Polymer for mRNA delivery, lipid / polymer hybrid nanoparticles using the polymer, and preparation method and application thereof

Abstract

The present invention discloses a polymer for mRNA delivery, lipid / polymer hybrid nanoparticles using the polymer, and their preparation and application. The method for preparing the cationic polymer of the present invention comprises synthesizing a precursor polymer with a terminal double bond through a Michael addition reaction using 1,6-hexanediol diacrylate (DAHE) and a hydrophilic amine as raw materials, and then performing an end-capping reaction with a hydrophobic amine. The present invention prepares a poly(β-amino ester) (PBAE) containing a hydrophilic backbone and a water-transporting end. By adding an auxiliary lipid such as (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP) to the PBAE polymer, the surface charge of the nanoparticles is increased within an appropriate range, thereby promoting cellular uptake of the nanoparticles and improving the mRNA delivery efficiency. The mRNA nanoformulation prepared using the cationic polymer and its formulation provided by the present invention can effectively improve the delivery efficiency of mRNA in cells and mice.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical preparations, and in particular to a polymer for mRNA delivery, lipid / polymer hybrid nanoparticles utilizing the polymer, and a preparation method and application thereof. Background Art

[0002] Messenger RNA (mRNA), a novel nucleic acid therapeutic, has recently become a hot topic of research. Synthetic mRNA encoding a specific protein, like natural mRNA, can express the corresponding functional protein in cells, thus holding broad application prospects in immunotherapy, regenerative medicine, infectious disease vaccines, and protein replacement / supplementation. Compared with plasmid DNA (pDNA) or small interfering RNA (siRNA)-based therapies, mRNA-based therapies offer numerous potential advantages: 1) mRNA expresses functional proteins directly in the cytoplasm, eliminating the need for nuclear entry like pDNA. Therefore, mRNA therapy eliminates the barrier of nuclear membrane traversal and can be highly effective in non-dividing or slowly dividing cells, such as dendritic cells; 2) mRNA does not need to integrate into cellular chromosomes, thus avoiding the risk of insertional mutagenesis; and 3) compared with RNA interference, mRNA translation of the target protein is simpler and more direct, without the off-target effects associated with siRNA. Furthermore, compared with corresponding recombinant proteins, the high charge density and flexibility of mRNA allow for higher loading efficiency in vectors; mRNA preparation is relatively simple, and biosafety risks are minimized.

[0003] However, mRNA vaccines also have major problems that limit their development. The mRNA molecule itself is extremely unstable and easily degraded by mRNA enzymes both inside and outside the body. Secondly, mRNA itself is negatively charged, making it difficult for cells to take up, resulting in low expression efficiency. Therefore, developing efficient mRNA delivery systems, improving mRNA expression efficiency, and enhancing vaccine-induced cellular and humoral immunity in vivo are key technologies for the development of mRNA therapy. However, the mRNA expression efficiency of cationic polymer-based delivery vectors reported in existing technologies is unsatisfactory. Therefore, it is urgent to develop a new cationic polymer-based mRNA delivery system and construct a stable, high-molecular-weight mRNA nanovaccine. Summary of the Invention

[0004] In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, to improve the stability of drug carriers and drug encapsulation efficiency, and to improve drug delivery efficiency and therapeutic effects, the present invention provides a polymer for mRNA delivery, lipid / polymer hybrid nanoparticles using the polymer, and a preparation method and application thereof. The present invention modifies PBAE by hydrophobic groups, thereby increasing the physical encapsulation effect of nucleic acids and promoting cell adsorption. At the same time, a cationic polymer / lipid hybrid strategy is used to combine lipids with good biocompatibility with structurally stable polymers. Auxiliary lipids such as (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP) are added to the PBAE polymer to increase the surface charge of the nanoparticles within an appropriate range, promote the uptake of nanoparticles by cells, and enhance the in vivo and in vitro delivery of mRNA.

