Bionic nano platform for collaborative treatment and preparation method and application thereof

Abstract

The invention relates to a bionic nano-platform for synergistic treatment, the bionic nano-platform comprises a core and a shell, the core comprises hollow calcium carbonate and manganese carbonate, the shell comprises a cell membrane and a fluorescent dye, and the shell covers the surface of the core. The preparation method comprises the following steps: synthesizing amorphous calcium carbonate; preparing hollow calcium carbonate; preparing hollow calcium carbonate-manganese carbonate nanoparticles; coating the cell membrane; and loading the fluorescent dye. The bionic nano platform for collaborative treatment can integrate multiple functions of gas release, photo-thermal heat production, immune regulation and control, in-vivo imaging and the like, and is used for collaborative treatment of liver cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a biomimetic nanoplatform for synergistic therapy, its preparation method, and its application. Background Technology

[0002] Liver cancer is one of the most common and deadliest malignant tumors worldwide, with its incidence and mortality rates showing an increasing trend year by year. Currently, treatment methods for liver cancer mainly include surgical resection, chemotherapy, radiotherapy, and interventional therapy. However, these traditional treatment methods have many limitations. For example, surgical resection is only suitable for patients with early-stage liver cancer. For patients with mid-to-late-stage liver cancer, due to tumor metastasis and spread, surgical resection often fails to completely remove tumor tissue. Chemotherapy and radiotherapy, while killing tumor cells, also cause severe damage to normal tissues and cells, leading to a series of serious side effects such as nausea, vomiting, hair loss, and decreased immunity. Although interventional therapy can treat tumors locally, its effectiveness is limited for some deep or multiple tumors. In recent years, with the development of nanotechnology, nanomedicine delivery systems have shown great application potential in the field of tumor treatment. Some nanoplatforms have been designed to load chemotherapeutic drugs, photothermal agents, etc., to improve drug targeting and therapeutic efficacy. For example, some studies have constructed nanosystems based on nanoliposomes, polymer nanoparticles, etc., to load chemotherapeutic drugs, delivering drugs to tumor tissue through passive or active targeting. Furthermore, photothermal therapy, as an emerging tumor treatment method, utilizes the thermal effect generated by photothermal agents under near-infrared light irradiation to kill tumor cells, offering advantages such as being non-invasive and highly effective. However, single treatment modalities often fail to achieve ideal therapeutic effects because tumor development is a complex process involving multiple signaling pathways and biological processes. Therefore, multimodal synergistic treatment strategies have become a current research hotspot in liver cancer treatment.

[0003] Current nanotherapy platforms and multimodal therapy strategies still have some shortcomings. On the one hand, many nanoplatforms lack good tumor targeting and immune evasion capabilities, resulting in low drug accumulation efficiency in tumor tissues and easy recognition and clearance by the immune system, affecting treatment efficacy. On the other hand, existing multimodal therapy strategies often simply combine different treatment modalities, lacking effective synergy between the various treatment mechanisms, making it difficult to achieve precise spatial and temporal coupling and fully leverage the advantages of multimodal therapy. Furthermore, some treatment methods may cause serious side effects during treatment, significantly impacting the patient's health. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a biomimetic nanoplatform for synergistic therapy. The biomimetic nanoplatform comprises a core and a shell, wherein the core comprises hollow calcium carbonate (hCaCO3) and manganese carbonate (Mn2(CO)). 10 MnCO (hereinafter referred to as MnCO) is a cell membrane and a fluorescent dye that cover the surface of the nucleus.

[0005] In one embodiment, the cell membrane includes a hepatocellular carcinoma cell membrane (HCCM); the fluorescent dye includes DiR.

[0006] A second aspect of the present invention also provides a method for preparing the above-mentioned biomimetic nanoplatform for synergistic therapy, comprising the following steps:

[0007] Synthesis of amorphous calcium carbonate (ACC): Calcium chloride (CaCl2) is dissolved, sealed, and placed in a closed, dry container containing ammonium bicarbonate (NH4HCO3). The mixture is reacted, centrifuged, washed, and dispersed in an organic solvent to obtain an amorphous calcium carbonate dispersion. Preparation of hollow calcium carbonate (hCaCO3): Dissolve trisodium phosphate (Na3PO4), add the amorphous calcium carbonate dispersion dropwise to the sodium phosphate solution, react, centrifuge, wash, and freeze-dry to obtain hollow calcium carbonate; Preparation of hollow calcium carbonate-manganese carbonate nanoparticles (hCaCO3-MnCO): Manganese carbonate was dissolved to obtain a manganese carbonate solution. A hollow calcium carbonate dispersion was added to the manganese carbonate solution, incubated, concentrated, centrifuged, and washed to obtain hollow calcium carbonate-manganese carbonate nanoparticles. Cell membrane coating: Cell membrane extraction, hollow calcium carbonate-manganese carbonate nanoparticles were dispersed in PBS, mixed with cell membrane, incubated, and extruded to obtain cell membrane coated nanoparticles (HCCM@hCaCO3-MnCO). Fluorescent dye loading: Fluorescent dye solution was added to the cell membrane-coated nanoparticle suspension, stirred in the dark, centrifuged, washed, and resuspended to obtain a biomimetic nanoplatform (HCCM@DiR@hCaCO3-MnCO).

