Doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses a doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticle as well as a preparation method and application thereof. The doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticle comprises a doxorubicin-doxorubicin dimer coupled prodrug and a ferroptosis activator ML210, wherein the doxorubicin-doxorubicin dimer coupled prodrug contains reduction response and is bridged by a disulfide bond; a doxorubicin dimer prodrug and a ferroptosis activator ML210 are co-assembled to form nanoparticles, so that the stability of the doxorubicin dimer prodrug is improved, meanwhile, the ferroptosis activator ML210 can temporarily increase the level of glutathione in tumor cells, disulfide bond breakage of the doxorubicin dimer prodrug in the cells is further promoted, and the curative effect of the doxorubicin dimer prodrug is enhanced; besides, the chemotherapeutic drug doxorubicin dimer prodrug and the ferroptosis activator ML210 have a synergistic anti-tumor effect, so that multi-way induction of tumor cell death is realized, a dual anti-tumor mechanism is realized, the anti-tumor efficiency is improved, the treatment effect is improved, meanwhile, through a co-assembly technology, the stability and the bioavailability of the drug are improved, and the curative effect is improved. The in-vivo tumor targeting property of the nano-material is enhanced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticle, and a preparation method and application thereof. Background Art

[0002] Doxorubicin, also known as doxorubicin, 14-hydroxydaunorubicin, adriamycin, etc., is a broad-spectrum chemotherapy drug in the field of anti-tumor drug research and development. Its mechanism of action is to interfere with cell division and inhibit cell proliferation by embedding DNA base pairs, thereby killing tumor cells in various growth cycles. It is suitable for the treatment of a variety of malignant tumors, such as acute leukemia, malignant lymphoma, breast cancer, etc., and is one of the commonly used anti-tumor drugs in clinical practice; however, the serious toxicity and side effects of doxorubicin, especially cardiac toxicity, are its most serious side effects, which limit its clinical application and may also cause adverse reactions such as bone marrow suppression, gastrointestinal reactions, and hair loss; in order to overcome this challenge, researchers have continuously explored improved forms of doxorubicin and found that two doxorubicin molecules are connected by specific chemical bonds to form a new drug form, namely, reduction-responsive doxorubicin dimer prodrug, which not only improves the drug loading and efficacy, but also has higher stability and safety, reduces the toxic side effects of traditional doxorubicin, and thus plays a more excellent role in anti-tumor treatment.

[0003] However, traditional reduction-responsive doxorubicin dimer prodrugs have encountered a series of challenges during their preparation and application. First, the nanoassemblies formed by traditional reduction-responsive doxorubicin dimer prodrugs have deficiencies in self-assembly performance, and it is difficult for them to form a stable and uniform dispersion system in solution, which affects the stability and bioavailability of the drug. Second, due to the low stability of the formed nanoassemblies, they are easily affected by environmental factors and disintegrate or aggregate, resulting in uncontrolled drug release, reducing the anti-tumor effect and limiting its research progress. Summary of the invention

[0004] One of the purposes of the present invention is to provide a doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles to solve the problems of poor self-assembly performance, low stability and poor anti-tumor effect of reduction-responsive doxorubicin dimer prodrug in the prior art; a second purpose is to provide a method for preparing doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles; a third purpose is to provide an application of doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles in the preparation of anti-tumor drugs.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticle comprises a chemotherapy drug doxorubicin dimer prodrug and a ferroptosis activator ML210.

[0007] According to the above technical means, the stability of doxorubicin dimer prodrug is increased by co-assembling doxorubicin dimer prodrug and ferroptosis activator ML210 to form nanoparticles. At the same time, ferroptosis activator ML210 can transiently increase the level of glutathione in tumor cells, further promote the cleavage of disulfide bonds of doxorubicin dimer prodrug in cells, and enhance the efficacy of doxorubicin prodrug. In addition, the chemotherapy drug doxorubicin dimer prodrug and ferroptosis activator ML210 have synergistic anti-tumor effects, thereby inducing tumor cell death through multiple pathways, realizing dual anti-tumor mechanisms, improving anti-tumor efficiency, and improving therapeutic effects. At the same time, through co-assembly technology, the stability and bioavailability of the drug are improved, and its tumor targeting in vivo is enhanced.

