Immune sensitization IRE treatment platform based on NK cell membrane coated manganese carbonate

Abstract

The invention relates to an immune sensitization IRE treatment platform based on NK cell membrane coated manganese carbonate. A manganese carbonate composite nano material is formed by coating the surfaces of manganese carbonate nano particles with cell membranes. According to the invention, higher tumor specificity enrichment is realized, non-target tissue accumulation and systemic toxicity risks are reduced, collaborative integration of tumor targeted delivery, immune activation and IRE ablation is realized, and compared with single IRE, anti-tumor immune response is significantly enhanced.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to an immunosensitization and immunotherapy (IRE) platform based on manganese carbonate encapsulated in NK cell membranes. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide, especially in its middle and late stages, where the lack of effective treatments leads to extremely poor prognoses. In my country, due to the high incidence of chronic liver disease, HCC poses a particularly serious threat to public health. Current clinical treatments include surgical resection, liver transplantation, local ablation (such as radiofrequency ablation, microwave ablation, and irreversible electroporation), and systemic therapy. While local ablation can effectively kill tumors, it still has limitations: thermal ablation is affected by the "heat sink effect," making it difficult to completely remove lesions adjacent to large blood vessels, easily leading to recurrence; arterial chemoembolization and radiotherapy often cause systemic toxicity due to drug or radiation effects, and are mostly limited to palliative control.

[0003] Irreversible electroporation (IRE) is a non-thermal ablation technique that has been widely used in recent years. It creates irreversible nanopores in the cell membrane using short-duration high-voltage pulses, disrupting the phospholipid bilayer and inducing cell death, while simultaneously triggering immunogenic cell death (ICD). Because it is independent of thermal effects, IRE can better preserve tumor-associated antigens and damage-associated molecular patterns (DAMPs), exhibiting superior immunogenicity compared to traditional thermal ablation. It also causes less damage to connective tissues such as blood vessels and bile ducts, making it particularly suitable for tumors near blood vessels. However, its immunomodulatory effects are limited: although IRE can induce ICD, the induction effect is insufficient to overcome the immunosuppressive characteristics of the tumor immune microenvironment (TME), making it difficult to generate a durable systemic anti-tumor immune response.

[0004] Current IRE technology still has the following shortcomings in terms of immunization:

[0005] Insufficient immune response: Although it can induce immunogenic cell death (ICD), the effect is limited and it is difficult to overcome the immunosuppressive characteristics of the tumor immune microenvironment (TME) and form a lasting systemic anti-tumor immunity.

[0006] Significant immunosuppressive microenvironment: The TME of HCC is rich in regulatory T cells (Tregs), M2 macrophages and myeloid-derived suppressor cells (MDSCs). At the same time, tumor cells themselves can also express a variety of immunosuppressive molecules, such as PD-1 / PD-L1, TIGIT / CD155, which further weakens the adaptive immune effect triggered by IRE.

[0007] Limitations of immune adjuvants: Current attempts to enhance IRE by combining Toll-like receptor (TLR) agonists, vaccines, or nucleic acid-based immune adjuvants to improve innate immune response are limited by low delivery efficiency, significant systemic side effects, and lack of tumor targeting, making clinical translation difficult;

[0008] Lack of targeted delivery platforms: The lack of platforms that can achieve precise delivery and effectively regulate immune responses at the tumor site leads to significant off-target effects and increases the risk of systemic toxicity.

[0009] Integration challenges in clinical applications: How to seamlessly integrate immune enhancement methods with commonly used clinical operating parameters and procedures in IREs, and achieve scalable preparation and stable quality control of materials, remains a technical bottleneck that urgently needs to be addressed.

[0010] In summary, existing technologies still have significant limitations in enhancing IRE-induced immune responses, improving the tumor microenvironment, and inducing systemic anti-tumor immunity. Therefore, there is an urgent need for a novel technological solution that combines tumor-targeted delivery with immune activation regulation to significantly enhance IRE-triggered anti-tumor immune effects while ensuring safety. Summary of the Invention

[0011] To address the shortcomings of existing technologies, the technical problem to be solved by this invention is to provide an immunosensitizing IRE therapeutic platform based on manganese carbonate encapsulated in NK cell membranes. This invention delivers nanomaterials that simultaneously activate the immune system from both innate and adaptive perspectives through tumor-targeted delivery. This material can be used in conjunction with irreversible electroporation (IRE) to synergistically enhance the anti-tumor immune response.

