Microwave ablation instrument and preparation method thereof

Abstract

The invention relates to the technical field of medical instruments, in particular to a microwave ablation instrument and a preparation method thereof. The microwave ablation instrument provided by the invention comprises a microwave needle body, a sodium alginate-calcium ion cross-linked network loaded on the surface of the microwave needle body and drug-loaded calcium carbonate adsorbed by the sodium alginate-calcium ion cross-linked network, the drug-loaded calcium carbonate comprises mesoporous CaCO3 and a demethylation drug loaded in the mesoporous CaCO3 and / or on the surface of the mesoporous CaCO3. The microwave ablation apparatus provided by the invention can reduce the risk of needle passage metastasis, significantly improve the local drug concentration of the tumor, respond to the tumor microenvironment, and improve the tumor treatment effect. Through targeted delivery, multi-mechanism cooperation and immune microenvironment regulation and control, a multi-dimensional treatment breakthrough from local ablation to system immune activation is realized, and the preparation is remarkably superior to the prior art in the aspects of curative effect accuracy, safety and long-acting recurrence / metastasis resistance.

Description

Technical Field

[0001] This invention relates to the field of medical device technology, and in particular to a microwave ablation device and its preparation method. Background Technology

[0002] Microwave ablation (MWA) is an interventional technique that uses the heat generated by the intense oscillation of polar molecules under microwave exposure to locally ablate tumors. Due to its minimally invasive nature, short-term clinical efficacy, and highly effective cell-killing ability, MWA is now widely used to treat several types of solid tumors, including hepatocellular carcinoma, lung cancer, and liver / lung metastases from colorectal cancer.

[0003] However, given the prevalence of tumors, microwave ablation technology still presents challenges in treating small or occult tumors. These tumors may be difficult to ablate effectively due to difficulties in imaging localization and the complexity of microwave needle puncture. Furthermore, even for larger tumors, incomplete treatment may occur if the margins are not clearly defined during ablation, leaving tumor residues and increasing the risk of disease recurrence and metastasis.

[0004] To overcome these limitations, some studies have proposed using adjuvant therapies to eliminate residual tumors and inhibit metastasis and recurrence, such as chemotherapy combined with microwave ablation, or immunotherapy combined with microwave ablation. Currently, microwave ablation combined with other treatments involves intravenous or intratumoral injection of relevant anti-tumor drugs, followed by microwave ablation. Both methods inevitably involve multiple invasive drug administrations, which can increase the risk of needle tract metastasis or reduce the amount of drug reaching the tumor. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a microwave ablation device and its preparation method. The microwave ablation device provided by the present invention can reduce the risk of needle tract metastasis, significantly increase the local drug concentration in the tumor, and respond to the tumor microenvironment to improve the tumor treatment effect.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] The present invention provides a microwave ablation device, comprising a microwave needle body, a sodium alginate-calcium ion crosslinking network loaded on the surface of the microwave needle body, and drug-loaded calcium carbonate adsorbed by the sodium alginate-calcium ion crosslinking network; the drug-loaded calcium carbonate comprises mesoporous CaCO3 and demethylated drugs loaded inside and / or on the surface of the mesoporous CaCO3.

[0008] Preferably, the demethylating agent includes one or more of zablaline, decitabine, azacitidine, and vorinostat.

[0009] Preferably, the particle size of the drug-loaded calcium carbonate is less than 200 nm.

[0010] This invention provides a method for preparing the microwave ablation device described above, comprising the following steps: mixing mesoporous CaCO3, demethylated drug and polar organic solvent to obtain a drug-loaded calcium carbonate dispersion;

[0011] The microwave needle body was modified by immersing it in an aqueous solution of sodium alginate to obtain a sodium alginate-modified microwave needle; the sodium alginate-modified microwave needle was then immersed in an aqueous solution of a soluble calcium salt for Ca2+ modification. 2+ Coordination and cross-linking are performed to form a sodium alginate-calcium ion cross-linking network, resulting in a microwave needle loaded with the sodium alginate-calcium ion cross-linking network.

[0012] The microwave needle loaded with sodium alginate-calcium ion crosslinking network is immersed in the drug-loaded calcium carbonate dispersion to obtain the microwave ablation device.

[0013] Preferably, the method for preparing the mesoporous CaCO3 includes: dissolving calcium chloride in ethanol to obtain a calcium chloride solution; introducing CO2 into the calcium chloride solution to react with the calcium chloride to generate mesoporous CaCO3; and obtaining mesoporous CaCO3 after solid-liquid separation.

[0014] Preferably, the soluble calcium salt includes calcium chloride; the concentration of the aqueous solution of the soluble calcium salt is 0.5-1.5 mol / L.

[0015] Preferably, the polar organic solvent includes ethanol and / or dimethyl sulfoxide.

[0016] Preferably, the concentration of the sodium alginate aqueous solution is 1-5 mg / mL.

[0017] Preferably, the modification time is 0.5-2 hours.

[0018] Preferably, before immersing the microwave needle body in an aqueous sodium alginate solution for modification, the microwave needle body is further subjected to sanding.

[0019] The present invention provides a microwave ablation device, comprising a microwave needle body, a sodium alginate-calcium ion crosslinking network loaded on the surface of the microwave needle body, and drug-loaded calcium carbonate adsorbed by the sodium alginate-calcium ion crosslinking network; the drug-loaded calcium carbonate comprises mesoporous CaCO3 and demethylated drugs loaded inside and / or on the surface of the mesoporous CaCO3.

