associated secretory phenotype (SASP), and improve the physical condition of aged mice, the present invention provides a prodrug nanoplatform that can achieve on-demand activation of ferroptosis to eliminate senescent cells, its preparation method, and its application in removing SnCs, reducing the level of senescence-associated secretory phenotype (SASP), and improving the physical condition of aged mice. The present invention discloses the following technical solutions:

[0007] A preparation method of a prodrug nanoplatform for activating ferroptosis to eliminate senescent cells, comprising the following steps:

[0008] (1) Mix dibenzocyclooctyne polyethylene glycol carboxylic acid, N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine, and calix[4]arene, and after reaction, dialyze to obtain an intermediate product DBCO-PEG-calix[4]arene;

[0009] (2) Mix the obtained DBCO-PEG-calix[4]arene with a ferritin-targeting polypeptide, and after click chemical reaction, dialyze to obtain the main body HK-PC of the prodrug nanoplatform;

[0010] (3) Mix the obtained HK-PC with a photosensitizer solution and a β-galactosidase-responsive probe solution to obtain the prodrug nanoplatform HK-PCGC.

[0011] Among them, in step (1), the weight-average molecular weight (Mw) of the dibenzocyclooctyne polyethylene glycol carboxylic acid is 4000-6000 Da, preferably 5000 Da; the mixed molar ratio of the dibenzocyclooctyne polyethylene glycol carboxylic acid, N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and calix[4]arene is 0.5-1.5:1-4:0.1-0.5:0.5-1.5. Preferably 1:3:0.3:1.

[0012] Among them, in step (1), the reaction is carried out under nitrogen, the reaction solvent is dimethyl sulfoxide, the reaction temperature is 50-65 °C, the reaction time is 20-32 h, preferably reacting at 60 °C for 24 h; for the dialysis, the cut-off molecular weight of the dialysis membrane is 3000-4000 Da, preferably 3500 Da.

[0013] Among them, in step (2), the ferritin-targeting polypeptide is HK-N3, and its structural formula is shown in Formula I:

[0014]

[0015] Among them, in step (2), the mixed molar ratio of the DBCO-PEG-calix[4]arene and the ferritin-targeting polypeptide is 1-2:1-2, preferably 1:1.

[0016] Among them, in step (2), for the click chemical reaction, the reaction solvent is pure water, the reaction is carried out at room temperature, the reaction time is 4-8 h, preferably 6 h; for the dialysis, the cut-off molecular weight of the dialysis membrane is 5000-7000 Da, preferably 6000 Da.

[0017] Preferably, in step (2), for the prepared host HK-PC, in an aqueous solution, its hydrophobic part will aggregate together to form the core of the micelle, while the hydrophilic part faces outward and contacts the aqueous phase to form a stable micelle structure. After the micelle is formed, this molecular recognition ability is retained and can bind to the hydrophobic photosensitizer and β-galactosidase-responsive probe in step (3).

[0018] Among them, in step (3), the photosensitizer is chlorin e6 (Ce6); the β-galactosidase-responsive probe is an adamantane-functionalized sugar probe (GD), preferably Thermo Fisher Galacton-Plus TM probe.

[0019] Among them, in step (3), the HK-PC is mixed with the photosensitizer solution and the β-galactosidase-responsive probe solution in the form of an HK-PC aqueous solution; the mixing volume ratio of the HK-PC aqueous solution, the photosensitizer solution and the β-galactosidase-responsive probe solution is 23:1:1.

[0020] The concentration of the HK-PC aqueous solution is 0.5-2 mg / mL, preferably 1 mg / mL;

[0021] The photosensitizer solution has a concentration of 0.5-2 mg / mL, and the solvent is isopropanol, preferably with a concentration of 1 mg / mL;

[0022] The β-galactosidase-responsive probe solution has a concentration of 0.5-2 mg / mL, and the solvent is dimethyl sulfoxide, preferably with a concentration of 1 mg / mL.

