A novel fusion vesicle and a preparation method and application thereof

Abstract

This invention aims to provide a hybrid nanovesicle system (CXCR4-HNV) formed by fusing CXCR4-engineered macrophage-derived small extracellular vesicles (sEVs) with lipid nanoparticles (LNPs), and co-loading siRNA targeting Siglec-15 and mRNA encoding interleukin-12 (IL-12) within it. This system is intended for precision immunotherapy of lung cancer bone metastases. Through the combination of CXCR4-mediated active targeting and the natural biocompatibility and macrophage affinity of sEVs, the fused vesicles accumulate in the CXCL12-high-expressing bone metastatic microenvironment and are efficiently taken up by tumor-associated macrophages due to homology affinity. Intravesical delivery of Siglec-15 siRNA can effectively silence the expression of the immunosuppressive molecule Siglec-15. At the same time, IL-12 mRNA is translated and secreted into functional IL-12 protein in the cell, which synergistically reverses the immunosuppressive tumor microenvironment and enhances the infiltration, activation and tumor killing ability of CD8⁺T cells, thereby achieving effective control and elimination of lung cancer bone metastases.

Description

Technical Field

[0001] This application relates to the fields of biomedicine and nanodelivery, and in particular to a targeted hybrid nanovesicle (HNV) formed by fusing CXCR4 engineered macrophage-derived small extracellular vesicles (sEVs) with lipid nanoparticles (LNPs). The vesicles co-load Siglec-15 siRNA and IL-12 mRNA dual nucleic acid drugs, and are particularly a targeted nucleic acid drug delivery platform for macrophage immunotherapy of lung cancer bone metastases. Background Technology

[0002] Bone metastasis is a common form of distant metastasis in advanced lung cancer, often caused by tumor cells spreading to the bone via the bloodstream and colonizing and proliferating in the bone microenvironment. Once bone metastasis occurs, patients may experience serious complications such as persistent bone pain, pathological fractures, spinal cord compression, and hypercalcemia, characterized by high morbidity, low cure rate, and a heavy medical burden. The pathological process of lung cancer bone metastasis mainly includes two stages: initial bone homing of tumor cells and the subsequent establishment of an immunosuppressive microenvironment. During this process, a large number of tumor-associated macrophages (TAMs) are recruited to the metastatic lesions and polarized into the M2 phenotype. By secreting anti-inflammatory factors, inhibiting T cell activation, promoting angiogenesis, and tissue remodeling, they form a highly immunosuppressive tumor microenvironment, which is a key factor leading to the failure of immunotherapy and the continued progression of the disease. Existing clinical interventions (such as bisphosphonates, RANKL inhibitors, radiotherapy, and systemic antitumor therapy) mainly focus on inhibiting bone destruction and controlling tumor burden, but they are difficult to effectively reprogram tumor-associated macrophages or restore antitumor immune responses, resulting in limited eradication effects on metastatic lesions. Studies have found that abnormal activation and functional deviation of macrophages in the lung cancer bone metastasis microenvironment are one of the main reasons hindering T cell-mediated tumor clearance.

[0003] 1. Targeting tumor microenvironment macrophages holds promise for improving immunosuppression in bone metastases. Clinically, bone metastases (such as lung cancer bone metastases) often exhibit significant immunosuppressive characteristics, with reduced T cell infiltration and functional exhaustion in their microenvironment, leading to poor efficacy of various antitumor drugs, including immune checkpoint inhibitors. Numerous studies have reported that tumor-associated macrophages (TAMs) are highly enriched in bone metastases and are often in an M2 polarized state. They not only secrete anti-inflammatory factors such as IL-10 and TGF-β but also directly inhibit T cell activation and cytotoxic function through multiple mechanisms, making them a key cell population driving local immunosuppression. However, due to the unique blood supply and poor drug permeability of bone tissue, coupled with the lack of efficient targeted delivery systems, existing therapies struggle to precisely deliver therapeutic drugs to macrophages in the bone metastasis microenvironment, severely hindering the implementation of immune microenvironment reprogramming strategies. To address this challenge, this invention provides a fusion vesicle delivery system that specifically targets macrophages in the tumor microenvironment. This system aims to precisely regulate macrophage function, reverse the immunosuppressive state, and thereby enhance the antitumor immune response, providing a new pathway for immunotherapy of bone metastases.

[0004] 2. Therapeutic targets: Siglec-15 and IL-12 In immunosuppressive tumor microenvironments such as lung cancer bone metastases, tumor-associated macrophages (TAMs) not only exhibit a significant increase in number but also profoundly influence anti-tumor immune responses through the expression of immunomodulatory molecules. Among these, Siglec-15, an immunosuppressive receptor specifically highly expressed on TAMs and some tumor cells, can directly inhibit T cell receptor signaling, weaken CD8⁺ T cell activation and effector function, and is one of the key molecules mediating immune escape. Simultaneously, the deficiency of IL-12 (interleukin-12) in the microenvironment further exacerbates the immunosuppressive state—IL-12, an important pro-inflammatory cytokine secreted by activated antigen-presenting cells (including M1 macrophages), can drive Th1 immune responses, enhance the cytotoxic activity of NK and CD8⁺ T cells, and promote the polarization of TAMs towards the anti-tumor M1 phenotype. Therefore, simultaneously blocking Siglec-15 signaling and restoring IL-12 expression in TAMs holds promise for synergistically relieving immunosuppression, reshaping the inflammatory microenvironment, and thereby effectively activating T cell-mediated anti-tumor immune responses. This invention is based on this dual regulatory strategy, which achieves precise intervention on the immunosuppressive network by targeting and delivering Siglec-15 siRNA and IL-12 mRNA to macrophages in the tumor microenvironment.

[0005] 3. Current Status of Existing Delivery Carriers Currently, lipid nanoparticles (LNPs), as one of the mainstream platforms for nucleic acid drug delivery, have been widely used in gene therapy due to their efficient nucleic acid encapsulation capabilities and advantages in promoting cellular uptake. However, issues such as immune recognition, non-specific distribution, and potential toxicity of LNPs in vivo, especially in the complex tumor microenvironment, limit their clinical application efficacy. In contrast, small extracellular vesicles (sEVs) are naturally secreted nanoscale membrane vesicles with excellent biocompatibility, low immunogenicity, and certain tissue targeting properties. The surface of sEVs is rich in various membrane proteins, which can mediate specific recognition and fusion with target cells, enabling them to cross biological barriers and achieve long-term circulation. Nevertheless, natural sEVs suffer from limited yield, high component heterogeneity, and low loading efficiency, making it difficult to meet the clinical needs for high-dose, controlled release. Summary of the Invention

[0006] To overcome the aforementioned limitations and combine the advantages of both, we propose constructing a hybrid nanovesicle (HNV) system. This system combines the natural membrane characteristics of sEVs with the engineerable structure of LNPs through physical fusion, retaining the biorecognition and targeting properties of sEVs while possessing the high loading capacity and structural stability of LNPs, thereby significantly improving the delivery efficiency and therapeutic safety of nucleic acid drugs. Specifically, the CXCR4-engineered macrophage-derived sEVs used in this invention, due to their high expression of CXCR4 molecules, possess chemotactic ability against the high expression of CXCL12 in bone metastases, enabling them to actively target tumor-associated macrophages (TAMs) in the lung cancer bone metastasis microenvironment. This invention further utilizes a hybrid fusion of small extracellular vesicles carrying the CXCR4 targeting molecule and lipid nanoparticles carrying Siglec-15 siRNA and IL-12 mRNA to form a composite carrier. Through this design, the fused vesicles not only possess high biocompatibility and high loading capacity, but also, through the CXCR4 molecule, endow them with high targeting specificity to the bone metastasis microenvironment, enabling them to efficiently accumulate in and be taken up by TAMs. The Siglec-15 siRNA delivered within the vesicles effectively silences the expression of the immunosuppressive molecule Siglec-15, while the IL-12 mRNA is translated intracellularly and secretes functional IL-12 protein, synergistically reversing the immunosuppressive state, enhancing the infiltration and killing function of CD8⁺ T cells, and ultimately achieving effective control of bone metastases.

