Application of miR-99b and SIRP alpha siRNA in preparation of medicine for treating hepatocellular carcinoma

Abstract

The invention discloses an application of a pharmaceutical composition containing miR-99b and SIRP alpha siRNA in preparation of a medicine for treating hepatocellular carcinoma and reversing a tumor immunosuppression microenvironment of the hepatocellular carcinoma. According to the pharmaceutical composition, two nucleic acid drugs are jointly delivered to tumor-related macrophages, and the dual effects of reprogramming M2-type macrophages into M1-type macrophages and down-regulating SIRP alpha proteins to block a CD47-SIRP alpha signal channel are synergistically exerted, so that the phagocytic function of the macrophages is enhanced, tumor growth and metastasis are inhibited, T cell infiltration is promoted, and the tumor-related macrophages can be effectively treated. Finally, immunosuppression is effectively reversed, anti-tumor immune response is activated, and targeted co-delivery of the pharmaceutical composition is achieved through lipid nanoparticles which are modified by mannose and contain disulfide bond PEGylated lipid.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to new pharmaceutical applications of nucleic acid drugs, and more specifically to the application of miR-99b and SIRPα siRNA in the preparation of drugs for treating hepatocellular carcinoma. Background Technology

[0002] Hepatocellular carcinoma is one of the most common malignant tumors worldwide, with a high incidence and poor prognosis, posing a serious threat to human health. Despite continuous advancements in surgical resection, local ablation, radiotherapy, chemotherapy, and targeted therapy, treatment outcomes remain unsatisfactory for patients in the middle and late stages. Tumor recurrence, metastasis, and treatment resistance remain the main causes of death for these patients.

[0003] In recent years, tumor immunotherapy, especially immune checkpoint inhibitors, has made groundbreaking progress in various cancers. However, in hepatocellular carcinoma (HCC), the overall response rate of single immune checkpoint inhibitors is limited. This has prompted researchers to focus on the key site shaping immunotherapy responses—the tumor microenvironment. The tumor microenvironment of HCC is a complex ecosystem with high immunosuppression, in which tumor-associated macrophages (TAMs), as the most abundant group of immune cells, play a crucial role. TAMs are highly plastic, mainly divided into the M1 phenotype with anti-tumor function and the M2 phenotype with pro-tumor function. During HCC progression, TAMs are usually polarized to the M2 phenotype, creating a microenvironment conducive to tumor growth and metastasis through various mechanisms such as secreting immunosuppressive cytokines (e.g., IL-10, TGF-β), promoting angiogenesis, and inhibiting T cell function, leading to poor efficacy of existing immunotherapies. Therefore, "reprogramming" M2-type TAMs into the M1 phenotype with anti-tumor activity is considered a highly promising strategy to reverse immunosuppression and improve the prospects for HCC treatment.

[0004] On the other hand, tumor cells evade immune surveillance by expressing "don't eat me" signals, which is another key immune escape mechanism. In liver cancer, the CD47 molecule, which is highly expressed on the surface of tumor cells, binds to the signal regulatory protein α on the surface of TAMs (tumor cells that metastasize to tumor cells), transmitting a strong inhibitory signal to macrophages, thereby actively inhibiting the phagocytosis of tumor cells by macrophages. Therefore, blocking the CD47-SIRPα pathway, like releasing the "brakes" on macrophages, is an important way to restore their ability to phagocytose and clear tumor cells.

[0005] Based on the above understanding, simultaneously targeting "M2-type TAMs reprogramming" and "CD47-SIRPα pathway blockade" is considered an ideal strategy for achieving synergistic anti-tumor immunotherapy. Nucleic acid drugs exhibit unique advantages in this regard: for example, miR-99b has been shown to effectively inhibit M2 polarization and promote M1-type macrophage activation by regulating signaling pathways such as NF-κB; while SIRPα siRNA can specifically knock down the expression of SIRPα in macrophages, fundamentally relieving CD47-mediated phagocytic inhibition. Theoretically, combining miR-99b with SIRPα siRNA can achieve the dual effects of "phenotype reversal" and "inhibition relief," synergistically activating the anti-tumor function of macrophages.

