Multifunctional manganese-based hybrid nanoplatform based on NK cell membrane modification and its preparation method and application

Abstract

The present invention relates to a multifunctional manganese-based hybrid nano-platform based on NK cell membrane modification, a preparation method and application thereof. By selecting a high-branched dendritic polymer PSeSeS modified with a diselenide bond as a carrier, the water solubility and stability are improved by partially modifying mPEG and phenylboronic acid, and the material is given specific reactivity to the tumor microenvironment. The terminal and tertiary amine groups of PSeSeS are further used to reduce potassium permanganate to generate MnO₂ nanoparticles, alleviate the hypoxic environment of the tumor, enhance photothermal therapy, and also generate reactive oxygen. Then, doxorubicin and cGAMP are loaded, combined with chemotherapy and immunotherapy, to enhance the immune system's ability to recognize and eliminate tumors; NK cell membrane modification is further used to improve targeting and in vivo stability. The nano-platform of the present invention overcomes the shortcomings of poor tumor microenvironment responsiveness, weak targeting, imprecise drug release and single treatment method in the prior art, and can significantly improve the effect and safety of liver cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a multifunctional manganese-based hybrid nano-platform based on NK cell membrane modification, and a preparation method and application thereof. Background Art

[0002] Liver cancer is a malignant tumor with high morbidity and mortality worldwide. It poses a serious threat to human health due to its invasiveness and high recurrence rate. Although traditional treatments, such as surgery, chemotherapy, and radiotherapy, have prolonged the survival of patients to a certain extent, the efficacy of these methods is limited due to the high heterogeneity of liver cancer cells and the complex tumor microenvironment. In particular, the drug resistance, systemic toxicity, hypoxia, and high glutathione (GSH) of chemotherapeutic drugs in the tumor microenvironment have greatly limited their clinical application. Therefore, there is an urgent need to develop a multifunctional nanoplatform that can target tumors and respond to changes in their microenvironment.

[0003] The rapid development of nanoscale drug delivery systems (NDDS) has improved the integration of immunotherapy with other treatment modalities. With the help of a variety of nanomaterials, NDDS can efficiently and precisely deliver antigens, adjuvants, and other immunoactive compounds to tumors or lymphatic tissues. This reduces side effects and enhances immune responses. Similarly, well-designed NDDS can promote the production of reactive oxygen species (ROS) through various dynamic treatments such as photodynamic, sonodynamic, and chemodynamic therapies. These dynamic nanodrugs fully synergize with immunotherapeutic agents to effectively kill cancer cells, produce immunogenic cell death (ICD), overcome the immunosuppression of the TME, and transform "cold tumors" into "hot tumors", thereby effectively regressing tumors. In particular, NDDSs containing transition metal (such as Mn, Cu, Fe, Co) ions or oxides have received increasing attention because they can respond to specific TME triggers, including glutathione (GSH), hydrogen peroxide (H 2 O 2 ) and acidic pH, and catalyzes the H in TME via a Fenton or Fenton-like reaction 2 O 2 The metal-based nanocatalysts can be converted into a large number of toxic oxidized hydroxyl radicals (•OH), thus achieving effective chemodynamic therapy (CDT). Many metal-based nanocatalysts can also perform tumor magnetic resonance (MR) imaging, thus promoting cancer treatment diagnosis.

[0004] In recent years, with the introduction of the concept of metal immunology, more and more evidence has shown that many transition metals exhibit inherent immunological functions; therefore, the application of metal-based NDDS in tumor treatment has become increasingly important. Among the many metal-based NDDS, Mn-based NDDS is particularly popular and has been widely used in the fields of high-efficiency MRI imaging, TME regulation, CDT, etc. Recent studies have emphasized that Mn 2+ Acts as an immunostimulant. 2+ It not only directly stimulates the cyclic guanosine monophosphate (GMP)-adenylic acid (AMP) synthase (cGAS)-stimulator of interferon genes (STING) pathway, but also promotes the efficacy of cGAMP on STING activation. This activation of the cGAS-STING pathway induces the production of type I interferon (IFN), thereby improving antigen presentation by dendritic cells (DCs) and enhancing anti-tumor T cell responses. With these advantages, innovative nanoplatforms such as amorphous porous manganese phosphate nanoparticles (NPs) biomineralized manganese dioxide (MnO 2 ), Mn 2+ Coordination micelles, and metallophenolic networks have been successfully developed for cGAS-STING related combined immunotherapy.

