A highly branched poly (β-amino ester) and its preparation method and application in mRNA delivery

Abstract

The present invention belongs to the technical field of biomedical materials, and discloses a highly branched poly (β-amino ester) and its preparation method and application in mRNA delivery. The present invention uses 1,4-butanediol diacrylate, 5-amino-1-pentanol, and 1,2-ethylenediamine as raw materials, and prepares a polymer with double bonds by addition reaction, and then end-capsulates by amino monomer to obtain a highly branched poly (β-amino ester) with terminal functionalization. The present invention optimizes the structure of the polymer by selecting raw materials and functionalized end-capping monomers, and the prepared highly branched poly (β-amino ester) has high biocompatibility, low cytotoxicity, and high mRNA delivery efficiency, which effectively solves the problem of low mRNA delivery efficiency of traditional cationic polymer carriers. The preparation method of the present invention has mild conditions, and raw materials are easily available, and low requirements for equipment, and is suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a highly branched poly (β-amino ester) and a preparation method thereof and application thereof in mRNA delivery. Background Art

[0002] mRNA has a large molecular weight, strong hydrophilicity, and biological activity thousands of times that of small molecule drugs, but its single-stranded structure makes it extremely unstable and easily degraded by nucleases; and mRNA itself carries a negative charge, so it is difficult to deliver it through the cell membrane, which also has a negative charge on the surface. Therefore, mRNA needs to be specially modified or packaged for delivery in order to achieve mRNA expression in cells.

[0003] At present, commonly used mRNA delivery carriers include lipid nanoparticles (LNP), polymer carriers, etc. Although these carriers have improved the delivery efficiency to a certain extent, they still need to be further optimized to improve specificity and stability. Moreover, mRNA delivery carriers also need to have good biocompatibility to ensure that they will not cause immune responses or other side effects in vivo. Lipid nanoparticles are one of the most advanced mRNA carriers, but their ingredients are complex and may cause immune responses or toxicity problems. Polymer carriers such as cationic polymers such as polyethyleneimine, poly (amino acids), and polyesters have the advantages of being able to tightly wrap mRNA and being easy to synthesize, and are widely used in the field of mRNA delivery. However, compared with LNP, cationic polymers generally show lower mRNA delivery efficiency, but the LNP synthesis process is cumbersome and also has higher cytotoxicity. Cationic polymers are still the most potential mRNA delivery carriers. Therefore, the improvement of the structure of cationic polymer carriers is a problem that needs to be solved urgently. Summary of the invention

[0004] The purpose of the present invention is to provide a highly branched poly (β-amino ester) and a preparation method and application thereof, so as to solve the problem of low mRNA delivery efficiency of existing cationic polymer carriers.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a highly branched poly (β-amino ester), the structural formula of the highly branched poly (β-amino ester) is as follows:

[0007]

[0008] Among them, n is 10 to 100.

[0009] The present invention also provides a method for preparing a highly branched poly(β-amino ester), comprising the following steps:

[0010] (1) reacting acrylic acid ester with 5-amino-1-pentanol in a solvent to obtain a base polymer;

[0011] (2) reacting a base polymer with 1,2-ethylenediamine in a solvent to obtain a polymer with a double bond;

[0012] (3) The polymer with double bonds is reacted with a capping agent in a solvent to obtain a highly branched poly(β-amino ester).

[0013] Preferably, in the above method for preparing a highly branched poly(β-amino ester), the acrylate in step (1) is 1,4-butanediol diacrylate; and the end-capping agent in step (3) is 3,6,9-trioxaundecane-1,11-diamine.

[0014] Preferably, in the above method for preparing a highly branched poly(β-amino ester), the molar ratio of the acrylic ester to 5-amino-1-pentanol in step (1) is 1-3:1-5.

[0015] Preferably, in the above method for preparing a highly branched poly(β-amino ester), the reaction in step (1) is carried out until the preset molecular weight reaches 3000 to 50000 Da; and the reaction temperature is 60 to 100°C.

