A ZnO porous hollow microsphere drug-carrying system and its application in the preparation of lymphoma drugs

Abstract

This invention discloses a ZnO porous hollow microsphere drug-loading system and its application in the preparation of lymphoma drugs. The drug-loading system uses 4-8 nm ZnO nanoparticles as units and PAA micelles as soft templates, and is formed by solvothermal assembly into porous hollow microspheres with a diameter of 80-140 nm. Doxorubicin is loaded through physical adsorption and electrostatic interaction, and the shell is formed by loosely stacked ZnO nanoparticles to create an interconnected porous structure. The resulting drug-loading system can achieve a drug loading capacity of up to 14.3 wt%, achieving stable and long-lasting sustained release based on the porous structure, with a low 24-hour release rate and no significant burst release. Animal experiments show that this drug-loading system can significantly inhibit lymphoma tumor growth, reduce cell proliferation activity, and increase apoptosis rate, with a tumor-suppressing effect superior to free doxorubicin. It also has no significant effect on mouse body weight, low systemic toxicity, good dispersibility and injectability, and shows promising application prospects in clinical chemotherapy for lymphoma.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine carrier technology, specifically relating to a ZnO porous hollow microsphere drug delivery system and its application in the preparation of lymphoma drugs. Background Technology

[0002] Lymphoma is a type of malignant proliferative tumor originating from the lymphohematopoietic system, characterized by high incidence, high recurrence rate, and strong invasiveness, seriously threatening human life and health. Doxorubicin (DOX), as an anthracycline broad-spectrum antitumor drug, is a core first-line drug in clinical lymphoma chemotherapy, achieving highly effective antitumor effects by embedding into the double helix of tumor cell DNA and inhibiting nucleic acid synthesis. However, the clinical application of doxorubicin faces significant technical bottlenecks: free doxorubicin is easily and rapidly metabolized and eliminated in the body, resulting in extremely low drug utilization and an inability to achieve slow and controllable release, making it difficult to maintain an effective drug concentration at the tumor site; at the same time, its own toxic side effects are strong, limiting the clinical dosage and failing to achieve the ideal antitumor therapeutic effect, greatly restricting its application in the clinical treatment of lymphoma.

[0003] To improve the delivery of doxorubicin and enhance drug loading and sustained-release performance, nanomedicine carriers have become a current research hotspot. Existing nanocarriers are mainly divided into two categories: organic and inorganic. Organic carriers (such as liposomes and polymer microspheres) suffer from drawbacks such as low mechanical strength, uncontrollable in vivo degradation, limited drug loading, and poor stability, making it difficult to meet the clinical requirements for long-acting drug delivery. Traditional inorganic nanocarriers (such as solid ZnO particles and SiO2 particles) have small specific surface areas and lack tunable pore structures and internal cavities, making it impossible to achieve efficient loading of doxorubicin or slow drug release, thus failing to solve the clinical application challenges of free doxorubicin.

[0004] ZnO-based nanomaterials have become highly promising inorganic drug carriers due to their excellent biocompatibility, biodegradability, and responsive degradation characteristics in the acidic microenvironment of tumors, as well as their fluorescent tracer advantages. However, existing ZnO-based drug carriers still have significant technical drawbacks: conventional ZnO nanoparticles (nanorings, nanosheets, and solid nanoparticles) have small specific surface areas, few drug adsorption sites, and low drug loading capacity; traditional hollow ZnO microspheres are mostly prepared using hard template methods, which are not only cumbersome in preparation and difficult to remove the template, easily causing damage to the carrier structure, but also have a dense outer shell structure lacking interconnected porous channels, which is not conducive to the efficient loading of doxorubicin, nor can it achieve slow and controllable drug release, seriously limiting the practical application of ZnO-based nanocarriers in the targeted delivery of anti-tumor drugs for lymphoma. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a ZnO porous hollow microsphere drug-carrying system and its application in the preparation of lymphoma drugs. By controllably assembling and constructing porous hollow ZnO sphere carriers, efficient loading and sustained release of doxorubicin can be achieved, thereby improving the anti-tumor therapeutic effect and reducing drug toxicity and side effects.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A ZnO porous hollow microsphere drug delivery system is disclosed. This system uses ZnO nanoparticles (NPs) with a particle size of 4-8 nm as the basic unit and PAA micelles as a soft template. ZnO porous hollow microspheres are formed through solvothermal assembly and serve as drug carriers. Doxorubicin is used as a model antitumor drug. Through physical adsorption and electrostatic interaction, doxorubicin is loaded into the internal hollow cavities of the ZnO porous hollow microspheres and the inter-pores between the ZnO NPs. The ZnO porous hollow microspheres have a diameter of 80-140 nm, and their shells are formed by the assembly and stacking of ZnO NPs, with inter-particle gaps, resulting in a porous structure.

