Degradable polycarbonate cation carrier and application thereof

Abstract

The invention discloses a degradable polycarbonate cationic carrier and application thereof, and belongs to the technical field of drugs, the degradable polycarbonate cationic carrier is a nano-micelle formed by self-assembly of diblock polymers, the diblock polymers are represented as A-B, the A block is mPEG, and the B block is PAMA, PDMA or PDEA; the molecular weight of the block A is 2000 to 5000, and the polymerization degree of the block B is 50 to 150. The degradable polycarbonate cation carrier has an mRNA delivery function, and is combined with mRNA through a positive and negative electricity interaction principle, so that the problem of gene waste caused by mRNA loading through a microfluidic method can be solved, and efficient delivery of antigen presenting cells (APC) is realized.

Description

Technical Field

[0001] The patent of this invention belongs to the field of pharmaceutical technology, and specifically relates to a degradable polycarbonate cationic carrier and its application. Background Art

[0002] RNA technology is developing rapidly as a new type of treatment. Among them, mRNA, as a single-stranded RNA (molecular weight: 300kDa ~ 5000kDa), once enters the target cell, will quickly express the target protein, achieving an efficient and short-term therapeutic effect. At present, the mRNA vaccine as an effective treatment for the new crown has been approved by the US FDA. mRNA has also entered the clinical trial stage in biological research fields such as genetic disease therapy, cancer therapy, protein replacement therapy and vaccines. These major breakthroughs have aroused the interest of a large number of scientific researchers in mRNA therapy.

[0003] Although mRNA drugs are currently growing rapidly in the market, there are still many challenges in achieving efficient mRNA therapeutic effects. First, unlike double-stranded gene drugs, mRNA is a single-stranded nucleic acid sequence. Its poor stability, easy degradation, electronegativity, and immunogenicity make it difficult for mRNA drugs to effectively exert their therapeutic effects. Secondly, the reticuloendothelial system and blood-brain barrier in mammals make it difficult for mRNA to reach the target site and exert its efficacy. Finally, even if mRNA reaches the target cell, it still faces the problem of lysosomal degradation. Therefore, it is necessary to develop efficient mRNA transport carriers.

[0004] At present, commonly used gene vectors mainly include viral vectors and non-viral vectors. Viral vectors have been successful in clinical practice, but there are still problems such as immunogenicity, low gene load, harmful genome integration, and high cost. Compared with viral vectors, non-viral vectors have received extensive attention from scientific researchers due to their modifiability and functionalization. At present, polymer cations, liposomes, and lipid nanoparticles (LNPs) lead the cutting-edge research of non-viral gene delivery vectors, and can successfully achieve effective delivery of genes in target cells. However, due to the non-degradability of many polymer cationic carriers and many cationic lipid components, some other side effects such as hemolysis and high inflammation can be caused. Therefore, it is urgent to develop efficient and non-toxic gene delivery vectors.

[0005] Therefore, it is very necessary to develop a new type of degradable gene delivery vector to solve the problems existing in cationic gene carriers, so as to achieve better gene delivery effects in vivo and in vitro. Summary of the invention

[0006] In order to solve the deficiencies in the prior art, the purpose of the present invention is to provide a degradable polycarbonate cationic carrier and its application. The present invention provides a diblock polymer with biodegradability, high biosafety, and can effectively load mRNA through positive and negative electrical interactions, namely, a diblock polymer of polyethylene glycol monomethyl ether / polycarboxylic acid betaine / polysulfonic acid betaine and polythymine, polyethylene glycol monomethyl ether-block-2-(tert-butylcarbonylamino)ethyl 5-methyl-2-oxygen-1,3-dioxane-5-carbonate (mPEG-b-PAMA), polyethylene glycol-block-poly 2-(dimethylamino)ethyl 5-methyl-2-oxygen-1,3-dioxane-5-carbonate (mPEG-b-PDMA) and polyethylene glycol-block-2-(diethylamino)ethyl 5-methyl-2-oxygen-1,3-dioxane-5-carbonate (mPEG-b-PDEA). The main chain of this series of diblock polymers is a carbonate bond, which can be degraded in the slightly acidic environment of the endosome or lysosome in the cell. Secondly, the side groups of the PAMA / PDMA / PDEA segments are primary or tertiary amines, which carry positive charges under physiological conditions and can achieve effective loading of single-stranded mRNA through the positive and negative charge interaction PTMA. The main chain of this diblock degradable polycarbonate cationic carrier is a carbonate bond, which can be degraded in vivo, which can effectively solve the in vivo toxic side effects caused by the inability to degrade traditional carbon chain cationic polymers, and at the same time, effectively overcome the problem of inflammation in the body. The PEG segment in this diblock polymer can effectively avoid protein adsorption and achieve long circulation of gene drugs in the body.

