Iron-based polypeptide probe for regulating mitochondrial function and application of iron-based polypeptide probe in tumor inhibition drugs

Abstract

The invention belongs to the technical field of nanoprobes, and relates to an iron-based polypeptide probe for regulating and controlling a mitochondrial function and application of the iron-based polypeptide probe in tumor inhibition drugs. The invention discloses an iron-based polypeptide probe for regulating and controlling a mitochondrial function, the iron-based polypeptide probe for regulating and controlling the mitochondrial function comprises superparamagnetic nano iron oxide SPION and a polypeptide ligand modified on the surface of the superparamagnetic nano iron oxide SPION, and the polypeptide ligand is an N-terminal succinylated D-type neuropeptide Y series polypeptide ligand S-DNPY; the iron-based polypeptide probe for regulating and controlling the mitochondrial function competitively inhibits a mitochondrial succinylation behavior under the action of SIRT5 enzyme and NAD + coenzyme on mitochondria, in-situ self-assembly is performed after succinylation is performed, iron-based polypeptide probe aggregation is realized, a mitochondrial membrane is damaged, and the mitochondrial function is regulated and controlled. And abnormal oxidation is triggered, and cell apoptosis and autophagy clearance of damaged mitochondria are induced. The invention also discloses a medicine for inhibiting tumors, which comprises the iron-based polypeptide probe for regulating and controlling the mitochondrial function.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanoprobes and relates to an iron-based polypeptide probe for regulating mitochondrial function and its application in tumor inhibition drugs. Background Art

[0002] Mitochondria are double-membrane organelles within cells, often referred to as the cell's "energy factory." They are present in most eukaryotic cells and have crucial functions, particularly in energy production, metabolic regulation, and cell death.

[0003] High expression of SIRT5 promotes tumor cell proliferation and affects breast cancer growth and metastasis by desuccinylation of multiple proteins and cytokines. Interference or silencing of SIRT5 in tumor cells leads to increased succinylation of IDH2 and other metabolic enzymes, significantly reduced NADPH and GSH levels, and enhanced induction of autophagy-related genes.

[0004] Highly specific probes for precisely regulating mitochondria to combat tumor cell proliferation have yet to be developed. This situation has severely hampered research efforts to delve deeper into the connection between mitochondria and tumor cell proliferation, and has also hindered the development of tumor treatment strategies based on mitochondrial regulation. Breakthrough innovations are urgently needed to fill this gap. Summary of the Invention

[0005] The purpose of the present invention is to address the above-mentioned problems existing in the prior art and propose an iron-based polypeptide probe for regulating mitochondrial function. Its surface-modified polypeptide ligand can effectively target the Y1R receptor on the MCF-7 cell membrane, regulating the mitochondrial oxidative stress process and cell apoptosis pathway.

[0006] One object of the present invention is achieved by the following technical solutions:

[0007] An iron-based polypeptide probe for regulating mitochondrial function, comprising superparamagnetic nano-iron oxide SPION with an average particle size of 0.1 to 5 μm and a polypeptide ligand modified on its surface, wherein the polypeptide ligand is an N-terminally succinylated D-type neuropeptide Y series polypeptide ligand S- D NPY;

[0008] The iron-based polypeptide probe regulating mitochondrial function, SIRT5 enzyme and NAD on mitochondria + Under the action of coenzyme, it competitively inhibits the desuccinylation behavior of mitochondria, and then self-assembles in situ after desuccinylation, realizing the aggregation of iron-based polypeptide probes, damaging the mitochondrial membrane, causing oxidative abnormalities and inducing cell apoptosis and autophagic clearance of damaged mitochondria.

[0009] Preferably, the polypeptide ligand is selected from any one or more of the following peptide segments and all amino acids are D-type: NPY, [Leu3]NPY, [Cys34]NPY, [Leu3,Cys34]NPY, [Leu17]NPY, [Leu3,Leu17]NPY, [Leu17,Cys34]NPY, [Leu3,Leu17,Cys34]NPY, [Leu10]NPY, [Leu3 ,Leu10]NPY, [Leu10,Cys34]NPY, [Leu3,Leu10,Cys34]NPY, [Leu10,Leu17]NPY, [Leu3,Leu10,L eu17]NPY, [Leu10,Leu17,Cys34]NPY, [Leu3,Leu10,Leu17,Cys34]NPY, [Asn6]NPY, [Leu3,Asn6] NPY, [Asn6,Cys34]NPY, [Leu3,Asn6,Cys34]NPY, [Asn6,Leu17]NPY, [Leu3,Asn6,Leu17]NPY, [A sn6,Leu17,Cys34]NPY, [Leu3,Asn6,Leu17,Cys34]NPY, [Asn6,Leu10]NPY, [Leu3,Asn6,Leu10]N PY, [Asn6,Leu10,Cys34]NPY, [Leu3,Asn6,Leu10,Cys34]NPY, [Asn6,Leu10,Leu17]NPY, [Leu3,A sn6,Leu10,Leu17]NPY, [Asn6,Leu10,Leu17,Cys34]NPY, [Leu3,Asn6,Leu10,Leu17,Cys34]NPY;Suc-NPY, [Leu3]Suc-NPY, [Cys34]Suc-NPY, [Leu3,Cys34]Suc-NPY, [Leu17]Suc-NPY, [Leu3,Leu17]Suc-NPY , [Leu17,Cys34]Suc-NPY, [Leu3,Leu17,Cys34]Suc-NPY, [Leu10]Suc-NPY, [Leu3,Leu10]Suc-NPY, [Leu10,Cy s34]Suc-NPY, [Leu3,Leu10,Cys34]Suc-NPY, [Leu10,Leu17]Suc-NPY, [Leu3,Leu10,Leu17]Suc-NPY, [Leu10, Leu17,Cys34]Suc-NPY, [Leu3,Leu10,Leu17,Cys34]Suc-NPY, [Asn6]Suc-NPY, [Leu3,Asn6]Suc-NPY, [Asn6,C ys34]Suc-NPY, [Leu3,Asn6,Cys34]Suc-NPY, [Asn6,Leu17]Suc-NPY, [Leu3,Asn6,Leu17]Suc-NPY, [Asn6,Leu 17,Cys34]Suc-NPY, [Leu3,Asn6,Leu17,Cys34]Suc-NPY, [Asn6,Leu10]Suc-NPY, [Leu3,Asn6,Leu10]Suc-NPY , [Asn6,Leu10,Cys34]Suc-NPY, [Leu3,Asn6,Leu10,Cys34]Suc-NPY, [Asn6,Leu10,Leu17]Suc-NPY, [Leu3,As n6,Leu10,Leu17]Suc-NPY, [Asn6,Leu10,Leu17,Cys34]Suc-NPY, [Leu3,Asn6,Leu10,Leu17,Cys34]Suc-NPY. ;

