A polypeptide-modified RNA nanodrug, and a preparation method and application thereof

Abstract

The application belongs to the field of genetic engineering and biological medicine technology, and specifically discloses a polypeptide-modified RNA nanomedicine and a preparation method thereof. The IF7C(RR)-modified cationic liposome delivery platform provided by the application can target lung tissues and efficiently act on B cells in the lung, weakens the immune suppression regulation network of the lung tissue through host-oriented immune regulation, and simultaneously enhances the tumor immune surveillance function mediated by T cells. + Mechanism research shows that knocking down ANXA1 can reverse the regulatory cytokine secretion profile of tumor-conditioned B cells, weaken their ability to polarize T cells to Tregs, restore the Th1 type immune characteristics of the body, and promote the proliferation and effector function activation of CD8 + T cells. The above synergistic effects enable the preparation to reset the pro-metastasis immune microenvironment of the lung tissue during the perioperative period, thereby effectively inhibiting the lung metastasis recurrence after breast cancer surgery.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and biomedicine, specifically relating to a polypeptide-modified cationic liposome, its preparation method, and its application. Background Technology

[0002] The formation of lung metastases in breast cancer depends not only on the tumor cells themselves but also on the significant influence of the pre-metastatic and metastatic lung microenvironment. The primary tumor can alter distal lung tissue through soluble factors, exosomes, and inflammatory signals, making it easier for circulating tumor cells to remain, survive, escape immune responses, and proliferate. Perioperative inflammation and transient immune dysfunction may further promote the colonization of residual or disseminated tumor cells in the lungs. Therefore, conventional treatments targeting only tumor cells are insufficient to adequately eliminate the pro-metastatic lung microenvironment.

[0003] B cells, especially those with immunomodulatory functions, can induce Foxp3 by secreting inhibitory cytokines such as IL-10, TGF-β, and IL-35. + Regulatory T cell formation, suppression of Th1 immune responses and restriction of CD8 + T cell proliferation and effector function are involved in the establishment and maintenance of the lung metastatic microenvironment. Annexin A1 (ANXA1) is a key molecule regulating tumor immune escape, and its signaling pathway is deeply involved in the immunosuppressive network. As a calcium-dependent phospholipid-binding protein, ANXA1 can upregulate PD-L1 by activating the IL-6 / STAT3 pathway, or promote IL-10 secretion through the FPR2 pathway, and synergistically remodel the immunosuppressive microenvironment with the TGF-β / Smad pathway. Targeted intervention has shown promising results, with anti-ANXA1 antibodies enhancing anti-tumor immunity and the derived peptide A11 inducing PD-L1 degradation through competitive binding to USP7. Although the central role of ANXA1 in tumor-immune interactions has been established, its specific regulatory mechanism on lung B cells in breast cancer lung metastases remains a mystery. Furthermore, although siRNA can specifically silence target genes according to sequence, naked siRNA has poor stability in the blood, is easily degraded by nucleases, and suffers from low cellular uptake efficiency, insufficient endosome escape, and unsatisfactory in vivo distribution. Therefore, there is an urgent need to develop delivery vectors that combine nucleic acid protection, lung tissue enrichment, and active recognition by lung B cells, so as to provide a feasible path for the clinical translation of targeted RNA therapeutics in the prevention and treatment of lung metastases in breast cancer. Summary of the Invention

[0004] The purpose of this invention is to provide a lung-targeting cationic liposome delivery system modified with IF7C(RR) peptide. This system stably encapsulates and intravenously delivers a small interfering RNA (siANXA1) targeting ANXA1 to lung B cells. By reducing ANXA1 expression in B cells, it influences the secretion of immunosuppressive cytokines such as IL-10, TGF-β, and IL-35 in the lung immune microenvironment, while simultaneously inhibiting their ability to induce Treg cell formation, thereby blocking the microenvironmental support for the proliferation of lung metastases. This system utilizes the lung tissue tropism of cationic liposomes and the specific binding of the IF7C(RR) peptide to ANXA1 to achieve a continuous delivery process of lung tissue enrichment, lung B cell uptake, and ANXA1 gene silencing. This reprograms tumor-conditioned immune-tolerant B cells into effector B cells that are conducive to anti-tumor immune responses, reshaping the lung immune microenvironment and inhibiting lung metastasis after breast cancer surgery.

[0005] Meanwhile, this invention provides a method for preparing and applying peptide-modified cationic liposomes.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0007] A peptide-modified cationic liposome consists of a cationic liposome shell modified with a peptide that specifically recognizes ANXA1, loaded with an siRNA core targeting ANXA1.

[0008] In a preferred embodiment of the present invention, the polypeptide that specifically recognizes ANXA1 is IF7C(RR), and its amino acid sequence is IFLLWQRCRR (SEQ ID NO:1). Other short peptides, cyclic peptides, antibodies, antibody fragments, single-domain antibodies, aptamers, or small molecule ligands capable of specifically recognizing ANXA1 may also be used to replace IF7C(RR); conserved amino acid substitutions, terminal modifications, or cyclization of IF7C(RR) may also be performed to improve stability and affinity.

[0009] In a preferred embodiment of the present invention, the raw materials for preparing the peptide-modified cationic liposomes that specifically recognize ANXA1 include DOTAP, cholesterol, and DSPE-PEG1000-IF7C(RR), with a molar ratio of (47-48):(47-48):(4-6), preferably 47.5:47.5:5. Taking the preparation of 10 mL of peptide-modified cationic liposomes (p-LP) with a total lipid concentration of 6 mM as an example, the total lipid molar amount is 60 μmol, of which DOTAP is 28.5 μmol, cholesterol is 28.5 μmol, and DSPE-PEG1000-IF7C(RR) is 3.0 μmol; other preparation batches can be scaled up or down proportionally according to the above ratio. Alternatively, the lipid components can be conventionally adjusted without changing the core technical concept of the present invention.

[0010] Specifically, the DSPE-PEG1000-IF7C(RR) is prepared by covalently coupling the thiol group of the cysteine ​​residue in the IF7C(RR) peptide with the terminal maleimide group of DSPE-PEG1000-Mal via a thiol-maleimide Michael addition reaction. In this embodiment of the invention, DSPE-PEG1000-IF7C(RR) was synthesized under contract and its structure was confirmed by 1H NMR spectroscopy and MALDI-TOF mass spectrometry. Alternatively, PEG-phospholipids of different molecular weights, other hydrophilic spacer arms, or cleavable linker arms can be used to connect the peptide to the liposome surface, as long as it does not significantly weaken ligand exposure and targeting ability.

[0011] In a preferred embodiment of the present invention, the sequence of the siRNA targeting ANXA1 is as follows:

[0012] Justice Chain: 5'-GCACAAAGCUAUCAUGGUUAAdTdT-3' (SEQ ID NO:2);

[0013] Antisense chain: 5'-UUAACCAUGAUAGCUUUGUGCdTdT-3' (SEQ ID NO:3).

[0014] In addition, other effective silencing sequences for ANXA1 mRNA can be selected, or corresponding siRNAs can be designed for human ANXA1 to meet clinical translation needs; the silencing efficiency and off-target effects of alternative sequences need to be verified. Similarly, chemically modified siRNA, shRNA, antisense oligonucleotides, miRNA regulators, or CRISPR interference systems can be used to inhibit ANXA1 expression, with reversible and short-term RNA silencing protocols being preferred.

[0015] In a preferred embodiment of the present invention, the composite ratio of the cationic liposome shell and the siRNA core is set such that 5 μL of liposomes with a total lipid concentration of 6 mM are added for every 1 μg of siRNA, and the charge ratio (N / P ratio) calculated according to the formulation is approximately 5:1.