[0023] Preferably, in step (3), the HK-PC, the photosensitizer solution, and the β-galactosidase-responsive probe solution are mixed and then subjected to a water bath sonication treatment to ensure that the hydrophobic photosensitizer and the β-galactosidase-responsive probe completely enter the hydrophobic cavity of the host molecule. The sonication treatment time is further preferably 30 min.

[0024] Preferably, in step (3), the generated HK-PCGC is in the form of nanoparticles (NPs), and the average particle size is 124 nm.

[0025] Preferably, in step (3), the β-galactosidase-responsive probe in the generated HK-PCGC prodrug nanoplatform is cleaved by β-galactosidase (β-gal) highly expressed in senescent cells, resulting in the release of fluorescent luminol. Through chemiluminescence resonance energy transfer (CRET), Ce6 is excited, and then Ce6 emits red light and generates a large amount of reactive oxygen species (ROS).

[0026] In a second aspect, the present invention provides a prodrug nanoplatform for activating ferroptosis to eliminate senescent cells prepared by the preparation method described in the first aspect.

[0027] In a third aspect, the present invention provides the use of the prodrug nanoplatform for activating ferroptosis to eliminate senescent cells described in the second aspect in the preparation of anti-aging drugs. Preferably, the prodrug nanoplatform can target and respond to senescent cells, and a large amount of reactive oxygen species (ROS) can be generated after being taken up by senescent cells. The ROS released by the prodrug nanoplatform drives the killing of senescent cells. Importantly, under the guidance of the ferritin-targeting polypeptide, the nanoparticles are promoted to reach ferritin and promote its autophagy degradation to release free iron, resulting in ferroptosis of senescent cells through an endogenous Fenton reaction with lipid peroxidation (LPO).

[0028] Beneficial effects:

[0029] The present invention provides a prodrug nanoplatform that can achieve on-demand activation of ferroptosis to eliminate senescent cells for anti-aging therapy. After internalization by cells, the prepared targeted prodrug nanoplatform (HK-PCGC) is enzymatically hydrolyzed by β-gal highly expressed in senescent cells (SnCs) to release the fluorescence - luminol; this fluorescent molecule further excites the photosensitizer through chemiluminescent resonance energy transfer, promoting the generation of a large amount of reactive oxygen species. The released superoxide ions drive the death of SnCs on the one hand and target ferritin on the other hand, promoting the release of free iron and triggering ferroptosis. The prodrug nanoplatform prepared by the present invention has the following beneficial effects:

[0030] (1) The HK-PCGC prodrug nanoplatform has a stable structure and good size uniformity;

[0031] (2) The HK-PCGC prodrug nanoplatform can target and respond to senescent cells;

[0032] (3) The HK-PCGC prodrug nanoplatform can target ferritin and promote its autophagic degradation to release free iron, leading to ferroptosis of senescent cells through the endogenous Fenton reaction with lipid peroxidation (LPO);

[0033] (4) The HK-PCGC prodrug nanoplatform can be used to remove SnCs, reduce the level of senescence-associated secretory phenotype (SASP), and improve the physical condition of old mice.

