Albumin manganese dioxide nanofomulation loaded with psoralen, its preparation method and application

Abstract

This invention discloses a psoralen-loaded albumin-manganese dioxide nanoparticle formulation, its preparation method, and its applications. The nanoparticle formulation is composed of psoralen, manganese dioxide, polyacrylamide hydrochloride, and albumin, wherein the mass ratio of psoralen, manganese dioxide, polyacrylamide hydrochloride, and albumin is 5-25:2.5:2.5-12.5:0.8-6. This nanoparticle formulation significantly improves the solubility and bioavailability of psoralen, and exhibits acid-sensitive and delayed-release properties. It also demonstrates good tumor targeting and can be used as a regulator of hypoxia and tumor-associated macrophages, improving the microenvironment of triple-negative breast cancer, and thus for the preparation of drugs for treating triple-negative breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a nano-formulation loaded with psoralen, its preparation method and application, specifically to an albumin-manganese dioxide nano-formulation loaded with psoralen, its preparation method and application. Background Technology

[0002] Breast cancer (BC) is one of the most common malignant tumors in women, ranking first in incidence among female malignant tumors. Triple-negative breast cancer (TNBC) is a type of breast cancer that is negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). This type of breast cancer is characterized by early onset, high invasiveness, high metabolic remodeling, and poor prognosis. Because TNBC lacks ER, PR, and HER2 expression, conventional endocrine therapy and targeted therapy are often ineffective. Taxanes and anthracyclines are common chemotherapy agents for TNBC, but they have significant toxic side effects.

[0003] Traditional research on anti-tumor drugs for breast cancer has primarily focused on the tumor cells themselves, such as altering multidrug resistance genes, inhibiting the upregulation of efflux pump proteins to reduce drug efflux, inhibiting metabolic activity to enhance drug activity, and regulating apoptosis gene expression to induce tumor cell apoptosis. However, the effects have been less than ideal, partly because tumor cell development stems not only from changes within the cells themselves but also from alterations in their surrounding microenvironment. With a deeper understanding of the biology of tumor-associated neoplasia (TNBC), treatment strategies based on the tumor microenvironment (TME) are gaining increasing attention. The TME refers to the local homeostatic environment composed of tumor cells, fibroblasts, mesenchymal cells, vascular endothelial cells, smooth muscle cells, inflammatory / immune cells, and the extracellular matrix. The tumor microenvironment differs significantly from the normal tissue microenvironment, primarily exhibiting characteristics such as hypoxia, weak acidity, reducing properties, and low vascular density. The tumor microenvironment plays a crucial role in tumor development and progression, including proliferation, immune escape, and distant metastasis. The immune microenvironment of breast cancer refers to the local internal environment jointly constituted by immune cells infiltrating the breast tumor and the active mediators they secrete, along with breast cancer cells. It is mainly mediated by tumor-associated macrophages (TAMs), tumor-associated phages (TANs), mammary gland cells (MDSCs), and regulatory T cells (Tregs) to create an immunosuppressive microenvironment. Tumor-associated macrophages (TAMs) are among the most abundant immune cells infiltrating breast cancer. Inhibiting the tumor-promoting phenotype of TAMs in the microenvironment, inducing TAMs to transform into a tumor-suppressing phenotype, or regulating the ratio and number of TAMs with both phenotypes holds promise for suppressing the growth of triple-negative breast cancer. However, research on TAM modulators for triple-negative breast cancer is currently limited.

[0004] Furthermore, cytokines are crucial signaling molecules in intercellular transduction and play a vital role in the regulation of the tumor microenvironment. Inflammatory cytokines are signaling molecules secreted by inflammatory cells. TNBC cells induce immune cells such as macrophages to transform into phenotypes favorable to their own growth through autocrine cytokines, or receive cytokines from other cells, activating their own growth-related signaling pathways and promoting their own development and metastasis. Regulating the expression of important immune / inflammatory factors in the tumor microenvironment is one of the strategies for treating breast cancer. However, current cytokine therapies based on monoclonal antibodies are not very effective and often have side effects. Therefore, it is necessary to find new therapeutic drugs that can simultaneously regulate multiple cytokines to significantly improve the tumor microenvironment.

[0005] Studies have found that psoralen (PSO) possesses immunomodulatory, anti-inflammatory, antibacterial, antioxidant, anti-osteoporosis, and anti-tumor effects. PSO inhibits cell proliferation by inducing G0 / G1 phase arrest in tumor cells and regulating the Wnt / β-catenin pathway; it inhibits tumor invasion and migration by suppressing the NF-κB signaling pathway and EMT; and it reverses multidrug resistance in tumors by inhibiting Bc1-2 gene protein expression, ABCB1 promoter activity, and P-gp transporter efflux. However, PSO has poor solubility, resulting in low bioavailability and limiting its anti-tumor applications. In recent years, a new technology has emerged in the field of pharmaceutical formulation: nanomedicine formulations. These formulations utilize nanotechnology to self-assemble or load traditional small-molecule chemotherapy drugs or large-molecule drugs such as proteins and nucleic acids onto nanocarriers, forming drugs with nanoscale structures. The particle size range of nanomedicines gives them unique size and surface effects, which can overcome the inherent limitations of traditional drugs, such as poor water solubility and strong cytotoxicity. They can improve drug efficacy, stability, solubility, in vivo circulation time, and tumor accumulation efficiency, and can target drugs to specific tissues and cells, thereby reducing the toxic side effects of drugs on the human body. However, there are currently no reports on combining psoralen with nanomedicine formulations to improve its solubility and bioavailability.

[0006] In view of this, providing a nanoformulation that can improve the solubility and bioavailability of psoralen for use in the preparation of drugs for the treatment of triple-negative breast cancer has become an urgent technical problem to be solved. Summary of the Invention

[0007] Therefore, the technical problem to be solved by this invention is that psoralen has low solubility and bioavailability. Thus, an albumin-loaded manganese dioxide nanoparticle formulation with improved solubility and bioavailability, as well as its preparation method and application, are proposed.

[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0009] The first aspect of the present invention provides an albumin-based manganese dioxide nanoparticle formulation loaded with psoralen, the nanoparticle formulation being composed of psoralen, manganese dioxide, polyacrylamide hydrochloride and albumin, wherein the mass ratio of psoralen, manganese dioxide, polyacrylamide hydrochloride and albumin is 5-25∶2.5∶2.5-12.5∶0.8-6.

[0010] A second aspect of the present invention provides a method for preparing the albumin-based manganese dioxide nanoparticles loaded with psoralen, comprising the following steps:

[0011] S1. Preparation of silica nanoparticle template: Tetraethyl orthosilicate is mixed with a solvent, ammonia is added and the mixture is ultrasonically reacted. The resulting reaction solution is centrifuged and the centrifuged precipitate is dispersed to obtain silica nanoparticle template solution.

[0012] S2. Preparation of manganese dioxide nanoparticles: Add potassium permanganate solution to the silica nanoparticle template solution, sonicate and centrifuge for the first time, disperse the precipitate in sodium carbonate solution, centrifuge for the second time, wash the precipitate, centrifuge for the third time to remove the precipitate, and obtain manganese dioxide nanoparticle solution.

[0013] S3. Preparation of polyacrylamide hydrochloride-manganese dioxide nanoparticle composite: Add polyacrylamide hydrochloride to the manganese dioxide nanoparticle solution, mix evenly and centrifuge, wash the centrifuged precipitate and disperse it to obtain polyacrylamide hydrochloride-manganese dioxide nanoparticle solution.

[0014] S4. Preparation of albumin-modified manganese dioxide nanoparticles: The polyacrylamide hydrochloride-manganese dioxide nanoparticle solution was added to the albumin solution, anhydrous ethanol was added, and the mixture was centrifuged. The precipitate was washed and dispersed to obtain the albumin-modified manganese dioxide nanoparticle solution.

[0015] S5. Preparation of albumin-modified manganese dioxide nanoparticles loaded with psoralen: Add psoralen solution to the albumin-modified manganese dioxide nanoparticle solution, stir and react, centrifuge, filter and centrifuge to obtain albumin-modified manganese dioxide nanoparticles loaded with psoralen.

[0016] A third aspect of the present invention provides the application of the psoralen-loaded albumin-manganese dioxide nanoparticle formulation in the role of a hypoxia and tumor-associated macrophage regulator.

[0017] The fourth aspect of this invention provides the application of the psoralen-loaded albumin-manganese dioxide nanoparticle formulation in the preparation of a drug for treating triple-negative breast cancer.

