Polydopamine-modified celecoxib and cisplatin-loaded dual drug-loaded microspheres as well as preparation method and application of polydopamine-modified celecoxib and cisplatin-loaded dual drug-loaded microspheres

Abstract

The invention belongs to the technical field of biological medicines, and discloses a polydopamine modified celecoxib and cisplatin loaded dual drug-loading microsphere, which takes a polydopamine modified polyvinyl alcohol microsphere as a carrier, and the surface of the microsphere is loaded with celecoxib and cisplatin. Celecoxib and cis-platinum loaded on the dual drug-loading microspheres prepared by the invention have a good synergistic effect, and can significantly inhibit the excessive growth of liver cancer in blood vessels; when being applied to the liver cancer TACE operation, the compound has relatively high safety and high operation success rate, and can remarkably inhibit tumor cell proliferation so as to improve the embolization chemotherapy effect. The invention also discloses a preparation method of the dual drug-loading microsphere.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polydopamine-modified dual-drug-loaded microsphere containing celecoxib and cisplatin, its preparation method, and its application. Background Technology

[0002] Liver cancer is the sixth most common cancer worldwide, with an estimated incidence exceeding one million cases by 2025, making it the fourth leading cause of cancer-related deaths globally. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, accounting for approximately 90% of primary liver cancers. Because cancer cells exhibit multiple mechanisms of disease progression and self-proliferation, monotherapy is often insufficient to treat advanced cancers. Single anticancer drugs rarely completely inhibit disease progression. Therefore, combination therapy to achieve a synergistic effect greater than the sum of its parts ("1+1>2") is an important approach in anti-tumor research.

[0003] Cisplatin, as a first-generation platinum-based chemotherapy drug, has a unique mechanism of action primarily through interfering with DNA replication, thereby inhibiting the rapid proliferation of tumor cells and inducing programmed cell death. However, patients receiving this treatment are prone to developing drug resistance, which affects treatment efficacy. When cisplatin is used in combination with celecoxib, Ki67 and PCNA levels are significantly higher than with cisplatin monotherapy. However, in some early clinical trials, combination therapy with celecoxib did not significantly improve progression-free survival.

[0004] Therefore, it is of great significance to provide a method that can effectively improve the synergistic effect of celecoxib and cisplatin in the treatment of liver cancer. Summary of the Invention

[0005] To address the problems and shortcomings of existing technologies, the present invention aims to provide polydopamine-modified dual-drug-loaded microspheres containing celecoxib and cisplatin, and a method for their preparation, thereby improving the synergistic effect of celecoxib and cisplatin in the treatment of liver cancer.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a polydopamine-modified dual-drug-loaded microsphere for celecoxib and cisplatin, using polydopamine-modified polyvinyl alcohol microspheres as a carrier, with celecoxib and cisplatin loaded on the surface of the microspheres.

[0007] This invention also provides a method for preparing polydopamine-modified dual-drug-loaded microspheres containing celecoxib and cisplatin. The method includes the following steps: (1) Polyvinyl alcohol microspheres were added to a dopamine solution prepared by Tris-HCl buffer, the pH was adjusted to 8-10, stirred at room temperature for 10 min-48 h, and polydopamine-modified polyvinyl alcohol microspheres were obtained after washing and centrifugation. (2) Dissolve cisplatin and celecoxib in PBS buffer to obtain a celecoxib / cisplatin blend solution; add the polydopamine-modified polyvinyl alcohol microspheres obtained in step (1) to the celecoxib / cisplatin blend solution, incubate for 30 min to 24 h, and centrifuge to obtain polydopamine-modified dual-drug-loaded microspheres.

[0008] Preferably, the polyvinyl alcohol microspheres in step (1) have a particle size of 100–300 μm.

[0009] Preferably, the concentration of Tris-HCl buffer in step (1) is 2 mg / mL and the pH is 8.5.

[0010] Preferably, the ratio of polyvinyl alcohol microspheres to dopamine solution in step (1) is (1-10) g / 100 mL, and the mass concentration of dopamine solution is 0.1%-10%.

[0011] Preferably, in step (2), the mass ratio of cisplatin to celecoxib is (0.4-2.5):1.

[0012] Preferably, in step (2), the ratio of polydopamine-modified polyvinyl alcohol microspheres to celecoxib / cisplatin blend solution is 10–100 mg / mL.

[0013] Furthermore, in step (1), the polyvinyl alcohol microspheres are prepared using an oil-in-water emulsion crosslinking method.

[0014] Furthermore, in step (2), the centrifugation speed is 3000 rpm and the centrifugation time is 10 min.

[0015] This invention also provides the application of polydopamine-modified dual-drug-loaded microspheres containing celecoxib and cisplatin in liver cancer treatment drugs.

[0016] Compared with the prior art, this application has the following advantages: 1. The dual-drug-loaded microspheres prepared in this invention are polyvinyl alcohol microspheres with a particle size of 100-300 μm, which are suitable for microsphere embolization of tumors with a lesion diameter of less than 3 cm, and also suitable for preliminary microsphere embolization of tumors with a lesion diameter of more than 5 cm.

