Targeting tumor-associated neutrophil biomimetic mRNA (messenger ribonucleic acid) nano vaccine as well as preparation method and application thereof

Abstract

The invention discloses a biomimetic mRNA (messenger ribonucleic acid) nano vaccine for targeting tumor-associated neutrophil as well as a preparation method and application of the biomimetic mRNA nano vaccine. The nano vaccine adopts a core-shell structure, wherein an inner core is a lipid nano particle loaded with mRNA (messenger ribonucleic acid) of a coding albumin-IL-36 gamma fusion protein; the shell is a tumor cell membrane of which the surface is over-expressed with a CD300LD protein extracellular domain. The vaccine can specifically target neutrophile granulocytes in a tumor microenvironment and reprogram the neutrophile granulocytes into an anti-tumor N1 phenotype, so that tumor growth is effectively inhibited, and the vaccine can cooperate with an immune checkpoint inhibitor to enhance the curative effect. The traditional Chinese medicine composition can significantly inhibit tumor growth and metastasis, improves the survival rate of tumor-bearing mice, has a synergistic effect with an immune checkpoint inhibitor, and is good in safety. The nano vaccine platform disclosed by the invention has the characteristic of modularization, and provides a new strategy for developing personalized immunotherapy aiming at cold tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a biomimetic mRNA nanovaccine targeting tumor-associated neutrophils, its preparation method, and its application. Background Technology

[0002] Tumor immunotherapy, particularly checkpoint inhibitor therapy targeting programmed cell death protein 1 (PD-1) and its ligand (PD-L1), has made significant progress in the treatment of various malignant tumors. However, this therapy has limited efficacy against so-called "cold tumors" such as hepatocellular carcinoma (HCC). These tumors typically exhibit a highly immunosuppressive tumor microenvironment (TME) and insufficient infiltration of effector T cells, making it difficult for the immune system to be effectively activated and attack tumor cells.

[0003] In recent years, the role of innate immune cells in anti-tumor immunity has received increasing attention. Neutrophils, as the most abundant type of leukocyte in circulation, are abundant in the tumor microenvironment (TME) and exhibit high plasticity. Studies have shown that tumor-associated neutrophils (TANs) can be divided into a pro-tumor N2 phenotype and an anti-tumor N1 phenotype. N1 phenotype neutrophils possess strong anti-tumor potential, capable of directly killing tumor cells by releasing reactive oxygen species (ROS), myeloperoxidase (MPO), neutrophil extracellular traps (NETs), and secreting pro-inflammatory cytokines, and further recruiting and activating adaptive immune cells, thus potentially reversing the "cold tumor" state. Therefore, specifically reprogramming N2-type TANs in the TME to N1-type has become a highly promising new therapeutic strategy. However, currently, there is a lack of effective means to precisely and efficiently target neutrophils within the TME and continuously regulate their phenotype, which severely limits the development of neutrophil-centered therapeutic strategies.

[0004] At the technology platform level, messenger RNA (mRNA) delivery technology based on lipid nanoparticles (LNPs) (such as its successful application in COVID-19 vaccines) provides new tools for tumor immunotherapy. Delivering mRNA encoding therapeutic proteins (such as cytokines) via LNPs allows for in situ, transient expression of target proteins in vivo, overcoming the problems of short half-life, high systemic toxicity, and high production costs associated with recombinant proteins. For example, interleukin-36γ (IL-36γ) is a potent pro-inflammatory cytokine that strongly activates myeloid immune cells, but systemic administration of it in recombinant protein form carries serious toxic risks. Local expression of IL-36γ using mRNA-LNPs offers the possibility of safely and effectively utilizing its immune-activating function.