[0006] However, translating this theoretical concept into an effective clinical treatment faces a core challenge: the lack of a delivery system capable of precisely, efficiently, and synergistically delivering two nucleic acid drugs to the same target cells (TAMs). Naked nucleic acid drugs are easily degraded, have low cellular uptake efficiency, and cannot achieve extrahepatic targeting. Although some nanocarriers have been developed for nucleic acid delivery, they generally suffer from the following problems: Insufficient targeting: Most vectors lack the ability to specifically recognize TAMs, especially the M2 subtype, resulting in the distribution of drugs in non-target tissues and cells, which not only reduces efficacy but may also increase off-target toxicity.

[0007] Synergy is difficult to guarantee: Simply mixing or injecting two drugs separately cannot ensure that they are taken up by the same cell and work synergistically within the same cell, making it difficult to achieve a synergistic effect of "1+1>2".

[0008] Limited penetration and responsive release capabilities in the tumor microenvironment: The carrier needs to overcome physiological barriers to reach the tumor site and intelligently release the drug under specific conditions of the TME (such as high concentrations of glutathione) to enhance uptake and efficacy. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method that overcomes the limitations of existing delivery systems and effectively co-delivers the synergistic therapeutic combination of miR-99b and SIRPα siRNA to tumor microenvironments (TAMs) in hepatocellular carcinoma, thereby fully leveraging the powerful functions of miR-99b and SIRPα siRNA in reversing immunosuppression and activating anti-tumor immunity in the preparation of drugs for treating hepatocellular carcinoma.

[0010] The technical solution adopted by this invention to solve its technical problem is: The use of a pharmaceutical composition in the preparation of a medicament for treating hepatocellular carcinoma, said pharmaceutical composition comprising therapeutically effective amounts of miR-99b and SIRPα siRNA.

[0011] Preferably, the pharmaceutical composition is configured to co-deliver miR-99b and SIRPα siRNA to tumor-associated macrophages in the tumor microenvironment.

[0012] Preferably, the treatment for hepatocellular carcinoma includes at least one of the following pharmacological effects: (a) Reprogramming M2 tumor-associated macrophages to M1 type; (b) Downregulate the expression of SIRPα protein in tumor-associated macrophages; (c) Block the CD47-SIRPα “Don’t eat me” signaling pathway; (d) Enhance the phagocytic activity of macrophages against hepatocellular carcinoma cells; (e) Inhibits the growth of hepatocellular carcinoma tumors; (f) Inhibits the metastasis of hepatocellular carcinoma; (g) Promotes the infiltration of CD3+ and CD8+ T cells in the tumor microenvironment.

[0013] Preferably, the miR-99b and SIRPα siRNA are co-encapsulated in the same delivery vector.

[0014] Preferably, the delivery carrier is lipid nanoparticles.

[0015] Preferably, the lipid nanoparticles are lipid nanoparticles that target M2 macrophages.

[0016] Preferably, the lipid nanoparticles are modified with mannose for specific uptake by M2 macrophages via mannose receptor-mediated endocytosis.

[0017] Preferably, the lipid nanoparticles contain PEGylated lipids with disulfide bonds, which are used to induce glutathione-responsive PEG shedding in the tumor microenvironment.

[0018] Another technical problem to be solved by the present invention is to provide a pharmaceutical composition for use in the preparation of a drug for reversing the tumor immunosuppressive microenvironment, the pharmaceutical composition comprising a therapeutically effective amount of miR-99b and SIRPα siRNA, and the reversal is achieved by reprogramming M2 type tumor-associated macrophages to M1 type and blocking the CD47-SIRPα signaling pathway.

[0019] Preferably, the tumor immunosuppressive microenvironment is the tumor microenvironment of hepatocellular carcinoma.

