Application of anti-tumor nano-drug with radiotherapy sensitization effect
Through the nanosensitizer AIBN@F127 encapsulated by azobisisobutyronitrile and Pluronic F127, combined with radiotherapy and immune checkpoint inhibitors, the drug resistance of triple-negative breast cancer to radiation therapy was solved, significantly improving the therapeutic effect and reduces damage to healthy tissue.
Patent Information
- Application Number
- CN202411185502.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-27
- Publication Date
- 2025-01-14
AI Technical Summary
The resistance of triple-negative breast cancer to traditional radiation therapy leads to poor treatment effects and is accompanied by damage to surrounding healthy tissues. The prior art lacks effective radiotherapy sensitizers to improve cancer cell sensitivity and reduce side effects.
Azobisisobutyronitrile and Pluronic F127 encapsulated nanosensitizer AIBN@F127 were used to generate azo radicals through X-ray activation, combining radiotherapy and immune checkpoint inhibitors to enhance tumor cytotoxicity and immune response.
Significantly inhibit the migration and growth of tumor cells, promote apoptosis, improve the targeted and safe nature of radiotherapy, enhance the ability of immunity to attack tumors, and jointly improve the therapeutic effect of breast cancer.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of an anti-tumor nano drug with radiotherapy sensitization effect. Background Art
[0002] Triple-negative breast cancer is an aggressive breast cancer subtype characterized by the loss of expression of estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2 (HER2), accounting for approximately 15% of all breast cancers. Current treatment options for TNBC include chemotherapy, surgery, and radiotherapy. Due to the endogenous H2O2, acidic pH, and hypoxic tumor microenvironment of TNBC, tumor cells have increased resistance to cancer treatments such as chemotherapy, radiotherapy (RT), and photodynamic therapy (PDT). This makes the treatment of TNBC more challenging than other subtypes of breast cancer.
[0003] Radiotherapy has many irreplaceable advantages and occupies an important position in cancer treatment. However, RT suffers from hypoxia-related radiation resistance induced by the complex tumor microenvironment. Nanomedicine has emerged as a promising approach to improve drug accuracy and reduce systemic toxicity. The development of nano-delivery platforms combining imaging angiography, magnetic / magnetic, drug chemotherapy, and immunotherapy has made the clinical treatment of TNBC more efficient and precise. By combining nanomedicine with radiotherapy, it is possible to improve the effectiveness of radiotherapy while minimizing damage to surrounding healthy tissues. This approach can help overcome the common radioresistance in TNBC and improve the overall response to treatment.
[0004] Azobisisobutyronitrile (AIBN) is an organic compound with the structural formula CAS No.: 78-67-1. Its working principle is that radiotherapy breaks its azo bond (N=N) to form azo free radicals to attack tumor cells. Studies have used similar drugs 2,2-azobis(2-methylpropionamide) hydrochloride (AIBA) to design nanosensitizer AIBA@MSNS based on sonodynamic therapy (SDT). It uses azo free radicals to induce tumor cell death under hypoxic conditions through non-oxygen free radical methods. Azobisisobutylimidazoline hydrochloride (AIBI) is combined with mesoporous copper sulfide (MCU) nanoparticles through infrared radiation. MCU uses near-infrared laser energy to induce photothermal therapy, converting light energy into heat to destroy uveal melanoma cells. The difference between radiotherapy and the other two is that radiotherapy has been used clinically to treat tumors. It is a common anti-tumor method with higher feasibility, and radiotherapy sensitizers have been used clinically. So far, there is no report on the combination of azobisisobutyronitrile and radiotherapy for tumor treatment.
