A tumor therapeutic hydrogel based on improving the tumor microenvironment to trigger gas therapy combined with chemotherapy

Abstract

This invention discloses a hydrogel for tumor treatment based on improving the tumor microenvironment to trigger gas therapy combined with chemotherapy, belonging to the field of biomedical technology. The hydrogel drug delivery system utilizes lipopolysaccharide (LPS) to induce local inflammatory stimulation, thereby altering the tumor microenvironment of breast cancer and promoting nitric oxide (NO) production. The NO donor arginine (L-Arg) and the chemotherapy drug doxorubicin (DOX) are transported using sodium alginate (ALG), achieving combined gas therapy and chemotherapy to inhibit breast tumor growth. The drug delivery system of this invention not only enhances the effect of chemotherapy but also simultaneously achieves precise drug delivery and reduces drug dosage. The preparation method of the drug delivery system of this invention is simple, safe, and without toxic side effects, showing promising application prospects in tumor treatment and possessing significant clinical value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a tumor therapeutic hydrogel based on improving the tumor microenvironment to trigger gas therapy combined with chemotherapy. Background Technology

[0002] The tumor microenvironment (TME) is a complex biological system composed of tumor tissue, endosomes, and lysosomes, characterized by hypoxia, weak acidity, high pressure, high reactive oxygen species, abundant reduced glutathione, growth factors, and proteolytic enzymes. It is a local homeostatic environment jointly constructed by tumor cells, stromal cells (composed of fibroblasts, blood vessels / lymphatic vessels, glial cells, and immune cells), and the extracellular matrix during tumor growth, playing a crucial role in tumor occurrence, development, invasion, metastasis, and the efficacy of antitumor drug therapy. Inflammation has a bidirectional regulatory effect on tumors; some studies have shown that it can promote tumor cell growth, while others have demonstrated that it can inhibit tumor metastasis.

[0003] NO, as one of the most common gaseous signaling molecules, plays a crucial regulatory role in cellular life activities such as vasomotor relaxation, inflammatory responses, immune responses, angiogenesis, anti-tumor activity, and nerve conduction. NO molecules not only maintain the physiological functions and activities of normal cells but are also closely related to the occurrence and development of tumors. Therefore, constructing a biocompatible hydrogel drug delivery system that can release NO gas and combining it with chemotherapy and gas therapy for targeted tumor treatment has become an important research direction. Summary of the Invention

[0004] In view of the shortcomings of existing technologies, this invention mainly provides a hydrogel drug delivery system for tumor treatment based on improving the tumor microenvironment to trigger gas therapy combined with chemotherapy. Based on improving the tumor microenvironment to trigger gas therapy combined with chemotherapy, the chemotherapeutic drugs DOX and L-Arg are carried by ALG to achieve the combined inhibition of breast tumor growth by gas therapy and chemotherapy. Local inflammatory stimulation is used to induce changes in the breast cancer tumor microenvironment to promote NO production, thereby achieving the effect of gas therapy sensitizing chemotherapeutic drugs. The composite hydrogel drug delivery system fixes the drug within the tumor, disperses it evenly, and releases it slowly, which can simultaneously achieve precise drug delivery and reduce the dosage of the drug.

[0005] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is as follows.

[0006] The present invention provides a pharmaceutical composition, characterized in that the composition comprises ALG, LPS, DOX and L-Arg.

[0007] Furthermore, the mixing ratio of ALG, LPS, DOX and L-Arg is 50:1:10:10.

[0008] Furthermore, the use of the pharmaceutical composition in the preparation of a medicament for treating tumors.

[0009] The present invention also provides the application of a hydrogel drug delivery system in the treatment of tumors, characterized in that the hydrogel drug delivery system comprises ALG, LPS, DOX and L-Arg.

[0010] Furthermore, the preparation method of the hydrogel drug delivery system specifically includes the following steps: a mixed solution containing ALG, LPS, DOX and L-Arg is ultrasonicated in a water bath for 10 min to obtain the composite hydrogel drug delivery system.

[0011] Furthermore, the mixing ratio of ALG, LPS, DOX, and L-Arg is 50:1:10:10.

[0012] Furthermore, the tumor is breast cancer.

[0013] The present invention also provides the application of a hydrogel drug delivery system composed of ALG, LPS, DOX and L-Arg in the treatment of tumors.

[0014] Furthermore, the mixing ratio of ALG, LPS, DOX and L-Arg is 50:1:10:10.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects.

[0016] (1) This invention proposes for the first time to select LPS as a local inflammatory stimulus, and uses this local inflammatory stimulus method to improve the tumor microenvironment, trigger NO generation, and realize the combined inhibition of breast tumor growth by gas therapy and chemotherapy.

[0017] (2) Since LPS, L-Arg and DOX are all administered in liquid form and are fluid, they are easily degraded when exposed to the biological environment for a long time, which can lead to unstable drug efficacy. In order to prolong the duration of drug action at the tumor site, improve drug stability and enhance drug efficacy, this invention proposes for the first time to use polysaccharide carbohydrate ALG extracted from kelp (brown algae such as kelp or Sargassum) as a carrier to prepare a hydrogel drug delivery system.

