Engineered dendritic cell membrane-bionic nanoparticles for delivering mrna and methods of making and using the same

Abstract

This invention belongs to the field of biomedical technology, specifically relating to engineered dendritic cell membrane biomimetic nanoparticles for mRNA delivery, their preparation method, and applications. Addressing the current lack of efficient and safe lymphatic organ-targeting carriers for mRNA delivery, this invention provides an engineered dendritic cell membrane biomimetic nanoparticle, its preparation method, and applications, offering a novel delivery carrier for mRNA. The biomimetic nanoparticles of this invention are prepared from dendritic cell membranes modified with antigen proteins, using a mixture of ionizable lipids and cofactor phospholipids. The delivery carrier retains the homing ability of dendritic cells, enabling efficient delivery of mRNA to lymphatic organs. The antigen proteins overexpressed on the dendritic cell membrane of the delivery system achieve combined mRNA and protein immunization, thereby inducing a stronger immune response in vivo.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to engineered dendritic cell membrane biomimetic nanoparticles for delivering mRNA, their preparation methods, and applications. Background Technology

[0002] Vaccine efficacy depends on effective delivery to immune-associated organs. Adaptive immune responses are primarily initiated in lymph nodes, which are crucial habitats for adaptive immune-associated lymphoid cells (APCs) and contain a large number of antigen-presenting cells (APCs). Therefore, delivering antigens to lymphoid organs may increase APC uptake, thereby improving vaccine efficacy. In vaccine development, mRNA vaccines are at the forefront. Lipid nanoparticles (LNPs) are the most advanced mRNA delivery system in clinical use, and currently, almost all approved mRNA vaccines for clinical use utilize LNPs. However, LNPs present several major challenges, including cytotoxicity, poor biodistribution, systemic hepatic accumulation, and lack of target specificity. It has been reported that after intramuscular injection, up to 10-20% of the injected dose of LNP-mRNA eventually enters the liver. Furthermore, non-immune cells at the injection site, including epithelial cells, fibroblasts, and adipocytes, also absorb a significant portion of the injected LNP-mRNA but do not participate in antigen presentation occurring in the draining lymph nodes. This unfavorable distribution leads to liver damage and reduces the immunogenicity of mRNA vaccines. Therefore, for mRNA vaccines, targeted delivery and expression of mRNA-encoded antigens in lymphoid organs is an effective strategy to improve the efficacy of mRNA vaccines and reduce side effects.

[0003] In recent years, biomimetic nanoparticles have become an effective method to improve the in vivo performance of nanodelivery systems. As natural analogs of liposomes, the phospholipid bilayer structure of cell membranes can endow nanocarriers with the inherent functions of cell membranes. This innovative approach can retain the physicochemical properties of nanocarriers while utilizing the multifaceted functions of the membrane, resulting in nanocarriers with better biocompatibility and lower immunogenicity, and the ability to evade clearance by the immune system. Dendritic cells (DCs) are the main antigen-presenting cells (APCs), crucial for stimulating T cell immune responses through antigen processing and presentation to T cells, which is essential for vaccine efficacy. Furthermore, DCs exhibit excellent lymph node homing ability due to the CCR7 receptor on their membranes. Nanoparticles coated with DC cell membranes exhibit antigen-presenting function and good homing ability due to their tissue-specific antigens or receptors. Therefore, biomimetic carriers based on dendritic cell membranes may be an effective strategy to improve the efficacy of mRNA vaccines and reduce side effects. As more and more research explores nanotechnology to simulate / fabricate cell membranes, and as the demand for more specific cell membrane functions increases, more and more researchers are beginning to reconstruct the basic building blocks of the cell membrane, such as phospholipid layers, peptides, proteins, and polysaccharides, to endow the cell membrane with various biological functions. In view of this, the present invention is proposed. Summary of the Invention

[0004] The technical problem this invention aims to solve is to effectively deliver mRNA to secondary lymphoid organs, including lymph nodes and the spleen, while simultaneously achieving combined mRNA and protein immunization, thereby improving the efficacy of mRNA vaccines and reducing side effects. The technical solution of this invention to solve the above-mentioned technical problem is to provide an engineered dendritic cell-inspired biomimetic targeting nanoparticle.

