Ferrocene-based drug dimer nanotherapeutic agent and its preparation method and application

Abstract

The present invention proposes a drug dimer nano-therapeutic agent based on ferrocene and its preparation method and application, which belongs to the field of drug development technology. The present invention proposes a drug delivery system based on ferrocene drug dimer, and especially develops a multi-mode ferroptosis nano-inducer (FSS), and explores its potential for inducing ferroptosis to treat colorectal cancer. Ferrocene-modified SAS prodrug and SN38 prodrug are prepared by chemically coupling SAS or SN38 with ferrocene dicarboxylic acid by bis(2-hydroxyethyl) disulfide through esterification reaction. The prodrug has the characteristic of responding to the specific release of high levels of GSH in the tumor microenvironment, and optimizes the pharmacokinetics and pharmacokinetic properties of clinically approved drugs. The clinical drug dimer based on ferrocene involved in the present invention has certain self-assembly characteristics, and the two drug dimers are co-assembled to prepare a nano-therapeutic agent with potential for combined application, which shows good application potential and prospect in the treatment of colorectal cancer.

Description

Technical Field

[0001] The present invention relates to technical fields such as materials science, synthesis of functional nanomaterials, regulation of assembly morphology, treatment of colorectal cancer, and drug delivery, and specifically to a multimodal ferroptosis nanoinducer, a preparation method, and applications thereof, and in particular to a method for preparing a spherical multimodal ferroptosis nanoinducer by assembling Fc-ss-SN38 and Fc-ss-SAS. Background Art

[0002] Colorectal cancer has rapidly become a major global health problem, with its etiology closely linked to the industrialization process. Emerging research has positioned ferroptosis as a powerful anti-tumor modality, introducing a key avenue for colorectal cancer management. However, glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) are significantly upregulated in colorectal cancer tissues compared with normal colon tissues, indicating that intrinsic resistance to ferroptosis weakens the therapeutic effect. To resolve this therapeutic impasse, it is necessary to enhance the sensitivity of colorectal cancer cells to ferroptosis. Four major sensitization strategies have been described: (1) modulation of iron metabolism, (2) regulation of ferroptosis-related lipid metabolism, (3) manipulation of amino acid metabolism, and (4) strict control of glutathione synthesis and degradation. The first two sensitization strategies discussed above enhance the lipid peroxide generation system, while the latter two strategies impair the lipid peroxide clearance system. In this context, the key to iron metabolism is to enhance the chelation of endogenous iron and the careful targeting of exogenous iron to tumor sites, thereby amplifying the efficacy of ferroptosis through local cellular iron overload. Furthermore, regulation of ferroptosis-related lipid metabolism is primarily achieved through lipid peroxidation catalyzed by enzymes such as arachidonic acid 12-lipoxygenase (ALOX12). Concurrently, amino acid metabolism is regulated, primarily by inhibiting glutaminase activity and limiting the uptake of specific amino acids, particularly cystine, thereby reducing intracellular reduced glutathione (GSH) concentrations and attenuating tumor resistance to ferroptosis. During lipid peroxidation, highly reactive lipid free radicals are generated, which enhance ferroptosis. Despite these advances, cancer cells demonstrate a remarkable ability to activate compensatory mechanisms after amino acid inhibition, aiming to restore GSH synthesis through alternative biochemical pathways. Consequently, increased ferroptosis sensitivity has shifted towards regulating GSH metabolism. This requires not only the consumption of excess reduced glutathione but also the inhibition of glutathione regeneration from oxidized glutathione (GSSG), which together reduce glutathione reserves within tumor cells and amplify the ferroptotic response.

[0003] These pathways intersect and mutually influence ferroptosis in tumor cells. However, integrating these pathways to enhance ferroptosis through single or dual approaches has been insufficient in maximizing therapeutic efficacy. We therefore propose an integrated multimodal strategy to enhance ferroptosis by simultaneously modulating the metabolism of iron ions, amino acids, and glutathione synthesis in tumor cells to significantly improve therapeutic indices. A pragmatic approach to bring this strategy closer to clinical application is to use existing clinical drugs as the primary reference for drug selection and combination therapy, namely drug repurposing, which utilizes established drugs with known safety profiles for new oncological uses. Drug repurposing has become an important and promising strategy in cancer therapy, providing important insights into drug selection and synergistic combinations. However, implementing drug repurposing in cancer therapy to achieve clinically potential multimodal ferroptosis-enhancing strategies remains a considerable challenge. Summary of the Invention

