Hyperbranched poly (beta-amino ester), preparation method, compound nanoparticles and application

Abstract

The invention provides hyperbranched poly (beta-amino ester), a preparation method, compound nanoparticles and application. A brand new A2 + B3 Michael addition strategy is adopted to synthesize a high-performance multi-reaction-site hyperbranched poly (beta-amino ester), the hyperbranched poly (beta-amino ester) and DNA or mRNA form compound nanoparticles, and DNA or mRNA delivery can be safely and efficiently mediated in cervical cells (HeLa), human embryo kidney cells (293T), kidney cells (COS-7) transformed from African green monkey SV40, kidney cells (Vero) and cartilage cells (SW1353). Compared with a mainstream commercial transfection reagent, the hyperbranched polymer monomer is wide in source, simple to synthesize, low in price and excellent in transfection performance, and has remarkable clinical application potential in the aspect of DNA or mRNA drug delivery.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials and relates to a type of hyperbranched poly (β-amino ester), a preparation method, composite nanoparticles and applications. Background Art

[0002] Gene therapy refers to the use of specific methods to deliver genetic materials such as DNA or RNA with specific functions into targeted tissues or cells for functional protein regulation to achieve the purpose of disease treatment. It has become one of the most promising therapies for treating various genetic diseases or acquired diseases. Because naked DNA and RNA are not easy to pass through the cell membrane, which is also negatively charged and hydrophobic, and are easily degraded by nucleases and restriction endonucleases in the blood or cytoplasm. Therefore, during the gene therapy process, it is necessary to use a carrier to compress and protect DNA and RNA. The lack of safe and efficient gene delivery vectors severely limits the clinical transformation of gene therapy.

[0003] At present, liposome nanoparticles are one of the most promising carriers for gene delivery, but their high price, poor serum tolerance, poor stability, complicated preparation process and complex ingredients limit their clinical application. Cationic polymers have attracted extensive attention due to their many advantages, such as wide sources of raw materials, flexible and diverse chemical compositions, easy to adjust topological structures, high gene loading efficiency and good serum stability. They mainly include polyethyleneimine (PEI) and poly(dimethylaminoethyl methacrylate) (PDMAEMA). Although cationic polymer carriers have shown certain application potential, they are prone to cytotoxicity due to their poor degradation performance and the monomer types are often relatively single.

[0004] Poly(β-amino ester) exhibits excellent gene transfection performance in various tissue cells such as fibroblasts, keratinocytes, cancer cells, stem cells, astrocytes and primary cells due to its widely available raw materials, easy synthesis, easy control of chemical composition and structure, and biodegradability. At present, branched poly(β-amino ester) mainly adopts A2+B'B'' (selecting trifunctional asymmetric monomers) strategy and A2+B3+C2 strategy. For the A2+B'B'' strategy (selecting trifunctional asymmetric monomers) (Japanese patent JP 2013-237853A), in order to slow down the polymerization reaction rate and inhibit gelation, trifunctional asymmetric monomers must be strictly selected and the connecting group must be cycloalkane, which results in very limited monomers available for the synthesis of branched poly(β-amino ester), which seriously hinders the commercial application of branched poly(β-amino ester). For the A2+B3+C2 strategy (China Publication Patent CN202411120468.4), in order to slow down the reaction rate and inhibit gelation, a certain proportion of difunctional symmetrical monomers is added to the reaction system to reduce the reaction probability of trifunctional monomers, which also causes uneven distribution of chemical components inside the branched poly (β-amino ester) molecular chain.

[0005] At the same time, since stepwise polymerization and branching strategies often produce branched poly(β-amino esters) with a wide molecular weight distribution, this significantly limits their application in efficient functional gene transfection and clinical transformation of gene therapy. Summary of the invention

[0006] In order to overcome the shortcomings of the above-mentioned prior art, the present invention adopts the A2+B3 Michael addition strategy to prepare a series of hyperbranched poly(β-amino esters) with narrow molecular weight distribution, increase the branching degree and terminal groups of the branched poly(β-amino esters), thereby enhancing its interaction with DNA and promoting the subsequent DNA delivery efficiency. At the same time, the narrow molecular weight distribution provides a guarantee for further batch stability, ultimately providing an important platform technology for clinical gene therapy.

