Engineering bionic nucleic acid nano-vesicle as well as preparation method and application thereof

Abstract

The invention discloses an engineered bionic nucleic acid nano-vesicle as well as a preparation method and application thereof, relates to the technical field of nano biomedicine, and aims at solving the problems that in-vivo targeting efficiency and immunogenicity of a traditional cationic lipid nano-carrier are limited due to deletion and cleavage obstacles of GSDMD expression in tumor cells. The technical key point of the invention is as follows: the engineered bionic nucleic acid nano-vesicle is provided and is prepared by wrapping a cationic lipid nucleic acid drug with an exosome derived from engineered macrophages; wherein the exosome from the engineered macrophage is the exosome from the macrophage with high expression of PD1, which is as shown in SEQ. ID. NO.1. The exosome from the engineered macrophage is the exosome from the macrophage with high expression of PD1; the lipid nucleic acid medicine is prepared by loading GSDMD-N mRNA (messenger Ribonucleic Acid) shown on the basis of SEQ.ID.NO.2 on a cationic liposome. The engineered bionic nucleic acid nano-vesicle is used for preparing an oral squamous cell carcinoma diagnostic kit and a therapeutic drug.

Description

Technical Field

[0001] This invention relates to the field of nanobiomedical technology, and more specifically, to an engineered biomimetic nucleic acid nanovesicle, its preparation method, and its application. Background Technology

[0002] Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors of the head and neck, accounting for approximately 90% of all oral malignancies. Despite significant breakthroughs in tumor immunotherapy, exemplified by immune checkpoint blockade (ICB), OSCC exhibits limited response rates and is prone to drug resistance. Furthermore, the tumor microenvironment (TME) is systemic and highly regulated, and tumors themselves are complex, adaptive, and heterogeneous. While conventional immunotherapy has achieved some success in cancer treatment, it still faces considerable challenges. Therefore, there is an urgent need to develop novel OSCC treatment strategies that can reshape the TME and activate anti-tumor immune responses.

[0003] Research has found that inducing pyroptosis in tumor cells can significantly enhance tumor immune surveillance and attack, providing a revolutionary strategy for cancer immunotherapy. Pyroptosis is a programmed necrotizing cell death mechanism mediated by the Gasdermin (GSDM) protein family. Gasdermin D (GSDMD) is the most widely studied member of the GSDM family. When GSDMD protein is cleaved by inflammatory caspases, its N-terminal domain (GSDMD-N) can oligomerize into pores on the cell membrane, leading to cell swelling and rupture, and releasing large amounts of pro-inflammatory factors and damage-associated molecules. This can not only directly eliminate tumor cells but also enhance the efficacy of immunotherapy. However, tumor cells do not express or express low levels of GSDMD protein, and the complex cleavage process of GSDMD limits pyroptosis in tumor cells. Therefore, how to avoid the loss of GSDMD expression and cleavage barriers inside tumor cells and achieve dual therapy of tumor pyroptosis and immune activation is an urgent problem to be solved.

[0004] mRNA therapy has wide applications in cancer immunotherapy and protein replacement therapy. mRNA nucleic acid drugs, through the construction of programmable delivery systems, can target and activate immune responses or repair tumor suppressor gene function within tumor cells, achieving specific intervention in malignant cells. This technology, with its highly customized coding capabilities, overcomes the limitations of traditional drugs on refractory targets, opening new avenues for precision tumor treatment. Combining targeted delivery vectors with mRNA therapy technology holds promise for targeted gene therapy of complex diseases. Nanoparticle drug delivery systems have attracted widespread attention in the biomedical field due to their unique advantages such as targeted delivery, multi-drug co-loading, and spatiotemporally controlled drug release. However, they are often recognized and rapidly cleared by the host's mononuclear macrophage system, resulting in short half-lives and insufficient tumor accumulation. To improve efficacy, researchers are committed to reducing non-specific uptake and constructing biomimetic nanocarriers. Studies have shown that exosomes, as endogenous vesicles, possess low immunogenicity, good biocompatibility, and the ability to cross biological barriers, making them ideal carriers for delivering nucleic acid drugs. Macrophage-derived exosomes express multiple membrane receptors on their surface, enabling them to recognize diverse endogenous and exogenous ligands. Therefore, there is an urgent need to develop an engineered exosome-loaded biomimetic nanocarrier to specifically deliver GSDMD-N mRNA into tumor cells, thereby improving tumor tissue targeting, inducing pyroptosis, and facilitating immune checkpoint blockade in tumor therapy. Summary of the Invention

[0005] The technical problem to be solved by this invention is:

[0006] Traditional cationic lipid nanocarriers have limited in vivo targeting efficiency and immunogenicity in addressing the loss of GSDMD expression and cleavage barriers within tumor cells.

[0007] Therefore, this invention provides an engineered biomimetic nucleic acid nanovesicle, its preparation method, and its application.

[0008] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0009] This invention provides an engineered biomimetic nucleic acid nanovesicle, which is prepared by encapsulating a cationic lipid nucleic acid drug on exosomes derived from engineered macrophages; wherein the exosomes derived from engineered macrophages are exosomes derived from macrophages that highly express PD1 as shown in SEQ.ID.NO.1; and the lipid nucleic acid drug is prepared by loading cationic liposomes with GSDMD-N mRNA as shown in SEQ.ID.NO.2.

[0010] This invention provides a method for preparing engineered biomimetic nucleic acid nanovesicles, comprising the following steps:

[0011] (1) Preparation of PD1-EVs:

[0012] Lentiviral stably transfected macrophages were constructed to obtain macrophages with high PD1 expression. After a series of differential centrifugations, exosomes PD1-EVs derived from macrophages with high PD1 expression, as shown in SEQ.ID.NO.1, were obtained. RAW-EVs exosomes derived from macrophages without high PD1 expression were obtained using the same method.

[0013] (2) Preparation of GSDMD-N mRNA:

[0014] GSDMD-N mRNA based on SEQ.ID.NO.2 was prepared by plasmid template preparation, in vitro transcription and modification, and purification and quality control of mRNA.

[0015] (3) Preparation of GSDMD-N mRNA@PEL:

[0016] An organic phase was prepared using D-Lin-MC3-DMA, DOPE, Cholesterol, and PEG2000-DMG. An aqueous phase was prepared by dissolving GSDMD-N mRNA in a buffer solution. The organic phase was then added dropwise to the aqueous phase using an organic phase injection method to assemble lipid nanoparticles GSDMD-N mRNA@LNP. GSDMD-N mRNA@LNP was then mixed with PD1-EVs and co-extruded sequentially through filters of different pore sizes using an extruder to obtain the biomimetic nanosystem GSDMD-N mRNA@PEL. The same method was used to obtain the biomimetic nanosystem GSDMD-N mRNA@EL, which combines GSDMD-N mRNA@LNP and RAW-EVs.