[0012] This invention provides a manganese carbonate composite nanomaterial, wherein the manganese carbonate composite nanomaterial consists of manganese carbonate nanoparticles coated with cell membranes.

[0013] Preferably, the manganese carbonate nanoparticles have a regular rhombohedral structure with an average particle size of about 100-200 nm; the cell membrane is an NK cell membrane.

[0014] Core: MnCO3 nanoparticles (rhombohedral, particle size 100–200 nm); decompose in the acidic tumor microenvironment (TME), releasing Mn. 2＋ It activates the cGAS–STING signaling pathway, promoting dendritic cell (DC) maturation and antigen presentation.

[0015] Outer layer: Enclosed by the natural NK cell membrane, retaining a variety of membrane receptors.

[0016] TIGIT protein can bind to CD155, which is highly expressed on the surface of tumor cells, competitively blocking the inhibitory effect of tumors on TIGIT on the surface of T cells, thereby enhancing CD8. ＋ T cell function; the NK membrane also endows particles with natural tumor recognition and homing capabilities, achieving targeted enrichment.

[0017] Mn 2＋ It mediates cGAS–STING pathway activation, induces type I interferon production, and enhances innate immunity. Intervention of the NK membrane-preserved TIGIT–CD155 axis can alleviate immunosuppression in the TME.

[0018] The platform integrates the triple effects of tumor-targeted delivery, immune activation, and IRE ablation, significantly amplifying the immune response.

[0019] This invention provides a method for preparing manganese carbonate composite nanomaterials, comprising:

[0020] (1) Mix hexadecyltrimethylammonium bromide (CTAB), cyclohexane, and pentanol, then add manganese source and water, mix well, and stir to obtain microemulsion A;

[0021] (2) Mix hexadecyltrimethylammonium bromide (CTAB), cyclohexane, and pentanol, then add water, NaHCO3, and NH4HCO3, and stir to obtain microemulsion B;

[0022] (3) Mix microemulsion A and microemulsion B, stir and react, purify, and obtain manganese carbonate nanoparticles;

[0023] (4) Manganese carbonate nanoparticles and NK cell membranes were mixed, sonicated, extruded, and centrifuged to obtain manganese carbonate composite nanomaterials.

[0024] The preferred embodiment of the above preparation method is as follows:

[0025] In step (1), the ratio of CTAB, cyclohexane, pentanol, water, and manganese source is 0.5-0.8 g: 18-25 mL: 0.8-1.2 mL: 0.5-0.8 mL: 3.0-3.5 mmol; the manganese source is MnCl2·4H2O.

[0026] In step (2), the ratio of CTAB, cyclohexane, pentanol, water, NaHCO3 and NH4HCO3 is 2.4-3.0 g: 70-90 mL: 3.5-4.5 mL: 2.4-3.0 mL: 6.0-6.5 mmol: 0.3-0.35 mmol.

[0027] In steps (1) and (2), the reaction is stirred for 1-2 hours; in step (3), the reaction is stirred for 0.5-1.5 hours.

[0028] In step (4), the extrusion is performed by extruding through a 400 nm and a 200 nm polycarbonate film 15-25 times each.

[0029] This invention provides the application of manganese carbonate composite nanomaterials in the preparation of antitumor drugs, wherein the manganese carbonate composite nanomaterials are used in combination with IRE.

[0030] This invention provides the application of manganese carbonate composite nanomaterials in the preparation of anti-liver cancer drugs, wherein the manganese carbonate composite nanomaterials are used in combination with IRE.

[0031] This invention provides the application of manganese carbonate composite nanomaterials in the preparation of IRE antitumor immunosensitizing reagents.

[0032] This invention utilizes manganese carbonate composite nanomaterials as a biomimetic nanoimmune platform. While achieving tumor-targeted delivery, it simultaneously enhances the immune effect through TIGIT-CD155 immune checkpoint intervention (adaptive immune activation) and cGAS–STING pathway activation (innate immune activation), synergistically strengthening the anti-tumor immune response induced by irreversible electroporation (IRE). Unlike existing strategies that solely rely on immune adjuvants or nanomaterials, this invention integrates tumor targeting and dual immune regulation functions, achieving a significant sensitization of IRE efficacy.