[0018] (3) The drug delivery system described in this invention can not only enhance the effect of chemotherapy, but also simultaneously deliver drugs accurately and reduce the dosage of drugs.

[0019] (4) The preparation method of the drug delivery system described in this invention is simple, safe and has no toxic side effects, and has good application prospects in tumor treatment. Attached Figure Description

[0020] Figure 1Concentration of ALG in the composite hydrogel drug delivery system. Experimental results of four different concentrations of ALG solutions. Injection of free DOX aqueous solution into Ca... 2+ Experimental results in buffer solution.

[0021] Figure 2 A schematic diagram of drug release in a composite hydrogel drug delivery system. (A) The composite hydrogel drug delivery system in a Ca-containing... 2+ Photographs showing drug release in an aqueous solution after shaking at 37°C for 24 hours. (B), (C), and (D) represent the drug release from the hydrogel drug delivery system in a solution containing Ca. 2+ The solution was shaken at 37°C for 24 hours in an aqueous solution, and the absorption spectra of DOX, L-Arg and LPS in the supernatant were detected by UV-vis.

[0022] Figure 3 In vitro NO release. (A) NO release by L-Arg at different pH values. (B) NO release by L-Arg at different ROS concentrations.

[0023] Figure 4 Effects of LPS stimulation on intratumoral ROS. (A) Changes in ROS content in 4T1 breast cancer cells after incubation with different concentrations of LPS. (B) Confocal image of ROS changes in 4T1 breast cancer cells at different time points after LPS stimulation, bar=20µm. (C) Changes in hydrogen peroxide content in tumors of tumor-bearing mice at different time points after LPS stimulation. (D) Confocal image of ROS changes in frozen sections of 4T1 breast tumors after LPS stimulation, bar=20µm. Values ​​are expressed as mean ± standard deviation (SD), (n=3), ** p <0.01, ns: no statistical significance.

[0024] Figure 5 In vitro toxicity assay of 4T1 breast cancer cells after gas therapy combined with chemotherapy. (A) Cell viability after incubation of 4T1 cells with DOX, L-Arg+ H2O2, and DOX+L-Arg+ H2O2, respectively. (B) Confocal micrographs of cell viability after incubation of 4T1 cells with DOX, L-Arg+ H2O2, and DOX+L-Arg+ H2O2, respectively, using the AM-PI live / dead cell assay, bar=100µm. Values ​​are expressed as mean ± standard deviation (SD), (n=3), *** p <0.001.

[0025] Figure 6Efficacy evaluation of the composite hydrogel drug delivery system in Balb / c mice bearing subcutaneous tumors. (A) Tumor images of mice after 15 days of treatment with different drug groups. (B) Tumor growth curves of mice after 15 days of treatment with different drug groups. (C) Tumor inhibition rate of mice after 15 days of treatment with different drug groups. Values ​​are expressed as mean ± standard deviation (SD), (n=6), *** p <0.001.

[0026] Figure 7 Safety evaluation. (A) Body weight change curves of mice treated with different drug groups over 15 days; (B) Histopathological H&E staining images of major organs (heart, liver, spleen, lung, kidney) of mice after 15 days of treatment with different drug groups, bar=100µm. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. The following embodiments are merely illustrative of the present invention and should not be construed as limiting the invention.

[0028] Example

[0029] Experimental materials and animals.

[0030] Main reagents.

[0031] .

[0032] Main instruments.

[0033] .

[0034] Experimental cell lines and animals.

[0035] The mouse breast cancer cell line (4T1) was purchased from Wuhan Pronosei Life Sciences Co., Ltd. Female Balb / c mice (6-8 weeks old, 18-22g, SPF grade) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. All animals were housed at the SPF Animal Experiment Center of Dalian Medical University. All animal housing and experimental procedures in this project complied with the "Regulations on Animal Protection and Use of Dalian Medical University".

[0036] Experimental methods.

[0037] Determine the concentration of ALG in the composite hydrogel drug delivery system.

[0038] To verify the gelling properties of ALG and determine the concentration of ALG in the composite hydrogel system, Coomassie Brilliant Blue was used as an indicator. 0.1 mL of ALG solution at different concentrations (1, 5, 10, and 20 mg / mL) was drawn into each solution using a 1 mL syringe and injected into 50 mL of a CaCl2 solution (Ca...2+ The ALG-injected Ca2+ was placed in a container with a concentration of 1.8 mmol / L, and photographs were taken at different time points during the drug administration process to observe the effects of ALG injection. 2+ The hydrogel formation state after solution preparation was determined to ascertain the concentration of ALG in the composite hydrogel system.

[0039] Synthesis of a composite hydrogel drug delivery system.