[0017] Further, the lentivirus described in step (1) was selected from HBLV-Pdcd1-3xflag-ZsGreen-PURO, and Raw264.7 cells were infected with the lentivirus HBLV-Pdcd1-3xflag-ZsGreen-PURO. 48 h after cell infection, uninfected cells were killed by adding and maintaining a puromycin concentration of 2-5 μg / ml, thereby ultimately obtaining a stable PD1 expression strain under the maintenance of puromycin.

[0018] Further, the organic phase described in step (3) is prepared with a molar ratio of D-Lin-MC3-DMA, DOPE, Cholesterol, and PEG2000-DMG of (48-55): (8-12): (35-40): (0.5-2).

[0019] Furthermore, in step (3), the molar ratio of the positive charge carried by D-Lin-MC3-DMA to the negative charge carried by GSDMD-N mRNA is 6:1.

[0020] Furthermore, in step (3), the volume ratio of the aqueous phase to the organic phase is 3:1.

[0021] Furthermore, in step (3), the mass ratio of GSDMD-N mRNA@LNP to PD1-EVs is (1-5):1.

[0022] Furthermore, the filter membranes with different pore sizes mentioned in step (3) are 800 nm, 400 nm, and 200 nm filter membranes, respectively.

[0023] This invention provides the application of the above-mentioned engineered biomimetic nucleic acid nanovesicles in the preparation of oral squamous cell carcinoma diagnostic kits and therapeutic drugs.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] This invention utilizes PD1-engineered macrophage-derived exosomes modified with cationic lipid nanoparticles loaded with GSDMD-N mRNA to construct an engineered biomimetic nucleic acid nanosystem, GSDMD-N mRNA@PEL, combining biomimetic nanotechnology and genetic engineering. On one hand, macrophage exosomes exhibit tumor tropism, actively targeting deep within tumors to deliver GSDMD-N mRNA and directly induce pyroptosis. On the other hand, they can block the binding of PD-L1 on tumor cell surface to PD-1 on T cell surface, improving the tumor microenvironment, thereby achieving efficient deep drug delivery and precise tumor treatment, providing a gene immunotherapy strategy.

[0026] The GSDMD-N mRNA@PEL nanotherapy agent of the present invention can be applied to a variety of tumor treatments or other disease treatments, and has great clinical application prospects. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the construction of GSDMD-N mRNA@PEL nanoparticles according to the present invention;

[0028] Figure 2 Western blot (WB) detection of PD1 protein levels on the surface of untreated macrophages, PD1-engineered macrophages, and exosomes derived from the above-mentioned cells in this embodiment of the invention (A); Western blot (WB) detection of exosome marker proteins on the surface of exosomes derived from untreated macrophages and PD1-engineered macrophages (B).

[0029] Figure 3 These are transmission electron microscopy (TEM) images of macrophage-derived exosome vesicles RAW-EVs and macrophage-derived exosome vesicles PD1-EVs that highly express PD1 in the embodiments of the present invention.

[0030] Figure 4 These are transmission electron microscopy (TEM) images of GSDMD-N mRNA@LNP and GSDMD-N mRNA@PEL nanoparticles in the embodiments of the present invention.

[0031] Figure 5 This is a particle size map of RAW-EVs and PD1-EVs detected using nanoparticle tracking analysis (NTA) in an embodiment of the present invention.

[0032] Figure 6 This is a Zeta potential map of RAW-EVs and PD1-EVs detected using nanoparticle tracking analysis (NTA) technology in an embodiment of the present invention.

[0033] Figure 7 This invention utilizes agarose gel electrophoresis to detect DNA plasmid patterns (A) and RNA quality control patterns (B) during the preparation of GSDMD-N mRNA.

[0034] Figure 8 The particle size (A) and potential diagram (B) of the GSDMD-N mRNA@LNP, PD1-EVs and GSDMD-N mRNA@PEL nanoparticles in the embodiments of the present invention are shown.

[0035] Figure 9 This is a laser confocal microscopy detection image of the GSDMD-N mRNA@PEL biomimetic nucleic acid nanoparticle fusion in an embodiment of the present invention;

[0036] Figure 10 The embodiment of this invention is for the stability detection of biomimetic nucleic acid nanoparticles;

[0037] Figure 11 Coomassie Brilliant Blue staining was used in this embodiment of the invention to detect protein retention in GSDMD-N mRNA@LNP, PD1-EVs and GSDMD-N mRNA@PEL.

[0038] Figure 12 The laser confocal detection image in this embodiment of the invention uses laser confocal microscopy to detect the lysosomal escape of GSDMD-N mRNA@PEL in SCC7 cells;

[0039] Figure 13 This is a diagram of the MTT assay showing the inhibitory effect of GSDMD-N mRNA@PEL on tumor cells in an embodiment of the present invention.

[0040] Figure 14This invention relates to the detection of pyroptosis in tumor cells induced by nanoparticles. (A) Bright-field microscopy was used to observe the pyroptosis process. (B) Scanning electron microscopy (SEM) was used to observe changes in pyroptosis. (C) Transmission electron microscopy (TEM) was used to observe changes in pyroptosis.

[0041] Figure 15 Figure A shows the expression of GSDMD-N protein on the cell surface detected by immunofluorescence staining in this embodiment of the invention; Figure B shows the results of Western blot detection of GSDMD-N protein expression level in cells after treatment with GSDMD-N mRNA@PEL.

[0042] Figure 16 The flow cytometry analysis in this embodiment of the invention is used to analyze the changes in HMGB1 (A) and CRT (B) expression levels in cells after treatment with GSDMD-N mRNA@PEL.

[0043] Figure 17 The laser confocal microscopy method in this embodiment of the invention was used to analyze the changes in the expression levels of HMGB1 (A) and CRT (B) in cells after treatment with GSDMD-N mRNA@PEL.

[0044] Figure 18 The present invention describes the detection of changes in ATP (A), LDH (B), and IL-1β levels in cell culture medium after GSDMD-N mRNA@PEL treatment.

[0045] Figure 19 This invention demonstrates how GSDMD-N mRNA@PEL induces the maturation of bone marrow-derived dendritic cells (BMDCs). Flow cytometry analysis was used to analyze the BMDC maturation phenotype.

[0046] Figure 20 The ELISA (Enzyme-Linked Immunosorbent Assay) in this embodiment of the invention was used to detect cytokines secreted by BMDCs. Compared with the control group, the secretion of key pro-inflammatory cytokines IL-12p70, TNF-α, IFN-γ and IL-6 in the culture supernatant of BMDCs in the GSDMD-N mRNA@PEL treatment group was significantly upregulated.