[0016] A method for preparing peptide-modified cationic liposomes includes the following steps:

[0017] DOTAP, cholesterol, and DSPE-PEG1000-IF7C(RR) were mixed and formed into a film, which was then hydrated and sonicated to obtain peptide-modified cationic liposomes.

[0018] The siRNA targeting ANXA1 is loaded into peptide-modified cationic liposomes to obtain the product.

[0019] As a preferred embodiment of the present invention, DOTAP, cholesterol, and DSPE-PEG1000-IF7C(RR) were mixed in a molar ratio of 47.5:47.5:5 and dissolved in anhydrous ethanol. The mixture was then rotary evaporated under reduced pressure at 37°C for 1 h to form a lipid film. RNase-free water was added, and the mixture was hydrated at 65°C for 40 min, with the total lipid concentration adjusted to 6 mM after hydration. After sonication in a 110 W water bath for 5 min, the mixture was filtered through a 0.22 μm sterile filter membrane to obtain peptide-modified cationic liposomes.

[0020] Applications of a polypeptide-modified cationic liposome include, but are not limited to:

[0021] (1) Application in the preparation of drugs for reprogramming lung B cell function;

[0022] (2) Application in the preparation of drugs that remodel the pulmonary immune microenvironment;

[0023] (3) Application in the preparation of drugs that inhibit lung metastasis and recurrence of breast cancer;

[0024] (4) Use in the preparation of drugs for treating lung metastases of tumors or lung immune-related diseases characterized by abnormal lung immunosuppressive B cells and ANXA1.

[0025] In a preferred embodiment of the present invention, the function of the reprogrammed lung B cells is to reprogram tolerant B cells into effector B cells that promote Th1 lymphocyte activation. The reprogrammed B cells can inhibit Foxp3. + Regulatory T cell differentiation, promotes Th1 cell activation, and relieves CD8 inhibition. + T cell proliferation was inhibited.

[0026] As a preferred embodiment of the present invention, the reshaping of the lung immune microenvironment includes reducing immunosuppressive Foxp3.+ Increased regulatory T cell ratio and CD8 + Cytotoxic T lymphocyte infiltration level.

[0027] A drug for inhibiting lung metastasis of breast cancer, comprising the peptide-modified cationic liposome.

[0028] In a preferred embodiment of the present invention, the content of polypeptide-modified cationic liposomes in the drug is 0.01 wt%-99.99 wt%.

[0029] In a preferred embodiment of the present invention, the drug further includes other antitumor pharmacodynamic components.

[0030] As a preferred embodiment of the present invention, the drug further includes pharmaceutically acceptable excipients, including but not limited to water for injection, buffers, osmotic pressure regulators, stabilizers, lyophilization protectants, antioxidants, and pH adjusters.

[0031] As a preferred embodiment of the present invention, the dosage form of the drug includes, but is not limited to, injection, lyophilized powder for injection, inhalation preparation, and intratracheal administration preparation.

[0032] As a preferred embodiment of the present invention, the route of administration of the drug includes, but is not limited to, intravenous administration, inhalation administration, intratracheal administration, or perioperative local delivery, in order to improve lung exposure.

[0033] The beneficial effects of this invention are:

[0034] The polypeptide-modified cationic liposomes provided by this invention have the following advantages:

[0035] (1) Dual targeting advantage: Combining the lung tissue tropism of DOTAP cationic liposomes with the active recognition of ANXA1 mediated by IF7C(RR) peptide not only improves the enrichment of the formulation in the lungs, but also enhances its ability to be taken up by lung tissue B cells.

[0036] (2) Unified delivery and therapeutic target: IF7C (RR) is used to recognize ANXA1, and the delivered siRNA is used to silence ANXA1 at the same time, so that the target recognition molecule and the therapeutic target molecule work together to improve cell selectivity and gene silencing efficiency.

[0037] (3) The physicochemical properties of the formulation are suitable for intravenous administration: the average hydrated particle size of the obtained particles is about 100-150 nm, the Zeta potential is about +20 mV, the polydispersity index is about 0.2, the siRNA encapsulation rate is about 80%, and the particle size, potential and polydispersity index are basically stable when stored at 4℃ for at least 14 days. The in vitro hemolysis rate is less than 5%.

[0038] (4) Host-guided immune reprogramming: The agent does not simply kill tumor cells, but silences lung B cells ANXA1, reduces immunosuppressive factors such as IL-10, TGF-β and IL-35, reduces Treg induction and restores Th1 and CD8. + T-cell effector function weakens the lung's pro-metastatic immune environment at its source.

[0039] (5) Reversible and sequence-specific: siRNA-mediated gene silencing has the characteristics of target sequence specificity, dose adjustable and relatively reversible, which helps to reduce the risks of long-term permanent gene modification.

[0040] (6) The efficacy is consistent with the model: The delivery system showed the effect of reducing lung metastasis burden and improving lung immune phenotype in both 4T1 and E0771 homologous breast cancer postoperative lung metastasis models, suggesting that its mechanism of action has a certain universality.

[0041] (7) Good biosafety: After intravenous administration to healthy mice, no significant abnormalities were found in the histology of major organs and the biochemical indicators of liver and kidney function.

[0042] The preparation process of peptide-modified cationic liposomes provided by this invention includes: coupling IF7C(RR) peptide with Mal-DSPE-PEG1000 to obtain DSPE-PEG1000-IF7C(RR) targeting lipids, which are then combined with DOTAP and cholesterol to prepare peptide-modified cationic liposomes. A delivery system is then obtained by electrostatically loading siANXA1. After intravenous administration, the cationic liposomes first accumulate in the lungs due to their inherent lung tissue tropism; the IF7C(RR) peptide on the liposome surface further binds to ANXA1 on the surface of lung tissue-related cells, promoting the anchoring of the formulation in B-cell-rich regions of the lungs and its absorption by B220. + B cells take up siRNA; after entering the cell, cationic liposomes promote siRNA endosome escape, releasing siANXA1 and reducing ANXA1 mRNA and protein expression through RNA interference; after ANXA1 silencing, the expression and secretion of immunosuppressive factors such as IL-10, TGF-β, and IL-35 in tumor-conditioned B cells decrease, while pro-inflammatory signals such as TNF-α and IL-6 are relatively enhanced; B cells induce Foxp3 + Treg and IL-10 + CD4 + The ability to form T cells is weakened, while IFN-γ is promoted. + Th1 cells form and relieve CD8 inhibition. + Inhibition of T cell proliferation. This reshapes the lung immune microenvironment, enhances cytotoxic T cell infiltration and granzyme B expression, and inhibits lung metastasis after breast cancer surgery.

[0043] The formulation provided by this invention can be used to prepare drugs for preventing or inhibiting lung metastasis of breast cancer, especially lung metastasis recurrence after breast cancer surgery, and has potential clinical application prospects and commercial value. Attached Figure Description

[0044] Figure 1 Preparation of siANXA1@p-LP and its mechanism of action in preventing lung metastasis of breast cancer.

[0045] Figure 2 The results are from the proton nuclear magnetic resonance (NMR) spectrum and mass spectrometry.

[0046] Figure 3 The physicochemical properties of siANXA1@p-LP are given.

[0047] Figure 4 This refers to the encapsulation of siANXA1@p-LP.

[0048] Figure 5 For the storage stability of siANXA1@p-LP.

[0049] Figure 6 The hemolysis status of siANXA1@p-LP.

[0050] Figure 7 These are stained sections of the major organs of a mouse.

[0051] Figure 8 The results are the biochemical analysis results of liver and kidney function in mouse blood.

[0052] Figure 9 The results are from an in vitro cell uptake experiment of siANXA1@p-LP.

[0053] Figure 10 This shows the distribution of siANXA1@p-LP in vivo.

