Preparation method of metal ion complexation mRNA delivery system as well as product and application of metal ion complexation mRNA delivery system

Abstract

The invention relates to a preparation method of a metal ion complexation mRNA delivery system and a product and application thereof, and the preparation method comprises the following steps: (1) mixing mRNA and metal ions in a solvent at 10-35 DEG C for 10-60 min to obtain nanoparticles; and (2) mixing the nanoparticles with a phospholipid membrane at 10-35 DEG C for 2-60 minutes, and wrapping the nanoparticles with the phospholipid membrane to obtain the metal ion complexation mRNA delivery system. Metal ions and mRNA are mixed and complexed to form complex nanoparticles, and mRNA loading capacity can be improved. The surfaces of the nanoparticles are coated with phospholipid membranes, so that the biocompatibility of the delivery system is improved, the in-vivo circulation time of the delivery system is prolonged, and meanwhile, fusion between the delivery system and a target cell membrane can be promoted. Besides, the mixing temperature of mRNA and metal ions is controlled to be 10-35 DEG C, the mixing time is controlled to be 10-60 min, the reaction condition is mild and rapid, the prepared nanoparticles are uniform in particle size, and safer and more effective mRNA delivery can be achieved.

Description

Technical Field

[0001] The present invention relates to the field of nanomaterials, and in particular to a preparation method of a metal ion complexed mRNA delivery system, and a product and application thereof. Background Art

[0002] Messenger RNA (mRNA) therapy is an emerging gene therapy with great application value. mRNA can not only directly kill tumor cells by encoding tumor suppressor genes, but also encode related antigens and translate them into proteins, promote DC cell maturation and activate antigen-specific T cells, and induce specific immune responses. mRNA does not require the support of plasmid backbone or viral packaging, is not immunogenic in itself, and has better safety. The mRNA production cycle is short, which can avoid the safety issues of plasmid or virus integration into the host genome. However, since mRNA is easily degraded by nucleases and difficult to enter cells directly, the delivery of mRNA to target cells is subject to various technical limitations. Therefore, the development of an effective mRNA drug targeted delivery system to protect mRNA from degradation is of great significance for the development of new mRNA therapies and vaccines.

[0003] The most widely used traditional delivery carrier at present is lipid nanoparticle LNP. As CN118319879A discloses a lipid nanoparticle, including ionizable lipid molecules, steroidal lipid molecules, neutral lipid molecules, PEG-lipid molecules, carotenoids or derivatives thereof, the lipid nanoparticles are used to encapsulate nucleic acid molecules to obtain lipid nanoparticles encapsulating nucleic acid molecules. However, LNP utilizes ionizable lipids to load mRNA through electrostatic interaction, and needs charge reversal to escape from lysosomes, and there are problems such as low cytoplasmic release efficiency, cell efflux effect and high toxicity. In addition, the drug based on LNP on the market needs to be transported and stored in a low temperature environment. Aggregation occurs when LNP is stored for a long time in solution, affecting the release and translation of subsequent mRNA.

[0004] Therefore, developing a method for preparing a stable mRNA delivery system to abandon the traditional gene loading mode through electrostatic interaction and achieve efficient cytoplasmic delivery and translation has become a technical problem that needs to be solved urgently. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a preparation method of a metal ion complex mRNA delivery system and its products and applications. The preparation method has mild and rapid reaction conditions, and the prepared metal ion complex mRNA delivery system can improve the efficiency of mRNA cytoplasmic delivery and achieve safer and more effective mRNA delivery.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a metal ion complexed mRNA delivery system, the preparation method comprising the following steps:

[0008] (1) mixing mRNA and metal ions in a solvent at 10 to 35° C. for 10 to 60 minutes to obtain nanoparticles;

[0009] (2) The nanoparticles are mixed with the phospholipid membrane at 10 to 35° C. for 2 to 60 minutes, and the nanoparticles are wrapped with the phospholipid membrane to obtain the metal ion complexed mRNA delivery system.