[0016] Preferably, in the above method for preparing a highly branched poly(β-amino ester), the molar ratio of the acrylic ester in step (1) to the 1,2-ethylenediamine in step (2) is 1-3:0.5-3.

[0017] Preferably, in the above method for preparing a highly branched poly(β-amino ester), the reaction temperature in step (2) is 80 to 100° C.; and the reaction time is 12 to 72 hours.

[0018] Preferably, in the above method for preparing a highly branched poly(β-amino ester), the molar ratio of the acrylic ester in step (1) to the end-capping agent in step (3) is 1-3:1-12.

[0019] Preferably, in the above method for preparing a highly branched poly(β-amino ester), the reaction temperature in step (3) is 25° C. and the reaction time is 12 to 72 hours.

[0020] The present invention also provides an application of a highly branched poly (β-amino ester) in preparing an mRNA delivery vector.

[0021] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention uses 1,4-butanediol diacrylate, 5-amino-1-pentanol, and 1,2-ethylenediamine as raw materials, and prepares a highly branched poly (β-amino ester) with double bonds through an addition reaction, and then caps it with an amino monomer to obtain a terminally functionalized highly branched poly (β-amino ester). The highly branched poly (β-amino ester) of the present invention optimizes the structure of the polymer by selecting the raw materials and the functionalized capping monomers. The prepared highly branched poly (β-amino ester) has high biocompatibility, low cytotoxicity, and high mRNA delivery efficiency, which effectively solves the problem of low mRNA delivery efficiency of traditional cationic polymer carriers.

[0023] (2) The highly branched poly (β-amino ester) of the present invention encapsulates mRNA to form composite nanoparticles. Through the relevant performance measurement of the composite nanoparticles, it can be known that the composite nanoparticles have high biocompatibility, can mediate efficient mRNA transfection, and have high stability. They are not easy to agglomerate during atomization, and can be used in the application field of atomization inhalation to administer drugs to the lungs, which is of great significance for the treatment of respiratory diseases.

[0024] (3) The preparation method of the present invention has mild conditions, readily available raw materials, low requirements on equipment, and is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the description of the embodiments or the prior art are briefly introduced below.

[0026] Figure 1 Schematic diagram of the synthesis process of the highly branched poly (β-amino ester) of the present invention;

[0027] Figure 2 is the GPC spectrum of the highly branched poly(β-amino ester) prepared in Example 1;

[0028] Figure 3 The highly branched poly (β-amino ester) prepared in Examples 1 to 4 1 H NMR spectrum;

[0029] Figure 4 The results of the test of the affinity efficiency of composite nanoparticles with different mass ratios to mRNA;

[0030] Figure 5 The particle size test results of composite nanoparticles with different mass ratios;

[0031] Figure 6 Zeta potential test results of composite nanoparticles with different mass ratios;

[0032] Figure 7The fluorescence detection results of the transfection of composite nanoparticles with different mass ratios are shown in FIG. 1 , wherein a is a composite nanoparticle with a mass ratio of 20:1, b is a composite nanoparticle with a mass ratio of 40:1, and c is a composite nanoparticle with a mass ratio of 60:1.

[0033] Figure 8 The activity diagram of A549 cells after transfection with composite nanoparticles of different mass ratios;

[0034] Fig. 9 The fluorescence detection results of composite nanoparticles with a mass ratio of 30:1 at different transfection times; a is transfection 24h, b is transfection 48h, c is transfection 72h, and d is transfection 96h;

[0035] Fig.10 The results of cell flow cytometry detection of the percentage of GFP-positive cells at different transfection times with composite nanoparticles of mass ratio 30:1;

[0036] Fig.11 The bar graph and fluorescence intensity results of the percentage of GFP-positive cells detected by cell flow cytometry at different transfection times for composite nanoparticles with a mass ratio of 30:1;

[0037] Fig.12 This is a graph of A549 cell activity at different transfection times of composite nanoparticles with a mass ratio of 30:1. DETAILED DESCRIPTION

[0038] The present invention provides a highly branched poly (β-amino ester), the structural formula of the highly branched poly (β-amino ester) is as follows:

[0039]

[0040] Among them, n is 10 to 100.

