Compound for enhancing targeting property of antitumor drug as well as preparation method and application of compound

Abstract

The invention belongs to the technical field of biological medicines, and relates to a compound for enhancing the targeting property of an antitumor drug as well as a preparation method and application of the compound. The invention provides a compound for enhancing the targeting property of an anti-tumor drug. The compound is composed of a carrier, the anti-tumor drug and a targeting drug, the targeted drugs are tinidazole and methimazole; in the compound, the proportion of the targeted drug is not less than 3% based on the carrier dosage; the carrier is albumin; the antitumor drug is fluvastatin. The invention provides an intervention strategy for synchronously targeting lipid raft metabolism and mitochondrial respiration so as to realize effective activation of T cells in a tumor microenvironment. The compound constructed by the invention is based on a dual response targeting mechanism of tinidazole and methimazole to a tumor microenvironment, so that the targeting property of fluvastatin to tumors is improved, and more solutions are provided for the fluvastatin to safely and effectively regulate and control a metabolic pathway of tumor cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to a complex for enhancing the targeting of anti-tumor drugs, a preparation method thereof, and an application thereof. Background Art

[0002] Immunotherapy (especially immune checkpoint inhibitors targeting the PD-1 / PD-L1 axis) has profoundly changed the treatment pattern of non-small cell lung cancer (NSCLC). Such therapies have significantly improved the overall survival and objective response rate of NSCLC patients, and their efficacy is related to the PD-L1 expression level. However, a large number of patients still face the problems of primary resistance or secondary resistance. This heterogeneity in immunotherapy response is mainly attributed to the immunosuppressive characteristics of the tumor microenvironment (TME), in which insufficient T cell infiltration and inhibition of cytotoxic T cell function constitute key obstacles. Breaking through the above bottlenecks is the core challenge for improving the efficacy of NSCLC immunotherapy. In NSCLC, the two core factors of impaired T cell function and insufficient infiltration are tumor hypoxia and the presence of cancer cell membrane lipid rafts. Hypoxia, as a common feature of solid tumors, not only inhibits T cell infiltration and function by stabilizing hypoxia-inducible factor 1α (HIF-1α), but also induces T cell metabolic reprogramming, leading to its exhausted state. In addition, hypoxia can up-regulate the expression of immunosuppressive molecules such as adenosine, further weakening the T cell activity in the tumor microenvironment (TME). In addition, lipid rafts, as cholesterol-rich microdomains in the plasma membrane, play a key regulatory role in the signal transduction of immune cells. These structures inhibit T cell activation and weaken the anti-tumor immune response by sequestering and inactivating key molecules (such as Lck, LAT) in the T cell receptor (TCR) signaling pathway. The interaction between lipid rafts and programmed death ligand 1 (PD-L1) can also promote immune escape by stabilizing PD-L1 on the cancer cell surface, inhibiting the killing effect mediated by T cells. Further studies have found that there is a two-way feedback loop between mitochondrial respiration, which is the main cause of hypoxia, and lipid raft formation, jointly exacerbating the immunosuppressive microenvironment. Lipid rafts promote mitochondrial respiratory function by altering the mitochondrial membrane fluidity through localizing energy substrate uptake channels and lipid accumulation. Enhanced energy metabolism, in turn, up-regulates the abundance of lipid rafts in tumor cells and stimulates the secretion of immunosuppressive cytokines such as TGF-β. In addition, lipid rafts regulate the release of mitochondrial metabolites (such as reactive oxygen species, lactate) into the tumor microenvironment (TME), further aggravating the acidic and hypoxic state, and inhibiting T cell infiltration and activity. Summary of the Invention

[0003] The object of the present invention is to solve the technical problems of insufficient T cell infiltration and inhibition of cytotoxic T cell function existing in current tumor treatment drugs. In this regard, the present invention provides a complex for enhancing the targeting of anti-tumor drugs, a preparation method thereof, and an application thereof to meet this need in the art.

[0004] On the one hand, the present invention relates to a complex for enhancing the targeting of anti-tumor drugs, which is composed of a carrier, an anti-tumor drug, and a targeting drug, and the targeting drug is tinidazole and methimazole;

[0005] In the complex, based on the amount of the carrier, the proportion of the targeting drug is not less than 3%;

[0006] The carrier is albumin;

[0007] The anti-tumor drug is fluvastatin.

[0008] Furthermore, in the complex for enhancing the targeting of anti-tumor drugs provided by the present invention, the mass ratio of tinidazole to methimazole is 2:1 to 1:2.

[0009] Furthermore, in the complex for enhancing the targeting of anti-tumor drugs provided by the present invention, the complex further includes a gold salt.

[0010] Furthermore, in the complex for enhancing the targeting of anti-tumor drugs provided by the present invention, the gold salt is chloroauric acid.

[0011] Furthermore, in the complex for enhancing the targeting of anti-tumor drugs provided by the present invention, the particle size of the complex is 80 to 120 nm.

[0012] On the other hand, the present invention relates to a preparation method of a complex for enhancing the targeting of anti-tumor drugs. Based on 10 mg of the amount of the carrier, it includes: mixing 0.5 to 2 mg of the anti-tumor drug and 0.3 to 1 mg of the targeting drug in a solvent to obtain a first solution;

[0013] Mixing 10 mg of the carrier and a disulfide bond reducing agent to obtain a second solution;

[0014] After fully mixing the first solution and the second solution, then adding 1 to 10×10 -3 mmol of the gold salt and mixing well.

[0015] Furthermore, in the preparation method of the complex for enhancing the targeting of anti-tumor drugs provided by the present invention, the mass ratio of the carrier to the disulfide bond reducing agent is 10:0.5 to 1.5.

[0016] Furthermore, in the preparation method of the complex for enhancing the targeting of anti-tumor drugs provided by the present invention, the disulfide bond reducing agent is tris(2-carboxyethyl)phosphine hydrochloride.

[0017] On the other hand, the present invention relates to a drug prepared by the described preparation method.

[0018] On the other hand, the present invention relates to the use of the described drug in the preparation of a tumor treatment drug, which supports the survival of T cells by improving the acidic and hypoxic microenvironment at the tumor site and inhibits the efflux of T cell inhibitory signals by blocking lipid raft formation.