[0007] The present invention is achieved through the following technical solutions The present invention discloses a type of hyperbranched poly (β-amino ester), a preparation method, composite nanoparticles and applications, and the structural formula thereof is as follows: ; R1 is a group on a triacrylate monomer, R2 is a group on an organic amine, R3 is a group on a capping agent monomer, and n is the number of repeating units, which ranges from 10 to 45.

[0008] Preferably, the molecular weight of the hyperbranched poly(β-amino ester) is in the range of 5.0 kg / mol to 40.0 kg / mol.

[0009] Preferably, the organic amine is 1-amino-3-propanol, 4-amino-1-butanol, 5-amino-1-pentanol, 6-amino-1-hexanol, butylamine, pentylamine, 1-(2-aminoethyl)piperazine, N-(2-aminoethyl)-morpholine, dodecylamine or octadecylamine.

[0010] ; Preferably, the triacrylate monomer is trimethylolpropane triacrylate and ethoxylated trimethylolpropane triacrylate, and the molecular weights thereof are 428, 692, and 912, respectively.

[0011] ; Preferably, the 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, 1,4-bis(3-aminopropyl)piperazine, 1,11-diamino-3,6,9-trioxaundecane, 2-methyl-1,5-pentanediamine, 1,2-ethylenediamine, 1,3-propylenediamine, 1,4-butylenediamine, and 1,6-hexanediamine.

[0012] ; The present invention also discloses a specific method for preparing a hyperbranched poly (β-amino ester) with a narrow molecular weight distribution by adopting a new type of A2+B3 Michael addition strategy, which comprises the following steps: 1) dissolving triacrylate monomers and organic amines in DMSO, and obtaining hyperbranched poly(β-amino ester) with a vinyl group at the end through Michael addition reaction; 2) reacting the hyperbranched poly(β-amino ester) having a vinyl group at the end obtained in step 1) with a capping agent to obtain a high-performance functionalized hyperbranched poly(β-amino ester); Preferably, in step 1), the molar ratio of the triacrylate monomer to the organic amine is 1:(0.4-1.8), and the reaction temperature is 60°C-90°C; Preferably, in step 2), the molar ratio of the end-capping agent, the amine, and the hyperbranched poly (β-amino ester) having a vinyl group at the end is (1-5):1, and the reaction is carried out at room temperature.

[0013] The invention provides a hyperbranched poly (β-amino ester) having a molecular weight range of 5.0 kg / mol to 40.0 kg / mol.

[0014] The present invention also provides a type of hyperbranched poly (β-amino ester) based composite nanoparticles, which are nanoparticles formed by the above-mentioned hyperbranched poly (β-amino ester) and DNA or mRNA.

[0015] The present invention also provides the use of the hyperbranched poly (β-amino ester) or the composite nanoparticle in preparing a gene carrier, wherein the hyperbranched poly (β-amino ester) is used to deliver genes to tissues or cells.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses a class of high-performance hyperbranched poly (β-amino ester) synthesized by a new A2+B3 Michael addition strategy, which is a class of hyperbranched poly (β-amino ester) with a new structure by direct reaction of branched monomers and small molecule amines. Compared with the A2+B'B'' strategy (selecting trifunctional asymmetric monomers) (Japanese patent JP 2013-237853A3), a wider range of commercial monomers can be selected to prepare branched poly (β-amino ester), and the prepared branched poly (β-amino ester) has better DNA and mRNA delivery efficiency and more commercial transformation potential.

[0017] Compared with the A2+B3+C2 strategy (China Publication Patent CN202411120468.4) adopted by existing branched polymers, star polymers or linear poly(β-amino esters), the branched poly(β-amino esters) prepared by the present invention under low molar ratio conditions have obvious structural differences and advantages, a wider chemical space and structural regularity, and thus have a relatively excellent affinity for DNA. The composite nanoparticles formed by the present invention and DNA are more evenly distributed, stable, biodegradable and biocompatible. The results of in vitro transfection show that the hyperbranched poly(β-amino ester) synthesized by the A2+B3 Michael addition strategy can efficiently deliver DNA and mRNA in a variety of tissue cell lines, especially in cancer cell lines, which provides an application basis for further cancer gene therapy. Compared with the commercial cationic polymer transfection reagents jetPEI and Lipo3000 mainly used in the current art, the hyperbranched poly(β-amino ester) disclosed in the present invention has greater clinical application potential.