[0005] Radiotherapy has been widely used in clinical practice. However, the efficacy of RT will be affected by the hypoxic microenvironment of the tumor, leading to tumor resistance to RT. At the same time, various serious side effects associated with RT remain one of the main challenges in clinical practice. How to make cancer cells more sensitive to RT while avoiding or minimizing damage to surrounding healthy tissues is an urgent problem that needs to be solved. In addition to many heavy metals (such as gold, platinum, and silver) that can amplify the radiation effect, azo radicals will also be an effective hypoxic tumor radiosensitizer. In recent years, the excellent efficacy of non-oxygen free radicals in the field of tumor treatment has attracted more and more attention. Studies have shown that azo radicals do not rely on the presence of oxygen. During the radiation process, the DNA damage caused by azo radicals is significantly enhanced. Summary of the invention
[0006] The purpose of the first aspect of the present invention is to provide the use of azobisisobutyronitrile in the preparation of a sensitizer, an anti-tumor drug with a sensitizing effect, or an anti-tumor drug carrier with a sensitizing effect.
[0007] The second aspect of the present invention aims to provide a radiosensitizer.
[0008] The third aspect of the present invention aims to provide a method for preparing the radiosensitizer of the second aspect of the present invention.
[0009] The purpose of the fourth aspect of the present invention is to provide the use of the radiosensitizer of the second aspect of the present invention.
[0010] The fifth aspect of the present invention is to provide a pharmaceutical composition.
[0011] The sixth aspect of the present invention aims to provide a reagent or a kit.
[0012] In order to achieve the above object, the technical solution adopted by the present invention is:
[0013] The first aspect of the present invention provides the use of azobisisobutyronitrile in preparing a sensitizer, preparing an anti-tumor drug with a sensitizing effect, or preparing an anti-tumor drug carrier with a sensitizing effect.
[0014] In some embodiments of the present invention, the sensitizer comprises a radiotherapy sensitizer.
[0015] In some embodiments of the present invention, the sensitizer is an X-ray radiotherapy sensitizer.
[0016] In some embodiments of the present invention, the azobisisobutyronitrile effectively inhibits tumor cell proliferation and migration and promotes tumor cell apoptosis through X-ray stimulation.
[0017] In some embodiments of the present invention, the tumor is at least one of esophageal cancer, head and neck cancer, nasopharyngeal cancer, ovarian cancer, lung cancer, gastric cancer, breast cancer, liver cancer, pancreatic cancer, colorectal cancer, skin cancer, malignant melanoma, bile duct cancer, bladder cancer or kidney cancer.
[0018] In some embodiments of the present invention, the tumor is breast cancer; further, it is triple-negative breast cancer.
[0019] The second aspect of the present invention provides a radiotherapy sensitizer, comprising azobisisobutyronitrile and a hydrogel, wherein the azobisisobutyronitrile is encapsulated in the hydrogel.
[0020] In some embodiments of the present invention, the hydrogel comprises Pluronic F127 and DSPE liposomes.
[0021] In some embodiments of the present invention, the particle size of the radiosensitizer is 125 to 225 nm.
[0022] In some preferred embodiments of the present invention, the particle size of the radiosensitizer is 150-200 nm.
[0023] The third aspect of the present invention provides a method for preparing the radiotherapy sensitizer of the second aspect of the present invention, comprising the following steps: dissolving azobisisobutyronitrile and hydrogel in the same volume of solvent respectively to obtain an azobisisobutyronitrile solution and a hydrogel solution;
[0024] Under ultrasonic conditions, the hydrogel solution is mixed with water and azobisisobutyronitrile solution is added to obtain a radiotherapy sensitizer.
[0025] In some embodiments of the present invention, the solvent is tetrahydrofuran.
[0026] In some embodiments of the present invention, the mass ratio of azobisisobutyronitrile to hydrogel is 1:8-15.
[0027] In some preferred embodiments of the present invention, the mass ratio of azobisisobutyronitrile to hydrogel is 1:8-12.
[0028] In some more preferred embodiments of the present invention, the mass ratio of azobisisobutyronitrile to hydrogel is 1:9-10.
[0029] In some embodiments of the present invention, after the azobisisobutyronitrile solution is added, it is volatilized for a certain period of time to remove tetrahydrofuran in the system.