[0008] In one embodiment, the incubation time is 25-35 minutes.

[0009] In one embodiment, the extrusion employs a decreasing aperture gradient extrusion.

[0010] Integrating functional units such as central control calcium carbonate, manganese salt, CO donor, cell membrane coating, or fluorescent dye in one go while maintaining their individual activities presents significant challenges. (1) Hollow calcium carbonate and manganese carbonate need to release Ca simultaneously within an acidic microenvironment window. 2+ CO and Mn 2+Otherwise, a spacetime misalignment will occur; this invention regulates Ca 2+ / Mn 2+ The proportions and shell thicknesses ensure that the release windows of each component highly overlap. (2) Hydrophobic near-infrared dye DiR is prone to non-specific aggregation or leakage on the surface of hollow carrier; In this invention, DiR is first pre-embedded into the lipid layer of liver cancer cell membrane, and then co-extruded with the carrier to achieve membrane-dye integrated fixation and avoid subsequent leakage. (3) Proteins are easily inactivated by shear force during cell membrane coating; the present invention adopts a decreasing pore size gradient extrusion process to complete the coating under mild conditions and maintain the activity of membrane proteins.

[0011] In one embodiment, the synthesis of the amorphous calcium carbonate includes the following steps: Calcium chloride solution was obtained by dissolving calcium chloride in a mixed solvent of ethanol and water. The calcium chloride solution was sealed and placed in a sealed dry container containing ammonium bicarbonate. The reaction was carried out, the product was collected by centrifugation, washed, and dispersed in ethanol to obtain an amorphous calcium carbonate dispersion. The mass ratio of calcium chloride to ammonium bicarbonate is 1:(30~60).

[0012] In one embodiment, the ethanol and water mixture is in a volume ratio of 150 to 250:1.

[0013] In one embodiment, the reaction time is 45-50 hours.

[0014] In one embodiment, the relative humidity is maintained at 70-80% during the reaction.

[0015] In one embodiment, the centrifugation time is 3 to 10 minutes.

[0016] In one embodiment, the preparation of the hollow calcium carbonate includes the following steps: Dissolve trisodium phosphate in water, add the amorphous calcium carbonate dispersion dropwise to the sodium phosphate solution, stir the reaction, collect the product by centrifugation, wash, and freeze-dry to obtain hollow calcium carbonate; The mass ratio of the amorphous calcium carbonate to the trisodium phosphate is 1:(6~10).

[0017] In one embodiment, the stirring reaction time is 25-35 minutes.

[0018] In one embodiment, the centrifugation time is 3 to 10 minutes.

[0019] In one embodiment, the preparation of the hollow calcium carbonate-manganese carbonate nanoparticles includes the following steps: Manganese carbonate was dissolved in methanol, and a methanol dispersion of hollow calcium carbonate was added to the manganese carbonate methanol solution. The mixture was incubated, vacuum dried and concentrated, centrifuged, and washed to obtain hollow calcium carbonate-manganese carbonate nanoparticles. According to the mass ratio, the manganese carbonate : the hollow calcium carbonate is (1~5):1.

[0020] In one embodiment, the incubation time is 5 to 15 minutes.

[0021] In one embodiment, the centrifugation time is 3 to 10 minutes.

[0022] In one embodiment, the cell membrane extraction during the cell membrane encapsulation includes the following steps: Collect cells, wash, add lysis buffer, let stand, homogenize, sonicate on ice, centrifuge to remove cell nuclei, centrifuge to collect membrane precipitate, and resuspend to obtain cell membrane suspension; According to the mass ratio, the hollow calcium carbonate-manganese carbonate nanoparticles: the cell membrane is (1~2):(1~2).

[0023] In one embodiment, the lysis buffer comprises tris-hydroxymethylaminomethane hydrochloride (Tris-HCl), ethylenediaminetetraacetic acid (EDTA), benzyl sulfonyl fluoride (PMSF), and a protease inhibitor.

[0024] In one embodiment, the settling time is 25-35 minutes.

[0025] In one embodiment, the sonication time is 1-5 min, the centrifugation time for removing cell nuclei is 8-15 min, and the centrifugation time for collecting membrane precipitates is 25-35 min.

[0026] In one embodiment, loading the fluorescent dye includes the following steps: A methanol solution of fluorescent dye was added to a suspension of cell membrane-coated nanoparticles, stirred in the dark, centrifuged, washed, and resuspended in PBS to obtain a biomimetic nanoplatform. The ratio of cell membrane-coated nanoparticles to fluorescent dye is (80~120):1 by mass.

[0027] In one embodiment, the stirring time in the dark is 0.5 to 2 hours.

[0028] In one embodiment, the centrifugation time is 8 to 15 minutes.

[0029] In a third aspect, the present invention also provides the application of the above-mentioned biomimetic nanoplatform for synergistic therapy, or the biomimetic nanoplatform for synergistic therapy obtained by the above preparation method, in the preparation of a drug for treating liver cancer.

[0030] In addition, the present invention also provides a drug for treating liver cancer, the drug comprising the above-mentioned biomimetic nanoplatform for synergistic treatment, or the biomimetic nanoplatform for synergistic treatment obtained by the above preparation method.

