Nanoparticles for cascade reaction to enhance radioimmunotherapy effect and application thereof

Abstract

The invention belongs to the technical field of biomedicine, and discloses a nano-particle for enhancing radioimmunotherapy effect through cascade reaction and application of the nano-particle, a preparation method of the nano-particle comprises the following steps: S1, synthesis of ZnO2; the preparation method comprises the following steps: dissolving ZnCl2 and PVP in absolute ethyl alcohol, adding ammonia water and H2O2, after the reaction is completed, collecting precipitates, washing, and dispersing the precipitates in absolute ethyl alcohol to prepare ZnO2; and S2, synthesis of ZnO2 (at) Cu: resuspending ZnO2 in water, sequentially adding ammonia water, a PAA solution, IPA, dopamine hydrochloride and a CuCl2 solution, stirring in a nitrogen atmosphere, centrifuging, washing with ethanol, and drying in vacuum to obtain the ZnO2 (at) Cu nano-particles, namely the nano-particles. According to the nanoparticles, the radiotherapy effect is enhanced through cascade reaction, copper death is induced, immune response is activated, effective treatment on tumors is achieved, and the problems of radiotherapy resistance and immunosuppressive tumor microenvironment are solved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to a nanoparticle for enhancing the effect of radioimmunotherapy by cascade reaction and its application. Background Art

[0002] Cancer is one of the main causes of death globally, and the incidence of breast cancer in women has been showing a steady upward trend in recent years. As an adjuvant treatment method after non-metastatic breast cancer surgery, radiotherapy plays an important role in eliminating residual tumor cells and preventing recurrence. However, radiation resistance severely limits the radiotherapy dose, and at the same time, the immunosuppressive tumor microenvironment hinders the effects of radiotherapy and immunotherapy. Therefore, it is necessary to design a treatment plan that can enhance the radiotherapy sensitivity of tumor tissues while protecting surrounding normal tissues.

[0003] In recent years, treatment strategies targeting ion metabolism have attracted much attention. Among them, copper ions play a key role in maintaining the intracellular redox balance after radiotherapy. Disrupting copper metabolism can trigger cuproptosis, which provides a new approach for cancer treatment. Summary of the Invention

[0004] The purpose of the present invention is to provide a nanoparticle for enhancing the effect of radioimmunotherapy by cascade reaction and its application. This nanoparticle enhances the radiotherapy effect through cascade reaction, induces cuproptosis, and activates the immune response to achieve effective treatment of tumors, overcoming the problems of radiotherapy resistance and immunosuppressive tumor microenvironment.

[0005] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0006] In the first aspect, the present invention provides a nanoparticle for enhancing the effect of radioimmunotherapy by cascade reaction, and its preparation method includes the following steps:

[0007] S1. Synthesis of ZnO2: Dissolve ZnCl2 and PVP in absolute ethanol, add ammonia water and H2O2. After the reaction is completed, collect the precipitate, wash it, and disperse it in absolute ethanol to obtain the ZnO2.

[0008] S2. Synthesis of ZnO2@Cu: Resuspend the ZnO2 in water, sequentially add ammonia water, PAA solution, IPA, dopamine hydrochloride, and CuCl2 solution, stir in a nitrogen atmosphere, and obtain ZnO2@Cu nanoparticles by centrifugation, ethanol washing, and vacuum drying, which are the nanoparticles.

[0009] In the second aspect, the present invention provides the application of the above nanoparticle in the preparation of radioimmunotherapy drugs.

[0010] In the third aspect, the present invention provides the application of the above nanoparticle in the preparation of drugs for treating tumors.

[0011] In the above technical solutions, the tumors include breast cancer, lung cancer, gastric cancer, colorectal cancer, oral cancer, liver cancer, cervical cancer, esophageal cancer, lymphoma, prostate cancer, thyroid cancer, brain tumor or pancreatic cancer.

[0012] In a fourth aspect, the present invention provides a drug containing the above-mentioned nanoparticles.