[0007] The preparation method of the above-mentioned ZnO porous hollow microsphere drug-carrying system includes the following steps: S1. Synthesis of ZnO NPs: Weigh 1.6-2.4 g of zinc acetate dihydrate (Zn(AC)2·2H2O) and dissolve it in 32-48 mL of anhydrous ethanol. Stir magnetically until completely dissolved to obtain a zinc ethanol solution. Simultaneously, weigh 0.64-0.96 g of sodium hydroxide and dissolve it in 8-12 mL of anhydrous ethanol. Stir until completely dissolved to obtain an alkaline ethanol solution. Stir at 700-900 r / min and slowly add the alkaline ethanol solution dropwise to the zinc ethanol solution. After the addition is complete, continue stirring for 4-6 min to obtain a clear solution. Add n-hexane to the clear solution and let it stand for 12-18 min to allow ZnO NPs to precipitate completely. After centrifugation, wash with anhydrous ethanol to obtain ZnO NPs.

[0008] S2. Preparation of ZnO porous hollow microspheres: S2.1. Preparation of dispersion: Add 24-36 mL of anhydrous ethanol to the ZnO NPs in step S1, and disperse by ultrasonication to form a uniform dispersion A; S2.2. Preparation of micelle solution: Weigh 0.16-0.24 g of polyacrylic acid (PAA) and dissolve it in 1.2-1.8 mL of ammonia water (mass fraction 25%-28%). Stir until completely dissolved, then add the solution dropwise to 24-36 mL of anhydrous ethanol. Stir at room temperature for 25-35 min to form PAA micelle solution, denoted as dispersion B. S2.3. Solvent-thermal assembly: Add all of dispersion B to dispersion A, and ultrasonically disperse and mix to obtain a mixed dispersion; transfer the mixed dispersion to a high-pressure reactor and carry out a solvothermal reaction at 120-160℃ for 5-7 hours; after the reaction, allow it to cool naturally to room temperature, centrifuge, wash successively with deionized water and anhydrous ethanol, dissolve PAA micelles in deionized water to remove the template, and vacuum dry to obtain ZnO porous hollow microspheres.

[0009] Using polyacrylic acid (PAA) micelles as a soft template, PAA molecules assemble into a uniform nanomicelle core-shell structure in an ammonia-ethanol system through hydrophobic and electrostatic interactions. Under solvothermal conditions, ZnO nanoparticles (NPs) are directionally adsorbed, arranged, and assembled on the surface of PAA micelles through electrostatic interactions and hydrogen bonding, forming a core-shell precursor with micelles as the core and ZnO nanoparticles as the shell. Subsequently, through a water washing process, the PAA micelles are completely removed by dissolving in deionized water, ultimately forming porous hollow microspheres with a hollow interior and a loosely packed shell of ZnO nanoparticles.

[0010] This structure has two key structural features: (1) The shell is assembled from 4-8 nm ZnO nanoparticles, and there are a large number of interconnected mesopores between the particles, which provide abundant physical adsorption sites and electrostatic binding sites for doxorubicin. (2) The internal hollow cavity can accommodate a large number of drug molecules, significantly increasing the drug loading capacity.

