Functional star-shaped poly (beta-amino ester) as well as preparation method and mRNA (messenger ribonucleic acid) delivery application thereof

Abstract

The invention provides functional star-shaped poly (beta-amino ester) as well as a preparation method and mRNA (messenger ribonucleic acid) delivery application thereof, and belongs to the technical field of biomedical materials. The functional star-shaped poly (beta-amino ester) has multiple terminal groups, a wide chemical space, narrow molecular weight distribution and excellent affinity to mRNA, composite nanoparticles formed by the functional star-shaped poly (beta-amino ester) and the mRNA are uniform in distribution and good in stability, and a carrier is biodegradable and good in biocompatibility. An in-vitro transfection result shows that the functional star-shaped poly (beta-amino ester) can efficiently deliver mRNA transfection in various tissue cell lines, and can still keep excellent cell activity after transfection, so that an important platformization technology is provided for further mRNA gene therapy.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials and relates to a class of functional star-shaped poly (β-amino ester) and a preparation method and mRNA delivery application thereof. Background Art

[0002] Gene therapy is the transfer of functional specific genetic material to target tissues or cells, thereby regulating the expression of specific proteins and ultimately achieving disease treatment. In particular, mRNA therapy has been applied to the treatment of genetic diseases, cancer and vaccine development. However, the current lack of safe and efficient gene delivery vectors has severely limited the clinical transformation of gene therapy.

[0003] In recent years, the number of clinical trials for gene therapy using non-viral vectors has increased significantly. More than 40 clinical trials have focused on the safety and efficacy of non-viral vectors, with research focuses mainly on gene therapy for pulmonary fibrosis, expression of therapeutic proteins, and development of antigen vaccines. Compared with traditional viral vectors, emerging non-viral vectors have the following advantages: (1) larger gene load, larger gene fragments can be delivered, and the application range is wider; (2) due to their non-chromosomal integration, they theoretically will not cause tumors and have less immunogenicity, and are safer; (3) during gene delivery, gene expression is limited to the target organ; (4) their design is flexible and the cost is low; (5) they are easy to prepare and easy to mass produce.

[0004] As a highly efficient and degradable cationic polymer carrier, poly(β-amino ester) has become one of the most promising new polymer carriers. At present, the research on gene delivery of poly(β-amino ester) mainly includes linear structure, hyperbranched structure and cyclic structure, and is mainly used for DNA delivery, which seriously limits its actual clinical application. Summary of the invention

[0005] In order to overcome the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a class of functional star-shaped poly (β-amino esters) and a preparation method and mRNA delivery drug application thereof.

[0006] The present invention is achieved through the following technical solutions:

[0007] A class of functional star-shaped poly(β-amino esters), wherein the structural formula of the functional star-shaped poly(β-amino ester) is as follows:

[0008]

[0009]

[0010] Among them, R1 is a group on a diacrylate monomer, R2 is a group on an organic amine, R3 is a group on a functional end-capping agent monomer, the organic amine is R2-NH2, the functional end-capping agent monomer is R3-NH2, and n=5-30.

[0011] Preferably, the diacrylate monomer is 1,4-butanediol diacrylate, 2,2-dithiodiethanol diacrylate, diethylene glycol diacrylate, tetraethylene glycol diacrylate, 1,1'-isopropyl bis(p-phenyleneoxy)di-2-propanol diacrylate or bisphenol A polyoxyethylene ether diacrylate.

[0012] Preferably, the organic amine is 4-amino-1-butanol, butylamine, pentylamine, hexylamine, 1-(2-aminoethyl)piperazine, N-(2-aminoethyl)-morpholine, dodecylamine or octadecylamine.

[0013] Preferably, the functionalized end-capping agent monomer is 1-(3-aminopropyl)-4-methylpiperazine, N-(3-aminopropyl)morpholine, N-(2-aminoethyl)morpholine, 2-methyl-2-morpholin-4-ylpropane-1-amine, 1-(2-aminoethyl)piperazine, 1,11-diamino-3,6,9-trioxaundecane, 1,2-ethylenediamine or 1,3-butylamine.

