Dual-effect microsphere loaded with idarubicin and taliquine as well as preparation method and application of dual-effect microsphere

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to idarubicin and taliquantel loaded dual-effect microspheres as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) preparing a dopamine solution by adopting a Tris-HCl buffer solution, adding polyvinyl alcohol microspheres into the dopamine solution, stirring at room temperature for 24-48 hours, washing, standing, and collecting precipitate to obtain polydopamine modified polyvinyl alcohol microspheres; and (2) adding the taliquine and the idarubicin into a PBS (Phosphate Buffer Solution) to obtain a taliquine-idarubicin mixed solution, adding the polydopamine modified polyvinyl alcohol microspheres into the taliquine-idarubicin mixed solution, incubating and centrifuging to obtain the dual-effect microspheres loaded with the idarubicin and the taliquin. The PTIMS serving as an embolism agent not only can embolize a tumor blood supply artery and realize local drug release in a tumor, but also can serve as a heat source for non-invasive photo-thermal ablation treatment after a TACE operation, so that drug release is further enhanced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a dual-functional microsphere loaded with idarubicin and taliquidar, a preparation method thereof, and an application thereof in the combined transcatheter arterial chemoembolization and photothermal ablation treatment of liver cancer. Background Art

[0002] Liver cancer is the sixth most common cancer in the world, among which hepatocellular carcinoma (HCC) is the most common type of liver cancer, accounting for about 90% of cases. Current treatments for HCC include liver transplantation, local ablation therapy, systemic therapy, and transcatheter arterial chemoembolization (TACE). Among them, TACE is an effective treatment for advanced HCC, and the performance of drug-loaded microspheres (DEM) plays a vital role in the prognosis of patients after TACE surgery, and is an indispensable component of TACE surgery. The ideal DEM should have the following characteristics: high drug delivery efficiency in tumor tissue, uniform and appropriate size, matching the diameter of blood vessels in the target area, and effective imaging capabilities that are conducive to postoperative efficacy monitoring. In recent decades, DC and Various DEMs have entered the market, gradually replacing traditional iodized oil embolic agents as the preferred option for TACE due to their ability to achieve high local drug concentrations, more complete embolization, and stable, sustained drug release. Currently, clinically used DEMs primarily rely on a positive and negative ion exchange mechanism for drug delivery. However, due to limitations in drug loading performance and the potential for multiple drug interactions, their drug delivery efficiency remains unsatisfactory.

[0003] Photothermal therapy (PTT) is a novel, minimally invasive approach for tumor thermal ablation in clinical practice. This process utilizes photothermal converters (PTAs) that, upon exposure to near-infrared (NIR) radiation, convert light energy into heat, elevating tissue temperature and inducing tumor cell apoptosis. Currently, PTAs, such as precious metal materials, carbon-based nanomaterials, and other two-dimensional materials (e.g., black phosphorus nanosheets, boron nitride, and MXenes), have demonstrated remarkable efficacy in PTT tumor treatment. However, high production costs, complex manufacturing processes, and potential biotoxicity have hindered the advancement of PTT. PDA, formed through the oxidation and self-polymerization of dopamine (DA) monomers, possesses excellent near-infrared photothermal conversion capabilities. Furthermore, PDA exhibits significant adhesion, excellent biocompatibility, and photothermally responsive drug release, attracting significant attention from the scientific community. Current PTT approaches are minimally invasive, precise, with a low incidence of adverse reactions and high specificity. Therefore, they can be effectively used for noninvasive photothermal ablation after transcatheter arterial pressure-activated cell death (TACE), increasing local tumor temperature while simultaneously enhancing local drug concentration and reducing tumor recurrence. This demonstrates significant potential when combined with TACE. On the one hand, PTT can induce tumor cell apoptosis, locally release tumor-specific antigens, initiate specific anti-tumor immune responses, and improve the tumor immune microenvironment. On the other hand, PTT also addresses the issue of tumor drug resistance after TACE. PTT enhances chemotherapy sensitivity by increasing temperature, thereby inhibiting the function of drug efflux pumps located on the tumor cell membrane.

[0004] Anthracyclines, such as doxorubicin, pirarubicin, and idarubicin (IDA), are effective tumor chemotherapy drugs that have been widely used in TACE and have good application prospects. Anthracyclines are cell cycle-specific chemotherapy drugs that show strong anti-proliferative effects on various types of tumors. In addition, anthracyclines can improve the tumor immune microenvironment by recruiting activated cytotoxic T lymphocytes to the tumor site. Among anthracyclines, IDA has attracted much attention due to its good lipophilicity, long biological half-life, and less cardiotoxicity. However, the existence of multidrug resistance (MDR) often makes single chemotherapy drugs ineffective. The main reason for drug resistance is that chemotherapy drugs (such as paclitaxel, doxorubicin, and IDA) are actively excreted through P-glycoprotein (P-gp) overexpressed on tumor cells. The occurrence of this phenomenon leads to a decrease in drug concentration in tumor cells, followed by drug resistance, which ultimately limits the efficacy of IDA. Tadalafil (TRQ) is a chemosensitizer and a specific inhibitor of P-gp. 32 TQR exhibits high affinity. By inducing P-gp to switch from a closed conformation to an open conformation, it hinders the binding of P-gp to its substrate and reduces the efflux of P-gp to anticancer drugs, thereby restoring the chemotherapy sensitivity of tumor cells.

[0005] Currently, there are relatively few studies investigating the combined use of P-gp inhibitors with TACE and PTT, with most ongoing research focusing on developing microspheres for combined TACE and photothermal ablation. However, challenges such as difficult-to-control temperature, side effects caused by sudden drug release, and tumor multidrug resistance continue to hinder progress in this field. Therefore, this study aimed to develop dual-action embolic microspheres containing idarubicin and taliquidar for combined TACE and photothermal ablation treatment of liver cancer. Summary of the Invention

[0006] In view of the problems and shortcomings in the prior art, the purpose of the present invention is to provide a dual-functional microsphere loaded with idarubicin and taliquidar and a preparation method thereof, and also to provide its application in the combined transcatheter arterial chemoembolization and photothermal ablation treatment of liver cancer.