[0010] Among them, the specific point values ​​in 10-35℃ can be selected from 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, etc., the specific point values ​​in 10-60min can be selected from 10min, 20min, 30min, 40min, 50min, 60min, and the specific point values ​​in 2-60min can be selected from 2min, 10min, 20min, 30min, 40min, 50min, 60min, etc.

[0011] The present invention mixes metal ions with mRNA, and the two are complexed to form complex nanoparticles. On the one hand, the metal ions can condense the originally loosely distributed mRNA to increase the load, and on the other hand, they can act as an adjuvant by stimulating immune-related pathways. The phospholipid membrane wrapping improves the biocompatibility of the delivery system, prolongs its circulation time in the body, and can also promote the fusion between the delivery system and the target cell membrane. In addition, the mixing temperature of mRNA and metal ions in step (1) is controlled at 10 to 35°C, and the time is controlled at 10 to 60 minutes. The reaction conditions are mild and fast, and the prepared nanoparticles have a uniform particle size, which can improve the efficiency of mRNA cytoplasmic delivery and achieve safer and more effective mRNA delivery.

[0012] Preferably, the metal ions in step (1) include Mn 2+ and / or Mo 4+ .

[0013] Preferably, the metal ion in step (1) is Mn 2+ and Mo 4+ .

[0014] In the present invention, Mn 2+ and Mo 4+ The compounding can enhance the complex stability between metal ions and mRNA, increase the mRNA encapsulation rate, and thus improve the mRNA delivery efficiency.

[0015] Preferably, the Mn2+ and Mo 4+ The molar ratio is (1~10):(1~10).

[0016] Among them, the specific point values ​​in the range of 1 to 10% may be selected from 1%, 3%, 5%, 7%, 9%, 10%, etc.

[0017] Based on Mn 2+ and Mo 4+ The potential interaction in terms of complex stability with mRNA can better improve the mRNA delivery efficiency when the two meet the above-mentioned specific ratio relationship.

[0018] Preferably, in step (1), the mass ratio of the mRNA to the metal ion is (1-10):(10-100).

[0019] Among them, the specific point values ​​​​from 1 to 10 can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., and the specific point values ​​​​from 10 to 100 can be selected from 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc.

[0020] Preferably, the mass ratio of metal ions to phospholipid membranes in the nanoparticles in step (2) is 1:(0.1-10).

[0021] Among them, the specific point values ​​between 0.1 and 10 can be selected from 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.

[0022] Preferably, the phospholipid membrane in step (2) comprises a natural membrane and / or an artificial synthetic lipid membrane.

[0023] Preferably, the natural membrane comprises any one or a combination of at least two of endothelial cell membrane, human embryonic kidney cell membrane, macrophage membrane, breast cancer cell membrane, colorectal cancer cell membrane or melanoma cell membrane.

[0024] Preferably, the raw materials for preparing the artificial synthetic lipid membrane include hydrogenated soybean lecithin, cholesterol and PEGylated lipid.

[0025] Preferably, the phospholipid membrane is a natural membrane.

[0026] The present invention creatively finds that when a natural membrane is selected to wrap the nanoparticles, the prepared metal ion complex mRNA delivery system has a higher delivery efficiency for mRNA.

[0027] Preferably, the solvent in step (1) comprises water or phosphate buffer.

[0028] Preferably, the mixing in step (1) further comprises centrifugation and resuspending the precipitate after centrifugation.

[0029] Preferably, the centrifugal speed is 1000-20000g, for example, 1000g, 3000g, 6000g, 9000g, 12000g, 15000g, 18000g, 20000g, etc.

[0030] Preferably, ultrasound is also performed during the mixing process in step (2).

[0031] Preferably, the mixing in step (2) further includes centrifugation and resuspending the precipitate after centrifugation.

[0032] Preferably, the centrifugal speed is 1000-20000g, for example, 1000g, 3000g, 6000g, 9000g, 12000g, 15000g, 18000g, 20000g, etc.