[0004] To address the shortcomings of current single or dual approaches to enhance ferroptosis by integrating ferroptosis pathways to maximize therapeutic efficacy, we proposed a comprehensive multimodal strategy to enhance ferroptosis by simultaneously modulating the metabolism of iron ions, amino acids, and glutathione synthesis in tumor cells. We prepared a multimodal ferroptosis nanoinducer to significantly improve therapeutic targets. A pragmatic approach to bringing this strategy closer to clinical application is to utilize existing clinical drugs as the primary guide for drug selection and combination therapy, a practice known as drug repurposing, where established drugs with known safety profiles are utilized for novel oncology applications.

[0005] The specific scheme adopted in the present invention is:

[0006] In the first aspect, the present invention provides a ferrocene-based drug dimer nanotherapeutic agent, which is formed by self-assembly of Fc-ss-SN38 and Fc-ss-SAS into nanoparticles, and by introducing DSPE-PEG 2k Prepared;

[0007] The Fc-ss-SN38 and Fc-ss-SAS are obtained by chemically coupling SN38 or SAS with ferrocenedicarboxylic acid via bis(2-hydroxyethyl) disulfide, and the structural formula is shown below:

[0008]

[0009] In a second aspect, the present invention provides a method for preparing the above-mentioned ferrocene-based drug dimer nanotherapeutic agent, comprising the following steps:

[0010] (1) Dissolve Fc-ss-SN38 and Fc-ss-SAS in tetrahydrofuran to obtain two prodrug solutions; 2k Dissolved in tetrahydrofuran to obtain DSPE-PEG2k solution;

[0011] (2) The prodrug solution obtained in step (1) and DSPE-PEG 2k The solutions were mixed, slowly added dropwise into pure water, and stirred at room temperature overnight to obtain the multimodal ferroptosis nanoinducer.

[0012] As a further optimization of the above preparation method, in step (1), the concentration of the prodrug solution is 1-5 mg / mL, more preferably 5 mg / mL; the DSPE-PEG 2k The concentration of the solution is 1-5 mg / mL, more preferably 5 mg / mL.

[0013] As a further optimization of the above preparation method, in step (2), the volume ratio of the Fc-ss-SN38 prodrug solution to the Fc-ss-SAS prodrug solution is 1:9, 1:4, 1:1, 4:1 or 9:1, more preferably 4:1.

[0014] As a further optimization of the above preparation method, in step (2), the reaction temperature is 4-60°C, more preferably 25°C; the reaction time is 12-48h, more preferably 24h.

[0015] As a further optimization of the above preparation method, the Fc-ss-SN38 or Fc-ss-SAS can be replaced with Fc-ss-AA, Fc-ART, Fc-ss-PTX, Fc-ss-IMI, Fc-ss-R848, Fc-ss-TRA, Fc-ss-CEL, Fc-ss-ATRA, etc. These ferrocene-based drug dimers also have self-assembly and co-assembly properties, and can be used to prepare a series of multifunctional nanotherapeutics.

[0016] In a third aspect, the present invention provides the use of the above-mentioned ferrocene-based drug dimer nanotherapeutic agent in any of the following:

[0017] (1) Multimodal induction of ferroptosis in colorectal cancer cells;

[0018] (2) Preparation of drugs for treating colorectal cancer.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. An innovative multimodal ferroptosis nanoinducer (FSS) was developed using the clinically relevant drugs SN38 and sulfasalazine.

[0021] 2. FSS exerts its effects by regulating multiple pathways, including iron metabolism, amino acid metabolism, and glutathione synthesis.

[0022] 3. FSS has the ability to release drugs in response to GSH and is effective in treating colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 TEM image of FSS provided in Example 1.

[0024] Figure 2 This is a statistical graph showing the cytotoxicity of FSS to CT26 cells provided in Application Example 1.

[0025] Figure 3 This is the statistical graph of Annexin V-FITC / PI double staining of CT26 cells by FSS provided in Application Example 1 and subsequent flow cytometry analysis.

[0026] Figure 4 The results of the FSS scratch test on CT26 cells provided in Application Example 1.

[0027] Figure 5 This is the analysis result of the effect of FSS on the MDA content of CT26 cells provided in Application Example 1.

