Nanometer delivery system for immune-chemical kinetics tumor treatment

Abstract

The invention relates to the technical field of medicines, in particular to a nano delivery system for treating tumors through immune-chemical kinetics. The hyaluronic acid coated copper peroxide quantum dots are prepared by adopting a one-step method, and hyaluronic acid is used as a stabilizer and a dispersing agent, so that the stability of peroxide can be improved, and the active targeting property is also realized. Meanwhile, the introduction of copper peroxide starts a Fenton-like reaction, and activates a chemical kinetics therapy. In addition, the copper peroxide also reacts with an immunotherapy drug metformin to generate nitric oxide, so that blood vessel normalization is promoted, single-group drug induction is realized, and a pleasant effect of multiple treatments is triggered. In-vivo and in-vitro experimental results show that HMSN-Met-HA-CuO2 has good biological safety and stable nitric oxide release ability, has considerable targeting ability to 4T1 tumor cells, and can effectively inhibit the growth of a tumor model.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a nanometer delivery system for immuno-chemodynamic therapy of tumors. Background Art

[0002] Single anti-cancer therapies can have significant side effects and cause significant pain to patients. Consequently, a growing number of researchers are focusing on combining or synergizing multiple treatments to improve efficacy while minimizing side effects. However, combining multiple treatments often comes with complex ingredients, difficult synthesis, and inconsistent results. Summary of the Invention

[0003] This study developed a nanoparticle platform that uses hyaluronic acid copper peroxide quantum dots to encapsulate metformin in hollow mesoporous silica drug-loaded particles (HMSN-Met@HA-CuO2). This platform uses nitric oxide to promote vascular normalization and hyaluronic acid to target the CD44 receptor, thereby enhancing drug retention and accumulation at the tumor site. This improves the tumor microenvironment and enhances the anti-tumor efficacy of chemokine-based combined immunotherapy.

[0004] In one aspect, the present invention provides a method for preparing a nano-delivery system for immuno-chemodynamic tumor therapy, wherein the method comprises the following steps:

[0005] 1) silica colloidal solution: ultrapure water and anhydrous ethanol are added to a reaction vessel, and then ammonia water is added and stirred. The mixture is added to the above mixed solution, and the above reaction system is stirred and reacted at 40° C. to obtain a silica solution. The supernatant is removed by centrifugation, and ultrapure water and anhydrous ethanol are added for ultrasonic dispersion and washing to remove unreacted raw materials to obtain silica particles. The silica particles are then uniformly dispersed in ultrapure water to obtain a silica colloidal solution for later use;

[0006] 2) Preparation of HMSN@CTAC Nanoparticles: CTAC and TEA were weighed and mixed with ultrapure water, stirred, and the silica colloidal solution prepared in step 1) was added, stirred, and the temperature was increased. TEOS was quickly added and stirred for reaction. The temperature was lowered, and solid sodium carbonate was added to selectively etch the silica nanoparticle template. The mixture was stirred, centrifuged, and washed with ultrapure water and anhydrous ethanol, respectively, to obtain HMSN@CTAC nanoparticles.

[0007] 3) Preparation of HMSN nanoparticles: Concentrated hydrochloric acid was dissolved in anhydrous ethanol to prepare a hydrochloric acid-ethanol solution. The HMSN@CTAC nanoparticles washed in step 2) were uniformly dispersed in the hydrochloric acid-ethanol solution by ultrasonication. After refluxing for 24 hours, the mixture was centrifuged and washed multiple times with ultrapure water and anhydrous ethanol. The extraction was repeated 2-3 times to ensure the removal of CTAC, thereby obtaining HMSN nanoparticles.

[0008] 4) HMSN-NH2 nanoparticles: HMSN nanoparticles were dispersed in anhydrous ethanol by ultrasonication, heated, and APTES was added while heating. The mixture was refluxed and centrifuged. The precipitate was collected and washed with water and ethanol respectively, and freeze-dried to obtain HMSN-NH2 nanoparticles.

[0009] 5) Preparation of HA-CuO2 nanoparticles: Hyaluronic acid (HA) was dissolved in ultrapure water, and NaOH solution was added and mixed. CuCl2 solution was then added, followed by slow dropwise addition of H2O2. The mixture was mixed, the pH was adjusted, and the mixture was washed with water to obtain HA-CuO2 nanoparticles.

[0010] 6) HMSN-Met nanoparticles: HMSN-NH2 nanoparticles were ultrasonically dispersed in PBS, Met was added, stirred, dialyzed, centrifuged, and the precipitate was collected to obtain HMSN-Met nanoparticles;

[0011] 7) HMSN@HA-CuO2 nanoparticles: HMSN-NH2 nanoparticles were ultrasonically dispersed in PBS, HA-CuO2 was added, stirred, dialyzed, centrifuged, and the precipitate was collected to obtain HMSN@HA-CuO2 nanoparticles;

[0012] 8) HMSN-Met@HA nanoparticles: HMSN-NH2 nanoparticles were ultrasonically dispersed in PBS, stirred with 20 mg Met, and HA was added to continue the reaction. The mixture was dialyzed and centrifuged, and the precipitate was collected to obtain HMSN-Met nanoparticles.

[0013] 9) HMSN-Met@HA-CuO2 nanoparticles: HMSN-NH2 nanoparticles were ultrasonically dispersed in PBS, Met was added, stirred, HA-CuO2 was added, dialyzed, centrifuged, and the precipitate was collected to obtain HMSN-Met@HA-CuO2.

[0014] Preferably, the volume ratio of TEOS in the reaction system of step 1) to ultrapure water in the dispersion system is 2 ml:60 mL.

[0015] Preferably, the volume ratio of ultrapure water: anhydrous ethanol: aqueous ammonia: TEOS in the reaction system of step 1) is 15 ml: 60 mL: 3 ml: 2 ml.

[0016] Preferably, in step 2), the weight-to-volume ratio of CTAC:TEA:ultrapure water:colloidal silica solution:TEOS is 6 g:60 mg:60 mL:20 ml:0.3 mL.

[0017] Preferably, the elevated temperature in step 2) is 80°C, the stirring reaction temperature in step 2) is 80°C, the cooling in step 2) is 50°C, and the final concentration of sodium carbonate added in step 2) is 0.6 mol / L. Preferably, the volume ratio of concentrated hydrochloric acid to anhydrous ethanol in step 3) is 1:9, and the reflux temperature is 70°C.

[0018] Preferably, in step 4), the weight-to-volume ratio of HMSN nanoparticles: anhydrous ethanol: APTES is 0.1 mg: 60 mL: 1 mL, and the heating and reflux temperature is 70°C.

[0019] Preferably, the concentration of the NaOH solution in step 5) is 0.01 M, and the weight-to-volume ratio of hyaluronic acid (HA): ultrapure water: NaOH solution: H₂O₂: CuCl₂ solution is 0.05 g:40 mL:5 mL:1 mL:5 mL. The weight-to-volume ratio of CuCl₂·2H₂O to water in the CuCl₂ solution is 0.136 g:5 mL, and the pH in step 5) is 10.

[0020] Preferably, in step 6), the weight-to-volume ratio of HMSN-NH2 nanoparticles:Met:PBS is 10 mg:20 mg:10 ml, and the PBS is a phosphate buffer solution with a pH of 7.

[0021] Preferably, the pH of the PBS phosphate buffer in step 7) is 7, and the weight-to-volume ratio of the HMSN-NH2 nanoparticles:PBS:HA-CuO2 is 10 mg:10 ml:10 mg.

[0022] Preferably, in step 8), the weight-to-volume ratio of HMSN-NH2 nanoparticles: PBS: Met: HA is 10 mg: 10 ml: 20 mg: 5 mg.

[0023] Preferably, the weight ratio of HMSN-NH2 nanoparticles:Met:HA-CuO2 in step 9) is 1:2:1, and the weight volume of the HMSN-NH2 nanoparticles and PBS must be 10 mg:10 ml.

[0024] On the other hand, the present invention provides a nano-delivery system for immuno-chemodynamic therapy of tumors prepared by the preparation method of the nano-delivery system for immuno-chemodynamic therapy of tumors.

[0025] The present invention adopts a one-step method to prepare hyaluronic acid-coated copper peroxide quantum dots. Hyaluronic acid acts as a stabilizer and dispersant, which can not only improve the stability of peroxide but also has active targeting. At the same time, the introduction of copper peroxide initiates a Fenton-like reaction and activates chemodynamic therapy. Copper peroxide also reacts with the immunotherapy drug metformin to generate nitric oxide, promotes vascular normalization, achieves a single group of "drug induction", and triggers the promising effect of multiple treatments. The results of in vivo and in vitro experiments show that HMSN-Met@HA-CuO2 has good biosafety, stable nitric oxide release ability, has considerable targeting ability for 4T1 tumor cells, and can effectively inhibit the growth of tumor models. This study proposed for the first time the goal of developing multiple functions of a single component to achieve multiple therapeutic effects of a simple component, providing a new idea for the further development of efficient anti-tumor treatment strategies based on simple components.