[0018] The technical solution of the present invention has the following advantages compared with the prior art:

[0019] The psoralen-loaded albumin-manganese dioxide nanoparticle formulation provided by this invention comprises psoralen, manganese dioxide, polyacrylamide hydrochloride, and albumin, wherein the mass ratio of psoralen, manganese dioxide, polyacrylamide hydrochloride, and albumin is 5-25:2.5:2.5-12.5:0.8-6. This nanoparticle formulation significantly improves the solubility and bioavailability of psoralen, and exhibits acid-sensitive and delayed-release properties. It also demonstrates good tumor targeting and can be used as a regulator of hypoxia and tumor-associated macrophages, improving the microenvironment of triple-negative breast cancer, and thus for the preparation of drugs to treat triple-negative breast cancer. Attached Figure Description

[0020] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0021] Figure 1 (a)-(b) are transmission electron microscopy images of H-MnO2-NPs;

[0022] (c)-(d) are transmission electron microscopy images of PSO / BSA-MnO2-NPs;

[0023] Figure 2 (a) is the particle size distribution diagram of PSO / BSA-MnO2-NPs; (b) is the Zeta potential diagram of PSO / BSA-MnO2-NPs.

[0024] Figure 3 It is an ultraviolet absorption spectrum;

[0025] Figure 4 It is a Fourier transform infrared spectrum;

[0026] Figure 5 It is a thermogravimetric analysis chart;

[0027] Figure 6 It is a differential scanning calorimetry (DSC) spectrum;

[0028] Figure 7 This is a graph showing the in vitro release detection of PSO / BSA-MnO2-NPs at different pH values;

[0029] Figure 8 These are laser confocal microscopy images of PSO / BSA-MnO2-NPs in MDA-MB-231 cells;

[0030] Figure 9 This is a flow cytometry image of PSO / BSA-MnO2-NPs in MDA-MB-231 cells;

[0031] Figure 10This is a graph showing the cytotoxicity test of PSO / BSA-MnO2-NPs against MDA-MB-231.

[0032] Figure 11 This is a graph showing the cytotoxicity test of BSA-MnO2-NPs against MDA-MB-231;

[0033] Figure 12 (a)-(g) are graphs showing the effect of PSO / BSA-MnO2-NPs on apoptosis in MDA-MB-231 cells;

[0034] Figure 13 This is a graph showing the cytotoxicity of CoCl2 against MDA-MB-231.

[0035] Figure 14 This is a graph showing the effect of CoCl2 on HIF-1α expression in MDA-MB-231 cells;

[0036] Figure 15 (a)-(j) are graphs showing the effect of PSO / BSA-MnO2-NPs on apoptosis in hypoxic MDA-MB-231 cells;

[0037] Figure 16 This is a graph showing the effect of PSO / BSA-MnO2-NPs on HIF-1α expression in hypoxic MDA-MB-231 cells;

[0038] Figure 17 (a)-(b) are flow cytometry results of molecular markers on the surface of THP-1 cells treated with different induction methods;

[0039] Figure 18 This is a graph showing the detection of cytokine levels secreted by THP-1 cells treated with different induction methods using ELISA.

[0040] Figure 19 These are confocal images of macrophage uptake of PSO / BSA-MnO2-NPs;

[0041] Figure 20 This is a flow cytometry image of macrophage uptake of PSO / BSA-MnO2-NPs;

[0042] Figure 21 (a)-(m) are graphs showing the effect of PSO / BSA-MnO2-NPs on apoptosis of MDA-MB-231 cells in the co-culture system;

[0043] Figure 22 This is a graph showing the effect of PSO / BSA-MnO2-NPs on the expression of immunosuppressive factors in macrophages. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0045] Unless otherwise specified, the reagents, methods, and equipment used in the embodiments of this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the embodiments of this invention are all commercially available.

[0046] Example 1

[0047] This embodiment provides an albumin-based manganese dioxide nanoparticle formulation loaded with psoralen, which is prepared by the following method:

[0048] S1. Preparation of silica nanoparticle (SiO2-NPs) template: 4.7 mL of anhydrous ethanol and 5.0 mL of ultrapure water were ultrasonically mixed for 10 min, 0.2 mL of tetraethyl orthosilicate (TEOS) was added, and the mixture was ultrasonically mixed for 20 min. 10.8 mL of 26% ammonia water was added, and the mixture was ultrasonically reacted for 60 min. The resulting solution was centrifuged at 13000 rpm for 10 min. The centrifuged precipitate was washed three times with ultrapure water and then dispersed with ultrapure water to obtain a SiO2-NPs solution.

[0049] S2. Preparation of hollow mesoporous manganese dioxide nanoparticles (H-MnO2-NPs): Add 50 mL of potassium permanganate

[0050] (KMnO4) solution was slowly added dropwise to SiO2-NPs solution (obtained in step S1), wherein the molar ratio of potassium permanganate to SiO2-NPs was 48:1. After sonication for 6 hours until homogeneous, the mixture was centrifuged at 13000 rpm for 10 minutes. The resulting precipitate was washed three times with ultrapure water and then dispersed in 20 mL of 2 mol / L sodium carbonate (Na2CO3) solution, wherein the molar ratio of Na2CO3 to SiO2-NPs was 8:1. The mixture was stirred at 200 rpm for 12 hours at 60 °C. The resulting product was centrifuged at 13000 rpm for 10 minutes. After washing the precipitate three times with ultrapure water, the mixture was centrifuged at 3500 rpm for 10 minutes. The precipitate was discarded to obtain H-MnO2-NPs.

[0051] S3. Preparation of polyacrylamide hydrochloride (PAH) coated manganese dioxide nanoparticles (PAH-MnO2-NPs): Add 5 mL of PAH solution with a concentration of 1.0 mg / mL to 5 mL of H-MnO2-NPs solution with a concentration of 0.5 mg / mL (obtained in step S2), mix and stir at 30 °C for 2 h, then centrifuge at 13000 rpm for 10 min, wash the obtained precipitate with ultrapure water 3 times and disperse it with ultrapure water to obtain PAH-MnO2-NPs solution.

[0052] S4. Preparation of bovine serum albumin (BSA) modified manganese dioxide nanoparticles (BSA-MnO2-NPs): 10 mL of PAH-MnO2-NPs solution with a concentration of 0.5 mg / mL (obtained in step S3) was added to 40 mL of BSA solution with a concentration of 0.05 mg / mL, and 5 mL of anhydrous ethanol was added dropwise. The mixture was stirred at 25 °C for 12 h, and then centrifuged at 13000 rpm for 10 min. The precipitate was washed three times with ultrapure water and then dispersed to obtain BSA-MnO2-NPs solution.

[0053] S5. Preparation of albumin-manganese dioxide nanoparticles loaded with psoralen (PSO / BSA-MnO2-NPs): 5 mL of PSO acetone solution was added dropwise to 20 mL of BSA-MnO2-NPs solution with a concentration of 2.0 mg / mL (obtained in step S4), wherein the amount of PSO used was 20 mg. The reaction was carried out at 40 °C and stirred at 100 rpm for 8 h. The acetone organic solvent was removed by stirring at 70 °C for 10 min. Then, the mixture was centrifuged at 3500 rpm for 10 min. After discarding the precipitate, the remaining solution was filtered through a 0.45 μm filter membrane to obtain PSO / BSA-MnO2-NPs.

[0054] The albumin-based manganese dioxide nanoparticle formulation loaded with psoralen provided in this embodiment significantly improves the solubility and bioavailability of psoralen, and exhibits acid sensitivity and delayed release characteristics, while also demonstrating good tumor targeting. It can be used as a hypoxia and tumor-associated macrophage regulator by inducing apoptosis in MDA-MB-231 cells, regulating the phenotype and cytokine secretion of tumor-associated macrophages in triple-negative breast cancer, and reducing the secretion of the immunosuppressive factors IL-10 and IL-6 from M2 macrophages. Furthermore, it can improve the hypoxia problem in the microenvironment of triple-negative breast cancer by inhibiting the expression of HIF-1α in CoCl2-induced MDA-MB-231 cells, catalyzing the decomposition of hydrogen peroxide to produce oxygen, and inhibiting the expression of hypoxia-inducible factors. Therefore, this nanoparticle formulation can be used to prepare drugs for treating triple-negative breast cancer, thereby improving the efficacy of triple-negative breast cancer treatment.

[0055] Example 2

[0056] This embodiment provides an albumin-based manganese dioxide nanoparticle formulation loaded with psoralen, which is prepared by the following method:

[0057] S1. Preparation of silica nanoparticle (SiO2-NPs) template: 4.1 mL of anhydrous ethanol and 4.0 mL of ultrapure water were ultrasonically mixed for 5 min, 0.4 mL of tetraethyl orthosilicate (TEOS) was added, and the mixture was ultrasonically mixed for 15 min. 2 mL of 25% ammonia water was added, and the mixture was ultrasonically reacted for 20 min. The resulting solution was centrifuged at 11000 rpm for 15 min. The centrifuged precipitate was washed 3 times with ultrapure water and then dispersed with ultrapure water to obtain SiO2-NPs solution.