[0007] In summary, the targeted nanovesicle delivery system based on the fusion of sEVs and LNPs combines high biocompatibility, specific targeting capability, and the synergistic regulatory effect of dual nucleic acid drugs. It effectively overcomes the limitations of existing siRNA therapy systems in terms of stability, targeting, and delivery efficiency, providing a novel, safe, and efficient nucleic acid drug treatment strategy for lung cancer bone metastases. This innovative delivery platform is expected to significantly improve the clinical prognosis of patients with bone metastases, demonstrating broad translational potential and application prospects.

[0008] This application provides a fusion vesicle, the raw materials of which include small extracellular vesicles and lipid nanoparticle-nucleic acid complexes, the mass ratio of the small extracellular vesicles to the lipid nanoparticle-nucleic acid complexes being (0.5-3):(0.5-3), the lipid nanoparticle-nucleic acid complexes including lipid nanoparticles and nucleic acids, the nucleic acids being loaded in the lipid nanoparticles, and the raw materials of the lipid nanoparticles including ionizable cationic lipids, cofactor phospholipids, cholesterol, and polyethylene glycol-modified lipids.

[0009] This application also provides a method for preparing the above-mentioned fusion vesicles, comprising the following steps: 1) The small interfering RNA of Siglec-15 and the messenger RNA encoding IL-12 were loaded into the lipid nanoparticles to obtain the lipid nanoparticle-nucleic acid complex. 2) The lipid nanoparticle-nucleic acid complex was fused with small extracellular vesicles to obtain fused vesicles; The raw materials for the lipid nanoparticles include ionizable cationic lipids, cofactor phospholipids, cholesterol, and polyethylene glycol-modified lipids.

[0010] This application also provides a method for delivering nucleic acids to cells for purposes other than disease diagnosis and treatment, comprising contacting the aforementioned fusion vesicles with the cells.

[0011] This application also provides the use of the above-mentioned fusion vesicles in the preparation of antitumor drugs.

[0012] The beneficial effects of this application include, but are not limited to: (1) By fusing small extracellular vesicles (sEVs) derived from macrophages with lipid nanoparticles (LNPs), a hybrid nanovesicle (HNV) is constructed, which combines the high biocompatibility and low immunogenicity of natural exosome membranes with the high nucleic acid loading capacity and structural stability of LNPs, thereby significantly improving the safety and in vivo circulation performance of the delivery system. (2) The surface of sEVs was modified by CXCR4 engineering strategy to give fusion vesicles the ability to actively target lung cancer bone metastases. The bone metastasis microenvironment highly expresses CXCL12, which can specifically recruit CXCR4 positive vesicles to accumulate in the lesion area and be efficiently taken up by tumor-associated macrophages (TAMs), significantly improving the drug delivery efficiency in target cells. (3) Siglec-15 siRNA and IL-12 mRNA are co-loaded in the vesicles to achieve dual immune regulation: siRNA silences the expression of the immunosuppressive molecule Siglec-15 in macrophages and relieves the inhibition of T cells; mRNA is translated and secreted into functional IL-12 protein in the cell, promoting TAMs to M1 polarization and activating CD8+ T cells, synergistically reshaping the anti-tumor immune microenvironment and enhancing the tumor clearance ability mediated by T cells.

[0013] In summary, the CXCR4 engineered sEVs-LNP fusion nanovesicle nucleic acid drug delivery system constructed in this invention combines precise targeting, dual-effect synergy, and good biosafety, providing a novel and efficient immunogene therapy strategy for immunosuppressive bone metastases such as lung cancer bone metastases. Attached Figure Description

[0014] This application will be further described by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, wherein: Figure 1 The results are shown as Western blots of the Siglec 15 siRNA sequence.

[0015] Figure 2 The image shown is a transmission electron microscope image of the LNP-nucleic acid complex. Scale bar: 200 nm.

[0016] Figure 3 The image shows the particle size analysis of the LNP-nucleic acid complex.

[0017] Figure 4 The diagram shows the zeta potential analysis of the LNP-nucleic acid complex.

[0018] Figure 5 The results are shown in the fluorescence microscopy of stable CXCR4 RAW264.7 cells. Scale bar: 100 μm.

[0019] Figure 6 The image shows the Western blot results of a macrophage cell line that stably and highly expresses CXCR4.

[0020] Figure 7 The diagram shows the particle size distribution of exosomes.

[0021] Figure 8 The image shows the surface zeta potential characteristics of exosomes.

[0022] Figure 9 The image shown is a transmission electron microscope image of CXCR4-modified exosomes. Scale bar: 100 nm.

[0023] Figure 10 The results are shown in the Western blot analysis of RAW cell-derived exosomes.

[0024] Figure 11 The image shows a fusion of Dil-labeled CXCR4-sEVs (red) and DiO-labeled lipid nanoparticles (LNP-dio, green), scale bar: 25µm.

[0025] Figure 12 The image shown is a transmission electron microscope image of CXCR4-HNV@siRNA Siglec-15+mRNA IL-12, scale bar: 50nm.

[0026] Figure 13 The results show that Western blot analysis of CXCR4-HNV@siRNA Siglec-15+mRNA showed that IL-12 could inhibit siglec 15 and promote IL-12 expression.

[0027] Figure 14 The results showed that the expression level of IL-12 mRNA in the fusion vesicle treatment group was significantly higher than that in the PBS control group (approximately 2.5 times higher), and there was no significant difference compared with the LNP group.

[0028] Figure 15 Displayed as confocal analysis of INOS, a marker of macrophage m1 upregulation in fusion vesicles, scale bar: 25um.

[0029] Figure 16 The diagram shows the experimental flowchart for indirectly evaluating the role of fusion vesicles.

[0030] Figure 17 The image shows the effect of macrophages in different treatment groups on T cell killing function after intervention with fusion vesicles, as detected by flow cytometry.

[0031] Figure 18 The image shows the targeting enrichment ability of hybrid nanovesicles detected using a mouse model of lung cancer bone metastases.

[0032] Figure 19 The image shows the targeting enrichment ability of hybrid nanovesicles detected using a mouse model of lung cancer bone metastases.

[0033] Figure 20 The images show tissue sections of three groups of mouse bone metastatic tumor samples: ree siRNA cy5.5, HNV@RNAscramble cy5.5, and CXCR4 HNV@RNAscramble cy5.5. Scale bar: 50um.

[0034] Figure 21 The graph shows the results of flow cytometry analysis of the proportion of M1 macrophages.

[0035] Figure 22 The image shows the expression of CD8+ T cells as detected by immunohistochemistry. Scale bar: 50um.

[0036] Figure 23 This shows a comparison of tumor size in mice with lung cancer bone metastases after treatment with different drugs.

[0037] Figure 24 This shows a comparison of tumor size in mice with lung cancer bone metastases after treatment with different drugs.

[0038] Figure 25 The images show histopathological analysis of the heart, liver, spleen, lungs, and kidneys. Scale bar: 50um.

[0039] Figure 26 The results are displayed as serum biochemical markers. Detailed Implementation

[0040] To more clearly illustrate the technical solutions of the embodiments in this specification, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some examples or embodiments of this specification. For those skilled in the art, these drawings can be applied to other similar scenarios without creative effort. Unless obvious from the context or otherwise specified, the same reference numerals in the drawings represent the same structures or operations.

[0041] As indicated in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not specifically refer to the singular and may also include the plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of expressly identified steps and elements, which do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

[0042] Flowcharts are used in this specification to illustrate the operations performed by the system according to embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed in exact order. Instead, the steps can be processed in reverse order or simultaneously. Furthermore, other operations can be added to these processes, or one or more steps can be removed from them.

[0043] This application provides a fusion vesicle, the raw materials of which include small extracellular vesicles and lipid nanoparticle-nucleic acid complexes, the mass ratio of the small extracellular vesicles to the lipid nanoparticle-nucleic acid complexes being (0.5-3):(0.5-3), the lipid nanoparticle-nucleic acid complexes including lipid nanoparticles and nucleic acids, the nucleic acids being loaded in the lipid nanoparticles, and the raw materials of the lipid nanoparticles including ionizable cationic lipids, cofactor phospholipids, cholesterol, and polyethylene glycol-modified lipids.

[0044] The term "lipid nanoparticle" in this application refers to a particle comprising multiple (e.g., more than one) lipid molecules physically bound together by intermolecular forces. LNPs can be, for example, microspheres (including monolayers and multilayers of small lipid vesicles, such as "liposomes"—layered lipid bilayers, which in some embodiments are substantially spherical, and in more specific embodiments may contain an aqueous core, such as a substantial portion containing RNA molecules), a dispersed phase in an emulsion, a microparticle, or an internal phase in a suspension. Emulsions, microparticles, and suspensions can be suitable compositions for local and / or external delivery.