[0005] Meanwhile, biomimetic nanotechnology, especially cell membrane coating technology, provides insights into improving the biocompatibility and targeting of nanocarriers. For example, coating nanoparticles with homologous tumor cell membranes can endow them with long-lasting circulation, immune escape, and homologous targeting capabilities to primary tumors and metastases. However, most existing biomimetic coating strategies rely on the inherent, but often insufficiently specific, targeting molecules of the tumor cell membrane itself, making it difficult to achieve efficient and precise recognition and binding of nanocarriers to a specific subset of immune cells (such as neutrophils) within the tumor microenvironment (TME).

[0006] In summary, existing technologies suffer from the following key shortcomings: firstly, there is a lack of efficient delivery systems capable of specifically recognizing and targeting neutrophils within the tumor microenvironment (TME); secondly, there is a lack of activation strategies that can continuously release polarization signals (such as IL-36γ) within neutrophils, thereby stably polarizing them into the N1 anti-tumor phenotype. These two core bottlenecks currently make it difficult to develop effective tumor nanovaccines that target neutrophil reprogramming.

[0007] Therefore, there is an urgent need in this field to develop a novel nanovaccine platform that integrates both specific targeting and efficient activation functions to achieve precise reprogramming of neutrophils within the tumor microenvironment (TME), thereby stimulating a strong innate and adaptive anti-tumor immune response and providing a new solution to overcome the resistance of "cold tumors" to existing immunotherapies. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a biomimetic mRNA nanovaccine targeting tumor-associated neutrophils, its preparation method, and its application. This vaccine can specifically target neutrophils in the tumor-associated neutrophil body (TME) and continuously deliver activation signals, polarizing them into an anti-tumor phenotype, thereby reshaping the immunosuppressive TME, enhancing the efficacy of immunotherapy, and is suitable for combined use with multiple therapies such as checkpoint inhibitors.

[0009] This invention is achieved through the following technical solution:

[0010] A biomimetic mRNA nanovaccine targeting tumor-associated neutrophils, wherein the nanovaccine is a nanoparticle with a core-shell structure, comprising a core and a shell; the core is a lipid nanoparticle loaded with mRNA encoding an albumin-IL-36γ fusion protein, and the shell is a tumor cell membrane coating the surface of the lipid nanoparticle, wherein the surface of the tumor cell membrane overexpresses the extracellular domain of the CD300LD protein.

[0011] Preferably, the nucleotide sequence of the mRNA encoding the albumin-IL-36γ fusion protein is shown in SEQ ID NO.3.

[0012] The above-mentioned method for preparing biomimetic mRNA nanovaccines includes the following steps:

[0013] Step 1) Prepare lipid nanoparticles loaded with mRNA encoding albumin-IL-36γ fusion protein;

[0014] Step 2) Isolate the cell membrane from tumor cells that stably overexpress the extracellular domain of the CD300LD protein;

[0015] Step 3) Mix the cell membrane obtained in Step 2) with the lipid nanoparticles obtained in Step 1), and use an extrusion method to coat the surface of the lipid nanoparticles with the cell membrane to obtain the final product.

[0016] Preferably, the tumor cells stably overexpressing the extracellular domain of the CD300LD protein in step 2) are obtained by constructing a lentiviral vector carrying the coding sequence of the extracellular domain of the CD300LD protein, transducing the target tumor cells, and selecting stable expression cell lines.

[0017] Preferably, in step 3), the mass ratio of the cell membrane to the lipid nanoparticles is 1:100.

[0018] A pharmaceutical composition characterized by comprising the above-described biomimetic mRNA nanovaccine and a pharmaceutically acceptable carrier.

[0019] The use of the above-mentioned biomimetic mRNA nanovaccines or the above-mentioned pharmaceutical compositions in the preparation of drugs for treating solid tumors.

[0020] Preferably, the solid tumor is liver cancer or melanoma.

[0021] Preferably, the drug is administered via intravenous injection.

[0022] Preferably, the drug is used in combination with one or more other cancer treatment methods selected from radiotherapy, chemotherapy, targeted therapy, or immunotherapy.