[0034] The present invention utilizes laser-free photothermal therapy (Laser-free PDT) to induce ferroptosis, thereby achieving the purpose of efficiently clearing senescent cells. Laser-free PDT can excite internal luminescence to exert a therapeutic effect without external light input. Technically, chemiluminescence has been demonstrated to have excellent ability to excite photosensitizers such as chlorin e6 (Ce6) through chemiluminescence resonance energy transfer (CRET), and subsequently enable it to generate cytotoxic singlet oxygen with a large Stokes shift. In the system of the present invention, a SA-β-Gal responsive probe is applied, and by combining a galactose moiety with adamantane-dioxetane, a senescent cell-responsive chemical reaction and subsequent CRET effect are achieved to realize specific killing. In addition, in order to precisely initiate the anti-aging treatment driven by ferroptosis, the photodynamic therapy system is also equipped with cleaved high molecular weight kininogen (HKa), which serves as a ferritin-targeting protein, directing the internalized photodynamic therapy complex to the ferritin cage, inducing an oxidative burst, and simultaneously triggering ferritin degradation to release ferrous ions. Finally, in order to improve the sensitivity and efficiency of the photodynamic therapy system, it is considered to combine a senescent cell-responsive signal sensor with a chemiluminescence resonance energy transfer mechanism in a limited space. For this purpose, the present invention utilizes a macrocyclic host molecule - calix[4]arene, whose main advantage lies in its ability to effectively load and assemble hydrophobic guest compounds to form various nanostructures. The macrocyclic substance also has characteristics such as low cytotoxicity, adjustable structure, easy functionalization, and responsiveness to external stimuli, making it a promising drug carrier platform. In summary, the senescent cell-clearing prodrug nanoplatform of the present invention ingeniously combines technologies such as senescent cell response, laser-free PDT, and endogenous ferroptosis induction to achieve the purpose of targeted clearance of senescent cells and improvement or treatment of senescent pathologies, and has broad application prospects. The target prodrug nanoplatform (HK-PCGC) prepared by the present invention does not require external radiation. The photodynamic cascade reaction and ferroptosis excitation technology triggered by senescent cells can achieve the effects of efficiently and targeted clearing senescent cells, reducing the degree of inflammatory senescence, and improving senescent-related pathological symptoms, and has no obvious impact on normal tissues. Therefore, it has the potential to be developed into drugs and technologies for anti-aging and prevention and treatment of senescent-related diseases. Brief Description of the Drawings

[0035] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0036] Figure 1 1H-NMR characterization diagram of HK-PEG-calix[4]arene (HK-PC). Among them, a is the calix[4]arene group, b is the dibenzocyclooctyne polyethylene glycol carboxyl group, and c is HK-N3. 1 1H-NMR) characterization diagram. Among them, a is the calix[4]arene group, b is the dibenzocyclooctyne polyethylene glycol carboxyl group, and c is HK-N3.

[0037] Figure 2 These are the characterization diagrams of HK-PC and HK-PCGC. Figure 2 In [Figure A], it is the particle size change diagram. Figure 2 In [Figure B], it is the potential change diagram.

[0038] Figure 3 These are the detection diagrams of the luminescence efficiency of luminol and Ce6 in the nano-prodrug. Figure 3 In [Figure A], it is the luminescence detection diagram of luminol and Ce6. Figure 3 In [Figure B], it is the fluorescence image of Ce6. Figure 3 In [Figure C], it is the quantitative analysis of the fluorescence intensity of Ce6.

[0039] Figure 4 These are the detection diagrams of the promotion of ferritin autophagy and ferroptosis by the HK-PCGC prodrug nano-platform. Figure 4 In [Figure A], it is the generation of free ferrous ions. Figure 4 In [Figure B], it is the Western blot detection of ferritin levels. Figure 4 In [Figure C], it is the detection of ferroptosis rate.

[0040] Figure 5 These are the effect diagrams of the effective removal of SnCs by HK-PCGC. Figure 5 In [Figure A], it is the SA-β-gal staining diagram; Figure 5 In [Figure B], it is the statistical analysis of the SA-β-gal ratio; Figure 5 In [Figure C], it is the change of senescence secretory phenotype.

[0041] Figure 6 These are the safety evaluation diagrams in the animal experiment application of HK-PCGC. Figure 6 In [Figure A], it is the HE section; Figure 6 In [Figure B], it is the blood biochemical indexes. Specific Embodiments

[0042] In the following examples, the experimental methods are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial sources unless otherwise specified.

[0043] The SA-β-Gal responsive probe GD used in the following examples is an adamantane-functionalized sugar probe, purchased from Thermo Fisher Galacton-Plus TM , with the product number T2118.

[0044] The polypeptide containing three nitrogen groups (HK-N3) used in the following examples is purchased from Wuhan Dangun Biotechnology Co., Ltd., and its structural formula is shown in Formula I:

[0045]

[0046] Unless otherwise specified, the PBS mentioned in the following examples is a 0.01 M PBS buffer solution with a pH of 7.2 - 7.4. In the present invention, w / v, unless otherwise specified, is g / mL.