[0014] The method for preparing the functional star-shaped poly(β-amino ester) comprises:

[0015] 1) subjecting a diacrylate monomer to a Michael addition reaction with an organic amine to obtain a linear poly(β-amino ester) having a vinyl group at the end;

[0016] 2) capping the linear poly(β-amino ester) having a vinyl group at the end obtained in step 1) with a functionalized capping agent monomer to obtain a linear poly(β-amino ester) having an amine group at the end;

[0017] 3) reacting the linear poly(β-amino ester) having an amine group at the end obtained in step 2) with a tetraacrylate monomer or a hexaacrylate monomer to obtain a star-shaped poly(β-amino ester) having a vinyl group at the end;

[0018] 4) reacting the star-shaped poly(β-amino ester) containing a vinyl group at the end obtained in step 3) with a functionalized end-capping agent monomer to obtain a functional star-shaped poly(β-amino ester).

[0019] Preferably, the tetraacrylate monomer is pentaerythritol tetraacrylate, and the hexaacrylate monomer is polydipentaerythritol hexaacrylate.

[0020] Preferably, in step 1), the molar ratio of the diacrylate monomer to the organic amine is (1-5):1; the reaction is carried out under a nitrogen atmosphere at a reaction temperature of 50-90°C.

[0021] Preferably, in step 3), the molar ratio of the tetraacrylate monomer or the hexaacrylate monomer to the linear poly(β-amino ester) having an amine group at the end is (0.025-1):(0.5-5), and the reaction temperature is 40°C-90°C.

[0022] The functional star-shaped poly (β-amino ester) is used in preparing a gene carrier, and the gene carrier is used for delivering genes to tissues or cells.

[0023] Preferably, the gene is mRNA.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The functional star-shaped poly(β-amino ester) disclosed in the present invention has a linear low molecular weight poly(β-amino ester) as an arm and a tetraacrylate or hexaacrylate monomer as a core molecule, and is a type of star-shaped poly(β-amino ester) with a completely new structure. The star-shaped poly(β-amino ester) has multiple terminal groups, a wider chemical space and a narrower molecular weight distribution, and has excellent affinity for mRNA. The composite nanoparticles formed by the star-shaped poly(β-amino ester) and mRNA have a relatively uniform distribution, good stability, and the carrier is biodegradable and has good biocompatibility. The results of in vitro transfection show that the star-shaped poly(β-amino ester) can efficiently deliver mRNA in a variety of tissue cell lines, which provides an application basis for further genetic diseases, cancer gene therapy and viral vaccine development. Compared with the commercial cationic polymer transfection reagents mainly used in the current art, the star-shaped poly(β-amino ester) disclosed in the present invention has a higher mRNA transfection efficiency and more clinical application potential.

[0026] The present invention also discloses a method for synthesizing the functional star-shaped poly(β-amino ester), wherein a linear poly(β-amino ester) with a vinyl group at the end is first prepared by using a Michael addition strategy by using a diacrylate and an organic amine, and then the prepared linear poly(β-amino ester) is subjected to an end-capping reaction with an end-capping agent monomer to prepare a linear poly(β-amino ester) with an amine group at the end, and then the linear poly(β-amino ester) is reacted with a tetraacrylate or hexaacrylate monomer (core molecule) to prepare a star-shaped poly(β-amino ester) with a vinyl group at the end, and finally reacted with a functional end-capping agent monomer to prepare a functional star-shaped poly(β-amino ester). The synthesis method has a wide range of monomer sources, a simple synthesis process, mild reaction conditions, low synthesis cost, and low requirements for reaction equipment, and is particularly suitable for industrial-scale production.

[0027] The present invention also discloses the use of the functional star-shaped poly (β-amino ester), which is confirmed by experiments to be a new type of mRNA delivery vector with high transfection efficiency and low toxicity. The accuracy and wide applicability of the invention have been verified in a variety of tissue cells including HeLa cells, SW1353 cells and HaCaT cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the synthesis of the functional star-shaped poly (β-amino ester) of the present invention; (a) is Example 1, and (b) is Example 2.

[0029] Figure 2 (a) is the gel permeation chromatography (GPC) curve of the linear poly(β-amino ester) with an amine group at the end in Example 1; its weight average molecular weight is 3000Da; (b) is the gel permeation chromatography (GPC) curve of the linear poly(β-amino ester) with an amine group at the end in Example 2; its weight average molecular weight is 4200Da.

