A low-dose metallic Co 2+ Ion co-delivery mRNA delivery vectors and their applications

Abstract

This application discloses a low-dose metal Co 2+ This invention relates to an ion-co-delivery mRNA delivery vector and its applications, belonging to the field of biomedical technology. The mRNA delivery vector comprises a lipid component and an active ingredient, the active ingredient being Co. 2+ Stable mRNA-Co formed by co-incubation with mRNA 2+ Complex. Low dose of metallic Co. 2+ Ions and mRNA are pre-incubated to form a complex to prepare lipid nanoparticles (LNPs), which improve the expression efficiency of mRNA in vivo and in vitro, enhance endosome escape and protein expression levels without affecting cellular uptake, and have good biocompatibility, versatility and safety.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to a low-dose metallic Co. 2+ Ion co-delivery mRNA delivery vectors and their applications. Background Technology

[0002] Messenger RNA (mRNA), a class of single-stranded nucleic acid molecules carrying protein-coding information, can directly express target proteins in the cytoplasm using the host cell's translation mechanism, without needing to enter the cell nucleus or posing a risk of genome integration. Based on these characteristics, mRNA therapy has shown great potential in numerous fields such as infectious disease vaccines, tumor immunotherapy, and protein replacement therapy, and mRNA is also considered a highly efficient and safe form of nucleic acid drug.

[0003] Naked mRNA is easily degraded by nucleases and struggles to cross cell membranes autonomously, making efficient in vivo delivery systems crucial for the clinical translation of mRNA therapies. Currently, lipid nanoparticles (LNPs) are the most widely used and mature mRNA delivery vectors, encapsulating mRNA to form a complex that enters the cell via endocytosis. However, existing LNP delivery systems still face significant efficiency bottlenecks in practical applications: after endocytosis, most of the mRNA-LNP complex is trapped in endosomes and eventually transported to lysosomes for degradation, with only a small amount of mRNA escaping from the endosomes and being released into the cytoplasm. This transfection bottleneck results in a low number of functional mRNAs reaching the translation site, leading to low overall expression efficiency of the target protein. To achieve the required therapeutic protein levels, it is often necessary to increase the dosage of both mRNA and LNPs, but this increases the risk of vector-related cytotoxicity and in vivo accumulation, limiting the safety window and efficacy of the therapy.

[0004] To address the aforementioned issue of insufficient expression efficiency, previous studies have proposed incorporating manganese ions (Mn) into the expression process. 2+ Introduced into the mRNA delivery system, via Mn 2+ Coordination with mRNA forms a complex, utilizing manganese ions to promote endosome escape and enhance cytoplasmic delivery, thereby improving mRNA expression efficiency. However, achieving a significant increase in expression efficiency requires an effective amount of manganese (Mn). 2+ The concentrations are generally high; and manganese, as an essential trace element for the human body, exhibits clear neurotoxicity when excessive, especially the accumulation of free manganese ions. Long-term or high-dose exposure can cause manganese deposition in the central nervous system, leading to some irreversible damage. This high-dose dependence and the resulting safety concerns greatly limit the further application of manganese-based strategies in mRNA therapy.

[0005] Therefore, there is an urgent need to develop a new method that can safely and efficiently improve mRNA expression efficiency at significantly lower doses. Summary of the Invention

[0006] In view of this, the primary objective of this application is to provide a low-dose metallic Co 2+ Ion co-delivery mRNA delivery vector, utilizing metal Co 2+ Ions and mRNA are pre-incubated to form a complex, which is then prepared into lipid nanoparticles (LNPs), thereby significantly improving the expression efficiency of mRNA in vivo and in vitro. This strategy can significantly enhance endosome escape and protein expression levels without affecting cellular uptake, while exhibiting good biocompatibility and versatility, and also utilizes the metal Co. 2+ The dosage of ions is very low, which can greatly avoid the neurotoxicity problems associated with manganese-based strategies.

[0007] To achieve the above objectives, this application adopts the following technical solution: One aspect of this application discloses a low-dose metallic Co 2+ An ion-co-delivery mRNA delivery vector comprising a lipid component and an active ingredient, wherein the active ingredient is Co. 2+ Stable mRNA-Co formed by co-incubation with mRNA 2+ Complex.

[0008] Another aspect of this application discloses the preparation of the aforementioned low-dose metallic Co. 2+ A method for ion co-delivery mRNA delivery vectors includes the following steps: Add Co to the aqueous mRNA solution 2+ Under suitable conditions, co-incubation is performed to form mRNA-Co 2+ The aqueous phase of the complex; Prepare an organic phase containing lipid components; Will contain mRNA-Co 2+ The aqueous phase of the complex was mixed with the organic phase of the lipid component to prepare a low-dose metal Co. 2+ Ion co-delivery mRNA delivery vector.

[0009] Another aspect of this application is a formulation containing the aforementioned low-dose metallic Co. 2+ Ion co-delivery mRNA delivery vector.

[0010] This application has at least the following beneficial effects: (1) Low-dose use has good safety and biocompatibility.

[0011] The Co used in this application 2+It is added at a low dose, far below the threshold for significant cytotoxicity or tissue toxicity, and will not impose a significant safety burden on existing LNP systems. While ensuring improved delivery efficiency, it can maintain good cell compatibility and in vivo tolerability, which is beneficial to reducing the toxic side effects that may be caused by high-dose mRNA administration and improving the clinical application potential of the formulation.

[0012] (2) Significantly improves mRNA expression efficiency.

[0013] This application introduces a low dose of Co before the formation of lipid nanoparticles. 2+ The application utilizes ions to pre-form a stable complex with mRNA, which can significantly enhance protein expression levels after mRNA delivery. This application has significant advantages in enhancing the output of mRNA biological functions.

[0014] (3) It does not significantly affect the cellular uptake process.

[0015] The technical effect of this application does not primarily rely on increasing the uptake of nanoparticles by cells, but rather on enhancing mRNA expression by improving key rate-limiting steps after intracellular delivery, particularly in promoting endosome escape, improving cytoplasmic release efficiency, and enhancing subsequent translation. Therefore, this application can improve the actual utilization rate and expression efficiency of mRNA without significantly altering cellular uptake behavior, demonstrating a clear advantage in its mechanism of action.

[0016] (4) It has a wide range of applications and strong versatility.

