Multifunctional nano-platform for ferroptosis therapy and preparation method and application thereof

Abstract

This invention discloses a multifunctional nanoplatform for the treatment of ferroptosis, its preparation method, and its applications. The multifunctional nanoplatform is BPN. pro BPN pro For DSPE-PEG 2000 -DPCP / DPPC@BP. This invention develops a multifunctional BPN. pro Nanoparticles are used to block the activity of multiple ferroptosis defense proteins in tumor cells, enabling potent ferroptosis therapy.

Description

Technical Field

[0001] This invention belongs to the field of anticancer drug technology, specifically relating to a multifunctional nanoplatform for ferroptosis treatment, its preparation method, and its application. Background Technology

[0002] Ferroprelation, which aims to induce excessive lipid peroxidation (LPO) in cells, has been shown to be a key mechanism for inhibiting various malignant tumors. Compared with traditional anticancer strategies, intracellular oxidative stress and subsequent membrane damage enable ferroprelation to overcome multidrug resistance (MDR) and fight cancer more effectively. Many metal ion ferroprelation nanotherapies based on the Fenton reaction attacking membrane phospholipids have flourished. To improve the eradication of solid tumors in the preclinical setting, combination therapy strategies integrating chemotherapy, immunotherapy, photodynamic therapy, and photothermal therapy with ferroprelation have been explored. For example, albumin nanoparticles loaded with Pt(IV) prodrugs and copper manganese oxides are currently used to combine ferroprelation therapy with chemotherapy. Furthermore, exosome inhibitors GW4869 and iron ions are encapsulated in semiconductor polymers to form metal-phenol networks for synergistic immuno-ferroprelation cancer therapy. However, the low biocompatibility of heavy metal ions hinders their practical application. In addition, Nature has reported that cancer cells can resist ferroprelation through highly efficient ferroptosis defense mechanisms. These mechanisms severely limit the efficacy of these ferroprelation therapies.

[0003] Specifically, cells rely on several key defense pathways / proteins to combat ferroptosis: for example, ferroptosis inhibitor protein 1 (FSP1) inhibits lipid peroxidation by producing panthenol (CoQH2) from ubiquinone. Glutathione peroxidase 4 (GPX4) utilizes glutathione (GSH) to detoxify lipid hydroperoxides. Furthermore, it is noteworthy that dihydroorotate dehydrogenase (DHODH) has recently been found to effectively inhibit lipid peroxidation free radicals using CoQH2. Ferroptosis therapies targeting these proteins show great potential. In recent years, there have been reports of using GPX4 or FSP1 inhibitors to combat cancer. However, the use of DHODH has been rarely reported. Moreover, since cells rely on several mechanisms / proteins to combat ferroptosis, inhibition of one is likely to be compensated for by a heavy reliance on another. To our knowledge, there are no reports of effective cancer treatment using inhibition of two key ferroptosis defense proteins. Here, we report the synergistic inhibition of two defense proteins, GPX4 and DHODH, to induce potent ferroptosis and cancer cell death.

[0004] Proteolytic targeting chimeras (PROTACs) have attracted considerable attention due to their ability to efficiently induce post-translational knockout of proteins via ubiquitination. A PROTAC comprises a unit targeting the E3 ubiquitin ligase and another unit for binding to the target protein. Mechanistically, PROTAC ubiquitinates and degrades the target protein via the ubiquitin-proteasome system (UPS). The PROTAC is then released and enters the next protein degradation cycle. Therefore, compared to traditional small molecule inhibitors, monoclonal antibodies, and nucleic acids, PROTACs offer higher therapeutic efficacy and lower dosage. To date, numerous anticancer strategies based on PROTAC protein degradation have been reported. However, PROTACs have rarely been reported for the degradation of ferroptosis defense proteins.

[0005] Currently, it is recognized in the field that some Bodipy derivatives have good photothermal effects and can be used for controlled drug release. The combination of Bodipy and small molecule drugs is becoming increasingly popular in imaging-assisted cancer treatment. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0007] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0008] Therefore, the object of the present invention is to overcome the shortcomings of the prior art and provide a multifunctional nanoplatform for the treatment of ferroptosis.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a multifunctional nanoplatform for the treatment of ferroptosis, comprising: a multifunctional nanoplatform being a BPN. pro The BPN pro For DSPE-PEG 2000 -DPCP / DPPC@BP.

[0010] Another object of the present invention is to provide a method for preparing a multifunctional nanoplatform for the treatment of ferroptosis.

[0011] To address the above problems, the present invention provides the following technical solution: a method for preparing a multifunctional nanoplatform for ferroptosis treatment, comprising the following steps: preparing a proteolytic targeted chimeric polypeptide, namely DPCP; preparing cathepsin B, namely CatB-cleavable DPCP;

[0012] Preparation of DSPE-PEG 2000 -DPCP copolymer: DPCP and DSPE-PEG 2000 -NH2 undergoes an amide condensation reaction to yield DSPE-PEG. 2000 -DPCP copolymer;

[0013] Preparation of BPN pro : fluoroboron dipyrrole (BP probe) and DSPE-PEG 2000 - DPCP and DPPC are mixed and nano-precipitated to obtain BPN. pro .

[0014] As a preferred embodiment of the preparation method of the multifunctional nanoplatform for ferroptosis treatment described in this invention, wherein: BPN is prepared pro In China, BP and DSPE-PEG 2000 - DPCP and DPPC are mixed in a mass ratio of 1:20:100 and nanoprecipitated.

[0015] In a preferred embodiment of the preparation method of the multifunctional nanoplatform for ferroptosis treatment described in this invention, BP is prepared by mixing BTPA (a precursor probe of BP) with compound 2. Compound 2 is 5-methyl-4-nitro-3-isoxazole carboxylic acid, which is added to anhydrous dichloromethane cooled in an ice bath. Subsequently, oxalyl chloride is added dropwise, followed by one drop of N,N-dimethylformamide. The ice bath is removed, and the reaction mixture is stirred overnight at room temperature. Then, the solvent is evaporated by rotary evaporation in a room temperature water bath to obtain an acyl chloride intermediate, which is immediately added dropwise to anhydrous dichloromethane cooled in an ice bath. Finally, a mixture of triethylamine and 11-azido-3,6,9-trioxaundecan-1-amine in anhydrous dichloromethane is added dropwise to the acyl chloride solution, and the reaction mixture is stirred at room temperature for 2 hours. After removing volatiles under reduced pressure, the crude product is purified by silica gel column chromatography using a mixture of ethyl acetate and petroleum ether at a volume ratio of 1:3 as the eluent to obtain compound 2.

[0016] In a preferred embodiment of the preparation method of the multifunctional nanoplatform for ferroptosis treatment described in this invention, the raw materials for the preparation of BP include BTPA, compound 2, and BP is prepared by dissolving BTPA, compound 2, and sodium ascorbate in a dichloromethane / methanol mixture, followed by degassing. Then, copper sulfate is dissolved in methanol and degassed. Finally, the copper sulfate solution is added to the reaction mixture and stirred overnight at room temperature. After removing volatiles under reduced pressure, the crude product is purified by silica gel column chromatography using an ethyl acetate / petroleum ether mixture as the eluent to obtain the BP probe.

[0017] As a preferred embodiment of the preparation method of the multifunctional nanoplatform for ferroptosis treatment described in this invention, the preparation process of BTPA is as follows: under a nitrogen atmosphere, compounds 5 and 4-(N,N-diphenylamino)benzaldehyde are dissolved in N,N-dimethylformamide. Then, the reaction mixture is heated to 120°C and acetic acid and piperidine are added dropwise after 15 minutes. After reacting for 24 hours, the crude product is washed with dichloromethane and brine, and the collected organic layer is dried with anhydrous sodium sulfate. After filtration and removal of volatiles under reduced pressure, the crude product is purified by silica gel column chromatography using a 1:2 volume ratio of dichloromethane / petroleum ether as the eluent to obtain compound BTPA.

[0018] In a preferred embodiment of the preparation method of the multifunctional nanoplatform for ferroptosis treatment described in this invention, compound 5 is obtained by dissolving compound 4 and 2,4-dimethylpyrrole in dichloromethane and adding trifluoroacetic acid. The reaction mixture is then stirred overnight at room temperature. Next, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone is added to the reaction mixture and stirred overnight at room temperature. Finally, N,N-diisopropylethylamine and a boron trifluoride diethyl ether complex are added dropwise and stirred overnight at room temperature. The crude product is washed with dichloromethane and brine, and the collected organic layer is dried over anhydrous sodium sulfate. After filtration and removal of volatiles under reduced pressure, the crude product is purified by silica gel column chromatography using a mixture of ethyl acetate and petroleum ether at a volume ratio of 1:90 as the eluent.