[0030] Figure 3 (a) is the GPC curve of the functional star-shaped poly (β-amino ester) in Example 1; its weight average molecular weight is 40,000 Da; (b) is the GPC curve of the functional star-shaped poly (β-amino ester) in Example 2; its weight average molecular weight is 34,000 Da.

[0031] Figure 4 (a) is the functional star-shaped poly (β-amino ester) with a weight average molecular weight of 40000Da in Example 1. 1 HNMR spectrum; (b) is the functional star-shaped poly (β-amino ester) with a weight average molecular weight of 34000Da in Example 2 1 HNMR spectrum.

[0032] Figure 5 (a) is the affinity test of the functional star-shaped poly (β-amino ester) for mRNA in Example 1; (b) is the affinity test of the functional star-shaped poly (β-amino ester) in Example 2, confirming its excellent mRNA affinity.

[0033] Figure 6 (a) is the particle size of the complex nanoparticles formed by the functional star-shaped poly (β-amino ester) compressed mRNA in Example 1; (b) is the particle size of the complex nanoparticles formed by the functional star-shaped poly (β-amino ester) compressed mRNA in Example 2.

[0034] Figure 7(a) is the zeta potential of the complex nanoparticles formed by the functional star-shaped poly (β-amino ester) compressed mRNA in Example 1; (b) is the zeta potential of the complex nanoparticles formed by the functional star-shaped poly (β-amino ester) compressed mRNA in Example 2.

[0035] Figure 8 (a) is the microscopic morphology of the complex nanoparticles formed by the functional star-shaped poly (β-amino ester) compressed mRNA in Example 1; (b) is the microscopic morphology of the complex nanoparticles formed by the functional star-shaped poly (β-amino ester) compressed mRNA in Example 2.

[0036] Fig. 9 In HeLa cells, SW1353 cells and HaCaT cells, (a) is a fluorescence photograph of the cells after transfection of mRNA encoding green fluorescent protein (GFP) with the functional star-shaped poly (β-amino ester) in Example 1; (b) is a fluorescence photograph of the cells after transfection of GFP mRNA with the functional star-shaped poly (β-amino ester) in Example 2.

[0037] Fig.10 (a) is the transfection result of the functional star-shaped poly (β-amino ester) in Example 1 in HeLa cells after transfection of GFP mRNA, which was quantitatively measured by flow cytometry; (b) is the transfection result of the functional star-shaped poly (β-amino ester) in Example 2 in SW1353 cells after transfection of GFP mRNA, which was quantitatively measured by flow cytometry.

[0038] Fig.11 (a) is the cell survival rate after mRNA transfection of the functional star-shaped poly (β-amino ester) in Example 1 at different mass ratios in HeLa cells; (b) is the cell survival rate after mRNA transfection of the functional star-shaped poly (β-amino ester) in Example 1 at different mass ratios in SW1353 cells. DETAILED DESCRIPTION

[0039] In order to further understand the present invention, the present invention is described below in conjunction with embodiments. These descriptions are only to further explain the features and advantages of the present invention and are not intended to limit the claims of the present invention.

[0040] The present invention discloses a class of functional star-shaped poly(β-amino ester), wherein the structural formula of the functional star-shaped poly(β-amino ester) is as follows:

[0041]

[0042]

[0043] In the formula, R1 is a group on a diacrylate monomer, R2 is a group on an organic amine, R3 is a group on a functional end-capping agent monomer, the organic amine is R2-NH2, the functional end-capping agent monomer is R3-NH2, and n=5-30.

[0044] The molecular weight of the star-shaped poly(β-amino ester) of the present invention is in the range of 7000 to 50000 Da, and the obtained product presents a star-shaped topological structure and a narrow molecular weight distribution.

[0045] The diacrylate monomers described in the present invention are 1,4-butanediol diacrylate, 2,2-dithiodiethanol diacrylate, diethylene glycol diacrylate, tetraethylene glycol diacrylate, 1,1'-isopropyl bis(p-phenyleneoxy)di-2-propanol diacrylate or bisphenol A polyoxyethylene ether diacrylate, and their structural formulas are shown below:

[0046]

[0047] The organic amine described in the present invention is 4-amino-1-butanol, butylamine, pentylamine, hexylamine, 1-(2-aminoethyl)piperazine, N-(2-aminoethyl)-morpholine, dodecylamine or octadecylamine, and their structural formulas are shown below:

[0048]