[0020] This invention loads demethylated drugs onto a microwave needle, achieving simultaneous and targeted release during ablation, significantly increasing local drug concentration in the tumor and avoiding the systemic diffusion problems associated with traditional intravenous / intratumoral injections. The integrated design reduces the need for multiple invasive procedures, eliminating the risk of needle tract metastasis associated with traditional multiple punctures. Furthermore, the microwave ablation device of this invention is loaded with mesoporous CaCO3, which adsorbs the demethylated drugs. The slightly acidic environment of the tumor (pH 6.0-6.8) triggers the decomposition of CaCO3, releasing the demethylated drugs and CaCO3. 2+ Simultaneously, CO2 is generated, in which Ca... 2+ It can deposit and induce calcium death in tumor cells, and the released demethylating drugs enhance immunotherapy, exhibiting multiple cell killing mechanisms. Meanwhile, CO2 bubbles can enhance ultrasound imaging contrast, and the high-dose cavitation effect enhances microwave energy deposition efficiency, thereby improving the therapeutic effect. Since the loaded demethylating drugs are released through tumor microenvironment response (pH-triggered CaCO3 decomposition), it can achieve precise spatiotemporal control and reduce off-target toxicity.

[0021] In addition, CaCO3 has high biocompatibility, and its degradation products (CaCO3, Ca ... 2+ CO2 can participate in physiological metabolism.

[0022] This invention integrates imaging monitoring, local ablation, drug / gas release, and immune modulation into a single operation. Through targeted delivery, multi-mechanism synergy, and immune microenvironment regulation, it achieves a multi-dimensional therapeutic breakthrough from local ablation to systemic immune activation. It is significantly superior to existing technologies in terms of efficacy precision, safety, and long-term anti-recurrence / metastasis effects. Attached Figure Description

[0023] Figure 1 Here are SEM (a) and TEM (b) images of calcium carbonate;

[0024] Figure 2 TEM and mapping images of calcium carbonate;

[0025] Figure 3 Particle size distribution (a) and zeta potential (b) of calcium carbonate;

[0026] Figure 4 The isothermal adsorption curve of calcium carbonate is shown.

[0027] Figure 5 This is a line graph showing the particle size variation of calcium carbonate over time under different pH conditions.

[0028] Figure 6 Particle size distribution diagrams for calcium carbonate and Zeb-CaCO3;

[0029] Figure 7TEM (a) and SEM images (b) of Zeb-CaCO3;

[0030] Figure 8 TEM and mapping images of Zeb-CaCO3;

[0031] Figure 9 The graph shows the release of Zeb from Zeb-CaCO3 at different pH (a) and different temperatures (b);

[0032] Figure 10 SEM images of WMA-needle@Alg / Zeb-CaCO3 at different magnifications;

[0033] Figure 11 TEM image of the material exfoliated from the surface of WMA-needle@Alg / Zeb-CaCO3;

[0034] Figure 12 Particle size distribution (a) and zeta potential (b) of the material exfoliated from the WMA-needle@Alg / Zeb-CaCO3 surface;

[0035] Figure 13 The graph shows the drug release at different pH and temperature conditions after WMA-needle@Alg / Zeb-CaCO3 was reused 1-3 times.

[0036] Figure 14 The graph shows the results of measuring the calcium carbonate content loaded on a microwave ablation device that can be reused 0-3 times.

[0037] Figure 15 Figure showing the results of the heating cycle test for reusing microwave ablation devices 0-3 times;

[0038] Figure 16 Figure 1: Results of CCK-8 cytotoxicity assay for different treatment groups;

[0039] Figure 17 Image showing cell staining results for different treatment groups;

[0040] Figure 18 Intracellular Ca in different treatment groups 2+ Content detection chart;

[0041] Figure 19 Graphs showing intracellular pH measurements in different treatment groups;

[0042] Figure 20 Figure 1 shows the results of cell marker detection in different treatment groups;

[0043] Figure 21The images show the results of animal treatment experiments. In the images, a is a photograph of the changes in tumor volume in each group of mice after treatment, b is a comparison of tumor volume in each group, and c is a comparison of tumor weight in each layer.

[0044] Figure 22 The results are shown in the flow cytometry analysis. Among them, a represents the analysis results of Treg cells in different groups, b represents the analysis results of CD86 expression in different groups, and c represents the analysis results of CD206 expression in different groups. Detailed Implementation

[0045] The present invention provides a microwave ablation device, comprising a microwave needle body, a sodium alginate-calcium ion crosslinking network loaded on the surface of the microwave needle body, and drug-loaded calcium carbonate adsorbed by the sodium alginate-calcium ion crosslinking network; the drug-loaded calcium carbonate comprises mesoporous CaCO3 and demethylated drugs loaded inside and / or on the surface of the mesoporous CaCO3.

[0046] The microwave ablation device provided by this invention includes a microwave needle body. In this invention, the microwave needle body is preferably made of medical-grade stainless steel or nickel-titanium alloy. The function of the microwave needle body is to provide microwave energy conduction and mechanical support. The microwave needle body generates high temperatures through high-frequency electromagnetic waves (microwaves), directly destroying diseased tissue. Its core principle is based on thermal effects and electromagnetic field effects; the microwave antenna focuses electromagnetic energy onto the corresponding tissue, causing the local temperature to rapidly rise to 60-100℃, leading to cell protein denaturation, membrane structure destruction, and irreversible coagulative necrosis.

[0047] The microwave ablation device provided by this invention includes a sodium alginate-calcium ion crosslinking network loaded on the surface of the microwave needle. In this invention, the sodium alginate-calcium ion crosslinking network serves to adsorb and fix drug-loaded calcium carbonate onto the surface of the microwave needle.

[0048] The microwave ablation device provided by this invention comprises drug-loaded calcium carbonate adsorbed by the sodium alginate-calcium ion crosslinking network; the drug-loaded calcium carbonate comprises mesoporous CaCO3 and a demethylating drug loaded inside and / or on the surface of the mesoporous CaCO3. In this invention, the particle size of the drug-loaded calcium carbonate is preferably less than 200 nm, more preferably 50-150 nm. This invention does not impose a special limitation on the loading amount of the demethylating drug, which can be adjusted by those skilled in the art according to actual needs.