[0047] Figure 21This invention demonstrates the therapeutic effect of GSDMD-N mRNA@PEL biomimetic nucleic acid nanoparticles on an oral squamous cell carcinoma-bearing mouse model. Figure (A) shows tumor tissue images of each group after treatment; (B) shows tumor growth curves; (C) shows tumor tissue weight analysis after treatment; and (D) shows changes in mouse body weight in each treatment group (G1: PBS group, G2: Flu mRNA@LNP group, G3: GSDMD-N mRNA@LNP group, G4: @GSDMD-N mRNA@EL group, G5: GSDMD-N mRNA@PEL group).

[0048] Figure 22 The images show sections of tumor tissue from mice in each treatment group in this invention, stained with hematoxylin-eosin (HE) and immunohistochemistry (IHC).

[0049] Figure 23 HE sections of the major organs (heart, liver, spleen, lung, and kidney) of mice in each treatment group in the embodiments of the present invention;

[0050] Figure 24 These are graphs showing the detection of blood biochemical indicators in mice of each treatment group in this embodiment of the invention.

[0051] Figure 25 This is a diagram of a nanoparticle hemolysis experiment in an embodiment of the present invention;

[0052] Figure 26 These are immunoflow cytometry analyses of tumor tissues from mice in each treatment group in this embodiment of the invention, including MHC II. + (Figure A) CD80+ CD86+ (Figure B), CD4 + (Figure C), CD8 + (Figure D) T cell levels (G1: PBS group, G2: Flu mRNA@LNP group, G3: GSDMD-N mRNA@LNP group, G4: @GSDMD-N mRNA@EL group, G5: GSDMD-N mRNA@PEL group);

[0053] Figure 27 Figure A shows the ELISA results of serum (Figure A) and tumor tissue (Figure B) of mice in each treatment group in this embodiment of the invention.

[0054] Figure 28 The figures show RNA sequencing diagrams of tumor tissues from mice in each treatment group in this invention embodiment; (A) is a differential expression volcano diagram; and (B) is a KEGG enrichment analysis diagram of differentially expressed genes.

[0055] Figure 29This is a GO enrichment analysis diagram of RNA sequencing of tumor tissues from mice in each treatment group in this embodiment of the invention;

[0056] Figure 30 This is a graph showing the GSEA enrichment analysis of RNA sequencing of tumor tissues from mice in each treatment group in this embodiment of the invention. Detailed Implementation

[0057] To enable those skilled in the art to better understand the present invention, exemplary embodiments or examples of the present invention will be described below in conjunction with the accompanying drawings. Obviously, the described embodiments or examples are merely some, not all, of the embodiments or examples of the present invention. All other embodiments or examples obtained by those skilled in the art based on the embodiments or examples of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0058] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0059] Example 1

[0060] This embodiment provides a method for preparing biomimetic nanonucleic acid vesicles.

[0061] (1) Preparation of exosome vesicles PD1-EVs derived from PD1-RAW264.7:

[0062] In this invention, Raw264.7 cells were infected with the lentivirus HBLV-Pdcd1-3xflag-ZsGreen-PURO. Forty-eight hours after infection, uninfected cells were killed by adding and maintaining a concentration of 2.0 μg / ml of Puromycin. With the maintenance of Puromycin, a stable strain with stable PD1 expression as shown in SEQ ID NO. 1 was finally obtained.

[0063] Exosomes derived from PD1-RAW264.7 cells were collected by ultracentrifugation. Cells were cultured to the appropriate density in complete medium (DMEM + 10% FBS), gently washed with PBS, and then cultured in serum-free medium for 24–48 h. The cell culture supernatant was transferred to centrifuge tubes and immediately placed on ice or in a 4°C freezer to inhibit protease activity. Centrifugation was performed at 300 g for 10 min at 4°C to remove suspended live cells. The supernatant was transferred to a new tube and centrifuged at 2000 g for 20 min at 4°C to remove dead cells and debris. The supernatant was transferred to an ultracentrifuge tube and centrifuged at 10000 g for 30 min at 4°C to remove organelles, apoptotic bodies, and other large particles. The supernatant was carefully collected and transferred to a new ultracentrifuge tube. Ultracentrifugation was performed at 100000–120000 g for 90 min at 4°C. At this point, exosomes formed a light white precipitate at the bottom of the tube. Carefully discard the supernatant and resuspend the precipitate in a large volume of pre-chilled PBS or sterile buffer. Centrifuge again at 100,000 - 120,000 g for 90 min at 4°C to remove contaminants. Carefully discard the supernatant and gently resuspend the precipitate in 200 μL of sterile PBS or a specific storage buffer (such as PBS containing protease inhibitors). (Operate on ice; briefly vortex and incubate at 4°C for 30 min to promote resuspending; this is the exosome suspension.) Aliquot the resuspended exosomes and store at -80°C for long-term storage, avoiding repeated freeze-thaw cycles.

[0064] (2) GSDMD-N mRNA synthesis.

[0065] Phase 1: DNA Template Preparation. A DNA sequence encoding the N-terminal domain from the mouse GSDMD gene shown in SEQ.ID.NO.2 is selected, and the self-inhibiting C-terminal domain sequence is removed to ensure that the translated protein has pore-forming activity without cleavage. A 5' cap analog binding site for post-transcriptional or co-transcriptional capping is designed at the front of the sequence. Highly efficient UTRs, 5' and 3' untranslated regions are added to enhance mRNA stability and translation ability. Codon optimization is performed on the coding sequence, using codons preferred by the host cell (mouse cells) to improve translation efficiency. A long Poly(A) sequence (typically 120-150 bases) is designed at the end of the sequence, or a structure capable of generating a Poly(A) tail is included in the template. The optimized sequence is cloned into a specialized in vitro transcription plasmid vector. This vector typically contains specific RNA polymerase promoters (most commonly the T7 promoter) and restriction endonuclease sites on both sides of the target sequence. The recombinant plasmid is transformed into *E. coli* for amplification, and then high-purity plasmid DNA is extracted and purified. The circular plasmid was digested with restriction endonucleases downstream of the Poly(A) sequence to cleave it into linear DNA. This is a crucial step to ensure that the transcribed mRNA has a precise 3' end and that no extraneous vector sequence is transcribed. After digestion, purification was performed to remove enzymes, buffer, and short fragments, yielding a pure linearized DNA template. The integrity, size, and purity of the linearized DNA template were analyzed by agarose gel electrophoresis.

[0066] Phase 2: In vitro transcription and co-transcriptional modification.