[0010] Preferably, the amino acid sequence of the neuropeptide Y series polypeptide ligand is shown in the following table:

[0011]

[0012]

[0013] Preferably, the S- DNPY is selected from any one or more of the following peptides and all amino acids are D-typed: Suc-NPY, [Leu3]Suc-NPY, [Cys34]Suc-NPY, [Leu3,Cys34]Suc-NPY, [Leu17]Suc-NPY, [Leu3,Leu17]Suc-NPY, [Leu17,Cys34]Suc-NPY, [Leu3,Leu17,Cys34]Suc-NPY, [Leu10]Suc-NPY, [Leu3,L eu10]Suc-NPY, [Leu10,Cys34]Suc-NPY, [Leu3,Leu10,Cys34]Suc-NPY, [Leu10,Leu17]Suc-NPY, [Leu3,Leu10,Le u17]Suc-NPY, [Leu10,Leu17,Cys34]Suc-NPY, [Leu3,Leu10,Leu17,Cys34]Suc-NPY, [Asn6]Suc-NPY, [Leu3,Asn6] Suc-NPY, [Asn6,Cys34]Suc-NPY, [Leu3,Asn6,Cys34]Suc-NPY, [Asn6,Leu17]Suc-NPY, [Leu3,Asn6,Leu17]Suc-N PY, [Asn6,Leu17,Cys34]Suc-NPY, [Leu3,Asn6,Leu17,Cys34]Suc-NPY, [Asn6,Leu10]Suc-NPY, [Leu3,Asn6,Leu10 ]Suc-NPY, [Asn6,Leu10,Cys34]Suc-NPY, [Leu3,Asn6,Leu10,Cys34]Suc-NPY, [Asn6,Leu10,Leu17]Suc-NPY, [Leu 3,Asn6,Leu10,Leu17]Suc-NPY, [Asn6,Leu10,Leu17,Cys34]Suc-NPY, [Leu3,Asn6,Leu10,Leu17,Cys34]Suc-NPY.

[0014] Preferably, the S- DNPY is selected from any one or more of the following peptides: D-Suc-NPY, D-[Leu3]Suc-NPY, D-[Cys34]Suc-NPY, D-[Leu3,Cys34]Suc-NPY, D-[Leu17]Suc-NPY, D-[Leu3,Leu17]Suc-NPY, D-[Leu17,Cys34]Suc-NPY, D-[Leu3,Leu17,Cys34]Suc-NPY, D-[Leu10]Suc-NPY, D-[Leu3,Leu10 ]Suc-NPY, D-[Leu10,Cys34]Suc-NPY, D-[Leu3,Leu10,Cys34]Suc-NPY, D-[Leu10,Leu17]Suc-NPY, D-[Leu3,Leu10,Leu17 ]Suc-NPY, D-[Leu10,Leu17,Cys34]Suc-NPY, D-[Leu3,Leu10,Leu17,Cys34]Suc-NPY, D-[Asn6]Suc-NPY, D-[Leu3,Asn6]Su c-NPY, D-[Asn6,Cys34]Suc-NPY, D-[Leu3,Asn6,Cys34]Suc-NPY, D-[Asn6,Leu17]Suc-NPY, D-[Leu3,Asn6,Leu17]Suc-NP Y, D-[Asn6,Leu17,Cys34]Suc-NPY, D-[Leu3,Asn6,Leu17,Cys34]Suc-NPY, D-[Asn6,Leu10]Suc-NPY, D-[Leu3,Asn6,Leu1 0]Suc-NPY, D-[Asn6,Leu10,Cys34]Suc-NPY, D-[Leu3,Asn6,Leu10,Cys34]Suc-NPY, D-[Asn6,Leu10,Leu17]Suc-NPY, D-[ Leu3,Asn6,Leu10,Leu17]Suc-NPY, D-[Asn6,Leu10,Leu17,Cys34]Suc-NPY, D-[Leu3,Asn6,Leu10,Leu17,Cys34]Suc-NPY.

[0015] Preferably, the iron-based polypeptide probe regulating mitochondrial function is activated by the SIRT5 enzyme and NAD + The desuccinylated polypeptide is desuccinylated under the action of a coenzyme and then self-assembled in situ to obtain a magnetic nanoparticle assembly modified with the desuccinylated polypeptide.

[0016] More preferably, the average particle size of the iron-based polypeptide probe for regulating mitochondrial function is 2 to 6 nm; and the hydrated particle size is 5 to 20 nm.

[0017] More preferably, the average particle size of the iron-based polypeptide probe for regulating mitochondrial function is 3 to 5 nm.

[0018] More preferably, the particle size of the desuccinylated polypeptide-modified magnetic nanoparticle assembly is greater than 100 nm.

[0019] Preferably, the iron-based polypeptide probe regulating mitochondrial function is located on the SIRT5 enzyme and NAD + Under the action of coenzyme, the level of cytochrome C (Cyt-C) in mitochondria is significantly reduced.

[0020] Preferably, the iron-based polypeptide probe for regulating mitochondrial function targets the Y1R receptor on the MCF-7 cell membrane.

[0021] Preferably, the iron-based polypeptide probe regulating mitochondrial function is activated by the SIRT5 enzyme and NAD + After desuccinylation under the action of coenzyme, it undergoes in situ self-assembly, causing mitochondrial membrane potential depolarization and cell apoptosis.

[0022] Preferably, the iron-based polypeptide probe regulating mitochondrial function is activated by the SIRT5 enzyme and NAD + Reduce NAD under the action of coenzyme + levels, thereby inducing cell apoptosis.

