A cerium-based iron death / pan-apoptosis nanoinducer and its preparation method and application in preparing drugs for treating breast cancer

Abstract

The present invention belongs to the field of pharmaceutical preparations and biomedicine technology, and relates to a cerium-based ferroptosis / pan-apoptosis nanoinducer, a preparation method thereof, and its application in the preparation of drugs for treating breast cancer. It comprises a near-infrared-triggered photothermal enhancement nanocarrier and MTO, wherein the MTO is encapsulated in CD nanoparticles of the nanocarrier. The nanocarrier is composed of CD nanoparticles coated with a membrane material; the CD nanoparticles are a metal polyphenol network structure formed by cerium ions and dopamine, and the dopamine in the metal polyphenol network structure is oxidatively polymerized into polydopamine; the membrane material is a cell membrane with homologous targeting to the target tumor cells. The cerium-based ferroptosis / pan-apoptosis nanoinducer provided by the present invention uses a near-infrared-triggered photothermal enhancement nanocarrier, which can achieve specific, space-time controllable tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations and biomedicine, and relates to a cerium-based ferroptosis / pan-apoptosis nano-inducer, a preparation method thereof, and an application thereof in preparing a drug for treating breast cancer. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Cerium (Ce)-based materials have the potential to simultaneously activate ferroptosis and pan-apoptosis, thereby circumventing the apoptotic resistance commonly seen in breast cancer (BC) chemotherapy. However, the limited and uncontrollable efficiency of Ce-catalyzed Fenton-like reactions and the complex ferroptosis defense system hinder the therapeutic efficacy of Ce-based systems. Summary of the Invention

[0004] In order to address the deficiencies of the prior art, the purpose of the present invention is to provide a cerium-based ferroptosis / pan-apoptosis nanoinducer, a preparation method thereof, and its application in the preparation of drugs for treating breast cancer. The cerium-based ferroptosis / pan-apoptosis nanoinducer provided by the present invention adopts near-infrared (NIR)-triggered photothermal enhanced nanocarriers, which can achieve specific and space-time controllable tumor treatment.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] In the first aspect, a near-infrared-triggered photothermal enhancement nanocarrier (named M@CD) is composed of CD nanoparticles coated with a membrane material; the CD nanoparticles are a metal polyphenol network (MPN) structure formed by cerium ions and dopamine (DA), and the dopamine in the metal polyphenol network structure is oxidatively polymerized into polydopamine (PDA); the membrane material is a cell membrane (M) with homologous targeting to the target tumor cells.

[0007] In the nanocarrier provided by the present invention, a metal polyphenol network structure is formed by the complex coordination of the phenol ligand DA and the cerium ion. Under the catalytic action of the cerium ion, DA is polymerized into PDA, which enables the nanocarrier to have an NIR-triggered photothermal effect. At the same time, the tumor microenvironment (TME) in which tumor cells are located is generally an acidic environment. The metal polyphenol network structure formed by the present invention affects the coordination relationship between DA and the cerium ion in the acidic environment, and can release the cerium ion, thereby mediating the excellent catalytic activity of the Fenton-like reaction, glutathione (GSH) consumption and O2 production capacity, and can generate a large amount of reactive oxygen species (ROS), thereby inducing ferroptosis / pan-apoptosis of tumor cells (especially breast cancer cells, such as 4T1 cells). Moreover, the released cerium ions change the structure of the CD nanoparticles, generating voids, which is conducive to the release of the encapsulated chemotherapy drugs.

[0008] In a second aspect, a method for preparing the above-mentioned near-infrared triggered photothermal enhancement nanocarrier comprises the following steps:

[0009] Mixing a tetravalent cerium salt solution and a dopamine solution, adjusting the pH to alkaline, and reacting to obtain CD nanoparticles (CD NPs); wherein the reaction includes a complexation reaction between cerium ions and dopamine, and a polymerization reaction in which tetravalent cerium ions oxidize dopamine;

[0010] After homologous targeting cell membranes are mixed with CD NPs, ultrasonic treatment is performed to allow the cell membranes to coat the CDNPs.