[0026] Compared with the prior art, the HMSN-Met@HA-CuO2 prepared in this invention has active targeting, low-dose release of NO, Cu 2+ , and activates the immune response of multifunctional nanomaterials; the present invention controls the particle size of copper peroxide quantum dots and increases the ability of nanoparticles to target tumor sites by encapsulating copper peroxide quantum dots with hyaluronic acid; the copper peroxide of the present invention can trigger a Fenton-like reaction and generate NO with metformin, while triggering chemodynamic therapy and gas therapy, achieving a "1+1>2" effect; the multifunctional nanoparticles HMSN-Met@HA-CuO2 of the present invention show excellent targeting ability, stable nitric oxide release, and significant chemodynamic combined immunotherapy efficacy both in vivo and in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the morphology characterization diagram of HMSN-Met@HA-CuO2.

[0028] Among them, a) is the TEM image of HMSN-NH2, HA-CuO2, magnified HA-CuO2 and HMSN-Met@HA-CuO2 (scale bar = 10 nm, 20 nm or 100 nm); b) is the SEM image of HMSN-NH2 (scale bar = 100 nm); c) is the SEM image of HMSN-Met@HA-CuO2 (scale bar = 100 nm); d) is the Zeta potential of HA, CuO2, HA-CuO2, HMSN, HMSN-NH2, Met, HMSN-Met, and HMSN-Met@HA-CuO2; e) is the infrared spectrum of HA, CuO2, HA-CuO2, HMSN, HMSN-NH2, Met, HMSN-Met, and HMSN-Met@HA-CuO2; f) is the Cu2p of HMSN-Met@HA-CuO2 nanoparticles. XPS spectra; g) C1s XPS spectrum of HMSN-Met@HA-CuO2 nanoparticles; h) O1s XPS spectrum of HMSN-Met@HA-CuO2 nanoparticles; i) N1s XPS spectrum of HMSN-Met@HA-CuO2 nanoparticles. j) Drug loading and encapsulation efficiency at different dose ratios.

[0029] Figure 2 a) UV-visible absorption spectra of PBS, H2O2, CuO and HMSN-Met@HA-CuO2 in KMnO4 (40 μM) solution; b) UV-visible absorption spectra of TMB in HMSN-Met@HA-CuO2 (200 μg / ml, pH=5.5) solution from 0 to 30 min; c) UV-visible absorption spectra of Cu 2+Acid-sensitive cumulative release curve; d) is the cumulative release curve of NO of HMSN-Met@HA-CuO2 (0.2, 0.5, 1.0, 2.0 mg / ml) and Met (2.0 mg / ml) + H2O2 (37°C, pH = 5.5); e) is the hemolysis rate of HMSN-Met@HA-CuO2 (50, 100, 200 μg / ml); f) is the hemolysis rate of Met, HMSN, HMSN-Met, HMSN@HA-CuO2, HMSN-Met@HA and HMSN-Met@HA-CuO2; g) is the hemolysis rate of different concentrations of HM Effect of SN-Met@HA-CuO2 (25, 50, 100, 150, 200 μg / ml) on the viability of L929 cells; h) Effect of different concentrations of HMSN-Met@HA-CuO2 (25, 50, 100, 150, 200 μg / ml) on the survival rate of 4T1 cells; i) Cell viability of 4T1 cells in HMSN, HMSN-Met, HMSN-Met@HA, HMSN@HA-CuO2 and HMSN-Met@HA-CuO2; j) Cell apoptosis of 4T1 cells after treatment with different formulas for 12 hours.

[0030] Figure 3 a) Representative histograms; b) The proportion of HMSN-Cy5@HA-CuO2 uptake by 4T1 cells under different incubation conditions (with and without HA, n = 3); c) Confocal images of 4T1 cells stained with DCFH-DA (scale bar = 50 μm); d) Relative ·OH levels in 4T1 cells after 4 h of treatment with PBS, HMSN, HMSN-Met, HMSN-Met@HA, HMSN@HA-CuO2, and HMSN-Met@HA-CuO2; e) Relative NO levels in 4T1 cells after 0, 2, 4, 8, 12, 24, and 48 h of treatment with HMSN-Met@HA-CuO2; f) Migration area and invasion assay (n = 3) quantified using ImageJ; g) Invasion assay images.

[0031] Figure 4 4T1 tumor-bearing mice treated with HMSN-Met@HA-CuO2 showed improved DC maturation. a) Evaluation of the therapeutic regimens for HMSN-Met@HA-CuO2-induced immune responses. b) Flow cytometry analysis of CD11c cells treated with different formulations. + Tumor-draining lymph node CD80 + CD86 + Representative flow chart of the results; c) Corresponding histograms (n=3). d) IFN-γ, e) TNF-α, f) IL-10 levels in tumor serum after different treatments were determined by ELISA; g) IL-4 secretion level (n=3). CD3 +CD4 + Lymphocytes (Ths) and CD3 + CD8 + Representative flow cytometry images of lymphocytes (CTLs). k) Scatter plots of Ths and CTLs in primary tumors. i) Proportions of Ths and j) CTLs in splenocytes (n = 3). l) Quantification of Ths and m) CTLs in tumor-infiltrating lymphocytes (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0032] Figure 5 a) Splenic CD4 + CD69 + and CD8 + CD69 + Representative flow cytometry analysis results of T cells. b) CD4 + CD69 + and c) CD8 + CD69 + T cell activation percentage (n=3). d) Splenic FOXP3 + CD4 + Representative flow cytometry scatter plots of lymphocytes. e) FOXP3 in tumors + CD4 + Lymphocytes. f) Spleen FOXP3 + CD4 + Lymphocytes and g) tumors FOXP3 + CD4 + Quantitative analysis of lymphocytes. h) Fluorescence images at different times after intravenous injection of HMSN-Cy5@HA-CuO2 (n=3), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (1): PBS; (2): HMSN-Met; (3): HMSN-Met@HA; (4): HMSN@HA-CuO2; (5): HMSN-Met@HA-CuO2.

[0033] Figure 6 a) Treatment regimen evaluating the antitumor effect of HMSN-Met@HA-CuO2. b) Body weight of 4T1 tumor-bearing mice after different treatments, c) tumor volume, d) tumor weight, and e) tumor image. f) H&E, TUNEL, and Ki67 staining of tumor tissue (sales bar = 100 μm). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0034] Figure 7a) Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), albumin (ALB), alkaline phosphatase (ALP), γ-glutamyltransferase (γ-GT), total bile acid (TBA), urea nitrogen (BUN), creatinine (CREA), and uric acid (UA) were measured 14 days after injection of PBS, HMSN, HMSN-Met, HMSN-Met@HA, HMSN@HA-CuO2, and HMSN-Met@HA-CuO2 nanoparticles. b) H&E analysis of major organs after 14 days of treatment with PBS, HMSN-Met, HMSN-Met@HA, HMSN@HA-CuO2, and HMSN-Met@HA-CuO2 nanoparticles. (Scale bar = 50 μm) DETAILED DESCRIPTION

[0035] TEOS: tetraethyl silicate;

[0036] CTAC: hexadecyltrimethylammonium chloride;

[0037] TEA: triethanolamine;

[0038] TEOS: tetraethyl silicate;

[0039] PBS: phosphate buffered saline, pH 7;

[0040] HA: hyaluronic acid;

[0041] Met: metformin.

[0042] Example 1 Preparation of HMSN-Met@HA-CuO2 Nanoparticles

[0043] Silica colloidal solution: Add 15 ml of ultrapure water and 60 ml of anhydrous ethanol to a reaction vessel, then add 3 ml of aqueous ammonia and stir at 40°C for 10 minutes. Add 2 ml of TEOS to the mixed solution and continue to react at 40°C with magnetic stirring for 1 hour. Centrifuge the silica colloidal solution at 12,000 rpm for 10 minutes, remove the supernatant, and ultrasonically disperse and wash with ultrapure water and anhydrous ethanol to remove unreacted raw materials. Repeat this alternating washing process 2-3 times before evenly dispersing in 60 ml of ultrapure water to obtain a silica colloidal solution for later use.

[0044] Preparation of HMSN@CTAC nanoparticles: Accurately weigh 6g of CTAC, 60mg of TEA and 60mL of ultrapure water and mix them evenly. After stirring at 30°C for 1h, add 20ml of the silica colloidal solution prepared in the previous step and magnetically stir for 1h to ensure uniform dispersion. Raise the temperature to 80°C, quickly add 0.3mL of TEOS, and continue stirring and reacting at 80°C for 1h. After lowering the temperature of the solution system to 50°C, add solid sodium carbonate to selectively etch the silica nanoparticle template, so that the final concentration of the sodium carbonate solution is 0.6mol / L. After magnetic stirring for 90min, collect the reaction solution at 12000r, centrifuge for 10min, and wash it twice with ultrapure water and anhydrous ethanol to remove soluble impurities to obtain HMSN@CTAC nanoparticles.

[0045] Preparation of HMSN nanoparticles: 5 mL of concentrated hydrochloric acid was dissolved in 45 mL of anhydrous ethanol to prepare a 10% hydrochloric acid-ethanol solution. The HMSN@CTAC nanoparticles washed in the previous step were evenly dispersed in the hydrochloric acid-ethanol solution by ultrasonication. After reflux at 70°C for 24 hours, the mixture was centrifuged and washed multiple times with ultrapure water and anhydrous ethanol. The extraction was repeated 2-3 times to ensure the removal of CTAC to obtain HMSN.