[0005] This invention provides an engineered dendritic cell membrane biomimetic nanoparticle, comprising an engineered dendritic cell membrane, ionizable lipids, and helper phospholipids. The engineered dendritic cell membrane is extracted from engineered dendritic cells, and the membrane surface of the engineered cells highly expresses viral antigen proteins. The expression of the viral antigen proteins can be achieved by introducing mRNA.

[0006] Furthermore, the engineered dendritic cell membrane is a cell membrane extracted from engineered dendritic cells prepared through genetic engineering or ligand-coupled pretreatment methods.

[0007] Furthermore, the dendritic cells are selected from at least one of immortalized mouse bone marrow-derived dendritic cells (DC2.4 cells) and bone marrow-isolated and induced-differentiated dendritic cells (BMDC).

[0008] Further, the ionizable lipid is at least one of YK009, MC3, SM102 or ALC-0315.

[0009] Further, the auxiliary phospholipid is at least one of 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-bis(docosahexaenoyl-sn-glycerol-3-phosphocholine), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC).

[0010] Further, the engineered dendritic cell membrane, the mixture of ionizable lipids and helper phospholipids, and the mRNA have a volume:volume:mass ratio of 1–10:0.44–1:1, wherein the mass ratio of ionizable lipids to helper phospholipids in the mixture of ionizable lipids and helper phospholipids is 0.5–5:1. Preferably, the ratio of ionizable lipids to helper phospholipids is 0.5–2:1.

[0011] The aforementioned engineered dendritic cell biomimetic targeted nanoparticles can be prepared by at least one of the following methods: ultrasonication, extrusion, or microfluidics.

[0012] The present invention also provides a nucleic acid delivery system comprising the above-described engineered dendritic cell membrane biomimetic nanoparticles and nucleic acid, wherein the nucleic acid is any one of plasmid, mRNA, DNA vaccine, or RNA vaccine. Preferably, the RNA vaccine is a COVID-19 mRNA vaccine.

[0013] Furthermore, the nucleic acid is mRNA, and the ratio between the dendritic cell membrane and mRNA in the engineered dendritic cell biomimetic targeting nanoparticle is dendritic cell membrane:mRNA (volume:mass) = 1 to 10:1.

[0014] Applications of the above-described engineered dendritic cell membrane biomimetic nanoparticles and nucleic acid delivery system in any of the following:

[0015] 1) Application in the preparation of in vitro mRNA transfection products;

[0016] 2) Applications in the preparation of in vivo mRNA delivery products;

[0017] 3) Application in the preparation of mRNA vaccine delivery products. The delivery is for non-disease treatment purposes.

[0018] In the experimental protocol, the transfection target includes cells or mammals, preferably HEK293T and DC2.4 cells, etc.

[0019] The engineered dendritic cell-inspired biomimetic targeting nanoparticles provided by this invention can serve as carriers for mRNA delivery, effectively protecting the mRNA and achieving efficient mRNA expression both in vitro and in vivo. The dendritic cell membrane in the delivery carrier of this invention endows the nanoparticles with lymph node homing properties, enabling targeted delivery of mRNA to lymphatic organs, which can improve the efficacy of mRNA vaccines and reduce side effects. Furthermore, the antigen protein overexpressed on the cell membrane of the delivery system achieves combined mRNA and protein immunization, thereby inducing a stronger immune response in vivo. Attached Figure Description

[0020] Figure 1 This is a diagram showing the detection results of engineered dendritic cell membrane antigen protein expression in this invention;

[0021] Figure 2 This is a characterization diagram of the particle size, potential, and morphology of the engineered dendritic cell membrane of this invention;

[0022] Figure 3 Figure 1 shows the results of particle size, potential, morphological characterization and protein abundance identification of engineered dendritic cell biomimetic targeting nanoparticles RBD-DCMNPs-mRNA.

[0023] Figure 4Figure 1 shows the results of cellular expression detection of RBD-DCMNPs-eGFP mRNA and DCMNPs-eGFP mRNA;

[0024] Figure 5 The images show the in vivo and ex vivo imaging results of RBD-DCMNPs-Fluc mRNA and DCMNPs-Fluc mRNA after intramuscular or tail vein injection in mice.

