Functional highly branched poly(beta-amino esters) with thioether-containing backbones, methods of making and uses thereof

Abstract

The application discloses a kind of main chain containing sulfide functional highly branched poly (beta-amino ester) and its preparation method and application, belong to the technical field of biomedical materials. By selecting different diacrylate monomers, branched monomers, small molecule mercaptan, small molecule amine monomer, highly branched poly (beta-amino ester) containing sulfide in main chain is prepared, and mRNA is efficiently delivered in HeLa, HepG2, COS-7, A549 and 293T cells. Compared with the mainstream commercial transfection reagent (jetPEI, Lipofectamine 2000), the polymer is cheap, easy to design and prepare, and has better transfection performance. It has significant clinical application potential in mRNA drug delivery.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical materials, and particularly relates to a kind of functional highly branched poly (beta-amino ester) containing thioether in main chain and a preparation method and application thereof. BACKGROUND

[0002] mRNA delivery has shown broad application prospects in the fields of infectious disease vaccines, tumor diseases, protein replacement and gene therapy. Compared with DNA, mRNA delivery has unique advantages, such as no transcription step, no need to enter the nucleus, no transgene insertion mutation, higher protein expression efficiency and faster kinetics. In addition, the dosage of mRNA is lower, generally 1 / 5 to 1 / 10 of DNA. However, the lack of safe and efficient mRNA delivery vectors seriously limits its clinical application. However, the lack of safe and efficient vectors limits the application of mRNA gene therapy in the clinic.

[0003] At present, commonly used non-viral vectors such as commercial lipid nanotechnology delivery lipid nanoparticles (LNP) have high transfection efficiency, but the problems of low temperature storage and poor stability greatly limit their application in commercialization; although the potential toxicity of inorganic nanoparticles can be adjusted by controlling the size and surface modification of inorganic nanoparticles, the in vivo degradation seriously limits its practical application.

[0004] Due to its wide source of monomers, easy to control structure and performance, and in vivo degradable, poly (beta-amino ester) as a highly efficient cationic polymer carrier has shown great application prospects in drug, protein and nucleic acid delivery. Highly branched poly (beta-amino ester) has shown excellent DNA delivery performance in vivo and in vitro due to its multiple terminal groups and three-dimensional topological structure, but there is no related report on mRNA delivery, which seriously limits its application in mRNA gene therapy. SUMMARY

[0005] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a kind of functional highly branched poly (beta-amino ester) containing thioether in main chain and a preparation method and application thereof, to solve the problem of lack of mRNA delivery vector.

[0006] In order to achieve the above purpose, the technical scheme is adopted as follows:

[0007] The application discloses a kind of functional highly branched poly (beta-amino ester) containing thioether in main chain, and its structural formula is as follows:

[0008]

[0009] Wherein, n=5~30;m=5~30;

[0010] R0 is a bisphenol A containing diacrylate monomer; R1 is a different branched monomer; R2 is a small molecule amine monomer, including monomers containing different numbers of amino groups; R3 is a different small molecule thiol; R4 is an amino containing end-capping agent.

[0011] Preferably, R0 has the formula of one of the following:

[0012]

[0013] R1 has the formula of one or more of the following:

[0014]

[0015] R2 has the formula of one of the following:

[0016]

[0017] R3 has the formula of one of the following:

[0018]

[0019] R4 has the formula of one of the following:

[0020]

[0021] Preferably, the functional highly branched poly(beta-amino ester) has a molecular weight in the range of 5,000 to 50,000 Da.

[0022] The application also discloses a preparation method of the functional highly branched poly(beta-amino ester) with a main chain containing a sulfide, which comprises the following steps: adding a bisphenol A containing diacrylate monomer, a branched monomer, a small molecule amine monomer and a small molecule thiol into dimethyl sulfoxide to perform a reaction; and then adding an end-capping agent and dimethyl sulfoxide to continue the reaction, so as to obtain the functional highly branched poly(beta-amino ester) with a main chain containing a sulfide.

