Diselenide-containing poly(beta amino ester) vectors for mRNA delivery and methods of making and using the same

Abstract

The application discloses a diselenide-containing poly-beta amino ester carrier for mRNA delivery and a preparation method and application thereof. The preparation of the diselenide-containing poly-beta amino ester carrier is a product obtained by reaction of a synthesized diselenide-1,6-hexanediol diacrylate monomer (DSeDA) and a hydrophobic amine as raw materials, and the product is used as a polymer intermediate, and the polymer intermediate is further reacted with a hydrophilic amine to obtain the diselenide-containing poly-beta amino ester carrier, wherein the hydrophobic amine raw material is one of C8, C12 and C16; the hydrophilic amine raw material is one of 2A1, 2A4 and 2A5; the linear cationic polymer product synthesized by the application introduces a diselenide bond into a main chain structure, and a tertiary amine N capable of being protonated into a cation exists in the main chain structure, the tertiary amine N can effectively combine a negatively charged nucleic acid through electrostatic interaction, so that the nucleic acid loading rate is improved; the hydrophobic alkane chain with a proper number and length can form a hydrophobic cavity when the nanometer particle is formed, the stability of the nanometer particle is improved, and the nanometer particle has a good application in delivery of mRNA molecules.

Description

Technical Field

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

[0002] Genetic medicines based on nucleic acid biomolecules are a rapidly emerging class of drugs in recent years. Nucleic acid therapies encompass a range of strategies, such as small interfering RNA (siRNA), messenger RNA (mRNA), and microRNA (miRNA), designed to modulate gene expression and provide avenues for treating cancer by directly targeting its genetic basis. In 2020, the FDA approved two mRNA-based COVID-19 vaccines, bringing global recognition to mRNA vaccines and sparking a wave of research into mRNA vaccines.

[0003] Compared to plasmid DNA (pDNA) or small interfering RNA (siRNA)-based therapies, mRNA-based therapies offer many potential advantages: 1) mRNA does not need to enter the cell nucleus to function. Once in the cytoplasm, mRNA initiates protein translation. In contrast, DNA must first enter the nucleus and then be transcribed into mRNA. This process makes DNA less efficient than mRNA, as its function depends on the destruction of the nuclear envelope during cell division. 2) Compared to DNA and viral vectors, mRNA does not insert into the genome but only transiently expresses the encoded protein, thus avoiding the risk of insertional mutagenesis. 3) Compared to RNA interference, mRNA translation of the target protein is simpler and more direct, without the off-target effects associated with siRNA. 4) mRNA for any target protein with a known sequence can be synthesized in a very short time, which is a significant advantage in vaccine research for outbreak-prone infectious diseases. Compared to proteins, the production, preparation, and purification of mRNA are simpler, faster, and less expensive. Advances in genetic engineering technology have made mRNA more suitable for personalized medicine.

[0004] The therapeutic potential of mRNA extends far beyond vaccines against infectious diseases; it holds potential for cancer vaccines, protein replacement therapy, gene editing therapy, and immunotherapy. However, its successful application requires overcoming various physiological barriers to effectively deliver mRNA to its target site, necessitating the use of delivery systems. Lipid and polymeric nanoparticles represent the primary approach for the clinical translation of gene-based medicines. These systems bypass biological barriers and utilize passive, active, and endogenous targeting mechanisms to facilitate intracellular delivery of nucleic acids within the correct cells of the target organ. Compared to other carriers, such as lipid nanoparticles, polymer-based delivery systems have generally received less attention, despite their unique ability to carefully tailor their chemical properties to promote mRNA protection, favorable pharmacokinetics, and potential for targeted delivery. Numerous polymeric nanoparticle-based RNA delivery systems have been developed to overcome these barriers to RNA delivery.