[0019] In a preferred embodiment of the preparation method of the multifunctional nanoplatform for ferroptosis treatment described in this invention, compound 4, 3-bromopropyne, is added to a solution of p-hydroxybenzaldehyde and potassium carbonate in N,N-dimethylformamide. The reaction mixture is then stirred overnight at 60°C. The crude product obtained by filtering out the potassium carbonate is then dissolved in dichloromethane and washed with brine. The collected organic layer is dried over anhydrous sodium sulfate. After filtration and removal of volatiles under reduced pressure, the crude product is purified by silica gel column chromatography using a mixture of ethyl acetate and petroleum ether as the eluent.

[0020] Another object of the present invention is to provide an application of a multifunctional nanoplatform for the treatment of ferroptosis.

[0021] To address the aforementioned technical problems, this invention provides the following technical solution: an application of a multifunctional nanoplatform for ferroptosis treatment, comprising: BPN pro BPN p1 and BPN p2 Incubate with cells.

[0022] As a preferred embodiment of the application of the multifunctional nanoplatform for ferroptosis therapy described in this invention, wherein: cells for ferroptosis therapy are placed in a solution of 0-200 μg / mL -1 In a multifunctional nanoplatform environment for the treatment of ferroptosis.

[0023] Beneficial effects of this invention:

[0024] Here, we propose a multifunctional nanoplatform BPN. pro This can be used for effective ferroptosis treatment by inhibiting the enzyme activities of DHODH and GPX4. Figure 1 As shown in a, in one embodiment, BP containing nitroisoxazole groups is encapsulated in thermoresponsive liposomes via a simple nanoprecipitation method, and the surface is modified with PROTAC peptide (DPCP) to form the final BPN. pro Nanoparticles. Here, DPCP consists of a cathepsin B (CatB) cleavable peptide, an E3 ligase-targeting peptide, and a bukelana unit targeting DHODH. Additionally, DSPE-PEG is used. 2000 -NH2 and DPPC (a thermosensitive phospholipid) were used to construct a thermoresponsive lipid shell for controlling BP release. For example... Figure 1 As shown in b, after reaching the tumor through high retention and permeability, as shown in the second implementation method, BPN pro The nanoparticles function as follows: i) Degrading DHODH. DPCP is released via the action of CatB. It specifically binds to DHODH and initiates the recruitment of the E3 ligase. Then, DHODH is degraded by UPS, and DPCP is released and enters another round of DHODH degradation. ii) Inhibiting GPX4. After near-infrared (NIR) light irradiation, BPN... pro The heat generated by the nanoparticles melts the lipid shell and releases BP. The nitroisoxazole group in the BP can be activated in cells as an electrophilic nitrile oxide group, which covalently binds to the selenocysteine ​​residue of GPX4 to inhibit its ferroptosis defense activity. In the absence of external metal ions, the inactivation of DHODH and GPX4 induces strong ferroptosis in tumor cells. In summary, a multifunctional BPN has been developed. pro Nanoparticles are used to block the activity of multiple ferroptosis defense proteins in tumor cells, enabling potent ferroptosis therapy. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0026] Figure 1 For BPN pro Schematic diagram of induced cancer ferroptosis therapy;

[0027] In the figure, Figure a shows BPN. pro The structure of BPN; Figure b shows the structure of BPN. pro Mediated ferroptosis therapy: (i) cancer-specific activation of DPCP to degrade DHODH; (ii) photothermal-induced release of BP, leading to GPX4 inactivation;

[0028] Figure 2 The synthetic route for compound BP in Example 1 is shown below;

[0029] Figure 3 BPN in Example 1 p1 and BPN p2 Structural diagram;

[0030] Figure 4 The values ​​used in Example 1 for calculating BP, DPCP, and DPCP control-1 and DPCP control-2 Absorbance standard curve for load capacity;

[0031] Figure 5 Compound 2 in Example 1 in CDCl3 1 1H NMR spectrum (500MHz);

[0032] Figure 6 Compound 2 in Example 1 in CDCl3 13 C NMR spectrum (125MHz);

[0033] Figure 7 The HRMS spectrum of compound 2 in Example 1;

[0034] Figure 8 Compound 4 in Example 1 in CDCl3 1 1H NMR spectrum (500MHz);

[0035] Figure 9 Compound 4 in Example 1 in CDCl3 13 C NMR spectrum (125MHz);

[0036] Figure 10Compound 5 in Example 1 in CDCl3 1 1H NMR spectrum (500MHz);

[0037] Figure 11 Compound 5 in Example 1 in CDCl3 13 C NMR spectrum (125MHz);

[0038] Figure 12 BTPA in CDCl3 in Example 1 1 1H NMR spectrum (500MHz);

[0039] Figure 13 BTPA in CDCl3 in Example 1 13 C NMR spectrum (125MHz);

[0040] Figure 14 The HRMS spectrum of BTPA in Example 1;

[0041] Figure 15 For example, BP in CDCl3 in Example 1 1 1H NMR spectrum (500MHz);

[0042] Figure 16 For example, BP in CDCl3 in Example 1 13 C NMR spectrum (125MHz);

[0043] Figure 17 The HRMS spectrum of BP in Example 1;

[0044] Figure 18 The chimeric peptide DPCP in CD3OD in Example 1 1 1H NMR spectrum (500MHz);

[0045] Figure 19 The HRMS spectrum of the chimeric peptide DPCP in Example 1;

[0046] Figure 20 The HPLC spectrum of the chimeric peptide DPCP in Example 1;

[0047] Figure 21 The chimeric peptide DPCP in CD3OD in Example 1 control-1 of 1 1H NMR spectrum (500MHz);

[0048] Figure 22 The chimeric peptide DPCP in Example 1 control-1 HRMS spectrum;

[0049] Figure 23 The chimeric peptide DPCP in Example 1 control-1 HPLC spectrum;

[0050] Figure 24 The chimeric peptide DPCP in CD3OD in Example 1 control-2 of 1 1H NMR spectrum (500MHz);

[0051] Figure 25 The chimeric peptide DPCP in Example 1 control-2 HRMS spectrum;

[0052] Figure 26 The chimeric peptide DPCP in Example 1 control-2 HPLC spectrum;

[0053] Figure 27 The proton NMR spectra of the three polypeptide and polymer conjugates in Example 1;

[0054] In the figure, Figure a shows the DSPE-PEG-DPCP in CD3OD. 1 1H NMR spectrum (500MHz); Figure b shows DSPE-PEG-DPCP in CD3OD. control-1 of 1 1H NMR spectrum (500MHz); Figure c shows DSPE-PEG-DPCP in CD3OD. control-2 of 1 The 1H NMR spectrum (500MHz) shows the characteristic peaks of the corresponding chimeric peptide and DSPE-PEG-NH2, respectively;

[0055] Figure 28 The synthetic route for the chimeric peptide DPCP conjugated with bukelane in Example 2 is shown below.

[0056] Figure 29 BPN in Example 2 pro BPN p1 and BPN p2 The representation;

[0057] Figure a shows a TEM image; Figure b shows a DLS image; Figure c shows the Zeta potential; Figure d shows the UV / Vis absorption of the material in PBS solution (pH 7.4); Figure e shows the fluorescence emission spectrum excited at 730 nm.

[0058] Figure 30 BPN in Example 2 pro The representation;

[0059] Figure a shows the diameter DLS study conducted over one week in 10% fetal bovine serum (FBS) and PBS (pH 7.4) buffer; Figure b shows the size distribution DLS study conducted over one week in 10% fetal bovine serum (FBS) and PBS (pH 7.4) buffer.

