Bi-selenium bond-containing poly-beta amino ester carrier for mRNA delivery and preparation method and application thereof

Abstract

The invention discloses a diselenide-bond-containing poly-beta amino ester carrier for mRNA delivery and a preparation method and application thereof, and the preparation method of the diselenide-bond-containing poly-beta amino ester carrier comprises the following steps: taking a synthesized diselene-1, 6-hexanediol diacrylate monomer (DSEDA) and hydrophobic amine as raw materials to react to obtain a product as a polymer intermediate; the raw material of the hydrophobic amine is one of C8, C12 and C16; the # imgabs0 # hydrophilic amine raw material is one of 2A1, 2A4 and 2A5; a diselenide bond is introduced into a main chain of the structure of the synthesized linear cationic polymer product, tertiary amine N capable of being protonated into cations exists in the main chain structure, and the tertiary amine N can be effectively combined with nucleic acid with negative charges through electrostatic interaction, so that the nucleic acid loading rate is increased; according to the invention, hydrophobic alkane chains with proper number and length are provided, a hydrophobic cavity can be formed when the nanoparticles are formed, the stability of the nanoparticles is improved, and the nanoparticles have good application in delivery of mRNA (messenger Ribonucleic Acid) molecules.

Description

Technical Field

[0001] The present invention relates to a diselenide bond-containing poly-β-amino ester (PBAE) carrier for mRNA delivery, and a preparation method and application thereof. Background Art

[0002] Genetic drugs based on nucleic acid biomolecules are a rapidly emerging class of drugs in recent years. Nucleic acid therapy includes a series of strategies, such as small interfering RNA (siRNA), messenger RNA (mRNA) and micro RNA (miRNA), which are designed to regulate gene expression and provide a way to treat cancer by directly targeting its genetic basis. In 2020, the FDA approved two mRNA-based COVID-19 vaccines, which made mRNA vaccines gain global recognition and set off a research boom in mRNA vaccines.

[0003] Compared with plasmid DNA (pDNA) or small interfering RNA-based therapies, mRNA-based therapies have many potential advantages: 1) mRNA does not need to enter the cell nucleus to function. Once it reaches the cytoplasm, mRNA initiates protein translation. In contrast, DNA needs to enter the nucleus first and then be transcribed into mRNA. This process makes DNA less efficient than mRNA because its function depends on the destruction of the nuclear envelope during cell division; 2) Compared with DNA and viral vectors, mRNA does not insert into the genome, but only expresses the encoded protein transiently, which can avoid the risk of insertional gene mutations; 3) Compared with RNA interference, the process of mRNA translation of target proteins is simpler and more direct, without off-target problems similar to siRNA. 4) For any target protein with a known sequence, its mRNA can be synthesized in a very short time, which is extremely advantageous in vaccine research for explosive infectious diseases. Compared with proteins, the growth, preparation and purification process of mRNA is simpler, faster and has lower production costs. The development of genetic engineering technology makes mRNA more suitable for personalized medicine.

[0004] The therapeutic power of mRNA extends far beyond vaccines against infectious diseases; they have potential in cancer vaccines, protein replacement therapy, gene editing therapy, and immunotherapy. However, their successful application requires overcoming various physiological barriers to effectively deliver mRNA to the target site, thus requiring the use of delivery systems. Lipid and polymer nanoparticles represent the main approach for the clinical translation of genetic medicines. These systems bypass biological barriers and promote intracellular delivery of nucleic acids within the correct cells of the target organ using passive, active, and endogenous targeting mechanisms. Compared with other carriers such as lipid nanoparticles, polymer-based delivery systems have generally received less attention, despite their unique ability to carefully tune their chemical properties to promote mRNA protection, have good pharmacokinetics, and have the potential for targeted delivery. Currently, many RNA delivery systems based on polymer nanoparticles have been developed to overcome these barriers in RNA delivery.

[0005] Polymer nanoparticles are one of the most promising nano-delivery materials for nucleic acid drugs due to their advantages such as simplicity of synthesis, structural diversity, synthetic scalability, high transfection rate, gene immunogenicity and good biocompatibility. Polyethyleneimine (PEI) is a polymer widely used for mRNA vaccine delivery in the early days, and its structure is often optimized to improve transfection efficiency. Dendritic macromolecules such as polyamidoamine (PAMAM) and polypropyleneimine (PPI) are also used for mRNA vaccine delivery. Poly-β-amino ester (PBAE) is a class of degradable cationic polymers with good biocompatibility and lower toxicity compared to PEI molecules. Based on the above, the introduction of redox-responsive diselenide bonds into the PBAE main chain is expected to develop a PBAE library that responds faster to stimuli and can effectively release nucleic acid cargo. Summary of the invention

[0006] The purpose of the present invention is to provide a diselenide bond-containing poly-β-amino ester carrier for mRNA delivery, and a preparation method and use thereof. The present invention discloses an efficient mRNA delivery carrier material to improve the stability of mRNA and the expression efficiency of mRNA in vivo and in vitro.

[0007] The structure of the diselenide bond-containing poly-β-amino ester carrier for mRNA delivery is selected from one of the following: .