[0005] The technical solution adopted in the present invention is as follows:

[0006] A method for preparing a polymer for mRNA delivery comprises the following steps:

[0007] 1) Using 1,6-hexanediol diacrylate DAHE and a hydrophilic amine as raw materials, heating and dissolving them at 80-95°C, and performing a Michael addition reaction for 40-60 hours. After the reaction, washing and drying are performed to obtain a precursor polymer with acrylates at both ends; wherein the hydrophilic amine raw material is one of A7, A8, A9, A13, and A15:

[0008] 2) The precursor polymer having acrylate esters at both ends obtained in step 1) is capped with a hydrophobic amine. The precursor polymer and the hydrophobic amine are stirred and reacted in an organic solvent at a reaction temperature of 50-65° C. for 20-55 hours, followed by drying to obtain a capped end product, which is referred to as polymer PBAE. The hydrophobic amine raw material is one of 2C4, 2C6, 2C8, 2C10, C8′, and C12′.

[0009] The reaction formula for preparing the polymer of the present invention is as follows:

[0010]

[0011] The structure of the hydrophilic main chain is one of the following:

[0012]

[0013] The hydrophobically terminated structure is one of the following:

[0014]

[0015] Furthermore, the structure of the polymer for mRNA delivery is selected from one of the following:

[0016]

[0017] Furthermore, in step 1), the molar ratio of DAHE to hydrophilic amine is 0.8-1.5:1, preferably 1-1.1:1; the reaction temperature in step 1) is 85-90° C., and the reaction time is 45-50 h.

[0018] Furthermore, in step 2), the molar ratio of the precursor polymer to the hydrophobic amine is 1:3-5, preferably 1:4, the reaction temperature is 55-60° C., and the reaction time is 45-50 h;

[0019] In step 2), the organic solvent is tetrahydrofuran, and the concentration of the hydrophobic amine in the organic solvent is 0.01-0.1 mol / L, preferably 0.03-0.0625 mol / L.

[0020] Furthermore, the polymer for mRNA delivery prepared by the method of the present invention has a molecular weight between 1.5 kDa and 37 kDa and a degree of polymerization between 10 and 52.

[0021] A method for preparing lipid / polymer hybrid nanoparticles comprises the following steps:

[0022] S1: dissolving luciferase mRNA in an acidic solution to obtain a first solution;

[0023] S2: dissolving the polymer prepared by the present invention and auxiliary lipids cholesterol, DOPE, PEG2000-DMG, and DOTAP in anhydrous ethanol to obtain a second solution;

[0024] S3: mixing the first solution and the second solution to obtain a mixed solution, and incubating the mixed solution on a shaking platform;

[0025] S4: The mixed solution after incubation was diluted with sterile PBS to obtain a suspension containing the prepared lipid / polymer hybrid nanoparticles.

[0026] Furthermore, in step S1, the acidic solution is a sodium citrate solution, 10 mM, pH = 4.2.

[0027] Furthermore, in step S2, the molar ratio of the polymer to cholesterol is 1:0.8-1.2, preferably 1:0.9-1.0;

[0028] The molar ratio of polymer to DOPE is 1:0.2-0.3, preferably 1:0.25;

[0029] The molar ratio of polymer to DOTAP is 1:0.2-0.3, preferably 1:0.25;

[0030] The molar ratio of polymer to PEG2000-DMG is 1:0.02-0.05, preferably 1:0.035-0.04;

[0031] The total mass of the polymer and DOTAP in the second solution of step S2 is 15-25 times, preferably 20 times, the mass of the luciferase mRNA in the first solution of step S1;

[0032] Wherein, the mass concentration of the polymer dissolved in anhydrous ethanol in step S2 is 0.5-2 g / L, preferably 1 g / L.

[0033] Furthermore, in step S3, the shaking incubation time is 0.5 to 1.5 hours, and in step S4, the mixed solution after incubation is diluted with PBS at a dilution ratio of 4 to 6 times, and the content of organic phase ethanol in the diluted solution is not higher than 10%.

[0034] The present invention also provides the use of the lipid / polymer hybrid nanoparticles in the preparation of mRNA vaccine drugs.