[0060] Figure 31 BPN in Example 3 pro BPN p1 and BPN p2 The representation;

[0061] Figure a shows the temperature change of the material in PBS solution (each sample concentration is 200 μg / mL). -1 ) and near-infrared light irradiation time (730nm, 1.0W cm) -2 The functional relationship between ) is shown in Figure b, which shows the relationship between ) with and without 15 minutes of illumination (730nm, 1.0Wcm). -2 In the case of BPN pro (200 μg mL) -1 PBS releases BP; Figure c shows BPN. pro BPN p1 and BPN p2 (100μg mL -1 ) with or without CatB (0.2 U mL) -1 HPLC chromatograms under the following conditions;

[0062] Figure 32 The five laser on / off cycles in Example 3 (730nm, 1.0W cm⁻¹) -2 15 minutes later, BPN pro BPN p1 and BPN p2 (200 μg mL each) -1 Photothermal stability;

[0063] Figure 33 BPN in Example 3 pro (200μg mL -1 ) Release BP without 730nm laser irradiation;

[0064] Figure 34 In vitro BPN in Example 4 pro mediated ferroptosis therapy;

[0065] In the figure, Figure a shows the BPN without laser irradiation. pro BPN p1 and BPN p2Cytotoxicity studies (n=3); Figure b shows laser irradiation (730nm, 1.0W cm⁻¹). -2 )15 minutes down BPN pro BPN p1 and BPN p2 Cytotoxicity studies (n=3); Figure c shows the results with materials (each sample was 40 μg / mL). -1 CLSM images of cells incubated for 24 hours; red fluorescence indicates cytoplasm, and blue fluorescence indicates nuclei stained with Hoechst 33342; Figure d shows the cells with the material (40 μg / mL per sample). -1 MFI (n=3) of cells incubated for 24 hours; p<0.001 between material group and control group; Figure e shows the results of buquina, DPCP, and BPN. pro BPN p1 or BPN p2 CLSM images of incubated cells; green fluorescence indicates DHODH antibody, and blue fluorescence indicates Hoechst 33342 stained cell nuclei; Figure f shows buquina, DPCP, and BPN. pro BPN p1 or BPN p2 The concentrations of MFI (buquina and DPCP) for incubating cells were both 10 μM, and the concentration of nanomaterials was 40 μg / mL. -1 The incubation time was 24 hours (n=3), and the pretreatment time for CA-074-Me, MLN4924, and BU-4061T was 6 hours; Figure g shows the proposed mechanism of DHODH degradation induced by CatB activation of DPCP; Figure h shows the Western blot analysis of intracellular GPX4 after different treatments; Figure i shows the proposed mechanism of BP-mediated covalent inhibition of GPX4; Figure j shows the effects of PBS (control) and BPN. p1 BPN p2 or BPN pro Incubation for 24 hours (nanoparticle concentration was 100 μg / mL) -1 Then, it was irradiated with NIR light for 15 minutes (730nm, 1.0W cm⁻¹). -2 Finally, the CLSM images of cells (n=3) were stained with PI / Calcein-AM, with red fluorescence and green fluorescence representing dead cells and live cells, respectively.

[0066] Figure 35 The DHODH immunofluorescence staining of cells after treatment with the three polypeptides in Example 4;

[0067] Figure a shows 4T1 cells with DPCP and DPCP. control-1 or DPCP control-2Confocal fluorescence images after 24 hours of incubation (10 μM each). Blue fluorescence shows Hoechst 33342, and green fluorescence shows DHODH antibody. Figure b shows the corresponding MFI (n = 3).

[0068] Figure 36 BPN in Example 5 pro In vitro anticancer mechanism and blood compatibility;

[0069] In the figure, Figure a shows the control (PBS) and BPN. p1 BPN p2 or BPN pro The cells used for incubation (all materials were at a concentration of 40 μg / mL) -1 The incubation period was 24 hours, followed by 15 minutes of light exposure (730nm, 1.0W cm⁻¹). -2 CLSM images of cells (n=3), "+Fer-1" group: cells pretreated with Fer-1 (100 μM) for 12 hours, green fluorescence showing C 11 -Bodipy 581 / 591 (10 μM) stained lipid peroxides; blue fluorescence represents Hoechst 33342 stained cell nuclei; Figure b shows the corresponding intracellular LPO levels of MFI and BPN. pro Compared with the control group: p < 0.001; Figure c shows BPN p1 BPN p2 and BPN pro Hemolytic activity (n=3), positive control: 1% Triton X-100 solution, inserted image: photographs of red blood cells incubated with different nanoparticles (centrifuged at 10,000 rpm);

[0070] Figure 37 In Example 5, 4T1 cells were compared with controls (PBS) and BPN without 730nm laser irradiation. p1 BPN p2 or BPN pro (40 μg mL each) -1 The confocal fluorescence image after 24 hours of incubation shows that the green fluorescence is C. 11 -Bodipy 581 / 591 LPO stained with (10 μM), the blue fluorescence is the cell nucleus stained with Hoechst;

[0071] Figure 38 For example, in Example 6, based on BPN pro Assessment of ferrodeogenesis;

[0072] Figure a shows the establishment of the tumor model and BPN. proTimeline of mediated ferroptosis therapy; Figure b shows tail vein injection of BPN. pro BPN p1 or BPN p2 (1mg mL -1 NIR-II images of mouse organs and tumors after administration of 200 μL; Figure c shows the tail vein administration of BPN. p1 BPN p2 and BPN pro (200μg mL -1 200 μL) 24 hours later, mice were exposed to light (730 nm, 1.0 W cm⁻¹) -2 Thermographic imaging 15 minutes later; Figure d shows the relative tumor volume changes in each group during the monitoring period (*p<0.05, **p<0.01); Figure e shows tumor images of each group after treatment; Figure f shows H&E staining of tumors in each group; Figure g shows immunofluorescence staining of tumors in each group, with green fluorescence indicating DHODH antibody and blue fluorescence indicating DAPI-stained cell nuclei; Figure h shows immunofluorescence staining of GPX4 in tumors, with red fluorescence indicating GPX4 antibody and blue fluorescence indicating DAPI-stained cell nuclei; Figure i shows the weight changes of mice in different treatment groups during the monitoring period.

[0073] Figure 39 NIR-II imaging of mouse tumors in Example 6;

[0074] In the figure, Figure a shows BPN injection via the tail vein. pro (1mg mL -1 NIR-II imaging of tumors at different time points after 200 μL (laser: 730 nm, ~89 mW cm⁻¹) -2 Exposure time: 200ms; Figure b shows the corresponding MFI;

[0075] Figure 40 In Example 6, saline and BPN were injected into the tail vein. p1 BPN p2 or BPN pro (200μgmL -1 ,200μL) and irradiated with (+L) or without 730nm laser (1.0W cm) -2 Histological H&E staining images of the heart, liver, spleen, lung and kidney of 4T1 tumor-bearing BALB / c mice on day 14 (15 minutes later).

[0076] Figure 41 Blood routine analysis of healthy BALB / c mice injected with different nanoparticles in Example 6 (n=3). Detailed Implementation

[0077] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0078] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0079] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0080] The instruments and raw materials used in the embodiments of this invention are as follows:

[0081] Unless otherwise specified, all chemicals were purchased from Anaiji Chemicals (China). Cell proliferation assay kits were purchased from Promega Biotechnology Co., Ltd. (Beijing, China). Cell culture media were purchased from Heklon Biochemicals Co., Ltd. (Shanghai, China). Trypsin, penicillin-streptomycin, and fetal bovine serum (FBS) were purchased from Gibco Life Technologies (USA). Buquina was purchased from Bidex Pharmaceuticals Co., Ltd. (Shanghai, China). Dipalmitoylphosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG) 2000 -NH2) was purchased from Yusi Pharmaceutical Technology Co., Ltd. (Chongqing, China). Lipid peroxidation detection probe (C 11 -Bodipy 581 / 591 The primary and secondary antibodies for glutathione peroxidase 4 (GPX4) were purchased from Anjiekai Biopharmaceutical Technology Co., Ltd. (Wuhan, China). The primary and secondary antibodies for dihydroorotate dehydrogenase (DHODH) were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China).

[0082] Instrument characterization: Proton and carbon NMR spectra were measured using a Bruker Avance III 500MHz spectrometer. Mass spectrometry was performed using a Bruker mass spectrometer (Bruker Daltonic flex analysis). An H-600 transmission electron microscope (Hitachi, Japan) was used to characterize the morphology of the nanoparticles. UV-Vis absorption spectra were recorded using a Cary 50 biospectrophotometer. FluoroLog-3 fluorescence spectrophotometer was used to measure fluorescence emission spectra at room temperature. Photothermal activity was determined using an RDXL4SD four-channel thermometer and a FLIR E6 infrared thermal imager. Confocal laser scanning microscopy (CLSM) images were obtained using a Nikon ECLIPSE Ti microscope. Cell viability data were recorded using a Synergy microplate reader (BioTek, USA). Near-infrared II images were acquired using a two-dimensional InGaAs camera.