[0023] Preferably, the iron-based polypeptide probe that regulates mitochondrial function can overactivate the autophagy pathway of damaged mitochondria, thereby promoting the induction of cell apoptosis.

[0024] Preferably, the iron-based polypeptide probe regulating mitochondrial function is located on the SIRT5 enzyme and NAD + Under the action of coenzymes, the mitochondrial membrane potential is depolarized; PINK1 aggregates on the mitochondrial outer membrane, recruits and activates Parkin, ubiquitinates mitochondrial proteins, and achieves overactivation of the autophagy pathway.

[0025] As an example, the iron-based polypeptide probe regulating mitochondrial function is composed of a polypeptide ligand S- D NPY is prepared by stirring and reacting activated superparamagnetic nano-iron oxide SPION.

[0026] More preferably, the activation comprises: mixing EDC and NHS with superparamagnetic nano-iron oxide SPION.

[0027] As a preference, the iron-based polypeptide probe regulating mitochondrial function comprises superparamagnetic nano-iron oxide SPION and its surface-modified N-terminal succinylated D-type neuropeptide Y series polypeptide ligand S- D The mass ratio of the added amount of NPY is 1:(2~6).

[0028] More preferably, the iron-based polypeptide probe regulating mitochondrial function comprises superparamagnetic nano-iron oxide SPION and its surface modified N-terminal succinylated D-type neuropeptide Y series polypeptide ligand S- D The mass ratio of NPY added is 1:4.

[0029] Preferably, the preparation method of the iron-based polypeptide probe for regulating mitochondrial function comprises: dissolving trivalent iron salt and divalent ferrous salt in dilute hydrochloric acid, vigorously stirring under a nitrogen atmosphere, then adding to the heated polymer solution and mixing evenly, adding concentrated ammonia water dropwise to adjust the pH value to 9-10, then heating the reaction, and after the reaction is completed, cooling, filtering, and purifying to obtain the product.

[0030] More preferably, the polymer in the polymer solution is polyacrylic acid PAA, and the molecular weight of the polyacrylic acid is 1000-6000.

[0031] More preferably, the mass ratio of the ferric salt, the ferrous salt, and the polymer in the polymer solution is (2-5):1:(15-40).

[0032] More preferably, the molar ratio of ferric iron to ferrous iron in the ferric iron salt and the ferrous iron salt is 1:(0.8-1.3).

[0033] More preferably, the concentration of hydrochloric acid is 0.1-5 mM.

[0034] More preferably, the heating temperature of the polymer solution is 60-90°C.

[0035] More preferably, the concentration of the concentrated ammonia water is 25-28%.

[0036] More preferably, the heating reaction temperature is 60-90° C., and the heating reaction time is 0.1-10 h.

[0037] Preferably, the iron-based polypeptide probe regulating mitochondrial function is imaged in MRI signal T1; the iron-based polypeptide probe regulating mitochondrial function is imaged in SIRT5 enzyme and NAD + The desuccinylation is carried out under the action of a coenzyme and then the desuccinylated polypeptide is self-assembled in situ to obtain a desuccinylated polypeptide-modified magnetic nanoparticle assembly. The desuccinylated polypeptide-modified magnetic nanoparticle assembly is imaged in MRI signal T2.

[0038] More preferably, the iron-based polypeptide probe regulating mitochondrial function is imaged as MRI signal T1 0 hours after entering the body; and imaged as MRI signal T2 3 to 12 hours later.

[0039] The second object of the present invention is achieved through the following technical solutions:

[0040] A tumor-inhibiting drug comprises 0.1-100 wt% of an iron-based polypeptide probe for regulating mitochondrial function.

[0041] Preferably, the surface of the iron-based polypeptide probe for regulating mitochondrial function is also loaded with a substance having therapeutic function.

[0042] Further preferably, the substance having therapeutic functions includes any one or a combination of two or more of substances used for photothermal therapy or photodynamic therapy, substances used for magnetic thermal therapy, substances used for sonodynamic therapy, substances used for chemotherapy, and substances used for radionuclide therapy.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] 1. The iron-based polypeptide probe for regulating mitochondrial function of the present invention undergoes desuccinylation and self-assembly after encountering succinate dehydrogenase on the inner membrane of the mitochondria at the tumor site, thereby regulating the mitochondrial oxidative stress process and cell apoptosis-related pathways. As a drug for inhibiting tumors, it can effectively kill tumor cells and improve the effect of tumor treatment.

[0045] 2. The polypeptide ligand of the iron-based polypeptide probe for regulating mitochondrial function of the present invention targets the Y1R receptor on the MCF-7 cell membrane. After entering the body, the iron-based polypeptide probe for regulating mitochondrial function acts precisely on MCF-7 cells, + Under the action of coenzyme, it competitively inhibits the desuccinylation behavior of mitochondria, and then self-assembles in situ after desuccinylation, causing mitochondrial membrane potential depolarization and initiation of cell apoptosis; reduces NAD + levels, thereby inducing cell apoptosis; it can also overactivate the autophagy pathway of damaged mitochondria, thereby promoting the induction of cell apoptosis.

[0046] 3. The iron-based polypeptide probe for regulating mitochondrial function of the present invention is activated by SIRT5 enzyme and NAD in mitochondria at the tumor site. + Under the action of the coenzyme, PINK1 undergoes desuccinylation and then self-assembles in situ, achieving the aggregation of iron-based polypeptide probes, damaging the mitochondrial membrane and causing depolarization of the mitochondrial membrane potential; after the mitochondrial membrane potential is depolarized, PINK1 aggregates on the mitochondrial outer membrane, recruits and activates Parkin, ubiquitinates mitochondrial proteins, and achieves overactivation of the autophagy pathway.

[0047] 4. The iron-based polypeptide probe for regulating mitochondrial function of the present invention can be used as a drug for inhibiting tumors and can be loaded with drugs on the surface to further enhance the effect of inhibiting tumors.