[0023] Preferably, the molar ratio of the reaction functional groups of the bisphenol A containing diacrylate monomer, the small molecule amine monomer and the small molecule thiol is 1: (0.5-0.7): (0.1-0.3); and the branched monomer accounts for 5%-50% of the total functional groups in terms of the functional groups.

[0024] Preferably, the bisphenol A containing diacrylate monomer, the branched monomer, the small molecule amine monomer and the small molecule thiol are added into dimethyl sulfoxide to perform a reaction at 60-120 ℃ for 6-48 h.

[0025] Preferably, before the end-capping agent is added, a gel permeation chromatography is used to monitor the highly branched poly(beta-amino ester) with a main chain containing a sulfide, and the reaction is terminated when the molecular weight of the polymer reaches 4,000-40,000 Da.

[0026] Preferably, the molar ratio of the end-capping agent to the excess bisphenol A-containing diacrylate functional group is (2-5):1.

[0027] Preferably, the conditions for adding the end-capping agent and dimethyl sulfoxide reaction are 25 DEG C for 48 h.

[0028] The application also discloses an application of the functional highly branched poly(beta-amino ester) with a main chain containing a thioether in preparation of an mRNA drug delivery carrier.

[0029] Preferably, the mass ratio of the functional highly branched poly(beta-amino ester) to the mRNA is (5-200):1.

[0030] Compared with the prior art, the application has the following beneficial effects:

[0031] The functional highly branched poly(beta-amino ester) with a main chain containing a thioether provided by the application is a biodegradable highly branched polymer with adjustable structure components, structure and performance, and is synthesized by using different monomer combinations, regulating branched structures and using different end-capping monomers. The functional highly branched poly(beta-amino ester) has good biodegradability and branched structure regulation performance due to the ester bond structure (R0 and the ester bond in part of R1, and the ester bond in the main bond formed after the reaction) in the main chain. The ionizable or positively charged groups (tertiary amines generated by the reaction and primary amines obtained by end-capping) can effectively compress mRNA, thereby realizing delivery. The functional highly branched poly(beta-amino ester) has high mRNA delivery efficiency and cell activity due to the special topological structure and chemical composition, and has certain clinical application potential. The effectiveness, accuracy and wide applicability of the functional highly branched poly(beta-amino ester) are verified in in-vitro cell experiments, which indicates that the functional highly branched poly(beta-amino ester) has good clinical transformation prospects and can be applied to preparation of an mRNA drug delivery carrier.

[0032] The preparation method of the functional highly branched poly(beta-amino ester) with a main chain containing a thioether provided by the application uses commercial diacrylate monomers, branched monomers, small molecule thiols, small molecule amines and amino-containing end-capping monomers, and the functional highly branched poly(beta-amino ester) with a main chain containing a thioether is prepared by a Michael addition method. The entire preparation process has the advantages of low cost (the cost of the synthesized functional highly branched poly(beta-amino ester) is lower than that of mainstream commercial transfection reagents (jetPEI, Lipofectamine 2000)), simple synthesis path, and easy regulation of chemical composition and structure performance.

[0033] Further, the molecular weight of the polymer can be controlled to be 5000-50000 Da by monitoring the molecular weight by gel permeation chromatography. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Synthesis scheme of the main chain thioether-containing hyperbranched poly(β-amino ester) of the present application;

[0035] Figure 2 Functionalized main chain thioether-containing hyperbranched poly(β-amino ester) prepared in Example 1 of the present application physical form chart;

[0036] Figure 3 Gel permeation chromatography (GPC) curve of the functionalized main chain thioether-containing hyperbranched poly(β-amino ester) prepared in Example 1 of the present application after purification;

[0037] Figure 4 Particle size test results of the complex of the functionalized main chain thioether-containing hyperbranched poly(β-amino ester) prepared in Example 1 of the present application and mRNA; 1 H NMR spectrum;

[0038] Figure 5 mRNA affinity performance test chart of the main chain thioether-containing hyperbranched poly(β-amino ester) prepared in Example 1 of the present application with a molecular weight of 15,000 Da;