[0008] Furthermore, the structural formula of the doxorubicin dimer prodrug is as follows:

[0009]

[0010] According to the above technical means, the doxorubicin dimer prodrug in the present invention is γDSSD (linker: 3,3'-dithiodipropionic acid), which is obtained by connecting two doxorubicin molecules through a linker. It can respond to specific stimuli (such as reducing environment, pH change, etc.) in the tumor microenvironment, thereby releasing the doxorubicin molecules, exerting anti-tumor effects, achieving precise drug delivery and targeted enrichment, and reducing toxic side effects on normal tissues. The doxorubicin dimer prodrug has broad application prospects in anti-tumor treatment, and compared with the existing doxorubicin dimer prodrug βDSSD (linker: 3,3'-dithiodipropionic acid), the length of the linker is extended, it has higher stability, and retains the anti-tumor activity of doxorubicin.

[0011] A method for preparing the above-mentioned doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles comprises the following steps:

[0012] S1, taking doxorubicin dimer prodrug, ferroptosis activator ML210 and surfactant, and fully dissolving them in an organic solvent to obtain a mixed solution;

[0013] S2. The mixed solution was added dropwise into deionized water under stirring, stirred for 3 minutes, and then placed in a dialysis bag for 12 hours to obtain doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles.

[0014] According to the above technical means, the doxorubicin dimer prodrug, the ferroptosis activator ML210 and the surfactant are fully dissolved in an organic solvent, and then the obtained mixed solution is dropped into deionized water, and finally dialyzed with a dialysis bag to obtain nanoparticles co-assembled with the doxorubicin dimer prodrug and the ferroptosis activator ML210. The nanoparticles not only achieve effective encapsulation of the two drug molecules, improve the solubility, stability and biocompatibility of the drugs, but also remove the organic solvent through dialysis treatment, further ensuring the purity and uniformity of the nanoparticles, providing an efficient and safe drug delivery system for subsequent anti-tumor treatment.

[0015] Furthermore, in step S1, the mass ratio of the doxorubicin dimer prodrug to the ferroptosis activator ML210 is 1:0 to 2:1.

[0016] According to the above technical means, in step S1, by precisely regulating the mass ratio of doxorubicin dimer prodrug and ferroptosis activator ML210 between 1:0 and 2:1, the drug composition of the co-assembled nanoparticles can be optimized to ensure that the nanoparticles have good stability and that the two active ingredients have an effective synergistic effect, thereby further enhancing the anti-tumor effect while maintaining the stability and biosafety of the drug.

[0017] Furthermore, the mass ratio of the doxorubicin dimer prodrug to the ferroptosis activator ML210 is 1:2.

[0018] According to the above technical means, when the mass ratio of doxorubicin dimer prodrug to ferroptosis activator ML210 is set to 1:2, the precise ratio can maximize the synergistic effect of the two drugs, which not only enhances the anti-tumor effect, but also may more effectively induce ferroptosis of tumor cells by increasing the content of ferroptosis activator. At the same time, the presence of doxorubicin dimer prodrug ensures the direct cytotoxic effect of chemotherapy drugs. The two complement each other and jointly improve the therapeutic effect, providing a new strategy for tumor treatment.

[0019] Furthermore, the surfactant in step S1 is distearoyl phosphatidylethanolamine-polyethylene glycol 2000.

[0020] According to the above technical means, distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) is used as a surfactant, which can give the co-assembled nanoparticles good hydrophilicity and biocompatibility, help the circulation and delivery of nanoparticles in the body, and improve the bioavailability and targeting of drugs.

[0021] Furthermore, the organic solvent in step S1 is dimethyl sulfoxide.

[0022] According to the above technical means, dimethyl sulfoxide (DMSO) is used as an organic solvent. DMSO has good solubility and can fully dissolve doxorubicin dimer prodrug, ferroptosis activator ML210 and surfactant DSPE-PEG2000 to form a uniform mixed solution; it is not only conducive to the subsequent formation and stabilization of nanoparticles, but DMSO is also miscible with water, which is easy to remove during the dialysis process, thereby ensuring the purity and quality of the final doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles.

[0023] Furthermore, the molecular weight of the dialysis bag in step S2 is 10 KDa.