[0007] The specific technical solutions of the present invention are as follows:

[0008] On the one hand, the present invention provides a two-block polymer, which is expressed as AB, wherein the A block is polyethylene glycol monomethyl ether (mPEG), and the B block is poly 2-(tert-butylcarbonylamino)ethyl 5-methyl-2-oxygen-1,3-dioxane-5-carbonate (PAMA), poly 2-(dimethylamino)ethyl 5-methyl-2-oxygen-1,3-dioxane-5-carbonate (PDMA) or poly 2-(diethylamino)ethyl 5-methyl-2-oxygen-1,3-dioxane-5-carbonate (PDEA); the molecular weight of the A block can be selected from 2000 to 5000, and the degree of polymerization of the B block can be controlled at 50 to 150.

[0009] Furthermore, PAMA, PDMA and PDEA are obtained by ring-opening polymerization of monomers 2-(tert-butylcarbonylamino)ethyl 5-methyl-2-oxo-1,3-dioxane-5-carbonate, 2-(dimethylamino)ethyl 5-methyl-2-oxo-1,3-dioxane-5-carbonate and 2-(diethylamino)ethyl 5-methyl-2-oxo-1,3-dioxane-5-carbonate, respectively, and the average relative molecular mass of PAMA / PDMA / PDEA is approximately 12355 to 31715.

[0010] Another aspect of the present invention provides a degradable polycarbonate cationic carrier, which is a nanomicelle formed by self-assembly of the above-mentioned diblock polymer. The nanomicelle can exist in the form of an aqueous solution or a freeze-dried powder.

[0011] Furthermore, the particle size of the nano micelle is 100 to 500 nm, and the potential is 0 mV to +50 mV.

[0012] Furthermore, the nanomicelles formed by the self-assembly of the mPEG and PAMA, PDMA or PDEA can exist in the form of an aqueous solution or a lyophilized powder. The preparation method of the aqueous solution and the lyophilized powder is: dissolving the diblock polymer in THF, slowly adding it dropwise into deionized water, naturally volatilizing, and fixing the volume to obtain a nanomicelle aqueous solution, and further freeze-drying the nanomicelle aqueous solution to obtain the nanomicelle lyophilized powder.

[0013] Another aspect of the present invention provides use of the above diblock polymer or the above degradable polycarbonate cationic carrier in preparing a delivery vector for mRNA.

[0014] Another aspect of the present invention provides an mRNA-loaded nanomicelle, which is prepared from the above-mentioned degradable polycarbonate cationic carrier and mRNA.

[0015] Furthermore, the mass ratio of the degradable polycarbonate cationic carrier fully loaded with mRNA is in the range of 10:1 to 30:1.

[0016] Further, the mRNA is an mRNA that translates a specific cytokine, a major histocompatibility complex, or a specific protein;

[0017] Preferably, the mRNA is an mRNA vaccine.

[0018] Another aspect of the present invention provides a method for preparing the mRNA-loaded nanomicelles, comprising the steps of: incubating the mRNA with the degradable polycarbonate cationic carrier at room temperature.

[0019] Furthermore, the mRNA-loaded nanomicelles can be in the form of an aqueous solution or a lyophilized powder. The preparation method of the aqueous solution and the lyophilized powder is: incubate the prepared diblock polymer nanomicelle aqueous solution with mRNA at room temperature for 20 minutes, and then freeze-dry to obtain the mRNA-loaded nanomicelle lyophilized powder.

[0020] Furthermore, the self-assembled nanomicelles and mRNA-loaded nanomicelle freeze-dried powder are redispersible.

[0021] Furthermore, the present invention provides a nanomicelle aqueous solution system, which is a system formed by self-assembly of a diblock polymer in water and redispersion of its nanomicelle freeze-dried powder, wherein the concentration of the nanomicelles is 1-2 mg / mL.