[0046] HMSN-NH2 nanoparticles: 0.1 mg of HMSN was dispersed in 60 mL of anhydrous ethanol by ultrasound (100 W, 30 min) and heated. When the mixture reached 70°C, 1 mL of APTES was added and refluxed at this temperature for 24 h. The precipitate was collected by centrifugation (12,000 rpm, 10 min) to obtain the HMSN-NH2 product. The product was washed three times with water and three times with ethanol, and then freeze-dried overnight.

[0047] Preparation of HA-CuO2 nanoparticles: Dissolve hyaluronic acid (0.050 g) in 40 mL of pure water, add 5 mL of 0.01 M NaOH solution, mix thoroughly, then add 0.136 g of CuCl2·2H2O dissolved in 5 mL of pure water. Then, slowly add 1 mL of H2O2 dropwise, mix thoroughly, adjust the pH to 10, and wash the mixture several times with water to obtain HA-CuO2 nanoparticles.

[0048] Preparation of HMSN-Met nanoparticles: 10 mg HMSN-NH2 nanoparticles were ultrasonically dispersed in 10 ml PBS (PH = 7), 20 mg Met was added and stirred at room temperature for 12 h, the reaction solution was transferred to a dialysis bag (1000 Da), 100 ml pure water was added and dialyzed for 24 h, and centrifuged at 12000 r for 10 min. The precipitate was collected to obtain HMSN-Met.

[0049] HMSN@HA-CuO2 nanoparticles: 10 mg HMSN-NH2 nanocarriers were ultrasonically dispersed in 10 ml PBS, 10 mg HA-CuO2 was added and stirred at room temperature for 12 h, then the reaction solution was transferred to a dialysis bag (1000 Da) and dialyzed with 100 ml pure water for 24 h. The solution was centrifuged at 12000 r for 10 min, and the precipitate was collected to obtain HMSN@HA-CuO2 nanoparticles.

[0050] HMSN-Met@HA nanoparticles: 10 mg HMSN-NH2 nanoparticles were ultrasonically dispersed in 10 ml PBS, 20 mg Met was stirred at room temperature for 3 h, 5 mg HA was added and the reaction was continued for 12 h. The reaction solution was transferred to a dialysis bag (1000 Da) and dialyzed with 100 ml pure water for 24 h. The solution was centrifuged at 12000 r for 10 min, and the precipitate was collected to obtain HMSN-Met.

[0051] HMSN-Met@HA-CuO2 nanoparticles: First, a UV spectrophotometer was used to establish a standard curve for the UV absorbance of Met (the standard solution concentration range was 2-12.5 μg / ml and had a good linear correlation within this concentration range) to facilitate subsequent quantitative analysis of Met. 10 mg of HMSN-NH2 nanoparticles were ultrasonically dispersed in 10 ml of PBS. 5 mg, 10 mg, and 20 mg of Met were added, respectively, and stirred at room temperature for 3 hours. 10 mg of HA-CuO2 was then added and stirred for 12 hours. The reaction solution was then transferred to a dialysis bag (1000 Da) and dialyzed with 100 ml of pure water for 24 hours. The solution was centrifuged at 12,000 r / min for 10 minutes, and the precipitate was collected to obtain HMSN-Met@HA-CuO2 nanoparticles with different ratios. The supernatant was taken to measure the drug loading, and three parallel experiments were performed to determine the optimal dosage ratio of HMSN:Met:HA-CuO2 was 1:2:1. HMSN-Met@HA-CuO2 nanoparticles were prepared according to this dosage ratio for subsequent experimental studies.

[0052] Example 2 Physicochemical Characterization of HMSN-Met@HA-CuO2 Nanoparticles

[0053] Particle size and potential detection: Take freshly prepared HMSN, HA, CuO2, HMSN-NH2, Met, HMSN-Met, HA-CuO2, and HMSN-Met@HA-CuO2, dilute them to 4 ml with pure water, and ultrasonically disperse them for 30 minutes to fully disperse them. Then use Nano-ZS particle size analyzer to detect their particle size and potential. All sample tests were repeated 5 times.

[0054] Morphology detection: The freshly prepared HMSN, HA-CuO2, and HMSN-Met@HA-CuO2 were ultrasonically dispersed in anhydrous ethanol. After sufficient dispersion, 50 μl of each was taken and slowly dropped onto the surface of the copper mesh. After the ethanol was allowed to evaporate, the morphology was observed using a transmission electron microscope.

[0055] Fourier transform infrared spectroscopy (FL-IR) analysis: Take dried HMSN, HMSN-NH2, HA, CuO2, HA-CuO2, HMSN-Met, HMSN-Met@HA-CuO2 samples and pre-dried potassium bromide at a mass ratio of 1:100, put them into an agate mortar and grind them thoroughly. Then put them on a tablet press and press them at a pressure of 10 MPa for one minute. After that, remove them and scan them using Fourier transform infrared spectroscopy and record the data from 400 cm -1 ~4000cm -1 spectrum.

[0056] Determination of drug loading capacity and encapsulation efficiency of HMSN-Met@HA-CuO2: HMSN-Met@HA-CuO2 was prepared with a dosage ratio of HMSN-NH2 and Met of 1:2. The dosage of HMSN-NH2 was 10 mg and that of Met was 20 mg. After the reaction was complete, the mixture was transferred to a dialysis bag (1000 Da) and dialyzed for 24 h. The amount of drug in the drug-loaded particles was determined by detecting the ultraviolet absorbance of the dialysate. The precipitate was collected at 8000 r for 10 min, washed with pure water, and freeze-dried overnight before being weighed to obtain the total mass of HMSN-Met@HA-CuO2. The drug loading capacity and encapsulation efficiency of HMSN-Met@HA-CuO2 were calculated by the following formula:

[0057]

[0058]

[0059] X-ray Photoelectron Spectroscopy (XPS) Analysis: XPS was used to characterize the surface elements and analyze the surface valence states of HMSN, HMSN@HA-CuO2, and HMSN-Met@HA-CuO2. XPS analysis included full spectra, C1s, O1s, Cu2p, and N1s spectra of the different materials. Binding energies were calibrated using C1s (248.8 eV). Nonlinear fitting software (CasaXPS) was used to analyze the data.

[0060] The present invention uses hollow mesoporous silica as a carrier, which is modified by amino to show positive charge, attracting the electronegative immunotherapy drug metformin, and wrapping HA-CuO2 quantum dots on the surface. The resulting product is HMSN-Met@HA-CuO2. TEM image (such as Figure 1a) It can be seen that HMSN-NH2 has a hollow mesoporous structure, and HA-CuO2 is a nanoparticle with good dispersion and uniform size. After magnification, its lattice fringes can be observed, confirming the successful synthesis of HA-CuO2 nanoparticles. The TEM image of HMSN-Met@HA-CuO2 shows that there is a uniform HA-CuO2 layer on the surface, and the nanoparticles are uniform and spherical in shape. The SEM image of HMMS-NH2 (such as Figure 1 b) shows that its surface size is uniform and the mesoporous structure is regular and orderly. The particle size (e.g. Figure 1 c). The particle size of HMSN-Met@HA-CuO2 increases and the mesoporous structure on the surface disappears, indicating that the HA-CuO2 layer is successfully coated. This is consistent with the TEM results. The potentials of HA, CuO2, HA-CuO2, HMSN, HMSN-NH2, Met, HMSN-Met and HMSN-Met@HA-CuO2 were measured using a Malvin Nano-ZS potential analyzer (e.g. Figure 1 d), respectively -27.57±0.76, 7.55±0.22, -12.5±0, 0.33, -28.50±0.37, 8.13±0.34, -7.41±0.74, -0.61±0.32 and -15.83±0.09, indicating the successful synthesis of the nanoparticles. This indicates that the nanoparticles prepared in this experiment have high electrostatic stability. In addition, HMSN-Met@HA-CuO2 nanoparticles are electronegative and are easily absorbed by the cell membrane after adsorption. FLIR images (such as Figure 1 e) shows that after amination, the hollow mesoporous silica has a wavelength of 3300 cm -1 The peak with medium intensity at 1650 cm-1 was observed, which confirmed the successful preparation of the aminated hollow mesoporous silica. -1 There is also a strong peak at 1350 cm -1 In addition, although the functional groups of HMSN-Met and HMMS-NH2 are similar, the absorption peak at 1220 cm -1 The CN stretching vibration absorption peak at the position is enhanced, confirming the successful loading of Met. Finally, the infrared spectrum of HMSN-Met@HA-CuO2 shows the above characteristic peaks. This proves the successful synthesis of HMSN-Met@HA-CuO2 nanoparticles. In addition, the XPS results of HMSN-Met@HA-CuO2 (such as Figure 1 f) shows that Cu 2+ The presence of C, O and N peaks in the spectrum (such as Figure 1g, h and i) belong to CC, CO, COC, O=C, OO, CN and NN respectively, indicating that all components in the nanoparticles are intact. In the experiment, the optimal dosing ratio was determined by controlling the mass ratio of nanocarriers to free drugs. Figure 1 As shown in Figure j, when the mass ratio of nanocarrier to free drug was 1:2, the coating efficiency and drug loading were optimal, and the drug loading was significantly different from that of the 1:1 experimental group. Therefore, the subsequent experimental materials were prepared with a mass ratio of carrier to free drug of 1:2.