[0019] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0020] Based on the pan-cancer data of the Cancer Genome Atlas (TCGA) database, the present invention quantitatively analyzes the hypoxia level of the patient cohort using the Buffa score and finds that the lipid raft characteristics, which are key factors in tumor progression, exhibit a microenvironment impact similar to hypoxia. Further analysis reveals that the co-occurrence characteristics of hypoxia and lipid rafts suggest that a dual-targeting strategy of jointly regulating lipid raft metabolism and mitochondrial respiration can effectively improve the insufficient response of NSCLC immunotherapy by reversing T cell functional defects and cytotoxicity inhibition. In this regard, the present invention constructs a complex that enhances the targeting of anti-tumor drugs. Based on the combination of tinidazole and methimazole, it can simultaneously respond to the two major characteristics of acidity and hypoxia in the tumor microenvironment, has a complementary and synergistic mechanism of action, eliminates the conflict of the mechanism of action of the two drugs or the interference of metabolic pathways with each other, and can achieve better tumor site targeting and tumor cell uptake effects. Experimental results show that when tinidazole and methimazole are co-modified on nanoparticles to form the complex that enhances the targeting of anti-tumor drugs provided by the present invention, under the pH 6.5 combined with hypoxia conditions that simulate the tumor microenvironment, its internalization efficiency in mouse Lewis lung cancer (LLC) cells is significantly higher than that under single acidic or single hypoxia conditions, and it has a stronger cytotoxic effect on LLC cells. On the other hand, to avoid the problem of toxicity superposition caused by the combined use of tinidazole and methimazole with fluvastatin, resulting in treatment interruption or inability to use, this application further verifies that while the drug exhibits excellent therapeutic effects during treatment, it has stable safety characteristics.

[0021] The technical solution provided by the present invention realizes a dual-responsive targeting mechanism for the acidic and hypoxic characteristics of the tumor microenvironment through the combination of tinidazole and methimazole, significantly improving the targeting specificity and efficacy of the anti-tumor drug fluvastatin. Tinidazole can selectively activate its targeting function under hypoxic conditions, while methimazole enhances the enrichment of the drug at the lesion site by responding to the acidic characteristics of the tumor microenvironment. The synergistic effect of the two breaks through the limitations of the single-targeting strategy in the prior art. There is no disclosure in the prior art of a targeting delivery system that combines tinidazole and methimazole for regulating the tumor microenvironment. Their combination not only accurately identifies tumor tissues through a dual-responsive mechanism, but also remodels the immunosuppressive microenvironment and activates T cell function through dual metabolic interventions of inhibiting lipid raft formation and mitochondrial respiration. In addition, this combination disrupts mitochondrial energy metabolism and lipid raft structure simultaneously by reducing the synthesis of coenzyme Q10 and cholesterol, blocking the tumor cell escape signaling pathway. This synergistic effect has not been reported in the prior art, providing more solutions for overcoming the immune therapy drug resistance of cancers such as non-small cell lung cancer. Brief Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0023] Figure 1 Hypoxia and abnormal lipid raft structure synergistically inhibit anti-tumor immunity and predict poor prognosis in lung adenocarcinoma (LUAD) (A) Kaplan-Meier survival curves based on the TCGA-LUAD cohort, showing that the Buffa hypoxia score has prognostic predictive value. (B) Gene set enrichment analysis (GSEA) shows significant activation of the oxidative phosphorylation pathway in the high Buffa hypoxia score group of the TCGA-LUAD cohort. (C) Differences in immune cell infiltration between the hypoxic group and the normoxic group in the TCGA-LUAD cohort. (D) Comparison of immune cell infiltration characteristics between the high / low lipid raft score groups in the TCGA-LUAD cohort. (E) Kaplan-Meier survival curves based on the TCGA-LUAD cohort to verify the prognostic predictive ability of the lipid raft score. (F-G) Compared with the low lipid raft score group, the oxidative phosphorylation (F) and hypoxia signals (G) are significantly activated in the high lipid raft score group in the TCGA-LUAD cohort. (H) Schematic diagram of the action mechanism of Abstatin.

[0024] Figure 2Physicochemical and pharmaceutical properties of Abstatin (A) Schematic diagram of the construction of Abstatin nanon preparation. (B) Morphological characteristics of Abstatin under transmission electron microscope (TEM). (C-D) Hydrodynamic diameter (C) and Zeta potential (D) of Abstatin. (E) Ultraviolet absorption spectra of human serum albumin (HSA, blue line), fluvastatin (FLU, green line), blank carrier (Mock, yellow line) and Abstatin (Abs, red line). (F) Fourier transform infrared (FT-IR) spectra of HSA, FLU, Mock and Abstatin. The characteristic peak of the benzene ring of fluvastatin is at 1400 cm-1, and the characteristic peak of the S-H bond of the Mock carrier is at 2400 cm-1. (G) Uptake efficiency of FITC-labeled Abstatin by LLC cells under different conditions detected by flow cytometry. (H) Detection of the activities of mitochondrial respiratory chain complexes I, II, III, IV and V. (I-J) Confocal fluorescence images (I) and relative fluorescence intensity quantification (J) of Filipin (cholesterol probe) and Alexa Fluor 488-labeled CTX-B (GM1 sphingolipid probe) after LLC cells were treated differently for 24 hours. (K) Representative images of pimonidazole (hypoxia probe) staining of tumor tissues in C57BL / 6 mice. Data are expressed as mean ± standard deviation, and one-way ANOVA was used for comparison between groups; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0025] Figure 3 Antitumor efficacy of Abstatin in an orthotopic mouse model of LUAD (A) Schematic diagram of the construction of the orthotopic mouse model of LUAD and the treatment intervention plan. (B-C) Representative gross photographs (B) and tumor number statistics (C) of the lung tissues of mice in different treatment groups. (D) Trend of body weight changes in each group of mice. (E-F) Representative images (scale bar: 100 μm) of hematoxylin-eosin (H&E) staining (E) and Masson staining (F) of lung tissues in different treatment groups. (G) Evaluation of the liver / kidney toxicity of Abstatin by alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine (CREA) levels. (H-I) H&E staining of liver (H) and kidney (I) tissues (scale bar: 100 μm). Data are expressed as mean ± standard deviation, and one-way ANOVA was used for comparison between groups; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0026] Figure 4Abstatin significantly enhances anti-tumor immunity in vivo by reprogramming hypoxia and lipid metabolism. (A) Hierarchical clustering heatmap of differentially expressed genes (DEGs) showing the gene expression pattern after Abstatin intervention. (B) The top 20 significantly enriched KEGG pathways of DEGs. (C-D) Gene set enrichment analysis (GSEA) shows significant inhibition of hypoxia (C) and oxidative phosphorylation (D) pathways after Abstatin treatment. (E) Hierarchical clustering heatmap of the top 20 significantly downregulated genes in the oxidative phosphorylation pathway. (F) Metabolites significantly downregulated after Abstatin intervention. (G) GSEA analysis indicates that Abstatin significantly inhibits lipid raft-related pathways. (H) Hierarchical clustering heatmap of the top 20 significantly downregulated genes in the membrane raft organization pathway. (I) Heatmap of the immune cell infiltration level in mouse tumor tissues after Abstatin treatment. (J) Abstatin significantly inhibits cell cycle-related biological processes. Data are presented as mean ± standard deviation, and one-way ANOVA was used for intergroup comparison; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0027] Figure 5 Abstatin treatment significantly enhances the efficacy of anti-PD-1 therapy in a humanized PDX model. (A) Schematic diagram of the construction of the humanized PDX model and treatment intervention protocol. (B-C) Gross photographs (B) and body weight change trends (C) of mice in each group (n = 5). (D-F) Gross photographs (D), weights (E), and growth curves (F) of tumors in mice in different treatment groups. (G-H) Representative images of hematoxylin-eosin (H&E) staining (G), Masson staining (H), and TUNEL staining (I) of tumor tissues under confocal laser scanning microscopy (CLSM) (scale bar: 100 μm). Data are presented as mean ± standard deviation, and one-way ANOVA was used for intergroup comparison (**p < 0.01)