[0005] Although manganese-based nanosystems have emerged as a powerful weapon for tumor immunotherapy due to their multiple functionalities in immune activation, TME remodeling, and MR imaging monitoring, there are few reports integrating all the advantages of manganese into one nanoplatform to enhance cancer therapy, especially through the combination of natural enzyme-based catalysts, metal-based catalysts, and immunostimulants. Summary of the invention

[0006] In view of the above technical deficiencies, the present invention provides a highly efficient nano-therapeutic platform that can respond to the tumor microenvironment, improve drug targeting, prolong in vivo circulation time and combine multiple treatment methods for the comprehensive treatment of liver cancer. The specific scheme is as follows.

[0007] The first aspect of the present invention provides a method for preparing a multifunctional manganese-based hybrid nanoplatform based on NK cell membrane modification, comprising the following steps:

[0008] 1) Synthesis of hyperbranched polymer PSeSeS;

[0009] 1.1 Dissolve selenocystamine dihydrochloride in pure water to obtain solution A, dissolve acryloyl chloride in dichloromethane to obtain solution B, and dissolve sodium hydroxide in water to obtain solution C; add solution B and solution C alternately dropwise to solution A under ice bath conditions, remove the ice bath after the addition is complete, and place the reaction solution at room temperature to continue the reaction. After the reaction is complete, add 10 times the volume of dichloromethane to extract, then wash, and finally remove the solvent by rotary evaporation, and vacuum dry to obtain a white powdery CSeC product;

[0010] 1.2 At room temperature, prepare a mixed solvent of pure water and methanol in a volume ratio of 1:3, add calcium chloride, then add the product CSeC of step 1.1 thereto, and stir until completely dissolved; then add piperazine solution obtained by dissolving piperazine in methanol for the first time, then condense and reflux at 50°C for 45-50h, add piperazine solution again and continue to react for 10-15h, after the reaction is completed, collect the product and adjust its pH to 4-6, then dialyze in pure water, and freeze-dry to obtain a white flocculent product PSeSeS;

[0011] 1.3 The PSeSeS dendrimer obtained in step 1.2 was reacted with mPEG-Mal under stirring at 30°C, and then freeze-dried in a dialysis bag to obtain PSeSeS-mPEG powder; the obtained PSeSeS-mPEG was then reacted with Br-PBA in DMSO solvent under stirring in a water bath, and finally the reaction mixture was dialyzed with water and freeze-dried to obtain SePP powder.

[0012] 2) Preparation of MSePP NPs;

[0013] KMnO 4 The aqueous solution is added to the SePP dendrimer solution obtained in step 1) and reacted under stirring at room temperature, and then dialyzed and freeze-dried to obtain dark brown powder of MSePP NPs;

[0014] 3) NK cell membrane modification;

[0015] The MSePP NPs obtained in step 2) were mixed with NK cell membrane fragments and NKCM@MSePP was prepared by extrusion method;

[0016] 4) Loading of DOX and cGAMP

[0017] The NKCM@MSePP obtained in step 3) is mixed with DOX and cGAMP at room temperature to prepare the nano-platform.

[0018] The present invention selects a highly branched dendrimer (PSeSeS) modified with a diselenide bond as a nanocarrier, and partially modifies the surface of PSeSeS with mPEG and phenylboronic acid (PBA), which not only improves the water solubility and stability of the nanomaterial, but also gives the material specific reactivity to the tumor microenvironment. The terminal and tertiary amine groups of PSeSeS are further used to reduce potassium permanganate to generate manganese dioxide (MnO 2 ) nanoparticles to relieve the hypoxic environment of the tumor, enhance photothermal therapy, and generate reactive oxygen species (ROS) through a Fenton-like reaction to further kill tumor cells. Then, the chemotherapy drug doxorubicin (DOX) and the STING signaling pathway agonist cGAMP are loaded, combined with chemotherapy and immunotherapy, and the immune system's ability to recognize and eliminate tumors is enhanced by inducing immunogenic cell death (ICD); NK cell membranes are further used to modify nanoparticles to improve the targeting and in vivo stability of nanomaterials.