[0045] Nucleic acids are polymers containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, and include DNA, RNA and their hybrids.

[0046] In some embodiments, the mass ratio of the small extracellular vesicles to the lipid nanoparticle-nucleic acid complex can be (0.75–2.75):(0.75–2.75). In some embodiments, the mass ratio of the small extracellular vesicles to the lipid nanoparticle-nucleic acid complex can be (1–2.5):(1–2.5). In some embodiments, the mass ratio of the small extracellular vesicles to the lipid nanoparticle-nucleic acid complex can be (1.25–2.25):(1.25–2.25). In some embodiments, the mass ratio of the small extracellular vesicles to the lipid nanoparticle-nucleic acid complex can be (1.5–2):(1.5–2). In some embodiments, the mass ratio of the small extracellular vesicles to the lipid nanoparticle-nucleic acid complex can be (1.75–2):(1.75–2).

[0047] In some embodiments, preferably, the mass ratio of the small extracellular vesicles to the lipid nanoparticle-nucleic acid complex can be 1:1.

[0048] In some embodiments, the small extracellular vesicles may be small extracellular vesicles with high CXCR4 expression on their surface.

[0049] In some embodiments, the surface potential of the small extracellular vesicles can be between -25 and -30 mV, exhibiting a negative charge.

[0050] In some embodiments, the small extracellular vesicles may express TSG101, CD81, and Alix proteins.

[0051] In some embodiments, the small extracellular vesicles may be expressed by macrophages. Preferably, in some embodiments, the macrophages may be macrophages that stably and highly express CXCR4. In some embodiments, the macrophages may be RAW 264.7 cells.

[0052] In some embodiments, the molar ratio of the lipid nanoparticles to the nucleic acid can be (3-5):(0.5-1).

[0053] In some embodiments, preferably, the molar ratio of the lipid nanoparticles to the nucleic acid can be (3.5-4.5):(0.8-1).

[0054] In some embodiments, more preferably, the molar ratio of the lipid nanoparticles to the nucleic acid can be 4:1.

[0055] In some embodiments, the nucleic acid may include a small interfering RNA targeting Siglec-15 and a messenger RNA encoding IL-12.

[0056] In some embodiments, the nucleotide sequence of the small interfering RNA targeting Siglec-15 may include at least one of the following three pairs: SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, and SEQ ID NO.5 and SEQ ID NO.6. In some embodiments, preferably, the nucleotide sequence of the small interfering RNA targeting Siglec-15 may be SEQ ID NO.5 and SEQ ID NO.6.

[0057] In some embodiments, the nucleotide sequence of the messenger RNA encoding IL-12 may be as shown in SEQ ID NO.7.

[0058] In some embodiments, the mass ratio of the small interfering RNA targeting Siglec-15 to the messenger RNA encoding IL-12 can be (0.5–3):(0.5–3). In some embodiments, the mass ratio of the small interfering RNA targeting Siglec-15 to the messenger RNA encoding IL-12 can be (0.75–2.75):(0.75–2.75). In some embodiments, the mass ratio of the small interfering RNA targeting Siglec-15 to the messenger RNA encoding IL-12 can be (1–2.5):(1–2.5). In some embodiments, the mass ratio of the small interfering RNA targeting Siglec-15 to the messenger RNA encoding IL-12 can be (1.25–2.25):(1.25–2.25). In some embodiments, the mass ratio of the small interfering RNA targeting Siglec-15 to the messenger RNA encoding IL-12 can be (1.5–2):(1.5–2). In some embodiments, the mass ratio of the small interfering RNA targeting Siglec-15 to the messenger RNA encoding IL-12 can be (1.75–2): (1.75–2).

[0059] In some embodiments, preferably, the mass ratio of the small interfering RNA targeting Siglec-15 to the messenger RNA encoding IL-12 can be 1:1.

[0060] In some embodiments, the raw materials of the lipid nanoparticles include an ionizable cationic lipid, cofactor phospholipid, cholesterol, and polyethylene glycol-modified lipid in a molar ratio of (40–60):(5–15):(30–45):(0.5–3). In some embodiments, the raw materials of the lipid nanoparticles include an ionizable cationic lipid, cofactor phospholipid, cholesterol, and polyethylene glycol-modified lipid in a molar ratio of (42–58):(6–14):(32–43):(0.75–2.75). In some embodiments, the raw materials of the lipid nanoparticles include an ionizable cationic lipid, cofactor phospholipid, cholesterol, and polyethylene glycol-modified lipid in a molar ratio of (44–56):(7–13):(34–41):(1–2.5). In some embodiments, the raw materials of the lipid nanoparticles include an ionizable cationic lipid, cofactor phospholipid, cholesterol, and polyethylene glycol-modified lipid in a molar ratio of (46–54):(8–12):(36–39):(1.25–2.25). In some embodiments, the raw materials for the lipid nanoparticles include an ionizable cationic lipid, cofactor phospholipid, cholesterol, and polyethylene glycol-modified lipid in a molar ratio of (48–52):(9–11):(38–39):(1.5–2). In some embodiments, the raw materials for the lipid nanoparticles include an ionizable cationic lipid, cofactor phospholipid, cholesterol, and polyethylene glycol-modified lipid in a molar ratio of (50–52):(10–11):(38–39):(1.75–2).

[0061] In some embodiments, preferably, the raw materials for the lipid nanoparticles may include ionizable cationic lipids, cofactor phospholipids, cholesterol, and polyethylene glycol-modified lipids in a molar ratio of 50:10:38.5:1.5.

[0062] In some embodiments, preferably, the ionizable cationic lipid can be DLin-MC3-DMA.

[0063] In some embodiments, preferably, the auxiliary phospholipid can be distearylphosphatidylcholine.

[0064] In some embodiments, preferably, the polyethylene glycol-modified lipid can be 1,2-dimyristic-rac-glycerol-methoxy polyethylene glycol 2000.

[0065] This application also provides a method for preparing the above-mentioned fusion vesicles, comprising the following steps: 1) The small interfering RNA of Siglec-15 and the messenger RNA encoding IL-12 were loaded into the lipid nanoparticles to obtain the lipid nanoparticle-nucleic acid complex. 2) The lipid nanoparticle-nucleic acid complex was fused with small extracellular vesicles to obtain fused vesicles; The raw materials for the lipid nanoparticles include ionizable cationic lipids, cofactor phospholipids, cholesterol, and polyethylene glycol-modified lipids.

[0066] In some embodiments, the preparation of the lipid nanoparticle-nucleic acid complex may include the following steps: 3) Dissolve the siRNA targeting Siglec-15 and the messenger RNA encoding IL-12 in citrate buffer as the aqueous phase; 4) Ionizable cationic lipids, cofactor phospholipids, cholesterol, and polyethylene glycol-modified lipids are dissolved in ethanol to form the ethanol phase; 5) Mix the aqueous phase and the ethanol phase to obtain the lipid nanoparticle-nucleic acid complex.

[0067] In some embodiments, the pH of the citrate buffer solution can be 3-5. In some embodiments, the pH of the citrate buffer solution can be 3.2-4.8. In some embodiments, the pH of the citrate buffer solution can be 3.4-4.6. In some embodiments, the pH of the citrate buffer solution can be 3.6-4.4. In some embodiments, the pH of the citrate buffer solution can be 3.8-4.2. In some embodiments, the pH of the citrate buffer solution can be 4.0-4.2.

[0068] In some embodiments, preferably, the pH of the citrate buffer solution can be 4.0. In some embodiments, more preferably, the citrate buffer solution can be an aqueous solution of citric acid. In some embodiments, even more preferably, the concentration of the citrate buffer solution can be 20 mM.

[0069] In some embodiments, the volume ratio of the ethanol phase to the water phase can be 1:3 to 1:5. In some embodiments, the volume ratio of the ethanol phase to the water phase can be 1:4.