[0025] Figure 6 Figure 1 shows the results of serum IgG antibody and neutralizing antibody detection in mice immunized with RBD-DCMNPs-mRNA and DCMNPs-mRNA COVID-19 vaccines. Detailed Implementation

[0026] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0028] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0029] The preparation of the cationic lipid material (YK009) in the following examples: The method described in Patent 202210034449.4, "A Cationic Lipid Compound, Compositions Containing the Same, and Uses," was used to prepare and synthesize 2-octyldecyl(decoxy)-4-oxobutyl(2-hydroxyethyl)amino)hexanoic acid ester (YK-009). Specifically, decyl(4-((2-hydroxyethyl)amino)butyrate) and 2-octyldecyl(6-bromohexanoic acid) were dissolved in acetonitrile. Potassium carbonate and potassium iodide were added to the above system, and the mixture was heated to 70°C and stirred for 20 hours. After cooling the reaction solution to room temperature, it was filtered. The filtrate was concentrated under vacuum to remove the solvent. The residue was purified by silica gel chromatography to obtain the target compound 2-octyldecyl-6-((4-(decoxy)-4-oxobutyl)(2-hydroxyethyl)amino)hexanoic acid ester.

[0030] Example 1: Preparation and Characterization of Engineered Dendritic Cell Membranes

[0031] This embodiment provides a process for preparing an engineered dendritic cell membrane.

[0032] (1) Constructing membrane protein expression plasmids

[0033] The expression plasmid pVAX1-XBB1.16 RBD for the spike protein (S) receptor-binding domain (RBD) of the Omicron XBB 1.16 mutant strain of COVID-19 was synthesized by Beijing Qingke Biotechnology Co., Ltd., and the membrane protein expression plasmid vector was pDisplay. TM (Invitrogen, V66020) The XBB 1.16RBD coding sequence (GenBank: WNV92758.1) and pDisplay were amplified by PCR. TM After PCR, the target fragment and pDisplay vector were recovered by agarose gel electrophoresis. TM The carrier fragment, pDisplay TM The vector fragment and the inserted target fragment were appropriately diluted to prepare a recombination reaction system, and the recombination reaction was carried out at 50℃ for 5 min.

[0034] Add the above recombinant product to the competent cell suspension, gently rotate the centrifuge tube to mix the contents, and let it stand in an ice bath for 30 minutes. Competent cells were placed in a 42°C water bath for 60 seconds, then the tubes were quickly transferred to an ice bath to cool the cells for 2 minutes. 500 μl of sterile LB medium (antibiotic-free) was added to the centrifuge tubes, mixed well, and incubated at 37°C and 150 rpm for 60 minutes using a shaker. The medium was then pipetted onto LB agar plates containing ampicillin and inverted at 37°C for 12 hours. Single colonies were selected and sequenced. After successful sequencing, the correct colonies were picked and inoculated into 5 ml of LB medium (antibiotic-containing), and incubated at 220 rpm and 37°C for 6-8 hours. Once the bacterial culture became turbid, it was inoculated into 200 mL of LB medium (antibiotic-containing), and incubated overnight at 220 rpm and 37°C. The recombinant plasmid pDisplay-XBB was extracted from the amplified bacterial cells using an endotoxin-free plasmid extraction kit (Tiangen Biotech (Beijing) Co., Ltd., DP117). 1.16RBD, specifically characterized by replacing pDisplay with the XBB 1.16RBD encoded sequence. TM The recombinant plasmid (Invitrogen, V66020) contains the recombinant sequence between the IgK leader sequence and the PDGFR transmembrane domain site, with other sequences remaining unchanged. This recombinant plasmid can express the XBB 1.16RBD protein on the cell membrane.