[0030] A fourth aspect of the present invention provides a use of a radiosensitizer in any one of (1) to (9):
[0031] (1) Application in the preparation of drugs for treating or preventing tumors;
[0032] (2) Enhance the cytotoxicity of X-rays to tumor cells;
[0033] (3) Preparation of products that enhance the cytotoxicity of X-rays to tumor cells;
[0034] (4) Reduce the migration ability of tumor cells;
[0035] (5) preparing products that reduce the migration ability of tumor cells;
[0036] (6) Promote apoptosis of tumor cells;
[0037] (7) preparing products that promote apoptosis of tumor cells;
[0038] (8) Preparation of products that inhibit tumor growth;
[0039] (9) Prepare products that enhance the therapeutic effect of immune checkpoint inhibitors on tumors.
[0040] In some embodiments of the present invention, the tumor is at least one of esophageal cancer, head and neck cancer, nasopharyngeal cancer, ovarian cancer, lung cancer, gastric cancer, breast cancer, liver cancer, pancreatic cancer, colorectal cancer, skin cancer, malignant melanoma, bile duct cancer, bladder cancer or kidney cancer.
[0041] In some embodiments of the present invention, the tumor is breast cancer; further, it is triple-negative breast cancer.
[0042] In some embodiments of the present invention, the immune checkpoint inhibitor comprises an antibody, a fusion protein, an aptamer or an immune checkpoint protein binding fragment thereof.
[0043] The fifth aspect of the present invention provides a pharmaceutical composition comprising the radiosensitizer and immune checkpoint inhibitor of the second aspect of the present invention.
[0044] In some embodiments of the present invention, the immune checkpoint inhibitor comprises an antibody, a fusion protein, an aptamer or an immune checkpoint protein binding fragment thereof.
[0045] In some embodiments of the present invention, the immune checkpoint inhibitor is an anti-immune checkpoint protein antibody or an antigen-binding fragment thereof.
[0046] In some embodiments of the present invention, the immune checkpoint inhibitor can be at least one selected from the following: anti-CTLA4 antibody, its derivative or its antigen-binding fragment; anti-PD-L1 antibody, its derivative or its antigen-binding fragment; anti-LAG-3 antibody, its derivative or its antigen-binding fragment; anti-OX40 antibody, its derivative or its antigen-binding fragment; anti-TIM3 antibody, its derivative or its antigen-binding fragment; and anti-PD-1 antibody, its derivative or its antigen-binding fragment.
[0047] In some embodiments of the present invention, the immune checkpoint inhibitor may be at least one selected from the following: ipilimumab, its derivatives or antigen-binding fragments thereof; tremelimumab, its derivatives or antigen-binding fragments thereof; nivolumab, its derivatives or antigen-binding fragments thereof; pembrolizumab, its derivatives or antigen-binding fragments thereof; pidilizumab, its derivatives or antigen-binding fragments thereof; atezolizumab, its derivatives or antigen-binding fragments thereof; durvalumab, its derivatives or antigen-binding fragments thereof; avelumab, its derivatives or antigen-binding fragments thereof; BMS-936559, its derivatives or antigen-binding fragments thereof; BMS-986016, its derivatives or antigen-binding fragments thereof;
[0048] GSK3174998, its derivatives or antigen-binding fragments thereof; TSR-022, its derivatives or antigen-binding fragments thereof; MBG453, its derivatives or antigen-binding fragments thereof; LY3321367, its derivatives or antigen-binding fragments thereof; and IMP321 recombinant fusion protein. Any antibody that can be used as an immune checkpoint inhibitor or other types of immune checkpoint inhibitors can be used without restriction.
[0049] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
[0050] In some embodiments of the invention, the components in the pharmaceutical composition can be administered separately, or some or all of them can be administered together. The components in the pharmaceutical composition can be administered substantially at different times, or some or all of them can be administered substantially at the same time. The components in the pharmaceutical composition can have the same or different administration cycles.