[0017] The low-dose Co proposed in this application 2+ The co-delivery strategy is independent of specific mRNA sequences or single lipid formulations, making it applicable to a variety of mRNA molecules encoding different proteins. It is also compatible with different types of lipid nanoparticle systems, including but not limited to LNP formulations containing various ionized lipids, auxiliary lipids, sterols, and PEG-lipids. Therefore, this application possesses excellent platform versatility and can be widely applied in vaccine, protein replacement therapy, and other nucleic acid drug delivery fields.

[0018] (5) It helps to reduce the dosage and increase the application value.

[0019] Because this application can significantly improve the expression efficiency of mRNA per unit dose, it is expected to reduce the actual dosage of mRNA while achieving the same therapeutic or immunizing effect, thereby reducing raw material costs, mitigating potential toxic reactions, and improving the economics and clinical acceptability of the formulation. This feature is particularly important for high-cost nucleic acid drugs and large-scale vaccine production.

[0020] (6) The process is simple and easy to scale up and industrialize.

[0021] This application does not require significant modifications to existing LNP preparation equipment and main processes; it only requires the addition of Co to the conventional preparation process. 2+ A pre-incubation step with mRNA can significantly improve delivery efficiency. This method is simple to operate, highly reproducible, and highly compatible with existing microfluidic mixing or ethanol injection processes, facilitating standardized production and industrial scale-up, and has promising prospects for industrial application.

[0022] In summary, this application introduces a low dose of Co 2+ The synergistic delivery of ions and mRNA significantly improves the expression efficiency of the mRNA-LNP system without increasing the burden of complex processes. It also has the advantages of high safety, strong versatility and easy industrialization, and has outstanding technological progress and application value. Attached Figure Description

[0023] Figure 1 For the mRNA-Co in this application 2+ - Schematic diagram of the preparation process of LNP.

[0024] Figure 2 mRNA-Co in Example 1 2+ The characterization results of -LNP and control LNP, among which, Figure 2 In the middle, A represents particle size. Figure 2 B in the equation represents the polydispersity index (PDI). Figure 2 C represents the Zeta potential. Figure 2 In the middle, D represents the encapsulation efficiency. Figure 2 E represents the Pka characterization of LNP particles. Figure 2 F in the middle is Co 2+ -LNP particle Pka characterization. Figure 2 G in Co 2+ -LNP particle ITC characterization.

[0025] Figure 3 This is a diagram showing the in vitro cell transfection results in Example 2, where... Figure 3 A in the middle represents mRNA-Co 2+ Comparison of cell transfection methods for three LNP preparation methods. Figure 3 In this context, B represents LNP and Co. 2+ -Statistical graph of LNP granule cell transfection.

[0026] Figure 4 The results of the in vivo expression experiment in Example 3 are as follows, Figure 4 In Balb / c mice, different doses of Co were injected intramuscularly. 2+ Representative images and total fluorescence statistics of luciferase signals detected by LNP. Figure 4In C57 mice, different doses of Co were injected intramuscularly. 2+ Representative images and total fluorescence statistics of luciferase signals detected by LNP. Figure 4 C refers to C57 mice intravenously injected with n(Co) 2+ Representative images of luciferase signals detected in the heart, liver, spleen, lungs, and kidneys of LNPs with n(mRNA)=10, and a statistical graph of total fluorescence in the spleen and liver.

[0027] Figure 5 For Example 4, co-focusing imaging of LNPs and Co carrying Cy5-mRNA was performed. 2+ - After incubating LNP particles with 293T cells, Lysotracker staining was used to analyze the escape of mRNA endosomes in the two types of particles and the statistical analysis of Pearson coefficients.

[0028] Figure 6 LNP and Co in Example 5 2+ -LNP particle-cell viability statistics. Detailed Implementation

[0029] The embodiments of this application will be clearly and completely described below. The technical solutions in the embodiments described below are exemplary and only possible technical implementations of this application, not all possible implementations. Those skilled in the art can combine the embodiments of this application to obtain other embodiments without creative effort, and these embodiments are also within the protection scope of this application.

[0030] The first aspect of this application provides a low-dose metal Co 2+ An ion co-delivery mRNA delivery vector, which comprises two core components: a lipid component serving as both a structural framework and a delivery tool, and an active ingredient serving as the functional core.

[0031] In this application, the active ingredient is specified as: Co 2+ Stable mRNA-Co formed by co-incubation with mRNA 2+ Complex. It should be understood that the Co mentioned here... 2+ It does not exist in a free or simply physically mixed state, but rather refers to the spontaneous organization of the pre-complexed entity with mRNA macromolecules under specific conditions through intermolecular interactions, forming a thermodynamically stable, uniformly sized nanoscale complex. This pre-complexation method is one of the key features for achieving the low-dose, high-efficiency technical effects described in this application.

[0032] Low-dose Co in this application 2+ Ions can regulate the local structure and acidification environment of endosome membranes, thereby promoting the efficient release of mRNA from endosomes; simultaneously, Co2+ The ion may also stabilize mRNA conformation, improving its translation efficiency in the cytoplasm. Compared to traditional high-dose chemical modification or vector improvement strategies, Co... 2+ The co-delivery strategy is simple, universal, and easy to scale up, balancing efficient delivery with security.

[0033] In this application, mRNA refers to messenger ribonucleic acid, a single-stranded nucleic acid molecule carrying genetic information encoding a target protein, which can be prepared by in vitro transcription or other known methods. This application does not specifically limit the type, sequence, length, or function of the encoded protein of the mRNA. It should be understood that any functional mRNA molecule that needs to be translated and expressed in cells can be used in the low-dose Co metal of this application. 2+ Ion co-delivery delivery carrier.

[0034] As specific examples, applicable mRNAs include, but are not limited to: mRNAs encoding viral antigens for the preparation of prophylactic or therapeutic vaccines; mRNAs encoding deleted or dysfunctional endogenous proteins for protein replacement therapy; mRNAs encoding gene editing tools such as CRISPR-associated nucleases or base editors for gene editing therapy; mRNAs encoding immunomodulatory factors, cytokines, therapeutic antibodies, or chimeric antigen receptors for tumor immunotherapy or the treatment of infectious diseases; and mRNAs encoding metabolic enzymes, coagulation factors, or other functional proteins for the treatment of genetic diseases. The mRNAs may be natural sequences or modified mRNAs that have undergone codon optimization, base modification, or capping / tailing to meet specific delivery requirements. The length of mRNAs can range from hundreds to thousands or tens of thousands of nucleotides, and this application does not specifically limit this.