[0020] The beneficial effects of this invention are as follows: This approach is not a simple combination of two drugs, but rather leverages the inherent complementarity and synergy of miR-99b and SIRPα siRNA in their mechanisms of action. miR-99b is responsible for reprogramming tumor-promoting "accomplices" (M2-type TAMs) into tumor-killing "warriors" (M1-type), thereby fundamentally altering the nature of the immune microenvironment. SIRPα siRNA, on the other hand, is responsible for removing the "brakes" (CD47-SIRPα signaling) imposed on macrophages by tumor cells, restoring their phagocytic function to normal. The synergy between the two creates a dual-mode of action: "modifying immune cells" and "disarming tumors," producing a synergistic anti-tumor effect far exceeding that of single-drug therapy, effectively overcoming the limitations of single-pathway treatment. This treatment strategy precisely targets the most abundant immune cells in the tumor microenvironment—tumor-associated macrophages. By setting the drug target as TAMs and using miR-99b and SIRPα siRNA to regulate their polarization state and phagocytic function respectively, this invention can reshape the tumor immune microenvironment at the core level, reversing the immunosuppressed "cold tumor" into an immune-activated "hot tumor", creating favorable conditions for subsequent immune responses (such as T cell infiltration and activation).

[0021] Experiments have confirmed that the pharmaceutical composition of this formulation can produce a series of interrelated positive pharmacological effects through the above-mentioned synergistic mechanism: at the cellular phenotype level: significantly increasing the proportion of M1 macrophages in tumor tissue while decreasing the proportion of M2 macrophages; at the signaling pathway level: effectively downregulating the expression of SIRPα on the surface of macrophages and blocking the CD47-SIRPα "don't eat me" signaling pathway; at the immune function level: greatly enhancing the phagocytic and clearance ability of macrophages against liver cancer cells; at the tumor behavior level: significantly inhibiting the in situ growth and distant metastasis of liver cancer tumors; at the systemic immune level: promoting the infiltration of cytotoxic T cells in tumor tissue and stimulating long-lasting anti-tumor immune memory.

[0022] Existing immune checkpoint inhibitors are ineffective or prone to resistance in many hepatocellular carcinoma patients, partly due to the suppressive myeloid immune cell population. This approach, by directly targeting and modifying this population, holds the promise of overcoming TAM-mediated immunosuppression, providing a novel treatment option with a different mechanism of action for patients insensitive to or resistant to existing immunotherapies, which has significant clinical implications. The core of this approach lies in the synergistic use of a specific combination of miR-99b and SIRPα siRNA, which can be achieved using various delivery systems known in the art. This provides flexibility in implementation. To achieve optimal efficacy, the combination can be co-encapsulated in an optimized delivery vector, such as mannose-modified lipid nanoparticles capable of GSH-responsive PEG shedding, thereby ensuring that both drugs are delivered simultaneously to the same target cells, maximizing their synergistic effect. Attached Figure Description

