A mitochondrion-targeted fenofibric acid, construction method and application thereof

Abstract

The present invention provides a mitochondrion-targeted fenofibric acid, and the mitochondrion-targeted fenofibric acid is a triphenylphosphine fenofibric acid conjugate. A method for constructing the mitochondrion-targeted fenofibric acid is to synthesize the mitochondrion-targeted fenofibric acid by coupling aminotriphenylphosphine with fenofibric acid. The specific steps include: dissolving fenofibric acid in dichloromethane containing triethylamine; then adding aminotriphenylphosphine, 1-hydroxybenzotriazole and EDCI to obtain a mixture; washing and rotary evaporation to obtain the mitochondrion-targeted fenofibric acid. The mitochondrion-targeted fenofibric acid of the present invention can efficiently inhibit respiratory chain complex I and generate a large amount of mtROS. Its cytotoxic effect is nearly 10 times that of fenofibrate, and it shows specific killing of tumor cells. The mitochondrion-targeted fenofibric acid can also cause calreticulin membrane translocation and IFN-I secretion, induce an in-situ tumor vaccine effect, and synergistically treat tumors with immune checkpoint blockade therapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a mitochondrion-targeted fenofibric acid, a construction method and an application thereof. Background Art

[0002] Although the current immune checkpoint blockade (ICB) therapy has brought about a huge change in the tumor treatment mode, new combination treatment strategies still need to be explored to enhance its efficacy in tumors with relatively low immunogenicity. In this regard, in situ tumor vaccination has received great attention. An in situ tumor vaccine refers to the fact that a local tumor can be transformed into a reservoir of tumor antigens and adjuvants after appropriate treatment, and initiate the formation of a systemic immune response against distant lesions in situ. Effective in situ vaccination should coordinate the following several immune events, including the in situ exposure and uptake of tumor antigens, the initiation of tumor-specific CD8 + T cell responses, and the formation of an immunologically active tumor microenvironment (TME). The phagocytosis of dying tumor cells by antigen-presenting cells (APCs) is the first step in activating anti-tumor immunity. Immunogenic tumor cell death can lead to the translocation of calreticulin (CRT) to the tumor cell surface, releasing an "eat me" signal to APCs, thus causing more efficient antigen uptake. On the other hand, type I interferon (IFN-I) is necessary for cross-presentation of exogenous antigens, and it is closely related to the generation of CD8 + T cell-dependent responses. IFN-I can cause the indigestion of exogenous antigens by maintaining a relatively high pH value in the phagosomes of APCs, enabling the antigens to escape into the cytoplasm, be degraded by proteasomes and then cross-presented. In addition, IFN-I can also enhance the functions of immune cells and relieve the immunosuppressive TME to achieve optimal anti-tumor immune initiation. Therefore, simultaneous induction of CRT exposure and IFN-I secretion is crucial for in situ vaccination.

[0003] Due to the presence of the endoplasmic reticulum-mitochondria biomembrane, mitochondrial reactive oxygen species (mtROS) can diffuse into the endoplasmic reticulum (ER) and potentially cause stress responses in it, which may further trigger a series of unfolded protein responses (UPR) including the exposure of CRT. Meanwhile, mitochondria contain various damage-associated molecular patterns (DAMPs). For example, cytochrome C, heme, and mitochondrial DNA (mtDNA) are typical inflammatory factors that stimulate multiple pattern recognition receptors (PRRs). It is worth mentioning that some studies have shown that mtDNA can escape from stressed mitochondria (such as oxidative damage) into the cytoplasm and activate the cGAS-STING pathway to synthesize and secrete IFN-I. Therefore, the strategy of increasing mtROS generation to synergistically promote the exposure of CRT and the leakage of mtDNA to promote IFN-I secretion may be feasible.

[0004] Fenofibrate (FF) is an FDA-approved lipid-lowering drug, and its principle is to reduce the lipid level in cells by activating the intracellular PPARα signaling pathway through its metabolite fenofibric acid (FFa) in vivo. In recent years, some in vitro studies have shown that unhydrolyzed FF can inhibit the activity of mitochondrial respiratory chain complex I to enhance the leakage of electrons in the electron transport chain, thereby promoting the generation of mitochondrial reactive oxygen species (mtROS) and killing tumor cells. However, the in vivo application effect is not satisfactory, and the reason may be related to the highly expressed esterases in vivo. These esterases will rapidly hydrolyze most of the FF into FFa, and since the carboxyl group in the fenofibric acid molecule is negatively charged and cannot be effectively accumulated in mitochondria, it cannot exert an inhibitory effect on complex I. This metabolic property potentially limits the generation of mtROS mediated by fenofibrate in vivo and makes related research only stay in the in vitro stage.