[0054] Figure 11 A flow cytometry gating strategy for quantitative detection of Cy5 fluorescence signal in lung B cells.

[0055] Figure 12 Flow cytometry analysis and immunofluorescence co-localization of Cy5-siANXA1@LP / p-LP uptake by lung B cells.

[0056] Figure 13 To detect the ANXA1 gene silencing efficiency in B cells treated with siANXA1@p-LP and stimulated with 4T1 conditioned medium.

[0057] Figure 14 Transcriptomic analysis of B cells treated with siANXA1@p-LP.

[0058] Figure 15Cytokine profile analysis of B cells treated with siANXA1@p-LP.

[0059] Figure 16 Cytokine profile analysis of B cell supernatant treated with siANXA1@p-LP.

[0060] Figure 17 CD4 in B cell and T cell co-culture system + Flow cytometry gating strategies for T cells.

[0061] Figure 18 B cells and CD4 were treated with different tumor conditioned media. + Schematic diagram of a T cell co-culture system.

[0062] Figure 19 The effect of siANXA1@p-LP treatment on functional Tregs in B cells.

[0063] Figure 20 For IFN-γ + CD4 + Representative flow cytometry scatter plots and quantitative analysis results of T cells.

[0064] Figure 21 CD8 + Schematic diagram and results of T cell proliferation detection experiment.

[0065] Figure 22 Experimental design and dosing timeline for lung metastasis after 4T1-Luc breast cancer surgery.

[0066] Figure 23 To enable dynamic detection of lung metastasis in small animals using in vivo imaging.

[0067] Figure 24 The therapeutic effect of siANXA1@p-LP on experimental lung metastases of 4T1-Luc breast cancer in mice was investigated.

[0068] Figure 25 Immunohistochemical staining of Ki67 in lung tissue sections (indicating tumor cell proliferation).

[0069] Figure 26 The expression level of ANXA1 in lung B cells was detected by flow cytometry.

[0070] Figure 27 Flow cytometry analysis of the lung immune microenvironment.

[0071] Figure 28 Experimental design and dosing timeline for E0771 breast cancer patients with postoperative lung metastasis.

[0072] Figure 29 To dynamically assess the effectiveness of treatments that inhibit lung metastasis.

[0073] Figure 30 The results are from in vitro observation of lung tissue and lung index measurement.

[0074] Figure 31 To verify the silencing efficiency of ANXA1 in B cells of lung tissue.

[0075] Figure 32 This is the result of immunophenotypic analysis of lung tissue by flow cytometry. Detailed Implementation

[0076] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments and experimental examples. However, those skilled in the art should understand that the embodiments are only used to illustrate the technical solution of the present invention and should not be regarded as limiting the scope of protection of the present invention. Based on the technical concept disclosed in the present invention, without changing the key components, key process conditions and their functional effects, scaling up or down the preparation batch proportionally, or making equivalent substitutions and process adjustments as is common in the art, all belong to reasonable modifications of the technical solution of the present invention.

[0077] Unless otherwise specified, the raw materials, reagents, instruments, etc. used in the following embodiments and experimental examples are all commonly used in the art or commercially available to the public. Unless otherwise specified, the experimental methods used in the experimental examples are conventional methods in the art. All terms and abbreviations used have their conventional meanings in the art.

[0078] Example 1

[0079] This embodiment provides a polypeptide-modified cationic liposome and its preparation method. Figure 1 A), specifically including:

[0080] (1) Synthesis of DSPE-PEG1000-IF7C(RR)

[0081] An IF7C(RR) polypeptide containing cysteine ​​residues, with the amino acid sequence IFLLWQRCRR, was used. The thiol group on the side chain of the cysteine ​​residues in this polypeptide underwent a thiol-maleimide Michael addition reaction with the terminal maleimide group of DSPE-PEG1000-Mal to obtain the amphiphilic conjugate DSPE-PEG1000-IF7C(RR). The DSPE-PEG1000-IF7C(RR) used in this example was synthesized by Xi'an Qiyue Biotechnology and its structure was confirmed by 1H NMR spectroscopy and MALDI-TOF mass spectrometry.

[0082] In the 1H NMR spectrum, the conjugate simultaneously exhibited characteristic proton signals originating from both DSPE-PEG1000-Mal and the IF7C(RR) peptide, while the characteristic maleimide resonance signal of DSPE-PEG1000-Mal completely disappeared, confirming that the thiol-maleimide reaction had been fully completed. Figure 2 A). Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MADS) showed a significant shift in the molecular weight of DSPE-PEG1000-Mal, further confirming the successful coupling reaction. Figure 2 B). The reaction route is as follows:

[0083] .

[0084] (2) Preparation of peptide-modified cationic liposomes

[0085] Taking the preparation of 10 mL of IF7C(RR) peptide-modified cationic liposomes p-LP with a total lipid concentration of 6 mM as an example, the total lipid molar amount was 60 μmol. According to the molar ratio of DOTAP:cholesterol:DSPE-PEG1000-IF7C(RR) = 47.5:47.5:5, 28.5 μmol of DOTAP, 28.5 μmol of cholesterol, and 3.0 μmol of DSPE-PEG1000-IF7C(RR) were respectively taken. The three lipids were dissolved separately in anhydrous ethanol and then combined to form a clear and homogeneous lipid-ethanol solution.

[0086] The above lipid ethanol solution was placed in a rotary evaporator and evaporated under reduced pressure at 37°C for 1 h until the organic solvent was almost completely removed and a continuous and uniform lipid film formed on the inner wall of the container. 10 mL of RNase-free water was added to wash away the lipid film, and the solution was hydrated in a water bath at 65°C for 40 min to obtain a crude lipid dispersion with a total lipid concentration of 6 mM. This dispersion was then treated with ultrasonication in a water bath at a power of 110 W for 5 min. Immediately after ultrasonication, the solution was filtered through a 0.22 μm sterile filter to obtain IF7C(RR) peptide-modified cationic liposomes p-LP.

[0087] In other embodiments of the present invention, the above-mentioned components and hydration volumes can be scaled up or down by the same proportion.

[0088] (3) Loading of siANXA1

[0089] siANXA1 and p-LP are loaded using an electrostatic recombination method. Taking 1 μg of siANXA1 as an example, 1 μg of siANXA1 is added to 1.0 μL of 1.5 M NaCl solution, and the volume is brought to 5.0 μL with RNase-free water to obtain the siRNA solution. Separately, 5.0 μL of the above-mentioned p-LP solution with a total lipid concentration of 6 mM is taken. The 5.0 μL of siRNA solution is added to the 5.0 μL of p-LP solution in portions, gently mixing with a pipette after each addition, avoiding vigorous vortexing. After mixing, the mixture is incubated at room temperature (20-25℃) for 15 min to obtain a total volume of 10.0 μL of siANXA1@p-LP, with an siRNA concentration of 0.1 mg / mL and a final NaCl concentration of 150 mM; the N / P ratio calculated according to this formulation is approximately 5:1.

[0090] In other embodiments of the present invention, when preparing a larger volume, each component is scaled up proportionally according to the above proportions.

[0091] Meanwhile, when preparing non-targeted cationic liposomes LP, DSPE-PEG1000-Mal was used to replace DSPE-PEG1000-IF7C(RR) in equal molar amounts. The remaining lipid molar ratios, film formation, hydration, sonication, filtration, and siANXA1 loading conditions were the same as those for p-LP, resulting in siANXA1@LP as a control.