[0011] S3. Doxorubicin loading: Take 0.10-0.14 g of the ZnO porous hollow microspheres obtained in step S2 and disperse them in 16-24 mL of phosphate buffer solution (PBS, pH=7.0-7.4) to form a homogeneous suspension. Add 0.015-0.025 g of doxorubicin hydrochloride to the suspension and stir and incubate at 4℃ in the dark for 10-14 h to allow doxorubicin to fully enter the cavity and pore structure of the porous hollow microspheres through physical adsorption and electrostatic interaction. Then, place the mixture in a dialysis bag and dialyze it with sterile physiological saline or PBS buffer (pH=7.0-7.4) at 4℃ for 24 h. Replace the dialysate every 6 h until no free doxorubicin can be detected in the dialysate to obtain a high-purity ZnO porous hollow microsphere drug-loaded system.

[0012] This invention also provides the application of the above-mentioned drug delivery system in the preparation of lymphoma drugs.

[0013] The beneficial effects of this invention are as follows: (1) The drug loading system of the present invention is assembled with 4-8 nm ZnO NPs to form a porous hollow microsphere carrier. The internal structure is hollow and there are a large number of interconnected pores between the shell particles, which can provide sufficient loading sites for doxorubicin. Experimental results show that the drug loading capacity can reach 14.3 wt%, which is higher than that of the solid ZnO nanoparticle drug loading system (<8 wt%), greatly improving the loading capacity of doxorubicin and solving the problem of low drug loading capacity of conventional ZnO-based carriers.

[0014] (2) The drug delivery system of the present invention relies on a special porous and hollow structure. The shell layer connects the pores and the internal cavity to form a multi-level drug release channel, which effectively slows down the drug release rate. Under normal physiological conditions, the drug release rate is only 20.2% in 24 hours, avoiding sudden drug release and achieving long-term stable sustained release of the drug, thereby improving the persistence of the drug's effect in the body.

[0015] (3) The drug delivery system of the present invention is used for the treatment of lymphoma. Compared with the drug delivery system of free doxorubicin and solid ZnO nanoparticles, the anti-tumor effect is significantly improved. It can effectively inhibit the growth of lymphoma tumors, reduce the tumor volume and weight, reduce the proliferation activity of tumor cells, increase the apoptosis rate of tumor cells, and the anti-tumor effect is more lasting and without obvious rebound.

[0016] (4) The drug delivery system of the present invention has excellent biosafety. It does not cause abnormal weight loss in the test mice during the treatment process and has no obvious systemic toxic side effects. It is superior to free doxorubicin. At the same time, the blank ZnO porous hollow microsphere drug delivery system has no anti-tumor cell toxicity and good biocompatibility.

[0017] (5) The ZnO porous hollow microsphere drug delivery system of the present invention has good dispersibility and good flowability, and can be smoothly injected by syringe. It has good clinical drug delivery operability, is suitable for clinical drug delivery treatment of lymphoma, and has high practical application value. Attached Figure Description

[0018] Figure 1 This is a model diagram illustrating the preparation of the ZnO porous hollow microsphere drug-carrying system of the present invention and its application in lymphoma drugs; Figure 2 In the figures (a) and (b), the transmission electron microscope (TEM) images and particle size statistics curves of ZnO NPs in Example 1 are shown respectively. Figure 3 (a) and (b) in the image are transmission electron microscope (TEM) and scanning electron microscope (SEM) images of the ZnO porous hollow microspheres prepared in Example 1, respectively. Figure 4 The XRD spectrum of the ZnO porous hollow microspheres prepared in Example 1; Figure 5 Transmission electron microscope image of ZnO microspheres in Comparative Example 1; Figure 6 In the figures (a) and (b), the N2 adsorption-desorption isotherms of ZnO microspheres in Example 1 and Comparative Example 1 are respectively. Figure 7 In the figures, (a) and (b) are the drug loading and drug release curves of the ZnO microsphere carriers prepared in Example 1 and Comparative Example 1, respectively. Figure 8 This is a photograph of the fluidity of the ZnO porous hollow microsphere drug-carrying system of Embodiment 1 of the present invention, injected by a syringe. Figure 9 These are comparative photographs of ex vivo tumor tissues from each experimental group of the present invention 21 days after administration; Figure 10 This is a growth curve of tumor volume change over time in mice of each experimental group in this invention; Figure 11 This is a histogram showing the weight of ex vivo tumors in each experimental group after treatment in this invention. Figure 12 This is a graph showing the weight change of mice in each experimental group during the treatment period in this invention. Figure 13 HE-stained images of tumor tissues from various experimental groups in this invention; Figure 14 This is an image of Ki-67 immunohistochemical staining in a tumor tissue section of the present invention; Figure 15 This is a photograph of a tumor tissue section stained with TUNEL in this invention; Figure 16 A quantitative map of cell damage rate in tumor tissue stained with HE; Figure 17 A quantitative graph of the Ki-67 proliferation index; Figure 18 A quantitative graph of TUNEL apoptosis rate. Detailed Implementation

