A metal ion-doped mRNA delivery platform and preparation method thereof

Abstract

The present invention relates to the field of nanomaterials, and in particular to a metal ion-doped mRNA delivery platform and a preparation method thereof. The method comprises mixing mRNA with divalent manganese ions and forming nuclei to obtain core particles; and then encapsulating the core particles with phospholipid molecules. The present invention utilizes the interaction between divalent manganese ions and mRNA to enrich the two into nuclei, and a large amount of mRNA can be encapsulated inside using a small dose of phospholipids. The reaction process is simple and efficient, and the conditions are mild. It not only reduces the amount of phospholipids, reduces the risk of side effects represented by inflammatory reactions, reduces the production of anti-drug antibodies, but also can enhance the in vivo immune effect with the help of the immune adjuvant effect of manganese ions. When using the same phospholipid dosage, the present invention can deliver a higher content of mRNA than commercial LNP products, achieve stronger in vivo expression and functional effects, and provide a new platform tool for building a safer and more effective mRNA delivery system.

Description

Technical Field

[0001] The present invention relates to the field of nanomaterials, and in particular to a metal ion-doped mRNA delivery platform and a preparation method thereof. Background Art

[0002] mRNA is a bridge in the process of genetic information transmission, which can encode proteins to make them effective in treating diseases. The mitigation of Covid-19 depends largely on the rapid development and use of mRNA vaccines. Currently commercialized vaccines include two products from Pfizer and Moderna. The carrier used is lipid nanoparticles (LNP) containing four phospholipids. The advantages of LNP include: clear components, high encapsulation rate, and low raw materials. The structure of mRNA encapsulated inside LNP is still unclear for the time being. Existing studies have proposed three models, including multilayer vesicles, nanostructured cores, and uniform core-shells. Another research group's work analyzed the actual mRNA-loaded LNPs based on multicolor spectroscopy technology, accounting for about 20%, and about 80% of LNPs have no mRNA load. Combined with the above work, it was found that there is still room for improvement in the mRNA drug loading in LNP. A higher drug loading corresponds to less phospholipid input, which can, on the one hand, reduce the cytotoxicity that may be caused by ionizable phospholipids, and may ultimately alleviate symptoms such as headache, fatigue and fever caused by the injection of mRNA vaccines; on the other hand, it can slow down the accelerated blood clearance effect caused by the production of Anti-PEG Antibody produced against PEG phospholipids, thereby reducing the efficacy of mRNA vaccines or mRNA drugs.

[0003] In addition, the effectiveness of mRNA vaccines and mRNA drugs still needs to be strengthened. Some studies have improved the effectiveness of vaccines and drugs and enhanced immune function by developing and screening new functional phospholipids, or by combining the delivery of vaccines with adjuvants, but none of them have solved the problems of low mRNA loading capacity and poor practicality of the delivery system. Summary of the invention

[0004] In order to solve the technical problem of low mRNA loading amount in the above-mentioned mRNA vaccines or mRNA drugs, the present invention provides a metal ion-doped mRNA delivery platform and a preparation method thereof.

[0005] Specifically, in the first aspect, the preparation method of the metal ion-doped mRNA delivery platform provided by the present invention comprises mixing mRNA with divalent manganese ions and nucleating to obtain core particles; and then encapsulating the core particles with phospholipid molecules. The present invention has developed a platform technology for co-delivery of antigens and manganese ion immune adjuvants with high loading capacity, mild and rapid reaction conditions, and ensures the delivery effect of mRNA while reducing phospholipids. The role of manganese ions in the system is to stimulate the STING pathway as an immune adjuvant, and on the other hand, it is responsible for condensing the mRNA originally loosely distributed in the LNP to increase the loading capacity. The first step reaction time of the overall technology is only 5min, and the reaction temperature is a relatively mild 65°C, highlighting the mild and rapid synthesis conditions. The final in vivo expression and tumor treatment experiments showed a more prominent effect compared to LNP. At the same time, the corresponding phospholipid antibodies in the body were measured to have a lower concentration than traditional LNPs, which is conducive to reducing side effects and has the prospect of further clinical application. In addition, the functional ionizable phospholipids and the delivered mRNA used in this platform technology can be replaced, proving that the mRNA delivery platform (including mRNA vaccines and mRNA drugs) technology of the present invention has a certain universality.

