A targeted drug for triple-negative breast cancer and its preparation and application

Abstract

The scheme discloses a triple-negative breast cancer targeting drug and preparation and application, proposes a combination drug of paclitaxel and celecoxib, and uses a polypeptide modified liposome to deliver the combination drug, realizes target co-delivery of the combination drug, and improves the effectiveness of ICD and the rejection of the anti-tumor immune response in triple-negative breast cancer through the joint action of paclitaxel and celecoxib in a reasonable proportion, and the experiment proves that the combination drug has good therapeutic effect and recurrence and metastasis inhibition effect.

Description

Technical Field

[0001] This invention belongs to the field of medicine, and in particular relates to a targeted drug for triple-negative breast cancer and its preparation and application. Background Technology

[0002] Triple-negative breast cancer (TNC), accounting for 15% to 20% of all breast cancers, is a malignant tumor lacking effective targeted therapy. It is typically highly aggressive and carries a high risk of recurrence and metastasis. Currently, chemotherapy is the primary systemic treatment for TNC, but patient prognosis is not ideal, with a median overall survival of only 13.3 months. While immunotherapy has shown efficacy in some tumor types, its effectiveness in TNC is not prominent. Immune checkpoint inhibitors do not offer higher overall survival rates than chemotherapy in TNC patients because the “cold” tumor microenvironment (TME) associated with TNC hinders immunotherapy. Therefore, strategies that can transform the “cold” TME into an immune “hot” TME may hold significant therapeutic potential for TNC.

[0003] Recently, a strategy of "inducing immunogenic cell death (ICD)" has been proposed to "activate" cold tumors. ICD is a specific form of apoptosis that enables the body to trigger a specific immune response against tumor cells. This response depends on the expression and release of various immunostimulatory damage-associated molecular patterns (DAMPs), including adenosine triphosphate (ATP), calreticulin (CRT), high-mobility group box 1 (HMGB1), and heat shock protein 70 (HSP70). These DAMPs activate dendritic cells (DCs), promoting their maturation and migration to lymph nodes. Mature DCs can more effectively present tumor antigens, thereby activating T cells and promoting a T-cell-dependent anti-tumor immune response. Recent studies have shown that chemotherapy drugs such as doxorubicin, paclitaxel (PTX), and oxaliplatin can induce ICD, thereby activating adaptive anti-tumor immunity. However, in the case of triple-negative breast cancer, the immunogenicity of the tumor cells killed by these chemotherapy drugs is often insufficient to trigger a strong anti-tumor immune response, leading to frequent tumor recurrence and less than ideal efficacy. In addition, chemotherapy-induced cell death can also trigger the massive release of immunosuppressive molecules (such as prostaglandin E2, PGE2), which can hinder the maturation of dendritic cells, weaken the immunogenicity of dying tumor cells, thereby affecting the effect of PTX-induced ICD and weakening the immune response to tumors. Summary of the Invention

[0004] The purpose of this invention is to address the above-mentioned problems by providing a targeted drug for triple-negative breast cancer, its preparation and application. It proposes a combination drug of paclitaxel and celecoxib, using peptide-modified liposomes to deliver the combination drug, achieving targeted co-delivery. Furthermore, the combined effect of a reasonable ratio of paclitaxel and celecoxib enhances the effectiveness of ICD in triple-negative breast cancer and reduces anti-tumor immune rejection, resulting in good therapeutic effects and inhibition of recurrence and metastasis.

[0005] A targeted drug for triple-negative breast cancer includes paclitaxel, celecoxib, and liposomes modified with a cyclic arginine-glycine-aspartic acid polypeptide, wherein paclitaxel and celecoxib are mixed in a molar ratio of 1:(1-30) and encapsulated by the liposomes to constitute a targeted drug for triple-negative breast cancer.

[0006] In the aforementioned targeted drugs for triple-negative breast cancer, paclitaxel and celecoxib are mixed in a molar ratio of 1:(2-20).

[0007] In the aforementioned targeted drugs for triple-negative breast cancer, paclitaxel and celecoxib are mixed in a molar ratio of 1:(2-10).

[0008] In the aforementioned targeted drugs for triple-negative breast cancer, paclitaxel and celecoxib are mixed in a molar ratio of 1:(4.4-5), such as 1:4.4, 1:5, etc.

[0009] In the aforementioned targeted drugs for triple-negative breast cancer, the liposomes selectively target tumor sites through specific interactions with integrins overexpressed in triple-negative breast cancer.