[0007] Based on the above purpose, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a dual-functional microsphere loaded with idarubicin and taliquidar, using polydopamine-modified polyvinyl alcohol microspheres as carriers, and the surface of the microspheres is loaded with idarubicin and taliquidar.

[0009] In a second aspect, the present invention provides a method for preparing dual-functional microspheres loaded with idarubicin and taliquidar, comprising the following steps:

[0010] (1) A dopamine solution was prepared using Tris-HCl buffer, polyvinyl alcohol (PVA) microspheres were added to the dopamine solution, stirred at room temperature for 24 to 48 hours, washed, allowed to stand, and the precipitate was collected to obtain polydopamine-modified polyvinyl alcohol microspheres (PMS);

[0011] (2) Adding tariquida and idarubicin into PBS buffer to obtain a tariquida-idarubicin mixed solution, adding the polydopamine-modified polyvinyl alcohol microspheres obtained in step (1) to the tariquida-idarubicin mixed solution, incubating and centrifuging to obtain dual-functional microspheres (PTIMS) loaded with idarubicin and tariquida.

[0012] Preferably, in step (1), the concentration of the dopamine solution is 0.1 to 0.5 wt%.

[0013] Preferably, in step (1), 1 to 3 g of polyvinyl alcohol microspheres are added to every 30 to 50 mL of dopamine solution.

[0014] Preferably, in the tariquida-idarubicin mixed solution of step (2), the concentration of tariquida is 1-5 mg / mL, and the concentration of idarubicin is 1-5 mg / mL.

[0015] Preferably, in step (2), 0.5 g of polydopamine-modified polyvinyl alcohol microspheres are added to every 5 to 10 mL of the talizine-idarubicin mixed solution.

[0016] Preferably, the pH of the Tris-HCl buffer in step (1) is 6.8 to 8.8.

[0017] Preferably, the standing time in step (1) is 5 to 10 minutes.

[0018] Preferably, the incubation time in step (2) is 12 to 24 hours.

[0019] Preferably, the centrifugal speed in step (2) is 2000-3000 rpm, and the time is 3-5 min.

[0020] Preferably, the washing in step (1) is performed using deionized water.

[0021] In a third aspect, the present invention provides the use of dual-functional microspheres loaded with idarubicin and taliquidar prepared by the preparation method of the first aspect or the second aspect in the combined treatment of liver cancer by transcatheter arterial chemoembolization and photothermal ablation.

[0022] Compared with the prior art, this application has the following advantages:

[0023] 1. In the present invention, IDA is used as a chemotherapy drug in TACE, which has a strong anti-tumor effect. TQR, as a P-gp inhibitor, can inhibit the expression of P-gp on the tumor cell membrane, prevent the efflux of IDA, and overcome the multidrug resistance of tumors. PDA has good biocompatibility and photothermal conversion ability, and has good stability when used as a heat source for photothermal ablation therapy. In the combined action mode of PTIMS of the present invention, TQR enhances the apoptotic effect of IDA, the inhibitory effect on cell migration and invasion, and the cell uptake of IDA, indicating that the addition of TQR can reduce the expression of P-gp, inhibit the active efflux of IDA from tumor cells, and increase the concentration of IDA in cells. In addition, due to the sensitivity of tumor cells to chemotherapy drugs induced by PTT, the effect of TQR is further enhanced under the action of PTT.

[0024] 2. The cumulative release of IDA on the 28th day of the PTIMS of the present invention was 205±13.1μg / mL, and the cumulative release of TQR was 285.8±22.1μg / mL, indicating that PTIMS has a stable drug release ability and meets the pharmacological requirements of clinical treatment. The cumulative release of IDA and TQR under near-infrared conditions increased to 255.8±19.9μg / mL and 329.6±20.0μg / mL, respectively, indicating that the photothermal conversion of PDA promoted the release of drugs.

[0025] 3. The present invention prepared PTIMS by simultaneously loading IDA and TQR onto PDA-coated PVA microspheres. When applied to the subcutaneous H22 tumor model of Balb / c mice, the TQR inhibited P-gp and the NIR enhanced tumor eradication and chemotherapy sensitivity, which was significantly different from the BC group (1079.18±50.44mm 3 ) and PTMS group (968.49±53.27mm 3 ) compared with the PIMS group (675.21±50.20mm 3 ), PTIMS group (321.43±58.09mm 3 ) and PTIMS+NIR group (110.08±49.33mm 3 ) group showed a significant decrease in tumor volume, the PTIMS+NIR group showed a greater degree of cell necrosis, and PTIMS+NIR significantly inhibited tumor growth.

[0026] 4. In the examples, the present invention was applied to a New Zealand rabbit VX2 liver xenograft tumor model. DSA-guided TACE was performed on the tumor. Preoperative angiography showed densely distributed blood vessels surrounding the left lobe VX2 tumor, and postoperative angiography revealed complete embolization of the surrounding vessels. Enhanced CT scans revealed the embolized area with a low-density area at 7 days and an enlarged area at 14 days, indicating successful tumor eradication.