[0061] Example 3 H2O2, TMB, NO, Cu 2+ Release and in vitro hemolysis

[0062] Potassium Permanganate Colorimetric Experiment: H2O2, CuO, and HMSN-Met@HA-CuO2 (copper peroxide at 0.2 mM each) were added to acidic potassium permanganate. The discoloration of the potassium permanganate by each material was observed and scanned using a UV spectrophotometer. Furthermore, the ability of the gradient concentration materials to discolor the acidic potassium permanganate was compared with that of hydrogen peroxide and photographed.

[0063] In order to evaluate the ability of HMSN-Met@HA-CuO2 to generate H2O2, we added H2O2, CuO and HMSN-Met@HA-CuO2 (the hydrogen peroxide content was 0.2 mM) into acidic potassium permanganate (40 μM) and detected the results using a UV spectrophotometer. Figure 2 As shown in a. Only the H2O2 and HMSN-Met@HA-CuO2 experimental groups completely faded the potassium permanganate, confirming that HMSN-Met@HA-CuO2 can produce H2O2.

[0064] TMB was used to detect hydrogen peroxide. When hydrogen peroxide was generated, the solution turned from colorless to blue. 200 μg / ml TMB solution was added to 200 μg / ml HMSN-Met@HA-CuO2 dispersion. The pH of the dispersion was 5.5. The full spectrum was scanned by UV spectrophotometer at 1, 2, 3, 4, 5, 10, 15, 20, 25, and 30. In vitro copper ion release from HMSN-Met@HA-CuO2: Atomic absorption spectroscopy was used to detect copper ion release and acid-sensitive release. First, the Cu 2+The standard curve (the concentration range of the standard solution is 0.5-2.5 μg / ml and has a good linear correlation within this concentration range) was prepared. 20 mg HMSN-Met was dispersed in 20 ml of release medium. The release conditions were pH = 7.4 and pH = 5.5 buffer solutions, respectively. The samples were shaken at 37°C and 400 r. After centrifugation at 8000 r for 5 min at 1, 2, 4, 6, 8, 12, and 24 h, one ml of supernatant was taken for testing and one ml of release solution was added. The supernatant was diluted five times and the copper ion release concentration at each time point was detected by atomic absorption spectroscopy.

[0065] Subsequently, TMB was used to detect the generation of ·OH by HMSN-Met@HA-CuO2 within 30 min, as shown in Figure 2 As shown in b, the ·OH production gradually increases with time. The content of copper ions in the nanomaterials was determined by atomic absorption spectrometry, where Cu 2+ The content is 37%.

[0066] In vitro copper ion release from HMSN-Met@HA-CuO2: Atomic absorption spectroscopy was used to detect copper ion release and acid-sensitive release. First, the Cu 2+ The standard curve (the concentration range of the standard solution is 0.5-2.5 μg / ml and has a good linear correlation within this concentration range) was prepared. 20 mg HMSN-Met was dispersed in 20 ml of release medium. The release conditions were pH = 7.4 and pH = 5.5 buffer solutions, respectively. The samples were shaken at 37°C and 400 r. After centrifugation at 8000 r for 5 min at 1, 2, 4, 6, 8, 12, and 24 h, one ml of supernatant was taken for testing and one ml of release solution was added. The supernatant was diluted five times and the copper ion release concentration at each time point was detected by atomic absorption spectroscopy.

[0067] The results of the study on the nanoparticle Cu under different acidic conditions were 2+ The release trend of Figure 2 c. The results showed that over time, Cu 2+ The release of Cu 2+ The release of HMSN-Met@HA-CuO2 nanoparticles was significantly increased, reflecting that HMSN-Met@HA-CuO2 nanoparticles can respond to the acidic tumor microenvironment.

[0068] In vitro NO release from HMSN-Met@HA-CuO2: HMSN-Met@HA-CuO2 was prepared into dispersions of 0.2 mg / ml, 0.5 mg / ml, 1.0 mg / ml, and 2.0 mg / ml using a buffer solution with a pH of 5.5. A mixture of 2.0 mg / ml Met and 200 μM H2O2 was set as the control group. Appropriate amounts of supernatant were collected at 1, 2, 4, 8, 12, 24, and 48 hours, and the nitric oxide concentration was detected using the Beyotime Nitric Oxide Detection Kit.

[0069] HMSN-Met@HA-CuO2 hemolysis experiment: 3 mL of rabbit blood was centrifuged in an anticoagulant tube (3000 rpm, 10 min) and the supernatant was discarded. PBS was then added and the cells were repeatedly pipetted and centrifuged again (3000 rpm, 10 min). This process was repeated until the supernatant was clear, and finally the sedimented red blood cells were obtained. The rabbit red blood cells were extracted and diluted with PBS to obtain a 2% red blood cell suspension. Then 1.25 mL of the red blood cell suspension was mixed with 1.25 mL of HMSN-Met@HA-CuO2 (50 , 100, and 200 μg / ml) as well as Met, HMSN, HMSN-Met, HMSN@HA-CuO2, HMSN-Met@HA, and HMSN-Met@HA-CuO2 (Met concentration was 30 μg / ml) were mixed, and NS (negative control (-)) and deionized water (positive control (+)) were set up. Next, all samples were incubated in a 37°C constant temperature water bath for 1 hour and then centrifuged at 3000 rpm for 10 minutes. The absorbance of the supernatant of each group was measured at 545 nm using a microplate reader. The hemolysis rate (%) was calculated as follows:

[0070]

[0071] The NO release was determined using the Bio-Tech NO detection kit. Figure 2 As shown in Figure d, the release of nitric oxide is dose-dependent and is only released at low doses in the presence of copper peroxide. In addition, when the hemolysis rate of a material is less than 5%, it is allowed to be used as a biomedical material in the human body.

[0072] The hemolysis rate of HMSN-Met@HA-CuO2 was tested in the range of 50μg / ml to 200μg / ml, which was less than 5% ( Figure 2 e). In addition, when the concentration of HMSN-Met@HA-CuO2 was 200 μg / ml, the hemolysis rate was only 4.05% ( Figure 2 f). This is a blood compatibility evaluation below the minimum standard, indicating that HMSN-Met@HA-CuO2 meets the standards of biomedical materials.

[0073] Example 4 Cytotoxicity Study

[0074] Cell culture: 4T1 (mouse breast cancer cells) were cultured in RPMI1640 medium containing 1% (V / V) penicillin-streptomycin and 10% (V / V) serum in a cell culture incubator at 37°C and 5% CO2.

[0075] Cell recovery: First prepare the instrument consumables needed for cell recovery and take out the 1640 complete medium from the 4℃ refrigerator for thawing. Then take out the frozen cells stored in liquid nitrogen or -80℃ refrigerator and immediately transfer them to a 37℃ constant temperature water bath for shaking and thawing. Be careful to keep the cryopreserved tube upright. After rapid thawing, disinfect and transfer to the clean bench. Transfer the liquid in the cryopreserved tube to a 15ml centrifuge tube and add double the volume of complete medium to dilute it. Centrifuge at 800rpm for 5min, discard the supernatant, add fresh complete medium to resuspend the cells, and then transfer the cells to a culture flask. Mark the culture flask with cell type, cell generation, operator and operation date, and then transfer it to an incubator for culture.

[0076] Cell culture medium replacement and passaging: Be alert to changes in culture medium color. If the culture medium changes from red to yellow, please replace the culture medium with fresh one. Observe the cell density under a microscope. When the intercellular gaps are large and there is a large growth space, consider changing the cell medium. Discard the original culture medium in the culture flask, add fresh complete culture medium, and return the culture flask to the incubator for continued culture. When the cell density reaches 80%, the cells need to be passaged. After discarding the original culture medium, add an appropriate amount of PBS to wash the cells. After discarding PBS, add an appropriate amount of trypsin to digest the cells and observe the cell digestion under a microscope. When the cells gradually change from adherent epithelial cells to single round cells and the cells fall off in pieces when the adherent cell side of the culture flask is tapped, immediately stop the digestion with an appropriate amount of culture medium, transfer the cell suspension to a 15ml centrifuge tube, centrifuge at 800rpm for 5min to collect the cells, discard the supernatant, add fresh complete culture medium to resuspend the cells, transfer them to a new culture flask at a ratio of 1:3, mark it, and return it to the incubator for continued culture.

[0077] Cell freezing: Transfer the overgrown T25 cell culture flask to a clean bench and discard the original culture medium. Add an appropriate amount of PBS to wash the cells. After discarding the PBS, add an appropriate amount of trypsin to digest the cells. After digestion, add complete culture medium to stop digestion. Collect the cell suspension into a 15ml centrifuge tube, centrifuge at 800rpm for 5min to collect the cells, discard the supernatant, add 1ml of cell rapid freezing solution to resuspend the cells, and then transfer them to a cell freezing tube and seal with sealing film. Label the body of the freezing tube with the cell name, cell generation, freezing time, and operator, and freeze it at -80℃. After storing at -80℃ overnight, it can be transferred to liquid nitrogen for long-term storage.