[0067] The purified linear DNA template was mixed with the following components: RNA polymerase (T7 RNA polymerase), nucleoside triphosphates: ATP, CTP, GTP, UTP, and transcription buffer (to provide a suitable pH and ionic environment). After incubation at 37°C for several hours, the RNA polymerase synthesized an mRNA strand using the DNA template, starting from the promoter. To mimic native mRNA and enhance translation initiation, a 5' cap was added to the mRNA during or after transcription. To reduce the immunogenicity of the mRNA itself (to prevent it from being recognized as viral RNA and cleared by the host's innate immune system), uridine (U) was replaced with pseudouridine or N1-methylpseudouridine; these modified nucleotides were added directly to the reaction system during transcription.

[0068] Phase 3: Purification and Quality Control.

[0069] To ensure the purity, integrity, and safety of the mRNA, the DNA template was degraded using DNase I. Lithium chloride (LiCl) was used for purification precipitation to initially remove impurities such as proteins and free nucleotides. Finally, rapid protein liquid chromatography (HPLC) was used for fine purification to remove residual DNA, truncated RNA fragments, double-stranded RNA byproducts, and excess nucleotides. Concentration was determined using NanoDrop, and the integrity, size, and purity of the mRNA were analyzed by agarose gel electrophoresis. Finally, sterility and endotoxin-free testing was performed to ensure the mRNA was suitable for subsequent cell experiments or in vivo administration.

[0070] (3) Preparation of cationic lipid nucleic acid drugs:

[0071] Stock solutions of 4-N,N-dimethylaminobutyric acid dilinoleyl-methyl ester (D-Lin-MC3-DMA), 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE), cholesterol, and dimyristoylglycerol-polyethylene glycol 2000 (PEG2000-DMG) were prepared with ethanol to concentrations of 20 mg / ml, 10 mg / ml, 20 mg / ml, and 10 mg / ml, respectively.

[0072] The mother liquors were ultrasonically mixed uniformly according to the molar ratio of 4-N,N-dimethylaminobutyric acid-dilinoleyl-methyl ester (D-Lin-MC3-DMA), 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (DOPE), cholesterol, and dimyristoylglycerol-polyethylene glycol 2000 (PEG2000-DMG) of 50:10:38.5:1.5 to prepare an organic phase.

[0073] A 1 mg / mL stock solution was prepared by dissolving the GSDMD-N mRNA shown in SEQ.ID.NO.2 in DEPC.

[0074] Based on the molar ratio of positive charge (nitrogen atom, N) in D-Lin-MC3-DMA to negative charge (phosphorus atom, P) in GSDMD-N mRNA of N / P = 6:1, and the volume ratio of organic phase to aqueous phase of 1:3, the GSDMD-N mRNA stock solution was dissolved in sodium citrate aqueous solution (pH = 4.5) to prepare an aqueous phase. The organic phase was added dropwise to the aqueous phase using the organic phase injection method, vortexed for 1 min and allowed to stand for 10 min to assemble into lipid nanoparticles GSDMD-N mRNA@LNP. These nanoparticles were then placed in dialysis bags and dialyzed in PBS buffer (4℃) for 12 h.

[0075] (4) Preparation of GSDMD-N mRNA@PEL:

[0076] The route for constructing GSDMD-N mRNA@PEL is as follows: Figure 1 As shown, GSDMD-N mRNA@LNP and PD1-EVs were mixed at a mass ratio of 3:1 and co-extruded sequentially through filter membranes (800 nm, 400 nm, 200 nm) using an extruder to obtain a GSDMD-N mRNA@PEL nanoparticle solution. The obtained nanoparticle solution was transferred to a ready-to-use dialysis bag with a molecular weight cutoff (MWCO) of 3.5 kDa and dialyzed in 1 L of pH 7.4 PBS buffer for 10 h to remove organic solvents and free small molecule compounds.

[0077] Primary product concentration: The obtained nanoparticles were transferred to an Amicon ultracentrifuge filter tube (MWCO 3.5 kDa) and nuclease-free water was added. After concentration by centrifugation at 2000 g at 4°C, PBS was added to adjust to physiological osmotic pressure.

[0078] Freeze-drying: After concentration, add 20% sucrose aqueous solution to make the final product solution concentration 10% sucrose aqueous solution. Transfer to freeze-drying storage tube and freeze in a freezer at -80℃ for 6 h. Then transfer to a freeze dryer and freeze-dry overnight at 4℃ and 200 mTorr. After packaging, store in a freezer at -20℃ or -80℃.

[0079] Comparative Example 1

[0080] The difference between this comparative example and Example 1 is that RAW264.7 cell-derived exosome vesicles RAW-EVs were prepared according to the method in step (1), and GSDMD-N mRNA@LNP and EVs were mixed at a mass ratio of 3:1 to prepare GSDMD-N mRNA@EL.

[0081] Example 2

[0082] This implementation provides characterization of biomimetic nanonucleic acid drugs.

[0083] (1) The expression level of PD1 on the surface of engineered RAW264.7 cells was quantitatively analyzed using Western blot. For example... Figure 2 As shown in Figure A, PD1 protein expression was increased in PD1-RAW264.7 cells and PD1-EVs. Figure 2 As shown in B, compared with RAW264.7 cell lysates, the exosomes derived from it specifically highly expressed CD63, CD81, Alix, and TSG101, but did not express Calnexin.

[0084] (2) TEM detection method: RAW-EVs, PD1-EVs, GSDMD-N mRNA@LNP, and GSDMD-N mRNA@PEL nanoparticle solutions were dropped onto a copper grid and incubated for 90 s. The droplets were then blotted dry with filter paper. Subsequently, the nanoparticles were stained with 0.2% phosphotungstic acid solution for 30 s, and the staining solution was carefully blotted dry. The morphology and particle size were observed under TEM. Figure 3 As shown, RAW-EVs and PD1-EVs exhibit a uniform vesicle size distribution (~100 nm) and a classic "cup-shaped" membrane structure, consistent with the globally recognized physical characteristics of exosomes in the extracellular vesicle research community. Figure 4 As shown, GSDMD-N mRNA@LNP exhibits a circular structure. GSDMD-N mRNA@PEL also exhibits a circular structure, with exosomes on the surface encapsulating lipid particles on the periphery. These particles are generally uniform in size and exhibit good dispersibility.

[0085] (3) The particle size and potential detection method is as follows: After diluting the RAW-EVs and PD1-EVs vesicle nanoparticle solutions, the particle size and potential of the exosomes are detected using nanoparticle tracking analysis (NTA). For example... Figure 5 As shown, the vesicle sizes of RAW-EVs and PD1-EVs are approximately 151.7 nm and 149.7 nm, respectively. Figure 6 The zeta potentials of RAW-EVs and PD1-EVs vesicles shown are approximately -29.2 mV and -25.1 mV, respectively, indicating that RAW-EVs and PD1-EVs vesicles conform to the typical physical characteristics of exosomes.