[0049] In this invention, the demethylating agent preferably includes one or more of zablaline, decitabine, azacitidine, and vorinostat. The demethylating agent can inhibit tumor methylation, enhance antigen exposure, and thus improve the efficacy of immunotherapy.

[0050] In this invention, the mesoporous CaCO3 is able to respond to the tumor microacidic environment, and upon being triggered to decompose, it releases demethylating drugs and Ca. 2+ Simultaneously, CO2 is generated, in which Ca... 2+ The device can induce calcium death in tumor cells through deposition, and the released demethylating drugs enhance immunotherapy, exhibiting multiple cell-killing mechanisms. CO2 bubbles can enhance ultrasound imaging contrast, while high-dose cavitation enhances microwave energy deposition efficiency, improving therapeutic efficacy. The microwave ablation device provided by this invention, through thermal triggering, allows microwave heating to promote vascular normalization and T-cell infiltration, which is beneficial for improving tumor treatment outcomes.

[0051] This invention provides a method for preparing the microwave ablation device described above, comprising the following steps: mixing mesoporous CaCO3, demethylated drug and polar organic solvent to obtain a drug-loaded calcium carbonate dispersion;

[0052] The microwave needle body was modified by immersing it in an aqueous solution of sodium alginate to obtain a sodium alginate-modified microwave needle; the sodium alginate-modified microwave needle was then immersed in an aqueous solution of a soluble calcium salt for Ca2+ modification. 2+ Coordination and cross-linking are performed to form a sodium alginate-calcium ion cross-linking network, resulting in a microwave needle loaded with the sodium alginate-calcium ion cross-linking network.

[0053] The microwave needle loaded with sodium alginate-calcium ion crosslinking network is immersed in the drug-loaded calcium carbonate dispersion to obtain the microwave ablation device.

[0054] Unless otherwise specified, all raw materials used in this invention are commercially available products well known in the art.

[0055] In this invention, the mesoporous CaCO3 is preferably obtained by self-preparation; the method for preparing the mesoporous calcium carbonate preferably includes the following steps: dissolving calcium chloride in ethanol to obtain a calcium chloride solution; introducing CO2 into the calcium chloride solution to react with the calcium chloride to generate mesoporous CaCO3, and obtaining mesoporous CaCO3 after solid-liquid separation.

[0056] In this invention, the calcium chloride is preferably anhydrous calcium chloride; the ethanol is preferably anhydrous ethanol. This invention does not impose any particular limitation on the concentration of the calcium chloride solution. In an embodiment of this invention, the concentration of CaCl2·2H2O in the calcium chloride solution is 1.5 mg / mL.

[0057] In this invention, the CO2 is preferably obtained in-house. Preferably, the calcium chloride solution and ammonium bicarbonate are heated separately in the same sealed vacuum environment. The CO2 generated by the thermal decomposition of the ammonium bicarbonate enters the calcium chloride solution and reacts with the calcium chloride to form mesoporous CaCO3. This invention does not specify a particular amount of ammonium bicarbonate; the generated CO2 only needs to react with CaCl2 to form calcium carbonate. In an embodiment of this invention, for 150 mg of CaCl2·2H2O, the amount of ammonium bicarbonate is 5 g.

[0058] In this invention, the heating temperature is preferably 30-80℃, and the reaction time is preferably 12-24h.

[0059] This invention does not have special requirements for the solid-liquid separation method; any solid-liquid separation method well-known in the art can be used, such as centrifugation. In the embodiments of this invention, the centrifugation speed is 8000 rpm and the time is 10 min. After the solid-liquid separation is completed, this invention preferably washes the solid obtained from the solid-liquid separation with anhydrous ethanol 2-3 times. The above method ensures that the obtained CaCO3 is mesoporous CaCO3.

[0060] After obtaining mesoporous CaCO3, the present invention mixes the mesoporous CaCO3, the demethylated drug, and the polar organic solvent to obtain a drug-loaded calcium carbonate dispersion.

[0061] In this invention, the demethylating agent preferably includes zablaline; the polar organic solvent preferably includes ethanol and / or dimethyl sulfoxide (DMSO), wherein the ethanol is preferably anhydrous ethanol.

[0062] The present invention does not impose a specific limitation on the amount of the demethylating agent, which can be adjusted according to the required loading. In the embodiments of the present invention, the mass ratio of the mesoporous calcium carbonate to the demethylating agent is 1:1.

[0063] In this invention, the mixing preferably includes: dispersing mesoporous calcium carbonate in an organic solvent, then adding a demethylating agent and stirring for 10-20 hours. In this invention, the stirring rate is preferably 300 rpm, and the stirring time can be 10 hours, 12 hours, 16 hours, or 20 hours.

[0064] In the mixing process of this invention, the demethylated drug is adsorbed by mesoporous CaCO3 and enters the interior of the mesoporous calcium carbonate or is adsorbed onto the surface of the mesoporous calcium carbonate to form drug-loaded calcium carbonate.

[0065] The present invention modifies the microwave needle by immersing the needle body in an aqueous solution of sodium alginate to obtain a sodium alginate-modified microwave needle.

[0066] Prior to the modification, the microwave needle body is preferably first sanded. In this invention, the sanding is preferably performed sequentially with 200-grit and 1000-grit sandpaper, each time for 2-5 times. This invention first uses 200-grit sandpaper for coarse sanding to quickly remove the oxide layer, contaminants, and processing residues from the surface of the microwave needle body, exposing the unoxidized metal substrate; then, 1000-grit sandpaper is used for fine sanding to refine the surface roughness, reduce scratch depth, and further clean the surface. By sanding, this invention imparts an appropriate rough surface to the microwave needle body, which can promote cell adhesion and subsequent modification with sodium alginate.