[0023] The beneficial effects of this invention are as follows:

[0024] (1) Precise targeting and low off-target risk: This invention is the first to use CD300LD protein as a specific target, which realizes efficient and precise delivery of nano-vaccines to neutrophils in the tumor microenvironment, significantly improving the targeting of treatment.

[0025] (2) Significant anti-tumor efficacy: In various mouse tumor models (hepatocellular carcinoma Hepa1-6, Hepa53.4 with poor immunogenicity, subcutaneous melanoma B16F10, and lung metastasis model), the nanovaccine of this invention can significantly inhibit tumor growth and metastasis and improve the survival rate to 85% when used as a single drug; it can also reprogram tumor-associated neutrophils to the anti-tumor N1 phenotype, activate the cGAS-STING pathway, and generate a systemic immune response. When used in combination with PD-L1 antibody, it can synergistically overcome drug resistance in "cold tumors".

[0026] (3) Good safety: In vitro and in vivo experiments show that the nano-vaccine of the present invention has no significant hemolytic activity or cytotoxicity, no adverse effects on major organs, and high biocompatibility.

[0027] (4) The platform is highly versatile: The nano-vaccine platform of the present invention has a modular design. By changing the outer shell membrane (target) and the core mRNA (load), it can be adapted to a variety of mRNA-based tumor immunotherapies, providing a universal tool for developing personalized vaccines. Attached Figure Description

[0028] Figure 1 Here is an electron microscope image of the CMNPs nanovaccine in Example 1;

[0029] Figure 2 This is a fluorescence image showing the colocalization of tdTomato (red) and neutrophil Ly6G (green) in the Cre-CMNPs and Cre-LNP groups in Example 2.

[0030] Figure 3 The results of the long-acting efficacy verification of CMNPs in Example 3;

[0031] Figure 4 The effect of CMNPs on inhibiting the growth of in situ hepatocellular carcinoma in Example 4: A represents liver weight; B represents the liver weight ratio;

[0032] Figure 5 Tumor volume (A) and survival curve (B) of mice in each group after CMNPs treatment of B16F10 subcutaneous tumors in Example 5.

[0033] Figure 6 For the biosafety evaluation of the CMNPs nanovaccine in Example 6: A is the change in body weight; B is the coefficient of each organ (heart, liver, spleen, lung, kidney); C is the serum alanine aminotransferase (ALT) level; D is the serum aspartate aminotransferase (AST) level; E is the serum alkaline phosphatase (ALP) level; F is the serum blood urea nitrogen (BUN) level; G is the serum creatine kinase (CK) level; H is the serum creatinine (CREA) level; I is the HE-stained sections of each organ (heart, liver, spleen, lung, kidney). Detailed Implementation

[0034] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0035] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.

[0036] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0037] The experimental animals involved in the following examples are:

[0038] Male Ai14 reporter mice were purchased from Wuhan Shulaibao Biotechnology Co., Ltd. and housed in the SPF-grade barrier facility of the Experimental Animal Center of Nanjing Medical University.

[0039] C57BL / 6 mice were purchased from the Experimental Animal Center of Nanjing Medical University and housed in the SPF-grade barrier facility of the Experimental Animal Center of Nanjing Medical University.

[0040] Example 1: Preparation of CMNPs nanovaccines

[0041] 1. Experimental materials

[0042] Preparation of lipid nanoparticles (LNPs): Using the ethanol injection method, ionizable lipids (ALC-0315), phospholipid helper (DSPC), cholesterol, and PEGylated lipids (DMG-PEG2000) were mixed in the ethanol phase at a molar ratio of 50:10:38.5:1.5. This mixture was then mixed with an aqueous phase containing albumin-IL-36γ fusion protein mRNA (synthesized by GenScript) (concentration of 1 mg / mL, HPLC concentration >90%, dissolved in sodium citrate buffer and packaged in enzyme-free tubes; 5 μg of albumin-IL-36γ fusion protein mRNA was used each time for the synthesis of 5 μg of vaccine). Subsequently, using a syringe pump, the lipid ethanol phase and the mRNA aqueous phase were instantaneously mixed at a volume ratio of 1:3 in a Y-type or T-type mixing chamber at a constant total flow rate (10 mL / min). The mixing process was carried out at room temperature (20~25℃). The self-assembly of lipids was driven by the miscibility of ethanol and water and pH changes to form a primary suspension of LNPs encapsulating mRNA, i.e., LNPs loaded with mRNA.