[0019] To make the above-mentioned objectives, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to examples. The following content is merely illustrative and explanatory of the concept of the present invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, as long as they do not deviate from the concept of the invention, they should all fall within the protection scope of the present invention.

[0020] Example 1 A method for preparing a ZnO porous hollow microsphere drug-carrying system. Figure 1 This is a model diagram illustrating the preparation of the ZnO porous hollow microsphere drug-carrying system of the present invention and its application in the treatment of lymphoma.

[0021] (1) Experimental materials: Zinc acetate dihydrate (Zn(AC)2·2H2O), sodium hydroxide (NaOH), n-hexane, anhydrous ethanol, polyacrylic acid (PAA, molecular weight Mv=5000), ammonia (25%-28%), doxorubicin hydrochloride (DOX·HCl), phosphate buffer (PBS, pH 7-7.4).

[0022] (2) Preparation method, including the following steps: S1. Synthesis of ZnO NPs: Weigh 2.0 g of zinc acetate dihydrate (Zn(AC)2·2H2O) and dissolve it in 40 mL of anhydrous ethanol. Stir magnetically until completely dissolved to obtain a zinc ethanol solution. Simultaneously, weigh 0.8 g of sodium hydroxide and dissolve it in 10 mL of anhydrous ethanol. Stir until completely dissolved to obtain an alkaline ethanol solution. Stir at 800 r / min and slowly add the alkaline ethanol solution dropwise to the zinc ethanol solution. After the addition is complete, continue stirring for 5 min to obtain a clear mixed solution. Add n-hexane to the clear solution and let it stand for 15 min to allow ZnO NPs to precipitate fully. Centrifuge the precipitate and wash it with anhydrous ethanol to obtain ZnO NPs.

[0023] S2. Preparation of ZnO porous hollow microspheres: S2.1. Preparation of dispersion: Add 30 mL of anhydrous ethanol to the ZnO NPs in step S1, and disperse by ultrasonication to form a uniform dispersion A; S2.2. Preparation of micelle solution: Weigh 0.2 g of polyacrylic acid (PAA) and dissolve it in 1.5 mL of ammonia water. Stir until completely dissolved, then add the solution dropwise to 30 mL of anhydrous ethanol. Stir at room temperature for 30 min to form PAA micelle solution, denoted as dispersion B. S2.3. Solvothermal Assembly: Add all of dispersion B to dispersion A, and ultrasonically disperse and mix to obtain a mixed dispersion; transfer the mixed dispersion to a high-pressure reactor and carry out a solvothermal reaction at 140℃ for 6 h; after the reaction is completed, allow it to cool naturally to room temperature, centrifuge, wash with deionized water and anhydrous ethanol in sequence, dissolve PAA micelles in deionized water to remove the template, and vacuum dry to obtain ZnO porous hollow microspheres.

[0024] S3. Doxorubicin loading: Take 0.12 g of the ZnO porous hollow microspheres obtained in step S2 and disperse them in 20 mL of phosphate buffer solution to form a homogeneous suspension. Add 0.02 g of doxorubicin hydrochloride to the suspension and incubate at 4°C in the dark for 12 h with stirring to allow doxorubicin to fully enter the cavities and pores of the porous hollow microspheres through physical adsorption and electrostatic interaction. Subsequently, place the mixture in a dialysis bag and dialyze it against sterile physiological saline or PBS buffer at 4°C for 24 h. Replace the dialysate every 6 h until no free doxorubicin is detected in the dialysate, thus obtaining a high-purity ZnO porous hollow microsphere drug-loaded system (denoted as DOX@ZnO PHMs).