[0005] To get rid of this limitation, this application conducts mitochondrial targeting modification on fenofibric acid to effectively improve its anti-tumor function, providing a theoretical basis and data support for the application of fenofibric acid in tumor treatment. Summary of the Invention

[0006] The present invention provides a method for constructing mitochondrial-targeted fenofibric acid with a simple preparation method and effective anti-tumor function improvement, as well as its construction method and application to solve the problems of the prior art.

[0007] The technical solution adopted by the present invention is as follows:

[0008] A mitochondrion-targeted fenofibric acid, which is a triphenylphosphine fenofibric acid conjugate with the chemical formula:

[0009]

[0010] A method for constructing mitochondrion-targeted fenofibric acid (Mito-FFa), which synthesizes mitochondrion-targeted fenofibric acid by coupling aminotriphenylphosphine and fenofibric acid.

[0011] Further, the specific steps include: dissolving fenofibric acid in dichloromethane containing triethylamine; then successively adding aminotriphenylphosphine, 1-hydroxybenzotriazole and EDCI to obtain a mixture, and reacting at room temperature for 12 h; then washing the above mixture 3 times with saturated NaCl solution, and rotary evaporating the washing solution to obtain mitochondrion-targeted fenofibric acid.

[0012] Further, the molar ratio of fenofibric acid to triethylamine, aminotriphenylphosphine, 1-hydroxybenzotriazole, and EDCI is 0.5:(1-2):(0.5-1):(0.3-1):(0.1-1).

[0013] Further, the molar ratio of fenofibric acid to triethylamine, aminotriphenylphosphine, 1-hydroxybenzotriazole, and EDCI is 0.5:1.5:0.75:0.6:0.7.

[0014] The application of mitochondrion-targeted fenofibric acid in the preparation of anti-tumor drugs.

[0015] Further, the mitochondrion-targeted fenofibric acid is used to prepare a drug for enhancing the immunogenicity of tumor cells.

[0016] Further, the mitochondrion-targeted fenofibric acid can inhibit respiratory chain complex I and enhance the generation of mitochondrial reactive oxygen species; on the one hand, mitochondrial reactive oxygen species cause ER oxidative stress and induce the externalization of calreticulin, promoting its phagocytosis by immune cells; on the other hand, mitochondrial reactive oxygen species increase the mitochondrial membrane permeability, promoting the leakage of mtDNA, thereby promoting the activation of the cGAS / STING signaling pathway in surrounding immune cells and inducing the synthesis and secretion of IFN-I.

[0017] Further, the anti-tumor drug prepared from the mitochondrion-targeted fenofibric acid is used in combination with immune checkpoint blockade therapy.

[0018] The beneficial effects obtained by the present invention are as follows: Mitochondrion-targeted fenofibric acid (Mito-FFa) can efficiently inhibit respiratory chain complex I and generate a large amount of mtROS in tumor cells. In vitro studies have shown that the cytotoxic effect of Mito-FFa is nearly 10 times that of fenofibrate, and it exhibits specific killing of tumor cells.

[0019] Advantages of Mito-FFa in inducing in-situ tumor vaccine effect:

[0020] 1. Simple synthesis method: The amide bond synthesis reaction is carried out between the carboxyl group of fenofibric acid and aminotriphenylphosphine with mitochondrial targeting function. The modification endows fenofibric acid with efficient mitochondrial accumulation function, which is the basis for its excellent immune activation effect.

[0021] 2. No safety risk: Since fenofibric acid is a drug approved by the FDA for marketing and has high in-vivo safety, and the mitochondrial targeting strategy mediated by TPP has also been widely used in the development of oral health products (MitoQ). Therefore, the intratumoral administration of Mito-FFa has extremely high safety. The results show that even when the dose of intratumoral injection of Mito-FFa is as high as 1600 μg / mouse (about 30 times the concentration corresponding to 50% tumor inhibition rate), the main organs, body weight, liver and kidney functions of the mice remain healthy.

[0022] 3. Excellent anti-tumor function: The constructed Mito-FFa is a potent immunogenic death inducer and has a strong ability to promote the externalization of calreticulin. Under their respective IC 50 s, Mito-FFa can induce 84.9% of live cells to expose calreticulin on the cell membrane, while the classical ICD inducer oxaliplatin is only 2.38%. At the same time, Mito-FFa can also cause oxidative damage to mitochondria and promote the cytoplasmic leakage of oxidative mitochondrial DNA, thereby promoting the secretion of IFN-I by surrounding immune cells. Such a high level of CRT externalization and IFN-I secretion play an important role in promoting tumor antigen phagocytosis, cross-presentation, DC maturation, CD8 + T cell priming and the alleviation of the tumor immunosuppressive microenvironment.