[0023] Figure 1 Figure 1 shows the in vitro study of the effect of TCD-LNPs on macrophage phenotypic reprogramming. (A) Schematic diagram of the mechanism by which TCD-LNPs promote M2 to M1 polarization through co-delivery of miR-99b and SIRPα siRNA; (B) Flow cytometry analysis of the changes in the ratio of CD86+ (M1) and CD206+ (M2) cells in macrophages after different TCD-LNP treatments; (C) Statistical analysis of the flow cytometry results in Figure (B); (D) Immunofluorescence staining showing the expression of CD68+CD86+ (M1) in macrophages after different treatments (scale bar: 50 μm); (E, G) Immunofluorescence staining showing the expression of NF-κB and TNF-α in macrophages after different treatments (scale bar: 50 μm); (F, H) Quantitative statistical analysis of the fluorescence intensity of NF-κB and TNF-α in Figures (E) and (G); (IP) Statistical graph of ELISA results of M1 / M2 related cytokines (NF-κB, TNF-α, IL-12, IL-6, iNOS, IL-10, TGF-β, Arg-1) secreted by macrophages treated with different TCD-LNPs; Figure 2This diagram illustrates the in vitro effects of TCD-LNPs on tumor cell growth and migration via macrophage reprogramming. (A) Schematic diagram of the experimental setup for co-culturing Hepa1-6 tumor cells with macrophages treated with different TCD-LNPs; (B) CCK-8 assay results showing the effect of different treatments on tumor cell proliferation at 24, 48, and 72 hours; (C) Confocal fluorescence microscopy images (scale bar: 50 μm) of tumor cells stained with calcein AM (live cells, green) / propidium iodide (dead cells, red); (D) Quantitative statistical analysis of cell viability in Figure (C); (E) Schematic diagram of the Transwell assay used to assess tumor cell migration ability; (F) Representative images of tumor cells migrating through Transwell chambers after treatment with different TCD-LNPs; (G) Microscopic images of tumor cell migration over 24 hours as shown by the scratch assay; (H) Quantitative analysis of tumor cell migration rate in Figure (G). Figure 3 Figure 1 shows a study on the enhancement of macrophage-mediated tumor cell phagocytosis by TCD-LNPs in vitro. (A) Immunofluorescence image showing the effect of different TCD-LNPs on macrophage SIRPα protein expression (scale bar: 50 μm); (B) Schematic diagram of TCD-LNPs-induced macrophage enhancement of Hepa1-6 tumor cells; (C) Confocal microscopy image showing the phagocytosis of Hepa1-6 tumor cells (DiI-labeled, red) by macrophages treated with different TCD-LNPs (DiO-labeled, green) (scale bar: 50 μm). Figure 1 (D) Image D provides a three-dimensional view of the phagocytic event; (D) Confocal microscopy Z-axis tomographic image of macrophages phagocytizing tumor cells; (E) Quantitative statistical analysis of SIRPα fluorescence intensity in Figure (A); (F) Statistical analysis of the number of tumor cells phagocytosed by macrophages in Figure (C); (G) Representative flow cytometry plot describing macrophage phagocytosis; (H) Quantitative analysis results of macrophage phagocytic efficiency by flow cytometry. Figure 4The diagram shows the in vivo targeting and antitumor effects of TCD-LNPs in the Hepa1-6 hepatocellular carcinoma mouse model. (A) In vivo imaging showing the distribution of different LNP formulations in tumor-bearing mice at different time points after intravenous injection; (B) Schematic diagram of the experimental timeline of TCD-LNPs treatment in Hepa1-6 tumor-bearing mice; (CF) Tumor growth curves and tumor weight statistics at the experimental endpoint for mice treated with PBS, mTCD-LNPs, sTCD-LNPs, and msTCD-LNPs; (G) TUNEL staining analysis of tumor tissue at the end of treatment to detect apoptosis (scale bar: 50 μm); (H) Ki-67 immunofluorescence staining analysis of tumor tissue at the end of treatment to detect cell proliferation (scale bar: 50 μm). Figure 5 This diagram illustrates the role of TCD-LNPs in in vivo macrophage reprogramming and its regulatory effect on the tumor microenvironment. (AB) Confocal images of immunofluorescence staining of macrophage markers CD86 (M1) and CD206 (M2) in tumor tissue (scale bar: 50 μm); (CD) Semi-quantitative analysis of CD86 and CD206 protein expression levels in (AB); (E) Immunofluorescence staining image of SIRPα expression in tumor tissue (scale bar: 50 μm); (F) Immunofluorescence staining image of CD3+ T cell infiltration in tumor tissue (scale bar: 50 μm); (G) Immunofluorescence staining image of CD8+ T cell infiltration in tumor tissue (scale bar: 50 μm); (HJ) Semi-quantitative analysis of SIRPα, CD3, and CD8 protein expression levels in (EG). Figure 6 Figure 1 shows a study on how TCD-LNPs promote macrophage reprogramming and inhibit tumor metastasis in vivo. (AD) ELISA analysis results of tumor-related cytokines (NF-κB, TNF-α, iNOS, Arg-1) in different treatment groups; (E) Schematic diagram of the mechanism by which TCD-LNPs inhibit lung cancer metastasis by reprogramming macrophages and enhancing phagocytosis; (FI) Heatmap showing the levels of metastasis-related cytokines (TGF-β, IL-10, IL-6, IL-12) in tumor tissues of different treatment groups; (J) H&E staining of lung tissue sections from mice in different treatment groups to assess lung metastasis (scale bar: 100 μm). Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments are not intended to limit the present invention.