[0070] In some embodiments, the mass ratio of siRNA targeting Siglec-15 to messenger RNA encoding IL-12 in the aqueous phase can be (0.5–3):(0.5–3). In some embodiments, the mass ratio of siRNA targeting Siglec-15 to messenger RNA encoding IL-12 in the aqueous phase can be (0.75–2.75):(0.75–2.75). In some embodiments, the mass ratio of siRNA targeting Siglec-15 to messenger RNA encoding IL-12 in the aqueous phase can be (1–2.5):(1–2.5). In some embodiments, the mass ratio of siRNA targeting Siglec-15 to messenger RNA encoding IL-12 in the aqueous phase can be (1.25–2.25):(1.25–2.25). In some embodiments, the mass ratio of siRNA targeting Siglec-15 to messenger RNA encoding IL-12 in the aqueous phase can be (1.5–2):(1.5–2). In some embodiments, the mass ratio of siRNA targeting Siglec-15 to messenger RNA encoding IL-12 in the aqueous phase can be (1.75–2):(1.75–2).

[0071] In some embodiments, preferably, the mass ratio of small interfering RNA targeting Siglec-15 to messenger RNA encoding IL-12 in the aqueous phase can be 1:1.

[0072] In some embodiments, the concentration of siRNA targeting Siglec-15 in the aqueous phase can be 0.05–2.0 mg / mL, and the concentration of messenger RNA encoding IL-12 can be 0.05–2.0 mg / mL.

[0073] For example, the concentration of siRNA targeting Siglec-15 in the aqueous phase can be 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, or 2.0 mg / mL. It also includes any range characterized by combinations of the above end values, which will not be elaborated here. For example, the concentration of messenger RNA encoding IL-12 can be 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, or 2.0 mg / mL. Any range characterized by combinations of the above end values ​​is also included, which will not be elaborated here.

[0074] In some embodiments, the fusion step may include mixing the ethanol phase with the aqueous phase, removing the ethanol by dialysis or tangential flow filtration, and replacing it with a neutral physiological buffer. Preferably, in some embodiments, the neutral physiological buffer may be PBS buffer or physiological saline.

[0075] In some embodiments, the small extracellular vesicles may be small extracellular vesicles with high CXCR4 expression on their surface.

[0076] In some embodiments, the surface potential of the small extracellular vesicles can be between -25 and -30 mV, exhibiting a negative charge.

[0077] In some embodiments, the small extracellular vesicles may express TSG101, CD81, and Alix proteins.

[0078] In some embodiments, the small extracellular vesicles may be expressed by macrophages. In some embodiments, preferably, the macrophages may be macrophages that stably and highly express CXCR4.

[0079] In some embodiments, the mass ratio of the small extracellular vesicles to the lipid nanoparticle-nucleic acid complex can be (0.75–2.75):(0.75–2.75). In some embodiments, the mass ratio of the small extracellular vesicles to the lipid nanoparticle-nucleic acid complex can be (1–2.5):(1–2.5). In some embodiments, the mass ratio of the small extracellular vesicles to the lipid nanoparticle-nucleic acid complex can be (1.25–2.25):(1.25–2.25). In some embodiments, the mass ratio of the small extracellular vesicles to the lipid nanoparticle-nucleic acid complex can be (1.5–2):(1.5–2). In some embodiments, the mass ratio of the small extracellular vesicles to the lipid nanoparticle-nucleic acid complex can be (1.75–2):(1.75–2).

[0080] In some embodiments, preferably, the mass ratio of the small extracellular vesicles to the lipid nanoparticle-nucleic acid complex can be 1:1.

[0081] In some embodiments, the lipid nanoparticle-nucleic acid complex may include lipid nanoparticles and nucleic acid, wherein the nucleic acid is loaded in the lipid nanoparticles. In some embodiments, preferably, the molar ratio of the lipid nanoparticles to the nucleic acid is (3-5):(0.5-1). In some embodiments, more preferably, the molar ratio of the lipid nanoparticles to the nucleic acid is (3.5-4.5):(0.8-1). In some embodiments, even more preferably, the molar ratio of the lipid nanoparticles to the nucleic acid is 4:1.

[0082] In some embodiments, after the aqueous and ethanol phases are mixed, the particles are further homogenized using an emulsification device. Preferably, in some embodiments, the emulsification device can be a liposome extruder.

[0083] This application also provides a method for delivering nucleic acids to cells for purposes other than disease diagnosis and treatment, comprising contacting the aforementioned fusion vesicles with the cells.

[0084] This application also provides the use of the above-mentioned fusion vesicles in the preparation of antitumor drugs.

[0085] In some embodiments, the tumor may be selected from lung cancer, bone metastases, nasopharyngeal carcinoma, gastric cancer, primary gallbladder cancer, leiomyosarcoma, liver cancer, oral cancer, head and neck cancer, adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, cervical endogenous adenocarcinoma, bile duct carcinoma, colonic adenocarcinoma, lymphoid tumors, and diffuse large B-cell tumors. Cellular lymphoma, esophageal cancer, glioblastoma multiforme, squamous cell carcinoma of the head and neck, chromophobe renal carcinoma, clear cell renal carcinoma, papillary renal carcinoma, acute myeloid leukemia, low-grade glioma of the brain, hepatocellular carcinoma, squamous cell carcinoma of the lung, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, colorectal cancer, malignant sarcoma, melanoma, testicular germ cell tumor, thyroid cancer, thymic carcinoma, endometrial cancer, uterine sarcoma, uveal melanoma, multiple myeloma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic myeloid leukemia, lymphoma, lung cancer, sarcoma, anal cancer, melanoma, retinoblastoma, or one or more of the following.

[0086] In some embodiments, preferably, the tumor may be selected from lung cancer and / or bone metastases. In some embodiments, more preferably, the bone metastases may be lung cancer bone metastases.

[0087] In some embodiments, the fusion vesicles can modulate the transformation of tumor-associated macrophages into M1 macrophages.

[0088] In some embodiments, the fusion vesicles can enhance the infiltration of CD8+ T cells within the tumor.

[0089] In some embodiments, the fusion vesicles can enhance T cell-mediated tumor clearance.

[0090] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0091] Example 1 - Selection of siRNA siglec15 sequence Qingke Biotechnology designed three siRNA Siglec15 sequences siRNA Siglec15 1 Sense (5'→3'): GCATGAAGTTCTACGAGAAdTdT (SEQ ID NO.1) Antisense (5'→3'): TTCTCGTAGAACTTCATGCdTdT (SEQ ID NO.2) siRNA Siglec15 2 Sense (5'→3'): CCAGTACTTCAGCAACTAAdTdT (SEQ ID NO.3) Antisense (5'→3'): TTAGTTGCTGAAGTACTGGdTdT (SEQ ID NO.4) siRNA Siglec15 3 Sense (5'→3'): GCACTACAGCTACTACAAAdTdT (SEQ ID NO.5) Antisense (5'→3'): TTTGTAGTAGCTGTAGTGCdTdT (SEQ ID NO.6) like Figure 1 Western blot results showed that the LNP-siRNA delivery system effectively knocked down Siglec15 protein expression. In the PBS group, the Siglec15 protein band intensity was significant, indicating high expression in the control group; while the Siglec15 expression level in the LNP-siRNA-scramble group (negative control) was similar to that in the PBS group, indicating that non-specific siRNA did not affect target gene expression. In contrast, all three siRNAs targeting Siglec15 (LNP-siRNA-Siglec151, 2, and 3) significantly reduced Siglec15 protein levels, with LNP-siRNA-Siglec153 showing the most significant effect, almost disappearing from the band, suggesting that sequence 3 has strong interference efficiency.

[0092] β-tubulin, used as an internal control protein, showed stable expression across all groups, verifying consistent loading amounts and ensuring the reliability of the experimental results. In summary, this lipid nanoparticle (LNP)-mediated siRNA delivery system exhibits good targeting and silencing efficiency, especially the LNP-siRNA-Siglec15 3 sequence. Sequence 3 will be selected for subsequent experiments.

[0093] Example 2 - Preparation and Identification of Lipid Nanoparticles: It is composed of ionizable cationic lipids, cofactor phospholipids, cholesterol, and polyethylene glycol-modified lipids, with the following molar percentages: ionizable cationic lipid (DLin-MC3-DMA) 50 mol%, distearate phosphatidylcholine (DSPC) 10 mol%, cholesterol 38.5 mol%, and 1,2-dimyristoyl-rac-glycerol-methoxy polyethylene glycol 2000 (DMG-PEG2000) 1.5 mol%. The nucleic acid payload consists of a small interfering RNA (siRNA) targeting Siglec-15 and a messenger RNA (mRNA) encoding interleukin-12 (IL-12), which are mixed in an aqueous solution at a mass ratio of 1:1 before preparation. Specifically, the mass concentration of Siglec-15 siRNA in the aqueous phase is 0.05 mg / mL to 2.0 mg / mL, the mass concentration of IL-12 mRNA is 0.05 mg / mL to 2.0 mg / mL, and the total mass of the two nucleic acids accounts for 5% to 15% of the total mass of the final lipid nanoparticles.