[0010] Among the aforementioned targeted drugs for triple-negative breast cancer, the mechanism of action involves liposome delivery of the drug to the tumor site. Paclitaxel induces immunogenic cell death in tumor cells, while celecoxib promotes dendritic cell maturation and antigen presentation by inhibiting the release of immunosuppressive molecules induced by paclitaxel, thereby activating anti-tumor immunity. Mature dendritic cells (DCs) can more effectively present tumor antigens, activating T cell-mediated anti-tumor immune responses.

[0011] Among the aforementioned targeted therapies for triple-negative breast cancer, the loadings of paclitaxel and celecoxib were 2.5% ± 0.5% and 5.6% ± 0.5%, respectively.

[0012] A method for preparing a targeted drug for triple-negative breast cancer includes the following components: soybean lecithin, cholesterol, phospholipid polyethylene glycol, palmitic acid modified with cyclic arginine-glycine-aspartic acid polypeptide (tumor-targeting ligand), paclitaxel, and celecoxib.

[0013] The mass fraction ratio of soybean lecithin, cholesterol, phospholipid polyethylene glycol, tumor-targeting ligand, paclitaxel and celecoxib is (50-70):(10-30):(10-30):(5-15):(1-4):(2-20);

[0014] Dissolve each component in chloroform and add it to a round-bottom flask;

[0015] Thin films are formed by rotary evaporation using a rotary evaporator;

[0016] Add an appropriate amount of deionized water and sonicate the mixture in a water bath at a set temperature for a set time.

[0017] The resulting solution is further processed through a liposome extruder with a set pore size.

[0018] In the above-mentioned methods for preparing targeted drugs for triple-negative breast cancer,

[0019] The set temperature is 0℃-6℃;

[0020] The set time is 10-30 minutes;

[0021] The specified aperture is 50-200 nanometers.

[0022] In the above-mentioned method for preparing targeted drugs for triple-negative breast cancer, the mass fraction ratio of soybean lecithin, cholesterol, phospholipid polyethylene glycol, tumor-targeting ligand, paclitaxel and celecoxib is 60:20:20:10:(2-3):(5-15), and the mass ratio of paclitaxel to celecoxib is 1:(2.2-2.5).

[0023] In the above-mentioned method for preparing targeted drugs for triple-negative breast cancer, the mass fraction ratio of soybean lecithin, cholesterol, phospholipid polyethylene glycol, tumor-targeting ligand, paclitaxel, and celecoxib is 60:20:20:10:2.86:7.14.

[0024] The set temperature is 4℃;

[0025] The set time is 20 minutes;

[0026] The specified aperture is 100 nanometers.

[0027] Application of the above-mentioned triple-negative breast cancer targeted drug in the preparation of a drug for the treatment of triple-negative breast cancer.

[0028] The application of the above-mentioned targeted drug or the targeted drug prepared by the above-mentioned preparation method in the preparation of a triple-negative breast cancer in situ tumor vaccine.

[0029] The advantages of this invention are:

[0030] This protocol combines paclitaxel and celecoxib in a specific ratio to form a drug combination, and develops liposomes modified with cyclic arginine-glycine-aspartic acid peptides. Using the provided preparation method, these liposomes are used to load the drug combination, thereby obtaining a targeted drug for treating triple-negative breast cancer. This targeted drug exhibits good therapeutic efficacy and can address the current lack of targeted therapies for triple-negative breast cancer, its poor treatment outcomes, and high recurrence rate after treatment.

[0031] This targeted drug significantly improves drug accumulation at the tumor site and has been demonstrated in trials to trigger a strong anti-tumor immune response in an orthotopic mouse model of triple-negative breast cancer, potentially leading to a new breakthrough in the treatment of triple-negative breast cancer. Attached Figure Description

[0032] Figure 1 This is a diagram illustrating the mechanism of action of the triple-negative breast cancer targeted drug of this invention;

[0033] Figure 2 This is a schematic diagram of the preparation process, physicochemical characterization, and tumor targeting experimental verification of the triple-negative breast cancer targeted drug of the present invention;

[0034] Figure 3 This is an experimental process and results diagram of the targeted drug for triple-negative breast cancer of this invention activating anti-tumor immunity and inhibiting the development of triple-negative breast cancer;

[0035] Figure 4 The experimental process and results of this invention's targeted drug for triple-negative breast cancer as an in situ tumor vaccine inhibiting the recurrence and metastasis of triple-negative breast cancer are shown in the figure.