[0049] The functional end-capping agent monomer of the present invention is 1-(3-aminopropyl)-4-methylpiperazine, N-(3-aminopropyl)morpholine, N-(2-aminoethyl)morpholine, 2-methyl-2-morpholin-4-ylpropane-1-amine, 1-(2-aminoethyl)piperazine, 1,11-diamino-3,6,9-trioxaundecane, 1,2-ethylenediamine or 1,3-butylamine, and their structural formulas are shown below:

[0050]

[0051] The present invention discloses a method for synthesizing a class of functional star-shaped poly(β-amino esters), and the synthesis route is shown in Figure 1 , including the following steps:

[0052] 1) subjecting a diacrylate monomer to a Michael addition reaction with an organic amine to obtain a linear poly(β-amino ester) having a vinyl group at the end;

[0053] 2) capping the linear poly(β-amino ester) having a vinyl group at the end obtained in step 1) with a functionalized capping agent monomer to obtain a linear poly(β-amino ester) having an amine group at the end;

[0054] 3) reacting the linear poly(β-amino ester) having an amine group at the end obtained in step 2) with a tetraacrylate monomer or a hexaacrylate monomer (core molecule) to obtain a star-shaped poly(β-amino ester) having a vinyl group at the end;

[0055] 4) reacting the star-shaped poly(β-amino ester) containing terminal vinyl groups obtained in step 3) with a functional end-capping agent monomer to perform a functional end-capping reaction to obtain a high-performance functional star-shaped poly(β-amino ester).

[0056] The tetraacrylate monomer of the present invention is pentaerythritol tetraacrylate, and the hexaacrylate monomer is polydipentaerythritol hexaacrylate.

[0057]

[0058] Specifically, the present invention discloses a method for synthesizing a class of functional star-shaped poly(β-amino esters), comprising the following steps:

[0059] 1) adding a diacrylate monomer and an organic amine to a solvent (e.g., dimethyl sulfoxide) and fully dissolving them by magnetic stirring, wherein the reaction molar ratio of the diacrylate monomer to the organic amine is (1-5):1; wherein the reaction is carried out at 50-90° C. under a nitrogen atmosphere to obtain a linear poly(β-amino ester) containing a vinyl group.

[0060] 2) During the reaction, the polymer molecular weight is monitored by GPC, and the reaction is terminated when the polymer molecular weight reaches a preset value of 2000 to 5000 Da. Functionalized end-capping agent monomers and solvents (e.g., dimethyl sulfoxide, tetrahydrofuran) are added to the reaction system, and the reaction is carried out at 25° C. for 24 to 48 hours. The molar ratio of the functionalized end-capping agent monomer to the star-shaped poly(β-amino ester) containing vinyl groups is (1 to 6):(0.5 to 2); the product is then purified in excess ether and fully freeze-dried for 48 to 72 hours to obtain a linear poly(β-amino ester) with a low molecular weight and narrow molecular weight distribution and an amine-terminated linear poly(β-amino ester).

[0061] 3) adding a linear poly(β-amino ester) having an amine group at the end and a tetraacrylate or hexaacrylate monomer (core molecule) into a solvent and fully dissolving the monomers by magnetic stirring, wherein the molar ratio of the tetraacrylate or hexaacrylate monomer to the linear poly(β-amino ester) having an amine group at the end is (0.025-1):(0.5-5), and reacting at 40° C. to 90° C.

[0062] 4) Use GPC spectrum to monitor the polymer molecular weight, and terminate the reaction when the polymer molecular weight approaches the set value of 7000-50000Da. Add a certain amount of functionalized end-capping agent monomer to the reaction system, react with the linear poly (β-amino ester) with an amine group at the end to cap the end, and after the reaction is completed, purify the product by precipitation method and freeze-dry for 48h-72h.

[0063] Example 1 (reaction scheme see Figure 1 (a))

[0064] Bisphenol A polyoxyethylene ether diacrylate (24mmol, 11.23g) and 5-amino-1-pentanol (20mmol, 2.06g) were added to dimethyl sulfoxide (DMSO) (13.3mL) for dissolution, and reacted at 90°C for 10h. After the system reached room temperature, 5 times the equivalent (compared to the equivalent of residual primary amine at the end of the obtained polymer) of N-(3-aminopropyl)morpholine was added and reacted for 72h to synthesize a linear poly(β-amino ester) with an amine terminal, whose molecular weight was 3000Da. Figure 2 (a) is the GPC curve of the linear poly(β-amino ester) with a molecular weight of 3000 prepared in this example, which preliminarily confirms that the linear poly(β-amino ester) was successfully synthesized.