[0031] In view of the shortcomings of the prior art, the present invention aims to solve the following technical problems: 1. Develop a biomimetic nanoplatform that enables synergistic gas-photothermal-immune therapy for liver cancer. This platform can efficiently and precisely couple the three treatment modalities of CO gas release, photothermal effect, and immune activation in space and time to achieve synergistic therapeutic effects superior to single therapies.

[0032] 2. Improve the targeting of the nanoplatform to the tumor site and its responsiveness in the tumor microenvironment, while endowing it with a certain immune escape ability, so as to improve the tumor enrichment of the drug and the therapeutic efficiency.

[0033] 3. Utilize the characteristics of the tumor microenvironment (such as acidity) to trigger the decomposition of the nanoplatform and the release of active substances, and further amplify the therapeutic effect through photothermal effect.

[0034] 4. Develop a multifunctional integrated biomimetic nanoplatform that can simultaneously perform functions such as gas release, photothermal heat generation, immunogenic cell death (ICD) induction, signaling pathway activation, and in vivo imaging.

[0035] Although existing technology knows Ca 2+ Overload, Mn 2+ -STING activation, CO mitochondrial damage, and cell membrane homology targeting act independently, but no synergistic cascade amplification was observed when these four factors were integrated. However, this invention discovers: (1) DiR-mediated 808nm photothermal heating can instantaneously open the TRPV1 channel and amplify Ca 2+ internal flow; (2) Ca 2+ Overload further promotes the degradation of manganese carbonate and releases CO; (3) CO and Mn 2+ It synergistically induces immunogenic cell death and significantly enhances anti-tumor immunity.

[0036] Furthermore, comparative experiments of this invention also found that after replacing DiR with other near-infrared dyes or changing the cell membrane source, the photothermal efficiency or the tumor cell targeting enrichment both decreased significantly, suggesting that the DiR-liver cancer cell membrane combination is irreplaceable for achieving homologous targeting and deep photothermal effects.

[0037] Compared with the prior art, the present invention has the following beneficial effects: 1. Excellent tumor targeting and immune evasion: HCCM modification endows the platform with excellent tumor targeting, enabling it to specifically accumulate in tumor tissue and increase the local concentration of therapeutic factors. Simultaneously, the camouflage effect of the cell membrane gives the platform the ability to evade the immune system, reducing the probability of being cleared by the immune system and prolonging its circulation time in the body.

[0038] 2. Multi-mode response and multi-mechanism synergy: The nanoplatform of this invention can responsively release Ca in the acidic tumor microenvironment. 2+ CO gas and Mn 2+ Furthermore, by combining the photothermal effect of DiR, multiple therapeutic mechanisms can be synergistically activated. These mechanisms are precisely coupled spatially and temporally, mutually reinforcing each other and significantly improving the efficacy of tumor treatment.

[0039] 3. Imaging function: The incorporation of DiR enables the platform to have fluorescence imaging capabilities, which facilitates real-time monitoring of the treatment process and evaluation of treatment effects, providing a basis for personalized treatment.

[0040] 4. High safety and good metabolism: The nanoplatform of this invention is prepared with biocompatible materials, which not only reduces the side effects on normal tissues and improves the safety of treatment, but also has good metabolic characteristics, which can be smoothly metabolized in the body and reduce the risk of long-term accumulation.

[0041] 5. Simple process: The synthesis process of this nanotechnology platform is relatively simple, without the need for complex equipment and cumbersome operating procedures, which reduces production costs and technical barriers. Attached Figure Description

[0042] Figure 1 (A) is the TEM image of ACC; (B) is the TEM image of hCaCO3; (C) is the TEM image of hCaCO3-MnCO. Figure 2 Figure 1 shows the results of experiments on the toxicity of different concentrations of HCCM@DiR@hCaCO3-MnCO to L929 cells. Figure 3 Figure 1 shows the results of the cytotoxicity experiment of different concentrations of HCCM@DiR@hCaCO3, HCCM@DiR@hCaCO3-MnCO, HCCM@DiR@hCaCO3+NIR and HCCM@DiR@hCaCO3-MnCO+NIR on Hepa1-6 cells. Figure 4 The effect of different treatment groups on intracellular ROS levels in Hepa1-6 cells; Figure 5 Fluo-4 probe was used to detect the effects of different treatment groups on intracellular Ca2+ in Hepa1-6 cells. 2+ The influence of level; Figure 6 To investigate the effects of different treatment groups on the levels of CRT and HMGB1 in Hepa1-6 cells. Detailed Implementation

[0043] This invention provides a biomimetic nanoplatform that integrates multiple functions such as gas release, photothermal heat generation, immune regulation, and in vivo imaging for the synergistic treatment of liver cancer.

[0044] The present invention is based on a biomimetic nanoplatform of hollow calcium carbonate (hCaCO3) loaded with manganese carbonate (MnCO3) and modified with liver cancer cell membranes (HCCM) doped with the fluorescent dye DiR. It is applied to the gas-photothermal-immunotherapy synergistic therapy of liver cancer and has the following characteristics: (1) For the first time, a single extrusion step was proposed to simultaneously complete cell membrane coating and near-infrared dye loading, simplifying the process; (2) For the first time, a quantifiable control strategy for the synergistic mechanism of "photothermal-calcium-gas-immunity" is presented; (3) Establish repeatable quality control indicators such as particle size, potential, and coating integrity.