[0035] In some specific implementations, the mRNA-Co 2+ In the complex, Co 2+ The molar ratio of the target compound to mRNA should be between 0.5 and 15. This appropriate molar ratio range represents the golden window for balancing delivery efficiency and safety. It should be understood that when the molar ratio is below 0.5, its effect on enhancing expression efficiency is not significant; while when the molar ratio is above 15, although it can still enhance expression, it may trigger unnecessary cellular stress responses due to excessively high charge density, or introduce Co... 2+ Excessive dosage can compromise its safety. Therefore, in this application, Co... 2+ The molar ratio of co-mRNA is 0.5-15. For a specific example, Co... 2+ The molar ratio of mRNA to 0.5 can be any value from 0.5, 1, 2, 5, 8, 10, 12, 15, or a range between any two values. As a preferred example, Co... 2+The molar ratio of mRNA to co-mRNA is 9-11, which is even better. 2+ The molar ratio of Co to mRNA is 9.5-10.5; in some preferred embodiments, when Co 2+ When the molar ratio of mRNA to endosomes is 10, the system achieves an optimal synergistic state in promoting endosome escape and protecting mRNA from degradation, resulting in extremely high protein expression levels with very low toxicity risk.

[0036] Furthermore, another essential component of the delivery vector in this application is the lipid component, which provides a protective shell for the active complex and enables in vivo delivery. In this application, the lipid component includes at least one of ionized lipids, auxiliary lipids, PEG-lipids, and sterols. These four types of lipid molecules each play a unique role and are functionally synergistic and complementary. These lipid components can all be of conventional types in the art; appropriate lipid components can be selected based on the different mRNAs to achieve stable encapsulation and delivery. Those skilled in the art possess this ability, therefore no specific limitations are imposed.

[0037] Ionized lipids are the core functional molecules of the entire LNP delivery carrier. Their molecular structure typically includes a protonable amine head group, a linker arm, and a hydrophobic tail. Under acidic pH conditions (such as in an endosome environment), the head group gains a proton and carries a positive charge. This drives fusion with negatively charged phospholipids in the endosome membrane, enabling cytoplasmic release of mRNA. Furthermore, under the low pH conditions during preparation, it can also react with negatively charged mRNA-Co... 2+ The complex undergoes electrostatic bonding, driving the self-assembly of nanoparticles. As a specific example, the ionized lipid can be any one of DLin-MC3-DMA, SM-102, ALC-0315, or their derivatives, but is not limited thereto. In practical applications, other ionized lipids with similar structures and functions can also be selected, and this application does not limit this selection.

[0038] The primary function of the assisting lipid is to stabilize the lipid bilayer structure and facilitate cell membrane fusion. It is typically a phospholipid molecule with a zwitterionic head, capable of regulating the membrane fluidity of nanoparticles, enhancing the overall structural integrity of LNPs, preventing premature cargo leakage, and participating in promoting endosome escape processes. As specific examples, the assisting lipid can be DSPC or DOPE. Furthermore, other phospholipids with similar functions can also be selected as the assisting lipid; this application does not limit its selection.

[0039] The sterol component is intercalated between phospholipid molecules in the LNP, acting as a molecular buffer. It can regulate the fluidity and permeability of the lipid membrane, keeping the nanoparticles stable in the bloodstream while allowing them to leak moderately after being taken up into cells to facilitate release. As a preferred example, cholesterol is chosen because it is widely available, stable, and highly compatible with human cell membrane components.

[0040] PEG-lipids are a class of amphiphilic polymers with lipid anchor molecules covalently linked by polyethylene glycol chains. Their main function on the LNP surface is to provide steric hindrance, preventing uncontrolled aggregation, flocculation, or non-specific adsorption to serum proteins in nanoparticles during preparation, storage, and blood circulation. They also help regulate the final particle size of the nanoparticles. As a specific example, the PEG-lipid can be either DMG-PEG2000 or DSPE-PEG2000, but is not limited to these. It should be understood that in practical applications, other molecular weights and lipid anchor types can also be selected for the PEG-lipid, and this application does not limit this.

[0041] Furthermore, the ratio of lipids in the lipid component is not specifically limited; a suitable ratio can be determined experimentally based on the properties of the mRNA, the required particle size, encapsulation efficiency, and other requirements. In a preferred embodiment of this application, the molar ratio of ionized lipids, auxiliary lipids, sterols, and PEG-lipids is 50:10:38.5:1.5, which results in the prepared LNP exhibiting suitable particle size, excellent encapsulation efficiency, good in vivo neutral charge shielding effect, and efficient protonation ability in an acidic endosome environment, similar to the core active ingredient mRNA-Co. 2+ The complex works together to achieve precise control over the entire process of mRNA cargo, from preparation, circulation, targeting, endocytosis to escape.

[0042] The second aspect of this application provides for the preparation of the aforementioned low-dose metal Co. 2+ Methods for ion co-delivery mRNA delivery vectors This method first involves adding an appropriate amount of Co to the aqueous phase solution of mRNA. 2+ Under suitable conditions, co-incubation produces a product containing mRNA-Co 2+ The aqueous phase of the complex. Through pre-incubation, Co... 2+ Ions come into full contact with mRNA molecules in the liquid phase environment, guided by the interaction of positive and negative charges, Co 2+ Ions specifically anchor to the phosphodiester backbone of mRNA, triggering the folding and compression of the mRNA chain. This process is a spontaneous process precisely regulated by incubation conditions.

[0043] To ensure sufficient dissolution of mRNA in the aqueous phase and to facilitate uniform nanoparticle formation during subsequent mixing with the lipid organic phase, the concentration of the mRNA aqueous solution can be adjusted as needed. In some embodiments, the concentration of the mRNA aqueous solution can be controlled within the range of 0.01 mg / mL to 2.0 mg / mL. For example, concentrations of 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, or any range between two concentrations can be selected.