[0035] The beneficial effects achieved by the present invention are:

[0036] The present invention innovatively adopts a new polymer molecule and optimizes the formulation of nanoparticles by incorporating auxiliary lipids such as DOTAP, thereby obtaining a new hybrid nanoparticle composition. The terminal modification of PBAE is changed from conventional hydrophilic groups to hydrophobic groups, thereby improving the physical encapsulation efficiency and delivery efficiency of nucleic acids. On the other hand, the addition of auxiliary lipids such as DOTAP increases the surface charge of the nanoparticles within an appropriate range, thereby solving the problem of insufficient cellular uptake of nanoparticles. The preparation strategy of this lipid / polymer hybrid nanoparticle (LPH) is simple, fast and easy to control. The nanoparticles have high transfection efficiency in vitro and good stability in vivo, and have high application value and very broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Represents the precursor polymer A7 1 H-NMR spectrum.

[0038] Figure 2 Represents the precursor polymer A8 1 H-NMR spectrum.

[0039] Figure 3 Represents the precursor polymer A9 1 H-NMR spectrum.

[0040] Figure 4 Represents the precursor polymer A13 1 H-NMR spectrum.

[0041] Figure 5Represents the precursor polymer A15 1 H-NMR spectrum.

[0042] Figure 6 It shows the molecular weight of precursor polymers A7, A8, A9, A11, A13, and A15.

[0043] Figure 7 Indicates A7-2C4, A7-2C6, and A7-2C10 1 H-NMR spectrum.

[0044] Figure 8 Indicates A8-C8', A8-2C8 1 H-NMR spectrum.

[0045] Figure 9 Indicates A9-2C10 1 H-NMR spectrum.

[0046] Figure 10 Indicates A13-C8', A15-C12' 1 H-NMR spectrum.

[0047] Figure 11 It shows the test results of in vitro mRNA delivery efficiency of LPH prepared with different polymer carriers.

[0048] Figure 12 represents the particle size and potential of LPH prepared with different polymer carriers;

[0049] Figure 13 It represents the encapsulation efficiency (Binding) of LPH prepared by different polymer carriers to Fluc mRNA (%);

[0050] Figure 14 It shows the expression of luciferase in mice 6 hours after tail vein injection of LPH prepared with different polymer carriers.

[0051] Figure 15 The figure shows the expression of luciferase in various organs of mice 8 hours after tail vein injection of LPH prepared with different polymer carriers.

[0052] Figure 16 The particle size potential of LPH prepared by polymer carrier A7-2C10 under various formulations is shown.

[0053] Figure 17 The graph shows the uptake of Cy5-mRNA-LPH prepared by polymer carrier A7-2C10 in various formulations by IGrov1 cells under fluorescence microscopy observation.

[0054] Figure 18The graph shows the uptake of Cy5-mRNA-LPH prepared by polymer carrier A7-2C10 in various formulations by IGrov1 cells after flow cytometry treatment. DETAILED DESCRIPTION

[0055] 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.

[0056] Example 1: Synthesis of A7-2C6

[0057] (1) In a 10 mL sample vial equipped with a stirrer, hydrophilic amine A7 (0.6683 g, 5.3387 mmol) and DAHE (1.3346 g, 5.8850 mmol) were added to a molar ratio of 1:1.1, with a total mass of approximately 2 g. The mixture was stirred and dissolved at 90°C for 48 hours. After cooling, the mixture was washed three times with n-hexane, purified, and dried in a vacuum drying oven overnight to obtain the precursor polymer A7.

[0058] (2) Use hydrophobic amine to cap the precursor polymer with acrylate at both ends. Precursor polymer A7 (0.1046 g, 0.0232 mmol), hydrophobic amine 2C6 (0.0173 g, 0.0933 mmol), and tetrahydrofuran (THF) (2 mL) were added to a 4 mL sample bottle, respectively, and the molar ratio of the precursor polymer to the hydrophobic amine was controlled to be 1:4. After stirring at 55°C for 48 hours, the mixture 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 A7-2C6.

[0059] In addition, the preparation methods of polymers A7-2C4 and A7-2C10 repeat the above experimental steps, except that the hydrophobic amine 2C6 is replaced by the same molar amount of hydrophobic amine 2C4 or hydrophobic amine 2C10.

[0060] Example 2: Synthesis of A8-2C8

[0061] (1) In a 10 mL sample vial equipped with a stirrer, hydrophilic amine A8 (0.6321 g, 5.4393 mmol) and DAHE (1.3638 g, 6.0273 mmol) were added to a molar ratio of 1:1.1, with a total mass of approximately 2 g. The mixture was stirred and dissolved at 90°C for 48 hours. After cooling, the mixture was washed three times with n-hexane, purified, and dried in a vacuum drying oven overnight to obtain the precursor polymer A8.