[0083] Example 1

[0084] This embodiment is used to illustrate BPN. pro Synthesis:

[0085] Prepare according to the following steps:

[0086] First, CatB-degradable DPCP was prepared. Next, DPCP and DSPE-PEG were... 2000 -NH2 undergoes an amide condensation reaction to yield DSPE-PEG. 2000 -DPCP copolymers. BP was also prepared in high yields via Knoevenagel condensation and CuI-mediated alkyne-azo click reaction. Figure 2 Finally, BP and DSPE-PEG were added. 2000 - DPCP and DPPC are mixed at a mass ratio of 1:20:100 and nanoprecipitated to obtain BPN. pro This is the optimal feed ratio for preparing thermoresponsive liposomes. Two control nanoparticles (BPN) p1 and BPN p2 It was also prepared using a similar method, using peptides that target DHODH only (DPCP). control-1 ) and peptides that target E3 ligase only (DPCP) control-2 ()( Figure 3 According to the UV / Vis absorption spectrum ( Figure 4 ), calculate BP, DPCP, DPCP control-1 and DPCP control-2The loading capacities were 0.6%, 5.9%, 3.3%, and 5.2%, respectively. Combined results from high-resolution mass spectrometry (HRMS), nuclear magnetic resonance (NMR) spectroscopy, and high-performance liquid chromatography (HPLC) confirmed the successful synthesis of all compounds, including BP, DPCP, and DPCP. control-1 DPCP control-2 DSPE-PEG 2000 -DPCP, DSPE-PEG 2000 -DPCP control-1 and DSPE-PEG 2000 -DPCP control-2 ( Figure 5-27 ).

[0087] BP and DSPE-PEG from Example 1 2000 If the mass ratio of DPCP and DPPC is changed to 1:20:50 during the mixing and nanoprecipitation process, BPN will appear. pro The decreased sensitivity to temperature results in poor thermal response performance.

[0088] BP and DSPE-PEG from Example 1 2000 If the mass ratio of DPCP and DPPC is changed to 1:20:300 during the mixing and nanoprecipitation process, BPN will be produced. pro The BPN is too sensitive to temperature, thus causing pro It cannot exist stably in biological applications.

[0089] Example 2

[0090] This embodiment describes the specific preparation process of each raw material in Example 1:

[0091] Synthesis of Compound 2: Compound 1, 5-methyl-4-nitro-3-isoxazolecarboxylic acid (1.0 g, 5.81 mmol), was added to anhydrous dichloromethane (30 mL) cooled in an ice bath. Subsequently, oxalyl chloride (1.0 mL, 11.6 mmol) was added dropwise, followed by one drop of N,N-dimethylformamide. The ice bath was removed, and the reaction mixture was stirred overnight at room temperature. The solvent was then rotary evaporated in a water bath at room temperature to obtain the acyl chloride intermediate, which was immediately added dropwise to anhydrous dichloromethane (35 mL) cooled in an ice bath. Finally, a mixture of triethylamine (1.1 mL, 14.5 mmol) and 11-azido-3,6,9-trioxaundecan-1-amine (1.27 g, 5.81 mmol) in anhydrous dichloromethane (15 mL) was added dropwise to the acyl chloride solution, and the reaction mixture was stirred at room temperature for 2 hours. After removing volatiles under reduced pressure, the crude product was purified by silica gel column chromatography using ethyl acetate / petroleum ether (v / v, 1:3) as eluent to give compound 2 (1.79 g), yield: 83%. 1 H NMR (500MHz, CDCl3): δ (ppm) 7.19 (s, 1H), 3.82-3.53 (m, 14H), 3.38 (t, J = 5.0Hz, 2H), 2.84 (s, 3H). 13 C NMR (125MHz, CDCl3): δ (ppm) 171.86, 156.60, 153.18, 70.59, 70.47, 70.35, 69.95, 69.18, 50.64, 39.92, 13.39. HRMS: calcd.m / z=372.14; found m / z=395.12733.

[0092] Synthetic steps of compound 4: 3-Bromopropyne (0.98 g, 8.2 mmol) was added to compound 3, namely p-hydroxybenzaldehyde (1.0 g, 8.2 mmol) and potassium carbonate (6.5 g, 47 mmol) in a solution of N,N-dimethylformamide (25 mL). The reaction mixture was then stirred overnight at 60 °C. The crude product obtained by filtering off the potassium carbonate was then dissolved in dichloromethane and washed with brine. The collected organic layer was dried over anhydrous sodium sulfate. After filtration and removal of volatiles under reduced pressure, the crude product was purified by silica gel column chromatography using ethyl acetate / petroleum ether (v / v, 1:30) as eluent to give compound 4 (0.71 g), yield: 57%. 1 H NMR (500MHz, CDCl3): δ (ppm) 9.95 (s, 1H), 7.87 (d, J = 8.7Hz, 2H), 7.10 (d, J = 8.6Hz, 2H), 4.79 (d, J = 2.4Hz, 2H), 2.57 (t, J = 2.4Hz, 1H).13 C NMR (125MHz, CDCl3): δ (ppm) 190.75, 162.34, 131.87, 130.57, 115.16, 77.54, 76.38, 55.93.

[0093] The synthesis of compound 5 was performed as follows: Compound 4 (1.0 g, 6.2 mmol) and 2,4-dimethylpyrrole (1.33 g, 14 mmol) were dissolved in dichloromethane (150 mL), and trifluoroacetic acid (0.05 mL) was added. The reaction mixture was then stirred overnight at room temperature. Next, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (1.41 g, 6.2 mmol) was added to the reaction mixture, and the mixture was stirred overnight at room temperature. Finally, N,N-diisopropylethylamine (3.62 g, 28 mmol) and the boron trifluoride diethyl ether complex (11.64 g, 82 mmol) were added dropwise, and the mixture was stirred overnight at room temperature. The crude product was washed with dichloromethane and brine, and the collected organic layer was dried over anhydrous sodium sulfate. After filtration and removal of volatiles under reduced pressure, the crude product was purified by silica gel column chromatography using ethyl acetate / petroleum ether (v / v, 1:90) as eluent to give compound 5 (0.75 g), yield: 32%. 1 H NMR (500MHz, CDCl3): δ (ppm) 7.19 (d, J = 8.6 Hz, 2H), 7.09 (d, J = 8.7 Hz, 2H), 5.98 (s,2H),4.76(d,J=2.4Hz,2H),2.56(d,J=2.4Hz,1H),2.55(s,6H),1.42(s,6H). 13 C NMR (125MHz, CDCl3): δ (ppm) 158.08, 155.34, 143.12, 141.48, 131.75, 129.22, 127.98, 121.16, 115.60, 78.03, 75.89, 56.01, 14.54.

[0094] Synthetic steps of BTPA: Compound 5 (0.38 g, 1.0 mmol) and 4-(N,N-diphenylamino)benzaldehyde (0.82 g, 3.0 mmol) were dissolved in N,N-dimethylformamide (10 mL) under a nitrogen atmosphere. The reaction mixture was then heated to 120 °C, and acetic acid (0.4 mL) and piperidine (0.4 mL) were added dropwise after 15 minutes. After reacting for 24 hours, the crude product was washed with dichloromethane and brine, and the collected organic layer was dried over anhydrous sodium sulfate. After filtration and removal of volatiles under reduced pressure, the crude product was purified by silica gel column chromatography using dichloromethane / petroleum ether (v / v, 1:2) as eluent to give compound BTPA (0.25 g), yield: 28%. 1 H NMR (500MHz, CDCl3): δ (ppm) 7.59 (d, J = 16.2 Hz, 2H), 7.47 (d, J = 8.7 Hz, 4H), 7. 30-7.26(m,7H),7.25-7.22(m,2H),7.17(d,J=16.2Hz,2H),7.13(d,J=7.4Hz,7 H),7.09(d,J=8.6Hz,2H),7.06(t,J=7.3Hz,4H),7.02(d,J=8.6Hz,4H),6.60( s,2H),4.76(d,J=2.4Hz,2H),2.56(t,J=2.4Hz,1H),1.47(s,6H),1.43(s,2H). 13 C NMR (125MHz, CDCl3): δ (ppm) 158.01, 152.46, 148.50, 147.14, 141.54, 137.29, 135.45, 133.59, 130.38, 129.80, 129.35, 128.50, 128.35, 1 24.98,123.53,122.48,117.50,117.37,115.45,78.09,75.87,56.02,34.66,26.90,25.27,20.70,14.80.HRMS:calcd.m / z=888.38; found m / z=888.37853.