[0013] The beneficial effects of the present invention are as follows:

[0014] The ZnO3@Cu nanoparticles of the present invention can release copper ions and ZnO2 in the tumor microenvironment, regulate the microenvironment, and increase the sensitivity of tumor cells to radiotherapy.

[0015] Copper ions induce cuproptosis by consuming GSH and generating ROS, and cooperate with radiotherapy to enhance the tumor treatment effect.

[0016] The combined application of the nanoparticles and radiotherapy can induce ICD, promote the maturation of DC and the activation of CD8 + T cell activation, effectively activate the immune response, and further inhibit tumor growth.

[0017] In vivo experiments have proved that ZnO2@Cu has good biosafety, providing a basis for its clinical application. Description of the Drawings

[0018] Figure 1 : Characterization of ZnO2@Cu, where: (A - B) Transmission electron microscope (TEM) images of ZnO2@Cu (A) and ZnO2 (B) nanoparticles; (C) Zeta potential of ZnO2@Cu and ZnO2; (D) Particle size change of ZnO2@Cu within 7 days; (E - F) X-ray photoelectron spectroscopy (XPS) full spectra of ZnO2@Cu (E) and ZnO2 (F); (G) Absorption spectra of 3,3',5,5'-tetramethylbenzidine (TMB) solution containing H2O2 over time after adding ZnO2@Cu.

[0019] Figure 2 : In vitro anti-tumor evaluation, where: (A) Consumption of glutathione (GSH) in 4T1 cells after different treatments; (B) Survival fraction of the colony formation assay after 4T1 cells were incubated with different formulations; (C) Western blot analysis of FDX1 expression in 4T1 cells after different treatments; (D - G) Immunofluorescence images of DLAT staining (D), DNA damage (E), reactive oxygen species (ROS) staining (F), and live / dead staining (G) under different treatment formulations.

[0020] Figure 3:In vitro immunogenic cell death (ICD) induction and immune activation, where: (A) Schematic diagram of in vitro ICD effect; (B-C) Immunofluorescence staining of CRT (B) and HMGB1 (C) in 4T1 cells after different treatments; (D) Release of HMGB1 in 4T1 cells; (E) Percentage of dendritic cell (DC) maturation after different treatments.

[0021] Figure 4 :In vivo anti-tumor performance, where: (A-C) Body weight fluctuations (A), tumor growth curves (B), and survival curves (C) of different treatment groups; (D-G) Immunofluorescence staining of FDX1 (D), ROS (E), TUNEL (F), and H&E (G) in different treatment groups.

[0022] Figure 5 :In vivo systemic anti-tumor immunity, where, (A) Immunofluorescence staining of CD8 + ; (B) Contents of TNF-α, (C) IL-6, and (D) IFN-γ in different treatment groups.

[0023] Figure 6 :In vivo biosafety, where (A-C) Damage indicators on the 15th day, including creatinine (CRE) (A), blood urea nitrogen (BUN) (B), and liver function indicators (C).

[0024] Figure 7 :Flow cytometry analysis of CD8 + T cells in the CD3 + T cell population.

[0025] Figure 8 :Flow cytometry analysis of mature DCs in tumor-draining lymph nodes. Detailed implementation manners

[0026] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the present invention to those skilled in the art. The present invention will only be defined by the claims.

[0027] The present invention provides a nanoparticle for enhancing the effect of radioimmunotherapy by cascade reaction, and its preparation method includes the following steps:

[0028] S1. Synthesis of ZnO2; Dissolve ZnCl2 and PVP in absolute ethanol, add ammonia water and H2O2, after the reaction is completed, collect the precipitate, wash it, and disperse it in absolute ethanol to obtain ZnO2.

[0029] S2. Synthesis of ZnO2@Cu: Resuspend ZnO2 in water, and successively add ammonia water, PAA solution, IPA, dopamine hydrochloride and CuCl2 solution, stir under a nitrogen atmosphere, and obtain ZnO2@Cu nanoparticles by centrifugation, ethanol washing and vacuum drying, which are the nanoparticles of the present invention.