[0048] 5. The iron-based polypeptide probe for regulating mitochondrial function of the present invention is used as a drug for inhibiting tumors, has low biological toxicity, obvious targeting effect, and no significant effect on other tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 TEM electron microscope image (A), particle size analysis image (B), and hydrated particle size statistical image (C) of the magnetic nanoparticles SPION in Example 1 of the present invention.

[0050] Figure 2 For S- in Examples 1, 4, and 5 of the present invention D TEM electron micrograph (A) and particle size analysis (B) of NPY-SPION.

[0051] Figure 3 is S- in Example 1 of the present invention D NPY, S- in Examples 1, 4, and 5 D HPLC profile of NPY-SPION.

[0052] Figure 4 This is a TEM image of a cell slice at 6 hours in Application Example 1 of the present invention.

[0053] Figure 5 This is the TEM image of the cell slice at 12 hours in Application Example 1 of the present invention.

[0054] Figure 6 (A) is a graph showing the absolute concentration of Fe in the material of MCF-7 cells in Application Example 2 of the present invention, and (B) is a graph showing the absolute concentration of Fe in the material of MCF-10A cells.

[0055] Figure 7 This is a graph showing the quantitative analysis of the fluorescence intensity of JC-1 using the ImageJ software in Example 3 of the present invention.

[0056] Figure 8 This is a graph showing ATP consumption of MCF-7 cells in Application Example 4 of the present invention.

[0057] Figure 9 NAD in Application Example 5 of the present invention + / NADH level analysis chart.

[0058] Figure 10 This is a diagram showing the loss of Cyt-C in mitochondrial cells in Application Example 6 of the present invention.

[0059] Figure 11This is a graph showing the expression of PINK1 in MCF-7 cells in Application Example 7 of the present invention.

[0060] Figure 12 The SPION and S- D Distribution of iron content of NPY-SPION in mouse heart, liver, spleen, kidney, lung, and tumor.

[0061] Figure 13 This is a diagram for evaluating the therapeutic effect on mice in Application Example 9 of the present invention. DETAILED DESCRIPTION

[0062] The technical solutions of the present invention are further described below through specific embodiments. It should be understood that the specific embodiments described herein are only used to help understand the present invention and are not used to specifically limit the present invention.

[0063] Unless otherwise specified, the raw materials used in the examples of the present invention are all commonly used raw materials in the art, and the methods used in the examples are all conventional methods in the art.

[0064] Herein, MCF-7 cells were cultured in DMEM medium (Gibco) containing 10% fetal bovine serum, and MCF-10A cells were cultured in Mesencult™ (Pricella, CM0525). All cells were cultured in a 37° C., 5% CO 2 incubator.

[0065] In the following, S- D NPY is D-[Leu3, Asn6, Leu10, Leu17, Cys34]Suc-NPY.

[0066] Example 1

[0067] (1) Under a nitrogen atmosphere, 0.1459 g of FeCl3·6H2O and 0.0554 g of FeCl2·4H2O were added to 2 ml of hydrochloric acid (concentration 1 mM) and stirred vigorously to obtain an iron precursor mixture. The mixture was then rapidly injected into 50 ml of a 5% aqueous solution of polymer PAA (molecular weight 3000) at 80°C. Concentrated ammonia (15 mL, 28%) was then added dropwise, and the pH was adjusted to 9-10. The temperature would then drop slightly. The mixture was then heated to 80°C for 1 h, cooled, and dialyzed for 72 h. The collected material was freeze-dried to obtain magnetic nanoparticles SPION. ICP analysis revealed an iron content of 66%.

[0068] Figure 1 The electron microscope image, particle size analysis graph and hydrated particle size statistics graph of magnetic nanoparticles SPION show that the average particle size of SPION is 4.4 nm and the hydrated particle size is 10 nm.

[0069] (2) Succinylated polypeptide S- D NPY is purified;

[0070] (3) 6 mg of EDC and 8 mg of NHS were added to the solution containing 6 mg of magnetic nanoparticles SPION in (1) and stirred for 5 h to activate the carboxyl groups, thereby obtaining magnetic nanoparticles after carboxyl group activation; 24 mg of succinylated polypeptide S- D NPY, the mass ratio of the SPION to the succinylated polypeptide is 1:4; stirring, dialysis, and freeze-drying to obtain an iron-based polypeptide probe S- D NPY-SPION.

[0071] Figure 2 For S- D From the electron microscope image and particle size diagram of NPY-SPION, we can see that the average particle size is 4.5 nm. Figure 3 For S- D NPY and S- D HPLC profile of NPY-SPION.

[0072] Example 2

[0073] (1) Under a nitrogen atmosphere, 0.1459 g of FeCl3·6H2O and 0.0776 g of FeSO4·4H2O were added to 2 ml of hydrochloric acid (concentration 1 mM) and stirred vigorously to obtain an iron precursor mixture. The mixture was then rapidly injected into 50 ml of a 5% aqueous solution of polymer PAA (molecular weight 1800) at 80°C. Concentrated ammonia (15 mL, 28%) was then added dropwise to adjust the pH to 9-10, at which point the temperature would drop slightly. The mixture was then heated to 80°C for 1 h, cooled, dialyzed for 72 h, and freeze-dried to obtain magnetic nanoparticles SPION.

[0074] (2) Succinylated polypeptide S- D NPY is purified;

[0075] (3) Same as Example 1(3).

[0076] Example 3

[0077] (1) Under a nitrogen atmosphere, 1 g of FeCl₃·6H₂O and 1 g of FeCl₂·4H₂O were added to 5 ml of 1 mM hydrochloric acid and stirred vigorously to obtain an iron precursor mixture. This mixture was then rapidly injected into 50 ml of a 10% aqueous solution of a polymer PAA (molecular weight 5000) at 80°C. Concentrated ammonia (30 mL, 28%) was then added dropwise, and the pH was adjusted to 9-10. The mixture was then heated to 80°C for 1 h, cooled, and dialyzed for 72 h to obtain a solution containing magnetic nanoparticles SPION.

[0078] (2) Succinylated polypeptide S- D NPY is purified;

[0079] (3) Same as Example 1(3).

[0080] Example 4

[0081] (1) Same as Example 1(1).

[0082] (2) Same as Example 1(2).

