Multi-mode synergistic interaction nanoparticle as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of breast cancer, and particularly relates to a multi-mode synergistic nanoparticle and a preparation method and application thereof.The nanoparticle comprises a zeolite imidazate framework material 8, artesunate and glucose oxidase are wrapped in the zeolite imidazate framework material 8, and the preparation method comprises the steps that artesunate is weighed to be dissolved in N, N-dimethyl formamide, and the mixture is stirred to be uniform; the preparation method comprises the following steps: dissolving artesunate and glucose oxidase in a N, N-dimethylformamide solution to obtain a medicine storage concentrated solution, weighing dimethylimidazole and dissolving in deionized water, weighing zinc nitrate hexahydrate and dissolving in deionized water, adding the weighed storage concentrated solution of glucose oxidase and artesunate into the dimethylimidazole solution and stirring, then adding the zinc nitrate hexahydrate solution, and uniformly stirring to obtain a medicine liquid; according to the preparation method, the drug resistance of a single chemotherapeutic drug is overcome, multi-mode synergistic treatment can be achieved, the tumor microenvironment can be adjusted or utilized at the same time, and the preparation method has important research significance and application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of breast cancer, and particularly relates to a multi-mode synergistic nanoparticle and a preparation method and application thereof. BACKGROUND

[0002] Breast cancer is a common malignant tumor in women, and has a high morbidity and mortality, seriously threatening the life and health of women. At present, in addition to surgical treatment, chemotherapy is still one of the main treatment methods for breast cancer. However, the toxic side effects and drug resistance of chemotherapeutic drugs seriously affect the treatment effect. Therefore, it is of great significance to find an efficient and low-toxic anti-breast cancer drug for improving the treatment effect and prolonging the survival period of patients. In recent years, researchers have found that traditional Chinese medicine has a long history of treating tumors, has the advantages of low toxicity and low cost, and some extracted components of traditional Chinese medicine have shown significant anti-tumor effects and have been successfully applied in clinical practice. This shows that developing Chinese herbal medicine as a new type of anti-tumor drug has great potential, and artemisinin is a typical representative. Artemisinin is an effective component extracted from the plant Artemisia annua. Its various derivatives (such as ART) not only have significant effects on severe and drug-resistant malaria, but also have immunomodulatory, anti-inflammatory, antioxidant and anti-tumor effects. However, although artemisinin and its derivatives show potential in anti-tumor, their clinical application still faces many challenges. For example, chemotherapeutic drugs generally lack tumor specificity and space-time controllability, and have poor water solubility, which easily leads to serious toxic side effects, limiting their wide application. Therefore, developing an efficient and safe drug delivery system has become an important research direction for the application of artemisinin and its derivatives in the field of tumor treatment. SUMMARY

[0003] In order to solve the problems in the prior art, the application provides a multi-mode synergistic nanoparticle and a preparation method and application thereof. By delivering ART and protein material GOx through ZIF-8, a stable nanoparticle is formed by self-assembly, which can realize multi-mode synergistic treatment, simultaneously regulate or utilize the tumor microenvironment, and has important research significance and application value. This platform not only overcomes the limitations of ART, but also enhances the anti-tumor effect through synergistic effect, providing a new idea and method for tumor treatment.

[0004] The technical problem of the application is solved by the following technical scheme:

[0005] The application aims to provide a multi-mode synergistic nanoparticle, which comprises zeolite imidazolate framework 8, and artemether and glucose oxidase are loaded in the zeolite imidazolate framework 8.

[0006] Artesunate (ART) not only has anti-malaria and anti-tumor activity, but also is an effective iron death inducer. However, its inherent physicochemical properties, such as poor water solubility, low biocompatibility and rapid in vivo metabolism, limit its clinical application. Glucose oxidase (GOx) has a significant deoxidation capacity. As a biological catalyst, GOx can consume the nutrients required by tumors in the tumor microenvironment, while increasing the levels of acidity, hypoxia and H2O2, which will delay tumor growth and division, and even high concentrations of hydrogen peroxide will cause tumor death. By reducing the glucose concentration in the tumor, GOx can effectively block the energy supply of tumor cells, thereby achieving starvation therapy.

[0007] Due to the unique microenvironment of tumor cells, it is found that zeolitic imidazolate framework 8 (ZIF-8) in ZIFs is stable under neutral conditions and has a low degradation rate, while in an acidic environment, the structure of ZIF-8 rapidly disintegrates, releasing more than 98% of Zn 2+ and the encapsulated guest molecules at the same time. This is because the acidic environment destroys the coordination bond between zinc and imidazole acid salt, causing the disintegration of ZIF-8 structure. Therefore, using ZIF-8 as a pH-responsive drug carrier can effectively reduce the non-specific release of drugs during blood circulation and promote the specific release of drugs at the tumor site, thereby achieving precise treatment of tumors. In addition, ZIF-8 also has high porosity, large specific surface area, good biodegradability, excellent mechanical and thermal stability, and adjustable surface properties, and can load a variety of proteins and maintain their biological activity.

[0008] The present application constructs a self-assembled nanoparticle based on the combination of GOx starvation therapy and ART chemotherapy. By self-assembly method, the drug is loaded in ZIF-8, which not only realizes high encapsulation efficiency, but also protects the protein from external stimulation. The present nanoparticle not only overcomes the drug resistance of single chemotherapy drugs, but also combines with starvation therapy, which plays a synergistic effect in inhibiting cancer, and further realizes deep and multi-directional inhibition of tumors.

[0009] Further, the mass ratio of artesunate and glucose oxidase is 3:1-2.5 (such as 3:1, 3:1.5, 3:2, 3:2.5).

[0010] Further, it also includes a red blood cell membrane, and the red blood cell membrane is coated on the surface of the zeolitic imidazolate framework 8.

[0011] The application discloses a preparation method of a multi-mode synergistic nanoparticle, and belongs to the technical field of medicine.

[0012] Further, under the condition of room temperature and the stirring speed of 1000-1500 rpm (for example, 1000 rpm, 1100 rpm, 1200 rpm, 1300 rpm, 1400 rpm or 1500 rpm), the prepared glucose oxidase and the concentrated solution of artesunate are added into the dimethylimidazole solution and stirred for 3-7 min (for example, 3 min, 4 min, 5 min, 6 min or 7 min), then the prepared zinc nitrate hexahydrate solution is added into the dimethylimidazole solution under the same stirring speed, and the stirring is continuously carried out at room temperature for 8-12 min (for example, 8 min, 9 min, 10 min, 11 min or 12 min).

[0013] Further, the method further comprises the following steps: the red blood cell membrane is subjected to ultrasonic treatment, the prepared ART / GOx@ZIF-8 is mixed with a red blood cell membrane solution, then the mixture is subjected to ultrasonic treatment, the mixture is extruded by using an extruder, filtration is carried out, and the excessive red blood cell membrane is removed by centrifugation, so that the red blood cell membrane coated nanoparticle is obtained.

[0014] Further, the ART / GOx@ZIF-8 is mixed with the red blood cell membrane solution at a ratio of 1:1, then the mixture is subjected to ultrasonic treatment for 50-70 s (for example, 50 s, 55 s, 60 s, 65 s or 70 s), the mixture is extruded back and forth by using an extruder for 8-12 times (for example, 8 times, 9 times, 10 times, 11 times or 12 times), the mixture is sequentially passed through polycarbonate membranes with the pore sizes of 400 nm, 200 nm and 100 nm, the excessive red blood cell membrane is removed by centrifugation, and the red blood cell membrane coated nanoparticle is obtained.

[0015] The application further discloses an application of the multi-mode synergistic nanoparticle or the preparation method of the multi-mode synergistic nanoparticle to the preparation of an antitumor product.

[0016] Compared with the prior art, the application has the beneficial technical effects that:

[0017] 1. The pH-responsive multimodal synergistic nanoparticles constructed by the present invention fully utilize the multiple effects of GOx starvation therapy, ART chemotherapy, Fenton reaction, etc. to overcome the drug resistance of single chemotherapy drugs. In addition, the traditional Chinese medicine ART is not only low-toxic and inexpensive, but also as an inducer of ferroptosis, it can induce ferritin autophagy and release unstable iron, thereby achieving multi-level and multi-mode tumor inhibition.