[0047] Example 1 Preparation of HK - PCGC NPs Prodrug Nanoparticle Platform

[0048] Preparation of the prodrug nanoparticle platform main body HK - PEG - calix[4]arene includes the following steps: (1) Preparation of the intermediate DBCO - PEG - calix[4]arene; (2) Preparation of the prodrug nanoparticle platform main body HK - PEG - calix[4]arene; (3) Generating the HK - PCGC NPs prodrug nanoparticle platform by combining the main body HK - PEG - calix[4]arene with the guests Ce6 and GD. The specific operations are as follows:

[0049] (1) Dissolve 68.3 mg of dibenzocyclooctyne polyethylene glycol carboxylic acid (DBCO - PEG - COOH, Mw: 5000 Da), 8.5 mg of N,N'-dicyclohexylcarbodiimide (DCC, a dehydrating agent that promotes the condensation reaction of carboxylic acid and alcohol), and 0.5 mg of 4 - dimethylaminopyridine (DMAP, a highly efficient nucleophilic acylation catalyst that can accelerate the reaction rate) in 4.5 mL of anhydrous dimethyl sulfoxide, and stir in a 60 °C water bath for 30 minutes to activate the carboxyl group. Subsequently, gradually add a solution of calix[4]arene (5.8 mg) in anhydrous dimethyl sulfoxide (0.5 mL) dropwise to the above solution, and stir in a 60 °C water bath under nitrogen protection for 24 hours. After the reaction is completed, add 90 mL of pure water and mix well, then centrifuge at 12000 rpm for 5 minutes to remove the precipitate. Transfer the supernatant to a dialysis membrane with a molecular weight cut - off of 3500 Da. After three rounds of water dialysis to remove unreacted substances, lyophilize the dialysis solution to obtain DBCO - PEG - calix[4]arene (DBCO - PEG - Calix[4]Arene), and its structural formula is as Figure 1 shown. The polymer is confirmed by hydrogen nuclear magnetic resonance ( 1 1H - NMR) in deuterated dimethyl sulfoxide.

[0050] (2) Dissolve 28 mg of the DBCO - PEG - calix[4]arene prepared in step (1) and 10 mg of the nitrogen - containing three - group polypeptide (ferritin - targeting polypeptide, HK - N3) in 20 mL of pure water, and stir at room temperature for 6 hours. Then transfer the solution to a dialysis membrane with a molecular weight cut - off of 6000 Da. After three rounds of water dialysis to remove unreacted substances, lyophilize the dialysis solution to obtain HK - PEG - Calix[4]Arene (HK - PEG - calix[4]arene, abbreviated as HK - PC). The polymer is confirmed by hydrogen nuclear magnetic resonance ( 1confirmed by 1H-NMR Figure 1 ) The chemical shift of calix[4]arene is approximately at 6.5 - 7 ppm, which corresponds to the proton peaks on the benzene ring of calix[4]arene. Meanwhile, the chemical shift of dibenzocyclooctyne polyethylene glycol carboxyl appears at approximately 7 - 8 ppm (proton peaks of dibenzocyclooctyne), while the chemical shift of HK-N3 is at approximately 6.5 - 7 ppm (proton peaks of the benzene ring of tryptophan).

[0051] (3) Prepare 500 μL of an aqueous solution containing 1 mg / mL of HK-PEG-calix[4]arene, then add a specified dose of chlorin e6 (Ce6, Ce6 concentration is 1 mg / mL) dissolved in isopropanol and a probe GD (GD concentration is 1 mg / mL) dissolved in dimethyl sulfoxide. The mixing volume ratio of the HK-PEG-calix[4]arene solution, Ce6 solution, and GD solution is 23:1:1. Subsequently, mix the solution thoroughly and sonicate it in a water bath for 30 minutes to ensure that the hydrophobic Ce6 and GD fully enter the hydrophobic cavity of the host molecule, generating HK-PCGC NPs nanoparticles. The Figure 2 results of A and B in Figure 2 confirmed the changes in particle size and potential of the prodrug, demonstrating the effectiveness of the synthesis reaction. The average particle size of HK-PCGC NPs nanoparticles shown in A in

[0052] Preparation of HK-PCG NPs nanoparticles: The same as the above steps, except that in step (3) above, the aqueous solution of HK-PEG-calix[4]arene is only mixed with the GD solution and then prepared into HK-PCG NPs nanoparticles according to the subsequent same steps.