[0078] Cytotoxicity evaluation of HMSN-Met@HA-CuO2 nanoparticles in normal cells and tumor cells

[0079] The mouse fibroblasts (L929) were counted using a cell science counter in suspension, and then the cells were seeded in a 96-well plate at a density of 5000 cells / well and cultured in an incubator overnight. The next day, the original culture medium was replaced with serum-free culture medium containing different concentrations of HMSN-Met@HA-CuO2 nanoparticles (0, 25, 50, 100, 150, 200 μg / ml). After 8 hours, the original culture medium was discarded and culture medium containing 10% CCK-8 solution was added. One hour later, the absorbance value of each well at 450 nm was calibrated using a microplate reader.

[0080] Mouse breast cancer cells (4T1) were counted using a cell science counter, and then the cells were seeded in a 96-well plate at a density of 5000 cells / well and cultured in an incubator overnight. The next day, the original culture medium was replaced with serum-free culture medium containing different concentrations of HMSN-Met@HA-CuO2 nanoparticles (0, 25, 50, 100, 150, 200 μg / ml) and different types of nanoparticles, namely HMSN, HMSN-Met, HMSN-Met@HA, HMSN@HA-CuO2, and HMSN-Met@HA-CuO2 (Met concentration was 30 μg / ml). On the third day, the original culture medium was discarded and culture medium containing 10% CCK-8 solution was added. One hour later, the absorbance value of each well at 450 nm was calibrated using a microplate reader. During the experiment, a blank group (culture medium containing 10% CCK-8) and a control group (no drug group) were set up. Cell viability was calculated according to the following formula:

[0081]

[0082] Flow cytometry was used to detect the apoptosis of tumor cells after incubation with HMSN, HMSN-Met, HMSN-Met@HA, HMSN@HA-CuO2, and HMSN-Met@HA-CuO2 (Met concentration was 30 μg / ml). 5The cells were seeded into six-well plates at a density of 100 μg / mL and placed back into the incubator for culture until the cells were fully adhered. PBS, HMSN, HMSN-Met, HMSN-Met@HA, HMSN@HA-CuO2, and HMSN-Met@HA-CuO2 (Met concentration was 30 μg / ml) were added respectively and co-cultured with the cells. After 12 hours, the cells were stained using the Annexin V-FITC / PI cell apoptosis detection kit. The specific steps were as follows: the original culture medium was collected into a 1.5 ml centrifuge tube, the cells were gently washed twice with cold PBS, and then the cells were digested with trypsin without ethylenediaminetetraacetic acid (EDTA) for 2 minutes. The cells were collected by centrifugation at room temperature (1000g, 5 minutes); the cells were resuspended with diluted 1× binding buffer, 5 μl Annexin V-FITC was added to each tube and gently mixed, and the cells were incubated at room temperature in the dark for 15 minutes. After that, 5 μl PI was added to each tube for staining for 15 minutes. After that, 400 μl 1× binding buffer was added to each tube, and the cells were immediately sieved and transferred to a flow tube for flow cytometry analysis.

[0083] The cytotoxicity of HMSN-Met@HA-CuO2 was evaluated using L929 and 4T1 cells. Figure 2 As shown in Figure g, with the increase of concentration, the cell survival rate decreased to varying degrees. After co-incubation with HMSN-Met@HA-CuO2 nanoparticles, the cell survival rate of L929 cells was greater than 80%, showing good biocompatibility. When the concentration of HMSN-Met@HA-CuO2 nanoparticles was increased to 100 μg / ml, the cell survival rate of 4T1 cells was less than 60%. Figure 2 h). In order to achieve the ideal therapeutic effect, we selected materials with a concentration of 200 μg / ml for subsequent experiments. In addition, at a concentration of 200 μg / ml, we evaluated the killing ability of different types of materials on 4T1 cells, such as Figure 2 As shown in i. Compared with the HMSN-Met@HA-CuO2 group, the cell survival rates of the HMSN group, HMSN-Met group, HMSN-Met@HA group and HMSN@HA-CuO2 group were significantly different, indicating that HMSN-Met@HA-CuO2 has a good killing effect on tumor cells. At the same time, there were significant differences in the cell survival rates of HMSN-Met@HA and HMSN-Met, which confirmed that HA targets the CD44 receptor, enhances the uptake of nanoparticles, and enhances the inhibitory effect on tumor cells. In addition, the AnnexinV-FITC / PI apoptosis detection kit was used to evaluate the toxicity of HMSN-Met@HA-CuO2 nanoparticles on 4T1 cells. As shown in Figure 2As shown in Figure j, the cell death rates induced by HMSN- and HMSN-Met were 16.1% and 19.7%, respectively, indicating that the non-carrier caused a small amount of cell death and that cell death increased after drug loading. The proportion of HMSN-Met@HA double-positive cells was 26.2%, indicating that HA has good pre-targeting properties. In addition, the proportions of HMSN@HA-CuO2 and HMSN-Met@HA-CuO2 cells in the double-positive area were 38.0% and 45.0%, respectively. This indicates that chemodynamic therapy has a strong killing ability on tumor cells. In addition, the introduction of Met further reduced the survival ability of tumor cells through the generation of ONOO-.

[0084] Example 5 Cellular uptake and cumulative ROS generation

[0085] Study on tumor cell uptake of HMSN-Met@HA-CuO2 nanoparticles

[0086] In vitro uptake of HMSN-Met@HA-CuO2 nanoparticles: Mouse breast cancer cells (4T1) were counted using a cell scientific counter and then 5×10 5 Cells were seeded at a density of 10 cells / well in a six-well plate and cultured overnight in an incubator. After 24 hours, once the cells had fully adhered, the culture medium was discarded and HMSN-Met@HA-CuO2 and HA+HMSN-Met@HA-CuO2 (200 μg / ml; cells were pretreated with hyaluronic acid aqueous solution (40 μg / ml) for 2 hours before HMSN-Met@HA-CuO2) were added and incubated for 4 hours. After incubation, the cells were harvested, filtered, and analyzed using flow cytometry.

[0087] HMSN-Met@HA-CuO2 nanoparticles cell scratch assay

[0088] 4T1 cells were cultured at 5 × 10 5 The cells were seeded at a density of 100 μg / ml in a 6-well plate and incubated overnight. After the cells were completely attached, three uniform scratches were made on the bottom of the 6-well plate using a 200 μl pipette tip. The detached cells were washed with PBS and then HMSN-Met@HA-CuO2 (200 μg / ml) was added to treat the cells. The cells were photographed and recorded using an optical microscope at 0, 6, 12, and 24 h.

[0089] Detection of ROS generated by HMSN-Met@HA-CuO2 nanoparticles in tumor cells

[0090] 5-(and 6)-Carboxy-2', 7'-dichlorodihydrofluorescein diacetate (Carboxy-H2DCFDA) probe was used to quantitatively measure the production of ROS in cells. 4T1 cells were cultured at 5×10 5 The cells were seeded at a density of 100 μg / ml in six-well plates and incubated overnight. After that, various preparations of PBS, HMSN, HMSN-Met, HMSN-Met@HA, HMSN@HA-CuO2, and HMSN-Met@HA-CuO2 (Met concentration was 30 μg / ml) were added to treat the cells for 12 h, and then incubated with Carboxy-H2DCFDA (10 μM, 1 ml) in the dark for 30 min. Carboxy-H2DCFDA was discarded and washed three times with PBS. The cells were digested and collected from the six-well plates, centrifuged at 1000 g for 5 min, and the supernatant was discarded. 300 μl PBS was added to resuspend the cells, filtered, and detected by flow cytometry.

[0091] Detection of OH generation by HMSN-Met@HA-CuO2 nanoparticles in tumor cells

[0092] 4T1 cells were cultured at 5 × 10 5 The cells were seeded at a density of 100 μg / ml in six-well plates and incubated overnight. After that, various preparations of PBS, HMSN, HMSN-Met, HMSN-Met@HA, HMSN@HA-CuO2, and HMSN-Met@HA-CuO2 (Met concentration was 30 μg / ml) were added to treat the cells for 12 h, and then incubated with ·OH probe (10 μM, 1 ml) in the dark for 30 min. After discarding the stain, the cells were washed three times with PBS. The cells were digested and collected from the six-well plates, centrifuged at 1000 g for 5 min, discarded the supernatant, and resuspended in 300 μl PBS. After filtration, the cells were detected by flow cytometry.

[0093] The premise for the anti-tumor effect of nanoparticles is that they can be absorbed by tumor cells. Low molecular weight hyaluronic acid has the ability to target CD44 receptors, which are overexpressed in mouse breast cancer cells (4T1). The targeting ability of HA was tested by pre-incubating HA with 4T1 cells to occupy a portion of CD44 receptors. The results of the test are as follows: Figure 3As shown in a and b. The uptake rate of the HMSN-Cy5@HA-CuO2+HA group pre-incubated with 4T1 cells for 4 hours was 8.45%, which was significantly lower than that of the HMSN-Cy5@HA-CuO2 group (37.3%), indicating that HMSN-Cy5@HA-CuO2 nanoparticles can effectively target tumors. HMSN-Met@HA-CuO2 can transport Cu in tumor cells through a Fenton-like reaction. 2+ and H2O2 to produce ROS and ·OH, which kill tumor cells. After incubating the nanomaterials with 4T1 cells, Hoechest33258 was used to locate the nuclear position, and DCFH-DA probe was used to detect ROS and ·OH. Figure 3 As shown in Figure c, the untreated cells are in good condition. The intercellular space in the HMSN, HMSN-Met and HMSN-Met@HA treated groups increased, but the cell morphology remained unchanged. The boundaries were clear and a small amount of ROS was produced because it was observed that in addition to a small amount of ROS in tumor cells, a small amount of reactive oxygen species was also produced when tumor cells died due to exposure to nanoparticles. The green fluorescence of the HMSN@HA-CuO2 and HMSN-Met@HA-CuO2 groups was significantly enhanced, indicating that CuO2 can induce ROS generation. Figure 3 As shown in (d), the amount of ·OH produced by HMSN@HA-CuO2 and HMSN-Met@HA-CuO2 increased significantly, indicating that CuO2 can induce a Fenton-like reaction and significantly increase the ·OH content produced in the tumor site.