[0095] Synthesis of the BP probe: BTPA (0.2 g, 0.23 mmol), compound 2 (0.26 g, 0.69 mmol), and sodium ascorbate (0.02 g, 0.07 mmol) were dissolved in dichloromethane / methanol (v / v, 1:10). The reaction mixture was then degassed by argon bubbling for 30 minutes at room temperature. Copper sulfate (0.02 g, 0.06 mmol) was then dissolved in methanol (1.0 mL) and degassed by argon bubbling for 30 minutes at room temperature. Finally, the copper sulfate solution was added to the original reaction mixture via syringe and stirred overnight at room temperature. After removing volatiles under reduced pressure, the crude product was purified by silica gel column chromatography using ethyl acetate / petroleum ether (v / v, 3:1) as eluent to obtain the BP probe (0.24 g), yield: 84%. 1 H NMR (500MHz, CDCl3): δ (ppm) 7.87 (s, 1H), 7.59 (d, J = 16.2Hz, 2H), 7.46 (d, J = 8.3Hz, 4H), 7 .28(d,J=7.7Hz,6H),7.25(s,1H),7.22(d,J=8.1Hz,2H),7.12(d,J=8.1Hz,10H),7.10(s,1 H),7.06(t,J=7.4Hz,5H),7.02(d,J=8.4Hz,4H),6.59(s,2H),5.22(s,2H),4.55(t,J=4.9 Hz,2H),3.89(t,J=4.9Hz,2H),3.68-3.56(m,14H),2.79(s,3H),1.47(s,4H),1.26(s,2H). 13 C NMR (125MHz, CDCl3): δ (ppm) 171.91, 158.78, 156.63, 153.13, 148.52, 147.10, 143.35, 130.32 ,129.87,129.35,128.50,124.99,124.20,123.55,122.43,117.50,117.30,115.25,70.47,70 .45,70.39,70.30,69.33,69.15,62.00,53.44,50.33,39.89,37.08,32.75,31.92,30.02,29. 69,29.65,29.35,27.07,22.69,19.73,14.82,14.13,13.39.HRMS:calcd.m / z=1260.52; found m / z=1260.52098.

[0096] DPCP, DPCP control-1 and DPCPcontrol-2 Synthesis: First, 2-chlorotriphenylmethyl chloro resin (1.14 mmol g) was used. -1 The resin was soaked in anhydrous DMF for 1 hour. Then, a mixed solution of DIPEA (10 equivalents) and Fmoc-Gly-OMe (4 equivalents) in DMF was added dropwise to the resin, and the mixture was stirred at room temperature under a nitrogen atmosphere for 3 hours. After washing the resin three times with DMF, the resin was reacted with MeOH / DPIA / DMF (v / v / v, 1:2:7) for 30 minutes to cap unreacted groups. The resin was then incubated with piperidine / DMF (v / v, 1:4) for 15 minutes to cleave the Fmoc protecting groups and perform amide condensation. The resin was then reacted with a series of Fmoc-protected amino acids (3 equivalents), HOBT (3.6 equivalents), HBTU (3.6 equivalents), and DIPEA (7.5 equivalents) for 2 hours to perform amino acid coupling. After amino acid coupling, the resin was reacted overnight with a mixture of bukelanatine (2 equivalents), HOBT (2.4 equivalents), HBTU (2.4 equivalents), and DIPEA (5 equivalents). The resin was washed sequentially with DMF, MeOH, and DCM, and then dried under vacuum for 30 minutes. Finally, the peptide DPCP was cleaved from the resin for 1.5 hours using TFA / H2O / 1,2-ethylenedithiol (v / v / v, 95:2.5:2.5). The crude product was purified by HPLC to obtain DPCP (70 mg), which was HOOC-GGLFGPIYPALASGSG-buquinatine, with the protein sequence shown in SEQ ID No. 1. The preparation process of DPCP is as follows. Figure 28 As shown. 1H NMR(500MHz,CD3OD,δ):8.17(dd,J=9.3,5.1Hz,1H),8.04(d,J=8.6Hz,1H),7.96(dd,J =9.5,2.8Hz,1H),7.78(dd,J=8.4,1.6Hz,3H),7.74-7.66(m,3H),7.59(td,J=7.8,1.8H z,1H),7.48-7.39(m,1H),7.31(td,J=7.5,1.2Hz,1H),7.28-7.13(m,7H),7.02(dd,J=8 .6,3.2Hz,2H),6.68(dd,J=8.5,2.0Hz,2H),4.68-4.52(m,5H),4.49-4.24(m,10H),4.1 8(q,J=7.3Hz,1H),4.03(d,J=16.8Hz,1H),3.99-3.83(m,9H),3.81-3.66(m,5H),3.64 -3.47(m,4H),3.19-3.11(m,1H),3.08-2.97(m,3H),2.88(dd,J=13.8,8.0Hz,1H),2.52 (s,3H),2.13(d,J=72.6Hz,4H),1.88(d,J=7.0Hz,6H),1.60(d,J=7.4Hz,9H),1.35(dd, J=52.3,7.1Hz,8H),1.15-1.03(m,1H),0.97-0.84(m,16H),0.79(t,J=7.4Hz,4H).HRMS m / z:[M] - calcd for C 91 H 114 F2N 17 O 21 ,1818.84; found,1818.83687. Control chimeric peptide DPCP control-1 and DPCP control-2 Prepared in a similar manner, DPCPcontrol-1 is HOOC-GGLFG-buquina, with the amino acid sequence shown in SEQ ID No. 2, and DPCPcontrol-2 is HOOC-GGLFGPIYPALASGSG-Ac, with the protein sequence shown in SEQ ID No. 3. It is worth noting that DPCP... control-1 It does not contain peptide fragments that target E3 ligases. 1HNMR (500MHz, CD3OD, δ): 8.13 (dd, J=9.3, 5.2Hz, 1H), 7.93 (dd, J=9.7, 2.8Hz, 1H), 7.76 (dd, J=8.2, 1.6Hz, 2H), 7.68 (d, J=8. 2Hz,2H),7.64(td,J=8.9,2.8Hz,1H),7.58(td,J=7.8,1.8Hz,1H),7.45-7.38(m,1H),7.34-7.27(m,5H),7.26-7.19(m,2H),4 .74(dd,J=8.4,6.0Hz,1H),4.36-4.28(m,1H),4.18(d,J=9.9Hz,2H),3.98-3.71(m,4H),3.31(p,J=1.6Hz,3H),3.21(dd,J=14 .0,6.0Hz,1H),3.01(dd,J=14.0,8.5Hz,1H),2.47(s,3H),1.75-1.52(m,3H),1.28(s,1H),0.89(dd,J=14.9,5.8Hz,7H).HRMS m / z:[M] - calcd forC 44 H 43 F2N6O7,805.32; found,805.31558.DPCP control-2 It was prepared without the bukelane unit. 1HNMR(500MHz,CD3OD,δ):7.25(s,6H),7.03(d,J=8.5Hz,2H),6.70(d,J=8.5 Hz,2H),4.67-4.56(m,2H),4.52(q,J=6.8Hz,1H),4.47-4.34(m,6H),4.28( td,J=7.3,4.0Hz,2H),4.22(q,J=7.3Hz,1H),4.03-3.85(m,11H),3.84-3.6 9(m,6H),3.67-3.43(m,4H),3.31(p,J=1.6Hz,4H),3.15(dd,J=14.1,6.6Hz, 1H),3.05(ddd,J=20.3,13.9,7.4Hz,2H),2.88(dd,J=13.8,8.0Hz,1H),2.2 3(ddd,J=12.5,10.5,5.0Hz,1H),2.07(d,J=10.3Hz,2H),2.01(s,3H),1.89( d,J=6.6Hz,6H),1.75-1.50(m,9H),1.41(d,J=7.3Hz,4H),1.31(d,J=7.0Hz ,4H),1.10(d,J=7.2Hz,1H),0.99-0.84(m,18H),0.80(t,J=7.4Hz,4H).HRMS m / z:[M] - calcd for C 70 H 103 N 16 O 21 ,1503.76;found,1503.75184.

[0097] DSPE-PEG-DPCP, DSPE-PEG-DPCP control-1 and DSPE-PEG-DPCP control-2 Synthesis: A mixture of DPCP (36 mg, 0.02 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (10 mg, 0.05 mmol), and hydroxysuccinimide (6 mg, 0.05 mmol) was dissolved in tetrahydrofuran (10 mL) and stirred at room temperature for 1 hour. Subsequently, this mixture was added dropwise to a solution containing DSPE-PEG. 2000 The solution of -NH2 (100 mg) in tetrahydrofuran (5 mL) was stirred at room temperature for 48 hours. Then, the crude product was further dialyzed through a dialysis membrane for 72 hours. Finally, the obtained DSPE-PEG was... 2000 -DPCP was freeze-dried in a vacuum. Additionally, the control polymer DSPE-PEG was used. 2000-DPCP control-1 and DSPE-PEG 2000 -DPCP control-2 It was also prepared using a similar method. The former was prepared via DSPE-PEG. 2000 -NH2 and DPCP control-1 The latter is prepared by DSPE-PEG 2000 -NH2 and DPCP control-2 The reaction was obtained.