[0022] Furthermore, the present invention provides the application of nano-micelles loaded with mRNA. The mRNA-loaded nano-micelles are injected subcutaneously into mice. The nano-micelles can successfully deliver mRNA to APC cells in the lymph nodes, thereby expressing corresponding cytokines or major histocompatibility complex (MHC), activating cytotoxic T cells, and performing tumor immunotherapy.

[0023] The beneficial effects of the present invention are:

[0024] 1. The present invention provides a diblock polymer mPEG-b-PAMA / mPEG-b-PDMA / mPEG-b-PDEA formed by polyethylene glycol monomethyl ether (mPEG) and poly 2-(tert-butylcarbonylamino)ethyl 5-methyl-2-oxy-1,3-dioxane-5-carbonate (PAMA), poly 2-(dimethylamino)ethyl 5-methyl-2-oxy-1,3-dioxane-5-carbonate (PDMA) or poly 2-(diethylamino)ethyl 5-methyl-2-oxy-1,3-dioxane-5-carbonate (PDEA), and a nanocarrier prepared from the diblock polymer, wherein mPEG acts as a hydrophilic segment to enhance the nanocarrier The stability of the aqueous solution reduces the interaction between proteins and nanocarriers in plasma and prolongs the circulation time of nanocarriers in the blood; PAMA, PDMA and PDEA act as cationic segments. Since the side groups of PAMA, PDMA and PDEA are primary amines or tertiary amines, they can bind to the phosphate groups on the mRNA through positive and negative electrical interactions, thereby achieving effective loading of mRNA. This mRNA loading form solves the problem of large amounts of mRNA loaded by microfluidic technology. At the same time, the main chain is a carbonate bond, which can be degraded in vivo, avoiding the in vivo toxicity and hemolysis problems caused by the inability of traditional carbon chain polymer cations to degrade, and can successfully deliver mRNA to APC cells.

[0025] 2. When the nanocarrier prepared by the diblock polymer is used for mRNA loading, it is highly dependent on the degree of polymerization of PAMA, PDMA or PDEA. If the degree of polymerization of PAMA, PDMA and PDEA is too low, mRNA cannot be successfully loaded. If the degree of polymerization of PAMA, PDMA and PDEA is too high, the surface charge of the nanomicelles will increase, which will cause additional hemolysis or inflammation risks. The present invention has found through experiments that when the degree of polymerization of the cationic segment PAMA / PDMA / PDEA is 100, the formed nanocarrier has good mRNA binding ability and exhibits excellent in vitro and in vivo mRNA delivery efficiency. When the nanomicelles mPEG formed by the self-assembly of the diblock polymer 2k - When the mass ratio of b-PAMA to mRNA is 10 / 1, mRNA can be fully loaded, mPEG 2k -b-PDMA and mPEG 2k When the mass ratio of -b-PDEA to mRNA was 30 / 1, mRNA could be completely loaded. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 .The nuclear magnetic resonance spectrum of the intermediate (5-ethyl-2-oxo-1,3-dioxy-5-yl)methyl 1H-imidazole-1-carbonate (TMPIC) prepared in Example 1. The positions of the nuclear magnetic peaks of various hydrogen protons in the spectrum prove the structural composition of TMPIC.

[0027] Figure 2 .Example 2 The nuclear magnetic resonance spectrum of the monomer 2-(tert-butylcarbonylamino)ethyl 5-methyl-2-oxo-1,3-dioxane-5-carbonate (AMA). The positions of the nuclear magnetic peaks of various hydrogen protons in the figure prove the structural composition of AMA.

[0028] Figure 3 Example 3 shows the nuclear magnetic resonance spectrum of the monomer 2-(dimethylamino)ethyl 5-methyl-2-oxo-1,3-dioxane-5-carbonate (DMA). The positions of the nuclear magnetic peaks of various hydrogen protons in the spectrum prove the structural composition of DMA.

[0029] Figure 4 Example 4 shows the nuclear magnetic resonance spectrum of the monomer 2-(diethylamino)ethyl 5-methyl-2-oxo-1,3-dioxane-5-carbonate (DEA). The positions of the nuclear magnetic peaks of various hydrogen protons in the spectrum prove the structural composition of DEA.