[0025] Example 1: Preparation of targeted lipid nanoparticles (msTCD-LNP) for co-delivery of miR-99b and SIRPα siRNA, materials and equipment: Lipids: DLin-MC3-DMA (Aladdin, Shanghai, China), DSPC (MedChemExpress, USA), cholesterol (Jizhi, Shanghai, China), DSPE-SS-PEG3000 (custom-synthesized by Ruixi Biotechnology, Xi'an, China), DSPE-PEG2000-mannose (MedChemExpress, USA). Nucleic acids: miR-99b mimic and SIRPα siRNA (GenePharma, Shanghai, China); Solvents and buffers: anhydrous ethanol (analytical grade), citrate buffer (10 mM, pH 4.0), 1× phosphate buffer (PBS, pH 7.4); Equipment: analytical balance, vortex mixer, syringe (1 mL), dialysis bag (MWCO: 3500 Da), 0.22 μm sterile filter membrane.

[0026] Preparation steps: a. Preparation of lipid solution: Accurately weigh the above lipids and dissolve them in anhydrous ethanol at a molar ratio of 50 / 10 / 38.5 / 0.75 / 0.75 to prepare a clear lipid ethanol solution with a total concentration of 12 mM.

[0027] b. Preparation of nucleic acid aqueous solution: Dissolve the therapeutic amount of miR-99b and SIRPα siRNA together in pre-cooled pH 4.0 citrate buffer, mix gently, and avoid generating bubbles.

[0028] C. Nanoparticle Assembly: At room temperature (25°C), two 1 mL syringes were used to draw 50 μL of lipid ethanol solution and 150 μL of nucleic acid aqueous solution, respectively. The two liquids were rapidly injected into the same container using a microfluidic device or manually, and immediately vortexed (≥1200 rpm) for 30 seconds. The volume ratio of lipid solution to nucleic acid aqueous solution was 1:3.

[0029] d. Incubation: Let the mixture stand at room temperature for 10 minutes to complete self-assembly.

[0030] e. Purification and sterilization: Transfer the assembled solution to a pretreated dialysis bag and dialyze in 1 L of 1×PBS at 4°C for 2 hours, changing the dialysate 3 times during the process. After dialysis, filter the solution through a 0.22 μm sterile membrane to obtain the msTCD-LNP suspension, which should be stored at 4°C protected from light.

[0031] Example 2: This embodiment aims to illustrate that the lipid composition can be adjusted within a certain range to achieve the purpose of this invention.

[0032] Scheme A (Highly Ionizable Lipids): LNPs were prepared according to a molar ratio of 60 / 8 / 30 / 1.0 / 1.0 (DLin-MC3-DMA / DSPC / cholesterol / DSPE-SS-PEG3000 / DSPE-PEG2000-mannose). This ratio aims to improve nucleic acid encapsulation efficiency and endosome escape efficiency.

[0033] Option B (High Cholesterol): LNPs were prepared in a molar ratio of 45 / 12 / 40 / 1.5 / 1.5. This ratio is designed to enhance the stability of the nanoparticles and the rigidity of the membrane, making it suitable for applications requiring long-term storage.

[0034] Preparation and characterization: The method was the same as in Example 1. The obtained LNPs were characterized, and the results showed that the average hydrated particle size of the LNPs prepared by schemes A and B were in the range of 70-130 nm, PDI < 0.25, and the encapsulation efficiency for both nucleic acids was > 80%, which met the formulation requirements.

[0035] Example 3 This embodiment aims to illustrate that there are reasonable alternatives to the key lipid components in this invention.

[0036] Option C (alternative to ionizable lipids): Replace DLin-MC3-DMA in Example 1 with an equimolar amount of SM-102. The remaining components, proportions, and preparation methods remain unchanged.