[0086] (4) such as Figure 7 As shown, agarose gel electrophoresis indicates that the electrophoretic pattern of the DNA quality control (linearized plasmid template) should show a single, clear, and sharp band at the expected molecular weight position, indicating that the template has been completely linearized and has not degraded. Figure 7 A). The electrophoretic pattern of in vitro transcribed mRNA should show a single, clear, and sharp band at the expected molecular weight position, indicating that the mRNA is intact and has not been degraded. Figure 7 B).

[0087] (5) After diluting the solutions of PD1-EVs, GSDMD-N mRNA@LNP, and GSDMD-N mRNA@PEL nanoparticles, the particle size and potential of exosomes were detected using nanoparticle tracking analysis (NTA). Figure 8 As shown, the particle size of GSDMD-N mRNA@PEL is larger than that of GSDMD-N mRNA@LNP. The zeta potential of GSDMD-N mRNA@PEL is close to that of PD1-EVs, indicating that GSDMD-N mRNA@PEL is successfully encapsulated by exosomes.

[0088] (6) Detection of fusion between GSDMD-N mRNA@LNP and PD1-EVs in GSDMD-N mRNA@PEL: Liposomes were bound with the red fluorescent dye Dio and exosomes with the green fluorescent dye Dil. Figure 9 As shown, the combination of liposome-labeled fluorescence and exosome-labeled fluorescence on the surface of the enveloped liposomes forms an orange-yellow fluorescence, indicating a high degree of fusion between exosomes and liposomes.

[0089] (7) Stability assay of GSDMD-N mRNA@PEL: To assess the physical stability of GSDMD-N mRNA@PEL, dynamic light scattering (DLS) was used to monitor its particle size for 7 days at 4°C. Figure 10 As shown, under storage conditions of 4°C, the average particle size of GSDMD-N mRNA@PEL is approximately 155 ± 1.5 nm, indicating that the particles are well dispersed and no obvious aggregation occurs.

[0090] (8) Coomassie Brilliant Blue Staining: To assess and compare protein changes in GSDMD-N mRNA@LNP, PD1-EVs, and GSDMD-N mRNA@PEL, Coomassie Brilliant Blue staining was used. All samples were thoroughly lysed with 1% Triton X-100 lysis buffer before detection to ensure lipid structure disruption and complete release of internal proteins. Figure 11 As shown, during the encapsulation process, the vast majority of exosome proteins were successfully integrated and retained in the final hybrid nanovesicles.

[0091] Example 3

[0092] This embodiment provides an experiment demonstrating the escape of GSDMD-N mRNA@PEL from cells via lysosomes.

[0093] Laser confocal microscopy was used to detect the escape of GSDMD-N mRNA@PEL from SCC7 tumor cells via lysosomes. To fully understand the uptake process of tumor cells, Cy5-labeled GSDMD-N mRNA was used.

[0094] After co-incubation with the lysosomal probe Lyso Tracker Deep Red (LTDR) for 2 h, as follows Figure 12 As shown, the nanomaterials are highly colocalized with LTDR and appear yellow. After 4 hours of incubation, the colocalization with lysosomes decreases, indicating the release of mRNA from the lysosomes.

[0095] It is evident that GSDMD-N mRNA@PEL can actively target oral squamous cell carcinoma cell line SCC7, releasing GSDMD-N mRNA into the cytoplasm via intracellular lysosome escape.

[0096] Example 4

[0097] This embodiment provides an evaluation of the cytotoxicity of nanoparticles.

[0098] The MTT assay was used to detect the toxic effects of drugs on SCC7 cells. Cells were seeded in 96-well plates at 3000 cells per well. After 24 h, the original culture medium was removed, and different concentrations of the drug (Flu mRNA@LNP, GSDMD-N mRNA@LNP, GSDMD-N mRNA@EL, and GSDMD-N mRNA@PEL) were added to the cells at 100 μL per well, with four parallel wells. FlumRNA served as the control mRNA (to exclude non-specific effects caused by the mRNA platform itself). Blank culture medium and untreated cells were also included as controls. After 48 h of drug-treated culture, 20 μL of MTT was added to each well, and the cells were incubated at 37°C for 4 h. After dissolving formazan in 150 μL of DMSO, the absorbance (OD) of each well was measured at 595 nm using a multi-mode microplate reader. Cell viability was calculated using the formula: Cell viability % = (Experimental group OD value - Blank group OD value) / (Control group OD value - Blank group OD value)

[0099] like Figure 13 As shown, the cell death rate increased in the biomimetic liposome treatment group, and the toxicity was in the following order: GSDMD-N mRNA@PEL > GSDMD-N mRNA@EL > GSDMD-N mRNA@LNP > Flu mRNA@LNP.

[0100] It is evident that GSDMD-N mRNA@PEL can significantly inhibit tumor growth and effectively kill oral squamous cell carcinoma cells SCC7.

[0101] Example 5

[0102] This embodiment provides the detection of pyroptosis in tumor cells induced by nanoparticles.

[0103] GSDMD-N mRNA@PEL successfully induced pyroptosis in tumor cells, and the evidence was fully validated at the molecular, subcellular, and cellular morphology levels.

[0104] (1) such as Figure 14 As shown in Figure A, the macroscopic process of pyroptosis was observed under a bright-field microscope: cell swelling, rounding, membrane bubbling, and eventual rupture. Figure 14 As shown in Figure B, SEM electron microscopy reveals characteristic "balloon-like" vesicle-like protrusions and pore structures on the cell membrane surface. Figure 14 As shown in C, TEM electron microscopy reveals the loss of cell membrane integrity and leakage of contents.

[0105] (2) such as Figure 15 Figure A shows that immunofluorescence staining reveals that the GSDMD-N protein is specifically located on the inner side of the cell membrane, forming a clear ring-shaped green signal, which is consistent with its classic function of oligomerizing after activation and forming pores on the cell membrane.

[0106] (3) Collect the treated cells, add RIPA protein lysis buffer, and extract total intracellular protein. Quantify the protein using quinoline carboxylic acid (BCA) standard, and separate equal volumes of protein using SDS-polyacrylamide gel electrophoresis. After electrophoresis, transfer the protein to a PVDF membrane. Block the PVDF membrane with blocking buffer, then incubate the PVDF membrane sequentially with primary antibodies (GSDMD-N and β-tublin) and horseradish peroxidase-conjugated secondary antibodies. Finally, soak the PVDF membrane in ECL luminescent substrate solution and photograph it using a gel imaging system. Figure 15 As shown in Figure B, Western blotting confirmed that GSDMD-N protein was specifically highly expressed in cells treated with GSDMD-N mRNA@LNP, GSDMD-N mRNA@EL, and GSDMD-N mRNA@PEL. The expression level of GSDMD-N protein was the highest in cells treated with GSDMD-N mRNA@PEL, while no band was observed in the PBS group and the Flu mRNA@LNP control group, indicating that the delivery system successfully translated the functional protein.