[0024] According to the above technical means, dialysis treatment using a dialysis bag with a molecular weight of 10KDa can effectively remove small molecule impurities and unassembled free drug molecules in the mixed solution, while allowing organic solvents (such as DMSO) and small molecule solutes (such as water molecules) to pass freely, thereby ensuring the purity and uniformity of the final doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles. The molecular weight cutoff value of 10KDa can also prevent the leakage of nanoparticles and ensure the integrity and stability of the nanoparticle structure.

[0025] A use of the above-mentioned doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles in the preparation of anti-tumor drugs.

[0026] An anti-tumor drug comprises the above-mentioned doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles.

[0027] Beneficial effects achieved by the present invention:

[0028] The present invention increases the stability of the doxorubicin dimer prodrug by co-assembling the doxorubicin dimer prodrug and the ferroptosis activator ML210 to form nanoparticles. At the same time, the ferroptosis activator ML210 can temporarily increase the level of glutathione in tumor cells, further promote the cleavage of the disulfide bond of doxorubicin-doxorubicin prodrug in the cells, and enhance the efficacy of the doxorubicin prodrug. In addition, the chemotherapy drug doxorubicin dimer prodrug and the ferroptosis activator ML210 have a synergistic anti-tumor effect, thereby inducing tumor cell death in multiple ways, achieving a dual anti-tumor mechanism, improving the anti-tumor efficiency, and improving the therapeutic effect. At the same time, through the co-assembly technology, the stability and bioavailability of the drug are improved, the tumor targeting in the body is enhanced, and the application prospect of doxorubicin in anti-tumor treatment is broadened. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The synthetic route of the doxorubicin dimer prodrug γDSSD of the present invention is shown in FIG.

[0030] Figure 2is a structural diagram of the doxorubicin dimer prodrug βDSSD of the present invention;

[0031] Figure 3 is a structural diagram of the doxorubicin dimer prodrug γDSSD of the present invention;

[0032] Figure 4 The stability diagram of βDSSD, γDSSD and different ratios of βDSSD:ML210 and γDSSD:ML210 in 10% fetal bovine serum-PBS of the present invention is shown;

[0033] Figure 5 is a particle size diagram of the doxorubicin dimer prodrug nanoparticle γDSSD of the present invention;

[0034] Figure 6 It is a transmission electron microscopy image of the doxorubicin dimer prodrug nanoparticle γDSSD of the present invention;

[0035] Figure 7 This is a particle size diagram of the doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticle γDSSDM of the present invention;

[0036] Figure 8 This is a transmission electron microscopic image of the doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles γDSSDM of the present invention;

[0037] Fig. 9 IC values ​​of the adriamycin solution, ML210 solution, adriamycin-ML210 mixed solution, βDSSD, βDSSDM, γDSSD, and γDSSDM of the present invention on 4T1 and L02 are shown in Table 1. 50 picture;

[0038] Fig.10 This is a graph showing the effects of the doxorubicin solution, ML210 solution, doxorubicin-ML210 mixed solution, βDSSD, βDSSDM, γDSSD, and γDSSDM of the present invention on 4T1 proliferation;

[0039] Fig.11 This is a graph showing the effect of adding doxorubicin solution, ML210 solution, doxorubicin-ML210 mixed solution, βDSSD, βDSSDM, γDSSD, γDSSDM and ferroptosis inhibitor Ferrostatin-1 on the proliferation effect of each group;

[0040] Fig.12 This is a graph showing changes in glutathione after ML210 treated 4T1 at different time points in the present invention;

[0041] Fig.13The effect of the ML210 solution, βDSSDM, and γDSSDM on the production of lipid peroxides in 4T1 cells treated with the present invention, and the effect of the addition of the ferroptosis inhibitor Ferrostatin-1 on the production of lipid peroxides in each group;

[0042] Fig.14 This is a graph showing the inhibition of tumors in 4T1 tumor-bearing mice by each treatment group;

[0043] Fig.15 This is a graph showing the effects of each treatment group on the body weight of 4T1 tumor-bearing mice. DETAILED DESCRIPTION

[0044] In order to better understand the technical scheme of the present invention, achieve the purpose and beneficial effect, the present invention is further specifically described by the following examples, but it should not be understood as limiting the present invention. Some non-essential improvements and adjustments made by those skilled in the art according to the above invention content are also considered to fall within the protection scope of the present invention. The experimental methods for which specific conditions are not indicated in the embodiments are usually carried out under conventional conditions or under the conditions recommended by the reagent manufacturer. Unless otherwise specified, the reagents and equipment used in the present invention are conventional reagents and equipment in the art.