[0045] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0047] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all test methods are conventional test methods in this field.

[0048] Example I. This invention provides a method for preparing a biomimetic nanoplatform, comprising the following steps: (1) Synthesis of ACC Dissolve 200 mg CaCl₂·2H₂O in 100 mL of an ethanol / water mixture (V / V = 200:1). Place the solution in a 250 mL wide-mouth bottle, seal the bottle opening with Parafilm, and prick 3-5 0.5 mm pinholes in the film. Place the bottle in a 500 mL desiccator containing 10 g NH₄HCO₃, seal, and incubate at 25°C for 48 h; maintain a relative humidity of 70-80% during this period. Centrifuge at 8000 rpm for 5 min to collect the white ACC, wash three times with ethanol, and finally disperse in ethanol at a concentration of 5 mg / mL. -1 , stored at 4℃ ≤7d.

[0049] In this embodiment, the mass ratio of calcium chloride to ammonium bicarbonate is 1:50.

[0050] (2) Synthesis of hCaCO3 Dissolve 20 mg Na3PO4 in 4 mL of deionized water. While magnetically stirring at 600 rpm, add 0.5 mL of ACC ethanol solution (5 mg / mL). -1 ) with 1 drop s -1 The solution was added dropwise to the Na3PO4 solution; the reaction was carried out at 25℃ for 30 min. After centrifugation at 8000 rpm for 5 min, the solution was washed three times with ethanol; the solution was freeze-dried for 12 h to obtain hCaCO3 powder, which was then stored at -20℃.

[0051] In this embodiment, the mass ratio of amorphous calcium carbonate to sodium phosphate is 1:8.

[0052] (3) Synthesis of hCaCO3-MnCO Weigh out 1.5 mg of manganese decacarbonyl dimanganese (Mn2(CO)). 10 (hereinafter referred to as MnCO) was dissolved in 1 mL of methanol and sonicated for 2 min (40 kHz, 100 W) to aid dissolution. 1 mL of hCaCO3 methanol dispersion (0.5 mg / mL) was then added. -1 Add MnCO3 solution; incubate at 25℃ and 100 rpm on a shaker for 10 min, then rotary evaporate under vacuum at 35℃ to a volume of 500 μL (pressure ≤100 mbar). Centrifuge at 8000 rpm for 5 min, wash three times with methanol; finally disperse in methanol at a concentration of 1 mg / mL. -1 Store at 4℃ away from light for ≤3 days.

[0053] In this embodiment, the mass ratio of manganese carbonate to hollow calcium carbonate is 3:1.

[0054] (4) HCCM extraction Collect 2×10 7Hepa1-6 cells were washed twice with PBS. 1 mL of ice-bath lysis buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.5 mM PMSF, and a 1× protease inhibitor cocktail) was added, and the cells were incubated for 30 min. Homogenization was performed three times at 20000 psi; followed by sonication in an ice-water bath for 3 seconds / 3 seconds, for a total of 3 min. Cells were denucleated at 2000g for 10 min; the supernatant was collected at 20000g for 30 min to collect the membrane pellet. The pellet was resuspended in 1 mL PBS, protein was determined using BCA, and the solution was adjusted to 1 mg / mL. -1 Prepare and use immediately.

[0055] (5) Synthesis of HCCM@DiR@hCaCO3-MnCO Resuspend 1 mg hCaCO3-MnCO in 1 mL PBS, and then add 1 mL HCCM (1 mg / mL). -1 Mix and pre-incubate at 37°C for 30 min. Extrude sequentially through 400 nm, 200 nm, and 100 nm polycarbonate membranes, 21 times each, at 37°C. Add 2 mL of the extrudate to 0.5 mg / mL of the mixture. -1 Add 0.1 mL of DiR methanol solution (0.1 mg / mL) to the solution. -1 Stir at 25℃ and 300rpm in the dark for 1 hour. Centrifuge at 12000g for 10 minutes and wash 3 times with PBS. Resuspend the final product HCCM@DiR@hCaCO3-MnCO in 1mL PBS and store at 4℃ in the dark. Use within 48 hours.

[0056] In this embodiment, the mass ratio of hollow calcium carbonate-manganese carbonate nanoparticles to cell membrane is 1:1; the ratio of cell membrane-coated nanoparticles to fluorescent dye is 100:1.

[0057] Example 1 Preparation of ACC 200 mg (1.36 mmol) of CaCl₂·2H₂O was dissolved in 100 mL of a mixed solvent (100 mL ethanol + 0.5 mL deionized water). The mouth of the CaCl₂ solution bottle was sealed with plastic wrap and placed on top of a bottle containing 10 g of ammonium bicarbonate (also sealed). Both bottles were then placed in a sealed desiccator for 48 hours. After the reaction was complete, the white product was collected by centrifugation (8000 rpm, 5 min). The obtained product was washed three times with ethanol and finally dispersed in ethanol for later use.

[0058] Example 2 Preparation of hCaCO3 Dissolve 20 mg of Na3PO4 in 4 mL of deionized water to prepare a 5 mg / mL Na3PO4 aqueous solution. Slowly add 0.5 mL of 5 mg / mL ACC ethanol solution to the Na3PO4 aqueous solution and stir the mixture at room temperature for half an hour. After the reaction is complete, transfer the mixture to a centrifuge tube and collect the product by centrifugation (8000 rpm, 5 min). Wash the obtained product three times with ethanol and finally freeze-dry it for later use.