[0006] The LNP-modified mRNA delivery platform provided by the present invention introduces metal ions, which, on the one hand, increases the mRNA loading capacity, increases the efficiency of phospholipid use and reduces the inflammatory side effects caused by some phospholipids (such as ionizable phospholipids or PEG-modified phospholipids); on the other hand, metal ions with immune adjuvant effects can enhance the immune response, thereby achieving the design goal of a safer and more effective mRNA delivery platform.

[0007] According to the present invention, mRNA vaccines / mRNA drugs are immune means with broad application prospects. Among the various mRNA delivery systems that have been developed, LNP is used to combat novel coronavirus infection because of its stability, safety and effectiveness. However, studies have found that some of the phospholipids used in LNP will produce certain inflammatory side effects, and a certain proportion of LNPs have a "no-load" phenomenon. Therefore, the present invention starts from increasing the mRNA loading capacity and increasing the efficiency of phospholipid use, using metal ions to first condense a large amount of mRNA into nuclei, and then uses the LNP formula ratio but a relatively reduced amount to wrap the kernel, in which manganese ions will also have the effect of an immune adjuvant to enhance the immune response, and ultimately produce a safer and more effective mRNA-based delivery platform technology.

[0008] Preferably, the length of the mRNA is 500 to 10000 nt, preferably 1000 to 6000 nt. In the present invention, the mRNA includes but is not limited to luciferase mRNA, spike mRNA, EGFP mRNA or OVA mRNA.

[0009] Preferably, the method comprises mixing the aqueous solution of mRNA with the divalent manganese ion solution using a pipette, vortexing the mixture, heating, centrifuging to remove the supernatant and resuspending with deionized water to obtain an aqueous solution containing the core particles; or mixing the aqueous solution of mRNA with the divalent manganese ion solution at the same flow rate using a microfluidic system, and vortexing, heating, centrifuging and resuspending the mixture to obtain an aqueous solution containing the core particles.

[0010] More preferably, the mixture of divalent manganese ions and mRNA is prepared using RNase-free water;

[0011] Preferably, the vortexing time is 1 to 30 minutes, preferably 6 minutes;

[0012] And / or, the heating method is metal bath heating or using a microfluidic internal heating device; preferably, the heating temperature is 37-85°C, preferably 60-70°C, more preferably 65°C; the heating time is 1-60 min, preferably 4-10 min, more preferably 5 min;

[0013] And / or, the centrifugal rotation speed is 1000-80000g, preferably 60000g.

[0014] Further preferably, in the mixture, the concentration of divalent manganese ions is 0.5 to 1000 mM, and the divalent manganese ion solution is preferably a solution of manganese chloride or manganese sulfate and the corresponding hydrate; and / or, in the mixture, the concentration of nucleotides is 50 to 10000 μM; and / or, in the mixture, the molar concentration ratio of divalent manganese ions to nucleotides in mRNA is (1 to 2000):1.

[0015] Preferably, the phospholipid molecules are composed of ionizable phospholipids, cholesterol, neutral lipids and PEGylated lipids; the phospholipid molecules are preferably composed of ionizable phospholipids, cholesterol, DSPC and DMG-PEG2000, and the ionizable phospholipids include but are not limited to SM-102, Dlin-MC3-DMA or ALC-0315.

[0016] Further preferably, the method comprises mixing an anhydrous ethanol solution containing phospholipid molecules and an aqueous solution containing the core particles, mixing repeatedly by blowing for several times, and dialyzing the mixed solution in PBS after standing; or using a microfluidic device to pass the anhydrous ethanol solution containing phospholipid molecules and the aqueous solution containing the core particles through a pipeline at a flow rate ratio of 1:(1-10), and dialyzing the obtained mixed solution in PBS.

[0017] More preferably, the volume ratio of anhydrous ethanol to the aqueous solution is 1:(1-50); preferably 1:(2-10);

[0018] Preferably, the mass ratio of the phospholipid molecule to the mRNA is (10-0.5):1, preferably 10:1;

[0019] Preferably, the standing time is 0.1 to 24 hours, preferably 1 hour.