[0025] (3) Structural and performance characterization (corresponding figures) Figure 2 TEM showed that the ZnO NPs had uniform particle size, with an average particle size of 5.98 nm and a narrow particle size distribution (4-8 nm), exhibiting monodispersity.

[0026] Figure 3 (a) and (b) clearly show, using TEM and SEM images respectively, that the prepared microspheres have a hollow porous structure with a diameter distribution of 80-140 nm and a porous morphology formed by ZnO NPs interstices on the surface.

[0027] Figure 4 XRD spectroscopy indicates that the product is ZnO with a hexagonal wurtzite structure and no impurity peaks.

[0028] Comparative Example 1 Solid ZnO nanoparticle drug delivery system: Steps S1 and S2 are replaced with commercially available 100 nm solid ZnO nanoparticles, and step S3 is the same as in Example 1.

[0029] TEM image ( Figure 5 The product is solid ZnO nanoparticles with no hollow porous structure.

[0030] Figure 6 N2 adsorption-desorption isotherms of ZnO microspheres in Example 1 and Comparative Example 1; Depend on Figure 6 As shown in (a), the sample from Example 1 exhibits a typical type IV isotherm and a distinct type H3 hysteresis loop, indicating that the material contains a large number of mesoporous and fissure-pore structures formed by the accumulation of ZnO nanoparticles. Figure 6 As shown in (b) of the paper, the solid ZnO nanoparticles in Comparative Example 1 exhibited extremely low adsorption capacity and no obvious hysteresis loop. The results confirm that the porous hollow microspheres of this invention possess abundant mesoporous channels, which can significantly enhance the drug loading and sustained-release performance of doxorubicin.

[0031] Figure 7In (a): the drug loading test results show that the drug loading in this embodiment can reach 14.3 wt%, which is significantly higher than that of the solid ZnO carrier in Comparative Example 1 (drug loading < 8 wt%).

[0032] Figure 7 (b) In vitro release experiments showed that the release rate was only 20.2% in a pH 7.4 (simulated blood) environment after 24 h, exhibiting good stability, while the release rate in the comparative example was as high as 63.6%.

[0033] Figure 8 This is a photograph of the fluidity of the ZnO porous hollow microsphere drug delivery system of this invention, when injected using a syringe.

[0034] Example 2 The preparation process in this embodiment is the same as in Example 1, only the parameters are replaced: S1, ZnO nanoparticle synthesis: 1.6 g zinc acetate dihydrate, 32 mL anhydrous ethanol; 0.64 g sodium hydroxide, 8 mL anhydrous ethanol; stirring speed 700 r / min, stirring for 4 min after dropwise addition, standing in n-hexane for 12 min, the rest is the same as in Example 1; S2. Preparation of ZnO porous hollow microspheres: ZnO nanoparticles were dispersed in 24 mL of anhydrous ethanol; PAA 0.16 g, ammonia water 1.2 mL, and anhydrous ethanol 24 mL were added and stirred for 25 min. The rest of the process was the same as in Example 1. S3. Drug loading: 0.10 g of ZnO porous hollow microspheres, 16 mL of PBS, and 0.015 g of doxorubicin hydrochloride were incubated in the dark for 10 h. The rest of the process was the same as in Example 1.

[0035] Example 3 The preparation process in this embodiment is the same as in Example 1, only the parameters are replaced: S1, ZnO nanoparticle synthesis: 2.4 g zinc acetate dihydrate, 48 mL anhydrous ethanol; 0.96 g sodium hydroxide, 12 mL anhydrous ethanol; stirring speed 900 r / min, stirring for 6 min after dropwise addition, standing in n-hexane for 18 min, the rest is the same as in Example 1; S2. Preparation of ZnO porous hollow microspheres: ZnO nanoparticles were dispersed in 36 mL of anhydrous ethanol; PAA 0.24 g, ammonia water 1.8 mL, and anhydrous ethanol 36 mL were added and stirred for 35 min. The rest of the process was the same as in Example 1. S3, Drug loading: 0.14 g of ZnO porous hollow microspheres, 24 mL of PBS, 0.025 g of doxorubicin hydrochloride, incubated in the dark for 14 h, the rest is the same as in Example 1.