[0039] Figure 6 Particle size test results of the complex of the main chain thioether-containing hyperbranched poly(β-amino ester) prepared in Example 1 of the present application with a molecular weight of 15,000 Da and mRNA;

[0040] Figure 7 Particle surface potential test results of the complex of the main chain thioether-containing hyperbranched poly(β-amino ester) prepared in Example 1 of the present application with a molecular weight of 15,000 Da and mRNA;

[0041] Figure 8 Microscopic morphology characterization chart of the complex of the main chain thioether-containing hyperbranched poly(β-amino ester) prepared in Example 1 of the present application with a molecular weight of 15,000 Da and mRNA;

[0042] Figure 9 Fluorescence chart of the cells of HeLa, HepG2, COS-7, A549 and 293T transfected with the mRNA encoding green fluorescent protein by the main chain thioether-containing hyperbranched poly(β-amino ester) prepared in Example 1 of the present application with a molecular weight of 15,000 Da after 24 h;

[0043] Figure 10 Activity evaluation results chart of the HepG2 cells after 24 h of transfection by the main chain thioether-containing hyperbranched poly(β-amino ester) prepared in Example 1 of the present application with a molecular weight of 15,000 Da;

[0044] Figure 11 Figure 1 shows the luciferase activity of HepG2 cells transfected with the backbone containing thioether highly branched poly (β-amino ester) having a molecular weight of 15,000 Da prepared for Example 1 of the present application after 24 h. DETAILED DESCRIPTION

[0045] In order to better understand the technical scheme of the present application, the technical scheme in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of the present application.

[0046] It should be noted that the terms "first", "second", and the like in the specification and claims of the present application and the above-described accompanying drawings are used to distinguish similar objects, and do not necessarily indicate a specific order or a chronological sequence. It should be understood that the data thus used can be interchanged under appropriate circumstances, so that the embodiments of the present application described herein can be implemented in an order other than that illustrated or described herein. In addition, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusion, for example, a process, method, system, product or device including a series of steps or units does not necessarily have to be limited to those steps or units clearly listed, but can include other steps or units not clearly listed or inherent to the process, method, product or device.

[0047] The present application will be further described in detail below in conjunction with the accompanying drawings:

[0048] The present application provides a synthesis method of a backbone containing thioether functional highly branched poly (β-amino ester), and the synthesis route is shown in Figure 1 , which comprises the following steps:

[0049] 1) A certain amount of diacrylate monomer, branched monomer, small molecule amine monomer and small molecule mercaptan are added to dimethyl sulfoxide, and reacted at 60-120℃ for 6-48h;

[0050] The molar ratio of the reaction functional groups of the diacrylate monomer, the small molecule amine monomer and the small molecule mercaptan is 1: (0.5-0.7) : (0.1-0.3) ; the functional groups of the branched monomer account for 5%-50% of the total functional groups.

[0051] The diacrylate monomer (R0) is a diacrylate monomer containing bisphenol A, including one of the following formulas:

[0052]

[0053] The branched monomer (R1) is one or more of the following acrylic ester, small molecule amine or thiol monomers:

[0054]

[0055]

[0056] The small molecule amine monomer includes monomers with different numbers of amino groups and molecular structures, and the structure is one of the following (R2):

[0057]

[0058] The small molecule thiol includes dithiols with different structures, and the structure is one of the following (R3):

[0059]

[0060] 2) Monitor the main chain containing sulfide highly branched poly (beta-amino ester) using gel permeation chromatography during the reaction, and terminate the reaction when the molecular weight of the polymer reaches 4,000-40,000 Da;

[0061] 3) A certain amount of functional capping agent and dimethyl sulfoxide is added to the reaction system of step 2), and the reaction is carried out at 25°C for 24-48h.

[0062] Wherein, the molar ratio of capping agent (R4) to excess acrylate functional group is (2-5):1; the capping agent is a small molecule amine monomer, and the structure is one of the following:

[0063]

[0064] 4) The product is purified by precipitation method, vacuum dried, and the main chain containing sulfide highly branched poly (beta-amino ester) is obtained, and the structure is as follows:

[0065]

[0066] Wherein, n=5-30; m=5-30.