[0041] The present invention also provides a method for preparing highly branched poly (β-amino ester), the synthesis process schematic diagram is as follows Figure 1 As shown, the following steps are included:

[0042] (1) reacting acrylic acid ester with 5-amino-1-pentanol in a solvent to obtain a base polymer;

[0043] (2) reacting a base polymer with 1,2-ethylenediamine in a solvent to obtain a polymer with a double bond;

[0044] (3) The polymer with double bonds is reacted with a capping agent in a solvent to obtain a highly branched poly(β-amino ester).

[0045] In the present invention, the acrylic acid ester in step (1) is preferably 1,4-butanediol diacrylate.

[0046] In the present invention, the solvent in step (1) is preferably dimethyl sulfoxide (DMSO).

[0047] In the present invention, the molar ratio of the acrylic acid ester to 5-amino-1-pentanol in step (1) is preferably 1-3:1-5, more preferably 1-2:2-4, and more preferably 1:2.

[0048] In the present invention, the volume ratio of the total mass of the acrylic ester and 5-amino-1-pentanol to the solvent in step (1) is preferably 100-500 mg:1 mL, more preferably 200-450 mg:1 mL, and more preferably 400 mg:1 mL.

[0049] In the present invention, the reaction in step (1) is preferably carried out until the preset molecular weight reaches 3000-50000Da, more preferably 10000-45000Da, and more preferably 44000Da; the reaction temperature is preferably 60-100°C, more preferably 65-80°C, and more preferably 70°C.

[0050] In the present invention, the molar ratio of the acrylic acid ester in step (1) to the 1,2-ethylenediamine in step (2) is preferably 1-3:0.5-3, more preferably 1-2:1-2, and more preferably 1:1.5.

[0051] In the present invention, the mass ratio of the 1,2-ethylenediamine to the volume ratio of the solvent in step (2) is preferably 500-1000 mg:1 mL, more preferably 550-800 mg:1 mL, and more preferably 600 mg:1 mL.

[0052] In the present invention, the solvent in step (2) is preferably dimethyl sulfoxide.

[0053] In the present invention, the reaction temperature in step (2) is preferably 80-100°C, more preferably 85-95°C, and more preferably 90°C; the reaction time is preferably 12-72h, more preferably 16-48h, and more preferably 24h.

[0054] In the present invention, the end-capping agent in step (3) is preferably 3,6,9-trioxaundecane-1,11-diamine.

[0055] In the present invention, the solvent in step (3) is preferably dimethyl sulfoxide.

[0056] In the present invention, the molar ratio of the acrylic acid ester in step (1) to the end-capping agent in step (3) is preferably 1-3:1-12, more preferably 1-2:4-8, and even more preferably 1:5.

[0057] In the present invention, the volume ratio of the end-capping agent in step (3) to the solvent is preferably 500-1000 mg:1 mL, more preferably 700-900 mg:1 mL, and more preferably 800 mg:1 mL.

[0058] In the present invention, the reaction temperature in step (3) is preferably 25° C.; the reaction time is preferably 12 to 72 h, more preferably 24 to 60 h, and more preferably 48 h.

[0059] In the present invention, after the reaction in step (3) is completed, the product is purified by precipitation method and then vacuum dried. The present invention does not limit the specific conditions of purification and vacuum drying, and the conditions can be selected according to the conventional conditions in the art.

[0060] The present invention also provides an application of a highly branched poly (β-amino ester) in preparing an mRNA delivery vector.