[0098] Synthesis of nanoparticles: BPN pro Nanoparticles through DSPE-PEG 2000 -DPCP, dipalmitoylphosphatidylcholine (DPPC), and BP were prepared by nanoprecipitation, with BP encapsulated in the hydrophobic core of liposomes through π-π stacking and hydrophobic interactions. Among them, BP, DSPE-PEG... 2000 -DPCP and DPPC were nanoprecipitated at a mass ratio of 1:20:100 to obtain BPN with good water solubility. pro This is the optimal feed ratio for preparing thermoresponsive liposomes. The specific steps are as follows: A mixture of DSPE-PEG-DPCP (1 mg), dipalmitoylphosphatidylcholine (DPPC; 5 mg), and BP (0.05 mg) was dissolved in chloroform (5 mL) and the solvent was removed under reduced pressure to form an organic film. The organic film was then hydrated in a 65°C water bath for 15 minutes and sonicated for 20 minutes. After filtration using a 0.22 μm polyvinylidene fluoride needle filter, the mixture was further purified five times (5000 rpm) through an ultrafiltration centrifuge tube (molecular weight cutoff: 50 kDa) to remove free drug. Finally, the concentrated BPN was... pro The suspension was freeze-dried and weighed. Two control nanoparticles (BPN) were used. p1 and BPN p2 CatB-cleavable peptides targeting only DHODH (denoted as DPCP) were used separately. control-1 ) and CatB-cleavable peptides that target only E3 ligases (denoted as DPCP) control-2 BP, DPCP, and DPCP were prepared using a similar method to replace DPCP. Based on the UV / Vis absorption spectra, the values ​​of BP, DPCP, and DPCP were calculated. control-1 and DPCP control-2 The load capacities were 0.6%, 5.9%, 3.3%, and 5.2%, respectively.

[0099] The prepared BPN pro BPN p1 and BPN p2 The characteristics such as Figure 29 As shown. Figure 29As shown in Figure a, these nanoparticles are spherical and uniformly distributed, as observed in transmission electron microscopy (TEM) images. Based on dynamic light scattering (DLS) results, BPN... pro BPN p1 and BPN p2 Having similar hydrodynamic dimensions (≈122nm) Figure 29 b). After one week of storage at room temperature in 10% fetal bovine serum (FBS) and phosphate-buffered saline (PBS), BPN pro The hydrodynamic dimensional changes are negligible. Figure 30 This indicates that BPN pro They exhibit good colloidal stability. Furthermore, these nanoparticles possess similar negative zeta potential values ​​( Figure 29 c). As observed in the UV / Vis spectrum, these nanoparticles exhibit absorption bands at 560 and 730 nm. Figure 29 d). After excitation with a 730 nm laser, the fluorescence spectrum showed a similar characteristic emission band at 760 nm, with a tail extending over 1000 nm. Figure 29 e). These data indicate that the binding of different peptides does not affect the physical properties of these materials.

[0100] Example 3

[0101] This embodiment is used to explore BPN. pro In vitro photothermal properties, drug release and CatB-specific activation

[0102] Under continuous irradiation by a 730nm laser, BPN pro BPN p1 and BPN p2 The temperature of the nanoparticle solution stabilized at approximately 45°C at t = 15 minutes. Figure 31 a). After five consecutive test cycles, the change in the photothermal curve is negligible. Figure 32 This confirms their excellent photothermal stability. These data indicate that the binding of different peptides does not affect the photothermal properties of these nanoparticles. Furthermore, photothermally triggered BP release was confirmed. Samples were analyzed by HPLC after continuous 730 nm illumination for 15 minutes. The results clearly show that BP is released from BPN. pro Released from ( Figure 31 b). Conversely, in the absence of light, even after prolonged incubation, the release of BP remains negligible. Figure 33 To evaluate BPN pro BPN p1 and BPN p2 Cancer-specific activation of these substances was investigated, and their interaction with CatB was analyzed using HPLC. Figure 31c). BPN p1 BPN p2 and BPN pro The elution peaks were observed at 7.7, 16.0, and 12.8 min, respectively. Conversely, in the treatment without CatB incubation, the HPLC chromatogram showed no signal. This confirms the presence of BPN. pro DPCP, BPN p1 DPCP control-1 and BPN p2 DPCP control-2 It can respond to CatB in-situ release. BPN pro BPN p1 and BPN p2 The characterization includes the temperature conditions in the PBS solution and the corresponding HPLC chromatograms of the experimental groups, as shown in the figure. Figure 31 As shown.

[0103] Example 4

[0104] This embodiment is used to detect BPN. pro Cellular uptake, in vitro DHODH and GPX4 inactivation, and in vitro therapeutic effects.

[0105] First, BPN was assessed using 4T1 mouse breast cancer cells via the MTS cell viability assay. pro BPN p1 and BPN p2 Cytotoxicity. Cells were incubated with different concentrations of nanoparticles, and cell viability values ​​all exceeded 91%. Figure 34 a) indicates that these nanoparticles possess negligible dark cell toxicity. However, in the presence of 730 nm light, the viability of 4T1 cells gradually decreased with increasing material concentration. Figure 34 b). In 100 μg mL -1 At nanoparticle concentrations, with BPN p1 or BPN p2 The incubated cells showed reduced cell viability (~43%).

[0106] Conversely, with BPN p1 and BPN p2 In comparison, BPN pro It exhibited significantly reduced cell viability (12%). BPN p1 and BPN p2 The similar decrease in cell viability can be attributed to photothermal release of BP, which induces ferroptosis in cells by inhibiting GPX4 activity. (BPN) pro The significant decrease in cell viability can be attributed to DPCP-induced DHODH degradation. The combined effect of GPX4 and DHODH inactivation induced a greater degree of ferroptosis. These data clearly demonstrate that BPN...pro Compared to BPN p1 and BPN p2 It has a stronger therapeutic effect.

[0107] Under laser irradiation, when the concentration of nanoparticles is changed to 1 μg / mL -1 This may lead to BPN. pro BPN p1 and BPN p2 Adverse consequences of insignificant cell-killing effect; when the concentration of nanoparticles is changed to 500 μg / mL -1 This may lead to BPN. pro BPN p1 and BPN p2 The adverse consequence of having similar cell-killing effects but being unable to distinguish which is stronger in terms of anti-cancer efficacy.

[0108] 4T1 cancer cells were placed in a BPN-containing environment. pro BPN p1 or BPN p2 Cells were cultured in BPN medium for 24 h, then nuclei were labeled with Hoechst 33342, and cell fluorescence imaging was performed using laser confocal scanning microscopy (CLSM). Compared with the PBS-treated control group, the control group treated with BPN... pro BPN p1 or BPN p2 The red fluorescence signal of the Bodipy probe can be clearly observed in the incubated cells. Figure 34 c). The relative mean fluorescence intensities (MFI) were 35.7, 35.2, and 32.6, respectively. Figure 34 d) This indicates that the nanoparticles exhibit similar cellular uptake. Cellular uptake is clearly unaffected by different peptide conjugations.

[0109] Due to BPN pro The presence of DPCP targeting the protein DHODH indicates that BPN... pro It can effectively induce the degradation of intracellular DHODH. Therefore, the expression level of intracellular DHODH was measured by immunofluorescence staining. Cells were then treated with buquina, DPCP, and BPN, respectively. pro BPN p1 and BPN p2 Incubate for 24 hours. Compared with the PBS-treated control group, the signal of DHODH antibody [secondary antibody labeled with Coralite488 (a green fluorescent probe)] was significantly different when using DPCP or BPN. pro The effect was significantly reduced in incubated cells (the volumes of DHODH antibody and its secondary antibody used were 5 μL and 500 μL, respectively). However, compared with buquina and BPN... p1 BPNp2 DPCP control-1 or DPCP control-2 The green fluorescence signal did not change significantly in the incubated cells. Figure 34 e and Figure 35 Compared to the PBS-treated group, the use of DPCP or BPN... pro The MFI of DHODH antibody in incubated cells decreased significantly by 81.7% and 86.8%, respectively. Conversely, in comparison with buquina and BPN... p1 BPN p2 DPCP control-1 or DPCP control-2 In the incubated group, the decrease in MFI of DHODH antibody was negligible. Figure 34 f and Figure 35 These data confirm that the efficient degradation of DHODH is attributed to BPN. pro Infixed DPCP.