[0017] 2. Polydopamine utilizes its adhesive properties to coat the surface of PVA particles, forming a PDA shell and endowing the PVA particles with polydopamine characteristics. The presence of numerous unique catechol structures on the polydopamine surface gives the drug-loaded microspheres a negative charge. This strong electrostatic repulsion results in highly stable embolic microspheres coated with PDA. This invention employs polydopamine surface modification of microspheres. Due to the adhesive properties of the polydopamine shell, when two drugs are loaded simultaneously, the encapsulation efficiency of each drug is above 55%, resulting in high overall drug loading efficiency that meets clinical needs.

[0018] 3. The dual-drug-loaded microspheres prepared in this invention exhibit a good synergistic effect between celecoxib and cisplatin. These microspheres significantly inhibit excessive angiogenesis in liver cancer by suppressing the expression of CD31 and VEGF; significantly improve the local immune microenvironment of liver cancer and reduce immunosuppression by inhibiting the expression of IL-10 and TGF-β; weaken local inflammatory responses by inhibiting COX-2 expression, converting cold tumors into hot tumors and providing favorable conditions for further immunotherapy; enable cisplatin to exert a sustained effect within tumor cells by upregulating CTR-1 and reducing MRP-2 expression, thus slowing the development of multidrug resistance; and promote T cell infiltration, especially CD4+ T cells, which play an important role in enhancing anti-tumor immunity.

[0019] 4. The dual-drug-loaded microspheres prepared in this invention have high safety and a high success rate in the application of TACE for liver cancer, and can significantly inhibit tumor cell proliferation, thereby improving the effect of embolization chemotherapy. Attached Figure Description

[0020] Figure 1 The images show the surface structure of the PMS samples prepared in Examples 1-4, as shown in the electron microscope (SEM) images.

[0021] Figure 2 A shows the surface structure electron microscope (SEM) images of the PVAMS, PMS, PCDMS, PCMS prepared in Example 2, and PDMS prepared in Comparative Example 1 and Comparative Example 2, respectively; B shows the particle size distribution of each sample.

[0022] Figure 3 This is the energy dispersive spectroscopy (EDS) analysis and elemental distribution map of PCDMS.

[0023] Figure 4 A shows the encapsulation efficiency of PCMS, PDMS, and PCDMS; B shows the cumulative release curves of celecoxib in PCMS and PCDMS; C shows the cumulative release curves of cisplatin in PDMS and PCDMS.

[0024] Figure 5 The image shows the experimental results of CCK-8.

[0025] Figure 6 The image shows the results of the scratch test.

[0026] Figure 7 A shows the weight change curves of mice in each group after treatment; B shows the tumor volume change curves of mice in each group after treatment.

[0027] Figure 8A shows typical images of immunohistochemical staining for CD31, VEGF, IL-10, and TGF-β, with a scale bar of 50 μm; B, C, D, and E are statistical analysis graphs of the percentage of positive areas for immunohistochemical staining for CD31, VEGF, IL-10, and TGF-β, respectively.

[0028] Figure 9 A, B, and C are statistical analysis graphs of the average values ​​of white blood cells, red blood cells, and platelets in the blood of experimental rabbits in each group 2 weeks after TACE; D, E, F, G, H, and I are curves showing the changes in liver and kidney function in experimental rabbits in each group after TACE; J is HE staining of the heart, spleen, lungs, and kidneys of experimental rabbits in each group 2 weeks after TACE, with a scale bar of 200 μm.

[0029] Figure 10 A is a schematic diagram of TACE surgery for rabbit VX2 orthotopic hepatocellular carcinoma; B is an enhanced CT scan image two weeks after the hepatocellular carcinoma model was constructed; C is a DSA image during the TACE surgery; D is an enhanced CT scan image one and two weeks after TACE surgery; E is a gross image of the left lobe of the liver two weeks after TACE surgery; F is HE staining and PCNA fluorescence staining images of each group two weeks after TACE surgery. Scale bar: 5000um, 200um, 100um.

[0030] Figure 11 A shows immunohistochemical and immunofluorescence staining images of CTR-1, MRP-2, Foxp3, CD4, and CD8 in each group 2 weeks after TACE, with a scale bar of 50 μm; B, C, and D are statistical analysis graphs of the percentage of positive areas for CTR-1, MRP-2, and Foxp3 immunohistochemical staining; E and F are statistical analysis graphs of the average fluorescence intensity of CD4 and CD8 immunofluorescence. Detailed Implementation