[0027] 5. The PTIMS of the present invention can be used as an embolic agent not only to embolize the tumor blood supply artery and achieve local drug release in the tumor, but also as a heat source for non-invasive photothermal ablation treatment after TACE, thereby further enhancing drug release. Due to the changes in the immune microenvironment at the embolization site after TACE and IDA promoting immunogenic cell death, the immune microenvironment of the tumor is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 a is the Raman spectrum results of PVA, PDA, TQR and IDA. Figure 1 b is the Raman spectrum results of PMS, PTMS, PIMS and PTIMS. Figure 1 c is the Zeta potential result diagram of each sample, Figure 1 d is the scanning electron microscope (SEM) image of the microspheres of each sample at different magnifications. Figure 1 e The microsphere particle size analysis results of each sample, Figure (f, g) are the scanning electron microscope-energy dispersive spectrum (EDS) element maps of each sample, scale: 2, 10, 20, 40 μm;

[0029] Figure 2 a is 808nm laser (0.5W / cm 2 and 0.8W / cm 2) Infrared thermal images of each sample at irradiation time of 0, 30, 60, 90, 120, 150, and 180 seconds, Figure 2 (b,c) are the temperature rise curves of each sample. Figure 2 d is PTIMS at 0.8W / cm 2 Temperature change results when 5 cycles of on / off are performed under irradiation conditions. Figure 2 (e, f) PTIMS at room temperature and 0.8 W / cm 2 Drug release results under intermittent irradiation conditions;

[0030] Figure 3 a is a schematic diagram of the anti-tumor mechanism of PTIMS in vitro anti-tumor effect chemotherapy drugs combined with PTT-mediated anti-tumor effect. Figure 3 b is a representative image of live-dead cell staining of H22 cells. Figure 3 c is the percentage of live cells and dead cells. Figure 3 d is the result of H22 cell viability at 1, 2, and 3 days of co-culture. Figure 3 (e, f) are the quantitative results of the scratch test and Transwell test. Figure 3 (g, h) Representative images of the scratch test and Transwell assay, scale bars: 40, 100, 300 μm, significant difference: NS, no statistical significance, *P < 0.05, **P < 0.01, ***P < 0.001;

[0031] Figure 4 (a, b) are representative images and statistical analysis results of Ki67 immunofluorescence staining of each sample. Figure 4 (c, d) are representative flow cytometry images (Annexin V-AbFluorTM488 / PI staining) and corresponding statistical analysis results. Figure 4 (e, g) Representative images and statistical analysis results of P-gp and IDA immunofluorescence staining, scale bars: 10 and 15 μm, significant difference: NS, no statistical significance, *P < 0.05, **P < 0.01, ***P < 0.001;

[0032] Figure 5 (a, b) are representative images and statistical analysis results of the photothermal performance of the H22 subcutaneous transplanted tumor model in Balb / C mice. Figure 5 c is the statistical analysis result of TNF-α immunohistochemical staining, Figure 5 d is a representative image of the tumor in each group 14 days after treatment. Figure 5 (e, f) are the statistical analysis results of Ki67 immunohistochemical staining and P-gp immunofluorescence staining. Figure 5(g, hi) Representative images of HE staining, TNF-α and Ki67 immunohistochemical staining, and P-gp immunofluorescence staining, scale bar: 100 μm, significant difference: NS, no statistically significant difference, *P<0.05, **P<0.01, ***P<0.001;

[0033] Figure 6 This is the tumor volume result of the subcutaneous H22 transplanted tumor model in Balb / C mice on day 14;

[0034] Figure 7 These are HE-stained images of the heart, liver, spleen, lung, and kidney of the Balb / C mouse subcutaneous H22 tumor model after treatment;

[0035] Figure 8 The figure shows the biochemical analysis results of ALT, AST, ALP, BUN, CERA and UA in the subcutaneous H22 transplanted tumor model of Balb / C mice;

[0036] Figure 9 a is a representative image of enhanced CT scan of New Zealand rabbit VX2 liver transplanted tumors on days 0, 7, and 14 by PTIMS. Figure 9 b is the DSA image before and after TACE. Figure 9 c is the result graph of red blood cell hemolysis rate, Figure 9 d is the HE staining image of embolized tissue. Figure 9 (e, f) are representative images of Ki67 and Cas-3 immunohistochemical staining and P-gp and CD8 immunofluorescence staining. Figure 9 (g, h, i, j) Statistical analysis of Ki67, Cas-3 immunohistochemical staining and P-gp, CD8 immunofluorescence staining results, scale bars: 2.5 cm, 1.5 cm, 250 μm, 100 μm, significant difference: NS, no statistical significance, *P < 0.05, **P < 0.01, ***P < 0.001;

[0037] Figure 10 This is a gross image of the New Zealand rabbit VX2 liver tumor model;

[0038] Figure 11 HE-stained images of the heart, lung, spleen, and kidney in a New Zealand rabbit VX2 liver tumor model 2 weeks after TACE.

[0039] Figure 12 The graph shows the biochemical analysis results of ALT, AST, BUN and CERA in the New Zealand rabbit VX2 liver tumor model. DETAILED DESCRIPTION

[0040] The following examples are intended only to further illustrate the present invention. It should be noted that all technical and scientific terms used herein have the same meanings as in the art to which the present invention pertains, unless otherwise specified. Experimental methods in the following examples, where specific conditions are not specified, were based on conventional techniques in the art or the conditions recommended by the manufacturer. Reagents and instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0041] In order to enable those skilled in the art to more clearly 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.

[0042] (1) Materials and instruments:

[0043] Polyvinyl alcohol embolic microspheres (100–300 μm) were purchased from Suzhou Hengrui Jialisheng Biomedical Technology Co., Ltd.;

[0044] Dopamine (MW = 189.64) and Tris-HCl buffer (pH = 8.8) were purchased from Beijing Solaibao Biotechnology Co., Ltd. (Beijing, China);

[0045] Idarubicin hydrochloride for injection (IDA·HCl) was purchased from Hanhui Pharmaceutical Co., Ltd. (Hangzhou, Zhejiang);

[0046] Taliquidar (TQR) was purchased from MedChemExpress Biotechnology;

[0047] RPIM 1640, PBS buffer, and fetal bovine serum were purchased from Beijing Solai Biotechnology Co., Ltd. (Beijing, China);

[0048] Trypsin-EDTA was purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. (Shanghai, China);

[0049] Live and dead cell double staining kit (KTA1001) and Annexin V-AbFluorTM 488 / PI apoptosis detection kit (KTA0002) were purchased from Aiboken Biotechnology Co., Ltd. (Wuhan);

[0050] Mouse hepatocellular carcinoma-22 (H22) cells were obtained from the Shanghai Cell Center of the Chinese Academy of Sciences;

[0051] Other chemical and biological reagents were of analytical grade.