[0092] Example 2

[0093] This embodiment provides an application of peptide-modified cationic liposomes (same as in Example 1), including:

[0094] (1) In the preparation of reprogrammed lung B cell function (reprogramming tolerant B cells into effector B cells that promote Th1 lymphocyte activation, the reprogrammed B cells can inhibit Foxp3) + Regulatory T cell differentiation, promotes Th1 cell activation, and relieves CD8 inhibition. + Application in drugs that inhibit T cell proliferation;

[0095] (2) In the preparation of remodeling the lung immune microenvironment (including reducing immunosuppressive Foxp3) + Increased regulatory T cell ratio and CD8 + Application in drugs that assess the level of cytotoxic T lymphocyte infiltration;

[0096] (3) Application in the preparation of drugs that inhibit lung metastasis of breast cancer.

[0097] Experimental Example

[0098] I. Experimental Materials

[0099] via MojoSortTM Primary B cells were isolated from the spleen tissue of BALB / c WT mice using a mouse B cell sorting kit. The isolated cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 U / mL penicillin-streptomycin. Mouse breast cancer cell lines 4T1 and E0771 were provided by the American Type Culture Collection (ATCC). 4T1-Luc and E0771-Luc are stable transfection cell lines expressing luciferase (LUC) and were cultured in DMEM complete medium containing 10% FBS and 100 U / mL penicillin / streptomycin. All cells were routinely cultured at 37°C under a constant temperature and humidity environment with 5% CO2.

[0100] BALB / c wild-type mice and C57BL / 6J wild-type mice were purchased from Beijing Spefolk Biotechnology Co., Ltd., and the housing environment strictly adhered to SPF-level cleanliness standards. All animal experimental procedures have been reviewed and approved by the Experimental Animal Ethics Committee of Zhengzhou University.

[0101] CHO-HP (Chol), DSPE-PEG1000-Mal (maleimide-based polyethylene glycol-modified distearate phosphatidylethanolamine), and DOTAP (1,2-dioleoyl-3-trimethylammonium propane) were purchased from Shanghai Aivito Pharmaceutical Technology Co., Ltd., while DSPE-PEG1000-IF7C(RR) was synthesized by Xi'an Qiyue Biotechnology. The MojoSort™ Mouse B Cell Isolation Kit was purchased from BioLegend. The siRNA and primer sequences are shown in Table 1.

[0102] Table 1. siRNA sequences and primer sequences (SEQ ID NO:2-17)

[0103]

[0104] II. Experimental Methods

[0105] 1. Physicochemical properties of liposomes

[0106] We compared IF7C(RR) peptide-modified siANXA1 liposome complexes with unmodified liposomes and systematically investigated their physicochemical properties, including appearance, particle size, potential, microstructure, siRNA encapsulation efficiency, storage stability, and biocompatibility.

[0107] Liposome particle size and potential detection: A suitable volume of the prepared liposome complex solution was measured, diluted, and its particle size and surface potential were measured using a laser particle size and potential analyzer. The results were recorded in detail.

[0108] Electron microscopy analysis: 10 μL of sample was added to a copper mesh, allowed to precipitate for 1 min, and the supernatant was removed with absorbent paper. 10 μL of uranium acetate was then added to the copper mesh, allowed to precipitate for 1 min, and the supernatant was removed with absorbent paper. The sample was dried at room temperature for several minutes. Transmission electron microscopy (TEM) was performed at 80-120 kV, and images were acquired and analyzed.

[0109] Non-denaturing PAGE gel electrophoresis assay: siRNA samples were double-stranded RNA (dsRNA). Naked siRNA, siANXA1@LP, or siANXA1@p-LP samples were taken, adjusted to 5 μL with RNase-free water (each sample containing 0.05 μg siRNA), and then 5 μL of 2×RNA Loading Buffer was added and mixed thoroughly. The mixture was kept on ice. 1×TBE was used as the electrophoresis buffer, with 10 μL loaded into each well. Electrophoresis was performed at 120 V for 60 min. After electrophoresis, the gel was stained for nucleic acids at room temperature in the dark for 15 min, washed with DEPC water, and images were acquired using a gel imaging system to evaluate the complexation / retention of free siRNA and liposomes with siRNA.

[0110] The RiboGreen method was used to determine the encapsulation efficiency: a standard curve of nucleic acid concentration versus fluorescence intensity was established using a serial dilution method. 200 μL of the nanocarrier suspension to be tested was placed in a 1.5 mL centrifuge tube, and an equal volume of 20% Triton X-100 solution was added. The tube was vortexed for 30 s to completely disintegrate the vesicle structure. After standing at room temperature for 15 min, the tube was serially diluted with TE buffer. RiboGreen reagent was added, and the fluorescence value was measured. The total nucleic acid concentration was calculated using the standard curve equation. An equal volume of the carrier suspension to be tested was taken, diluted directly with TE buffer without surfactant treatment, and the fluorescence intensity was measured. To eliminate interference from nanoparticle scattering, some samples were centrifuged at 2500 g for 20 min, and the supernatant was used for analysis. The concentration of free nucleic acid and the nucleic acid encapsulation efficiency were calculated.

[0111] 2. Safety assessment of liposomes

[0112] In vitro hemolysis experiments were performed using siANXA1@LP and siANXA1@p-LP, respectively. Simultaneously, healthy BALB / c WT mice were treated with the same tail vein injection regimen as in the therapeutic experiments, divided into four groups: PBS, siNC@p-LP, siANXA1@LP, and siANXA1@p-LP. At the experimental endpoint, major organs (liver, spleen, lungs, kidneys, and heart) were collected for hematoxylin-eosin (H&E) staining. To more comprehensively assess physiological toxicity, key serum biochemical indicators reflecting liver and kidney function were measured.

[0113] 3. Uptake of liposome complexes by primary B cells and B cells in lung tissue

[0114] B cells from the spleen of BALB / c WT mice were selected as model cells. Fluorescence tracing experiments used Cy5-labeled siANXA1, with Cy5 conjugated to the 5′ end of the positive strand of siANXA1. The fluorescent tracer formulation contained only Cy5-siANXA1 and was not mixed with unlabeled siANXA1. The total siRNA dosage and liposome complexation ratio were consistent with the non-fluorescent formulation. B cells were cultured at 5 × 10⁻⁶ cells / year. 6 B cells were seeded at a density of 1 B cell per 35 mm confocal glass dish in poly-L-lysine-treated culture dishes and cultured overnight. After incubation, PBS, Cy5-siANXA1@LP, or Cy5-siANXA1@p-LP were added, and the cells were incubated for 2, 4, and 6 h, respectively. After incubation, the cells were washed twice with PBS, stained with Hoechst 33342, and observed under a confocal microscope. Flow cytometry quantification assays were performed using B cells at a density of 1 × 10⁻⁶ cells / dish. 6 Each well was seeded with one cytogen per well in a 24-well plate, treated for the same duration, washed, and Cy5 fluorescence was detected.

[0115] To investigate the in vivo distribution characteristics of liposome formulations, BALB / c WT mice bearing 4T1 tumors were injected via tail vein. At 6 h and 24 h, in vivo imaging, flow cytometry, and immunohistochemistry were used to investigate the uptake of B lymphocytes by the siANXA1@p-LP delivery system in the lungs. First, 4T1 cells in logarithmic growth phase were collected, resuspended in sterile PBS, and 1×10⁶ cells were subcutaneously seeded at the right mammary pad of each mouse. 6 Four T1 tumor cells were collected, and the tumor volume was measured using calipers. After 10 days, the subcutaneous tumor volume in mice reached 200 mm. 3 Surgical resection was performed on both sides. Mice were injected via tail vein with PBS or different cationic liposome complexes labeled with Cy5 (siRNA at a dose of 0.5 mg / kg). After a period of time, the mice were sacrificed after perfusion of the heart with PBS, and heart, liver, kidney, spleen, and lung tissues were collected for fluorescence imaging analysis. After imaging, half of the lung tissue was thoroughly digested with collagenase and other digestive enzymes. The resulting cell suspension was filtered, centrifuged, and the supernatant was discarded. Red blood cells were lysed with erythrocyte lysate, neutralized with PBS, and resuspended after centrifugation with an appropriate volume of staining buffer. 100 µL of the cell suspension was added, and flow cytometry antibody was added for staining for 20 min. The cells were washed twice with staining buffer, stained with a live / dead dye, resuspended, and analyzed using flow cytometry. After fluorescence detection, the other half of the lung tissue was embedded in embedding medium, sectioned, and stained.