[0035] (2) Extraction of engineered dendritic cell membranes (RBD-DCM) with high expression of antigen protein XBB 1.16RBD

[0036] Immortalized mouse bone marrow-derived dendritic cells (DC2.4, from Beina Biotechnology) were seeded in 100mm wells and incubated at 37°C in a 5% CO2 incubator until the cells reached 80-90% confluence. Transfection with the recombinant plasmid pDisplay-XBB1.16RBD was then performed. Forty-eight hours after transfection, the culture medium was discarded, and cells were scraped off using a cell scraper. The collected dendritic cells were washed three times with pre-chilled PBS. The cell pellet was resuspended in hypotonic lysis buffer and repeatedly frozen and thawed to lyse the cells. Unly lysed cells were removed by centrifugation at 700g for 15 min at 4°C. The supernatant was further centrifuged at 50,000 rpm for 30 min at 4°C to collect the cell membrane. The collected cell membrane pellet was washed with PBS and centrifuged again. The collected cell membrane was resuspended in sterile, enzyme-free water, sonicated on ice, filtered through a 0.2μm filter, aliquoted, and stored at -80°C for later use. Unengineered dendritic cell membranes (DCMs) do not require transfection with recombinant plasmids; the extraction method is the same as above.

[0037] (3) Verification of membrane protein expression

[0038] Western blotting (WB) was used to detect membrane protein expression: a pre-cast protein electrophoresis gel (Bolt) was used. TMTotal protein (30 μg) was separated by 4-to-12% Bis-Tris electrophoresis (200 V, 22 min) on a Mini Protein Gel (Invitrogen, NW04120BOX). The separated proteins on the gel were transferred to an iBlot 2 Transfer Stacks PVDF (Invitrogen, IB24001) membrane under gradient voltage (20 V, 1 min; 23 V, 4 min; 25 V, 2 min), and then incubated at room temperature in 1×TBST containing 5% skim milk powder at 20 rpm for 1 h. A 1:2000 dilution of mouse monoclonal antibody against the SARS-CoV-2 spike protein receptor-binding domain (RBD) was added, along with Omicron Reactive mouse MAb (Sinochem Biotech, 40592-MM117), and incubated at room temperature at 20 rpm for 2 h. The membrane was washed with 1×TBST at 60 rpm for 10 min at room temperature, repeated three times to completely remove primary antibody residue. Secondary antibody was prepared using horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody diluted 1:10000 (Goat Anti-Mouse IgG Secondary Antibody (HRP) (Yiqiao Shenzhou, SSA007), at 20 rpm for 1 h at room temperature. The membrane was washed with 1×TBST at 60 rpm for 10 min at room temperature, repeated three times to completely remove secondary antibody residue. The membrane was incubated with an ECL chemiluminescence ultrasensitive colorimetric kit (Yisheng Biotechnology, 36208ES60) at room temperature in the dark for 3 min, and the HRP-labeled antibody-bound antigen was detected in a chemiluminescence analyzer. Bands and protein markers were visualized by exposure using PageRuler. TM Prestained Protein Ladder (Invitrogen, 26617) band alignment was used to verify the expression of the target antigen protein. Antibodies on the membrane were removed using membrane regeneration buffer (Soluble, SW3020), and the membrane was blocked again before incubation with membrane protein internal control antibodies to check the consistency of protein loading. The primary antibody was Na+ / K+-ATPaseα1 Rabbit Polyclonal Antibody (Abbkin, ABL1141), and the secondary antibody was horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody dilution (1:10000), Goat Anti-Rabbit IgG Secondary Antibody (HRP) (SSA004, Yiqiao Shenzhou).

[0039] (4) Characterization of engineered dendritic cell membranes

[0040] Morphological characterization results of the prepared dendritic cell membranes showed that the particle size and potential of the extracted dendritic cell membranes were detected by a Litesizer 500 (Anton Paar) nanoparticle size analyzer, and the morphology of the cell membranes was characterized by a transmission electron microscope (TEM-7650).

[0041] The results are as follows Figure 1 As shown, the expression of the target protein was successfully detected on the engineered dendritic cell membrane.

[0042] The results are as follows Figure 2 As shown in Figures A and 2B, the dendritic cell membrane has a particle size of approximately 124.5 nm and a potential of approximately -8.5 mV. The morphology of the dendritic cell membrane was observed using transmission electron microscopy, and the results are as follows: Figure 2 As shown in Figure C, most dendritic cell membranes are spherical, with particle sizes close to the measured values.