[0031] The following embodiments are only for further elaboration of the present invention. It should be noted that all techniques and scientific terms used in this invention, unless otherwise stated, have the same meaning as those in the technical field to which this invention pertains. Experimental methods in the following embodiments that do not specify specific conditions all employ conventional techniques in this technical field or follow the conditions recommended by the manufacturer; reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0033] Example 1: The preparation of polydopamine-modified dual-drug-loaded microspheres containing celecoxib and cisplatin includes the following steps: (1) Polyvinyl alcohol (PVA) microspheres were prepared by water-in-oil emulsion crosslinking method. Liquid paraffin containing Span 80 (1.8%, w / v) was used as the continuous phase (oil phase), and an aqueous solution of polyvinyl alcohol (PVA) (10%, w / v) was selected as the aqueous dispersion phase. 10 mL of the dispersion phase was added to 50 mL of the continuous phase at 60 °C, and the mixture was magnetically stirred for 30 min to form an emulsion. Then, 1.8 mL of 25 wt% glutaraldehyde (GA) was added and stirred for 10 min. Next, 1.5 mL of 1 mol / L hydrochloric acid was added as a catalyst, and the mixture was crosslinked for 5 h. PVA microspheres were collected by centrifugation and washed three times with ethanol and deionized water. The lyophilized microspheres were filtered through a 150-mesh standard sieve. The obtained PVA microspheres had a particle size of 100–300 μm.

[0034] (2) Preparation of polydopamine-modified PVA microspheres Dopamine hydrochloride powder was dissolved in Tris-HCl buffer (concentration of 2 mg / mL, pH of 8.5) to prepare a 0.1% dopamine solution. 0.5 g of PVA microspheres obtained in step (1) was added to 50 mL of dopamine solution, the pH was adjusted to 8.5, and the mixture was stirred at room temperature for 48 h. The microspheres were washed with deionized water to remove unreacted dopamine. After centrifugation, polydopamine-modified PVA microspheres were obtained and labeled as 0.1% PMS.

[0035] (3) Loading celecoxib and cisplatin 20 mg of cisplatin powder and 50 mg of celecoxib powder were dissolved in 10 mL of PBS buffer to prepare a celecoxib / cisplatin blend solution. 100 mg of the PMS obtained in step (2) was added to the celecoxib / cisplatin blend solution, and the mixture was incubated on a shaker for 24 h. After centrifugation at 3000 rpm for 10 min, the supernatant was removed to obtain polydopamine-modified dual-drug-loaded microspheres of celecoxib and cisplatin (named PCDMS). The microspheres used polydopamine-modified polyvinyl alcohol microspheres as carriers, and the surface of the microspheres was loaded with celecoxib and cisplatin.

[0036] Example 2: The content is basically the same as that of Example 1, except that the concentration of the dopamine solution prepared in step (2) is 0.2%, and the polydopamine-modified PVA microspheres are denoted as 0.2%PMS.

[0037] Example 3: The content is basically the same as that of Example 1, except that the concentration of the dopamine solution prepared in step (2) is 0.5%, and the polydopamine-modified PVA microspheres are denoted as 0.5%PMS.

[0038] Example 4: The content is basically the same as that of Example 1, except that the concentration of the dopamine solution prepared in step (2) is 1%, and the polydopamine-modified PVA microspheres are denoted as 1%PMS.

[0039] To verify the loading of drug-loaded microspheres, the PMS samples prepared in step (2) of Examples 1 to 4 of this invention were subjected to physicochemical characterization tests, as follows.

[0040] SEM images of the PVA microspheres prepared in Examples 1-4 were obtained using a JSM-7401F microscope (JEOL, Japan) equipped with an energy dispersive spectroscopy (EDS) system. The results are as follows: Figure 1 As shown.

[0041] Figure 1 The results showed that after modifying PVA microspheres with 0.1% polydopamine solution, the surface of the microspheres was covered with less polydopamine and the surface was relatively flat, which could not meet the drug loading requirements. After modifying PVA microspheres with 0.2% polydopamine solution, the microspheres maintained their normal morphology, appearing as spherical or near-spherical shapes with a rough, wrinkled, and uneven surface, which could achieve multiple drug loading functions, meeting the ideal requirements of TACE surgery. After modifying PVA microspheres with 0.5% and 1% polydopamine solutions, the PVA microspheres lost their normal morphology, aggregated into clusters, and significantly increased in size, with a thicker polydopamine shell, which could not meet the drug loading requirements and seriously affected the TACE surgery effect.

[0042] Comparative Example 1: The content of Comparative Example 1 is basically the same as that of Example 2. The difference is that the specific process of step (3) is as follows: 50 mg of celecoxib powder is dissolved in 10 mL of PBS buffer to prepare celecoxib solution; 100 mg of PMS obtained in step (2) is added to celecoxib solution, and after incubation on a shaker for 24 h, it is centrifuged at 3000 rpm for 10 min, and the supernatant is removed to obtain polydopamine-modified celecoxib-loaded microspheres (named PCMS).

[0043] Comparative Example 2: The content of Comparative Example 2 is basically the same as that of Example 2. The difference is that the specific process of step (3) is as follows: 20 mg of cisplatin powder is dissolved in 10 mL of PBS buffer to prepare cisplatin solution; 100 mg of PMS obtained in step (2) is added to cisplatin solution, and after incubation on a shaker for 24 h, it is centrifuged at 3000 rpm for 10 min, and the supernatant is removed to obtain polydopamine-modified cisplatin-loaded drug-loaded microspheres (named PDMS).