[0009] The method for preparing a diselenide bond-containing poly-β-amino ester carrier for mRNA delivery comprises the following steps:

[0010] 1) Selenium powder, sodium borohydride and 6-bromo-1-hexanol are used as raw materials, and the selenium powder is weighed into a beaker. After the selenium powder is fully dispersed with ultrapure water, sodium borohydride is added in portions. The reaction conditions are an ice water bath and the reaction time is 25-40 min. After the reaction, a reddish brown liquid product is obtained, and the product does not need to be purified; the obtained reddish brown liquid product is dropped into a 6-bromo-1-hexanol solution dissolved in solvent B, and the reaction is condensed and refluxed under N2 protection. The reaction temperature is 50°C ± 5°C and the reaction time is 24h ± 4h. After the reaction, a yellow liquid is obtained as the selenium-containing monomer 1, which is purified by silica gel column chromatography;

[0011] 2) Using the purified selenium-containing monomer 1 and acryloyl chloride as raw materials, the selenium-containing monomer 1 and triethylamine are dissolved in solvent B, and acryloyl chloride is added dropwise to react under a N2 atmosphere. The reaction temperature is room temperature and the reaction time is 24h±4h. After the reaction is completed, a yellow oil is obtained as the selenium-containing monomer 2, and the product is purified by silica gel column chromatography;

[0012] 3) Selenium-containing monomer 2 and hydrophobic amine are used as raw materials, and selenium-containing monomer 2 and hydrophobic amine are added to a reaction bottle, the reaction temperature is 90°C ± 5°C, and the reaction time is 48h. Solvent-free reaction, after the reaction is completed, a yellow-brown viscous product is obtained, and the polymer intermediate is purified by n-hexane precipitation;

[0013] 4) Using the purified polymer intermediate and hydrophilic amine as raw materials, the purified polymer intermediate and hydrophilic amine are dissolved in solvent C, the reaction temperature is 55°C ± 5°C, the reaction time is 24h ± 4h, and after the reaction is completed, the product is dried in vacuum to obtain a yellow-brown viscous product, that is, the preparation is completed; the reaction formula for preparing the poly-β-amino ester molecule of the present invention is as follows:

[0014]

[0015] Wherein, the structural formula of the hydrophobic amine (R1-NH2) is one of C8, C12, and C16:

[0016]

[0017] The hydrophilic amine (R2-NH2) has a structural formula of one of 2A1, 2A4, and 2A5;

[0018]

[0019] From the above structural formula, it can be seen which hydrophobic amines and hydrophilic amines are used to prepare the diselenide bond-containing poly β-amino ester carrier of the present invention. For example, the raw material hydrophobic amine and hydrophilic amine used to prepare the poly β-amino ester carrier 2A1-C12 are C12 and 2A1, respectively.

[0020] Furthermore, in step 1), the molar ratio of selenium powder to sodium borohydride is 1:0.8-2, preferably 1:1, the molar ratio of selenium powder to 6-bromo-1-hexanol is 1:1.8-3, preferably 1:2, and solvent B is THF.

[0021] Furthermore, in step 1), the eluent for purification by silica gel column chromatography is a mixed solution of ethyl acetate and normal hexane in a volume ratio of 1:0.5-2, preferably a mixed solution of ethyl acetate and normal hexane in a volume ratio of 1:1.

[0022] Furthermore, in step 2), the molar ratio of the purified selenium-containing monomer 1 to acryloyl chloride is 1:2.5-4, preferably 1:3-4, the molar ratio of the purified selenium-containing monomer 1 to triethylamine is 1:2.5-4, preferably 1:3-4, the solvent B is THF, the reaction temperature in step 2) is room temperature, the reaction time is 24h±4h, the product after the reaction is filtered, the filtrate is dried, the obtained product is dissolved in dichloromethane, mixed and extracted with saturated brine, and the product is purified by silica gel column chromatography, and the eluent volume ratio of the silica gel column chromatography purification is 50:0.5-2 of n-hexane-ethyl acetate mixed solution.

[0023] Furthermore, in step 3), the molar ratio of the hydrophobic amine to the selenium-containing monomer 2 is 1:1-1.2, preferably 1:1-1.05, and the polymer intermediate product is purified by n-hexane precipitation.

[0024] Furthermore, in step 4), the molar ratio of the purified polymer intermediate to the hydrophilic amine is 1:3-5, preferably 1:4, the solvent C is anhydrous THF, and the final product does not need to be purified.

[0025] The diselenide bond-containing poly-β-amino ester carrier provided by the invention has good delivery ability in the process of delivering mRNA molecules.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1) The poly-β-amino ester carrier material synthesized by the present invention introduces diselenide bonds, key amines and hydrophobic units that are responsive to redox-sensitive stimuli. Existing studies have shown that nanomaterials containing diselenide bonds can achieve the purpose of controlling the release of cargo by responding to GSH when entering the tumor microenvironment. This is an advantage of the synthesis of our present invention; in addition, key amines and hydrophobic units are necessary groups for forming efficient mRNA delivery carriers. In the selection of hydrophobic amines, the present invention uses C8, C12, and C16 respectively, and effectively regulates the hydrophobicity and cationic density of the material and the number of hydrophobic chains during the material synthesis process. In terms of hydrophilic amines, the present invention uses hydrophilic amines 2A1, 2A4, and 2A5 to effectively regulate the hydrophilicity of the material.