[0028] Figure 6Biological safety assessment of Abstatin in the PDX model. (A) Assessment of the hepatotoxicity of Abstatin by the levels of total bilirubin (TBIL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin (ALB) and H&E staining images of liver tissues (scale bar: 50 μm). (B) Representative H&E staining images of the heart tissues of PDX mice (scale bar: 50 μm). (C) Assessment of the nephrotoxicity of Abstatin by the levels of blood urea nitrogen (BUN), creatinine (CREA) and H&E staining images of kidney tissues (scale bar: 50 μm). (D-E) Representative H&E staining images of the spleen (D) and lung (E) tissues of PDX mice (scale bar: 50 μm). (F) Detection of the levels of white blood cells (WBC), granulocytes (Gran), red blood cells (RBC) and platelets (PLT) in the peripheral blood of mice after treatment.

[0029] Figure 7 Abstatin enhances anti-PD-1 efficacy and activates anti-tumor T cell immune responses. (A) The top 20 significantly enriched KEGG pathways of differentially expressed genes (DEGs) after the combination treatment of Abstatin and anti-PD-1. (B-C) The combination treatment significantly promotes tumor cell apoptosis (B) and inhibits the cell cycle-related pathways (C). (D-E) Gene set enrichment analysis (GSEA) and heatmap show that the combination treatment significantly inhibits the biological processes related to lipid rafts (D) and hypoxia (E) in the PDX mouse model. (F-G) The levels of coenzyme Q10 (F) and cholesterol (G) in the tumor tissues of the combination treatment group are significantly decreased. (H) GSEA analysis shows that the combination treatment enhances T cell activation, T cell-mediated cytotoxicity and T cell immune response pathways. (I) Heatmap of the immune cell infiltration levels in the tumor tissues of mice after the combination treatment of Abstatin and anti-PD-1 and anti-PD-1 monotherapy. (J) Flow cytometry shows the proportions of cytotoxic T lymphocytes (CTLs) and regulatory T cells (Tregs) in the tumor tissues of different treatment groups. (K) Quantitative analysis of CD8+ GZMB+ (granzyme B positive) and CD25+ FOXP3+ (FoxP3 positive) cells in the tumor tissues (n = 3). Data are presented as mean ± standard deviation. Student's t-test was used for comparison between two groups, and one-way ANOVA was used for comparison among multiple groups; *p < 0.05; **p < 0.01; ***p < 0.001.

[0030] Figure 8 Ranking of the hypoxia status in the Cancer Genome Atlas (TCGA) pan-cancer cohort. The hypoxia status was evaluated using the Buffa hypoxia score and ranked according to the interquartile range (IQR).

[0031] Figure 9 Computer simulation of the binding conformations of albumin and different statins.

[0032] Figure 10 Particle size distribution of Abstatin solution for transmission electron microscopy (TEM) analysis. The histogram shows the diameter distribution of 99 randomly measured particles by ImageJ, and the Gaussian fitting curve (red) indicates an average diameter of 106.6 nm.

[0033] Figure 11 For stability tests. (A) Colloidal stability of Abstatin in PBS (pH 7.4 and 6.5) containing 20% fetal bovine serum (FBS) detected by DLS. (B) Hemolysis analysis of different concentrations of Abstatin (mean ± standard deviation, n = 3). After standing for 3 hours, the supernatant hemoglobin was observed by centrifugation (the inset is a physical photo). The results showed that the hemolysis rate of each concentration (0.06 - 0.5 mg / ml) was less than 5%.

[0034] Figure 12 For immunofluorescence (IF) detection of the expression of Caveolin-1 in LLC cells after different treatments (scale bar: 20 μm).

[0035] Figure 13 Physical photos of lung tissues of mice with orthotopic models of lung adenocarcinoma (LUAD) after different treatments.

[0036] Figure 14 For the tumor suppression effect of different doses of Abstatin in an orthotopic model of lung adenocarcinoma (LUAD) constructed with LLC-luc cells. (A) Bioluminescence images of mice in different doses of Abstatin treatment groups on days 7, 18, and 28. (B) Physical photos and quantity statistics of lung tumors in each group of mice (mean ± standard deviation). (C) H&E staining images of lung tumors in mice in different doses of Abstatin treatment groups (scale bar: 50 μm).

[0037] Figure 15 For ICP-MS quantification of the distribution of Abstatin in the main organs of mice with orthotopic LUAD models, and organ fluorescence signals at 4, 8, 12, 24, 48, and 72 hours after tail vein injection of Abstatin.