[0065] Then, a linear poly(β-amino ester) (13.2 g) with an amine terminal and polydipentaerythritol hexaacrylate (0.2 g) were dissolved in an organic solvent DMSO, wherein the concentration of the linear poly(β-amino ester) with an amine terminal was 300 mg / mL. The polymerization reaction was carried out at 60° C. under magnetic stirring, and the molecular weight of the polymer was detected by GPC. Then, an excess of N-(3-aminopropyl)morpholine was added for end-capping reaction, and finally, the polymer was precipitated in an excess of ether and freeze-dried to finally obtain a high-performance functional star-shaped poly(β-amino ester) with a weight average molecular weight of 40,000 Da.

[0066] Figure 3 (a) is the GPC curve of the purified high-performance functional star-shaped poly(β-amino ester) with a molecular weight of 40,000 Da prepared in this example, which confirms that the high-performance functional star-shaped poly(β-amino ester) was successfully synthesized. Figure 4 (a) is the H NMR spectrum of the purified high-performance functional star-shaped poly(β-amino ester) with a molecular weight of 40,000 Da prepared in this example. The H NMR spectrum shows characteristic peaks of benzene rings at 7.2 ppm to 7.6 ppm, which once again confirms that the functional star-shaped poly(β-amino ester) was successfully synthesized.

[0067] Example 2 (reaction scheme see Figure 1 (b))

[0068] 1,4-Butanediol diacrylate (24mmol, 4.752g) and 5-amino-1-pentanol (20mmol, 2.06g) were added to dimethyl sulfoxide (DMSO) (15.90mL) for dissolution, reacted at 60°C for 12h, added dimethyl sulfoxide (DMOS) (the concentration in the total reaction system was 30%), and after the system reached room temperature, 5 times (compared to the equivalent of residual primary amines at the end of the obtained polymer) of 1,11-diamino-3,6,9-trioxa-undecane was added, and reacted at room temperature for 48h to synthesize a linear poly (β-amino ester) with an amino group at the end, and its molecular weight was 4200Da.

[0069] Then, a linear poly(β-amino ester) (6.8 g) with an amine terminal and pentaerythritol tetraacrylate (0.18 g) were dissolved in an organic solvent DMSO, wherein the concentration of the linear poly(β-amino ester) with an amine terminal was 400 mg / mL. The polymerization reaction was carried out at 60°C under magnetic stirring, and the molecular weight of the polymer was detected by GPC. Then, an excess of 1,11-diamino-3,6,9-trioxa-undecane was added for end-capping reaction, and finally, the functional star-shaped poly(β-amino ester) was precipitated in an excess of ether and freeze-dried to obtain a molecular weight of 34000 Da.

[0070] Figure 2 (b) is the GPC curve of the linear poly(β-amino ester) with a molecular weight of 4200 Da prepared in this example. As the reaction proceeds, it is also confirmed that the linear poly(β-amino ester) is successfully synthesized.

[0071] Figure 3 The GPC curve of the functional star-shaped poly(β-amino ester) with a molecular weight of 34,000 Da prepared in this example can confirm that the linear functional star-shaped poly(β-amino ester) was successfully synthesized. Figure 4 This is the H-NMR spectrum of the functional star-shaped poly(β-amino ester) with a molecular weight of 34,000 Da prepared in this example after purification. The characteristic peaks of the H-NMR spectrum again confirmed that the polymer was successfully synthesized.

[0072] Example 3

[0073] The mass ratio of the high-performance functional star-shaped poly (β-amino ester) with a molecular weight of 40000Da to mRNA obtained in Example 1 is 20:1, 40:1, and 60:1, respectively, wherein the amount of mRNA used is 0.1 μg, and RiboGreen is used to test the affinity of the functional star-shaped poly (β-amino ester) to mRNA. First, the star-shaped poly (β-amino ester) is added to the mRNA solution encoding green fluorescent protein, vortexed at high speed for 30 seconds, and then allowed to stand for 20 minutes to form composite nanoparticles. Then, the composite nanoparticle solution is diluted to 100 μL using TE buffer solution, and then 100 μL RiboGreen working solution is added, and the excited fluorescence intensity is detected under the conditions of an excitation wavelength of 480 nm and an emission wavelength of 520 nm. Further, a similar method is used to prepare composite nanoparticles, and the particle size and surface potential thereof are tested using dynamic light scattering (DLS), and the composite nanoparticles are cleaned and freeze-dried at the same time, and their microscopic morphology is characterized using TEM.