[0011] In the present invention, the complexation reaction and polymerization reaction proceed simultaneously. Through the complexation reaction, the cerium ion and dopamine are coordinated and complexed to form a metal polyphenol network structure skeleton. At the same time, the tetravalent cerium ion is oxidizing and can directly oxidize dopamine to polymerize, thereby polymerizing the dopamine in the skeleton structure into polydopamine, which gives the CD NP a photothermal effect. At the same time, this structure can also achieve the encapsulation of chemotherapy drugs, thereby achieving the purpose of drug loading.

[0012] In a third aspect, a cerium-based ferroptosis / pan-apoptosis nanoinducer comprises the above-mentioned near-infrared-triggered photothermal enhancement nanocarrier and mitoxantrone (MTO), wherein the MTO is encapsulated in the CD nanoparticles of the nanocarrier.

[0013] The chemotherapeutic drug used in this study is MTO. Mechanistically, MTO is involved in glutathione (GSH) depletion, lipid metabolism reprogramming, reactive oxygen species (ROS) production, and regulation of ferroptosis-related proteins by intervening in the cGAS-STING, P53, and Nrf2 axes, thereby exhibiting multiple ferroptosis-sensitizing effects. MTO is also a polyphenolic compound, facilitating its encapsulation by CD nanoparticles.

[0014] This invention combines a nanocarrier (M@CD) with MTO, resulting in M@CD / MTO potentiating ferroptosis and pan-apoptosis activation. Experimental studies have shown that M@CD / MTO induces severe cellular oxidative stress, leading to pan-apoptosis, by inhibiting the normal function of the mitochondrial electron transport chain. Furthermore, the potent ferroptosis mediated by M@CD / MTO is primarily due to its dual-pronged regulation of the GSH / GPX4 axis and lipid metabolism. More specifically, in addition to its direct GSH depletion effect, M@CD / MTO also manipulates the GSH / GPX4 axis by modulating related signaling pathways. Excessive Ce ions and MTO increase ferroptosis sensitivity by activating the cGAS-STING and P53 pathways, thereby inhibiting the downstream solute carrier family 7, member 11 (SLC7A11), which is responsible for transporting cysteine ​​into cells as a raw material for GSH synthesis. Furthermore, M@CD / MTO inhibits GPX4 expression through MTO-mediated downregulation of the upstream nuclear factor erythroid 2-related factor 2 (Nrf2). From the perspective of lipid metabolism, M@CD / MTO can inhibit SLC47A1, thereby enriching lipids containing polyunsaturated fatty acids and achieving robust ferroptosis; in addition, the PDA-mediated photothermal heating effect is conducive to PUFA-supported lipid metabolic reprogramming. In addition to the above-mentioned M@CD / MTO-mediated ferroptosis sensitization mechanism through regulation of the GSH / GPX4 axis and lipid metabolism, there are also some other supporting mechanisms, such as ferritin 1 (FPN1) inhibition and lysosomal damage caused by iron overload. In addition to the direct cell killing effect of M@CD / MTO-induced ferroptosis / pan-apoptosis, M@CD / MTO also helps reshape the TME from an immunosuppressive state to an immune-activated state and enhances systemic anti-tumor immunity by effectively inducing ICD, thereby strongly inhibiting primary tumor growth and lung metastasis.

[0015] In a fourth aspect, a method for preparing the above-mentioned cerium-based ferroptosis / pan-apoptosis nanoinducer comprises the following steps:

[0016] A tetravalent cerium salt solution and a dopamine solution containing MTO are mixed and uniformly mixed, and the pH is adjusted to alkaline for reaction to obtain CD / MTO nanoparticles (CD / MTO NPs); wherein the reaction includes a complexation reaction between cerium ions and dopamine, and a polymerization reaction in which tetravalent cerium ions oxidize dopamine;

[0017] After the homologous targeted cell membrane is mixed with CD / MTO NP, ultrasonic treatment is performed to allow the cell membrane to coat the CD / MTO NP.

[0018] In the present invention, MTO is added during the preparation process of CD nanoparticles, which is beneficial to the loading of MTO.

[0019] In a fifth aspect, a use of the above-mentioned cerium-based ferroptosis / pan-apoptosis nanoinducer in the preparation of drugs for treating breast cancer.

[0020] Specifically, the drug of the present invention is used for photothermal-enhanced chemotherapy / chemodynamic combination therapy.