[0051] In some embodiments of the present invention, the components of the pharmaceutical composition can be independently administered in various suitable ways, including but not limited to, oral or parenteral (through intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral administration routes, such as by injection or infusion). In some embodiments, the components of the pharmaceutical composition can be independently administered orally or by injection, such as intravenous injection or intraperitoneal injection.
[0052] In some embodiments of the present invention, the components in the pharmaceutical composition can each independently be in a suitable dosage form, including but not limited to tablets, lozenges, pills, capsules (e.g., hard capsules, soft capsules, enteric-coated capsules, microcapsules), elixirs, granules, syrups, injections (intramuscular, intravenous, intraperitoneal), emulsions, suspensions, solutions, dispersions and sustained-release preparations for oral or parenteral administration.
[0053] The sixth aspect of the present invention provides a reagent or a kit, comprising the radiotherapy sensitizer of the second aspect of the present invention. The beneficial effects of the present invention are:
[0054] The present invention uses azobisisobutyronitrile to prepare a sensitizer (X-ray radiotherapy sensitizer) for the first time, and uses Pluronic F127 (F127) to encapsulate azobisisobutyronitrile to obtain the radiotherapy sensitizer AIBN@F127. After X-ray irradiation, tumor cells take up AIBN@F127 and are activated, generating azo free radicals in situ, which has a strong anti-cancer effect. It can effectively kill TNBC cells, thereby mobilizing a strong anti-tumor immune ability.
[0055] Specifically, in vivo and in vitro experiments have shown that the radiosensitizer AIBN@F127 can enhance the toxic effect of X-rays on tumor cells, significantly inhibit the migration ability of tumor cells, promote tumor cell apoptosis, and inhibit tumor growth. At the same time, AIBN@F127+RT combined with PD-L1 inhibitors can synergistically improve the response rate of cancer cells and the therapeutic effect on breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS
[0056] Figure 1 Transmission electron microscope image of AIBN@F127.
[0057] Figure 2 This is the infrared spectrum of AIBN@F127.
[0058] Figure 3 Dynamic light scattering diagram of AIBN@F127.
[0059] Figure 4 The stability results of AIBN@F127.
[0060] Figure 5 The cytotoxicity evaluation results of AIBN@F127 at different concentrations and 4Gy x-ray irradiation; A is the cytotoxicity evaluation result of 4T1 cells, B is the cytotoxicity evaluation result of MDA-MD-231 cell line, and C is the colony experiment result.
[0061] Figure 6 The migration ability of 4T1 cells under different treatment conditions.
[0062] Figure 7 Cell apoptosis of 4T1 cells under different treatment conditions.
[0063] Figure 8 The ability of different treatment groups to release carbon free radicals stimulated by radiotherapy in a hypoxic environment.
[0064] Fig. 9The results of evaluating the combined enhancement effect of AIBN@F127 through intracellular carbon radical generation and DNA damage.
[0065] Fig.10 The therapeutic effects of different treatment groups on tumors; A is the experimental treatment flow chart, and B is the volume and weight of the tumor in 4T1 breast tumor model mice after treatment with different treatment groups. In the figure, ** represents P < 0.01, *** represents P < 0.005, and **** represents P < 0.001.
[0066] Fig.11 HE staining of major organs in 4T1 breast tumor model mice after treatment in different treatment groups.
[0067] Fig.12 These are the flow cytometry results of tumor tissues in 4T1 breast tumor model mice after treatment with different treatment groups. In the figure, *** represents P < 0.005, and **** represents P < 0.001.
[0068] Fig.13 These are the results of immunohistochemistry, HE staining, and TUNEL staining of tumor tissues in 4T1 breast tumor model mice after treatment in different treatment groups.
[0069] Fig.14 The therapeutic effect of AIBN@F127+RT combined with PD-L1 inhibitor on breast cancer; A is the experimental treatment flow chart, B is the tumor volume of mice in different treatment groups, and C is the tumor weight of mice in different treatment groups. In the figure, ** represents P < 0.01, *** represents P < 0.005, and **** represents P < 0.001. DETAILED DESCRIPTION
[0070] The present invention is further described in detail below through specific examples.