[0030] The nanoparticles of the present invention can be used to enhance the effect of radioimmunotherapy and treat tumors, and synergistically enhance the effect through multiple mechanisms. The specific uses are as follows:

[0031] Enhancing the radiotherapy effect: In the tumor microenvironment, ZnO2@Cu will decompose and release copper ions and ZnO2. Copper ions can consume glutathione (GSH) in tumor cells, weaken the self-repair ability of tumor cells, and thus enhance the sensitivity of tumor cells to radiotherapy. At the same time, ZnO2 can produce a large amount of H2O2, providing a material basis for subsequent reactions. During radiotherapy, the two cooperate with each other to cause greater damage to tumor cells.

[0032] Inducing cuproptosis: The released copper ions will trigger cuproptosis, and the process includes causing the aggregation of lipoylated proteins and the loss of Fe-S clusters, triggering severe cytotoxic stress, and promoting the death of tumor cells. And copper ions are reduced to Cu by consuming FDX1 + ,Cu + reacts with H2O2 produced by ZnO2 to generate toxic reactive oxygen species (ROS), further inducing cell death and exacerbating the death process of tumor cells.

[0033] Activating the immune response: While ZnO2@Cu and radiotherapy act together to induce cuproptosis in tumor cells, it will promote immunogenic cell death (ICD). This will cause tumor cells to release damage-associated molecular patterns (DAMPs), such as calreticulin (CRT), high-mobility group protein B1 (HMGB1) and adenosine triphosphate (ATP), etc. These DAMPs can promote the maturation of dendritic cells (DCs), activate CD8 + T cells, trigger a systemic immune response, and enhance the body's immune clearance ability against tumor cells.

[0034] Example 1

[0035] A nanoparticle for enhancing the effect of radioimmunotherapy by cascade reaction, and its preparation method includes the following steps:

[0036] Synthesis of zinc oxide (ZnO2):

[0037] Zinc chloride (ZnCl2, 135 mg) and polyvinylpyrrolidone (PVP, 690 mg) were dissolved in 30 mL of absolute ethanol under ultrasonic conditions. 2 mL of ammonia water (10 mol / L) was added, and 2 mL of 30% hydrogen peroxide was slowly added dropwise with stirring (dropwise speed = 200 μL / min), and the reaction was continued for 24 hours. Subsequently, the precipitate was collected, washed with ethanol, and then redispersed and stored in absolute ethanol.

[0038] Synthesis of zinc dioxide@copper (ZnO2@Cu):

[0039] Zinc dioxide (ZnO2, 5 mg) was resuspended in 5 mL of ultrapure water and sonicated for 10 minutes. 75 μL of ammonia water (2 mol / L) and 50 μL of polyacrylic acid (PAA) solution with a concentration of 200 μg / mL were added in sequence, and the mixture was stirred for 10 minutes. 25 mL of isopropyl alcohol (IPA) was slowly added dropwise (dropwise speed = 100 μL / min), followed by the addition of 250 μL of ammonia water and 125 μL of dopamine hydrochloride (concentration of 50 μg / mL). Then, a solution containing copper chloride (CuCl2, 1.0 mg / mL, concentration of 10 mmol / L) was added, and the resulting mixture was stirred under a nitrogen atmosphere for 1 hour. Zinc dioxide@copper (ZnO2@Cu) nanoparticles were obtained by centrifugation, ethanol washing, and vacuum drying.

[0040] Example 2

[0041] A nanoparticle for enhancing the effect of cascade reaction in radioimmunotherapy, and its preparation method includes the following steps:

[0042] Synthesis of zinc dioxide (ZnO2):

[0043] Zinc chloride (ZnCl2, 125 mg) and polyvinylpyrrolidone (PVP, 670 mg) were dissolved in 30 mL of absolute ethanol under ultrasonic conditions. 2 mL of ammonia water (10 mol / L) was added, and 2 mL of 30% hydrogen peroxide was slowly added dropwise with stirring (dropwise speed = 200 μL / min), and the reaction was continued for 24 hours. Subsequently, the precipitate was collected, washed with ethanol, and then redispersed and stored in absolute ethanol.