[0033] Beneficial effects

[0034] (1) Significantly improved tumor targeting and safety:

[0035] Encapsulated by NK cell membranes, the nanoparticles possess natural tumor recognition and homing capabilities; the TIGIT retained by the NK membrane can bind to CD155 on the surface of tumor cells, thereby blocking its immunosuppressive effect on TIGIT on T cells and enhancing CD8. ＋ T cell function is enhanced, and adaptive immune effects are improved. Compared with immune adjuvants or free ion delivery that lack targeting, this invention achieves higher tumor-specific enrichment and reduces the risk of non-target tissue accumulation and systemic toxicity.

[0036] (2) Synergistic amplification of immune effects

[0037] MnCO3 decomposes and releases Mn in the acidic tumor microenvironment. 2＋ It activates the cGAS–STING pathway, promotes dendritic cell maturation, drives macrophage polarization towards the M1 type, enhances innate immune responses, and strengthens CD8. ＋Effector function of T cells; synergistic effect of NK membrane and TIGIT-CD155 axis intervention effectively alleviates the tumor immunosuppressive microenvironment;

[0038] The platform achieves synergistic integration of "tumor-targeted delivery + immune activation + IRE ablation", which significantly enhances the anti-tumor immune response compared to IRE alone.

[0039] (3) Advantages in clinical application

[0040] It is directly compatible with existing IRE clinical equipment and parameters without additional modifications; the nanoparticle preparation process can be mass-produced, and the NK membrane encapsulation process is stable and controllable, possessing good potential for clinical translation.

[0041] (4) Overall effect

[0042] Technically: It significantly enhances the immune response of IREs, improves the tumor immune microenvironment, and reduces the risk of recurrence and metastasis;

[0043] Economically: No additional expensive equipment or complex adjuvant systems are required; the source of materials is controllable; and the cost is low.

[0044] In society: It is expected to improve patients' survival rate and quality of life, and promote a new model of immunosensitization therapy for solid tumors. Attached Figure Description

[0045] Figure 1 Transmission electron microscopy and energy dispersive spectroscopy (EDS) elemental distribution of NKM@MnCO3 nanoparticles; from left to right: high-resolution TEM image, elemental distribution of Mn, O, C, P, N and elemental superposition diagram;

[0046] Figure 2 SDS-PAGE protein electrophoresis was used to analyze the coating of NK cell membranes on MnCO3 nanoparticles; lane I: NK92 cell lysate; lane II: purified NK cell membrane (NKM); lane III: NKM-coated MnCO3 nanoparticles (NKM@MnCO3); lane IV: uncoated MnCO3 nanoparticles.

[0047] Figure 3 Laser confocal microscopy was used to evaluate the targeting ability of MnCO3@NKM against liver cancer cells;

[0048] Figure 4 Flow cytometry was used to assess the effects of different treatments on the expression of CRT on the surface of tumor cells.

[0049] Figure 5 In vitro flow cytometry was used to assess the effects of different treatments on dendritic cell (DC) activation.

[0050] Figure 6 Tumor mass in mice 15 days after treatment;

[0051] Figure 7 Tumor mass in mice 15 days after treatment;

[0052] Figure 8 CD8 in tumor tissues of mice in different treatment groups ＋ Flow cytometry results of T cell proportion;

[0053] Figure 9 IFN-γ in tumor tissues of mice in different treatment groups ＋ CD8 ＋ Flow cytometry results of T cell proportion;

[0054] Figure 10 Gzmb in tumor tissues of mice in different treatment groups ＋ CD8 ＋ Flow cytometry results of T cell proportion;

[0055] Figure 11 CD25 in tumor tissues of mice in different treatment groups ＋ FoxP3 ＋ CD4 + Flow cytometry results of T cell proportion;

[0056] Figure 12 Dendritic cells (CD80) in tumor tissues of mice in different treatment groups + CD86 + Flow cytometry results of DC (dendritic cell) maturity ratio;

[0057] Figure 13 CD80 in tumor tissues of mice in different treatment groups ＋ Flow cytometry results of the proportion of M1 macrophages;

[0058] Figure 14 CD206 in tumor tissues of mice in different treatment groups ＋ Flow cytometry results of the proportion of M2 macrophages;

[0059] Figure 15 Flow cytometry results of the M1 / M2 macrophage ratio in tumor tissues of mice in different treatment groups. Detailed Implementation

[0060] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0061] Example 1

[0062] I. Synthesis of MnCO3 Nanoparticles

[0063] MnCO3 nanoparticles were prepared using a reverse microemulsion method, and the steps are as follows:

[0064] (1) Weigh 0.667 g of hexadecyltrimethylammonium bromide (CTAB) and dissolve it in a mixture of 20 mL cyclohexane and 1 mL pentanol. Then add 0.667 mL of deionized water (containing 3.33 mmol MnCl2·4H2O) to obtain solution A.