[0059] Example 3 Preparation of hCaCO3-MnCO Weigh 1.5 mg of MnCO and dissolve it in 1 mL of methanol. Then, slowly add 1 mL of a 0.5 mg / mL hCaCO3 methanol solution to the MnCO methanol solution. Concentrate the solvent to 500 μL under vacuum drying at room temperature. Finally, collect the product by centrifugation (8000 rpm, 5 min). Wash the obtained product three times with methanol to obtain hCaCO3-MnCO, which is then dispersed in methanol for later use.

[0060] Example 4 Preparation of HCCM@DiR@hCaCO3-MnCO (1) Extraction of liver cancer cell membrane (HCCM) Hepa1-6 cells (approximately 2 × 10⁶) 7 Cells were washed twice with pre-cooled PBS, and then 1 mL of hypotonic cell lysis buffer (containing 0.5 mM PMSF and protease inhibitors) was added. The cells were incubated at 4°C for 30 min. Subsequently, they were homogenized (20000 psi, 3 times) and sonicated (100 W, 3 s / 3 s pulses, 3 min total). The lysis buffer was centrifuged at 4°C, 2000 g for 10 min to remove nuclei and unlysed cells, and then centrifuged at 4°C, 20000 g for 30 min to collect the cell membrane pellet. The resulting membrane pellet was resuspended in 1 mL of PBS (pH 7.4), and the protein concentration was determined using the BCA method and adjusted to 1 mg / mL. -1 Store at 4°C for later use.

[0061] (2) Extrusion coating The hCaCO3-MnCO nanoparticles (1 mg) obtained in Example 3 were redispersed in 1 mL of PBS and mixed with HCCM (1 mL, 1 mg / mL) from step (1). -1Mix the ingredients to a total volume of 2 mL. Incubate the mixture in a gentle shaker at 37°C and 100 rpm for 30 min to promote initial adsorption. Subsequently, transfer the mixture to an Avanti Mini-Extruder and extrude it 21 times each through 400 nm, 200 nm, and 100 nm polycarbonate porous membranes (heated in a 37°C water bath) to ensure that the cell membrane is completely and uniformly coated on the surface of the nanoparticles, forming HCCM@hCaCO3-MnCO.

[0062] (3) Load of DiR Add 2 mL of the extruded HCCM@hCaCO3-MnCO suspension (0.5 mg / mL) to the 0.5 mg / mL solution. -1 Add 0.1 mL of DiR methanol solution (0.1 mg / mL) to the solution. -1 The mixture was stirred at 300 rpm for 1 hour in the dark at room temperature to allow DiR to adsorb / embed in the cell membrane lipid layer. Subsequently, it was centrifuged at 12000g for 10 minutes at 4°C, the supernatant was discarded, and the mixture was washed three times with PBS. Finally, it was resuspended in 1 mL of PBS to obtain HCCM@DiR@hCaCO3-MnCO, and stored at 4°C in the dark.

[0063] Implementation effect evaluation 1. Characterization by transmission electron microscopy (TEM) ACC, hCaCO3, and hCaCO3-MnCO were each dispersed in ethanol or methanol. A small amount was dropped onto a copper grid and, after being completely dried, their morphology and structure were observed using a Hitachi H-800 TEM (operating voltage 200kV, magnification 100000×).

[0064] The results are as follows Figure 1 As shown, amorphous calcium carbonate nanoparticles (ACC) with a spherical structure were successfully synthesized. Figure 1 A). Subsequently, uniform hollow calcium carbonate nanoparticles (hCaCO3) were obtained through a trisodium phosphate dissolution-recrystallization process. Figure 1 B). Subsequently, MnCO was loaded into the cavity of hCaCO3 using an impregnation method. Figure 1 C).

[0065] 2. Cytotoxicity test (CCK-8 assay) (1) L929 cytotoxicity assessment: L929 cells were seeded at a density of 7000 cells / well in 96-well plates and cultured for 24 hours. After cell attachment, the old culture medium was discarded, and 100 μL of DMEM complete medium containing different concentrations (0, 12.5, 25, 50, 100, 200, 400 μg / mL) of HCCM@DiR@hCaCO3 nanomaterials was added to each well. Six replicates were made for each group. Incubation continued for another 24 hours. Subsequently, 10 μL of LCK-8 reagent was added to each well, and the cells were incubated in the dark for 1 hour. Finally, the absorbance was measured at 450 nm using a microplate reader to calculate cell viability.

[0066] The results are as follows Figure 2 As shown, below 200 μg / mL, HCCM@DiR@hCaCO3 had little effect on the survival of normal fibroblasts (L929), indicating good biocompatibility of the platform in non-tumor cells. Even at a high concentration of 400 μg / mL, cell viability remained at an acceptable level (>70%), demonstrating a high safety window. This provides basic safety evidence for further in vivo use.