[0018] The present invention also discloses a method for synthesizing the hyperbranched poly(β-amino ester), wherein a hyperbranched poly(β-amino ester) with a vinyl group at the end is prepared by using triacrylate and a small molecule organic amine at a low molar ratio and a Michael addition strategy, and then the prepared hyperbranched poly(β-amino ester) with a vinyl group at the end is subjected to an end-capping reaction with an organic amine to prepare a high-performance functionalized hyperbranched poly(β-amino ester). The synthesis method has a wide range of monomer sources, a simple synthesis process, a low synthesis cost, and low equipment requirements, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 Schematic diagram of the synthesis of hyperbranched poly (β-amino ester) with different molecular weights in Examples 1 to 8 of the present invention.

[0021] Figure 2 is the hyperbranched poly (β-amino ester) with different molecular weights in Examples 1 to 8 of the present invention 1 H NMR spectrum.

[0022] Figure 3 This is a gel permeation chromatography (GPC) curve of hyperbranched poly (β-amino ester) with different molecular weights in Examples 1 to 8 of the present invention.

[0023] Figure 4 The affinity of hyperbranched poly(β-amino ester) with different molecular weights in Examples 1 to 8 of the present invention to DNA confirms its excellent DNA affinity.

[0024] Figure 5 The particle sizes of the composite nanoparticles formed by DNA compression by hyperbranched poly (β-amino ester) of different molecular weights in Examples 1 to 8 of the present invention confirm its excellent DNA compression performance.

[0025] Figure 6 The zeta potential of the composite nanoparticles formed by the hyperbranched poly (β-amino ester) compressed DNA of different molecular weights in Examples 1 to 8 proves that it can effectively shield the negative charge of DNA itself and promote subsequent cellular uptake.

[0026] Figure 7 The microscopic morphology of the composite nanoparticles formed by the hyperbranched poly (β-amino ester) compressed DNA in Example 5 confirms that the formed composite nanoparticles have excellent stability.

[0027] Figure 8 The qualitative evaluation (fluorescence results) of the GFP DNA transfection performance mediated by hyperbranched poly(β-amino ester)s of different molecular weights in Examples 4 to 7 of the present invention in HeLa cells confirmed their excellent DNA transfection efficiency.

[0028] Fig. 9The quantitative evaluation of the GFP DNA transfection performance mediated by hyperbranched poly(β-amino ester)s of different molecular weights in Examples 4 to 7 of the present invention in HeLa cells (flow cytometry results) confirmed their excellent DNA transfection efficiency.

[0029] Fig.10 The cell activity after DNA transfection mediated by hyperbranched poly(β-amino ester)s of different molecular weights in Examples 4 to 7 of the present invention was evaluated in HeLa cells. The results showed that the cells could still maintain a high activity after transfection, confirming its excellent biosafety.

[0030] Fig.11 This is a qualitative evaluation of the GFP DNA transfection performance mediated by hyperbranched poly(β-amino ester)s of different molecular weights in Examples 4 to 7 of the present invention in 293T cells (fluorescence results). The results of fluorescence microscopy show that all hyperbranched poly(β-amino esters) exhibit excellent DNA transfection efficiency.

[0031] Fig.12 The results are quantitative evaluation of the GFP DNA transfection performance mediated by hyperbranched poly(β-amino ester)s of different molecular weights in Examples 4 to 7 of the present invention in 293T cells (flow cytometry results), confirming their excellent DNA transfection efficiency.

[0032] Fig.13 The cell activity after DNA transfection mediated by hyperbranched poly (β-amino ester) of different molecular weights in Examples 4 to 7 of the present invention was evaluated in 293T cells. The results showed that the cell activity after transfection exceeded 90%, confirming its excellent cell activity.

[0033] Fig.14 This is a qualitative evaluation of the GFP DNA transfection performance mediated by hyperbranched poly(β-amino ester)s of different molecular weights in Examples 4 to 7 of the present invention in SW1353 cells (fluorescence results). The results of fluorescence microscopy show that all hyperbranched poly(β-amino esters) exhibit excellent DNA transfection efficiency.

[0034] Fig.15 The results are a quantitative evaluation of the GFP DNA transfection performance mediated by hyperbranched poly(β-amino ester)s of different molecular weights in Examples 4 to 7 of the present invention in SW1353 cells (flow cytometry results), confirming their excellent DNA transfection efficiency.

[0035] Fig.16 The cell activity after DNA transfection mediated by hyperbranched poly (β-amino ester) of different molecular weights in Examples 4 to 7 of the present invention was evaluated in SW1353 cells. The results showed that the cell activity after transfection exceeded 90%, confirming its excellent cell activity.

