Temperature-sensitive hydrogel drug sustained-release stent for jointly loading antihypertensive drug and chemical / immunotherapy drug and application of temperature-sensitive hydrogel drug sustained-release stent

Abstract

The invention relates to a thermo-sensitive hydrogel drug sustained-release stent for jointly loading a hypotensive drug and a chemical / immunotherapy drug, which is prepared from a thermo-sensitive material and loads three drug components, namely losartan, oxaliplatin and an immune checkpoint inhibitor. The temperature-sensitive hydrogel can form gel in situ under the triggering of body temperature, and meanwhile, the acting time of the medicine on a target part is prolonged. The antihypertensive drug losartan potassium can reduce tumor interstitial substances, reduce tumor solid stress, relieve vascular compression in tumors and improve tumor hypoxia. Meanwhile, the chemotherapeutic drug oxaliplatin can induce tumor immunogenic cell death, and the tumor immunosuppressive microenvironment is effectively relieved. The immune checkpoint inhibitor can be used for accurately blocking the combination of the PD-L1 and the PD-1 and activating the T cell mediated anti-tumor immune response. According to the present invention, the potent anti-tumor immune response can be triggered, the excellent tumor inhibition effect can be achieved, the strong far-end effect can be even induced, and the growth of the far-end tumor can be effectively inhibited.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical medical technology, in particular to a temperature-sensitive hydrogel drug sustained-release stent loaded with antihypertensive drugs and chemotherapeutic / immunotherapeutic drugs and application thereof. BACKGROUND

[0002] Immunotherapy is a new cancer treatment method, and PD-1 / PD-L1 immune checkpoint inhibitors have been widely approved by the US Food and Drug Administration (FDA) for clinical treatment of various types of cancer. These PD-1 / PD-L1 immune checkpoint inhibitors (ICIs) can block PD-1 or PD-L1 on the cell surface, eliminate immune suppression signals, re-activate T cells infiltrating tumors, and destroy cancer cells. However, there is an extremely complex immune suppression environment in the tumor microenvironment, such as accumulation of immunosuppressive cells and overexpression of immunosuppressive cytokines, which leads to decreased efficacy of PD-1 / PD-L1 ICIs. Clinical evidence shows that a large number of patients do not respond to PD-1 / PD-L1 ICIs or develop resistance after being given PD-1 / PD-L1 ICIs treatment. The patient response rate of PD-1 / PD-L1 ICIs is only between 20-40%. Therefore, there is an urgent need in the clinic to develop a new strategy to counteract the tumor immunosuppressive microenvironment to improve the therapeutic effect of PD-1 / PD-L1 ICIs.

[0003] Oxaliplatin (Oxpt) is an anticancer drug widely used in the clinical treatment of various cancers. It mainly forms cross-linking with cancer cell DNA, thereby effectively blocking the replication and transcription of cancer cells to exert its strong anticancer effect. Recently, emerging evidence shows that Oxpt can induce immunogenic cell death by releasing various key immunogenic signals, such as calreticulin (CRT), ATP and HMGB1. These signals can activate and recruit dendritic cells (DCs) and cytotoxic T lymphocytes, triggering a series of effective anti-tumor immune responses, effectively reversing the "cold" immunosuppressive microenvironment in the tumor to a "hot" immune response microenvironment. However, uncontrolled tumor cell growth and abnormal vascular generation can lead to hypoxia in the tumor microenvironment. Increasing evidence shows that the hypoxic microenvironment can weaken the anti-tumor immune response by reducing immune effector cells and recruiting immunosuppressive cells. Therefore, in order to further improve the effectiveness of tumor immunotherapy, it is imperative to explore innovative strategies that can reverse the hypoxic state of tumors. SUMMARY

[0004] To address the aforementioned technical problems, this invention provides a thermosensitive hydrogel drug-release scaffold loaded with both antihypertensive drugs and chemo / immunotherapy drugs. The thermosensitive hydrogel scaffold is prepared from thermosensitive materials and loaded with three drug components: losartan, oxaliplatin, and an immune checkpoint inhibitor (PD-L1 antibody (aPDL1)). The thermosensitive hydrogel can gel in situ upon temperature triggering, simultaneously prolonging the drug's action time at the target site. The clinical antihypertensive drug losartan potassium can reduce tumor stroma, decrease tumor solid stress, and relieve intratumoral vascular compression, thereby improving tumor hypoxia. Simultaneously, the chemotherapeutic drug oxaliplatin can induce immunogenic cell death in tumors, effectively alleviating the tumor's immunosuppressive microenvironment. The immune checkpoint inhibitor aPDL1 can precisely block the binding of PD-L1 to PD-1, activating T cell-mediated antitumor immune responses. This invention can trigger a potent antitumor immune response, achieving excellent tumor suppression effects and even inducing strong distant effects, effectively inhibiting the growth of distant tumors.

[0005] The first objective of this invention is to provide the application of a thermosensitive hydrogel drug-release stent loaded with a combination of an antihypertensive drug and a chemo / immunotherapy drug in the preparation of an antitumor drug, wherein the antitumor drug is used to prevent or treat primary tumors, distant tumors, or metastatic tumors, and the liquid formulation for preparing the thermosensitive hydrogel drug-release stent loaded with the combination of the antihypertensive drug and the chemo / immunotherapy drug comprises:

[0006] The antihypertensive drug mentioned is losartan;

[0007] Chemotherapy drug, wherein the chemotherapy drug is oxaliplatin;

[0008] Immunotherapy drugs, wherein the immunotherapy drugs are PD-1 / PD-L1 immune checkpoint inhibitors;

[0009] Thermosensitive gel matrix, wherein the thermosensitive gel matrix gels in vivo (e.g., at 36-37°C) and does not gel at room temperature.