[0005] Polymer nanoparticles have the advantages of simplicity of synthesis, structural diversity, synthetic scalability, high transfection rate, gene immunogenicity and good biocompatibility, and are one of the most promising nucleic acid drug nanodelivery materials. Polyethyleneimine (PEI) is a polymer that was widely used in the early days of mRNA vaccine delivery, and its structure is often optimized to improve transfection efficiency. Dendrimers such as polyamidoamine (PAMAM) and polypropyleneimine (PPI) have also been 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, a preparation method and use thereof, and 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:

[0008] The method for preparing a diselenide bond-containing poly-β-amino ester carrier for mRNA delivery comprises the following steps:

[0009] 1) Selenium powder, sodium borohydride, and 6-bromo-1-hexanol are used as raw materials. The selenium powder is weighed into a beaker, and after the selenium powder is fully dispersed with ultrapure water, sodium borohydride is added portionwise. The reaction conditions are an ice-water bath and the reaction time is 25-40 minutes. 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 added dropwise to a 6-bromo-1-hexanol solution dissolved in solvent B. The reaction is condensed and refluxed under nitrogen protection. The reaction temperature is 50°C ± 5°C and the reaction time is 24h ± 4h. After the reaction, a yellow liquid is obtained, which is the selenium-containing monomer 1, which is purified by silica gel column chromatography;

[0010] 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 under a nitrogen atmosphere to react at room temperature for 24 h ± 4 h. After the reaction, a yellow oil is obtained as the selenium-containing monomer 2, and the product is purified by silica gel column chromatography;

[0011] 3) Selenium-containing monomer 2 and a hydrophobic amine were added to a reaction flask at 90°C ± 5°C for 48 hours. A solvent-free reaction was performed to obtain a yellow-brown viscous product, which was then purified by n-hexane precipitation.

[0012] 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 vacuum dried to obtain a yellow-brown viscous product, indicating that the preparation is complete; the reaction formula for preparing the poly (β-amino ester) molecule of the present invention is as follows:

[0013]

[0014] The structural formula of the hydrophobic amine (R1-NH2) is one of C8, C12, and C16:

[0015]

[0016] The hydrophilic amine (R2-NH2) has a structural formula of one of 2A1, 2A4, and 2A5;

[0017]

[0018] 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 hydrophobic amine and hydrophilic amine used to prepare the poly-β-amino ester carrier 2A1-C12 are C12 and 2A1, respectively.

[0019] 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.

[0020] Furthermore, in step 1), the eluent for purification by silica gel column chromatography is a mixture of ethyl acetate and n-hexane in a volume ratio of 1:0.5-2, preferably a mixture of ethyl acetate and n-hexane in a volume ratio of 1:1.

[0021] Furthermore, in step 2), the molar ratio of the purified selenium-containing monomer 1 and acryloyl chloride is 1:2.5~4, preferably 1:3~4, and the molar ratio of the purified selenium-containing monomer 1 and triethylamine is 1:2.5~4, preferably 1:3~4, and the solvent B is THF. The reaction temperature in step 2) is room temperature, the reaction time is 24h±4h, and the product after the reaction is filtered, the filtrate is dried, and the obtained product is dissolved in dichloromethane, mixed and extracted with saturated brine, and the product is purified by silica gel column chromatography. The eluent volume ratio of the purified silica gel column chromatography is a mixture of n-hexane and ethyl acetate of 50:0.5-2.

[0022] 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.

[0023] Furthermore, in step 4), the molar ratio of the purified polymer intermediate to the hydrophilic amine is 1:3-5, preferably 1:4, and the solvent C is anhydrous THF. The final product does not need to be purified.

[0024] The diselenide bond-containing poly-β-amino ester carrier provided by the present invention has good delivery ability in the process of delivering mRNA molecules.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1) The poly-β-amino ester carrier material synthesized in this invention incorporates a redox-sensitive stimulus-responsive diselenide bond, a key amine, and a hydrophobic unit. Existing research has demonstrated that nanomaterials containing diselenide bonds can achieve controlled cargo release in response to GSH upon entry into the tumor microenvironment, a significant advantage of our invention. Furthermore, the key amine and hydrophobic unit are essential for efficient mRNA delivery. The hydrophobic amines used in this invention are C8, C12, and C16, respectively, to effectively control the material's hydrophobicity, cation density, and number of hydrophobic chains during synthesis. Regarding the hydrophilic amines, the hydrophilic amines 2A1, 2A4, and 2A5 are used to effectively control the material's hydrophilicity.

[0027] 2) Compared with the molecules for mRNA delivery reported in the prior art, 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 to negatively charged nucleic acids through electrostatic interactions, thereby increasing the nucleic acid loading rate; with an appropriate number and length of hydrophobic alkane chains, it can form a hydrophobic cavity when forming nanoparticles, improve 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, which can be better used in tumor treatment, immunotherapy and other aspects.