[0038] Figure 16 For the efficacy comparison of Abstatin with the lipid raft inhibitor methyl-β-cyclodextrin (MβCD) and the mitochondrial inhibitor metformin in an orthotopic LUAD model constructed with LLC-luc cells. (A) Bioluminescence images of mice in different treatment groups on days 7, 18, and 28. (B) Physical photos and quantity statistics of lung tumors in each group of mice. (C) Serum levels of IL-4, IL-6, and IFN-γ in each group of mice. Abs: Abstatin; MβCD: methyl-β-cyclodextrin; Met: metformin (data are presented as mean ± standard deviation).

[0039] Figure 17 Efficacy of Abstatin in a mouse model of LUAD bone metastasis. (A) Bioluminescence images of mice in different treatment groups on days 7, 14, and 21. (B) Micro-CT images of the tibia of mice in each group (scale bar: 1 cm).

[0040] Figure 18 Is the plasma concentration-time curve of Abstatin (72 hours). Mice in the LUAD orthotopic model were injected with 2 mg / kg Abstatin via the tail vein, and the data are expressed as mean ± standard deviation.

[0041] Figure 19 Is the biosafety of different doses of Abstatin in the LUAD orthotopic mouse model. (A) Levels of white blood cells (WBC), lymphocytes (Lym), neutrophils (Neu), platelets (PLT), and red blood cells (RBC) in the blood of mice after different treatments. (B) Levels of serum IL-4, IL-6, and IFN-γ in mice in different treatment groups. (C) Hematoxylin-eosin (H&E) staining images of the heart, liver, spleen, and kidney (scale bar: 50 μm). The data are expressed as mean ± standard deviation.

[0042] Figure 20 Is the biosafety of Abstatin with an extended treatment of 1 week in the LUAD orthotopic mouse model. (A) Bioluminescence images of mice in different treatment groups on days 28, 31, and 35. (B) Levels of serum IL-4, IL-6, and IFN-γ in mice in different doses of Abstatin treatment groups. (C) H&E staining images of lung tumors (scale bar: 50 μm). (D) Levels of WBC, Lym, Neu, PLT, and RBC in the blood of mice in different doses of Abstatin treatment groups. (E) H&E staining images of the heart, liver, spleen, and kidney (scale bar: 50 μm). The data are expressed as mean ± standard deviation. Detailed implementation manners

[0043] Next, the technical solutions of the present invention will be described in conjunction with the embodiments. However, the present invention is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified. The %, as used in the following embodiments, is mass percentage unless otherwise specified. The ratios, as used in the following embodiments, are mass ratios unless otherwise specified.

[0044] Example 1

[0045] The present invention relates to the preparation process of Abstatin.

[0046] Human serum albumin (HSA, 10 mg) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 1 mg) were separately dissolved in 1 mL of PBS buffer. Fluvastatin (0.5 - 2 mg), tinidazole, and methimazole were dissolved in DMSO. The mass ratio of tinidazole to methimazole was 2:1 - 1:2, and the total dosage was 0.3 - 1 mg. Subsequently, 500 μL of the HSA solution was mixed with 500 μL of the TCEP solution and sonicated for 10 minutes. 3.5 mL of PBS buffer was added to terminate the reaction. 10 μL of the small molecule mixture containing fluvastatin, tinidazole, and methimazole drugs was added to the solution and sonicated for 20 minutes. Finally, 0.1 - 1 mL of HAuCl4 solution (10 mM) was added and sonicated for 5 minutes.

[0047] Within the above dosage ranges, there were no significant differences in the pharmaceutical properties of the drugs. The preparation process of Abstatin used in the following examples is as follows:

[0048] Human serum albumin (HSA, 10 mg) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 1 mg) were separately dissolved in 1 mL of PBS buffer. Fluvastatin (1 mg), tinidazole (0.2 mg), and methimazole (0.1 mg) were dissolved in DMSO. Subsequently, 500 μL of the HSA solution was mixed with 500 μL of the TCEP solution and sonicated for 10 minutes. 3.5 mL of PBS buffer was added to terminate the reaction. 10 μL of the small molecule mixture was added to the solution and sonicated for 20 minutes. Finally, 0.5 mL of HAuCl4 solution (10 mM) was added and sonicated for 5 minutes.

[0049] Example 2

[0050] This example provides the construction principle of Abstatin.

[0051] The key intersection of hypoxia and lipid raft formation is 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase). As a core component of the mevalonate pathway, this enzyme is responsible for synthesizing cholesterol, an essential substance for lipid raft formation, and coenzyme Q10, a key factor in mitochondrial respiration. In NSCLC, the expression and activity of HMG-CoA reductase are generally upregulated, promoting enhanced cholesterol synthesis and hyperactive mitochondrial function, thereby maintaining tumor growth and shaping an immunosuppressive microenvironment. Statins inhibit HMG-CoA reductase and show potential to sensitize immunotherapy by disrupting lipid raft structure and regulating cholesterol metabolism. However, the dose required to achieve tumor suppression far exceeds the standard for lipid-lowering therapy and may cause serious adverse reactions such as myopathy and rhabdomyolysis. Such risks highlight the necessity of developing targeted delivery strategies that can achieve effective therapeutic concentrations at the tumor site while minimizing systemic toxicity. Abstatin aims to simultaneously target hypoxia and lipid raft formation, overcome NSCLC immune resistance, reshape the tumor microenvironment, and restart T cell anti-tumor function.

[0052] Based on the pan-cancer data of the Cancer Genome Atlas (TCGA) database, this application used the Buffa score to quantitatively analyze the hypoxia levels of patient cohorts. The results showed that lung adenocarcinoma (LUAD), the main subtype of NSCLC, exhibited one of the highest degrees of hypoxia characteristics. In-depth analysis of 498 LUAD patients found that patients in the hypoxia group divided based on the hypoxia score had a significantly lower overall survival rate compared to the normoxia group, along with the activation of the oxidative phosphorylation pathway ( Figure 1 A, Figure 1 B, and Figure 8 ). This phenomenon is consistent with the recently discovered mechanism by which tumor cells promote mitochondrial respiration and improve energy acquisition efficiency through metabolic reprogramming. Notably, the enhanced mitochondrial respiration induced by metabolic reprogramming in LUAD may exacerbate local oxygen depletion. Further analysis showed that the level of immune cell infiltration in hypoxic patients was significantly lower than that in normoxic patients, which partially explained the low response rate of LUAD to immunotherapy ( Figure 1 C).