[0043] CD300LD-ECD overexpression lentivirus (Hanheng Biotechnology), B16F10 and Hepa1-6 cell lines (ATCC) (using DMEM culture medium with 10% FBS), and cell membrane separation kit (Invent).

[0044] The amino acid sequence of the albumin-IL-36γ fusion protein is shown in SEQ ID NO.1 as follows:

[0045] MKWVTFLLLLFVSGSAFSRGVFRREAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDTLAGGSGGGGSGGGRETPDFGEVFDLDQQVWIFRNQALVTVPRSHRVTPVSVTILPCKYPESLEQDKGIAIYLGIQNPDKCLFCKEVNGHPTLLLKEEKILDLYHHPEPMKPFLFYHTRTGGTSTFESVAFPGHYIASSKTGNPIFLTSKKGEYYNINFNLDIKS。

[0046] The amino acid sequence of the extracellular domain (ECD) of the CD300LD protein is shown in SEQ ID NO.2 as follows:

[0047] QDSVTGPEEVSGQEQGSLTVQCRYSSYWKGYKKYWCRGVPQRSCDILVETDKSEQLVKKNRVSIRDNQRDFIFTVTMEDLRMSDAGIYWCGITKGGPDPMFKVNVNIDQAPKSSMMTTTATVLKSIQPSAENTGKEQVTQSKEVTQSRPHTRSLLSSIY.

[0048] The nucleotide sequence encoding the albumin-IL-36γ fusion protein mRNA is shown in SEQ ID NO.3, as follows:

[0049]

[0050] 2. Experimental Procedure

[0051] (1) LNPs loaded with albumin-IL-36γ fusion protein mRNA (IL-36γ-LNP) were prepared by ethanol injection method, as follows:

[0052] First, ionizable lipids (ALC-0315), phospholipids assisting phospholipids (DSPC), cholesterol, and PEGylated lipids (DMG-PEG2000) were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 to form the lipid-ethanol phase. Then, the mRNA encoding the target protein (albumin-IL-36γ fusion protein) was dissolved in citrate buffer (or 10 mM citrate buffer containing 1% EDTA) at pH 4.0 to form the mRNA-aqueous phase.

[0053] Using a syringe pump or microfluidic device, the lipid ethanol phase and the mRNA aqueous phase were instantaneously mixed at a volume ratio of 1:3 in a Y-type or T-type mixing chamber at a constant total flow rate (10 mL / min). The mixing process was carried out at room temperature (20–25 °C), and the self-assembly of lipids was driven by the miscibility of ethanol and water and pH changes to form a primary suspension of LNPs encapsulating mRNA.

[0054] Subsequently, the initial LNP suspension was transferred to a dialysis bag with a molecular weight cutoff of 100 kDa and dialyzed against 1×PBS (pH 7.4) at 4°C for 24–48 h to remove ethanol, replace the buffer, and stabilize the particle structure. After dialysis, the LNP suspension was concentrated using an ultrafiltration centrifuge tube (100 kDa molecular weight cutoff) and then filtered through a 0.22 μm sterile filter membrane for sterilization. The obtained LNP was stored at 4°C for a short period (1–2 weeks) or at -80°C for a long period, avoiding repeated freeze-thaw cycles.

[0055] The encapsulation efficiency of IL-36γ-LNP was quantified by RiboGreen fluorescence method, and the result was >90%. The hydration size, polydispersity index (PDI), and zeta potential of the particles were determined by dynamic light scattering, and typical values ​​are as follows:

[0056] Particle size: 175.6±2.6 nm; PDI<0.2; Zeta potential: 1.81±0.5 mV.