[0071] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.
[0072] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.
[0073] The BALB / c mice (female, 5 weeks old) used in the examples were purchased from the Experimental Animal Center of Southern Medical University.
[0074] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.
[0075] Example 1 Preparation of Nanosensitizer AIBN@F127
[0076] In this embodiment, 2,2′-azobis(isobutyronitrile) (AIBN) encapsulated with Pluronic F127 (denoted as F127, purchased from Shanghai McLean Company, with the item number Lot#: C13859796) was used to prepare the nanosensitizer AIBN@F127. The specific preparation process is as follows: 0.005 mg AIBN and 0.05 mg F127 were dissolved in 0.5 mL tetrahydrofuran, respectively. The tetrahydrofuran containing F127 was slowly added to 5 mL ddH2O (deionized water) under an ultrasonic environment, and then the tetrahydrofuran containing AIBN was added to the above solution under an ultrasonic environment, and evaporated for one day (the tetrahydrofuran was evaporated), and the solution was left with 5 mL, and the nanosensitizer AIBN@F127 was obtained.
[0077] Example 2 Structural characterization of nanosensitizer AIBN@F127
[0078] In this example, transmission electron microscopy, infrared spectroscopy and dynamic light scattering (DLS) were used to characterize the AIBN@F127 prepared above.
[0079] Transmission electron microscopy images Figure 1 As shown in Figure 1, AIBN@F127 is a spherical particle. Figure 2 As shown in the figure, the successful preparation of AIBN@F127 NPs was confirmed, and the characteristic peaks of AIBN and F127 were present. Figure 3 As shown, the particle size of AIBN@F127 is 160 nm.
[0080] AIBN@F127 was further stored in deionized water at 4°C for 30 days, during which the particle size was measured and the stability was investigated. Figure 4 As shown, there was no significant change in particle size after at least one month in deionized water.
[0081] Example 3: Verification of the effect at the cellular level
[0082] In this example, triple-negative breast cancer tumors (4T1 cells and MDA-MD-231 cell lines) were used to evaluate the cytotoxicity of AIBN@F127 at different concentrations and 4 Gy X-ray irradiation.
[0083] 1. Cell Viability Assay
[0084] 4T1 cells and MDA-MD-231 cells (denoted as 231 cells) were incubated in a 96-well plate for 24 h. 3The cells were placed in a 96-well plate at different densities. The cells were cultured in fresh medium at 37°C. After 24 h of attachment, AIBN@F127 was treated at concentrations of 0, 25, 50, 100, 200, and 300 μg / mL, respectively. After further incubation for 12 h in the RT group and AIBN@F127+RT group, the cells were irradiated with X-rays (4 Gy) and cultured for 24 h. The medium was discarded and 100 μL of fresh medium containing 10% Cell Counting Kit-8 reagent (FDbio Science) was added. After incubation for 1 to 4 h, the absorbance at 450 nm was measured using a microplate reader (Bio-Rad), and the cell viability was calculated according to the manufacturer's instructions.
[0085] The results showed that the half inhibitory concentration (IC 50 ) was 100 mg / L. Compared with AIBN@F127 or 4 Gy irradiation alone, 4 Gy X-ray combined with AIBN@F127 produced greater tumor cytotoxicity ( Figure 5 (A~B).
[0086] 2. Colony Assay
[0087] 4T1 cells were seeded in triplicate in six-well plates (1000 cells per well), then divided into four groups (n=3) and incubated at 37°C, 5% CO2 for 3 days. After the cells adhered, they were treated with complete medium containing AIBN@F127 (100 μg / mL). The cells were then irradiated with 4 Gy x-rays. The six-well plates were further incubated for 7 to 10 days until visible colonies were formed. Finally, the cells were fixed with 4% formalin for 15 minutes and stained with 0.5% crystal violet (Macklin) for 30 minutes. Finally, the colony survival rate of more than 50 cells was calculated to evaluate the effects of different treatments.