[0110] To elucidate the degradation pathway of DHODH, UPS was selectively inhibited using corresponding enzyme inhibitors. First, cells were pre-incubated with a 26S proteasome inhibitor (BU-4061T), a NEDD8 activator inhibitor (MLN4924), and a CatB inhibitor (CA-074-Me), respectively. MLN4924 inhibits E3 ligase activity by suppressing Neddylation of its core subunit (Cullin protein), while BU-4061T inhibits 26S proteasome activity by covalently binding to the catalytic subunit. These cells, treated with the various inhibitors, were then subjected to BPN. pro Incubate together. Compared to the control group incubated with PBS, BPN pro The green fluorescence and MFI in the incubated cells no longer decreased. Figure 34 According to literature reports, such as Figure 34 As shown in g, the proposed BPN pro The mechanism of DHODH degradation is as follows: First, CatB cleaves and releases DPCP. Then, DPCP specifically binds to DHODH and initiates the recruitment of the E3 ligase. Next, the E2 ubiquitin conjugate is recruited. Under the action of the NEDD8 activator, ubiquitin is directly transferred from the E2 conjugate to DHODH to form a ubiquitin chain. Finally, the 26S proteasome is recruited and degrades DHODH. DPCP is then released and enters the next DHODH degradation cycle.

[0111] Since BP contains a nitroisoxazole group that can covalently bind to a selenocysteine ​​residue, it is speculated that BPN... proIt can effectively downregulate the activity of intracellular GPX4. Therefore, Western blotting was used to analyze the protein level of GPX4 in 4T1 cancer cells after different treatments. For comparison, control nanoparticles (BTPAN) were prepared using BP without nitroisoxazole groups via the same method. pro In the absence of light, BPN pro and BTPAN pro No significant downregulation of GPX4 was caused. Figure 34 h). Compared with the control group (PBS) and BTPAN pro Compared to the previous group, after laser irradiation, BPN pro Intracellular GPX4 levels were significantly downregulated. This indicates that photothermal-induced BP release significantly inhibited GPX4 in 4T1 cancer cells due to the nitroisoxazole group in BP. According to literature reports, such as... Figure 34 As shown in i, the proposed intracellular activation mechanism of BP-mediated GPX4 inhibition is as follows: After photothermal-induced release of BP into the cell, firstly, the nitroisoxazole group undergoes ring-opening hydrolysis to form an unstable ketone intermediate; subsequently, the ketone intermediate undergoes Retro-Claisen-like condensation to release acetic acid and form a nitroketoxime; next, it undergoes ring-closure dehydration to form a furazan intermediate; then, the furazan intermediate undergoes a ring-opening tautomerism to form a nitrile oxide; finally, the electrophilic group of the nitrile oxide covalently binds to the selenocysteine ​​residue of GPX4 to form an adduct, ultimately achieving effective inhibition of GPX4.

[0112] Given the effective degradation of DHODH and the inhibition of GPX4, Calcein-AM / PI (cell survival / death indicator) staining experiments were used to further confirm the presence of BPN. pro Therapeutic effects on 4T1 cancer cells. For example... Figure 34 As shown in j, without NIR light irradiation, using BPN p1 BPN p2 or BPN pro The incubated cells clearly showed a green fluorescent signal from Calcein-AM. Conversely, a red fluorescent signal from PI was clearly observed in the CLSM images of cells incubated with these nanoparticles. Among them, BPN showed a red fluorescent signal from PI under near-infrared light illumination. p1 and BPN p2 The released BP triggered LPO, inducing cell death. As expected, BPN... pro The most effective ferrodeogenesis therapy was achieved through further degradation of DHODH.

[0113] Example 5

[0114] This example illustrates the potential mechanism of ferroptosis treatment:

[0115] To clarify BPN pro The mechanism of mediated ferroptosis therapy, using C 11 -Bodipy 581 / 591 The levels of lipid peroxides (a biomarker of ferroptosis) in 4T1 cancer cells incubated with nanoparticles were detected. 11 -Bodipy 581 / 591 It is a widely used LPO-specific sensor that can be oxidized by intracellular lipid peroxides and displays a bright green fluorescent signal. Compared with the PBS-treated control group, the BPN... p1 or BPN p2 The CLSM images of the incubated group showed approximately 7.8 times stronger green fluorescence signal after 730 nm laser irradiation. Figure 36 ab). This indicates that BPN p1 and BPN p2 Significant LPO was induced in cells. This was because photothermal-triggered BP release effectively inhibited GPX4 and led to the accumulation of lipid hydroperoxides. Conversely, BPN was observed in the absence of light irradiation, as seen in CLSM images. p1 and BPN p2 The group showed a weak green fluorescence signal similar to the control group. Figure 37 This confirms that photothermally released BP has the ability to promote intracellular LPO. Furthermore, after 730nm laser irradiation, it interacts with BPN... p1 and BPN p2 Compared to the previous group, BPN can be clearly observed from the CLSM images. pro The group exhibited a fairly strong green fluorescence signal ( Figure 36 ab), this indicates that BPN pro Higher LPO levels were induced in cells. This is because of the interaction with BPN. p1 DPCP targeting only DHODH control-1 and BPN p2 DPCP targeting only the E3 ligase control-2 In comparison, BPN pro DPCP in the formula can simultaneously target DHODH and E3 ligase. DPCP then achieves the degradation and clearance of DHODH via UPS, ultimately leading to a decrease in CoQH2 and the accumulation of LPO. Interestingly, it is similar to BPN. p1 and BPN p2 Compared to the group, BPN in the absence of light irradiation pro The group showed a similar weak green fluorescence signal ( Figure 37This is because the ferroptosis defense activity of GPX4 is preserved, and it plays a role in detoxifying lipid peroxides in the absence of photothermal-induced BP release. Compared with the control group, BPN pro The group showed a green fluorescence signal approximately 12.1 times stronger after irradiation with a 730nm laser. Figure 36 b). This indicates a connection with BPN. p1 and BPN p2 In comparison, BPN pro It possesses the strongest ability to induce ferroptosis. This is because BPN pro GPX4 inhibition and DHODH degradation-induced ferroptosis induced maximal LPO levels in cells. Pretreatment of cells with the ferroptosis inhibitor (Fer-1) followed by laser irradiation reduced BPN levels. pro The green fluorescence in the CLSM images of the incubated cells decreased by approximately 7-fold. These data clearly confirm that BPN... pro The potent anticancer effect is attributed to ferroptosis induced by high LPO levels. Furthermore, mouse erythrocytes did not exhibit significant hemolytic activity after incubation with these nanoparticles. Figure 36 c). This indicates that these materials have good blood compatibility and can be used for subsequent in vivo biological studies.

[0116] Example 6

[0117] This embodiment is used to illustrate BPN. pro Biodistribution, thermal imaging, treatment efficacy assessment, DHODH and GPX4 downregulation, and biosafety:

[0118] Considering BPN pro Building on its excellent performance at the cellular level, we will next explore its in vivo anticancer efficacy using 4T1 tumor-bearing BALB / c mice. First, a tumor model was established by subcutaneously inoculating mice with 4T1 cells. (The text continues with further details about cell-level performance and in vivo anticancer efficacy.) Figure 38 As shown in Figure a, 7 days later, BPN was administered via the tail vein. pro BPN p1 or BPN p2 The drug was injected into tumor-bearing mice. The tumor site was then irradiated with a 730nm laser. Finally, tumor growth rate was recorded continuously for 14 days.

[0119] To determine the optimal time for laser irradiation, during BPN injection... pro Near-infrared II (NIR-II) imaging experiments were performed at different time points afterward. It was observed that the tumor site was brightest at 24 hours. Figure 39 This indicates that the nanoparticles achieved maximum tumor accumulation at 24 hours. Therefore, the optimal time for light irradiation was determined to be 24 hours after material injection. Furthermore, biodistribution studies showed that BPN… proBPN p1 and BPN p2 It mainly accumulates in the spleen and liver, followed by tumors. Figure 38 b).