[0094] Messenger RNA encoding interleukin-12 (SEQ ID NO.7): The preparation process includes: dissolving the lipid components in the above molar ratio in the ethanol phase, and simultaneously dissolving Siglec-15 siRNA and IL-12 mRNA mixed in a 1:1 mass ratio in an acidic citrate buffer at pH 4.0 as the aqueous phase; under rapid vortex mixing conditions, the ethanol phase and the aqueous phase are instantaneously mixed at a volume ratio of 1:3 to 1:5. Taking advantage of the positively charged protonated properties of ionizable cationic lipids under low pH conditions, the two nucleic acid molecules are electrostatically adsorbed and efficiently encapsulated. Subsequently, the ethanol is removed by dialysis or tangential flow filtration and replaced with PBS, and the co-loaded lipid nanoparticles (LNP-nucleic acid complex) are self-assembled.

[0095] To characterize the prepared LNP-nucleic acid complex, its morphology was observed using transmission electron microscopy (TEM); its particle size distribution was determined by nanoparticle tracking analysis (NTA); and its surface charge was detected using a Zeta potentiometer.

[0096] As mentioned above Figures 2 to 4 As shown, the prepared LNP has the characteristics of uniform particle size (~150 nm), good dispersibility, moderately positive Zeta potential (+14.5 mV), good physical stability and potential cellular uptake capacity.

[0097] Example 3 - Preparation of sEVs To obtain small extracellular vesicles (sEVs) with high CXCR4 expression, macrophages (RAW264.7) stably transfected with the CXCR4 gene via lentivirus were first selected. Single-clonal cell lines stably expressing CXCR4 were obtained through puromycin selection. The cells were then replaced with complete medium containing exosome-depleted fetal bovine serum (FBS) and cultured for 48–72 hours to collect conditioned medium containing sEVs. The collected medium was then centrifuged sequentially at 300 ×g (10 min), 2,000 ×g (20 min), and 10,000 ×g (30 min) to remove live cells, dead cells, and large debris. Finally, the supernatant was ultracentrifuged at 100,000 ×g for 70 minutes at 4°C. The supernatant was discarded, and the pellet was resuspended in pre-cooled PBS and centrifuged again at 100,000 ×g. Centrifuge at ×g for 70 minutes and wash once. The final sEVs precipitate is a high-purity, CXCR4-positive small extracellular vesicle that can be stored at −80°C for later use. The obtained CXCR4-sEVs are analyzed for particle size distribution and zeta potential by NTA (nanoparticle tracking analysis), their morphology is photographed by TSM (transmission electron microscopy), and the expression of typical marker proteins (such as Alix, CD81, TSG101) and CXCR4-specific domains is verified by WB.

[0098] Results analysis: such as Figure 5 As shown, a stable macrophage cell line with high expression of CXCR4 (LV-CXCR4-pur-OE) was successfully constructed. A strong and uniform GFP signal was visible under a fluorescence microscope, indicating that the lentivirus transfection efficiency was high and the cells were in good condition. Figure 6 Western blot further confirmed that CXCR4 protein (approximately 37 kDa) was significantly upregulated in these cells, while almost no expression was observed in the control group. The bands of the internal control β-tubulin were consistent, indicating that the sample loading was uniform and the results were reliable. This stable cell line not only verified the successful overexpression of CXCR4, but also laid a key foundation for the subsequent isolation and acquisition of small extracellular vesicles (sEVs) displaying CXCR4 on their surface. This endows the constructed fusion vesicles with the ability to actively target the lung cancer bone metastasis microenvironment (highly expressing CXCL12), providing a reliable cell source and delivery platform for achieving macrophage-specific uptake and immune microenvironment remodeling.

[0099] Results analysis, Figure 7 and Figure 8 The particle size distribution and surface Zeta potential characteristics of exosomes are shown respectively: Figure 7 The NTA results showed that the vesicles exhibited a single-peak distribution, with the main peak concentrated in the range of 80–120 nm, which is consistent with the typical size range of small extracellular vesicles (sEVs), indicating that the sample is homogeneous and has high purity. Figure 8 Zeta potential assays showed that the surface potentials of both ordinary exosomes (EV) and CXCR4-modified exosomes (CXCR4 EV) were between -25 and -30 mV, exhibiting stable negative charge, indicating good colloidal stability. Furthermore, the expression of CXCR4 did not significantly alter the surface charge characteristics of the exosomes, verifying their structural integrity and biocompatibility. This lays the physicochemical foundation for their subsequent application as targeted delivery carriers in the lung cancer bone metastasis microenvironment.

[0100] Figure 10 Western blot results showed that typical exosome marker proteins TSG101, CD81, and Alix were detected in both RAW cell-derived exosomes (CON-sEVs and CXCR4-sEVs), and their expression levels in CXCR4-sEVs were comparable to those in the control group, indicating that the isolated vesicles had good exosome specificity. Meanwhile, the endoplasmic reticulum marker Calnexin was not detected in any samples, suggesting high exosome purity and no significant cell debris contamination. CXCR4 was significantly expressed in CXCR4-sEVs, with a molecular weight of approximately 37 kDa, confirming successful engineering and effective anchoring of CXCR4 on the exosome surface, providing a molecular basis for achieving targeted bone transfer microenvironment.

[0101] Example 4 - Preparation and Characterization of HNV HNV was obtained by combining LNP and sEVs. The synthesized and extracted LNP and sEVs were used, and their concentrations were determined (protein concentration represented by BCA assay, and lipid mass represented by a phospholipid quantification kit (e.g., Stewart method)). The initial fusion ratio was sEVs : LNP = 1 : 1 (w / w). Both were diluted to the same volume with PBS for mixing. In a sterile laminar flow hood, the 1:1 sEVs and LNP suspensions were added to a sterile EP tube. Gently pipette to mix (avoid vigorous vortexing to prevent air bubbles or structural damage), and incubate at room temperature for 10-15 minutes.

[0102] Clean the extruder assembly with anhydrous ethanol, rinse with sterile PBS, and finally sterilize with 75% ethanol and irradiate with UV light in a laminar flow hood for 30 minutes (or use sterile disposable components). Take two 200 nm polycarbonate membranes (PCMembrane), moisten them, and place them on either side of the central channel of the extruder. Place a support disc on each side of the membrane.

[0103] Tighten both ends of the squeezer to ensure no leakage. First, draw 1 mL of sterile PBS buffer and push the syringe to pass the buffer through the membrane, repeating 3-5 times. Remove air from the membrane to prevent air bubbles from obstructing sample passage and wet the pores. Empty the PBS from the syringe. Draw the pre-mixed sEVs + LNP mixture into one of the airtight syringes and connect it to one end of the squeezer. Connect the other empty syringe to the other end of the squeezer. Push the syringe plunger at a constant, slow rate (approximately 0.5-1 mL / min) to pass the mixture through the 200 nm membrane into the empty syringe. Squeeze back and forth 11-21 times. Remove the squeezer and replace it with a 100 nm PC membrane. Repeat the above steps of "wetting -> loading sample -> squeezing back and forth 11-21 times". Carefully aspirate the liquid from the final syringe into a sterile EP tube, label it HNV, aliquot, and store at 4°C for short-term storage (within 1 week) or -80°C for long-term storage (avoid repeated freeze-thaw cycles).

[0104] The successful construction of HNV was verified by labeling LNPs with the fluorescent dye DiO and sEVs with Dil.

[0105] like Figure 11Confocal fluorescence images showed that Dil-labeled CXCR4-sEVs (red) and DiO-labeled lipid nanoparticles (LNP-dio, green) exhibited highly overlapping fluorescence signals during the fusion process (shown as yellow areas in the merged image), indicating that the two successfully achieved physical fusion in vitro to form a two-component hybrid vesicle. The uniform distribution of red and green signals and the obvious co-localization phenomenon indicate that CXCR4-sEVs and LNP have good compatibility and binding efficiency, providing direct visual evidence for constructing fusion vesicles with both targeting and nucleic acid delivery functions.