[0057] (2) Hepa1-6 and B16F10 cells were infected with CD300LD-ECD lentivirus (MOI=20, infection for 24 h), as follows:

[0058] First, seed Hepa1-6 cells or B16F10 cells into 6-well plates, with approximately 3 × 10⁶ cells per well. 5Cells were cultured overnight at a concentration of 50% ( / mL). Lentiviral MOI=20 and Polybrene concentration of 1 μg / mL were added, followed by transfection according to the 24-hour inoculation procedure specified in the Hanheng Biotechnology instructions. After transfection, GFP was observed under a fluorescence microscope to confirm an infection efficiency >90%, and Western blotting confirmed high expression of CD300LD protein in transfected cells.

[0059] (3) Collect overexpressing cells and use the membrane protein separation kit (Invent Biotechnology Minute™ Plasma Membrane / Protein Separation and Cell Components Kit) to extract their cell membrane components according to the instructions. Then, calculate the cell membrane concentration according to the BCA protein assay method for storage at -80℃.

[0060] (4) Cell membrane sonication and liposome extrusion coating

[0061] ① Cell membrane ultrasound pretreatment

[0062] The isolated tumor cell membrane suspension (protein concentration determined by the BCA method) was placed in pre-chilled 1.5 mL centrifuge tubes and sonicated under ice bath conditions. A probe-type sonicator (Sonics VCX 750) was used, with an output power of 30W, a cycle time of 2s on / 1s off, and a total processing time of 2 minutes. Throughout the sonication process, the sample was kept immersed in an ice-water mixture to avoid localized overheating that could lead to membrane protein denaturation or aggregation. The sonication aimed to further homogenize cell membrane fragments, reduce their size, and promote membrane flexibility, thereby enhancing their fusion efficiency with the lipid nanoparticle surface.

[0063] ②Mixing and extrusion coating of tumor cell membrane with LNP

[0064] The sonicated cell membrane suspension was mixed with mRNA-loaded IL-36γ-LNP (based on ALC-0315 lipid mass) at a mass ratio of 1:100 in pre-cooled 1×PBS (pH 7.4), with the total volume not exceeding 80% of the extruder's injection chamber volume. The mixture was transferred to the liposome extruder (Avanti® Mini-Extruder), fitted with a 200 nm polycarbonate membrane, and extruded at 4°C. Pre-extrusion was performed 2-3 times to initially homogenize the system, followed by 11 final extrusions at a slow, uniform pusher speed (approximately 1 mL / min). After each of the 1st, 3rd, 5th, 7th, 9th, and 11th extrusions, a small sample was taken and its hydrated particle size and polydispersity index (PDI) were measured using a dynamic light scattering analyzer. When the particle size change was less than 5 nm after 2-3 consecutive extrusions and the PDI ≤ 0.2, membrane coating was considered complete, and the particles achieved a uniform and stable core-shell structure. The resulting cell membrane-coated mRNA nanovaccines (CMNPs) were stored at 4°C for later use or used in subsequent experiments within 24 hours.

[0065] 3. Experimental Results

[0066] like Figure 1 As shown, transmission electron microscopy revealed that CMNPs possessed a clear core-shell structure. The particle size of CMNPs was 184.9 ± 3.9 nm; the zeta potential was -8.48 ± 0.5 mV; the mRNA encapsulation efficiency was 90.08% ± 0.75%; the cell membrane coverage efficiency was 89% ± 3.5%; and the PDI was 0.209 ± 0.02.

[0067] Example 2: Validation of CMNP targeting and efficacy (using the orthotopic Hepa1-6 liver cancer model as an example)

[0068] 1. Model Establishment

[0069] Male Ai14 mice weighing approximately 22 g and 6-7 weeks old were injected into the liver via orthotopic hepatic injection. The liver was first dissected using sterilized scissors and forceps, and then 1×10⁻⁶ insulin was injected into the center of the left lobe of the liver using an insulin syringe. 6 Mice were randomly divided into two groups using Hepa1-6 cells: a Cre-LNP group and a Cre-CMNPs group.