[0061] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0062] Example 1

[0063] This embodiment provides a highly branched poly (β-amino ester), the preparation method of which comprises the following steps:

[0064] (1) 1,4-butanediol diacrylate and 5-amino-1-pentanol are added in a molar ratio of 1:2, and the total mass concentration of the two in DMSO is 400 mg / mL. The mixture is reacted at 70° C. During the reaction, the weight average molecular weight (Mw) in each time period is detected by gel permeation chromatography (GPC). When the Mw is 44000 Da, the mixture is cooled to room temperature to stop the reaction, thereby obtaining a base polymer;

[0065] (2) adding 1,2-ethylenediamine and DMSO to the base polymer system, wherein the molar ratio of 1,2-ethylenediamine to 1,4-butanediol diacrylate is 1.5:1, and the mass volume ratio of 1,2-ethylenediamine to DMSO is 600 mg:1 mL, and reacting at 90° C. for 24 hours to obtain a polymer with double bonds;

[0066] (3) 3,6,9-trioxaundecane-1,11-diamine (122) and DMSO were added to a polymer system with double bonds. 122 was added at a molar ratio of 5:1 to 1,4-butanediol diacrylate, and the mass volume ratio of 122 to DMSO was 800 mg:1 mL. The reaction was carried out at 25° C. for 48 h. After the reaction was completed, the product was purified by a precipitation method and vacuum dried to obtain a highly branched poly (β-amino ester).

[0067] The GPC spectrum of the highly branched poly(β-amino ester) prepared in this example is as follows: Figure 2 As shown by Figure 2 It can be seen that the Mw of the highly branched poly(β-amino ester) is 51,000 Da.

[0068] The highly branched poly (β-amino ester) prepared in this example 1 H NMR spectrum Figure 3 As shown by Figure 3 It can be seen that the end-capping agent 122 reacts with the double bond and is successfully grafted onto the end of the polymer.

[0069] The highly branched poly (β-amino ester) prepared in this example and mRNA were diluted in a sodium citrate buffer solution, and the highly branched poly (β-amino ester) polymer and mRNA mass ratios of 20:1, 40:1, and 60:1 were uniformly mixed, and incubated statically for 30 minutes to form composite nanoparticles for subsequent detection.

[0070] mRNA affinity: The composite nanoparticles of each mass ratio were placed in PicoGreen working solution, the fluorescence intensity was tested, and then the mRNA affinity efficiency was calculated. The results are as follows: Figure 4 As shown. Figure 4 It can be seen that the mRNA affinity efficiency of the composite nanoparticles with three mass ratios can reach more than 90%, and they have excellent mRNA affinity.

[0071] Composite nanoparticle size: Composite nanoparticles of various mass ratios were dispersed in water and the particle size was measured using dynamic light scattering (DLS). Figure 5 As shown. Figure 5 It can be seen that the particle sizes of the composite nanoparticles with three mass ratios are between 150 and 200 nm, with high stability and not easy to agglomerate.

[0072] Zeta potential: Zeta potential diagram of composite nanoparticles of various mass ratios is shown in Figure 6 The Zeta potential of the composite nanoparticles with three mass ratios is between +10 and +20 mV, which is positive.

[0073] Transfection efficiency and cytotoxicity: mRNA delivery experiments were conducted in A549 cells. A549 cells were inoculated in a 96-well plate at 10,000 cells per well and cultured for 24 hours. Composite nanoparticles of various mass ratios were transferred to 96-well plates for transfection, respectively. The amount of mRNA used in each well was 50 ng. Green fluorescent protein (GFP) was used as a reporter gene to qualitatively and quantitatively evaluate the efficiency of transfection. After incubation for 24 hours, the expression of GFP was observed using a fluorescence microscope. The fluorescence results are shown in Figure 2. Figure 7 As shown, the activity of A549 cells after transfection was Figure 8 As shown. Figure 7 It can be seen that the composite nanoparticles with three mass ratios can mediate efficient mRNA transfection. Figure 8 It can be seen that the cell activity after transfection of cells with composite nanoparticles of three mass ratios was above 80%.