[0053] Preparation of the control group: Meanwhile, the present invention also prepared non-ferritin-targeted control groups m-PCG and m-PCGC. The synthesis method of m-PCGC is similar to the method described in steps (1) - (3) above, except that the DBCO-PEG-COOH described in step (1) is replaced with methoxypolyethylene glycol carboxylic acid (Mw: 5000, 68.3 mg), and step (2) is not included. The synthesis method of m-PCG is similar to the method described in steps (1) - (3) above, except that the DBCO-PEG-COOH described in step (1) is replaced with methoxypolyethylene glycol carboxylic acid (Mw: 5000, 68.3 mg) to obtain m-PEG-calix[4]arene, step (2) is not included, and in step (3), the aqueous solution of m-PEG-calix[4]arene is only mixed with the GD solution for subsequent operations.

[0054] Example 2 Detection of CRET efficiency and senescence response efficiency of HK-PCGC NPs prodrug nanoplatform

[0055] (1) To verify the chemiluminescence resonance energy transfer (CRET) between GD and Ce6, 200 μL of an aqueous solution containing HK-PCGC (or other nanoparticles prepared in Example 1) was added to a 96-well plate, and then 5 U / mL of β-galactosidase (β-Gal) was added. The induced chemiluminescence intensity was detected. GD in the nanoplatform was enzymatically cleaved by β-gal to release the fluorescence molecule luminol; this fluorescent molecule further excited Ce6 to produce fluorescence through the chemiluminescence resonance energy transfer CRET. As Figure 3 shown in the luminescence detection graph A in

[0056] (2) Non-senescent or senescent A549 cells (1×10^5 cells per well, and the senescent cells were constructed by stimulating the cells with 0.5 μM mitomycin for 2 days, see the reference Adv Mater. 2024 Oct; 36(41): e2409329 for details) were treated with HK-PCGC NPs (equivalent Ce6 concentration of 10 μM) for 4 hours. Then the cells were collected, fixed with 4% paraformaldehyde and stained with DAPI. Imaging was performed using a laser confocal scanning microscope (laser A1R, Nikon, Japan) for characterization. As Figure 3 shown in B and C in

[0057] Example 3 Detection of the promotion of ferritin autophagy and ferroptosis by the HK-PCGC HK prodrug nanoplatform

[0058] (1) The senescent A549 cells were seeded in a T75 cell culture flask and cultured for 24 hours. Then, different nanodrugs (m-PCGC, HK-PCGC, or HK-PCGC+Fer-1, and the addition concentration of the nanodrugs was calculated according to the concentration of Ce6, which was 6 μg / mL) were added to the corresponding cell cultures and cultured for another 4 hours, while adding only an equal volume of PBS as a control. Subsequently, the cells in the culture flask were collected and resuspended in 1 mL of PBS to obtain a cell suspension for subsequent analysis. Specifically, 800 μL of the cell suspension was ultrasonically treated, lysed, and centrifuged in an ice bath to obtain the supernatant, which was then transferred to a 30KDa ultrafiltration centrifugal tube and centrifuged at 3500 g for 20 minutes. The filtrate was collected, and the content of ferrous ions was detected according to the instructions of the ferrous ion detection kit (Solarbio, BC5415). The remaining 200 μL of the cell suspension was centrifuged to obtain a cell pellet, which was incubated in 50 μL of RIPA lysis buffer for lysis. After centrifugation, the lysate was recovered, and the total protein was quantified using the BCA assay. The content of free ferrous ions was expressed as the iron content per milligram of total protein. As Figure 4 shown in A of