[0010] Losartan (Los) is a potent angiotensin II receptor blocker (ARB) widely used in the clinical treatment of hypertension. This invention discovers that Los can reduce solid stress in tumor tissue by inhibiting the production of extracellular matrix (collagen and hyaluronic acid) within tumor cells. The growth of the tumor matrix exacerbates the complexity of the tumor microenvironment, leading to significantly increased solid stress, vascular compression, reduced tumor blood supply, and ultimately, worsened tumor hypoxia. Therefore, Los can be used to alleviate intratumoral hypoxia. Furthermore, as an antihypertensive drug, systemic administration of Losartan may pose certain risks. The product of this invention maximizes drug efficacy through synergistic effects with other drugs and a sustained-release mechanism, while also reducing potential risks.

[0011] Furthermore, it includes at least one of the following:

[0012] (1) The immunotherapy drug is an anti-PD-L1 antibody;

[0013] (2) The thermosensitive gel matrix is ​​Pluronic F127.

[0014] Furthermore, the liquid formulation includes at least one of the following:

[0015] (1) The concentration of antihypertensive drugs is 50-100 mg / mL;

[0016] (2) The concentration of chemotherapy drugs is 0.1-10 mg / mL;

[0017] (3) The concentration of immunotherapy drugs is 0.1-10 mg / mL;

[0018] (4) The mass percentage of the thermosensitive gel matrix is ​​22%-26%.

[0019] Furthermore, in the liquid formulation, the concentration of the antihypertensive drug is 70 mg / mL, the concentration of the chemotherapy drug is 0.15 mg / mL, the concentration of the immunotherapy drug is 0.6 mg / mL, and the mass percentage of the thermosensitive gel matrix is ​​24%. This invention, after determining the drug combination based on drug function, optimized the concentration to achieve synergistic effects of several drugs. As is well known, synergy refers to the total effect being greater than the simple sum of the individual effects when two or more drugs are used together, and its essence is "concentration-dependent." The same drug combination, at different concentration ratios, may exhibit different effects such as synergy, additive effects, or even antagonism. We found that too low a concentration may not achieve the efficacy of a single drug, while too high a concentration may lead to antagonism or toxicity accumulation. Only at appropriate concentrations can synergy be generated through complementary mechanisms (such as pathway cross-linking). Therefore, concentration optimization here is also one of our innovations. In addition, the gelation temperature of thermosensitive hydrogels is affected by the loaded drug during the preparation process. After loading the drug, the gelation temperature of the prepared thermosensitive hydrogel changes. We explored the gelation of specific drugs and determined that in vivo gelation can be achieved when the F127 concentration is 24%.

[0020] Furthermore, the tumor is a solid tumor, including but not limited to breast cancer, lung cancer, kidney cancer, liver cancer, colon cancer, intestinal cancer, bladder cancer, pancreatic cancer, etc. It performs best in the treatment of breast cancer and can even be used to treat distant tumors.

[0021] A second object of the present invention is to provide a liquid formulation for forming in vivo a thermosensitive hydrogel drug-releasing scaffold that combines a loaded antihypertensive drug and a chemo / immunotherapy drug, the liquid formulation comprising:

[0022] The antihypertensive drug is losartan, with a concentration of 50-100 mg / mL;

[0023] Chemotherapy drug, wherein the chemotherapy drug is oxaliplatin, at a concentration of 0.1-10 mg / mL;

[0024] Immunotherapy drugs, wherein the immunotherapy drugs are PD-1 / PD-L1 immune checkpoint inhibitors, at a concentration of 0.1-10 mg / mL;

[0025] Thermosensitive gel matrix, wherein the thermosensitive gel matrix gels in vivo (e.g., at 36-37°C) at a concentration of 22%-26%.

[0026] Furthermore, it includes at least one of the following:

[0027] (1) The liquid preparation is administered via in situ injection;

[0028] (2) The immunotherapy drug is an anti-PD-L1 antibody;

[0029] (3) The thermosensitive gel matrix is ​​Pluronic F127.

[0030] A third objective of the present invention is to provide a pharmaceutical composition comprising losartan, oxaliplatin, a PD-1 / PD-L1 immune checkpoint inhibitor, and a thermosensitive gel matrix (Pluronic F127), wherein the thermosensitive gel matrix can form a gel at 36-37°C.

[0031] A fourth object of the present invention is to provide an antitumor drug, comprising the liquid formulation or pharmaceutical composition.

[0032] A fifth object of the present invention is to provide the use of the liquid formulation or pharmaceutical composition in the preparation of an antitumor drug, wherein the tumor includes breast cancer.