[0036] Figure 5 This is a graph showing the results of a study on the effect of different concentrations of PTX and the ratio of CXB to PTX on the efficacy of the triple-negative breast cancer targeted drug of this invention.

[0037] Figure 6 This invention relates to the detection of tumor apoptosis in 4T1 cells after treatment with different drug groups, including (A) flow cytometry analysis of apoptotic cells in different groups; and (B) quantitative analysis of the proportion of apoptotic cells.

[0038] Figure 7 These are the H&E (hematoxylin-eosin) staining and Ki-67 staining results of tumor samples after treatment with different drug groups in this embodiment of the invention.

[0039] Figure 8 These are the flow cytometry results of immune cells after treatment with different drug groups in the antitumor immune response test of this invention: (A) the proportion of mature dendritic cells in the tumor; (B) the proportion of effector T cells in the tumor; and (C) the proportion of effector memory T cells in the spleen.

[0040] Figure 9 The diagram shows the biosafety assessment results of the triple-negative breast cancer targeted drug in the embodiments of the present invention. (A) Blood biochemical analysis of different groups (n=4); (B) H&E staining of ex vivo organs (heart, liver, spleen, lung and kidney) of different groups. Detailed Implementation

[0041] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0042] like Figure 1 As shown, this scheme provides a targeted drug for triple-negative breast cancer. This drug mainly comprises three components: paclitaxel (PTX), celecoxib (CXB), and liposomes modified with a cyclic arginine-glycine-aspartic acid peptide. Paclitaxel (PTX) and celecoxib (CXB) are combined in a molar ratio of 1:(1-30) and encapsulated in liposomes to constitute a targeted drug for treating triple-negative breast cancer. The molar ratios can be 1:2, 1:5, 1:10, etc. In this embodiment, a 1:5 ratio is preferred, i.e., a 1:2.5 mass ratio. Furthermore, when co-encapsulated in a 1:5 molar ratio, the final drug loadings of PTX and CXB are preferably 2.5% and 5.6%, respectively.

[0043] like Figure 2 As shown, the preparation method of the aforementioned targeted drug is as follows:

[0044] The components include: soybean lecithin (Phosphol ipid), cholesterol (Cholesterol), phospholipid polyethylene glycol (DSPE-PEG), palmitic acid C16-cRGD modified with cyclic arginine-glycine-aspartic acid peptide as a tumor-targeting ligand, paclitaxel (PTX), and celecoxib (CXB).

[0045] Dissolve all components in chloroform and add to a round-bottom flask.

[0046] Subsequently, a thin film was formed by rotary evaporation using a rotary evaporator.

[0047] Next, add an appropriate amount of deionized water and sonicate the mixture in a temperature-controlled water bath at a set temperature for a set time. Deionized water is not a key component, and its specific amount is determined by those skilled in the art based on the needs, such as the amount to be prepared. There are no specific restrictions here, as long as it can be used to prepare the required targeted drug.

[0048] Finally, the resulting solution is further processed through a liposome extruder with a set pore size to obtain the targeted drug.

[0049] The mass fraction ratio of soybean lecithin, cholesterol, phospholipid polyethylene glycol, tumor-targeting ligand, paclitaxel, and celecoxib is (50-70):(10-30):(10-30):(5-15):(1-4):(2-20). In various experiments conducted on this scheme, the mass fractions of soybean lecithin, cholesterol, phospholipid polyethylene glycol, tumor-targeting ligand, paclitaxel, and celecoxib in the prepared targeted drug are 60:20:20:10:2.86:7.14, respectively. The set temperature during the preparation process is 4℃, the set time is 20 minutes, and the set pore size is 100 nanometers.

[0050] The targeted drug synthesized using the above method and proportions exhibits a uniform spherical morphology, as shown in the transmission electron microscope (TEM) image. Figure 2 B). The hydration diameter and Zeta potential of the liposomes were 65.8 ± 6.1 nm and -28.5 ± 0.5 mV, respectively. Figure 2 C and D). The stability of PTX / CXB@Lip-cRGD (a combination drug of paclitaxel (PTX) and celecoxib (CXB) encapsulated in liposomes modified with cRGD (i.e., a targeted drug prepared by the above method)) was tested in DMEM culture medium containing 10% fetal bovine serum. No significant change in particle size was observed over 72 hours. Figure 2 E). Therefore, the targeted drug prepared by the above method and proportions meets the stability requirements.