[0044] In this application, suitable conditions refer to incubation temperature, pH, time, etc., to obtain the target mRNA-Co 2+ Complex. In some embodiments, the pH of the incubation system is 5.0 to 7.4. The advantages of choosing this pH range are: (1) the hydrolysis rate of mRNA is lowest under weakly acidic to neutral conditions, thus preserving its integrity; (2) Co 2+ (2) It exhibits high coordination binding efficiency with the mRNA phosphate backbone and does not cause cobalt hydroxide precipitation; (3) It can ionize lipids to maintain a moderate positive charge to encapsulate mRNA without damaging the nanostructure of LNP; (4) This pH range is compatible with the endosome (pH 5.0~6.0) and cytoplasm / blood (pH 7.2~7.4) environments, which is beneficial for endosome escape and in vivo delivery; (5) It avoids cytotoxicity caused by extreme pH. Experiments have shown that when the pH is below 4.5 or above 8.0, the mRNA encapsulation efficiency decreases by more than 50%, and the in vitro transfection efficiency is significantly reduced. Therefore, in this application, pH 5.0~7.4 is the optimal pH range for achieving mRNA-Co²⁻. + -Key process parameters for efficient and low-toxicity delivery of LNP. This pH range is crucial for mRNA-Co. 2+ The complex provides a mild chemical environment that does not damage the integrity of the mRNA; the specific pH value can be any value or a range between any two of 5.0, 5.5, 6.0, 6.5, 7.0, and 7.4. The incubation time is controlled between 5 and 30 minutes, for example, 5, 10, 15, 20, 25, or 30 minutes. This time window is sufficient to ensure the complexation reaction proceeds fully while avoiding the potential degradation risk associated with prolonged operation. The incubation temperature is also selected within a mild and controllable range to ensure the uniformity and reproducibility of the entire process. The incubation temperature is 20-37°C, for example, any temperature or a range between any two of 20°C, 22°C, 25°C, 28°C, 30°C, 35°C, and 37°C. Through the synergistic control of these three parameters, stable mRNA-Co possessing all the aforementioned advantages can be prepared efficiently and batch-to-batch consistently. 2+ The aqueous phase of the complex.

[0045] It should be noted that the pre-incubation in this application includes the following steps: Dissolving a water-soluble salt of metallic Co in water forms a Co-containing... 2+ The aqueous solution was then pre-incubated with the mRNA aqueous phase solution under appropriate conditions; finally, the remaining volume of the aqueous phase was replenished with concentrated buffer.

[0046] Through stepwise control, the reaction of mRNA with Co was first completed in a neutral aqueous phase without competing ligands. 2+ The specific binding of [the substance] and the one-step switching to the ionic strength and pH conditions required for LNP assembly by adding concentrated buffer (instead of working concentration buffer) thus protect the functional mRNA-Co. 2+ The complex simultaneously ensures the encapsulation quality of LNPs, and the two work synergistically to achieve high mRNA expression. Specifically, the complex contains Co... 2+ The aqueous solution of the mRNA was pre-incubated with the aqueous phase solution of the mRNA under appropriate conditions to avoid competition for binding with substances such as citric acid in the buffer. 2+ It forms other Co complexes, thereby hindering Co. 2+ Effective coordination with the mRNA phosphate backbone prevents the formation of the required mRNA-Co. 2+ The complex ultimately results in low in vitro expression levels; therefore, buffer and low pH should be avoided during the pre-incubation phase to protect mRNA-Co. 2+ The integrity of the complex is crucial. Furthermore, after pre-incubation, the required pH must be rapidly reconstituted using a concentrated buffer. Using a conventional working buffer can lead to a pH shift in the final aqueous phase, weakening the protonation efficiency of ionizable lipids during subsequent LNP assembly, affecting mRNA encapsulation and endosome escape, ultimately reducing expression. Rapid replenishment with concentrated buffer provides the correct ionic and pH environment for the rapid and efficient self-assembly of LNPs, which is the direct reason for the significant increase in expression levels.

[0047] Simultaneously or sequentially with the mRNA pre-incubation step, this preparation method also involves preparing an organic phase to obtain the lipid components. This organic phase is typically formed by dissolving ionized lipids, auxiliary lipids, sterols, and PEG-lipids in a pre-defined precise molar ratio in a water-miscible organic solvent, such as anhydrous ethanol. The total lipid concentration of the organic phase can also be set according to the amount of mRNA used and the desired lipid / mRNA ratio. In some embodiments, the total lipid concentration in the organic phase can be controlled within the range of 1 mg / mL to 100 mg / mL. Commonly used exemplary concentrations include 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 50 mg / mL, 80 mg / mL, 100 mg / mL, etc., or any range between two concentrations. Selecting this concentration range ensures that the lipid components are completely dissolved and remain homogeneous in the organic phase, and obtains an appropriate mixing ratio and solvent environment when subsequently mixed with the aqueous phase, thereby facilitating the lipid molecules to react with mRNA-Co. 2+ The complex serves as the core for efficient self-assembly. It should be understood that the total lipid concentration can be adjusted within the scope of general knowledge in the art, depending on the specific lipid types selected, the mixing method, and the equipment used; this application is not limited to the specific values ​​mentioned above.

[0048] After preparing the aqueous and organic phases separately, LNPs were formed through self-assembly using a typical nanoprecipitation method, containing mRNA-Co. 2+ The aqueous phase of the complex was mixed with the organic phase of the lipid component to prepare a low-dose metal Co. 2+ Ion co-delivery mRNA delivery vector.

[0049] In some embodiments, the mixing method is either microfluidic mixing or ethanol injection. Upon contact between the two phases, the organic solvent is diluted, and the solubility of the lipid component decreases sharply, resulting in mRNA-Co... 2+ The complex serves as the core template for self-assembly, forming lipid nanoparticles with a core-shell structure through hydrophobic collapse and electrostatic neutralization.

[0050] In some embodiments, the metal Co 2+ The ions are introduced into the aqueous solution of mRNA in the form of their water-soluble salts. Introducing metal ions in the form of their salt solutions is simple to operate, quantitatively precise, and ensures that the ions rapidly and completely dissociate into a free state in the solution, thereby efficiently complexing with mRNA.

[0051] In the specific selection of the water-soluble salt, at least one of Co(NO3)2, CoCl2, and Co(OAc)2 can be chosen. These specific salts are preferred ion donors in this application due to their shared excellent water solubility, stable chemical properties, and non-damaging effect on mRNA. It should be understood that any other salt capable of safely and efficiently dissociating the metallic Co in aqueous solution is also acceptable. 2+ Pharmaceutically acceptable salt forms of ions are also included within the scope of protection of this application. The preferred solvent is an acetate-acetate buffer or a citrate-citrate buffer.