[0043] Example 2: Preparation and Characterization of RBD-DCMNPs-mRNA

[0044] (1) Preparation of XBB 1.16RBD mRNA: Using pVAX1-XBB1.16 RBD plasmid as a template, a template suitable for in vitro mRNA synthesis was obtained by linearization and purification through enzyme digestion. Subsequently, XBB 1.16RBD mRNA was obtained by reconstitution with purified sterile enzyme-free water using the T7 in vitro transcription kit (Novizan, DD4201-01) and the mRNA capping kit (Novizan, DD4109-01, DD4110-01).

[0045] (2) The ionizable lipid used in this invention is YK009. Other ionizable lipids that can be used, but are not limited to this, include at least one of the commonly used cationic lipids MC3, SM102, and ALC-0315 in the art for preparing cationic liposomes. For the auxiliary phospholipid, this invention uses dioleoylphosphatidylethanolamine (DOPE), a commonly used auxiliary lipid in the art, to improve the delivery efficiency of the delivery system. The specific operation is as follows: Accurately weigh 1 mg of the ionizable lipid YK009 and dissolve it in 100 μl of anhydrous ethanol, sonicate for 15 min until completely dissolved, and set aside. Simultaneously, accurately weigh 1 mg of the auxiliary lipid DOPE and dissolve it in 100 μl of anhydrous ethanol, sonicate for 15 min until completely dissolved, and set aside. Mix the ionizable lipid and auxiliary phospholipid at a mass ratio of 5:6. Mix the ionizable lipid / auxiliary phospholipid mixture with XBB 1.16RBD mRNA at a volume:mass ratio of 0.44 μl:1 μg. Eight μl of engineered dendritic cell membrane (RBD-DCM) highly expressing antigen protein XBB 1.16RBD was added to the aforementioned mixture of ionizable lipids, helper phospholipids, and mRNA. The mixture was then sonicated for 15 min. Finally, it was extruded using a 200 nm pore size lipid nanoextruder to obtain dendritic cell biomimetic targeting nanoparticles.

[0046] The particle size and potential of the prepared dendritic cell biomimetic targeting nanoparticles were detected by a Litesizer 500 nanoparticle size analyzer, and the morphology of the RBD-DCMNPs-XBB 1.16RBD mRNA was characterized by a transmission electron microscope (TEM-7650).

[0047] SDS-PAGE analysis was performed on the protein expression profiles of engineered dendritic cell membranes and engineered dendritic cell biomimetic targeted nanoparticles.

[0048] The results are as follows Figure 3 As shown in A and 3B, the dendritic cell-inspired targeted nanoparticles RBD-DCMNPs-XBB from South Africa, containing 1.16RBD mRNA, have a particle size of approximately 187.3 nm. The potential is approximately -6.7 mV, and the dispersion index (PDI) is approximately 0.208.

[0049] The results are as follows Figure 3 As shown in Figure C, the RBD-DCMNPs-XBB 1.16RBD mRNA nanoparticles are uniformly spherical.

[0050] The results are as follows Figure 3 As shown in D, the protein profiles of the cell membrane and the RBD-DCMNPs-XBB 1.16RBD mRNA nanoparticles are similar, and the membrane proteins are retained when the nanoparticles are prepared by membrane coating.

[0051] Example 3: Evaluation of the in vivo and in vitro delivery efficacy of RBD-DCMNPs-mRNA

[0052] I. In vivo and in vitro delivery efficacy of RBD-DCMNPs-eGFP mRNA

[0053] 1. Following the method in part (2) of Example 2, replace XBB 1.16RBD mRNA with eGFP mRNA (Trilink, L-7201) to prepare RBD-DCMNPs-eGFP mRNA (eGFP mRNA = 1.5 μg).

[0054] 2. Following the method in Part (2) of Example 2, the engineered dendritic cell membrane RBD-DCM was replaced with an unengineered dendritic cell membrane (DCM), and XBB 1.16RBD mRNA was replaced with eGFP mRNA (Trilink, L-7201) to prepare dendritic cell membrane (DCM) biomimetic nanoparticles DCMNPs-eGFP mRNA (eGFP mRNA = 1.5 μg).

[0055] The prepared RBD-DCMNPs-eGFP mRNA and the control DCMNPs-eGFP mRNA (eGFP mRNA = 1.5 μg) were transfected into cells coated with 2 × 10⁻⁶ mRNA. 5 In 24-well plates containing HEK293T or DC2.4 cells, DCMNPs-eGFP mRNA (mRNA = 1.5 μg) served as a control. Green fluorescent protein expression was observed and photographed using a fluorescence microscope 24 h after transfection.