[0037] Option D (alternative PEG lipids): Replace DSPE-SS-PEG3000 and DSPE-PEG2000-mannose in Example 1 with equimolar amounts of DSPE-SS-PEG2000 and DSPE-PEG5000-mannose. All other components, proportions, and preparation methods remain unchanged.

[0038] Performance verification: The LNPs prepared by schemes C and D were verified to have good particle size distribution and encapsulation efficiency, and showed preferential targeting of M2 macrophages in in vitro cell uptake experiments.

[0039] Example 4 This example aims to illustrate that the ratio of miR-99b to SIRPα siRNA and the total loading amount are adjustable.

[0040] Scheme E (equimolar loading): When preparing the nucleic acid aqueous solution, the molar ratio of miR-99b to SIRPα siRNA is 1:1, while the total amount of nucleic acid remains unchanged.

[0041] Option F (focusing on reprogramming): Adjust the nucleic acid molar ratio to miR-99b : SIRPα siRNA = 2 : 1.

[0042] Option G (High Loading): While keeping the total lipid concentration constant, increase the amount of nucleic acid feed by 20% to prepare LNPs with high loading.

[0043] Preparation and evaluation: The method was the same as in Example 1. Testing showed that all schemes successfully assembled LNPs. In vitro biological evaluation indicated that different ratios of combinations exhibited synergistic effects, with the equimolar ratio (Scheme E) showing the best overall performance in most experiments.

[0044] Example 5 This embodiment provides various pharmaceutically acceptable dosage forms of the pharmaceutical compositions of the present invention.

[0045] Lyophilized powder for injection: Take the msTCD-LNP suspension prepared in Example 1, add 5% (w / v) trehalose as a lyophilization protectant, dissolve thoroughly, and dispense into vials at 1.0 mL / vial. Place in a lyophilizer and proceed with pre-freezing, primary drying, and secondary drying to prepare a white lyophilized powder. Reconstitute with water for injection before use.

[0046] Intravenous infusion solution: The msTCD-LNP suspension prepared in Example 1 was mixed with 5% glucose injection and diluted to a total nucleic acid concentration of 0.2 mg / mL. After sterilization by 0.22 μm filtration, it was filled into 100 mL infusion bags to prepare a dosage form that can be directly used for intravenous drip.

[0047] Experimental Example 1 In vitro validation of TCD-LNPs-induced macrophage phenotypic reprogramming Experimental objective: To verify that TCD-LNPs can reprogram M2 macrophages into the M1 phenotype with anti-tumor function.

[0048] Experimental methods: IL-4-induced M2-like macrophages were divided into five groups and treated with PBS, mTCD-LNP, sTCD-LNP, msTCD-LNP and non-responsive control CD-LNP for 24 hours, respectively. The expression of cell surface markers CD86 (M1) and CD206 (M2) was detected by flow cytometry. The expression levels of intracellular NF-κB and TNF-α and the secretion of M1 / M2-related cytokines in the culture supernatant were detected by immunofluorescence staining and ELISA kits.

[0049] The experimental results are as follows: Schematic diagram of the mechanism of action: The mechanism by which TCD-LNPs reprogram macrophages through co-delivery of miR-99b and SIRPα siRNA is as follows: Figure 1 As shown in Figure A.

[0050] Phenotypic transformation: Flow cytometry ( Figure 1 BC) and immunofluorescence staining ( Figure 1 D) The results showed that the msTCD-LNP treatment group most effectively increased the proportion of CD86+ M1 cells (to 56.6%) and decreased the proportion of CD206+ M2 cells (to 8.03%).

[0051] Molecular mechanisms and cytokines: Immunofluorescence ( Figure 1 EH) showed that msTCD-LNP significantly increased the expression of NF-κB and TNF-α. ELISA results ( Figure 1 IP) confirmed that it promotes the secretion of M1-related factors and inhibits the secretion of M2-related factors.

[0052] Experimental conclusions: TCD-LNPs can efficiently reprogram M2 macrophages to the M1 phenotype. This process is activated by the NF-κB pathway and is accompanied by the establishment of a pro-inflammatory cytokine profile. The co-delivery of the two drugs and the GSH-responsive design are crucial to this synergistic effect.