[0036] Fig.17 The qualitative evaluation (fluorescence results) of the GFP DNA transfection performance mediated by hyperbranched poly(β-amino ester)s of different molecular weights in Examples 4 to 7 of the present invention in COS-7 cells confirmed their excellent DNA transfection efficiency.

[0037] Fig.18 The results are quantitative evaluation of the GFP DNA transfection performance mediated by hyperbranched poly(β-amino ester)s of different molecular weights in Examples 4 to 7 of the present invention in COS-7 cells (flow cytometry results), confirming their excellent DNA transfection efficiency.

[0038] Fig.19 The cell activity after DNA transfection mediated by hyperbranched poly(β-amino ester)s of different molecular weights in Examples 4 to 7 of the present invention was evaluated in COS-7 cells. The results showed that the cell activity after transfection exceeded 92%, confirming its excellent cell activity.

[0039] Fig. 20 The results of fluorescence microscopy showed that all hyperbranched poly(β-amino esters) exhibited excellent DNA transfection efficiency.

[0040] Fig.21 The quantitative evaluation of the GFP DNA transfection performance mediated by hyperbranched poly(β-amino ester)s of different molecular weights in Examples 4 to 7 of the present invention in Vero cells (flow cytometry results) confirmed their excellent DNA transfection efficiency.

[0041] Fig. 22 The cell activity after DNA transfection mediated by hyperbranched poly (β-amino ester) of different molecular weights in Examples 4 to 7 of the present invention was evaluated in Vero cells. The results showed that the cell activity after transfection exceeded 89%, confirming its excellent cell activity.

[0042] Fig.23 For comparative example 1, under normal conditions, the reaction molar ratio of triacrylate monomer to organic amine is 1:1.5, and the reaction is carried out at 90°C. The polymerization reaction produces gel in a very short time, and the physical state of the polymer in the gel state is as follows.

[0043] Fig.24 For comparative example 2, under normal conditions, the reaction molar ratio of triacrylate monomer to organic amine is 1:1.8, and the reaction is carried out at 90°C. The polymerization reaction produces gel in a very short time, and the physical state of the polymer in the gel state is as follows. DETAILED DESCRIPTION

[0044] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0045] It should be noted that the process equipment or devices not specifically specified in the following embodiments are all conventional equipment or devices in the art.

[0046] It should be noted that the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to define the scope of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the present invention without substantially changing the technical content.

[0047] Example 1: Trimethylolpropane triacrylate (16 mmol, 4.741 g) and 3-amino-1-propanol (20 mmol, 1.51 g) were dissolved in dimethyl sulfoxide (DMSO) (6.601 mL), reacted at 90°C for 4 h, and dimethyl sulfoxide (DMOS) was added (the total mass of the monomers accounted for 10% of the total reaction system). After the system reached room temperature, 5 times the equivalent (compared to the equivalent of the residual primary amine at the end of the obtained polymer) of propylene diamine was added, and reacted at room temperature for 48 hours to synthesize a functionalized hyperbranched poly (β-amino ester) with a molecular weight of 19.3 kg / mol, namely HPAE-1 ( Figure 1 ). Figure 3 The GPC curve of the functionalized hyperbranched poly(β-amino ester) prepared in this example confirms that the linear poly(β-amino ester) is successfully synthesized with a molecular weight of 19.3 kg / mol and a dispersion index of 2.6. Figure 2 The purified functionalized hyperbranched poly(β-amino ester) with a molecular weight of 19.3 kg / mol prepared in this example showed a hydrogen NMR spectrum. The characteristic peaks of the H NMR spectrum once again confirmed that the polymer was successfully synthesized.

[0048] Example 2: Trimethylolpropane triacrylate (16 mmol, 4.741 g) and 4-amino-1-butanol (19 mmol, 1.86 g) were dissolved in dimethyl sulfoxide (DMSO) (6.601 mL), reacted at 90°C for 4 h, and dimethyl sulfoxide (DMOS) was added (the total mass of the monomer accounted for 20% of the total reaction system). After the system reached room temperature, 5 times the equivalent (compared to the equivalent of the residual primary amine at the end of the obtained polymer) of 2-methyl-1,5-pentanediamine was added, and reacted at room temperature for 48 hours to synthesize a functionalized hyperbranched poly (β-amino ester) with a molecular weight of 24.3 kg / mol, namely HPAE-2 ( Figure 1 ). Figure 3 The GPC curve of the functionalized hyperbranched poly(β-amino ester) prepared in this example confirms that the linear poly(β-amino ester) is successfully synthesized with a molecular weight of 24.3 kg / mol and a dispersion index of 3.0. Figure 2 The purified functionalized hyperbranched poly(β-amino ester) with a molecular weight of 24.3 kg / mol prepared in this example showed a hydrogen NMR spectrum. The characteristic peaks of the H NMR spectrum once again confirmed that the polymer was successfully synthesized.