[0062] (2) Use a hydrophobic amine to cap the precursor polymer with acrylates at both ends. Add the precursor polymer A8 (0.1032 g, 0.0159 mmol), the hydrophobic amine 2C8 (0.0153 g, 0.0635 mmol) to a 4 mL sample vial, controlling the molar ratio of the precursor polymer to the hydrophobic amine to be 1:4, and tetrahydrofuran (THF) (2 mL). After stirring at 55°C for 48 hours, dry in a fume hood for two days, and then dry in a vacuum drying oven for one day to obtain the final product A8-2C8.

[0063] In addition, the preparation method of polymer A8-C8' repeats the above experimental steps, except that the hydrophobic amine 2C8 is replaced by the same molar amount of hydrophobic amine C8'.

[0064] Example 3: Synthesis of A9-2C10

[0065] (1) In a 10 mL sample vial equipped with a stirrer, hydrophilic amine A9 (0.8123 g, 4.6872 mmol) and DAHE (1.1806 g, 5.2177 mmol) were added to a molar ratio of 1:1.1, with a total mass of approximately 2 g. The mixture was stirred and dissolved at 90°C for 48 hours. After cooling, the mixture was washed three times with n-hexane, purified, and dried in a vacuum drying oven overnight to obtain the precursor polymer A9.

[0066] (2) Use hydrophobic amine to cap the precursor polymer with acrylate at both ends. Precursor polymer A9 (0.1078 g, 0.0117 mmol), hydrophobic amine 2C10 (0.0142 g, 0.0478 mmol), and tetrahydrofuran (THF) (2 mL) were added to a 4 mL sample bottle, respectively, and the molar ratio of the precursor polymer to the hydrophobic amine was controlled to be 1:4. After stirring at 55°C for 48 hours, the mixture 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 A9-2C10.

[0067] Example 4: Synthesis of A13-C8'

[0068] (1) In a 10 mL sample vial equipped with a stirrer, hydrophilic amine A13 (0.5342 g, 5.9928 mmol) and DAHE (1.4781 g, 6.5325 mmol) were added to a molar ratio of 1:1.1, with a total mass of approximately 2 g. The mixture was stirred and dissolved at 90°C for 48 hours. After cooling, the mixture was washed three times with n-hexane, purified, and dried in a vacuum drying oven overnight to obtain the precursor polymer A13.

[0069] (2) Use hydrophobic amine to cap the precursor polymer with acrylate at both ends. Precursor polymer A13 (0.1097 g, 0.0233 mmol), hydrophobic amine C8' (0.0136 g, 0.0949 mmol), and tetrahydrofuran (THF) (2 mL) were added to a 4 mL sample bottle, respectively, and the molar ratio of the precursor polymer to the hydrophobic amine was controlled to be 1:4. After stirring at 55°C for 48 hours, the mixture 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 A13-C8'.

[0070] Example 5: Synthesis of A15-C12'

[0071] (1) In a 10 mL sample vial equipped with a stirrer, hydrophilic amine A15 (0.5931 g, 5.7488 mmol) and DAHE (1.4208 g, 6.2792 mmol) were added to a molar ratio of 1:1.1, with a total mass of approximately 2 g. The mixture was stirred and dissolved at 90°C for 48 hours. After cooling, the mixture was washed three times with n-hexane, purified, and dried in a vacuum drying oven overnight to obtain the precursor polymer A15.

[0072] (2) Use hydrophobic amine to cap the precursor polymer with acrylate at both ends. Precursor polymer A15 (0.1021 g, 0.0309 mmol), hydrophobic amine C12' (0.0248 g, 0.1246 mmol), and tetrahydrofuran (THF) (2 mL) were added to a 4 mL sample bottle, respectively, and the molar ratio of the precursor polymer to the hydrophobic amine was controlled to be 1:4. After stirring at 55°C for 48 hours, the mixture 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 A15-C12'.