[0070] 2. Dosing regimen

[0071] On day 5 post-tumor inoculation, mice in both groups were injected intravenously with 5 μg of Cre-CMNPs and 5 μg of Cre-LNPs prepared in Example 1, respectively. The tail vein administration was single-dose, and the tracing model was considered successfully established when luminescence of tdTomato protein in the liver was observed via in vivo imaging on day 7 (48 h post-administration). Subsequently, mice were sacrificed on day 7, and liver tumors were harvested. Liver tissues from both groups were paraffin-embedded, sectioned, and stained with anti-tdTomato antibody (Arigo, ARG55724) to assess protein expression. Furthermore, liver tumor sections were stained with anti-Ly6G (clone 1A8, Bio X Cell) antibody at a dilution of 1:500 for intracellular tracking to assess the targeting of Cre-CMNPs to neutrophils.

[0072] 3. Experimental Results

[0073] like Figure 2 As shown, in the Ai14 reporter mouse model, after injection of CMNPs loaded with Cre-mRNA, immunofluorescence confirmed that compared with the Cre-LNP group, the tdTomato fluorescence signal (red fluorescence) in the liver tumor region of the Cre-CMNPs group was significantly higher than that of the Ly6G group. + Neutrophils (green fluorescence) showed more colocalization and stronger signal.

[0074] Example 3: Verification experiment on the extension of the in vivo half-life of IL-36γ by albumin fusion

[0075] This embodiment aims to demonstrate that, compared with the conventional recombinant IL-36γ protein, the albumin-IL-36γ fusion protein encoded by mRNA and delivered via CMNPs in this invention has a significantly prolonged duration of efficacy in vivo.

[0076] 1. Laboratory animals and grouping

[0077] Male C57BL / 6 mice aged 6-8 weeks were selected. The liver was surgically exposed, and 1×10⁻⁶ mg / L was injected orally into the left lobe of the liver. 6 An orthotopic liver cancer model was constructed using mouse Hepa1-6 liver cancer cells.

[0078] C57BL / 6 mice that underwent orthotopic Hepa1-6 tumor reconstruction were randomly divided into two groups (n=3), as follows:

[0079] Recombinant proteome: 10 μg of recombinant mouse IL-36γ protein was injected intraperitoneally.

[0080] CMNPs group: CMNPs nanovaccines prepared in Example 1 by tail vein injection of a 2 μg dose (calculated as IL-36γ).

[0081] 2. Sample collection and testing

[0082] (1) Blood was collected from the posterior orbital venous plexus of mice at 6, 9, 12, 24 and 48 h after administration. Serum was obtained after the blood was allowed to stand and centrifuged.

[0083] (2) Detection method

[0084] Using a commercially available mouse IL-36γ-ELISA kit (BY-EM223515, Nanjing Boyan Biotechnology), the concentration of IL-36γ in serum was measured at each time point, strictly following the instructions.

[0085] 3. Results and Analysis

[0086] like Figure 3 As shown, the serum IL-36γ concentration in the recombinant protein group decreased rapidly after administration, indicating that it was rapidly cleared and had a short half-life. In stark contrast, the serum IL-36γ concentration in the CMNPs group remained at a significantly higher level during the observation period, and the rate of decrease was slow, indicating that the albumin fusion strategy effectively prolonged the retention time of IL-36γ in the bloodstream, and its in vivo half-life was significantly prolonged compared to the recombinant protein (P < 0.001).

[0087] The experimental results of this embodiment show that by fusing IL-36γ with albumin and having its encoding mRNA delivered by CMNPs, the in vivo circulation time of IL-36γ can be significantly prolonged, solving the technical problem of short half-life of recombinant cytokines. This demonstrates that the present invention successfully solves the key technical problem of "short half-life and need for frequent high-dose administration" of recombinant cytokines in the prior art, laying a pharmacokinetic foundation for achieving sustained and long-lasting immune cell activation.