[0052] (2) Sample preparation

[0053] Example 1:

[0054] The invention discloses a dual-functional microsphere (PTIMS) loaded with idarubicin and tariquidar, which uses polyvinyl alcohol microspheres modified with polydopamine as carriers and the surface of the microspheres is loaded with idarubicin and tariquidar.

[0055] The preparation method comprises the following steps:

[0056] (1) A 0.2% dopamine solution was prepared using a Tris-HCl buffer solution with a pH of 8.8. 2 g of polyvinyl alcohol (PVA) microspheres were added to 50 ml of the dopamine solution and stirred at room temperature for 48 h. The unreacted dopamine was then washed with deionized water to remove the solution. After washing, the solution was allowed to stand for 10 min to collect the precipitate, thereby obtaining polydopamine-modified polyvinyl alcohol microspheres (PMS).

[0057] (2) 10 mg of tariquida and 10 mg of idarubicin were mixed and added to 10 ml of PBS buffer to obtain a tariquida-idarubicin mixed solution, and then 0.5 g of polydopamine-modified polyvinyl alcohol microspheres obtained in step (1) were added to the tariquida-idarubicin mixed solution, incubated on a shaker for 24 h, and then centrifuged at 3000 rpm for 5 min to obtain dual-functional microspheres (PTIMS) loaded with idarubicin and tariquida.

[0058] Comparative Example 1:

[0059] The invention discloses a microsphere (PTMS) loaded with tariquidal, which uses polyvinyl alcohol microspheres modified with polydopamine as carriers and the surface of the microspheres is loaded with tariquidal.

[0060] The preparation method is basically the same as that of Example 1, except that idarubicin is not added.

[0061] Comparative Example 2:

[0062] The invention discloses idarubicin-loaded microspheres (PIMS), which are characterized in that polyvinyl alcohol microspheres modified with polydopamine are used as carriers and idarubicin is loaded on the surface of the microspheres.

[0063] The preparation method is basically the same as that of Example 1, except that talizine is not added.

[0064] (3) Performance testing

[0065] 1. Physical and chemical properties test

[0066] The PMS, PTIMS obtained in Example 1, the PTMS obtained in Comparative Example 1, the PIMS obtained in Comparative Example 2, polyvinyl alcohol microspheres (PVA), talizquidar (TQR), and idarubicin (IDA) were used as samples for physical and chemical property tests. Figure 1 shown.

[0067] The specific test methods are as follows:

[0068] Raman spectroscopy (Thermo Scientific DXR 3Xi, USA) was used to analyze the fluorescence intensity of the samples in the range of 500 to 2200 cm -1 The bond vibration was tested; the zeta potential was measured using a Nano-2S90 Zeta Sizer (Malvern Instruments, UK); SEM images and EDS analysis were performed using a JSM-7401F microscope equipped with an EDS system, and the surface morphology and elemental composition were analyzed.

[0069] By Raman spectroscopy test ( Figure 1 a, b) The results show that the characteristic peaks of PDA are 1369 and 1568 cm -1 , corresponding to the deformation and expansion vibrations of catecholamines in PDA, PDA characteristic peaks appeared in PMS, PTMS, PIMS and PTIMS, indicating that PVA microspheres were coated in PDA, while no obvious characteristic peaks were observed in PVA, TQR and IDA.

[0070] Further Zeta potential test was conducted. Figure 1 As shown in Figure c, the zeta potentials of PVA, PDA, and TQR are negative, while that of IDA is positive. The zeta potential of PMS (-22.73 ± 0.48 mV) is more negative than that of PVA (-5.10 ± 0.32 mV), indicating that the PMS surface is successfully coated with PDA. The zeta potentials of PIMS and PTIMS, respectively, are (-9.92 ± 0.36) mV and (-10.55 ± 0.52) mV, both showing a positive trend compared to that of PMS, indicating that the PVA microspheres are loaded with IDA.

[0071] Scanning electron microscopy (SEM) Figure 1 As shown in Figure d, all samples are uniformly spherical or ellipsoidal. The outer surface of PVA is smooth, while that of PMS is rough, exhibiting an irregular, inhomogeneous, and grooved structure. The surface microstructures of PTMS, PIMS, and PTIMS are similar to those of PMS, with the presence of drug crystal particles. This is due to the insolubility of TQR and the excellent adhesion properties of PDA, resulting in the presence of a large number of crystals on this surface.

[0072] Particle size analysis ( Figure 1 e) shows that the PVA microspheres are mainly concentrated around 110 μm, while the particle size of other microspheres is about 140 μm.

[0073] Through EDS element map ( Figure 1f, g) Analysis shows that since PVA is mainly composed of C and O, PDA and TQR are mainly composed of C, N, and O, and IDA is mainly composed of Cl, the PTIMS surface is distributed with C (35.117%), N (7.641%), O (40.862%), and Cl (16.38%), indicating that PDA and the payloads TQR and IDA are successfully encapsulated.

[0074] 2. In vitro photothermal properties test

[0075] The PMS, PTIMS obtained in Example 1, the PTMS obtained in Comparative Example 1, the PIMS obtained in Comparative Example 2, and polyvinyl alcohol microspheres (PVA) were used as samples for in vitro photothermal property testing. Figure 2 shown.