[0028] 2) Compared with the molecules for mRNA delivery reported in the prior art, the linear cationic polymer product synthesized in the present invention introduces a diselenide bond into the main chain structure, and there is a tertiary amine N that can be protonated into a cation in the main chain structure, which can effectively bind negatively charged nucleic acids through electrostatic interactions, thereby increasing the nucleic acid loading rate; with an appropriate number and length of hydrophobic alkane chains, when forming nanoparticles, a hydrophobic cavity can be formed, which improves the stability of the nanoparticles and has a good use in delivering mRNA molecules. Slight changes in the structure can affect the properties of the compound. This application is expected to develop a responsive delivery carrier with the help of the introduction of diselenide, so as to be better applied to tumor treatment, immunotherapy and other aspects.

[0029] 3) Compared with the molecules used for mRNA delivery reported in the prior art, the poly (β-amino ester) molecules of the present application do not require the addition of additional auxiliary components when preparing nucleic acid nanoparticles. The in vitro and in vivo delivery effect tests and physical property characterizations do not require the addition of additional auxiliary components. The preparation of the entire experimental nanoparticles only has two components, the polymer material and the encapsulated mRNA, which can have a certain transfection effect and has a low application cost.

[0030] Elemental selenium is an essential trace element for the human body. The bond energy of the Se-Se bond is relatively low, the bond is relatively active, and it is easier to respond to redox stimulation, making selenium-containing polymers a special type of material. The linear cationic polymer product synthesized by the present invention introduces a diselenide bond into the main chain structure, and there is a tertiary amine N that can be protonated into a cation in the main chain structure, which can effectively bind negatively charged nucleic acids through electrostatic interactions, thereby increasing the nucleic acid loading rate; the hydrophobic alkane chain with an appropriate number and length can form a hydrophobic cavity when forming nanoparticles, improve the stability of the nanoparticles, and has a good use in delivering mRNA molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 DSSe 1 H-NMR spectrum.

[0032] Figure 2 DSeDA 1 H-NMR spectrum.

[0033] Figure 3 Indicates DSeDA-C8 1 H-NMR spectrum.

[0034] Figure 4 Indicates 2A1-C8 1 H-NMR spectrum.

[0035] Figure 5 Indicates DSeDA-C12 1 H-NMR spectrum.

[0036] Figure 6 Indicates 2A1-C12 1 H-NMR spectrum.

[0037] Figure 7 Indicates DSeDA-C16 1 H-NMR spectrum.

[0038] Figure 8 Indicates 2A1-C16 1 H-NMR spectrum.

[0039] Fig. 9 Indicates 2A5-C8 1 H-NMR spectrum.

[0040] Fig.10 Indicates 2A5-C12 1 H-NMR spectrum.

[0041] Fig.11 Indicates 2A5-C16 1 H-NMR spectrum.

[0042] Fig.12 Indicates 2A4-C8 1 H-NMR spectrum.

[0043] Fig.13 Nanoparticle size and potential characterization diagram.

[0044] Fig.14 represents the mRNA loading rate of the nanoparticles.

[0045] Fig.15 Table 2 shows the in vitro delivery effect of nanoparticles after 48 hours of cell transfection.

[0046] Fig.16 Represents the in vivo delivery effect of nanoparticles 6 hours after tail vein injection.

[0047] Fig.17 Shows the organ distribution of nanoparticles 6 hours after tail vein injection. DETAILED DESCRIPTION

[0048] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0049] Example 1: Synthesis of 2A1-C8

[0050] 1) Weigh selenium powder (126.65 mmol) into a 500 mL beaker, then add 100 mL of ultrapure water into the beaker. After the selenium powder is fully dispersed, add sodium borohydride (126.65 mmol) to the selenium powder aqueous solution in portions. The reaction is reacted in an ice-water bath for 30 min to obtain a reddish brown Na2Se2 aqueous solution. Weigh 6-bromo-1-hexanol (253.3 mmol) into a 500 mL three-necked round-bottom flask, add 100 mL of THF into the round-bottom flask, and condense under N2 Under reflux conditions, Na2Se2 aqueous solution (synthesized in the previous step) was added to the reaction through a constant pressure dropping funnel, stirred at 50°C, and reacted for 24 hours. After the reaction, the solvent was removed by rotary evaporator to obtain a yellow liquid as selenium-containing monomer 1, recorded as DSSe, which was purified by silica gel column chromatography, loaded with n-hexane, and dry loaded. The product was purified using an eluent of n-hexane: ethyl acetate = 1:1 (v / v) to obtain a yellow liquid, recorded as DSSe, and the yield of the product was 48% (the yield was calculated based on 6-bromo-1-hexanol). 1 H-NMR spectrum Figure 1 shown.