[0053] When exploring other regulatory mechanisms of the LUAD immune microenvironment, this application found that the lipid raft characteristics, a key factor in tumor progression, showed a similar microenvironment impact to hypoxia. After stratifying LUAD patients based on the lipid raft score, a significant lack of immune cell populations with anti-tumor activity was observed in the high lipid raft score group ( Figure 1 D). Clinical prognosis analysis further confirmed a statistically significant association between an increased lipid raft score and a shortened overall survival of patients ( Figure 1 E). Notably, compared to the low lipid raft score group, patients with a high lipid raft score showed a double enrichment of hypoxia characteristics and the oxidative phosphorylation signaling pathway.Figure 1 F and Figure 1 G), revealing a positive regulatory relationship between hypoxia and lipid raft characteristics. The co-occurrence characteristics of hypoxia and lipid rafts suggest that a dual-targeting strategy of jointly regulating lipid raft metabolism and mitochondrial respiration can effectively improve the insufficient response of NSCLC immunotherapy by reversing T cell function defects and cytotoxicity inhibition.

[0054] The synergistic promotion of immunosuppression by hypoxia and lipid raft formation prompted this application to explore an intervention strategy that simultaneously targets lipid raft metabolism and mitochondrial respiration to effectively activate T cells in the tumor microenvironment. Cholesterol, as the main structural component of lipid rafts, has a key intersection with the key substrate coenzyme Q10 of mitochondrial respiration in the biosynthetic pathway - the enzymatic conversion reaction of HMG-CoA to mevalonic acid. It is worth noting that the HMG-CoA reductase that catalyzes this reaction is the common rate-limiting enzyme of the two synthetic pathways ( Figure 1 H). Statins can exert antitumor effects by inhibiting the activity of HMG-CoA reductase. To overcome the safety problems of statins in tumor treatment, this application was inspired by the albumin-paclitaxel system and developed a novel statin delivery system with albumin as the carrier ( Figure 1 H). Computational screening results showed that fluvastatin has a high binding affinity with albumin and is an ideal candidate drug for albumin-mediated delivery ( Figure 9 ). This strategy can achieve targeted enrichment of more statin molecules in tumor sites while reducing the dosage of albumin.

[0055] The core challenge in the antitumor application of statins lies in the safety risks they pose. This problem stems from the dual regulatory effects of statins on lipid metabolism and mitochondrial respiration, which are crucial for normal cell function. Therefore, improving the tumor-targeting specificity of drugs has become the key breakthrough point. To achieve this goal, this application integrated methimazole and tinidazole (targeting functional modules) into the albumin-bound fluvastatin system to construct an intelligent nanomedicine Abstatin that can respond to the acidic, hypoxic, and reducing characteristics of the tumor microenvironment ( Figure 2 A). Transmission electron microscopy (TEM) and dynamic light scattering (DLS) analyses showed that Abstatin has a stable particle size of approximately 100 nm and a zeta potential of 42.39 mV, which are beneficial for its targeting of tumor tissues and interaction with tumor cells ( Figure 2 B, Figure 2 C, Figure 2 D and Figure 3) To verify the complete assembly of the nanoparticles, blank control particles (mock) without fluvastatin were prepared by the same process in this application. Fourier transform infrared spectroscopy (FT-IR) and ultraviolet spectroscopy (UV) detection confirmed the presence of characteristic peaks of fluvastatin, albumin, and the connection peak of the targeting module, thus confirming the successful synthesis of the nanoformulation ( Figure 2 E and Figure 2 F). In addition, Abstatin exhibits excellent colloidal stability and blood compatibility, enabling it to have high-efficiency tumor targeting ability and maintain long-term therapeutic efficacy ( Figure 11 )

[0056] The Abstatin nanoformulation constructed based on the tumor microenvironment-responsive design can be selectively internalized by tumor cells. The experimental results show that under the combined condition of pH 6.5 and hypoxia simulating the tumor microenvironment, the internalization efficiency of Abstatin in mouse Lewis lung cancer (LLC) cells is significantly higher than that under single acidic or single hypoxia conditions ( Figure 2 G). Further studies have shown that Abstatin has a stronger cytotoxic effect on LLC cells ( Figure 10 ). These findings confirm that Abstatin has excellent pharmacological properties, laying a theoretical foundation for the safe and effective regulation of tumor cell metabolic pathways by fluvastatin.

[0057] Example 3

[0058] This example provides the functional verification of Abstatin.

[0059] To verify whether Abstatin can simultaneously inhibit lipid raft formation and improve the hypoxia state induced by mitochondrial oxidative phosphorylation, LLC cells were treated with Abstatin and unencapsulated fluvastatin respectively. As Figure 2 shown in H, Abstatin can significantly reduce the activities of mitochondrial respiratory chain complexes I and V in cells. In the mitochondrial respiratory chain, complex I serves as the starting site of electron transfer and directly uses coenzyme Q10 as the substrate; while complex V serves as the terminal component of the respiratory chain and is the main site for ATP generation during mitochondrial respiration. The above results indicate that Abstatin inhibits the activity of HMG-CoA reductase through fluvastatin, not only reducing the biosynthesis of coenzyme Q10, but also directly damaging mitochondrial respiratory function and oxygen consumption level. Further in the LUAD in situ mouse model, pimonidazole staining of hypoxic probes in lung tumor tissues confirmed that Abstatin has a significant hypoxia-relieving effect ( Figure 2 K).