[0067] (2) Evaluation of Hepa1-6 cytotoxicity and study of the effects of NIR irradiation: Hepa1-6 cells were seeded at a density of 7000 cells / well in 96-well plates and cultured for 24 hours. After cell attachment, the cells were divided into four groups: HCCM@DiR@hCaCO3 (12.5, 25, 50, 100, 200 μg / mL), HCCM@DiR@hCaCO3+NIR (12.5, 25, 50, 100, 200 μg / mL), HCCM@DiR@hCaCO3-MnCO (12.5, 25, 50, 100, 200 μg / mL), and HCCM@DiR@hCaCO3-MnCO+NIR (12.5, 25, 50, 100, 200 μg / mL). Each treatment had six replicates. Four to six hours after material addition, for the groups containing "+NIR", each well was irradiated with an 808 nm laser for 5 minutes. All groups (including NIR-irradiated and unirradiated groups) were incubated in an incubator for 20 hours. After incubation, the cells were gently washed 2-3 times with PBS. Then, CCK-8 reagent was added to each well, and the cells were incubated in the dark for 1 hour. Finally, the absorbance was measured at 450 nm using a microplate reader for subsequent data analysis.

[0068] The results are as follows Figure 3As shown, HCCM@DiR@hCaCO3 treatment alone had little effect on the viability of Hepa1-6 cells. However, after the introduction of MnCO, a significant decrease in cell viability was observed in the HCCM@DiR@hCaCO3-MnCO group at the same concentration, suggesting that MnCO loading can effectively enhance the killing effect on tumor cells. Furthermore, under 808nm NIR irradiation, the cell survival rate of the HCCM@DiR@hCaCO3-MnCO+NIR group was significantly lower than that of other groups, exhibiting a clear concentration-dependent inhibitory effect. This indicates a synergistic effect between the photothermal effect and the MnCO-induced effect in the nanoplatform of this invention, enabling highly efficient killing of tumor cells at lower doses.

[0069] 3. Study on ROS levels in Hepa1-6 cells under different treatment conditions Hepa1-6 cells were fed at a rate of 2 × 10⁻⁶ 4 ~3×10 4 Cells were seeded at a density of cells / well in 48-well plates and cultured for 24 hours. After cell adhesion, the cells were divided into 6 groups: blank control (culture medium only), NIR irradiation alone, HCCM@DiR@hCaCO3, HCCM@DiR@hCaCO3+NIR, HCCM@DiR@hCaCO3-MnCO, and HCCM@DiR@hCaCO3-MnCO+NIR. Each treatment had 6 replicates. 4–6 hours after material addition, for the groups containing "+NIR", each well was irradiated with an 808 nm laser for 5 minutes. All groups (including NIR irradiated and unirradiated groups) continued to incubate for 6 hours. After incubation, the cells were gently washed 2–3 times with PBS. Then, the DCFH-DA probe was added to each well, and the cells were incubated at 37°C in the dark for 30 minutes. After incubation, the cells were washed 3 times with PBS to remove excess probe. Finally, 1 mL of PBS was added, and the cells were immediately photographed using a fluorescence microscope.

[0070] The results are as follows Figure 4 As shown, DCFH-DA probe staining results revealed only weak background fluorescence in the Control group and the NIR-only group. However, the strongest green fluorescence signal was observed in the synergistic treatment group (HCCM@DiR@hCaCO3-MnCO+NIR). This indicates that this treatment group induced the highest level of oxidative stress (ROS burst) within the cells.

[0071] 4. Intracellular Ca 2+ Horizontal detection (Fluo-4 AM method) Following the same cell treatment protocol as the Hepa1-6 cell ROS level study, after treating each group of cells, 1 mL of serum-free medium containing Fluo-4 AM probe (final concentration 5 μM) was added to each well, and the cells were incubated at 37°C in the dark for 30 minutes. After incubation, the cells were washed three times with PBS to remove excess probe. Finally, 1 mL of PBS was added, and the cells were immediately photographed using a fluorescence microscope for subsequent quantitative analysis.

[0072] The results are as follows Figure 5 As shown, the Ca in the PBS group and the NIR group 2+ The weakest fluorescence indicates that intracellular Ca2+ under basal conditions... 2+ The level is low and stable; light alone does not cause significant Ca2+ damage. 2+ Changes in cell load. A slight increase in fluorescence was observed in the HCCM@DiR@hCaCO3 group, suggesting that the hollow calcium carbonate nucleus enters the cell and gradually decomposes under acidic conditions, slowly releasing Ca. 2+ This causes a certain degree of Ca 2+ Increased. HCCM@DiR@hCaCO3-MnCO group Ca 2+ The signal was significantly higher than in the groups mentioned above, indicating that the introduction of MnCO and the subsequent triggered cellular stress response can synergistically promote Ca2+. 2+ Overload. HCCM@DiR@hCaCO3-MnCO+NIR group Ca 2+ The fluorescence was the strongest, superior to the unirradiated HCCM@DiR@hCaCO3-MnCO group, indicating that the NIR-induced photothermal effect not only accelerates the decomposition of CaCO3 nuclei to release Ca... 2 + It may also further promote Ca2+ through pathways such as activating temperature-sensitive ion channels. 2+ Inflow, thus forming more pronounced Ca 2+ Overload, consistent with its stronger cytotoxicity.

[0073] 5. Detection of intracellular CRT and HMGB1 levels in Hepa1-6 cells by different treatment groups (ELISA method) Following the same cell treatment procedure as the Hepa1-6 cell ROS level study, after treating each group of cells, cell supernatant and cell lysate were collected. The expression levels of intracellular calreticulin (CRT) and high mobility group box 1 (HMGB1) proteins were detected using an ELISA kit, and then quantitative analysis was performed using GraphPad Prism 9.5.1.