[0058] S2. Preparation of hollow mesoporous manganese dioxide nanoparticles (H-MnO2-NPs): 40 mL of potassium permanganate (KMnO4) solution was slowly added dropwise to the SiO2-NPs solution (obtained in step S1), wherein the molar ratio of potassium permanganate to SiO2-NPs was 12:1. After sonication for 1 h until homogeneous, the mixture was centrifuged at 11000 rpm for 15 min. The resulting precipitate was washed three times with ultrapure water and then dispersed in 15 mL of 1.5 mol / L sodium carbonate (Na2CO3) solution, wherein the molar ratio of Na2CO3 to SiO2-NPs was 2:1. The mixture was stirred at 100 rpm for 24 h at 40 °C. The resulting product was centrifuged at 11000 rpm for 15 min. The precipitate was washed three times with ultrapure water and then centrifuged at 3000 rpm for 15 min. The precipitate was discarded to obtain H-MnO2-NPs.

[0059] S3. Preparation of polyacrylamide hydrochloride (PAH) coated manganese dioxide nanoparticles (PAH-MnO2-NPs): Add 4 mL of 0.5 mg / mL PAH solution to 6 mL of 0.4 mg / mL H-MnO2-NPs solution (obtained in step S2), mix and stir at 25 °C for 1 h, then centrifuge at 11000 rpm for 15 min, wash the obtained precipitate three times with ultrapure water and disperse it with ultrapure water to obtain PAH-MnO2-NPs solution.

[0060] S4. Preparation of bovine serum albumin (BSA) modified manganese dioxide nanoparticles (BSA-MnO2-NPs): 8 mL of PAH-MnO2-NPs solution with a concentration of 0.6 mg / mL (obtained in step S3) was added to 35 mL of BSA solution with a concentration of 0.02 mg / mL, and 4 mL of anhydrous ethanol was added dropwise. The mixture was stirred at 35 °C for 25 h, and then centrifuged at 11000 rpm for 15 min. The precipitate was washed three times with ultrapure water and then dispersed to obtain BSA-MnO2-NPs solution.

[0061] S5. Preparation of albumin-containing manganese dioxide nanoparticles loaded with psoralen (PSO / BSA-MnO2-NPs): 4 mL of PSO acetone solution was added dropwise to 17 mL of BSA-MnO2-NPs solution with a concentration of 1.5 mg / mL (obtained in step S4), wherein the amount of PSO used was 5 mg. The reaction was stirred at 150 rpm at 25 °C for 4 h, and stirred at 75 °C for 15 min to remove the acetone organic solvent. Then, the mixture was centrifuged at 3000 rpm for 15 min, the precipitate was discarded, and the remaining solution was filtered through a 0.45 μm filter membrane to obtain PSO / BSA-MnO2-NPs.

[0062] Example 3

[0063] This embodiment provides an albumin-based manganese dioxide nanoparticle formulation loaded with psoralen, which is prepared by the following method:

[0064] S1. Preparation of silica nanoparticle (SiO2-NPs) template: 14 mL of anhydrous ethanol and 9.7 mL of ultrapure water were ultrasonically mixed for 20 min, 0.8 mL of tetraethyl orthosilicate (TEOS) was added, and the mixture was ultrasonically mixed for 25 min. 16.5 mL of 28% ammonia water was added, and the mixture was ultrasonically reacted for 120 min. The resulting solution was centrifuged at 15000 rpm for 5 min. The centrifuged precipitate was washed 3 times with ultrapure water and then dispersed with ultrapure water to obtain SiO2-NPs solution.

[0065] S2. Preparation of hollow mesoporous manganese dioxide nanoparticles (H-MnO2-NPs): 60 mL of potassium permanganate (KMnO4) solution was slowly added dropwise to the SiO2-NPs solution (obtained in step S1), wherein the molar ratio of potassium permanganate to SiO2-NPs was 96:1. After sonication for 1 h until homogeneous, the mixture was centrifuged at 14000 rpm for 5 min. The resulting precipitate was washed three times with ultrapure water and then dispersed in 25 mL of 2.5 mol / L sodium carbonate (Na2CO3) solution, wherein the molar ratio of Na2CO3 to SiO2-NPs was 10:1. The mixture was stirred at 300 rpm for 3 h at 70 °C. The resulting product was centrifuged at 14000 rpm for 5 min. The precipitate was washed three times with ultrapure water and then centrifuged at 4000 rpm for 5 min. The precipitate was discarded to obtain H-MnO2-NPs.

[0066] S3. Preparation of polyacrylamide hydrochloride (PAH) coated manganese dioxide nanoparticles (PAH-MnO2-NPs): Add 8 mL of PAH solution with a concentration of 2.5 mg / mL to 8 mL of H-MnO2-NPs solution with a concentration of 0.8 mg / mL (obtained in step S2), mix and stir at 50 °C for 6 h, then centrifuge at 14000 rpm for 5 min, wash the obtained precipitate with ultrapure water 3 times and disperse it with ultrapure water to obtain PAH-MnO2-NPs solution.

[0067] S4. Preparation of bovine serum albumin (BSA) modified manganese dioxide nanoparticles (BSA-MnO2-NPs): 15 mL of PAH-MnO2-NPs solution with a concentration of 0.8 mg / mL (obtained in step S3) was added to 45 mL of BSA solution with a concentration of 0.15 mg / mL, and 8 mL of anhydrous ethanol was added dropwise. The mixture was stirred at 50 °C for 36 h, and then centrifuged at 14000 rpm for 5 min. The precipitate was washed three times with ultrapure water and then dispersed to obtain BSA-MnO2-NPs solution.

[0068] S5. Preparation of albumin-containing manganese dioxide nanoparticles loaded with psoralen (PSO / BSA-MnO2-NPs): 8 mL of PSO acetone solution was added dropwise to 25 mL of BSA-MnO2-NPs solution with a concentration of 2.5 mg / mL (obtained in step S4), wherein the amount of PSO used was 25 mg. The reaction was carried out at 50 °C and 250 rpm for 24 h with stirring. The acetone organic solvent was removed by stirring at 65 °C for 20 min. Then, the mixture was centrifuged at 5000 rpm for 5 min, the precipitate was discarded, and the remaining solution was filtered through a 0.45 μm filter membrane to obtain PSO / BSA-MnO2-NPs.

[0069] Experimental Example

[0070] The test samples in the following experimental examples were all prepared using the method provided in Example 1.

[0071] 1. The PSO content in PSO / BSA-MnO2-NPs was determined by high performance liquid chromatography (HPLC).

[0072] Chromatographic conditions: Column: Agilent ZORBAX SB C18 (4.6 mm × 250 mm, 5 μm); Column temperature: 30 ℃; Detection wavelength: 245 nm; Mobile phase: methanol-water (55:45); Flow rate: 1.0 L / min; Injection volume: 10 μL.

[0073] PSO Standard Curve: Accurately weigh 6.25 mg of PSO standard and dilute to 25 mL with methanol to obtain a PSO standard solution concentration of 250 μg / mL. Dilute this standard solution to 0.41, 1.0, 2.5, 6.4, 16, 40, 100, and 250 μg / mL, respectively, filter through a 0.45 μm organic filter membrane, and inject into high-performance liquid chromatography (HPLC). Detect the peak area of ​​different concentrations of PSO standard solution. Perform linear regression with PSO standard solution concentration as the x-axis and peak area corresponding to different concentrations as the y-axis to plot the PSO standard curve and derive the linear formula.

[0074] Determination of PSO content in PSO / BSA-MnO2-NPs: Take 0.5 mL of PSO / BSA-MnO2-NPs dispersion, dilute to volume with methanol, sonicate for 30 min, centrifuge at 12000 rpm for 15 min, take the supernatant and filter it through a 0.45 μm aqueous filter membrane, inject it into high performance liquid chromatography, and determine the PSO content in PSO / BSA-MnO2-NPs.

[0075] Determination of PSO encapsulation efficiency in PSO / BSA-MnO2-NPs:

[0076] Take an appropriate amount of PSO / BSA-MnO2-NPs dispersion and determine the PSO content as W using the method described above. 总 Separately, add the PSO / BSA-MnO2-NPs dispersion to an ultrafiltration centrifuge tube, centrifuge at 12000 rpm for 30 min, dilute the filtrate with methanol to a final volume, and inject the sample into a high-performance liquid chromatography (HPLC) chamber to determine the total free PSO content (W). 游 The encapsulation ratio is calculated using the formula: Encapsulation ratio = (W 总 -W 游 ) / W 总 ×100%.

[0077] Linear regression analysis was performed on the peak areas of PSO standard solutions at different concentrations determined by HPLC and their corresponding concentrations to obtain the standard curve of PSO standard solutions (equation: y = 70.326x + 14.946(R²)). 2 =1), indicating a good linear relationship between the peak area and concentration of PSO in the HPLC determination range of 0.4-250 μg / mL. The encapsulation efficiency was measured to be (73.8±0.12)%.