[0083] (3) Compared with Example 1(3), the difference is that the mass ratio of the SPION to the succinylated polypeptide is 1:2.

[0084] Example 5

[0085] (1) Same as Example 1(1).

[0086] (2) Same as Example 1(2).

[0087] (3) Compared with Example 1(3), the difference is that the mass ratio of the SPION to the succinylated polypeptide is 1:6.

[0088] Application Example 1

[0089] Changes in mitochondrial morphology.

[0090] After MCF-7 cells were cultured to the logarithmic growth phase, SPION, S- D NPY, S- D After incubation with NPY-SPION for 6 and 24 hours, cells were collected and fixed with 2.5% glutaraldehyde for 2-4 hours. After washing with PBS, cells were transferred to 1% osmium tetroxide for 1-2 hours. Following fixation, cells were dehydrated using a gradient of 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol, followed by replacement with 100% acetone. Subsequently, cell samples were infiltrated in a 1:1 mixture of acetone and epoxy resin for 2 hours, transferred to pure resin overnight at 4°C, pre-embedded at 37°C for 4 hours, and polymerized at 60°C for 24-48 hours. After embedding, 70-90 nm ultrathin sections were cut using an ultramicrotome and mounted on nickel or copper grids. Prior to staining, cells were stained with 2% uranyl acetate for 10-15 minutes, washed with distilled water, and then stained with 2.6% lead citrate for 10 minutes. Cells were then washed thoroughly and dried before use. Finally, transmission electron microscopy (TEM) was used at 80-120 kV to observe mitochondrial morphology and analyze membrane integrity, cristae structure, and possible damage or autophagy.

[0091] like Figure 4 、 5The TEM images show that S- D After 6 hours, NPY-SPION was widely distributed on the mitochondrial membrane, and some mitochondrial structures began to be destroyed. This shows that S- D NPY-SPION can quickly enter the mitochondria and cause a certain degree of damage. When the action time is extended to 24 hours, S- D NPY-SPION caused significant morphological changes in the mitochondria of cells. The mitochondrial membrane was damaged and the cristae were blurred and indistinguishable. This change further indicated that the mitochondrial function of the cells had been significantly damaged. In sharp contrast, SPION and S- D NPY is difficult to distinguish within the cells and maintains a low level within the cells, indicating that it is relatively evenly distributed within the cells and does not cause significant accumulation or damage.

[0092] Application Example 2

[0093] The SPION and S- D NPY-SPION was added to the culture medium of MCF-7 cells (human breast cancer cells) and MCF-10A cells (human normal mammary epithelial cells) and incubated for 0, 2, 4, 8, 12, and 24 hours, and the absolute concentration of Fe in the intracellular material was determined by inductively coupled plasma mass spectrometry (ICP-MS).

[0094] like Figure 6 As shown in (A), after 4 hours of culture, the iron content in MCF-7 cells increased significantly, indicating that most of the SPIONs and S- D NPY-SPION has been successfully taken up by MCF-7 cells. After 12 hours of culture, S- D The iron content in MCF-7 cells in the NPY-SPION group increased 5.8 times compared with the initial level, while that in the SPION group was only 3.3 times, indicating that S- D The intracellular uptake of NPY-SPION is nearly 2 times higher than that of SPION. D The uptake of NPY-SPION in MCF-7 cells was more significant. Figure 6 (B) MCF-10A cells were resistant to S- D The uptake of NPY-SPION was lower than that of MCF-7 cells. D The iron content in MCF-10A cells of NPY-SPION increased only 2.6 times compared with the initial level and had no significant difference from the SPION group. This difference was mainly attributed to the high expression of Y1R receptor on the MCF-7 cell membrane, indicating that S- DTargeting of Y1R by NPY is crucial in this process.

[0095] Application Example 3

[0096] The mitochondrial membrane potential of MCF-7 cells was detected using JC-1 kit.

[0097] Experimental groups and control groups were set up, wherein the SPION and S- D NPY, S- D NPY-SPION were added to MCF-7 culture medium at a concentration of 1 mM; the concentration of the control group was 0 mM.

[0098] The samples of experimental groups 1 to 3 and the control group were incubated with MCF-7 for 4 hours and 8 hours, respectively, and then stained with JC-1 for 20 minutes at 37°C in the dark. After washing three times with PBS, the cells were imaged using a confocal microscope LSCM (TCS SP5 II, Leica, Germany). The excitation / emission wavelengths of JC-1 monomers were 488 / 525 nm, and the excitation / emission wavelengths of JC-1 aggregates were 560 / 595 nm.

[0099] ImageJ software was used to quantify and statistically analyze the fluorescence intensity. Figure 7 The quantitative data showed that in S- D In the NPY-SPION group, the ratio of JC-1 aggregates to JC-1 monomers was reduced 15-fold compared with the control group. These results indicate that the S- D NPY-SPION self-assembly causes mitochondrial membrane potential depolarization, which is considered an early indicator of cell apoptosis.

[0100] Application Example 4

[0101] ATP in MCF-7 cells was measured using an Elabscience kit.

[0102] Experimental groups and control groups were set up, wherein the SPION and S- D NPY, S- D NPY-SPION were added to MCF-7 culture medium at a concentration of 1 mM; the concentration of the control group was 0 mM.

[0103] Incubate the cells with MCF-7 cells in test groups 1 to 3 and the control group for 4 and 8 hours, respectively. Aspirate the culture medium and add 200 μl of lysis buffer per well of a 6-well plate to lyse the cells. Centrifuge at 12,000 rpm for 5 minutes at 4°C and collect the supernatant.

[0104] The colorimetric working solution was incubated at 37°C for 1 hour before use. The ATP standard consisted of a 10 mmol / L stock solution and a 1 mmol / L working solution.

[0105] During the experiment, each sample group was placed in an EP tube, and the control or assay working solution was added. After incubation at 37°C for 30 minutes, a protein precipitant was added, mixed, and centrifuged. The supernatant was used for the color development reaction. During the color development reaction, the color development agent working solution and the terminator were added in sequence. After shaking the plate and letting it rest, the OD value was measured at a wavelength of 636nm.