[0067] In this invention, the concentration of the sodium alginate aqueous solution is preferably 1-5 mg / mL, and in specific embodiments it can be 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, or 5 mg / mL. In this invention, the modification time is preferably 0.5-2 h, and in specific embodiments it can be 0.5 h, 1 h, 1.5 h, or 2 h. This invention physically attaches sodium alginate to the microwave needle body by immersing it in the sodium alginate aqueous solution. After the immersion is complete, the microwave needle body is removed, washed with water, and a sodium alginate-modified microwave needle is obtained.

[0068] After obtaining the sodium alginate-modified microwave needle, the present invention immerses the sodium alginate-modified microwave needle in an aqueous solution of soluble calcium salt for Ca2+ treatment. 2+ Coordination and cross-linking are performed to form a sodium alginate-calcium ion cross-linking network, resulting in a microwave needle loaded with the sodium alginate-calcium ion cross-linking network.

[0069] In this invention, the soluble calcium salt preferably includes calcium chloride; the concentration of the aqueous solution of the soluble calcium salt is preferably 0.5-1.5 mol / L, and in specific embodiments it can be 0.5, 1, or 1.5 mol / L. In this invention, the soaking time in the aqueous solution of the soluble calcium salt is preferably 2-4 hours, and in specific embodiments it can be 2, 3, or 4 hours. This invention achieves Ca... 2+ The adsorption of sodium alginate promotes cross-linking, forming a sodium alginate-calcium ion cross-linked network. After impregnation, the microwave needle body is removed and washed with water to obtain a microwave needle loaded with the sodium alginate-calcium ion cross-linked network.

[0070] After obtaining the microwave needle loaded with a sodium alginate-calcium ion crosslinking network, the present invention immerses the microwave needle loaded with the sodium alginate-calcium ion crosslinking network into the drug-loaded calcium carbonate dispersion to obtain the microwave ablation device.

[0071] In this invention, the impregnation is preferably carried out under stirring conditions; the impregnation time is preferably 20-30 hours, and in a specific embodiment, it is 24 hours. This invention adsorbs drug-loaded calcium carbonate onto a sodium alginate-modified microwave needle through impregnation, and after removal from the solution, a microwave ablation device is obtained.

[0072] The microwave ablation device prepared by this invention can be reused. After use, it can be re-polished and sterilized, and the above preparation process can be repeated to obtain a microwave ablation device that can be used clinically again. Compared with existing microwave ablation needles, it can reduce medical waste and lower medical costs.

[0073] The microwave ablation device and its preparation method provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0074] Example 1

[0075] (1) Weigh 150mg CaCl2·2H2O and dissolve it in 100mL of anhydrous ethanol and place it in container 1. At the same time, put 5g of NH4HCO3 in another blank container 2. Place container 1 and container 2 in a closed vacuum environment at 80℃ and react for 24h. Take out the white calcium carbonate solution in container 1 and place it in a centrifuge tube for centrifugation (8000rpm, 10min). After centrifugation, a white calcium carbonate precipitate is obtained. Then wash it with anhydrous ethanol 2-3 times and store the precipitate.

[0076] (2) Disperse 30 mg of calcium carbonate in 10 mL of anhydrous ethanol, add 30 mg of Zeb (mass ratio of 1:1 to calcium carbonate), stir at 300 rpm for 12 h, and the drug Zeb enters the interior of the mesoporous calcium carbonate to obtain a drug-loaded calcium carbonate dispersion.

[0077] (3) The microwave needle (WMA-needle) was sanded twice with 200-mesh and 1000-mesh sandpaper, then immersed in a 5 mg / mL sodium alginate aqueous solution for 30 min. After removal, it was washed twice with water to obtain a sodium alginate-modified microwave needle. This needle was then immersed in a 1 mol / L CaCl2 solution for 3 h to achieve Ca... 2+ The adsorption and cross-linking of sodium alginate were carried out, and the product was washed twice with water to obtain a microwave needle (denoted as WMA-needle@Alg) loaded with a sodium alginate-calcium ion cross-linking network.

[0078] (4) The microwave needle modified with sodium alginate was immersed in a drug-loaded calcium carbonate dispersion and stirred for 12 hours to obtain a microwave ablation device, denoted as WMA-needle@Alg / Zeb-CaCO3.

[0079] Comparative Example 1

[0080] The difference from Example 1 is that the drug Zeb is not added, but otherwise it is the same as Example 1. The resulting microwave ablation device is denoted as WMA-needle@Alg / CaCO3.

[0081] Comparative Example 2

[0082] 30 mg of calcium carbonate was dispersed in 10 mL of anhydrous ethanol, and 30 mg of Zeb (mass ratio of 1:1 to calcium carbonate) was added. The mixture was stirred at 300 rpm for 12 h. The drug Zeb entered the mesoporous calcium carbonate, and a drug-loaded calcium carbonate dispersion was obtained. After centrifugation, the mixture was washed once with anhydrous ethanol to obtain drug-loaded calcium carbonate, denoted as Zeb-CaCO3.

[0083] Structural and performance testing:

[0084] The synthesized calcium carbonate was observed using scanning electron microscopy and transmission electron microscopy, and the results are as follows: Figure 1 As shown, by Figure 1 It can be seen that the prepared calcium carbonate is a nearly spherical particle with a particle size of about 100 nm.

[0085] A representative calcium carbonate particle was selected for energy dispersive spectroscopy (EDS) scanning, and the results are as follows: Figure 2 As shown, by Figure 2 It can be seen that calcium carbonate is nearly spherical with a particle size of about 100 nm and has pores, which are mesoporous.

[0086] Dilute calcium carbonate to 0.1 mg / mL with anhydrous ethanol, sonicate for 60 s to ensure uniform dispersion, and pipette 1 mL of sample into a plastic cuvette. Set the particle size analyzer parameters to refractive index 1.0 and temperature 25℃, and run 3–5 times (taking the average value) to obtain the particle size distribution of calcium carbonate. Pipette 0.5–1 mL of sample into the Zeta potential cell, select "Zeta Potential Measurement" mode, and run 5–10 times (taking the average value) to obtain the Zeta potential of calcium carbonate. The particle size distribution and Zeta potential results of calcium carbonate are shown below. Figure 3 As shown. By Figure 3 It can be seen that the particle size of calcium carbonate is mostly concentrated in 100 nm, and the Zeta potential is 3 mV.