[0060] Based on the characteristics that Filipin and cholera toxin B subunit (CTxB) specifically bind to cholesterol and GM1 sphingolipid respectively, these two probes have become the gold standard method for observing cell membrane lipid rafts. Through this technical system, the present application found that both Abstatin and its active ingredient fluvastatin can significantly reduce the lipid raft content in tumor cells, and the inhibitory effect of Abstatin is slightly better than that of free fluvastatin ( Figure 2 I and Figure 2 J). This slight difference may be due to the limitation of the penetration efficiency of free drugs in the complex tissue microenvironment. Further, immunofluorescence confocal imaging with an antibody against caveolin-1, a key component of the lipid raft structure and a core target protein for lipid metabolism homeostasis, showed that Abstatin treatment can significantly reduce the abundance of the lipid raft domain in the tumor cell membrane ( Figure 11 ). Based on the characteristic that cholesterol is a key binding medium for various functional proteins and glycolipids within lipid rafts, the present application proposed a scientific hypothesis: Abstatin disrupts the compositional structure of tumor cell membrane lipid rafts by reducing cholesterol content, ultimately leading to the loss of integrity and a decrease in the abundance of the lipid raft membrane domain.

[0061] Subsequently, an orthotopic mouse model of LUAD was established by tail vein injection of LLC cells to preliminarily evaluate the in vivo function of Abstatin. To verify whether Abstatin can improve the immune microenvironment to enhance the efficacy of anti-PD-1 therapy, the model mice were randomly divided into five groups (n = 5 per group, and the sample size was determined based on the effect size of similar tumor models in pre-experiments and literature reports): control group: intraperitoneal injection of PBS, HSA group: human serum albumin (2 mg / kg), anti-PD-1 monotherapy group: 5 mg / kg, Abstatin monotherapy group: 2 mg / kg, combination therapy group: anti-PD-1 (5 mg / kg) + Abstatin (2 mg / kg) ( Figure 3 A). After continuous administration 9 times, euthanasia was performed to evaluate the lung tumor burden. Compared with the control group and the HSA group, the number of lung tumors in the Abstatin monotherapy group and the combination therapy group was significantly reduced ( Figure 3 B, Figure 3 C and Figure 13 ). The tumor inhibitory effect of the combination therapy group was more significant than that of the anti-PD-1 monotherapy group, and this result was verified by H&E and Masson staining ( Figure 3 E and Figure 3 F).

[0062] To further evaluate the in vivo dose effect of Abstatin, PBS and three gradient doses of Abstatin (2 mg / kg, 8 mg / kg, 16 mg / kg) were injected into the tail vein of orthotopic model mice of LUAD. Under the same treatment regimen, different doses of Abstatin all showed a lung tumor inhibitory effect ( Figure 14) ICP-MS analysis confirmed that Abstatin could effectively infiltrate tumor tissues in vivo ( Figure 15 ), elucidating the material basis for its potent antitumor effect. The above findings systematically demonstrated that Abstatin could stably induce a persistent antitumor effect in vivo.

[0063] To verify the dual mechanism of action of Abstatin, this application comparatively analyzed the therapeutic effects of Abstatin, the lipid raft inhibitor methyl-β-cyclodextrin (MβCD), and the mitochondrial respiration inhibitor metformin. As Figure 16 shown, compared with the treatment groups using single lipid raft or mitochondrial respiration inhibitors alone, the number and volume of lung metastases in the Abstatin treatment group were significantly reduced, suggesting that dual-target inhibition of lipid rafts and mitochondrial respiration by Abstatin could produce a synergistic antitumor effect.

[0064] Furthermore, a mouse model of lung adenocarcinoma metastasis expressing luciferase (LLC-luc cells) was used to evaluate the efficacy of Abstatin. Figure 17 It was shown that high-intensity bioluminescence signals were presented in the hind limb regions of the PBS and HSA control group mice, indicating active tumor metastasis progression; while the tumor-related luminescence signals in the tibia regions of the Abstatin treatment group mice were significantly weakened, and pathological examinations confirmed that the degree of bone destruction induced by tumors in this group was significantly alleviated compared with the control group (extensive osteolytic lesions). The above evidence systematically revealed the potent antitumor effect of Abstatin.

[0065] Abstatin demonstrated stable safety characteristics during the treatment process. Mice receiving single-agent treatment and combination treatment with Abstatin did not show significant weight loss, and their body weight levels were comparable to those of the other three groups ( Figure 3 D). Compared with the control group, there were no significant differences in the levels of the main liver and kidney function physiological indicators - alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA) - in the two treatment groups of mice ( Figure 3 G). Hematoxylin-eosin (HE) staining of liver and kidney tissue sections did not show morphological or structural abnormalities ( Figure 3 H and Figure 3 I). The blood drug concentration of mice in the standard treatment dose group was relatively stable, further corroborating this conclusion ( Figure 18 ). In addition, in the LUAD orthotopic model, high-dose Abstatin in different dose treatment groups did not have an adverse effect on organ structure and did not induce systemic inflammation ( Figure 19 ). Extended observation for 1 week of mice receiving the same treatment regimen showed that the volume of lung tumor lesions did not increase significantly, there were no abnormalities in organ structure, and no evidence of systemic inflammatory response was detected ( Figure 20)。In summary, it is shown that Abstatin is fully non-toxic to major metabolic organs in a mouse model.

[0066] To analyze the specific effects of Abstatin in vivo, the present application performed RNA sequencing analysis on tumor tissues of the Abstatin treatment group and the PBS control group in a LUAD orthotopic mouse model. The results showed that there were significant differences in gene expression between the two groups, especially in the pathways related to mitochondrial respiration and lipid raft-enriched lipid metabolism ( Figure 4 A and Figure 4 B). Gene set enrichment analysis (GSEA) indicated that compared with the control group, Abstatin treatment could synergistically down-regulate the hypoxia signaling pathway and the oxidative phosphorylation pathway, and this finding was consistent with the performance at the cellular level ( Figure 4 C, Figure 4 D and Figure 4 E). Further metabolomics analysis showed that the levels of coenzyme Q10 and NADH, the direct substrates of oxidative phosphorylation, were significantly reduced in tumor tissues ( Figure 4 F). The above results together confirmed that Abstatin effectively alleviated the in vivo hypoxia state by inhibiting tumor mitochondrial respiration function.

[0067] Similarly, metabolite analysis found that after Abstatin treatment, the levels of cholesterol and sphingosine were significantly reduced, and these two components are the basic components of lipid rafts ( Figure 4 F). This result indicated that Abstatin had an inhibitory effect on cholesterol biosynthesis at the tumor site. In addition, compared with the control group, the Abstatin treatment group showed a significant enrichment of inhibitory signals in the processes of lipid metabolism, sphingolipid metabolism, and glycerophospholipid biosynthesis pathways ( Figure 4 G). Due to the reduced synthesis and metabolism of various structural components of lipid rafts, a down-regulation of membrane raft organization was observed ( Figure 4 G and Figure 4 H). In summary, Abstatin achieved lipid raft inhibition by interfering with the synthesis and metabolism of these key components.