[0078] 2. Detection of PSO / BSA-MnO2-NPs morphology by transmission electron microscopy (TEM)

[0079] Take an appropriate amount of H-MnO2-NPs and PSO / BSA-MnO2-NPs dispersions, dilute them appropriately, drop them onto a copper grid, stain with 2% phosphotungstic acid for 1 min, air dry, and observe the morphology of the nanoparticles under a transmission electron microscope. The detection results are as follows: Figure 1As shown in (a)-(d), under transmission electron microscopy (TEM) imaging, H-MnO2-NPs (average particle size 40.72±3.09nm) exhibit a uniform spherical morphology with visible surface pores and porous structure, and the shell thickness is 3.65±0.29nm; PSO / BSA-MnO2-NPs (average particle size 174.01±5.38nm) are uniform spheres.

[0080] 3. Determination of PSO / BSA-MnO2-NPs particle size and zeta potential

[0081] Take an appropriate amount of H-MnO2-NPs and PSO / BSA-MnO2-NPs dispersion, dilute it appropriately, add 1 mL to the sample cell, and measure its zeta potential and particle size distribution using a nanoparticle size analyzer.

[0082] like Figure 2 As shown in (a), PSO / BSA-MnO 2- The particle size of the NPs was (178.3±0.76) nm, and the polydispersity index (PDI) was 0.135±0.030 (PDI<0.3), indicating that the PSO / BSA-MnO2-NPs had uniform particle size and good dispersibility and stability. Figure 2 As shown in (b), the Zeta potential of PSO / BSA-MnO2-NPs is +(25.6±0.60)mV.

[0083] 4. Ultraviolet absorption detection

[0084] The ultraviolet spectrophotometer scanned BSA-MnO2-NPs, PSO, and PSO / BSA-MnO2-NPs respectively, and detected their absorption wavelengths. The test results are as follows: Figure 3 As shown, the results indicate that PAH has no strong absorption peak. After adding BSA and reacting with PAH-MnO2-NPs, the characteristic peak of H-MnO2-NPs at 310 nm disappears, indicating that BSA coats PAH-MnO2-NPs to form BSA-MnO2-NPs. After loading PSO to form PSO / BSA-MnO2-NPs, the maximum absorption peak in the ultraviolet spectrum is the characteristic peak of PSO.

[0085] 5. Fourier Transform Infrared (FTIR) Scanning Detection

[0086] Weigh appropriate amounts of HA-MnO2-NPs, PSO, BSA-MnO2-NPs, and PSO / BSA-MnO2-NPs lyophilized powders, and mix them thoroughly with dried potassium bromide powder in a mortar at a weight ratio of 1:10. Use a Fourier transform infrared spectrophotometer to measure the mixture at 4000–400 cm⁻¹. -1 The samples were scanned, and the infrared spectral absorption of each sample was analyzed.

[0087] Test results are as follows Figure 4 As shown, the peaks of H-MnO2-NPs are: 3465, 3053, 2359, 2326, and 1689 cm⁻¹. -1 Among them, 1456 is the characteristic peak of H-MnO2-NPs, and BSA-MnO2-NPs have characteristic peaks at 2924, 2360, 2326, and 1661 cm⁻¹. -1 There are peaks, including 2924, 2360, and 2326 cm. -1 Characteristic peaks for methyl and methylene groups, 1532 cm⁻¹ -1 The peak is characteristic of BSA, and at 1465 cm⁻¹ -1 The absence of peaks near the characteristic peaks of H-MnO2-NPs indicates that BSA is fully deposited on the surface of H-MnO2-NPs, forming BSA-MnO2-NPs by coating hollow manganese dioxide nanoparticles. Free PSO is present at 1722 cm⁻¹. -1 It has a characteristic peak at 1722 cm⁻¹, while PSO / BSA-MnO₂-NPs have a peak at 1722 cm⁻¹. -1 Nearby (1724cm) -1 The presence of a characteristic peak at this location indicates that PSO / BSA-MnO2-NPs contains PSO.

[0088] 6. Thermogravimetric analysis (TG)

[0089] Accurately weigh appropriate amounts of PSO, PSO / BSA-MnO2-NPs, BSA-MnO2-NP, and physical mixtures of BSA-MnO2-NP and PSO, and analyze the changes of the samples at each stage within the temperature range of 0 to 600℃.

[0090] Thermogravimetric analysis results are as follows Figure 5 As shown in the figure, the onset and termination temperatures of weight loss in the physical mixture of free PSO and BSA-MnO2-NPs are similar to those of free PSO (140-300℃), while...

[0091] The weight loss onset temperature (50℃) of PSO / BSA-MnO2-NPs is earlier, and the weight loss rate at 50-510℃ is lower than that of a physical mixture of free PSO and BSA-MnO2-NPs. This indicates that the PSO in PSO / BSA-MnO2-NPs is loaded within BSA-MnO2-NPs, which is not equivalent to a simple physical mixture of free PSO and BSA-MnO2-NPs.

[0092] 7. Differential Scanning Calorimetry (DSC)

[0093] Accurately weigh appropriate amounts of PSO, PSO / BSA-MnO2-NPs, BSA-MnO2-NP, and physical mixtures of BSA-MnO2-NP and PSO, and analyze the changes of the samples at each stage within the temperature range of 0 to 600℃.

[0094] DSC analysis revealed the morphology of PSO within the PSO / BSA-MnO2-NPs structure. PSO exhibited a distinct characteristic peak at 275.3℃, indicating its crystalline form. The characteristic peak of the blank BSA-MnO2-NPs spectrum was at 260.1℃, which did not overlap with the characteristic peak influencing PSO. Figure 6 As can be seen from the data, PSO / BSA-MnO2-NPs have no characteristic peak at 275.3℃, indicating that PSO exists in an amorphous state in PSO / BSA-MnO2-NPs.

[0095] 8. Stability Test

[0096] The stability of PSO / BSA-MnO2-NPs in various aqueous media was tested over 30 days: ultrapure water (DDI), physiological saline (pH 7.4), and DMEM medium containing 10% fetal bovine serum. PSO / BSA-MnO2-NPs samples prepared in triplicate were stored at 4°C. The stability of PSO / BSA-MnO2-NPs over 4 weeks was evaluated using PSO / BSA-MnO2-NP particle size and encapsulation efficiency as evaluation indicators.

[0097] The test results are shown in Table 1:

[0098] Table 1

[0099]

[0100]

[0101] The results showed that the particle size and encapsulation efficiency of PSO / BSA-MnO2-NPs were relatively stable within 2 weeks in ultrapure aqueous solution and DMEM solution containing 10% FBS, after which the stability gradually decreased. When stored at 4℃ in ultrapure aqueous solution for 4 weeks, the encapsulation efficiency decreased from (72.9±0.44)% to (66.5±1.50)%, the particle size increased to (182.5±1.44) nm, and precipitation occurred. In DMEM solution containing 10% FBS, the encapsulation efficiency of PSO / BSA-MnO2-NPs did not change significantly within 1 week of storage. The particle size was large, and its stability decreased afterward. By week 4, the particle size reached (215.8±4.18) nm, and the encapsulation efficiency was (62.5±0.66)%, resulting in precipitation. However, after one week in physiological saline solution, the stability decreased sharply, and aggregation occurred. By week 4, the particle size reached (502.3±65.9) nm, and the encapsulation efficiency was (32.2±1.79)%. This indicates that PSO / BSA-MnO2-NPs are suitable for storage in ultrapure water. When using them, they should be prepared into the appropriate concentration using physiological saline or serum-containing culture medium.

[0102] 9. Detection of PSO release from PSO in PSO / BSA-MnO2-NPs

[0103] Dispersions of PSO / BSA-MnO2-NPs and PSO of the same concentration were placed in dialysis bags with a molecular weight of 10,000-14,000. The constant temperature shaker was set at 100 r / min and 37 °C. In vitro release experiments were conducted in release media at pH 7.4, pH 6.5, and pH 5.5. Samples were taken at 0.5, 1, 2, 4, 8, 12, 24, 36, and 48 h, and an equal volume of fresh release media was added. The cumulative release rate of PSO / BSA-MnO2-NPs and PSO in the release media was calculated.

[0104] To investigate the sustained-release effect of nanoparticles under different pH conditions, the release of PSO from PSO / BSA-MnO2-NPs was monitored for 48 hours in PBS (pH 7.4, 6.5, 5.5). The test results are as follows: Figure 7 As shown, under pH 7.4 conditions, PSO's cumulative release rate exceeded 60% within 4 hours and was almost completely released within 24 hours. In contrast, PSO / BSA-MnO2-NPs exhibited delayed release characteristics, with a cumulative release exceeding 90% after 30 hours. Compared to pH 7.4 conditions, the cumulative release of PSO in PSO / BSA-MnO2-NPs was even higher at pH 6.5 or 5.5, indicating that PSO release from PSO / BSA-MnO2-NPs is accelerated under weakly acidic conditions, which ensures rapid drug release under tumor microenvironment conditions.

[0105] 10. Detection of the inhibitory effect of psoralen-loaded albumin-manganese dioxide nanoparticles on the growth of MDA-MB-231 breast cancer cells.