[0051] In addition, to observe the biodistribution of the drug in vivo, the near-infrared fluorescent dye IR780 was used to replace the CXB encapsulated in the liposomes in the experiment. PTX / IR780@Lip (a combination of PTX and IR780 encapsulated in ordinary liposomes) or PTX / IR780@Lip-cRGD (a combination of PTX and IR780 encapsulated in cRGD liposomes) were injected via the tail vein into 4T1 orthotopic tumor model mice, and imaging was performed using a small animal in vivo imaging system. Figure 2 F and Figure 2 As shown in G, the fluorescence intensity at the tumor site in the PTX / IR780@Lip-cRGD group gradually increased, reaching a peak at 24 hours post-injection, approximately twice that of the PTX / IR780@Lip group. Furthermore, consistent results were obtained from in vitro fluorescence imaging of major organs and tumors in mice sacrificed 48 hours post-injection. Figure 2 (H and I). These results demonstrate the tumor-targeting ability of the prepared targeted drug.

[0052] Figure 2In the diagram, (A) is a flowchart of the preparation of PTX / CXB@Lip-cRGD using rotary evaporation; (B) is a transmission electron microscope (TEM) image of PTX / CXB@Lip-cRGD, scale bar = 100 nm; (C) is the hydration diameter of different drug groups (n = 3); (D) is the zeta potential of different drug groups (n = 3); (E) is the hydration diameter of PTX / CXB@Lip-cRGD in DMEM medium containing 10% fetal bovine serum. Stability test of D, lasting 72 hours (n=3); (F) in vivo fluorescence imaging of 4T1 tumor-bearing mice after injection of PTX / IR780@Lip or PTX / IR780@Lip-cRGD, and (G) quantitative analysis of fluorescence intensity in tumor tissue at 24 hours (n=3); (H) ex vivo fluorescence imaging of major organs and tumor tissue of mice 48 hours after injection, and (I) quantitative analysis of fluorescence intensity (n=3), *p<0.05.

[0053] The mechanism of action of the targeted drugs provided in this protocol is as follows: liposomes modified with cRGD selectively target tumor tissue through specific interactions with integrins overexpressed in triple-negative breast cancer. This allows the encapsulated paclitaxel (PTX) and celecoxib (CXB) to accumulate in triple-negative breast cancer tumor cells, triggering a strong anti-tumor immune response. After the drugs are targeted to the tumor site using liposomes, PTX induces immunogenic cell death in tumor cells, while CXB promotes the maturation of dendritic cells (DCs) by inhibiting the release of PTX-induced immunosuppressive molecules. Mature DCs can more effectively present tumor antigens and activate T cells.

[0054] To verify the effectiveness and practicality of the targeted drugs provided in this protocol in the treatment of triple-negative breast cancer and as an in situ vaccine for triple-negative breast cancer, a series of experiments were conducted using 4T1 cells (mouse triple-negative breast cancer cells). The experiment involved preparing 4T1 cells, a number of mice, and multiple groups of the test drugs.

[0055] The PTX / CXB@Lip-cRGD group uses cRGD-modified liposome-encapsulated paclitaxel (PTX) and celecoxib (CXB) combination drugs (i.e. targeted drugs prepared by the above preparation method);

[0056] PBS group, control group;

[0057] Free PTX / CXB group: PTX and CXB combination drugs not encapsulated by liposomes;

[0058] The PTX / CXB@Lip group consists of a combination of PTX and CXB drugs encapsulated in conventional liposomes.

[0059] CXB@Lip-cRGD group, CXB encapsulated in cRGD liposomes;

[0060] PTX@Lip-cRGD group, PTX encapsulated in cRGD liposomes.

[0061] The above-mentioned experimental drugs meet the principle of controlling variables. For example, in the PTX / CXB@Lip-cRGD group and the PTX / CXB group, each drug has the same amount of PTX and CXB; in the CXB@Lip-cRGD group and the PTX@Lip-cRGD group, each drug has the same amount of CXB and PTX as the PTX / CXB@Lip-cRGD group, and the preparation methods are similar, etc.