[0067] The functional main chain containing sulfide highly branched poly (beta-amino ester) prepared by the application is used for mRNA delivery, including the following steps:

[0068] 1) Cell culture: HeLa, HepG2, COS-7, A549 and 293T cells are cultured under standard culture conditions, and the cell density is 0.5x10 4 -2.0x10 4Cells were seeded at a density of 1 cell / well in 96-well plates.

[0069] 2) When the cell density reached 60% to 90%, the solution of the main chain containing sulfide highly branched poly (β-amino ester) was vortexed with the mRNA solution containing the coding green fluorescent protein or the solution of luciferase mRNA at a high speed for 15 to 60 s, and incubated for 10 to 45 min, and then transferred to the cells for transfection. The mass ratio of polymer:mRNA was (5 to 200):1, and the mRNA mass in each well was 10 to 300 ng.

[0070] 3) After 24 to 72 hours of transfection, the transfected green fluorescent protein cells were observed and photographed under a fluorescence microscope.

[0071] 4) After 24 to 72 hours of transfection, luciferase working solution was added, and the intensity of luminescence was quickly detected in the enzyme label instrument to test the luciferase activity of the transfected cells and the activity of the transfected cells.

[0072] 5) After 24 to 72 hours of transfection, alamarBlue solution was added after removing the cell supernatant, and incubated in the incubator for 30 min to test the cell activity.

[0073] Example 1

[0074] 1. Synthesis of functional main chain containing sulfide highly branched poly (β-amino ester)

[0075] 1) Bisphenol A ethoxylate diacrylate (EO / phenol = 1.5), trimethylolpropane triacrylate, 2-aminoethyl morpholine, 3-methylamino propylamine and 1,2-ethanedithiol were added to dimethyl sulfoxide at a molar ratio of 1:0.2:0.7:0.1:0.2, and a "one-pot" Michael addition reaction was carried out at 60°C for 6h;

[0076] 2) Gel permeation chromatography was used to monitor the main chain containing sulfide highly branched poly (β-amino ester) during the reaction, and the reaction was terminated when the molecular weight of the polymer reached 4,000 to 40,000 Da;

[0077] 3) Then 4-morpholine propylamine and dimethyl sulfoxide were added to the reaction system of step 2), and reacted at 25°C for 48h to obtain a reaction product; wherein the molar ratio of 4-morpholine propylamine to acrylate functional group is 3:1;

[0078] 4) The reaction product was purified by precipitation method, and after vacuum drying, a functional main chain containing sulfide highly branched poly (β-amino ester) with a molecular weight of 15000 Da was obtained, and the structure formula is as follows:

[0079]

[0080] The physical phase diagram of the branched poly(β-amino ester) obtained in this example at 25°C is shown in Figure 2 ; the GPC curve of the branched poly(β-amino ester) prepared in this example is shown in Figure 3 ; the 1 H NMR spectrum of the branched poly(β-amino ester) prepared in this example is shown in Figure 4 .

[0081] 2. mRNA affinity performance test of the synthesized functional highly branched poly(β-amino ester)

[0082] First, the highly branched poly(β-amino ester) containing a sulfide in the main chain was added to the mRNA (TriLink, L-7601-1000) solution at a mass ratio of 10:1, 20:1, 30:1 and 40:1, respectively, where the amount of mRNA was 0.3 μg, and high-speed vortexed for 15-60 s, and then stood for 10-45 min to form a complex nanoparticle solution. Then, the complex nanoparticle solution was diluted to 100 μL using a TE buffer solution, and 100 μL of RiboGreen working solution (0.5%) was added. Under the conditions of excitation wavelength of 480 nm and emission wavelength of 520 nm, the fluorescence intensity of the excitation was detected to test the affinity performance of the highly branched poly(β-amino ester) containing a sulfide in the main chain to mRNA. Further, a similar method was used to prepare a complex nanoparticle solution, where the mass ratio of the highly branched poly(β-amino ester) containing a sulfide in the main chain to mRNA was 10:1, 20:1, 30:1 and 40:1, and the complex nanoparticle solution was diluted to 1 mL using deionized water. Finally, DLS was used to test the particle size and surface potential, and the complex nanoparticle solution was freeze-dried, and TEM was used to characterize the micro-morphology.