[0028] Figure 6 This is the analysis result of the effect of FSS on the Fe(II) content in CT26 cells provided in Application Example 1.

[0029] Figure 7 This is the analysis result of the effect of FSS on the cysteine ​​content in CT26 cells provided in Application Example 1.

[0030] Figure 8 This is the analysis result of the effect of FSS on the GSH content in CT26 cells provided in Application Example 1.

[0031] Figure 9 The results of the in vivo anti-tumor effect of FSS on CT26 subcutaneous tumor-bearing mice provided in Application Example 2 are shown in FIG.

[0032] Figure 10 The figure shows the analysis results of the effect of FSS on Ki-67 expression in tumors of CT26 subcutaneous tumor-bearing mice provided in Application Example 2.

[0033] Figure 11 This is the analysis result of the effect of FSS on the in vivo tumor Tunel expression in CT26 subcutaneous tumor-bearing mice provided in Application Example 2.

[0034] Figure 12 This is a graph showing the analysis results of the effect of FSS on the in vivo tumor GPX4 protein expression in CT26 subcutaneous tumor-bearing mice provided in Application Example 2.

[0035] Figure 13The figure shows the analysis results of the effect of FSS on the in vivo tumor Nrf2 protein expression in CT26 subcutaneous tumor-bearing mice provided in Application Example 2.

[0036] Figure 14 This is the result diagram of the in vivo anti-tumor effect of FSS on CT26 colon orthotopic tumor-bearing mice provided in Application Example 2.

[0037] Figure 15 The figure provides the analysis results of the effect of FSS on Ki-67 expression in tumors of CT26 orthotopic colon tumor-bearing mice in Application Example 2.

[0038] Figure 16 This is the analysis result of the effect of FSS on the in vivo tumor Tunel expression in CT26 colon orthotopic tumor-bearing mice provided in Application Example 2.

[0039] Figure 17 This is a graph showing the analysis results of the effect of FSS on the in vivo tumor GPX4 protein expression in CT26 colon orthotopic tumor-bearing mice provided in Application Example 2.

[0040] Figure 18 The figure shows the analysis results of the effect of FSS on the in vivo tumor Nrf2 protein expression in CT26 colon orthotopic tumor-bearing mice provided in Application Example 2. DETAILED DESCRIPTION

[0041] In this study, we identified SN38, an active metabolite of the widely used chemotherapy drug irinotecan, and sulfasalazine (SAS), a treatment for ulcerative colitis, as candidate drugs for our multimodal ferroptosis-enhanced CRC therapy. By conjugating ferrous iron to SN38 and SAS, respectively, via disulfide bonds, we synthesized for the first time two prodrugs that can self-assemble into GSH-responsive nanomedicines, thereby creating an innovative multimodal ferroptosis nanoinducer (i.e., a ferrocene-based drug dimer nanotherapeutic). First, the camptothecin derivative SN38 not only inhibits topoisomerase I, hindering tumor cell proliferation and inducing apoptosis, but also regulates key proteins in ferroptosis: it downregulates the nuclear transcription factor erythroid 2-related factor 2 (Nrf2), reduces GSH production, and inhibits GPX4 activity, thereby attenuating lipid peroxide decomposition and promoting ferroptosis. Concurrently, SN38 upregulates p53 and enhances the expression of arachidonic acid ALOX12, thereby promoting the accumulation of lipid peroxides and promoting the ferroptosis process. SAS is an FDA-approved ferroptosis inducer that acts by inhibiting SLC7A11 and reducing cysteine ​​influx (a GSH precursor), thereby promoting ferroptosis. Thus, upon entering tumor cells, nanocomposite FSS modulates the lipid peroxide generation and clearance system by disrupting amino acid and iron metabolism and reducing GSH levels, thereby significantly increasing the accumulation of intracellular lipid peroxides. This multimodal effect significantly enhances ferroptosis in colorectal cancer cells. In summary, the multimodal ferroptosis inducer FSS represents a novel and promising approach for the development of colorectal cancer treatment by utilizing induced ferroptosis to enhance the efficacy of chemotherapy.

[0042] The present invention provides a multi-mode ferroptosis nano-inducer, wherein the raw materials for preparing the multi-mode ferroptosis nano-inducer include: Fc-ss-SN38, Fc-ss-SAS, DSPE-PEG 2k and solvents.