[0030] Figure 5 Example 5 Two-block polymer polyethylene glycol monomethyl ether 2000-block-poly 2-(tert-butylcarbonylamino)ethyl 5-methyl-2-oxo-1,3-dioxane-5-carbonate (mPEG 2k-b-PAMA) NMR spectrum, the positions of the NMR peaks of various hydrogen protons in the figure prove that mPEG 2k -Structural composition of b-PAMA.

[0031] Figure 6 Example 6 Two-block polymer polyethylene glycol monomethyl ether 2000-block-poly 2-(dimethylamino)ethyl 5-methyl-2-oxo-1,3-dioxane-5-carbonate (mPEG 2k -b-PDMA) NMR spectrum, the positions of the NMR peaks of various hydrogen protons in the figure prove that mPEG 2k -Structural composition of b-PDMA.

[0032] Figure 7 Example 7 Two-block polymer polyethylene glycol monomethyl ether 2000-block-poly 2-(diethylamino)ethyl 5-methyl-2-oxo-1,3-dioxane-5-carbonate (mPEG 2k -b-PDEA) NMR spectrum, the positions of the NMR peaks of various hydrogen protons in the figure prove that mPEG 2k -Structural composition of b-PDEA.

[0033] Figure 8 Example 8 Nanomicelle particle size and potential characterization. The results in the figure prove that the diblock polymer can be successfully assembled into nanomicelles, and the surface charge is positive.

[0034] Fig. 9 Example 9 Characterization of the ability of nanomicelles to bind mRNA. The results in the figure prove that mPEG 2k -b-PAMA can completely bind to mRNA at a mass ratio of 10:1, mPEG 2k -b-PDMA and mPEG 2k -b-PDEA can completely bind to mRNA at a mass ratio of 30:1. And the three have the ability to bind to mRNA under different mass ratios.

[0035] Fig.10 Example 10 Characterization of the transfection ability of nanomicelles loaded with mEGFP gene in DC2.4 cells. The results in the figure prove that the three vectors can deliver the target gene to DC2.4 cells and show a certain gene expression ability. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with the implementation method.

[0037] Example 1: Synthesis of intermediate (5-ethyl-2-oxo-1,3-dioxy-5-yl)methyl 1H-imidazole-1-carbonate (TMPIC)

[0038] Disperse trimethylolpropane (TMP, 50g, 0.37mol) in acetone (600mL), add carbonyldiimidazole (CDI, 140g, 0.86mol) in batches under argon protection, react at room temperature for 1 hour after the addition of CDI is complete, stop stirring, let stand for 1 hour, filter, wash with ether, and dry to obtain white solid TMPIC (67.5g, 0.27mol), with a yield of 78%. The NMR spectrum is shown in Figure 1 shown.

[0039] Example 2: Synthesis of Monomer 2-(tert-butylcarbonylamino)ethyl 5-methyl-2-oxo-1,3-dioxane-5-carbonate (AMA)

[0040] Take TMPIC (3g, 11.79mmol) and dissolve it in 190mL acetone. Under argon atmosphere, add cesium fluoride (0.36g, 2.37mmol) and N-(tert-butyloxycarbonyl)ethanolamine (1.9g, 11.79mmol), and stir the reaction at room temperature for 16h. Remove acetone by rotary evaporation, add 100mL dichloromethane to dissolve the remaining solid in the bottle, wash twice with DD water, retain the dichloromethane phase, add anhydrous magnesium sulfate to dry, filter, and vacuum rotary evaporation to remove dichloromethane to obtain AMA monomer (3.27g) with a yield of 88%. The nuclear magnetic resonance spectrum is as follows Figure 2 shown.

[0041] Example 3: Synthesis of Monomer 2-(Dimethylamino)ethyl 5-methyl-2-oxo-1,3-dioxane-5-carbonate (DMA)

[0042] Take TMPIC (3g, 11.79mmol) and dissolve it in 190mL acetone. Under argon atmosphere, add cesium fluoride (0.36g, 2.37mmol) and N,N-dimethylethanolamine (1.05g, 11.79mmol), and stir the reaction at room temperature for 16 hours. Remove acetone by rotary evaporation, add 100mL dichloromethane to dissolve the remaining solid in the bottle, wash twice with DD water, retain the dichloromethane phase, add anhydrous magnesium sulfate to dry, filter, and vacuum rotary evaporation to remove dichloromethane to obtain DMA monomer (2.51g) with a yield of 82%. The nuclear magnetic resonance spectrum is shown as follows: Figure 3 shown.