[0076] The specific test methods are as follows:

[0077] 0.2 g of sample was irradiated by 808 nm NIR with power set to 0.5 and 0.8 W / cm 2 To evaluate the photothermal performance, an infrared thermal imaging camera (Testo, Shanghai, China) was used to obtain thermal images and record the temperature changes of each sample in real time; an 808 nm, 0.8 W / cm 2 The photothermal stability of the composite was evaluated by heating under near-infrared light and natural cooling for 5 cycles.

[0078] 0.2 g of sample was placed in 10 mL of PBS buffer for drug release. The temperature of the constant temperature oscillator was fixed at 37 °C, the speed was 50 r / min, and the wavelength was 808 nm and the power was 0.8 W / cm 2 The effect of photothermal on drug release was studied by irradiating with near-infrared light for 5 minutes every 2 days. Samples were collected at 1 hour, 6 hours, 12 hours, 1 day, 3 days, 7 days, 14 days, and 28 days, and an equal amount of PBS buffer was added to each sample. The absorbance at each point was measured and the cumulative drug release percentage was calculated. The test results are shown in Figure 2. Figure 2 shown.

[0079] Figure 2 a is a representative image of real-time temperature monitoring. It can be seen that the microspheres with surface modification of polydopamine have photothermal conversion ability, and the photothermal conversion ability is not affected after loading drugs.

[0080] like Figure 2 As shown in (b, c), the temperature of PVA increased slightly at 27.8℃ and 27.7℃ respectively under near-infrared light irradiation. In contrast, the temperature of the other groups of samples continued to increase. 2 When the temperature of PMS, PTMS, PIMS and PTIMS increases to 40, 40, 41.1 and 40.2℃, respectively, the temperature of 0.8W / cm2 The temperatures rose to 45.7, 47.4, 45.7 and 46.4℃ respectively.

[0081] In order to achieve effective tumor inhibition through PTT (42-48°C), 0.8 W / cm 2 The subsequent experiments were carried out with the near-infrared power. After 5 consecutive cycles of irradiation ( Figure 2 d), PTIMS has remarkable photothermal stability, which can sustain a temperature rise of up to 46.1°C.

[0082] pass Figure 2 (e, f) Evaluation of drug release behavior of PTIMS. On day 28, the cumulative release of IDA was 232.5±20.0μg / mL (PIMS) and 205±13.1μg / mL (PTIMS), and the cumulative release of TQR was 298.3±19.6μg / mL (PIMS) and 285.8±22.1μg / mL (PTIMS), indicating that PTIMS has stable drug release capacity and meets the pharmacological requirements of clinical treatment. 2 The release of IDA and TQR was detected under intermittent near-infrared to evaluate the photothermal responsive drug release ability of PTIMS. The cumulative release amounts of IDA and TQR increased to 255.8±19.9μg / mL and 329.6±20.0μg / mL under near-infrared, respectively, indicating that the photothermal conversion of PDA promoted the release of drugs.

[0083] 3. In vitro anti-tumor effect test

[0084] The PTIMS obtained in Example 1, the PTMS obtained in Comparative Example 1, and the PIMS obtained in Comparative Example 2 were used as samples. Each sample was immersed in PBS buffer for 24 hours, and the extracts (supernatants) of each sample were collected after centrifugation. H22 cells were treated with the extracts of each sample and divided into the following five groups according to different treatment methods: PTIMS group, PTMS group, PIMS group, BC group (PBS buffer), and PTIMS+NIR group (PTIMS+NIR irradiation) for in vitro anti-tumor effect testing. The test results are shown in FIG. Figure 3 、 Figure 4 shown.

[0085] The specific test methods are as follows:

[0086] 3.1CCK-8 detection and live and dead cell staining

[0087] The effects of different groups on H22 cell activity were evaluated by CCK-8 assay and live / dead cell staining.

[0088] 3.2 Cell apoptosis detection

[0089] The digested H22 cells (1×10 5 / mL) were inoculated into 6-well plates and cultured at 37°C in 5% CO2. The cells were then divided into groups and treated. After treatment, they were incubated for 24 h, washed with PBS buffer, digested into single-cell suspensions, stained with Annexin V-AbFluorTM488 and PI, and detected by flow cytometry (NOVOCYte3130, ACEA, USA).

[0090] 3.3 Scratch test

[0091] The digested H22 cells (1×10 5 100 μg / mL) were inoculated into 6-well plates and cultured overnight at 37°C, 5% CO₂ (cell confluence reached over 90%). Wounds were scratched using a 10 μL sterile pipette tip and rinsed with PBS buffer to remove free cells. The cells were then grouped and treated. After treatment, images were taken under an optical microscope at 0 and 24 hours. Wound healing rate = (W1 - W2) / W0 × 100%, where W1 is the scratch area at 0 hours, W2 is the scratch area at 24 hours, and W0 is the scratch area at 0 hours.

[0092] 3.4 Transwell assay

[0093] H22 cells (4×10 4 After treatment, the cells were seeded in the upper chamber and incubated at 37°C in 5% CO2 for 48 h. Non-migrated cells remaining on the upper surface of the filter membrane were removed with a cotton swab, and the migrated cells on the lower surface of the filter membrane were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet solution for 30 min. The cells were then photographed under an optical microscope.

[0094] 3.5 Immunofluorescence staining

[0095] H22 cells were incubated on coverslips and then grouped and treated. After treatment, they were fixed with 4% paraformaldehyde and treated with 0.1% Triton-100. The expression of Ki67 and P-gp was analyzed. The cells were then treated with goat serum for 30 minutes at 25°C and incubated with anti-Ki67 and P-gp primary antibodies and FITC- and Cy3-labeled secondary antibodies. Cell nuclei were stained with DAPI. IDA uptake was detected using a laser confocal microscope (CLSM, Zeiss LSM 800, Germany) at Ex. 534 nm / Em. 572 nm.