[0088] The results showed that colony experiments indicated that AIBN@F127 had enhanced X-ray properties ( Figure 5 Middle C).
[0089] After further cell counting, 2×10 5 The cells were suspended in 300 μL serum-free medium and inoculated in the upper chamber of the cells. 750 μL of complete medium containing 10% fetal bovine serum was added to the lower chamber. After 12 h of culture, AIBN@F127 and RT were added according to the different treatments of each group. After 24 h of culture, the chamber was removed, washed and dried. The cells were fixed with methanol for 15 min, dried at room temperature, and stained with hematoxylin for 15 min. After washing, the impermeable cells were removed with a cotton swab and dried again at room temperature. Three fields of view were randomly selected under a 200× optical lens, and the number of cells on the lower surface of the filter membrane was counted and the average value was taken.
[0090] The results showed that AIBN@F127+4Gy significantly impaired the migration ability of treated cells compared with other treatment groups ( Figure 6 ).
[0091] 3. Apoptosis Assay
[0092] To further verify the synergistic therapeutic effect of AIBN@F127 in vitro, Annexin V-FITC / PI staining and flow cytometry were used to detect cell apoptosis after different treatments.
[0093] The specific detection process is as follows: 4T1 cells were cultured in a 100 mm dish for 24 h and then treated with AIBN@F127 (100 μg / mL) for 12 h. The culture medium was then supplemented with culture medium. The cells in the RT group were irradiated with X-rays (4 Gy) and then cultured for 12 h. The apoptosis assay was performed using the Annexin V-FITC / PI apoptosis kit (E-CK-A211B-50Assays, Elabscience).
[0094] The results showed that AIBN@F127 combined with X-ray irradiation (4 Gy) induced significant apoptosis of 4T1 cells compared with the control group ( Figure 7 ).
[0095] WB experiments were further used to demonstrate the effect of the combined AIBN@F127+RT group on cell apoptosis. Specifically, 4T1 cells were cultured overnight in 35 mm tissue culture dishes and then treated with PBS or AIBN@F127 at 100 μg / mL for 12 h, followed by irradiation with 0 or 4 Gy. After 24 h, whole cell lysates were extracted from Western cell lysis buffer and RIPA buffer, and protease inhibitors and phosphatase inhibitors were added. The total protein concentration was determined using the bis-cholic acid method (Fdbio Science). The protein samples were separated on SDS-PAGE gels using a Western blotting instrument and then electrotransferred to a 0.22 mm PVDF membrane. The protein membrane was blocked in 5% bovine serum albumin (BSA) for 1 h and matched with the corresponding primary antibodies (anti-caspase-3, anti-bcl-2, anti-parp). The primary antibodies were incubated overnight at 4 °C. After washing three times with TBST, the membrane was incubated with the secondary antibody at room temperature for 1 h. It was then washed three times with TBST. The bands were then visualized on a BLT GelView 6000Pro using luminescent reagents and quantified using ImageJ software.
[0096] Radiotherapy stimulates the ability to release carbon free radicals in a hypoxic environment. Under hypoxic (1% O2) or normoxic (21% O2) conditions, radiotherapy-stimulated carbon free radicals can oxidize DCFH-DA to produce stronger fluorescence ( Figure 8 ).
[0097] The combined enhancement effect of AIBN@F127 was evaluated by intracellular carbon radical generation and DNA damage, respectively. 2',7'-dichlorofluorescein diacetate (DCFH-DA), a fluorescent probe (green fluorescence). Phosphorylated H2AX histone (i.e., γ-H2AX) was used as a key marker for radiation-induced DNA double-strand breaks, and the formation of γ-H2AX foci was further detected by immunofluorescence (green fluorescence). Compared with the control group, AIBN@F127+RT caused a higher level of γ-h2ax-positive foci in the cell nucleus, indicating that AIBN@F127 induced cancer cells that were highly sensitive to X-ray irradiation. Western blot results showed that the γ-H2AX level was significantly reduced in the AIBN@F127+RT treatment group ( Fig. 9 ).