[0020] The preparation method provided by the present invention is gentle and rapid, and enables manganese ions and mRNA to achieve supramolecular self-assembly into nanoparticles through coordination and electrostatic interaction. The preparation method of the metal ion-doped mRNA delivery platform provided in a further preferred embodiment of the present invention is as follows:

[0021] A1) mixing the divalent manganese ion solution and the aqueous solution containing mRNA, vortexing for 5-10 minutes, preferably vortexing for 8 minutes, placing in a metal bath and heating at 45-100° C., preferably at 65° C., for 2-30 minutes, preferably for 5 minutes, and cooling to room temperature after heating;

[0022] A2) centrifuging the solution obtained in step A1) at a speed of 60,000 g for 5 to 30 min, preferably for 20 min, removing the supernatant after centrifugation, and dispersing the precipitate in water to obtain an aqueous solution containing the core particles;

[0023] B1) dissolving phospholipid molecules in anhydrous ethanol in proportion, injecting the aqueous solution containing the core particles into the anhydrous ethanol using a pipette, wherein the weight ratio of the phospholipids in the anhydrous ethanol to the weight ratio of the core particles in the aqueous solution is (15-1):1, preferably 10:1, and the volume ratio of anhydrous ethanol to the aqueous solution is 1:(2-10), preferably 1:3, and repeatedly mixing by blowing 100-500 times with a pipette, preferably 200 times; standing for 0.5-3 hours, preferably 1 hour, and dialyzing the mixed solution in PBS, preferably 0.5-2 hours.

[0024] The second aspect of the present invention provides a metal ion-doped mRNA delivery platform, which is obtained by the preparation method of the above-mentioned metal ion-doped mRNA delivery platform.

[0025] Preferably, the mRNA and the divalent manganese ions in the inner core particle contain coordination bonds and electrostatic interactions.

[0026] Preferably, in the metal ion-doped mRNA delivery platform, the mass proportion of divalent manganese ions is 1% to 5%, the mass proportion of mRNA is 5% to 90%, and the mass proportion of phospholipid molecules is 15% to 94%; preferably, the mass proportion of mRNA is 5% to 20%, and the mass proportion of phospholipid molecules is 75% to 95%.

[0027] The present invention also provides a method for preparing the metal ion-doped mRNA delivery platform or an application of the metal ion-doped mRNA delivery platform, including any of the following:

[0028] 1) Application in the preparation of mRNA vaccines and / or mRNA drugs;

[0029] 2) Application in the preparation of drugs for enhancing immunity, immunotherapy and tumor treatment.

[0030] The beneficial effects of the present invention are at least:

[0031] 1) The present invention can produce mRNA vaccines and mRNA therapeutic drug platforms with higher loading, more effectiveness and safety, and the overall synthesis conditions are mild and rapid, and the biological activity of mRNA can be guaranteed to the maximum extent;

[0032] 2) All reactants used in the mRNA delivery platform of the present invention have good biocompatibility, no toxic side effects, and have been approved by the FDA, with strong prospects for clinical transformation;

[0033] 3) The mRNA delivery platform of the present invention combines antigen: mRNA and adjuvant: Mn 2+ Through coordination bonds and electrostatic interactions, the nanoparticles are combined and the antigen and immune adjuvant are co-delivered to increase the antibody titer and improve the immunotherapy effect.

[0034] 4) The mRNA delivery platform of the present invention compacts mRNA through metal ions, changes its originally loose structure in LNP, improves phospholipid utilization, increases loading capacity, further reduces the titer of anti-drug antibodies brought by PEG phospholipid components, increases the immune efficiency of mRNA vaccines and mRNA drugs, further improves vaccine and drug efficacy, and improves system applicability and immunotherapy effects. The mRNA delivery platform constructed with the participation of metal ions provided by the present invention has mild and rapid reaction conditions, can deliver immune adjuvants and antigens together into the body, increases antibody titer, is safe and has no side effects, and reduces the titer of anti-drug antibodies to slow down their accelerated clearance effect in the blood, improves the efficiency of the delivery platform, and has a strong prospect for clinical application transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. It is obvious that the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1The morphology (a) and particle size and dispersion characterization (b) of Mn-mRNA and Lipid@Mn-mRNA (L@Mn-mRNA) in the embodiments of the present invention, scale bar = 200 nm;