[0074] Figure 5 (a) is a test of the affinity performance of the high-performance functional star-shaped poly (β-amino ester) with a molecular weight of 40,000 Da prepared in Example 1 to mRNA. This example confirms that when the mass ratios are 20:1, 40:1 and 60:1, respectively, the binding efficiency of the high-performance functional star-shaped poly (β-amino ester) with a molecular weight of 40,000 Da prepared in Example 1 to mRNA is more than 90%, and as the mass ratio increases, the binding efficiency also increases, confirming its excellent mRNA affinity performance.

[0075] Figure 5 (b) is a test of the affinity performance of the high-performance functional star-shaped poly (β-amino ester) with a molecular weight of 34,000 Da prepared in Example 2 for mRNA. This example confirms that when the mass ratios are 20:1, 40:1 and 60:1, respectively, the binding efficiency of the high-performance functional star-shaped poly (β-amino ester) with a molecular weight of 34,000 Da prepared in Example 2 for mRNA is more than 90%, confirming its excellent mRNA affinity performance.

[0076] Figure 6 (a) is a particle size test of the composite nanoparticles formed by the high-performance functional star-shaped poly (β-amino ester) with a molecular weight of 40000Da and mRNA prepared in Example 1. The DLS results confirmed that the high-performance functional star-shaped poly (β-amino ester) with a molecular weight of 40000Da prepared in Example 1 can effectively compress mRNA, and the particle size of the composite nanoparticles is less than 320nm, which can meet the subsequent cell uptake.

[0077] Figure 6(b) is a particle size test of the composite nanoparticles formed by the high-performance functional star-shaped poly (β-amino ester) with a molecular weight of 34000Da and mRNA prepared in Example 2. The DLS results confirmed that the high-performance functional star-shaped poly (β-amino ester) with a molecular weight of 34000Da prepared in Example 2 can effectively compress mRNA, and the particle size of the composite nanoparticles is less than 300nm, which can meet the subsequent cell uptake.

[0078] Figure 7 (a) is the zeta potential test of the composite nanoparticles formed by the high-performance functional star-shaped poly (β-amino ester) with a molecular weight of 40000Da and mRNA prepared in Example 1. The DLS results confirmed that the high-performance functional star-shaped poly (β-amino ester) with a molecular weight of 40000Da prepared in Example 1 can effectively shield the negative potential of mRNA itself, and the potential on the surface of the composite nanoparticles is greater than +10mV, which provides a prerequisite for further mRNA delivery.

[0079] Figure 7 (b) is the zeta potential test of the composite nanoparticles formed by the high-performance functional star-shaped poly (β-amino ester) with a molecular weight of 34000Da and mRNA prepared in Example 2. The DLS results confirmed that the high-performance functional star-shaped poly (β-amino ester) with a molecular weight of 34000Da prepared in Example 2 can effectively shield the negative potential of mRNA itself, and the potential on the surface of the composite nanoparticles is greater than +15mV, which provides a prerequisite for further mRNA delivery.

[0080] Figure 8 (a) is a microscopic morphology characterization of the composite nanoparticles formed by the high-performance functional star-shaped poly (β-amino ester) with a molecular weight of 40,000 Da and mRNA prepared in Example 1. TEM results confirm that the composite nanoparticles formed by the high-performance functional star-shaped poly (β-amino ester) with a molecular weight of 40,000 Da and mRNA prepared in Example 1 have uniform particle size distribution and a relatively stable structure, which confirms its excellent stability and provides an important guarantee for in vitro and in vivo gene delivery.

[0081] Figure 8 (b) is a microscopic morphology characterization of the composite nanoparticles formed by the high-performance functional star-shaped poly (β-amino ester) with a molecular weight of 34000Da and mRNA prepared in Example 2. TEM results confirm that the composite nanoparticles formed by the high-performance functional star-shaped poly (β-amino ester) with a molecular weight of 34000Da and mRNA prepared in Example 2 have uniform particle size distribution and a relatively stable structure, which confirms its excellent stability and provides an important guarantee for in vitro and in vivo gene delivery.