[0033] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0034] To improve the therapeutic efficacy of PD-1 / PD-L1 ICIs and reverse tumor hypoxia, this invention provides a thermosensitive hydrogel drug-release scaffold that combines antihypertensive drugs and chemo / immunotherapy drugs. The thermosensitive hydrogel F127, after precise injection into tumor tissue, responds rapidly to body temperature, forming a stable gel structure and providing an ideal platform for sustained and stable drug release. This invention also optimizes the formulation, achieving an organic synergy of several drugs: Los significantly reduces tumor matrix formation, effectively reduces tumor solid stress, alleviates intratumoral vascular compression, and ultimately reverses the hypoxia-mediated tumor immunosuppressive microenvironment; Oxpt induces tumor immunogenic cell death, recruits and activates immune cells, triggering a strong anti-tumor immune response; aPDL1 precisely blocks the interaction between PD-L1 and PD-1, successfully releasing immunosuppression on effector T cells and further activating T cell-mediated anti-tumor immune responses. All components in this invention have been approved by the FDA and have demonstrated good biocompatibility and safety, ensuring the safety of this system in clinical applications. Attached Figure Description

[0035] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0036] Figure 1 The gelation temperature test results of F127 hydrogel (a) and F127@Oxpt-Los-BSA hydrogel (b) under different F127 concentrations (18%, 20%, 22%, 24%, 26%, 28%) in Example 1 of the present invention are as follows:

[0037] Figure 2 The results are the rheological test results of F127 and F127@Oxpt-Los-BSA at 4°C and 37°C respectively in Embodiment 1 of the present invention, where a is the modulus change of 24% F127 at 4°C, b is the modulus change of 24% F127 at 37°C, c is the modulus change of 24% F127@Oxpt-Los-BSA at 4°C, and d is the modulus change of 24% F127@Oxpt-Los-BSA at 37°C.

[0038] Figure 3 The cumulative release test results of RhB (a), Los (b) and BSA (c) of F127@RhB-Los-BSA hydrogel at 37°C in Example 2 of this invention are shown.

[0039] Figure 4 This is the cytotoxicity test result of Oxpt on 4T1 cells in Example 3 of the present invention;

[0040] Figure 5 This is a flow cytometry analysis and quantitative results of CRT expression (a) and HMGB1 release (b) on the cell surface of 4T1 cells after Oxpt treatment in Example 3 of the present invention, as well as the amount of ATP released (c).

[0041] Figure 6 The images (ac) and percentages (eg) of CD45, CD8 cells and DCs in mice after different treatments were analyzed by flow cytometry in Example 4 of this invention, as well as the representative flow cytometry images (d) and quantitative results (h) of mature DCs in lymph nodes.

[0042] Figure 7 These are the immunohistochemical staining and quantitative results of Hyaluronic acid (a), Collagen I (b), and α-SMA (c) in tumor tissues of tumor-bearing mice after different treatments in Example 5 of this invention;

[0043] Figure 8 These are the quantitative values ​​of the solid stress of the tumor after different treatments in Example 5 of the present invention;

[0044] Figure 9 These are the perfusion vessel fraction (a) and relative vessel area fraction (b) of 4T1 tumors after different treatments in Example 5 of the present invention.

[0045] Figure 10 These are the immunohistochemical staining and quantitative results of HIF-1α (a) and Hypoxyprobe (b) in tumor tissues of tumor-bearing mice after different treatments in Example 5 of this invention;

[0046] Figure 11The images (af) and the percentages (gl) of MDSCs, M1, M2, CD3, CD8 and Tregs cells in the tumor of mice after different treatments are representative images (af) and corresponding cell percentages (gl) in Example 6 of this invention, obtained by flow cytometry analysis.

[0047] Figure 12 This is a graph showing the weight change of breast cancer-bearing mice after treatment with the thermosensitive hydrogel sustained-release stent in Example 7 of this invention.

[0048] Figure 13 These are the blood routine test results (a) and blood biochemistry test results (b) of breast cancer-bearing mice treated with the thermosensitive hydrogel sustained-release scaffold in Example 7 of this invention.

[0049] Figure 14 This is a primary tumor growth curve of breast cancer-bearing mice treated with the thermosensitive hydrogel sustained-release scaffold in Example 7 of the present invention.

[0050] Figure 15 This is a graph showing the survival time of breast cancer-bearing mice after treatment with the thermosensitive hydrogel sustained-release scaffold in Example 7 of this invention.

[0051] Figure 16 The CD8+ levels in the primary tumors of different groups of breast cancer-bearing mice treated with the thermosensitive hydrogel sustained-release scaffold in Example 7 of this invention are... + Representative flow cytometry images of T cells and Tregs (a, b) and corresponding quantitative results (ce);

[0052] Figure 17 This is a curve of distal tumor growth in breast cancer-bearing mice treated with a thermosensitive hydrogel sustained-release scaffold in Example 8 of the present invention.

[0053] Figure 18 The CD8+ levels in the distal tumors of different groups of breast cancer-bearing mice treated with the thermosensitive hydrogel sustained-release scaffold in Example 8 of this invention are... + Representative flow cytometry images (a, b) and corresponding quantitative results (ce) of T cells and Tregs. Detailed Implementation

[0054] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0055] The technical solution involved in this invention is as follows:

[0056] This invention provides a thermosensitive hydrogel drug-releasing stent that combines a load of an antihypertensive drug and a chemo / immunotherapy drug. The thermosensitive hydrogel stent is made of a thermosensitive material and is loaded with an antihypertensive drug and a chemo / immunotherapy drug.

[0057] Preferably, the temperature-sensitive polymer material is Pluronic F127. During the preparation of the temperature-sensitive hydrogel, its gelation temperature is affected by the loaded drug. After the drug is loaded, the gelation temperature of the prepared temperature-sensitive hydrogel also changes.

[0058] Preferably, the antihypertensive drug is losartan potassium. Losartan potassium can reduce tumor stroma, decrease tumor solid stress, and relieve intratumoral vascular compression, thereby improving tumor hypoxia.