[0073] Example 6: Determination of polymer structure and molecular weight

[0074] The precursor polymers A7, A8, A9, A13 and A15 prepared in Examples 1-5 were tested respectively by dissolving 10 mg of the dried precursor polymer sample in deuterated chloride, transferring the sample to a nuclear magnetic resonance tube, and performing nuclear magnetic resonance spectroscopy ( 1 H nuclearmagnetic resonance, 1 H NMR) was used to characterize the structure and molecular weight of the cc-PBAE precursor polymer. 1 The precursor polymers A7, A8, A9, A13, and A15 were characterized by H NMR. 1 The H-NMR spectra are as follows Figure 1-5 As shown, 1In HNMR, 6.4ppm, 6.1ppm, and 5.8ppm are the proton peaks of the double bonds in the precursor polymer, indicating that the double bonds have not been completely reacted; 4.1ppm, 1.6ppm, and 1.4ppm are the proton peaks of the main chain -O-CH2-CH2-CH2-. According to the spectrum analysis, the molecular weights of the precursor polymers A7, A8, A9, A13, and A15 are as follows: Figure 6 shown.

[0075] The final polymer products prepared in Examples 1-5 were tested respectively by dissolving 10 mg of the dried final polymer product sample in deuterated chloride, transferring it to a nuclear magnetic resonance tube, and analyzing it by hydrogen nuclear magnetic resonance spectroscopy ( 1 H nuclearmagnetic resonance, 1 H NMR) was used to characterize the structure of the final cc-PBAE polymer. 1 H-NMR spectra are summarized in Figure 7 , polymers A8-C8' and A8-2C8 prepared in Example 2 1 H-NMR spectra are summarized in Figure 8 , polymer A9-2C10 1 H-NMR spectrum Figure 9 As shown, polymers A13-C8', A15-C12' 1 H-NMR spectra are summarized in Figure 10 .

[0076] Example 7: In vitro delivery efficiency of LPH

[0077] The synthesized polymer carrier PBAE was dissolved in anhydrous ethanol with DOTAP, DOPE, cholesterol, and PEG2000-DMG at a concentration of 1 g / L. The five components were mixed at a molar ratio of PBAE:DOTAP:DOPE:Chol:PEG2000-DMG = 40:10:10:38.5:1.5. Luciferase mRNA (Fluc mRNA) was dissolved in a sodium citrate solution (10 mM, pH = 4.2). The prepared ethanol solution was mixed with a sodium citrate solution (10 mM, pH = 4.2) at a volume ratio of 1:1. The mass ratio of (DOTAP + PBAE) to luciferase mRNA (Fluc mRNA) was approximately 20:1. After incubation, the solution was diluted fivefold with PBS to obtain a composite nanoparticle solution (containing 2.5 mg of luciferase mRNA per liter of composite nanoparticle solution). The composite nanoparticles were designated LPH.

[0078] After IGrov1 cells were cultured in DMEM medium until they were adherent, the composite nanosolution prepared above was added (the addition amount was 12.5 / 25 / 50 ng per 10,000 cells). After transfection under this condition for 48 hours, the medium was removed and luciferin, a substrate of luciferase, was added. The in vitro mRNA delivery efficiency was tested by the luminescence value.

[0079] Through the above operation process, the A7-2C4, A7-2C6, A7-2C8, A8-C8', A8-2C8, A9-2C10, A13-C8', and A15-C12' polymer molecules synthesized in Examples 1-6 of the present invention were used as delivery vectors, and the approved lipid molecule ALC-0315 was used as a positive control group of the delivery vector. They were mixed with auxiliary lipids and mRNA encoding luciferase to prepare nanoparticles, which were added to IGrov1 cells. After 48 hours of transfection, the in vitro delivery efficiency of mRNA was evaluated by the expression of luciferase in the cells. The in vitro mRNA delivery efficiency test results of different delivery vectors are shown in FIG. Figure 11 . Figure 11 It shows that the polymer carrier synthesized by the present invention has a comparable mRNA delivery efficiency to that of ALC0315, the main delivery carrier in the new crown vaccine preparation, and its luminescence value shows good dose responsiveness under different mRNA doses.

[0080] Example 8: Particle size, surface charge, and mRNA loading efficiency of LPH

[0081] Hybrid nanoparticles were prepared according to the method described in Example 7. The particle size and surface potential of LPH were measured by dynamic light scattering (DLS). The particle size and surface charge test results of nanoparticle suspensions prepared from polymers with different structures are summarized in Figure 12 middle.