[0044] Synthesis of zinc dioxide@copper (ZnO2@Cu):

[0045] Redissolve zinc dioxide (ZnO2, 6 mg) in 8 mL of ultrapure water and sonicate for 10 minutes. Sequentially add 75 μL of ammonia water (2 mol / L) and 50 μL of polyacrylic acid (PAA) solution with a concentration of 200 μg / mL, and stir for 10 minutes. Slowly add 25 mL of isopropyl alcohol (IPA, dropping rate = 100 μL / min) dropwise, then add 250 μL of ammonia water and 125 μL of dopamine hydrochloride (concentration of 50 μg / mL). Next, add a solution containing copper chloride (CuCl2, 1.0 mg / mL, concentration of 10 mmol / L), and stir the resulting mixture under a nitrogen atmosphere for 1 hour. Zinc dioxide@copper (ZnO2@Cu) nanoparticles are obtained by centrifugation, ethanol washing, and vacuum drying.

[0046] Example 3

[0047] A nanoparticle for enhancing the effect of cascade reaction in radioimmunotherapy, and its preparation method includes the following steps:

[0048] Synthesis of zinc dioxide (ZnO2):

[0049] Dissolve zinc chloride (ZnCl2, 135 mg) and polyvinylpyrrolidone (PVP, 700 mg) in 30 mL of absolute ethanol under ultrasonic conditions, add 2 mL of ammonia water (10 mol / L), and slowly add 3 mL of 30% hydrogen peroxide dropwise (dropping rate = 200 μL / min) under stirring, and continue the reaction for 24 hours. Then collect the precipitate, wash it with ethanol, and redisperse it in absolute ethanol.

[0050] Synthesis of zinc dioxide@copper (ZnO2@Cu):

[0051] Redissolve zinc dioxide (ZnO2, 8 mg) in 10 mL of ultrapure water and sonicate for 10 minutes. Sequentially add 75 μL of ammonia water (2 mol / L) and 60 μL of polyacrylic acid (PAA) solution with a concentration of 200 μg / mL, and stir for 10 minutes. Slowly add 30 mL of isopropyl alcohol (IPA, dropping rate = 100 μL / min) dropwise, then add 250 μL of ammonia water and 135 μL of dopamine hydrochloride (concentration of 50 μg / mL). Next, add a solution containing copper chloride (CuCl2, 1.0 mg / mL, concentration of 10 mmol / L), and stir the resulting mixture under a nitrogen atmosphere for 1 hour. Zinc dioxide@copper (ZnO2@Cu) nanoparticles are obtained by centrifugation, ethanol washing, and vacuum drying.

[0052] The nanoparticles of the present invention and their applications will be described in detail below in conjunction with the examples.

[0053] Use the nanoparticles prepared in Example 1 for the following experiments.

[0054] Characterization of Nanoparticles

[0055] As Figure 1 shown, the morphology of the nanoparticles was observed using transmission electron microscopy (TEM), showing that ZnO2 was spherical nanoparticles with an average diameter of 210.5 nm, and ZnO2@Cu had a coating on the surface of ZnO2. Elemental analysis was carried out by X-ray photoelectron spectroscopy (XPS), confirming the presence of copper element in ZnO2@Cu. Dynamic light scattering (DLS) was used to evaluate the stability, and the results showed that the average diameter of ZnO2@Cu remained stable within one week.

[0056] Its ability to generate ROS was verified by testing its reaction with 3,3',5,5'-tetramethylbenzidine (TMB) under simulated tumor microenvironment conditions.

[0057] In Vitro Experiments

[0058] Cell culture: The 4T1 murine breast cancer cell line was used and cultured in RPMI-1640 medium.