[0106] 10.1 Preparation of experimental solutions

[0107] (1) Preparation of DMEM complete culture medium: Mix 90mL of DMEM incomplete culture medium, 10mL of fetal bovine serum, and 1mL of penicillin-streptomycin, and filter through a 0.22μm sterile filter membrane.

[0108] (2) Preparation of thiazolyl blue (MTT) solution: Accurately weigh 250.0 mg of MTT powder, place it in a 50 mL centrifuge tube, add sterile phosphate buffer (PBS) under light protection to prepare a solution with a concentration of 5 mg / mL, shake slowly on a shaker for 2 h, filter through a 0.22 μm sterile filter membrane, dispense and store at -20℃ for later use.

[0109] (3) Preparation of PSO solution: Dissolve PSO in DMEM complete medium containing 0.1% dimethyl sulfoxide (DMSO) to prepare a PSO solution with a concentration of 200 mg / mL. Then dilute with DMEM complete medium to the required concentration for the experiment and filter through a 0.22 μm sterile filter membrane.

[0110] (4) Preparation of PSO / BSA-MnO2-NPs and BSA-MnO2-NPs stock solutions: Dilute the PSO / BSA-MnO2-NPs and blank BSA-MnO2-NPs dispersions with DMEM complete medium to the required concentration for the experiment, and filter them through a 0.22μm sterile filter membrane.

[0111] 10.2 Cell Culture

[0112] MDA-MB-231 cells were cultured in DMEM complete medium at 37°C in a 5% CO2 incubator.

[0113] 10.3 Cell uptake detection

[0114] (1) Preparation of BSA-MnO2-NPs (C6 / BSA-MnO2-NPs) loaded with coumarin 6

[0115] Method for determining coumarin 6 (C6) content: C6 is autofluorescent and can be detected by fluorescence analysis. A fluorescence analysis method for quantitative detection of C6 was established. Accurately weigh 0.1 mg of C6 standard and place it in a 25 mL volumetric flask. Dilute to volume with methanol. Take 0.5 mL of the diluted C6 standard solution and place it in a 5 mL volumetric flask. Dilute to volume with methanol. The concentration of the obtained C6 standard solution is 400 ng / mL. Dilute the C6 standard solution to 12.5, 25, 50, 100, and 200 ng / mL, respectively. Take the same volume of each diluted C6 standard solution and quantify the fluorescein C6 using an ELISA reader. The excitation wavelength is 488 nm and the emission wavelength is 525 nm. Plot a standard curve.

[0116] Preparation of C6 / BSA-MnO2-NPs: Following the preparation method of PSO / BSA-MnO2-NPs described above, C6 (dissolved in acetone) was used to replace PSO. An appropriate amount of C6 / BSA-MnO2-NPs was taken, diluted with methanol, sonicated for 30 min, centrifuged at 12000 rpm for 15 min, and the fluorescein content was determined using the same method as described above.

[0117] (2) MDA-MB-231 cell uptake assay

[0118] Laser confocalization: Seed 15×10⁶ cells / mL in a 15mm confocal dish. 4 MDA-MB-231 cells were cultured in an incubator for 24 hours. After removing the old culture medium, the cells were washed three times with PBS and treated for 2 hours in the dark with a medium containing C6 / BSA-MnO2-NPs (C6 concentration of 200 nM). A control group with 200 nM free C6 and a blank control group were set up. The cells were incubated for 2 hours with 2 mL of fresh culture medium added accordingly. After removing the old culture medium, the cells were washed three times with pre-cooled PBS, fixed with 500 μL of 4% paraformaldehyde for 10 min, and washed three times with pre-cooled PBS. Then, 4',6-diamidinyl-2-phenylindole (DAPI) staining solution was added, and the cells were allowed to stand at room temperature for 5 min and washed three times with pre-cooled PBS. Finally, 500 μL of PBS was added, and the cell uptake was observed under a laser confocal microscope.

[0119] Flow cytometry: After digestion and centrifugation, MDA-MB-231 cells were seeded at 30 × 10⁴ cells / well in 6-well plates and cultured for 24 h. The old culture medium was removed, and the cells were washed three times with PBS. Under dark conditions, the cells were treated with medium containing C6 / BSA-MnO₂-NPs (C6 concentration of 200 ng / mL) for 2 h. A control group with free C6 concentration of 200 ng / mL and a blank control group were set up. The cells were replaced with 2 mL of fresh culture medium and incubated for 2 h accordingly. After removing the old culture medium, the cells were washed three times with pre-cooled PBS, digested with 0.25% EDTA-free trypsin, and digestion was terminated by adding medium containing 10% fetal bovine serum. The cells were collected by centrifugation, washed three times with pre-cooled PBS, resuspended in 500 μL of PBS, centrifuged at 2000 rpm for 5 min, and the uptake level of nanoparticles by the cells was quantitatively analyzed by flow cytometry (excitation wavelength 488 nm). The average uptake (measured by average fluorescence intensity) was calculated from three parallel experiments.

[0120] The test results are shown in Figures 8-9. The measured standard curve for C6 is y = 53.08x + 1783310.84 (R²). 2 =0.99), the C6 content in the prepared C6-labeled BSA-MnO2-NPs nanoparticles was determined by a full-wavelength microplate reader, and diluted to the same concentration for cell uptake experiments; Figure 8 The results showed that C6-labeled nanoparticles exhibited green fluorescence after endocytosis, mainly distributed in the cytoplasm, while the cell nucleus showed blue fluorescence after DAPI staining, indicating that the nanoparticles could penetrate the cell membrane. Further flow cytometry was used to quantitatively analyze the uptake in both cell types, with uptake intensity expressed as the average fluorescence intensity of the cells. Figure 9 The average fluorescence intensity of MDA-MB-231 cells incubated with C6 / BSA-MnO2-NPs medium was significantly higher than that of free C6 (p<0.01), indicating that BSA-MnO2-NPs promotes the uptake of drugs by cells.

[0121] 10.4 Cytotoxicity Test

[0122] MDA-MB-231 cells were administered at 5 × 10⁻⁶ 3Cells were seeded per well in 96-well plates and cultured in a cell culture incubator. After 24 hours of culture, the old culture medium was removed, and 200 μL / well of different concentrations of PSO (50, 200, 300, 400, 500, 600 μM), blank BSA-MnO2-NPs (0.375, 0.625, 1.25, 2.5, 5, 10, 20 μg / mL), and PSO / BSA-MnO2-NPs (50, 200, 300, 400, 500, 600 μM as PSO) were added. A control group without drugs and a blank group were also included. Each group had 6 replicates and was placed in an incubator for further culture. After 48 hours, add 20 μL of MTT solution per well; after 4 hours of culture, remove the supernatant, add 150 μL of DMS solution per well, place on a microplate reader, record the OD value at 490 nm, and calculate cell viability and IC50.

[0123] Cell viability (%) = (OD value of drug group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0124] like Figure 10-11 As shown, the viability of MDA-MB-231 cells decreased with increasing PSO concentration, indicating that PSO and PSO / BSA-MnO2-NPs enhanced the inhibitory effect on cell proliferation. The calculated IC50 value of PSO was 335.6 μM, and the IC50 value of PSO / BSA-MnO2-NPs was 169.9 μM, indicating that PSO / BSA-MnO2-NPs had a stronger effect on MDA-MB-231 cells than PSO. When the concentration of blank BSA-MnO2-NPs was 0.25-2 μg / mL, the cell survival rate of MDA-MB-231 cells was greater than 80%, indicating that its toxicity to MDA-MB-231 cells was negligible.

[0125] 10.5 Apoptosis Test

[0126] Take MDA-MB-231 cells at 3×10 5Cells were seeded in 6-well plates and, after cell attachment, the supernatant was removed. Then, 2 mL of culture medium containing PSO (400 μM), blank BSA-MnO2-NPs (1.0 μg / mL), or PSO / BSA-MnO2-NPs (100, 200, and 400 μM, calculated as PSO) was added. A blank treatment group served as the control group. After culturing for 48 h, the old culture medium was removed, and cells were digested with trypsin without EDTA. Fresh complete culture medium was added to terminate the digestion. Cells were collected by centrifugation, washed with ice-cold PBS, and centrifuged at 1000 rpm for 5 min. The supernatant was then discarded, and 500 μL of Binding Buffer was added to suspend the cells in a flow cytometer. 5 μL each of Annexin-V / APC and PI staining solution were added, mixed well, and incubated at room temperature in the dark for 15 min. The cells were then loaded onto a flow cytometer to detect apoptosis.