[0051] 2) Weigh the purified monomer DSSe (5 g, 13.8781 mmol) into a 500 mL three-necked round-bottom flask, add 80 mL THF to the flask, add triethylamine (41.6343 mmol) under stirring, dilute acryloyl chloride (41.6343 mmol) with 20 mL THF, transfer to a constant pressure funnel, slowly drop acryloyl chloride solution under N2 atmosphere, react at room temperature, react for 24 hours, and after the reaction, a yellow oily substance is obtained as selenium-containing monomer 2, recorded as DSeDA. After the reaction, the product is filtered, the filtrate is dried and the obtained product is dissolved in dichloromethane, mixed with saturated brine and extracted, and then purified by silica gel column chromatography; n-hexane is loaded into the column, and the product is purified by using an eluent of n-hexane: ethyl acetate = 50:1 (v / v), which is a yellow oily substance, recorded as DSeDA (the yield is calculated based on the monomer DSSe), and the product yield is 42%. 1 H-NMR spectrum Figure 2 shown.

[0052] 3) Weigh the purified monomer DSeDA (2 g, 4.27 mmol) and hydrophobic amine C8 (4.067 mmol) into a 20 mL brown reaction bottle and react without solvent at 90 °C for 48 h. After the reaction, a yellow-brown viscous product is obtained. The crude product is purified by n-hexane precipitation and dried, and is recorded as DSeDA-C8. 1 H-NMR spectrum Figure 3As shown, δ=6.4ppm, δ=6.13ppm, δ=5.83ppm are the displacements of three hydrogen atoms on the terminal carbon-carbon double bond, and the peak area at each displacement is 1 hydrogen; δ=0.88ppm is the hydrogen atom on the side chain hydrophobic amine -CH3, and its peak area is approximately 60 hydrogens. The ratio of the two values ​​indicates that the degree of polymerization of DSeDA-C8 is 20, and the average molecular weight is about 12,000.

[0053] 4) Weigh the purified polymer intermediate DSeDA-C8 (80 mg) and the hydrophilic amine 2A1 into a 4 mL brown reaction bottle, control the feed molar ratio of the polymer intermediate to the hydrophilic amine to be 1:4, the reaction solvent is anhydrous THF, the reaction temperature is 55°C, the reaction time is 24 h, and after the reaction, dry it in a fume hood for two days and then dry it in a vacuum drying oven for one day to obtain the final product, recorded as 2A1-C8. 1 H-NMR spectrum Figure 4 As shown, Figure 3 In contrast, the NMR peak at the hydrogen displacement on the carbon-carbon double bond disappeared, indicating that the hydrophilic amine 2A1 successfully participated in the reaction and completed the end-capping of the polymer intermediate. After end-capping with the hydrophilic amine, the NMR analysis showed that the double bonds at both ends of the purified polymer intermediate obtained in the previous step disappeared, indicating that the end-capping rate was almost 100%.

[0054] Example 2: Synthesis of 2A1-C12

[0055] The monomer DSeDA was prepared according to the method of Example 1.

[0056] 1) Weigh the purified monomer DSeDA (2 g, 4.27 mmol) and hydrophobic amine C12 (4.067 mmol) into a 20 mL brown reaction bottle and react without solvent at 90 °C for 48 h. After the reaction, a yellow-brown viscous product was obtained. The crude product was purified by n-hexane precipitation and recorded as DSeDA-C12. 1 H-NMR spectrum Figure 5 As shown, δ=6.4ppm, δ=6.13ppm, δ=5.83ppm are the displacements of three hydrogen atoms on the terminal carbon-carbon double bond, and the peak area at each displacement is 1 hydrogen; δ=0.88ppm is the hydrogen atom on the side chain hydrophobic amine -CH3, and its peak area is approximately 54 hydrogens. The ratio of the two values ​​indicates that the degree of polymerization of DSeDA-C12 is 18 and the molecular weight is approximately 11800.

[0057] 2) Weigh the purified polymer intermediate DSeDA-C12 (80 mg) and the hydrophilic amine 2A1 into a 4 mL brown reaction bottle, control the feed molar ratio of the polymer intermediate to the hydrophilic amine to be 1:4, the reaction solvent is anhydrous THF, the reaction temperature is 55°C, the reaction time is 24 h, and after the reaction, dry it in a fume hood for two days and then dry it in a vacuum drying oven for one day to obtain the final product, recorded as 2A1-C12. 1 H-NMR spectrum Figure 6 As shown, Figure 5 In comparison, the NMR peak at the hydrogen displacement on the carbon-carbon double bond disappeared, indicating that the hydrophilic amine 2A1 successfully participated in the reaction and completed the end-capping of the polymer intermediate.

[0058] Example 3: Synthesis of 2A1-C16

[0059] The monomer DSeDA was prepared according to the method of Example 1.

[0060] 1) Weigh the purified monomer DSeDA (2 g, 4.27 mmol) and hydrophobic amine C16 (4.067 mmol) into a 20 mL brown reaction bottle and react without solvent at 90 °C for 48 h. After the reaction, a yellow-brown viscous product is obtained. The crude product is purified by n-hexane precipitation and is recorded as DSeDA-C16. 1 H-NMR spectrum Figure 7 As shown, δ=6.4ppm, δ=6.13ppm, δ=5.83ppm are the displacements of three hydrogen atoms on the terminal carbon-carbon double bond, and the peak area at each displacement is 1 hydrogen; δ=0.88ppm is the hydrogen atom on the side chain hydrophobic amine -CH3, and its peak area is approximately 24 hydrogens. The ratio of the two values ​​indicates that the degree of polymerization of DSeDA-C16 is 8 and the molecular weight is approximately 5700.