[0045] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0046] Example 1: Synthesis of doxorubicin dimer prodrug γDSSD

[0047] Synthesis route see Figure 1 , the specific steps are as follows:

[0048] S1. Weigh doxorubicin hydrochloride (1.2 g, 2.07 mmol) into a 100 mL round-bottom flask, add 30 mL of dichloromethane and stir to dissolve, then add triethylamine (288.7 μL, 2.07 mmol) and stir at room temperature for 30 min.

[0049] S2. Weigh 3,3'-dithiodipropionic acid (210.3 mg, 1.0 mmol) into a 150 mL round-bottom flask, add 30 mL of dichloromethane and 10 mL of dimethylformamide and stir to dissolve, then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 439.3 mg, 2.30 mmol) and 4-dimethylaminopyridine (DMAP, 293.3 mg, 2.4 mmol), activate in an ice bath for 1 h, heat to 25 ° C, and continue stirring at the same temperature for 30 min.

[0050] S3, then add the adriamycin suspension dropwise to the above reaction solution and stir the reaction at the same temperature for 12 hours, add the same amount of EDCI and DMAP as in step S2, and continue the reaction for 12 hours; after the reaction, remove dichloromethane and dimethylformamide under reduced pressure, dissolve the residue with 300 mL of dichloromethane, wash with saturated brine, and collect the product through silica gel column chromatography (eluent, dichloromethane:methanol=125:1-60:1), and concentrate under reduced pressure to obtain a red target product (753.6 mg), with a yield of 59.6% and a purity of 96.81%, i.e., adriamycin dimer prodrug γDSSD, whose structural formula is as follows: Figure 3 shown.

[0051] Example 2: Preparation of doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles with different ratios

[0052] The co-assembled nanoparticles were prepared by precipitation method, and the ratio of doxorubicin dimer prodrug and ferroptosis activator ML210 was screened by single factor optimization method, which specifically included the following steps:

[0053] S1. Take the doxorubicin dimer prodrug synthesized under the conditions of Example 1 and the ferroptosis activator ML210 (CAS No.: 1360705-96-9) in a mass ratio of 1:0, 1:0.5, 1:1, 1:2, 1:4 and 2:1, and stearoyl phosphatidylethanolamine-polyethylene glycol 2000 with a mass of 25% of the mass of the doxorubicin dimer prodrug, and fully dissolve them in 400 μL of dimethyl sulfoxide to obtain mixed solutions of various ratios;

[0054] S2. The mixed solutions of each ratio were added dropwise to 4 mL of deionized water under stirring, stirred for 3 min, and then placed in a dialysis bag with a molecular weight of 10 KDa for dialyzation for 12 h to obtain doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles of different ratios.

[0055] Example 3: Stability determination of co-assembled nanoparticles of doxorubicin dimer prodrug-ferroptosis activator at different ratios

[0056] 1. Experimental Materials

[0057] Structural formula Figure 2 The doxorubicin dimer prodrug βDSSD, the doxorubicin dimer prodrug γDSSD synthesized under the conditions in Example 1, doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles of different proportions prepared under the conditions in Example 2, ferroptosis activator ML210, 10% fetal bovine serum, and PBS (pH 7.4).

[0058] 2. Experimental methods

[0059] 150 μL of nanoparticles formed by βDSSD and γDSSD and doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles with different ratios (the mass ratio of doxorubicin dimer prodrug and ferroptosis activator ML210 was 1:0, 1:0.5, 1:1, 1:2, 1:4 and 2:1), were dispersed in 1.5 mL of PBS containing 10% fetal bovine serum and pH 7.4, and incubated at 37°C for 12 h. Each group was taken out and photographed, and the particle size and polydispersity index were measured using a particle size analyzer. The changes in particle size and polydispersity index among the groups before and after incubation were compared.