[0083] The affinity performance test results are shown in Figure 5 , Figure 5 It is confirmed that the affinity efficiency of the highly branched poly(β-amino ester) containing a sulfide in the main chain with a molecular weight of 15,000 Da prepared in Example 1 to mRNA is more than 90% when the mass ratio is 10:1, 20:1, 30:1 and 40:1, confirming its excellent mRNA affinity performance. The DLS analysis results are shown in Figure 6 and Figure 7 , Figure 6 The results confirm that the highly branched poly(β-amino ester) containing a sulfide in the main chain with a molecular weight of 15,000 Da prepared in Example 1 can effectively compress mRNA, and as the mass ratio increases, the particle size of the formed complex nanoparticles is smaller, all less than 250 nm, further confirming that the formed complex nanoparticles are more conducive to cell uptake.Figure 7 The results prove that the backbone containing sulfide of the highly branched poly(β-amino ester) with a molecular weight of 15,000 Da prepared in Example 1 can effectively shield the negative potential of mRNA itself, and the Zeta potential of the nano-surface of the formed complex is higher as the mass ratio increases, and is all positive. The TEM characterization results are shown in Figure 8 , Figure 8 The results prove that the complex nanoparticles formed by the backbone containing sulfide of the highly branched poly(β-amino ester) with a molecular weight of 15,000 Da prepared in Example 1 and mRNA have uniform particle size distribution and relatively stable structure, proving good stability.

[0084] 3. mRNA delivery experiment of the backbone containing sulfide of the highly branched poly(β-amino ester) prepared in Example 1 is carried out outside cells

[0085] HeLa, HepG2, COS-7, A549, 293T cells are cultured under standard culture conditions, and are inoculated in a 96-well plate at a density of 2.0×10 4 cells / well. When the cell density reaches 50%-80%, transfection is carried out. The corresponding mass of the backbone containing sulfide of the highly branched poly(β-amino ester) is added to the mRNA solution, wherein the mass ratio of the backbone containing sulfide of the highly branched poly(β-amino ester) to the green fluorescent protein-encoding mRNA (TriLink, L-7601-1000) is 20:1, the amount of the green fluorescent protein-encoding mRNA per well is 50 ng, high-speed vortex is carried out for 15-60 s, then standing is carried out for 10-45 min, static incubation is carried out for 20 min, complex nanoparticles are formed, and then the 96-well plate is transferred for transfection. After 24 h of continuous culture, the cells transfected with green fluorescent protein are observed and photographed under a fluorescence microscope.

[0086] The transfection performance evaluation results are shown in Figure 9 , Figure 9 It can be seen that the backbone containing sulfide of the highly branched poly(β-amino ester) prepared in the application can exhibit higher green fluorescent protein expression in various tissue cells (HeLa, HepG2, COS-7, A549 and 293T cells) due to multiple terminal groups, flexible chemical composition and good biocompatibility.

[0087] 4. The branched poly(β-amino ester) prepared in Example 1 is tested for mRNA transfection efficiency and cell activity outside cells

[0088] HepG2 cells are cultured under standard culture conditions, and are inoculated in a 96-well plate at a density of 2.0×10 4The cells were seeded in 96-well plates at a density of 1 cell / well, and when the cell density reached 50%-80%, transfection was performed. The corresponding amount of the backbone containing sulfide highly branched poly (beta-amino ester) solution was added to the luciferase-encoding mRNA solution, wherein the mass ratio of the backbone containing sulfide highly branched poly (beta-amino ester) to the luciferase-encoding mRNA (TriLink, L-7202-1000) was 10:1, 20:1, 30:1 and 40:1, and the amount of luciferase-encoding mRNA per well was 50 ng. High-speed vortex was performed for 15-60 s, and then the solution was left to stand for 10-45 min. After 20 min of static incubation to form complex nanoparticles, the solution was transferred to a 96-well plate for transfection. Then, after 24 h of continuous culture, for the luciferase-encoding mRNA-transfected cells, the HepG2 cells were washed three times with PBS under light shielding conditions, and then 50 μL of CellTiter-Flour TM reagent (Promega, E7120) and DMEM medium were added to the UC-3 cells, and incubation was performed for 30 min. Cell activity was detected at an excitation wavelength of 400 nm and an emission wavelength of 500 nm. Finally, 25 μL of One-Glo TM reagent (Promega, E7120) was added, and the relative luminescence intensity was detected to determine the transfection efficiency of luciferase.