[0056] The results are as follows Figure 4 As shown, green fluorescent protein expression was detected in both RBD-DCMNPs-eGFP mRNA and DCMNPs-eGFP mRNA after transfection into HEK293T or DC2.4 cells. Da .

[0057] II. In vivo and in vitro delivery efficacy of RBD-DCMNPs-Fluc mRNA

[0058] 1. Following the method in part (2) of Example 2, replace XBB 1.16RBD mRNA with Fluc mRNA (Trilink, L-7202) to prepare RBD-DCMNPs-Fluc mRNA (Fluc mRNA = 10 μg).

[0059] 2. Following the method in Part (2) of Example 2, the engineered dendritic cell membrane RBD-DCM was replaced with an unengineered dendritic cell membrane (DCM), and XBB 1.16RBD mRNA was replaced with Fluc mRNA (Trilink, L-7202) to prepare dendritic cell membrane (DCM) biomimetic nanoparticles DCMNPs-Fluc mRNA (eGFP mRNA = 1.5 μg).

[0060] RBD-DCMNPs-Fluc mRNA (Fluc mRNA = 10 μg) and DCMNPs-Fluc mRNA (Fluc mRNA = 10 μg) were injected intramuscularly or intravenously into female Balb / c mice. In vivo imaging and in vitro imaging of important tissues and organs were performed at different time points after injection to detect the expression efficiency distribution of luciferase in mice.

[0061] The results are as follows Figure 5 As shown in Figure A, in vivo imaging results indicate that strong bioluminescent signals were detected in mice at different time points after intramuscular injection of RBD-DCMNPs-Fluc mRNA. A bioluminescent signal was still detected 72 hours after injection, and there was no significant difference in the bioluminescent signals between the RBD-DCMNPs-Fluc mRNA and DCMNPs-Fluc mRNA groups. These results suggest that engineered cell membranes do not affect the in vivo mRNA delivery efficiency of the cell membrane biomimetic nanoparticles. Figure 5 The in vitro imaging results shown in Figure B indicate that RBD-DCMNPs-Fluc mRNA and DCMNPs-Fluc mRNA can be expressed in lymph nodes of mice after intramuscular injection.

[0062] The results are as follows Figure 5 As shown in Figure C, in vivo imaging results indicate that strong bioluminescent signals were detected in both RBD-DCMNPs-Fluc mRNA and DCMNPs-Fluc mRNA after tail vein injection at different time points, and there was no significant difference in the intensity of the two signals. This suggests that engineered cell membranes do not affect the in vivo mRNA delivery efficiency of the cell membrane biomimetic nanoparticles. The bioluminescent signal essentially disappeared 72 hours after injection. Figure 5 As shown in Figure D, after tail vein injection, RBD-DCMNPs-Fluc mRNA and DCMNPs-Fluc mRNA were mainly concentrated in the liver, spleen, lungs, and lymph nodes, with the spleen showing the highest expression level.

[0063] The above results demonstrate that surface-engineered dendritic cell-inspired nanoparticles can deliver mRNA to lymphatic organs.

[0064] Example 4: Serum antibody detection after RBD-DCMNPs-mRNA immunization of mice

[0065] BALB / c mice (female, 6-8 weeks old, 16-18g, Beijing Vital River Pharmaceutical Co., Ltd.) were randomly divided into three groups: an RBD-DCMNPs-RBD mRNA (RBD-mRNA = 10μg) vaccine immunization group, a DCMNPs-RBD mRNA (RBD-mRNA = 10μg) vaccine immunization group, and a negative control group (n=5 per group, fed normally). Mice were immunized via intramuscular injection. Immunization was performed three times, and on day 10 after the third immunization, sufficient mouse serum was obtained by orbital blood collection.

[0066] 1. The ELISA method was used to detect the specific binding antibody against the SARS-CoV-2 XBB1.16 mutant strain RBD antigen in the serum of immunized mice. The specific detection method is as follows:

[0067] (1) Coating: The RBD protein of the XBB1.16 mutant strain (Yiqiao Shenzhou, 40592-V08H136) was diluted to 1 ng / μl with carbonate buffer (50 mM, pH 9.6, filtered through a 0.22 μm filter). 100 μg of RBD protein dilution was added to each well of a 96-well plate, sealed, and incubated overnight at 4°C.