[0107] In summary, this fully demonstrates that GSDMD-N mRNA@PEL can efficiently and precisely trigger pyroptosis in tumor cells.

[0108] Example 6

[0109] This embodiment provides the detection of immunogenic death of tumor cells induced by nanoparticles.

[0110] To verify the immunogenicity of tumor cell death induced by GSDMD-N mRNA@PEL, the expression levels of HMGB1 and CRT in cells were detected. Healthy SCC7 cells were used at a concentration of 1×10⁻⁶ cells. 5Cells were seeded at a density of [number] cells / well in 6-well plates and incubated overnight at 37°C to allow cell adhesion. Nanoparticles were added to each group, with three replicates per group, and the plates were incubated at 37°C in the dark for 48 hours. Cells were collected, washed three times with PBS, and fixed with 4% PFA at room temperature for 15 minutes. HMGB1 assay: Cells were infiltrated with 0.1% Triton X-100 at room temperature for 15 minutes, then incubated with the Alexa Fluor® 488 Rabbit monoclonal antibody HMGB1 at 4°C for 12 hours. CRT assay: Cells were incubated with the Alexa Fluor® 488 Rabbit monoclonal antibody CRT at 4°C for 12 hours. Flow cytometry analysis was performed under the following conditions: λex = 488 nm, λem = 525 ± 30 nm.

[0111] The levels of CRT and HMGB1 in the cells treated with the above method were detected using laser confocal microscopy.

[0112] The culture medium after the cells were treated with the above drugs was collected. The ATP release content in the cell culture medium was detected using an ATP kit, the LDH release content in the cell culture medium was detected using an LDH kit, and the IL-1β content in the cell culture medium was detected using an ELISA kit.

[0113] like Figure 16 As shown in Figure AB, flow cytometry revealed that HMGB1 expression decreased and CRT expression increased in cells after treatment with GSDMD-N mRNA@PEL.

[0114] like Figure 17 As shown in AB, laser confocal microscopy further revealed that HMGB1 expression decreased and CRT content increased in cells after GSDMD-N mRNA@PEL treatment.

[0115] like Figure 18 As shown in AB, the release of ATP and LDH is increased. Figure 18 As shown in Figure C, the IL-1β content in the cell culture medium increased. These results indicate that GSDMD-N mRNA@PEL induces immunogenic cell death.

[0116] Example 7

[0117] This embodiment provides the detection of the maturation of bone marrow-derived dendritic cells (BMDCs) induced by in vitro nanoparticles.

[0118] (1) Bone marrow cells were extracted from the femur and tibia of C57BL / 6 mice and cultured in GM-CSF-containing medium for 7 days to induce immature BMDCs. SCC7 cells were treated with different groups of nanoparticles for 48 h and co-incubated with the above BMDCs for 48 h. Cells were collected. Cells were washed 1-2 times with pre-cooled flow cytometry staining buffer. Cells were resuspended in staining buffer, and fluorescent antibodies against CD11c, CD80, and CD86 molecules were added for surface staining. Cells were incubated at 4°C in the dark for 30 min. Cells were washed twice with staining buffer to remove unbound antibodies. Finally, cells were resuspended with an appropriate amount of buffer and sieved for flow cytometry. Data were obtained using flow cytometry. Viable cells were first delineated using FSC-A / SSC-A, and then dendritic cell populations were further delineated by CD11c positivity. The mean fluorescence intensity (MFI) of CD80 and CD86 molecules in the target cell population was analyzed and compared with the negative control group to calculate the upregulation factor.

[0119] (2) Mature BMDCs secrete cytokines. After collecting the cell supernatant, the expression levels of IL-6, IL-12p70, IFN-γ and TNF-α were detected using an ELISA kit to corroborate the flow cytometry results from a functional perspective.

[0120] like Figure 19 As shown, flow cytometry analysis revealed that, compared with the PBS group and the Flu mRNA@LNP group, the proportion of highly expressed co-stimulatory molecules CD80 and CD86 in the CD11c+ cell population of BMDCs co-cultured with pyroptosis tumor cells was significantly increased, with GSDMD-N mRNA@PEL inducing the highest proportion of BMDC maturation.

[0121] like Figure 20 As shown, ELISA detection revealed that the levels of key activation-related cytokines IL-12p70, TNF-α, IFN-γ, and IL-6 secreted by BMDCs in the co-culture supernatant were also significantly upregulated.

[0122] The above results demonstrate that GSDMD-N mRNA@PEL can effectively activate BMDCs by inducing pyroptosis in tumor cells, enabling them to acquire a mature phenotype and function, which provides the necessary conditions for subsequent activation of anti-tumor T cell immune responses in vivo.

[0123] Example 8

[0124] This embodiment provides an in vivo evaluation of the antitumor effects of the nanoparticles. (G1: PBS group, G2: Flu mRNA@LNP group, G3: GSDMD-N mRNA@LNP group, G4: @GSDMD-N mRNA@EL group, G5: GSDMD-N mRNA@PEL group).

[0125] A CDX model of OSCC was established in tumor-bearing mice to evaluate the antitumor effect of GSDMD-N mRNA@PEL. C57BL / 6 mice, 4-6 weeks old, were housed in an SPF-grade barrier system. Feed and water were sterilized and provided to the animals freely in the animal facility. A CDX model was established in C57BL / 6 mice when the tumor volume reached approximately 150 mm. 3 Mice were randomly divided into four groups: PBS group, FlumRNA@LNP group, GSDMD-N mRNA@LNP group, GSDMD-N mRNA@EL group, and GSDMD-N mRNA@PEL group. Each group consisted of four mice. Administered the drug 1 mg / kg every three days via a tail vein injection (150 μL per mouse). Tumor volume and body weight were recorded every other day. Mice were sacrificed after 21 days. Tumor tissue and major organs were collected for hematoxylin-eosin staining (HE) and immunohistochemistry (IHC).

[0126] Fresh mouse whole blood was collected and prepared into a 4% (v / v) erythrocyte suspension using PBS. 100 μL of the erythrocyte suspension was added to each 1.5 mL centrifuge tube; an equal volume of nanoparticle solution was added to each experimental group, an equal volume of PBS was added to the negative control group, and an equal volume of ddH2O was added to the positive control group. The final volume of each system was 200 μL. All sample tubes were gently shaken to mix and incubated in a 37°C water bath for 6 h. After incubation, the tubes were centrifuged at 3000 rpm for 10 min, and 200 μL of the supernatant was added to a 96-well plate. The absorbance at 540 nm was measured using a microplate reader.