[0018] 2. The present invention utilizes ZIF-8 to specifically cleave and release drug-loaded substances in an acidic environment. At the same time, its advantages such as high porosity and huge surface area can stably encapsulate drugs and improve the effective release of anti-tumor drugs in tumor sites or tumor tissues, thereby achieving specific targeting and precise treatment of tumors. The Zn released by itself 2+ It can further inhibit the glycolysis of tumor cells and form a synergistic anti-cancer effect with starvation therapy.

[0019] 3. Based on the heterogeneity of TNBC in the process of ferroptosis, this invention innovatively combines ART (iron ion inducer), GOx and Zn 2+ ①In ART and Zn 2+ Under the synergistic effect of ferric ions, the iron ion level is effectively induced to increase, thereby activating and enhancing the ferroptosis process. ② The induced generation of unstable divalent iron (Fe 2+ ) can undergo a Fenton reaction with H2O2 produced by GOx, further enhancing the inhibitory effect on tumor cells. This strategy not only achieves efficient activation of ferroptosis but also enhances the anti-tumor effect through chemodynamic therapy, providing a new approach for the treatment of TNBC.

[0020] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are specifically listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the preparation of RA / G@Z nanoparticles of the present invention and their anti-tumor treatment.

[0022] Figure 2 Single-factor investigation of RA / G@Z. (a) Particle size distribution of A@Z containing different amounts of ART; (b) PDI and particle size distribution of ART / GOx@ZIF-8 containing different amounts of GOx; (c) Cell proliferation after incubation with ART / GOx@ZIF-8 containing different amounts of GOx.

[0023] Figure 3(a) Particle size distribution of the nanoparticles; (b) Zeta potential change.

[0024] Figure 4 (a) TEM image of ZIF-8; (b) TEM image of R-A / G@Z.

[0025] Figure 5 (a) XRD patterns of ZIF-8, A@Z, G@Z and R-A / G@Z nanoparticles; (b) Elemental analysis of R-A / G@Z.

[0026] Figure 6 SDS-PAGE analysis of red blood cell membrane, R-A / G@Z and GOx protein (a: marker; b: red blood cell membrane; c: R-A / G@; d: GOx).

[0027] Figure 7 (a) Standard curve of GOx; (b) Standard curve of ART.

[0028] Figure 8 Stability test results of R-A / G@Z.

[0029] Figure 9 ART release curves of R-A / G@Z in pH = 7.4 and pH = 5.5 environments.

[0030] Figure 10 Confocal detection of 4T1 cell uptake after treatment with different nanoparticles, blue is DAPI labeled nucleus, green is C6, and red is Cy3-BSA.

[0031] Figure 11 Pharmacodynamic test analysis of nanoparticles. After Free ART, R-Z, R-G@Z, R-A@Z and R-A / G@Z were incubated with 4T1 cells for 24 h, cell viability was detected.

[0032] Figure 12 (a) DCFH-DA probe detection of ROS production in 4T1 cells after incubation with Free ART, R-Z, R-G@Z, R-A@Z and R-A / G@Z nanoparticles; (b) Green fluorescence intensity quantification chart.

[0033] Figure 13 (a) JC-1 probe detection of mitochondrial membrane potential change in 4T1 cells after incubation with nanoparticles; (b) Mitochondrial membrane potential fluorescence intensity quantification chart.

[0034] Figure 14(a) FNP1, p53, SLC7A11 and GPX4 protein expression levels. I: PBS; II: Free ART; III: R-ZIF-8; IV: R-G@Z; V: R-A@Z; VI: R-A / G@Z; (b) FNP1, p53, SLC7A11 and GPX4 protein normalized quantitative analysis.

[0035] Figure 15 Fig. 6 is a graph of hemolysis experiment results, (a) hemolysis rate of red blood cell suspension after incubation with R-A / G@Z at different concentrations; (b) hemolysis rate of red blood cell suspension after incubation with Free ART, R-Z, R-G@Z, R-A@Z and R-A / G@ at a concentration of 500 pg / mL.

[0036] Figure 16 Fig. 7 is a graph of biodistribution in tumor-bearing mice, (a) fluorescence imaging of tumor-bearing mice after injection of Free Cy5.5 or BSA-Cy5.5@ZIF-8 for 2, 4, 6, 8, 12, 24 h (Tu: tumor; He: heart; Li: liver; Sp: spleen; Lu: lung; Ki: kidney); (b) quantification of tumor fluorescence intensity in mice.

[0037] Figure 17 Fig. 8 is a graph of subcutaneous injection of 4T1 cells to construct a breast cancer model and its treatment scheme.

[0038] Figure 18 Fig. 9 is a graph of monitoring tumor inhibition in mice in each group in the in vivo tumor inhibition experiment.

[0039] Figure 19 Fig. 10 is a graph of tumor inhibition in mice in the in vivo tumor inhibition experiment, (a) change in tumor size over time in 4T1 breast cancer models; (b) tumor resection on day 21 after treatment in each group.

[0040] Figure 20 Fig. 11 is a graph of images of mice treated differently after 19 days in the in vivo tumor inhibition experiment.

[0041] Figure 21 Fig. 12 is a graph of H&E and TUNEL staining of tumor tissues collected from mice treated differently, scale bar: 50 pm.

[0042] Figure 22 Fig. 13 is a graph of body weight change of 4T1 tumor-bearing mice after drug treatment.

[0043] Figure 23 Fig. 14 is a graph of H&E staining of main organs (heart, liver, spleen, lung, kidney) collected from mice treated differently, scale bar: 50 pm. DETAILED DESCRIPTION

[0044] The technical solutions of the present application will be further described in detail below in combination with specific embodiments. It should be understood that the following embodiments are only illustratively described and explained, and should not be interpreted as limiting the scope of protection of the present application. Any technology achieved based on the above description of the present application is covered within the scope of protection intended by the present application.

[0045] In addition, unless otherwise specifically stated, the various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or prepared by existing methods.

[0046] Preparation of A / G@Z

[0047] Drug-loaded ART / GOx@ZIF-8 (A / G@Z) was prepared by self-assembly method. ART was weighed and dissolved in N,N-dimethylformamide (DMF) solution to obtain a drug stock solution. 283 mg of 2-methylimidazole (2-MIM) was weighed and dissolved in 1 mL of deionized water, and 15 mg of zinc nitrate hexahydrate (Zn(NO3)2·6H2O) was weighed and dissolved in 100 μL of deionized water. Under the condition of stirring speed of 1200 rpm and room temperature, the weighed GOx and ART stock solution were added to the 2-MIM solution and stirred for 5 min, then the Zn(NO3)2·6H2O solution was added dropwise to the above mixture solution under the condition of stirring at 1200 rpm, and stirred at room temperature for 10 min. Drug-loaded A / G@Z nanoparticles were spontaneously formed to obtain a white suspension. Centrifugation was performed to obtain a white precipitate. Water washing was performed three times to remove unreacted reactants and drugs adsorbed on the surface of ZIF-8 with weak force. The final product was collected by centrifugation.

[0048] Extraction and purification of red blood cell membrane

[0049] 1 mL of fresh blood was collected from the mouse orbit and placed in a 1.5 mL EP tube containing sodium heparin. Centrifugation was performed at 2000 rpm for 10 min at 4°C to remove the upper plasma and the white material at the interface. Washing was performed with pre-cooled PBS buffer three times (2000 rpm, 10 min) until the supernatant was colorless. The red blood cells were mixed with pre-cooled 0.25×PBS at a ratio of 1:10, and hypotonic treatment was performed at 4°C for 1 h. Centrifugation was performed at 12000 rpm for 30 min to remove the supernatant hemoglobin. Washing was repeated three times until the supernatant was colorless to obtain the red blood cell membrane (RBCM).

[0050] Preparation of R-A / G@Z

[0051] Fresh and pure red blood cell membrane was subjected to ultrasonic treatment (150 W, 2 min), and the prepared A / G@Z was mixed with the red blood cell membrane solution at a ratio of 1:1, then the mixture was subjected to ultrasonic treatment for 60 s, and was extruded back and forth 10 times with a micro-extruder, and was sequentially passed through 400 nm, 200 nm and 100 nm polycarbonate membranes, and the excess red blood cell membrane was removed by centrifugation, and the obtained nanoparticles were red blood cell membrane-coated nanoparticles (R-A / G@Z).