[0043] In the present invention, Fc-ss-SN38 and Fc-ss-SAS are self-assembled to form spherical particles, and DSPE-PEG is introduced into the spherical particles. 2k The stability of the nanoparticles was enhanced to form a multimodal ferroptosis nanoinducer. This multimodal ferroptosis nanoinducer enabled the assembled spherical particles to exhibit excellent anti-colorectal cancer properties. The effectiveness of the prepared multimodal ferroptosis nanoinducer was further confirmed, providing guidance for the subsequent preparation of multimodal ferroptosis nanoinducers.

[0044] The structural formulas of Fc-ss-SN38 and Fc-ss-SAS are as follows:

[0045]

[0046] The preparation process of Fc-ss-SN38 (or Fc-ss-SAS) is as follows:

[0047] (1) Bis(2-hydroxyethyl) disulfide and tert-butyldimethylsilyl chloride (TBSCl) were added to a DMF solution containing imidazole. After the reaction, the mixture was purified by column chromatography to obtain a colorless liquid compound 1;

[0048] (2) Ferrocene dicarboxylic acid was dissolved in DCM, EDC was added and stirred for 10 min, then DMAP and compound 1 were added. After the reaction was completed, column chromatography was performed to obtain compound 2 as a light yellow oil;

[0049] (3) Add triethylamine trihydrofluoride to the THF solution of compound 2, stir at room temperature, and after the reaction is completed, column chromatography is performed to obtain compound 3 as a yellow oil;

[0050] (4) p-Nitrophenyl chloroformate was added to a DCM solution of compound 3 containing DIPEA. After the reaction was completed at room temperature, column chromatography was performed to obtain compound 4;

[0051] (5) SN38 (or SAS) is added to a DCM solution of compound 4 containing DIPEA. After the reaction is completed at room temperature, column chromatography is performed to obtain compound 5, namely Fc-ss-SN38 (or Fc-ss-SAS).

[0052] Preferably, the multimodal ferroptosis nanoinducer is formed into spherical particles by self-assembly of Fc-ss-SN38 and Fc-ss-SAS, and DSPE-PEG is introduced into the spherical particles. 2k The stability of the nanoparticles is enhanced.

[0053] Preferably, the multimodal ferroptosis nanoinducer is a nanosphere.

[0054] Preferably, the particle size of the nanospheres is about 150 nm.

[0055] Preferably, the Fc-ss-SN38, Fc-ss-SAS and DSPE-PEG 2k The purity is 98% or higher, for example, 98%, 98.5%, 99%, 99.5%, etc.

[0056] Preferably, the solvent is water, preferably deionized water.

[0057] The present invention assembles Fc-ss-SN38 and Fc-ss-SAS into nanoparticles, and introduces DSPE-PEG 2k Enhance the stability of nanoparticles and prepare multimodal ferroptosis nanoinducers. This method uses ferrocenedicarboxylic acid (FCCa) modified SN38 / SAS and DSPE-PEG2k The multimodal ferroptosis nanoinducer is prepared in deionized water using ferroptosis as raw material. The preparation method of the present invention is simple and easy to operate, and the preparation is completed in an environmentally friendly aqueous solution without the introduction of other components. The prepared multimodal ferroptosis nanoinducer is uniform in size and evenly dispersed.

[0058] Preferably, in step (1), the concentration of the Fc-ss-SN38 or Fc-ss-SAS prodrug solution is 1-5 mg / ml, for example, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, etc., preferably 5 mg / ml.

[0059] Preferably, in step (1), the DSPE-PEG 2k The concentration of the solution is 1-5 mg / ml, for example, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, etc., preferably 5 mg / ml.

[0060] Preferably, in step (2), the volume ratio of the Fc-ss-SN38 prodrug solution to the Fc-ss-SAS prodrug solution is 1:9; 1:4; 1:1; 4:1; 9:1, preferably 4:1.

[0061] Preferably, in step (2), the reaction temperature is 4-60°C (for example, it can be 4°C, 5°C, 6°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, etc.), preferably 25°C, and the reaction time is 12-48h (for example, it can be 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, 35h, 40h, 45h, 48h, etc.), preferably 24h.