[0127] like Figure 21 As shown in AC, there was no significant difference in tumor size between the PBS group and the Flu mRNA@LNP group, while the GSDMD-NmRNA@EL and GSDMD-N mRNA@PEL groups showed significant inhibition of tumor growth compared to other groups, and no signs of tumor recurrence were observed during the 21-day monitoring period. Figure 21 As shown in D, the weight of mice in all groups did not change significantly during the treatment process, and there was no sharp decrease or increase in weight in a short period of time.

[0128] like Figure 22As shown, HE staining in the PBS and Flu mRNA@LNP groups revealed that tumor cells maintained a dense and active growth state, while the tumor tissues in the GSDMD-N mRNA@LNP, GSDMD-N mRNA@EL, and GSDMD-N mRNA@PEL groups exhibited increased intercellular spaces, reduced cell volume, deeply stained cytoplasm, and nuclear fragmentation. The Ki67 proliferation index reflected the proliferative status of tumor tissue. In the PBS and Flu mRNA@LNP control groups, Ki67-positive cell nuclei showed a high-density, diffuse brown distribution, indicating that tumor cells were in a highly proliferative state. In contrast, the number of Ki67-positive cells was significantly reduced in the GSDMD-N mRNA@LNP, GSDMD-N mRNA@EL, and GSDMD-N mRNA@PEL treatment groups, and they were mostly scattered or focally distributed, suggesting that the proliferative activity of tumor cells was effectively inhibited.

[0129] like Figure 23 As shown, no obvious pathological changes were found in the major organs of mice in each treatment group, indicating that the drug dosage used was reasonable and the various treatment methods had high biosafety.

[0130] like Figure 24 As shown, all blood biochemical indicators of mice in each treatment group were normal, and the nanomaterials did not cause liver and kidney toxicity side effects.

[0131] like Figure 25 As shown, the supernatant of all nanomaterial-treated groups was clear and transparent, similar to the negative control group, with no obvious red color. The hemolysis rates of GSDMD-N mRNA@EL and GSDMD-N mRNA@PEL were both less than 5%, which meets the requirement of less than 5% in the national pharmaceutical industry standard "YY / T 1532-2017 Hemolysis Test of Nanomaterials for Biological Evaluation of Medical Devices", indicating that they have good in vitro biological safety.

[0132] Example 9

[0133] This embodiment provides an evaluation of the antitumor immune-enhancing effect of nanoparticles. (G1: PBS group, G2: Flu mRNA@LNP group, G3: GSDMD-N mRNA@LNP group, G4: @GSDMD-N mRNA@EL group, G5: GSDMD-N mRNA@PEL group).

[0134] To investigate the role of GSDMD-N mRNA@PEL in the tumor immune microenvironment, tumors from each treatment group were collected, digested into single cells, and subjected to staining for immune-related indicators, followed by flow cytometry analysis. Simultaneously, the levels of TNF-α, IFN-γ, and IL-6 in tumor tissues and animal serum from each treatment group were detected by ELISA after treatment.

[0135] The collection method is as follows: Tumor tissue was immersed in a six-well plate containing PBS, and the connective tissue surrounding the tumor was removed. The tissue was then washed twice with PBS and placed in the six-well plate. The tumor tissue was then cut into pieces approximately 1 mm in size using scissors. 3 Small tissue fragments were added to six-well plates in serum-free high-glucose medium (DMEM) containing collagenase IV (200 U / mL) and D Nase I (40 U / mL), and then incubated in a 37°C water bath for 1 h for digestion. Cells with 100 μm pores were placed above 50 mL centrifuge tubes, and the digestion solution containing the tumor tissue fragments was slowly added into a cell strainer. Simultaneously, DMEM serum-free medium containing 2 mM EDTA was added dropwise to terminate the digestion. If large tissue fragments were still visible after filtration, the cells were filtered again to obtain a cell suspension. The suspension was centrifuged at 2000 rpm for 5 min, the supernatant was discarded, and the resulting pellet was the cells extracted from the tumor tissue. The cell pellet was resuspended in PBS buffer for subsequent antibody fluorescence staining.

[0136] The staining methods are as follows: In the immunocellular analysis, cells were first co-incubated with anti-CD16 / 32 antibody (4°C, 30 min) to block the non-specific binding of Fc receptors. For the analysis of mature dendritic cells, the cell suspension was further stained with anti-CD45-FITC, anti-CD11c-PE-cy7, anti-MHC II-APC / Fire™750, anti-CD80-PE, and anti-CD86-APC under light-protected conditions (4°C, 30 min). For the analysis of tumor-infiltrating lymphocytes, anti-CD45-FITC, anti-CD3-APC-cy7, anti-CD4-PE-cy7, and anti-CD8-APC were stained under the same conditions (light-protected, 4°C, 30 min). After staining, cells were centrifuged at 350 g for 5 min at 4°C with FACS buffer (1×PBS containing 3% FBS), washed once, and finally analyzed by flow cytometry using 7-AAD-Percp-Cy5.5 reagent.

[0137] ELISA assay was performed as follows: Blood was collected from the heart of tumor-bearing mice, incubated at room temperature for 30 min, and centrifuged at 2000 g for 15 min at 4°C. The supernatant was collected, aliquoted, and stored at -80°C. Tumor tissue was weighed and washed with pre-chilled PBS. PBS containing protease inhibitors was added at a volume ratio of 1:9, and homogenized on ice. The homogenate was centrifuged at 10000 g for 15 min at 4°C. The supernatant was collected, aliquoted, and stored at -80°C. A mouse-specific TNF-α, IFN-γ, and IL-6 ELISA kit was used. Standards were serially diluted. Standards and samples (serum 1:2 dilution; tissue homogenate diluted according to pre-experimental dilutions) were added to pre-coated 96-well plates, 100 μL per well, with 3 replicates. The plates were incubated at room temperature for 2 h. The liquid was discarded, and the plates were washed 4 times with washing buffer and patted dry. 100 μL of biotinylated detection antibody was added to each well, and the plates were incubated at room temperature for 1–2 h. The plates were washed 4 times. Add 100 μL of streptavidin-HRP to each well and incubate at room temperature in the dark for 30 min. Wash the plate 4 times. Add 100 μL of TMB substrate solution to each well and incubate at room temperature in the dark for 15-30 min. Add 50-100 μL of stop solution to each well. Immediately measure the absorbance at 450 nm using a microplate reader, with 570 nm or 540 nm as the reference wavelength.