[0052] Experimental Example 1: Single-factor investigation of R-A / G@Z

[0053] (1) Exploring the optimal dosage of ART

[0054] Under the condition that other conditions remain unchanged, the mass of ART was set to 1, 2, 3, 4, 5 and 6 mg, and the ART was dissolved in a DMF solution to obtain a concentrated drug stock solution. The concentrated drug stock solution of ART was added to the 2-MIM solution and stirred for 5 min, and then the Zn(NO3)2·6H2O solution was added dropwise to the above obtained mixture under the condition of stirring at 1200 rpm, and the mixture was stirred at room temperature for 10 min to obtain A@Z nanoparticles. The effect of different mass of ART on the particle size of the nanoparticles was investigated.

[0055] (2) Exploring the optimal dosage of GOx

[0056] According to the mass of ART fixed in the above experiment, under the condition that other conditions remain unchanged, the mass of GOx was set to 1, 1.5, 2 and 2.5 mg, and the weighed GOx and the concentrated drug stock solution of ART were added to the 2-MIM solution and stirred for 5 min, and then the Zn(NO3)2·6H2O solution was added dropwise to the above obtained mixture under the condition of stirring at 1200 rpm, and the mixture was stirred at room temperature for 10 min to form drug-loaded A / G@Z nanoparticles spontaneously. The effect of different mass of GOx on the particle size of the nanoparticles was investigated, and the weight ratio between ART and GOx was determined by optimizing the synergistic anti-tumor effect.

[0057] As shown in Figure 2 (a), the particle size of the nanoparticles was positively correlated with the mass of ART, and when the mass of ART reached 4 mg, the particle size of the nanoparticles suddenly increased to 527.27 nm. Through the preliminary pre-experiment and literature research, it was found that the nanoparticles had a relatively good effect on uptake into cells at about 150-250 nm, and therefore the mass of ART was selected to be 3 mg based on the particle size and PDI of the nanoparticles for subsequent experiments.

[0058] According to the mass of the known ART, the mass of GOx was changed. Cells cultured in the medium supplied with glucose (10 mM) as a carbon source were incubated with synthetic ART / GOx@ZIF-8 for 24 h, and the weight ratio between ART and GOx was 3:1, 3:1.5, 3:2 and 3:2.5, respectively. As shown in Figure 2 (c), with the increase of the proportion of GOx, the cell proliferation inhibition rate showed a significant dose-dependent enhancement. It is worth noting that when the mass ratio reached 3:2, the inhibition reached a stable plateau. In addition Figure 2 (b), when the weight ratio between ART and GOx was 3:2, the particle size was about 240.05 nm, and the PDI was 0.212, so the W ART :W GOx = 3:2 was used to synthesize ART / GOx@ZIF-8 and the following experiments were carried out.

[0059] Experimental Example 2: Particle size, potential and morphology of R-A / G@Z

[0060] ZIF-8, A / G@Z, R-A / G@Z were diluted into a solution of a certain concentration, and the particle size and Zeta potential were measured by 90Plus PALS high sensitivity Zeta potential and particle size analyzer, and the appearance characteristics were observed by 200kV JEM-2100 transmission electron microscope (JEOL, Japan).

[0061] The 90Plus PALS high sensitivity Zeta potential and particle size analyzer detected the particle size distribution of the carrier ZIF-8 and the co-delivery nanoparticles R-A / G@Z, and the Zeta potential change of the nanoparticles. The particle size distribution results are shown in Figure 3 (a), first detected the size of the carrier nanoparticles was about 164 nm, and the PDI was 0.193, indicating that the carrier material could form a relatively stable dispersed nanoparticles in aqueous solution. Through the self-assembly method, the size of the nanoparticles A / G@Z was 242.14 nm (PDI: 0.239), and after the red blood cell membrane was attached to its surface, the particle size was about 256.43 nm (PDI: 0.245). The particle size difference between A / G@Z and R-A / G@Z was about 14 nm, which was consistent with the reported thickness of the red blood cell bilayer membrane (14-16 nm), which preliminarily proved that the red blood cell had been attached to the surface of the nanoparticles.

[0062] The Zeta potential results are shown in Figure 3(b) shows, the surface of ZIF-8 is modified by 2-MIM which is deprotonated, and the strong negative charge of 2-MIM can coordinate with metal ions, making ZIF-8 positively charged. The isoelectric point (pi) of GOx is about 4.9, thus making G@Z charge reversed and negatively charged; the red blood cell membrane is attached to the surface of A / G@Z due to the ionization of sialic acid residues on the membrane, and the nanoparticles tend to be negatively charged.

[0063] Experimental Example 3: Transmission Electron Microscopy

[0064] As shown in Figure 4 (a), the TEM image clearly shows that the ZIF-8 particles are uniform hexagons with smooth surfaces. As shown in Figure 4 (b), compared with the obvious three-dimensional structure of ZIF-8, the morphology of R-A / G@Z gradually becomes irregular, and there is a clear single-layer membrane outline, with clear boundaries, showing a characteristic core-shell structure, further proving that the red blood cell membrane is successfully coated on the surface of the nanoparticles.

[0065] Experimental Example 4: Crystal Structure Identification and Elemental Analysis

[0066] In order to confirm the successful synthesis of the nanoparticles, in addition to the characterization of the morphology and dispersity of the materials, the crystal structure of the synthesized ZIF-8, G@Z, A@Z and R-A / G@Z nanoparticles was investigated by XRD, and the experimental results are shown in Figure 5 (a). The blank ZIF-8 and the drug-loaded ZIF-8 have a certain crystallinity, and the XRD spectrum data are consistent with the reported ZIF-8 crystal structure data, proving that the crystal structures of the samples are consistent with ZIF-8 and the drug loading does not cause damage to the integrity of the ZIF-8 crystal structure. In addition, elemental analysis was also performed on the nanoparticles, as shown in Figure 5 (b), the elements C, N and Zn are concentratedly distributed, and similar regions appear with the Merge graph, indicating that the nanoparticles are successfully prepared.

[0067] Experimental Example 5: Verification of Membrane Protein Retention after Cell Membrane Coating Nanoparticles

[0068] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the protein expression of the red blood cell membrane-coated nanoparticles. The red blood cell membrane and the nanoparticles were mixed with RIPA lysis buffer containing 1 mM PMSF, and lysed at 4°C for 1 h, then centrifuged at 12000 rpm for 10 min, and the supernatant was taken. The protein concentration was determined using a BCA protein quantification kit, 25 μg of protein was mixed with the loading buffer, and separated by 10% SDS-PAGE. The gel was stained with Coomassie brilliant blue, and after destaining, the Bio-Rad gel imaging system was used for detection and photography.

[0069] By using SDS-PAGE analysis, as shown in Figure 6 Figure 6, it can be seen that RBCM, R-A / G@Z and GOx all have obvious protein bands, and the protein electrophoretic bands of RBCM and R-A / G@Z are highly similar, and R-A / G@Z and GOx also have similar protein bands, which can indicate that RBCM and GOx are successfully loaded on the nanoparticles.

[0070] Experimental Example 6: Determination of drug loading of R-A / G@Z

[0071] According to the BCA method, the content of GOx in the nanoparticles was detected, the supernatant of R-A / G@Z obtained after centrifugation was taken out, and the OD value was determined according to the BCA method, then the corresponding concentration was calculated according to the standard curve, and finally the GOx encapsulation efficiency and drug loading were calculated according to the following formula: EE(%) = W 包 / W 总 × 100%; DL% = W 包 / W 纳米粒 × 100%.

[0072] ART content in the nanoparticles was also determined by high performance liquid chromatography. The liquid chromatography detection conditions of ART are as follows:

[0073] Chromatographic column: SinoPark C18 column (150 mm x 4.6 mm, 5 μm); instrument: Shimadzu LC-20AD; column temperature: 30°C; mobile phase: acetonitrile-phosphate buffer (pH 3.0) = 44:56 (V / V); retention time: 12 min; flow rate: 1 mL / min; detection wavelength: 216 nm; injection volume: 10 μL.

