Cerium-based ferroptosis / pan-apoptosis nano inducer, preparation method thereof and application of cerium-based ferroptosis / pan-apoptosis nano inducer in preparation of breast cancer treatment drugs

Abstract

The invention belongs to the technical field of pharmaceutical preparations and biological medicines, and relates to a cerium-based ferroptosis / pan-apoptosis nano inducer, a preparation method thereof and application of the cerium-based ferroptosis / pan-apoptosis nano inducer in preparation of medicines for treating breast cancer. Comprising a near-infrared triggered photo-thermal enhanced nano-carrier and MTO (Methanol To Olefins), and the MTO is encapsulated in CD (Compact Disc) nano-particles of the nano-carrier. The nano-carrier is formed by coating CD nano-particles with a membrane material; the CD nanoparticles are of a metal polyphenol network structure formed by cerium ions and dopamine, and dopamine in the metal polyphenol network structure is oxidized and polymerized into polydopamine; the membrane material is a cell membrane with homologous targeting property with a target tumor cell. According to the cerium-based ferroptosis / pan-apoptosis nano inducer provided by the invention, a near-infrared triggered photo-thermal enhanced nano carrier is adopted, so that specific and space-time controllable tumor treatment can be realized.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations and biomedicine, and relates to a cerium-based ferric death / 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 the 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 a person 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 of cells 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 ferric death / pan-apoptosis nanoinducer and a preparation method thereof and an application thereof in the preparation of drugs for treating breast cancer. The cerium-based ferric death / pan-apoptosis nanoinducer provided by the present invention adopts a near-infrared (NIR) triggered photothermal enhanced nanocarrier, which can achieve specific, 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 enhanced 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 complexation and coordination of the phenol ligand DA and the cerium ion. Under the catalytic action of the cerium ion, DA is polymerized into PDA, so that the nanocarrier has a photothermal effect triggered by NIR. Meanwhile, the tumor microenvironment (TME) where the 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 ability, 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, after the cerium ions are released, the structure of the CD nanoparticles is changed to generate gaps, which is conducive to the release of the encapsulated chemotherapeutic drugs.

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

[0009] The tetravalent cerium salt solution and the dopamine solution are mixed and uniformly mixed, and the pH is adjusted to alkaline for reaction to obtain CD nanoparticles (CD NP); 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 the homologous targeted cell membrane is mixed with CD NP, ultrasonic treatment is performed so that the cell membrane is coated with CDNP to obtain the product.

[0011] In the present invention, the complexation reaction and the polymerization reaction are carried out simultaneously, and the cerium ions are coordinated and complexed with dopamine through the complexation reaction, thereby forming a skeleton of the metal polyphenol network structure, and the tetravalent cerium ions are oxidizing and can directly oxidize dopamine to polymerize, so that the dopamine in the skeleton structure is polymerized into polydopamine, so that the CD NP has a photothermal effect. At the same time, the structure can also realize the encapsulation of chemotherapeutic drugs, thereby achieving the purpose of drug loading.

[0012] In a third aspect, a cerium-based iron death / 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 the present invention is MTO. Mechanistically, MTO involves glutathione (GSH) consumption, lipid metabolism reprogramming, reactive oxygen species (ROS) production, and regulation of ferroptosis-related proteins by intervening in cGAS-STING, P53 and Nrf2 axes, thereby having multiple ferroptosis-sensitizing effects. At the same time, MTO is also a polyphenolic compound, which is conducive to the encapsulation of CD nanoparticles.

[0014] The present invention combines a nanocarrier (M@CD) with MTO, and M@CD / MTO has an enhanced ferroptosis / pan-apoptosis activation effect. Experiments have shown that M@CD / MTO causes severe cellular oxidative stress by inhibiting the normal function of the mitochondrial electron transport chain, leading to pan-apoptosis. In addition, the strong ferroptosis mediated by M@CD / MTO is mainly due to its two-pronged regulation of the GSH / GPX4 axis and lipid metabolism. More specifically, in addition to the direct GSH depletion effect, M@CD / MTO also manipulates the GSH / GPX4 axis by regulating related signaling pathways. Excessive Ce ions and MTO can increase the sensitivity of ferroptosis 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. In addition, M@CD / MTO also inhibits the expression of GPX4 through the 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 strong 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 the 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 to 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 iron death / pan-apoptosis nano-inducer comprises the following steps:

[0016] The tetravalent cerium salt solution and the 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 NP); wherein the reaction includes a complexation reaction between cerium ions and dopamine, and a polymerization reaction of tetravalent cerium ions oxidizing dopamine;

[0017] After the homologous targeted cell membrane is mixed with CD / MTO NP, ultrasonic treatment is performed so that the cell membrane covers 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] A fifth aspect is the use of the above-mentioned cerium-based ferric death / pan-apoptosis nano-inducer in the preparation of drugs for the treatment of breast cancer.

[0020] Specifically, the drug described in the present invention is used for photothermal-enhanced chemotherapy / chemodynamic combined 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+ The MPN structure formed by ) and polydopamine 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 achieving 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 of tumor cells and reversal of the immunosuppressive microenvironment, creating more possibilities for the use of Ce-based nanomaterials in the field of cancer treatment.

[0023] 2. The nano-inducer (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. The present invention also comprehensively explores the various ferroptosis sensitization mechanisms mediated by MTO in the treatment system, providing a substantial supplement to the MTO-mediated ferroptosis synergistic mechanism. The present invention sets a precedent for the activation of ferroptosis / pan-apoptosis and provides a new BC treatment model. In addition, the molecular mechanism research provides data support and theoretical basis for the combined treatment of BC, especially the combined treatment based on ferroptosis activation. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings in the specification, 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 3Zeta 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 350-900nm.

[0031] Figure 6 SDS-PAGE profiles 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).

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

[0035] Fig.10 These are real-time fluorescence images of the mouse in vivo 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] Fig.11 Photos of tumors in tumor-bearing mice after drug administration (a) and H&E staining results (b) (scale bar: 100 μm).

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

[0038] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those 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 exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates 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 hinders the therapeutic effect of Ce-based systems, the present invention proposes a cerium-based ferroptosis / pan-apoptosis nanoinducer, a preparation method thereof, and an application thereof in the preparation of drugs for treating 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] The tetravalent cerium salt solution and the dopamine solution are mixed and uniformly mixed, and the pH is adjusted to alkaline for reaction to obtain CD NP; wherein the reaction includes a complexation reaction of cerium ions and dopamine, and a polymerization reaction of tetravalent cerium ions oxidizing dopamine;

[0046] After the homologous targeted cell membrane is mixed with CD NP, ultrasonic treatment is performed so that the cell membrane is coated with CDNP to obtain the product.

[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 prepare a tetravalent cerium salt solution. Ce(SO4)2 is easily added to water to produce a basic salt, so adding a sulfuric acid solution can avoid the basic salt. Specifically, the sulfuric acid solution is dilute sulfuric acid, and the concentration of the dilute sulfuric acid is 0.1 to 10 mmol / L.

[0049] In some embodiments, the solvent of the dopamine solution is an ethanol aqueous solution. Ethanol can be mixed with water in any proportion, specifically, the volume ratio of ethanol to water is 1:4.5-5.5, and the dissolution effect is better under this condition.

[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 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 crushing the target tumor cells, centrifuging to collect the membrane precipitate, i.e., cell membrane fragments; resuspending the membrane precipitate, ultrasonicating, and physically extruding. Specifically, a pre-cooled hypotonic lysis buffer is used for ultrasonication to achieve target tumor cell lysis and crushing. More specifically, an ultrasonic cell crusher is used to crush the cells at least 20 times. Specifically, after the cells are crushed, centrifuge at 2500-3500rcf for 8-12 minutes to collect the supernatant; the obtained supernatant is first centrifuged at 9000-11000rcf for 8-12 minutes, and then the supernatant is further centrifuged at 90000-110000rcf 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 nano-inducer, comprising the above-mentioned near-infrared triggered photothermal enhancement nano-carrier and mitoxantrone, wherein the MTO is encapsulated in the CD nanoparticles of the nano-carrier.