[0044] (a) Performance Testing 1. Structural characterization of drug-loaded microspheres To verify the loading capacity of the drug-loaded microspheres, physicochemical characterization tests were performed on the PVAMS, PMS, and PCDMS prepared in Example 2 of this invention, the PCMS prepared in Comparative Example 1, and the PDMS prepared in Comparative Example 2, as detailed below.

[0045] SEM images and EDS dispersion maps of each sample were obtained using a JSM-7401F microscope (JEOL, Japan) equipped with an energy dispersive spectroscopy (EDS) system, allowing for analysis of the sample surface morphology and elemental composition. Fourier transform infrared spectroscopy was performed on the samples, with a scanning range of 500–4000 cm⁻¹. X-ray photoelectron spectroscopy was used to analyze the properties and chemical state of the samples, with a measurement range of 1200–0 cm⁻¹. The results are as follows: Figure 2 and Figure 3 As shown.

[0046] Figure 2 Results showed that PVA microspheres were uniformly spherical with smooth and intact surfaces; PMS microspheres were ellipsoidal or quasi-spherical with rough, wrinkled, and uneven surfaces; PCMS, PDMS, and PCDMS had similar morphologies to PMS, and all had drug crystals on their surfaces. Particle size statistical analysis revealed (see...) Figure 2 B), PVA particles are concentrated at around 100 μm, while PMS, PCMS, PDMS and PCDMS gradually increase in diameter, all concentrated at around 120 μm. Among them, PCDMS has the largest increase in diameter, which is attributed to the surface modification of polydopamine and the adsorption of drugs.

[0047] Figure 3 The EDS results showed that C, N, O, Pt, Cl, S and F were uniformly distributed on the PCDMS surface. This result confirmed that the polyvinyl alcohol surface was coated with polydopamine and successfully loaded with celecoxib and cisplatin.

[0048] 2. Drug loading and release characteristics of drug-loaded microspheres To verify the drug loading and release characteristics of the drug-loaded microspheres, the drug loading and release characteristics of the PCDMS prepared in Example 2 of this invention, the PCMS prepared in Comparative Example 1, and the PDMS samples prepared in Comparative Example 2 were tested as follows.

[0049] (1) Drug loading characteristic test method: Weigh 20 mg celecoxib and 10 mg cisplatin, and prepare celecoxib solution of 2 mg / mL and cisplatin solution of 1 mg / mL respectively using physiological saline solution. Use pipettes to successively pipette 0.02, 0.05, 0.1, 0.5, 1.0 and 5.0 mL of the two solutions to prepare two groups of solutions with concentrations of 2, 5, 10, 20, 100 and 500 μg / mL respectively. After measuring the absorbance of each sample with a UV spectrophotometer, a standard curve is prepared. The PCDMS prepared in Example 2, the PCMS prepared in Comparative Example 1 and the PDMS prepared in Comparative Example 2 are used as test samples. Vortex mix thoroughly, let stand for 24 h, and then centrifuge at 12000 rpm for 10 min to separate the precipitate and the supernatant. Take 160 μL of the supernatant and measure the absorbance of each sample using a UV spectrophotometer to obtain the content of celecoxib and cisplatin (W1). Then, determine the embedding ratio (ER): ER (%) = [ (W-W1) / W]×100, where W represents the dosage (mg).

[0050] (2) Drug release characteristic test method: The precipitate separated after centrifugation, i.e., the drug-loaded microspheres, was placed in a 15 ml clean centrifuge tube, and 10 mL of drug release medium was added. The drug release medium was deionized water with pH=7.4. The temperature of the constant temperature shaker was fixed at 37℃ and the rotation speed was set to 100 rpm. Samples were taken sequentially at 6 h, 12 h, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d, 8 d, 10 d, 12 d, and 14 d. The same volume of blank drug release medium was added immediately after each sampling. After sampling, the absorbance of each sample was measured, and the cumulative drug release percentage of each group of drug-loaded microspheres was calculated. The encapsulation efficiency and cumulative release curve of each sample are shown in the figure. Figure 4 As shown.

[0051] Figure 4 Figure A shows that when only one drug, celecoxib (PCMS) or cisplatin (PDMS), is loaded onto the PMS, the encapsulation efficiencies of celecoxib and cisplatin are 70.79±2.48% and 74.97±1.18%, respectively. When both celecoxib and cisplatin (PCPDMS) are loaded onto the PMS, the encapsulation efficiencies of celecoxib and cisplatin are 58.31±2.45% and 62.47±2.96%, respectively. These results indicate that dual-loading of drugs slightly affects the drug loading efficiency of the PMS, but the encapsulation efficiencies of both drugs remain above 50%, and the drug loading efficiency is maintained at a high level. Figure 4Figures B and C show that at day 14, the cumulative release rates of celecoxib were 92.54±2.6% (PCMS) and 88.1±1.38% (PCDMS), respectively; while the cumulative release rates of cisplatin were 88.69±2.59% (PDMS) and 84.4±2.56% (PCDMS), respectively. These results indicate that the simultaneous loading of celecoxib and cisplatin onto PMS results in a synergistic effect greater than the sum of its parts (1+1>2), demonstrating stable in vitro release and a long-term, slow release that meets the pharmacological requirements for clinical treatment.