[0074] The absorbance of a series of concentrations of GOx and ART standard solution was determined by using a microplate reader and high performance liquid chromatography, respectively, and a standard curve was drawn using the average absorbance (A) as the ordinate and the GOx / ART concentration (C) as the abscissa, and a linear regression equation was obtained.

[0075] As shown in Figure 7 (a), according to the regression equation Y = 1.214X + 0.005302, the correlation coefficient R 2 = 0.991, indicating that the GOx solution has a good linear relationship in the range of 0.05-0.5 mg / mL. The encapsulation efficiency (EE) % = (total drug amount - free drug amount) / total drug amount x 100%. The encapsulation efficiency of GOx is 74.3%, and the drug loading is 12.4%.

[0076] As shown in 7(b), according to the regression equation Y = 664416X - 60660, the correlation coefficient R 2= 0.9998, indicating that the ART solution was in good linear relationship in the range of 0.1-10 mg / mL. The encapsulation efficiency of ART was 100%, and the drug loading was 20%.

[0077] Experimental Example 7: Investigation of the stability of R-A / G@Z

[0078] R-A / G@Z was dissolved in PBS buffer and stored at 4°C for 72 h. Then it was characterized by 90Plus PALS high sensitivity Zeta potential and particle size analyzer, and the particle size, potential and dispersion of the nanoparticles at different time points (0, 6, 12, 24, 48, 60 and 72 h) within 72 h were observed, and its appearance, whether there was precipitation and other physical phenomena were observed.

[0079] To evaluate the stability of the drug carrier in the physiological environment, the present application selected PBS as the simulation system for investigation. The red blood cell membrane-wrapped nanoparticles were dispersed in PBS, and after being stored at 4°C for 72 h, the solution still remained clear and transparent. Further test results showed that the particle size and potential of the nanoparticles did not change significantly during the storage period, indicating that the red blood cell membrane-wrapped nanoparticles had good stability. Figure 8 The particle size change of the nanoparticles at different time points was shown, further verifying its stability.

[0080] Experimental Example 8: Evaluation of the degradation performance of pH-responsive R-A / G@Z

[0081] 3 mL of R-A / G@Z solution was transferred to a dialysis bag with a molecular weight cut-off of 3500 Da, and then placed in a 50 mL centrifuge tube containing 17 mL of phosphate buffer (pH = 7.4 and pH = 5.5), and incubated at 100 rpm and 37°C. At the predetermined reaction time points (0, 0.1, 0.3, 1, 2, 4, 8, 12, 24 h), 3 mL of solution was taken from the centrifuge tube and 3 mL of new phosphate buffer was added. The sample was taken out from the centrifuge tube and its concentration was determined using Shimadzu LC-20AD. The cumulative release amount of ART can be obtained from the following formula:

[0082]

[0083] Controllable release efficiency of the drug is one of the key parameters for evaluating the performance of the nano-drug delivery system. Therefore, the present application systematically investigated the drug release behavior of R-A / G@Z under different pH conditions. As shown in Figure 9As shown, R-A / G@Z exhibits significant pH-responsive release characteristics in the environment of pH = 5.5: the rapid release of the drug is observed within the first 4 hours, followed by sustained release and reaching a final release rate of 98% after 24 hours, which is significantly higher than the release amount under the condition of pH = 7.4. The above studies show that the ZIF-8-based nano-drug delivery system has excellent pH-responsive drug release capability, can realize effective controllable release of the drug, and is beneficial to enhancing the killing ability of the drug on cancer cells.

[0084] The R-A / G@Z nanoparticles are successfully synthesized by a self-assembly method. Through single-factor investigation, the optimal preparation prescription and process of the nanoparticles are finally determined as follows: the mass of ART is 3 mg, and the mass of glucose oxidase is 2 mg. Next, the preparation and a series of characterizations of the red blood cell membrane-coated nanoparticles are deeply researched. TEM and XRD show that R-A / G@Z has a good crystal form, and the particle size is 255 nm. The drug loading amounts of ART and GOx in R-A / G@Z are 20% and 12.4% respectively, and R-A / G@Z has pH-responsive drug release characteristics. The nanoparticle encapsulation efficiency is higher than 90% and has good sustained-release performance, which is measured by high performance liquid chromatography. At the same time, sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting are used to characterize the membrane protein on the nanoparticles, and the results show that the composition of the membrane protein on the nanoparticles is basically consistent with that of the natural cells. In this experiment, PBS is selected to investigate the stability of the drug carrier in the physiological environment, and the results show that the particle size, encapsulation efficiency and other parameters do not change obviously within 72 h, and the drug carrier is relatively stable.

[0085] Experimental Example 9: In-vitro functional evaluation of multi-mode synergistic nanoparticles

[0086] Cell lines: 4T1 cells (mouse breast cancer cells), Hela cells (human cervical cancer cells), BT549 (human breast ductal carcinoma cells), CT26 (mouse colon cancer cells), MDA-MB-231 (human breast cancer cells), MCF-7 (human breast cancer cells) are from the ATCC cell library (American Type Culture Collection, Manassas, USA). The cells are placed in a 37℃ constant temperature incubator with the conditions of 5% CO2 and 95% relative humidity. The complete culture medium used for 4T1 cells, BT549 cells and CT26 cells is RPMI1640 culture medium containing 10% (by volume) heat-inactivated fetal bovine serum and 1% (by volume) ampicillin-streptomycin double-antibiotic, and the complete culture medium used for Hela cells, MDA-MB-231 cells and MCF-7 cells is DMEM culture medium containing 10% (by volume) heat-inactivated fetal bovine serum and 1% (by volume) ampicillin-streptomycin double-antibiotic.

[0087] Working solution: 1 x electrophoresis solution: Clycine 18.77 g, Tris 3.03 g, SDS 1 g, add ultrapure water to dissolve, constant volume to 1000 mL.

[0088] 1 x transfer solution: Clycine 5.8 g, Tris 2.9 g, add 200 mL of methanol to dissolve, add ultrapure water to constant volume to 1000 mL.

[0089] 1 x TBST buffer: Pour the purchased TBS powder into a 5000 mL beaker, dissolve it in 2000 mL of ultrapure water.

[0090] Blocking solution: Precisely weigh 5 g of skimmed milk powder, dissolve it in 100 mL of TBST solution.

[0091] Cryopreservation solution: Prepare 10 mL of cryopreservation solution, add 7 mL of RPMI 1640 or DMEM medium, 1 mL of DMSO, and 2 mL of serum.

[0092] 4.5% Potassium permanganate solution: Precisely weigh 4.5 g of potassium permanganate, add distilled water to constant volume to 100 mL.

[0093] (1) Cell uptake experiment

[0094] 4T1 cells were seeded in a 6-well plate at a density of 2 x 10 5 After washing the cells with PBS, Cy3, C6 and ZIF-8 were prepared into different complexes: Cy3@Z, C6@Z, Cy3 / C6@Z, then diluted with the corresponding RPMI 1640 basic medium, and the different complexes were incubated with the cells at 37°C for 4 h, then washed with pre-cooled PBS buffer for 3 times. Fixed with 4% paraformaldehyde for 10 min, then discarded the fixing solution and washed with pre-cooled PBS buffer for 3 times. Added DAPI to stain the cell nucleus for 10 min. After washing with pre-cooled PBS buffer for 3 times, immediately observed the endocytosis situation using a fluorescence inverted microscope.

[0095] Effective cell uptake is the premise of achieving synergistic therapy, therefore, in order to evaluate the uptake of R-A / G@Z by tumor cells, green fluorescent dye C6 was used to simulate the chemotherapeutic drug ART, and red fluorescent dye Cy3-labeled BSA was used to simulate GOx to track its intracellular uptake. After co-incubating ZIF-8 single-loaded C6 and Cy3-BSA (C-6@ZIF-8, Cy3-BSA@ZIF-8) nanoparticles and co-delivery nanoparticles with 4T1 cells for 4 h, the cell uptake was observed using a fluorescence inverted microscope. The results are as follows Figure 10As shown, green and red fluorescence was observed around the cells incubated with drug-loaded nanoparticles, indicating that ZIF-8 could effectively deliver ART and GOx into cells.