[0088] Example 4: Therapeutic evaluation of CMNPs nanovaccines in an orthotopic hepatocellular carcinoma model

[0089] This embodiment aims to quantitatively evaluate the inhibitory effect of CMNPs nanovaccine on the growth of hepatocellular carcinoma (HCC) using objective physiological indicators—liver weight / body weight ratio (liver weight ratio) and absolute liver weight—and directly demonstrate its therapeutic efficacy.

[0090] 1. Establishment of experimental animal and tumor models

[0091] Male C57BL / 6 mice aged 6-8 weeks were selected. The liver was surgically exposed, and 1×10⁻⁶ mg / L was injected orally into the left lobe of the liver. 6 An orthotopic liver cancer model was constructed using mouse Hepa1-6 liver cancer cells.

[0092] 2. Experimental grouping and dosing regimen

[0093] Five days after the tumors began to grow, the tumor-bearing mice were randomly divided into three groups (n=5 per group), as follows:

[0094] PBS control group: 250 μL of phosphate-buffered saline (PBS) was injected via the tail vein.

[0095] IL-36γ-LNP group: 5 μg of lipid nanoparticles without cell membrane coating and loaded only with albumin-IL-36γ fusion protein mRNA were injected via tail vein.

[0096] CMNPs treatment group: 5 μg of the CMNPs nanovaccine prepared in Example 1 was injected via tail vein.

[0097] All groups received the medication once on day 5 and once on day 8 after tumor inoculation.

[0098] 3. Sample collection and data processing

[0099] Mice were euthanized on day 9 after the first administration (i.e., day 14 after tumor inoculation). The entire liver of each mouse was completely removed and weighed. After being gently rinsed with pre-cooled PBS and blotted dry with filter paper, the liver weight was measured using a precision electronic balance. The final weight of each mouse was recorded.

[0100] The formula for calculating liver weight ratio is as follows:

[0101] Liver weight percentage (%) = (Total liver weight / Mouse body weight) × 100%

[0102] 4. Results and Analysis

[0103] (1) Absolute liver weight

[0104] like Figure 4 As shown in Figure A, the mean whole liver weight of mice in the CMNPs treatment group was significantly lower than that in the PBS control group and the IL-36γ-LNP group (P < 0.01). This indicates that CMNPs treatment can directly alleviate pathological liver weight gain caused by increased tumor burden.

[0105] (2) Liver weight ratio

[0106] like Figure 4 As shown in Figure B, the liver weight ratio in the CMNPs treatment group was significantly lower than that in the other two groups (P < 0.001). This indicator eliminates the interference of individual weight differences and more accurately reflects the tumor load relative to the body. Its significant decrease strongly demonstrates that CMNPs can effectively inhibit the growth of liver cancer in vivo.

[0107] Example 5: The therapeutic effect of CMNPs on "cold tumor" B16F10

[0108] 1. Experimental Procedure

[0109] Male C57BL / 6 mice aged 6-8 weeks and weighing approximately 22 g were selected, and each mouse was subcutaneously injected with 5 × 10⁵ g of the solution. 5 B16F10 cells. Divided into three groups of 5 cells each: control group, IL-36γ-LNP group, and CMNPs group.

[0110] The subcutaneous tumor model was treated with 5 μg of CMNPs prepared in Example 1 via tail vein on days 5 and 8 after inoculation.

[0111] 2. Experimental Results

[0112] like Figure 5 As shown, the CMNPs group increased the survival rate of mice to 45 days, while the control group only had a survival rate of 22 days, indicating that CMNPs can significantly inhibit the growth of subcutaneous tumors and prolong the survival of mice.

[0113] The experimental results of this embodiment show that the CMNPs nanovaccine is a novel strategy for tumor immunotherapy that is highly efficient, safe, and targeted, and has broad prospects for clinical application.