[0036] Application: The ZnO porous hollow microsphere drug-carrying system prepared in Example 1 is used in the preparation of lymphoma therapeutic drugs. (1) Experimental materials: Cells and animals: Human non-Hodgkin lymphoma cell line SU-DHL-4, purchased from Shanghai Guandao Bioengineering Co., Ltd., product number: SU-DHL-4; SPF-grade female BALB / c nude mice (6–8 weeks old), purchased from Shanghai SLAC Laboratory Animal Co., Ltd., animal production license number: SCXK (Shanghai) 2022-0004; Reagents: Doxorubicin hydrochloride (DOX·HCl), purchased from MedChemExpress (MCE) company, product number: HY-15142, ZnO porous hollow microspheres (self-made), Ki-67 proliferation detection kit (immunohistochemistry method), purchased from Beijing Solarbio Science & Technology Co., Ltd., product number: KHA022, TUNEL apoptosis detection kit (in situ end labeling method), purchased from Wuhan Eclite Biotechnology Co., Ltd., product number: E-CK-A324.

[0037] (2) Establishment of nude mouse lymphoma model: On the 35th day before the experiment, SPF-grade nude mice were purchased and adaptively raised in the experimental environment for 5 days, corresponding to the time interval from Figure 1 the 35th day before the experiment to the 30th day before the experiment. On the 30th day before the experiment, the human non-Hodgkin lymphoma cell line SU-DHL-4 in the logarithmic growth phase was collected, and the cell concentration was adjusted to 5×10 7 / mL, and 100 μL of the cell suspension was subcutaneously injected into the right back of each nude mouse. After inoculation, the mice were normally raised and observed. When the subcutaneous tumor volume of the nude mice grew to 80-100 mm³, drug administration treatment was initiated.

[0038] (3) Grouping and drug administration: The successfully modeled nude mice were randomly divided into 4 groups: Group 1 (PBS control group): 100 μL of sterile PBS was injected peritumorally.

[0039] Group 2 (blank control group, ZnO microsphere group): 100 μL of blank ZnO porous hollow microsphere suspension was injected peritumorally.

[0040] Group 3 (pure drug group, DOX group): An aqueous doxorubicin solution (dose 5.0 mg / kg) was injected peritumorally.

[0041] Group 4 (DOX@ZnO PHMs group): A suspension of a porous hollow microsphere drug-loading system with the same doxorubicin equivalent dose was injected peritumorally.

[0042] Tumor suppression effect (refer to Figure 9 , Figure 10 , Figure 11 ): The tumor volume of the mice was measured every 3 days to dynamically monitor the tumor suppression effect of each group ( Figure 10The results showed that the tumors in the PBS group exhibited exponential growth, with an average final volume of 1800 mm² at the end of the experiment. 3 The tumor growth trend in the blank control group (ZnO PHMs, empty vector) was not significantly different from that in the PBS group, confirming that the blank vector itself had no anti-tumor activity; in the pure drug group (DOX, free doxorubicin), tumor growth was inhibited to some extent, with a final volume of approximately 900 mm². 3 However, tumor growth rebounded significantly in the later stages of the experiment; while tumor growth was significantly inhibited in the DOX@ZnO PHMs (drug delivery system of this invention) group, with a final volume of only about 280 mm². 3 The tumor-suppressing effect was far superior to that of the free drug group.

[0043] Mice were sacrificed 21 days after drug administration, and tumors were removed and weighed (reference). Figure 11 The results showed that the tumor weight in the PBS group was approximately 0.82 g, and in the blank control group (ZnO PHMs group) approximately 0.76 g, with no statistically significant difference between the two groups. The tumor weight in the pure drug group (DOX group) decreased to approximately 0.57 g. The DOX@ZnO PHMs group had the lowest tumor weight, only approximately 0.38 g, which was completely consistent with the tumor volume detection results. (Ex vivo tumor photographs are included.) Figure 9 The results also clearly show that the DOX@ZnO PHMs group had the smallest tumor volume, further confirming that the drug delivery system of this invention has excellent in vivo tumor suppression effect.