[0028] 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 testing and physical property characterization do not require the addition of additional auxiliary components. The preparation of the entire experimental nanoparticles only requires two components: polymer material and encapsulated mRNA, which can achieve a certain transfection effect and has low application cost.

[0029] Elemental selenium is an essential trace element for the human body. The low bond energy of the Se-Se bond makes it more active and more responsive to redox stimuli, making selenium-containing polymers a unique class of materials. The linear cationic polymer product synthesized by the present invention incorporates a diselenide bond into the main chain structure, and the main chain structure contains a tertiary amine N that can be protonated to form a cation. This effectively binds negatively charged nucleic acids through electrostatic interactions, thereby increasing the nucleic acid loading rate. The hydrophobic alkane chains with an appropriate number and length can form a hydrophobic cavity when forming nanoparticles, improving the nanoparticle's stability and having excellent applications in delivering mRNA molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 DSSe 1 H-NMR spectrum.

[0031] Figure 2 DSeDA 1 H-NMR spectrum.

[0032] Figure 3 Indicates DSeDA-C8 1 H-NMR spectrum.

[0033] Figure 4 Indicates 2A1-C8 1 H-NMR spectrum.

[0034] Figure 5 Indicates DSeDA-C12 1 H-NMR spectrum.

[0035] Figure 6 Indicates 2A1-C12 1 H-NMR spectrum.

[0036] Figure 7 Indicates DSeDA-C16 1 H-NMR spectrum.

[0037] Figure 8 Indicates 2A1-C16 1 H-NMR spectrum.

[0038] Figure 9 Indicates 2A5-C8 1 H-NMR spectrum.

[0039] Figure 10 Indicates 2A5-C12 1 H-NMR spectrum.

[0040] Figure 11 Indicates 2A5-C16 1 H-NMR spectrum.

[0041] Figure 12 Indicates 2A4-C8 1 H-NMR spectrum.

[0042] Figure 13 Nanoparticle size and potential characterization diagram.

[0043] Figure 14 represents the mRNA loading rate of nanoparticles.

[0044] Figure 15 Table 4 shows the in vitro delivery effect of nanoparticles after 48 hours of cell transfection.

[0045] Figure 16 Shows the in vivo delivery effect of nanoparticles 6 hours after tail vein injection.

[0046] Figure 17 Shows the organ distribution of nanoparticles 6 hours after tail vein injection. DETAILED DESCRIPTION

[0047] The present invention will be further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0048] Example 1: Synthesis of 2A1-C8

[0049] 1) Selenium powder (126.65 mmol) was weighed into a 500 mL beaker, and then 100 mL of ultrapure water was added to the beaker. After the selenium powder was fully dispersed, sodium borohydride (126.65 mmol) was added to the selenium powder aqueous solution in portions. The reaction was reacted in an ice-water bath for 30 min to obtain a reddish-brown Na2Se2 aqueous solution; 6-bromo-1-hexanol (253.3 mmol) was weighed into a 500 mL three-necked round-bottom flask, and 100 mL of THF was added to the round-bottom flask. The mixture was stirred under N2 condensation. 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 evaporation to obtain a yellow liquid as selenium-containing monomer 1, recorded as DSSe. It was purified by silica gel column chromatography, loaded with n-hexane, dry loaded, and the eluent n-hexane: ethyl acetate = 1:1 (v / v) was used to purify the product as a yellow liquid, recorded as DSSe, and the product yield was 48% (calculated based on 6-bromo-1-hexanol). 1 H-NMR spectrum Figure 1 shown.

[0050] 2) Weigh the purified monomer DSSe (5 g, 13.8781 mmol) into a 500 mL three-necked round-bottom flask, add 80 mL of THF to the flask, add triethylamine (41.6343 mmol) under stirring, dilute acryloyl chloride (41.6343 mmol) with 20 mL of THF, transfer to a constant pressure funnel, slowly add acryloyl chloride solution dropwise under N2 atmosphere, react at room temperature, and react for 24 hours. After the reaction, a yellow oily substance is obtained as selenium-containing monomer 2, which is recorded as DSeDA. The product after the reaction is filtered, the filtrate is dried and the obtained product is dissolved in dichloromethane, mixed and extracted with saturated brine, and purified by silica gel column chromatography; the column is loaded with n-hexane and the product is purified using an eluent of n-hexane:ethyl acetate = 50:1 (v / v) as an eluent to obtain a yellow oily substance, recorded as DSeDA (yield calculated based on monomer DSSe), with a product yield of 42%. 1 H-NMR spectrum Figure 2 shown.