[0114] Example 6 Safety evaluation of CMNPs

[0115] 1. Experimental Procedure

[0116] An orthotopic liver cancer model was constructed using the method described in Example 4 and the cells were randomly divided into two groups of 5 cells each: a control group (PBS treatment) and a CMNPs group.

[0117] Peripheral blood serum was collected from mice receiving different treatment regimens, and blood urea nitrogen (BUN) and creatinine (CREA) were measured using aspartate aminotransferase (AST / GOT) test kits, alanine aminotransferase (ALT / GPT) test kits, alkaline phosphatase (ALP / AKP) assay kits, and gamma-glutamyl transferase (γ-GT / GGT) test kits, according to the manufacturer's instructions.

[0118] In the safety evaluation of mice, body weight changes were monitored at fixed time points to assess systemic toxicity. After sacrifice at the experimental endpoint, major organs such as the heart, liver, spleen, lungs, and kidneys were completely removed, weighed, and organ coefficients (organ wet weight / final body weight × 100%) were calculated to screen for target organ toxicity. Subsequently, the organs were fixed in formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). The morphological changes of the tissues were observed under a microscope to systematically evaluate the potential toxic effects of the nanovaccine on major organs.

[0119] 2. Experimental Results

[0120] like Figure 6As shown, there were no significant differences in serum biochemical analysis results, body weight results, and HE staining among all groups.

[0121] The experimental results of this embodiment show that the CMNPs nanovaccine exhibits good biocompatibility in tumor treatment.

[0122] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A biomimetic mRNA nanovaccine targeting tumor-associated neutrophils, characterized in that, The nanovaccine is a nanoparticle with a core-shell structure, including a core and a shell; the core is a lipid nanoparticle loaded with mRNA encoding albumin-IL-36γ fusion protein, and the shell is a tumor cell membrane covering the surface of the lipid nanoparticle, wherein the surface of the tumor cell membrane overexpresses the extracellular domain of CD300LD protein.

2. The biomimetic mRNA nanovaccine targeting tumor-associated neutrophils according to claim 1, characterized in that, The nucleotide sequence of the mRNA encoding the albumin-IL-36γ fusion protein is shown in SEQ ID NO.

3.

3. A method for preparing a biomimetic mRNA nanovaccine as described in claim 1 or 2, characterized in that, Includes the following steps: Step 1) Prepare lipid nanoparticles loaded with mRNA encoding albumin-IL-36γ fusion protein; Step 2) Isolate the cell membrane from tumor cells that stably overexpress the extracellular domain of the CD300LD protein; Step 3) Mix the cell membrane obtained in Step 2) with the lipid nanoparticles obtained in Step 1), and use an extrusion method to coat the surface of the lipid nanoparticles with the cell membrane to obtain the final product.

4. The preparation method according to claim 3, characterized in that, Step 2) The tumor cells that stably overexpress the extracellular domain of the CD300LD protein are obtained by constructing a lentiviral vector carrying the coding sequence of the extracellular domain of the CD300LD protein, transducing the target tumor cells, and selecting stable expression cell lines.

5. The preparation method according to claim 3, characterized in that, In step 3), the mass ratio of the cell membrane to the lipid nanoparticles is 1:

100.

6. A pharmaceutical composition, characterized in that, It comprises a biomimetic mRNA nanovaccine as described in claim 1 or 2, and a pharmaceutically acceptable carrier.

7. The use of the biomimetic mRNA nanovaccine of claim 1 or 2 or the pharmaceutical composition of claim 6 in the preparation of a medicament for treating solid tumors.

8. The application according to claim 7, characterized in that, The solid tumor is either liver cancer or melanoma.

9. The application according to claim 7, characterized in that, The drug is administered via intravenous injection.

10. The application according to claim 7, characterized in that, The drug is used in combination with one or more other cancer treatment methods selected from radiotherapy, chemotherapy, targeted therapy, or immunotherapy.

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