[0033] In a second aspect, the present invention provides a metal ion complex mRNA delivery system prepared by the method for preparing the metal ion complex mRNA delivery system as described in the first aspect.

[0034] In a third aspect, the present invention provides an application of the metal ion complex mRNA delivery system as described in the second aspect in the preparation of mRNA vaccines and / or mRNA drugs.

[0035] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The present invention mixes metal ions with mRNA, and the two are complexed to form complex nanoparticles. On the one hand, the metal ions can condense the originally loosely distributed mRNA to increase the load, and on the other hand, they can act as an adjuvant by stimulating immune-related pathways. The phospholipid membrane wrapping improves the biocompatibility of the delivery system, prolongs its circulation time in the body, and can also promote the fusion between the delivery system and the target cell membrane. In addition, the mixing temperature of mRNA and metal ions is controlled at 10 to 35°C, and the time is controlled at 10 to 60 minutes. The reaction conditions are mild and fast, and the prepared nanoparticles have a uniform particle size, which can improve the efficiency of mRNA cytoplasmic delivery and achieve safer and more effective mRNA delivery.

[0038] Furthermore, the metal ion Mn 2+ and Mo 4+The compounding can enhance the complex stability between metal ions and mRNA, increase the mRNA encapsulation rate, and thus improve the mRNA delivery efficiency.

[0039] Furthermore, when a natural membrane is selected to encapsulate the nanoparticles, the prepared metal ion complexed mRNA delivery system has a higher delivery efficiency for mRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is a transmission electron microscopy characterization result diagram of the nanoparticles prepared in step (1) of Example 1;

[0041] Figure 2 This is a transmission electron microscopy characterization result diagram of the nanoparticles prepared in step (1) of Example 2;

[0042] Figure 3 This is a transmission electron microscopy characterization result diagram of the nanoparticles prepared in step (1) of Comparative Example 1;

[0043] Figure 4 This is a transmission electron microscopy characterization result diagram of the nanoparticles prepared in step (1) of Comparative Example 2;

[0044] Figure 5 is a graph showing the particle size and Zeta potential characterization results of Mn-PTEN-NP@M prepared in Example 1;

[0045] Figure 6 is a graph showing the particle size and Zeta potential characterization results of Mn-PTEN-NP@L prepared in Example 3;

[0046] Figure 7 This is a test diagram of the transfection effect of different metal ion complexed mRNA delivery systems in Test Example 4;

[0047] Figure 8 This is a test diagram of the mRNA delivery effect of different metal ion complexed mRNA delivery systems in Test Example 4;

[0048] Fig. 9 is a schematic diagram of the treatment process of mice in different test groups in Application Example 1;

[0049] Fig.10 This is a comparison chart of the tumor volumes of mice in different test groups in Application Example 1;

[0050] Fig.11 This is a comparison chart of tumor weights of mice in different test groups in Application Example 1;

[0051] Fig.12 is the immunohistochemical image of tumor sections of mice in different experimental groups in Application Example 1;

[0052] Fig.13 This is a comparison chart of the antibody titers of mice in different test groups in Application Example 2. DETAILED DESCRIPTION

[0053] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0054] Example 1

[0055] This embodiment provides a metal ion complexed mRNA delivery system, which is prepared by the following method:

[0056] (1) 100 μg of Mn 2+ (tetrahydrate manganese chloride aqueous solution, 10 mg / ml) and 10 μg of PTEN mRNA (1 mg / ml, 10 μl, purchased from Trilink Biotechnologies, USA) were mixed in 100 μl of water, pipetted evenly, and incubated at 25° C. for 30 min. After the incubation, the mixture was centrifuged at 12000 g for 10 min to obtain nanoparticles Mn-PTEN-NP.