[0019] Preferably, in the step 1.1, selenocystamine dihydrochloride: acryloyl chloride: sodium hydroxide = 2.0-3.0 g: 2.5-4 mL: 1.5-2.0 g; solution A: solution B: solution C = 10-15 mL: 5-8 mL: 4-5 mL, and the reaction time is 12 h to 24 h.

[0020] Preferably, in step 1.2, the mixed solvent of pure water and methanol: calcium chloride: CSeC: piperazine = 8–12 mL: 0.2–0.25 g: 0.5–0.8 g: 150-180 μL; in the piperazine solution, piperazine: methanol = 150-180 μL: 300-350 μL; the amount of piperazine solution added for the first time is: 150-180 μL; the amount added additionally is: 300-350 μL.

[0021] Preferably, in step 1.3, PSeSeS dendrimer: mPEG-Mal: water = 40–60 mg: 40–50 mg: 8–12 mL; reaction time is: 20–30 hours.

[0022] Preferably, in step 1.3, the molecular weight cutoff of the dialysis bag is: 8000-12000 Da; and the dialysis time is: 2-4 days.

[0023] Preferably, in step 1.3, PSeSeS-mPEG:Br-PBA:DMSO=40-60mg:10–15 mg:18–22 mL, and the reaction temperature is: 60–80°C.

[0024] Preferably, in step 1.4, KMnO 4: SePP dendrimer = 5.0–10.0 mg: 40–60 mg, KMnO 4 The concentration of the aqueous solution is: 0.2–0.3 mg / mL, the addition rate is: 0.8–1.2 mL / min; in the SePP dendrimer solution, SePP dendrimer: water = 40–60 mg: 8–12 mL; the reaction time is: 0.5–2 hours; the dialysis time is: 0.5–2 days.

[0025] Preferably, in step 4), NKCM@MSePP: DOX: cGAMP = 100 mg: 5~20 mg: 2~3.5 mg.

[0026] Another aspect of the present invention also provides a multifunctional manganese-based hybrid nanoplatform based on NK cell membrane modification prepared by the preparation method.

[0027] Another aspect of the present invention also provides the use of a multifunctional manganese-based hybrid nanoplatform based on NK cell membrane modification in the preparation of liver cancer drugs.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. Enhanced responsiveness of the tumor microenvironment.

[0030] By introducing diselenide bonds into the dendrimer (PSeSeS) structure, these bonds are reductively cleaved under the high glutathione (GSH) concentration in tumor cells, leading to the disintegration of the nanocarrier and the release of the payload. 2 ) Nanoparticles can release reactive oxygen species (ROS) in a reducing environment, further promoting the breakage of diselenide bonds, releasing drugs, and enhancing the synergistic effect of photothermal therapy and chemotherapy. Therefore, the nanocarrier of the present invention has stronger responsiveness in the tumor microenvironment, can effectively cope with the highly reducing conditions in the tumor, and accurately release the loaded drugs, thereby improving the therapeutic effect of the drugs and reducing side effects.

[0031] 2. Improved biocompatibility and immune escape ability.

[0032] By partially modifying the surface of PSeSeS with polyethylene glycol (mPEG) and phenylboronic acid (PBA), this modification not only improves the water solubility and biocompatibility of the nanocarrier, but also enhances the immune escape ability, so that the nanocarrier can circulate in the body for a long time and effectively reach the tumor site, reducing the early clearance of the drug. As a result, the nanoplatform of the present invention has better biocompatibility and prolonged blood circulation time, thereby improving the stability and effectiveness of the drug in the body and reducing nonspecific toxicity.

[0033] 3. Tumor targeting is significantly enhanced.