[0052] (ii) In vitro experiments of drug-loaded microspheres To determine the in vitro cytotoxicity of the dual-drug-loaded microspheres PCDMS prepared in this invention, human liver cancer cells SMMC-7721 were selected as seed cells for CCK-8 assay and scratch assay.

[0053] The PVAMS samples (PVA group), PMS samples (PMS group), and PCDMS samples (PCDMS group) prepared in Example 2 of this invention, the PCMS samples (PCMS group) prepared in Comparative Example 1, and the PDMS samples (PDMS group) prepared in Comparative Example 2 were soaked in 4 mL of PBS solution for 24 h and then centrifuged. The supernatant of each group was collected as the extraction solution for later use. At the same time, a blank control group (Control group) was set up, which consisted of 4 mL of PBS solution.

[0054] CCK-8 assay method: Digested human liver cancer cells SMMC-7721 were seeded in 96-well plates (100 μl / well) and incubated at 37°C with 5% CO2. After complete cell attachment, the cells were washed three times with PBS solution, and 25 μl of the above-mentioned extraction solutions were added to each well. Each group was then divided into three incubation groups for 24 h, 48 h, and 72 h. After incubation for 24, 48, and 72 h, 10 μl of CCK-8 reagent was added to each well, and incubation was continued for 3 h for color development. The optical density (OD) value was detected at 450 nm using a microplate reader (BioTek, USA). The experimental results are as follows: Figure 5 As shown.

[0055] Scratch assay method: Digested human liver cancer cells SMMC-7721 were seeded in 24-well plates and incubated at 37°C with 5% CO2. When the cells reached confluence of more than 90%, a straight line was drawn in the center of the cell growth area at the bottom of the culture dish using a 200 μL sterile pipette tip. The cells were washed three times with PBS solution to remove the scratched cells, and fresh culture medium and the above-mentioned extraction solutions were added. After culturing for another 48 h, photographs were taken. The experimental results are as follows: Figure 6 As shown.

[0056] from Figure 5 The results showed that in SMMC-7721 cells, the celecoxib-loaded sample (PCMS) had limited inhibitory effect on tumor cells, and the inhibitory ability weakened further with increasing drug concentration over time. The cisplatin-loaded sample (PDMS) and the sample simultaneously loaded with celecoxib and cisplatin (PCDMS) showed better inhibitory ability on tumor cell proliferation, exhibiting a typical time-dependent effect, meaning that cell proliferation gradually decreased with longer drug treatment time. The sample simultaneously loaded with celecoxib and cisplatin (PCDMS) was significantly superior to the other groups in both its ability to inhibit tumor cell proliferation and the duration of drug treatment, with statistically significant differences (p<0.05). This indicates that celecoxib and cisplatin have a good synergistic effect; the simultaneous loading of celecoxib and cisplatin on PMS significantly enhanced the ability of PCDMS to inhibit tumor cell proliferation, especially with increasing time, where the difference in inhibitory ability widened considerably.

[0057] from Figure 6 The results showed no significant difference in healing speed between the Control group and the PVA and PMS groups. Compared with the Control group, the healing speed of the PCMS group was slightly slower, while the healing speed of the PDMS and PCDMS groups was significantly slower. PCDMS significantly inhibited cell migration. These results indicate that microspheres loaded with celecoxib and cisplatin alone have a certain inhibitory effect on SMMC-7721, while PCDMS loaded with both celecoxib and cisplatin can work synergistically to significantly inhibit the migration ability of liver cancer cells.

[0058] (iii) Animal experiments with drug-loaded microspheres Experimental Example 1: In vivo animal experiment on subcutaneous H22 xenograft tumors in mice loaded with drug-eluting microspheres (1) Experimental animals and grouping Twenty-five 5-week-old female Balb / c mice were randomly divided into five groups: PVA group, PMS group, PCMS group, PDMS group, and PCDMS group.

[0059] (2) Construction and administration regimen of subcutaneous H22 xenograft model in Balb / c mice The right axilla of each mouse was prepared and disinfected. 1×10⁻⁶ mmol / L was injected subcutaneously into the axilla of each mouse. 8 H22 cell suspension. Tumor growth was monitored daily until the tumor volume reached 100 mm. 3 This indicates the successful establishment of a Balb / c mouse subcutaneous H22 xenograft model. Each mouse was treated by injecting a saline solution containing 100 mg of the corresponding group sample into its tumor. The specific treatment regimen is shown in Table 1. (3) Measurement scheme After drug administration, mouse body weight and tumor volume were measured every 2 days. After 14 days, the mice underwent surgical dissection and exploration. After skin incision, the tumor was separated layer by layer from surrounding tissue and muscle, and photographs were taken for evaluation. A portion of the separated tumor was used for HE staining, VEGF staining, CD31 staining, IL-10 staining, and TGF-β staining. The experimental results are as follows: Figures 7-8 As shown.