[0023] 4. Synergistic effect with immune checkpoint blockade therapy: Mito-FFa effectively activates antigen-specific CD8 + T cells in vivo. The powerful anti-tumor immune response of CD8 + T cells makes the combination of Mito-FFa and anti-PD-L1 show extremely high synergistic effect. The data show that the in-situ tumors of 54% (4 / 7) mice have completely regressed. At the same time, the treatment of Mito-FFa + aPD-L1 also almost cleared the lung metastases of the mice and effectively extended the survival period of the mice. Brief Description of the Drawings

[0024] Figure 1 It is the synthesis route diagram of Mito-FFa of the present invention;

[0025] Figure 2Mass spectrum of Mito-FFa of the present invention;

[0026] Figure 3 1H NMR spectrum of Mito-FFa of the present invention;

[0027] Figure 4 Schematic diagram of the effect of Mito-FFa of the present invention in inducing in-situ tumor vaccine to combat primary and metastatic tumors;

[0028] Figure 5a Schematic diagram of Mito-FFa targeting mitochondria in Example 1 of the present invention to improve the mitochondrial accumulation ability of FF;

[0029] Figure 5b Different accumulations of Mito-FFa and FF in whole tumor cells and their mitochondria in Example 1 of the present invention; Data are presented as mean ± SD (n = 3, one-way ANOVA);

[0030] Figure 5c Using PRISM to calculate the IC50 of each cell line in Example 1 of the present invention;

[0031] Figure 5d Dose-cytotoxicity curves of FF and Mito-FFa in mouse triple-negative breast cancer 4T1 cells in Example 1 of the present invention; Data are presented as mean ± SD (n = 3);

[0032] Figure 5e Dose-cytotoxicity curves of FF and Mito-FFa in mouse breast cancer MDA-MB-231 cells in Example 1 of the present invention; Data are presented as mean ± SD (n = 3);

[0033] Figure 5f Dose-cytotoxicity curves of FF and Mito-FFa in mouse colorectal cancer CT26 cells in Example 1 of the present invention; Data are presented as mean ± SD (n = 3);

[0034] Figure 5g Dose-cytotoxicity curves of Mito-FFa in the above three kinds of tumor cells and normal liver AML-12 cells in Example 1 of the present invention; Data are presented as mean ± SD (n = 3);

[0035] Figure 5h Inhibitory effects of FF and Mito-FFa on mitochondrial respiratory chain complex I in Example 1 of the present invention; Data are presented as mean ± SD (n = 3, one-way ANOVA);

[0036] Figure 5iFluorescence quantification of mtROS induced by Example 1 FF and Mito-FFa of the present invention; Data are represented as mean ± SD (n = 3, one-way ANOVA);

[0037] Figure 5j Fluorescence images of mtROS induced by Example 1 FF and Mito-FFa of the present invention; Scale bar = 50 μm;

[0038] Figure 5k Flow cytometry characterization of mitochondrial superoxide in Example 1 of the present invention;

[0039] Figure 6a Growth curves of 4T1 tumors after intratumoral injection of normal saline and different doses of Mito-FFa in Example 2 of the present invention; Data are represented as mean ± SD (n = 7, one-way ANOVA);

[0040] Figure 6b Body weight curves of mice under different treatments in Example 2 of the present invention; Data are represented as mean ± SD (n = 7, one-way ANOVA);

[0041] Figure 6c H&E sections of the hearts, livers, spleens, lungs, and kidneys of mice after single intratumoral injection of PBS and Mito-FFa (1600 μg / mouse) in Example 2 of the present invention; Scale bar = 400 μm;

[0042] Figure 6d Alanine aminotransferase content in the bodies of mice after single intratumoral injection of PBS and Mito-FFa (1600 μg / mouse) in Example 2 of the present invention (n = 5, one-way ANOVA);

[0043] Figure 6e Aspartate aminotransferase content in the blood of mice after single intratumoral injection of PBS and Mito-FFa (1600 μg / mouse) in Example 2 of the present invention (n = 5, one-way ANOVA);

[0044] Figure 6f Urea content in the blood of mice after single intratumoral injection of PBS and Mito-FFa (1600 μg / mouse) in Example 2 of the present invention (n = 5, one-way ANOVA);

[0045] Figure 6g Creatinine content in the blood of mice after single intratumoral injection of PBS and Mito-FFa (1600 μg / mouse) in Example 2 of the present invention (n = 5, one-way ANOVA);

[0046] Figure 7a Schematic diagram of mtROS-induced ER stress in Example 3 of the present invention;

[0047] Figure 7b Protein blot of CHOP and GADPH in Example 3 of the present invention;