[0062] Figure 3 An experiment was conducted to investigate the activation of anti-tumor immunity and inhibition of triple-negative breast cancer development by the targeted drugs provided in this protocol. (A) is a schematic diagram of the treatment process; (B) is a photograph of the tumors in 4T1 tumor-bearing mice (n=4) treated with different drug groups on day 21; (C) is the tumor growth inhibition curve of mice after treatment with different drug groups (n=4); (D) is the PGE2 level in mouse tumor tissue after treatment with different drug groups (n=3). (EG) is a flow cytometry analysis of mouse immune cells after treatment with different drug groups; (E) is the proportion of mature DCs in tumor tissue; (F) is the CD8+ level in tumor tissue. + T cell proportion; (G) is the proportion of effector memory T cells in the spleen (n=3); (HJ) are the levels of cytokines in the tumor after treatment with different drug groups: (H) TNF-α, (I) IFN-γ, (J) IL-10, *p<0.05, **p<0.01, ns indicates no significant difference.

[0063] In this experiment, mice were divided into six groups (PBS group, Free PTX / CXB group, CXB@Lip-cRGD group, PTX@Lip-cRGD group, PTX / CXB@Lip group, and PTX / CXB@Lip-cRGD group), and received the designated treatment every four days after the initial treatment, for a total of five treatments. Figure 3 A). For example Figure 3 B and Figure 3 As shown in Figure C, the PTX / CXB@Lip-cRGD group exhibited the highest tumor growth inhibition rate, reaching 81.1% at 21 days post-treatment. The Free PTX / CXB group and the PTX / CXB@Lip group showed only moderate inhibitory effects, demonstrating the necessity and effectiveness of targeted ligand modification.

[0064] Compared with the PTX@Lip-cRGD group, the PTX / CXB@Lip-cRGD group showed a 21% increase in tumor growth inhibition rate, demonstrating the effectiveness of the combination drug. This is because CXB inhibited PGE2 synthesis, as indicated by the decrease in intratumoral PGE2 levels. Figure 3 D), while the inhibition of PGE2 can promote the maturation of dendritic cells (DCs), thereby enhancing the induction of ICD and improving the efficiency of tumor suppression.

[0065] Furthermore, histological analysis using H&E and Ki-67 staining also confirmed the antitumor effect of PTX / CXB@Lip-cRGD. Figure 7 ).

[0066] To verify the induction of ICD after treatment with different drug groups, this embodiment further investigated the immunological characteristics, including DC maturation and T cell activation. Figure 3 EG showed changes in the proportion of mature DCs and CD8+ T cells, as well as changes in the levels of related immunostimulatory cytokines TNF-α and IFN-γ. It can be seen that, compared with other groups, the proportion of mature DCs in tumors treated with PTX / CXB@Lip-cRGD was significantly higher. Figure 3 E and Figure 8 A). Similarly, the PTX / CXB@Lip-cRGD group showed an increase in CD8+ T cells at the tumor site ( Figure 3 F and Figure 8 B) and effector memory T cells (CD44+CD62L-) in the spleen Figure 3 G and Figure 8 The population with C) showed a significant increase. Furthermore, PTX / CXB@Lip-cRGD treatment led to increased levels of immunostimulatory cytokines (including TNF-α and IFN-γ) and decreased levels of the immunosuppressive cytokine IL-10. Figure 3 These results all indicate that the targeted drug provided by this protocol can activate a strong anti-tumor immune response, demonstrating the effectiveness and practicality of this targeted drug in stimulating anti-tumor immunity to inhibit the development of triple-negative breast cancer.

[0067] Furthermore, Figure 4 A trial was conducted to investigate the efficacy of the aforementioned targeted drugs as an in situ vaccine in inhibiting recurrence and lung metastasis in triple-negative breast cancer. Figure 3 This experiment was conducted based on previous studies activating anti-tumor immunity and inhibiting the development of triple-negative breast cancer. The primary tumor was removed 15 days after treatment, and 4T1 tumor cells were inoculated into the contralateral breast fat pad on day 36. Tumor cell development was monitored until day 50. Figure 4 A). Figure 4In the diagram, (A) is a schematic diagram of the experiment verifying the distal immune effect induced by PTX / CXB@Lip-cRGD. (B) is a curve showing the tumor-free incidence rate in mice after treatment with different drug groups (n=10). (C) is a representative photograph and histological analysis of lung tumor metastasis in mice after treatment with different drug groups; the dashed circles represent tumor metastasis. (D) is a statistical analysis of lung tumor nodules (n=10). (E) is a schematic diagram of the anti-tumor immune process induced by PTX / CXB@Lip-cRGD. After administration, PTX / CXB@Lip-cRGD selectively targets the tumor site through the specific interaction between cRGD and the overexpressed integrins in triple-negative breast cancer. Once it reaches the tumor site, PTX can induce ICD in tumor cells, while CXB promotes the maturation of DCs by inhibiting PTX-induced PGE2 release. This process promotes the activation of effector and memory T lymphocytes, effectively inhibiting the development of triple-negative breast cancer. At the same time, as an in situ vaccine strategy, it inhibits the formation of distant tumors and lung metastasis, **p<0.01.