[0021] The beneficial effects of the present invention are:

[0022] 1. The nano-inducer (M@CD / MTO) provided by the present invention uses M@CD as the carrier, wherein the cerium ion (Ce 3+ / Ce 4+ ) and polydopamine form an MPN structure that not only has a photothermal effect, but also has an acidic response, and is wrapped with a homologous targeting cell membrane, enabling homologous targeting, thereby enabling specific, space-time controllable tumor treatment; at the same time, after MPN is loaded with MTO, it can synchronously trigger ferroptosis and pan-apoptosis of tumor cells (especially BC cells), and can be used for photothermally enhanced ferroptosis / pan-apoptosis activation and immunosuppressive microenvironment reversal of tumor cells, creating more possibilities for the use of Ce-based nanomaterials in the field of cancer treatment.

[0023] 2. The nanoinducer (M@CD / MTO) provided by the present invention has a high permeability and long retention (EPR) effect and a tropism effect on homologous tumor cells, thereby being able to accurately deliver cerium ions (Ce 3+ / Ce 4+ ) and MTO.

[0024] 3. This invention also comprehensively explores the various ferroptosis-sensitizing mechanisms mediated by MTO in the therapeutic system, providing substantial supplementation to the MTO-mediated ferroptosis-enhancing mechanism. This invention sets a precedent for the activation of ferroptosis / pan-apoptosis and provides a new treatment model for BC. Furthermore, the molecular mechanism research provides data support and theoretical foundation for combination therapies for BC, particularly those based on ferroptosis activation. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0026] Figure 1 Schematic diagram of the synthesis of M@CD / MTO NPs.

[0027] Figure 2 is the particle size distribution of CD / MTO NP and M@CD / MTO NP.

[0028] Figure 3is the Zeta potential of M, CD / MTO NP and M@CD / MTO NP.

[0029] Figure 4 Transmission electron microscopy images of CD / MTO NP and M@CD / MTO NP.

[0030] Figure 5 Ultraviolet absorption spectra of M, MTO, CD, and M@CD / MTO at 350-900 nm.

[0031] Figure 6 SDS-PAGE images of cell lysate, cell membrane, CD / MTO NP, and M@CD / MTO NP.

[0032] Figure 7 The release curves of the formulations in PBS (pH 7.4 and 5.5) are shown.

[0033] Figure 8 The results of uptake of free MTO and M@CD / MTO by 4T1 cells after treatment for different time periods (scale bar: 30 μm).

[0034] Figure 9 The lipid peroxide (LPO) production of different preparations in 4T1 cells under different treatment conditions.

[0035] Figure 10 These are real-time fluorescence images of mice after tail vein injection of different preparations and fluorescence images of organs and tumors in vitro 24 hours later. a) In vivo imaging of free MTO and M@CD / MTO; b) Distribution of free MTO and M@CD / MTO in tumors and major organs 24 hours later.

[0036] Figure 11 Photos of tumors in tumor-bearing mice after drug administration (a) and H&E staining results (b) (scale bar: 100 μm).

[0037] Figure 12 This is a technical principle diagram of the present invention. DETAILED DESCRIPTION

[0038] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0039] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0040] In view of the limited and uncontrollable efficiency of Ce-catalyzed Fenton-like reactions and the complex cell ferroptosis defense system that hinder the therapeutic effect of Ce-based systems, the present invention proposes a cerium-based ferroptosis / pan-apoptosis nanoinducer, a preparation method thereof, and its application in the preparation of drugs for the treatment of breast cancer.

[0041] A typical embodiment of the present invention provides a near-infrared-triggered photothermal enhancement nanocarrier, which is composed of CD nanoparticles coated with a membrane material; the CD nanoparticles are a metal polyphenol network structure formed by cerium ions and dopamine, and the dopamine in the metal polyphenol network structure is oxidatively polymerized into polydopamine; the membrane material is a cell membrane with homologous targeting to the target tumor cells.

[0042] The nanocarrier provided by the present invention can achieve specific, space-time controllable tumor treatment through the selection of materials and the formed structure.

[0043] In some embodiments, the cell membrane is from a 4T1 cell membrane.