[0040] After determining the ALG concentration, an ALG concentration of 20 mg / mL was prepared, sonicated in a water bath for 10 min, and used as a stock solution for later use at 4℃. A mixed solution containing ALG, LPS, DOX, and L-Arg was prepared and sonicated in a water bath for 10 min to obtain the composite hydrogel drug delivery system (final concentrations: ALG 5 mg / mL; LPS 0.1 mg / mL; DOX 1 mg / mL; L-Arg 1 mg / mL). 0.1 mL of free DOX aqueous solution and the composite hydrogel drug delivery system mixture were drawn separately using a 1 mL syringe and injected into 50 mL of a CaCl2 solution (Ca... 2+ The composite hydrogel drug delivery system was synthesized by taking photographs at different time points in a beaker containing 1.8 mmol / L.

[0041] Drug release in a composite hydrogel drug delivery system.

[0042] To test the LPS+DOX+L-Arg+ALG composite hydrogel system in the presence of Ca 2+ To investigate drug release in solution, mixtures of LPS and ALG, DOX and ALG, and L-Arg and ALG were prepared. 1 mL of each mixture was drawn up using a syringe and injected into a solution containing 4 mL of CaCl2 (Ca...). 2+ The solution (1.8 mmol / L) was placed in a transparent glass bottle and incubated at 37°C on a shaker for 24 hours. Photos were taken at 0 h and 24 h for each group. The supernatant was then collected, and the UV-Vis absorbance of each sample was measured using a microplate reader to determine drug release. Additionally, each group was compared with free samples to verify the feasibility of ALG hydrogel as a drug delivery carrier.

[0043] Detection of NO release in vitro.

[0044] NO release under different ROS concentrations: L-Arg, as a natural NO donor, can release NO through the release of H2O2 and... 1 The oxidation of ROS, including O2, to NO was achieved. Different concentrations of H2O2 solutions (0, 50, 200, 1000, 10000 µmol / L) were added to L-Arg solutions and incubated in a 37°C shaker for 24 h. 50 µL of each solution was then transferred to a 96-well plate.

[0045] NO release at different pH values: Since the TME itself is a weakly acidic environment, to better verify the ability of L-Arg to release NO as a NO donor, the release of NO from L-Arg at different time points was examined under neutral and weakly acidic environments. HCl was used to simulate the weakly acidic environment of the tumor, and the pH of the HCl aqueous solution was measured to be 6.5 using a pH meter. Separately, purified water was taken, and its pH was measured to be 7.4 using a pH meter. 10 mL each of dilute hydrochloric acid solution with a pH of 6.5 and purified water with a pH of 7.4 were used to prepare a mixed solution of L-Arg and H2O2 (concentrations of L-Arg 100 mg / mL and H2O2 10 mmol / L, respectively), and vortexed for 1 min after preparation. 50 µL of the solution was transferred to 96-well plates at 12, 24, and 48 h. The experiment was repeated in triplicate.

[0046] The NO detection kit was used to detect the added standard and sample, and the absorbance was measured at 540 nm using an ELISA reader. The concentration of nitric oxide in the sample was calculated based on the standard curve.

[0047] Cell culture.

[0048] 4T1 breast cancer cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin at 37°C in an incubator with 5% CO2 and 95% humidity. After passage, subsequent cell experiments were performed when the cells were in the logarithmic growth phase.

[0049] The effect of acute inflammatory stimulation on intracellular ROS in tumor cells.

[0050] H2O2 is the main ROS. To determine the effect of LPS acute inflammatory stimulation on intracellular ROS in tumor cells, 4T1 cells were incubated with LPS solutions of different concentrations, and the intracellular H2O2 content was measured after 24 hours.

[0051] 4T1 mouse breast cancer cells were passaged into 6-well plates. Cells in logarithmic growth phase (1.0 × 10⁻⁶ cells) were cultured. 6 LPS solutions at concentrations of 0.25, 0.5, and 1 μg / mL were added to each cell / well and incubated.

[0052] 24 hours later, the H2O2 content in the cells was measured according to the H2O2 detection kit procedure. After collecting the cells, lysis buffer was added to fully lyse them. The supernatant was used for subsequent assays. Quantitative amounts of standards and H2O2 detection reagents were added to 96-well plates according to the procedure. The absorbance of the samples was measured at 560 nm using a microplate reader. The concentration of H2O2 in the samples was calculated based on a pre-defined standard curve.

[0053] Changes in intracellular ROS content in 4T1 cells at different time points after inflammatory stimulation.

[0054] The DCFH-DA fluorescent probe was used to detect the generation of free radicals in 4T1 breast cancer cells. DCFH-DA powder was weighed and dissolved directly in 100 μL of anhydrous DMSO, and a 10 mmol / L DCFH-DA stock solution was prepared with serum-free medium. Before administration, the DCFH-DA working solution was diluted to 20 μmol / L with serum-free medium. LPS powder was weighed and a 10 μg / mL stock solution was prepared. Before administration, a 0.25 μg / mL working solution was prepared with medium.