[0096] like Figure 3 As shown in a, PTIMS promotes tumor cell apoptosis and inhibits tumor cell migration and invasion by increasing temperature and photothermal responsive drug release.

[0097] The results of live-dead cell staining assay ( Figure 3 b, c) show that there were no red dead cells in the BC and PTMS groups, with the percentages of live cells being 99.61±0.55% and 99.80±0.28%, respectively. In contrast, red dead cells appeared in the PIMS, PTIMS, and PTIMS+NIR groups, with percentages of 82.80±6.26%, 67.89±5.74%, and 42.34±3.29%, respectively, indicating that TQR enhanced the apoptotic effect of IDA and further amplified it under NIR, while TQR itself had no apoptotic effect.

[0098] CCK-8 assay ( Figure 3 d) shows that compared with the BC and PTMS groups, the PIMS, PTMS, and PTIMS+NIR groups exhibited significant cytotoxicity at day 2. Meanwhile, cell viability decreased significantly in a time-dependent manner, reaching 29.06±4.85% (PIMS group), 14.83±2.53% (PTIMS group), and 5.13±0.94% (PIMS+NIR group) at day 3, respectively.

[0099] Scratch test Figure 3 As shown in (e, g), the migration area decreased from 78.14±3.70% (BC group) and 78.27±4.32% (PTMS group) to 62.88±2.46% (PIMS group), 22.73±3.27% (PTIMS group) and 10.76±3.41% (PTIMS group). Figure 3 As shown in (f,h), the expression of IDA in H22 cells increased from 392.00±22.38 in the BC group and 388.33±21.85 in the PTMS group to 247.67±24.14 in the PIMS group, 149.67±26.89 in the PTIMS group, and 19.33±4.64 in the PTIMS+NIR group. These results indicate that TQR does not inhibit H22 cell migration and invasion. TQR enhances the inhibitory effect of IDA on H22 cell migration and invasion, which is further enhanced under PTT.

[0100] Ki67 immunofluorescence staining was used to detect the anti-proliferative ability of cells. Figure 4 As shown in (a, b), the MFI of Ki67 decreased from 100.00±7.17% in the BC group and 99.08±5.78% in the PTMS group to 76.06±6.29% in the PIMS group, 52.55±6.07% in the PTIMS group, and 28.63±4.17% in the PTIMS+NIR group. Figure 4 As shown in (c, d), it is consistent with the results of Ki67 immunofluorescence staining, further confirming the above conclusion.

[0101] Immunofluorescence staining of P-gp and IDA Figure 4 As shown in (e, g), the MFI of P-gp was 100.00±4.40% in BC, 21.92±2.89% in the PTMS group, 54.33±3.89% in the PIMS group, 15.88±2.38% in the PTIMS group, and 5.06±1.46% in the PTIMS+NIR group. This indicates that TQR treatment reduced P-gp expression, with a significant decrease in the PTIMS+NIR group. The MFI of IDA was 37.23±3.89% in the PIMS group, 67.29±7.48% in the PTIMS group, and 100±7.50% in the PTIMS+NIR group, indicating that TQR treatment significantly enhanced cellular uptake of IDA, which was further enhanced by PTT. Therefore, TQR treatment reduced P-gp expression, inhibited the active efflux of IDA from tumor cells, and increased intracellular IDA concentration. Moreover, this effect was further enhanced under the action of PTT, which was related to the PTT-induced sensitivity of tumor cells to chemotherapeutic drugs.

[0102] 4. Effect Testing on H22 Subcutaneous Transplanted Tumors in Mice

[0103] Female Balb / C mice (6 weeks old) were purchased from the Experimental Animal Center of Hualan Biological Co., Ltd. and raised by the Animal Experiment Center of Henan Province (SPF grade). The PTIMS obtained in Example 1, the PTMS obtained in Comparative Example 1, and the PIMS obtained in Comparative Example 2 were used as samples to treat mice. The mice were divided into 5 groups according to different treatment methods: BC group (injected with equal volume of PBS buffer), PTMS group, PIMS group, PTIMS group, and PTIMS+NIR group (after intratumoral injection of PTIMS, 0.8W / cm2 NIR irradiation for 5 minutes every 2 days) to test the effect on H22 subcutaneous transplanted tumors in mice. The test results are shown in Figure 2. Figures 5 to 8 shown.

[0104] The specific test methods are as follows:

[0105] 4.1 Construction of H22 subcutaneous transplanted tumor model in Balb / C mice

[0106] 150 μL (containing 1×10 7 Resuspended H22 cells in PBS buffer were injected into the right anterior dorsal flank of Balb / C mice. When the tumor volume reached 100 mm 3 At 0 days, 0.2 g of each sample was injected into the tumor of each group of mice, and the data were recorded as day 0. Tumor volume was measured every 2 days. All implantation procedures were approved by the Animal Care Committee of the First Affiliated Hospital of Zhengzhou University (ZZU-LA20210423

[02] ) and followed the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

[0107] 4.2 In vivo photothermal performance

[0108] According to the results of in vitro photothermal performance test, 0.8W / cm 2 In vivo evaluation was performed using near-infrared light. During the first NIR treatment after sample injection, mice in the BC and PTIMS+NIR groups received NIR irradiation to evaluate the photothermal properties of PTIMS+NIR. Temperature change data were recorded using the same method as previously described.

[0109] 4.3 Histological and biochemical analysis

[0110] On day 14, mice were sacrificed, and tumor tissues were collected, fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. HE staining, immunofluorescence staining (for P-gp), and immunohistochemical staining (for Ki67 and TNF-α) were performed according to standard procedures. Histological changes were observed under a microscope. Hearts, livers, spleens, lungs, and kidneys were collected from each group for HE staining, and serum samples were collected for analysis of ALT, AST, ALP, BUN, CREA, and UA.

[0111] exist Figure 5 In (a, b), the local temperature of the PTIMS group rapidly increased to 46.1°C, demonstrating that PTIMS has good in vivo photothermal conversion performance.