[0044] A second embodiment of the present invention provides a method for preparing the above-mentioned near-infrared-triggered photothermal enhancement nanocarrier, comprising the following steps:

[0045] Mixing a tetravalent cerium salt solution and a dopamine solution, adjusting the pH to alkaline, and reacting to obtain CD NPs; wherein the reaction includes a complexation reaction between cerium ions and dopamine, and a polymerization reaction in which tetravalent cerium ions oxidize dopamine;

[0046] After homologous targeting cell membranes are mixed with CD NPs, ultrasonic treatment is performed to allow the cell membranes to coat the CDNPs.

[0047] The tetravalent cerium salt described in the present invention refers to a compound whose cation is a tetravalent cerium ion, such as Ce(SO4)2.

[0048] In some embodiments, Ce(SO4)2 is added to a sulfuric acid solution to form a tetravalent cerium salt solution. Ce(SO4)2, when added directly to water, easily forms a basic salt, so adding a sulfuric acid solution can avoid the formation of a basic salt. Specifically, the sulfuric acid solution is dilute sulfuric acid, with a concentration of 0.1 to 10 mmol / L.

[0049] In some embodiments, the solvent of the dopamine solution is an ethanol-water solution. Ethanol can be mixed with water in any proportion. Specifically, the volume ratio of ethanol to water is 1:4.5-5.5, which has a better dissolution effect.

[0050] In some embodiments, the molar ratio of the tetravalent cerium salt to dopamine is 1:2.5-3.5.

[0051] In some embodiments, the pH is adjusted to an alkaline pH of 8.0 to 9.0, preferably 8.8 to 9.2.

[0052] In some embodiments, the pH is adjusted to alkaline and the reaction time is 3.5 to 4.5 hours.

[0053] In some embodiments, after adjusting the pH to alkaline for reaction, centrifugation, washing, and freeze-drying are sequentially performed. The purpose of freeze-drying is to maintain the microscopic morphology of the CD nanoparticles.

[0054] In some embodiments, the ultrasonic treatment time is 20 to 40 minutes.

[0055] In some embodiments, the cell membrane is obtained by: lysing and disrupting the target tumor cells, centrifuging to collect the membrane precipitate, i.e., cell membrane fragments; resuspending the membrane precipitate, sonicating, and physically squeezing it. Specifically, sonication is performed using a pre-cooled hypotonic lysis buffer to achieve lysis and disruption of the target tumor cells. More specifically, the cells are disrupted at least 20 times using an ultrasonic cell disruptor. Specifically, after cell disruption, the cells are centrifuged at 2500-3500 rcf for 8-12 minutes to collect the supernatant; the resulting supernatant is first centrifuged at 9000-11000 rcf for 8-12 minutes, and the supernatant is further centrifuged at 90,000-110,000 rcf for 1 hour to finally obtain cell membrane fragments.

[0056] In some embodiments, the mass ratio of cell membrane to CD NP is 1:35-45.

[0057] A third embodiment of the present invention provides a cerium-based ferroptosis / pan-apoptosis nanoinducer, comprising the above-mentioned near-infrared-triggered photothermal enhancement nanocarrier and mitoxantrone, wherein the MTO is encapsulated in the CD nanoparticles of the nanocarrier.

[0058] The present invention uses mitoxantrone as a chemotherapeutic agent and an ICD inducer. In some embodiments, the drug loading of the nanocarrier and the encapsulated mitoxantrone is 9.00-11.00%, specifically 9.05-10.73%.

[0059] A fourth embodiment of the present invention provides a method for preparing the above-mentioned cerium-based ferroptosis / pan-apoptosis nanoinducer, comprising the following steps:

[0060] A tetravalent cerium salt solution and a dopamine solution containing MTO are mixed and uniformly mixed, and the pH is adjusted to alkaline for reaction to obtain CD / MTO NPs; wherein the reaction includes a complexation reaction between cerium ions and dopamine, and a polymerization reaction in which tetravalent cerium ions oxidize dopamine;

[0061] After the homologous targeted cell membrane is mixed with CD / MTO NP, ultrasonic treatment is performed to allow the cell membrane to coat the CD / MTO NP.

[0062] The parameters of this embodiment are the same as those of the second embodiment of the present invention except for the addition of MTO.

[0063] In some embodiments, the molar ratio of the tetravalent cerium salt, dopamine, and MTO is 1:2.5-3.5:0.30-0.40.