[0059] Preferably, the chemoimmunotherapy drugs are oxaliplatin and aPDL1, respectively. Oxaliplatin can induce immunogenic cell death in tumors, effectively alleviating the immunosuppressive tumor microenvironment. Meanwhile, the immune checkpoint inhibitor aPDL1 can precisely block the binding of PD-L1 to PD-1, activating T cell-mediated anti-tumor immune responses.

[0060] Preferably, the preparation method of the F127 drug delivery system (F127@Oxpt-Los-aPDL1) is as follows:

[0061] (1) Weigh out Pluronic F127 powder, add Pluronic F127 powder to ddH2O, place on ice and stir continuously until completely dissolved;

[0062] (2) Add aPDL1 to a final concentration of 0.6 mg / mL and mix thoroughly;

[0063] (3) Add Los and Oxpt sequentially to reach concentrations of 70 mg / mL and 0.15 mg / mL respectively, until completely dissolved.

[0064] Preferably, the concentration of Pluronic F127 is 24%.

[0065] The drug-release scaffold of the present invention can be used for anti-tumor purposes. It has been verified that it can at least inhibit the growth of primary subcutaneous breast cancer tumors or inhibit the growth of distal secondary breast cancer tumors or prolong the survival time of breast cancer-bearing mice.

[0066] The materials involved in this invention are as follows:

[0067] aPDL1 antibody: InVivoMAb anti-mouse PD-L1 (B7-H1), BE0101, BioXcell.

[0068] Example 1: Preparation and Characterization of Thermosensitive Hydrogel Drug-Release Scaffold

[0069] Preparation of F127@Oxpt-Los-aPDL1 hydrogel: Weigh an appropriate amount of F127 powder and add it to ddH2O. Place the solution on ice and stir continuously until completely dissolved. Initially add aPDL1 to a final concentration of 0.6 mg / mL and mix thoroughly. Subsequently, add Los and Oxpt sequentially to reach concentrations of 70 mg / mL and 0.15 mg / mL, respectively, until completely dissolved to obtain F127@Oxpt-Los-aPDL1 hydrogel.

[0070] Gel behavior study of hydrogel drug delivery system: Different concentrations of F127 and F127@Oxpt-Los-BSA (bovine serum albumin BSA was used to replace aPDL1 antibody) were prepared. The gelation temperature of F127 and F127@Oxpt-Los-BSA was detected using the inverted observation method, increasing by 0.5℃ each time. 0.1 mL of each concentration of F127 and F127@Oxpt-Los-BSA solution was added to a 1.5 mL EP tube and placed in a water bath at a pre-set specific temperature for 5 minutes. Subsequently, these tubes were inverted for 30 seconds to measure gel formation. The temperature at which no flow was observed in the sample was recorded as the gel formation temperature. The test results are as follows: Figure 1 As shown, the critical gelation temperature decreases with increasing concentrations of F127 and F127@Oxpt-Los-BSA, while drug encapsulation leads to a relative increase in the gelation temperature of F127. To ensure the drug-release stent is suitable for in vivo injection and to shorten the in vivo gelation time, a 24% concentration of F127 hydrogel was selected for thermally triggered in situ gelation (F127 concentration is 24% in the following text).

[0071] Rheological properties of hydrogels before and after gelation: The rheological properties of empty and Oxpt-Los-BSA-encapsulated F127 hydrogels at different temperatures were analyzed using frequency scanning mode. At 4℃, the elastic modulus (G') of both was less than the viscous modulus (G"), exhibiting liquid properties. Figure 2 a and 2c). Conversely, at 37°C, the elastic modulus (G') of both is greater than the viscous modulus (G"), indicating the formation of a hydrogel. Figure 2 (b and 2d).

[0072] Example 2: Characterization of drug release behavior in a thermosensitive hydrogel drug-release scaffold

[0073] To investigate the sustained-release capacity of the F127 scaffold, the release profiles of Rhodamine B (RhB, used to replace Oxpt), Los, and BSA were quantitatively determined. A 24% concentration of F127@RhB-Los-BSA was prepared according to the above method. 400 μL of F127@RhB-Los-BSA solution was added to a centrifuge tube and incubated at 37°C to form a gel. Three samples were repeated. Subsequently, 3 mL of phosphate-buffered saline (PBS) solution was slowly added, maintaining the temperature at 37°C. 20 μL of supernatant was collected at different time points, and the concentrations of RhB, Los, and BSA in the supernatant were detected using a microplate reader, high-performance liquid chromatography (HPLC), and a BCA protein assay kit, respectively. Release efficiency curves were then plotted. Figure 3 As shown, RhB, Los and BSA were continuously released over 72 hours, demonstrating a good sustained-release effect.

[0074] Example 3: Detection of Oxpt's in vitro cytotoxicity and in vitro induced immunogenic cell death (ICD)

[0075] In vitro cytotoxicity assay of Oxpt: 4T1 cells were collected and prepared into a cell suspension, and seeded into 96-well plates at 200 μL per well. After 24 hours, a predetermined concentration of Oxpt solution was added to the cells in each well. 4T1 cells were incubated with Oxpt for 48 hours. Then, the old culture medium was discarded, and 100 μL of MTT (5 mg / mL) was added to each well. The cells were incubated at 37°C for another 4 hours. The MTT was then removed, and 200 μL of DMSO was added to each well. The cells were incubated at room temperature for 30 minutes, with shaking every 5 minutes. Finally, the OD values ​​of each sample well were measured at 570 nm using a microplate reader, and statistical analysis was performed. Figure 4 As shown, the viability of 4T1 cells decreased significantly with increasing Oxpt concentration, indicating that Oxpt has a significant cytotoxic effect on cells.