[0044] Safety evaluation: Tumor-bearing nude mice were administered the drug in groups as described above. During a 21-day experimental period, the activity and mental state of the mice in each group were observed and recorded daily to systematically determine whether the drug caused systemic toxicity. Weight change was the core quantitative indicator for assessing drug toxicity. During the experiment, the live weight of mice in each group was measured and recorded every 3 days using an electronic balance for 21 consecutive days. Finally, a dynamic growth curve of mouse weight over time was plotted (see reference). Figure 12 ).

[0045] During the experiment, mice in the PBS group, blank control group, ZnO microsphere group, and DOX@ZnO PHMs group showed steady weight gain and normal condition; mice in the pure drug group (DOX group) showed significant weight loss after 7 days of administration, and exhibited cardiotoxicity and systemic wasting symptoms, indicating that the drug delivery system of this invention can significantly reduce the systemic toxic side effects of doxorubicin.

[0046] Cell proliferation and apoptosis analysis (reference) Figures 13-18 ): Twenty-one days after drug administration, mice in each group were sacrificed, and tumor tissues were collected for relevant testing and analysis. The quantitative analysis methods for relevant pathological sections are as follows: Hematoxylin and eosin (HE) stained sections of tumor tissue were observed under an optical microscope (magnification of ×400). Abnormal tumor cells were manually identified (main characteristics included: large and deeply stained nuclei, significant nuclear atypia, disordered cell arrangement, and tissue structure destruction), and the cell abnormality rate was calculated accordingly. Ki-67 proliferation index and TUNEL apoptosis rate were quantitatively analyzed using ImageJ software: Five high-magnification microscopic fields were randomly selected from each group, and the number of Ki-67 positive cells, TUNEL positive cells, and the total number of cells in the field were counted under a uniform image grayscale threshold. Relevant quantitative pathological indicators were calculated using the following independent mathematical formulas: Abnormal cell rate (%) = (Number of abnormal cells / Total number of cells) × 100% Ki-67 proliferation index (%) = (Number of Ki-67 positive cells / Total number of cells) × 100% TUNEL apoptosis rate (%) = (Number of TUNEL-positive cells / Total number of cells) × 100% Figure 13 These are HE-stained images of tumor tissues from various experimental groups in this invention. Figure 16 A quantitative map of cell damage rate in tumor tissue stained with HE; Figure 13 and Figure 16 The results showed that the abnormal cell rate was approximately 2.6% in the control group PBS group, approximately 11.5% in the blank control group ZnO microsphere group, approximately 17% in the pure drug group DOX group, and approximately 34.5% in the DOX@ZnO PHMs group. This indicates that the drug delivery system of the present invention can achieve precise spatiotemporal delivery of doxorubicin, thereby inducing a stronger, more lasting, and more thorough killing effect on the tumor site, which is a reliable pathological marker of improved efficacy.

[0047] Figure 14 This is an image of Ki-67 immunohistochemical staining in a tumor tissue section from this invention. Figure 17 A quantitative graph of the Ki-67 proliferation index; Figure 14 and Figure 17 The results showed that the proliferation index of the control group PBS group was (45.0±3.2)%, the pure drug group DOX group decreased to (25.0±2.1)%, and the DOX@ZnO PHMs group decreased to (18.0±1.5)%, which could significantly inhibit the proliferation of tumor cells. Figure 15 This is a photograph of a tumor tissue section stained with TUNEL in this invention. Figure 18 A quantitative graph of TUNEL apoptosis rate. Figure 15 and Figure 18The results showed that the apoptosis rate of the DOX@ZnO PHMs group was (35.0±0.6)%, significantly higher than that of the pure drug group DOX (16.0±0.4)%. These results confirm that the drug delivery system of this invention can enhance the therapeutic effect of lymphoma by improving tumor pathology, inhibiting cell proliferation, and promoting apoptosis.