[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 iron death / pan-apoptosis nano-inducer, comprising the following steps:

[0060] The tetravalent cerium salt solution and the dopamine solution containing MTO are mixed and uniformly mixed, and the pH is adjusted to alkaline for reaction to obtain CD / MTO NP; wherein the reaction includes a complexation reaction of cerium ions and dopamine, and a polymerization reaction of tetravalent cerium ions oxidizing dopamine;

[0061] After the homologous targeted cell membrane is mixed with CD / MTO NP, ultrasonic treatment is performed so that the cell membrane covers 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 nano-inducer in the preparation of a drug for treating breast cancer.

[0065] Specifically, the drug described in the present invention is used for photothermal-enhanced chemotherapy / chemodynamic combined 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 in conjunction with 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 or raw materials used in the present invention can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in the present invention are used in a conventional manner in the art or in accordance with the product instructions. In addition, any method and material similar or equivalent to the described content can be applied to the method of 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 Bio-Tech Biotechnology Co., Ltd.).

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

[0070] 4T1 cells were harvested with PBS solution containing 2mM EDTA, and washed three times with PBS by centrifugation (1000rcf, 10 minutes). The collected cells were then suspended in precooled hypotonic lysis buffer (every 10mL 20mM Tris-HCl pH7.4, 10mM KCl, 2mM MgCl2 and 1 mini protease inhibitor without EDTA), and ultrasonic cell crusher was used to break at least 20 times, and then centrifuged at 3000rcf for 10 minutes. Supernatant was collected and centrifuged at 10000rcf for 10 minutes, and then supernatant was further centrifuged at 100000rcf for 1 hour. The membrane precipitate collected was washed once with 10mM Tris-HCl (pH=7.5), centrifuged and resuspended in PBS. Finally, the cell membrane was ultrasonically treated for 30s, and physically extruded 5 times by 400nm polycarbonate membrane, to obtain 4T1 cell membrane vesicles (M). The obtained M was stored at 4°C 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 (the volume ratio of water to ethanol was 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 stirred for 4 hours. CD / MTO NPs were obtained by centrifugation (10000 rpm, 15 minutes) and washing twice with deionized water. Finally, 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 min to obtain membrane-coated M@CD / MTO or membrane-coated M@CD NP. (Synthesis schematic diagram 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, 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 profile of M, further proving the successful encapsulation of M.

[0075] Experimental Example 4

[0076] Take the MTO solution, the M obtained in Example 1, the CD NP obtained in Example 2, and the M@CD / MTO obtained in Example 3, and perform ultraviolet scanning in the wavelength range of 350-900nm. Figure 5 As shown, 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 both 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 the figure. Figure 7 As shown, within 24 h, M@CD / MTO released about 30.67% of the drug at pH 7.4 and about 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 cell 4T1 was used as the test cell line. 4T1 cells were cultured in RPMI-1640 medium containing 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 slides 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 fixation with 4% paraformaldehyde for 10 minutes and staining with DAPI, and washing 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 NP increases with time, indicating that the uptake of the drug is time-dependent. Moreover, at each time point, the fluorescence of M@CD / MTO NP is stronger than that of free MTO, indicating that the preparation of M@CD / MTO NP significantly increases the uptake of the drug 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 10 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, incubated for 4 hours, and then NIR (808 nm, 1.5 W / cm 2 , 1 minute per well), and then incubate for 4 hours. Then add C11BODIPY 581 / 591 fluorescent probe (10μmol / L) and incubate at 37°C for 30 minutes. Finally, seal the slides and observe the accumulation of LPO 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 as follows Fig. 9 As shown in Figure 3, compared with the control group, both MTO- and M@CD-treated cells showed some LPO accumulation, which may be due to the GSH consumption and ROS production capacity of MTO and M@CD. M@CD / MTO treatment led to a significant increase in LPO accumulation due to the combined effects of MTO and M@CD. Cells treated with M@CD / MTO+L showed the highest LPO level, which may be due to the enhanced ROS production and GSH consumption under photothermal warming.