[0076] Detection of Oxpt-induced immune cell death (ICD) in vitro: To detect Oxpt-triggered ICD, we examined characteristic markers of ICD in Oxpt-treated 4T1 cells, including expression of cell surface calreticulin (CRT) and the release of high-mobility group box 1 (HMGB1) and ATP in 4T1 cells. First, 4T1 cells were seeded in 12-well plates and cultured. The next day, 1 mL of fresh medium containing 0.15 mg / mL Oxpt was added to each well, and the cells were incubated for 8 hours. Then, fresh medium was replaced, and the cells were cultured for another 24 hours. To detect CRT, 4T1 cells were collected, incubated with anti-CRT primary antibody, fixed with 4% paraformaldehyde for 15 minutes, and then incubated with Alexa Fluor 488-bound fluorescent secondary antibody at room temperature for 30 minutes. Finally, flow cytometry analysis was performed. To detect HMGB1, 4T1 cells were collected and resuspended in cold PBS. These cells were then infiltrated and fixed with osmosis buffer and 100% cold methanol. After centrifugation, the supernatant was collected, and the cells were resuspended in 100 μL of incubation buffer (0.5 g BSA dissolved in 100 mL PBS) and incubated for 10 minutes. Finally, the cells were incubated with anti-HMGB1 primary antibody and Alexa Fluor 488-bound fluorescent secondary antibody at room temperature for 1 hour and 30 minutes, respectively, before flow cytometry analysis. ATP release from Oxpt-treated cells was analyzed using an enhanced ATP assay kit. After the same treatment procedure as described above, the cell culture supernatant was collected, and ATP levels were measured using a microplate reader according to the manufacturer's instructions. Figure 5 As shown in Figures a and b, Oxpt treatment significantly increased the expression of CRT on the surface of 4T1 tumor cells and promoted the release of HMGB1 from 4T1 cells. Results Figure 5 The results showed that Oxpt treatment significantly enhanced ATP release from 4T1 cells. These findings confirm that Oxpt is a potent immunogenic inducer of cell death.

[0077] Example 4: Oxpt induces accumulation of immune cells and maturation of dendritic cells (DCs) in vivo.

[0078] Establishment of a subcutaneous 4T1 breast cancer tumor-bearing mouse model: 4T1 breast cancer cells were cultured in 1640 cell culture medium, digested, centrifuged at 900 rpm, 4°C for 3 minutes to collect cells, resuspended in PBS, and subcutaneously inoculated into Balb / c mice. The tumor volume in the mice grew to approximately 150 mm. 3 Experiments were conducted at that time.

[0079] Grouping and administration of tumor-bearing mice: 4T1 tumor-bearing mice were randomly divided into three groups: untreated, F127, and F127@Oxpt. Mice in the untreated group received no treatment. Mice in the F127 group received an intratumoral injection of 50 μL of F127 (24%). Mice in the F127@Oxpt group received an intratumoral injection of 50 μL of F127@Oxpt (F127: 24%, Oxpt: 0.15 mg / mL). When the tumor size reached 150 mm... 3 The injection was administered according to the above protocol, and a second injection was given on the 6th day.

[0080] To investigate the effects of Oxpt on immune cells, tumors and lymph nodes from mice treated with the above methods were collected for flow cytometry analysis. Tumor tissue was minced in PBS solution. Then, with the aid of digestion fluid, the tumor tissue was further processed into single-cell suspensions. Cells were resuspended in FACS buffer and stained with Biolegend's fluorescently conjugated antibodies against CD45, CD3, CD4, CD8, CD11c, and IA / IE, according to the manufacturer's instructions. Lymphoid tissue was also processed to prepare single-cell suspensions. Cells were resuspended in FACS buffer and stained with Biolegend's fluorescently conjugated antibodies against CD45, CD11c, CD80, and CD86, according to the manufacturer's instructions. These tumor or lymphoid tissue cells were then analyzed by flow cytometry.

[0081] result Figure 6 The results showed that, compared with the untreated group and the F127-only group, F127@Oxpt significantly increased total tumor immune cells (CD45). + CD8 + Infiltration of cytotoxic T lymphocytes and dendritic cells (DCs). Furthermore, F127@Oxpt treatment significantly promoted the maturation of DCs in lymph nodes. These results collectively confirm that Oxpt treatment can induce an immunogenic microenvironment within the tumor.

[0082] Example 5: Los reduces tumor solid stress and improves vascular perfusion.

[0083] Establishment of a subcutaneous 4T1 breast cancer tumor-bearing mouse model: 4T1 breast cancer cells were cultured in 1640 cell culture medium, digested, centrifuged at 900 rpm, 4°C for 3 minutes to collect cells, resuspended in PBS, and subcutaneously inoculated into Balb / c mice. The tumor volume in the mice grew to approximately 150 mm. 3 Experiments were conducted at that time.

[0084] Grouping and administration of tumor-bearing mice: 4T1 tumor-bearing mice were randomly divided into three groups: untreated, F127, and F127@Los. Mice in the untreated group received no treatment. Mice in the F127 group received an intratumoral injection of 50 μL of 24% F127. Mice in the F127@Los group received an intratumoral injection of 50 μL of F127@Los (F127: 24%, Los: 70 mg / mL). Tumors were treated when they reached a size of 150 mm. 3 When necessary, injections should be administered according to the above-mentioned groupings.