[0098] Example 4
[0099] In this example, 4T1 cells were subcutaneously injected into mice to establish a primary 4T1 breast tumor model. After the model was successfully constructed, the mice were randomly divided into 4 groups, with n = 4 in each group, including a control group (no treatment), a group using AIBN@F127 alone, a group treated with X-rays, a group irradiated (4 Gy), and a group simultaneously given AIBN@F127 NPs and 4 Gy X-rays (i.e., AIBN@F127+RT group, AIBN@F127+4Gy group). Mice were treated with AIBN@F127 (at a dose of 25 mg / kg) or combined with radiotherapy (RT) every two days. Fig.10 (A).
[0100] The results are as follows Fig.10 As shown in Figure B, the tumor volume of the control group continued to increase, while the tumor growth of the AIBN@F127+RT group was significantly slowed down, and the effect was significantly better than that of AIBN@F127 or X-ray treatment alone, indicating that the combined treatment of RT and AIBN@F127 can significantly improve the therapeutic effect of breast tumors. The organs of mice in each group were further stained with HE. The results are shown in Figure 2. Fig.11 As shown, no obvious damage was found in the major organs of the AIBN@F127+RT group, indicating that the combined treatment of RT and AIBN@F127 is relatively safe.
[0101] Tumor tissues of mice in each group were taken and flow cytometry was performed. The results are as follows: Fig.12As shown, the combined use of AIBN@F127+RT in the tumor increased immune cell infiltration. After inoculation of 4T1 tumor cells, the tumor volume in the control group continued to increase, while free RT or AIBN@F127 had a certain inhibitory effect on tumor growth. In contrast, tumor growth in the AIBN@F127+RT group was significantly reduced. Modulating the immunosuppressive TME has great potential in enhancing anti-tumor T cell immunity. Compared with the control group (2.11% CD4 + T cells and 3.4% CD8 + Compared with AIBN@F127+RT, the content of effector T cells in tumor tissue increased significantly (7.14% CD4 + T cells and 7.19% CD8 + T cells).
[0102] Immunohistochemistry, HE staining, and TUNEL staining were performed on tumor tissue sections of mice in each group. Immunohistochemistry: Soak in a concentration gradient of xylene and ethanol successively. Use 0.1M sodium citrate solution (pH 6.0) to evaporate the antigen. Use 3% H202 solution to remove endogenous peroxidase activity, block with 5% bovine serum albumin solution, and incubate at room temperature for 60 minutes. According to the instructions, add an appropriate proportion of primary antibody and incubate overnight at 4°C. Incubate with the second antibody for 30 minutes and develop color with DAB. The sections were re-stained with hematoxylin for 5 seconds, dehydrated again with different concentrations of ethanol, sealed with xylene transparent neutral glue, and scanned with a slice scanner.
[0103] Through immunohistochemical analysis of the tumor microenvironment, the results are as follows Fig.13 As shown in Figure 2, the expression levels of PCNA and CD8 in the AIBN@F127+RT treatment group were found to be higher than those in the control group. Histological examination of tumor tissue sections showed that the tumor cells were regular in shape, uniform in size, and densely arranged. In addition, TUNEL staining showed that the tumor cells treated with AIBN@F127+RT had obvious apoptosis. In summary, these results indicate that AIBN@F127+RT treatment effectively inhibited tumor growth in vivo.