[0087] Isothermal adsorption tests were performed on the synthesized calcium carbonate, and isothermal adsorption curves were plotted. The results are as follows: Figure 4 As shown. By Figure 4 It can be seen that the specific surface area of ​​the synthesized calcium carbonate is 16.2860 m². 2 / g, with an average pore size of 4.4360nm.

[0088] Calcium carbonate was suspended in 3 mL of PBS solution with pH values ​​of 5.0, 6.5, and 7.4, respectively, and placed in dialysis bags with a molecular weight cutoff of 3500 kDa, sealing both ends with dialysis clamps. The dialysis bags containing calcium carbonate were completely immersed in 10 mL of PBS solution with the same pH value and incubated in a shaker at 150 rpm and 37°C under light-protected conditions. At specific time intervals, 1 mL of dialysis solution was collected from the system, and the particle size was measured. The solution was then returned to the original container. The results are as follows: Figure 5 As shown, by Figure 5 It is known that an acidic environment can accelerate the decomposition of calcium carbonate, and the lower the pH, the more complete the decomposition of calcium carbonate.

[0089] The particle size of drug-loaded calcium carbonate (CaCO3-Zeb) was measured and compared with that of calcium carbonate. The results are as follows: Figure 6 As shown. By Figure 6 It can be seen that the particle size of CaCO3-Zeb is larger than that of pure calcium carbonate particles, and the particle size distribution is more concentrated.

[0090] Scanning electron microscopy and transmission electron microscopy were performed on CaCO3-Zeb, and the results are shown in the figure. Figure 7 , Figure 7 Image a is a TEM image, and image b is a SEM image. Representative particles were selected for energy dispersive spectroscopy (EDS) scanning, and the results are shown in the figure. Figure 8 .Depend on Figure 7 and Figure 8 It can be seen that after drug loading, the elements are evenly distributed in calcium carbonate, and the morphology remains basically unchanged, but the particle size increases slightly.

[0091] Drug release assays were performed on CaCO3-Zeb to investigate the effects of temperature and pH on drug release. The specific experimental procedure was as follows: 10 mg of CaCO3-Zeb was accurately weighed and added to a dialysis bag, which was then clamped shut at both ends. PBS buffer at different pH values ​​was added to centrifuge tubes. The centrifuge tubes were placed in a 37°C water bath at 100 rpm, ensuring the dialysis bag was completely immersed in the PBS. At predetermined time points, 3 mL of PBS buffer was collected to measure the OD value. The Zeb concentration was obtained based on the linear relationship between Zeb concentration and the OD value of the UV absorption peak, and the release amount was then calculated. The results are shown below. Figure 9 .Depend on Figure 9 As shown in section a, the more Zeb is released from CaCO3-Zeb as the temperature increases, with the Zeb release reaching 15% at 45℃ for 30 minutes; Figure 9 As shown in b, the more acidic the environment, the more drug is released. At pH 5.0, the drug release reaches a plateau of about 30% after 10 hours.

[0092] The WMA-needle@Alg / Zeb-CaCO3 was observed under scanning electron microscopy at different magnifications, and the results are as follows: Figure 10 As shown. By Figure 10 It can be seen that nanoparticles were successfully loaded onto the surface of the microwave needle.

[0093] Calcium carbonate was peeled off from the surface of WMA-needle@Alg / Zeb-CaCO3 using a blade, and observed under a transmission electron microscope. The results are as follows: Figure 11 As shown, particle size and zeta potential were characterized, and the results are shown in [Figure number missing]. Figure 12 .Depend on Figure 11 and Figure 12 It can be seen that the morphology and particle size of calcium carbonate stripped from WMA-needle@Alg / Zeb-CaCO3 are basically unchanged compared with the pure calcium carbonate prepared in step (1). The particle size is less than 200nm, concentrated at about 100nm, and the potential is 1.331mV. This shows that the loading method of the present invention does not affect the morphology and function of calcium carbonate.

[0094] The reuse process is as follows: The used WMA-needle@Alg / Zeb-CaCO3 was cleaned, dried, and then sanded twice each with 200-mesh and 1000-mesh sandpaper. It was then immersed in a 5 mg / mL sodium alginate aqueous solution for 30 minutes, removed, and washed twice with water to obtain a sodium alginate-modified microwave needle. The sodium alginate-modified microwave needle was then immersed in a drug-loaded calcium carbonate dispersion and stirred for 12 hours to obtain a microwave ablation device. The reused microwave ablation device was wrapped with a dialysis membrane, clamped at both ends, and placed in PBS buffer solutions of different pH values ​​and temperatures, specifically PBS buffer solutions (pH 5.0, 6.5, and 7.4 at 37°C) and PBS buffer solutions (pH 7.4) at temperatures of 25°C, 37°C, and 45°C. 1 mL of the dialysis solution was taken at specific time points for UV measurement. The results are shown in [Figure 1]. Figure 13 .Depend on Figure 13 It can be seen that the drug release decreases with increasing reuse frequency. The calcium carbonate content loaded on the reused microwave ablation needles was determined. Specifically, the microwave ablation needles were soaked in concentrated hydrochloric acid for 2-4 hours to ensure that all calcium carbonate was dissolved into calcium ions. The solution was then diluted to the concentration required for ICP testing, and the calcium carbonate content on the microwave ablation needles after different reuse frequencies was measured. The results are as follows: Figure 14 As shown. By Figure 14 It can be seen that the amount of calcium carbonate loaded is reduced after repeated use, but the calcium carbonate loading can still reach 30μg after three reuses, indicating that the microwave ablation needle can be reused.