[0037] Figure 2 The cellular uptake of LNP (black group) and Lipid@Mn-mRNA (red group) in DC2.4 cells at 4h (a) and 10h (b) and quantitative statistics (c) were analyzed by flow cytometry in the examples of the present invention;

[0038] Figure 3 The bioluminescence intensity and quantitative statistics (b) of LNP (black group) and Lipid@Mn-mRNA (red group) in small animals at 6h and 24h (a) were analyzed using the small animal imaging system (IVIS) in the examples of the present invention;

[0039] Figure 4 The expression of p-TBK1 and p-IRF3 related proteins in the STING related pathway of each group was verified by immunoblotting (a) and quantitative statistics (b) and (c) in the examples of the present invention;

[0040] Figure 5 The changes in tumor volume over time after different groups of mice were given drugs after being tumor-bearing in the examples of the present invention;

[0041] Figure 6 The figures are the changes in the titers of Anti-PEG IgG (A) and IgM (B) antibodies in mice before and after administration in the examples of the present invention. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] If no specific technology or conditions are specified in the embodiments of the present invention, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the devices, instruments, reagents, etc. is not specified, they are all conventional products that can be purchased through regular channels. The raw materials used in the present invention can be easily purchased in the domestic product market.

[0044] In the embodiment of the present invention, by optimizing and transforming the LNP delivery platform, reducing the amount of carrier phospholipids, the immune adjuvant metal ions, manganese ions and antigen mRNA are delivered to the body, and the reaction process temperature is relatively mild, only 65 ° C, and the reaction process is very rapid, only 5 minutes. At the same time, it has been verified that the delivery platform can deliver mRNA of different lengths and can be delivered using different functional phospholipids, with good interchangeability and versatility. All reaction raw materials used in the present invention are FDA-approved products, non-toxic and harmless, with good biocompatibility, and have a wide range of clinical application prospects.

[0045] Example 1. Synthesis and characterization of Mn-mRNA core structure and L@Mn-mRNA core-shell structure

[0046] In this example, the mRNA is luciferase mRNA (1912 nt) and the ionizable phospholipid is SM-102. Specifically, for a reaction volume of 200 μL, a 600 μL EP tube was selected. First, 50 μL of 6 mM Mn 2+ Solution (tetrahydrate manganese chloride aqueous solution) and 130 μL enzyme-free water were mixed, and then 20 μL luciferase mRNA with a concentration of 1 mg / mL was mixed with the former, and then placed on a vortexer for 6 minutes, and then heated at 65 ° C in a metal bath for 5 minutes. After the reaction was completed and returned to room temperature, the unreacted product was removed at a speed of 60000g, and Mn-mRNA particles were dispersed with 200 μL enzyme-free water. Then SM-102, cholesterol, DSPC and DMG-PEG2000 were mixed with anhydrous ethanol as solvent according to the ratio in the preparation (molar ratio 50:38.5:10:1.5), and the volume ratio of anhydrous ethanol: aqueous phase = 1:3, the mass ratio of phospholipid: mRNA = 10:1, and the aqueous phase was quickly injected into the ethanol phase using a pipette, and the mixture was repeatedly blown 100 times, and then left at room temperature for 1 hour. Finally, a dialysis cup with a molecular weight cutoff of 10 kDa was used to replace the excess ethanol in the system to obtain L@Mn-mRNA.

[0047] After obtaining Mn-mRNA and L@Mn-mRNA, the surface morphology was characterized by biological transmission electron microscopy. It was found that Mn-mRNA was a monodispersed spherical particle with a smooth surface, and L@Mn-mRNA had an additional film on this basis. At the same time, after coating with phospholipids, the particle size increased from 65.66nm in the core to 89.01nm in the end. The surface potential increased from -14.6mV of NP to +1.06mV of L@NP. Figure 1 The morphology (a) and particle size and dispersion characterization (b) of Mn-mRNA and L@Mn-mRNA are shown, scale bar = 200 nm.