[0065] (2) Weigh 2.668 g of CTAB and disperse it in a mixture of 80 mL of cyclohexane and 4 mL of pentanol. Then add 2.668 mL of deionized water (containing 6.33 mmol NaHCO3 and 0.33 mmol NH4HCO3) to obtain solution B.

[0066] (3) Solution A reacted under magnetic stirring for 2 h to form a stable microemulsion; Solution B was also treated in the same way.

[0067] (4) Mix the two microemulsions and continue stirring for 1 h to induce the precipitation of MnCO3 nanoparticles.

[0068] (5) The precipitate was collected by centrifugation and washed three times with anhydrous ethanol and two times with deionized water.

[0069] (6) The purified product can be used directly or stored after freeze-drying.

[0070] II. Extraction of NK cell membrane

[0071] NK cell membranes were obtained from NK92 cells, following these steps:

[0072] (1) Collect NK92 cell pellet and resuspend it in hypotonic lysis buffer (200 mL deionized water, 1.7115 g sucrose, 1 mM KCl, 2 mL Tris-HCl, pH 7.4).

[0073] (2) Incubate in an ice bath for 30 min to promote osmotic pressure lysis.

[0074] (3) Use a Dounce homogenizer to homogenize 40 times to break the cells.

[0075] (4) Centrifuge at 500 × g, 4 ℃ for 10 min to remove unlysed cells and nuclei; repeat homogenization and centrifugation 3 times to improve membrane yield.

[0076] (5) Combine all supernatants and centrifuge at 21,000 × g, 4 ° C for 10 min to collect the membrane precipitate.

[0077] (6) The resulting membrane precipitate was washed twice with PBS and stored at –80 °C for later use.

[0078] III. Preparation of MnCO3@NKM Nanoparticles

[0079] (1) Mix MnCO3 nanoparticles with NK cell membranes at the same protein concentration (0.2 mg / mL) and sonicate for 5 min to disperse them evenly.

[0080] (2) The mixture was extruded through 400 nm and 200 nm polycarbonate membranes 20 times each to promote membrane coating and obtain a complete membrane-encapsulated structure.

[0081] (3) The obtained MnCO3@NKM nanoparticles were centrifuged at 2,000 × g and 4 ° C for 15 min to remove unbound cell membranes and impurities.

[0082] (4) The final product can be resuspended in PBS and used directly, or stored at 4 °C for later use.

[0083] 1. Material Characterization Methods

[0084] Morphology: TEM was used to observe the morphology of MnCO3 nanoparticles and determine their size distribution; EDS was used to confirm their elemental composition.

[0085] Protein characterization: SDS-PAGE comparison of the consistency of MnCO3@NKM and NK cell membrane protein bands;

[0086] 2. Validation of in vitro tumor targeting and immune activation

[0087] Targeting assessment: CLSM confocal microscopy was used to observe the differences in uptake of MnCO3 and MnCO3@NKM in H22 hepatocellular carcinoma cells and RAW264.7 macrophages, demonstrating that the NK membrane confers tumor specificity.

[0088] Validation of immunogenic cell death (ICD): Flow cytometry analysis of CRT expression confirmed that MnCO3@NKM can induce tumor ICD.

[0089] Dendritic cell (DC) activation: After co-incubating tumor cells with the material, they were then co-cultured with BMDCs, and the changes in the DC maturation markers CD80 / CD86 were detected by flow cytometry.

[0090] 3. Animal model establishment: BALB / c mice were subcutaneously injected with H22 hepatocellular carcinoma cells (1 × 10⁻⁶) into the right axilla. 6 A subcutaneous HCC model was established by suspending tumor cells in 100 μL PBS. The tumor volume was increased to 100–150 mm². 3 Then processing begins.

[0091] 4. Grouping and drug administration: Mice were randomly divided into six groups (n=5): PBS, MnCl2@NKM, MnCO3@NKM, PBS+IRE, MnCl2@NKM+IRE, and MnCO3@NKM+IRE.