[0048] Figure 7c Representative flow cytometry image of PI- / CRT+ 4T1 tumor cells in Example 3 of the present invention;

[0049] Figure 7d Representative flow cytometry quantification of PI- / CRT+ 4T1 tumor cells in Example 3 of the present invention; Data are expressed as mean ± SD (n = 3, one-way ANOVA);

[0050] Figure 7e Representative flow cytometry phagocytosis (%) of BMDC in Example 3 of the present invention; Data are expressed as mean ± SD (n = 3, one-way ANOVA)

[0051] Figure 7f Representative flow cytometry image showing the phagocytic ability of BMDC in Example 3 of the present invention; Data are expressed as mean ± SD (n = 3, one-way ANOVA);

[0052] Figure 8a Schematic diagram of Mito-FFa mediating the secretion of IFN-I by peripheral immune cells in Example 4 of the present invention;

[0053] Figure 8b qPCR quantification of DNA in the cytoplasmic extract of 4T1 tumor cells in Example 4 of the present invention; Data are expressed as mean ± SD (n = 3, one-way ANOVA);

[0054] Figure 8c Schematic diagram of the co-culture system of Raw 264.7 and cells treated with Mito-FFa in Example 4 of the present invention;

[0055] Figure 8d Protein blot of p-STING, STING, p-IRF3, IRF3 and GADPH in Example 4 of the present invention; Figure 8e Detection of IFN-β level in the co-culture medium of Raw264.7 / 4T1 tumor cells by ELISA in Example 4 of the present invention; Data are expressed as mean ± SD (n = 3, one-way ANOVA)

[0056] Figure 8f Detection of ATP secretion by a luciferin-based ATP detection kit in Example 4 of the present invention; Data are expressed as mean ± SD (n = 3, one-way ANOVA);

[0057] Figure 8gThis is a representative flow cytometry dot plot of mature DCs in Example 4 of the present invention; data are presented as mean ± SD (n = 3, one-way ANOVA).

[0058] Figure 8h This is a representative flow cytometry quantitative plot of mature DCs in Example 4 of the present invention; data are presented as mean ± SD (n = 3, one-way ANOVA).

[0059] Figure 9a This is the primary tumor growth curve of 4T1 tumors after administration of saline, Mito-FFa, Mito-FFa + αPD-L1, and Mito-FFa + αCD8a in Example 5 of the present invention; data are presented as mean ± SD (n = 7, one-way ANOVA).

[0060] Figure 9b This is the representative flow cytometry dot plot of mature DCs (CD80 + CD86 + / CD11c + ) in tumor-draining lymph nodes in Example 5 of the present invention;

[0061] Figure 9c This is the proportion of mature DCs in tumor-draining lymph nodes after intratumoral injection of Saline and Mito-FFa (60 μg / mouse) in Example 5 of the present invention; data are presented as mean ± SD (n = 5, one-way ANOVA).

[0062] Figure 9d This is the percentage of tumor-infiltrating CD8+ T cells detected by flow cytometry in Example 5 of the present invention; data are presented as mean ± SD (n = 3 in the Mito-FFa + αPD-L1 group, n = 7 in other groups, one-way ANOVA).

[0063] Figure 9e This is the quantification of immunohistochemical staining of infiltrating CD8+ T cells in Example 5 of the present invention; scale bar = 100 μm, data are presented as mean ± SD (n = 3, one-way ANOVA).

[0064] Figure 9f This is a representative image of immunohistochemical staining of infiltrating CD8+ T cells in Example 5 of the present invention; scale bar = 100 μm;

[0065] Figure 9g This is the IFN-γ level in tumor tissues of different groups in Example 5 of the present invention; data are presented as mean ± SD (n = 3 in the Mito-FFa + αPD-L1 group, n = 7 in other groups, one-way ANOVA).

[0066] Figure 10aBody weights of each 4T1 tumor-bearing mouse during treatment in Example 6 of the present invention (n = 7).

[0067] Figure 10b Lungs fixed with Bouin's solution in Example 6 of the present invention.

[0068] Figure 10c Image of the H&E-stained section of the lung fixed with Bouin's solution in Example 6 of the present invention.

[0069] Figure 10d Quantification of lung lesions in different groups in Example 6 of the present invention; data are presented as mean ± SD (n = 7, one-way ANOVA).