[0094] Example 6 Cumulative NO production and cell migration

[0095] 4T1 cells were cultured at 2 × 10 5 The cells were seeded at a density of 100 μg / ml in a 12-well plate and incubated overnight. After the cells were completely attached, HMSN-Met@HA-CuO2 (200 μg / ml) was added to treat the cells for 0, 2, 4, 8, 12, 24, and 48 h, respectively. The cells were lysed with cell lysis buffer and collected into a 1.5 ml centrifuge tube. The cells were centrifuged at 1000 g for 15 min, and 50 μl of supernatant was transferred to a 96-well plate. 50 μl of NO detection reagent I and 50 μl of NO detection reagent II were added. After incubation in the dark for 15 min, the absorbance of each well at 540 nm was detected using a microplate reader.

[0096] NO is widely used in gas therapy. Abnormal blood vessels in the tumor site often lead to hypoxia and insufficient blood perfusion in the tumor site, resulting in poor tumor treatment effects. Nitric oxide-induced vascular normalization can effectively alleviate the root cause of tumor malignancy and drug resistance, allowing drugs to accumulate effectively in the tumor site. Figure 3As shown in Figure 5, low doses of NO were produced at 0, 2, 4, 8, 12, 24, and 48 h, indicating that HMSN-Met@HA-CuO2 continuously produced low doses of NO in cells, thereby promoting effective drug treatment. The anti-tumor cell invasion ability of HMSN-Met@HA-CuO2 was evaluated by scratch test. Figure 3 f. Quantitative results of scratch area showed that the scratch area of ​​cells at 0 h was the same, with no significant difference. Then, the scratch area of ​​cells incubated with HMSN-Met@HA-CuO2 for 6 h or 12 h, and at 24 h, was significantly different from that of the control group. Figure 3 As shown in (g), the scratch area of ​​the control group healed after 24 h, while the cells in the HMSN-Met@HA-CuO2 group only migrated slightly, indicating that HMSN-Met@HA-CuO2 has a good ability to resist tumor cell invasion.

[0097] Example 7 Immune Effects and Therapeutic Mechanisms

[0098] 4T1 tumor-bearing mice, when the tumor grows to 100mm 3When the nude mice were about 100 μl, they were randomly divided into 5 groups (3 mice in each group) and injected with 100 μl of PBS, HMSN-Met, HMSN-Met@HA, HMSN-Met@HA-CuO2, and HMSN-Met@HA-CuO2 (equivalent to Met concentration of 4 mg / kg) through the tail vein. On the 7th day, the mice in each group were killed by cervical dislocation and immersed in 75% alcohol for 2 minutes. The tumor-draining lymph nodes were collected in PBS buffer, and the spleen and tumor tissues were also collected. For lymph nodes, the lymph nodes were ground on a cell sieve with a 5 ml syringe piston, rinsed with PBS and collected in a 15 ml ep tube, centrifuged (450 g, 5 min), the supernatant was discarded, and stained with CD86-PE (0.5 μl), CD80-percpcy5.5 (0.5 μl), and CD11c-BV605 (0.5 μl) antibodies at 4 ° C for 30 min. Finally, after washing twice with PBS, 300 μl of cell fixative was added and resuspended, and stored at 4 degrees. Before being put on the machine, it was filtered into a flow tube with a 200 mesh sieve, and the expression levels of CD80, CD86, and CD11c were determined using a flow cytometer. For the spleen, transfer the spleen to 5 ml of culture medium (six-well plate), grind, collect the grinding liquid, transfer to a 15 ml centrifuge tube, centrifuge (450g, 5min), add 3 ml of red blood cell lysis buffer to each tube, lyse for 2 minutes, add 5 ml of PBS, centrifuge (450g, 5min), add 3 ml of PBS, divide into 3 ep tubes, centrifuge, precipitate and stain for later use. For the tumor, pick out about 100 mg of non-marginal, non-necrotic tissue from the tumor, weigh it, and digest it with digestion solution. 1 ml of digestion solution is required for every 100 mg of tumor tissue. Cut the tumor tissue into 1.5 ml ep tubes and add digestion solution. Shake at 37 degrees Celsius for 1 hour, centrifuge (450g, 5min), and collect the supernatant to -80°C (retain for ELISA). The specific operation steps of the ELISA experiment are as follows: take out the supernatant stored in the -80℃ refrigerator and thaw it in the 4℃ refrigerator. Be careful to avoid repeated freezing and thawing; use a 96-well plate dedicated to ELISA, add 50μl capture antibody / coating buffer to each well, cut a large piece of sealing film to cover the 96-well plate, and incubate at 4℃ overnight. Try to avoid adding liquid to the outermost edge of the 96-well plate; aspirate the buffer in the wells and rinse the plate 3 times with washing buffer squeezed out from a wash bottle. Leaving a certain soaking time (about 1 minute) during each wash process can improve the washing effect; tap the plate on absorbent paper to remove all buffer; block the wells with 100μl 1× ELISA / ELISPO Diluent (blocking buffer) diluent, cover with sealing film, and incubate at room temperature for 1 hour; prepare standard solution: add the corresponding volume of deionized water according to the label on the standard bottle, vortex gently for 10-30 minutes to ensure that the standard is completely dissolved. The standard must be used immediately after dissolution and must not be stored for next use; aspirate the blocking buffer in the wells and wash at least once according to the previous method;1. Dilution of the highest concentration standard to draw a standard curve, with a total of at least 8 points, and two parallel wells; add 50μl of 1× ELISA / ELISPO Diluent to the wells, make the first well blank, add the highest concentration standard to the first empty well at 100μl / well, transfer 50μl of the highest concentration standard from the first well to the second well, mix the liquid in the second well by multiple aspiration and spitting, then transfer 50μl of the liquid to the third well, and so on until at least 7 points are diluted. After mixing the liquid in the last well, aspirate and discard 50μl of the liquid. In addition, add another well after the last well, which contains only 50μl of 1× ELISA / ELISPO Diluent. Be careful not to scratch the bottom of the well during the whole process, and do not mix too vigorously to avoid bubbles; add 50μl of sample to the well containing 50μl of 1× ELISA / ELISPO Diluent diluent wells, cover the 96-well plate with sealing film, and incubate at room temperature for 2 hours (or incubate at 4°C overnight for maximum sensitivity); prepare detection antibodies: dilute detection antibodies (1000× and 250×) to 1× with 1× ELISA / ELISPO Diluent diluent and wash the plate, repeat 3-5 times; add 50μl of 1× ELISA / ELISPO Diluent diluted detection antibody to all wells, seal the plate and incubate at room temperature for 1 hour; prepare Streptavidin-HRP: dilute with 1× ELISA / ELISPO Dilute HRP (100×) to 1× with Diluent Diluent; aspirate and wash the plate as described above, repeat 3-5 times; add 50 μl of diluted Streptavidin-HRP to all wells, seal the plate, and incubate at room temperature for 30 minutes; aspirate and wash the plate as described above, ensuring a 1-2 minute soak time, and repeat the wash 5-7 times; add 50 μl of 1× TMB solution to each well and incubate at room temperature for 15 minutes; add 50 μl of stop solution to each well and read the plate at 450 nm using a multi-function full-wavelength microplate reader. If wavelength subtraction is available, subtract the 570 nm value from the 450 nm value and analyze the data.

[0099] Collect the precipitate, grind it in a 6-well plate, centrifuge it (450g, 5min), resuspend it in PBS, divide it into 2 tubes, centrifuge it, and stain the precipitate for later use. The staining steps are as follows:

[0100] (1) Cell pellets collected from spleen or tumor were stained with CD3-FITC (1 μl), CD4-percpcy5.5 (0.5 μl), and CD8-PE (0.5 μl) antibodies at 4°C for 30 min. After washing twice with PBS, the cells were resuspended in 300 μl of cell fixative and stored at 4°C. Before loading, the cells were filtered through a 200-mesh sieve into a flow cytometer and antigen-specific T cells were measured using a flow cytometer.