[0095] When using a photothermal camera to detect thermal cycling at 30-60℃, the time required for the microwave ablation needle to naturally cool back to 30℃ after heating to 60℃ and stopping output is measured. The control group consists of microwave needles that have not undergone any treatment, while C0-C3 consist of microwave needles that have been reused 1-3 times. Results are shown in […]. Figure 15(The horizontal axis represents time, and the vertical axis represents the temperature range of the temperature increase). From Figure 15 It can be seen that reusing the device 1-3 times does not affect the heating and cooling effect.

[0096] CCK-8 Cytotoxicity Test:

[0097] 4T1 cells were cultured in a medium containing 89% DMEM, 10% FBS (serum), and 1% PS (penicillin-streptomycin mixture) by volume, incubated in a CO2 incubator, and passaged at a ratio of 1:3.

[0098] The cells were allowed to proliferate to the logarithmic growth stage. The cells were observed to have normal morphology under a microscope. The adherent 4T1 cells in the culture flask were digested with trypsin and transferred to 15mL centrifuge tubes. The cells were centrifuged at 1000r / min for 3min.

[0099] Discard the supernatant from the centrifuge tube, resuspend the cells in culture medium, pipette to homogenize the cells, and add 100 μL of the cell suspension to each well of a 96-well plate. Count the cells in each well using a cell counter to ensure approximately 1 × 10⁶ cells per well. 5 ;

[0100] The 96-well plate was incubated in a CO2 incubator for 24 hours.

[0101] After the cells were fully adhered to the culture vessel, they were divided into five groups: G1 (control) (untreated cells), G2 (Zeb-CaCO3), G3 (WMA-needle), G4 (WMA-needle@Alg / CaCO3), and G5 (WMA-needle@Alg / Zeb-CaCO3). After irradiation at 5W for 1 min, the cells were cultured for another 24 h.

[0102] Discard the culture medium in each well, wash the plate 3 times with PBS, and then replace it with culture medium containing 10% CCK-8 by adding 200 μL to each well.

[0103] After incubating the 96-well plate in a CO2 incubator for 1.5 hours, remove the plate, take it out, remove the 96-well plate cover, place it in the sample area of ​​a multi-functional microplate reader, and measure the absorbance of each well at a wavelength of 450 nm.

[0104] See results Figure 16 .Depend on Figure 16 It can be seen that the cell survival rate of group G1 was 100%, that of group G2 was 73.225%, that of group G3 was 70.13%, that of group G4 was 44.426%, and that of group G5 was 35.61%. Group G5 cells had a stronger killing ability than the other groups.

[0105] Cell staining experiment:

[0106] 4T1 cells were seeded into culture plates and, after adhesion, were divided into five groups: G1 (blank, untreated cells), G2 (Zeb-CaCO3), G3 (WMA-needle), G4 (WMA-needle@Alg / CaCO3), and G5 (WMA-needle@Alg / Zeb-CaCO3). After irradiation at 5W for 1 min, the cells were cultured for 12 h. Staining procedure: Remove the culture medium and gently wash the cells 1-2 times with PBS (to remove serum esterase interference); add the staining working solution, specifically a mixture of Calcein-AM and PI (1 μM Calcein-AM + 2 μg / mL PI in 1 mL of medium), cover the cells, and incubate at 37°C in the dark for 15-30 minutes (excessive incubation may lead to false positives). Stop staining: Discard the staining solution and wash the cells 1-2 times with PBS.

[0107] Observation under a fluorescence microscope: Live cells: green fluorescence (Calcein-AM, FITC channel); Dead cells: red fluorescence (PI, TRITC / Cy3 channel). Staining results are shown below. Figure 17 ,Depend on Figure 17 It is evident that G5 cells showed the highest number of dead cells and the lowest number of surviving cells, indicating that G5 cells exhibit the strongest cytotoxicity. This is because WMA-needle achieves a progressive increase in efficacy by introducing CaCO3 and Zeb-CaCO3 components layer by layer: the basic WMA-needle constructs the therapeutic framework, generating high temperatures through microwave ablation to kill tumor cells; the CaCO3 component induces calcium death in tumor cells; and the Zeb component in Zeb-CaCO3 achieves anti-methylation, inhibiting microtubule dynamics, blocking tumor cell mitosis, inducing apoptosis, and simultaneously reshaping the tumor vascular microenvironment, reducing metastatic potential. The components work synergistically, resulting in a stepwise increase in overall efficacy.

[0108] Intracellular Ca 2+ Content detection:

[0109] Cell treatment: 4T1 cells were seeded into culture plates and, after attachment, were treated and grouped into five groups: G1 (blank group, untreated cells), G2 (Zeb-CaCO3), G3 (WMA-needle), G4 (WMA-needle@Alg / CaCO3), and G5 (WMA-needle@Alg / Zeb-CaCO3). After irradiation at 5W for 1 min, the cells were cultured for 12 h.

[0110] Take an appropriate amount of Fluo-4AM stock solution and dilute it with PBS to prepare a 0.5-5 μM working solution;

[0111] For the cultured cells to be tested, remove the culture medium and wash three times with PBS;

[0112] Add Fluo-4AM at 37℃ and incubate for 10-60 min to load the fluorescent probe; the working solution should be sufficient to fully cover the cells; then remove the culture medium and wash three times with PBS. After washing, you can consider incubating for another 20-30 minutes to ensure that Fluo-4AM is completely converted into Fluo-4 in the cells.

[0113] Fluo-4 fluorescence was detected using laser confocal microscopy to determine changes in intracellular calcium ion concentration. Results are shown below. Figure 18 ,Depend on Figure 18 It can be seen that the fluorescence of groups G4 and G5 is the strongest, indicating that the calcium ion content is the highest. Increased calcium ion content can cause calcium overload, leading to tumor cell death, and thus has the ability to kill tumors.