[0082] Example 4

[0083] HeLa cells, SW1353 cells and HaCaT cells were expressed at 1.2×10 4 The cells were inoculated in a 96-well plate at a density of 10 cells / well, and the cells were cultured at 37°C overnight. The sodium acetate buffer solution of the high-performance functional star-shaped poly (β-amino ester) prepared in the example was mixed with a sodium acetate buffer solution of 0.1 μg GFP mRNA, and the mass ratio of the star-shaped poly (β-amino ester) to mRNA was 20:1, 40:1, and 60:1, respectively. After standing for 15 minutes, it was added to the serum culture medium, mixed thoroughly, and then slowly added to the cells. The cells were then cultured for 24 hours, and the cells transfected with green fluorescent protein mRNA were observed under a fluorescence microscope.

[0084] Fig. 9 (a) is a qualitative evaluation of the mRNA transfection performance of the high-performance functional star-shaped poly(β-amino ester) with a molecular weight of 40,000 Da prepared in Example 1. Fig. 9 (a) It can be found that in HeLa cells, SW1353 cells and HaCaT cells, due to the excellent mRNA affinity and compressibility of the functional star-shaped poly (β-amino ester) at a mass ratio of 40:1 and 60:1, the functional star-shaped poly (β-amino ester) exhibited a higher mRNA transfection efficiency, proving that it can meet the requirements of efficient mRNA delivery in a variety of tissue cells.

[0085] Fig. 9 (b) is a qualitative evaluation of the mRNA transfection performance of the high-performance functional star-shaped poly(β-amino ester) with a molecular weight of 34,000 Da prepared in Example 2. Fig. 9 (b) It can be found that in HeLa cells, SW1353 cells and HaCaT cells, due to the excellent mRNA affinity and compressibility of the functional star-shaped poly (β-amino ester) at a mass ratio of 40:1 and 60:1, the functional star-shaped poly (β-amino ester) exhibited a higher GFP mRNA transfection efficiency, proving that it can meet the requirements of efficient mRNA delivery in a variety of tissue cells.

[0086] Fig.10 (a) is a quantitative evaluation of the mRNA transfection performance of the high-performance functional star-shaped poly(β-amino ester) with a molecular weight of 40,000 Da prepared in Example 1. Fig.10 (a) shows that in HeLa cells, the highest transfection efficiency of GFP mRNA mediated by the high-performance functional star-shaped poly (β-amino ester) with a molecular weight of 40,000 Da is over 52%.

[0087] Fig.10 (b) is a quantitative evaluation of the mRNA transfection performance of the high-performance functional star-shaped poly(β-amino ester) with a molecular weight of 34,000 Da prepared in Example 2. Fig.10 (b) shows that in SW1353 cells, the highest transfection efficiency of GFP mRNA mediated by the high-performance functional star-shaped poly (β-amino ester) with a molecular weight of 34,000 Da is over 40%.

[0088] Example 5

[0089] The cells were 1.2×10 4 The cells were inoculated in a 96-well plate at a density of 10 cells / well, and the cells were cultured at 37°C overnight. The sodium acetate buffer solution of the high-performance functional star-shaped poly (β-amino ester) prepared in the example was mixed with 0.1 μg of sodium acetate buffer solution encoding green fluorescent protein mRNA, and the mass ratio of the star-shaped poly (β-amino ester) to mRNA was 20:1, 40:1 and 60:1, respectively. After standing for 15 minutes, it was added to the serum culture medium, mixed thoroughly and then slowly added to the cells. The cells were then cultured for 24 hours, and the cell activity after transfection with luciferase-encoding DNA was quantitatively evaluated using Alamar Blue solution (concentration of 10%).

[0090] Fig.11 (a) is the evaluation of the activity of cells after mRNA transfection by the high-performance functional star-shaped poly(β-amino ester) with a molecular weight of 40,000 Da prepared in Example 1. Fig.11 It can be found that in HeLa cells, the high-performance functional star-shaped poly (β-amino ester) with a molecular weight of 40,000 Da still maintains more than 95% of cell activity after transfection. This is mainly due to the large number of degradable ester bonds and good biocompatibility in its main chain. Therefore, it exhibits high cell activity, providing the necessary conditions for subsequent clinical applications.