[0064] A fifth embodiment of the present invention provides a use of the above-mentioned cerium-based ferroptosis / pan-apoptosis nanoinducer in the preparation of a drug for treating breast cancer.

[0065] Specifically, the drug of the present invention is used for photothermal-enhanced chemotherapy / chemodynamic combination therapy.

[0066] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0067] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents and raw materials used in the present invention can be purchased through conventional channels. Unless otherwise specified, the reagents and raw materials used in the present invention are used in a conventional manner in the art or according to the product instructions. In addition, any methods and materials similar to or equivalent to those described herein can be applied to the present invention.

[0068] Experimental materials: cerium sulfate (Ce(SO4)2), dopamine hydrochloride (DA·HCl), mitoxantrone dihydrochloride (MTO·2HCl, Shanghai Aladdin Biochemical Technology Co., Ltd.); lipid peroxide (LPO) detection probe (C11 BODIPY581 / 591, Shanghai Maokang Biotechnology Co., Ltd.); 4% paraformaldehyde, 4',6-diamidino-2-phenylindole (DAPI, Shanghai Beyotime Biotechnology Co., Ltd.).

[0069] Example 1 Preparation of 4T1 cell membrane vesicles (M)

[0070] 4T1 cells were harvested in the PBS solution containing 2mM EDTA, and washed three times with PBS by centrifugation (1000rcf, 10 minutes). The cells collected were then suspended in precooled hypotonic lysis buffer (every 10mL 20mM Tris-HCl pH 7.4, 10mM KCl, 2mM MgCl2 and 1 mini protease inhibitor that does not contain EDTA), and ultrasonic cell disruptor was used to break at least 20 times, then centrifuged 10 minutes under 3000rcf. Supernatant was collected and centrifuged 10 minutes under 100000rcf, and then supernatant was further centrifuged 1 hour under 100000rcf. The membrane precipitate collected was washed once with 10mM Tris-HCl (pH=7.5), centrifuged and resuspended in PBS. Finally, cell membrane was ultrasonicated for 30s, and physically extruded 5 times through a 400nm polycarbonate membrane to obtain 4T1 cell membrane vesicles (M). The gained M was stored at 4 ℃ until used again.

[0071] Example 2 Preparation of CD / MTO Nanoparticles (CD / MTO NPs)

[0072] Under vigorous stirring in a 40°C water bath, 33.22 mg of Ce(SO4)2 was dissolved in 1.5 mL of dilute sulfuric acid (1 mmol / L) to obtain solution A. 56.89 mg of DA·HCl and 18.11 mg of MTO·2HCl were dissolved in 7.5 mL of a water-ethanol mixture (water to ethanol volume ratio of 5:1) to obtain solution B. Solutions A and B were stirred for 15 minutes until completely mixed. The pH was adjusted to 8.5 with 2 mmol / L NaOH solution, and then the reaction was stirred for 4 hours. CD / MTO NPs were obtained by centrifugation (10,000 rpm, 15 minutes) and washed twice with deionized water. Finally, the CD / MTONPs were freeze-dried and stored at 4°C for later use. If MTO was not added in this step, blank carrier CD NPs were obtained.

[0073] Example 3 Preparation of M@CD / MTO Nano-Inducer

[0074] M and CD / MTO NP (or CD NP) were uniformly mixed (the mass ratio of M to CD / MTO NP (or CD NP) was about 1:40) and subjected to water bath ultrasonic treatment for 30 minutes to obtain membrane-coated M@CD / MTO or membrane-coated M@CD NP. (Synthesis schematic can be seen Figure 1 ). Take the M obtained in Example 1, the CD / MTO obtained in Example 2, and the M@CD / MTO obtained in Example 3 to measure the particle size and potential, as shown in FIG. Figure 2 、 3As shown in the figure, compared with CD / MTO, the particle size of M@CD / MTO increased by about 15.2 nm, and the potential of M@CD / MTO was close to that of membrane vesicle M, indicating the successful encapsulation of M. Figure 4 The transmission electron microscopy image more intuitively proves that M is attached to the surface of CD / MTO to form M@CD / MTO bionic nanoparticles with uniform particle size. Figure 6 This indicates that M@CD / MTO retains the protein map of M, further proving the successful encapsulation of M.