[0059] Evaluation of the synergistic effect of cuproptosis and radiotherapy: As Figure 2 shown, the intracellular GSH content was detected in different treatment groups (PBS, RT, ZnO2@Cu, ZnO2+RT, ZnO2@Cu+RT). The results showed that the GSH content in the ZnO2@Cu+RT group was significantly decreased, indicating that it promoted cuproptosis. Western blot analysis of FDX1 expression and DLAT aggregation showed that FDX1 was significantly down-regulated and DLAT aggregation was obvious in the ZnO2@Cu+RT group, indicating that this group effectively triggered cell cuproptosis.

[0060] Cell death detection: As Figure 2 shown, γ-H2AX staining, ROS generation detection (using DCFH-DA), cell viability detection (using calcein-AM / PI staining) and colony formation assay were carried out. The results showed that the ZnO2@Cu+RT group had severe DNA damage, increased ROS generation, decreased cell viability and stronger inhibitory effect on tumor cells, indicating that ZnO2@Cu enhanced the sensitivity of tumor cells to radiation.

[0061] Detection of immunogenic cell death (ICD) induction: As Figure 3 shown, immunofluorescence staining of CRT and HMGB1 was performed on cells in different treatment groups. It was found that the secretion of CRT and HMGB1 in the ZnO2@Cu+RT group was enhanced. The treated 4T1 cells were co-cultured with dendritic cells (DC), and the maturation of DC was analyzed by flow cytometry. The results showed that the maturation of DC in the ZnO2@Cu+RT group was significantly enhanced, indicating that this group promoted ICD.

[0062] In Vivo Experiments

[0063] Animal model establishment: Female Balb / c mice at 5 - 6 weeks of age were used, and a tumor model was constructed by subcutaneous injection of 4T1 cells.

[0064] Therapeutic effect evaluation: As Figures 4-5 shown, the mice were divided into PBS, RT, ZnO2@Cu, ZnO2+RT, and ZnO2@Cu+RT groups. After treatment, body weight, tumor volume, and survival time were monitored. The results showed that the ZnO2@Cu+RT group had a significant tumor inhibitory effect and the longest survival time. Immunofluorescence staining of FDX1, ROS detection, TUNEL, and H&E staining of tumor tissues were performed to confirm that this group induced cuproptosis and obvious tumor apoptosis and necrosis. As Figures 6-7 shown, CD8 + T cells were detected by immunofluorescence staining, and CD8 + T cells in the CD3 + T cell population were analyzed by flow cytometry, and the DC maturation level and the secretion of cytokines (IL-6, TNF-α, and IFN-γ) in serum were detected. The results showed that the immune response was enhanced in the ZnO2@Cu+RT group.

[0065] Toxicity detection: As Figure 8 shown, blood biochemical indexes and histological changes of major organs were detected after injecting ZnO2@Cu into healthy mice. The results showed no obvious systemic toxicity.

[0066] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of this invention.

Claims

1. A nanoparticle for enhancing the effect of cascade reaction in radioimmunotherapy, characterized in that: The preparation method comprises the following steps: S1. Synthesis of ZnO2: Dissolve ZnCl2 and PVP in absolute ethanol, add ammonia water and H2O2. After the reaction is completed, collect the precipitate, wash it, and disperse it in absolute ethanol to obtain the ZnO2. S2. Synthesis of ZnO2@Cu: Resuspend the ZnO2 in water, and successively add ammonia water, PAA solution, IPA, dopamine hydrochloride, and CuCl2 solution. Stir under a nitrogen atmosphere, and obtain ZnO2@Cu nanoparticles by centrifugation, ethanol washing, and vacuum drying, which are the nanoparticles.

2. Use of the nanoparticles according to claim 1 in the preparation of radioimmunotherapy drugs.

3. Use of the nanoparticles according to claim 1 in the preparation of drugs for treating tumors.

4. The application according to claim 3, wherein: The tumors include breast cancer, lung cancer, gastric cancer, colorectal cancer, oral cancer, liver cancer, cervical cancer, esophageal cancer, lymphoma, prostate cancer, thyroid cancer, brain tumor, or pancreatic cancer.

5. A drug, characterized in that: It contains the nanoparticles according to claim 1.

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