[0096] (2) Efficacy experiment of R-A / G@Z

[0097] 4T1 cells were seeded in 96-well plates at a density of 6 x 10 3 cells / well and incubated overnight in a 37°C incubator with 5% CO2. Then, 100 μL of RPMI 1640 complete medium containing different complexes (PBS, Free ART, R-ZIF-8, R-G@Z, R-A@Z, R-A / G@Z) was added to the 96-well plates, respectively. After incubation in the incubator for 24 h, the medium in the well plates was removed, and 100 μL of 10% CCK-8 solution was added to each well. After incubation in the incubator for another 2 h, the absorbance at 450 nm of each well was measured using a microplate reader, and the cell viability was calculated according to the following formula: Cell viability (%) = OD 450 (experimental group) - OD 450 (blank group) / OD 450 (negative control) - OD 450 (blank group) x 100%.

[0098] To compare the in vitro anti-tumor activity of R-ZIF-8, R-G@Z, R-A@Z, R-A / G@Z nanoparticles and free ART, the CCK8 method was used to investigate the proliferation inhibition ability of each group of nanoparticles on 4T1 cells, as shown in Figure 11 As shown, the activity of 4T1 cells gradually decreased as the concentration of ART in each group of nanoparticles increased from 5 μg / mL to 75 μg / mL. Compared with free drug ART, the cell activity decreased significantly after incubation with R-A@Z, which loaded ART alone. In addition, when the concentration of ART was 75 μg / mL, the nanoparticles co-loaded with ART and GOx (R-A / G@Z) could significantly reduce the activity of 4T1 cells compared with the nanoparticles loaded with ART alone. This indicates that nanoparticles co-delivering ART and GOx can be effectively endocytosed by breast cancer 4T1 cells, and can play a synergistic role in inhibiting and killing tumor cells in the cell.

[0099] (3) Evaluation of intracellular ROS generation ability experiment

[0100] The 2',7'-dichlorofluorescein diacetate (DCFH-DA) reactive oxygen species detection kit was used to detect the production of ROS. 4T1 cells were seeded in 96-well plates at a density of 5 x 10 4Cells were seeded at a density of 100 μg / mL in a 24-well plate and cultured overnight in a 37°C incubator containing 5% CO2. RPMI 1640 complete medium containing different complexes (PBS, Free ART, R-ZIF-8, RG@Z, RA@Z, RA / G@Z) was then added to the 24-well plate, with three replicates per experimental group. After incubation in a constant temperature incubator for 24 hours, the medium was removed from the plate, and DFCH-DA diluted 1:1000 in serum-free RPMI 1640 (final concentration: 10 μM) was added to each well. The plates were incubated in a 37°C incubator for 20 minutes, and then gently washed 2-3 times with serum-free RPMI 1640 to remove any DFCH-DA that had not entered the cells. The plates were then placed under an inverted microscope for observation and imaging.

[0101] The fluorescent probe DCFH-DA was used to observe the generation of intracellular ROS under a fluorescence inverted microscope. The probe could be oxidized by intracellular ROS to produce enhanced green fluorescence, while GOx could significantly increase the concentration of intracellular H2O2. At the same time, ART could increase the intracellular Fe 2+ The concentration of Fe 2+ It can react with H2O2 to produce a large amount of ROS. Studies have shown that Zn released by ZIF-8 2+ Increase intracellular Zn 2+ concentration, thereby inducing the production of ROS. Figure 12 As shown in the figure, cells treated with RA / G@Z showed the highest green fluorescence intensity. In contrast, the fluorescence intensity of other groups decreased. This may be attributed to the synergistic production of a large amount of ROS by ART, GOx, and ZIF-8, which made the RA / G@Z group have the strongest fluorescence, further proving that the constructed RA / G@Z nanoparticles can achieve a synergistic effect.

[0102] (4) Mitochondrial membrane potential detection

[0103] Mitochondria are the site of energy generation in animal cells. Energy accumulates in the inner mitochondrial membrane, causing the proton concentration on both sides to be inconsistent, thus generating membrane potential. When the mitochondrial membrane potential is high, JC-1 will accumulate in the mitochondrial matrix, producing red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot accumulate in the mitochondrial matrix, and produces green fluorescence. Therefore, the JC-1 probe can be used to detect changes in mitochondrial membrane potential. 4T1 cells in the logarithmic growth phase were taken and 5×10 cells were placed in each well. 4The cell density of 1.5x105 / mL was inoculated in a 24-well plate and cultured overnight in an incubator. PBS, Free ART, R-ZIF-8, R-G@Z, R-A@Z, and R-A / G@Z (keeping the concentration of ART consistent, 10 μg / mL) diluted with blank culture medium were added. After 24 h of incubation, the culture medium was removed, and the culture medium and JC-1 staining working solution were added again. After mixing well, the plate was placed in an incubator at 37°C for 20 min. After incubation, the supernatant was aspirated, and the cells were washed twice with JC-1 staining buffer. New culture medium was added again, and the plate was observed under a fluorescence microscope.

[0104] Mitochondrial membrane potential is an important indicator for evaluating the normal function of mitochondria, and its stability is crucial for maintaining the normal physiological function of cells. In order to determine whether the nanoparticles interfere with mitochondrial function after being delivered into mitochondria, the change of mitochondrial membrane potential was further detected by using JC-1 probe. As shown in FIG. 6, the green fluorescence of mitochondria in tumor cells treated with nanoparticles gradually increased, and the red fluorescence gradually weakened, indicating that the mitochondrial membrane potential gradually decreased, which showed that the nanoparticles caused the decrease of mitochondrial membrane potential and destroyed the integrity of mitochondria. Figure 13

[0105] (5) Western blot experiment

[0106] Extraction of total protein in cells: the culture medium in the well plate after 48 h of culture was discarded, and the cells were washed with pre-cooled PBS buffer for 3 times. Then, cell lysis solution prepared by mixing RIPA and protease inhibitor at a ratio of 1:100 was added to the cell well plate, and the amount of addition was determined according to the amount of cells (50-300 μL). The plate was placed on ice and shaken for 30 min. Then, the cells were collected using a cell scraper, and the cell fragments and lysis solution were collected into a 1.5 mL centrifuge tube. The tube was then placed in a pre-cooled centrifuge and centrifuged at 4°C and 12000 rpm for 10 min. Then, the supernatant was collected into a new centrifuge tube, which was the total protein in the cells.

[0107] BCA method for determining protein concentration: according to the BCA protein quantification kit, the BCA standard was diluted to form a concentration gradient. The diluted protein standard and sample were added to a 96-well plate, and 3 replicate wells were set for each group. Then, the prepared BCA working solution was added to the corresponding wells, and the plate was incubated in a 37°C oven for 30 min. The absorbance value of each well at 562 nm was measured by a microplate reader, and a standard concentration curve was drawn. The OD value of the sample was substituted into the standard curve to obtain the protein concentration of the sample to be tested. According to the determination result, the final concentration of the sample was adjusted, the solution used was cell lysis solution, and then 5xLoading buffer was added in proportion. The protein was denatured by boiling at 100°C for 10 min. After cooling to room temperature, the sample was aliquoted and stored at 80°C for standby use. ​

[0108] SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis:

[0109] ① Choose the appropriate percentage of acrylamide according to the molecular size of the protein to be separated. In this experiment, 10% SDS-PAGE gel was used.

[0110] ② Loading: The loading amount of each group of sample protein was 25 μg, and 5 μL of protein Marker was added to the loading holes at both ends.

[0111] ③ Electrophoresis: After adding the electrophoresis solution, the inner layer was higher than the outer layer to ensure the voltage difference. First, fix the voltage at 80 V, then increase the voltage to 120 V after the concentrated gel is run out, and end the electrophoresis when the blue indicator band runs to the bottom of the separation gel.