[0060] (4) Measurement results and analysis Figure 7 As shown in Figure A, there was no significant difference in body weight changes among the groups of mice. However, Figure 7 Figure B shows that, in terms of tumor volume, the tumor volume of mice treated with PDMS and PCDMS was significantly reduced, showing statistical differences compared with PVA, PMS, and PCMS, with the PCDMS group showing the most significant tumor volume reduction.

[0061] CD31 and VEGF are important biomarkers for liver cancer metastasis and invasion. This invention discovers (through immunohistochemistry) Figure 8 The expression of CD31 and VEGF was abundant in the PVA and PMS groups (A, B, and C), and the cisplatin-loaded PDMS group also showed high expression of CD31 and VEGF. However, the celecoxib-loaded PCMS and the PCDMS group simultaneously loaded with celecoxib and cisplatin showed inhibition of CD31 and VEGF expression. The inhibition was most significant in the PCDMS group. These results indicate that the combined use of celecoxib and cisplatin can significantly inhibit excessive angiogenesis in hepatocellular carcinoma.

[0062] IL-10 and TGF-β dominate the tumor environment of various cancers, primarily responsible for immunosuppression. This invention, through immunohistochemical staining analysis, reveals (…). Figure 8 The expression of IL-10 was high in the PVA, PMS, and PDMS groups (A, D, and E), while low in the PCMS and PCDMS groups, with the lowest expression in the PCDMS group; TGF-β expression showed similar results. These results indicate that the application of cisplatin and celecoxib can significantly improve the local immune microenvironment of hepatocellular carcinoma and reduce immunosuppression.

[0063] Experimental Example 2: In vivo animal experiment on the inhibition of rabbit VX2 liver transplant tumors by drug-loaded microspheres (1) Experimental animals and grouping The experimental rabbits were randomly divided into 5 groups: PVA group, PMS group, PCMS group, PDMS group and PCDMS group.

[0064] (2) Construction of rabbit VX2 intrahepatic xenograft model and drug administration regimen The VX2 tumor tissue was cut into pieces approximately 1 mm in size. 3 The size of the tumor was determined by resuspending it in physiological saline, and 0.5 mL of the resuspended solution was inoculated into the lateral thigh muscle of each group of experimental rabbits. Two weeks after tumor implantation, an elastic mass with a diameter of 2-3 cm could be palpated on the lateral thigh of each group of experimental rabbits, indicating successful tumor-bearing breeding rabbit preparation. Before tumor removal, the tumor-bearing rabbits were routinely fasted for 8 hours, and then anesthetized by intramuscular injection of 0.2 mg / kg xylazine hydrochloride and 5 mg / kg sodium pentobarbital, with the injection volume depending on body weight. After the anesthesia took effect, the VX2 tumor tissue located in the lateral thigh muscle was exposed and dissected, and the removed VX2 tumor tissue was placed in a sterile culture dish. The surrounding muscle, blood vessels, fascia, and necrotic tissue of the VX2 tumor tissue were carefully removed, and the tissue was washed three times with physiological saline. The viable VX2 tumor tissue was selected, cut into small pieces, and inserted into the tip of a 21G puncture needle for later use.

[0065] After anesthesia, rabbits were fixed in a supine position on a rabbit table, and the skin preparation area was disinfected and prepared strictly according to surgical aseptic techniques. A 2-3 cm longitudinal incision was made along the midline of the abdomen, and the abdominal wall was dissected layer by layer to expose the liver. The left lobe of the liver was pulled out using hemostatic forceps, and a puncture needle was inserted 1 cm into the liver parenchyma. After inserting a bolus, the puncture needle was withdrawn, and the puncture site was sealed with gelatin sponge. The left lobe of the liver was gently returned to the abdomen, and the muscle and skin incisions were sutured layer by layer, and the wound was disinfected. Ceftazidime injection (0.1 mL / Kg) was administered intramuscularly for 3 consecutive days postoperatively to prevent infection. Two weeks after intrahepatic implantation in the rabbits, enhanced CT (CTA) was used to evaluate whether the growth of the VX2 tumor tissue met the criteria for tumorigenesis, including the location and size of the intrahepatic VX2 tumor. Enhanced CT in the arterial phase showed peripheral enhancement of the VX2 tumor with low-density shadows inside, suggesting necrosis and liquefaction within the tumor (Figure 5B), indicating that the tumor met the criteria. The experimental rabbits were anesthetized as above, and an indwelling venous catheter (22G) was inserted into the marginal ear vein for scanning using a Siemens dual-element spiral CT scanner.