[0120] After determining the maximum tumor accumulation time of the nanoparticles, in vivo ferroptosis therapy was performed. BPN was administered via tail vein injection. pro BPN p1 or BPN p2 24 hours later, a laser (730nm, 1.0W cm⁻¹) was used. -2 The tumors of mice were irradiated. After 15 minutes of irradiation, the tumor temperature was observed to gradually rise to approximately 48 degrees Celsius. Figure 38 c). At the same irradiation time point, BPN pro BPN p1 or BPN p2 The tumor temperature values ​​of the groups were similar. This may be because these nanoparticles have similar tumor accumulation and photothermal properties. Subsequently, BPN... pro The anticancer effects were explored. Changes in relative tumor volume in different treatment groups were shown in the following figures. Figure 38 As shown in d. Compared with the control group treated with physiological saline, BPN injection in the absence of light exposure... p1 BPN p2 or BPN pro Tumor growth was not significantly inhibited in the group with BPN. p1 and BPN p2 The group achieved better therapeutic effects under laser irradiation. Compared with the control group treated with saline, the relative tumor volume was reduced by 2.5 times and 2.3 times, respectively. This clearly demonstrates the contribution of photothermal-induced BP release to the treatment. BPN was observed under light irradiation. pro Tumor growth was significantly inhibited in the treatment group. Compared with the control group treated with saline, the relative tumor volume was reduced by 20.4 times. The downregulation of GPX4 and DHODH activity is attributed to BPN. pro The main reason for the excellent therapeutic effect. After completing the efficacy evaluation, tumors were removed from the mice for photographing. Figure 38 e). In the absence of light exposure, the tumor sizes in the saline injection group and the nanoparticle injection group were similar. BPN p1 and BPN p2 Tumors in the injection group shrank after light exposure. In contrast, BPN... pro The injection group showed the smallest tumor size after light irradiation. Subsequently, the tumors were stained with hematoxylin and eosin (H&E). Compared to other treatment groups, the light-irradiated BPN... pro The tumors in this group showed significant nuclear shrinkage. Figure 38 f). These data clearly confirm BPN proExcellent tumor treatment results.

[0121] Intratumoral DHODH and GPX4 were detected using DHODH antibody (with a secondary antibody labeled Coralite488) and GPX4 antibody (with a secondary antibody labeled Cyanine3 (a red fluorescent probe)). BPN pro The green fluorescence of the group was significantly reduced ( Figure 38 This indicates that DHODH has significantly degraded. Figure 38 h shows immunofluorescence staining of GPX4. BPN under light illumination. pro The group showed very weak red fluorescence, indicating significant interference with GPX4 within the tumor. During the 14-day monitoring period, there were no abnormal changes in body weight in any group. Figure 38 i). The heart, liver, spleen, lungs, and kidneys maintain their normal physiological morphology ( Figure 40 Blood routine tests showed that systemic administration of BPN... pro Subsequently, these blood parameters in healthy mice showed no abnormal changes. Figure 41 These data undoubtedly demonstrate that the proposed anti-cancer strategy has good efficacy and good biosafety.

[0122] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A multifunctional nanoplatform for the treatment of ferroptosis, characterized in that: The multifunctional nanoplatform is a thermoresponsive liposome BPN. pro The BPN pro For DSPE-PEG 2000 -DPCP / DPPC@BP; DPCP consists of a cathepsin B cleavable peptide, an E3 ligase targeting peptide, and a bukelana unit targeting DHODH, and its structural formula is shown in formula (1): Equation (1); BP is prepared by Knoevenagel condensation and CuI-mediated alkyne azo click reaction, and its structural formula is shown in formula (2): Equation (2); The preparation method of the multifunctional nanoplatform for ferroptosis treatment includes the following steps: Preparation of protein hydrolysis-targeting chimeric polypeptide DPCP: Preparation of cathepsin B-cleavable DPCP; Preparation of DSPE-PEG 2000 -DPCP copolymer: DPCP and DSPE-PEG 2000 -NH2 undergoes an amide condensation reaction to yield DSPE-PEG. 2000 -DPCP copolymer; Preparation of BPN pro : BP, DSPE-PEG 2000 -DPCP and dipalmitoylphosphatidylcholine (DPPC) were mixed and nanoprecipitated to obtain BPN. pro .

2. The multifunctional nanoplatform for ferroptosis treatment according to claim 1, characterized in that: The preparation of BPN pro In China, BP and DSPE-PEG 2000 - DPCP and DPPC are mixed in a mass ratio of 1:20:100 and nanoprecipitated.

3. The multifunctional nanoplatform for ferroptosis treatment according to claim 1, characterized in that: The BP was prepared by mixing BTPA and compound 2. The preparation method of compound 2 was as follows: 5-methyl-4-nitro-3-isoxazole carboxylic acid was added to anhydrous dichloromethane cooled in an ice bath. Then, oxalyl chloride was added dropwise and 1 drop of N,N-dimethylformamide was added. The ice bath was removed and the reaction mixture was stirred overnight at room temperature. Then, the solvent was evaporated by rotary evaporation in a water bath at room temperature to obtain an acyl chloride intermediate, which was immediately added dropwise to anhydrous dichloromethane cooled in an ice bath. Finally, a mixture of triethylamine and 11-azido-3,6,9-trioxaundecan-1-amine in anhydrous dichloromethane was added dropwise to the acyl chloride intermediate solution, and the reaction mixture was stirred at room temperature for 2 hours. After removing volatiles under reduced pressure, the crude product was purified by silica gel column chromatography using ethyl acetate / petroleum ether at a volume ratio of 1:3 as the eluent to obtain compound 2. The structural formula of BTPA is shown in equation (3): Equation (3); The structural formula of compound 2 is shown in formula (4): Equation (4).

4. The multifunctional nanoplatform for ferroptosis treatment according to claim 3, characterized in that: The preparation process of BTPA is as follows: under a nitrogen atmosphere, compounds 5 and 4-(N,N-diphenylamino)benzaldehyde are dissolved in N,N-dimethylformamide. Then, the reaction mixture is heated to 120 °C and acetic acid and piperidine are added dropwise after 15 minutes. After reacting for 24 hours, the crude product is washed with dichloromethane and brine, and the collected organic layer is dried with anhydrous sodium sulfate. After filtration and removal of volatiles under reduced pressure, the crude product is purified by silica gel column chromatography using a 1:2 volume ratio of dichloromethane / petroleum ether as the eluent to obtain compound BTPA. The structural formula of compound 5 is shown in formula (5): Equation (5).

5. The multifunctional nanoplatform for ferroptosis treatment according to claim 4, characterized in that: The preparation method of compound 5 is as follows: compound 4 and 2,4-dimethylpyrrole are dissolved in dichloromethane and trifluoroacetic acid is added. Then, the reaction mixture is stirred overnight at room temperature. Next, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone is added to the reaction mixture and stirred overnight at room temperature. Finally, N,N-diisopropylethylamine and boron trifluoride diethyl ether complex are added dropwise and stirred overnight at room temperature. The crude product is washed with dichloromethane and brine, and the collected organic layer is dried with anhydrous sodium sulfate. After filtration and removal of volatiles under reduced pressure, the crude product is purified by silica gel column chromatography using a mixed solution of ethyl acetate / petroleum ether at a volume ratio of 1:90 as the eluent. The structural formula of compound 4 is shown in formula (6): Equation (6).

6. The multifunctional nanoplatform for ferroptosis treatment according to claim 5, characterized in that: The preparation method of compound 4 is as follows: 3-bromopropyne is added to a solution of N,N-dimethylformamide containing p-hydroxybenzaldehyde and potassium carbonate; then, the reaction mixture is stirred overnight at 60 °C; then, the crude product obtained by filtering out potassium carbonate is dissolved in dichloromethane and washed with brine; the collected organic layer is dried with anhydrous sodium sulfate; after filtration and removal of volatiles under reduced pressure, the crude product is purified by silica gel column chromatography using a mixed solution of ethyl acetate / petroleum ether as eluent.

7. The multifunctional nanoplatform for ferroptosis treatment according to claim 3, characterized in that: In the preparation of BP, the raw materials include BTPA and compound 2. BP is obtained by dissolving BTPA, compound 2 and sodium ascorbate in a dichloromethane / methanol mixture, followed by degassing to obtain a reaction mixture. Then, copper sulfate is dissolved in methanol and degassed. Finally, the copper sulfate solution is added to the reaction mixture and stirred overnight at room temperature. After removing volatiles under reduced pressure, the crude product is purified by silica gel column chromatography using an ethyl acetate / petroleum ether mixture as the eluent to obtain BP.

8. The use of the multifunctional nanoplatform for ferroptosis therapy as described in claim 1 in the preparation of a medicament for ferroptosis therapy of tumors.

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