[0068] Given the dual harmful effects of hypoxia and lipid raft formation on the survival of T cells in the tumor microenvironment, the present application systematically explored the potential mechanism of Abstatin in regulating the tumor immune landscape. Immune cell infiltration analysis showed that the number of T cells (especially CD8+ T cells) in the tumor tissues of the Abstatin treatment group showed a specific increase ( Figure 4 I). This significant improvement in the immune microenvironment may be the key mechanism for Abstatin to exert its tumor suppressive effect, and this conclusion was supported by the experimental data of tumor cell cycle progression arrest and proliferation inhibition ( Figure 4 J).

[0069] Based on the significant effect of Abstatin in enhancing the immune microenvironment in a mouse model, this application further constructs a human-derived tumor xenograft (PDX) model to evaluate its immunotherapeutic synergy. After transplanting PDX tumors into the axillary region of NOD / SCID mice, immune reconstitution is completed with human peripheral blood mononuclear cells (PBMCs). The experimental groups are divided into: PBS control group, anti-PD-1 monotherapy group (5 mg / kg), Abstatin monotherapy group (2 mg / kg), and Abstatin combined with anti-PD-1 group (same dose), and administered intraperitoneally every 4 days ( Figure 5 A and Figure 5 B). During the 13-day dosing cycle, no abnormal weight loss was observed in the mice of each group ( Figure 5 C). Monitoring of tumor volume and weight showed that the combination treatment group exhibited a more significant tumor control effect compared to the anti-PD-1 monotherapy group, and its inhibition rate was approximately twice that of the monotherapy group ( Figure 5 D, Figure 5 E and Figure 5 F). The results of H&E and Masson staining further confirmed that the pathological damage of the tumor tissue in the combination treatment group was the most significant ( Figure 5 G-H). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay showed that the number of apoptotic cells in the tumor tissue of the combination treatment group was significantly increased compared to the anti-PD-1 monotherapy group ( Figure 5 I). The above findings jointly verified the synergistic efficacy of Abstatin monotherapy and combination treatment regimens in inhibiting tumor progression.

[0070] Both Abstatin monotherapy and its combination treatment with anti-PD-1 showed good safety profiles. The levels of liver function indicators - aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin (ALB), and total bilirubin (TBIL) - in the treatment group mice were not significantly different from those in the control group. Combining with the H&E staining results of the liver tissues of each group of mice, it indicated that Abstatin treatment did not induce hepatotoxicity ( Figure 6 A). In addition, the H&E staining of kidney tissues and renal function indicators (creatinine CREA, blood urea nitrogen BUN) were within the normal range, suggesting normal renal function ( Figure 6 C). Pathological examinations of the heart, spleen, and lung tissues showed that no structural changes occurred in the mice of each group ( Figure 6 B, Figure 6 D and Figure 6 F), and there were also no significant fluctuations in blood routine parameters ( Figure 12 ). These findings fully demonstrated that Abstatin has excellent safety profiles at therapeutic doses.

[0071] To analyze the underlying mechanism by which the combination therapy of Abstatin and anti-PD-1 inhibits tumor progression in the LUAD humanized PDX model, this application conducted a systematic analysis. RNA sequencing results showed that the combination therapy had a significant regulatory effect on the signaling pathways related to metabolic reprogramming and energy acquisition ( Figure 7 A). Further studies showed that the combination therapy regimen synergistically enhanced the anti-tumor efficacy by enhancing tumor cell apoptosis and arresting the cell cycle progression ( Figure 7 B and Figure 7 C). Notably, compared with the control group, the tumor tissues in the combination therapy group still showed enrichment of plasma membrane raft signals and hypoxia characteristics ( Figure 7 D and Figure 7 E). Metabolomic analysis showed that the levels of coenzyme Q10 and cholesterol in the combination therapy group were significantly reduced ( Figure 7 F and Figure 7 G). Combining the above results, it was confirmed that Abstatin effectively reprogrammed mitochondrial respiration and lipid raft formation, thus creating favorable conditions for activating anti-tumor immunity by anti-PD-1 therapy.

[0072] When analyzing the effects of the tumor tissues in the combination therapy group, this application found that in the mice receiving the combination therapy of Abstatin and anti-PD-1, the signaling pathways related to T cell-mediated anti-tumor immunity were significantly upregulated, including T cell activation, T cell-mediated cytotoxicity, and T cell immune response pathways ( Figure 7 H). Therefore, this application further analyzed and compared the immune cell infiltration characteristics between the combination therapy group and the anti-PD-1 monotherapy group, and observed an increase in the proportion of various immune-promoting cells including CD8+ T cells in the combination therapy group ( Figure 7 I). To verify this immune enhancement effect, this application performed flow cytometry analysis on the tumor tissues of all groups. The results showed that the proportion of regulatory T cells (Tregs) with immunosuppressive properties in the tumor tissues of the combination therapy group was reduced, while the proportion of cytotoxic T cells was significantly increased, and this effect was particularly obvious in the anti-PD-1 monotherapy group ( Figure 7 J and Figure 7 K). Taken together, these findings indicate that Abstatin enhances the sensitivity of LUAD tumors to anti-PD-1 therapy by improving the immunosuppressive microenvironment.

[0073] As one of the cancer types with the lowest response rate to immunotherapy represented by anti-PD-1, improving the immunosuppressive microenvironment of non-small cell lung cancer (NSCLC) has become a key challenge. A large number of studies have shown that compared with other cancer types, the degree of hypoxia in the NSCLC tumor microenvironment is more significant. This hypoxic state may lead to T cell inactivation, thereby promoting immune escape. In this application, the lung adenocarcinoma (LUAD) cohort in the TCGA database was selected for analysis because it has the highest incidence and the worst prognosis in NSCLC. The analysis showed that in the tumor tissues of LUAD patients with significant hypoxic characteristics, the oxidative phosphorylation pathway, the core process of mitochondrial respiration, was significantly upregulated, accompanied by a severe deficiency in the infiltration of immune cells at the tumor site. As the core organ for gas exchange in the body, the lung tissue itself has relatively high mitochondrial activity, and the energy acquisition of lung tumors is more inclined to rely on oxidative phosphorylation. This upregulation of oxidative phosphorylation driven by metabolic reprogramming may be an important reason for the severe hypoxia in the LUAD microenvironment. However, treatment methods such as direct oxygen supply aimed at alleviating hypoxia are not only difficult to maintain long-term but may also inadvertently promote tumor progression. Therefore, there is an urgent need to find alternative therapeutic targets that can synergistically inhibit hypoxia and jointly regulate T cell function.