[0061] 2) Weigh the purified polymer intermediate DSeDA-C16 (80 mg) and the hydrophilic amine 2A1 into a 4 mL brown reaction bottle, control the feed molar ratio of the polymer intermediate to the hydrophilic amine to be 1:4, use anhydrous THF as the reaction solvent, the reaction temperature is 55°C, the reaction time is 24 h, and after the reaction, dry in a fume hood for two days and then dry in a vacuum drying oven for one day to obtain the final product, recorded as 2A1-C16. 1 H-NMR spectrum Figure 8 As shown, Figure 7 In comparison, the NMR peak at the hydrogen displacement on the carbon-carbon double bond disappeared, indicating that the hydrophilic amine 2A1 successfully participated in the reaction and completed the end-capping of the polymer intermediate.

[0062] Example 4: 2A5-C8

[0063] The purified polymer intermediate DSeDA-C8 was prepared according to the method of Example 1.

[0064] The purified polymer intermediate DSeDA-C8 (80 mg) and the hydrophilic amine 2A5 were weighed into a 4 mL brown reaction bottle, and the molar ratio of the polymer intermediate to the hydrophilic amine was controlled to be 1:4. The reaction solvent was anhydrous THF, the reaction temperature was 55 ° C, the reaction time was 24 h, and after the reaction, it was dried in a fume hood for two days and then dried in a vacuum drying oven for one day to obtain the final product, which was recorded as 2A5-C8. 1 H-NMR spectrum Fig. 9 As shown, Figure 3 In comparison, the NMR peak at the hydrogen displacement on the carbon-carbon double bond disappeared, indicating that the hydrophilic amine 2A5 successfully participated in the reaction and completed the end-capping of the polymer intermediate.

[0065] Example 5: Synthesis of 2A5-C12

[0066] The purified polymer intermediate DSeDA-C12 was prepared according to the method of Example 2.

[0067] The purified polymer intermediate DSeDA-C12 (80 mg) and the hydrophilic amine 2A5 were weighed into a 4 mL brown reaction bottle, and the molar ratio of the polymer intermediate to the hydrophilic amine was controlled to be 1:4. The reaction solvent was anhydrous THF, the reaction temperature was 55 ° C, the reaction time was 24 h, and after the reaction, it was dried in a fume hood for two days and then dried in a vacuum drying oven for one day to obtain the final product, which was recorded as 2A5-C12. 1 H-NMR spectrum Fig.10 As shown, Figure 5 In comparison, the NMR peak at the hydrogen displacement on the carbon-carbon double bond disappeared, indicating that the hydrophilic amine 2A5 successfully participated in the reaction and completed the end-capping of the polymer intermediate.

[0068] Example 6: Synthesis of 2A5-C16

[0069] The purified polymer intermediate DSeDA-C16 was prepared according to the method of Example 3.

[0070] Weigh the purified polymer intermediate DSeDA-C16 (80 mg) and the hydrophilic amine 2A5 into a 4 mL brown reaction bottle, control the feed molar ratio of the polymer intermediate to the hydrophilic amine to be 1:4, the reaction solvent is anhydrous THF, the reaction temperature is 55 ° C, the reaction time is 24 h, and after the reaction, dry it in a fume hood for two days and then dry it in a vacuum drying oven for one day to obtain the final product, recorded as 2A5-C16. 1 H-NMR spectrum Fig.11 As shown, Figure 7In comparison, the NMR peak at the hydrogen displacement on the carbon-carbon double bond disappeared, indicating that the hydrophilic amine 2A5 successfully participated in the reaction and completed the end-capping of the polymer intermediate.

[0071] Example 7: Synthesis of 2A4-C8

[0072] The purified polymer intermediate DSeDA-C8 was prepared according to the method of Example 1.

[0073] The purified polymer intermediate DSeDA-C8 (80 mg) and the hydrophilic amine 2A4 were weighed into a 4 mL brown reaction bottle, and the molar ratio of the polymer intermediate to the hydrophilic amine was controlled to be 1:4. The reaction solvent was anhydrous THF, the reaction temperature was 55 ° C, the reaction time was 24 h, and after the reaction, it was dried in a fume hood for two days and then dried in a vacuum drying oven for one day to obtain the final product, which was recorded as 2A4-C8. 1 H-NMR spectrum Fig.12 As shown, Figure 3 In comparison, the NMR peak at the hydrogen displacement on the carbon-carbon double bond disappeared, indicating that the hydrophilic amine 2A4 successfully participated in the reaction and completed the end-capping of the polymer intermediate.

[0074] 1. The structure of the diselenide bond-containing poly-β-amino ester carrier of the present invention is confirmed by nuclear magnetic resonance spectroscopy. The nuclear magnetic resonance spectrum is as follows: Figure 1-Figure 12 shown.