[0106] Figure 12 The image shown is a transmission electron microscope image of CXCR4-HNV@siRNA Siglec-15+mRNA IL-12, scale bar: 50nm. The results show that CXCR4-HNV@siRNA Siglec-15+mRNA IL-12 can inhibit siglec 15 and promote IL-12 expression.

[0107] like Figure 13 The results showed that Siglec-15 siRNA delivered via CXCR4-sEVs-HNV (fusion vesicles) significantly inhibited Siglec-15 protein expression in macrophages (Western blot showed a significant decrease in its band), while the LNP@siRNASiglec-15 / mRNA IL-12 control group also showed a similar effect, indicating that both vectors can effectively deliver siRNA to achieve gene silencing; simultaneously, as Figure 14 As shown, the expression level of IL-12 mRNA in the fusion vesicle treatment group was significantly higher than that in the PBS control group (approximately 2.5 times higher), and there was no significant difference compared with the LNP group. This suggests that the fusion vesicles can efficiently deliver IL-12 mRNA and achieve intracellular translation, thereby synergistically achieving dual regulation of downregulation of immunosuppressive factors and upregulation of pro-inflammatory factors, verifying the dual-function role of this system in reshaping the tumor immune microenvironment.

[0108] Example 5 - Validation of CXCR4-HNV@siRNA Siglec-15+mRNA IL-12 function like Figure 15Confocal images showed significant differences in the expression levels of the M1 marker iNOS (red fluorescence) in macrophages after different treatments: iNOS was almost not expressed in the PBS and negative control vesicle (HNV@RNAscramble) groups; delivery of Siglec-15 siRNA or IL-12 mRNA alone could partially induce iNOS upregulation; while the fusion vesicles co-loaded with siRNA and mRNA (HNV@siRNA Siglec-15+mRNA IL-12) further enhanced iNOS expression; especially in the CXCR4-HNV@siRNA Siglec-15+mRNA IL-12 group with CXCR4 targeting modification, the iNOS fluorescence signal was the strongest and widely distributed, indicating that the targeted fusion vesicles can efficiently reprogram macrophages to transform into the anti-tumor M1 phenotype and achieve synergistic reversal of the immunosuppressive microenvironment.

[0109] Example 6 - Co-culturing macrophages and T cells after fusion vesicle action to detect T cell killing ability. like Figure 16 As shown, macrophages were first co-incubated with each group of drugs for 48 hours to induce them to secrete specific factors into the culture supernatant under drug stimulation. Subsequently, this "macrophage supernatant" was collected and added to a new culture system containing spleen-derived T cells for further co-culture for 48 hours. The aim was to study the effect of soluble factors released by drug-regulated macrophages on T cell function (such as activation, proliferation, or differentiation), thereby indirectly evaluating the immunomodulatory effect of nanoparticles mediated by the immune microenvironment.

[0110] like Figure 17As shown, flow cytometry results demonstrate the effect of different treatment groups (PBS group, HNV@RNAscramble group, HNV@siRNA Siglec-15, HNV@mRNA IL-12, HNV@siRNA Siglec-15+mRNA IL-12, CXCR4-HNV@siRNA Siglec-15+mRNA IL-12; note: in all the above treatment groups, the concentration of HNV in the fusion vesicles was 1 mg / ml, the concentration of siRNA was 0.5 mg / ml, and the concentration of mRNA was 1 mg / ml) on the T cell killing function of macrophages after intervention with fusion vesicles. Annexin V / PI double staining was used to assess the apoptosis of lung cancer LLC tumor cells. The results showed that the apoptosis rate of lung cancer LLC tumor cells in the PBS group and the HNV@RNAscramble group (control) was extremely low (0% and 0.93%, respectively), indicating that the immune response was not activated. Delivery of Siglec-15 siRNA alone (10.2%) or IL-12 alone... mRNA (9.68%) partially promoted T cell-mediated killing; while the fusion vesicles co-loaded with siRNA and mRNA (HNV@siRNA Siglec-15+mRNA IL-12) significantly improved the killing efficiency (18.8%); in the CXCR4-HNV@siRNA Siglec-15+mRNA IL-12 group, the apoptosis rate of lung cancer LLC tumor cells reached the highest (19.7%), indicating that this targeting system not only effectively reprogrammed macrophages to M1 type, but also significantly enhanced their ability to activate CD8+ T cells, thereby synergistically enhancing the anti-tumor immune effect, verifying the excellent performance of this fusion vesicle in enhancing T cell killing function.

[0111] Example 7 - After tail vein injection of CXCR4-HNV@Siglec 15 siRNA + IL-12 mRNA into C57 mice, it was able to target tumor microenvironment macrophages. Establishing a C57BL / 6 mouse bone metastasis model: First, 6-8 week old female C57BL / 6 mice were selected, anesthetized, fixed on the operating table, and the right hind limb knee joint area was disinfected. Then, the skin was incised next to the patella to expose the distal femur, and a 27G sterile needle was used to drill through the bone cortex in the intercondylar fossa of the femur to enter the medullary cavity. Subsequently, tumor cells (LLC mouse lung cancer cells, approximately 1×10^5 cells / mouse) suspended in PBS were slowly injected into the medullary cavity through a microinjector. After removing the needle, the needle hole was sealed with bone wax to prevent cell extravasation, and the incision was sutured. Four weeks later, in vivo drug distribution experiments were conducted.

[0112] like Figure 18As shown, using the aforementioned mouse model of lung cancer bone metastases, hybrid nanovesicles (HNVs) with different ratios were used to encapsulate cy5.5 fluorescently labeled siRNA—including unmodified groups (sEVs:LNP = 1:1, 1:5, 1:2, 2:1, 5:1), targeted groups constructed from CXCR4 engineered exosomes (CXCR4 sEVs:LNP, in the same ratio), and free cy5.5. The fluorescently labeled siRNA control group was injected into mice with lung cancer bone metastases via tail vein injection. Twelve hours after administration, the mice were euthanized and major organs (bone metastases, liver, spleen, kidney, heart, lungs, etc.) were isolated. The fluorescence signal intensity in each tissue was detected using an IVIS in vivo imaging system. The experiment showed that compared with free siRNA and untargeted HNV, CXCR4-modified fusion vesicles (CXCR4-HNV) showed significantly enhanced fluorescence signal at the tumor site and a longer duration, especially exhibiting excellent targeting enrichment ability at sEVs:LNP ratios of 1:1 to 5:1. Considering the complexity of exosome raw material extraction and the tendency of fluorescence at the tumor site to saturate at sEVs:LNP ratios of 1:1 to 5:1, a sEVs:LNP ratio of 1:1 was used in subsequent treatment experiments.

[0113] like Figure 19 As shown, using the aforementioned mouse model of lung cancer bone metastases, hybrid nanovesicles (HNVs) encapsulating Cy5.5 fluorescently labeled siRNA in different ratios—including a free Cy5.5 fluorescently labeled siRNA control group, an unmodified HNV group (sEVs:LNP = 1:1, 1:5, 1:2, 2:1, 5:1), and a CXCR4-engineered exosome-based HNV-targeting group (CXCR4sEVs:LNP, in the same ratio)—were injected intravenously into tumor-bearing mice. Subsequently, longitudinal fluorescence imaging (IVIS system) was performed on the live mice at 6, 12, and 24 hours post-administration. By dynamically monitoring the change in fluorescence signal intensity at the tumor site (femoral region) over time, the pharmacokinetic behavior, tumor-targeting accumulation efficiency, and retention capacity of each formulation in vivo were evaluated. The experiment showed that at 12 hours… Around 1:1, the fluorescence effect was strongest. Compared with free siRNA and untargeted HNV, CXCR4-modified fusion vesicles (CXCR4-HNV) showed significantly enhanced fluorescence signal at the tumor site and a longer duration. In particular, at sEVs:LNP ratios of 1:1 to 5:1, they showed excellent targeting enrichment ability. Considering the complexity of exosome raw material extraction and the tendency of fluorescence at the tumor site to saturate at sEVs:LNP ratios of 1:1 to 5:1, a 1:1 sEVs:LNP ratio was used in subsequent treatment experiments.