[0074] This application shows that the formation of lipid rafts in tumors not only affects the prognosis of patients as previously reported but also shows a significant upregulation of both oxidative phosphorylation and hypoxia signals in the population with high lipid raft scores. There is a subtle and significant positive correlation between these two factors, which together lead to the inhibition of the infiltration and activation of immune cells (especially T cells) at the tumor site. In addition, numerous previous studies have elucidated a significant interaction between mitochondrial respiration and lipid raft formation, especially in biological processes such as integrating signal transduction, metabolic reprogramming, and exosome communication. These interactions synergistically regulate the secretion of tumor-promoting or immunosuppressive cytokines and chemokines by tumor cells, impair T cell function, and establish a feedback loop that promotes the development of an immunosuppressive microenvironment. Combining the data of this application, this evidence indicates that combined targeting of mitochondrial respiration inhibition and lipid raft disruption can be a strategic approach to alleviating the immunosuppressive tumor microenvironment (TME). Based on this, this application developed the albumin-bound statin Abstatin, which achieves a synergistic enhancement of the anti-tumor immune response by simultaneously inhibiting mitochondrial respiration and disrupting lipid raft structure. Specifically, this drug improves the acidic hypoxic microenvironment at the tumor site to support T cell survival, while inhibiting lipid raft formation to block the efflux of T cell inhibitory signals. This dual mechanism of action aims to simultaneously increase the infiltration level and cytotoxic function of T cells at the LUAD tumor site, and the experimental data of the mouse model confirmed the significant enhancement effect of this strategy on the anti-tumor immune response.

[0075] In recent years, although progress has been made in the drug development for the immunoresistance of non-small cell lung cancer (NSCLC), its clinical application still faces three key challenges: (1) the long-term efficacy is limited due to a single target; (2) the combination therapy of multiple targets requires the combination of multiple components, which is likely to increase the metabolic burden and the complexity of the medication regimen; (3) the tumor targeting is insufficient, and increasing the dose to ensure the efficacy is likely to cause systemic toxicity. To address the above problems, this application locks in two key processes that have a co-directional regulatory effect on T cell function based on mechanism analysis - mitochondrial respiration and lipid raft formation, and discovers that the two converge in the enzymatic reaction catalyzed by HMG-CoA reductase. Based on this, this application develops Abstatin with fluvastatin as the core component, which can simultaneously inhibit the key substrates required for mitochondrial respiration and lipid raft formation. In addition, Abstatin achieves precise targeting through tumor microenvironment-responsive design, forming a differential distribution with normal tissues. Studies on the LUAD in situ model and patient-derived xenograft (PDX) model show that monotherapy with Abstatin or combination therapy with anti-PD-1 can significantly improve the sensitivity of LUAD to immunotherapy, and it has excellent safety characteristics. The strategy proposed in this application undoubtedly provides the translational potential of multi-target synergistic intervention for immunotherapy sensitization. Generally speaking, the innovative strategy developed in this application not only provides more paradigms for the functional remodeling of existing drugs, but can also be used as an adjuvant treatment method to significantly improve the clinical efficacy of existing anti-tumor regimens through multi-mechanism synergy.

[0076] As described above, the basic principles, main features and advantages of the present invention are preferably described. The above embodiments and the description are only for describing the preferred embodiments of the present invention. The present invention is not limited by the above embodiments. Without departing from the spirit and scope of the present invention, various changes and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the present invention.

Claims

1. A composite for enhancing the targeting of an anti-tumor drug, the composite consisting of a carrier, an anti-tumor drug and a targeting drug, characterized in that: The targeted drugs are tinidazole and methimazole; In the complex, the targeted drug accounts for no less than 3% based on the amount of carrier used; The carrier is albumin; The anti-tumor drug is fluvastatin.

2. The composite for enhancing the targeting of anti-tumor drugs according to claim 1, characterized in that: The mass ratio of metronidazole to methimazole is 2:1 to 1:

2.

3. The composite for enhancing the targeting of anti-tumor drugs according to claim 1, characterized in that: The complex also includes a gold salt.

4. The composite for enhancing the targeting of anti-tumor drugs according to claim 3, characterized in that: The gold salt is chloroauric acid.

5. The composite for enhancing the targeting of anti-tumor drugs according to claim 4, characterized in that: The particle size of the composite is 80-120 nm.

6. A method for preparing a composite for enhancing the targeting of an anti-tumor drug according to any one of claims 1 to 5, characterized in that: Taking the dosage of the carrier as 10 mg, the method comprises: mixing 0.5 to 2 mg of the anti-tumor drug and 0.3 to 1 mg of the targeted drug in a solvent to obtain a first solution; Mixing 10 mg of the carrier and a disulfide bond reducing agent to obtain a second solution; After the first solution and the second solution are fully mixed, 1 to 10×10 -3 mmol gold salt is thoroughly mixed.

7. The method for preparing the composite for enhancing the targeting of anti-tumor drugs according to claim 6, characterized in that: The mass ratio of the carrier to the disulfide bond reducing agent is 10:0.5-1.

5.

8. The method for preparing the composite for enhancing the targeting of anti-tumor drugs according to claim 7, characterized in that: The disulfide bond reducing agent is tris(2-carboxyethyl)phosphine hydrochloride.

9. A drug, characterized in that The method is prepared by the method according to any one of claims 6 to 8.

10. Use of the drug according to claim 9 in the preparation of a drug for treating tumors, characterized in that: The tumor therapeutic drug improves the acidic hypoxic microenvironment of the tumor site to support T cell survival, while inhibiting lipid raft formation to block the outflow of T cell inhibitory signals.

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