[0051] 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 was obtained. The crude product was purified by n-hexane precipitation and dried, and was recorded as DSeDA-C8. 1 H-NMR spectrum Figure 3As shown, δ = 6.4 ppm, δ = 6.13 ppm, and δ = 5.83 ppm represent the displacements of three hydrogen atoms on the terminal carbon-carbon double bond, with the peak area at each displacement corresponding to one hydrogen atom. δ = 0.88 ppm represents the hydrogen atom on the side chain hydrophobic amine -CH3, with a peak area corresponding to approximately 60 hydrogen atoms. The ratio of these two values ​​indicates that DSeDA-C8 has a degree of polymerization of 20 and an average molecular weight of approximately 12,000.

[0052] 4) Weigh the purified polymer intermediate DSeDA-C8 (80 mg) and the hydrophilic amine 2A1 into a 4 mL brown reaction bottle, control the 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, which is recorded as 2A1-C8. 1 H-NMR spectrum Figure 4 As shown, Figure 3 In contrast, the NMR peaks at the hydrogen shifts on the carbon-carbon double bond disappear, 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, NMR analysis revealed that the double bonds at both ends of the purified polymer intermediate obtained in the previous step had completely disappeared, indicating a near 100% end-capping efficiency.

[0053] Example 2: Synthesis of 2A1-C12

[0054] Monomer DSeDA was prepared according to the method of Example 1.

[0055] 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 = 6.4 ppm, δ = 6.13 ppm, and δ = 5.83 ppm represent the displacements of three hydrogen atoms on the terminal carbon-carbon double bond, with the peak area at each displacement corresponding to one hydrogen atom. δ = 0.88 ppm represents the hydrogen atom on the side chain hydrophobic amine -CH3, with a peak area corresponding to approximately 54 hydrogen atoms. The ratio of these two values ​​indicates that the degree of polymerization of DSeDA-C12 is 18, and the molecular weight is approximately 11,800.

[0056] 2) Weigh the purified polymer intermediate DSeDA-C12 (80 mg) and hydrophilic amine 2A1 into a 4 mL brown reaction bottle, control the 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 in a vacuum drying oven for one day to obtain the final product, which is 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.

[0057] Example 3: Synthesis of 2A1-C16

[0058] Monomer DSeDA was prepared according to the method of Example 1.

[0059] 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 was obtained. The crude product was purified by n-hexane precipitation and recorded as DSeDA-C16. 1 H-NMR spectrum Figure 7 As shown, δ = 6.4 ppm, δ = 6.13 ppm, and δ = 5.83 ppm represent the displacements of three hydrogen atoms on the terminal carbon-carbon double bond, with the peak area at each displacement corresponding to one hydrogen atom. δ = 0.88 ppm represents the hydrogen atom on the side chain hydrophobic amine -CH3, with a peak area corresponding to approximately 24 hydrogen atoms. The ratio of these two values ​​indicates that DSeDA-C16 has a degree of polymerization of 8 and a molecular weight of approximately 5700.

[0060] 2) Weigh the purified polymer intermediate DSeDA-C16 (80 mg) and the hydrophilic amine 2A1 into a 4 mL brown reaction bottle, control the 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 in a vacuum drying oven for one day to obtain the final product, which is 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.

[0061] Example 4: 2A5-C8

[0062] The purified polymer intermediate DSeDA-C8 was prepared according to the method of Example 1.

[0063] The purified polymer intermediate DSeDA-C8 (80 mg) and hydrophilic amine 2A5 were weighed and put into a 4 mL brown reaction bottle. 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 Figure 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.

[0064] Example 5: Synthesis of 2A5-C12

[0065] The purified polymer intermediate DSeDA-C12 was prepared according to the method of Example 2.

[0066] The purified polymer intermediate DSeDA-C12 (80 mg) and hydrophilic amine 2A5 were weighed and put into a 4 mL brown reaction bottle. 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 Figure 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.

[0067] Example 6: Synthesis of 2A5-C16

[0068] The purified polymer intermediate DSeDA-C16 was prepared according to the method of Example 3.