[0059] , the HK-PCGC nanodrug could significantly increase the content of ferrous ions in cells. In this example, HK-PCGC+Fer-1 represents the simultaneous addition of HK-PCGC and the ferroptosis inhibitor (Ferrostatin-1) with an addition concentration of 1 μM. Figure 4 (2) 200 μL of the cells in the culture flask obtained in step (1) were collected and incubated (incubated on ice) in 200 μL of lysis buffer containing a protease inhibitor mixture (purchased from Beyotime, and the formula includes 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium azide). An equal volume of the sample was separated by 10% SDS-PAGE gel electrophoresis. The separated proteins were then transferred to a PVDF membrane and blocked with 5% skim milk. Then the membrane was incubated with ferritin antibody overnight at 4 °C, and then incubated with an appropriate horseradish peroxidase-conjugated secondary antibody. An electrochemiluminescence (ECL) detection substrate was added, and the target band was visualized using a ChemiDoc imaging system (Bio-Rad, USA). As Figure 4 shown in B of

[0060] (3) Cell death was detected by the lactate dehydrogenase (LDH) assay (cytotoxicity detection kit). For this purpose, 8×10 3 senescent A549 cells were seeded and co-incubated with HK-PCGC NPs (or other nanoplatforms prepared in Example 1) (refer to Step 1 of this Example), and appropriate controls were set up, including a background control containing only the culture medium and a maximum enzyme activity control of cells without drug treatment. One hour before the predetermined time, 150 μL of LDH release reagent was added to the "maximum enzyme activity control". All cell cultures were harvested as planned and centrifuged at 400 g for 5 minutes. Then, 120 μL of the supernatant was collected, mixed with 60 μL of LDH detection working solution, and incubated in the dark at room temperature for 30 minutes. The absorbance was measured at 490 nm. The iron death induced by nanoparticles with or without 1 μM Fer-1 was detected according to the above steps. The cytotoxicity (%) was calculated as follows: Cytotoxicity (%) = (Absorbance of treated sample - Absorbance of background blank control) / (Absorbance of maximum enzyme activity control - Absorbance of background blank control) × 100. As shown in C of Figure 4 , after treatment with HK-PCGC nanodrugs, the cell mortality rate increased to 90%.

[0061] Example 4 Detection of the effective removal of SnCs by HK-PCGC

[0062] 18-month-old C57BL / 6 mice (25 - 30 g, female) were intravenously infused (the nanodrugs prepared in Example 1 were dissolved in PBS and infused in the form of a drug solution), twice every two weeks (two times in a row on two consecutive days at the beginning of every two weeks), and given the drug HK-PCGC NPs (equivalent Ce6 dose of 5 mg / kg), and the classical anti-aging drugs dasatinib and quercetin (D: 0.7 mg / kg, Q: 2.6 mg / kg) were used as positive controls, and were administered intravenously, with a dosing frequency of twice every two weeks (two times in a row on two consecutive days at the beginning of every two weeks). After 8 weeks, the main organs of the senescent mice (including the liver, lung, and kidney) were obtained to detect the number of SA-β-gal positive cells and the changes in the senescent secretory phenotype. The results showed that the HK-PCGC nanodrugs could effectively remove SnCs and significantly reduce the senescent phenotypes of the liver, lung, and kidney ( Figure 5 A and B in). The heat map analysis of the differentially expressed genes (DGs) in the lung and liver showed that HK-PCGC attenuated the expression of genes related to inflammatory cytokines, chemokines, proteases, and senescence-associated secretory phenotype (SASP) in the lungs and livers of old mice ( Figure 5 C in). Figure 5 young in represents 8-week-old C57BL / 6 mice.

[0063] Safety Evaluation in the Application of HK-PCGC Animal Experiment in Example 5

[0064] 18-month-old C57BL / 6 mice were intravenously infused in the manner described in Example 4, twice every two weeks, with HK-PCGC NPs (equivalent Ce6 dose of 5 mg / kg), and the classical anti-aging drugs dasatinib and quercetin (D: 0.7 mg / kg, Q: 2.6 mg / kg) were used as positive controls. After 8 weeks, pathological sections were taken from the major organs of the aging mice (including the heart, spleen, liver, lungs, and kidneys), and blood samples were taken for routine toxicological examinations. The results showed that there were no obvious toxic and side effects in the major organs of the mice ( Figure 6 A in). As Figure 6 shown in B in, in terms of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), nanoparticle treatment had no significant effect on liver function; measured by creatinine (CR) and uric acid (UA), there was also no significant effect on kidney function; and there was also no significant effect on lipid metabolism factors involving triglyceride (TG) and cholesterol (TC).