[0138] like Figure 26 As shown in AD, in flow cytometry analysis, dendritic cells in the GSDMD-N mRNA@PEL treatment group exhibited a significantly activated state compared to the PBS group and the Flu mRNA@LNP control group. This was manifested in a significant upregulation of the expression levels of MHC class II molecules and co-stimulatory molecules (CD80, CD86) on the dendritic cell surface, suggesting an effective enhancement of their antigen-presenting capacity. This mature dendritic cell phenotype can more effectively initiate and regulate adaptive immune responses. Further analysis of immune cell subsets showed that CD4+... + T cells and CD8 + The proportion of infiltrating T cells increased significantly. This indicates that dendritic cell activation induced by GSDMD-NmRNA@PEL successfully promoted the recruitment, expansion, and functional differentiation of antigen-specific T cells, thereby enhancing anti-tumor immune surveillance and killing capabilities.

[0139] like Figure 27 As shown, GSDMD-N mRNA@PEL promotes serum ( Figure 27 A) and tumor tissue ( Figure 27 B) promotes the secretion of immune factors TNF-α, IFN-γ and IL-6, thereby improving the tumor immune microenvironment.

[0140] Example 10

[0141] To further investigate the effect of GSDMD-N mRNA@PEL on gene expression patterns, single-cell RNA-seq was performed on mouse tumors from the Flu mRNA@LNP control group and the GSDMD-N mRNA@PEL treatment group.

[0142] Transcriptome analysis of six samples was performed using high-throughput sequencing technology, yielding a total of 41.56 Gb of clean data. Each sample's clean data reached 5.60 Gb, with Q30 base percentages of 95.4% or higher. The clean reads of each sample were aligned with a specified reference genome, achieving an alignment efficiency exceeding 94.5%. Based on the alignment results, alternative splicing prediction analysis, gene structure optimization analysis, and novel gene discovery were conducted, identifying 2049 new genes, of which 466 received functional annotation. In this embodiment, Fold Change ≥ 2 and FDR < 0.01 were used as the differentially expressed gene screening criteria. In each comparison group, differentially expressed gene lists, functional enrichment analysis of differentially expressed genes, and GSEA analysis results were obtained. The experimental procedure included sample testing, library construction and quality control, and sequencing.

[0143] like Figure 28 As shown in Figure A, after treatment with GSDMD-N mRNA@PEL, 993 genes were upregulated and 1266 genes were downregulated in tumor tissue.

[0144] like Figure 28 B and Figure 29 As shown, KEGG and GO enrichment analyses revealed that GSDMD-N mRNA@PEL treatment activated multiple immune response pathways and cell death pathways.

[0145] like Figure 30 As shown, GSEA enrichment analysis revealed that GSDMD-N mRNA@PEL treatment activated pathways related to inducing tumor immune responses, antigen presentation pathways, and inflammatory factor release pathways.

[0146] In summary, the nanovesicles of this invention possess the triple advantages of molecular targeting and blocking of PD1 / PD-L1, excellent delivery characteristics of exosomes, and direct induction of tumor cell pyroptosis. They can precisely target and deliver GSDMD-N mRNA to OSCC cells, effectively inducing tumor cell pyroptosis, releasing a large number of inflammatory factors and tumor antigens, thereby strongly activating dendritic cells and recruiting T lymphocytes, ultimately reversing the immunosuppressive microenvironment of OSCC. This provides a novel and highly efficient pyroptosis-immune synergistic therapeutic strategy for conquering solid tumors represented by OSCC.

[0147] This invention utilizes a PD1-engineered biomimetic lipid nanodelivery system to directly target and deliver mRNA encoding the GSDMD-N protein into tumor cells, bypassing the limitations of endogenous signaling pathways in tumor cells. Within hours of delivery, the GSDMD-N mRNA is translated into the functional GSDMD-N protein, initiating pyroptosis much faster than upregulating endogenous gene expression through small molecule drugs or gene editing, releasing a strong "immune alarm signal," thereby achieving highly efficient and specific killing of tumors.

[0148] While the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and all such changes and modifications will fall within the scope of protection of the present invention.

Claims

1. An engineered biomimetic nucleic acid nanovesicle, characterized in that, The drug was prepared by encapsulating cationic lipid nucleic acid drugs on exosomes derived from engineered macrophages; wherein the exosomes derived from engineered macrophages are exosomes derived from macrophages that highly express PD1 as shown in SEQ.ID.NO.1; the lipid nucleic acid drug was prepared by loading cationic liposomes with GSDMD-N mRNA as shown in SEQ.ID.NO.

2.

2. A method for preparing an engineered biomimetic nucleic acid nanovesicle formulation, characterized in that, Includes the following steps: (1) Preparation of PD1-EVs: Lentiviral stably transfected macrophages were constructed to obtain macrophages with high PD1 expression. After centrifugation, exosomes PD1-EVs derived from macrophages with high PD1 expression as shown in SEQ.ID.NO.1 were obtained. (2) Preparation of GSDMD-N mRNA: GSDMD-N mRNA based on SEQ.ID.NO.2 was prepared by plasmid template preparation, in vitro transcription and modification, and purification and quality control of mRNA. (3) Preparation of GSDMD-N mRNA@PEL: An organic phase was prepared using D-Lin-MC3-DMA, DOPE, Cholesterol, and PEG2000-DMG. An aqueous phase was prepared by dissolving GSDMD-N mRNA in a buffer solution. The organic phase was then added dropwise to the aqueous phase using an organic phase injection method to assemble lipid nanoparticles GSDMD-N mRNA@LNP. GSDMD-N mRNA@LNP was then mixed with PD1-EVs and co-extruded sequentially through filter membranes of different pore sizes using an extruder to obtain the biomimetic nucleic acid nanosystem GSDMD-N mRNA@PEL.

3. The method according to claim 2, characterized in that, The lentivirus mentioned in step (1) is selected from HBLV-Pdcd1-3xflag-ZsGreen-PURO.

4. The method according to claim 3, characterized in that, The organic phase described in step (3) is prepared with a molar ratio of D-Lin-MC3-DMA, DOPE, Cholesterol, and PEG2000-DMG of (48-55): (8-12): (35-40): (0.5-2).

5. The method according to claim 4, characterized in that, In step (3), the molar ratio of the positive charge of D-Lin-MC3-DMA to the negative charge of GSDMD-N mRNA is 6:

1.

6. The method according to claim 5, characterized in that, In step (3), the volume ratio of the aqueous phase to the organic phase is 3:

1.

7. The method according to claim 6, characterized in that, In step (3), the mass ratio of GSDMD-N mRNA@LNP to PD1-EVs is (1-5):

1.

8. The method according to claim 7, characterized in that, The filter membranes with different pore sizes mentioned in step (3) are 800 nm, 400 nm and 200 nm filter membranes, respectively.

9. The application of the engineered biomimetic nucleic acid nanovesicles according to claim 1 in the preparation of oral squamous cell carcinoma diagnostic kits and therapeutic drugs.

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