[0112] ④ Membrane transfer: Soak the PVDF membrane in 100% methanol for 10 min in advance, and soak the sandwich clamp, filter paper and sponge in the membrane transfer solution. Cut off the excess part of the gel after electrophoresis, open the membrane transfer clamp, and place the sponge, four layers of filter paper, gel, PVDF membrane, four layers of filter paper, and sponge in turn. This process should avoid the generation of air bubbles. A small triangle can be cut on the PVDF membrane at any corner to facilitate the distinction between the front and back. Then place the membrane transfer clamp in the membrane transfer tank containing 1x membrane transfer solution and transfer at a constant current of 200 mA for 90 min. After the membrane transfer is completed, take out the PVDF membrane and place it in 1x TBST buffer for washing 3 times, each for 5 min.

[0113] ⑤ Blocking: Use tweezers to transfer the PVDF membrane to the blocking solution and gently shake on the shaker for 1 h.

[0114] ⑥ Incubate the primary antibody: After the blocking is completed, transfer the PVDF membrane to 1x TBST buffer and wash 3 times, each for 5 min. Then place it in the pre-prepared primary antibody and incubate at 4°C overnight. After incubation, wash in 1x TBST buffer (10 min x 3).

[0115] ⑦ Incubate the secondary antibody: Place the membrane in the pre-prepared secondary antibody and incubate at room temperature for 2 h. After incubation, wash in 1x TBST buffer (10 min x 3).

[0116] ⑧ Development: Soak the PVDF membrane in ECL developing solution (A liquid: B liquid = 1:1) and observe and take a picture using the chemiluminescence gel imaging system.

[0117] The expression of FNP1, p53, SLC7A11 and GPX4 proteins in 4T1 cells was evaluated by Western blot (WB) analysis. The analysis results are as follows Figure 14As shown, the gene expression of FNP1, SLC7A11 and GPX4 in the administration group was inhibited to a certain extent compared with the PBS group, and the protein band in the R-A / G@Z nanoparticle administration group was obviously lighter, indicating that the inhibition effect was the best. At the same time, compared with the PBS group, the gene expression of p53 in the administration group was obviously increased, and the protein band in the R-A / G@Z nanoparticle administration group was deeper, which indicated that the nanoparticles could be effectively delivered into cells. And further proved that Zn 2+ can inhibit FNP1 protein, promote Fe 2+ generation, and can induce the up-regulation of p53 protein, inhibit SLC7A11 and GPX4 protein, and cause lipid peroxidation.

[0118] In order to preliminarily explore the feasibility of R-A / G@Z nanomedicine in the treatment of breast cancer, mouse breast cancer cells 4T1 were selected and a series of experiments were carried out to evaluate the function of co-delivery nanoparticles in vitro. First of all, the cell uptake ability of nanoparticles was observed by fluorescence inverted microscope. The endocytosis experiment results show that R-A / G@Z nanoparticles can be taken up by cells, which lays a foundation for subsequent cell level efficacy research. In order to study the effect of nanoparticles on cell apoptosis, mitochondrial membrane potential and oxidative stress, CCK8, ROS generation and mitochondrial membrane potential change experiments prove that after the nanoparticles enter 4T1 cells, they can effectively release ART and GOx and Zn 2+ , ART small molecules can inhibit cell proliferation and induce cell apoptosis, and cooperate with GOx and Zn 2+ to play a role in inhibiting tumor cell growth. Finally, through the FerroOrange fluorescent probe, it is verified that ART and ZIF-8 can induce the increase of Fe 2+ in vivo, which shows that ART can play its anti-tumor effect through iron-dependent mechanism in the tumor microenvironment, and the acidic degradation characteristics of ZIF-8 further promote the release of Fe 2+ , thereby enhancing the ferroptosis effect. The accumulation of Fe 2+ not only can directly induce tumor cell death, but also can destroy the redox balance of tumor cells through ROS production, further enhancing the anti-tumor effect. This finding provides an important basis for the multi-modal synergistic treatment mechanism of R-A / G@Z nanoparticles.

[0119] In summary, R-A / G@Z nanoparticles can effectively deliver chemotherapeutic drugs ART and GOx, have good cell uptake ability, and can effectively induce breast cancer cell apoptosis.

[0120] Experimental Example 10: In vivo anti-tumor effect of multi-modal synergistic nanoparticles and evaluation of their safety

[0121] Blab / c female mice were selected to construct tumor-bearing mouse models by subcutaneous injection of 4T1 cells, for evaluating the in vivo biodistribution, anti-tumor effect and biological safety of the nanoparticles. Near-infrared fluorescent dye Cy5.5 was used to simulate hydrophobic drug ART, combined with in vivo imaging technology, to observe the changes of Cy5.5 in mice, so as to determine the distribution of the drug in the mice. At the same time, fluorescence imaging was performed on the isolated tumors and main organs to investigate the enrichment of the nanoparticles in various tissues after blood circulation. During the entire treatment period, the size of the mouse tumor was detected at regular intervals to evaluate the growth inhibition effect of the nanoparticles on the tumor. During the entire treatment period, the body weight changes of the mice were detected regularly, combined with H&E staining and TUNEL staining of the main organs (heart, liver, spleen, lung, kidney) to evaluate the biological safety of the nanoparticles and their toxic side effects on the mice.

[0122] Establishment of 4T1 tumor-bearing mouse model: 4T1 cells in logarithmic growth phase were dispersed in PBS buffer at 1 × 10 7 6-8-week-old female Balb / c mice were used as model animals, the hair near the right rear leg of the mice was shaved, and then 100 μL of 4T1 cell suspension was subcutaneously inoculated into the right rear leg of the mice using an insulin needle, to establish a mouse breast cancer model. The inoculation process was strictly performed under sterile conditions. The mice were continuously raised in a constant temperature and humidity SPF environment, and the growth of the tumor was observed daily.

[0123] (1) In vitro hemolysis experiment

[0124] Biocompatibility is also one of the key points for evaluating nanoparticle drugs. Fresh blood of mice was collected, and 1 mL of blood was taken from the mouse orbit and placed in a 1.5 mL EP tube containing heparin sodium. Centrifugation was performed at 4°C and 2000 rpm for 10 min, and the upper plasma and interface white material were carefully removed. Then, the sample was washed with 4°C pre-cooled PBS buffer solution for three times, until the supernatant was no longer red. The centrifugation condition was 2000 rpm for 10 min. A 2% red blood cell suspension (v / v) was prepared using PBS buffer solution.

[0125] To the centrifuge tube, add 2% of the red blood cell suspension and Free ART, R-ZIF-8, R-G@Z, R-A@Z, R-A / G@Z at a concentration of 500 μg / mL, and R-A / G@Z at different concentrations (5, 10, 50, 100, 500, 1000 μg / mL), use PBS as the negative control group, and deionized water as the positive control group, and prepare 3 parallel samples for each group. After mixing, incubate at 37°C for 3 h, centrifuge at 3000 rpm for 10 min after incubation, collect the supernatant, and take 100 μL of each sample to a 96-well plate. Measure the absorbance value at 577 nm per well using a microplate reader. Calculate the corresponding hemolysis rate. Hemolysis rate % = [(sample to be tested - negative) / (positive - negative)] x 100%.

[0126] As shown in Figure 15 (a), the red blood cell membranes in the positive control group are obviously ruptured, and the red blood cell membranes in the negative control group are well preserved. After R-A / G@Z at a series of concentrations is incubated with the red blood cell suspension for a certain period of time and then centrifuged, no red blood cell membrane rupture occurs even at a concentration as high as 1000 μg / mL, and the hemolysis rate is still less than 5%. In addition, Free ART, R-Z, R-G@Z, R-A@Z and R-A / G@Z nanoparticles at a concentration of 500 μg / mL were also incubated with the red blood cell suspension, as shown in Figure 15 (b), no obvious red blood cell membrane rupture occurred, and the hemolysis rate was still less than 5%, proving that they have good biological safety and meet the needs of intravenous injection.

[0127] (2) Drug distribution study in tumor-bearing mice

[0128] The constructed mouse 4T1 model was used to evaluate the distribution of co-delivery nanoparticles in mice. When the tumor tissue of the mouse grew to 100 mm 3 , a hydrophobic near-infrared dye Cy5.5 and Cy5.5-labeled R-A / G@Z (ART concentration of 6 mg / kg, Cy5.5 concentration of 1 μg / μL) were prepared, and tail vein injection was used for administration, with an injection volume of 200 μL.