[0062] Preferably, the method for preparing the multimodal ferroptosis nanoinducer comprises the following steps:

[0063] (1) Dissolve the Fc-ss-SN38 prodrug solution and the Fc-ss-SAS prodrug solution with a purity of more than 98% in tetrahydrofuran to obtain a 1-5 mg / ml prodrug solution; 2k Dissolve in tetrahydrofuran to obtain 1-5 mg / ml DSPE-PEG 2k solution;

[0064] (2) The prodrug solution obtained in step (1) and DSPE-PEG 2kThe solutions are mixed in a volume ratio of 1:9; 1:4; 1:1; 4:1; 9:1, and reacted at 4-60° C. for 12-48 hours to obtain the multimodal ferroptosis nanoinducer.

[0065] The structural formulas of other ferrocene-based drug dimers Fc-ss-AA, Fc-ART, Fc-ss-PTX, Fc-ss-IMI, Fc-ss-R848, Fc-ss-TRA, Fc-ss-CEL, and Fc-ss-ATRA are shown below:

[0066]

[0067] These ferrocene-based drug dimers also have self-assembly and co-assembly properties, and new nanotherapeutics based on such drug dimers can be developed to improve the shortcomings of the original drugs in clinical applications.

[0068] The present invention further provides a use of the multimodal ferroptosis nanoinducer in the treatment of colorectal cancer.

[0069] In the present invention, the specific steps of the multimodal ferroptosis nanoinducer in the treatment of colorectal cancer are:

[0070] (A) The multimodal ferroptosis nanoinducer was added to the culture system of colorectal cancer cells and incubated for 24 hours. Cytotoxicity assay, wound healing assay, flow cytometry apoptosis assay, and detection of ferroptosis-related indicators (malondialdehyde, glutathione, and iron ion content) were performed.

[0071] (B) The multimodal ferroptosis nanoinducer was administered to tumor-bearing mice via tail vein injection for in vivo anti-colorectal cancer and drug safety evaluation.

[0072] Preferably, in step (A), the amount of the multimodal ferroptosis nanoinducer added to the colorectal cancer cell culture system is 0.03%-10% of the total culture system liquid, which is 1:(50-200), for example, it can be 0.03%, 0.06%, 0.12%, 0.25%, 0.5%, 1%, 5%, 10%, etc.

[0073] Preferably, in step (A), the cell culture temperature is 25° C., and the cell culture time is 24 hours.

[0074] Preferably, in step (B), the dosage for the tumor-bearing mice is 100 μL.

[0075] Preferably, in step (B), the tumor-bearing mice are of balb / c breed and female gender, and the tumor-bearing methods are subcutaneous tumor-bearing and cecal in situ tumor-bearing.

[0076] In the present invention, the multimodal ferroptosis nanoinducer achieves excellent anti-tumor effects in the treatment of colorectal cancer, and enhances the death of colorectal cancer cells by promoting cancer cell ferroptosis in a multimodal manner.

[0077] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. It should be understood by those skilled in the art that the specific embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0078] The sources of the components in the following examples are as follows: SN38 (purity 98%, Sigma), SAS (purity 98%, Sigma), ferrocene dicarboxylic acid (purity 98%, Sigma), bis(2-hydroxyethyl) disulfide, DSPE-PEG 2k (purity 98%, Sigma), THF (analytical grade, Sigma).

[0079] Example 1

[0080] This embodiment provides a multimodal ferroptosis nanoinducer (FSS), which is prepared by the following preparation method:

[0081] (1) Weigh 5 mg of Fc-ss-SN38 and pour it into a 2 mL plastic centrifuge tube, add 1 ml of tetrahydrofuran, and shake for 5 min to promote sample dissolution. If there is any undissolved part, ultrasonicate for 3 min to completely dissolve it to avoid the presence of insoluble matter, and prepare a 5 mg / ml mother solution; weigh 5 mg of Fc-ss-SAS and pour it into a 2 mL plastic centrifuge tube, add 1 ml of tetrahydrofuran, and shake for 5 min to promote sample dissolution. If there is any undissolved part, ultrasonicate for 3 min to completely dissolve it to avoid the presence of insoluble matter, and prepare a 5 mg / ml mother solution; weigh 1 mg of DSPE-PEG 2k Pour into a 2 mL plastic centrifuge tube and add 1 mL of tetrahydrofuran. Shake for 5 minutes to promote sample dissolution. If there is any undissolved part, sonicate for 3 minutes to completely dissolve it to avoid the presence of insoluble matter, and prepare a 1 mg / mL stock solution.