[0127] like Figure 12 As shown in (a)-(g), where, Figure 12 (a)-(f) show the test results for blank, BSA-MnO2-NPs (1.0 μg / ml), PSO (200 μM), PSO (400 μM), PSO (800 μM), PSO / BSA-MnO2-NPs (100 μM), PSO / BSA-MnO2-NPs (200 μM), and PSO / BSA-MnO2-NPs (400 μM), respectively. The apoptosis rate of MDA-MB-231 cells treated with 200 μM free PSO was (8.55 ± 0.21)%, while the apoptosis rate of the medium concentration PSO / BSA-MnO2-NPs (200 μM, based on PSO) treatment group was (44.50 ± 0.10)%. PSO / BSA-MnO2-NPs significantly enhanced the apoptosis-inducing effect of PSO on MDA-MB-231 cells. The apoptosis rate in the blank BSA-MnO2-NPs treatment group was (8.93±1.20)%, which was significantly increased compared with the blank control group. This indicates that blank BSA-MnO2-NPs also have a certain apoptosis-inducing effect. After preparing PSO / BSA-MnO2-NPs by loading PSO, it may synergistically enhance the apoptosis-inducing effect of PSO.

[0128] 11. Detection of the effect of psoralen-loaded albumin-manganese dioxide nanoparticles on improving hypoxia in MDA-MB-231 breast cancer cells.

[0129] 11.1 Preparation of experimental solutions

[0130] (1) Prepare phosphate buffer (PBST) (10×): Dissolve 4.0g KCl + 16.0g NaCl + 72.6g disodium hydrogen phosphate dodecahydrate + 10mL Tween 20 + 4.8g potassium dihydrogen phosphate in ultrapure water to a final volume of 2000mL.

[0131] (2) Preparation of electrophoresis solution (10×): Dissolve 45g of Tris-base, 15g of sodium dodecyl sulfate (SDS), and 216g of glycine in ultrapure water to 1500mL.

[0132] (3) Preparation of transfer solution (1×): Dissolve 5.8g Tris-base + 2.9g glycine in ultrapure water to 800mL, and then add 200mL methanol.

[0133] (4) Prepare 1640 complete medium: THP-1 medium: 90mL 1640 incomplete medium (containing 0.05mmol / Lβ-mercaptoethanol) + 10mL fetal bovine serum + 1mL double antibiotics.

[0134] (5) Hypoxia model construction: using 200-400 μmol·L -1 A hypoxic cell model was induced using cobalt chloride (CoCl2) as a hypoxia inducer, and the induction conditions were investigated.

[0135] 11.2 MTT assay for CoCl2 cytotoxicity

[0136] Because CoCl2 may be toxic to tumor cells, the MTT assay was used to determine a safe dose (cell viability >90%) before inducing hypoxia: MDA-MB-231 cells were injected at a dose of 4 × 10⁻⁶. 3 Cells were seeded per well in 96-well plates and incubated in a cell culture incubator. After 24 hours of culture, the old culture medium was removed, and 200 μL / well of different concentrations of CoCl2 (12.5, 25, 50, 100, 200, 400, 800 μM) were added. A control group without CoCl2 and a blank group were also included, with 6 replicates in each group. The plates were then incubated for further culture. After 48 hours, 20 μL / well of MTT solution was added. After 4 hours of culture, the supernatant was removed, and 150 μL / well of DMSO solution was added. The plates were then placed on a microplate reader, and the OD value was recorded at 490 nm. Cell viability and IC50 were calculated.

[0137] Cell viability (%) = (OD value of drug group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0138] To eliminate the influence of CoCl2's own cytotoxicity on subsequent experiments, an appropriate hypoxic modeling dose needs to be selected. The cytotoxicity data of CoCl2 on MDA-MB-231 cells at 24h and 48h are as follows: Figure 13As shown, 200 μM CoCl2 showed low cytotoxicity to MDA-MB-231 cells within 48 h. 200 μM CoCl2 is also a commonly used dose for inducing in vitro cell hypoxia, therefore it was selected as the hypoxia-inducing dose.

[0139] 11.3 Western blot evaluation of hypoxia models

[0140] MDA-MB-231 cells were administered at 4 × 10⁻⁶ 3 Cells were seeded in 96-well plates and cultured in a cell culture incubator. After 24 hours of culture, the old culture medium was removed, and 200 μL of culture medium was added to each well. A normal control group (complete culture medium) and a CoCl2 group (containing 200 μM CoCl2 complete culture medium) were established. After 24 hours of culture at 37°C and 5% CO2, the supernatant was discarded, and the cells were washed twice with PBS. Pre-prepared cell lysis buffer was added, and cells were lysed for 5-10 seconds. Cells were scraped off, and the lysate containing cells was transferred to centrifuge tubes. The cells were centrifuged at 10,000 rpm for 5 minutes at 4°C. The collected supernatant was the extracted total cell protein. The supernatant was diluted, and 20 μL of the diluted solution was used for BCA assay to determine the total protein concentration. The total cell protein was added to SDS-PAGE loading buffer at a ratio of 4:1 (V / V), denatured in a 100°C metal bath for 3-5 minutes, and stored at -80°C.

[0141] Prepare linear solutions of protein standards and standard protein solutions according to the kit instructions to create protein quantitative standard curves; prepare protein sample solutions in the same way, mix thoroughly with BCA working solution, incubate at 37°C for 20 min, measure OD value at 562 nm using an ELISA reader, calculate the total protein concentration of each sample, and determine the subsequent loading amount of sample protein.

[0142] Protein sample solutions from each group were separated by SDS-PAGE electrophoresis, transferred to PVDF membranes using a transfer apparatus, blocked with 5% skim milk powder at 37°C for 1 hour, incubated with primary antibody at 4°C overnight, washed with PBST 4 times (15 min / time), added with secondary antibody containing horseradish peroxidase and incubated at 37°C for 1 hour, washed with PBST 4 times (15 min / time), ECL developing solution was added to the membrane, exposed using a chemiluminescence imaging system, and grayscale values ​​were calculated using ImageJ software.

[0143] Western blotting was used to detect HIF-1α expression in the hypoxia model group and the control group. The results are as follows: Figure 14 As shown, the detection results indicate that HIF-1α expression was significantly increased in the MDA-MB-231 cell hypoxia model group (p<0.001).

[0144] 11.4 Effect of PSO / BSA-MnO2-NPs on Apoptosis in Hypoxic MDA-MB-231 Cells

[0145] MDA-MB-231 cells were administered at 4 × 10⁻⁶ 5 Cells were seeded into 6-well plates. After cell attachment, the supernatant was removed, and the cells were washed twice with PBS. A complete medium containing 200 μM CoCl2 was prepared. Based on the 200 μM CoCl2 medium, 2 ml of medium containing PSO (400 μM), blank BSA-MnO2-NPs (1.0 μg / mL), and PSO / BSA-MnO2-NPs (100, 200, and 400 μM, respectively, based on PSO) were prepared. A drug-free COCl2 treatment group and a drug-free plain medium blank group were also set up. After culturing for 48 h, the cells were washed with PBS and digested with EDTA-free trypsin. The cells were collected by centrifugation, washed with pre-cooled PBS, and centrifuged at 1000 rpm for 5 min. Then discard the supernatant, add 300 μL of Binding Buffer to suspend the cells, and place them in a flow cytometer; add 5 μL each of Annexin-V / FITC and PI staining solution, mix well, and incubate at room temperature in the dark for 15 min; finally, detect cell apoptosis on a flow cytometer.

[0146] Flow cytometry analysis was used to compare the effects of Ctrol, CoCl2, PSO (400 μM), CoCl2+PSO (400 μM), BSA-MnO2-NPs (2 μg / ml), and CoCl2+PSO / BSA-MnO2-NPs (100 μM, 200 μM, 400 μM) on apoptosis in MDA-MB-231 cells. The results are as follows: Figure 15 As shown in (a)-(j), where, Figure 15 (a)-(d): Ctrol, CoCl2, PSO (400μM), CoCl2+PSO (400μM), (e): CoCl2+BSA-MnO2-NPs (1.0μg / mL); (f)-(h), CoCl2+

[0147] The results showed that the apoptosis rate of MDA-MB-231 cells under the hypoxia model induced by CoCl2 was not significantly different from that of normally cultured cells. Under the hypoxia model induced by CoCl2, the apoptosis rate of MDA-MB-231 cells induced by PSO decreased, indicating that hypoxia can inhibit the apoptosis-inducing effect of PSO. Under hypoxia conditions, PSO / BSA-MnO2-NPs can significantly increase the apoptosis rate of MDA-MB-231 cells induced by PSO.

[0148] Detection of the effect of 11.5PSO / BSA-MnO2-NPs on improving hypoxia

[0149] MDA-MB-231 cells were seeded at 3 × 10⁵ cells / well in 6-well plates. After cell attachment, the supernatant was removed. Complete medium containing 200 μM COCl₂ was prepared. Based on the 200 μM COCl₂ medium, 2 mL of medium containing PSO (400 μM), blank BSA-MnO₂-NPs (1.0 μg / mL), and PSO / BSA-MnO₂-NPs (200 μM, based on PSO) were prepared. A drug-free CoCl₂ treatment group and a drug-free plain medium blank group were also set up. After culturing for 48 h, total protein was extracted and protein concentration was measured. Protein concentration was determined by BCA method, HIF-1α protein was measured by Western blot, and the band density values ​​were calculated by Image Pro Plus 5.0 software.