[0082] Hybrid nanoparticles were prepared according to the method described in Example 7, and the free mRNA concentration in the nanoparticle suspension was determined using the Quant-iT RiboGreen Assay Kit to quantify the loading efficiency of LPH on Fluc mRNA. The specific implementation steps were as follows: 100 μL of nanoparticle solution diluted 10 times with TE buffer (mRNA concentration of 0.25 mg / L) was added to a 96-well black plate, followed by the addition of 100 μL of RiboGreen reagent diluted 800 times with TE buffer, and the plate was gently pipetted and then analyzed using a microplate reader (λ ex =485nm, λ em=535nm) to detect fluorescence intensity. The free mRNA content in each well can be obtained by using the standard curve of mRNA and RiboGreen fluorescence intensity. Combined with the total amount of mRNA in the added nanoparticle solution, the loading efficiency of LPH on mRNA can be calculated. The results of the LPH loading efficiency test of each polymer carrier on mRNA are summarized in Figure 13 .

[0083] Example 9: In vivo mRNA delivery effect test of LPH

[0084] The synthesized polymer carrier PBAE was dissolved in anhydrous ethanol with DOTAP, DOPE, cholesterol, and PEG2000-DMG. The concentration of each polymer carrier in anhydrous ethanol was 20 g / L, and the molar ratio of the five was PBAE:DOTAP:DOPE:Chol:PEG2000-DMG = 40:10:10:38.5:1.5. Luciferase mRNA (Fluc mRNA) was dissolved in sodium citrate solution (10 mM, pH = 4.2). The ethanol solution and sodium citrate solution (10 mM, pH = 4.2) were mixed at a controlled volume ratio of 1:1.

[0085] The mass ratio of (DOTAP+PBAE) to luciferase mRNA (Fluc mRNA) was approximately 20:1. After incubation, the solution was diluted 5-fold with PBS to obtain a composite nanoparticle solution (containing 50 mg of luciferase mRNA per liter of composite nanoparticle solution).

[0086] Naked mRNA group: Luciferase mRNA (Fluc mRNA) was dissolved in sodium citrate solution (10 mM, pH = 4.0), incubated, and diluted with PBS to obtain an mRNA nanoparticle solution (50 mg luciferase mRNA per liter of nanoparticle solution).

[0087] Nanoparticle solutions prepared from the polymer molecule or naked mRNA groups were used to test the in vivo mRNA delivery efficacy of 6-week-old female BALB / c mice weighing approximately 20g. The mRNA injection dose was 0.5mg / kg, and the injection volume per mouse was 0.2mL. The corresponding lipid-mRNA nanoparticle solution or mRNA nanoparticle solution was injected into the mice via the tail vein. Six hours after administration, the luciferase expression efficiency in the mice was measured using a small animal imaging device.

[0088] According to the above process, when the polymer carriers were A7-2C4, A7-2C6, A7-2C8, A8-C8', A8-2C8, A9-2C10, A13-C8', and A15-C12', the expression results of luciferase in mice were shown in Figure 2. Figure 14As shown. Figure 14 It can be seen that 6 hours after the injection of 9 hybrid nanoparticle solutions, most of the mice showed fluorescence expression of varying intensities, among which the LPH prepared by the polymer molecule A13C8' showed a strong delivery effect in mice.

[0089] According to the above process, when the injected drug is a naked mRNA nanoparticle solution, the expression of luciferase in mice is measured. Figure 14 As shown. Figure 14 It can be seen that there is no expression of luciferase after naked mRNA is injected into the tail vein of mice.

[0090] Example 10: Distribution of LPH in organs in vivo

[0091] After 6 hours of in vivo mRNA delivery test of LPH according to Example 9, the mice in the LPH experimental group and the naked mRNA group were dissected and their organs were imaged (the organs dissected were liver, spleen, lung, heart and kidney). The expression results of luciferase in each organ of the mice are shown in the figure. Figure 15 .according to Figure 15 As a result, A7-2C4, A7-2C6, A7-2C8, A8-C8', A8-2C8, A9-2C10, A13-C8', and A15-C12' synthesized in Examples 1-6 of the present invention can deliver mRNA to the lungs and spleen to express a large amount of luciferase.