[0114] Intracellular pH detection:

[0115] Cell treatment: 4T1 cells were seeded into culture plates and, after attachment, were divided into five groups: G1 (untreated), G2 (Zeb-CaCO3), G3 (WMA-needle), G4 (WMA-needle@Alg / CaCO3), and G5 (WMA-needle@Alg / Zeb-CaCO3). After irradiation at 5W for 1 min, the cells were cultured for 12 h. Then, the following steps were performed:

[0116] 1. Take an appropriate amount of BCECFAM stock solution and dilute it with PBS to a working solution of 0.5-5 μM;

[0117] 2. For the cultured cells to be tested, remove the culture medium and wash three times with PBS;

[0118] 3. Add BCECFAM at 37℃ and incubate for 10-60 min to load the fluorescent probe; the working solution volume should be sufficient to fully cover the cells;

[0119] 4. Then wash three times with PBS;

[0120] 5. Use laser confocal microscopy to detect the fluorescence of BCECFAM to determine changes in intracellular pH.

[0121] See results Figure 19 ,Depend on Figure 19 It can be seen that group G5 showed the least fluorescence, indicating the highest pH. This is because group G5 had the best therapeutic effect, directly killing tumor cells, reducing the number of tumor cells, and increasing lactic acid and protons (H+). + The secretion of glycolysis decreased; after tumor regression, the oxidative metabolism of normal cells replaced glycolysis, reducing lactic acid production; activated T cells and other factors could indirectly inhibit tumor growth and reduce acidic metabolites. These results indicate that the acidic tumor microenvironment was alleviated to the greatest extent possible after treatment.

[0122] Cell marker detection:

[0123] 4T1 cells were seeded into culture plates and, after attachment, were divided into five groups: G1 (blank group, untreated cells), G2 (Zeb-CaCO3), G3 (WMA-needle), G4 (WMA-needle@Alg / CaCO3), and G5 (WMA-needle@Alg / Zeb-CaCO3). After irradiation at 5W for 1 min, the cells were cultured for 12 h. The results were then analyzed according to the kit instructions (Beyotime HIF1α / Ki67 / CD31 kit). The test results are shown below. Figure 20 HIF1α is a core transcription factor in the cellular response to hypoxia, stably expressed under hypoxic conditions. It is a poor prognostic marker: high expression of HIF1α suggests tumors adapted to hypoxia, highly invasive, prone to metastasis, and resistant to radiotherapy and chemotherapy. Figure 20 It can be seen that the G5 group showed the best improvement in hypoxia.

[0124] Ki67 is present in proliferating cells (G1 / S / G2 / M phases) and not expressed in quiescent cells (G0 phase); a high Ki67 index (e.g., >30% in breast cancer) indicates rapid tumor growth, poor differentiation, and a poor prognosis. Figure 20 It can be seen that the proliferation of tumor cells in group G5 is reduced.

[0125] CD31 is a marker of vascular endothelial cells used to quantify tumor angiogenesis. High CD31 expression suggests abundant tumor blood vessels, ample nutrient supply, and a tendency for metastasis. Figure 20 It can be seen that G5 tumors have reduced angiogenesis.

[0126] Animal therapy experiments:

[0127] Methods: Flux-labeled 4T1 cells (1×10⁻⁶) were used to... 6 The vaccine was subcutaneously injected into the right leg of BABL / C mice. The volume was increased to 1000 mm². 3 The corresponding experiments can be performed at this time. BABL / C mice were fully anesthetized (sodium pentobarbital, 60 mg / kg) and placed in a supine position. They were divided into five groups: G1 (blank group, untreated cells), G2 (Zeb-CaCO3), G3 (WMA-needle), G4 (WMA-needle@Alg / CaCO3), and G5 (WMA-needle@Alg / Zeb-CaCO3), with four mice in each group. The microwave ablation needle was inserted into the center of the tumor perpendicular to its long axis. The temperature was controlled at 70±5℃ and maintained for 5 minutes. The tumor volume of the mice was monitored, and their body weight was measured. When the tumor volume of the mice exceeded 2000 mmHg, the tumor was considered closed. 3 The default death value is calculated using the following formula for mouse volume: (length × width) 2) / 2. Weigh the tumor. See results below. Figure 21 In this diagram, a shows photographs of tumor volume changes in mice after treatment (4 columns corresponding to 4 mice in each group), b shows a comparison of tumor volume in each group, and c shows a comparison of tumor weight in each group. Figure 21 It can be seen that the tumors in group G5 were the smallest in weight and volume, and the treatment effect was the best.

[0128] Flow cytometry analysis of mouse tumors and spleen:

[0129] The experimental procedure is as follows:

[0130] (1) Sample processing:

[0131] Spleen: Grind on a sieve, collect the cell suspension, centrifuge (300g, 5min), wash and resuspend the cells.

[0132] Tumors: Transfer the tissue to a small culture dish (60×15mm) or a 6-well plate, add tissue dissociation solution (abs9482) to cut the tissue into small pieces, incubate in a shaker water bath at 37°C for 45 min, press the tissue through a 70 μm sieve using the plunger of a syringe, centrifuge (300g, 5 min), discard the supernatant, treat with 1× cleavage red (abs9101), wash and resuspend the cells.

[0133] (2) Cell division:

[0134] 10 per tube 6 For both left and right cells, the initial protocol requires 1 blank control, 14 single-stain controls (one tube per antibody dye), and n fully stained tubes. For the actual experiment, only 1 blank control, 1 single-stain control (for both live and dead cells), and n fully stained tubes are needed.

[0135] (3) Comparison settings

[0136] Blank control: One tube is prepared for each sample. Except for not staining with antibody dye, the other treatments are the same as for fully stained tubes. Only the fixation and membrane rupture operation is performed.

[0137] Single staining control: Each tube of cells is stained with only one antibody or dye, and the staining is confirmed according to the antigen expression characteristics (surface or intracellular).