[0150] Western blotting was used to detect the expression of HIF-1α in the hypoxia model group (CoCl2), CoCl2+PSO / BSA-MnO2-NPs (200 μM), CoCl2+PSO (200 μM), and CoCl2+BSA-MnO2-NPs (1.0 μg / mL). Figure 16 As shown, compared with the hypoxia model group and the free PSO group, the expression of HIF-1α in the PSO / BSA-MnO2-NPs group was significantly reduced, indicating that PSO / BSA-MnO2-NPs can alleviate CoCl2-induced hypoxia in MDA-MB-231 cells.

[0151] 12. Detection of the regulation of tumor-associated macrophage phenotype by albumin-manganese dioxide nanoparticles loaded with psoralen.

[0152] 12.1 Preparation of macrophage cell differentiation induction and conditioned medium (CM)

[0153] 2×10 6Human mononuclear leukocyte cell line THP-1 was seeded at a density of 10 cells / mL and cultured in culture flasks for 24 h. A THP-1 blank control group and a PMA treatment group were established. The treatment group was treated with 200 nM phorbol ester (PMA). Cells were cultured at 37°C in a 5% CO2 cell culture incubator for 24 h to induce cell adhesion and differentiation into M0 macrophages. Cell morphology in both groups was observed under an inverted microscope. Differentiated M0 macrophages were washed three times with PBS to remove residual PMA. An M0 blank control group and an IL-4 treatment group were established. The treatment group was treated with 20 nM phorbol ester (PMA). IL-4 treatment was used to induce M0 cells to differentiate into M2 macrophages by culturing them in a 37℃, 5% CO2 cell culture incubator for 24 h. Cell morphology in both groups was observed under an inverted microscope. Differentiated M2 macrophages were washed three times with PBS to remove residual IL-4, then cultured in DMEM containing 10% fetal bovine serum at 37℃, 5% CO2 for 24 h. The supernatant was collected, centrifuged at 1500 rpm for 15 min, and filtered through a 0.22 μm filter to remove cells and debris, yielding M2 macrophage conditioned medium (CM2). Differentiated M0 macrophages were washed three times with PBS to remove residual PMA. An M0 blank control group and a lipopolysaccharide (LPS) treatment group were established. The treatment group received 20 nM IL-4... LPS treatment was used to induce M0 cells to differentiate into M1 macrophages by culturing them in a 37℃, 5% CO2 cell culture incubator for 24 h. The morphology of the two groups of cells was observed under an inverted microscope. Conditioned culture media for non-polarized macrophages produced by M0 and M1 macrophages (CM0 and CM1, respectively) were prepared using the same method. Cells cultured in normal complete culture medium served as controls.

[0154] 12.2 Macrophage Phenotypic Identification

[0155] Flow cytometry: Macrophages were assessed by flow cytometry 24 hours after IL-4 stimulation of PMA-induced adherent THP-1 cells. Immunophenotyping. 2 × 10⁶ M₀ nonpolarized and M₂ polarized cells were collected. Cells were digested with 0.25% trypsin without EDTA and washed twice with pre-chilled PBS. The cell suspension was then aliquoted into sterile Eppendorf tubes and incubated on ice with an Fc receptor blocker for 10 min. The cells were then washed twice with pre-chilled PBS. Fluorescently labeled anti-Human CD86 (PE-labeled) and CD206 antibody (APC-labeled) were added on ice and incubated at 4°C in the dark for 30 min. Cells without any antibody pretreatment were used as blank controls. Cells were washed twice with pre-chilled PBS to remove excess antibody, resuspended in 300 μL PBS, and analyzed by flow cytometry.

[0156] Cytokine assay: Macrophages were assessed by cytokine levels 24 hours after IL-4 stimulation. Immunophenotype. Induction with 2 × 10⁶ THP-1 molecules yielded M0 nonpolarized [immunophenotype / immunophenotype]. M1 polarization M2 polarization Collect the supernatant, centrifuge at 10,000 rpm, and determine the concentrations of cytokines TNF-α, IL-6, and IL-10 in the supernatant according to the ELISA kit instructions to investigate the effect of PSO / BSA-MnO2-NPs on cytokine production in cells.

[0157] like Figure 17 As shown in (a)-(b), the proportion of THP-1 cells induced by PMA and IL-4 positively expressing the M2 macrophage marker CD206 was significantly higher than that of THP-1 cells induced by PMA alone (p<0.001). At the same time, the proportion of THP-1 cells induced by PMA and IL-4 positively expressing the M1 macrophage marker CD86 was significantly lower than that of THP-1 cells induced by PMA and LPS (p<0.001), indicating that PMA and IL-4 successfully induced THP-1 cells to differentiate into M2 macrophages.

[0158] like Figure 18 As shown, in THP-1 cells induced by PMA and IL-4, the levels of immunosuppressive cytokines IL-6 and IL-10 were significantly higher than those induced by PMA alone. Meanwhile, the inflammatory factor TNF-α in THP-1 cells induced by PMA and IL-4 was significantly lower than that in THP-1 cells induced by PMA alone. According to reports, M2 macrophages mainly secrete immunosuppressive cytokines; therefore, the cytokine level measurements are consistent with the cell phenotype, verifying that the THP-1 cells induced by PMA and IL-4 have an M2 phenotype.

[0159] 12.3 Detection of macrophage uptake of PSO / BSA-MnO2-NPs

[0160] Laser confocal microscopy detection: 30×10⁻⁶ seeds were inoculated in a 15mm confocal dish. 4THP-1 cells were induced to differentiate into M2 macrophages using the aforementioned method. The old culture medium was removed, and the cells were washed three times with PBS. Under light-protected conditions, the cells were treated with a medium containing C6 / BSA-MnO2-NPs (C6 concentration 200 ng / mL) for 2 h. A control group with 200 ng / mL free C6 and a blank control group were established, and 2 mL of fresh culture medium was added and incubated for 2 h accordingly. The old culture medium was removed, and the cells were washed three times with pre-cooled PBS. 500 μL of 4% paraformaldehyde was added for fixation for 10 min, followed by three washes with pre-cooled PBS. DAPI staining solution was added, and the cells were allowed to stand at room temperature for 5 min, followed by three washes with pre-cooled PBS. Finally, 500 μL of PBS was added, and cell uptake was observed under a laser confocal microscope.

[0161] Flow cytometry analysis: THP-1 cells were analyzed at 30 × 10⁻⁶ cells / cells. 4 Cells were seeded per well in 6-well plates and cultured for 24 h. M2 macrophages were induced to differentiate according to method 2.2. The old culture medium was removed, and the cells were washed three times with PBS. Under dark conditions, the cells were treated with medium containing C6 / BSA-MnO2-NPs (C6 concentration 200 ng / mL) for 2 h. A free C6 control group (200 ng / mL) and a blank control group were also included, with 2 mL of fresh culture medium added for 2 h of incubation. The old culture medium was removed, and the cells were washed three times with pre-cooled PBS. Digestion was performed using 0.25% EDTA-free trypsin, and the digestion was terminated by adding medium containing 10% fetal bovine serum. Cells were collected by centrifugation, washed three times with pre-cooled PBS, resuspended in 500 μL PBS, and centrifuged at 2000 rpm for 5 min. The uptake level of nanoparticles by the cells was quantitatively analyzed using flow cytometry (excitation wavelength 488 nm). The average uptake (measured by average fluorescence intensity) was calculated from three parallel experiments.

[0162] like Figures 19-20 As shown, C6-labeled nanoparticles, after being internalized by M2 macrophages, exhibited green fluorescence, mainly distributed in the cytoplasm. The cell nucleus, stained with DAPI, showed blue fluorescence. The presence of fluorescence in M2 macrophages indicates that the nanoparticles can penetrate the cell membrane. Further quantitative analysis of the uptake by the two cell types was performed using flow cytometry. The uptake intensity was expressed as the average fluorescence intensity of the cells. Combined with the flow cytometry results, it was found that the average fluorescence intensity of M2 macrophages incubated with C6 / BSA-MnO2-NPs medium was significantly higher than that of free C6, indicating that BSA-MnO2-NPs promote drug uptake by M2 macrophages.