[0070] Figure 10e Survival curve of mice bearing 4T1 breast tumors in Example 6 of the present invention. Detailed implementation mode

[0071] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0072] During specific implementation: Method for constructing mitochondrially targeted fenofibric acid

[0073] Preparation of 4-aminobutyltriphenylphosphonium bromide and Mito-FFa: Synthesis of 4-aminobutyltriphenylphosphonium bromide (TPP-NH2): (4-Bromobutyl)triphenylphosphonium bromide (1 g, 2.512 mmol) was dissolved in ammonia methanol solution (7N, 100 mL, 700 mmol) and incubated in a sealed flask at room temperature for 3 days. Then the solvent was evaporated and the residue was a viscous liquid. After vacuum drying, a colorless foam was obtained. This product could be used in subsequent reactions without further purification.

[0074] Fenofibric acid (160 mg, 0.5 mmol) was dissolved in dichloromethane DCM (20 mL) containing triethylamine (160 μl, 1.5 mmol). Subsequently, TPP-NH2 (369 mg, 0.75 mmol), 1-hydroxybenzotriazole (Hobt, 81 mg, 0.60 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDCI (135 mg, 0.7 mmol) were added in sequence and reacted at room temperature for 12 h. Then the mixture was washed 3 times with saturated NaCl solution and Mito-FFa was obtained by rotary evaporation. Yield: 90%. All the above chemical reagents were purchased from Shanghai J&K Chemical Technology Co., Ltd.

[0075] AsFigure 3 As shown, the 1H NMR spectrum analysis of Mito-Ffa, MS: C39H38ClNO3P+: Cal. 634.23, found 634 (M+). 1H NMR (400 MHz, DMSO-d6) δ 8.23 - 8.20 (m, 1H), 7.91 - 7.86 (m, 3H), 7.80 - 7.72 (m, 11H), 7.69 - 7.60 (m, 7H), 6.93 - 6.89 (m, 2H), 3.60 - 3.53 (m, 2H), 3.17 - 3.12 (m, 2H), 1.64 - 1.60 (m, 2H), 1.46 - 1.44 (m, 8H).

[0076] Example 1: Mito-FFa inhibits tumor cell growth by interfering with mitochondrial function

[0077] To determine whether Mito-FFa could be more effectively localized within mitochondria, this application detected Mito-FFa in intact cells or isolated mitochondria by HPLC 12 hours after administration (the final concentration of Mito-FFa in the cell culture medium was 8 μM). Compared with FF, the uptake of Mito-FFa in intact cells and its accumulation in mitochondria increased by 1.3-fold and 4.5-fold, respectively ( Figure 5a -b).

[0078] The anti-proliferative effects of FF and Mito-FFa in various cell lines.

[0079] Compared with FF, due to TPP-mediated mitochondrial targeting ( Figure 5c -f), the IC50 values of Mito-FFa for breast 4T1 tumor cells, breast MDA-MB-231 tumor cells, and colon CT26 tumor cells decreased by 9.9, 19.9, and 9.6-fold, respectively. Then, normal alpha mouse liver 12 (AML12) cells (a kind of hepatocyte) were used to detect whether the cytotoxicity of Mito-FFa was tumor cell-specific. The IC of Mito-FFa for AML12 cells detected at 104.7 μM 50 was much higher than that of the above three tumor cell lines ( Figure 5c , g). The significant difference in the cytotoxicity of Mito-FFa between tumor cells and AML12 cells may be due to the hyperpolarized mitochondrial membrane potential (MMP) of tumor cells, which promotes the selective accumulation of TPP conjugates within mitochondria.

[0080] The reason for the induction of tumor cell death by Mito-FFa in 4T1 tumor cells in this application is the key to realizing the further application of Mito-FFa.

[0081] First, at their respective IC50 concentrations, the inhibition rates of Mito-FFa and FF on respiratory chain complex I were 86.1% and 27.1% ( Figure 5h ), indicating the highly specific effect of Mito-FFa on mitochondria. Among them, respiratory chain complex I is a metabolic enzyme that can connect electrons to transport from NADH to ubiquinone for proton transmembrane transfer. A large number of studies have shown that inhibiting complex I will lead to more electron leakage and reaction with O2, thus generating more mtROS. Therefore, in this application, tumor cells were treated with Mito-FFa and FF at their IC50 concentrations, and Mito Red CM-H2XRos (a mitochondrial-specific probe that can only emit fluorescence after being oxidized by ROS, purchased from Yeasen Biotech) was used to detect the level of mtROS. As Figure 5i shown in -j, only weak red fluorescence could be detected in 4T1 breast tumor cells treated with fenofibrate (80 μM). However, even at a concentration much lower than FF (8 μM), the red fluorescence brightness of mtROS induced by Mito-FFa was significantly higher, which should be due to its more effective mitochondrial accumulation and inhibitory effect on complex I. To further clarify the types of mtROS induced by Mito-FFa, flow cytometry analysis based on MitoSOX (purchased from Yeasen Biotech) was used to detect mitochondrial superoxide anions. Figure 5k The results shown confirmed the generation of mitochondrial superoxide anions. The excessive generation of mtROS may lead to mitochondrial depolarization. The weak damage effect of FF on mitochondria may indicate that it does not mainly inhibit the growth of tumor cells by acting on mitochondria, which is consistent with previous studies.