[0082] (2) Take 80 μL Fc-ss-SN38 mother solution, 20 μL Fc-ss-SAS, 100 μL DSPE-PEG 2k The mother liquor was placed in a 1.5 mL plastic centrifuge tube and shaken for 3 minutes to mix the three solutions evenly; the mixed solution was slowly dropped into pure water and stirred at room temperature overnight to obtain the multimodal ferroptosis nanoinducer.

[0083] in, Figure 1 The TEM image of FSS provided in Example 1 is Figure 1It can be seen that the multimodal ferroptosis nanoinducers are spherical particles with uniform particle size.

[0084] Application Example 1

[0085] We performed MTT assay to investigate the cytotoxicity of FSS to CT26 cells. Figure 2 ). The results showed that FSS exhibited superior cytotoxicity. In addition, Figure 3 Annexin V-FITC / PI double staining and subsequent flow cytometry analysis showed that the apoptosis induction rate of the FSS-treated group was 40%, indicating that FSS can significantly enhance tumor cell death. Then, in order to observe the ability of FSS to inhibit the proliferation of CT26 cells, a cell scratch test was performed. Figure 4 As shown in the figure, the cell migration rate of the FSS-treated group at 24h and 48h was lower than that of the control group, indicating that FSS exhibited significant anti-tumor cell proliferation ability. The above results show that FSS has a significant effect of inducing apoptosis and inhibiting cancer cell proliferation, indicating that FSS has superior anti-tumor potential in vitro.

[0086] Based on the significant anti-tumor effect of FSS in vitro, we further explored the ferroptosis-related molecular mechanism of FSS-induced cell death in CT26 cells. The exploration project mainly included the intracellular oxidative stress marker MDA ( Figure 5 )、Fe(II)( Figure 6 ), ferrous ions ( Figure 7 ) and GSH( Figure 8 ) content. The MDA content in the FSS-treated group was 16 times higher than in the control group. Fe(II) and total iron levels were also higher in the FSS-treated group, demonstrating that FSS significantly induced iron overload in CT26 cells. Cysteine ​​and GSH levels were also lower in the FSS-treated group than in the control group. These results suggest that FSS can cascade down GSH production.

[0087] Application Example 2

[0088] To verify the antitumor effect of FSS in the subcutaneous CT26 tumor model, FSS was injected intravenously through the tail vein, and the changes in tumor volume were monitored. Figure 9 As shown, the tumor volume of the FSS-treated group increased the least, indicating that the use of FSS had a significant inhibitory effect on tumor growth. After the treatment was completed, the mice were humanely killed and the tumors were removed for observation and measurement. Compared with the control group (1493.12 mg), the average tumor weight of the FSS-treated group (496 mg) was the smallest, and the use of FSS once again demonstrated an excellent in vivo anti-tumor effect. In order to observe the internal conditions of the tumor more microscopically, sections were prepared from the tumor tissue, as shown in Figure 2. Figure 10 and Figure 11As shown, Ki67 and TUNEL results revealed that the FSS-treated group had the fewest Ki-67-positive cells, while more TUNEL-positive cells were found in the other groups. Semi-quantitative results showed that the number of Ki67-positive cells in the FSS-treated group was approximately one-third that of the PBS-treated control group, while the number of TUNEL-positive cells in the FSS-treated group was three times higher than that in the PBS-treated control group. These results demonstrate that FSS significantly triggers necrosis and apoptosis and exerts the most potent inhibitory effect, demonstrating its potent anti-tumor properties.

[0089] Based on the ability of FSS to trigger ferroptosis in monolayers of CT26 cells, the biological mechanisms of ferroptosis in animal models were further investigated by evaluating specific markers in subcutaneous CT26 tumor-bearing mice. Figure 12 As shown in the figure, the fluorescence signal of GPX4 in the FSS-treated group was weaker than that in the control group, indicating that the FSS-treated group had the most severe reduction in the ability to clear cellular lipid peroxides in vivo compared with the control group. In addition, the fluorescence signal of Nrf2 was the weakest in the FSS-treated group compared with the control group, further indicating that FSS caused the downregulation of Nrf2 in vivo ( Figure 13 These results indicate that FSS inhibits tumor growth through robust ferroptosis in tumors, suggesting that FSS promotes tumor cell death and achieves antitumor effects through a multimodal cascade amplifying ferroptosis in subcutaneous CT26 tumor-bearing mouse models.