[0034] The present invention enhances the targeting ability of nanoparticles at the tumor site by modifying nanoparticles with NK cell membranes and relying on the specific recognition mechanism between natural receptors on the NK cell membranes and tumor cell surface antigens. This strategy has better targeting and recognition capabilities than traditional antibody or small molecule ligand modification. The resulting nanocarrier has stronger tumor targeting and can be more effectively enriched at the tumor site, thereby improving treatment efficiency and reducing the impact on healthy tissues.

[0035] 4. Multiple therapeutic synergistic effects.

[0036] The nanocarrier of the present invention can simultaneously load the chemotherapy drug doxorubicin (DOX) and the STING signaling pathway agonist cGAMP. 2 The ROS generation of nanoparticles enhances the effect of photothermal therapy, doxorubicin performs chemotherapy to kill tumor cells, and cGAMP activates the STING pathway to initiate an immune response, thereby achieving a synergistic effect of multiple therapies. The nanoplatform of the present invention can significantly improve the anti-tumor effect through the synergistic effect of chemotherapy, photothermal therapy and immunotherapy, and reduce the limitations of a single treatment method.

[0037] 5. Precise drug release.

[0038] The present invention realizes precise drug release through the tumor microenvironment responsiveness of the diselenide bond, and effectively controls the release of drugs at the tumor site by combining the redox properties of manganese dioxide. At the same time, NK cell membrane modification further improves the enrichment of drugs at the tumor site, allowing the drug to more effectively target tumor tissues, accurately release the loaded drug at the tumor site, improve treatment efficiency, and reduce systemic toxic side effects.

[0039] 6. Long-term immune activation.

[0040] By loading the STING signaling pathway agonist cGAMP, it can stimulate the body's innate immune response and initiate anti-tumor immune response. Combined with NK cell membrane-modified nanocarriers, it enhances the immune system's recognition and clearance of tumors, continuously activates the body's immune system, and effectively enhances tumor immune surveillance and clearance functions, achieving long-term inhibition of tumor recurrence, thereby achieving long-term anti-tumor effects.

[0041] The nanoplatform of the present invention overcomes the shortcomings of the prior art, such as poor tumor microenvironment responsiveness, weak targeting, imprecise drug release and single treatment method, and can significantly improve the effect and safety of liver cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is the release curve of DOX and manganese ions;

[0043] Figure 2 is the photothermal curve of MSePP NPs;

[0044] Figure 3 Endocytosis diagram of Bel-7402 cells treated with different experimental groups;

[0045] Figure 4 The cytotoxicity of different experimental groups to Bel-7402 cells. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0047] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0048] Embodiment 1:

[0049] This embodiment provides a multifunctional manganese-based hybrid nanoplatform NKCM@MSePP / DOX / cGAMP based on NK cell membrane modification, and the preparation method thereof is as follows:

[0050] 1) Preparation of hyperbranched polymer (PSeSeS)

[0051] 1.1 Dissolve 2.50 g of selenocystamine dihydrochloride in 11 mL of pure water to obtain solution A, dissolve 3.30 mL of acryloyl chloride in 3.30 mL of dichloromethane to obtain solution B, and dissolve 1.76 g of sodium hydroxide in 4.40 mL of water to obtain solution C.

[0052] Solution B and solution C were added dropwise alternately to solution A under ice bath conditions and stirred rapidly. After the addition was completed, the ice bath was removed and the reaction solution was placed at room temperature to continue the reaction for 6 h. After the reaction was completed, 10 times the volume of dichloromethane was added to extract it and washed with pure water 3 times. Finally, the solvent dichloromethane was removed by rotary evaporation at 45 ° C, and vacuum dried overnight to obtain a white powdery CSeC product.