[0092] The contents described in this specification are merely an enumeration of the 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.

[0093] Example 11: Verification of DOTAP-promoted cellular uptake in LPH

[0094] The synthesized polymer carrier A7-2C10 was dissolved with DOTAP, DOPE, cholesterol, and PEG2000-DMG in anhydrous ethanol. The solubility concentration of the polymer carrier A7-2C10 in ethanol was 1 g / L, and the molar ratio of the five was (DOTAP+polymer carrier A7-2C10):DOPE:Chol:PEG2000-DMG=50:10:38.5:1.5. The molar ratios of DOTAP and polymer carrier A7-2C10 were set to 1:1, 4:1, and 8:1, respectively. The mass ratio of (DOTAP+polymer carrier A7-2C10) to luciferase mRNA (Fluc mRNA) was approximately 20:1. The prepared composite LPH nanoparticles were recorded as D / P1-1, D / P4-1, and D / P8-1, respectively.

[0095] Polymer nanoparticles prepared with a 20:1 mass ratio of polymer A7-2C10 to mRNA were designated PBAE. LPH without DOTAP was designated DOTO. The molar ratio of A7-2C10, DOPE, cholesterol, and PEG2000-DM was (A7-2C10:DOPE:Chol:PEG2000-DMG) = 50:10:38.5:1.5. All other preparation conditions were based on the method described in Example 7.

[0096] The particle size and surface potential of the composite nanoparticles were measured by dynamic light scattering (DLS). Figure 16 ,The results showed that the incorporation of auxiliary lipids such as DOTAP effectively increased the surface charge of nanoparticles.

[0097] The cellular uptake of Cy5-mRNA-complex nanoparticles was analyzed by fluorescence microscopy and flow cytometry. The complex nanoparticle solution was prepared according to the above method, with the only difference being that "Cy5-mRNA (i.e., Cy5-labeled mRNA) was used instead of luc mRNA." Other conditions remained unchanged, and the prepared LPH was recorded as Cy5-mRNA-complex nanoparticles.

[0098] IGROV 1 cells were seeded into 6-well plates at a density of 100,000 cells / well. After culturing for 24 hours, 2 mL of fresh DMEM medium was replaced. The above five Cy5-mRNA-loaded composite nanoparticle solutions were added at a dose of 150 μL per well (dose of 250 ng mRNA / well). After incubation for 2 hours or 6 hours, the DMEM medium was removed, the cells were washed with PBS, and 1 mL of 4% paraformaldehyde was added for fixation for 30 minutes. After removing the fixative, the cells were washed with PBS, and DAPI (10 μg / mL) was added for staining for 15 minutes. The cells were washed with PBS and observed under a fluorescence microscope. The results are shown in FIG. Figure 17 As shown, the Cy5 fluorescence signal was stronger after treatment with DOTAP-incorporated LPH.

[0099] IGROV 1 cells were seeded in 6-well plates at a density of 300,000 cells / well. After culturing for 24 hours, 2 mL of fresh DMEM medium was replaced. Cy5-mRNALPH (250 ng mRNA / well) was added at a dose of 150 μL per well. After 6 hours of incubation after administration, the cells were digested and collected by centrifugation and resuspended in PBS. Fluorescence histograms were recorded using a flow cytometer and data were processed and analyzed using FlowJo software. The results are shown in Figure 2. Figure 18 As shown, the average Cy5 fluorescence signal of cells treated with DOTAP-incorporated LPH was higher, and the proportion of Cy5 signal-positive cells was higher.

Claims

1. A method for preparing a polymer for mRNA delivery, characterized in that The following steps are involved: 1) Using 1,6-hexanediol diacrylate (DAHE) and a hydrophilic amine as raw materials, heating and dissolving the mixture at 80-95°C for 40-60 hours, washing and drying the mixture after the reaction to obtain a precursor polymer with acrylates at both ends; wherein the hydrophilic amine raw material is one of A7, A8, A9, A13, and A15: ； 2) The precursor polymer having acrylate esters at both ends obtained in step 1) is capped with a hydrophobic amine. The precursor polymer and the hydrophobic amine are reacted in an organic solvent with stirring at a reaction temperature of 50-65° C. for 20-55 hours, followed by drying to obtain a capped end product, designated as polymer PBAE; wherein the hydrophobic amine raw material is one of 2C4, 2C6, 2C8, 2C10, C8', and C12': 。 2. The method for preparing a polymer for mRNA delivery according to claim 1, wherein In step 1), the molar ratio of DAHE to hydrophilic amine is 0.8-1.5:1; the reaction temperature in step 1) is 85-90° C., and the reaction time is 45-50 h.