[0091] Fig.11 (b) is the evaluation of the activity of cells after mRNA transfection by the high-performance functional star-shaped poly(β-amino ester) with a molecular weight of 34,000 Da prepared in Example 2. Fig.11 (b) It can be found that in HeLa cells, the high-performance functional star-shaped poly (β-amino ester) with a molecular weight of 34,000 Da still maintains more than 91% cell activity after transfection. This is mainly due to the large number of degradable ester bonds and good biocompatibility in its main chain. Therefore, it exhibits high cell activity, providing the necessary conditions for subsequent clinical applications.

[0092] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A class of functional star-shaped poly(β-amino esters), It is characterized in that The functional star-shaped poly(β-amino ester) has a structural formula of the following Formula 1 or Formula 2: Among them, R 1 is a group on the diacrylate monomer, R 2 is a group on the organic amine, R 3 is a group on the functionalized end-capping agent monomer, and the organic amine is R 2 -NH 2 , the functionalized end-capping agent monomer is R 3 -NH 2 , n=5~30.

2. The functional star-shaped poly(β-amino ester) according to claim 1, It is characterized in that The diacrylate monomer is 1,4-butanediol diacrylate, 2,2-dithiodiethanol diacrylate, diethylene glycol diacrylate, tetraethylene glycol diacrylate, 1,1'-isopropyl bis(p-phenyleneoxy)di-2-propanol diacrylate or bisphenol A polyoxyethylene ether diacrylate.

3. The functional star-shaped poly(β-amino ester) according to claim 1, It is characterized in that The organic amine is 4-amino-1-butanol, butylamine, pentylamine, hexylamine, 1-(2-aminoethyl)piperazine, N-(2-aminoethyl)-morpholine, dodecylamine or octadecylamine.

4. The functional star-shaped poly(β-amino ester) according to claim 1, It is characterized in that The functionalized end-capping agent monomer is 1-(3-aminopropyl)-4-methylpiperazine, N-(3-aminopropyl)morpholine, N-(2-aminoethyl)morpholine, 2-methyl-2-morpholin-4-ylpropane-1-amine, 1-(2-aminoethyl)piperazine, 1,11-diamino-3,6,9-trioxaundecane, 1,2-ethylenediamine or 1,3-butylamine.

5. A method for preparing the functional star-shaped poly(β-amino ester) according to any one of claims 1 to 4, It is characterized in that include: 1) subjecting a diacrylate monomer to a Michael addition reaction with an organic amine to obtain a linear poly(β-amino ester) having a vinyl group at the end; 2) capping a linear poly(β-amino ester) having a vinyl group at the end with a functionalized capping agent monomer to obtain a linear poly(β-amino ester) having an amine group at the end; 3) reacting a linear poly(β-amino ester) having an amine group at the end with a tetraacrylate monomer or a hexaacrylate monomer to obtain a star-shaped poly(β-amino ester) having a vinyl group at the end; 4) reacting the star-shaped poly(β-amino ester) containing a vinyl group at the end with a functionalized end-capping agent monomer to obtain a functional star-shaped poly(β-amino ester).

6. The method for preparing the functional star-shaped poly(β-amino ester) according to claim 5, It is characterized in that The tetraacrylate monomer is pentaerythritol tetraacrylate, and the hexaacrylate monomer is polydipentaerythritol hexaacrylate.

7. The method for preparing the functional star-shaped poly(β-amino ester) according to claim 5, It is characterized in that In step 1), the molar ratio of the diacrylate monomer to the organic amine is (1-5):1; the reaction is carried out under a nitrogen atmosphere at a reaction temperature of 50-90°C.

8. The method for preparing the functional star-shaped poly(β-amino ester) according to claim 5, It is characterized in that In step 3), the molar ratio of the tetraacrylate monomer or the hexaacrylate monomer to the linear poly(β-amino ester) having an amine group at the end is (0.025-1):(0.5-5), and the reaction temperature is 40°C-90°C.

9. Use of the functional star-shaped poly(β-amino ester) according to any one of claims 1 to 4 in the preparation of a gene carrier, It is characterized in that The gene vector is used to deliver genes to tissues or cells.

10. Use of the functional star-shaped poly (β-amino ester) according to claim 9 in preparing a gene carrier, It is characterized in that The gene is mRNA.

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