[0069] The purified polymer intermediate DSeDA-C16 (80 mg) and hydrophilic amine 2A5 were weighed and put into a 4 mL brown reaction bottle. 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-C16. 1 H-NMR spectrum Figure 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.

[0070] Example 7: Synthesis of 2A4-C8

[0071] The purified polymer intermediate DSeDA-C8 was prepared according to the method of Example 1.

[0072] The purified polymer intermediate DSeDA-C8 (80 mg) and hydrophilic amine 2A4 were weighed and put into a 4 mL brown reaction bottle. 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 Figure 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.

[0073] 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 spectrum is as follows: Figures 1-12 shown.

[0074] 2. Nanoparticle Preparation and Characterization

[0075] The mRNA used for delivery was firefly luciferase-encoding mRNA (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, and 2A4-C8) were dissolved in THF to prepare stock solutions of specific concentrations, all at 5g / L. 2g / L of Luc mRNA was evenly dispersed in 10mM citric acid-sodium hydrogen phosphate buffer (pH 4.2) to prepare the Luc mRNA stock solution.

[0076] 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 incubated for 1 hour to obtain a nanoparticle composite solution. 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.

[0077] Nanoparticle solutions were prepared by the above-mentioned method, and the particle size and surface potential of the nanoparticles were measured by dynamic light scattering (DLS). Figure 13 As shown in the figure, we can see that the particle size of nanoparticles made of different materials is within 400nm, and 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. Appropriate particle size and potential can promote the uptake of nanoparticles.

[0078] 3. Material loading efficiency of luc mRNA

[0079] The Luc mRNA loading efficiency of the nanoparticles was determined using RiboGreen RNA quantification reagent (purchased from Thermo Fisher Scientific). A standard curve of Luc mRNA concentration versus fluorescence intensity was first generated. Luc mRNA dilutions of 0 ng / μL, 0.05 ng / μL, 0.1 ng / μL, 0.2 ng / μL, 0.3 ng / μL, 0.4 ng / μL, and 0.5 ng / μL were prepared, and 100 μL of each concentration was added to a 96-well black plate, with each well in triplicate. Each well was then incubated with 100 μL of a 2000-fold diluted RiboGreen detection reagent in 1×TE buffer. The plate was shaken in the dark for 5 minutes, and fluorescence values ​​were measured using a fluorescence microplate reader (λex = 485 nm, λem = 535 nm). A standard curve was plotted with relative fluorescence intensity as the ordinate and Luc mRNA concentration (ng / μL) as the abscissa.

[0080] 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 black board. Each nanoparticle was plated in triplicate, and 100 μL of a 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 preparing the standard curve. 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 as follows: Figure 14 shown.

[0081] 4. In vitro mRNA delivery efficiency

[0082] Human ovarian cancer cells (IGROV 1) were used for in vitro mRNA delivery. IGROV 1 cells were cultured in DMEM high-glucose medium supplemented with 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin. The culture conditions were 5% CO2 and 37°C in a cell culture incubator. Luc mRNA was also used for in vitro mRNA delivery.

[0083] After IGROV 1 cells were cultured to adherence, 1×10 4 After 6-12 hours of culture, cells were attached to the surface of the culture medium, and then 200 μL of fresh DMEM medium was replaced. Nanoparticles were prepared according to the preparation method described in step 2, "Nanoparticle Preparation and Characterization," with 20 μL of the prepared nanoparticle solution (25 ng of Luc mRNA / well) added to each well. After incubation of the nanoparticles and cells for 48 hours, intracellular firefly luciferase expression was measured using a firefly luciferase assay kit (purchased from Titan Technology Co., Ltd.), and cell viability was assessed using the MTT assay. The commercially available reagent MC3 was used as a positive control to select lipid nanoparticles with high cellular delivery efficiency.

[0084] A blank control group was also established. IGROV 1 cells were cultured in DMEM medium until adherent. A Luc mRNA solution containing no nanoparticles and a final Luc mRNA concentration of 1.25 ng / μL was then added to each well at a dose of 20 μL. Transfection was continued under these conditions for 48 hours to test the in vitro mRNA delivery efficiency.