3. The method for preparing a polymer for mRNA delivery according to claim 2, wherein In step 1), the molar ratio of DAHE to hydrophilic amine is 1-1.1:

1.

4. The method for preparing a polymer for mRNA delivery according to claim 1, wherein In step 2), the molar ratio of the precursor polymer to the hydrophobic amine is 1:3-5, the reaction temperature is 55-60°C, and the reaction time is 45-50 h; In step 2), the organic solvent is tetrahydrofuran, and the concentration of the hydrophobic amine in the organic solvent is 0.01-0.1 mol / L.

5. The method for preparing a polymer for mRNA delivery according to claim 4, characterized in that In step 2), the molar ratio of the precursor polymer to the hydrophobic amine is 1:4; in step 2), the concentration of the hydrophobic amine in the organic solvent is 0.03-0.0625 mol / L.

6. A polymer for mRNA delivery prepared by the method according to any one of claims 1 to 5, characterized in that: Its molecular weight is between 1.5 kDa and 37 kDa, and its degree of polymerization is between 10 and 52.

7. A method for preparing lipid / polymer hybrid nanoparticles, characterized in that The following steps are involved: S1: dissolving luciferase mRNA in an acidic solution to obtain a first solution; S2: Dissolving the polymer of claim 6 and auxiliary lipids cholesterol, DOPE, PEG2000-DMG, and DOTAP in anhydrous ethanol, respectively, and obtaining a second solution by combining the five components; S3: mixing the first solution and the second solution to obtain a mixed solution, and incubating the mixed solution on a shaking platform; S4: The mixed solution after incubation was diluted with sterile PBS to obtain a suspension containing the prepared lipid / polymer hybrid nanoparticles.

8. The method for preparing lipid / polymer hybrid nanoparticles according to claim 7, characterized in that In step S1, the acidic solution is a sodium citrate solution, 10 mM, pH = 4.

2.

9. The method for preparing lipid / polymer hybrid nanoparticles according to claim 7, wherein In step S2, The molar ratio of polymer to cholesterol is 1:0.8-1.2; The molar ratio of polymer to DOPE is 1:0.2-0.3; The molar ratio of polymer to DOTAP is 1:0.2-0.3; The molar ratio of polymer to PEG2000-DMG was 1:0.02–0.05; Wherein, the mass concentration of the polymer dissolved in anhydrous ethanol in step S2 is 0.5-2 g / L; The total mass of the polymer and DOTAP in the second solution of step S2 is 15-25 times the mass of the luciferase mRNA in the first solution of step S1.

10. The method for preparing lipid / polymer hybrid nanoparticles according to claim 9, characterized in that In step S2, The molar ratio of polymer to cholesterol is 1:0.9-1.0; The molar ratio of polymer to DOPE was 1:0.25; The molar ratio of polymer to DOTAP was 1:0.25; The molar ratio of polymer to PEG2000-DMG was 1:0.035-0.04; Wherein, the mass concentration of the polymer dissolved in anhydrous ethanol in step S2 is 1 g / L; The total mass of the polymer and DOTAP in the second solution of step S2 is 20 times the mass of the luciferase mRNA in the first solution of step S1.

11. The method for preparing lipid / polymer hybrid nanoparticles according to claim 10, characterized in that The shaking incubation time in step S3 is 0.5 to 1.5 h. In step S4, the mixed solution after incubation is diluted with sterile PBS at a dilution ratio of 4 to 6 times. The content of organic phase ethanol in the diluted solution is not higher than 10%.

12. A lipid / polymer hybrid nanoparticle prepared according to the method of any one of claims 7 to 11.

13. Use of the lipid / polymer hybrid nanoparticles according to claim 12 in the preparation of mRNA vaccine drugs.

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