[0075] 2. Nanoparticle Preparation and Characterization

[0076] The mRNA for delivery was mRNA encoding firefly luciferase (Luc mRNA, purchased from Celgene Pharmaceuticals (Shanghai Co., Ltd.)). Seven synthesized poly-β-amino ester materials (2A1-C8, 2A1-C12, 2A1-C16, 2A5-C8, 2A5-C12, 2A5-C16, 2A4-C8) were dissolved in THF to prepare a specific concentration of mother solution, and the concentration of the material mother solution was 5g / L. 2g / L Luc mRNA was evenly dispersed in 10mM citric acid-sodium hydrogen phosphate buffer (pH=4.2) to prepare a Luc mRNA mother solution.

[0077] According to the mass ratio of delivery vector (poly β-amino ester) to luciferase mRNA of 30:1, the material mother solution and the Luc mRNA mother solution were mixed, and the nanoparticle composite solution was obtained after incubation for 1 hour. The concentration of Luc mRNA in the composite solution was 1.25 ng / μL, and the concentration of the delivery vector was 37.5 ng / μL.

[0078] Nanoparticle solutions were prepared by the above method, and the nanoparticle size and surface potential were measured by dynamic light scattering (DLS). Fig.13 As shown in the figure, we can see that the particle size of nanoparticles made of different materials is all within 400nm, the potential is 10-17mV, the particle size and potential of the prepared nanoparticles are within the range that can be used as nanoparticles for in vivo delivery, and the appropriate particle size and potential can promote the uptake of nanoparticles.

[0079] 3. Material loading luc mRNA efficiency

[0080] The Luc mRNA loading efficiency of the nanoparticles was detected using RiboGreen RNA quantitative reagent (purchased from Thermo Fisher Scientific). First, a standard curve of Luc mRNA concentration corresponding to fluorescence intensity was made, and 0ng / μL, 0.05ng / μL, 0.1ng / μL, 0.2ng / μL, 0.3ng / μL, 0.4ng / μL, and 0.5ng / μL of Luc mRNA dilutions were prepared respectively, and 100μL of each concentration was added to a 96-well black board, and each concentration was repeated three times; 100μL of RiboGreen detection reagent diluted 2000 times was added to each well, and the dilution buffer was 1×TE. The plate was vibrated for 5 minutes under light-proof conditions, and the fluorescence value (λex=485nm, λem=535nm) was measured by a fluorescence microplate reader. The relative fluorescence intensity value was used as the ordinate and the abscissa was the Luc-mRNA concentration (ng / μL) to draw a standard curve.

[0081] For the determination of the loading rate of each poly-β-amino ester carrier, we followed the sample preparation method of step 2 "nanoparticle preparation and characterization" above. For each poly-β-amino ester material, 80 μL of nanoparticle complex solution (containing 100 ng of Luc mRNA) was prepared and diluted 5 times with 10 mM citric acid-disodium hydrogen phosphate buffer (pH = 4.2). 100 μL of the diluted nanoparticle solution was added to each well of a 96-well blackboard. Each nanoparticle was plated in triplicate wells, and 100 μL of 2000-fold diluted RiboGreen detection reagent was added to each well. The fluorescence value of each material was detected using the same detection conditions as when the standard curve was prepared. The free Luc mRNA content was calculated based on the standard curve, thereby calculating the material-loaded Luc mRNA loading rate. The mRNA loading rate of each material is shown in Figure 2. Fig.14 shown.

[0082] 4. In vitro mRNA delivery efficiency

[0083] Human ovarian cancer cells (IGROV 1) were selected as cells for in vitro mRNA delivery. The culture medium for IGROV 1 cells was DMEM high-glucose medium supplemented with 10% (v / v) FBS and 1% (v / v) double-antibody (penicillin-streptomycin). The culture conditions of the cell culture incubator were 5% CO2 and 37° C. Luc mRNA was still selected for in vitro mRNA delivery.

[0084] After IGROV 1 cells were cultured to adherence, 1×10 4 After 6h-12h of culture, 200μL of fresh DMEM medium was replaced; the preparation of nanoparticles was still carried out according to the preparation method of step 2 "nanoparticle preparation and characterization" above, and the prepared nanoparticle solution (25ng Luc mRNA / well) was added at a dose of 20μL per well. After the nanoparticles and cells were incubated for 48h, the firefly luciferase expression level in the cells was detected using a firefly luciferase detection kit (purchased from Titan Technology Co., Ltd.), and the cell viability was detected by MTT method. The commercial reagent MC3 was used as a positive control to select lipid nanoparticles with higher cell-level delivery efficiency.

[0085] In addition, a blank control group was set up, specifically: after IGROV 1 cells were cultured in DMEM medium until they were attached, 20 μL of Luc mRNA solution "without nanoparticles and with a final concentration of 1.25 ng / μL of Luc mRNA" was added to each well, and transfection was performed under this condition for 48 hours to test the in vitro mRNA delivery efficiency.

[0086] The steps for preparing the nanoparticle solution when the commercial reagent MC3 is used as the positive control are as follows: the lipid carrier MC3 and the auxiliary lipids cholesterol, DSPC and DMG-PEG 2000 are mixed and dissolved in anhydrous ethanol at a molar ratio of 50:38.5:10:1.5 to obtain an ethanol solution. Luc mRNA 10mM citric acid-sodium hydrogen phosphate buffer (pH=4.2) is diluted to prepare a Luc mRNA mother solution. The control ethanol solution is mixed with the Luc mRNA mother solution, and the mixture is incubated for 1h to obtain a nanoparticle solution, in which the mass ratio of MC3 to Luc mRNA is 30:1, and the concentration of Luc mRNA is 1.25ng / μL.