[0112] exist Figure 5 d and Figure 6 Compared with the BC group (1079.18±50.44mm 3 ) and PTMS group (968.49±53.27mm 3 ) compared with the PIMS group (675.21±50.20mm 3 ), PTIMS group (321.43±58.09mm 3 ) and PTIMS+NIR group (110.08±49.33mm 3 ) were significantly reduced, and the tumor volume growth of the PTIMS+NIR group was most significantly inhibited.

[0113] The histological evaluation of the ex vivo tumor tissues was performed by comparing the HE staining results of the BC group and the PTMS group ( Figure 5 g), it can be observed that the incidence of nuclear lysis and nuclear pyknosis is higher in the PIMS group, PTIMS group, and PTIMS+NIR group, and the PTIMS+NIR group exhibits a greater degree of cell necrosis.

[0114] like Figure 5As shown in (c, e, h), the TNF-α positive staining area was 6.42±0.77% in the BC group, 9.39±0.94% in the PTMS group, 18.60±1.32% in the PIMS group, 32.81±3.09% in the PTIMS group, and 46.00±2.28% in the PTIMS+NIR group; the number of Ki67+ cells was 4437±313 in the BC group, 3901±383 in the PTMS group, 2596±101 in the PIMS group, 1793±90% in the PTIMS group, and 921±106 in the PTIMS+NIR group.

[0115] P-gp immunofluorescence staining ( Figure 5 f, i) show that P-gp expression levels decreased from 100±4.52% in the BC group and 82.24±4.97% in the PIMS group to 20.03±1.84% in the PTMS group, 20.40±3.62% in the PTIMS group, and 6.90±1.49% in the PTIMS+NIR group. These results indicate that PTIMS+NIR significantly inhibited tumor growth, which was associated with TQR inhibiting P-gp and NIR enhancing tumor eradication and chemotherapy sensitivity.

[0116] Organ tissue samples (heart, liver, spleen, lung and kidney) and fresh plasma were collected from each group for histological and biochemical analysis. HE staining ( Figure 7 ) The results showed that compared with the other experimental groups, the BC group had no significant changes in multiple organs, and the biochemical indicators ALT, AST, ALP, BUN, CREA and UA were also within the standard range ( Figure 8 ).

[0117] 5. Test of the inhibitory effect on VX2 liver transplanted tumor in New Zealand rabbits

[0118] Twenty New Zealand male rabbits were purchased from Hualan Biological Products Co., Ltd. The PTIMS obtained in Example 1, the PTMS obtained in Comparative Example 1, and the PIMS obtained in Comparative Example 2 were used as samples to treat the rabbits. The rabbits were divided into four groups according to the different treatment methods: BC group (injected with equal volume of PBS buffer), PTMS group, PIMS group, and PTIMS group. The inhibitory effect on VX2 liver transplanted tumors in New Zealand rabbits was tested. The test results are shown in Figure 2. Figures 9 to 12 shown.

[0119] The specific test methods are as follows:

[0120] 5.1 Establishment of a Rabbit VX2 Liver Transplantation Tumor Model

[0121] A rabbit VX2 liver transplant tumor model was constructed, and all procedures were approved by the Animal Care Committee of the First Affiliated Hospital of Zhengzhou University (ZZU-LA20210423

[02] ) and followed the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

[0122] 5.2 Hemolysis Assessment

[0123] The hemolytic potential of each group of microspheres was evaluated, including normal saline (BC) group, pure water (PC) group, PVA group, PTMS group, PIMS group and PTIMS group.

[0124] 5.3TACE

[0125] Two weeks after the VX2 tumor mass was implanted into the liver of the experimental rabbit, enhanced CT was used to evaluate whether the tumor tissue growth met the tumor formation criteria (marked as 0d), including the location and size of the tumor. When the tumor met the specified criteria, transcatheter arterial chemoembolization was performed under digital subtraction angiography (DSA). A 2 cm incision was made in the right groin of the experimental rabbit to expose the femoral artery. A 5f catheter was inserted into the femoral artery, followed by a 2.7f microcatheter and a 0.021-inch guidewire. According to the grouping, the microcatheter was moved to the tumor-feeding artery and tumor embolization was performed. After treatment, angiography was repeated to confirm that the tumor-feeding artery was completely embolized. The microcatheter, guidewire and catheter were then removed, and the femoral artery was sutured. All animals were injected with antibiotics for 3 consecutive days after surgery. The PTIMS group underwent enhanced CT scans 7 and 14 days after TACE to evaluate the embolization effect.

[0126] 5.4 Histological and biochemical analysis

[0127] Fourteen days after TACE, rabbits were sacrificed, and tumor tissues were collected, fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. Hematoxylin and eosin staining, immunofluorescence staining (for P-gp and CD8), and immunohistochemistry (for Ki67 and Cas-3) were performed according to standard procedures. Histological changes were observed microscopically. In addition, hearts, spleens, lungs, and kidneys were collected from each group for hematoxylin and eosin staining, and serum samples were collected for analysis of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN), and creatinine (CRE).

[0128] The microspheres should be introduced into the hepatic artery through a microcatheter and in direct contact with the blood. Caution is required to prevent unnecessary iatrogenic hemolysis. The results of the hemolysis assay are shown in Figure 9 c. The hemolysis rates (HR) of PIMS, PTMS, and PTIMS were all below 5% (P < 0.05), comparable to those of commercially available PVA microspheres, with no significant hemolysis.

[0129] like Figure 9As shown in a, enhanced CT at 0 days showed significant enhancement in the arterial phase around the tumor, and low-density liquefied necrosis areas were visible inside the tumor, confirming the successful establishment of the New Zealand rabbit VX2 liver transplant tumor. TACE treatment of the tumor was performed under DSA guidance ( Figure 9 b) Preoperative angiography showed dense blood vessels around the VX2 tumor in the left lobe of the liver, and postoperative angiography showed complete embolization of the blood vessels around the tumor. Figure 9 a) shows that a low-density area appeared in the embolized area at 7 days and an enlarged area appeared at 14 days, indicating successful tumor eradication.