[0057] (2) Collect 1.0×10 7 J774A.1 macrophages were resuspended in 1 ml of pre-cooled TM buffer (pH 7.4, 0.01 M Tris, 0.001 M MgCl2). The cells were squeezed out, and 0.25 ml of 1 M sucrose was added to the cell homogenate, and then gradient centrifugation was performed to obtain cell membranes: first centrifugation at 2000 rpm for 10 min and the supernatant was taken, then centrifugation at 5000 rpm for 10 min and the supernatant was taken again, and finally centrifugation at 12000 rpm for 20 min to obtain the precipitate to obtain the macrophage membrane.

[0058] (3) The nanoparticles prepared in step (1) and the macrophage membrane prepared in step (2) were mixed at a mass ratio of metal ions to cell membrane proteins of 1:1, and then the mixture was ultrasonicated in a 25°C water bath for 5 minutes, and the mixture was extruded through polycarbonate porous membranes with pore sizes of 800 μm, 400 μm and 200 μm in sequence using an extruder, and repeatedly extruded 10 times. Next, the repeatedly extruded mixture was centrifuged at 12000 rpm, and the precipitate was collected to obtain the metal ion complexed mRNA delivery system Mn-PTEN-NP@M.

[0059] Example 2

[0060] This embodiment provides a metal ion complex mRNA delivery system, which is different from the embodiment 1 only in that the Mn in step (1) is 2+The solution was mixed with mRNA in 100 μl of PBS instead of in 100 μl of water.

[0061] Example 3

[0062] This embodiment provides a metal ion complexed mRNA delivery system, which differs from Embodiment 1 only in that the macrophages are replaced with artificial synthetic lipid membranes, which are prepared by the following method:

[0063] (1) 100 μg of Mn 2+ (tetrahydrate manganese chloride aqueous solution, 10 mg / ml) and 10 μg of PTEN mRNA (1 mg / ml, 10 μl) were mixed in 100 μl of water, pipetted evenly, and incubated at 25° C. for 30 min. After the incubation, the mixture was centrifuged at 12000 g for 10 min to obtain nanoparticles Mn-PTEN-NP.

[0064] (2) 100 μg of lipid (hydrogenated soybean lecithin: cholesterol: DSPE-mPEG2000 mass ratio of 56:39:5) was mixed with 600 μl of anhydrous ethanol as solvent, and the mixture was incubated at 25°C for 30 min to obtain an artificial synthetic lipid membrane.

[0065] (3) The nanoparticles prepared in step (1) were dispersed in 200 μl of water, and were quickly introduced into the artificial synthetic lipid membrane prepared in step (2), and mixed by pipetting 100 times. The mixture after mixing by pipetting was allowed to stand at 25° C. for 60 min, and the excess ethanol was removed by a 10 kDa dialysis bag to obtain the metal ion complexed mRNA delivery system Mn-PTEN-NP@L.

[0066] Example 4

[0067] This example provides a metal ion complex mRNA delivery system, which differs from Example 1 only in that PTEN mRNA is replaced with EGFP mRNA of equal mass (purchased from Trilink Biotechnologies, USA) to obtain Mn-EGFP-NP@M.

[0068] Example 5

[0069] This example provides a metal ion complex mRNA delivery system, which differs from Example 3 only in that PTEN mRNA is replaced with an equal mass of EGFP mRNA to obtain Mn-EGFP-NP@L.

[0070] Example 6

[0071] This example provides a metal ion complex mRNA delivery system, which differs from Example 1 only in that PTEN mRNA is replaced with RBD mRNA of equal mass (purchased from Trilink Biotechnologies, USA) to obtain Mn-RBD-NP@M.

[0072] Example 7

[0073] This example provides a metal ion complexed mRNA delivery system, which differs from Example 3 only in that PTEN mRNA is replaced with RBD mRNA of equal mass to obtain Mn-RBD-NP@L.

[0074] Example 8

[0075] This example provides a metal ion complex mRNA delivery system, which differs from Example 1 in that PTEN mRNA is replaced with RBD mRNA of equal mass, and 100 μg of Mn 2+ (aqueous solution of manganese chloride tetrahydrate, 10 mg / ml) was replaced with 50 μg of Mn 2+ (aqueous solution of manganese chloride tetrahydrate, 10 mg / ml) and 50 μg of Mo 4+ (ammonium molybdate aqueous solution, 10 mg / ml) to obtain Mn-Mo-RBD-NP@M.