[0129] Subsequently, in vivo fluorescence imaging of the mice was performed using a live imaging instrument at predetermined reaction time points (2, 4, 6, 8, 12, 24 h). After 24 h of injection, the mice were sacrificed, and the main organs (heart, liver, spleen, lung and kidney) and tumor were separated in vitro, washed with PBS buffer to remove surface bloodstains, and the fluorescence signal was observed using a live imaging instrument.

[0130] Tumor tissue enrichment effect of nanodrugs is an important indicator for evaluating their targeting ability. Female BALB / c mice were selected as experimental objects for in vivo biodistribution imaging study to explore the distribution of chemotherapy drugs in the nanoparticles in the breast cancer subcutaneous tumor-bearing mouse model. As shown in Figure 16 , in the 4T1 tumor-bearing mouse model, after tail vein injection of Free Cy5.5 and BSA-Cy5.5@ZIF-8 nanoparticles, both showed significant differences in tumor tissue enrichment. No obvious fluorescence signal was observed at the tumor site of the mice injected with Free Cy5.5, while Cy5.5 fluorescence was detected at the tumor site of the mice injected with BSA-Cy5.5@ZIF-8 nanoparticles at 2h, and the fluorescence intensity remained at a high level after 24h, while the Free Cy5.5 group had no obvious fluorescence signal at this time.

[0131] Further analysis of the in vitro fluorescence photos of the main internal organs (heart, liver, spleen, lung, kidney) and tumor tissues after 24h injection found that the fluorescence intensity of the tumor site of the BSA-Cy5.5@ZIF-8 nanoparticle group was significantly higher than that of the Free Cy5.5 group. These results showed that the encapsulated Cy5.5 nanoparticles could effectively enter the tumor tissue through the enhanced permeability and retention effect (EPR effect) of the tumor tissue, and showed a longer retention time.

[0132] (3) In vivo tumor inhibition effect

[0133] The constructed mouse 4T1 model was used to evaluate the tumor inhibition effect of co-delivery nanoparticles. When the tumor volume grew to 100mm 3 , the mice were randomly divided into 6 groups, 5 mice in each group. Group 1: PBS group; Group 2: Free ART group; Group 3: R-ZIF-8 group; Group 4: R-G@Z group; Group 5: R-A@Z group; Group 6: R-A / G@Z group. The injection interval was 3 days, and the dosage was ART equivalent dose of 6mg / kg, a total of 7 times, 200μL each time.

[0134] From the first tail vein injection, the body weight and tumor volume of the mice were recorded every three days. The volume (V) of the mouse tumor was calculated by the following equation: V = 0.5 × (LS 2 ). Where S: short diameter of tumor tissue, L: long diameter of tumor tissue.

[0135] After the last administration for 24h, the mice were sacrificed, and the main organs (heart, liver, spleen, lung and kidney) and tumors of the mice were isolated in vitro, washed with PBS buffer to remove surface bloodstains, and stored in 4% paraformaldehyde for subsequent histopathological analysis.

[0136] In the above experiments, the co-delivery system showed good cellular endocytosis and cytotoxicity, and could accumulate in large quantities at the tumor site. In order to further study its effect on tumor inhibition, a subcutaneous breast cancer tumor-bearing mouse model was constructed. When the tumor grew to 100 mm 3 After the size was measured, the mice were randomly divided into 6 groups for drug administration: PBS group, FreeART group, ZIF-8 group, RG@Z group, RA@Z group, and RA / G@Z group, with 7 mice in each group. The modeling and drug administration schemes are as follows Figure 17 As shown, the ART equivalent dose was 6 mg / kg and administered 7 times every three days. The tumor growth of the mice was measured every three days, and the growth curve is shown in Figure 2. Figures 18-20 As shown in the figure, compared with the PBS group, the other five groups all had a certain degree of tumor growth inhibition effect. However, during the entire anti-tumor treatment period, the volume of mice in the Free ART, R-ZIF-8, RG@Z, and RA@Z groups continued to grow, showing a weak tumor growth inhibition effect. The tumor volumes after the end of treatment were 772.4, 588.6, 463.2, and 217.2 mm, respectively. 3 In contrast, RA / G@Z had the best tumor inhibition effect, with the tumor volume after treatment being 88.2 mm 3 , indicating that the co-delivery system loaded with ART and GOx has a good tumor growth inhibition effect. This result is consistent with the previous in vitro experimental results. The significant tumor inhibition effect of RA / G@Z is attributed to its oxidation reaction and the generation of a large amount of ROS. It also proves that RA / G@Z can achieve a certain accumulation effect in tumor tissue, while penetrating tumor cells to release ART and GOx to achieve a synergistic effect of inhibiting tumor growth.

[0137] (4) Histopathological analysis

[0138] Hematoxylin and eosin (H&E) staining: Tumor tissue and other organs (heart, liver, spleen, lung, and kidney) were fixed in 4% paraformaldehyde, dehydrated, and fixed at 65°C for 12 hours. The dehydrated tissues were then embedded in paraffin and sectioned into 5-μm sections using a microtome.

[0139] Staining was performed using the hematoxylin-eosin (H&E) method. The specific steps are as follows: sections are first dewaxed and rehydrated, followed by staining with hematoxylin and eosin solutions, sequentially. After staining, sections are dehydrated and transparentized, and finally mounted with neutral gum. The stained sections are observed and analyzed under an optical microscope.

[0140] TUNEL staining: Paraffin sections of tumor tissue treated as described above were washed three times in 1× PBS buffer, then incubated in permeabilization buffer (Proteinase K working solution) at 37°C for 30 minutes. After incubation, they were washed three times with 1× PBS buffer. At 37°C in the dark, 50 μL of TdT enzyme reaction solution (45 μL Equilibration Buffer, 1 μL biotin-11-dUTP, and 4 μL TdT Enzyme) was added to the sections. The sections were incubated for 60 minutes, followed by three washes with 1× PBS buffer. After aspirating the sections, 50 μL of Streptavidin-TRITC labeling solution was added at 37°C in the dark for 30 minutes, followed by three washes with 1× PBS buffer. At room temperature in the dark, DAPI staining solution was added to counterstain the nuclei, incubated for 10 minutes, and then the staining solution was removed. Finally, the sections were mounted with a mounting medium composed of a 6:4 ratio of glycerol to PBS. Confocal laser scanning microscopy (CLSM) was used to observe fluorescence images of the sections, where blue fluorescence marked cell nuclei and red fluorescence marked apoptotic cells.

[0141] The isolated tumor tissues were stained with hematoxylin and eosin (H&E staining). Figure 21 As shown in the figure. In the blank control group, the tumor cells were structurally intact, tightly arranged and evenly distributed, with a dense, regular oval morphology and no obvious abnormalities or necrosis. In contrast, the tumor tissue in the RA / G@Z group showed a significant increase in fibrous structures, enlarged intercellular spaces, and irregular cell morphology. H&E staining results showed that RA / G@Z nanoparticles were more toxic to 4T1 tumor tissue, causing abnormal tumor tissue morphology and cell shrinkage.

[0142] To further evaluate the apoptosis of tumor tissue, TUNEL staining was performed. TUNEL staining specifically detects apoptotic cells by attaching labeled dUTP to the 3'-OH end of DNA breaks in apoptotic cells under the action of terminal deoxyribonucleotide transferase. The TUNEL staining results are shown in Figure 2. Figure 21 As shown, green fluorescence signals represent apoptotic cells. While there was almost no green fluorescence signal in the blank control group, a large area of ​​green fluorescence signal appeared in the RA / G@Z-treated group, indicating a significant increase in apoptotic cells in the tumor tissue. This result further confirms the strong pro-apoptotic effect of RA / G@Z nanoparticles on 4T1 tumor tissue.

[0143] (5) In vivo safety evaluation

[0144] weight changes

[0145] The above results have demonstrated the desired anti-cancer performance of the prepared R-A / G@Z at the cell and animal levels. Finally, the in vivo biological safety of R-A / G@Z was studied. Firstly, during the treatment of breast cancer mice, the body weight of mice was measured every 3 days to evaluate the systemic toxicity of nanoparticles to mice. The results are shown in Figure 22 As shown in the whole treatment process, the body weight of mice in the administration group was basically consistent with that of mice in the PBS group, and no obvious fluctuation occurred. This result shows that the nanoparticles have no significant effect on the drinking and eating behavior of mice, indicating that they have low systemic toxicity.