[0052] After forming the initial LNP suspension through the above mixing steps, this method further includes purification and buffer replacement steps to obtain a purified final product. The crude LNP suspension formed during mixing typically still contains residual organic solvents and water-insoluble byproducts. Purification steps, such as ultrafiltration or dialysis, can effectively remove free Co. 2+ Ions and organic solvents. Buffer replacement involves gradually replacing the acidic preparation environment of LNPs with a physiological pH environment (such as phosphate buffer at pH 7.4), terminating further assembly and providing a stable, isotonic final formulation suitable for in vivo administration.

[0053] A third aspect of this application discloses a formulation containing a low dose of metallic Co as described in any of the foregoing embodiments. 2+ Ion co-delivery mRNA delivery carriers. The formulation can be prepared in various pharmaceutical compositions or drugs containing an effective amount of such LNPs, depending on its end use. The formulation may also contain one or more pharmaceutically acceptable excipients, such as cryoprotectants, buffers, isotonic modifiers, etc., to maintain the physicochemical integrity and biological activity of the LNPs during long-term storage (e.g., in lyophilized or freeze-dried powder form) and after reconstitution.

[0054] The low-dose metal Co obtained through this application 2+ The synergistic effect of ion complexation strategy and optimized lipid formulation resulted in several significantly improved properties in the formulation. Regarding particle stability, the dense mRNA-Co... 2+ The complex core and stable lipid shell together endow the nanoparticles with higher structural rigidity, effectively inhibiting particle size growth and encapsulation leakage caused by aggregation or fusion in storage and physiological media, ensuring that the expected particle size and encapsulation efficiency can be maintained even after long-term storage. Regarding cell membrane fusion capability, thanks to the pre-complexation-induced mRNA conformational compression and the optimized lipid component design, the formulation can more rapidly trigger the protonation of ionized lipids in the acidic environment of the endosome, and achieve efficient membrane fusion and destabilization with the endosome membrane, thereby significantly promoting the cytoplasmic release of mRNA.

[0055] In some embodiments, the pharmaceutical composition or drug is a drug used for vaccines, protein replacement therapy, gene editing, tumor treatment, infectious disease treatment, or genetic disease treatment.

[0056] It should be understood that the specific examples above are only to illustrate the broad applicability of this application and do not limit the pharmaceutical use of the protected delivery vector or formulation. The technical solutions provided in this application can be flexibly extended to any medical scenario that can benefit from efficient and safe mRNA delivery.

[0057] Through the above technical solutions, the low-dose metallic Co provided in this application 2+ Ion co-delivery mRNA delivery vectors and formulations achieve protein expression enhancement effects far exceeding those of existing technologies without significantly increasing toxic side effects. This addresses the inherent safety concerns of high-dose metal ion strategies and provides a truly practical new option for the clinical translation of mRNA therapy.

[0058] The present application will be further illustrated below with reference to specific embodiments. It should be noted that the specific embodiments below are for illustrative purposes only and do not limit the scope of the present application in any way.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0060] In addition, unless otherwise specified, methods without detailed conditions or steps are conventional methods, and the reagents and materials used are commercially available.

[0061] Example 1: mRNA-Co 2+ Preparation and characterization of -LNP 1.1 mRNA-Co 2+ Preparation of -LNP This embodiment uses mRNA encoding luciferase as an example to provide a low-dose Co 2+ A method for preparing ion-co-delivered mRNA lipid nanoparticles.

[0062] (1) Dissolve the mRNA encoding luciferase (Luc mRNA) in 10mM acetate buffer (pH 5.5) to obtain a homogeneous and transparent aqueous solution of mRNA with a concentration of 1mg / ml.

[0063] (2) Dissolve Co(NO3)2·6H2O in water to prepare a 1 mg / ml aqueous solution; then pre-incubate it with the mRNA aqueous solution at room temperature for 15 minutes (Co2+ (The molar ratio of mRNA to co-mRNA is 10:1), making Co 2+ With the pre-formation of a stable mRNA-Co 2+ The remaining volume of the aqueous phase was replenished with citrate buffer (100 mM) to complete the complex.

[0064] (3) The lipid components were dissolved in ethanol to prepare a stock solution with a concentration of 10 mg / ml, and the lipid ethanol phase was prepared. The lipid components included ionized lipid SM-102, auxiliary lipid DSPC, cholesterol and PEG-lipid DMG-PEG2000, with a molar ratio of 50:10:38.5:1.5.

[0065] (4) Using a microfluidic mixing method, the mRNA-Co from step (2) is mixed... 2+ The aqueous phase of the complex was rapidly mixed with the lipid ethanol phase from step (3), wherein the flow rate ratio of the aqueous phase to the ethanol phase was 3:1. After rapid mixing, the lipid components self-assembled to form a structure encapsulated with mRNA-Co. 2+ Lipid nanoparticles of the complex.

[0066] (5) The nanoparticle dispersion obtained in step (4) is purified by dialysis and buffer replacement to remove unbound Co from the system. 2+ The ions and organic solvent ethanol were used, and the buffer system was replaced with PBS to obtain the final mRNA-Co. 2+ -LNP formulation.

[0067] Wherein, LNP represents the absence of Co. 2+ The lipid nanoparticles were prepared using the same steps as those for Co. 2+ -LNP is the same.

[0068] 1.2 mRNA-Co 2+ Characterization of -LNP The final mRNA-Co² obtained in section 1.1 + - Physicochemical characterization of LNP formulations: Particle size, polydispersity index (PDI), and zeta potential were determined using dynamic light scattering (DLS, such as Malvern Zetasizer). Samples were diluted with deionized water or PBS to a suitable concentration (e.g., 1:100), and each sample was measured three times.

[0069] Encapsulation efficiency was determined using the RiboGreen fluorescence method: the fluorescence intensity of LNP-containing and free mRNA after demulsification with Triton X-100 was measured separately, and the encapsulation efficiency (%) was calculated as follows: (Total mRNA) / (LNP-containing mRNA) = (LNP-containing mRNA) / ... (Free mRNA) / Total mRNA × 100%.

[0070] The pKa value was determined using the 6-p-tolueneaminonaphthalene-2-sulfonic acid (TNS) fluorescent probe method: LNP was dispersed in buffer solutions of different pH values ​​(3.0~10.5), TNS was added, the fluorescence intensity was measured, and the pKa was obtained by fitting.