[0066] The drug administration and procedure for the experimental rabbits utilized transcatheter arterial chemoembolization (TACE). After the tumor met the criteria, the skin was prepared in the right groin area of ​​the experimental rabbit, and a 2 cm longitudinal incision was made along the course of the artery after disinfection. The femoral artery in experimental rabbits is relatively thin and often runs alongside the femoral vein and femoral nerve within the fascia. The femoral artery was bluntly dissected using a glass needle to avoid injury to the vein and nerve. After exposing the right femoral artery, the proximal end was temporarily clamped, and the distal end of the femoral artery was lifted with a No. 5 suture. A small incision was made obliquely towards the proximal end using ophthalmic scissors, and a 5-F catheter sheath was inserted and fixed. A 5-F catheter was introduced under DSA and inserted along the celiac trunk to the T12 and L1 vertebrae. Angiography was used to locate the opening of the celiac artery. Subsequently, a 2.7F microcatheter and a 0.021-inch guidewire were introduced, with the guidewire preceding the catheter. After entering the celiac artery, angiography was used to determine the opening and course of the hepatic artery; typically, the rabbit celiac artery divides into three branches, with the central, obliquely ascending branch being the hepatic artery. After entering the hepatic artery, the tumor-feeding artery was superselected, and embolization therapy was performed on the tumor according to the group. Post-treatment angiography confirmed complete embolization of the tumor-feeding artery. The microcatheter, microguidewire, and guiding catheter were withdrawn. The femoral artery was ligated, and the muscle and skin incisions were sutured layer by layer. The sutured wounds were disinfected. All experimental rabbits received intramuscular injections of ceftazidime (0.1 mL / Kg) for three consecutive days after TACE to prevent infection.

[0067] (3) Measurement scheme Blood samples were drawn from rabbits on days 7 before TACE and on days 1, 3, 7, and 14 after TACE to measure red blood cells, white blood cells, platelets, and liver and kidney function indicators such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin (ALB), total bilirubin (TBIL), blood urea nitrogen (BUN), and serum creatinine (Cr). Enhanced CT scans were performed two weeks after the establishment of the rabbit intrahepatic VX2 tumor model and one and two weeks after TACE. After the rabbit livers were completely removed, tumor tissues from each group were fixed with paraformaldehyde and then stained with HE, PCNA, CTR-1, MRP-2, CD4, CD8, and Foxp3. The experimental results are as follows: Figure 8 , Figure 9 and Figure 10 As shown.

[0068] (4) Measurement results and analysis Figure 9 Figures A, B, and C show that 14 days after TACE, the white blood cell, red blood cell, and platelet counts in each group were within the normal range or slightly higher than the theoretical normal value, indicating that cisplatin did not cause significant bone marrow suppression. Figure 9The results of D, E, F, and G showed that ALT, AST, ALB, and TBIL in the five groups of experimental rabbits increased significantly within one week after surgery. This is because a large amount of tumor tissue and hepatocytes undergo ischemic necrosis in the short term after TACE, resulting in transient abnormal liver function. All groups returned to normal levels two weeks after surgery. Figure 9 The H and I results showed that renal function-related indicators Cr and BUN also showed transient increases, which returned to normal levels after 2 weeks, except for the PDMS group. Figure 8 The results showed that HE staining revealed no pathological changes in other tissues. These results indirectly confirm that the dual-drug-loaded microspheres PCDMS prepared in this invention have high safety in TACE for liver cancer.

[0069] Figure 10 The B-line showed that two weeks after the establishment of the VX2 tumor model in the liver of the experimental rabbit, a follow-up CT scan was performed. The enhanced CT arterial phase showed that the VX2 tumor edge was enhanced and the internal low-density shadow suggested necrosis and liquefaction inside the tumor. Figure 10 The CT scan showed that the tumor VX2 in the left lobe of the liver had a dense distribution of blood vessels around it, presenting a typical "ball-hugging sign". After microsphere embolization, the tumor's blood supply artery was occluded, the "ball-hugging sign" disappeared, and the tumor was no longer visible on the screen. Figure 10 The D-scan showed that enhanced CT scans were performed 1 week and 2 weeks after TACE. Two weeks after the surgery, a large area of ​​low density shadow was visible in the embolized area, and the tumor enhancement disappeared, indicating that the tumor was completely necrotic on imaging. Figure 10 The E-scan showed that, upon gross examination, there was a significant necrotic area in the left lobe of the liver, which completely covered the VX2 tumor. After cutting along the longitudinal axis of the tumor, the tumor parenchyma was found to be fish-flesh-like, with ischemic necrosis inside. Figure 10 F-staining revealed that, after HE staining, embolic microspheres were concentrated within hepatic vessels. Erythrocyte congestion, hepatocyte cytoplasmic depigmentation, vacuolation, and enlarged nuclei—signs of cell necrosis—were observed in the local tissue spaces. To further evaluate the embolization effect of PCDMS on tumors, tumor proliferation was detected by PCNA fluorescence staining. Compared with the PVA group, PCNA expression was significantly reduced in the PCMS, PDMS, and PCDMS groups, with the most significant reduction in the PCDMS group. These results indicate that PCDMS significantly increases the success rate of TACE surgery, and that cisplatin combined with celecoxib can significantly inhibit tumor cell proliferation, thereby improving the efficacy of embolization chemotherapy.