[0055] 4T1 breast cancer cells in logarithmic growth phase were passaged into confocal culture dishes and cultured at 1.5 × 10⁻⁶ m². 5 (1 cell / well) After the cell count reached 80% of the confocal dish, the dish was washed with PBS, and 1 mL of 0.25 μg / mL LPS working solution was added to each dish. Then, grouped experiments were performed. The experiment was divided into 4 groups (n=3): LPS administration for 2 h, 12 h, and 24 h, and a PBS blank control group.

[0056] After drug administration, the supernatant in the confocal dish was discarded at the corresponding time, and the cells were washed twice with PBS.

[0057] Add 1 mL of 20 μmol / L DCFH-DA working solution to each group of confocal dishes, incubate in an incubator for 20 min in the dark, and then stain with DAPI for 10 min in the dark. After staining, take pictures of the confocal dishes under a confocal microscope.

[0058] Changes in hydrogen peroxide content within tumors of Balb / c tumor-bearing mice at different time points after inflammatory stimulation.

[0059] First, a subcutaneous tumor model was established: Balb / c mice, weighing 18-22g, were housed in an animal testing center for one week to acclimatize. 4T1 breast cancer cells were inoculated into the right shoulder of each mouse. 4T1 cells in logarithmic growth phase were digested with trypsin, resuspended in PBS, and the cell suspension was transferred to a cell counting chamber for microscopic counting. The cell suspension was centrifuged at 1000 rpm for 4 minutes, and the supernatant was discarded. The cells were washed once with PBS to remove residual serum. After centrifugation again, an appropriate amount of PBS was added until the cell density reached 1×10⁻⁶ cells / mL. 7 Cells / mL. Mice were shaved on their left shoulder and inoculated with 4T1 breast cancer cells. Each tumor model mouse was subcutaneously inoculated with 0.1 mL of cell suspension, i.e., 1 × 10⁻⁶ cells / mL. 7 4 T1 cells were inoculated into mice. After inoculation, mice were observed and raised in an animal testing center. Tumor size was measured, and mice were monitored until the tumor reached 100 mm. 3 At that time, the experiment was conducted.

[0060] After establishing the subcutaneous tumor model, mice were subjected to inflammatory stimulation. Lipopolysaccharide (LPS) was used as a pro-inflammatory drug, and mice were injected intratumorally with LPS at a dose of 1 mg / mL, 20 µL / mouse. Mice were divided into groups, and one group of mice was perfused at 6, 12, 24, and 48 h after administration. The hydrogen peroxide content in the tumor was measured.

[0061] Tumor tissue was placed on ice, shredded, and mixed thoroughly. 10 mg of each tumor sample was weighed, and the specified amount of lysis buffer was added. The mixture was vortexed and homogenized to break down and lyse the cells. The concentration of H2O2 in the sample was determined according to the procedure outlined in the H2O2 detection kit.

[0062] Changes in ROS in frozen sections of 4T1 breast tumors after inflammatory stimulation.

[0063] Subcutaneous tumor model establishment: The method is the same as in 2.8 Subcutaneous tumor model establishment.

[0064] Drug administration: After establishing the subcutaneous tumor model, mice were divided into two groups (n=3): an LPS administration group and a PBS control group. Lipopolysaccharide (LPS) was used as a pro-inflammatory drug, and mice were injected intratumorally with LPS at 1 mg / mL, 20 µL / mouse. The control group was injected intratumorally with PBS solution.

[0065] Cryosectioning: 24 hours after drug administration, mice were perfused, tumors were removed, and immediately placed in liquid nitrogen for 5 minutes, then frozen at -80°C for 12 hours. The tumors were embedded in embedding medium, frozen, and sectioned using a cryostat. The sectioned tumors were placed on absorbent slides for staining.

[0066] Staining: After the frozen sections were thawed, they were stained with DCFH-DA (20 μmol / L) working solution diluted with PBS. 100 μL of DCFH-DA (20 μmol / L) working solution was added to each sample and stained in the dark for 20 min. Then, DAPI staining solution was added and stained in the dark for 10 min.

[0067] Photography: After the slides are mounted, they are photographed using a confocal fluorescence microscope.

[0068] Detection of drug toxicity to 4T1 breast cancer cells in a composite hydrogel drug delivery system.

[0069] 4T1 breast cancer cells in logarithmic growth phase were added to 96-well plates for incubation (approximately 1 × 10⁶ cells per well). 4(Number of cells / well), and the drug was added after 24 hours. The concentrations of DOX were 0.5, 1, 2, and 5 μg / mL; L-Arg concentrations were 50, 100, 200, and 400 μg / mL; LPS concentrations were 0.5, 1, 2, and 5 μg / mL; and H2O2 concentrations were 12.5, 50, 100, and 400 μmol / L. Since NO is released from L-Arg in the presence of free radicals, the cytotoxicity of NO can be demonstrated by co-administering L-Arg and H2O2. The L-Arg concentration was always 100 μg / mL, and the H2O2 concentrations were 10, 20, 50, and 100 μmol / L.