[0085] To investigate the specific effects of Los on the tumor stroma, tumor tissue was collected on day 3 post-drug administration. Immunohistochemical staining was used to analyze the major components of the tumor stroma, including hyaluronic acid, collagen I, and α-smooth muscle actin (α-SMA). Figure 7 As shown, after F127@Los treatment, the contents of the three main matrix components mentioned above were significantly reduced compared with the control group and the F127 group, indicating that Los can significantly reduce the matrix content in the tumor.

[0086] To further investigate the impact of Los (Los) on the biomechanical properties of the tumor microenvironment, tumors collected after the above treatment were washed three times with PBS. Then, the tumor was cut along its long axis to 80% of its shortest dimension and immersed in PBS for 10 minutes. The degree of incision opening was measured and photographed. Figure 8 As shown, F127@Los significantly reduced the level of solid stress in 4T1 tumor tissue. These results indicate that Los reduces the tumor matrix content, thereby reducing solid stress within the tumor.

[0087] Subsequently, the ameliorative effect of Los on tumor vascular structure was investigated in detail. On day 3 post-administration, 100 μL of DyLight 594-conjugated lectin was slowly administered via the tail vein 7 minutes before tumor tissue collection. The tumors were then collected and prepared into frozen sections. Immunofluorescence staining was performed using anti-CD31 antibody as a vascular marker. CD31-positive areas were considered blood vessels. Vascular perfusion fraction was quantified as the ratio of the co-positive area of ​​lectin and CD31 to the total CD31-positive area. Staining statistical results were also presented. Figure 9 The results showed that F127@Los treatment significantly increased the proportion of perfused blood vessels within the tumor, without significantly affecting the total number of blood vessels, indicating that Los can effectively relieve vascular compression by reducing solid stress within the tumor.

[0088] Finally, tumors were collected on day 3 post-drug administration, and the hypoxic status within the tumors was evaluated by immunohistochemical staining using hypoxia-inducible factor-1α (HIF-1α) and hypoxia probes. Figure 10As shown, the level of HIF-1α in tumor tissue of the F127@Los group was significantly decreased, as was the proportion of hypoxic areas. These results indicate that Los can effectively improve tumor hypoxia by enhancing intratumoral vascular perfusion and thus improving oxygen delivery.

[0089] These results validate that Los effectively modulates the abnormal mechanical microenvironment that compresses tumor blood vessels by inhibiting the production of tumor-associated matrix, thereby improving tumor vascular perfusion and alleviating tumor tissue hypoxia.

[0090] Example 6: Los reprogramming of the tumor immunosuppressive microenvironment

[0091] Establishment of a subcutaneous 4T1 breast cancer tumor-bearing mouse model: 4T1 breast cancer cells were cultured in 1640 cell culture medium, digested, centrifuged at 900 rpm, 4°C for 3 minutes to collect cells, resuspended in PBS, and subcutaneously inoculated into Balb / c mice. The tumor volume in the mice grew to approximately 150 mm. 3 Experiments were conducted at that time.

[0092] Grouping and administration of tumor-bearing mice: 4T1 tumor-bearing mice were randomly divided into three groups: untreated, F127, and F127@Los. Mice in the untreated group received no treatment. Mice in the F127 group received an intratumoral injection of 50 μL of 24% F127. Mice in the F127@Los group received an intratumoral injection of 50 μL of F127@Los (F127: 24%, Los: 70 mg / mL). Tumors were treated when they reached a size of 150 mm. 3 The injection was administered according to the above protocol, and a second injection was given on the 6th day.

[0093] To investigate the potential impact of Los on the tumor immunosuppressive microenvironment, mouse tumors after the aforementioned treatment were collected for flow cytometry analysis. Tumors were collected, minced in PBS solution, and used for flow cytometry analysis. The tumor tissue was then further processed into single-cell suspensions. Cells were resuspended in FACS buffer and stained with Biolegend's fluorescently conjugated antibodies against CD45, CD3, CD4, CD8, CD11b, Gr-1, F4 / 80, CD86, CD206, and FOXP3 according to the manufacturer's instructions. These cells were then analyzed by flow cytometry. Results showed that compared to the untreated group and the F127 group, the F127@Los group had a significantly reduced number of immunosuppressive cells in the tumor tissue, including myeloid-derived suppressor cells (MDSCs). Figure 11 (a and g), M2-like tumor-associated macrophages (TAMs) Figure 11 (c and i) and immunosuppressive Tregs ( Figure 11In stark contrast, the number of M1-like TAMs with antitumor effects within the tumor was significantly increased (f and l). Figure 11 (b and h). Furthermore, after F127@Los injection, compared with the untreated group and the F127 group, intratumoral CD3 levels were significantly increased. + T lymphocytes ( Figure 11 (d and j) and cytotoxic CD8 + The percentage of T lymphocytes has significantly improved. Figure 11 (e and k). These results clearly demonstrate that Los has the ability to successfully reprogram the immune microenvironment from an immunosuppressive state to an immunostimulatory state, which may be attributed to Los-induced tumor stroma suppression and relief of the hypoxic microenvironment within the tumor.

[0094] Example 7: Evaluation of the antitumor efficacy of thermosensitive hydrogel drug-release scaffold

[0095] Establishment of a subcutaneous 4T1 breast cancer tumor-bearing mouse model: 4T1 breast cancer cells were cultured in 1640 cell culture medium, digested, centrifuged at 900 rpm, 4°C for 3 minutes to collect cells, resuspended in PBS, and subcutaneously inoculated into Balb / c mice. The tumor volume in the mice grew to approximately 150 mm. 3 Experiments were conducted at that time.