[0089] The cell activity evaluation results are shown in Table 1. Figure 10 From the cell survival rate results, it can be found that due to the good biocompatibility and degradability of the backbone containing sulfide highly branched poly (beta-amino ester) and the extremely low amount used, the HepG2 cells after transfection can still maintain a high cell activity, which indicates that the functional backbone containing sulfide highly branched poly (beta-amino ester) has obvious advantages in mRNA delivery. The transfection performance evaluation results are shown in Table 2. Figure 11 The transfection results show that due to the multiple end groups, flexible chemical composition and good biocompatibility, the backbone containing sulfide highly branched poly (beta-amino ester) can efficiently mediate the transfection of luciferase-encoding mRNA in HepG2 cells.

[0090] The above content only illustrates the technical idea of the present application, and cannot limit the protection scope of the present application. Any modification made according to the technical idea of the present application on the basis of the technical solution falls within the protection scope of the claims of the present application.

Claims

1. A class of functional highly branched poly(β-amino esters) having a main chain containing a thioether, characterized in that, The structural formula is as follows: 。 2. Process for the preparation of a class of main chain thioether containing functional highly branched poly(β-amino esters) according to claim 1, characterized in that, The bisphenol A ethoxylate diacrylate, the trimethylolpropane triacrylate, the 2-aminoethyl morpholine, the 3-methyl amino propylamine and the 1,2-ethanedithiol are added into the dimethyl sulfoxide to react; the 4-morpholine propylamine and the dimethyl sulfoxide are further added to continue the reaction, and a functional highly branched poly (beta-amino ester) containing a sulfide in a main chain is prepared; The molar ratio of the bisphenol A ethoxylate diacrylate, the trimethylolpropane triacrylate, the 2-aminoethyl morpholine, the 3-methyl amino propylamine and the 1,2-ethanedithiol is 1:0.2:0.7:0.1:0.

2.

3. Process for the preparation of a class of functional highly branched poly(β- amino esters) containing thioethers according to claim 2, characterized in that, The bisphenol A ethoxylate diacrylate, the trimethylolpropane triacrylate, the 2-aminoethyl morpholine, the 3-methyl amino propylamine and the 1,2-ethanedithiol are added into the dimethyl sulfoxide to react at 60-120 ℃ for 6-48 h.

4. Process for the preparation of a class of functional highly branched poly(β- amino esters) containing thioethers according to claim 2, characterized in that, Before the 4-morpholine propylamine is added, the highly branched poly (beta-amino ester) containing a sulfide in a main chain is monitored by using gel permeation chromatography, and the reaction is terminated when the molecular weight of the polymer reaches 4,000-40,000 Da.

5. Process for the preparation of a class of functional highly branched poly(β- amino esters) containing thioethers according to claim 2, characterized in that, The molar ratio of the 4-morpholine propylamine to the acrylate functional group is (2-5):

1.

6. Process for the preparation of a class of functional highly branched poly(β- amino esters) containing thioethers according to claim 2, characterized in that, The reaction conditions for adding the 4-morpholine propylamine and the dimethyl sulfoxide are as follows: reaction at 25 ℃ for 48 h.

7. Use of the functional highly branched poly (beta-amino ester) containing a sulfide in a main chain in the preparation of an mRNA drug delivery carrier.

8. Use according to claim 7, characterized in that, The mass ratio of the functional highly branched poly (beta-amino ester) to the mRNA is (5-200):1.

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