[0085] The procedure for preparing the nanoparticle solution using the commercial reagent MC3 as a positive control is as follows: the lipid carrier MC3 is mixed with the auxiliary lipids cholesterol, DSPC, and DMG-PEG 2000 in a molar ratio of 50:38.5:10:1.5 and dissolved in anhydrous ethanol to produce an ethanol solution. Luc mRNA is diluted in 10mM citric acid-disodium hydrogen phosphate buffer (pH 4.2) to prepare a Luc mRNA stock solution. The control ethanol solution is then mixed with the Luc mRNA stock solution and incubated for 1 hour to produce a nanoparticle solution with a mass ratio of MC3 to Luc mRNA of 30:1 and a Luc mRNA concentration of 1.25ng / μL.

[0086] The poly (beta) 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. 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 the mRNA. The in vitro mRNA delivery efficiency and cell viability test results of different delivery vectors are shown in FIG. Figure 15 , Figure 15 The cell viability (i.e., number of surviving cells) of each group is the 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. Figure 15 It shows that the mRNA delivery efficiency of the poly (beta) 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.

[0087] 5. In vivo mRNA delivery effect test

[0088] The experimental subjects were 5-week-old female BALB / c mice weighing approximately 20 grams.

[0089] Seven synthesized poly(β-amino ester) materials (2A1-C8, 2A1-C12, 2A1-C16, 2A5-C8, 2A5-C12, 2A5-C16, and 2A4-C8) were dissolved in THF to prepare specific concentrations of 20 g / L. Luc mRNA was evenly dispersed in 10 mM citric acid-sodium hydrogen phosphate buffer (pH 4.2) to prepare a Luc mRNA stock solution. The stock solutions were mixed at a mass ratio of 30:1 between the delivery vehicle (poly(β-amino ester)) and the luciferase mRNA. After incubation for 1 hour, the nanoparticle complex solution was obtained. The final Luc mRNA concentration in the complex solution was 50 ng / μL (40 times the experimental concentration in cells). All other sample preparation parameters were similar.

[0090] 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 groups were prepared using poly (β-amino ester) molecules 2A1-C8, 2A1-C12, 2A1-C16, 2A5-C8, 2A5-C12, 2A5-C16, and 2A4-C8.

[0091] 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 naked mRNA group was still administered with a dose of 0.5 mg / kg per mouse. The Luc mRNA was diluted to 50 ng / μL with 10 mM citric acid-disodium hydrogen phosphate buffer (pH = 4.2). Each mouse was injected with 200 μL of the diluted Luc mRNA solution with a concentration of 50 ng / μL through the tail vein. Six hours after administration, the mice were anesthetized and the small animal in vivo imaging system was used to perform imaging to evaluate the mRNA delivery effect in vivo.

[0092] According to the above process, the expression results of luciferase in mice were measured. Figure 16 As shown. Figure 16 Six hours after injection of seven poly(β-amino ester)-mRNA composite nanoparticle solutions, 2A1-C8, 2A1-C12, 2A1-C16, and 2A4-C8 all showed strong fluorescence in mice, while 2A5-C8, 2A5-C12, and 2A5-C16 showed poor in vivo delivery. The results indicate that a backbone containing hydrophobic amines with a medium carbon chain length (12 carbon atoms) exhibited better in vivo delivery, while the end-capping hydrophilic amines 2A1 and 2A4 were more effective than 2A5. This suggests the importance of selecting end-capping amines and backbone-building amines when constructing vectors. Overall, the synthesized materials demonstrated excellent mRNA delivery in vivo. Injection of naked mRNA into the tail vein of mice did not reveal any luciferase expression.

[0093] 6. Organ Distribution of Nanoparticles

[0094] Six hours after administration, the experimental group and the naked mRNA group were dissected and imaged (the organs removed were liver, spleen, lung, heart and kidney). The expression results of luciferase in each organ of the mice are shown in the figure. Figure 17 .according to Figure 17 As a result, the poly (β-amino ester) molecules 2A1-C8, 2A1-C12, 2A1-C16, 2A5-C12, and 2A4-C8 synthesized in the present invention can deliver mRNA to the spleen and express a large amount of luciferase.