[0070] 24 hours after drug administration, 10 μL of CCK-8 detection solution was added to each well, and the mixture was incubated in the dark for 1.5 hours. The absorbance (OD value) at 450 nm was then measured using a microplate reader.

[0071] Cell viability (%) = [(As - Ab) / (Ac - Ab)] × 100.

[0072] The OD value of each repeating well is taken as the mean ± SD.

[0073] As, Ab, and Ac represent the OD values ​​of the experimental wells, blank wells, and control wells, respectively.

[0074] Detection of cytotoxicity of gas therapy combined with chemotherapy in 4T1 breast cancer cells.

[0075] 4T1 breast cancer cells in logarithmic growth phase were added to 96-well plates for incubation (approximately 1 × 10⁶ cells per well). 4 (Number of doses per well), and the drug was added after 24 hours. The chemotherapy group was administered DOX 1 μg / mL; the gas administration group was administered L-Arg 100 μg / mL and H2O2 100 μmol / L; and the combined administration group was administered DOX 1 μg / mL, L-Arg 100 μg / mL and H2O2 100 μmol / L.

[0076] 24 hours after drug administration, 10 μL of CCK-8 detection solution was added to each well, and the cells were incubated in the dark for 1.5 hours. The absorbance (OD value) at 450 nm was then measured using a microplate reader. Cell viability was calculated in the same manner as in Experiment 2.10.

[0077] Apoptosis experiment.

[0078] The Calcein-AM / PI assay was used to qualitatively evaluate the apoptosis of 4T1 breast cancer cells induced by chemotherapy, gas therapy, and chemotherapy combined with gas therapy.

[0079] 4T1 breast cancer cells in logarithmic growth phase were passaged and cultured in confocal culture dishes (1.5 × 10⁻⁶). 5Cells per well were counted. Once the cell count reached 80% of the confocal dish, group experiments were conducted. The experiment consisted of four groups: DOX+NO group, NO group, DOX group, and blank control group (n=3). After discarding the supernatant, each group was treated with DOX+L-Arg+H2O2, L-Arg+H2O2, DOX, and culture medium, respectively. After incubation at 37℃ for 12 hours, cells were stained with Calcein-AM / PI reagent for 15 minutes. Following staining, the confocal dish was photographed under a confocal microscope; live cells showed green fluorescence, while dead cells showed red fluorescence.

[0080] Efficacy evaluation of composite hydrogel drug delivery system in Balb / c mice with subcutaneous tumors.

[0081] Establishment of a subcutaneous tumor model: The experimental method is the same as in section 2.8, "Establishment of a Subcutaneous Tumor Model".

[0082] Grouping and Administration: Balb / c mice bearing subcutaneous tumors were randomly divided into 6 groups (n=6): PBS group, LPS+DOX+L-Arg group, LPS+L-Arg+ALG group, DOX+ALG group, DOX+L-Arg+ALG group, and LPS+DOX+L-Arg+ALG group (final concentrations: ALG 5 mg / mL; LPS 0.1 mg / mL; DOX 1 mg / mL; L-Arg 1 mg / mL). The prepared reagents for each group were injected into the center of the subcutaneous tumor at a volume of 50 μL. The length and width of the subcutaneous tumors in each group were measured every other day throughout the treatment process. On the last day of measurement, the tumors in each group were removed and weighed.

[0083] Calculation: The subcutaneous tumor volume of each group of experimental mice was calculated based on the tumor length and width measured during treatment, and the tumor inhibition rate was calculated using the following formula:

[0084] Tumor volume (mm) 3 = (long diameter × short diameter) 2 ) / 2;

[0085] Tumor inhibition rate (%) = (tumor volume in PBS group - tumor volume in treatment group) × 100 / tumor volume in PBS group.

[0086] H&E staining of tumor tissue.

[0087] On day 15 of treatment with the composite hydrogel drug delivery system, mice were perfused, tumors were removed, and placed in formalin. After tissue dehydration, clearing, paraffin embedding, sectioning and baking, dewaxing, Mayer's hematoxylin staining, eosin staining, gradient elution, and mounting, the tissues were examined under a microscope. Cell nuclei appeared blue, while cytoplasm, erythrocytes, muscle fibers, and collagen fibers appeared in varying degrees of red, allowing for observation of tissue morphology and structure.

[0088] Testing.

[0089] On day 15 of treatment with the composite hydrogel drug delivery system, mice were perfused, tumors were removed, and placed in formalin. Tissue dehydration, clearing, paraffin embedding, sectioning, baking, and dewaxing were performed following the same procedures as H&E staining. Biotin-labeled solution, working solution, and chromogenic solution were prepared according to the TUNEL assay kit instructions, and the samples were then labeled and chromogenic. After these procedures, the nuclei were counterstained with DAPI, and the slides were mounted with a mounting solution containing an anti-fluorescence quencher. The stained sections were observed under a fluorescence microscope and photographed.

[0090] Efficacy and safety evaluation of composite hydrogel drug delivery system in Balb / c mice with subcutaneous tumors.