[0068] The above experimental results show that the tumor recurrence rate and the number of lung metastatic nodules in the PTX / CXB@Lip-cRGD group were significantly lower than those in other groups. In the PBS group, tumors began to appear approximately one week after re-inoculation, showing an incidence rate of 100%. Figure 4 B). The tumor incidence rate in the PTX / CXB@Lip-cRGD group was significantly reduced to 50%, much lower than the 90% observed in the PTX@Lip-cRGD group. This result indicates that the combination drug elicited a strong immune memory response to tumor recurrence.

[0069] In addition, such as Figure 4 C and Figure 4 As shown in Figure D, the average number of lung metastatic nodules in the PTX / CXB@Lip-cRGD group was 2.2, while the average number in the PBS group and the PTX@Lip-cRGD group was 10.6 and 6.2, respectively. By assessing the number of metastatic nodules in lung tissue, it can be concluded that the anti-metastatic effect of PTX / CXB@Lip-cRGD is also very prominent.

[0070] In summary, this protocol utilizes the provided preparation method to prepare a cRGD-modified liposome encapsulating a specific ratio of PTX and CXB drugs, which can serve as a targeted drug for the treatment of triple-negative breast cancer. This targeted drug can effectively accumulate in triple-negative breast cancer cells to induce ICD, promote DC maturation through the release of DAMPs and the blocking of the immunosuppressive molecule PGE2 production. This mechanism not only activates effector T cells but also memory T cells, ultimately inhibiting tumor recurrence and metastasis. Figure 4 E).

[0071] In addition, in vivo biocompatibility analysis was performed in this embodiment. Healthy BALB / c mice (n=4) were treated with PBS or PTX / CB@Lip-cRGD every four days for a total of four times (PTX dose was 10 mg / kg, CXB dose was 25 mg / kg). On day 21, blood samples were collected for blood biochemical analysis, including measurements of blood urea nitrogen (BUN), aspartate aminotransferase (AST), total bilirubin (TBIL), creatinine (CR), and alanine aminotransferase (ALT). Furthermore, histological examination of the mice's major organs (heart, liver, spleen, lungs, and kidneys) was performed using H&E staining. The results showed that the use of targeted drugs did not cause significant changes in blood biochemical parameters in the major organs of normal mice. Figure 9 A), and in the major organs ( Figure 9 The absence of significant pathological changes in B) indicates that the drug has good biocompatibility.

[0072] Furthermore, to investigate the effects of the concentration of the combined drug PTX and different ratios of CXB and PTX on efficacy, this embodiment further conducted in vitro experiments, exposing 4T1 cells to different ratios of PTX and CXB for 24 hours. Figure 5 As shown, (A) shows the effect of different concentrations of PTX on the expression level of COX-2 gene in 4T1 cells (n=3); (B) shows the secretion of PGE2 by 4T1 cells under different concentrations of PTX (n=3); (C) shows the expression level of COX-2 gene in 4T1 cells after treatment with a mixture of PTX and CXB at different molar ratios (n=3); (D) shows the secretion of PGE2 by 4T1 cells under different drug treatments (n=3); (E) shows the CRT membrane translocation of 4T1 cells after treatment with different drugs and (F) quantitative analysis of CRT-positive cells (n=3); (G) shows the release of extracellular ATP by 4T1 cells after treatment with different drugs (n=3); (H) shows the expression of HSP70 by 4T1 cells after treatment with different drugs and (I) quantitative analysis of HSP70-positive cells (n=3). *p<0.05, ns indicates no significant difference.

[0073] from Figure 5 As can be seen, the use of PTX alone led to a dose-dependent increase in COX-2 mRNA expression. Figure 5 A), the release of PGE2 also increased in a dose-dependent manner. Figure 5 B).