[0048] Example 2. Comparison of the uptake of L@Mn-mRNA and LNP in DC2.4 cells using Cy-5 labeled luciferase mRNA as a reporter gene

[0049] The same method as in Example 1 was used, except that the mRNA in this example was Cy-5 labeled luciferase mRNA, and the ionizable phospholipid was SM-102 phospholipid. The prepared L@Mn-mRNA and LNP loaded with mLUC were incubated with DC2.4 cells respectively, and the cells were collected after 10 hours. The cells were analyzed by fluorescence quantitative analysis using flow cytometry. The results showed that the fluorescence intensity of L@Mn-mRNA was twice that of LNP. The L@Mn-mRNA prepared by the platform of the present invention had a more efficient cell uptake ability ( Figure 2 ).

[0050] Example 3. Comparison of in vivo expression of L@Mn-mRNA and LNP using luciferase mRNA as reporter gene

[0051] In this example, the mRNA is luciferase mRNA, and the ionizable phospholipid is SM-102 phospholipid. The L@Mn-mRNA prepared in Example 1 and the LNP loaded with mLUC were injected into the tail base of BALB / c mice, and the injection site was imaged using a small animal imaging system for bioluminescence at 6h and 24h after injection. After quantitative analysis, the bioluminescence intensity of L@Mn-mRNA at the injection site was twice that of LNP, which may be due to better cellular uptake ( Figure 3 ).

[0052] Example 4. Verification of L@Mn-mRNA in vivo from Mn 2+ Western blot characterization of activated STING-related immune pathways

[0053] The same method as in Example 1 was used, except that the mRNA in this example was OVA mRNA, and the ionizable phospholipid was SM-102 phospholipid. First, primary bone marrow-derived dendritic cells (BMDC) were extracted from 6-week-old male mice. After incubating L@Mn-mRNA and LNP with BMDC for 24 hours, the cells were lysed to extract intracellular proteins for western blotting characterization of STING-related immune pathways. Quantitative analysis was performed on the downstream gene TBK1 and the corresponding phosphorylated p-TBK1 of the STING-related pathway, and the downstream gene IRF3 and the corresponding phosphorylated p-IRF3. The results of western blotting demonstrated that L@Mn-mRNA activated the STING-related pathway, such as Figure 4 As shown, 1 is the PBS group, 2 is the LNP group, 3 is the half-dose group of L@Mn-mRNA, and 4 is the L@Mn-mRNA group.

[0054] Example 5. Evaluation of the anti-tumor effect of L@Mn-mRNA as a therapeutic vaccine in vivo

[0055] Same as Example 4, except that the mRNA was OVA mRNA and the ionizable phospholipid was SM-102 phospholipid. First, a certain number of melanoma B16-OVA cells were injected into the right back of BALB / c mice to establish a B16-OVA tumor model. One week later, L@Mn-mRNA (mRNA dose 10 μg) / L@Mn-mRNA (5 μg) / LNP (10 μg) / PBS were injected subcutaneously in the groin of both sides of the mice. The volume of the mouse tumor was measured ( Figure 5 ), proving that the L@Mn-mRNA in the present invention has a good therapeutic effect as a therapeutic vaccine.

[0056] Example 6. Verification that the L@Mn-mRNA vaccine platform of the present invention produces lower anti-PEG antibody titers after administration than LNP

[0057] In this example, the mRNA is OVA mRNA, and the ionizable phospholipid is SM-102 phospholipid. Based on Example 5, serum samples of mice in each group were taken before treatment and after the final treatment, and the titers of Anti-PEG IgG and Anti-PEG IgM antibodies before and after administration of each mouse were measured using an ELISA kit. Figure 6 As shown, the vaccine platform of the present invention will produce lower Anti-PEG antibody titers than LNP while having better therapeutic effects, further avoiding the accelerated blood clearance effect and the toxic side effects caused by phospholipids.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a metal ion-doped mRNA delivery platform, characterized in that: The method comprises mixing an aqueous solution of mRNA with a divalent manganese ion solution by a pipette, vortexing the mixture, heating it, centrifuging it to remove the supernatant and resuspending it with deionized water to obtain an aqueous solution containing core particles; or mixing an aqueous solution of mRNA with a divalent manganese ion solution at the same flow rate by a microfluidic system, vortexing it, heating it, centrifuging it and resuspending it to obtain an aqueous solution containing core particles; and then wrapping the core particles with phospholipid molecules; the heating temperature is 37-85°C and the heating time is 1-60 minutes.