[0106] according to Figure 8 The results show that after 4 hours, the D Significant ATP depletion was observed in MCF-7 cells treated with NPY-SPION. Significant ATP depletion is often accompanied by mitochondrial dysfunction, leading to damage of the electron transport chain, electron leakage and excessive generation of reactive oxygen species (ROS). Due to insufficient ATP supply, Na+ / K+-ATPase and Ca 2 The functions of key ion pumps such as Ca+-ATPase are impaired, and intracellular ion homeostasis is disrupted. 2 Overload further exacerbates mitochondrial damage. Excessive accumulation of ROS not only oxidatively damages proteins, lipids, and DNA but also triggers an oxidative stress cascade, leading to loss of mitochondrial membrane potential and activation of apoptotic signaling. Ultimately, ATP depletion, ROS accumulation, and ion homeostasis imbalance work together to induce cell death and potentially trigger inflammation and tissue damage.

[0107] Application Example 5

[0108] Experimental groups and control groups were set up, wherein the SPION and S- D NPY, S- D NPY-SPION were added to MCF-7 culture medium at a concentration of 1 mM; the concentration of the control group was 0 mM.

[0109] The experimental groups 1 to 3 and the control group were incubated with MCF-7 cells for 4 hours and 8 hours respectively. + Lyse cells with NADH extract. Centrifuge at 12,000 × g for 5–10 minutes at 4°C and remove the supernatant. Prepare NADH standard solution and alcohol dehydrogenase working solution. Measure NAD by absorbing 20 μL of the sample to be tested into a 96-well plate. + The sample to be tested is absorbed in a centrifuge tube and heated in a 60°C water bath for 30 minutes to decompose NAD + The individual NADH contents were determined by aspirating 20 μL of the sample to be measured into a 96-well plate.

[0110] according to Figure 9 The results showed that autophagy dysfunction leads to the accumulation of intracellular NAD + Levels of NAD+ are reduced, which in turn induces apoptosis. This loss is due to the loss of mitochondrial quality control in mitophagy, leading to increased ROS production and DNA damage. Excessive activation of PARP / SIRT depletes cellular NAD+ levels, disrupts mitochondrial membrane potential, and ultimately leads to cell death. D NPY affects intracellular NAD + level, and the NAD+ / NADH ratio dropped to 0.58 of the control group after 8 hours. D In the NPY-SPION group, the ratio further decreased to 0.27. These results indicate that S- D NPY overactivates SIRT5, aggravating NAD + Consume, make S- D NPY-SPION further inhibited cell activity.

[0111] Application Example 6

[0112] Cyt-C in MCF-7 cell mitochondria was determined using an Elabscience kit.

[0113] Experimental groups and control groups were set up, wherein the SPION and S- D NPY, S- D NPY-SPION were added to MCF-7 culture medium at a concentration of 1 mM; the concentration of the control group was 0 mM.

[0114] The samples of test groups 1 to 3 and control group were incubated with MCF-7 cells. 2×10 7 Treat the cells with mitochondrial isolation reagent. Then centrifuge at 1000×rpm at 4°C for 10 minutes and remove the supernatant. Centrifuge at 11000×rpm at 4°C for 10 minutes. Take 100μL of the sample to be tested and the standard and add them to the ELISA plate and incubate at 37°C for 90 minutes. Drain the liquid in the wells without washing. Add 100μL of biotinylated antibody working solution to each well, coat the plate, and incubate at 37°C for 1 hour. Drain the liquid in the wells, add 350μL of washing solution to each well, soak for 1 minute, absorb in the ELISA plate or discard, and repeat 3 times. Add 100μL of HRP enzyme conjugate working solution to each well, coat the plate, and incubate at 37°C for 30 minutes. Add 90μL of substrate solution (TMB) to each well, coat the plate, and incubate at 37°C without light for 15 minutes. After adding 50μL of stop solution to each well, immediately measure the optical density (OD value) of each well at a wavelength of 450nm using a microplate reader.

[0115] according to Figure 10The results showed that compared with the control group, S- D The Cyt-C level in the mitochondria of the NPY-SPION group was significantly reduced, and dropped to 0.73 of the control group after 8 hours, while the Cyt-C level in the mitochondria of the S- D NPY and SPION did not cause significant changes, with values ​​of 0.93 and 1.01, respectively, relative to the control group. This change indicates that S- D The NPY-SPION group caused the depolarization of the mitochondrial membrane, which released Cyt-C from the mitochondria into the cytoplasm, thereby triggering a cascade of apoptotic signaling.

[0116] Application Example 7

[0117] Immunofluorescence testing.

[0118] The expression of PINK1 in MCF-7 cells was detected using primary antibody (PINK1) and fluorescent secondary antibody (CoraLite594–conjugated Goat Anti-Rabbit IgG (H+L)).

[0119] Experimental groups and control groups were set up, wherein the SPION and S- D NPY, S- D NPY-SPION was added to MCF-7 cells for incubation at a concentration of 1 mM; the concentration of the control group was 0 mM.

[0120] Experimental groups 1 to 3 and the control group were incubated with MCF-7 cells for 8 hours, then washed three times with PBS. The cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature, washed three times with PBS for 5 minutes each, and then permeabilized with 0.5% Triton X-100 for 10 minutes at room temperature and washed again three times with PBS. Subsequently, the cells were blocked with 5% BSA for 30 minutes to reduce nonspecific binding. The primary antibody was then added, incubated for 1 hour at room temperature, and then incubated overnight at 4°C. The next day, the cells were washed three times with PBS for 5 minutes each. Fluorescently labeled secondary antibodies were then added, incubated in the dark for 1 hour at room temperature, and washed three times with PBS for 5 minutes each. Nuclei were then stained with Hoechst 33342, incubated at room temperature for 10 minutes in the dark, and washed three times with PBS. Images were acquired using a confocal LSCM microscope (TCS SP5 II, Leica, Germany), with appropriate excitation wavelengths selected. Fluorescence signals were analyzed using software such as ImageJ. The excitation / emission wavelengths for Hoechst 33342 are 350 / 461 nm. The excitation / emission wavelengths of the fluorescent secondary antibody were 594 / 618 nm.