[0095] 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 considered 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: ； The preparation method of the carrier comprises the following steps: 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 the reaction is carried out in an ice-water bath to generate a reddish-brown liquid product containing Na2Se2, which does not require purification. The resulting reddish-brown liquid product is added dropwise to a solution of 6-bromo-1-hexanol dissolved in solvent B, and the reaction is condensed and refluxed under nitrogen protection. After the reaction, solvent B is removed by rotary evaporation to obtain a yellow liquid as selenium-containing monomer 1, which is 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 purified selenium-containing monomer 1 and acryloyl chloride as raw materials, the selenium-containing monomer 1 and triethylamine are dissolved in solvent B. Acryloyl chloride is added dropwise to the mixture under a nitrogen atmosphere. After the reaction, a yellow liquid is obtained as selenium-containing monomer 2, which is then 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. Selenium-containing monomer 2 and hydrophobic amine are charged into a reaction flask and reacted without solvent. After the reaction, a yellow-brown viscous product is obtained. 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: ； 4) Using the purified polymer intermediate and hydrophilic amine as raw materials, the purified polymer intermediate and hydrophilic amine are dissolved in solvent C and reacted. After the reaction, the product is vacuum dried to obtain a yellow-brown viscous product, thereby completing the preparation; wherein the hydrophilic amine raw material is one of 2A1, 2A4, and 2A5; 。 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 the reaction is carried out in an ice-water bath to generate a reddish-brown liquid product containing Na2Se2, which does not require purification. The resulting reddish-brown liquid product is added dropwise to a solution of 6-bromo-1-hexanol dissolved in solvent B, and the reaction is condensed and refluxed under nitrogen protection. After the reaction, solvent B is removed by rotary evaporation to obtain a yellow liquid as selenium-containing monomer 1, which is 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 purified selenium-containing monomer 1 and acryloyl chloride as raw materials, the selenium-containing monomer 1 and triethylamine are dissolved in solvent B. Acryloyl chloride is added dropwise to the mixture under a nitrogen atmosphere. After the reaction, a yellow liquid is obtained as selenium-containing monomer 2, which is then 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. Selenium-containing monomer 2 and hydrophobic amine are charged into a reaction flask and reacted without solvent. After the reaction, a yellow-brown viscous product is obtained. 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: ； 4) Using the purified polymer intermediate and hydrophilic amine as raw materials, the purified polymer intermediate and hydrophilic amine are dissolved in solvent C and reacted. After the reaction, the product is vacuum dried to obtain a yellow-brown viscous product, thereby completing the preparation; wherein the hydrophilic amine raw material is one of 2A1, 2A4, and 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, the molar ratio of selenium powder to 6-bromo-1-hexanol is 1:1.8-3, 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 3, characterized in that In step 1), the molar ratio of selenium powder to sodium borohydride is 1:1, and the molar ratio of selenium powder to 6-bromo-1-hexanol is 1:

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 1), the reaction time for preparing Na2Se2 in an ice-water bath is 25-40 minutes; the reaction temperature for preparing the 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 an ethyl acetate-n-hexane mixture with a volume ratio of 1:0.5-2.

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 2), the molar ratio of the purified selenium-containing monomer 1 and acryloyl chloride is 1:2.5~4, the molar ratio of the purified selenium-containing monomer 1 and triethylamine is 1:2.5~4, 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, and after mixed extraction with saturated brine, the product is purified by silica gel column chromatography, and the eluent for purification by silica gel column chromatography is a mixture of n-hexane and ethyl acetate with a volume ratio of 50:0.5-2.

7. The method for preparing a diselenide bond-containing poly-β-amino ester carrier for mRNA delivery according to claim 6, characterized in that In step 2), the molar ratio of the purified selenium-containing monomer 1 to acryloyl chloride is 1:3-4, and the molar ratio of the purified selenium-containing monomer 1 to triethylamine is 1:3-4.

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 3), the molar ratio of the hydrophobic amine to the selenium-containing monomer 2 is 1:1-1.2, and the polymer intermediate product is purified by precipitation with n-hexane.

9. The method for preparing a diselenide bond-containing poly-β-amino ester carrier for mRNA delivery according to claim 8, characterized in that In step 3), the molar ratio of the hydrophobic amine to the selenium-containing monomer 2 is 1:1-1.

05.

10. 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 48 hours.

11. 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, the solvent C is anhydrous THF, and the final product does not need to be purified.

12. The method for preparing a diselenide bond-containing poly-β-amino ester carrier for mRNA delivery according to claim 11, characterized in that In step 4), the molar ratio of the purified polymer intermediate to the hydrophilic amine is 1:

4.

13. 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.

14. 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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