[0043] Example 4: Synthesis of Monomer 2-(Diethylamino)ethyl 5-methyl-2-oxo-1,3-dioxane-5-carbonate (DEA)

[0044] Take TMPIC (3g, 11.79mmol) and dissolve it in 190mL acetone. Under argon atmosphere, add cesium fluoride (0.36g, 2.37mmol) and N,N-diethylethanolamine (1.38g, 11.79mmol), and stir the reaction at room temperature for 16h. Remove acetone by rotary evaporation, add 100mL dichloromethane to dissolve the remaining solid in the bottle, wash twice with DD water, retain the dichloromethane phase, add anhydrous magnesium sulfate to dry, filter, and vacuum rotary evaporation to remove dichloromethane to obtain DEA monomer (2.67g) with a yield of 79%. The nuclear magnetic resonance spectrum is as follows Figure 4 shown.

[0045] Example 5: Diblock polymer polyethylene glycol monomethyl ether 2000-block-poly 2-(tert-butylcarbonylamino)ethyl 5-methyl-2-oxo-1,3-dioxane-5-carbonate (mPEG 2k Synthesis of -b-PAMA

[0046] Weigh mPEG 2k (63.5mg, 0.032mmol), DBU (439mg, 0.032mmol), TU (13.2mg, 0.030mmol), monomer AMA (1.11g, 3.2mmol), dissolved in 2mL anhydrous dichloromethane, reacted for 24h, added two drops of acetic acid to terminate the reaction, rotary evaporated to remove dichloromethane, added dichloromethane and trifluoroacetic acid mixture 4mL (volume ratio 1 / 1), stirred at room temperature for 4h, rotary evaporated to obtain the target product. The nuclear magnetic resonance spectrum is shown as follows Figure 5 shown.

[0047] Example 6: Diblock polymer polyethylene glycol monomethyl ether 2000-block-poly 2-(dimethylamino)ethyl 5-methyl-2-oxo-1,3-dioxane-5-carbonate (mPEG 2k Synthesis of -b-PDMA)

[0048] Weigh mPEG 2k (63.5mg, 0.032mmol), DBU (439mg, 0.032mmol), TU (13.2mg, 0.030mmol), monomer DMA (0.8804g, 3.2mmol), dissolved in 2mL anhydrous dichloromethane, reacted for 24h, added two drops of acetic acid to terminate the reaction, rotary evaporation, remove dichloromethane, and obtain the target product. The NMR spectrum is shown as Figure 6 shown.

[0049] Example 7: Diblock polymer polyethylene glycol monomethyl ether 2000-block-poly 2-(diethylamino)ethyl 5-methyl-2-oxo-1,3-dioxane-5-carbonate (mPEG 2k Synthesis of -b-PDEA

[0050] Weigh mPEG 2k (63.5mg, 0.032mmol), DBU (439mg, 0.032mmol), TU (13.2mg, 0.030mmol), monomer DEA (0.970g, 3.2mmol), dissolved in 2mL anhydrous dichloromethane, reacted for 24h, added two drops of acetic acid to terminate the reaction, rotary evaporation, remove dichloromethane, and obtain the target product. The NMR spectrum is shown as Figure 7 shown.

[0051] Example 8: Preparation and characterization of nanomicelles

[0052] Accurately weigh mPEG 2k -b-PAMA / mPEG 2k -b-PDMA / mPEG 2k -b-PDEA (20 mg) was dissolved in 1 mL of tetrahydrofuran and slowly dropped into deionized water (10 mL) to evaporate naturally until the tetrahydrofuran evaporated completely. The volume was fixed to obtain a nanoparticle aqueous solution with a concentration of 2 mg / mL.

[0053] The nanoparticle solution was transferred to the sample cell and the mPEG was detected by dynamic light scattering (DLS) at a temperature of 25°C and an angle of 173°. 2k -b-PAMA / mPEG 2k -b-PDMA / mPEG 2k -b-PDEA nanomicelles particle size and potential. Figure 8 shown.