[0116] 4. Cytokine profile analysis of B cells after ANXA1 silencing with siANXA1@p-LP

[0117] Using sorted B lymphocytes as model cells, after tumor supernatant treatment (4T1-CM treatment), the silencing effect of different treatment groups (G1: PBS, G2: 4T1-CM+PBS, G3: 4T1-CM+siNC@p-LP, G4: 4T1-CM+siANXA1@jet, G5: 4T1-CM+siANXA1@LP, G6: 4T1-CM+siANXA1@p-LP) on ANXA1 expression in B cells was investigated. Simultaneously, the effects of ANXA1 silencing on other immune-regulating cytokines were studied using whole-transcriptome sequencing (bulk RNA-seq), qPCR, and enzyme-linked immunosorbent assay (ELISA). The Trizol method for separating and extracting total RNA, reverse transcription for preparing cDNA, quantitative real-time PCR analysis, whole-transcriptome sequencing, extraction of cellular proteins, Western blotting, and ELISA for cytokine detection are all standard methods in this field.

[0118] 5. Research on reprogramming tumor-conditioned medium (T-CM)-induced tolerant B cells into effector B cells that promote Th1 lymphocyte activation using siANXA1@p-LP.

[0119] Spleens from BALB / c WT mice were selected for B cell sorting. After stimulation with tumor supernatant, siANXA1@p-LP was added for treatment, and T cell sorting was performed simultaneously. After co-incubation, Th1 and CD8 were detected by flow cytometry. + T cell proliferation.

[0120] Isolation of B and T cells from mouse spleen: Healthy BALB / c WT mice were euthanized, and the spleens were removed and homogenized in PBS. The cell homogenate was collected, centrifuged at 400 g for 5 min, the supernatant was discarded, and the cells were lysed with erythrocyte lysis buffer. The homogenate was then neutralized with PBS, centrifuged again, washed with PBS, and purified using MojoSort. TM Resuspend cells in buffer and incubate on ice for 30 min. Filter spleen cells using a 70 µm cell filter, centrifuge at 400 g for 5 min, resuspend cells, and adjust cell density to 1 × 10⁶ cells / mL. 8 100 μL / mL. 7Add cell suspension of 10 μL / cell to a new tube, add 10 µL of Biotin-Antibody Cocktail, mix well by pipetting, and incubate on ice for 15 min. Resuspend streptavidin nanobeads by vortexing at maximum speed, add 10 µL, mix well, and incubate on ice for 15 min. Add 2.5 mL of MojoSort to the above system. TM Buffer the cells and let them stand on a magnetic rack for 5 minutes. Collect the supernatant, which is the target cell. To improve cell purity, repeat this step twice. Centrifuge the collected supernatant, collect the cell pellet, resuspend it in culture medium, and then incubate it in a cell culture incubator.

[0121] Reprogrammed B cells suppress Foxp3 + Detection of regulatory T cell differentiation: Sorted B cells were evenly seeded into 24-well plates at a density of 800,000 cells per well. Different treatments (G1: PBS, G2: siNC@p-LP, G3: siANXA1@LP, G4: siANXA1@p-LP) were added for 48 h. Sorted CD4+ cells were then... + T cells were co-cultured with the above-treated B cells, and after 5 days, Foxp3 was analyzed by flow cytometry. + Regulatory T cell detection.

[0122] Assay for the effect of reprogrammed B cells on Th1 cell activation: Sorted B cells were seeded into 24-well plates, with 8 × 10⁶ cells per well. 5 Different treatments (G1: PBS, G2: siNC@p-LP, G3: siANXA1@LP, G4: siANXA1@p-LP) were added and treated at 37°C for 48 h. After sorting, CD4... + T cells were co-cultured with B cells treated with the above different methods for 5 days, and CD4 counts were analyzed by flow cytometry. + Measurement of changes in IFN-γ expression in T cells.

[0123] Reprogrammed B cells relieve CD8 + Detection of T cell proliferation inhibition: Spleens from BALB / c tumor-bearing mice were used for B cell sorting, and the sorted B cells were seeded into 24-well plates. Equal amounts of different treatments (G1: PBS, G2: siNC@p-LP, G3: siANXA1@LP, G4: siANXA1@p-LP) were added to the wells, and the plates were incubated at 37°C for 48 h. The sorted CD8 cells were resuspended in PBS. + T cells were resuspended in 1 mL of buffer to adjust the cell concentration. CD8 cells were sorted using CFSE labeling. +T cells were incubated with CFSE at a final working concentration of 5 μM at 37°C for 5 min, then neutralized with 10 volumes of 1×PBS buffer, centrifuged at 400g for 5 min, the supernatant was discarded, and the precipitate was resuspended in a culture medium containing CD3 / CD28 magnetic beads and IL2. The treated CD8... + T cells were co-cultured with B cells in a 24-well plate for 72 h before flow cytometry was used to detect CFSE proliferation.

[0124] 6. Study on the immunomodulatory and antitumor effects of siANXA1@p-LP in the treatment of postoperative lung metastases in a mouse 4T1 breast cancer tumor model.

[0125] Based on 4T1-Luc subcutaneous tumor mice, an experimental lung metastasis model was established after surgical resection. Following this, the mice were treated with three injections of siANXA1@p-LP. The results were monitored using in vivo imaging, statistical analysis of lung metastases via HE staining of lung tissue at the experimental endpoint, and the expression of ANXA1 in B cells and the immunosuppressive effect on Foxp3. + Regulatory T cells and CD8 + Using T cells as evaluation indicators, this study investigated the antitumor effect of siANXA1@p-LP by silencing ANXA1 expression.

[0126] Experimental postoperative lung metastasis treatment study based on 4T1 tumor model mice: BALB / c mice underwent subcutaneous orthotopic tumor bearing: Logarithmically growing 4T1-Luc tumor cells were collected, and the cell pellet was resuspended in sterile PBS buffer and counted on a clean bench. 1 × 10⁻⁶ cells were subcutaneously inoculated into the left mammary pad of each mouse. 6 Four T1-Luc cells were used, and the tumor volume was measured using calipers. After approximately 10 days, when the subcutaneous tumor volume reached 200 mm, the tumor was successfully treated. 3 Surgical dissection was performed on both sides. Establishment of a breast cancer lung metastasis model: Seven days after subcutaneous tumor inoculation, when 4T1-Luc cells were in the logarithmic growth phase, cells were collected and the cell pellet was resuspended in sterile PBS. 1×10⁻⁶ cells were injected into each animal via the tail vein. 6 Tumor cells. Drug treatment: On days 3, 6, and 9 after tumor implantation, mice were injected with different drugs via the tail vein (G1: PBS, G2: siNC@p-LP, G3: siANXA1@LP, G4: siANXA1@p-LP). Imaging observation: On day 12, mice were injected intraperitoneally with luciferase substrate at a dose of 150 mg / kg body weight, and in vivo imaging was performed 5 minutes later. Experimental endpoint treatment: After imaging, the animals were sacrificed, and the lung tissue was used for paraffin section pathological analysis and flow cytometry detection.