[0060] 3. Experimental results

[0061] Visual observation showed that since all groups of βDSSD produced precipitation after incubation, the particle size and polydispersity index were not measured. Figure 4 As shown in Table 1, although γDSSD did not produce precipitation in 10% fetal bovine serum-PBS, the polydispersity index of γDSSD measured by the particle size analyzer increased significantly, indicating that the nanoparticles in the system were not uniform; and the particle size and polydispersity index of each co-assembled nanoparticle added with ML210 changed little before and after incubation for 12 hours, proving that the addition of the ferroptosis activator ML210 further improved the stability of γDSSD in 10% fetal bovine serum-PBS;

[0062] Table 1 Particle size and polydispersity index of different ratios of γDSSD:ML210 before and after 12h incubation

[0063]

[0064] Example 4: Experiment on cell inhibition rate of nanoparticles co-assembled with different ratios of doxorubicin dimer prodrug and ferroptosis activator

[0065] 1. Experimental Materials

[0066] Breast cancer cell line (4T1) derived from Balb / c mouse breast solid tumor, and different ratios of γDSSDM described in Example 2 (the mass ratios of doxorubicin dimer prodrug γDSSD and ferroptosis activator ML210 are 1:0, 1:0.5, 1:1, 1:2, 1:4 and 2:1).

[0067] 2. Experimental methods

[0068] 4T1 adherent cells were counted after digestion and 2×10 3The cells were inoculated at a density of 100 μL / well in a 96-well plate. After 12 h of adherence culture, the culture medium was discarded and fresh culture medium containing different concentration gradients of γDSSDM in Example 2 at different ratios was added. After 48 h of incubation, the drug-containing culture medium was discarded, and 100 μL of fresh culture medium and 10 μL of CCK8 (Cell Counting Kit-8) were added to each well. The cells were incubated at 37°C for 2 h. The absorbance of each well was measured at 450 nm using an ELISA reader, and the IC values ​​of the solution and the preparation were calculated by the inhibition rate using GraphPad software. 50 , where inhibition rate (%) = 100-(AB / A0-B), B is the absorbance value of the blank well, A is the absorbance value of the drug-treated group, and A0 is the absorbance value of the control group; at the same time, the combined index = X×AB combination / A alone +Y×AB combination / B alone , X is the molar ratio of drug A in the mixed solution, Y is the molar ratio of drug B in the mixed solution, AB combination IC for combined use 50 , A alone IC for drug A alone 50 , B alone IC for drug B alone 50 .

[0069] 3. Experimental results

[0070] As shown in Table 2, when the combination index is <1, it indicates that the doxorubicin dimer prodrug γDSSD and the ferroptosis activator ML210 have a combined anti-tumor effect. From the results, it can be seen that the difference in the combination index values ​​of the ratio of 1:2 and the ratio of 1:4 is small, and the stability of the 1:2 nanoparticles is also better. Therefore, it is finally determined that when the ratio of γDSSD and ML210 is 1:2, the combined anti-tumor effect of γDSSD and ML210 is better and the stability is better.

[0071] Table 2 Combination index of different ratios of γDSSD:ML210 on 4T1 cells

[0072]

[0073] Example 5: Preparation of doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles According to the optimal ratio conditions determined in Example 4, a precipitation method was used, which specifically includes the following steps:

[0074] S1. Take 4 mg of doxorubicin dimer prodrug, 8 mg of ferroptosis activator ML210 (CAS No.: 1360705-96-9) in a mass ratio of (1:2) and 1 mg of stearoyl phosphatidylethanolamine-polyethylene glycol 2000, and fully dissolve them in 400 μL of dimethyl sulfoxide to obtain a mixed solution;

[0075] S2. The mixed solution was added dropwise to 4 mL of deionized water under stirring, stirred for 3 min, and then placed in a dialysis bag with a molecular weight of 10 KDa for 12 h to obtain doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles.

[0076] Example 6: Particle size, polydispersity index, zeta potential and drug loading determination

[0077] 1. Experimental Materials

[0078] Nanoparticles (γDSSD) formed by self-assembly of doxorubicin dimers synthesized under the conditions in Example 1 and nanoparticles (γDSSDM) co-assembled of doxorubicin dimer prodrug-ferroptosis activator prepared under the conditions in Example 5.

[0079] 2. Experimental methods

[0080] S1. Take 200 μL of γDSSD and γDSSDM and dilute them to 2 mL with deionized water. Use a particle size analyzer to measure the particle size, polydispersity index particle size and Zeta potential.