[0096] Grouping and administration of tumor-bearing mice: 4T1 tumor-bearing mice were randomly divided into five groups: untreated group, F127@Los group, F127@Oxpt group, F127@Oxpt-aPDL1 group, and F127@Oxpt-Los-aPDL1 group. Mice in the untreated group received no treatment. The remaining four groups of mice were injected intratumorally with 50 μL of F127@Oxpt, F127@Los, F127@Oxpt-aPDL1, or F127@Oxpt-Los-aPDL1 (F127: 24%, Los: 70 mg / mL, Oxpt: 0.15 mg / mL, aPDL1: 0.6 mg / mL) on days 0 and 6, respectively.

[0097] To assess the safety of the thermosensitive hydrogel drug-release scaffold, the weight of mice in each group was measured and recorded every two days after the first drug injection, and blood samples were collected from each group. Complete blood count indicators were measured using whole blood, and biochemical indicators were measured using serum. The results showed that the thermosensitive hydrogel drug-release scaffold did not induce a significant decrease in body weight in tumor-bearing mice. Figure 12 Furthermore, both hematological and blood biochemical parameters fluctuated within the normal range. Figure 13 (F127@Oxpt-Los-aPDL1 group). These findings indicate that the thermosensitive hydrogel drug-release scaffold has no significant toxic side effects in mice.

[0098] After the first drug injection, the tumor volume of the mice was monitored every two days. The length and width of the tumor were measured using calipers, and the total tumor volume was calculated as (length × width). 2 ) / 2. Tumor volume monitoring statistics are as follows Figure 14 As shown, F127@Oxpt-aPDL1 treatment significantly delayed tumor growth compared to the untreated group. Furthermore, when Los was added, F127@Oxpt-Los-aPDL1 exhibited a stronger tumor growth inhibitory effect than F127@Oxpt-aPDL1, possibly because Los significantly reduced the tumor stroma, alleviated intratumoral hypoxia, and thus reversed the tumor immunosuppressive microenvironment. Additionally, as... Figure 15 As shown, compared with the untreated group, the survival rate of mice in the F127@Oxpt-Los-aPDL1 group was significantly improved, indicating that the combined treatment of F127@Oxpt-aPDL1 and Los has a significant therapeutic effect on tumors.

[0099] To further explore the mechanism of the antitumor effect of F127@Oxpt-Los-aPDL1, flow cytometry was used to analyze the cytotoxic CD8+ in tumor tissues of different groups of mice. + Tumor infiltration of T lymphocytes and Tregs. Results Figure 16 The results showed that the F127@Oxpt-Los-aPDL1 treatment group significantly increased intratumoral CD8 counts compared to the untreated group. + T cell infiltration was observed, while the number of Tregs in tumor tissue was significantly reduced compared to untreated cases. Therefore, the antitumor immune balance index (cytotoxic CD8+) in the F127@Oxpt-Los-aPDL1 group was significantly lower. + The ratio of T lymphocytes to Tregs was significantly higher in the untreated group than in the untreated group. Figure 16 (e). These results indicate that F127@Oxpt-Los-aPDL1 effectively triggers T cell-mediated anti-tumor immunity, achieving excellent tumor growth inhibition.

[0100] Example 8: Evaluation of the effect of thermosensitive hydrogel drug-release scaffold on inhibiting distal tumors

[0101] A subcutaneous bilateral 4T1 breast cancer tumor-bearing mouse model was established: 4T1 breast cancer cells were cultured in 1640 cell culture medium, digested, centrifuged at 900 rpm, 4°C for 3 minutes to collect the cells, resuspended in PBS, and subcutaneously inoculated into the right dorsal region of Balb / c mice. Seven days after inoculation of the primary tumor (right dorsal region), a second tumor was inoculated into the left dorsal region to simulate a distal tumor.

[0102] Grouping and administration of tumor-bearing mice: 4T1 tumor-bearing mice were randomly divided into five groups: untreated, F127@Los, F127@Oxpt, F127@Oxpt-aPDL1, and F127@Oxpt-Los-aPDL1. Mice in the untreated group received no treatment. On days 0 and 6, F127@Los, F127@Oxpt, F127@Oxpt-aPDL1, and F127@Oxpt-Los-aPDL1 (F127: 24%, Los: 70 mg / mL, Oxpt: 0.15 mg / mL, aPDL1: 0.6 mg / mL) were injected into the primary tumor at a dose of 50 μL per mouse.

[0103] Following the first drug injection, the distal tumor volume in mice was monitored every two days. The length and width of the tumor were measured using calipers, and the total tumor volume was calculated as (length × width). 2 ) / 2. Tumor volume monitoring statistics are as follows Figure 17 As shown, compared with the untreated group, F127@Oxpt-aPDL1 treatment effectively inhibited the growth of distal tumors, completely eliminating the distal tumor in one mouse. Furthermore, when Los was added, the inhibitory effect of F127@Oxpt-Los-aPDL1 on distal tumors was further enhanced compared to F127@Oxpt-aPDL1, even completely eliminating the distal tumor in three mice.