[0075] Experimental Example 4

[0076] Take MTO solution, M obtained in Example 1, CD NP obtained in Example 2, and M@CD / MTO obtained in Example 3, and perform UV scanning in the wavelength range of 350-900nm. Figure 5 As shown in Figure 3, M@CD / MTO contains the characteristic absorption peaks of CD and MTO, indicating the successful encapsulation of MTO in the Ce-based nanoplatform.

[0077] Example 5

[0078] The drug release behavior of M@CD / MTO at pH 7.4 and 5.5 was determined by dynamic membrane dialysis (MWCO = 14kDa) to verify the pH sensitivity of MTO release in M@CD / MTO. Take a 15mL centrifuge tube and add 10mL of release medium, add the preparation to the dialysis bag, tie the two ends of the dialysis bag, and immerse it in the release medium. The centrifuge tube containing the sample was placed in a constant temperature oscillator at 37°C and 100r / min and incubated. At the test time point, 1mL of release medium was taken out and an equal amount of fresh release medium was added. The concentration of MTO in the culture medium was determined by HPLC. The cumulative release percentage of each sampling point was calculated, and finally the drug release curve was drawn. The results are shown in Figure 2. Figure 7 As shown in the results, within 24 h, M@CD / MTO released approximately 30.67% of the drug at pH 7.4 and approximately 55.38% of the drug at pH 5.5, showing good pH-responsive drug release behavior, which provides a guarantee for tumor site-specific drug release.

[0079] Example 6

[0080] Mouse breast cancer 4T1 cells were used as the test cell line. 4T1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U / mL), and streptomycin (100 μg / mL). All cells were cultured at 37°C in an atmosphere of 5% CO2.

[0081] 4T1 cells were cultured at 10 5The cells were seeded at a density of 10 cells / well in a 12-well culture plate containing a slide and cultured for 24 hours. The cells were then treated with MTO solution (2 μg / mL) or M@CD / MTO NP (containing MTO 2 μg / mL) for 0.5, 1, 2, or 4 hours. After being fixed with 4% paraformaldehyde for 10 minutes and stained with DAPI and washed with PBS, the slides were collected, sealed with anti-fluorescence quenching liquid, and observed under a high-speed spinning disk confocal microscope (Dragonfly 200, Andor, England). Figure 8 As shown in the figure, the fluorescence of MTO and M@CD / MTO NPs increased with time, indicating that drug uptake is time-dependent. Moreover, at each time point, the fluorescence of M@CD / MTO NPs was stronger than that of free MTO, indicating that the preparation of M@CD / MTO NPs significantly increased the uptake of drugs by 4T1 cells, which is conducive to the full therapeutic effect of the drug.

[0082] Example 7

[0083] 4T1 cells were cultured at 10 5 The cells were seeded at a density of 100 cells / well in a 12-well culture plate containing a slide and cultured for 24 hours. Then, the culture medium containing different preparations was added and incubated for 4 hours. Then, NIR (808 nm, 1.5 W / cm 2 , 1 minute per well) and then incubated for 4 hours. Then C11BODIPY 581 / 591 fluorescent probe (10μmol / L) was added and incubated at 37°C for 30 minutes. Finally, the slides were sealed and the accumulation of LPO was observed under a high-speed spinning disk confocal microscope (Dragonfly 200, Andor, England). Group I: control group, Group II: MTO, Group III: M@CD, Group IV: M@CD+L, Group V: M@CD / MTO, Group VI: M@CD / MTO+L. The results are shown in Figure 2. Figure 9 As shown in the figure, compared with the control group, cells treated with MTO and M@CD showed some LPO accumulation, which may be due to the GSH consumption and ROS production capacity of MTO and M@CD. Due to the combined effects of MTO and M@CD, M@CD / MTO treatment led to a significant increase in LPO accumulation. Cells treated with M@CD / MTO+L showed the highest LPO levels, which may be due to the enhanced ROS production and GSH consumption under photothermal heating.

[0084] Example 8

[0085] Female Balb / c mice (6-8 weeks old) were purchased from Beijing Weitonglihua Animal Technology Co., Ltd. All animal experiments were performed in strict accordance with the protocol approved by the Experimental Animal Center of Shandong University. To obtain the 4T1 xenograft tumor-bearing mouse model, 2×10 64T1 cells were injected subcutaneously into the right axilla of each mouse. 3 Tumor volume was calculated using the following formula, where a represents the maximum length (mm) and b represents the minimum length (mm).