[0138] (4) Live and dead staining

[0139] Prepare the FVS dye stock solution according to the instructions, aliquot and store at -20°C, avoiding repeated freeze-thaw cycles. For each tube, resuspend the cells in (1 mL DPBS + 1 μL FVS780) stock solution for staining. For larger sample sizes, premix the solution; for example, for 20 samples, prepare 20 μL FVS780 + 20 mL DPBS, and resuspend the cells in 1 mL of the premix in each tube. Incubate at 4°C in the dark for 30 minutes. Centrifuge to remove the supernatant, add 1 mL of staining buffer (1% FBS in PBS), and centrifuge at 300g for 5 minutes. After removing the supernatant, wash once more with 1 mL of stain buffer.

[0140] (5) FCR blockade (553141)

[0141] After premixing 2 μL (1 μg) / test + Stain buffer (554656) 40 μL / test, add 40 μL of the premixed solution to each tube to resuspend the cells, vortex thoroughly, and incubate at 4°C for 20 min.

[0142] (6) Surface staining

[0143] Surface staining: Add premixed antibody, mix well, and incubate at 4°C in the dark for 30 minutes.

[0144] Washing: Wash and resuspend with 1 mL of stain buffer, 300 g for 5 min, wash 1-2 times.

[0145] (7) Fixation and perforation & intracellular staining (follow the instructions for Fixation and Perforation Kit 562574).

[0146] (8) Flow cytometry sample preparation: Resuspend cells in 350 μL of stain buffer, and analyze the data using a flow cytometer. Results are shown below. Figure 22 . Figure 22 In the text, "****" represents p < 0.0001, indicating that the difference is highly significant.

[0147] Treg cell analysis: by detecting CD4 + CD25 + FoxP3 + Identification of regulatory T cells (Tregs) using biomarkers. Figure 22 Figure a shows the changes in the proportion of Treg cells in different groups (G1-G5). As can be seen from the G1 to the G5 groups, the number of Treg cells increases. Treg cells are a type of immunosuppressive T cell that maintains autoimmune tolerance and suppresses excessive immune responses. An increase in Treg cells can suppress anti-tumor immunity (such as CD8+). + T cell and NK cell function).

[0148] CD86 expression analysis (see...) Figure 22 (b) CD86, as a marker of immune-activated macrophages (M1 type) and a co-stimulatory molecule for T cells, plays a role in CD11b. + F4 / 80 + The positive rate in the cell population showed an increasing trend from group G1 to group G5 (reaching 15.56% in group G5). High expression of CD86 indicates enhanced immune activation of macrophages, and high expression of CD86 can promote T cell activation (via CD28 signaling) and enhance anti-tumor immune response.

[0149] CD206 expression analysis (see...) Figure 22 c): CD206, as a marker associated with immunosuppressive macrophages (M2 type), has a significant role in CD11b... + F4 / 80 + The positive rate in the cell population was highest in group G1 (20.02%), and significantly decreased in subsequent groups (G2-G5). CD206 + A decrease in macrophages indicates a polarization of M2 macrophages (pro-tumor) towards M1 macrophages (anti-tumor), which is beneficial for enhancing the killing function of T cells.

[0150] In conclusion, Figure 22 This suggests that from G1 to G5, the immune microenvironment may shift from a pro-tumor (high CD206, low CD86) to an anti-tumor (low CD206, high CD86) state.

[0151] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A microwave ablation device, characterized in that, The device includes a microwave needle body, a sodium alginate-calcium ion crosslinking network loaded on the surface of the microwave needle body, and drug-loaded calcium carbonate adsorbed by the sodium alginate-calcium ion crosslinking network; the drug-loaded calcium carbonate includes mesoporous CaCO3 and demethylated drugs loaded inside and / or on the surface of the mesoporous CaCO3.

2. The microwave ablation device according to claim 1, characterized in that, The demethylating agent includes one or more of zabralline, decitabine, azacitidine, and vorinostat.

3. The microwave ablation device according to claim 1, characterized in that, The drug-loaded calcium carbonate has a particle size of less than 200 nm.

4. The method for preparing the microwave ablation device according to any one of claims 1-3, characterized in that, Includes the following steps: Mesoporous CaCO3, demethylated drug, and polar organic solvent were mixed to obtain a drug-loaded calcium carbonate dispersion; The microwave needle body was modified by immersing it in an aqueous solution of sodium alginate to obtain a sodium alginate-modified microwave needle. The sodium alginate-modified microwave needle was immersed in an aqueous solution of soluble calcium salt for Ca... 2+ Coordination and cross-linking are performed to form a sodium alginate-calcium ion cross-linking network, resulting in a microwave needle loaded with the sodium alginate-calcium ion cross-linking network. The microwave needle loaded with sodium alginate-calcium ion crosslinking network is immersed in the drug-loaded calcium carbonate dispersion to obtain the microwave ablation device.

5. The preparation method according to claim 4, characterized in that, The method for preparing mesoporous CaCO3 includes: dissolving calcium chloride in ethanol to obtain a calcium chloride solution; introducing CO2 into the calcium chloride solution to react with the calcium chloride to generate mesoporous CaCO3; and obtaining mesoporous CaCO3 after solid-liquid separation.

6. The preparation method according to claim 4, characterized in that, The soluble calcium salt includes calcium chloride; the concentration of the aqueous solution of the soluble calcium salt is 0.5-1.5 mol / L.

7. The preparation method according to claim 4, characterized in that, The polar organic solvents include ethanol and / or dimethyl sulfoxide.

8. The preparation method according to claim 4, characterized in that, The concentration of the sodium alginate aqueous solution is 1-5 mg / mL.

9. The preparation method according to claim 4 or 8, characterized in that, The modification process takes 0.5-2 hours.

10. The preparation method according to claim 4, characterized in that, Before immersing the microwave needle body in an aqueous sodium alginate solution for modification, the process also includes sanding the microwave needle body.

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