[0074] Composite nanoparticles with a mass ratio of highly branched poly(β-amino ester) polymer to mRNA of 30:1 were prepared by the same method as above, and transfection was performed according to the above method. After incubation for 24h, 48h, 72h, and 96h, the fluorescence results were as follows Fig. 9 The results of flow cytometry detection of the percentage of GFP-positive cells are shown in Fig.10 As shown, the bar graph of the percentage of GFP positive cells and the fluorescence intensity detected by cell flow cytometry are shown in Fig.11 As shown, A549 cell viability Fig.12 As shown. Figures 9 to 11 It can be seen that the composite nanoparticles with a mass ratio of 30:1 at different transfection times can mediate efficient mRNA transfection, with a transfection efficiency between 50 and 60% and a fluorescence intensity of up to 8.7×10 4 RLU. Fig.12 It can be seen that the cell activity at different transfection times was above 90%.

[0075] Example 2

[0076] This embodiment provides a highly branched poly (β-amino ester), the preparation method of which comprises the following steps:

[0077] (1) 1,4-butanediol diacrylate and 5-amino-1-pentanol are added in a molar ratio of 1:1, and the total mass concentration of the two in DMSO is 500 mg / mL. The reaction is carried out at 60° C. During the reaction, GPC is used to detect the Mw in each time period. When the Mw is 38000 Da, the reaction is stopped by cooling to room temperature to obtain a base polymer;

[0078] (2) adding 1,2-ethylenediamine and DMSO to the base polymer system, wherein the molar ratio of 1,2-ethylenediamine to 1,4-butanediol diacrylate is 0.5:1, and the mass volume ratio of 1,2-ethylenediamine to DMSO is 800 mg:1 mL, and reacting at 90° C. for 36 hours to obtain a polymer with double bonds;

[0079] (3) 3,6,9-trioxaundecane-1,11-diamine (122) and DMSO were added to a polymer system with double bonds. The molar ratio of 122 to 1,4-butanediol diacrylate was 3:2, and the mass volume ratio of 122 to DMSO was 800 mg:1 mL. The reaction was carried out at 25°C for 24 h. After the reaction was completed, the product was purified by a precipitation method and vacuum dried to obtain a highly branched poly (β-amino ester).

[0080] The Mw of the highly branched poly(β-amino ester) was 49000 Da as determined by GPC.

[0081] Highly branched poly(β-amino ester) 1 H NMR spectrum Figure 3 As shown, the end-capping agent 122 reacts with the double bond and is successfully grafted onto the end of the polymer.

[0082] Example 3

[0083] This embodiment provides a highly branched poly (β-amino ester), the preparation method of which comprises the following steps:

[0084] (1) 1,4-butanediol diacrylate and 5-amino-1-pentanol are added in a molar ratio of 3:4, and the total mass concentration of the two in DMSO is 300 mg / mL. The reaction is carried out at 80° C. During the reaction, GPC is used to detect the Mw in each time period. When the Mw is 45000 Da, the reaction is stopped by cooling to room temperature to obtain a base polymer;

[0085] (2) adding 1,2-ethylenediamine and DMSO to the base polymer system, wherein the molar ratio of 1,2-ethylenediamine to 1,4-butanediol diacrylate is 2:3, and the mass volume ratio of 1,2-ethylenediamine to DMSO is 600 mg:1 mL, and reacting at 90° C. for 48 hours to obtain a polymer with double bonds;

[0086] (3) 3,6,9-trioxaundecane-1,11-diamine (122) and DMSO were added to a polymer system with double bonds. 122 was added at a molar ratio of 5:3 to 1,4-butanediol diacrylate, and the mass volume ratio of 122 to DMSO was 1000 mg:1 mL. The reaction was carried out at 25°C for 52 h. After the reaction was completed, the product was purified by a precipitation method and vacuum dried to obtain a highly branched poly (β-amino ester).

[0087] The Mw of the highly branched poly(β-amino ester) was 57,000 Da as determined by GPC.