[0130] The embolization effect of PTIMS was further evaluated by histological analysis. Figure 10 and Figure 9 As shown in (d), no microspheres were observed in the BC group, while brown-black PDA-coated microspheres were observed in the PTMS, PIMS, and PTIMS groups, indicating that vascular embolization of the VX2 liver transplanted tumor was successful after TACE.

[0131] Immunohistochemical staining ( Figure 9 e, g, h) The results showed that the number of Ki67+ cells in the BC group, PTMS group, PIMS group, and PTIMS group were 312±26, 297±22, 220±10, and 117±7 per mm, respectively. 2 , the Cas-3 positive areas were 15.39±0.80%, 18.58±2.18%, 34.80±1.71%, and 57.91±1.28% in the BC group, PTMS group, PIMS group, and PTIMS group, respectively.

[0132] Immunofluorescence staining was used to evaluate the expression of P-gp and immune microenvironment in tumor tissues. Figure 9 As shown in (f, i, j), the relative MFIs of P-gp in the BC, PTMS, PIMS, and PTIMS groups were 100.00±6.37%, 1.87±0.37%, 84.13±5.77%, and 1.16±0.45%; the relative MFIs of CD8 in the BC, PTMS, PIMS, and PTIMS groups were 100.00±26.98%, 213.96±31.09%, 335.09±38.97%, and 505.72±36.15%, respectively. These results suggest that TQR can inhibit P-gp expression, thereby reducing IDA efflux and increasing IDA accumulation within tumor cells, enhancing the antiproliferative and pro-apoptotic effects of IDA. Furthermore, alterations in the immune microenvironment at the embolization site after TACE and the promotion of immunogenic cell death by IDA improve the tumor immune microenvironment.

[0133] Considering the importance of biosafety as a key parameter in the development of new medical devices, pathological changes and biochemical indicators (ALT, AST, BUN and CREA) of the heart, spleen, lung and kidney were evaluated. Figure 11 and Figure 12 As shown, no obvious pathological changes were found in each group by HE staining, and the biochemical indicators of each group were within the normal range on day 0 and day 14.

[0134] In summary, the dual-effect microspheres loaded with IDA and TQR prepared in this study are used for TACE combined with non-invasive photothermal ablation therapy. PTIMS as a drug carrier has temperature-responsive drug release and enhances the tumor immune microenvironment, which can effectively overcome the MDR of the tumor, showing superior therapeutic effects and has great potential for clinical application.

[0135] The above embodiments are specific implementation methods of the present invention, but the implementation methods of the present invention are not limited to the above embodiments. Any other combination, change, modification, substitution, and simplification that does not exceed the design concept of the present invention shall fall within the scope of protection of the present invention.

Claims

1. A dual-functional microsphere loaded with idarubicin and tariquidar, characterized in that: Poly(vinyl alcohol) microspheres modified with polydopamine were used as carriers, and idarubicin and talizine were loaded on the surface.

2. The method for preparing the dual-functional microspheres loaded with idarubicin and talizine as claimed in claim 1, characterized in that: The following steps are involved: (1) A dopamine solution was prepared using Tris-HCl buffer, polyvinyl alcohol microspheres were added to the dopamine solution, stirred at room temperature for 24 to 48 hours, washed, allowed to stand, and the precipitate was collected to obtain polydopamine-modified polyvinyl alcohol microspheres; (2) Add tariquidar and idarubicin into PBS buffer to obtain a tariquidar-idarubicin mixed solution, add the polydopamine-modified polyvinyl alcohol microspheres obtained in step (1) into the tariquidar-idarubicin mixed solution, incubate and centrifuge to obtain dual-functional microspheres loaded with idarubicin and tariquidar.

3. The method for preparing the dual-functional microspheres loaded with idarubicin and talizine as claimed in claim 2, characterized in that: In step (1), the concentration of the dopamine solution is 0.1-0.5 wt %.

4. The method for preparing the dual-functional microspheres loaded with idarubicin and talizine as claimed in claim 3, characterized in that: In step (1), 1 to 3 g of polyvinyl alcohol microspheres are added to every 30 to 50 mL of dopamine solution.

5. The method for preparing the dual-functional microspheres loaded with idarubicin and talizine as claimed in claim 2, characterized in that: In the tariquida-idarubicin mixed solution of step (2), the concentration of tariquida is 1-5 mg / mL, and the concentration of idarubicin is 1-5 mg / mL.

6. The method for preparing the dual-functional microspheres loaded with idarubicin and talizine as claimed in claim 5, characterized in that: In step (2), 0.5 g of polydopamine-modified polyvinyl alcohol microspheres are added to every 5 to 10 mL of the talizine-idarubicin mixed solution.

7. The method for preparing dual-functional microspheres loaded with idarubicin and talizine as claimed in claim 4, characterized in that: The pH of the Tris-HCl buffer in step (1) is 6.8 to 8.

8.

8. The method for preparing dual-functional microspheres loaded with idarubicin and tariquidar according to claim 6, characterized in that: The incubation time in step (2) is 12 to 24 hours.

9. The method for preparing dual-functional microspheres loaded with idarubicin and talizquidar according to claim 8, characterized in that: The centrifugal speed in step (2) is 2000-3000 rpm, and the time is 3-5 min.

10. Use of the dual-functional microspheres loaded with idarubicin and tariquidar according to claim 1 or the dual-functional microspheres loaded with idarubicin and tariquidar prepared by the preparation method according to any one of claims 2 to 9 in the combined transcatheter arterial chemoembolization and photothermal ablation treatment of liver cancer.

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