[0076] Comparative Example 1

[0077] This embodiment provides a metal ion complex mRNA delivery system, which differs from Embodiment 1 only in that the incubation temperature in step (1) is replaced from 25°C to 4°C.

[0078] Comparative Example 2

[0079] This embodiment provides a metal ion complex mRNA delivery system, which differs from Embodiment 1 only in that the incubation temperature in step (1) is replaced from 25°C to 37°C.

[0080] Comparative Example 3

[0081] This comparative example provides a commercial PTEN mRNA delivery carrier LNP, which is prepared using the Moderna formula: the ratio of lipid to mRNA is calculated according to the nitrogen-phosphorus ratio of 6:1. The four lipids are prepared with ethanol and mixed according to SM-102: DSPC: Cholesterol: DMG-PEG2k = 50:10:38.5:1.5 (molar ratio) as the oil phase; 20μg mRNA is prepared with 1ml sodium acetate solution as the aqueous phase. The microfluidic chip parameters are set: 1.2ml total volume; 3:1 flow rate ratio; 12ml / min total speed; the first 0.1ml volume is not needed, and the last 0.05ml volume is not needed, and the LNP solution is collected. The collected LNP solution is ultrafiltered with a 100KD ultrafiltration tube, 3ml PBS is added, and centrifuged 3 times at 6000rpm for 10min to remove ethanol and sodium acetate to prepare PTEN-LNP.

[0082] Comparative Example 4

[0083] This comparative example provides a commercial EGFP mRNA delivery vector LNP, which differs from comparative example 3 only in that PTEN mRNA is replaced with EGFP mRNA of equal mass to prepare EGFP-LNP.

[0084] Comparative Example 5

[0085] This comparative example provides a commercial EGFP mRNA delivery vector LNP, which differs from comparative example 3 only in that PTEN mRNA is replaced with RBD mRNA of equal mass to prepare RBD-LNP.

[0086] Test Example 1

[0087] Validation of the structural properties of nanoparticles and metal ion complexed mRNA delivery systems

[0088] (1) The morphology of the nanoparticles prepared in step (1) of Example 1, Example 2, Comparative Example 1, and Comparative Example 2 was characterized by transmission electron microscopy. The results were as follows: Figures 1 to 4 shown.

[0089] Depend on Figure 1 and Figure 2 From the comparison, it can be seen that when metal ions and mRNA are mixed in PBS, there are a large number of lamellar structures in addition to nanoparticles in the system. It is speculated that the lamellar structures are formed by the coordination of metal ions and phosphate groups. The presence of a large number of lamellar structures will further affect the wrapping of nanoparticles by the phospholipid membrane, and thus affect the mRNA loading rate.

[0090] Depend on Figure 1 and Figures 3-4 It can be seen from the comparison that at 25℃, Mn 2+It can form nanoparticles with uniform particle size with mRNA, with a particle size of about 40nm; when the mixing temperature is lowered to 4°C, the particle size of the prepared nanoparticles is too large, which will directly affect the delivery efficiency of mRNA; when the mixing temperature is raised to 37°C, the prepared nanoparticles will aggregate, affecting the interaction with the phospholipid membrane, thereby reducing the delivery efficiency of mRNA.

[0091] (2) The hydrated particle size of the nanoparticles prepared in step (1) of Example 1 was measured using a dynamic light scattering instrument, and the hydrated particle size of the nanoparticles was found to be 98.53±10.33.

[0092] (3) The particle size and Zeta potential of the metal ion complexed mRNA delivery system Mn-PTEN-NP@M prepared in Example 1 were characterized by dynamic light scattering. Figure 5 The particle size and Zeta potential of the metal ion complexed mRNA delivery system Mn-PTEN-NP@L prepared in Example 3 were characterized by dynamic light scattering. Figure 6 As shown, the particle size is 153.2±4.5 nm. The particle size of the delivery system formed by the artificial synthetic lipid membrane is larger than that of the delivery system formed by the natural membrane.