[0054] Example 9: Detection of the ability of nanomicelles to bind mRNA

[0055] mRNA and mPEG 2k -b-PAMA, mPEG 2k -b-PDMA or mPEG 2k -b-PDEA nanomicelle solution according to Fig. 9 The mass ratio shown was incubated at room temperature for 20 minutes, and the loading buffer was added and transferred to the sample well of the agarose plate. The agarose gel plate was transferred to the electrophoresis tank for running and the binding ability of mRNA was detected by ultraviolet. The results are shown in Fig. 9 shown.

[0056] Example 10: Detection of GFP expression in DC2.4 cells in vitro

[0057] mEGFP gene (mRNA expressing green fluorescent protein) and mPEG 2k -b-PAMA, mPEG2k -b-PDMA or mPEG 2k -b-PDEA nanomicelle solutions were incubated at room temperature for 20 min at mass ratios of 10:1, 30:1 and 30:1 to obtain nanomicelles loaded with mEGFP gene. 5 ) were plated on a 24-well plate overnight and incubated with the nanomicelles loaded with mEGFP gene for 4 h in the presence or absence of serum, then the culture medium was replaced with new complete culture medium and cultured for another 20 h. The expression of green fluorescent protein was observed by confocal fluorescence microscopy. Fig.10 shown.

[0058] The above embodiment only selects polyethylene glycol monomethyl ether 2000 and AMA / DMA / DEA with a polymerization degree of 100 as representatives. Obviously, the above embodiment is only an example for clear description, and is not a limitation of the implementation method. For ordinary technicians in the field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from this are still within the protection scope of the invention.

Claims

1. A diblock polymer, characterized in that The two-block polymer is represented as AB, wherein the A block is polyethylene glycol monomethyl ether, and the B block is poly 2-(tert-butylcarbonylamino)ethyl 5-methyl-2-oxygen-1,3-dioxane-5-carbonate, poly 2-(dimethylamino)ethyl 5-methyl-2-oxygen-1,3-dioxane-5-carbonate or poly 2-(diethylamino)ethyl 5-methyl-2-oxygen-1,3-dioxane-5-carbonate; the molecular weight of the A block is 2000-5000, and the degree of polymerization of the B block is 50-150.

2. A degradable polycarbonate cationic carrier, characterized in that: The carrier is a nanomicelle formed by self-assembly of the diblock polymer according to claim 1.

3. The degradable polycarbonate cationic carrier according to claim 2, characterized in that: The particle size of the nano micelle is 100-500nm, and the potential is 0mV-+50mV.

4. The degradable polycarbonate cationic carrier according to claim 2, characterized in that: The nano micelles are in the form of aqueous solution or freeze-dried powder.

5. The degradable polycarbonate cationic carrier according to claim 4, characterized in that: The preparation of the nanomicelle aqueous solution and the nanomicelle lyophilized powder comprises the steps of: dissolving the diblock polymer of claim 1 in THF, slowly dropping it into deionized water, volatilizing it naturally, and fixing the volume to obtain the nanomicelle aqueous solution, and further freeze-drying the nanomicelle aqueous solution to obtain the nanomicelle lyophilized powder.

6. A nanomicelle loaded with mRNA, characterized in that: The nano micelle is prepared from the degradable polycarbonate cationic carrier according to claim 2 and mRNA.

7. The nano micelle according to claim 6, characterized in that The mass ratio of the degradable polycarbonate cationic carrier fully loaded with mRNA according to claim 2 is in the range of 10:1 to 30:

1.

8. The nano micelle according to claim 6, characterized in that The mRNA is an mRNA that translates a specific cytokine, a major histocompatibility complex, or a specific protein; Preferably, the mRNA is an mRNA vaccine.

9. The nano micelle according to claim 6, characterized in that The nano micelles are in the form of aqueous solution or freeze-dried powder.

10. The method for preparing the mRNA-loaded nanomicelles according to claim 6, characterized in that: The method comprises the steps of: incubating mRNA and the nanomicelle aqueous solution self-assembled by the diblock polymer according to claim 1 according to claim 2 at room temperature to obtain the nanomicelle aqueous solution loaded with mRNA, and then freeze-drying to obtain the nanomicelle lyophilized powder loaded with mRNA.

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