[0053] 1.2 Preparation of GSH-responsive hyperbranched polymer (PSeSeS)

[0054] PSeSeS is prepared by Michael addition reaction of CSeC and piperazine. The specific steps are: prepare 10 mL of a mixed solvent of pure water and methanol (the volume ratio of pure water to methanol is 1 / 3) at room temperature, add 0.222 g of calcium chloride, then continue to add 0.64 g of BAC and stir until completely dissolved. Then dissolve 160 μL of piperazine in 300 μL of methanol and add it to the above reaction solution. Subsequently, condense and reflux at 50 °C for 48 h, and then add 320 μL of piperazine to continue the reaction for 12 h. After the reaction is completed, collect the product and adjust its pH to 4-6 with hydrochloric acid, then dialyze in pure water for 2 days (MWCO = 1000), and freeze-dry to obtain the final white flocculent product PSeSeS.

[0055] 1.3 Preparation of PSeSeS-mPEG-PBA (SePP)

[0056] First, PSeSeS dendrimer (50 mg in 10 mL water) was reacted with mPEG-Mal (48.1 mg in 10 mL water) under magnetic stirring at 30 °C for 24 h. Then, the mixture was dialyzed against water for 3 days (2 L, 9 times) using a dialysis bag with a MWCO of 10000 Da and lyophilized to obtain PSeSeS-mPEG powder. Then the obtained PSeSeS-mPEG (50 mg in 10 mL DMSO) was reacted with Br-PBA (11.7 mg in 5 mL DMSO) in a 70 °C water bath under magnetic stirring for 24 h. The reaction mixture was dialyzed against water for 3 days (2 L, 9 times) using a dialysis bag with a MWCO of 10000 Da and finally lyophilized to obtain SePP powder.

[0057] 2) Preparation of MSePP NPs

[0058] KMnO4 (8.0 mg, 0.25 mg / mL aqueous solution) was added to the SePP dendrimer solution (50 mg, 10 mL water) at a rate of 1 mL / min using a syringe pump and reacted under magnetic stirring at room temperature for 1 hour. Afterwards, the mixture was dialyzed against water for 1 day (2 L, three times) using a dialysis bag with a molecular weight cutoff (MWCO) of 10 000 Da and then freeze-dried to obtain a dark brown powder of MSePP NPs.

[0059] 3) NK cell membrane modification

[0060] First, NK cell membrane fragments were mixed evenly in deionized water. Then MSePP NPs (w / w=1:1) were added, and the mixture of cell membrane fragments and MSePP NPs was co-extruded through polycarbonate filters with different pore sizes (1 μm, 0.8 μm, 0.45 μm) using an Avanti liposome extruder. Finally, the extruded mixture was centrifuged at 8000 rpm for 10 minutes to collect NKCM@MSePP.

[0061] 4) Loading of DOX and cGAMP

[0062] The prepared 100 mg NKCM@MSePP was mixed with 20 mg DOX in 100 μL PBS at room temperature for 30 min to prepare the NKCM@MSePP / DOX complex. To further load cGAMP, the obtained MSePP / DOX complex was mixed with 3 mg cGAMP in 100 μL PBS for 30 min and then filtered to obtain NKCM@MSePP / DOX / cGAMP.

[0063] Comparative Example 1

[0064] This comparative example provides a nano-platform MSePP / DOX / cGAMP, and its preparation method refers to Example 1. Compared with Example 1, the difference is that step 3) NK cell membrane modification step is missing.

[0065] Test Example 1: Release of DOX and manganese ions

[0066] To investigate the release behavior of DOX and Mn ions under different reducing conditions, the prepared NKCM@MSePP / DOX / cGAMP nanoparticles were dispersed in PBS buffer with an initial DOX concentration of 100 µg / mL and a Mn ion concentration of 10 µg / mL, and 0 mM GSH (control group) and 10 mM GSH (experimental group) were added to simulate different reducing conditions in the tumor microenvironment. The samples were placed in a 10,000 Da molecular weight cutoff (MWCO) dialysis bag and gently stirred in a shaker at 37°C. The external solution was collected at different time points (0.5, 1, 3, 5, 7, 16, 24, 48, 72 hours), and the release of DOX (at 480 nm) was detected by UV-Vis spectrophotometer and Mn was detected by inductively coupled plasma mass spectrometry (ICP-MS). 2+ The experimental results will compare the release of DOX and Mn in the presence or absence of GSH. 2+ The cumulative release of the nanoparticles was used to evaluate the responsiveness and intelligent release ability of the nanoparticles to the reducing environment. Figure 1As shown in the figure, in the presence of GSH, DOX and manganese ions can be released faster and more.