[0093] Test Example 2

[0094] Verification of the stability of the metal ion complexed mRNA delivery system prepared in Example 1

[0095] The Mn-PTEN-NP@M precipitate (mRNA 10 μg) prepared in Example 1 was resuspended in 100 μl PBS to obtain a Mn-PTEN-NP@M resuspension, and the resuspension was placed at 4°C for 0 day, 10 days, and 30 days, respectively; the Mn-PTEN-NP@M (mRNA 10 μg) prepared in Example 1 was lyophilized, and the Mn-PTEN-NP@M powder obtained by lyophilization was placed at 25°C for 10 days and 30 days, respectively, and the particle size and potential of the Mn-NP@M under different storage conditions were tested. The results are shown in Table 1. The particle size and potential did not change significantly, indicating that the Mn-PTEN-NP@M prepared in Example 1 can be stably stored for at least 30 days in either liquid or powder state, providing a good foundation for the subsequent application of the delivery system.

[0096] Table 1

[0097] sample Size(nm) Zeta potential (mV) Resuspension - Day 0 136.2±10.3 -26.9±2.2 Resuspension - 10 days 145.2±7.1 -22.0±2.1 Resuspension-30 days 168.4±16.1 -26.1±0.1 Freeze-dried powder-10 days 150.1±15.9 -26.8±0.4 Freeze-dried powder-30 days 170.6±4.5 -26.0±0.7

[0098] Test Example 3

[0099] Verification of the mRNA encapsulation efficiency of the metal ion complexed mRNA delivery system prepared in Example 1

[0100] The encapsulation efficiency of mRNA by Mn-PTEN-NP@M prepared in Example 1 was tested by Nanodrop instrument. 2 μl of supernatant was added to the instrument to obtain the concentration of unencapsulated mRNA in the supernatant. The encapsulation efficiency was calculated by subtraction method to be 98.0±1.1%, indicating that the metal ion complexed mRNA delivery system prepared in Example 1 has excellent encapsulation effect on mRNA.

[0101] Test Example 4

[0102] Verification of transfection efficiency of metal ion complexed mRNA delivery system

[0103] (1) 1×10 5 HCT116 cells were inoculated into 6-well plates and cultured for 12 h. The Mn-PTEN-NP@M prepared in Example 1 and the PTEN-LNP prepared in Comparative Example 3 were added to the cells and incubated for 36 h. The final concentration of PTEN mRNA was 1 μg / ml. The expressed PTEN protein was detected by Western Blot. The results are shown in Figure 2. Figure 7 As shown, it is shown that the metal ion complexed mRNA delivery system prepared by the present invention produces a better transfection effect than commercial LNP, and the expression of PTEN protein is higher.

[0104] (2) DC2.4 cells were inoculated into a confocal culture dish and cultured for 12 h to allow the cells to adhere to the glass bottom of the confocal culture dish. The nanoparticles Mn-EGFP-NP prepared in step (1) of Example 4, Mn-EGFP-NP@M prepared in Example 4, and Mn-EGFP-NP@L prepared in Example 5 were added to the cells and incubated for 36 h, and the final concentration of EGFPP mRNA was 3 nM.

[0105] The above cells were imaged using a laser scanning confocal microscope. Figure 8 As shown, the metal ion complex mRNA delivery system wrapped in phospholipid membrane produced better transfection effect than nanoparticles formed by metal ion and mRNA complex, and the PTEN protein expression was higher; at the same time, the transfection effect of natural membrane Mn-EGFP-NP@M was better than that of artificial synthetic lipid membrane Mn-EGFP-NP@L, indicating that natural membrane has a better delivery effect on mRNA.