[0104] Example 5
[0105] The immune enhancement provided by immunogenic tumor cell death induced by AIBN@F127+RT may create a favorable environment for improving immune checkpoint inhibitor therapy. Therefore, this example studies whether AIBN@F127+RT combined with PD-L1 inhibitors can synergistically improve the response rate of cancer cells and the therapeutic effect on breast cancer. The details are as follows:
[0106] 4T1 tumors treated with AIBN@F127+RT combined with PD-L1 inhibitors in vivo ( Fig.14Middle A). The mice were randomly divided into 4 groups (n=4 in each group), namely 1) control group; 2) PD-L1 inhibitor treatment group (anti-PD-L1 antibody), PD-L1; 3) X-ray + PD-L1 inhibitor treatment group, 4Gy+PD-L1; 4) AIBN@F127 + X-ray irradiation treatment group, AIBN@F127+4Gy; 5) AIBN@F127 + PDL-1 inhibitor combined with X-ray irradiation treatment group, AIBN@F127+4Gy+PD-L1. In the experiment, we inoculated triple-negative breast cancer (TNBC) cells (4T1 cells) as the primary tumor on the right side of each mouse. One week later, the AIBN@F127+4Gy group and the AIBN@F127+4Gy+PD-L1 group were locally injected with 0.5mg kg -1 The mice were treated with 4 Gy of AIBN@F127 NPs and 4 Gy of X-ray irradiation was given on days 0, 2, 4, 6, and 8. Subsequently, PD-L1 inhibitors were intravenously injected into each tumor at a dose of 75 μL per mouse on days 1, 3, 5, and 7.
[0107] The results showed that AIBN@F127-based radiotherapy combined with PD-L1 inhibitor treatment can significantly inhibit tumor growth. In the AIBN@F127+4Gy+PD-L1 treatment group, tumor growth was significantly inhibited throughout the treatment period ( Fig.14 (B~C).
[0108] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.
Claims
1. Application of azobisisobutyronitrile in the preparation of sensitizers, anti-tumor drugs with sensitizing effects, or anti-tumor drug carriers with sensitizing effects.
2. The use according to claim 1, characterized in that: The sensitizer includes a radiotherapy sensitizer; Preferably, the sensitizer is an X-ray radiotherapy sensitizer.
3. A radiotherapy sensitizer, comprising azobisisobutyronitrile and a hydrogel, wherein the azobisisobutyronitrile is encapsulated in the hydrogel.
4. The radiosensitizer according to claim 3, characterized in that The hydrogel comprises PluronicF127 and DSPE liposome.
5. The radiosensitizer according to claim 3, characterized in that: The particle size of the radiosensitizer is 125-225 nm.
6. The method for preparing the radiosensitizer according to any one of claims 3 to 5, comprising the following steps: Dissolving azobisisobutyronitrile and hydrogel in the same volume of solvent respectively to obtain an azobisisobutyronitrile solution and a hydrogel solution; Under ultrasonic conditions, the hydrogel solution is mixed with water, and an azobisisobutyronitrile solution is added to obtain a radiotherapy sensitizer.
7. The preparation method according to claim 6, characterized in that: The solvent is tetrahydrofuran; Preferably, the mass ratio of azobisisobutyronitrile to hydrogel is 1:8-15.
8. Use of the radiosensitizer according to any one of claims 3 to 5 in any one of (1) to (9): (1) Application in the preparation of drugs for treating or preventing tumors; (2) Enhance the cytotoxicity of X-rays to tumor cells; (3) Preparation of products that enhance the cytotoxicity of X-rays to tumor cells; (4) Reduce the migration ability of tumor cells; (5) preparing products that reduce the migration ability of tumor cells; (6) Promote apoptosis of tumor cells; (7) preparing products that promote apoptosis of tumor cells; (8) Preparation of products that inhibit tumor growth; (9) Prepare products that enhance the therapeutic effect of immune checkpoint inhibitors on tumors; Preferably, the tumor is at least one of esophageal cancer, head and neck cancer, nasopharyngeal cancer, ovarian cancer, lung cancer, gastric cancer, breast cancer, liver cancer, pancreatic cancer, colorectal cancer, skin cancer, malignant melanoma, bile duct cancer, bladder cancer or kidney cancer.
9. A pharmaceutical composition comprising the radiosensitizer according to any one of claims 3 to 5 and an immune checkpoint inhibitor; Preferably, the immune checkpoint inhibitor comprises an antibody, a fusion protein, an aptamer or an immune checkpoint protein binding fragment thereof.
10. A reagent or kit comprising the radiosensitizer according to any one of claims 3 to 5.