[0081] S2. Take 200 μL of γDSSD and γDSSDM respectively and add them to ultrafiltration centrifuge tubes with a molecular weight of 1000Da. Centrifuge at 10000 rpm / min for 10 min and collect the liquid at the bottom of the centrifuge tube; take 100 μL of the nanoparticles before and after centrifugation respectively and add 4.9 mL of methanol to ultrasonically destroy the nanoparticle structure to release the drug; use high performance liquid chromatography to determine the drug loading capacity respectively. The chromatographic conditions are as follows: chromatographic column: C18 column (150 mm×4.6 mm, 5 μm); mobile phase: methanol: 0.01 mol / L diammonium phosphate solution-acetic acid (70:30:0.5, V / V / V); flow rate: 1.0 mL / min; detection wavelength: 232 nm; column oven: 40℃; injection volume: 20 μL.

[0082] 3. Experimental results

[0083] like Figure 5-Figure 8 As shown in Table 3, the γDSSDM prepared under the conditions of Example 5 of the present invention has successfully assembled the adriamycin dimer prodrug and the ferroptosis activator ML210 into one, and compared with γDSSD, the γDSSDM obtained by the present invention is more evenly dispersed and more stable;

[0084] Table 3 Particle size, dispersion index, zeta potential and drug loading of γDSSD and γDSSDM

[0085]

[0086] Drug loading 1 is the drug loading of doxorubicin dimer prodrug; drug loading 2 is the drug loading of ferroptosis activator ML210.

[0087] Example 7: Anti-tumor experiment

[0088] 1. Experimental Materials

[0089] Breast cancer cell line (4T1) derived from Balb / c mouse breast solid tumor, human normal liver cells (L02), doxorubicin, ferroptosis activator ML210, doxorubicin-ML210 mixed solution, doxorubicin dimer prodrug self-assembled nanoparticles γDSSD, doxorubicin dimer prodrug self-assembled nanoparticles βDSSD, and co-assembled nanoparticles γDSSDM and βDSSDM prepared under the conditions of Example 5.

[0090] 2. Experimental methods

[0091] 4T1 and L02 adherent cells were counted after digestion and 2×10 3 pcs / well and 5×10 3 The cells were inoculated at a density of 100 μL / well in a 96-well plate. After 12 h of adherence culture, the culture medium was discarded and fresh culture medium containing different concentration gradients of doxorubicin solution (DOX Sol), ML210 solution (ML210 Sol), doxorubicin-ML210 mixed solution (Mix Sol, the mass ratio of doxorubicin to ML210 was 1:2), βDSSD, βDSSDM, γDSSD and γDSSDM was given. The culture medium without drug solution was added as a control. After incubation for 48 h, the culture medium containing drug solution was discarded, and 100 μL culture medium and 10 μL CCK8 (CellCounting Kit-8) were added to each well. The plates were incubated at 37°C for 2 h. The absorbance of each well was measured at 450 nm using an enzyme reader, and the IC of the solution and preparation was calculated by the inhibition rate using graphpad software. 50 , where inhibition rate (%) = 100-(AB / A0-B), B is the absorbance value of the blank well, A is the absorbance value of the drug-treated group, and A0 is the absorbance value of the control group; calculate the tumor selection index = IC 50 Tumor cells / IC 50 Normal cells.

[0092] 3. Experimental results

[0093] like Fig. 9 As shown in Table 4, the IC of 4T1 calculated by comparison 50 , βDSSD compared with doxorubicin solution, IC 50 The IC of γDSSD was reduced by 100-fold compared with that of doxorubicin solution. 50The IC values ​​of βDSSD and γDSSD were reduced by 207 times. 50 The difference comes from the change of the intermediate connecting chain. After the doxorubicin is simply synthesized into dimers, the anti-tumor effect of the parent drug will be significantly reduced. After ML210 is loaded, the IC 50 The stability of the adriamycin solution is comparable to that of the adriamycin solution, which is due to the combined anti-tumor effect of the two, and the regulatory effect of ML210 on intracellular glutathione is conducive to the cleavage of the adriamycin prodrug (see Example 9); Fig. 9 As shown in Table 5, the IC 50 and the calculated tumor selectivity index, IC for normal cells 50 The larger the tumor selectivity index, the higher the safety. Compared with doxorubicin solution and mixed solution, both βSSDM and γDSSDM showed higher safety, and γDSSDM was safer.