[0087] The poly-β-amino ester molecules 2A1-C8, 2A1-C12, 2A1-C16, 2A5-C8, 2A5-C12, 2A5-C16, 2A4-C8 synthesized in Examples 1-7 of the present invention or the commercially available positive control reagent MC3 were used as delivery vectors, and the delivery vectors were mixed with mRNA encoding luciferase to prepare nanoparticles, which were co-incubated with IGROV 1 cells. After 48 hours of transfection, the expression of luciferase in the cells was detected by a microplate reader to evaluate the in vitro delivery efficiency of mRNA. The in vitro mRNA delivery efficiency and cell viability test results of different delivery vectors are shown in Fig.15 , Fig.15 The cell viability (i.e., number of surviving cells) of each group is a relative value compared to the cell viability of the blank control group. The efficiency of in vitro mRNA delivery can be reflected by the fluorescence intensity value in the test results. Fig.15 It shows that the mRNA delivery efficiency of the poly (β-amino ester) vectors synthesized in Examples 1-7 of the present invention is 5-18 times that of the commercially available positive control reagent MC3.

[0088] 5. In vivo mRNA delivery effect test

[0089] The experimental subjects were 5-week-old female BALB / c mice weighing about 20 grams.

[0090] The seven synthesized polyβ-amino ester materials (2A1-C8, 2A1-C12, 2A1-C16, 2A5-C8, 2A5-C12, 2A5-C16, 2A4-C8) were dissolved in THF to prepare a specific concentration of the mother solution, and the concentration of the material mother solution was 20g / L. Luc mRNA was evenly dispersed in 10mM citric acid-sodium hydrogen phosphate buffer (pH=4.2) to prepare Luc mRNA mother solution. According to the mass ratio of the delivery carrier (polyβ-amino ester) to luciferase mRNA of 30:1, the material mother solution was mixed with the Luc mRNA mother solution, and the nanoparticle composite solution was obtained after incubation for 1h. The final concentration of Luc mRNA in the composite solution was 50ng / μL (40 times the cell experiment concentration), and the other sample preparation parameters were similar.

[0091] The mice were randomly divided into 8 groups, 2 mice in each group, 7 of which were experimental groups and 1 was a naked mRNA group. The nanoparticles in the experimental group were prepared using poly-β-amino ester molecules 2A1-C8, 2A1-C12, 2A1-C16, 2A5-C8, 2A5-C12, 2A5-C16, and 2A4-C8.

[0092] Each mouse in the experimental group was injected with 200 μL of different nanoparticle solutions loaded with Luc mRNA through the tail vein (the mRNA dose was 0.5 mg / kg), that is, each mouse was administered 10 μg. The dose of each mouse in the naked mRNA group was still 0.5 mg / kg, and Luc mRNA was diluted to 50 ng / μL with 10mM citric acid-sodium hydrogen phosphate buffer (pH = 4.2). Each mouse was injected with 200 μL of Luc mRNA solution with a diluted concentration of 50 ng / μL through the tail vein. The mice were anesthetized 6 hours after administration, and the small animal in vivo imaging system was used for imaging to evaluate the delivery effect of mRNA in vivo.

[0093] According to the above process, the expression results of luciferase in mice were measured. Fig.16 As shown. Fig.16 It can be seen that 6 hours after the injection of the composite nanoparticle solution formed by 7 poly-β-amino ester molecules and mRNA, 2A1-C8, 2A1-C12, 2A1-C16, and 2A4-C8 mice all had strong fluorescence expression in vivo, and 2A5-C8, 2A5-C12, and 2A5-C16 had poor in vivo delivery effects; the results showed that the main chain with a medium carbon chain (containing 12 carbons) had a better in vivo delivery effect, and the end-capped hydrophilic amines 2A1 and 2A4 were more effective in the in vivo delivery process than 2A5, which inspired us to select the importance of end-capping amines and amines involved in the main chain construction when constructing the carrier. In general, the synthesized material has an excellent mRNA delivery effect in vivo. After naked mRNA was injected into the tail vein of mice, there was no expression of luciferase.

[0094] 6. Organ distribution of nanoparticles

[0095] The experimental group and naked mRNA group were dissected and imaged 6 hours after administration (the organs removed were liver, spleen, lung, heart and kidney). The expression results of luciferase in each organ of the mouse are shown in the figure. Fig.17 .according to Fig.17 As a result, the poly-β-amino ester molecules 2A1-C8, 2A1-C12, 2A1-C16, 2A5-C12, and 2A4-C8 synthesized in the practice of the present invention can deliver mRNA to the spleen and thereby express a large amount of luciferase.

[0096] The contents described in this specification are merely an enumeration of implementation forms of the inventive concept, and the protection scope of the present invention should not be regarded as being limited to the specific forms described in the embodiments.