[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. 64T1 cells were injected subcutaneously into the right axilla of each mouse. 3 Tumor volume was calculated by 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 NPs (containing MTO 2 mg / kg) through 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 NPs, mice were killed 24 hours after injection, and the fluorescence intensity of major organs (heart, liver, spleen, lung, kidney) and tumors were recorded under the IVIS spectral system. Fig.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 was beneficial for tumor-specific drug delivery, which may be attributed to the EPR effect and 4T1 cell membrane-mediated homologous targeted tumor recruitment. Fig.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 Fig.11 As shown in the figure, both the free MTO group and the nanocarrier M@CD group could moderately inhibit tumor growth compared with the control group. When NIR radiation induced a mild photothermal effect of the M@CD nanocarrier, tumor growth was more effectively inhibited. In addition, the relative tumor volume of the M@CD / MTO group was much lower than that of the free MTO group, indicating that the nanoplatform could enhance the intratumoral delivery and distribution of MTO, and further improve the efficiency of tumor growth inhibition by combining the DNA damage activity and multi-pathway ferroptosis induction ability of MTO with the ferroptosis induced by the Fenton-like reaction of Ce ions. The M@CD / MTO+L group had the smallest relative tumor volume during the treatment, indicating that the combination of chemotherapy, CDT-induced ferroptosis, and mild photothermal heating had the strongest tumor inhibitory effect. Correspondingly, the H&E staining images showed that the tumors in the M@CD / MTO+L treatment group exhibited the strongest pathological damage, further proving that the combination of chemotherapy, CDT-induced ferroptosis, and mild photothermal heating had the strongest tumor inhibitory effect.

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

[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A near-infrared triggered photothermal enhancement nanocarrier, characterized in that: The method is composed of CD nanoparticles coated with membrane materials; the CD nanoparticles are metal polyphenol network structures formed by cerium ions and dopamine, and 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 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: The tetravalent cerium salt solution and the dopamine solution are mixed and uniformly mixed, and the pH is adjusted to alkaline for reaction to obtain CD NP; wherein the reaction includes a complexation reaction of cerium ions and dopamine, and a polymerization reaction of tetravalent cerium ions oxidizing dopamine; After the homologous targeted cell membrane is mixed with CD NP, ultrasonic treatment is performed so that the cell membrane covers the CD NP.

4. The preparation method according to claim 3, characterized in that: Adding Ce(SO4)2 into a sulfuric acid solution to prepare a tetravalent cerium salt solution; Or, the solvent of the dopamine solution is 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, characterized in that: The molar ratio of the tetravalent cerium salt to dopamine is 1:2.5-3.5; Or, adjust the pH to an alkaline pH of 8.0 to 9.0, preferably 8.8 to 9.2; Or, 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 NP is 1:35-45.

6. A cerium-based iron death / pan-apoptosis nanoinducer, characterized in that: It comprises the near-infrared triggered photothermal enhancement nanocarrier as claimed in claim 1 or 2 and mitoxantrone, wherein the MTO is encapsulated in the CD nanoparticles of the nanocarrier.

7. The cerium-based iron death / pan-apoptosis nano-inducing agent according to claim 6, characterized in that: The drug loading of the nano-carrier and the encapsulated mitoxantrone is 9.00-11.00%, preferably 9.05-10.73%.

8. A method for preparing the cerium-based iron death / pan-apoptosis nano-inducer according to claim 6, characterized in that: The steps include: The tetravalent cerium salt solution and the dopamine solution containing MTO are mixed and uniformly mixed, and the pH is adjusted to alkaline for reaction to obtain CD / MTO NP; wherein the reaction includes a complexation reaction of cerium ions and dopamine, and a polymerization reaction of tetravalent cerium ions oxidizing dopamine; After the homologous targeted cell membrane is mixed with CD / MTO NP, ultrasonic treatment is performed so that the cell membrane covers the CD / MTO NP.

9. The preparation method according to claim 8, characterized in that: The molar ratio of tetravalent cerium salt, dopamine and MTO is 1:2.5-3.5:0.30-0.

40.

10. Use of the cerium-based ferroptosis / pan-apoptosis nano-inducer according to claim 6 or 7 in the preparation of a drug for treating breast cancer.

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