[0049] Example 3: Trimethylolpropane triacrylate (16 mmol, 4.741 g) and 5-amino-1-pentanol (18 mmol, 1.850 g) were dissolved in dimethyl sulfoxide (DMSO) (29.379 mL), reacted at 90°C for 4 h, and dimethyl sulfoxide (DMOS) was added (the total mass of the monomers accounted for 20% of the total reaction system). After the system reached room temperature, 3 times the equivalent (compared to the equivalent of the residual primary amine at the end of the obtained polymer) of 3,6,9-trioxaundecane-1,11-diamine was added, and reacted at room temperature for 48 hours to synthesize a functionalized hyperbranched poly (β-amino ester) with a molecular weight of 24.8 kg / mol, namely HPAE-3 ( Figure 1 ). Figure 3 The GPC curve of the functionalized hyperbranched poly(β-amino ester) prepared in this example confirms that the linear poly(β-amino ester) is successfully synthesized with a molecular weight of 24.8 kg / mol and a dispersion index of 2.9. Figure 2 The purified functionalized hyperbranched poly(β-amino ester) with a molecular weight of 24.8 kg / mol prepared in this example showed a hydrogen NMR spectrum. The characteristic peaks of the H NMR spectrum confirmed that the polymer was successfully synthesized.

[0050] Example 4: Trimethylolpropane triacrylate (16 mmol, 4.741 g) and 6-amino-1-hexanol (14 mmol, 1.640 g) were dissolved in dimethyl sulfoxide (DMSO) (14.88 mL), reacted at 90° C. for 4 h, and dimethyl sulfoxide (DMOS) was added (the total mass of the monomers accounted for 20% of the total reaction system). After the system reached room temperature, 3 times the equivalent (compared to the equivalent of the residual primary amine at the end of the obtained polymer) of 3,6,9-trioxaundecane-1,11-diamine was added, and reacted at room temperature for 48 hours to synthesize a functionalized hyperbranched poly (β-amino ester) with a molecular weight of 16.5 kg / mol, namely HPAE-4 ( Figure 1 ). Figure 3 The GPC curve of the functionalized hyperbranched poly(β-amino ester) prepared in this example confirms that the linear poly(β-amino ester) is successfully synthesized with a molecular weight of 16.5 kg / mol and a dispersion index of 2.2. Figure 2 The purified functionalized hyperbranched poly(β-amino ester) with a molecular weight of 16.5 kg / mol prepared in this example showed a hydrogen NMR spectrum. The characteristic peaks of the H NMR spectrum confirmed that the polymer was successfully synthesized.

[0051] Example 5: Trimethylolpropane triacrylate (16 mmol, 4.741 g) and 5-amino-1-pentanol (22 mmol, 2.266 g) were dissolved in dimethyl sulfoxide (DMSO) (7.007 mL), reacted at 90 ° C for 4 h, and dimethyl sulfoxide (DMOS) was added (the total mass of the monomers accounted for 30% of the total reaction system). After the system reached room temperature, 5 times the equivalent (compared to the equivalent of the residual primary amine at the end of the obtained polymer) of propylene diamine was added, and the reaction was carried out at room temperature for 48 hours to synthesize a functionalized hyperbranched poly (β-amino ester) with a molecular weight of 20.1 kg / mol, namely HPAE-5 ( Figure 1 ). Figure 3 The GPC curve of the functionalized hyperbranched poly(β-amino ester) prepared in this example confirms that the linear poly(β-amino ester) is successfully synthesized with a molecular weight of 20.1 kg / mol and a dispersion index of 3.0. Figure 2 The purified functionalized hyperbranched poly(β-amino ester) with a molecular weight of 20.1 kg / mol prepared in this example was analyzed by hydrogen NMR spectrum. The characteristic peaks of the H NMR spectrum confirmed that the polymer was successfully synthesized.