[0070] Figure 11 Figures A and B show that CTR-1 (drug efflux protein) expression was higher in the PCDMS group and lower in the PDMS group, with a statistically significant difference between the two groups (p<0.05). MRP-2 is an important drug efflux protein, which is crucial for the sustained action of cisplatin in tumor cells. Figure 11Figures A and C show that the combined application of celecoxib to inhibit COX-2 significantly affected MRP-2 expression in liver cancer cells, with the PDMS group showing a significantly higher level than the PCDMS group (p<0.05). This result indicates that cisplatin can rapidly (<14 days) induce drug resistance in tumor cells through local application, while the slow release of celecoxib-loaded PCDMS at the cisplatin site can upregulate CTR-1 and reduce MRP-2 expression by inhibiting the COX-2 pathway, allowing cisplatin to exert a sustained effect within tumor cells and slowing the development of multidrug resistance. Foxp3+ T cells are immunosuppressive cells. Figure 11 Figures A and D show that the PVA, PMS, and PDMS groups accumulated a greater number of regulatory T cells, while the PCMS and PCDMS groups loaded with celecoxib showed some differences, exhibiting weaker immunosuppression and a more active immune response. Celecoxib application can reduce local inflammation by inhibiting COX-2 expression, modulating the tumor immune microenvironment, and converting cold tumors into hot tumors, thus providing favorable conditions for further immunotherapy. CD4+ T cells and CD8+ T cells are common immune killer cells in the tumor microenvironment. Figure 11 Figures A, E, and F show that the PCDMS group significantly promoted T cell infiltration, especially CD4+ T cells, which play an important role in enhancing anti-tumor immunity.

[0071] The above embodiments are specific implementations of the present invention, but the implementation of the present invention is not limited to the above embodiments. Any other combination, change, modification, substitution, or simplification that does not exceed the design concept of the present invention shall fall within the protection scope of the present invention.

Claims

1. A polydopamine-modified dual-drug-loaded microsphere containing celecoxib and cisplatin, characterized in that, Polydopamine-modified polyvinyl alcohol microspheres were used as carriers, and celecoxib and cisplatin were loaded on the surface of the microspheres.

2. The method for preparing polydopamine-modified dual-drug-loaded microspheres of celecoxib and cisplatin as described in claim 1, characterized in that, Includes the following steps: (1) Polyvinyl alcohol microspheres were added to a dopamine solution prepared by Tris-HCl buffer, the pH was adjusted to 8-10, stirred at room temperature for 10 min-72 h, and polydopamine-modified polyvinyl alcohol microspheres were obtained after washing and centrifugation. (2) Dissolve cisplatin and celecoxib in PBS buffer to obtain a celecoxib / cisplatin mixed solution; add the polydopamine-modified polyvinyl alcohol microspheres obtained in step (1) to the celecoxib / cisplatin mixed solution, incubate for 30 min to 24 h, and centrifuge to obtain polydopamine-modified dual drug-loaded microspheres.

3. The method for preparing polydopamine-modified dual-drug-loaded microspheres of celecoxib and cisplatin as described in claim 2, characterized in that, In step (1), the ratio of the amount of polyvinyl alcohol microspheres to the amount of dopamine solution is (1-10) g / 100 mL, and the mass concentration of the dopamine solution is 0.1%-10%.

4. The method for preparing polydopamine-modified dual-drug-loaded microspheres of celecoxib and cisplatin as described in claim 2, characterized in that, In step (2), the mass ratio of cisplatin to celecoxib is (0.4-2.5):

1.

5. The method for preparing polydopamine-modified dual-drug-loaded microspheres of celecoxib and cisplatin as described in claim 4, characterized in that, In step (2), the ratio of the polydopamine-modified polyvinyl alcohol microspheres to the celecoxib / cisplatin blend solution is 10–100 mg / mL.

6. The method for preparing polydopamine-modified dual-drug-loaded microspheres of celecoxib and cisplatin as described in claim 2 or 3, characterized in that, In step (1), the concentration of the Tris-HCl buffer is 2 mg / mL and the pH is 8.

5.

7. The method for preparing polydopamine-modified dual-drug-loaded microspheres of celecoxib and cisplatin as described in claim 2, characterized in that, In step (1), the polyvinyl alcohol microspheres are prepared by water-in-oil emulsion crosslinking method.

8. The method for preparing polydopamine-modified dual-drug-loaded microspheres of celecoxib and cisplatin as described in claim 2, characterized in that, In step (1), the polyvinyl alcohol microspheres have a particle size of 100–300 μm.

9. The use of the polydopamine-modified dual-drug-loaded microspheres of celecoxib and cisplatin as described in claim 1, or the polydopamine-modified dual-drug-loaded microspheres of celecoxib and cisplatin prepared by any of the methods described in claims 2-8, in the treatment of liver cancer.

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