[0068] (2) Washing: After coating overnight, pour out the 96-well plate to remove the protein coating solution, add 200 μL of washing buffer (containing 0.2% Tween-20 and 1×TBS) to each well, gently shake by hand for 30 seconds and then pat dry on paper. Repeat 6 times.

[0069] (3) Blocking: Add 200 μl of blocking solution (1×TBS containing 2% BSA) to each well and incubate at 37°C for 2 h;

[0070] (4) Washing the plate: Pour out the 96-well plate to remove the blocking solution, add 200 μl of washing solution (containing 0.2% Tween-20 and 1×TBS) to each well, gently shake by hand for 30 seconds, and then pat dry on paper. Repeat 6 times.

[0071] (5) Incubation with primary antibody: The serum from immunized mice was serially diluted 10-fold with antibody diluent (washing buffer containing 0.5% BSA) to obtain 10... -1 Up to 10 -6 For serum at different dilutions, add 100 μl of serum diluent to each well and incubate at 37°C for 2 hours;

[0072] (6) Washing the plate: Pour out the 96-well plate to remove the serum diluent, add 200 μL of washing buffer (containing 1×TBS of 0.2% Tween-20) to each well, gently shake by hand for 30 seconds, and then pat dry on paper. Repeat 6 times.

[0073] (7) Incubation of secondary antibody: horseradish peroxidase-labeled goat anti-mouse IgG (H+L) (Beyotime, A0216) was diluted 250 times with antibody dilution buffer (washing buffer containing 0.5% BSA) to obtain secondary antibody dilution buffer. 100 μl of secondary antibody dilution buffer was added to each well and incubated at 37°C for 1 h.

[0074] (8) Washing the plate: Pour out the 96-well plate to remove the secondary antibody dilution solution, add 200 μl of washing buffer (containing 0.2% Tween-20 and 1×TBS) to each well, gently shake by hand for 30 seconds, and then pat dry on paper. Repeat 6 times.

[0075] (9) Color development: Add 100 μl of TMB substrate (Tiangen) to each well and incubate at room temperature in the dark for 20 min;

[0076] (10) Terminate color development: Add 50 μL of 2M H2SO4 to each well and detect the OD value of A450 on a microplate reader;

[0077] (11) Determination of serum binding antibody IgG titer: If the OD / negative control OD of a certain dilution is ≥2.1, the OD / negative control OD of the next dilution is <2.1. This dilution factor is the antibody titer corresponding to the serum sample (if the negative control OD is <0.05, it is calculated as 0.05).

[0078] 2. Determination of NT50 titer of neutralizing antibody against South African mutant pseudovirus:

[0079] The antibody neutralizing activity of immune serum was evaluated using the SARS-CoV-2 XBB1.16 mutant pseudovirus. The SARS-CoV-2 XBB1.16 mutant pseudovirus used in the evaluation was provided by the National Institutes for Food and Drug Control (NIFDC). The evaluation methodology followed the literature "Quantitative Detection Method for Neutralizing Antibodies in Clinical Serum and Corresponding Biological Products Based on Pseudoviruses." Specific detection methods are as follows:

[0080] Mouse immune serum was serially diluted 3-fold from 1 / 30 using DMEM complete medium to obtain 6 different serum dilutions, which were then mixed with 2×10⁻⁶ DMEM complete medium. 4 TCID50 pseudoviruses were incubated at 37°C. A control group without pseudoviruses and a control group without serum samples were also included. After one hour of incubation, 2 × 10⁻⁶ cells were added to each control group. 4 293 T-ACE2 cells were cultured at 37°C under 5% CO2. Since the pseudovirus expresses firefly luciferase upon entering the cells, it reacted with a luminescent substrate after 24 hours, and luminescence was detected. The percentage of pseudovirus inhibition was calculated by comparing the luminescence value with that of the pseudovirus control group. The half-maximal inhibitory dilution (MCD) was calculated by determining the serum dilution factor required for 50% pseudovirus inhibition. The MCD, or half-neutralizing dilution (NT50), represents the neutralizing activity of serum antibodies against the pseudovirus.