[0101] (2) Cell pellets collected from spleen or tumor were stained with CD4-percpcy5.5 (0.5ul) antibody at 4°C in the dark for 30 minutes and washed twice with PBS. Freshly prepared 0.1ml FOXP3 fixation and permeabilization working solution was added to each sample and vortexed to disperse the cells. The cells were incubated at room temperature in the dark for 30 minutes, and 1ml of freshly prepared 1× permeabilization solution was added for washing. The supernatant was discarded after centrifugation. FOXP3-AF488 (0.5ul) antibody was added and incubated at room temperature in the dark for 30 minutes. 1ml of freshly prepared 1× permeabilization solution was added for washing. The supernatant was discarded after centrifugation. Finally, 300ul of PBS was added for resuspending and stored at 4°C. The cells were filtered into a flow tube with a 200-mesh sieve before being loaded onto the flow cytometer, and regulatory T cells were measured using a flow cytometer.

[0102] (3) Cell pellets collected from the spleen, spleen single cell suspension centrifuged (450g, 5min), discarded the supernatant, stained with CD69-AF700 (1ul), CD4-FITC (0.5ul), CD8-PE (0.5ul) antibodies at 4°C in the dark for 30min, washed once with 1ml PBS, resuspended in 300μL cell fixative, and stored at 4°C. Filtered with a 200-mesh sieve into a flow cytometer before loading, CD8 was measured using a flow cytometer. + Antigen presentation efficiency of T cells.

[0103] The treatment plan of HMSN-Met@HA-CuO2 immune effect is as follows Figure 4 As shown in a. Flow cytometry was used to detect CD86 in the tumor-draining lymph nodes of mice + 、CD80 + cells with CD11c + The ratio of cells. Figure 4 As shown in b and c, CD80 in PBS group, HMSN-Met group and HMSN@HA-CuO2 group + CD86 + The cell numbers were similar and no significant differences were found. However, CD80 + CD86 + The number of cells was significantly different from that of HMSN-Met@HA-CuO2 group (CD11c +Compared with the three groups, the CD80 + CD86 + The number of cells increased significantly (CD11c + The HMSN-Met@HA-CuO2 group showed the best results in DC activation and maturation, likely due to the introduction of copper oxide, which improved the tumor microenvironment, effectively promoted lymph node migration, enhanced drug bioavailability, and created a favorable immune environment for subsequent DC homing prediction.

[0104] The cytokine IFN-γ is produced by activated CD4 + and CD8 + An effective molecule secreted by T cells, it plays an important role in enhancing immune response and preventing tumor development. IFN-γ can directly kill tumor cells, increase the expression of MHC-I, and make tumor cells sensitive to CD8 + T cell-mediated lytic reaction is sensitive. ELISA kits were used to detect the levels of IFN-γ, TNF-α, IL-4, and IL-10 cytokines in tumor supernatants on day 7 after different treatments. Figure 4 As shown in Figures df, significant differences in IFN-γ, TNF-α, and IL-10 cytokine levels were observed between the PBS, HMSN-Met, HMSN@HA-CuO2, and HMSN-Met@HA-CuO2 groups. No significant differences were observed between the HMSN-Met@HA and HMSN-Met@HA-CuO2 groups, likely due to the identical Met and HA content in the targeted 4T1 cells, resulting in no significant differences in immune responses. Significant differences in TNF-α and IL-10 levels were observed between the HMSN-Met and HMSN-Met@HA groups, suggesting that HA targeting the overexpressed CD44 receptor on the 4T1 cell surface can enhance drug uptake and optimize therapeutic efficacy. Figure 4 gThe results showed that there were significant differences in the expression levels of IL-4 between the HMSN-Met, HMSN-Met@HA, HMSN-Met@HA-CuO2 groups and the PBS group, indicating that the presence of Met caused an immune response in the tumor site and could induce immunotherapy.

[0105] Lymphocytes are divided into T lymphocytes (CD3 + ), B lymphocytes (CD19 + ) and NK cells (CD16 + CD56 + CD3 -), T lymphocytes can be divided into helper T cells, Th cells (CD3 + CD4 + CD8 - ) and cytotoxic T lymphocytes (CTL / Tc) (CD3 + CD4 - CD8 + These cells play a role in maintaining and regulating adaptive immune responses, while Tc cells can directly kill tumor cells. This study detected CD3 in spleen and tumor tissues. + CD4 + and CD3 + CD8 + T cells to determine the generation of antigen-specific T cells. Figure 4 As shown in h and i, CD3 + CD4 + The number of T cells (accounting for 18.0%) was significantly higher than that in the PBS, HMSN-Met, HMSN-Met@HA and HMSN@HA-CuO2 groups (accounting for 10.1%, 12.5%, 14.3% and 11.8%, respectively). The number of CD3 + CD4 + The number of T cells was significantly higher than that in the PBS group. Figure 5 a and b show that CD3+CD8+T cells and CD3 + CD8 + The expression of T cells (accounting for 10.6%) was significantly higher than that of PBS, HMSN-Met, HMSN-Met@HA and HMSNt@HA-CuO2 groups (3.85%, 6.72%, 8.20% and 5.26% respectively). + CD4 + The number of T cells was higher than that in the HMSN-Met group, confirming that the nanoparticles induced an effective immune response. Figure 4 lm shows that CD3 of HMSN-Met@HA-CuO2 group + CD4 + T cells (13.4%) and CD3 + CD8 + T cells (15.0%) were higher than those in the PBS group. + CD4 + T cells (2.13%, 7.01%, 9.40%, 6.95%) and CD3 + CD8 +T cells (1.96%, 6.52%, 8.51%) also existed at different levels in HMSN-Met, HMSN-Met@HA and HMSNt@HA-CuO2 groups. + CD8 + T cells and CD3 + CD4 + The high expression of T cells confirmed that HMSN-Met@HA-CuO2 nanoparticles have significant advantages in enhancing immune response therapy.

[0106] The upregulation of early activation signal (CD69) on the cell surface is a sign of T cell activation. + and CD8 + The number of activated T cells. Figure 5 As shown in ac, CD4 + CD69 + T cells and CD8 + CD69 + The number of T cells was lower in the PBS group (10.3% and 10.1%, respectively), the number of HMSN-Met cells was lower in the PBS group (14.6% and 13.8%, respectively), and the number of HMSN-Met@HA cells was lower in the PBS group (17.5% and 18.0%, respectively). + CD69 + T cells and CD8 + CD69 + The T cells in the HMSN-Met@HA-CuO2 group increased significantly (24.1% and 28.9%, respectively). + CD69 + T cells and CD8 + CD69 + The proportion of T cells was only 13.3% and 12.7%, which did not increase significantly. Therefore, Met can effectively activate CD4 + and CD8 + T cells promote the proliferation and activation of antigen-specific T cells. Tregs typically express Foxp3. Their primary function is to maintain normal immune tolerance and control inflammatory responses. Increased Tregs indicate that immune responses may be suppressed. Figure 5 d and f show that FOXP3 in spleen cells of treatment group + CD4 + The number of cells decreased significantly, and FOXP3 in tumor cells + CD4 + Cells also showed the same excellent effect ( Figure 5e and g). Thus, HMSN-Met@HA-CuO2 reduced the percentage of Treg cells, thereby alleviating tumor-related immunosuppression.

[0107] Example 8 Antitumor Efficacy

[0108] The experiment was conducted in strict compliance with animal welfare and ethical standards. The acceptance unit is the Laboratory Animal Welfare and Ethics Committee of Zhejiang Ocean University, acceptance number: 2022050. Four-week-old female Balb / c mice were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. All samples and instruments were irradiated with ultraviolet light for 1 hour before the experiment. At the end of the experiment, all mice were killed by cervical dislocation. After 7 days of adaptive feeding, 4T1 cells (2×10 cells per mouse) were injected subcutaneously into the right lower abdomen of the mice. 6 cells), when the tumor grows to 70 mm 3 When the size of the nude mice was about 100 μl, the nude mice were randomly divided into 5 groups (5 mice in each group) and injected with 100 μl of PBS, HMSN-Met, HMSN-Met@HA, HMSN@HA-CuO2, and HMSN-Met@HA-CuO2 (equivalent to Met concentration of 4 mg / kg) through the tail vein every 48 hours. The injection was stopped on the 8th day. The tumor size and body weight of the mice were recorded every 2 days. The formula for calculating tumor volume is: tumor volume = (tumor length × tumor width) 2 On day 14, mice were sacrificed, tumor tissues were collected and stored in 4% tissue fixative. After dehydration, embedding, and freezing, sections were cut into 5-μm sections. Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) apoptosis detection kit, hematoxylin and eosin (H&E) staining, and Ki67 staining were used to assess apoptosis in tumor tissues of each group.

[0109] Study on the tumor targeting efficacy of HMSN-Met@HA-CuO2 in vivo

[0110] In order to study the ability of nanoparticles to migrate to systemic organs and target tumors after tail vein injection, 4T1 tumor-bearing mice were injected with HMSN-Cy5@HA-CuO2 (equivalent Cy5 concentration was 0.1 mg / kg) through the tail vein. The hearts, livers, spleens, lungs, kidneys, and tumors of the tumor-bearing mice were removed at 0, 4, 8, 12, and 24 hours, respectively, and the distribution and intensity of the Cy5 fluorescence signal were detected using a small animal in vivo imaging device.