[0114] like Figure 20Three groups of mouse bone metastasis tumor samples were collected and sectioned. Confocal microscopy analysis of the tissue sections showed that Cy5.5-labeled siRNA was mainly located in F4 / 80 positive macrophages, and the co-localization degree of siRNA with macrophages in the CXCR4-HNV group was significantly higher than that in the control group. This demonstrates that the system can efficiently target and be taken up by tumor-associated macrophages, achieving precise delivery and immune regulation of nucleic acid drugs in the bone metastasis microenvironment.

[0115] Example 8 - Analysis of tumor microenvironment immune cells after drug treatment in each group Treatment of C57 / BL6 lung cancer bone metastases in mice: The above-mentioned C57 / BL6 lung cancer bone metastases were randomly divided into six groups: PBS control group, negative control vesicle group (HNV@RNAscramble), single-drug treatment group (tail vein injection of HNV containing 10 μg Siglec-15 siRNA or 10 μg IL-12 mRNA, respectively), non-targeted co-loaded drug group (HNV@siRNA Siglec-15+mRNA IL-12, containing 10 μg siRNA and 10 μg mRNA), and CXCR4-targeted co-loaded drug group (CXCR4-HNV@siRNA Siglec-15+mRNA). IL-12 (dose as before); the treatment regimen was to administer the drug via the tail vein every 3 days starting from the day after tumor colonization was confirmed (day 7 post-inoculation), for a total of 4 treatments (days 7, 10, 13, and 16). Mice were euthanized 48 hours after the last administration (day 18), and femoral and lung tissues were collected for biological analysis to systematically evaluate the inhibitory effect on bone metastases and the ability to regulate the immune microenvironment in each group.

[0116] Bone metastasis tumor tissues from lung cancer in mice of each group were collected and subjected to immunological flow cytometry analysis, such as... Figure 21The analysis results showed that the proportion of M1 macrophages (defined by the CD86⁺CD206⁻ phenotype) in the tumor microenvironment changed significantly after different treatments: the proportion of M1 in the PBS group and the HNV@RNAscramble group was extremely low (0.55% and 0.34%, respectively), indicating a basic state of immunosuppression; delivery of Siglec-15 siRNA alone increased the proportion of M1 to 27.2%, suggesting that silencing immune checkpoints helps promote the transformation of macrophages to an anti-tumor phenotype; delivery of IL-12 mRNA alone further increased it to 29.3%; while the fusion vesicles co-loaded with siRNA and mRNA (HNV@siRNA Siglec-15+mRNA IL-12) increased the proportion of M1 to 44.3%, showing a synergistic effect; CXCR4-HNV@siRNA Siglec-15+mRNA modified with CXCR4 targeting also showed a significant increase. In the IL-12 group, the proportion of M1 was the highest at 57.6%, significantly higher than in other groups. This demonstrates that the targeting system can not only efficiently deliver bifunctional nucleic acids, but also accurately enrich bone metastases and effectively reprogram tumor-associated macrophages to the M1 phenotype, thereby reshaping the immunosuppressive microenvironment and enhancing the anti-tumor immune response.

[0117] like Figure 22 Immunohistochemical results showed that the infiltration of CD8+ T cells in tumor tissue significantly increased with increasing treatment intensity. CD8+ T cells were sparsely distributed in PBS and the control group, but significantly increased in the HNV@siRNA Siglec-15+mRNA IL-12 group. Particularly in the CXCR4-HNV@siRNA Siglec-15+mRNA IL-12 group, CD8+ T cells were highly aggregated around tumor nests, exhibiting dense brownish-red positive staining, indicating that it effectively recruited and activated the anti-tumor T cell response.

[0118] In summary, this fusion vesicle system can not only effectively regulate the transformation of tumor-associated macrophages to the M1 phenotype, but also significantly enhance the infiltration of CD8+ T cells in tumors, thereby synergistically constructing an anti-tumor immune microenvironment and providing strong support for achieving efficient and durable immunotherapy.

[0119] Example 9 - Comparison of tumor treatment effects in different groups of mice like Figure 23 , Figure 24As shown, the fusion vesicle (HNV)-mediated gene therapy strategy demonstrated significant efficacy in inhibiting tumor growth. In tumor tissue images, the tumors in the PBS group and the HNV@scramble group were significantly larger, darker red, and firmer, indicating active tumor proliferation. In contrast, the tumors in the HNV@siRNA Siglec-15, HNV@mRNA IL-12, and combination therapy groups (HNV@siRNASiglec-15 + mRNA IL-12) showed significantly smaller volumes, with some exhibiting shrinkage or softening, indicating that gene intervention effectively inhibited tumor progression. In particular, the CXCR4-targeted modified HNV@siRNASiglec-15 + mRNA IL-12 group exhibited the smallest tumor size, further demonstrating that the targeted delivery system enhanced drug accumulation efficiency at the tumor site.

[0120] Correspondingly, the tumor volume growth curve over time also confirmed the above results: the tumors in the PBS group and the HNV@scramble group grew rapidly, showing an exponential upward trend; both the HNV@siRNA Siglec-15 and HNV@mRNA IL-12 groups showed certain inhibitory effects; while the HNV@siRNA Siglec-15+mRNA IL-12 group, which combined siRNA and mRNA, showed a stronger anti-tumor effect; the most significant was the CXCR4-HNV@siRNA Siglec-15+mRNA IL-12 group, whose tumor growth was almost completely inhibited, and the tumor volume at 25 days was much lower than that of other groups, suggesting that this targeted bifunctional gene delivery system has a synergistic effect.

[0121] In summary, the fusion vesicle system not only delivers functional nucleic acids efficiently, but also significantly enhances tumor-specific targeting capabilities through CXCR4 targeting modification, thereby effectively inhibiting tumor growth and demonstrating good therapeutic potential.

[0122] Example 10 - Safety Study of Fusion Vesicles As mentioned above Figure 25 and Figure 26As shown, based on histopathological analysis and biochemical index detection results, the fusion vesicle (HNV) group exhibited good safety in multiple organs. HE staining revealed that, compared with the PBS control group, no significant tissue damage or inflammatory response was observed in major organs such as the heart, liver, spleen, lungs, and kidneys in any of the HNV treatment groups (including HNV@scramble, HNV@siRNA Siglec-15, HNV@mRNA IL-12, HNV@siRNA Siglec-15+mRNA IL-12, and CXCR4 HNV@siRNA Siglec-15+mRNA IL-12). For example, liver cells were neatly arranged without obvious necrotic foci; the glomeruli and tubules of the kidneys were intact; the alveolar structures were clear without significant edema or exudation; and the cardiac muscle fiber striations were clear without signs of degeneration or necrosis.

[0123] Meanwhile, serum biochemical indicators further validated its safety: key indicators such as AST, ALT, TP, ALB, UREA, and CREA showed no significant differences among the experimental groups, indicating that the HNV vector did not cause significant liver or kidney damage or systemic metabolic disorders. Particularly noteworthy is that even in the combined treatment group co-loaded with siRNA and mRNA, no enhanced toxicity was observed, suggesting that the system has good biocompatibility and in vivo tolerability.

[0124] In summary, the fusion vesicle delivery system demonstrates excellent safety profiles from both histological and biochemical functional assessment perspectives, providing strong support for subsequent clinical translation.

[0125] The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this specification. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification and therefore remain within the spirit and scope of the exemplary embodiments described herein.

[0126] Furthermore, this specification uses specific terms to describe embodiments thereof. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "an embodiment," "one embodiment," or "an alternative embodiment" in different locations throughout this specification do not necessarily refer to the same embodiment. Moreover, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0127] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are modified in some examples with the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed depending on the characteristics required by individual embodiments. In some embodiments, numerical parameters should take into account specified significant digits and employ a general method of digit reservation. Although the numerical ranges and parameters used to confirm their breadth of range in some embodiments of this specification are approximate values, in specific embodiments, such values ​​are set as precisely as feasible.

[0128] Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments described herein. Other variations may also fall within the scope of this specification. Therefore, alternative configurations of the embodiments described herein are intended to be illustrative rather than limiting, and should be considered consistent with the teachings of this specification. Accordingly, the embodiments described herein are not limited to those explicitly introduced and described herein.

Claims

1. A fusion vesicle, characterized in that, The raw materials of the fusion vesicles include small extracellular vesicles and lipid nanoparticle-nucleic acid complexes, with a mass ratio of (0.5-3):(0.5-3) for the small extracellular vesicles and lipid nanoparticle-nucleic acid complexes. The lipid nanoparticle-nucleic acid complexes include lipid nanoparticles and nucleic acids, with the nucleic acids loaded in the lipid nanoparticles. The raw materials of the lipid nanoparticles include ionizable cationic lipids, cofactor phospholipids, cholesterol, and polyethylene glycol-modified lipids.