[0106] Application Example 1

[0107] Evaluation of anti-tumor drug effects using metal ion complexed mRNA delivery system

[0108] (1) Construction of breast cancer mouse model: 20 mice (BALB / c female mice, 6 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd.) were selected and 1×10 5 4T1 cells were injected into the fat pad of each mouse to establish an orthotopic breast cancer model.

[0109] (2) Construction of antibody-coupled delivery system: The αPD-L1 antibody was coupled to the cell membrane of the metal ion complexed mRNA delivery system of Example 1 through a bifunctional maleimide molecule, and Mn-PTEN-NP@PM was obtained at a mass ratio of PTEN mRNA to αPD-L1 of 1:2.

[0110] (3) Experimental grouping and intervention method: The 20 mice were equally divided into 4 groups, with 5 mice in each group, namely the control group, mRNA+αPD-L1 group, Mn-PTEN-NP group, and Mn-PTEN-NP@PM group. On the 8th day of the orthotopic breast cancer model, each experimental group continued to be treated as follows. The treatment flow diagram is shown in the figure. Fig. 9 As shown:

[0111] Control group: PBS was injected intravenously every three days for a total of four times;

[0112] mRNA+αPD-L1 group: PTEN mRNA (0.5 mg / kg) and αPD-L1 (1 mg / kg) were intravenously injected once every three days for a total of four times;

[0113] Mn-PTEN-NP group: Mn-PTEN-NP prepared in step (1) of Example 1 was intravenously injected (PTEN mRNA injection amount was kept at 0.5 mg / kg), once every three days, for a total of four times;

[0114] Mn-PTEN-NP@PM group: Mn-PTEN-NP@PM was injected intravenously (the injection amount of PTEN mRNA was kept at 0.5 mg / kg), once every three days, for a total of four times.

[0115] (4) Evaluation of anti-tumor effects

[0116] (4.1) Starting from the start of drug administration, the larger diameter (L) and smaller diameter (S) of the tumor of mice in each experimental group were monitored every 2 days using a vernier caliper, and the formula V = 0.5 × L × S was used. 2 The tumor volume was calculated. Fig.10 As shown, it was shown that Mn-PTEN-NP@PM reduced the growth rate of tumor.

[0117] (4.2) On the 20th day of establishing the orthotopic breast cancer model (i.e., 3 days after the last drug administration), the mice were killed, and the tumors were collected and weighed. The tumors of the mice in each experimental group were collected and fixed in 4% paraformaldehyde for immunofluorescence staining to evaluate the expression effect of PTEN. The results are shown in Fig.11 As shown, it was shown that Mn-PTEN-NP@PM inhibited tumor growth, with an inhibition rate of up to 81.3%.

[0118] (4.3) The collected tumors were sectioned and stained. The tumor tissues were fixed in 4% paraformaldehyde, dehydrated, embedded, and then sectioned using a microtome. The tumor sections were dewaxed, hydrated, heat antigen repaired, immunohistochemical circles were drawn, nonspecific sites were blocked, and antibody staining was performed at room temperature. The results were observed under a microscope. The expression of PTEN in each experimental group was evaluated. The results are as follows: Fig.12 As shown, this indicates that the metal ion complex mRNA delivery system can effectively deliver PTEN mRNA to the tumor site and achieve the restoration of PTEN in the tumor.

[0119] Application Example 2

[0120] Evaluation of mRNA vaccine antibody titers using metal ion complexed mRNA delivery system

[0121] The RBD mRNA delivery systems prepared in Examples 6 to 8 and Comparative Example 5 were used to immunize mice by intramuscular injection, each injection containing 5 μg of RBD mRNA, and the immunization program included a primary immunization at week 0 and two booster immunizations at weeks 3 and 6. Blood was collected through the ocular vein, and serum samples were collected on day 56 for antibody titer determination.