[0104] To elucidate the in vitro mechanism of action of F127@Oxpt-Los-aPDL1 on distal tumors, flow cytometry was used to analyze the cytotoxic CD8+ in distal tumor tissues of different groups of mice. + Tumor infiltration of T lymphocytes and Tregs. Flow cytometry results showed that, compared with the untreated group, F127@Oxpt-Los-aPDL1 treatment resulted in increased distal tumor cell cytotoxicity (CD8+). + The proportion of T lymphocytes increased significantly ( Figure 18 In cases a and c), the proportion of Tregs decreased significantly ( Figure 18 (b and d). Furthermore, the antitumor immune balance index (cytotoxic CD8+) of the F127@Oxpt-Los-aPDL1 group was [not specified]. + The ratio of T lymphocytes to Tregs was significantly higher in the untreated group than in the untreated group. Figure 18 (e). These results indicate that F127@Oxpt-Los-aPDL1 treatment can induce potent T-cell-mediated tumor killing, achieving therapeutic effects against distant tumors.

[0105] In summary, this invention constructs an in-situ injectable thermosensitive hydrogel drug-release scaffold, denoted as F127@Oxpt-Los-aPDL1, which can remodel the tumor immunosuppressive microenvironment and enhance aPDL1-mediated anti-tumor immune responses. This F127-based hydrogel system utilizes its unique thermosensitive properties to promote rapid gelation upon exposure to physiological temperatures, thereby ensuring the retention and sustained release of the loaded therapeutic agent within the tumor microenvironment. The Los carried by the F127 hydrogel reduces tumor solid stress, improves vascular perfusion, and promotes the relief of hypoxia-induced tumor immunosuppression. The loaded Oxpt can recruit and activate anti-tumor immune cells within the tumor by triggering immunogenic cell death, creating an immunogenic microenvironment within the tumor. aPDL1 effectively inhibits the interaction between PD-L1 and PD-1, successfully activating anti-tumor effector T cells, achieving potent tumor immunotherapy. In a 4T1 mouse mammary tumor model, we demonstrated that F127@Oxpt-Los-aPDL1 not only inhibited the growth of the primary tumor but also the growth of distant tumors. This invention demonstrates the great potential of this thermosensitive hydrogel drug-release scaffold (F127@Oxpt-Los-aPDL1) in reshaping the tumor immunosuppressive microenvironment and enhancing anti-tumor immunity, indicating a broad prospect for the application of mechanical modulation in tumor immunotherapy.

[0106] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of a thermosensitive hydrogel drug-releasing scaffold loaded with both an antihypertensive drug and a chemo / immunotherapy drug in the preparation of antitumor drugs, characterized in that, The antitumor drug is used to prevent or treat primary tumors, distant tumors, or metastatic tumors. The liquid formulation used to prepare the thermosensitive hydrogel drug-releasing stent, which combines the antihypertensive drug and the chemotherapy / immunotherapy drug, includes: The antihypertensive drug mentioned is losartan; Chemotherapy drug, wherein the chemotherapy drug is oxaliplatin; Immunotherapy drugs, wherein the immunotherapy drugs are PD-1 / PD-L1 immune checkpoint inhibitors; A thermosensitive gel matrix that gels in vivo.

2. The application according to claim 1, characterized in that, The immunotherapy drug is an anti-PD-L1 antibody. And / or, the thermosensitive gel matrix is ​​Pluronic F127.

3. The application according to claim 2, characterized in that, The liquid formulation contains at least one of the following characteristics: (1) The concentration of antihypertensive drugs is 50-100 mg / mL; (2) The concentration of chemotherapy drugs is 0.1-10 mg / mL; (3) The concentration of immunotherapy drugs is 0.1-10 mg / mL; (4) The mass percentage of the thermosensitive gel matrix is ​​22%-26%.

4. The application according to claim 3, characterized in that, In the liquid formulation, the concentration of the antihypertensive drug is 70 mg / mL, the concentration of the chemotherapy drug is 0.15 mg / mL, the concentration of the immunotherapy drug is 0.6 mg / mL, and the mass percentage of the thermosensitive gel matrix is ​​24%.

5. The application according to any one of claims 1-4, characterized in that, The tumors are solid tumors, including breast cancer.

6. A liquid formulation, characterized in that, The liquid formulation is used to form a thermosensitive hydrogel drug-releasing scaffold in vivo, which combines a loaded antihypertensive drug and a chemotherapy / immunotherapy drug, wherein the liquid formulation comprises: The antihypertensive drug is losartan, with a concentration of 50-100 mg / mL; Chemotherapy drug, wherein the chemotherapy drug is oxaliplatin, at a concentration of 0.1-10 mg / mL; Immunotherapy drugs, wherein the immunotherapy drugs are PD-1 / PD-L1 immune checkpoint inhibitors, at a concentration of 0.1-10 mg / mL; Thermosensitive gel matrix, wherein the thermosensitive gel matrix forms a gel in vivo, and the concentration is 22%-26%.

7. The liquid formulation according to claim 6, characterized in that, The liquid formulation contains at least one of the following characteristics: (1) The liquid preparation is administered via in situ injection; (2) The immunotherapy drug is an anti-PD-L1 antibody; (3) The thermosensitive gel matrix is ​​Pluronic F127.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes losartan, oxaliplatin, a PD-1 / PD-L1 immune checkpoint inhibitor, and a thermosensitive gel matrix, which can form a gel at 36°C-37°C.

9. An antitumor drug, characterized in that, Includes the liquid formulation of claim 6 or 7 or the pharmaceutical composition of claim 8.

10. The use of the liquid formulation of claim 6 or 7 or the pharmaceutical composition of claim 8 in the preparation of an antitumor drug, characterized in that, The tumors include breast cancer.

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