[0121] The PINK1 / Parkin pathway is central to mitochondrial quality control. Figure 11The results show that after mitochondrial membrane depolarization, PINK1 accumulates on the mitochondrial outer membrane, where it recruits and activates Parkin, ubiquitinating mitochondrial proteins and initiating autophagy clearance. Although this process plays a key role in maintaining cellular homeostasis, its disorder can lead to cell apoptosis. The immunofluorescence imaging results and quantitative fluorescence intensity analysis of PINK1 showed that S- D After NPY-SPION treatment, the expression level of PINK1 was significantly upregulated, increasing by 2.1 times compared with the control group. This phenomenon indicates that S- D NPY-SPION may accelerate the clearance of damaged mitochondria by activating the PINK1 / Parkin-mediated mitophagy pathway, thereby affecting cellular energy metabolism and survival. Furthermore, upregulation of PINK1 may be closely associated with increased apoptosis, as under conditions of excessive stress, mitophagy fails to effectively clear damaged mitochondria, potentially inducing further activation of apoptotic pathways, such as the caspase family cascade, ultimately leading to cell death.

[0122] Application Example 8

[0123] In vivo toxicity test and in vivo distribution test.

[0124] MCF-7 orthotopic tumor-bearing mice were randomly divided into three groups, with 5 mice in each group.

[0125] The three groups of mice were injected with PBS and SPION, S- D NPY-SPION (3 mg / kg).

[0126] The body weight of mice was recorded every 3 days. Blood was collected for biochemical and routine blood tests. The heart, liver, spleen, kidney, lung and other major organs of mice were preserved in 10% formalin solution and stained with hematoxylin-eosin (H&E) for histological analysis to evaluate S- D Toxicity of NPY-SPIONs.

[0127] according to Figure 12 The results showed that in the SPION-treated group, a large amount of Fe was retained by the spleen 24 hours after injection, forming a significant enrichment phenomenon, indicating that SPINO was mainly cleared by the reticuloendothelial system in the body. Only a small amount of Fe entered the tumor tissue, indicating that the efficiency of SPION entering the tumor through the enhanced permeability and retention effect (EPR effect) was low. In contrast, S- D The accumulation of Fe in the tumor site of the NPY-SPION treated group was significantly enhanced, and the retention in the spleen was relatively low, indicating that S- DNPY has good biocompatibility and can effectively reduce the nonspecific clearance of the probe by the reticuloendothelial system. More importantly, compared with the SPION group, S- D The accumulation of NPY-SPION in tumor tissue increased significantly, showing that S- D The targeted modification of NPY plays a key role in promoting the delivery of the probe to the tumor site. This result further indicates that S- D NPY may enhance the uptake of SPIONs in tumor cells through a Y1 receptor (Y1R)-mediated targeted recognition mechanism, thereby improving their retention in tumor sites. D NPY may also promote the deep penetration and cellular uptake of SPIONs by regulating the tumor microenvironment (TME), further enhancing their accumulation in tumors.

[0128] Application Example 9

[0129] In vivo tumor suppression assay.

[0130] Experimental group and control group were set up, wherein experimental group 1 was a 1mM tumor-inhibiting drug sample 1 obtained by mixing SPION and PBS in Example 1, and experimental group 2 was a 1mM tumor-inhibiting drug sample 1 obtained by mixing SPION and PBS in Example 1. D 1 mM tumor-inhibiting drug sample 2 was obtained by mixing NPY-SPION with PBS, and the control group was PBS solution.

[0131] Mice bearing orthotopic MCF-7 tumors were randomly divided into three groups of five. The experimental and control groups were injected orally on days 1 and 8, respectively, at a dose of 3 mg / kg. The inhibitory effects on tumors were observed. Tumor size and mouse weight were recorded every two days.

[0132] according to Figure 13 The results showed that compared with the control group with rapid tumor growth, S- D The tumor growth in the NPY-SPION group was almost completely inhibited, and the tumor growth trend in the SPION group was similar to that in the control group. D NPY-SPION exhibited significantly enhanced anti-tumor activity, demonstrating its significant in vivo therapeutic efficacy. Furthermore, no significant changes in body weight or histological abnormalities in major organs were observed in any of the groups.

[0133] Application Example 10

[0134] MRI imaging test.

[0135] Will U- D NPY was prepared according to the steps in Example 1. D NPY-SPION.

[0136] The S- D NPY-SPION was placed in TRIS buffer (pH = 7.4), and 0.005 equivalents of SIRT5 and 2 equivalents of NAD were added. + The whole process was incubated at 37 ° C to obtain S- D NPY-SPION+SIRT5、U- D NPY-SPION+SIRT5.

[0137] T1-weighted and T2-weighted imaging were performed using a T1 fast spin echo sequence with a repetition time (TR) of 500 ms and an echo time (TE) of 18.2 ms to obtain T1-weighted images, and a T2 fast spin echo sequence with a TR of 2000 ms and an echo time of 40 ms to obtain T2-weighted images.

[0138] S- D NPY-SPION maintains good dispersion and stably provides T1-weighted MRI signals, showing excellent positive contrast imaging capabilities.

[0139] S- D NPY-SPION+SIRT5 and U- D Due to the strong peptide-guided self-assembly ability, NPY-SPION gradually forms aggregates as the material concentration increases, enhancing the magnetic dipole interaction between particles, further inducing local magnetic field inhomogeneity, accelerating the transverse relaxation of water protons, and gradually enhancing the T2-weighted MRI dark signal, all showing negative contrast imaging capabilities.

[0140] And S- D The particle size change of NPY-SPION+SIRT5 showed the same trend as that of U- D The hydrated particle size increased significantly, and the dark signal of T2-weighted MRI gradually increased, showing negative contrast imaging capabilities. D NPY-SPION interacts with SIRT5 and NAD + After mixed incubation, desuccinylation, aggregation and self-assembly occurred.

[0141] The SPIONs and S- D NPY-SPION was tested according to the methods of Examples 1 to 10, and all showed the same effect trend as Example 1. Among them, compared with Examples 1, 4, and 5, Example 1 has better tumor inhibition performance.