2. The fusion vesicle as described in claim 1, characterized in that, The mass ratio of the small extracellular vesicles to the lipid nanoparticle-nucleic acid complex is 1:1; And / or, the small extracellular vesicles are small extracellular vesicles with high CXCR4 expression on their surface; And / or, the surface potential of the small extracellular vesicles is between -25 and -30 mV, exhibiting negative charge; And / or, the small extracellular vesicles express TSG101, CD81 and Alix proteins; And / or, the small extracellular vesicles are expressed by macrophages, preferably macrophages that stably and highly express CXCR4.

3. The fusion vesicle as described in claim 1, characterized in that, The molar ratio of the lipid nanoparticles to the nucleic acid is (3-5):(0.5-1), preferably (3.5-4.5):(0.8-1).

4. The fusion vesicle as described in claim 1, characterized in that, The nucleic acid includes a small interfering RNA targeting Siglec-15 and a messenger RNA encoding IL-12.

5. The fusion vesicle as described in claim 4, characterized in that, The nucleotide sequence of the small interfering RNA targeting Siglec-15 includes at least one of the following three pairs: SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6; And / or, the nucleotide sequence of the messenger RNA encoding IL-12 is shown in SEQ ID NO.

7.

6. The fusion vesicle as described in claim 4, characterized in that, The mass ratio of the small interfering RNA targeting Siglec-15 to the messenger RNA encoding IL-12 is (0.5-3):(0.5-3), preferably, the mass ratio of the small interfering RNA targeting Siglec-15 to the messenger RNA encoding IL-12 is 1:

1.

7. The fusion vesicle as described in claim 1, characterized in that, The raw materials of the lipid nanoparticles include ionizable cationic lipids, cofactor phospholipids, cholesterol and polyethylene glycol-modified lipids in a molar ratio of (40-60):(5-15):(30-45):(0.5-3). Preferably, the raw materials of the lipid nanoparticles include ionizable cationic lipids, cofactor phospholipids, cholesterol and polyethylene glycol-modified lipids in a molar ratio of 50:10:38.5:1.

5. And / or, the ionizable cationic lipid is DLin-MC3-DMA; And / or, the auxiliary phospholipid is distearylphosphatidylcholine. And / or, the PEGylated lipid is 1,2-dimyristoyl-rac-glycerol-methoxy polyethylene glycol 2000.

8. The method for preparing fusion vesicles according to any one of claims 1 to 7, characterized in that, Includes the following steps: 1) The small interfering RNA of Siglec-15 and the messenger RNA encoding IL-12 were loaded into the lipid nanoparticles to obtain the lipid nanoparticle-nucleic acid complex. 2) The lipid nanoparticle-nucleic acid complex was fused with small extracellular vesicles to obtain fused vesicles; The raw materials for the lipid nanoparticles include ionizable cationic lipids, cofactor phospholipids, cholesterol, and polyethylene glycol-modified lipids.

9. The preparation method according to claim 8, characterized in that, The preparation of the lipid nanoparticle-nucleic acid complex includes the following steps: 3) Dissolve the siRNA targeting Siglec-15 and the messenger RNA encoding IL-12 in citrate buffer as the aqueous phase; 4) Ionizable cationic lipids, cofactor phospholipids, cholesterol, and polyethylene glycol-modified lipids are dissolved in ethanol to form the ethanol phase; 5) Mix the aqueous phase and the ethanol phase to obtain the lipid nanoparticle-nucleic acid complex.

10. The preparation method according to claim 9, characterized in that, The citrate buffer solution has a pH of 3 to 5, preferably a pH of 4.0, more preferably an aqueous solution of citric acid, and even more preferably a concentration of 10 to 50 mM, preferably 20 mM.

11. The preparation method according to claim 9, characterized in that, The volume ratio of the ethanol phase to the water phase is 1:3 to 1:

5.

12. The preparation method according to claim 9, characterized in that, The mass ratio of siRNA targeting Siglec-15 to messenger RNA encoding IL-12 in the aqueous phase is (0.5-3):(0.5-3), preferably, the mass ratio of small interfering RNA targeting Siglec-15 to messenger RNA encoding IL-12 in the aqueous phase is 1:

1.

13. The preparation method according to claim 9, characterized in that, The concentration of siRNA targeting Siglec-15 in the aqueous phase is 0.05–2.0 mg / mL, and the concentration of messenger RNA encoding IL-12 is 0.05–2.0 mg / mL.

14. The preparation method according to claim 8, characterized in that, The fusion step includes mixing the ethanol phase with the aqueous phase, removing the ethanol by dialysis or tangential flow filtration, and replacing it with a neutral physiological buffer. Preferably, the neutral physiological buffer is PBS buffer.

15. The preparation method according to claim 8, characterized in that, The small extracellular vesicles are small extracellular vesicles with high expression of CXCR4 on their surface; And / or, the surface potential of the small extracellular vesicles is between -25 and -30 mV, exhibiting negative charge; And / or, the small extracellular vesicles express TSG101, CD81 and Alix proteins; And / or, the small extracellular vesicles are expressed by macrophages, preferably macrophages that stably and highly express CXCR4.

16. The preparation method according to claim 15, characterized in that, The macrophages that stably express CXCR4 are obtained by transferring the CXCR4 gene into RAW 264.7 cells and then screening them; preferably, the transfer is mediated by lentivirus.

17. The preparation method according to claim 8, characterized in that, The mass ratio of the small extracellular vesicles to the lipid nanoparticle-nucleic acid complex is (0.5-3):(0.5-3), preferably, the mass ratio of the small extracellular vesicles to the lipid nanoparticle-nucleic acid complex is 1:1; And / or, the lipid nanoparticle-nucleic acid complex comprises lipid nanoparticles and nucleic acid, wherein the nucleic acid is loaded in the lipid nanoparticles. Preferably, the molar ratio of the lipid nanoparticles to the nucleic acid is (3.5-4.5):(0.8-1), and more preferably, the molar ratio of the lipid nanoparticles to the nucleic acid is 4:

1.

18. The preparation method according to claim 8, characterized in that, After the aqueous phase and the ethanol phase are mixed, the particles need to be homogenized by an emulsification device, preferably a liposome extruder.

19. A method for delivering nucleic acids to cells for purposes other than disease diagnosis and treatment, comprising contacting the fusion vesicles of any one of claims 1 to 7 with the cells.

20. The use of the fusion vesicles as described in any one of claims 1 to 7 in the preparation of antitumor drugs.

21. The application as described in claim 20, characterized in that, The tumors mentioned are selected from lung cancer, bone metastases, nasopharyngeal carcinoma, gastric cancer, primary gallbladder cancer, leiomyosarcoma, liver cancer, oral cancer, head and neck cancer, adrenocortical carcinoma, urothelial carcinoma of the bladder, breast cancer, cervical squamous cell carcinoma, cervical endometrial adenocarcinoma, bile duct carcinoma, colon adenocarcinoma, lymphoid tumors, and diffuse large B-cell tumors. The tumor is selected from one or more of the following: lymphoma, esophageal cancer, glioblastoma multiforme, squamous cell carcinoma of the head and neck, chromophobe renal carcinoma, clear cell renal carcinoma, papillary renal carcinoma, acute myeloid leukemia, low-grade glioma of the brain, hepatocellular carcinoma, squamous cell carcinoma of the lung, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, colorectal cancer, malignant sarcoma, melanoma, testicular germ cell tumor, thyroid cancer, thymic carcinoma, endometrial cancer, uterine sarcoma, uveal melanoma, multiple myeloma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, chronic myeloid leukemia, lymphoma, lung cancer, sarcoma, anal cancer, melanoma, and retinoblastoma. Preferably, the tumor is selected from lung cancer and / or bone metastases. More preferably, the bone metastases are lung cancer bone metastases.

22. The application as described in claim 20, characterized in that, The fusion vesicles regulate the transformation of tumor-associated macrophages into M1-type macrophages; And / or, the fusion vesicles enhance the infiltration of CD8+ T cells within the tumor; And / or, the fused vesicles enhance T cell-mediated tumor clearance.

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