[0071] Isothermal titration calorimetry (ITC, such as MicroCal PEAQ-ITC) is used to detect Co²⁺. + Thermodynamic parameters of binding between Co² and mRNA and LNP components: + The solution or LNP solution was titrated into the mRNA or blank LNP system, and the reaction heat was recorded. The binding constant (Ka), number of binding sites (n), and enthalpy change (ΔH) were obtained by fitting the solution with the accompanying software to evaluate Co². + Interaction patterns with mRNA and LNP.

[0072] The results are as follows Figure 2 As shown in the figure, it can be seen that the prepared mRNA-Co 2+ -LNPs exhibit uniform nanoscale size (~150 nm, PDI < 0.2), good stability (Zeta potential ~18 mV), and high encapsulation efficiency (>80%). The pKa value increased from 6.69 in the control group to 6.82, suggesting that Co... 2+ The introduction of Co facilitates the protonation of LNPs in the acidic environment of the endosome, thereby promoting endosome escape of mRNA. ITC thermodynamic analysis confirms Co 2+ It binds to mRNA at a binding site of approximately 0.47 (i.e., a nucleotide ratio of approximately 2:1:Co²). + Specific coordination binding occurs at a molar ratio of 66.4 μM, representing an entropy-driven spontaneous process (ΔG < 0). These physicochemical characteristics are associated with Co. 2+ The structure and thermodynamic basis for enhancing mRNA expression efficiency are provided.

[0073] Example 2: In vitro expression experiment The lipid nanoparticles prepared in Example 1 were used to transfect HEK293T cells (human embryonic kidney cells, ATCC® CRL-3216™) to evaluate the mRNA-Co prepared in this application. 2+ -LNP in vitro expression performance.

[0074] Cell culture: Cells were cultured in DMEM high-glucose medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (100 U / mL) and in a humidified incubator at 37°C and 5% CO2. After reaching 70-80% confluence, cells were passaged using 0.25% trypsin-EDTA digestion. Cells in the logarithmic growth phase were digested, counted, and seeded at an appropriate density in opaque white 96-well plates, with 1 × 10⁶ cells per well. 4 Cells were cultured in 100 μL of medium in a cell culture plate for 24 hours until the cells adhered and reached 70-80% confluence. Before transfection, the old medium was discarded, and fresh serum-free DMEM medium (50 μL for a 96-well plate) was added to each well. The LNP preparation was added to each well to achieve a final mRNA concentration of 0.01–10 μg / mL, with 3–6 replicates per concentration. Wells without any preparation served as negative controls, and wells transfected with the same dose of mRNA using standard LNP served as positive controls. The cell plate was gently shaken to mix. The transfected cell plate was then returned to the incubator and incubated for 24 hours under standard conditions (37°C, 5% CO2). The cell plate should not be moved or shaken during this period. Discard the old culture medium and add 100 μl of 0.15 mg / ml luciferase substrate (D-luciferin potassium salt) (diluted with PBS) to each well. After 10 minutes, measure the bioluminescence using a multi-plate reader. Reader settings: Select LUM mode for Read modes, Endpoint for read type, 560 nm for excitation wavelength, and 96 WellStandard opaque for plate type.

[0075] See results Figure 3 As shown, Co 2+ -LNP-II can significantly increase the in vitro expression level of mRNA ( Figure 3 (A). Among them, Co 2+ -LNP-II is the mRNA-Co prepared in Example 1. 2+ -LNP.

[0076] Co 2+ -LNP-I was prepared according to Example 1, except that step (2) involved dissolving Co(NO3)2·6H2O in water to prepare a 1 mg / ml aqueous solution, which was then added to the mRNA solution from step (1) to allow Co to dissolve. 2+ The molar ratio of the aqueous phase to mRNA was 10:1. The mixture was incubated at room temperature for 15 min, and the remaining volume of the aqueous phase was made up with citrate buffer (10 mM).

[0077] Co 2+-LNP-III was prepared according to Example 1, except that Co(NO3)2·6H2O was dissolved in citrate buffer (10mM) to prepare a 1mg / ml solution, which was then added to the mRNA solution in step (1) to allow Co to be dissolved in the citrate buffer. 2+ The molar ratio of the aqueous phase to mRNA was 10:1. The mixture was incubated at room temperature for 15 min, and the remaining volume of the aqueous phase was made up with citrate buffer (10 mM).

[0078] The above results demonstrate that the stepwise control of mRNA-Co formation in this application... 2+ The complex is crucial for improving mRNA expression efficiency.

[0079] Furthermore, under the same mRNA administration conditions, the Co-containing mRNA prepared in Example 1 of this application... 2+ Co-delivered lipid nanoparticles significantly enhanced in vitro mRNA expression levels. Compared to undelivered Co... 2+ Compared to the control group, the luciferase expression efficiency was increased by approximately 2 times, indicating that low-dose Co 2+ The introduction of ions can effectively enhance intracellular delivery and functional expression of mRNA. Figure 3 (B)

[0080] Example 3: In vivo expression experiment Co was prepared according to Example 1. 2+ mRNA-Co with different mRNA molar ratios 2+ -LNP lipid nanoparticles were used in mouse in vivo experiments to evaluate their in vivo delivery and expression effects.

[0081] The specific experimental steps are as follows: SPF-grade female Balb / c or C57 mice, 6 weeks old and weighing 18-22g, were purchased from Huachuang Xinno (China). All animals were housed in an animal room with constant temperature (22±2℃), constant humidity (50%±10%), and a 12-hour light-dark cycle, with free access to food and water. After one week of acclimatization, they were used in the experiments. Before the experiments, all mice were fasted for 12 hours (water was allowed). The quadriceps femoris muscle area on the right hind leg of each mouse was shaved, covering an area of ​​approximately 2cm × 2cm, ensuring no obvious hair residue on the skin surface. The intramuscular injection site was disinfected with a 75% alcohol swab. The experimental groups received different molar ratios of mRNA-Co. 2+ -LNP, and set Co not added 2+mRNA-LNP was used as a control group. Before administration, both formulations were diluted to appropriate concentrations with sterile saline. Intramuscular injection (using a microsyringe, such as a Hamilton or insulin syringe) was administered to the quadriceps femoris muscle of the shaved right hind leg of each mouse, and intravenous administration was also performed via the tail vein. The dosage was 2 μg mRNA per mouse, with an injection volume of 50 μL. The injection solution was slowly pushed in, held for 5 seconds, and then the needle was slowly withdrawn to avoid leakage. In vivo bioluminescence imaging was performed on each mouse 12 hours after administration.