[0142] In summary, the iron-based polypeptide probe for regulating mitochondrial function of the present invention undergoes desuccinylation and self-assembly after encountering succinate dehydrogenase on the inner membrane of the mitochondria at the tumor site, thereby regulating the mitochondrial oxidative stress process and cell apoptosis-related pathways. As a drug for inhibiting tumors, it can effectively kill tumor cells and improve the effect of tumor treatment.

[0143] The various aspects, embodiments, and features of the present invention should be considered in all respects as illustrative and not limiting, the scope of which is defined solely by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0144] In the preparation method of the present invention, the order of the steps is not limited to the order listed. Persons skilled in the art will appreciate that variations in the order of the steps are within the scope of the present invention without inventive effort. Furthermore, two or more steps or actions may be performed simultaneously.

[0145] Finally, it should be noted that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit its implementation. Persons skilled in the art may make various modifications, additions, or substitute similar methods for the described specific embodiments. It is not necessary and impossible to provide comprehensive examples of all implementations here. However, obvious variations or modifications arising from the essential spirit of the present invention remain within the scope of protection of the present invention, and interpreting them as any additional limitations would be contrary to the spirit of the present invention.

Claims

1. An iron-based polypeptide probe for regulating mitochondrial function, characterized in that: The iron-based polypeptide probe for regulating mitochondrial function comprises superparamagnetic nano-iron oxide SPION with an average particle size of 0.1 to 5 μm and a polypeptide ligand modified on its surface, wherein the polypeptide ligand is a polypeptide ligand S- D NPY; The iron-based polypeptide probe regulating mitochondrial function, SIRT5 enzyme and NAD on mitochondria + Under the action of coenzyme, it competitively inhibits the desuccinylation behavior of mitochondria, and then self-assembles in situ after desuccinylation, realizing the aggregation of iron-based polypeptide probes, damaging the mitochondrial membrane, causing oxidative abnormalities and inducing cell apoptosis and autophagic clearance of damaged mitochondria.

2. The iron-based polypeptide probe for regulating mitochondrial function according to claim 1, characterized in that The S- D NPY is selected from any one or more of the following peptides: D-Suc-NPY, D-[Leu3]Suc-NPY, D-[Cys34]Suc-NPY, D-[Leu3,Cys34]Suc-NPY, D-[Leu17]Suc-NPY, D-[Leu3,Leu17]Suc-NPY, D-[Leu17,Cys34]Suc-NPY, D-[Leu3,Leu17,Cys34]Suc-NPY, D-[Leu10]Suc-NPY, D-[Leu3,Leu10 ]Suc-NPY, D-[Leu10,Cys34]Suc-NPY, D-[Leu3,Leu10,Cys34]Suc-NPY, D-[Leu10,Leu17]Suc-NPY, D-[Leu3,Leu10,Leu17 ]Suc-NPY, D-[Leu10,Leu17,Cys34]Suc-NPY, D-[Leu3,Leu10,Leu17,Cys34]Suc-NPY, D-[Asn6]Suc-NPY, D-[Leu3,Asn6]Su c-NPY, D-[Asn6,Cys34]Suc-NPY, D-[Leu3,Asn6,Cys34]Suc-NPY, D-[Asn6,Leu17]Suc-NPY, D-[Leu3,Asn6,Leu17]Suc-NP Y, D-[Asn6,Leu17,Cys34]Suc-NPY, D-[Leu3,Asn6,Leu17,Cys34]Suc-NPY, D-[Asn6,Leu10]Suc-NPY, D-[Leu3,Asn6,Leu1 0]Suc-NPY, D-[Asn6,Leu10,Cys34]Suc-NPY, D-[Leu3,Asn6,Leu10,Cys34]Suc-NPY, D-[Asn6,Leu10,Leu17]Suc-NPY, D-[ Leu3,Asn6,Leu10,Leu17]Suc-NPY, D-[Asn6,Leu10,Leu17,Cys34]Suc-NPY, D-[Leu3,Asn6,Leu10,Leu17,Cys34]Suc-NPY.

3. The iron-based polypeptide probe for regulating mitochondrial function according to claim 1, characterized in that The iron-based peptide probe regulating mitochondrial function is expressed in SIRT5 enzyme and NAD + After desuccinylation under the action of coenzyme, it undergoes in situ self-assembly, causing depolarization of mitochondrial membrane potential, reducing NAD+ levels, and over-activating the autophagy pathway of damaged mitochondria.

4. The iron-based polypeptide probe for regulating mitochondrial function according to claim 1, characterized in that The iron-based polypeptide probe that regulates mitochondrial function targets the Y1R receptor on the MCF-7 cell membrane.

5. The iron-based polypeptide probe for regulating mitochondrial function according to claim 1, characterized in that The superparamagnetic nano iron oxide SPION and its surface modified N-terminal succinylated D-type neuropeptide Y series polypeptide ligand S- D The mass ratio of the added amount of NPY is 1:(2~6).

6. The iron-based polypeptide probe for regulating mitochondrial function according to claim 1, characterized in that The preparation method of the iron-based polypeptide probe for regulating mitochondrial function comprises: dissolving a trivalent ferrous salt and a divalent ferrous salt in dilute hydrochloric acid, vigorously stirring under a nitrogen atmosphere, then adding the solution to a heated polymer solution and mixing evenly, dropwise adding concentrated ammonia water to adjust the pH value to 9-10, then heating the solution for reaction, cooling, filtering, and purifying the solution after the reaction is completed.

7. The iron-based polypeptide probe for regulating mitochondrial function according to claim 1, characterized in that The mass ratio of the ferric salt, the ferrous salt and the polymer in the polymer solution is (2-5):1:(15-40).

8. The iron-based polypeptide probe for regulating mitochondrial function according to claim 1, characterized in that The average particle size of the iron-based polypeptide probe for regulating mitochondrial function is 2 to 6 nm.

9. A drug for inhibiting tumors, characterized in that: The method comprises 0.1 to 100 wt % of the iron-based polypeptide probe for regulating mitochondrial function according to any one of claims 1 to 8.

10. The tumor-suppressing drug according to claim 9, characterized in that: The surface of the iron-based polypeptide probe that regulates mitochondrial function is also loaded with substances having therapeutic functions.

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