[0127] Bioluminescence Imaging: Preparation of Fluorescein Stock Solution: Dissolve D-fluorescein potassium salt in calcium- and magnesium-free PBS buffer to prepare a fluorescein stock solution with a concentration of 15 mg / mL. Filter the solution through a 0.22 μm filter and aliquot it into light-protected centrifuge tubes. Store the solution at -20°C. Fluorescein Potassium Salt Injection: Inject the above-mentioned fluorescein stock solution intraperitoneally into mice at a dose of 150 mg / kg of mouse body weight. Mouse Anesthesia: Five minutes after injection of fluorescein potassium salt, place the mice in an anesthesia chamber and anesthetize them with isoflurane. Imaging Detection: Place the anesthetized mice in the imaging chamber of the small animal in vivo imaging system. Continuously administer an appropriate amount of gaseous anesthetic to maintain the anesthesia state. After adjusting the appropriate exposure time, perform bioluminescence imaging.

[0128] 7. Anti-tumor metastasis effect of siANXA1@p-LP in the E0771 breast cancer postoperative lung metastasis model

[0129] Using E0771-Luc subcutaneous tumor-bearing mice as the research subjects, an experimental lung metastasis model was further constructed after surgical resection of the subcutaneous tumor. After the model was established, the mice were treated with different preparations (G1: PBS, G2: siNC@p-LP, G3: siANXA1@LP, G4: siANXA1@p-LP). In vivo bioluminescence imaging was performed on days 1, 3, 8, and 13 to dynamically track the progression of lung metastasis. At the experimental endpoint (day 13), lung organs were harvested from the mice, and the tumor burden was analyzed by lung index (lung weight / body weight ratio). At the same time, the expression level of ANXA1 in B cells and the immunosuppressive Foxp3 were detected. + Regulatory T cells and CD8 + This study investigated relevant T cell indicators to explore the anti-tumor effect of siANXA1@p-LP by silencing ANXA1 expression.

[0130] Experimental Treatment of Postoperative Lung Metastasis Based on E0771 Tumor Model Mice: Construction of Subcutaneous Orthotopic Tumors: E0771-Luc cells in logarithmic growth phase were digested, then resuspended in sterile PBS, and seeded (1×10⁻⁶ cells) at the left mammary pad of each mouse. 6 (each tumor individually), measuring the size of the tumor. After 10 days, when the subcutaneous tumor volume reached 200 mm... 3Surgical resection was performed on both sides. Establishment of the experimental lung metastasis model: On day 7 after orthotopic inoculation, 1 million E0771-Luc tumor cells were injected into each mouse via the tail vein. Drug treatment: After tumor bearing, the model mice were injected via the tail vein with different formulations of liposome complexes (G1: PBS, G2: siNC@p-LP, G3: siANXA1@LP, G4: siANXA1@p-LP), for a total of 3 injections. Small animal in vivo imaging: Luciferase substrate was injected intraperitoneally at 150 mg / kg mouse body weight, and small animal in vivo imaging was performed 5 minutes later. After imaging, the mice were euthanized, and lung tissue was removed; one portion of the lung tissue was embedded in paraffin and stained, while the other portion was used for flow cytometry analysis. Immunohistochemical staining and flow cytometry analysis are standard methods in this field.

[0131] III. Experimental Results

[0132] 1. Physicochemical characterization of the siANXA1@p-LP delivery system

[0133] Both siANXA1@LP and siANXA1@p-LP formed homogeneous emulsion dispersions in water and exhibited a significant Tyndall effect, suggesting the presence of nanoscale particles in the system. Figure 3 A). Dynamic light scattering analysis results show that siANXA1@LP and siANXA1@p-LP have similar intensity-weighted particle size distribution characteristics, with an average hydrated particle size of approximately 100-150 nm and low polydispersity indices (approximately 0.2), indicating that both have narrow particle size distributions. Figure 3 B, C). Both liposome complexes exhibited a positive Zeta potential of approximately +20 mV, with similar values. This potential characteristic is beneficial for siRNA complexation and cellular uptake. Figure 3 D).

[0134] Transmission electron microscopy results showed that both siANXA1@LP and siANXA1@p-LP were smooth, nearly spherical vesicles with no obvious aggregation. Figure 4 A). In the non-denaturing PAGE electrophoresis experiment, both liposome formulations completely retained siANXA1; calculations showed that the encapsulation efficiency of siRNA by siANXA1@LP and siANXA1@p-LP was approximately 80% ( Figure 4 B, C).

[0135] During storage, the particle size, polydispersity index, and potential of siANXA1@p-LP remained essentially unchanged for at least 14 days, indicating that it has good colloidal stability. Figure 5 ).

[0136] 2. The siANXA1@p-LP delivery system exhibits good biocompatibility.

[0137] Blood was collected from healthy mice via orbital sampling and prepared into a 2% erythrocyte suspension. This suspension was then co-incubated with the prepared cationic liposome complex at 37°C for 3 hours. In vitro incubation results showed that the hemolysis rates of siANXA1@LP and siANXA1@p-LP were extremely low (<5%), similar to the hemolysis rate of the saline negative control group, and significantly lower than that of the deionized water positive control group. Figure 6 ).

[0138] All experimental groups (PBS group, siNC@p-LP group, siANXA1@LP group, and siANXA1@p-LP group) demonstrated good systemic safety. Histopathological evaluation further validated the drug's biosafety. Sections of major organs such as the heart, liver, spleen, lungs, and kidneys from the experimental mice showed clear and intact microstructures, with no obvious tissue lesions, acute cell damage, or abnormal inflammatory cell infiltration observed. Figure 7 Serum biochemical analysis of liver and kidney function showed high consistency in the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine (CR) among the groups, and no abnormal increases in these indicators due to drug administration were observed. Figure 8 The above results indicate that siANXA1@p-LP has good systemic safety.

[0139] 3. Cy5-siANXA1@p-LP uptake and targeted enrichment by B cells in vivo and in vitro

[0140] After co-incubating Cy5-siANXA1@LP (G1) with B cells for 2 h, only a weak Cy5 fluorescence signal was observed under confocal microscopy, and this signal gradually increased after 4 h and 6 h of incubation. Conversely, at all detection time points, Cy5-siANXA1@p-LP (G2) induced significantly stronger Cy5 fluorescence signals in cells than Cy5-siANXA1@LP, and the fluorescence was distributed in a perinuclear punctate pattern, consistent with the endocytic uptake characteristics of liposomes. Figure 9 A). Flow cytometry results showed that, compared with the PBS control group (G0), the Cy5 fluorescence signal of both liposome formulations shifted to the right with increasing incubation time; after 2-6 h of incubation, the mean fluorescence intensity (MFI) of Cy5 in cells significantly increased, and at all detection time points, the Cy5 MFI of the Cy5-siANXA1@p-LP treatment group was higher than that of the Cy5-siANXA1@LP treatment group ( Figure 9 B, C).

[0141] Fluorescence imaging results showed that Cy5 fluorescence signals could be detected in the lung, liver, and spleen tissues of both groups of mice 6 h after injection. The fluorescence signal in the lung tissue of the Cy5-siANXA1@p-LP(G2) treatment group was visibly stronger. Figure 10 A). Quantitative analysis of radiation efficiency showed that among all the organs tested, the lung tissue had the highest Cy5 fluorescence enrichment level; at 6 h after injection, the fluorescence signal of the lung tissue in the Cy5-siANXA1@p-LP treatment group was significantly higher than that in the Cy5-siANXA1@LP (G1) treatment group, while there was no significant difference in the fluorescence signal of the liver and spleen tissues between the two groups. Figure 10 B). 24 h after injection, the overall Cy5 fluorescence intensity in each organ decreased, but it was still detectable. Statistical analysis of the proportion of fluorescence signal in each organ to the total detected signal showed that the fluorescence proportion in lung tissue was the highest among all organs; and at both 6 h and 24 h after injection, the fluorescence proportion in the lung tissue of mice treated with Cy5-siANXA1@p-LP was higher than that in the Cy5-siANXA1@LP treatment group (B). Figure 10 C).