[0111] A 4T1 breast cancer mouse model was established to study the anti-tumor and anti-tumor effects of HMSN-Met@HA-CuO2 ( Figure 6 a) HMSN-Cy5@HA-CuO2 was injected into the tail vein of mice to observe the distribution of nanoparticles throughout the body. The results obtained using a small animal vivisection device at 4, 8, 12, and 24 hours are shown in Figure 2. Figure 5 h. The results showed that HMSN-Met@HA-CuO2 was mainly enriched in the tumor site at 12 hours, reached a peak at 12 hours, and then began to be metabolized through the liver and kidneys. Various indicators of mice were monitored during the treatment process, and the results were shown as follows Figure 6 As shown in b, the body weight of mice did not decrease significantly, and the tumor volume ( Figure 6 c) was gradually controlled during the treatment. Tumor weight and images of mice ( Figure 6 d and e) show that the tumor mass of the treated mice was significantly reduced. It is impressive that HMSN-Met@HA-CuO2 exhibited a better tumor ablation effect. Figure 6 f. The cells in the PBS group were closely arranged, with clear boundaries, full cytoplasm, and obvious nuclei. The cells in the HMSN-Met group began to shrink, with a small amount of cavities appearing. The cells in the HMSN-Met@HA group ruptured and ablated, with unclear boundaries. The nuclei in the HMSN@HA-CuO2 group shrank, with blurred boundaries. The cells in the HMSN-Met@HA-CuO2 group ablated over a large area, resulting in loss of cavities and cytoplasm. In addition, the TUNEL and Ki67 results ( Figure 6 f) showed that HMSN-Met@HA-CuO2 could better play its role in destroying tumor cell DNA and inducing lipid peroxidation in tumor cells, thereby leading to tumor cell apoptosis.

[0112] Example 9 Biosafety Study of HMSN-Met@HA-CuO2 Nanoparticles

[0113] On the 14th day, blood was collected from the orbits of mice in each group for liver and kidney function test. All mice were killed and the main organs (heart, liver, spleen, lung, and kidney) were collected and stored in 4% tissue fixative for more than 6 hours. The tissues were trimmed, dehydrated, embedded, frozen, and cut into 5 μm thick slices for hematoxylin andeosin (H&E) staining.

[0114] Orbital blood was collected from mice for biochemical index detection. The results are shown in Figure 7 a. All biochemical indicators of mice in each experimental group were within the normal range. H&E staining results are shown in Figure 2. Figure 7 The results showed that HMSN-Met@HA-CuO2 could be safely injected intravenously without causing serious adverse reactions or toxic side effects in vivo or in vitro.

[0115] Finally, it should be noted that the above is merely one specific embodiment of the present invention. Obviously, the present invention is not limited to the above embodiment and is susceptible to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention are considered to be within the scope of protection of the present invention.

Claims

1. A method for preparing a nano-delivery system for immuno-chemodynamic tumor therapy: characterized in that, The method comprises the following steps: 1) silica colloidal solution: ultrapure water and anhydrous ethanol are added to a reaction vessel, and then ammonia water is added and stirred. The mixture is added to the above mixed solution, and the above reaction system is stirred and reacted at 40° C. to obtain a silica solution. The supernatant is removed by centrifugation, and ultrapure water and anhydrous ethanol are added for ultrasonic dispersion and washing to remove unreacted raw materials to obtain silica particles. The silica particles are then uniformly dispersed in ultrapure water to obtain a silica colloidal solution for later use; 2) Preparation of HMSN@CTAC Nanoparticles: CTAC and TEA were weighed and mixed with ultrapure water, stirred, and the silica colloidal solution prepared in step 1) was added, stirred, and the temperature was increased. TEOS was quickly added and stirred for reaction. The temperature was lowered, and solid sodium carbonate was added to selectively etch the silica nanoparticle template. The mixture was stirred, centrifuged, and washed with ultrapure water and anhydrous ethanol, respectively, to obtain HMSN@CTAC nanoparticles. 3) Preparation of HMSN nanoparticles: Concentrated hydrochloric acid was dissolved in anhydrous ethanol to prepare a hydrochloric acid-ethanol solution. The HMSN@CTAC nanoparticles washed in step 2) were uniformly dispersed in the hydrochloric acid-ethanol solution by ultrasonication. After refluxing for 24 hours, the mixture was centrifuged and washed multiple times with ultrapure water and anhydrous ethanol. The extraction was repeated 2-3 times to ensure the removal of CTAC, thereby obtaining HMSN nanoparticles. 4) HMSN-NH2 nanoparticles: HMSN nanoparticles were dispersed in anhydrous ethanol by ultrasonication, heated, and APTES was added while heating. The mixture was refluxed and centrifuged. The precipitate was collected and washed with water and ethanol respectively, and freeze-dried to obtain HMSN-NH2 nanoparticles. 5) Preparation of HA-CuO2 nanoparticles: Hyaluronic acid (HA) was dissolved in ultrapure water, and NaOH solution was added and mixed. CuCl2 solution was then added, followed by slow dropwise addition of H2O2. The mixture was mixed, the pH was adjusted, and the mixture was washed with water to obtain HA-CuO2 nanoparticles. 6) HMSN-Met nanoparticles: HMSN-NH2 nanoparticles were ultrasonically dispersed in PBS, Met was added, stirred, dialyzed, centrifuged, and the precipitate was collected to obtain HMSN-Met nanoparticles; 7) HMSN@HA-CuO2 nanoparticles: HMSN-NH2 nanoparticles were ultrasonically dispersed in PBS, HA-CuO2 was added, stirred, dialyzed, centrifuged, and the precipitate was collected to obtain HMSN@HA-CuO2 nanoparticles; 8) HMSN-Met@HA nanoparticles: HMSN-NH2 nanoparticles were ultrasonically dispersed in PBS, stirred with 20 mg Met, and HA was added to continue the reaction. The mixture was dialyzed and centrifuged, and the precipitate was collected to obtain HMSN-Met nanoparticles. 9) HMSN-Met@HA-CuO2 nanoparticles: HMSN-NH2 nanoparticles were ultrasonically dispersed in PBS, Met was added, stirred, HA-CuO2 was added, dialyzed, centrifuged, and the precipitate was collected to obtain HMSN-Met@HA-CuO2.

2. The method for preparing a nano-delivery system for immuno-chemodynamic tumor therapy according to claim 1, characterized in that The volume ratio of TEOS in the reaction system of step 1) to ultrapure water in the dispersion system is 2 ml:60 ml; the volume ratio of ultrapure water: anhydrous ethanol: ammonia water: TEOS in the reaction system of step 1) is 15 ml:60 ml:3 ml:2 ml.

3. The method for preparing a nano-delivery system for immuno-chemodynamic tumor therapy according to claim 1, characterized in that In the step 2), the weight-to-volume ratio of CTAC:TEA:ultrapure water:colloid silica solution:TEOS is 6 g:60 mg:60 mL:20 ml:0.3 mL; the elevated temperature in the step 2) is 80° C., the stirring reaction temperature in the step 2) is 80° C., the cooling in the step 2) is 50° C., and the final concentration of sodium carbonate added in the step 2) is 0.6 mol / L.

4. The method for preparing a nano-delivery system for immuno-chemodynamic tumor therapy according to claim 1, characterized in that In the step 3), the volume ratio of concentrated hydrochloric acid to anhydrous ethanol is 5:45, and the reflux temperature is 70°C.

5. The method for preparing a nano-delivery system for immuno-chemodynamic tumor therapy according to claim 1, characterized in that In the step 4), the weight-to-volume ratio of HMSN nanoparticles: anhydrous ethanol: APTES is 0.1 mg: 60 mL: 1 mL, and the heating and reflux temperature is 70°C.

6. The method for preparing a nano-delivery system for immuno-chemodynamic tumor therapy according to claim 1, characterized in that The concentration of the NaOH solution in step 5) is 0.01 M, and the weight-to-volume ratio of hyaluronic acid (HA): ultrapure water: NaOH solution: H₂O₂: CuCl₂ solution is 0.050 g:40 mL:5 mL:1 mL:5 mL. The weight-to-volume ratio of CuCl₂·2H₂O to water in the CuCl₂ solution is 0.136 g:5 mL. The pH in step 5) is 10.

7. The method for preparing a nano-delivery system for immuno-chemodynamic tumor therapy according to claim 1, characterized in that In step 6), the weight-to-volume ratio of HMSN-NH2 nanoparticles:Met:PBS is 10 mg:20 mg:10 ml, and the PBS is a phosphate buffer solution with a pH of 7.

8. The method for preparing a nano-delivery system for immuno-chemodynamic tumor therapy according to claim 1, characterized in that In step 7), PBS is a phosphate buffer solution with a pH of 7, and the weight-to-volume ratio of the HMSN-NH2 nanoparticles: PBS: HA-CuO2 is 10 mg: 10 ml: 10 mg.

9. The method for preparing a nano-delivery system for immuno-chemodynamic tumor therapy according to claim 1, characterized in that In the step 8), the weight-to-volume ratio of HMSN-NH2 nanoparticles: PBS: Met: HA is 10 mg: 10 ml: 20 mg: 5 mg; in the step 9), the weight ratio of HMSN-NH2 nanoparticles: Met: HA-CuO2 is 1:2:1, and the weight-to-volume ratio of the HMSN-NH2 nanoparticles and PBS must be 10 mg: 10 ml.

10. The nano-delivery system for immuno-chemodynamic tumor therapy prepared according to the method according to any one of claims 1 to 9.

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