[0053] Experiment Example 2 TCD-LNPs enhance macrophage phagocytosis and inhibit tumor cell growth and migration. Experimental objective: To verify the ability of macrophages reprogrammed by TCD-LNPs to phagocytose tumor cells and their inhibitory effect on tumor cell proliferation and migration.

[0054] Experimental methods: Co-cultivation model: Schematic diagram of the experimental setup as shown below Figure 2 As shown in Figure A.

[0055] Phagocytosis assay: Macrophages treated with different LNPs were labeled with DiO (green) and Hepa1-6 tumor cells were labeled with DiI (red). After co-incubation, the phagocytic efficiency was quantitatively analyzed by confocal microscopy and flow cytometry.

[0056] Antitumor activity: via the CCK-8 method ( Figure 2 B), Live / Dead Staining ( Figure 2 CD), Transwell migration experiment ( Figure 2 EF) and scratch test ( Figure 2 GH) was used to assess its inhibitory effects on tumor cell viability, apoptosis, and migration.

[0057] The experimental results are as follows: Enhanced phagocytosis: such as Figure 3 As shown in A and 5E, TCD-LNPs effectively downregulated SIRPα expression in macrophages. (Schematic diagram of phagocytosis experiment) Figure 3 B) Confocal images ( Figure 3 CD) and flow cytometry results ( Figure 3 GH) showed that the phagocytic efficiency of the msTCD-LNP group was as high as 46.3%, which was 10.75 times that of the control group.

[0058] Inhibition of proliferation and migration: In the co-culture system, the msTCD-LNP group showed the highest inhibition rate on the proliferation of Hepa1-6 cells. Figure 2 B), and can most effectively induce tumor cell apoptosis (B) Figure 2 CD) and inhibit migration ( Figure 2 EH).

[0059] Experimental conclusion: TCD-LNPs significantly enhanced the phagocytic and clearance capacity of macrophages against tumor cells through a synergistic effect—namely, miR-99b-driven M2 to M1 phenotype reversal and SIRPαsiRNA-mediated "don't eat me" signaling blockade—and effectively inhibited the growth and migration of tumor cells.

[0060] Experimental Example 3 In vivo targeting, anti-tumor and anti-metastatic effects of TCD-LNPs Experimental objective: To evaluate the in vivo distribution, targeting ability, anti-tumor efficacy, and metastasis inhibition effect of TCD-LNPs in a Hepa1-6 hepatocellular carcinoma mouse model.

[0061] The experimental method is as follows: In vivo targeting: DIR-labeled PBS, CD-LNP (non-responsive), and TCD-LNP were injected via tail vein, and their distribution in tumor-bearing mice was observed within 96 hours using an in vivo imaging system. Figure 4 A).

[0062] Antitumor efficacy: Tumor-bearing mice were randomly divided into PBS, mTCD-LNP, sTCD-LNP, and msTCD-LNP groups, and treated intravenously (experimental timeline as follows). Figure 4 (As shown in B). Tumor volume was measured periodically, and tumor weight was taken at the endpoint. Figure 4 CF), and TUNEL staining was performed ( Figure 4 G) and Ki-67 immunofluorescence analysis ( Figure 4 H).

[0063] Immune microenvironment and metastasis: Immunofluorescence staining of tumor tissue was performed to analyze macrophage phenotype (CD86 / CD206, Figure 5 AD), SIRPα expression ( Figure 5 E, 5H) and T cells (CD3+ / CD8+, Figure 5 FG, 5I-J) infiltration. Cytokine levels in tumor tissue were detected by ELISA. Figure 6 AI, and lung metastasis was assessed by H&E staining of lung tissue. Figure 6 J).

[0064] The experimental results are as follows: Excellent targeting: such as Figure 4 As shown in Figure A, in the TCD-LNP group, the fluorescence signal at the tumor site remained strong 96 hours after injection, while the fluorescence in the liver was almost completely cleared.