[0090] Application Example 3

[0091] In order to more objectively simulate the entire process of tumor occurrence, development and invasion in vivo, an orthotopic mouse colon cancer tumor model was constructed to more objectively evaluate the anti-tumor effect of FSS.

[0092] like Figure 14 As shown in the figure, after the treatment was completed, the mice were humanely killed and the tumors were removed for observation and measurement. Compared with the control group (1621.17 mg), the average tumor weight of the FSS-treated group (435 mg) was the smallest, and the use of FSS once again demonstrated an excellent in vivo anti-tumor effect. Figure 15 and Figure 16 As shown in Figure 2, the results of Ki67 and TUNEL showed that the FSS-treated group had fewer Ki-67 positive cells and more TUNEL positive cells than the control group. Figure 17 As shown in the figure, the fluorescence signal of GPX4 in the tumor of the FSS-treated group was weaker than that in the control group, revealing that the lipid peroxide clearance ability of the cells in the FSS-treated group was most seriously decreased compared with the control group. Secondly, the fluorescence signal of Nrf2 was weaker in FSS compared with the control group, further indicating that FSS caused Nrf2 downregulation in the orthotopic CT26 tumor-bearing mouse model ( Figure 18 ).

[0093] The above results further illustrate the objective fact that FSS exerts its powerful anti-tumor efficacy through the cascade amplification of ferroptosis effects in the real process of tumor growth, development and invasion in orthotopic models.

[0094] The applicant declares that the present invention illustrates the multimodal ferroptosis nanoinducer and its preparation method and application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement to the present invention, the equivalent replacement of the raw materials of the product of the present invention, the addition of auxiliary ingredients, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims

1. A ferrocene-based drug dimer nanotherapeutic agent, characterized in that: The ferrocene-based drug dimer nanotherapeutic agent is formed by self-assembly of Fc-ss-SN38 and Fc-ss-SAS to form nanoparticles, and by introducing DSPE-PEG 2k Prepared; The Fc-ss-SN38 and Fc-ss-SAS are obtained by chemically coupling SN38 or SAS with ferrocenedicarboxylic acid via bis(2-hydroxyethyl) disulfide, and the structural formula is shown below: 。 2. A method for preparing the ferrocene-based drug dimer nanotherapeutic agent according to claim 1, characterized in that: The preparation method comprises the following steps: (1) Dissolve Fc-ss-SN38 and Fc-ss-SAS in tetrahydrofuran to obtain two prodrug solutions; 2k Dissolved in tetrahydrofuran to obtain DSPE-PEG 2k solution; (2) The prodrug solution obtained in step (1) and DSPE-PEG 2k The solutions were mixed, slowly dropped into pure water, and stirred at room temperature overnight to obtain the ferrocene-based drug dimer nanotherapeutic agent.

3. The preparation method according to claim 2, characterized in that In step (1), the concentration of the prodrug solution is 1-5 mg / mL; the DSPE-PEG 2k The concentration of the solution is 1-5 mg / mL.

4. The preparation method according to claim 3, characterized in that In step (1), the concentration of the prodrug solution is 5 mg / mL; the DSPE-PEG 2k The concentration of the solution was 5 mg / mL.

5. The preparation method according to claim 4, characterized in that In step (2), the volume ratio of the Fc-ss-SN38 prodrug solution to the Fc-ss-SAS prodrug solution is 1:9, 1:4, 1:1, 4:1 or 9:

1.

6. The preparation method according to claim 5, characterized in that The volume ratio of the Fc-ss-SN38 prodrug solution to the Fc-ss-SAS prodrug solution is 4:

1.

7. The preparation method according to claim 2, characterized in that In step (2), the reaction time is 12-48 h.

8. The preparation method according to claim 7, characterized in that In step (2), the reaction time is 24 h.

9. Use of the ferrocene-based drug dimer nanotherapeutic agent according to claim 1 or the ferrocene-based drug dimer nanotherapeutic agent prepared by the preparation method according to any one of claims 2 to 8 in the preparation of a therapeutic drug for multimodally inducing ferroptosis of colorectal cancer cells.

10. Use of the ferrocene-based drug dimer nanotherapeutic agent according to claim 1 or the ferrocene-based drug dimer nanotherapeutic agent prepared by the preparation method according to any one of claims 2 to 8 in the preparation of a drug for treating colorectal cancer.

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