[0067] Test Example 2: Photothermal Performance

[0068] In order to test the photothermal performance of MSePP NPs nanoparticles, the MSePP NPs nanoparticles obtained in step 2) were used to disperse different concentrations of MSePP NPs nanoparticles (31.25, 62.5, 125, 250 µg / mL) in 10 mL of deionized water, and then each solution was irradiated for 5 minutes using an 808 nm near-infrared laser (power density 1 W / cm²), and the temperature rise curve was plotted every 10 seconds. Figure 2 As shown, MSePP NPs have good photothermal properties. When the concentration is 62.5 μg / mL, the temperature rises by 49.7 °C under laser irradiation.

[0069] Test Example 3: Cell endocytosis

[0070] To evaluate the endocytosis efficiency of MSePP nanoparticles with or without NK cell membrane modification, Bel-7402 hepatoma cells were first cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO. 2 Cultured in an incubator. When the cells grew to 70–80% confluence, they were replaced with NKCM@MSePP / DOX / cGAMP or MSePP / DOX / cGAMP-treated cells for different treatment times (0.5, 1, 2, 4, and 6 hours). After incubation, the cells were washed three times with PBS, and the fluorescence intensity was quantified by up-flow cytometry using Flowjo to compare the endocytosis efficiency of the differently modified nanoparticles. Figure 3 As shown, the fluorescence of the NKCM@MSePP / DOX / cGAMP group was stronger than that of the MSePP / DOX / cGAMP group at different times.

[0071] Test Example 4: Cytotoxicity

[0072] The CCK-8 method for detecting cell activity was used to evaluate the toxic effects of different components on human liver cancer cells Bel-7402. The specific steps are as follows: First, human liver cancer cells Bel-7402 were inoculated in a 96-well plate at a density of 5000 cells / well, and then placed in a carbon dioxide incubator to culture overnight. Subsequently, the original culture medium was aspirated and replaced with fresh complete culture medium extract containing NKCM@MSePP / DOX or NKCM@MSePP, with 5 parallels for each concentration. A portion of the wells in the NKCM@MSePP / DOX group were treated with light (808 nm, 1W / cm 2, 5min), and then cultured in an incubator for 24 h. After culture, the cells were washed once with PBS and 100 μL of fresh culture medium (containing 10% CCK-8) was added to each well. Incubated in an incubator for a period of time, the absorbance at a wavelength of 450 nm was detected and recorded using a microplate reader, and the cell survival rate was calculated using the following formula: Cell survival rate (%) = (absorbance of the experimental group - absorbance of the blank group) / (absorbance of the negative control group - absorbance of the blank group) × 100%.

[0073] The results are as follows Figure 4 As shown, the NKCM@MSePP / DOX+NIR group was able to kill tumor cells better.

[0074] From the above, it can be seen that the NKCM@MSePP / DOX / cGAMP nano-platform of the embodiment of the present invention, by introducing a diselenide bond into the PSeSeS structure, can release DOX and manganese ions faster and more at a higher glutathione (GSH) concentration, thereby enhancing the synergistic effect of photothermal therapy and chemotherapy; the photothermal effect is good; by modifying the NK cell membrane, the targeting ability of nanoparticles at the tumor site is enhanced by means of the specific recognition mechanism of the natural receptors on the NK cell membrane and the tumor cell surface antigens. The nano-platform of the present invention can significantly improve the anti-tumor effect of liver cancer through the synergistic effect of chemotherapy, photothermal therapy and immunotherapy, reducing the limitations of a single treatment method.

[0075] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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.