[0086]

[0087] Example 9

[0088] 4T1 tumor-bearing mice were randomly divided into two groups (n=3) and injected with MTO (2 mg / kg) or M@CD / MTO NP (containing MTO 2 mg / kg) via the tail vein. For in vivo imaging, mice were anesthetized by intraperitoneal injection of chloral hydrate and imaged using an IVIS spectral system (PerkinElmer, USA) at predetermined time points (1, 3, 8, 12, and 24 hours). To study the in vivo distribution of M@CD / MTO NP, mice were sacrificed 24 hours after injection, and the fluorescence intensity of major organs (heart, liver, spleen, lung, kidney) and tumors was recorded under the IVIS spectral system. Figure 10 As shown in (a), compared with the free MTO group, the M@CD / MTO group showed stronger fluorescence at the tumor site at all time points, indicating that the fabricated nanoplatform is conducive to tumor-specific drug delivery, which may be attributed to the EPR effect and 4T1 cell membrane-mediated homologous targeted tumor recruitment. Figure 10 b Consistent with the in vivo imaging results, the fluorescence signal of the tumor in the M@CD / MTO group was significantly stronger, and the quantitative results showed that the average fluorescence intensity of the tumor in the M@CD / MTO group was 1.76 times that of the free MTO group.

[0089] Example 10

[0090] 4T1 tumor-bearing mice were randomly divided into 6 groups (n=5) and injected with different preparations (MTO equivalent to 2 mg / kg) through the tail vein. Group I: control group (normal saline injected through the tail vein), Group II: MTO group (Ce6 injected through the tail vein), Group III: M@CD group (M@CD injected through the tail vein), Group IV: M@CD+L group (M@CD injected through the tail vein and laser irradiated 8 hours later), Group V: M@CD / MTO group (M@CD / MTO injected through the tail vein), Group VI: M@CD / MTO+L group (M@CD / MTO injected through the tail vein and laser irradiated 8 hours later). The laser radiation was set at 808 nm, 1.5 W / cm 2, 5 minutes. The drug was administered on days 0, 3, 6, 9, and 12. The tumor volume and body weight of the mice were recorded every two days. After 14 days, the mice were killed, and the tumors in each group were collected and weighed. The eyeballs were removed to collect blood and the cytokines in the blood were measured by ELISA. The main organs (heart, liver, spleen, lung, kidney) and tumors of each group were dissected and the sections were subjected to H&E staining. In addition, the tumors were subjected to Tunel, CD4 and CD8 immunofluorescence staining to further study the therapeutic effect. In addition, the spleen, lymph nodes and tumors of each group were ground and centrifuged to extract lymphocytes, tumor-associated macrophages and tumor-infiltrating lymphocytes. As Figure 11 As shown, both the free MTO group and the nanocarrier M@CD group modestly inhibited tumor growth compared to the control group. When NIR irradiation induced a mild photothermal effect from the M@CD nanocarrier, tumor growth was more effectively inhibited. Furthermore, the relative tumor volume in the M@CD / MTO group was much lower than that in the free MTO group, indicating that this nanoplatform enhanced the intratumoral delivery and distribution of MTO. Furthermore, the combination of MTO's DNA-damaging activity and multi-pathway ferroptosis-inducing ability with ferroptosis induced by the Fenton-like reaction of Ce ions further improved the efficiency of tumor growth inhibition. The M@CD / MTO+L group had the smallest relative tumor volume during treatment, indicating that the combination of chemotherapy, CDT-induced ferroptosis, and mild photothermal heating had the strongest tumor inhibitory effect. Correspondingly, H&E staining revealed that tumors in the M@CD / MTO+L treatment group exhibited the greatest pathological damage, further demonstrating that the combination of chemotherapy, CDT-induced ferroptosis, and mild photothermal heating had the strongest tumor inhibitory effect.