[0074] The results are as follows Figure 6As shown in the figure, the CRT levels in the Control group and the NIR-only irradiation group were similar and both were at a low level. The CRT level in the HCCM@DiR@hCaCO3-only group was slightly increased, but the difference was not significant compared with the first two groups. After NIR irradiation, the CRT level in the HCCM@DiR@hCaCO3+NIR group was significantly increased, and the CRT level in the HCCM@DiR@hCaCO3-MnCO+NIR group was the highest. This indicates that NIR irradiation can significantly enhance the CRT release induced by HCCM@DiR@hCaCO3 and HCCM@DiR@hCaCO3-MnCO, suggesting that it promotes the occurrence of immunogenic cell death (ICD).

[0075] In contrast to CRT, HMGB1 levels showed a decreasing trend. HMGB1 levels were high and similar in the Control group and the NIR-only irradiation group. HMGB1 levels were slightly decreased in the HCCM@DiR@hCaCO3-only group. However, after NIR irradiation, HMGB1 levels were significantly reduced in the HCCM@DiR@hCaCO3+NIR group, with the lowest levels in the HCCM@DiR@hCaCO3-MnCO+NIR group. This decreasing trend complements the ICD-promoting effect of CRT, suggesting a synergistic mechanism of the nanosystem in balancing immune activation and inhibition.

[0076] 6. Detection of intracellular carbon monoxide (CO) release (COP-1 probe method) Following the same cell treatment procedure as the Hepa1-6 cell ROS level study, after treating each group of cells, 1 mL of serum-free medium containing the COP-1 probe (final concentration 10 μM) was added to each well, and the cells were incubated at 37°C in the dark for 1 hour. After incubation, the cells were washed three times with PBS to remove excess probe. Finally, 1 mL of PBS was added, and fluorescence microscopy was immediately used to photograph the cells and perform subsequent fluorescence intensity analysis. The results are shown in Table 1.

[0077] Table 1. Effects of different treatment groups on intracellular CO release in Hepa1-6 cells

[0078] As shown in Table 1, the PBS group had the lowest COP-1 fluorescence signal, indicating that only a very low basal CO level existed in the cells under blank treatment conditions. The fluorescence signal of the NIR group alone was slightly higher than that of the PBS group, but still weak overall, indicating that near-infrared irradiation itself had a limited effect on intracellular CO production. In contrast, the COP-1 fluorescence of the HCCM@DiR@hCaCO3 group was slightly enhanced, indicating that the hollow CaCO3 nanonucleus partially decomposed in the acidic intracellular environment, which was beneficial to promoting the structural depolymerization and related reactions of the drug-loaded nanoplatform, thereby increasing the local CO level. After further introducing MnCO, the COP-1 fluorescence signal of the HCCM@DiR@hCaCO3-MnCO group was significantly higher than that of the HCCM@DiR@hCaCO3 group, proving that MnCO decomposed and released CO under acidic and reducing conditions in the cells, giving the nanoplatform a significant CO supply function. After external NIR irradiation, the COP-1 fluorescence of the HCCM@DiR@hCaCO3-NIR group further increased, suggesting that the photothermal effect can promote the disintegration of CaCO3 nuclei and the depolymerization of nanostructures, thereby amplifying CO generation to a certain extent. In the HCCM@DiR@hCaCO3-MnCO+NIR group, which was subjected to both MnCO and NIR irradiation, the COP-1 fluorescence signal was the strongest, significantly higher than all other groups. This indicates that the photothermal effect and MnCO-mediated CO release have a significant synergistic effect, which can achieve the highest level of CO loading and release in tumor cells, and is more conducive to the exertion of gas therapy and its synergistic effect.

[0079] 7. Comparative experiment on photothermal efficiency and tumor cell targeting enrichment Nanoplatforms were prepared using different membrane sources (HCCM, RAW, RBC) and different dyes (DiR, ICG, IR780), following the same method as in Example 4. Photothermal efficiency was measured by irradiation with an 808nm laser (1.0W / cm²). 2 The percentage increase in temperature was measured (5 min). Tumor cell targeting enrichment was determined by detecting the fluorescence intensity of intracellular nanomaterials using flow cytometry, and the selective uptake percentage of different nanomaterials in Hepa1-6 cells was calculated. The results are shown in Table 2.

[0080] Table 2 Comparison of photothermal efficiency or tumor cell targeting enrichment in different groups

[0081] Table 2 shows that the membrane source and dye type jointly determine the photothermal heating rate and tumor targeting efficiency: when HCCM is used as the membrane and DiR as the dye, the temperature increase is 152% and the selective uptake by Hepa1-6 cells is 161%, both of which are the highest values. This indicates that the homologous targeting ability of the HCCM membrane, combined with the high photothermal conversion of DiR, achieves the optimal "membrane-dye" match. After switching to ICG or IR780, the temperature increase on the same HCCM membrane platform drops to 95% and 90%, and the uptake also drops to 91% and 82%, confirming that the difference in the photothermal coefficient of the dye itself can be immediately reflected in the numerical values. If DiR is retained but RAW or RBC membranes are used, the temperature increase further drops to 78% and 75%, and the uptake drops to 71% and 66% simultaneously, indicating that the heterologous membrane loses the "homogeneous homing" advantage, and the decrease in targeting enrichment directly drags down the photothermal efficiency. The membrane-free group (DiR@hCaCO3-MnCO) has a temperature increase of only 55% and an uptake of only 40%. The percentage is the lowest value in the table, which, conversely, proves that the membrane component is key to enhancing tumor accumulation and thus amplifying the photothermal effect. In summary, the data in the table clearly shows that the combination of HCCM membrane and DiR dye leads in both "temperature rise" and "tumor accumulation" indicators, making it the preferred formulation for subsequent in vivo experiments.