2. The method for preparing the metal ion-doped mRNA delivery platform according to claim 1, characterized in that: The length of the mRNA is 500 to 10000 nt.

3. The method for preparing the metal ion-doped mRNA delivery platform according to claim 2, characterized in that: The length of the mRNA is 1000 to 6000 nt.

4. The method for preparing the metal ion-doped mRNA delivery platform according to claim 1, characterized in that: The mixture of divalent manganese ions and mRNA was prepared using RNase-free water.

5. The method for preparing the metal ion-doped mRNA delivery platform according to claim 1, characterized in that: The vortexing time is 1 to 30 minutes; and / or, the heating method is metal bath heating or using a microfluidic internal heating device.

6. The method for preparing the metal ion-doped mRNA delivery platform according to claim 1, characterized in that: The heating temperature is 60-70° C.; the heating time is 4-10 min; and / or the centrifugal speed is 1000-80000 g.

7. The method for preparing the metal ion-doped mRNA delivery platform according to claim 1, characterized in that: In the mixture, the concentration of divalent manganese ions is 0.5 to 1000 mM; and / or, in the mixture, the concentration of nucleotides is 50 to 10000 μM; and / or, in the mixture, the molar concentration ratio of divalent manganese ions to nucleotides in mRNA is (1 to 2000):

1.

8. The method for preparing the metal ion-doped mRNA delivery platform according to claim 1, characterized in that: The phospholipid molecules are composed of ionizable phospholipids, cholesterol, neutral lipids and PEGylated lipids.

9. The method for preparing the metal ion-doped mRNA delivery platform according to claim 8, characterized in that: The phospholipid molecules are composed of ionizable phospholipids, cholesterol, DSPC and DMG-PEG2000, and the ionizable phospholipids include but are not limited to SM-102, Dlin-MC3-DMA or ALC-0315.

10. The method for preparing the metal ion-doped mRNA delivery platform according to claim 8, characterized in that: The method comprises mixing an anhydrous ethanol solution containing phospholipid molecules and an aqueous solution containing core particles, blowing and beating the mixture several times to mix repeatedly, and dialyzing the mixed solution in PBS after standing; or using a microfluidic device to pass the anhydrous ethanol solution containing phospholipid molecules and the aqueous solution containing core particles through a pipeline at a flow rate ratio of 1: (1 to 10), and dialyzing the obtained mixed solution in PBS.

11. The method for preparing the metal ion-doped mRNA delivery platform according to claim 10, characterized in that: The volume ratio of anhydrous ethanol to the aqueous solution is 1:(1-50).

12. The method for preparing the metal ion-doped mRNA delivery platform according to claim 11, characterized in that: The volume ratio of anhydrous ethanol to the aqueous solution is 1:(2-10).

13. The method for preparing the metal ion-doped mRNA delivery platform according to claim 11, characterized in that: The mass ratio of the phospholipid molecules to the mRNA is (10-0.5):1; and / or the standing time is 0.1-24h.

14. A metal ion-doped mRNA delivery platform, characterized in that: Obtained by the preparation method of the metal ion-doped mRNA delivery platform according to any one of claims 1-13.

15. The metal ion-doped mRNA delivery platform according to claim 14, characterized in that: The mRNA and divalent manganese ions in the inner core particles contain coordination bonds and electrostatic interactions; and / or, in the metal ion-doped mRNA delivery platform, the mass proportion of divalent manganese ions is 1% to 5%, the mass proportion of mRNA is 5% to 90%, and the mass proportion of phospholipid molecules is 15% to 94%.

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