[0092] The administration was via tail vein injection, once every 3 days for a total of 3 times; the dose of MnCO3 was 5 mg / kg, and the dose of MnCl2·4H2O was 8.61 mg / kg (equivalent to 215.25 μg / mouse), while maintaining consistent NK cell membrane concentration. IRE was performed 16 h after injection.

[0093] The IRE parameters were as follows: using a BTX ECM 830 pulse generator, the voltage was set to 1000 V, the pulse width to 100 μs, and the pulse count to 90. Each tumor site was treated for 3 rounds.

[0094] Follow-up and evaluation indicators: Regularly monitor mouse body weight and tumor volume (V = 1 / 2 × length × width). 2 At the experimental endpoint, survival status, tumor weight, and histological changes (HE, TUNEL, immunohistochemistry) were recorded.

[0095] (iv) Testing and Results

[0096] 1. Material Characterization: Transmission electron microscopy (TEM) and energy dispersive spectroscopy (EDS) results showed that the MnCO3 nanoparticles exhibited a regular rhombohedral structure with an average particle size of approximately 100–200 nm, and that Mn, C, and O elements were uniformly distributed. Further detection of N and P elemental signals, originating from protein and phospholipid components of the NK cell membrane, confirmed that the NK cell membrane had successfully coated the MnCO3 surface (e.g., ...). Figure 1 (As shown).

[0097] SDS-PAGE protein electrophoresis results showed that the protein band distribution of MnCO3@NKM was highly consistent with that of the NK cell membrane, while naked MnCO3 showed almost no bands, further proving that NK cell membrane-related proteins were completely preserved (e.g., Figure 2 (As shown).

[0098] 2. Targeting Validation: Laser confocal microscopy (CLSM) results showed that MnCO3@NKM uptake efficiency in H22 hepatocellular carcinoma cells was significantly higher than that of naked MnCO3, while uptake was limited in RAW264.7 macrophages, demonstrating good tumor specificity. This result proves that NK cell membrane encapsulation endows the nanoplatform with selective recognition and targeting of tumor cells, providing a prerequisite for subsequent immune activation effects (such as...). Figure 3 (As shown).

[0099] 3. Validation of immunogenic cell death (ICD)

[0100] Flow cytometry was used to detect the exposure level of calreticulin (CRT) on the surface of tumor cells under different treatment conditions. The results showed that CRT expression in tumor cells treated with MnCO3@NKM was significantly higher than that in the MnCO3 and PBS control group (e.g., [missing data]). Figure 4 (As shown in the figure). These results suggest that MnCO3@NKM can effectively induce immunogenic cell death (ICD) and promote the exposure of tumor antigens and damage-associated molecular patterns (DAMPs), thereby providing necessary signals for the recognition and activation of downstream immune cells.

[0101] 4. Assessment of dendritic cell (DC) activation

[0102] Further co-culture experiments were conducted on tumor cells and bone marrow-derived dendritic cells (BMDCs). The specific method involved treating tumor cells with different materials for 24 hours, followed by co-incubation with BMDCs; subsequently, flow cytometry was used to detect the functional status of BMDCs. Results showed that the MnCO3@NKM treatment group significantly upregulated the maturation markers CD80 and CD86 of BMDCs, and the proportion of double-positive DCs significantly increased (e.g., ...). Figure 5 (As shown). This result indicates that MnCO3@NKM can promote DC maturation, enhance antigen uptake and presentation capabilities, thereby providing a basis for CD8 cell proliferation. ＋ This lays the foundation for the activation of the T-cell immune response.

[0103] 5. Tumor growth and survival: The tumor volume in the MnCO3@NKM+IRE group was significantly reduced. By day 15 of treatment, both the tumor volume and weight were lower than those in the control group. Some mice achieved complete remission and their survival was significantly prolonged.

[0104] 6. Immunological assessment:

[0105] CD8 ＋ Increased T cell infiltration (approximately 22%, compared to approximately 8% in controls), accompanied by enhanced IFN-γ and Granzyme B expression; decreased Treg proportion (approximately 4%, compared to approximately 16% in controls); and significantly increased DC maturation rate (CD80). ＋ / CD86 ＋ (Expression upregulated); the macrophage M1 / M2 ratio increased from about 0.35 to 2.3, suggesting a shift in the immune environment toward pro-inflammatory tendencies.