[0076] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a multifunctional manganese-based hybrid nanoplatform based on NK cell membrane modification, characterized in that it comprises the following steps: 1) Preparation of hyperbranched polymer PSeSeS 1.1 Dissolve 2.50 g of selenocystamine dihydrochloride in 11 mL of pure water to obtain solution A, dissolve 3.30 mL of acryloyl chloride in 3.30 mL of dichloromethane to obtain solution B, and dissolve 1.76 g of sodium hydroxide in 4.40 mL of water to obtain solution C; Solution B and solution C were added dropwise alternately to solution A under ice bath conditions and stirred rapidly. After the addition was completed, the ice bath was removed and the reaction solution was placed at room temperature to continue the reaction for 6 h. After the reaction was completed, 10 times the volume of dichloromethane was added to extract it and washed with pure water 3 times. Finally, the solvent dichloromethane was removed by rotary evaporation at 45°C, and vacuum dried overnight to obtain a white powdery CSeC product. 1.2 Prepare 10 mL of a mixed solvent of pure water and methanol at room temperature, wherein the volume ratio of pure water to methanol is 1 / 3, and add 0.222 g of calcium chloride, and then continue to add 0.64 g of CSeC obtained in step 1.1, and stir until completely dissolved; then dissolve 160 μL of piperazine in 300 μL of methanol and add it to the above reaction solution; then, condense and reflux at 50 °C for 48 h, and then add 320 μL of piperazine and continue to react for 12 h; after the reaction is completed, collect the product and adjust its pH to 4-6 with hydrochloric acid, and then dialyze it in pure water using a dialysis bag with MWCO = 1000 for 2 days; freeze-dry to obtain the final white flocculent product PSeSeS; 1.3 First, 50 mg of the PSeSeS dendrimer obtained in step 1.2 was dissolved in 10 mL of water, and 48.1 mg of mPEG-Mal was dissolved in 10 mL of water. The two were mixed and reacted at 30 °C for 24 hours under magnetic stirring. Then, the mixture was dialyzed with water for 3 days, 2 L, 9 times using a dialysis bag with a MWCO of 10,000 Da, and lyophilized to obtain PSeSeS-mPEG powder. Then, 50 mg of the obtained PSeSeS-mPEG powder was dissolved in 10 mL of DMSO, and 11.7 mg of Br-PBA was dissolved in 5 mL of DMSO. The two were mixed and reacted in a 70 °C water bath under magnetic stirring for 24 hours. The reaction mixture was dialyzed with water for 3 days, 2 L, 9 times using a dialysis bag with a MWCO of 10,000 Da, and finally lyophilized to obtain SePP powder. 2) Preparation of MSePP NPs A 0.25 mg / mL KMnO4 aqueous solution containing 8.0 mg KMnO4 was added to a SePP dendrimer solution at a rate of 1 mL / min using a syringe pump. The SePP dendrimer solution was obtained by dissolving 50 mg SePP in 10 mL water, and reacted under magnetic stirring at room temperature for 1 hour. After that, the mixture was dialyzed against water for 1 day, 2 L, three times using a dialysis bag with a molecular weight cutoff of 10,000 Da, and then freeze-dried to obtain a dark brown powder of MSePP NPs. 3) NK cell membrane modification First, NK cell membrane fragments were mixed evenly in deionized water, and then MSePP NPs were added at w / w=1:

1. The mixture of cell membrane fragments and MSePP NPs was co-extruded through polycarbonate filter membranes with pore sizes of 1 μm, 0.8 μm, and 0.45 μm using an Avanti liposome extruder. Finally, the extruded mixture was centrifuged at 8000 rpm for 10 minutes to collect NKCM@MSePP. 4) Loading of DOX and cGAMP The prepared 100 mg NKCM@MSePP was mixed with 20 mg DOX in 100 μL PBS at room temperature for 30 min to prepare the NKCM@MSePP / DOX complex, and then the obtained MSePP / DOX complex was mixed with 3 mg cGAMP in 100 μL PBS for 30 min and filtered to obtain NKCM@MSePP / DOX / cGAMP.

2. A multifunctional manganese-based hybrid nanoplatform based on NK cell membrane modification prepared by the preparation method as described in claim 1.

3. Use of the multifunctional manganese-based hybrid nanoplatform based on NK cell membrane modification as described in claim 2 in the preparation of drugs for treating liver cancer.

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