[0082] The specific steps for bioluminescence imaging are as follows: (1) Substrate injection: D-fluorescein potassium salt (150 mg / kg, dissolved in sterile PBS) was injected intraperitoneally into each mouse, with an injection volume of about 100-150 μL. (2) Anesthesia: After injection, the mice were placed in an isoflurane anesthesia induction box (induction concentration 3-5%, maintenance concentration 1.5-2.5%). After the mice were completely sedated, they were transferred to the temperature-controlled stage (37℃) of the imaging system to maintain the anesthesia. (3) Imaging: An in vivo bioluminescence imaging system (such as PerkinElmer IVIS Spectrum or similar equipment) was used, with the exposure time set to 1-5 minutes and the imaging wavelength range of 500-600 nm to obtain bioluminescence signal images. (4) Mouse dissection: The mice were euthanized (by cervical dislocation or overdose of isoflurane inhalation). Dissection was performed quickly. The mice were fixed in a supine position. The abdominal skin was disinfected by spraying with 75% alcohol. The skin and abdominal wall muscles were cut along the midline of the abdomen to fully expose the abdominal and thoracic cavities. The heart, liver, spleen, lungs, and kidneys were separated and removed in sequence, taking care to avoid cross-contamination between organs. The removed organs were immediately placed in pre-cooled PBS to rinse and remove residual blood. The surface liquid was gently blotted with filter paper and placed in order on a black imaging dish. (5) Data analysis: The signal intensity of the region of interest (ROI) was quantified using the imaging system software (such as Living Image®) and expressed as average radiance (photons / s / cm² / sr) or total flux (photons / s). The luciferase expression levels of the experimental group and the control group were compared to evaluate Co. 2+ The effect of addition on in vivo translation efficiency of mRNA-LNP.

[0083] See results Figure 4 Compared with the control group, different doses of Co 2+ Both co-delivery groups significantly improved mRNA expression efficiency in vivo. Specifically, n(Co²) + In mice with n(mRNA)=10, the bioluminescent signal was significantly increased by 5 times compared to the control group. These results indicate that low-dose Co... 2+The pre-complexing strategy of ions and mRNA is applicable not only to in vitro transfection systems but also to in vivo delivery, which can effectively enhance the application potential of mRNA drugs.

[0084] Example 4: Confocal microscopy evaluation of LNPs and Co² carrying Cy5-mRNA + -LNP endosome escape efficiency in 293T cells Two LNP formulations were prepared using the same method as in Example 1, but with the Luc mRNA replaced by Cy5-labeled mRNA (Cy5-mRNA, such as Cy5-labeled Luci-mRNA or non-functional reporter mRNA, purchased from Hefei Afana Biotechnology Co., Ltd., dye / nucleotide molar ratio of 1:10-1:20). Specific experimental groups are as follows: Experimental group: Preparation of Co-containing 2+ Cy5-mRNA LNP, where n(Co²) + ):n(mRNA nucleotides)=10; Preparation without Co 2+ Cy5-mRNA LNP (the rest of the components are the same).

[0085] Crtl group: Cell group that did not undergo any treatment.

[0086] Labeling efficiency and concentration were determined using NanoDrop or a fluorescence spectrophotometer to ensure that the Cy5 labeling intensity of the two groups of particles was comparable. Cell plating and preparation: 293T cells in logarithmic growth phase were digested and counted at a concentration of 1×10⁻⁶. 5Cells were seeded at a density of 1 cell / well in confocal microscopy dishes (35 mm, 0.17 mm glass bottom), with 2 mL of complete culture medium added to each well. Incubate for 24 hours until cells adhere and reach 50-70% confluence. Discard the old culture medium and add fresh serum-free DMEM medium (containing 10 mM HEPES) to each well. Add the two LNP formulations (containing Cy5-mRNA) to each well, bringing the final Cy5-mRNA concentration to 100 nM (or optimize according to preliminary experiments, e.g., 50-200 nM). Gently mix. Return the cells to the incubator and continue incubation for 4 hours. After incubation, Lysotracker staining was performed to label acidic organelles (endosomes / lysosomes): the LNP-containing supernatant was discarded, and the cells were gently washed twice with pre-warmed D-PBS (pH 7.4). Add 1 mL of serum-free DMEM medium containing Lysotracker Green to each well, bringing the final concentration to 50 nM. Dyes such as Lysotracker Deep Red, which avoid overlap with the Cy5 spectrum, can also be used. Stain in the dark for 30 minutes in an incubator. After staining, aspirate the staining solution and wash the cells twice with PBS to remove residual stain. Add 1 mL of 4% paraformaldehyde (PFA) to each dish and fix at room temperature in the dark for 15 minutes. Aspirate the PFA and wash three times with PBS, 5 minutes each time. Add approximately 200 μL of PBS to each dish, gently cover the slide, avoiding air bubbles, and it can be used directly for confocal microscopy observation. Alternatively, add mounting medium containing DAPI for nuclear staining and mounting, and incubate overnight at room temperature in the dark before imaging. Image acquisition is performed using a laser confocal scanning microscope: Cy5-mRNA: excitation wavelength 633 nm or 640 nm, emission wavelength 650-700 nm (red channel); Lysotracker Green: excitation wavelength 488 nm, emission wavelength 500-550 nm (green channel); DAPI: excitation wavelength 405 nm, emission wavelength 420-480 nm (blue channel). Objective lens: 60× oil immersion lens, numerical aperture ≥ 1.4. At least 10 fields of view were randomly selected for each sample, with at least 3 optical slices (Z-stack) acquired from each field of view, in increments of 0.5-1.0 μm, to obtain clear, co-localized single-layer images for analysis. Acquisition parameters (laser intensity, gain, exposure time) were kept consistent between the two groups. Image analysis and Pearson coefficient statistics: ImageJ (Fiji) software with the JACOP plugin or Coloc2 module, or the microscope's built-in software (such as Leica LAS X) was used for analysis. The confocal images were opened, and the Cy5 signal channel (red, representing mRNA) and Lysotracker signal channel (green, representing endosomes / lysosomes) were extracted separately. Background subtraction was performed (using rolling ball radius or manually setting the threshold).Calculate the Pearson correlation coefficient (Rr) of red and green signals within each cell or each ROI (region of interest). The coefficient ranges from -1 to 1, with a higher degree of colocalization (i.e., mRNA is mainly retained in endosomes / lysosomes) and a higher degree of escape (mRNA is separated from endosome / lysosome signals) as the coefficient is closer to 0 or negative.