[0091] Weight measurement of Balb / c mice with subcutaneous tumors: The weight of Balb / c mice was measured every other day after administration of different treatment components.

[0092] Biosafety testing: To examine the biosafety of different treatment groups on animals, mice were perfused on day 15 after administration, and major organs such as heart, liver, spleen, lung, and kidney of tumor-bearing mice were collected for H&E staining. The sections were analyzed using a tissue scanner to observe tissue morphology and structure and determine the biosafety of the composite hydrogel drug delivery system.

[0093] Experimental results.

[0094] ALG gelling ability and synthesis of composite hydrogel drug delivery system.

[0095] The final concentration of ALG in the hydrogel drug delivery system was selected as 5 mg / mL. The ALG in the composite hydrogel can react with the normal concentration of Ca2+ within the tumor. 2+ The reaction rapidly forms a network-structured hydrogel.

[0096] The concentration of ALG in the composite hydrogel drug delivery system.

[0097] like Figure 1 As shown in (A), the research group selected four different concentrations of ALG solution (1, 5, 10, and 20 mg / mL) for experiments. When ALG with a concentration of 1 mg / mL was injected into Ca... 2+ After 0.5 hours, the mixture rapidly diffused to the bottom of the entire beaker, exhibiting weak gelling properties and failing to provide adequate drug support. When the ALG concentration was 5 mg / mL, the formed hydrogel had moderate firmness, and its morphology did not significantly change within 0.5 hours. At ALG concentrations of 10 mg / mL and 20 mg / mL, the viscosity of ALG itself was too high, hindering injection, and the mixed drug... 2+When ejected from the solution, the needle tip rapidly gelled, resulting in a relatively hard gel that was unsuitable as a drug carrier in vivo. Therefore, the concentration of ALG in the composite hydrogel drug delivery system was determined to be 5 mg / mL.

[0098] Synthesis of a composite hydrogel drug delivery system.

[0099] like Figure 1 As shown in (B), when free DOX aqueous solution is injected into Ca 2+ In the buffer solution, the red DOX aqueous solution dissolved rapidly. When the composite hydrogel drug delivery system was injected into Ca... 2+ In the solution, red, blocky gel tissue was visible, and no obvious diffusion of red DOX solution was observed. After 24 hours, the formed hydrogel structure was dense and could be grasped with surgical forceps. ALG was observed to be present in the Ca-containing... 2+ A network-like hydrogel structure rapidly forms in the solution, immobilizing the drug within the gel and preventing its escape. After 24 hours, a red color appears in the solution, indicating that free DOX is gradually released from the hydrogel. This demonstrates that ALG in the composite hydrogel is similar to the normal concentration of Ca in the tumor. 2+ The reaction rapidly forms a network structure. Utilizing the unique biological properties of hydrogels, anti-tumor drugs are injected into the tumor, which uniformly disperses and fixes them within the tumor, allowing for slow release to reduce the dosage and minimize side effects.

[0100] Drug release in a composite hydrogel drug delivery system.

[0101] like Figure 2 As shown in (A), after a 24-hour constant-temperature shaker experiment, the composite hydrogel drug delivery system solution changed from colorless and transparent to red, demonstrating that the drug was released from ALG and Ca. 2+ Released during cross-linking. To further investigate the drug release capacity, the supernatant of each experimental group was measured using UV-Vis. Figure 2 As shown in (B)-(D), at 0 h, there were almost no UV-Vis absorption peaks due to the lack of drug release. After 24 h, the supernatants of DOX+ALG, L-Arg+ALG, and LPS+ALG solutions all showed varying degrees of absorbance, indicating that small amounts of DOX, L-Arg, and LPS were released from ALG. However, the absorbance was much lower than that of free ALG, DOX, and L-Arg. These results demonstrate that the gel has a non-compact structure and can serve as a drug delivery carrier.

[0102] In vitro NO release.

[0103] L-Arg, as a natural NO donor, can be used through methods including H2O2 and... 1The oxidation of ROS, including O2, into NO. Since the TME itself is a weakly acidic environment, in order to better test the ability of L-Arg to release NO as a NO donor, the release of NO by L-Arg at different time points was tested under neutral (pH 7.4) and weakly acidic (pH 6.5) environments.

[0104] like Figure 3 As shown in (A), under acidic conditions, the NO release in 12 hours is 3.75 times that under neutral conditions, the NO release in 24 hours is 7.52 times that under neutral conditions, and the NO release in 48 hours is 3.55 times that under neutral conditions. This proves that it is feasible to use L-Arg as a NO donor in the weakly acidic environment of tumors.

[0105] like Figure 3 As shown in (B), the amount of NO released by L-Arg increases with the increase of H2O2 concentration in the presence of ROS.

[0106] The effect of LPS stimulation on intratumoral ROS.

[0107] Both 4T1 breast cancer cells and 4T1 subcutaneous tumors showed significantly increased ROS levels after LPS stimulation.