[0065] Significant anti-tumor effects: Tumor growth was significantly inhibited in the msTCD-LNP treatment group, with minimal tumor volume and weight. Figure 4 CF), and tumor tissue has the highest rate of apoptosis ( Figure 4 G), lowest proliferation index ( Figure 4 H).

[0066] Remodeling the immune microenvironment: In the msTCD-LNP group of tumor tissues, M1 macrophages increased and M2 macrophages decreased. Figure 5 AD), SIRPα expression is downregulated ( Figure 5 E, H), while CD3+ and CD8+ T cell infiltration significantly increased ( Figure 5 FG, IJ).

[0067] Effective anti-metastasis: The msTCD-LNP group can increase the level of anti-metastatic cytokines and decrease the level of pro-metastatic factors in tumors. Figure 6 AI), its anti-transfer mechanism is illustrated in the diagram. Figure 6 E. H&E staining of lung tissue ( Figure 6 J) showed that it had the smallest number and area of ​​lung metastases.

[0068] Experimental conclusion: The TCD-LNPs of this invention can efficiently target tumor tissues in vivo, exert excellent effects in inhibiting tumor growth and metastasis by reshaping the immune microenvironment and direct killing, and the msTCD-LNPs delivered in dual-drug combination show the strongest synergistic therapeutic advantage.

[0069] The above embodiments of the present invention are not intended to limit the scope of protection of the present invention. The implementation of the present invention is not limited thereto. All other modifications, substitutions or alterations made to the above structure of the present invention based on the above content of the present invention, in accordance with ordinary technical knowledge and common practice in the field, without departing from the basic technical idea of ​​the present invention, shall fall within the scope of protection of the present invention.

Claims

1. The use of a pharmaceutical composition in the preparation of a medicament for treating hepatocellular carcinoma, characterized in that, The pharmaceutical composition contains therapeutically effective amounts of miR-99b and SIRPα siRNA.

2. The application according to claim 1, characterized in that, The pharmaceutical composition is configured to co-deliver miR-99b and SIRPα siRNA to tumor-associated macrophages in the tumor microenvironment.

3. The application according to claim 1 or 2, characterized in that, The treatment of hepatocellular carcinoma includes at least one of the following pharmacological effects: (a) Reprogramming M2 tumor-associated macrophages to M1 type; (b) Downregulate the expression of SIRPα protein in tumor-associated macrophages; (c) Block the CD47-SIRPα "Don't eat me" signaling pathway; (d) Enhance the phagocytic activity of macrophages against hepatocellular carcinoma cells; (e) Inhibits the growth of hepatocellular carcinoma tumors; (f) Inhibits the metastasis of hepatocellular carcinoma; (g) Promotes the infiltration of CD3+ and CD8+ T cells in the tumor microenvironment.

4. The application according to claim 1 or 2, characterized in that, The miR-99b and SIRPα siRNA were co-encapsulated in the same delivery vector.

5. The application according to claim 4, characterized in that, The delivery carrier is lipid nanoparticles.

6. The application according to claim 5, characterized in that, The lipid nanoparticles are lipid nanoparticles that target M2 macrophages.

7. The application according to claim 6, characterized in that, The lipid nanoparticles are modified with mannose and are specifically taken up by M2 macrophages through mannose receptor-mediated endocytosis.

8. The application according to claim 5, characterized in that, The lipid nanoparticles contain PEGylated lipids with disulfide bonds, which are used to induce glutathione-responsive PEG shedding in the tumor microenvironment.

9. The use of a pharmaceutical composition in the preparation of a medicament for reversing the tumor immunosuppressive microenvironment, characterized in that, The pharmaceutical composition comprises therapeutically effective amounts of miR-99b and SIRPα siRNA, and the reversal is achieved by reprogramming M2 tumor-associated macrophages to M1 type and blocking the CD47-SIRPα signaling pathway.

10. The application according to claim 9, characterized in that, The tumor immunosuppressive microenvironment mentioned is the tumor microenvironment of hepatocellular carcinoma.

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