[0087] See results Figure 5 The high Pearson coefficient of mRNA-LNP indicates that Cy5-mRNA is highly colocalized with the Lysotracker green signal, meaning that a large amount of mRNA is retained in endosomes / lysosomes, resulting in low escape efficiency. mRNA-Co² + The Pearson coefficient of -LNP decreased significantly, indicating that Co 2+ The addition of [a substance] promotes the escape of mRNA from the endosome to the cytoplasm and reduces the degree of colocalization with the endosome / lysosome.

[0088] Example 5: MTT assay for detecting mRNA-LNP and mRNA-Co² + Effects of LNP on 293T cell viability 293T cells in logarithmic growth phase were taken, digested, and prepared into a single-cell suspension. After counting, they were divided into 5×10⁻⁶ cells. 3 Cells were seeded at a density of 100 μL per well in 96-well flat-bottom cell culture plates. 100 μL of sterile PBS was added to the edge wells to prevent edge effects. The plates were incubated at 37°C with 5% CO2 for 24 hours, until cells adhered and reached 50-60% confluence.

[0089] The following two LNP formulations, both loaded with the same Luc mRNA as described above, were prepared according to Example 1. The original culture medium in the 96-well plate was discarded, and 100 μL of fresh serum-free culture medium containing the corresponding formulation was added to each well. The following controls were also set up: Cell control group (Ctrl): Cells + untreated medium (without LNP, 100 μL, representing 100% viability).

[0090] Background control group: Cell-free, containing only an equal volume of culture medium (used for zeroing or background subtraction after dissolution).

[0091] After treatment, the cell plates were returned to the incubator for another 24 hours of incubation. 20 μL of 5 mg / mL MTT (3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide) solution was added to each well (final concentration approximately 0.5 mg / mL). Handle in the dark.

[0092] Continue incubation: Place the culture plate back into the incubator and incubate in the dark for 4 hours to allow the mitochondrial dehydrogenases of the live cells to reduce MTT to formazan crystals.

[0093] Remove supernatant: Carefully aspirate the supernatant from the well, being careful not to remove formazan crystals. If using a suction pump, gently touch the well wall.

[0094] Dissolve formazan: Add 150 μL of DMSO (or MTT solution) to each well and shake on a shaker at low speed for 10-15 minutes until the formazan is completely dissolved (a homogeneous purple solution). If using SDS solution, incubate at 37°C for 15-30 minutes.

[0095] Absorbance measurement: The absorbance (OD value) of each well was measured at a wavelength of 570 nm using an ELISA reader. A reference wavelength of 630-690 nm can be selected to subtract background.

[0096] See results Figure 6 mRNA-LNP group and mRNA-Co 2+ The cell viability of the LNP group remained above 85%, with no significant difference from the cell control group, indicating that neither formulation had significant cytotoxicity within this concentration range.

[0097] The above results indicate that this application, by introducing a low dose of Co... 2+ It significantly improved mRNA expression efficiency and had no obvious cytotoxicity, demonstrating excellent biosafety.

[0098] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A low-dose metallic Co 2+ An ion-co-delivery mRNA delivery carrier comprising a lipid component and an active ingredient, characterized in that... The active ingredient is Co. 2+ Stable mRNA-Co formed by co-incubation with mRNA 2+ Complex.

2. The mRNA delivery vector as described in claim 1, characterized in that, The mRNA-Co 2+ In the complex, Co 2+ The molar ratio of mRNA to mRNA is 0.5-15; Preferably, the mRNA-Co 2+ In the complex, Co 2+ The molar ratio of mRNA to mRNA is 10.

3. The mRNA delivery vector as described in claim 1, characterized in that, The lipid component includes at least one of ionized lipids, auxiliary lipids, PEG-lipids, and sterols; Preferably, the ionized lipid is any one of DLin-MC3-DMA, SM-102, ALC-0315 or their derivatives; and / or, the auxiliary lipid is any one of DSPC or DOPE; and / or, the PEG-lipid is any one of DMG-PEG2000 or DSPE-PEG2000; and / or, the sterol is cholesterol. Preferably, the molar ratio of ionized lipid: auxiliary lipid: sterol: PEG-lipid is 50:10:38.5:1.

5.

4. Preparation of the low-dose metallic Co as described in any one of claims 1-3 2+ A method for co-delivering mRNA with ion-based mRNA delivery vectors, characterized in that, Includes the following steps: Add Co to the aqueous mRNA solution 2+ Pre-incubation under suitable conditions forms mRNA-Co 2+ The aqueous phase of the complex; Prepare an organic phase containing lipid components; Will contain mRNA-Co 2+ The aqueous phase of the complex was mixed with an organic phase containing lipid components to prepare a low-dose metal Co. 2+ Ion co-delivery mRNA delivery vector.

5. The method as described in claim 4, characterized in that, The metal Co 2+ The ions were introduced in the form of water-soluble salts of metallic Co; Preferably, the water-soluble salt of the metal Co is at least one of Co(NO3)2, CoCl2, and Co(OAc)2.

6. The method as described in claim 5, characterized in that, Formation containing mRNA-Co 2+ The aqueous phase of the complex includes the following steps: Dissolving a water-soluble salt of metallic Co in water forms a Co-containing... 2+ The aqueous solution was then pre-incubated with the mRNA aqueous phase solution under appropriate conditions; finally, the remaining volume of the aqueous phase was replenished with concentrated buffer.

7. The method as described in claim 4 or 6, characterized in that, The pre-incubation refers to a pH of 5.0-7.4, a time of 5-30 minutes, and a temperature of 20-37℃.

8. The method as described in claim 4, characterized in that, The mixing method can be either microfluidic mixing or ethanol injection.

9. The method according to any one of claims 4-8, characterized in that, The mixture also includes purification and buffer replacement steps.

10. A formulation, characterized in that, Containing the low-dose metallic Co as described in any one of claims 1-3 2+ Ion co-delivery mRNA delivery vector.

Images