[0108] The effect of LPS acute inflammatory stimulation on ROS in tumor cells.

[0109] H2O2 is the main ROS. To determine the effect of LPS acute inflammatory stimulation on intracellular ROS in tumor cells, 4T1 cells were incubated with LPS solutions of different concentrations, and the intracellular H2O2 content was measured after 24 hours.

[0110] like Figure 4 As shown in (A), inflammatory stimulation can increase the intracellular hydrogen peroxide content. Before inflammatory stimulation, the average intracellular hydrogen peroxide content in 4T1 cells was 4.86 μmol / L. After incubation with 0.25, 0.5, and 1 μg / L LPS for 24 h, the average intracellular hydrogen peroxide content in 4T1 cells was 10.99, 10.93, and 10.30 μmol / L, respectively. It can be seen that after incubating 4T1 cells with different concentrations of LPS for 24 h, the intracellular H2O2 content did not change significantly, but compared with 4T1 cells without LPS, the H2O2 content was significantly increased.

[0111] Changes in hydrogen peroxide content in 4T1 breast cancer cells at different time points after inflammatory stimulation.

[0112] like Figure 4As shown in (B), a small amount of green fluorescence signal was observed in the PBS control group 4T1 breast cancer cells, indicating a low level of free radicals. After 2 hours of LPS inflammatory stimulation, no significant change in green fluorescence was observed in the confocal image, indicating no significant change in the concentration of free radicals within the 4T1 breast cancer cells. After 12 hours of LPS inflammatory stimulation, a very bright green fluorescence signal appeared under the confocal microscope, indicating a significant increase in the concentration of free radicals within the 4T1 breast cancer cells after 12 hours of LPS inflammatory stimulation. After 24 hours of LPS inflammatory stimulation, relatively bright green fluorescence was still observed under the confocal microscope, indicating that the concentration of free radicals within the 4T1 breast cancer cells remained high after 12 hours of LPS inflammatory stimulation.

[0113] Changes in hydrogen peroxide content within tumors of Balb / c tumor-bearing mice at different time points after inflammatory stimulation.

[0114] like Figure 4 As shown in (C), the intratumoral H2O2 content changes over time after inflammatory stimulation. The intratumoral H2O2 content initially increases gradually, from 1.16 μmol / L at 6 h, 1.97 μmol / L at 12 h, reaching a peak of 3.54 μmol / L at 24 h after drug administration. The content then decreases to 1.16 μmol / L after 48 h. Based on the temporal changes in intratumoral H2O2 content, subsequent combination chemotherapy can be administered. The increase in H2O2 content not only promotes the conversion of L-Arg to NO, enhancing the therapeutic effect of gaseous therapy, but also acts as a signaling molecule triggering oxidative stress, increasing stress and damage to cancer cells, leading to enhanced cytotoxicity and apoptosis.

[0115] Changes in ROS in frozen sections of 4T1 breast tumors after LPS inflammatory stimulation.

[0116] Previous studies have investigated the ROS production of 4T1 breast cancer cells in response to LPS inflammatory stimulation. To further verify the oxidative stress induced by local LPS inflammatory stimulation, this study continues using 4T1 breast tumor tissue. Figure 4 As shown in (D), using DCFH-DA as a probe, tumor tissue was taken from 4T1 breast cancer-bearing mice after 24 hours of LPS inflammatory stimulation and frozen sections were prepared. After staining and observation, it was found that after inflammatory stimulation, green fluorescence covered the tumor sections, while the green fluorescence of the tumor in the control group was relatively weak, which proved that the ROS content in the tumor increased significantly after LPS stimulation.

[0117] Detection of cytotoxicity of composite hydrogel drug delivery system against 4T1 breast cancer cells.

[0118] The results of CCK8 cell viability and apoptosis assays showed that when gas therapy was used in combination with chemotherapy, the effect of inhibiting tumor cell growth was significantly enhanced.

[0119] In vitro toxicity test of gas therapy combined with chemotherapy on 4T1 breast cancer cells.

[0120] like Figure 5 As shown in (A): When 4T1 breast cancer cells were incubated with the chemotherapy drug DOX alone, the survival rate of 4T1 breast cancer cells was 80.43%. When 4T1 breast cancer cells were incubated with L-Arg and H2O2 to simulate gas therapy alone, the survival rate of 4T1 breast cancer cells was 94.09%. When 4T1 breast cancer cells were incubated with gas therapy and chemotherapy simultaneously, the survival rate of 4T1 breast cancer cells was 55.40%. It can be seen that when gas therapy and chemotherapy are used in combination, the effect of inhibiting tumor cell growth is significantly enhanced.

[0121] Apoptosis experiment.

[0122] like Figure 5 (B) shows that after incubation with low concentrations of DOX and NO (reaction of L-Arg with H2O2), some cells died (red). At the same dosage, the combined use of the two significantly increased the cell death rate. This demonstrates at the cellular level that the combined use of gas therapy and chemotherapy enhances tumor-killing activity. The results are consistent with the cytotoxicity test results of gas therapy combined with chemotherapy on 4T1 breast cancer cells.