[0142] The targeting of Cy5-labeled liposomes to mouse lung B cells was detected by flow cytometry. Following tail vein administration, single-cell suspensions of mouse lung tissue were prepared at predetermined time points, and B220 cells were analyzed. + B cell population gate analysis ( Figure 11 The results showed that at 6 h and 24 h after injection, compared with the Cy5-siANXA1@LP treatment group (G1), the Cy5 fluorescence histograms of the Cy5-siANXA1@p-LP treatment group (G2) showed a significant rightward shift, and the Cy5 MFI was significantly increased. Figure 12 A) indicates that the uptake efficiency of Cy5-siRNA by B cells in lung tissue is significantly enhanced. Further observation of Cy5 uptake was performed using immunofluorescence staining of lung tissue sections. + Spatial distribution characteristics of cells in lung tissue. Only trace amounts of Cy5 fluorescence signal were detected in the lung tissue of mice in the PBS-treated group (G0), while scattered Cy5-positive cells were observed in the lung tissue of mice in the Cy5-siANXA1@LP-treated group; and a large number of Cy5 cells were present in the lung tissue of mice in the Cy5-siANXA1@p-LP-treated group. + Cells, and most of these cells are related to B220 + Signal colocalization suggests that Cy5-labeled liposomes can target and accumulate in B-cell-rich regions of lung tissue. Figure 12 B).

[0143] 4. siANXA1@p-LP silences ANXA1 expression and induces the transformation of tumor conditional B cells from a regulatory phenotype to a pro-inflammatory phenotype.

[0144] In vitro experimental results showed that, compared with PBS treatment alone, 4T1 cell conditioned medium (4T1-CM) treatment significantly upregulated the mRNA and protein expression levels of ANXA1, while siNC@p-LP treatment had no significant regulatory effect on ANXA1 expression. Figure 13 Conversely, all ANXA1 siRNA formulations reduced the mRNA and protein expression levels of ANXA1, with siANXA1@p-LP-mediated gene knockdown showing the most significant effect. Its effect was comparable to or even better than JetPRIME transfection, and significantly superior to unmodified liposomes.

[0145] Whole transcriptome sequencing results showed that differentially expressed genes between the two groups were mainly enriched in immune-related KEGG signaling pathways, including Th1 / Th2 cell differentiation, B cell receptor, TNF, chemokines, cytokine-cytokine receptor, JAK-STAT, MAPK, NOD-like receptor, calcium signaling, and PI3K-Akt pathway, suggesting that ANXA1 knockdown can globally remodel the signaling regulatory network in tumor conditional B cells. Figure 14 A). Analysis of heatmaps and volcano plots revealed extensive remodeling of the cellular transcriptome after ANXA1 silencing: the expression levels of classic regulatory / anti-inflammatory genes (including Il10, Tgfb1, Ebi3, Il4, Il13, and Prdm1) were significantly reduced, while the expression levels of genes associated with cytotoxicity or pro-inflammatory responses (such as Gzmb, Tnf, Il12b (p35), Il1b, Cd40, and Ifngr1) were relatively increased. Figure 14 B, C).

[0146] Subsequently, the expression of key immune regulatory cytokines was validated at the mRNA and protein levels. Consistent with transcriptome sequencing results, compared with the PBS control group, 4T1-CM treatment significantly induced the mRNA expression levels of IL10, Tgfb1, and p35, and significantly increased the secretion of IL-10, TGF-β, and IL-35, while siNC@p-LP treatment had minimal effect on the above indicators. Figure 15 AC, Figure 16AC). All ANXA1 siRNA treatments significantly reduced the mRNA and protein expression levels of these cytokines, with the lowest expression levels of IL-10, TGF-β, and IL-35 observed after siANXA1@p-LP treatment. Conversely, 4T1-CM treatment only slightly upregulated the expression of TnF and IL-6, while ANXA1 silencing further increased the mRNA and protein levels of TNF-α and IL-6, with the highest expression levels observed in the JetPRIME-transfected siANXA1 group (siANXA1@jet) and the siANXA1@p-LP group. Figure 15 DE, 16D-E).

[0147] 5. siANXA1@p-LP induces the transformation of tumor conditional B cells from tolerant to effector cells that promote Th1 lymphocyte production.

[0148] In vitro experimental results showed that treatment with 4T1 cell conditioned medium (4T1-CM) could induce primary B cells to acquire strong induction ability and promote the induction of naïve CD4+. + T cells differentiate into immunomodulatory phenotypes ( Figure 17 CD4 + T cells were co-cultured with 4T1-CM-induced B cells treated with PBS or siNC@p-LP ( Figure 18 ), CD4 + Foxp3 in T cell population + The proportion of regulatory T cells remains at a high level (approximately 8%-11%), and most of them are CD4+. + T cells can secrete IL-10. However, knocking down ANXA1 in tumor conditional B cells significantly weakened their tolerogenic activity. Compared with two control groups, B cells treated with siANXA1@LP or siANXA1@p-LP induced Treg cells and IL-10 levels. + CD4 + The number of T cells was significantly reduced in all groups, with the siANXA1@p-LP treatment group showing the most significant reduction. Figure 19 A, B).

[0149] Conversely, knockdown of ANXA1 reduces the secretion of interferon-γ (IFN-γ) by CD4+. + The proportion of T cells showed a gradual upward trend, reaching the highest level in the siANXA1@p-LP treatment group. Figure 20 ), prompt CD4 + The immune response of T cells is shifting from a regulatory approach to one that is more Th1-oriented.

[0150] To investigate the effect of ANXA1-silenced B cells on CD8 +The effect on T cell proliferation was investigated by introducing carboxyfluorescein diacetate succinimide (CFSE) labeling technology. Figure 21 A). CD8 + When T cells are co-cultured with B cells treated with PBS or siNC@p-LP, only a small number of CD8 cells are observed. + T cells proliferate and divide, which is related to the 4T1-CM-induced B cells' response to CD8. + The inhibitory effect on T cell proliferation was consistent with this. However, ANXA1 knockdown largely reversed this inhibitory effect: after co-culturing B cells treated with siANXA1@LP, proliferating CD8 cells with low CFSE expression (CFSElow) showed increased activity. + The proportion of T cells was significantly increased; B cells treated with siANXA1@p-LP showed further enhanced CD8+. + T cell proliferation capacity ( Figure 21 B).

[0151] 6. siANXA1@p-LP inhibits lung metastasis after 4T1 breast cancer surgery in mice and remodels the lung immune microenvironment.

[0152] A 4T1-Luc breast cancer postoperative lung metastasis model was constructed to evaluate the in vivo therapeutic effect of peptide-modified siANXA1 liposomes. Figure 22 The results showed that mice treated with PBS and siNC@p-LP experienced rapid metastasis progression, as evidenced by a sustained increase in bioluminescent signals over time; conversely, the non-targeted siANXA1@LP group only showed moderate metastasis inhibition, while siANXA1@p-LP treatment significantly inhibited the growth of tumor metastases. Figure 23 This potent anti-metastatic activity was further validated experimentally: after treatment with siANXA1@p-LP, the number and area of ​​metastatic nodules in mouse lung tissue were significantly reduced. Figure 24 AC), and the proportion of Ki-67 positive tumor proliferating cells in the lungs was also significantly reduced ( Figure 25 ).