[0074] When CXB and PTX are used together, the level of COX-2 mRNA is significantly reduced. Figure 5C), and the inhibitory effect was maximized when the molar ratio of PTX to CXB was 1:5. Simultaneously, the use of the PTX / CXB combination significantly reduced the level of PGE2 released from 4T1 cells compared to the control group and PTX alone. Figure 5 D). These results indicate that CXB can pharmacologically inhibit PGE2 biosynthesis in PTX-induced triple-negative breast cancer cells.

[0075] In addition, to evaluate the efficacy of this combination drug in inducing ICD, this embodiment also examined the expression of immunostimulatory DAMPs. Figure 5 E- Figure 5 As can be seen from G, the membrane translocation of the CRT ( Figure 5 E and Figure 5 F), HSP70 expression ( Figure 5 H and Figure 5 I) and the release of ATP ( Figure 5 G) did not change significantly, indicating that inhibiting PGE2 did not affect PTX-induced DAMP exposure. Furthermore, the combination of PTX and CXB did not significantly differ from PTX alone in cell death rate. Figure 6 These findings indicate that CXB can inhibit PTX-induced PGE2 release without affecting the toxicity of PTX to 4T1 triple-negative breast cancer cells or the expression of immunostimulatory DAMPs, providing a pharmacological explanation for the effectiveness of targeted drugs, while also achieving the optimal molar ratio of PTX to CXB of 1:5.

[0076] The specific embodiments described in this example are merely illustrative of the present invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A triple negative breast cancer targeting drug, characterized in that, The targeting drug comprises paclitaxel, celecoxib and liposomes modified by a cyclic arginine-glycine-aspartic acid polypeptide, and the paclitaxel and the celecoxib are mixed in a molar ratio of 1: (4.4-5) and are encapsulated by the liposomes to form the targeting drug for triple-negative breast cancer; The liposomes selectively target tumor sites through specific interaction with integrins overexpressed in triple-negative breast cancer; The mechanism of action of the targeting drug is that, after the drug is targeted and delivered to the tumor site by the liposomes, the paclitaxel induces immunogenic cell death of tumor cells, while the celecoxib promotes the maturation and antigen presentation of dendritic cells by inhibiting the release of immunosuppressive molecules induced by paclitaxel, thereby activating T cell-mediated anti-tumor immunity. 2.The triple negative breast cancer targeting drug according to claim 1, characterized in that, In the targeting drug, the drug loading amounts of paclitaxel and celecoxib are 2.5%±0.5% and 5.6%±0.5%, respectively.

3. A method of preparing a triple negative breast cancer targeting drug, characterized by, The targeting drug comprises components: soybean lecithin, cholesterol, phospholipid polyethylene glycol, palmitic acid modified by a cyclic arginine-glycine-aspartic acid polypeptide, paclitaxel and celecoxib; The mass ratio of soybean lecithin, cholesterol, phospholipid polyethylene glycol, tumor targeting ligand, paclitaxel and celecoxib is (50-70):(10-30):(10-30):(5-15):(1-4):(2-20), and the mass ratio of paclitaxel and celecoxib is 1: (2.2-2.5); each component is dissolved in chloroform and added to a round-bottom flask; A rotary evaporator is used to form a thin film; An appropriate amount of deionized water is added, and the mixture is ultrasonically treated in a water bath at a set temperature for a set time; The obtained solution is further treated by a liposome extruder with a set pore size.

4. The method for preparing a targeted drug for triple-negative breast cancer according to claim 3, characterized in that, The mass ratio of soybean lecithin, cholesterol, phospholipid polyethylene glycol, tumor targeting ligand, paclitaxel and celecoxib is 60:20:20:10:(2-3): (5-15); The set temperature is 0-6°C; The set time is 10-30 minutes; The set pore size is 50-200 nanometers.

5. The method for preparing a targeted drug for triple-negative breast cancer according to claim 4, characterized in that, The mass ratio of soybean lecithin, cholesterol, phospholipid polyethylene glycol, tumor targeting ligand, paclitaxel and celecoxib is 60:20:20:10:2.86:7.14; The set temperature is 4°C; The set time is 20 minutes; The set nanometer is 100 nanometers.

6. Use of the triple-negative breast cancer targeting drug of claim 1 or 2 in the preparation of a triple-negative breast cancer tumor treatment drug.

7. Use of the targeting drug of any one of claims 1-2 or the targeting drug prepared by the preparation method of any one of claims 3-5 in the preparation of a triple-negative breast cancer in situ vaccine.

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