[0065] Therefore, the HK-PCGC constructed in the present invention can effectively remove senescent cells, reduce the level of senescence-associated secretory phenotype (SASP), improve the physical function of old mice, and show minimal adverse effects.

[0066] In summary, the prodrug nanoparticle platform capable of activating ferroptosis on demand constructed in the present invention can effectively target and respond to clear senescent cells, and has good therapeutic effects in anti-aging treatment.

[0067] The present invention provides a prodrug nanoparticle platform capable of activating ferroptosis on demand to clear senescent cells, and ideas and methods for its preparation and application. There are many specific methods and ways to implement this technical solution. The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be realized by the prior art.

Claims

1. A preparation method of a prodrug nano-platform for activating ferroptosis to eliminate senescent cells, characterized in that, The following steps are involved: (1) dibenzocyclooctyne polyethylene glycol carboxylic acid, N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and calix[4]arene are mixed, reacted and dialyzed to obtain an intermediate product DBCO-PEG-calix[4]arene; (2) mixing the DBCO-PEG-calix[4]arene with the ferritin targeting peptide, and performing a click chemistry reaction followed by dialysis to obtain the prodrug nanoplatform HK-PC; (3) Mixing the HK-PC with a photosensitizer solution and a β-galactosidase response probe solution to obtain a prodrug nanoplatform HK-PCGC.

2. The preparation method according to claim 1, characterized in that In step (1), the weight average molecular weight of the dibenzocyclooctyne polyethylene glycol carboxylic acid is 4000-6000 Da; the mixing molar ratio of the dibenzocyclooctyne polyethylene glycol carboxylic acid, N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and calix[4]arene is 0.5-1.5:1-4:0.1-0.5:0.5-1.

5.

3. The preparation method according to claim 1, wherein In step (1), the reaction is carried out under nitrogen, the reaction solvent is dimethyl sulfoxide, the reaction temperature is 50-65° C., and the reaction time is 20-32 h; and the dialysis has a dialysis membrane with a molecular weight cutoff of 3000-4000 Da.

4. The preparation method according to claim 1, wherein In step (2), the ferritin targeting polypeptide is HK-N3, and its structural formula is shown in Formula I:

5. The preparation method according to claim 1, characterized in that, In step (2), the mixing molar ratio of the DBCO-PEG-calix[4]arene and the ferritin targeting polypeptide is 1-2:1-2.

6. The preparation method according to claim 1, characterized in that, In step (2), the click chemistry reaction is carried out with pure water as the reaction solvent at room temperature for 4 to 8 hours; and the dialysis is carried out with a dialysis membrane having a molecular weight cutoff of 5000 to 7000 Da.

7. The preparation method according to claim 1, characterized in that, In step (3), the photosensitizer is dihydrochlorin e6; and the β-galactosidase response probe is an adamantane functionalized sugar probe.

8. The preparation method according to claim 1, characterized in that, In step (3), the HK-PC is mixed with the photosensitizer solution and the β-galactosidase response probe solution in the form of a HK-PC aqueous solution, and the concentration of the HK-PC aqueous solution is 0.5 to 2 mg / mL; The photosensitizer solution has a concentration of 0.5-2 mg / mL and the solvent is isopropanol; The β-galactosidase response probe solution has a concentration of 0.5 to 2 mg / mL and the solvent is dimethyl sulfoxide; The mixed volume ratio of the HK-PC aqueous solution, the photosensitizer solution and the β-galactosidase response probe solution is 23:1:

1.

9. A prodrug nanoplatform for activating ferroptosis and eliminating senescent cells prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the prodrug nanoplatform for activating ferroptosis and eliminating senescent cells as claimed in claim 9 in the preparation of anti-aging drugs.

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