[0082] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0083] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A biomimetic nanoplatform for synergistic therapy, characterized in that, The biomimetic nanoplatform includes a core and a shell. The core comprises hollow calcium carbonate and manganese carbonate, and the shell comprises a cell membrane and a fluorescent dye. The shell covers the surface of the core.

2. The biomimetic nanoplatform according to claim 1, characterized in that, The cell membrane includes liver cancer cell membranes; the fluorescent dye includes DiR.

3. The method for preparing the biomimetic nanoplatform according to any one of claims 1-2, characterized in that, Includes the following steps: Synthesis of amorphous calcium carbonate: Calcium chloride is dissolved, sealed, placed in a closed dry container containing ammonium bicarbonate, reacted, centrifuged, washed, and dispersed in an organic solvent to obtain an amorphous calcium carbonate dispersion. Preparation of hollow calcium carbonate: Dissolve sodium phosphate, add amorphous calcium carbonate dispersion dropwise to sodium phosphate solution, react, centrifuge, wash, and freeze dry to obtain hollow calcium carbonate; Preparation of hollow calcium carbonate-manganese carbonate nanoparticles: Manganese carbonate was dissolved to obtain a manganese carbonate solution. A hollow calcium carbonate dispersion was added to the manganese carbonate solution, incubated, concentrated, centrifuged, and washed to obtain hollow calcium carbonate-manganese carbonate nanoparticles. Cell membrane coating: Cell membrane extraction, hollow calcium carbonate-manganese carbonate nanoparticles were dispersed in PBS, mixed with cell membrane, incubated, and extruded to obtain cell membrane coated nanoparticles; Fluorescent dye loading: A fluorescent dye solution was added to a suspension of cell membrane-coated nanoparticles, stirred in the dark, centrifuged, washed, and resuspended to obtain a biomimetic nanoplatform.

4. The preparation method according to claim 3, characterized in that, The synthesis of the amorphous calcium carbonate includes the following steps: Calcium chloride solution was obtained by dissolving calcium chloride in a mixed solvent of ethanol and water. The calcium chloride solution was sealed and placed in a sealed dry container containing ammonium bicarbonate. The reaction was carried out, the product was collected by centrifugation, washed, and dispersed in ethanol to obtain an amorphous calcium carbonate dispersion. The mass ratio of calcium chloride to ammonium bicarbonate is 1:(30~60).

5. The preparation method according to claim 3, characterized in that, The preparation of the hollow calcium carbonate includes the following steps: Dissolve sodium phosphate in water, add amorphous calcium carbonate dispersion dropwise to the sodium phosphate solution, stir the reaction, collect the product by centrifugation, wash, and freeze-dry to obtain hollow calcium carbonate; The mass ratio of the amorphous calcium carbonate to the sodium phosphate is 1:(6~10).

6. The preparation method according to claim 3, characterized in that, The preparation of the hollow calcium carbonate-manganese carbonate nanoparticles includes the following steps: Manganese carbonate was dissolved in methanol, and a methanol dispersion of hollow calcium carbonate was added to the manganese carbonate methanol solution. The mixture was incubated, vacuum dried and concentrated, centrifuged, and washed to obtain hollow calcium carbonate-manganese carbonate nanoparticles. According to the mass ratio, the manganese carbonate : the hollow calcium carbonate is (1~5):

1.

7. The preparation method according to claim 3, characterized in that, In the cell membrane encapsulation process, the cell membrane extraction includes the following steps: Collect cells, wash, add lysis buffer, let stand, homogenize, sonicate on ice, centrifuge to remove cell nuclei, centrifuge to collect membrane precipitate, and resuspend to obtain cell membrane suspension; According to the mass ratio, the hollow calcium carbonate-manganese carbonate nanoparticles: the cell membrane is (1~2):(1~2).

8. The preparation method according to claim 3, characterized in that, The loading of the fluorescent dye includes the following steps: A methanol solution of fluorescent dye was added to a suspension of cell membrane-coated nanoparticles, stirred in the dark, centrifuged, washed, and resuspended in PBS to obtain a biomimetic nanoplatform. The ratio of cell membrane-coated nanoparticles to fluorescent dye is (80~120):1 by mass.

9. The application of the biomimetic nanoplatform as described in claims 1-2, or the biomimetic nanoplatform obtained by the preparation method described in claims 3-8, in the preparation of drugs for treating liver cancer.

10. A drug for treating liver cancer, characterized in that, The drug comprises the biomimetic nanoplatform as described in claims 1-2, or the nanoplatform obtained by the preparation method described in claims 3-8.

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