[0082] N.S. indicates no significant difference, *p < 0.05; **p < 0.01; ***p < 0.001.

[0083] In summary, the mitochondrial-targeted fenofibric acid Mito-FFa can more effectively induce the generation of mtROS and exert excellent anti-tumor effects.

[0084] Example 2 Anti-tumor effect of Mito-FFa in vivo

[0085] The anti-tumor effect of Mito-FFa in a murine triple-negative breast cancer model (4T1) was evaluated. First, 4T1 tumor cells (5×10 5per mouse), when the tumor volume reached approximately 100 mm 3 At that time, the mice were randomly divided into 4 groups. After single intratumoral injection of different doses of (20 μg, 60 μg, and 200 μg / mouse) Mito-FFa, as Figure 6a shown, the treatments with 20 μg, 60 μg, and 200 μg / mouse Mito-FFa significantly slowed down the growth of the established 4T1 tumors, and the tumor growth inhibition (TGI) rates were 47.6%, 68.1%, and 100%, respectively.

[0086] On the other hand, the present application also evaluated the biosafety of intratumoral injection of Mito-FFa. It was found that during the administration period, no significant change in the body weight of the mice was observed ( Figure 6b ). Even when the dose was sharply increased to 1600 μg / mouse, the results of histological analysis also showed that the main organs of the mice ( Figure 6c ) and serum biochemical indexes, such as alanine aminotransferase content (ALT, Figure 6d ), aspartate aminotransferase (AST, Figure 6e ), urea content (UREA, Figure 6f ), creatinine content (CREA, Figure 6g ) still remained at healthy levels. These results indicate that local administration of Mito-FFa has excellent anti-tumor effects while also having high biosafety.

[0087] N.S. indicates no significant difference, *p < 0.05; **p < 0.01; ***p < 0.001.

[0088] Example 3 Mito-FFa induces endoplasmic reticulum stress, increases CRT exposure and phagocytosis

[0089] Some mitochondria are physically connected to the ER to form the mitochondria-associated ER membrane (MAM), which participates in basic biological processes. For example, the research by M. Booth et al. shows that mtROS can diffuse from the MAM to the ER, thereby inducing ER oxidative stress ( Figure 7a ). Therefore, the present application studied whether Mito-FFa-mediated mtROS would cause ER stress and subsequent CRT exposure. The expression of CHOP protein (YT0912, ImmunoWay) after Mito-FFa treatment indicated the occurrence of the unfolded protein response (UPR) and endoplasmic reticulum stress ( Figure 7b ).

[0090] Then, this application compared the ability of Mito-FFa, FF, and oxaliplatin (OxPt, purchased from Sino-Platinum Metals, a mature and classic ICD inducer) to induce CRT exposure at their respective IC50 concentrations. Figure 7c ,d Flow cytometry data based on CRT fluorescent antibody (ab196158, abcam) showed that OxPt (40 μM) could expectedly induce CRT exposure. However, when treated with Mito-FFa (8 μM), 84.9% of viable tumor cells (Pi - ) had CRT exposure, while only 1% of cells had CRT exposure when treated with FF. Moreover, compared with OxPt treatment, Mito-FFa-induced CRT exposure was enhanced by 35.7-fold. CRT exposed on the surface of tumor cells releases an "eat me" signal to APCs and promotes their phagocytosis by APCs through binding to CD91 on APCs. Therefore, the promoting effect of Mito-FFa on APC phagocytosis was evaluated by flow cytometry and CLSM. To reduce non-specific phagocytosis, we used bone-marrow derived dendritic cells (BMDCs) for phagocytosis comparison. As Figure 7e , fj showed, cells treated with Mito-FFa were most effectively phagocytosed by BMDCs. Therefore, these results indicate that the high level of CRT exposure induced by Mito-FFa significantly promotes the phagocytosis of tumor cells by APCs, thus ensuring sufficient uptake of tumor antigens.

[0091] N.S. indicates no significant difference, *p < 0.05; **p < 0.01; ***p < 0.001.