[0094] Table 4 IC of each group on 4T1 cells 50

[0095]

[0096] Table 5 IC of each group on L02 cells 50 Tumor Selectivity Index

[0097]

[0098] Example 8: Antiproliferation assay

[0099] 1. Experimental Materials

[0100] Breast cancer cell line (4T1) derived from Balb / c mouse breast solid tumor, doxorubicin, ferroptosis activator ML210, doxorubicin-ML210 mixed solution (mass ratio of doxorubicin and ML210 is 1:2), doxorubicin dimer prodrug self-assembly γDSSD, doxorubicin dimer prodrug self-assembly βDSSD, ferroptosis inhibitor Ferrostatin-1 (Fer-1, CAS No.: 347174-05-4), and co-assembly γDSSDM and βDSSDM prepared under the conditions of Example 5.

[0101] 2. Experimental methods

[0102] 4T1 adherent cells were counted after digestion and 1×10 2The cells were inoculated at a density of 100 / well in a 96-well plate. After culturing for 12 hours, the culture medium was discarded and fresh culture medium containing 50nM doxorubicin solution (DOX Sol), ML210 solution (ML210 Sol), doxorubicin-ML210 mixed solution (Mix Sol), βDSSD, βDSSDM, γDSSD, γDSSDM, βDSSDM+1μM Fer-1 and γDSSDM+1μM Fer-1 were given. Culture medium without drug solution was added as a control. The cells were incubated for 2, 3, 4 and 5 days, respectively. The culture medium containing drug solution was discarded, 100μL culture medium and 10μL CCK8 (Cell Counting Kit-8) were added to each well, and the plates were incubated at 37°C for 2 hours. The absorbance of each well was measured at 450nm using a microplate reader.

[0103] 3. Experimental results

[0104] By comparing the absorbance values ​​of each group, Fig.10 As shown in the figure, compared with the control group, ML210 solution, βDSSD, and γDSSD had less effect on proliferation, while doxorubicin solution, doxorubicin-ML210 mixed solution, βDSSDM, and γDSSDM all showed significant proliferation inhibition effects; Fig.11 As shown, the addition of ferroptosis inhibitor Fer-1 can inhibit the anti-proliferative effect of βDSSDM and γDSSDM, indicating that the effect of βDSSDM and γDSSDM on proliferation is dependent on the occurrence of ferroptosis.

[0105] Example 9: Experiment on increasing intracellular glutathione levels by ML210

[0106] 1. Experimental Materials

[0107] Breast cancer cell line (4T1) derived from Balb / c mouse mammary solid tumor, ferroptosis activator ML210 and glutathione assay kit.

[0108] 2. Experimental methods

[0109] 4T1 adherent cells were counted after digestion and 1×10 6 The cells were inoculated at a density of 100 cells / well in a 6-well plate, and after culturing for 12 hours, the culture medium was discarded; 1 μM ML210 solution was added and incubated for 1 hour, 3 hours, 6 hours, 9 hours and 12 hours respectively; after the incubation, the cells were collected and the glutathione content of each group was determined according to the instructions of the glutathione determination kit.

[0110] 3. Experimental results

[0111] like Fig.12As shown, according to the mechanism of action of ML210, the inhibition of ML210 on its target protein GPX4 will regulate the intracellular glutathione content; the results showed that when cells were treated with ML210 for different time periods, intracellular glutathione showed a trend of first increasing and then decreasing, and the brief increase in glutathione can promote the breakage of the γDSSD prodrug released from the cells, thereby inducing tumor cell death in multiple ways and improving the anti-tumor efficiency.

[0112] Example 10: Lipid peroxide detection

[0113] 1. Experimental Materials

[0114] Breast cancer cell line (4T1) derived from Balb / c mouse breast solid tumor, ferroptosis activator ML210, ferroptosis inhibitor Ferrostatin-1 (Fer-1, CAS No.: 347174-05-4), co-assembly γDSSDM and βDSSDM prepared under the conditions of Example 5, and a lipid peroxide detection kit.

[0115] 2. Experimental methods

[0116] 4T1 adherent cells were counted after digestion and 5×10 4 The cells were inoculated at a density of cells / well in a 12-well plate. After 12 hours of adherence culture, the culture medium was discarded. 10 μM ML210 solution, γDSSDM and βDSSDM, ML210 solution + 5 μM Fer-1, βDSSDM + 5 μM Fer-1 and γDSSDM + 5 μM Fer-1 were added and incubated for 6 hours. After the cells were treated with a lipid peroxide detection kit, the lipid peroxides in the cells were determined by flow cytometry.