[0163] 12.4 Effect of PSO / BSA-MnO2-NPs on apoptosis in co-cultured MDA-MB-231 cells

[0164] MDA-MB-231 cells were seeded at 4 × 10⁵ cells / well in 6-well plates. After cell attachment, the supernatant was removed, and the cells were washed twice with PBS. Then, 2 mL each of macrophage conditioned medium CM0 and CM2 containing PSO (400 μM), blank BSA-MnO₂-NPs (1.0 μg / mL), and PSO / BSA-MnO₂-NPs (100, 200, and 400 μM, respectively, based on PSO) were added. The drug-free conditioned medium CM0 group, drug-free conditioned medium CM2 group, and drug-free plain medium blank group were set as control groups. After culturing for 48 h, the cells were washed with PBS, digested with trypsin without EDTA, collected by centrifugation, washed with pre-cooled PBS, and centrifuged at 1000 rpm for 5 min. Then discard the supernatant, add 300 μL of Binding Buffer to suspend the cells, and place them in a flow cytometer; add 5 μL each of Annexin-V / FITC and PI staining solution, mix well, and incubate at room temperature in the dark for 15 min; finally, detect cell apoptosis on a flow cytometer.

[0165] Apoptosis rates were analyzed by flow cytometry under CM2 and CM2+400μM PSO conditions. The results are as follows: Figure 21 As shown in (a)-(m), where, Figure 21 (a)-(f): Blank, CM2, PSO (400μM), CM2+PSO (400μM), BSA-MnO2-NPs (1.0μg / mL), CM2+BSA-MnO2-NPs (1.0μg / mL); Figure 21 (g), (i), (k): PSO / BSA-MnO2-NPs (100μM, 200μM, 400μM); Figure 21 (h), (j), (l): CM2+PSO / BSA-MnO2-NPs (100μM, 200μM, 400μM). Under CM2 co-culture conditions, the apoptosis rate of MDA-MB-231 cells was not significantly different from that of normally cultured cells (p>0.05); high concentration of PSO (800μM) significantly reduced the apoptosis rate of MDA-MB-231 cells (p<0.05); low, high, and medium concentrations of PSO / BSA-MnO2-NPs significantly increased the apoptosis rate of MDA-MB-231 cells (p<0.001), and also enhanced cell apoptosis compared with free PSO (p<0.001), indicating that CM2 co-culture is a favorable condition for MDA-MB-231 cells to resist apoptosis, and PSO / BSA-MnO2-NPs can reverse the apoptosis resistance of MDA-MB-231 cells induced by CM2 co-culture conditions.

[0166] 12.5PSO / BSA-MnO2-NPs affect the expression of macrophage immunosuppressive factors.

[0167] M2 macrophages were administered at a rate of 2 × 10⁻⁶. 6 Cells were seeded in 6-well plates. After cell attachment, the supernatant was removed. Then, 2 mL of 1640 complete culture medium containing PSO (400 μM), blank BSA-MnO2-NPs (1.0 μg / mL), or PSO / BSA-MnO2-NPs (100, 200, and 400 μM, calculated as PSO) was added. The drug-free treatment group served as the control group. After culturing for 48 h, the supernatant was collected and centrifuged at 10,000 rpm for 5 min. The concentrations of cytokines IL-6, IL-10, and TNF-α in the supernatant were measured according to the ELISA kit instructions to investigate the effect of PSO / BSA-MnO2-NPs on IL-6, IL-10, and TNF-α.

[0168] Macrophage-derived immunosuppressive factors such as IL-6 and IL-10 can participate in the process of resisting apoptosis in breast cancer cells. ELISA analysis was performed on the effects of the blank control group, PSO (400 μM), BSA-MnO2-NPs (1.0 μg / ml), and PSO / BSA-MnO2-NPs (100 μM, 200 μM, 400 μM) groups on cytokine levels in induced differentiated M2 macrophages. The results are as follows: Figure 22 As shown, PSO / BSA-MnO2-NPs significantly reduced the levels of immunosuppressive factors IL-6 and IL-10 in macrophages, but had no significant effect on the inflammatory factor TNF-α, indicating that PSO / BSA-MnO2-NPs promote apoptosis in MDA-MB-231 cells by reducing the levels of macrophage-derived immunosuppressive factors.

[0169] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A manganese dioxide nanoparticle formulation loaded with psoralen, characterized in that, The nano-formulation is composed of psoralen, manganese dioxide, polyacrylamide hydrochloride, and albumin, wherein the mass ratio of psoralen, manganese dioxide, polyacrylamide hydrochloride, and albumin is 5-25:2.5:2.5-12.5:0.8-6; the nano-formulation is prepared by the following steps: S1. Preparation of silica nanoparticle template: Tetraethyl orthosilicate is mixed with a solvent, ammonia is added and the mixture is ultrasonically reacted. The resulting reaction solution is centrifuged and the centrifuged precipitate is dispersed to obtain silica nanoparticle template solution. S2. Preparation of manganese dioxide nanoparticles: Add potassium permanganate solution to the silica nanoparticle template solution, sonicate and centrifuge for the first time, disperse the precipitate in sodium carbonate solution, centrifuge for the second time, wash the precipitate, centrifuge for the third time to remove the precipitate, and obtain manganese dioxide nanoparticle solution. S3. Preparation of polyacrylamide hydrochloride-manganese dioxide nanoparticle composite: Add polyacrylamide hydrochloride to the manganese dioxide nanoparticle solution, mix evenly and centrifuge, wash the centrifuged precipitate and disperse it to obtain polyacrylamide hydrochloride-manganese dioxide nanoparticle solution. S4. Preparation of albumin-modified manganese dioxide nanoparticles: The polyacrylamide hydrochloride-manganese dioxide nanoparticle solution was added to the albumin solution, anhydrous ethanol was added, and the mixture was centrifuged. The precipitate was washed and dispersed to obtain the albumin-modified manganese dioxide nanoparticle solution. S5. Preparation of albumin-modified manganese dioxide nanoparticles loaded with psoralen: Add psoralen solution to albumin-modified manganese dioxide nanoparticle solution, stir and react, centrifuge, filter and centrifuge to obtain albumin-modified manganese dioxide nanoparticles loaded with psoralen.

2. The albumin-based manganese dioxide nanoparticle formulation loaded with psoralen according to claim 1, characterized in that, The average particle size of the nano-formulation is 150-250 nm.

3. The albumin-based manganese dioxide nanoparticle formulation loaded with psoralen according to claim 1 or 2, characterized in that, The manganese dioxide is hollow mesoporous manganese dioxide; the albumin is bovine serum albumin.

4. The albumin-based manganese dioxide nanoparticle formulation loaded with psoralen according to claim 3, characterized in that, In step S1, the solvent is prepared by ultrasonically mixing anhydrous ethanol and ultrapure water, with a volume ratio of anhydrous ethanol to ultrapure water of 4.1-14:4-9.

7. The anhydrous ethanol and ultrapure water are ultrasonically reacted for 5-20 minutes. The volume ratio of tetraethyl orthosilicate to ultrapure water is 0.2-0.8:4-9.

7. The concentration of ammonia is 25-28%, with a volume ratio of ammonia to tetraethyl orthosilicate of 2-16.5:0.2-0.

8. After adding ammonia, the ultrasonic reaction time is 20-120 minutes. The centrifugation speed is 11000-15000 rpm, and the centrifugation time is 5-15 minutes.

5. The albumin-based manganese dioxide nanoparticle formulation loaded with psoralen according to claim 3, characterized in that, In step S2, the molar ratio of potassium permanganate to silica nanoparticles is 12-96:1, the molar ratio of sodium carbonate to silica nanoparticles is 2-10:1, the ultrasonic treatment time is 1-9 hours, the precipitate is dispersed in sodium carbonate solution and stirred at 100-300 rpm at 40-70℃ for 3-24 hours, the first and second centrifugation treatments are performed at 11000-14000 rpm for 5-15 minutes, and the third centrifugation treatment is performed at 3000-4000 rpm for 5-15 minutes.

6. The albumin-based manganese dioxide nanoparticle formulation loaded with psoralen according to claim 3, characterized in that, In step S3, the concentration of polyacrylamide hydrochloride is 0.5-2.5 mg / mL, the mixing is carried out at 25-50℃ for 1-6 h, and the centrifugation speed is 11000-14000 rpm for 5-15 min. In step S4, the concentration of albumin is 0.02-0.15 mg / mL, the volume ratio of anhydrous ethanol to albumin solution is 2-8:40, and after adding anhydrous ethanol, the step further includes stirring at 25-50℃ for 12-36 h, and the centrifugation speed is 11000-14000 rpm for 5-15 min.

7. The albumin-based manganese dioxide nanoparticle formulation loaded with psoralen according to claim 3, characterized in that, In step S5, the solvent in the psoralen solution is acetone, the stirring reaction temperature is 25-50℃, the time is 4-24h, the stirring speed is 100-250rpm, the centrifugation speed is 3000-5000rpm, the centrifugation time is 5-15min, and the filtration uses a 0.45μm filter membrane.

8. The use of an albumin-based manganese dioxide nanoparticle formulation loaded with psoralen as described in any one of claims 1-7 in the preparation of a breast cancer cell inhibitor under hypoxic conditions.

9. The use of an albumin-containing manganese dioxide nanoparticle formulation loaded with psoralen as described in any one of claims 1-7 in the preparation of a drug for treating triple-negative breast cancer.

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