[0092] Example 4 Mito-FFa induces Ox-mtDNA leakage and activates the cGAS-STING signaling pathway, promoting IFN-I secretion

[0093] Excessive mtROS induced by Mito-FFa may enhance mitochondrial membrane permeability and induce the release of mtDNA into the cytoplasm to activate the cGAS-STING signaling pathway, thereby promoting IFN-I secretion ( Figure 8a) To verify this speculation, the present application first separated the treated 4T1 cells, purified DNA from the cytoplasmic extract, and performed qPCR analysis. Among them, histone-H3 (purchased from abcam), Hsp-60 (purchased from abcam), and GAPDH (purchased from abcam) were used as markers for measuring nuclear, mitochondrial, and cytoplasmic components, respectively. qPCR analysis showed that more mtDNA (represented by Dloop2 and CytB) rather than nuclear DNA (represented by Tert) was enriched in the cytoplasm of 4T1 tumor cells treated with 8 uM Mito-FFa for 6 hours ( Figure 8b ), which confirmed that Mito-FFa treatment mediated the leakage of mtDNA.

[0094] Next, the present application explored whether the mtDNA escaping from stressed mitochondria would activate the cGAS-STING pathway. When co-cultured with Mito-FFa-treated 4T1 cells for 24 hours, the phosphorylation levels of STING and interferon regulate factor 3 (IRF-3) in Raw264.7 increased significantly ( Figure 8c , d). WB antibodies against P-STING (D8F4W), STING (D1V5L), P-IRF-3 (D6O1M), and IRF-3 (D83B9) were all purchased from Cell Signaling Technology. Meanwhile, when co-incubated with Mito-FFa-treated 4T1 cells, the level of IFN-β induced in Raw264.7 cells was much higher than that in other treatment groups ( Figure 8e ). In addition, it was also observed that Mito-FFa treatment caused more ATP (another ICD marker for immune activation) to be released extracellularly ( Figure 8f ). Since IFN-β and ATP are effective pro-inflammatory mediators that can recruit and activate DCs, Mito-FFa-treated 4T1 cells were then added to immature BMDCs to evaluate their potential adjuvant effect. As Figure 8g and h showed, more mature BMDCs (CD80 + CD86 + / CD11c + ) were observed in the Mito-FFa group after 24 hours of co-culture. Therefore, these results indicate that Mito-FFa can induce mtDNA leakage and promote the secretion of IFN-I by surrounding immune cells to promote DC maturation.

[0095] N.S. indicates no significant difference, *p < 0.05; **p < 0.01; ***p < 0.001.

[0096] Example 5 In situ tumor vaccination induced by Mito-FFa

[0097] This application evaluated the in situ vaccination effect of Mito-FFa in poorly immunogenic 4T1 tumors. When the tumor volume reached approximately 100 mm 3 , Mito-FFa (60 μg / mouse) was injected intratumorally. As Figure 9a shown, single intratumoral injection of Mito-FFa significantly slowed the growth of established 4T1 tumors, with a tumor growth inhibition (TGI) rate of 68.1%.

[0098] Next, to investigate whether Mito-FFa-treated tumors exhibited an immune-activated phenotype, this application collected tumor draining lymph nodes (TDLNs) from Mito-FFa-treated mice to detect DC cell maturation for evaluating the immune priming status. Compared with the PBS group, Mito-FFa treatment significantly promoted DC maturation, as manifested by increased expression of CD80 + / 86 + ([[]] Figure 9b Figure 9b , c). Meanwhile, both flow cytometry and IHC analysis confirmed that a large number of CD8+ T cells were recruited into Mito-FFa-treated tumors ([[]] Figure 9d Figure 9d -f), and this led to a significant increase in the level of IFN-γ ([[]] Figure 9g Figure 9g ). To demonstrate whether these infiltrating CD8 + T cells were involved in the antitumor effect of Mito-FFa. Therefore, CD8 + T cells in mice were depleted by anti-CD8a antibody (αCD8a, purchased from Biolegend). The results showed that the therapeutic effect of Mito-FFa was significantly weakened (TGI 68.7% vs 25.1%), indicating that CD8 + T cells were closely involved in the inhibition of in situ tumors by Mito-FFa ([[]] Figure 9a Figure 9a ).

[0099] Successful ICB treatment depends on a strong CD8+ T cell response. Therefore, this application explored the synergistic effect of Mito-FFa and PD-L1 blockade therapy (αPD-L1, purchased from Biolegend). Excellent synergistic effects were observed, with 57% (4 / 7) complete responses in 96.1% TGI ([[]] Figure 9a Figure 9a ). In addition, αPD-L1 treatment also significantly enhanced Mito-FFa-induced CD8+ T cell infiltration and IFN-γ secretion(Figure 9e -g). Therefore, these results indicate that Mito-FFa, as a potential ICD inducer, can effectively convert "cold tumors" into "hot tumors" and initiate CD8 + T cell-dependent immune responses and cooperate with αPD-L1 to regress orthotopic tumors.

[0100] N.S. indicates no significant difference, *p < 0.05; **p < 0.01; ***p < 0.001.