[0122] The specific method for antibody titer determination is as follows: RBD monomer protein was diluted to 3 μg / ml using ELISA coating solution (Solarbio), then added to a 96-well ELISA plate and incubated at 4°C for 12 hours. Next, the plate wells were blocked for 1 hour using skim milk (BD) prepared with 5% PBS. Serum samples were diluted in 3-fold proportions starting from a concentration of 1:20 using 5% skim milk as the dilution medium (the final concentrations were 1:20, 1:60, 1:180, 1:540, 1:1620, 1:4860, 1:14580, 1:43740, and 1:131220, respectively). After washing with PBS, the diluted serum samples were added to the wells and incubated at 37°C for 2 hours. After that, wash again with PBST, add goat anti-mouse IgG (Gene-Protein Link, P03S01M) diluted 1:2000, and continue incubation at 37°C for 1.5 hours. After five PBST washes, add 60μl of TMB solution (Beyotime) to each well. The reaction is finally terminated with 2M hydrochloric acid, and the absorbance (OD450) is measured at a wavelength of 450nm using an enzyme reader. The antibody titer of the serum is defined as the maximum dilution ratio when the serum reaction value reaches 2.5 times the average value of the blank well. The results are shown in Fig.13 As shown in the figure, the antibody titers from high to low are Mn-Mo-RBD-@M, Mn-RBD-NP@M, Mn-RBD-NP@L, and RBD-LNP. This shows that the metal ion complexed mRNA delivery systems prepared by the present invention all produce higher antibody titers than LNP. 2+ and Mo 4+ Complexing with mRNA and encapsulating mRNA through natural membranes can further enhance the immune effect of mRNA.

[0123] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0124] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0125] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing a metal ion complexed mRNA delivery system, characterized in that: The preparation method comprises the following steps: (1) mixing mRNA and metal ions in a solvent at 10 to 35° C. for 10 to 60 minutes to obtain nanoparticles; (2) The nanoparticles are mixed with the phospholipid membrane at 10 to 35° C. for 2 to 60 minutes, and the nanoparticles are wrapped with the phospholipid membrane to obtain the metal ion complexed mRNA delivery system.

2. The preparation method according to claim 1, characterized in that: The metal ions in step (1) include Mn 2+ and / or Mo 4+ .

3. The preparation method according to claim 1 or 2, characterized in that: The metal ion in step (1) is Mn 2+ and Mo 4 + ; Preferably, the Mn 2+ and Mo 4+ The molar ratio is (1~10):(1~10).

4. The preparation method according to any one of claims 1 to 3, characterized in that: The mass ratio of mRNA to metal ion in step (1) is (1-10):(10-100); Preferably, the mass ratio of metal ions to phospholipid membranes in the nanoparticles in step (2) is 1:(0.1-10).

5. The preparation method according to any one of claims 1 to 4, characterized in that: The phospholipid membrane in step (2) includes a natural membrane and / or an artificial synthetic lipid membrane; Preferably, the natural membrane comprises any one or a combination of at least two of an endothelial cell membrane, a human embryonic kidney cell membrane, a macrophage cell membrane, a breast cancer cell membrane, a colorectal cancer cell membrane or a melanoma cell membrane; Preferably, the raw materials for preparing the artificial synthetic lipid membrane include hydrogenated soybean lecithin, cholesterol and PEGylated lipid; Preferably, the phospholipid membrane is a natural membrane.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The solvent in step (1) includes water or phosphate buffer.

7. The preparation method according to any one of claims 1 to 6, characterized in that: The mixing in step (1) further comprises centrifugation and resuspending the precipitate after centrifugation; Preferably, the centrifugal speed is 1000-20000g.

8. The preparation method according to any one of claims 1 to 7, characterized in that: Ultrasound is also performed during the mixing process of step (2); Preferably, the mixing in step (2) further comprises centrifugation and resuspending the precipitate after centrifugation; Preferably, the centrifugal speed is 1000-20000g.

9. A metal ion complex mRNA delivery system prepared by the method for preparing the metal ion complex mRNA delivery system according to any one of claims 1 to 8.

10. Use of the metal ion complex mRNA delivery system as claimed in claim 9 in the preparation of mRNA vaccines and / or mRNA drugs.

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