[0117] 3. Experimental results

[0118] like Fig.13 As shown in the figure, after the cells were treated with ferroptosis activators ML210, γDSSDM and βDSSDM, the content of intracellular lipid peroxides increased significantly, which is one of the indicators of cell ferroptosis. After ML210, γDSSDM and βDSSDM were incubated with the ferroptosis inhibitor Fer-1, the content of intracellular lipid peroxides was significantly lower than that without the addition of Fer-1, indicating that this process depends on the occurrence of ferroptosis.

[0119] Example 11: In vivo anti-tumor effect

[0120] 1. Experimental Materials

[0121] Breast cancer cell line (4T1) derived from Balb / c mouse breast solid tumor, Balb / c female mice (18-22g), doxorubicin, doxorubicin-ML210 mixed solution (mass ratio of doxorubicin to ML210 is 1:2), and co-assembled γDSSDM prepared under the conditions of Example 5.

[0122] 2. Experimental methods

[0123] After digestion of 4T1 adherent cells, mice were subcutaneously inoculated with the cell suspension and the tumor volume was observed. When the tumor volume increased to 80-100 mm 3 The 4T1 tumor-bearing mice were randomly divided into 4 groups (n=4) and injected with normal saline, doxorubicin solution (DOX Sol), doxorubicin-ML210 mixed solution (Mix Sol), and γDSSDM through the tail vein. The dosage was 5 mg / kg (the same dosage of doxorubicin in each group). The drugs were administered once every three days for four consecutive times. During the entire treatment period, the tumor volume and mouse weight were measured every day.

[0124] 3. Experimental results

[0125] like Fig.14 As shown in the figure, compared with the control group, the tumor volume growth of the drug-treated group slowed down, and γDSSDM had the most obvious inhibitory effect on tumor growth. On the one hand, because γDSSDM has good stability, more nanoparticles can accumulate in the tumor through the EPR effect, and under the redox conditions of the tumor site, doxorubicin and ML210 are released to achieve a combined anti-tumor effect;

[0126] like Fig.15 As shown, the body weight of mice in the γDSSDM group remained basically unchanged, while the body weight of mice in the doxorubicin solution and doxorubicin-ML210 mixed solution treatment groups decreased significantly, indicating that direct injection of doxorubicin can cause serious toxic side effects on mice, while nanoformulations can reduce its toxic side effects to a certain extent.

[0127] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticle, characterized in that: Including the chemotherapy drug doxorubicin dimer prodrug and the ferroptosis activator ML210.

2. The doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles according to claim 1, characterized in that: The structural formula of the doxorubicin dimer prodrug is shown below:

3. A method for preparing doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles according to claim 1 or 2, characterized in that: The following steps are involved: S1, taking doxorubicin dimer prodrug, ferroptosis activator ML210 and surfactant, and fully dissolving them in an organic solvent to obtain a mixed solution; S2. The mixed solution was added dropwise into deionized water under stirring, stirred for 3 minutes, and then placed in a dialysis bag for 12 hours to obtain doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles.

4. The method for preparing doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles according to claim 3, characterized in that: The mass ratio of the doxorubicin dimer prodrug and the ferroptosis activator ML210 in step S1 is 1:0 to 2:

1.

5. The method for preparing doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles according to claim 4, characterized in that: The mass ratio of the doxorubicin dimer prodrug to the ferroptosis activator ML210 is 1:

2.

6. The method for preparing doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles according to claim 3, characterized in that: The surfactant in step S1 is distearoyl phosphatidylethanolamine-polyethylene glycol 2000.

7. The method for preparing doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles according to claim 3, characterized in that: The organic solvent in step S1 is dimethyl sulfoxide.

8. The method for preparing doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles according to claim 4, characterized in that: The molecular weight of the dialysis bag in step S2 is 10 KDa.

9. Use of the doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles according to any one of claims 1 to 8 in the preparation of anti-tumor drugs.

10. An anti-tumor drug, characterized in that: The invention comprises the doxorubicin dimer prodrug-ferroptosis activator co-assembled nanoparticles as described in any one of claims 1 to 8.

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