[0081] The results are as follows Figure 6 As shown in Figure A, high-titer antigen-specific binding antibody IgG was detected in mice immunized with both the RBD-DCMNPs-RBD mRNA (RBD-mRNA = 10 μg) vaccine and the DCMNPs-RBD mRNA (RBD-mRNA = 10 μg) vaccine. The IgG antibody level in the RBD-DCMNPs-RBD mRNA group was significantly higher than that in the DCMNPs-RBD mRNA (RBD-mRNA = 10 μg) vaccine immunization group, with average IgG antibody titers of 1:24200 and 1:362000, respectively.

[0082] The results are as follows Figure 6 As shown in B, high titers of neutralizing antibodies were detected in mice immunized with both the RBD-DCMNPs-RBD mRNA (RBD-mRNA = 10 μg) vaccine and the DCMNPs-RBD mRNA (RBD-mRNA = 10 μg) vaccine. The neutralizing antibody level in the RBD-DCMNPs-RBD mRNA group was significantly higher than that in the DCMNPs-RBD mRNA (RBD-mRNA = 10 μg) vaccine group, with average neutralizing antibody titers of 1:5448 and 1:42074, respectively.

[0083] The above results indicate that engineered cell membrane biomimetic nanoparticles RBD-DCMNPs-RBD mRNA can achieve dual immunity of mRNA and protein, and can induce a stronger humoral immune response compared with unengineered cell membrane biomimetic nanoparticles DCMNPs-RBD mRNA.

[0084] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. An engineered dendritic cell membrane-bionic nanoparticle, characterized in that, It includes engineered dendritic cell membranes, ionizable lipids, and auxiliary phospholipids. The engineered dendritic cell membranes are extracted from engineered dendritic cells, and the membrane surface of the engineered cells expresses viral antigen proteins.

2. The engineered dendritic cell membrane-bionic nanoparticle of claim 1, wherein, The engineered dendritic cell membrane is a cell membrane extracted from engineered dendritic cells prepared through genetic engineering or ligand-coupled pretreatment methods.

3. The engineered dendritic cell membrane-bionic nanoparticle of claim 2, wherein, The dendritic cells are selected from at least one of immortalized dendritic cells and dendritic cells isolated from bone marrow and induced to differentiate.

4. The engineered dendritic cell membrane-bionic nanoparticle of claim 1, wherein, The ionizable lipid is at least one of YK009, MC3, SM102 or ALC-0315.

5. The engineered dendritic cell membrane-bionic nanoparticle of claim 1, wherein, The auxiliary phospholipid is at least one of 1,2-distearate-sn-glycerol-3-phosphocholine, 1,2-bis(docosahexaenoyl)-sn-glycerol-3-phosphocholine, 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine, and 1,2-dioleoyl-sn-glycerol-3-phosphocholine.

6. The engineered dendritic cell membrane-bionic nanoparticle of claim 1, wherein, The volume-to-volume-to-mass ratio of the engineered dendritic cell membrane, the mixture of ionizable lipids and helper phospholipids, and mRNA is 1–10. The mass ratio of ionizable lipids to phospholipids in the mixture of ionizable lipids and phospholipids is 0.5 to 5:

1.

7. A nucleic acid delivery system, characterized in that, The invention includes engineered dendritic cell membrane biomimetic nanoparticles and nucleic acids as described in any one of claims 1-6, wherein the nucleic acid is any one of plasmid, mRNA, DNA vaccine or RNA vaccine.

8. The nucleic acid delivery system of claim 7, wherein, The nucleic acid is mRNA, and the ratio between the dendritic cell membrane and mRNA in the engineered dendritic cell biomimetic targeting nanoparticles is dendritic cell membrane volume: mRNA mass = 1 to 10:

1.

9. The use of the engineered dendritic cell membrane biomimetic nanoparticles according to any one of claims 1-6 and the nucleic acid delivery system according to claim 7 or 8 in any of the following: 1) Application in the preparation of in vitro mRNA transfection products; 2) Applications in the preparation of in vivo mRNA delivery products; 3) Application in the preparation of mRNA vaccine delivery products.

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