Claims

1. A diselenide bond-containing poly-β-amino ester carrier for mRNA delivery, wherein the structure is selected from one of the following:

2. The method for preparing a diselenide bond-containing poly-β-amino ester carrier for mRNA delivery according to claim 1, characterized in that The following steps are involved: 1) Selenium powder, sodium borohydride and 6-bromo-1-hexanol are used as raw materials. After the selenium powder is fully dispersed in solvent A, sodium borohydride is added and reacted in an ice-water bath to generate a reddish-brown liquid product containing Na2Se2. The product does not need to be purified; The The obtained reddish brown liquid product was added dropwise to a 6-bromo-1-hexanol solution dissolved in solvent B, and condensed and refluxed under N2 protection. After the reaction, the solvent B was removed by rotary evaporation to obtain a yellow liquid as selenium-containing monomer 1, which was then purified by silica gel column chromatography; The selenium-containing monomer 1 is denoted as DSSe, and its structural formula is as follows: 2) Using the purified selenium-containing monomer 1 and acryloyl chloride as raw materials, the selenium-containing monomer 1 and triethylamine are dissolved in solvent B, and acryloyl chloride is dropped into the mixture solution under a N2 atmosphere. After the reaction is completed, a yellow liquid is obtained as the selenium-containing monomer 2, and the product is purified by silica gel column chromatography; The selenium-containing monomer 2 is denoted as DSeDA, and its structural formula is as follows: 3) Selenium-containing monomer 2 and hydrophobic amine are used as raw materials, and the selenium-containing monomer 2 and the hydrophobic amine are placed in a reaction bottle, and reacted without solvent. After the reaction is completed, a yellow-brown viscous product is obtained, and the crude product is purified by precipitation. The purified polymer intermediate is recorded as DSeDA-C; wherein the hydrophobic amine raw material is one of C8, C12, and C16; The polymer intermediate is denoted as DSeDA-C, and its structural formula is as follows: In the structural formula of DSeDA-C, the substituent R1 is a C8 alkyl group, a C12 alkyl group or a C16 alkyl group, and its structural formula is as follows: C8 C12 C16 4) using the purified polymer intermediate and the hydrophilic amine as raw materials, dissolving the purified polymer intermediate and the hydrophilic amine in solvent C, reacting, and vacuum drying after the reaction to obtain a yellow-brown viscous product, and the preparation is completed; wherein the hydrophilic amine raw material is one of 2A1, 2A4, and 2A5; 2A1 2A4 2A5 3. The method for preparing a diselenide bond-containing poly-β-amino ester carrier for mRNA delivery according to claim 2, characterized in that In step 1), the molar ratio of selenium powder to sodium borohydride is 1:0.8-2, preferably 1:1, the molar ratio of selenium powder to 6-bromo-1-hexanol is 1:1.8-3, preferably 1:2, the solvent A is ultrapure water, and the solvent B is THF.

4. The method for preparing a diselenide bond-containing poly-β-amino ester carrier for mRNA delivery according to claim 2, characterized in that In step 1), the reaction time for preparing Na2Se2 under ice-water bath conditions is 25-40 min; the reaction temperature for preparing selenium-containing monomer 1 is 50°C±5°C, the reaction time is 24h±4h, and the eluent for purification by silica gel column chromatography is a mixed solution of ethyl acetate and n-hexane in a volume ratio of 1:0.5-2.

5. The method for preparing a diselenide bond-containing poly-β-amino ester carrier for mRNA delivery according to claim 2, characterized in that In step 2), the molar ratio of the purified selenium-containing monomer 1 to acryloyl chloride is 1:2.5-4, preferably 1:3-4, the molar ratio of the purified selenium-containing monomer 1 to triethylamine is 1:2.5-4, preferably 1:3-4, the solvent B is THF, the reaction temperature in step 2) is room temperature, the reaction time is 24h±4h, the product after the reaction is filtered, the filtrate is dried by spin drying, the obtained product is dissolved in dichloromethane, mixed and extracted with saturated brine, and the product is purified by silica gel column chromatography, and the eluent volume ratio of the silica gel column chromatography purification is 50:0.5-2 of n-hexane-ethyl acetate mixed solution.

6. The method for preparing a diselenide bond-containing poly-β-amino ester carrier for mRNA delivery according to claim 2, characterized in that In step 3), the molar ratio of the hydrophobic amine to the selenium-containing monomer 2 is 1:1-1.2, preferably 1:1-1.05, and the polymer intermediate product is purified by n-hexane precipitation.

7. The method for preparing a diselenide bond-containing poly-β-amino ester carrier for mRNA delivery according to claim 2, characterized in that Step 3) The reaction temperature is 90°C ± 5°C and the reaction time is 48h.

8. The method for preparing a diselenide bond-containing poly-β-amino ester carrier for mRNA delivery according to claim 2, characterized in that In step 4), the molar ratio of the purified polymer intermediate to the hydrophilic amine is 1:3-5, preferably 1:4, the solvent C is anhydrous THF, and the final product does not need to be purified.

9. The method for preparing a diselenide bond-containing poly-β-amino ester carrier for mRNA delivery according to claim 2, characterized in that In step 4), the reaction temperature is 55°C ± 5°C, and the reaction time is 24h ± 4h.

10. Use of the diselenide bond-containing poly-β-amino ester carrier for mRNA delivery according to claim 1 in delivering mRNA molecules.

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