[0101] Example 6 Mito-FFa-induced CD8+ T cell responses cooperate with αPD-L1 to inhibit 4T1 tumor metastasis

[0102] This application evaluated the ability of Mito-FFa-mediated immune responses to inhibit metastasis after resection of primary tumors. On the 16th day of treatment in Example 5, the 4T1 tumors of tumor-bearing mice in each group were resected, and then their spontaneous metastasis was monitored for a long time. Due to the increase in metastasis burden, the body weights of mice in the PBS group gradually decreased ( Figure 10a ), and all died before the 52nd day ( Figure 10e ). However, the lifespan of mice in the Mito-FFa group was significantly prolonged, and 3 / 7 (42.8%) of the mice had stable or increased body weights and survived until the 120th day ( Figure 10a , e). Meanwhile, a large number of metastatic nodules were observed in the lungs of PBS-treated mice, while only sporadic lesions were observed in the Mito-FFa group ( Figure 10b -d). It has been previously determined that local treatment with Mito-FFa can initiate CD8+ T cells. In this model, when CD8+ T cells were immunodepleted with αCD8a, the anti-tumor metastatic activity of Mito-FFa also disappeared, showing a survival curve comparable to that of PBS treatment ( Figure 10a -e). On the other hand, Mito-FFa showed excellent synergy with aPD-L1, resulting in a long-term survival rate of 71.4% (5 / 7) in mice ( Figure 10b , f), and a significant reduction in metastatic lesions in normal organs including the lungs ( Figure 10a -e). These results indicate that the CD8+ T cell responses induced by Mito-FFa through in-situ tumor vaccination (especially in combination with ICB treatment) can inhibit the systemic metastasis of highly immunosuppressive solid tumors.

[0103] N.S. indicates no significant difference, *p < 0.05; **p < 0.01; ***p < 0.001.

[0104] In summary, the present application has developed an efficient mitochondrion-targeted fenofibric acid, which can serve as an effective immunogenic cell death inducer for in situ tumor vaccines. Due to the high accumulation of Mito-FFa in mitochondria, it can more effectively inhibit Complex I, thereby generating more mtROS to induce ER stress and mtDNA leakage. ER stress and mtDNA leakage significantly promote the externalization of CRT and IFN-I secretion, and enhance phagocytosis and antigen presentation. Our results show that a single intratumoral injection of Mito-FFa can effectively induce CD8 + T cell-dependent anti-tumor immune responses against primary and metastatic tumors and significantly enhance the efficacy of PD-L1 blockade therapy. These results suggest that Mito-FFa has the potential to be a good partner for current ICB therapies.

[0105] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Use of mitochondrion-targeted fenofibric acid in the preparation of an anti-tumor drug, characterized in that: The mitochondrial-targeted fenofibric acid is a triphenylphosphine fenofibric acid conjugate, and its chemical formula is: The mitochondrial-targeted fenofibric acid is used to prepare a drug for enhancing the immunogenicity of tumor cells.

2. Use of the mitochondrion-targeted fenofibric acid according to claim 1 in the preparation of an anti-tumor drug, characterized in that: The mitochondrial-targeted fenofibric acid is synthesized by coupling aminotriphenylphosphine with fenofibric acid.

3. Use of mitochondrion-targeted fenofibric acid according to claim 2 in the preparation of an anti-tumor drug, characterized in that: The specific steps include: dissolving fenofibric acid in dichloromethane containing triethylamine; then successively adding aminotriphenylphosphine, 1-hydroxybenzotriazole and EDCI to obtain a mixture, and reacting at room temperature for 12 h; then washing the above mixture 3 times with saturated NaCl solution, and rotary evaporating the washing solution to obtain the mitochondrial-targeted fenofibric acid.

4. Use of mitochondrion-targeted fenofibric acid according to claim 3 in the preparation of an anti-tumor drug, characterized in that: The molar ratio of the fenofibric acid to triethylamine, aminotriphenylphosphine, 1-hydroxybenzotriazole, and EDCI is 0.5:(1-2):(0.5-1):(0.3-1):(0.1-1).

5. Use of mitochondrion-targeted fenofibric acid according to claim 4 in the preparation of anti-tumor drugs, characterized in that: The molar ratio of the fenofibric acid to triethylamine, aminotriphenylphosphine, 1-hydroxybenzotriazole, and EDCI is 0.5:1.5:0.75:0.6:0.

7.

6. The use of mitochondrion-targeted fenofibric acid according to claim 1 in the preparation of an anti-tumor drug, characterized in that: The anti-tumor drug prepared from the mitochondrial-targeted fenofibric acid is used in combination with immune checkpoint blockade therapy.

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