[0048] In summary, the ZnO porous hollow microsphere drug-loading system constructed in this invention successfully prepared hollow microspheres with high specific surface area and multi-level porous structure using the PAA micelle soft template method. Compared with free doxorubicin, it has a higher drug loading capacity, better sustained-release effect, more significant tumor-suppressing effect in lymphoma treatment, and lower systemic toxicity, showing good prospects for clinical translation.

Claims

1. A method for preparing a ZnO porous hollow microsphere drug-carrying system, characterized in that, Includes the following steps: S1. Synthesis of ZnO nanoparticles: Zinc acetate dihydrate was dissolved in anhydrous ethanol to obtain a zinc ethanol solution; sodium hydroxide was dissolved in anhydrous ethanol to obtain an alkaline ethanol solution; the alkaline ethanol solution was added dropwise to the zinc ethanol solution under stirring, and after stirring, n-hexane was added and allowed to stand to precipitate. After centrifugation and washing with anhydrous ethanol, ZnO nanoparticles were obtained. S2. Preparation of ZnO porous hollow microspheres: ZnO nanoparticles were dispersed in anhydrous ethanol to obtain dispersion A; polyacrylic acid was dissolved in ammonia and then added dropwise to anhydrous ethanol with stirring to prepare PAA micelle solution, which is dispersion B; dispersion B and dispersion A were mixed and ultrasonically homogenized, and subjected to a solvothermal reaction. After the reaction, the mixture was cooled, centrifuged, washed to remove the PAA micelle template, and vacuum dried to obtain ZnO porous hollow microspheres; S3. Loading of doxorubicin: ZnO porous hollow microspheres were dispersed in phosphate buffer solution, doxorubicin hydrochloride was added, and the mixture was stirred and incubated in the dark. Then, the free doxorubicin was removed by dialysis to obtain the ZnO porous hollow microsphere drug loading system. The ZnO porous hollow microsphere drug-carrying system is assembled using ZnO nanoparticles with a particle size of 4-8 nm as units and PAA micelles as soft templates. The ZnO porous hollow microspheres have a diameter of 80-140 nm. Doxorubicin is used as a model drug and is loaded into the porous hollow ZnO microspheres. The shell of the microspheres is formed by the assembly and stacking of ZnO NPs, with gaps between the particles, forming a porous structure. In step S2, the ZnO nanoparticles are dispersed in 24-36 mL of anhydrous ethanol; the amount of polyacrylic acid is 0.16-0.24 g, dissolved in 1.2-1.8 mL of ammonia water with a mass fraction of 25%-28%, and then added dropwise to 24-36 mL of anhydrous ethanol and stirred for 25-35 min.

2. The preparation method according to claim 1, characterized in that: In step S1, the amount of zinc acetate dihydrate added is 1.6-2.4 g, corresponding to 32-48 mL of anhydrous ethanol; the amount of sodium hydroxide added is 0.64-0.96 g, corresponding to 8-12 mL of anhydrous ethanol; the stirring speed is 700-900 r / min; after the addition is completed, the stirring is carried out for 4-6 min; and the hexane standing time is 12-18 min.

3. The preparation method according to claim 1, characterized in that: In step S2, the solvothermal reaction temperature is 120-160℃, and the reaction time is 5-7 h.

4. The preparation method according to claim 3, characterized in that: The solvothermal reaction temperature was 140℃, and the reaction time was 6 h.

5. The preparation method according to claim 1, characterized in that: In step S3, the amount of ZnO porous hollow microspheres is 0.10-0.14 g, dispersed in 16-24 mL of phosphate buffer solution with pH=7.0-7.4; the amount of doxorubicin hydrochloride is 0.015-0.025 g, and incubated at 4℃ in the dark for 10-14 h.

6. The preparation method according to claim 1, characterized in that: In step S3, the dialysis time is 24 hours, and the dialysate is changed every 6 hours. The dialysate is sterile physiological saline or PBS buffer with pH=7.0-7.

4.

7. A ZnO porous hollow microsphere drug-carrying system prepared by the preparation method according to any one of claims 1-6.

8. The use of the ZnO porous hollow microsphere drug-carrying system of claim 7 in the preparation of drugs for treating lymphoma.

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