[0146] Histopathological analysis

[0147] In addition to evaluating the in vivo biological safety of nanoparticles from the changes in the body weight of mice, after the treatment of mice was completed, the main organs of mice, including the heart, liver, spleen, lung and kidney, were collected. Then H&E staining was used to evaluate the biological toxicity of R-A / G@Z. As shown in Figure 23 There was no obvious difference between the administration group and the PBS group, showing good biocompatibility of the nanoparticles, and no obvious pathological changes were observed, which further confirmed that under the given dose, the nanoparticles in each group did not show serious toxicity, indicating that a nanomedicine preparation with low biological toxicity was successfully constructed.

[0148] The present application constructed a tumor-bearing mouse model by subcutaneously injecting 4T1 cells, and systematically investigated the biodistribution, anti-tumor effect and biological safety of nanoparticles in vivo. Firstly, the fluorescence signal of Cy5 in mice at different time points was tracked using in vivo imaging technology, and ex vivo tumor and main organs were subjected to fluorescence imaging analysis. The results showed that the R-A / G@Z nanodelivery system could effectively enrich in the tumor site.

[0149] During the treatment, the volume changes of the orthotopic tumors of mice were regularly monitored, and it was found that the R-A / G@Z nanoparticles significantly inhibited the growth of tumors, showing good anti-tumor effect. At the same time, by monitoring the body weight changes of mice and combining with H&E staining analysis of main organs (heart, liver, spleen, lung, kidney), it was confirmed that the nanoparticles had good biological safety and did not cause obvious toxic damage to normal tissues of mice.

[0150] In summary, the R-A / G@Z nanoparticles prepared in the present application not only exhibit significant anti-tumor activity, but also have good biocompatibility, and have certain feasibility for the treatment of breast cancer.

[0151] Cell lines and experimental animals used in this study: 4T1 cells (mouse breast cancer cells) were obtained from the ATCC cell bank. All experimental animals and related procedures were performed with the approval of the Animal Experimentation Management Committee of Chongqing University of Technology. Healthy female Blab / c mice aged 6-8 weeks (weighing approximately 20 g) were purchased from Hunan Slake Laboratory Animal Co., Ltd. and housed uniformly in a standard SPF-grade clean environment. All mice had free access to food and water throughout the experiments.

[0152] In order to verify the feasibility of the nanomedicine of the present invention in the treatment of breast cancer, the present invention uses 4T1 cells and mouse breast cancer models to explore at the cellular level and animal experimental level to evaluate the application potential of the nanomedicine of the present invention in the targeted treatment of breast cancer, and comprehensively evaluates the effectiveness of the nanomedicine in the treatment of breast cancer at the cellular level and animal experimental level.

[0153] The present invention successfully prepared nanoparticles RA / G@Z that can effectively inhibit tumor cells to overcome the drug resistance of a single chemotherapy drug. After systemic administration, RA / G@Z can accurately release ART, GOx and Zn in the weakly acidic tumor microenvironment. 2+ Among them: ①ART increases intracellular Fe 2+ 2. GOx activates the endogenous ferroptosis pathway horizontally and reacts with H2O2 produced by GOx to produce Fenton reaction, thus achieving chemokinetic therapy; 3. GOx continuously consumes glucose and oxygen in tumor cells, destroys energy metabolism homeostasis, and induces starvation therapy effect; 4. Zn released by acidolysis of ZIF-8 carrier 2+ It not only significantly increases ROS levels through the mitochondrial pathway, but also further increases intracellular Fe by regulating key iron metabolism proteins (such as FPN1). 2+ This multimodal treatment strategy based on the regulation of the tumor microenvironment has excellent potential for tumor treatment and provides a new approach to utilizing the tumor microenvironment to improve synergistic cancer treatment.

[0154] The present application realizes co-loading of ART and GOx by constructing a pH-responsive nano delivery system based on ZIF-8, not only solves the problems of poor water solubility and insufficient tumor targeting of ART, but also enhances the precise delivery of drugs through an acid-responsive release mechanism, and reduces the systemic toxic side effects. The present application innovatively combines chemotherapy with starvation therapy, uses GOx to consume glucose in the tumor microenvironment, cuts off the energy supply of the tumor, and at the same time induces tumor cell apoptosis through ART, to exert a synergistic anti-tumor effect, providing a new strategy for overcoming breast cancer chemotherapy resistance. In addition, the construction of the nano platform of the present application provides a reference for the delivery of other hydrophobic anti-tumor drugs, and promotes the development of modern dosage forms of active ingredients of traditional Chinese medicine. From the perspective of clinical transformation, the present application lays a foundation for the development of an efficient and low-toxic breast cancer treatment plan, and is expected to improve the quality of life and prolong the survival period of patients, and has a broad application prospect.

[0155] The above-mentioned embodiment numbers of the present application are only for description, and do not represent the advantages and disadvantages of the embodiments.

[0156] The embodiments of the present application are described above in combination with the drawings, but the present application is not limited to the above-mentioned specific embodiments, and the above-mentioned specific embodiments are only illustrative and not limiting, and those skilled in the art can make many forms under the inspiration of the present application without departing from the scope of the present application and the scope protected by the claims, which all belong to the protection of the present application.

Claims

1. A multi-mode synergistic nanoparticle, characterized in that: The invention comprises a zeolite imidazolate framework material 8, wherein artesunate and glucose oxidase are contained in the zeolite imidazolate framework material 8.

2. The multimodal synergistic nanoparticle according to claim 1, characterized in that: The mass ratio of artesunate to glucose oxidase is 3:1-2.

5.

3. The multimodal synergistic nanoparticle according to claim 1, characterized in that: It also includes red blood cell membranes, which are coated on the surface of the zeolite imidazolate framework material 8.

4. The method for preparing multimodal synergistic nanoparticles according to any one of claims 1 to 3, characterized in that: The method comprises: weighing artesunate and dissolving it in an N,N-dimethylformamide solution to obtain a concentrated drug stock solution; weighing dimethylimidazole and dissolving it in deionized water; weighing zinc nitrate hexahydrate and dissolving it in deionized water; adding the weighed glucose oxidase and the concentrated artesunate stock solution to the dimethylimidazole solution and stirring; then adding the zinc nitrate hexahydrate solution and continuing to stir to obtain a suspension; centrifuging to obtain a precipitate; washing with water and centrifuging to collect the final product ART / GOx@ZIF-8.

5. The method for preparing multimodal synergistic nanoparticles according to claim 4, characterized in that: Under room temperature conditions with a stirring speed of 1000-1500 rpm, the weighed glucose oxidase and artesunate stock concentrated solution are added to the dimethylimidazole solution and stirred for 3-7 minutes. Subsequently, the zinc nitrate hexahydrate solution is added at the same speed and stirring is continued at room temperature for 8-12 minutes.

6. The method for preparing multimodal synergistic nanoparticles according to claim 4, wherein: Also includes: The erythrocyte membrane is ultrasonically treated, and the prepared ART / GOx@ZIF-8 is mixed with the erythrocyte membrane solution. The mixture is then ultrasonically treated, extruded, filtered, and the excess erythrocyte membrane is removed by centrifugation. The resulting nanoparticles are erythrocyte membrane-coated nanoparticles.

7. The method for preparing multimodal synergistic nanoparticles according to claim 6, characterized in that: ART / GOx@ZIF-8 was mixed with red blood cell membrane solution in a ratio of 1:1, and then the mixture was ultrasonically treated for 50-70 seconds and squeezed back and forth 8-12 times using an extruder, passed through 400nm, 200nm, and 100nm polycarbonate membranes in turn, and the excess red blood cell membrane was removed by centrifugation. The resulting nanoparticles were red blood cell membrane-coated nanoparticles.

8. Use of the multimodal synergistic nanoparticles according to any one of claims 1 to 3 or the method for preparing the multimodal synergistic nanoparticles according to any one of claims 4 to 7 in the preparation of anti-tumor products.

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