[0153] The study then investigated whether the therapeutic effect depended on the aforementioned immune regulatory mechanism. Experimental results showed that siANXA1@p-LP administration specifically silenced ANXA1 expression in mouse lung B cells. Figure 26 This leads to the remodeling of the lung immune microenvironment. Specifically, this immunomodulatory effect manifests as the presence of immunosuppressive Foxp3 in lung tissue. + The proportion of regulatory T cells was significantly reduced, and Foxp3... + The proportion of Treg decreased to approximately 1.03% ( Figure 27 A), and CD8a +The infiltration level of cytotoxic T lymphocytes was significantly increased, rising to approximately 11.6%. Figure 27 B). The above data indicate that siANXA1@p-LP can effectively improve the immunosuppressive microenvironment of lung tissue and revitalize the body's anti-tumor immune response.

[0154] 7. siANXA1@p-LP inhibits E0771 postoperative lung metastasis model of breast cancer and remodels the lung immune microenvironment.

[0155] To verify the reliability of the above research results, a second homologous E0771-Luc breast cancer lung metastasis model was constructed in C57BL / 6J WT mice. Figure 28 In vivo dynamic bioluminescence monitoring results showed that, compared with the PBS group and the non-targeted control group, the siANXA1@p-LP formulation could continuously delay the growth of lung metastases in mice. Figure 29 Gross observation revealed that the siANXA1@p-LP treatment group had very few tumor foci on the lung surface, while the lung tissues of all control group mice showed a significantly heavier metastatic burden. Figure 30 A). Furthermore, the mice receiving this targeted therapy had the lowest lung index among all experimental groups ( Figure 30 B).

[0156] At the mechanistic level, targeted silencing of ANXA1 in B cells was also successfully achieved in the E0771 breast cancer lung metastasis model. Figure 31 This gene intervention again induced a favorable immunophenotype in mouse lung tissue: a decreased proportion of regulatory T cells and Foxp3. + The proportion of Treg decreased from approximately 26.7% to approximately 6.86%. Figure 32 A), and CD8 + Increased T cell infiltration level ( Figure 32 B). Furthermore, CD8+ was found in the lung tissue of mice treated with siANXA1@p-LP. + The expression level of granzyme B in T cells was significantly increased, and the proportion of granzyme B-positive cells reached approximately 42%. Figure 32 (C) This suggests that the treatment both promotes the recruitment of effector T cells and enhances their cytotoxic function. In summary, the above results indicate that siANXA1@p-LP exerts a potent anti-tumor metastasis effect by regulating B cell-mediated lung immune microenvironment remodeling.

[0157] Lung B cells are key regulatory factors in the lung metastasis microenvironment, and this invention employs a delivery system-based intervention strategy to functionally reprogram this cell population through ANXA1 silencing. The resulting IF7C(RR)-modified cationic liposome delivery platform can target lung tissue and efficiently act on pulmonary B cells, weakening the immunosuppressive regulatory network of lung tissue through host-guided immune modulation while enhancing T cell-mediated tumor immune surveillance. Mechanistic studies show that knocking down ANXA1 can reverse the regulatory cytokine secretion profile of tumor-conditional B cells and weaken their CD4-inducing ability. + The ability of T cells to polarize into Tregs, while restoring the body's Th1 immune characteristics and promoting CD8. + T cell proliferation and effector function activation. The aforementioned synergistic immunomodulatory effects demonstrate that this agent can effectively and persistently inhibit the growth of postoperative lung metastases in different homologous breast cancer models. This result confirms that resetting the pro-metastatic immune microenvironment of lung tissue during the perioperative period can effectively inhibit postoperative lung metastasis recurrence in breast cancer. However, existing interventions for breast cancer lung metastases mostly target tumor cells directly, making it difficult to precisely regulate the pre-metastatic microenvironment and immunosuppressive host cells in the metastatic microenvironment. At the same time, naked siRNA has poor in vivo stability, is easily degraded, and has limited cellular uptake and endosome escape efficiency. Although conventional nanodelivery systems can accumulate in the lungs, they lack sufficient cell selectivity for lung B cells, thus making it difficult to efficiently silence annexin A1 (ANXA1) in lung B cells and reverse its pro-metastatic immunomodulatory function.

[0158] This invention elucidates the core mechanism of ANXA1 silencing-mediated functional reprogramming of lung tissue B cells, clarifies the key pathway of B cell remodeling of the lung immune microenvironment, and confirms the core regulatory role of lung tissue B cells in the formation and development of metastatic lesions. Their functional state directly affects the pro-metastatic / anti-metastatic tendency of the lung immune microenvironment, providing a new direction for the screening of intervention targets for lung metastasis.

[0159] Although the technical solution of the present invention has been described in detail above with general descriptions, specific embodiments, and experimental examples, it should be noted that the embodiments and experimental examples are only used to illustrate the technical solution and technical effects of the present invention, and should not be regarded as any limitation on the scope of protection of the present invention. Simple modifications, alterations, or improvements made based on the technical concept of the present invention are all within the scope of protection claimed by the present invention.

Claims

1. A polypeptide-modified cationic liposome, characterized in that: It consists of a cationic liposome shell modified with a peptide that specifically recognizes ANXA1, loaded with an siRNA core that targets ANXA1.

2. The polypeptide-modified cationic liposome according to claim 1, characterized in that: The polypeptide that specifically recognizes ANXA1 is IF7C(RR), and its amino acid sequence is IFLLWQRCRR.

3. The polypeptide-modified cationic liposome according to claim 1, characterized in that: The raw materials for preparing the peptide-modified cationic liposomes that specifically recognize ANXA1 include DOTAP, cholesterol and DSPE-PEG1000-IF7C(RR), with a molar ratio of (47-48):(47-48):(4-6).

4. The polypeptide-modified cationic liposome according to claim 3, characterized in that: The DSPE-PEG1000-IF7C(RR) is prepared by covalent coupling of the thiol group of the cysteine ​​residue contained in the IF7C(RR) polypeptide with the maleimide group at the end of DSPE-PEG1000-Mal via a thiol-maleimide Michael addition reaction.

5. The polypeptide-modified cationic liposome according to claim 1, characterized in that: The sequence of the siRNA targeting ANXA1 is as follows: Chain of Justice: 5'-GCACAAAGCUAUCAUGGUUAAdTdT-3'; Antonym chain: 5'-UUAACCAUGAUAGCUUUGUGCdTdT-3'.

6. The polypeptide-modified cationic liposome according to claim 1, characterized in that: The ratio of the cationic liposome shell to the siRNA core is 5 μL of liposomes with a total lipid concentration of 6 mM for every 1 μg siRNA added.

7. A method for preparing polypeptide-modified cationic liposomes as described in any one of claims 1-6, characterized in that: Includes the following steps: DOTAP, cholesterol, and DSPE-PEG1000-IF7C(RR) were mixed and formed into a film, which was then hydrated and sonicated to obtain peptide-modified cationic liposomes. The siRNA targeting ANXA1 is loaded into peptide-modified cationic liposomes to obtain the product.

8. The application of a polypeptide-modified cationic liposome as described in any one of claims 1-6, characterized in that: Including but not limited to: (1) Application in the preparation of drugs for reprogramming lung B cell function; (2) Application in the preparation of drugs that remodel the pulmonary immune microenvironment; (3) Application in the preparation of drugs that inhibit lung metastasis and recurrence of breast cancer; (4) Use in the preparation of drugs for treating lung metastases of tumors or lung immune-related diseases characterized by abnormal lung immunosuppressive B cells and ANXA1.

9. A drug for inhibiting lung metastasis of breast cancer, characterized in that: Includes peptide-modified cationic liposomes as described in any one of claims 1-6.

10. The medicament according to claim 9, characterized in that: The content of polypeptide-modified cationic liposomes in the drug is 0.01 wt%-99.99 wt%.

Images