[0123] Efficacy evaluation of composite hydrogel drug delivery system in Balb / c mice with subcutaneous tumors.

[0124] Figure 6 (A) is a photograph of the in situ tumor removed on day 15 of treatment, clearly showing the final tumor suppression effect. All treatment groups showed tumor suppression, with the LPS+DOX+L-Arg+ALG composite hydrogel drug delivery system exhibiting the most significant effect. This indicates that the experimental hypothesis of using the LPS+DOX+L-Arg+ALG composite hydrogel drug delivery system to induce TME changes through LPS, triggering the drug delivery system, combining chemotherapy and gas therapy for precise drug release, and promoting NO gas therapy to enhance chemotherapy sensitization, is feasible. Figure 6 (B) shows the tumor growth volume changes in 4T1 breast cancer-bearing mice after drug administration in different treatment groups. As shown, the changes in tumor volume 15 days after drug administration are basically consistent with the results of the in situ tumor images taken on day 15 of treatment. After 15 days of treatment, compared with the PBS control group, the tumor volume of all drug-treated mice was smaller, and tumor growth was slowed. Among them, the LPS+DOX+L-Arg+ALG composite hydrogel drug delivery system showed the slowest tumor growth and the smallest tumor volume. This indicates that all treatment groups had a tumor-suppressing effect. Figure 6(C) Tumor inhibition rates in 4T1 breast cancer-bearing mice after different treatment groups were shown. The tumor inhibition rates of the LPS+DOX+L-Arg group, LPS+L-Arg+ALG group, DOX+ALG group, DOX+L-Arg+ALG group, and LPS+DOX+L-Arg+ALG group were 19.43%, 23.70%, 35.13%, 51.18%, and 85.10%, respectively. It is evident that the LPS+DOX+L-Arg+ALG composite hydrogel drug delivery system exhibited the highest tumor inhibition rate. The LPS+DOX+L-Arg drug delivery group had the lowest tumor inhibition rate, indicating the loss of ALG and Ca2+. 2+ The three-dimensional network hydrogel structure formed by the reaction leads to rapid drug metabolism in the body, thus failing to achieve a good therapeutic effect. This also demonstrates that ALG is incompatible with the normal concentration of Ca within the tumor. 2+ The three-dimensional network hydrogel structure formed by the reaction plays a crucial role in the composite hydrogel drug delivery system. This structure can fix therapeutic drugs within the tumor, disperse them uniformly, and release them slowly, while also achieving precise drug delivery and reducing the required dosage.

[0125] Efficacy and safety evaluation of composite hydrogel drug delivery system in Balb / c mice with subcutaneous tumors.

[0126] To verify the biosafety of the composite hydrogel drug delivery system, the body weight of tumor-bearing mice was measured every other day during the experiment, and H&E staining was performed on important organs such as the heart, liver, spleen, lungs, and kidneys. Figure 7 (A) shows no significant difference in body weight among the tumor-bearing mice in each treatment group, suggesting that no drug-induced systemic toxicity led to weight loss. Figure 7 As shown in (B), no obvious pathological changes were observed in the cellular and tissue structures of the heart, liver, spleen, lungs, and kidneys. This suggests that the composite hydrogel drug delivery system has good biocompatibility.

[0127] In summary, the LPS+DOX+L-Arg+ALG composite hydrogel drug delivery system not only has significant anti-tumor effects, but also exhibits good biocompatibility.

Claims

1. A pharmaceutical composition for treating breast cancer, characterized in that, The composition comprises ALG, LPS, DOX, and L-Arg; the mixing ratio of ALG, LPS, DOX, and L-Arg is 50:1:10:10; the preparation method of the drug composition includes the following steps: preparing a mixed solution containing ALG, LPS, DOX, and L-Arg and sonicating in a water bath for 10 min to obtain a composite hydrogel drug delivery system; the drug composition interacts with the tumor microenvironment Ca through ALG. 2+ Cross-linking forms a three-dimensional network structure for loading and sustained release of LPS, DOX, and L-Arg; LPS stimulates the tumor microenvironment to produce ROS, and activates L-Arg to release NO under weakly acidic conditions, thereby achieving the combined drug action of gas therapy and chemotherapy.

2. The application of a hydrogel drug delivery system in the preparation of a medicament for treating breast cancer, characterized in that, The hydrogel drug delivery system includes ALG, LPS, DOX, and L-Arg; the mixing ratio of ALG, LPS, DOX, and L-Arg in the drug delivery system is 50:1:10:10; the preparation method of the drug includes the following steps: preparing a mixed solution containing ALG, LPS, DOX, and L-Arg and sonicating it in a water bath for 10 min to obtain the composite hydrogel drug delivery system.

3. The application as described in claim 2, characterized in that, The hydrogel drug delivery system induces NO production in the breast cancer tumor microenvironment and enhances the chemotherapeutic effect of DOX.

Images