[0088] Highly branched poly(β-amino ester) 1 H NMR spectrum Figure 3 As shown, the end-capping agent 122 reacts with the double bond and is successfully grafted onto the end of the polymer.

[0089] Example 4

[0090] This embodiment provides a highly branched poly (β-amino ester), the preparation method of which comprises the following steps:

[0091] (1) 1,4-butanediol diacrylate and 5-amino-1-pentanol are added in a molar ratio of 3:5, and the total mass concentration of the two in DMSO is 500 mg / mL. The reaction is carried out at 100° C. During the reaction, GPC is used to detect the Mw in each time period. When the Mw is 40,000 Da, the reaction is stopped by cooling to room temperature to obtain a base polymer;

[0092] (2) adding 1,2-ethylenediamine and DMSO to the basic polymer system, wherein the molar ratio of 1,2-ethylenediamine to 1,4-butanediol diacrylate is 3:1, and the mass volume ratio of 1,2-ethylenediamine to DMSO is 1000 mg:1 mL, and reacting at 90° C. for 60 h to obtain a polymer with double bonds;

[0093] (3) 3,6,9-trioxaundecane-1,11-diamine (122) and DMSO were added to a polymer system with double bonds. The molar ratio of 122 to 1,4-butanediol diacrylate was 4:1, and the mass volume ratio of 122 to DMSO was 1000 mg:1 mL. The reaction was carried out at 25°C for 48 hours. After the reaction was completed, the product was purified by a precipitation method and vacuum dried to obtain a highly branched poly (β-amino ester).

[0094] The Mw of the highly branched poly(β-amino ester) was 55000 Da as determined by GPC.

[0095] Highly branched poly(β-amino ester) 1 H NMR spectrum Figure 3 As shown, the end-capping agent 122 reacts with the double bond and is successfully grafted onto the end of the polymer.

[0096] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A highly branched poly(β-amino ester), characterized in that The structural formula of the highly branched poly(β-amino ester) is shown below: Among them, n is 10 to 100.

2. The method for preparing a highly branched poly(β-amino ester) according to claim 1, characterized in that: The following steps are involved: (1) reacting acrylic acid ester with 5-amino-1-pentanol in a solvent to obtain a base polymer; (2) reacting a base polymer with 1,2-ethylenediamine in a solvent to obtain a polymer with a double bond; (3) The polymer with double bonds is reacted with a capping agent in a solvent to obtain a highly branched poly(β-amino ester).

3. The method for preparing a highly branched poly (β-amino ester) according to claim 2, characterized in that: The acrylate in step (1) is 1,4-butanediol diacrylate; and the end-capping agent in step (3) is 3,6,9-trioxaundecane-1,11-diamine.

4. The method for preparing a highly branched poly(β-amino ester) according to claim 3, characterized in that: In step (1), the molar ratio of the acrylic acid ester to 5-amino-1-pentanol is 1-3:1-5.

5. The method for preparing a highly branched poly (β-amino ester) according to claim 2 or 3, characterized in that: The reaction in step (1) is carried out until the preset molecular weight reaches 3000-50000Da; the reaction temperature is 60-100°C.

6. The method for preparing a highly branched poly(β-amino ester) according to claim 5, characterized in that: The molar ratio of the acrylic acid ester in step (1) to the 1,2-ethylenediamine in step (2) is 1-3:0.5-3.

7. A method for preparing a highly branched poly (β-amino ester) according to claim 2 or 6, characterized in that: The reaction temperature in step (2) is 80-100° C. and the reaction time is 12-72 hours.

8. The method for preparing a highly branched poly(β-amino ester) according to claim 7, characterized in that: The molar ratio of the acrylic ester in step (1) to the end-capping agent in step (3) is 1-3:1-12.

9. The method for preparing a highly branched poly(β-amino ester) according to claim 8, characterized in that: The reaction temperature of step (3) is 25° C. and the reaction time is 12 to 72 hours.

10. Use of a highly branched poly (β-amino ester) according to claim 1 in preparing an mRNA delivery vector.

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