[0106] like Figure 1 As shown, the transmission electron microscopy and energy dispersive spectroscopy (EDS) elemental distribution of NKM@MnCO3 nanoparticles are presented. The results show that Mn, C, and O elements are uniformly distributed in the nanoparticles, indicating that the MnCO3 particles have a complete structure. At the same time, P and N element signals were detected, which originated from the phospholipids and protein components of the NK cell membrane, proving that the cell membrane coating was successful.

[0107] like Figure 2 As shown, SDS-PAGE protein electrophoresis analysis was performed to analyze the coating of NK cell membranes on MnCO3 nanoparticles. The results showed that the protein band distribution of NKM@MnCO3 (lane III) was highly consistent with that of NK cell membrane (lane II), while naked MnCO3 (lane IV) showed almost no protein signal, indicating that NK cell membrane-related proteins were successfully retained and coated on the surface of MnCO3 nanoparticles.

[0108] like Figure 3 As shown, laser confocal microscopy was used to evaluate the targeting ability of MnCO3@NKM on hepatocellular carcinoma cells. H22 hepatocellular carcinoma cells and RAW264.7 macrophages were co-incubated with MnCO3 or MnCO3@NKM, respectively, and fluorescence was detected using DAPI (blue, labeling cell nuclei) and Cy5.5 (red, labeling nanoparticles). The results showed that naked MnCO3 had limited uptake in H22 cells, while the fluorescence signal of MnCO3@NKM was significantly enhanced in H22 tumor cells; compared with RAW264.7 macrophages, the uptake advantage of MnCO3@NKM in tumor cells was more significant. These results indicate that NK cell membrane encapsulation endows the nanoplatform with selective targeting and enhanced uptake ability of hepatocellular carcinoma cells.

[0109] like Figure 4As shown, flow cytometry was used to assess the effects of different treatments on the expression of calreticulin (CRT) on the surface of tumor cells. The levels of CRT on the surface of tumor cells in the PBS control group, MnCO3 group, and MnCO3@NKM group were detected by flow cytometry. The results showed that the CRT expression induced by the MnCO3@NKM group (purple peak) was significantly higher than that in the MnCO3 group (blue peak) and the PBS control group (green peak), suggesting that MnCO3@NKM can effectively enhance immunogenic cell death (ICD), which is beneficial for promoting downstream antigen presentation and immune response activation.

[0110] like Figure 5 As shown, in vitro flow cytometry was used to assess the effects of different treatments on dendritic cell (DC) activation. The expression of surface maturation markers CD80 and CD86 in dendritic cells was detected by flow cytometry in the PBS control group, MnCO3 group, and MnCO3@NKM group. The results showed that CD80 expression was significantly higher in the MnCO3@NKM group (right figure). ＋ CD86 ＋ The proportion of dendritic cells (DCs) was significantly higher in the MnCO3 group and the PBS control group, suggesting that MnCO3@NKM can effectively promote dendritic cell maturation and enhance antigen presentation ability, thereby facilitating the activation of downstream T cell immune responses.

[0111] After confirming the significant inhibitory effect of MnCO3@NKM combined with IRE on tumor growth, the immune response in the tumor microenvironment was further evaluated. Flow cytometry analysis revealed that this group of mice exhibited a more active anti-tumor immune status.

[0112] Enhanced T cell effector function: In treatment groups V (IRE + MnCl2@NKM) and VI (IRE + MnCO3@NKM), CD8+ ＋ The proportion of T cells was significantly higher in the VI group than in the PBS control group (8.3 ± 3.2%), with the highest proportion in the VI group (22.0 ± 5.9%). Figure 8 ). VI group CD8 ＋ The expression levels of IFN-γ and granzyme B in T cells were significantly upregulated, suggesting enhanced cytotoxic function. Figure 9 and 10 ).

[0113] Immunosuppressive remission: Immunosuppressive regulatory T cells (CD4+) in the VI (IRE + MnCO3@NKM) group ＋ CD25 ＋ FoxP3 ＋The percentage of patients with PBS significantly decreased (4.2 ± 2.8%), which was lower than that in the PBS group (16.5 ± 7.2%). Figure 11 This indicates that the degree of immunosuppression in the tumor microenvironment has been reduced.