[0091] like Figure 12 As shown in the figure, under the catalysis of Ce ions, DA polymerizes to produce PDA, which gives the developed nanoinducer the NIR-triggered photothermal heating property; this self-heating effect can enhance the ferroptosis / pan-apoptosis induced by M@CD / MTO and destroy the dense TME to improve drug permeability. 3+ and Ce 4+ ), M@CD mediates the catalytic activity of the Fenton-like reaction, GSH consumption, and O2 production capacity in an acidic environment, generating a large amount of ROS, thereby triggering ferroptosis / pan-apoptosis of 4T1 cells. This effect is further enhanced by combining M@CD with MTO (M@CD / MTO). In addition to the direct cell killing effect of ferroptosis / pan-apoptosis induced by M@CD / MTO, M@CD / MTO also helps reshape the TME from an immunosuppressive state to an immunostimulatory state, and enhances systemic anti-tumor immunity by effectively inducing ICD, thereby effectively inhibiting primary tumor growth and lung metastasis.

[0092] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A near-infrared-triggered photothermal enhancement nanocarrier, characterized by: The method is composed of CD nanoparticles coated with a membrane material; the CD nanoparticles are a metal polyphenol network structure formed by cerium ions and dopamine, and the dopamine in the metal polyphenol network structure is oxidatively polymerized into polydopamine; the membrane material is a cell membrane with homologous targeting to the target tumor cells.

2. The near-infrared-triggered photothermal enhancement nanocarrier according to claim 1, characterized in that: The cell membrane is derived from 4T1 cell membrane.

3. A method for preparing the near-infrared-triggered photothermal enhancement nanocarrier according to claim 1, characterized in that: The steps include: Mixing a tetravalent cerium salt solution and a dopamine solution, adjusting the pH to alkaline, and reacting to obtain CD NPs; wherein the reaction includes a complexation reaction between cerium ions and dopamine, and a polymerization reaction in which tetravalent cerium ions oxidize dopamine; After homologous targeted cell membranes are mixed with CD NPs, ultrasonic treatment is performed to allow the cell membranes to coat the CD NPs.

4. The preparation method according to claim 3, wherein: Add Ce(SO4)2 to sulfuric acid solution to prepare tetravalent cerium salt solution; Alternatively, the solvent of the dopamine solution is an ethanol aqueous solution; Alternatively, after adjusting the pH to alkaline and reacting, centrifugation, washing, and freeze-drying are sequentially performed.

5. The preparation method according to claim 3, wherein: The molar ratio of tetravalent cerium salt to dopamine is 1:2.5~3.5; Alternatively, adjust the pH to an alkaline pH of 8.0 to 9.0; Alternatively, the pH is adjusted to alkaline and the reaction time is 3.5 to 4.5 hours; Alternatively, the mass ratio of cell membrane to CD NPs was 1:35–45.

6. The preparation method according to claim 5, characterized in that: Adjust the pH to an alkaline pH of 8.8-9.

2.

7. A cerium-based iron death / pan-apoptosis nanoinducer, characterized by: The method comprises the near-infrared triggered photothermal enhancement nanocarrier according to claim 1 or 2 and mitoxantrone, wherein the MTO is encapsulated in the CD nanoparticles of the nanocarrier.

8. The cerium-based iron death / pan-apoptosis nanoinducer according to claim 7, characterized in that: The drug loading of nanocarriers and encapsulated mitoxantrone was 9.00~11.00%.

9. The cerium-based iron death / pan-apoptosis nanoinducer according to claim 8, characterized in that: The drug loading of nanocarriers and encapsulated mitoxantrone ranged from 9.05 to 10.73%.

10. A method for preparing the cerium-based ferroptosis / pan-apoptosis nanoinducer according to claim 7, characterized in that: The steps include: A tetravalent cerium salt solution and a dopamine solution containing MTO are mixed and uniformly mixed, and the pH is adjusted to alkaline for reaction to obtain CD / MTO NPs; wherein the reaction includes a complexation reaction between cerium ions and dopamine, and a polymerization reaction in which tetravalent cerium ions oxidize dopamine; After the homologous targeted cell membrane is mixed with CD / MTO NP, ultrasonic treatment is performed to allow the cell membrane to coat the CD / MTO NP.

11. The preparation method according to claim 10, characterized in that: The molar ratio of tetravalent cerium salt, dopamine and MTO is 1:2.5~3.5:0.30~0.

40.

12. Use of the cerium-based ferroptosis / pan-apoptosis nanoinducer according to any one of claims 7 to 9 in the preparation of a drug for treating breast cancer.

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