[0114] Dendritic cell maturation: DC maturation (CD80) ＋ CD86 ＋ The proportion of mature DCs in both the V (IRE + MnCl2@NKM) group (23.7 ± 7%) and the VI (IRE + MnCO3@NKM) group (29.3 ± 11.1%) was significantly higher than that in the PBS control group (8.3 ± 5.2%). The proportion of mature DCs was significantly increased in the MnCO3@NKM combined with IRE treatment group. These results suggest that DC function is maturing, and antigen-presenting capacity is enhanced, which is beneficial for further activating downstream T cell responses. Figure 12 ).

[0115] Macrophage remodeling: The macrophage phenotype of the VI (IRE + MnCO3@NKM) group was significantly remodeled, with CD80... ＋ The proportion of M1 type increased ( Figure 13 ),

[0116] CD206 ＋ The proportion of M2 type decreased ( Figure 14 This resulted in a significantly higher M1 / M2 ratio in the VI (IRE + MnCO3@NKM) group (2.3 ± 1.3), far exceeding that in the PBS group (0.3 ± 0.1). Figure 15 This indicates that the tumor microenvironment exhibits stronger immune stimulation characteristics.

[0117] Follow-up results showed that tumor growth was significantly inhibited in the MnCO3@NKM + IRE group, with almost no significant recurrence during the observation period, and both tumor volume and weight were significantly reduced. Simultaneously, no significant decrease in animal body weight was observed, suggesting that this treatment regimen has good safety. This effect is attributed to: tumor targeting conferred by the NK cell membrane and intervention through the TIGIT-CD155 axis immune checkpoint; Mn 2＋ Release activates the cGAS–STING pathway, promoting DC maturation, macrophage M1 polarization, and CD8 activation. ＋ Enhanced T cell effects.

[0118] In summary, this invention achieves immunosensitization of IRE therapy through tumor-targeted delivery and immune activation regulation, significantly improving the anti-tumor effect.

Claims

1. A manganese carbonate composite nanomaterial, characterized in that, The manganese carbonate composite nanomaterial consists of manganese carbonate nanoparticles coated with cell membranes.

2. The manganese carbonate composite nanomaterial according to claim 1, characterized in that, The manganese carbonate nanoparticles have a regular rhombohedral structure with an average particle size of about 100-200 nm; the cell membrane is an NK cell membrane.

3. A method for preparing manganese carbonate composite nanomaterials, comprising: (1) Mix hexadecyltrimethylammonium bromide (CTAB), cyclohexane, and pentanol, then add manganese source and water, mix well, and stir to obtain microemulsion A; (2) Mix hexadecyltrimethylammonium bromide (CTAB), cyclohexane, and pentanol, then add water, NaHCO3, and NH4HCO3, and stir to obtain microemulsion B; (3) Mix microemulsion A and microemulsion B, stir and react, purify, and obtain manganese carbonate nanoparticles; (4) Manganese carbonate nanoparticles and NK cell membranes were mixed, sonicated, extruded, and centrifuged to obtain manganese carbonate composite nanomaterials.

4. The preparation method according to claim 3, characterized in that, In step (1), the ratio of CTAB, cyclohexane, pentanol, water, and manganese source is 0.5-0.8g: 18-25 mL: 0.8-1.2 mL: 0.5-0.8 mL: 3.0-3.5 mmol; the manganese source is MnCl2·4H2O.

5. The preparation method according to claim 3, characterized in that, In step (2), the ratio of CTAB, cyclohexane, pentanol, water, NaHCO3 and NH4HCO3 is 2.4-3.0 g: 70-90 mL: 3.5-4.5 mL: 2.4-3.0 mL: 6.0-6.5 mmol: 0.3-0.35 mmol.

6. The preparation method according to claim 3, characterized in that, In steps (1) and (2), the reaction is stirred for 1-2 hours; in step (3), the reaction is stirred for 0.5-1.5 hours.

7. The preparation method according to claim 3, characterized in that, In step (4), the extrusion is performed by extruding through 400 nm and 200 nm polycarbonate films 15-25 times each.

8. The application of a manganese carbonate composite nanomaterial in the preparation of antitumor drugs, characterized in that, The manganese carbonate composite nanomaterial is used in combination with IRE.

9. The application of a manganese carbonate composite nanomaterial in the preparation of anti-liver cancer drugs, characterized in that, The manganese carbonate composite nanomaterial is used in combination with IRE.

10. Application of a manganese carbonate composite nanomaterial in the preparation of IRE antitumor immunosensitizing reagent.

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