[0052] Example 6: Trimethylolpropane triacrylate (16 mmol, 4.741 g) and 4-amino-1-pentanol (24 mmol, 2.136 g) were dissolved in dimethyl sulfoxide (DMSO) (27.508 mL), reacted at 90°C for 4 h, and dimethyl sulfoxide (DMOS) was added (the total mass of the monomers accounted for 20% of the total reaction system). After the system reached room temperature, 5 times the equivalent (compared to the equivalent of the residual primary amine at the end of the obtained polymer) of propylene diamine was added, and reacted at room temperature for 48 hours to synthesize a functionalized hyperbranched poly (β-amino ester) with a molecular weight of 13.7 kg / mol, namely HPAE-6 ( Figure 1 ). Figure 3 The GPC curve of the functionalized hyperbranched poly(β-amino ester) prepared in this example confirms that the linear poly(β-amino ester) was successfully synthesized with a molecular weight of 13.7 kg / mol and a dispersion index of 1.8. Figure 2 The purified functionalized hyperbranched poly(β-amino ester) with a molecular weight of 13.7 kg / mol prepared in this example was analyzed by hydrogen NMR spectrum. The characteristic peaks of the H NMR spectrum confirmed that the polymer was successfully synthesized.

[0053] Example 7: Trimethylolpropane triacrylate (16 mmol, 4.741 g) and 4-amino-1-pentanol (24 mmol, 2.136 g) were dissolved in dimethyl sulfoxide (DMSO) (27.508 mL), reacted at 90°C for 4 h, and dimethyl sulfoxide (DMOS) was added (the total mass of the monomer accounted for 10% of the total reaction system). After the system reached room temperature, 4 times the equivalent (compared to the equivalent of the residual primary amine at the end of the obtained polymer) of 3,6,9-trioxaundecane-1,11-diamine was added, and reacted at room temperature for 24 hours to synthesize a functionalized hyperbranched poly (β-amino ester) with a molecular weight of 16.1 kg / mol, namely HPAE-7 ( Figure 1 ). Figure 3 The GPC curve of the functionalized hyperbranched poly(β-amino ester) prepared in this example confirms that the linear poly(β-amino ester) is successfully synthesized with a molecular weight of 16.1 kg / mol and a dispersion index of 1.8. Figure 2 The purified functionalized hyperbranched poly(β-amino ester) with a molecular weight of 16.1 kg / mol prepared in this example was analyzed by hydrogen NMR spectrum. The characteristic peaks of the H NMR spectrum confirmed that the polymer was successfully synthesized.

[0054] Example 8: Trimethylolpropane triacrylate (10 mmol, 2.981 g) and 4-amino-1-pentanol (15 mmol, 1.335 g) were dissolved in dimethyl sulfoxide (DMSO) (6.477 mL), reacted at 90°C for 4 h, and dimethyl sulfoxide (DMOS) was added (the total mass of the monomers accounted for 10% of the total reaction system). After the system reached room temperature, 4 times the equivalent (compared to the equivalent of the residual primary amine at the end of the obtained polymer) of 2-methyl-1,5-pentanediamine was added, and reacted at room temperature for 72 hours to synthesize a functionalized hyperbranched poly (β-amino ester) with a molecular weight of 14.2 kg / mol, namely HPAE-8 ( Figure 1 ). Figure 3 The GPC curve of the functionalized hyperbranched poly(β-amino ester) prepared in this example confirms that the linear poly(β-amino ester) is successfully synthesized with a molecular weight of 14.2 kg / mol and a dispersion index of 1.4. Figure 2 The purified functionalized hyperbranched poly(β-amino ester) with a molecular weight of 14.2 kg / mol prepared in this example showed a hydrogen NMR spectrum. The characteristic peaks of the H NMR spectrum confirmed that the polymer was successfully synthesized.

[0055] Example 9: Preparation of composite nanoparticles formed by hyperbranched poly (β-amino ester) of different molecular weights in Examples 1 to 8 (whose molecular weights are 19.3 kg / mol, 24.3 kg / mol, 24.8 kg / mol, 16.5 kg / mol, 20.1 kg / mol, 13.7 kg / mol, 16.1 kg / mol and 14.2 kg / mol, respectively) and DNA (Biyuntian D2815). When the mass ratio of hyperbranched poly (β-amino ester) polymer to DNA is 60:1, the amount of DNA is 1.5 μg, and PicoGreen is used to test the DNA affinity of the composite nanoparticles. First, the degraded polymer is quickly added to the GFP DNA (Biyuntian D2815) solution, vortexed at high speed for 30 seconds, and then allowed to stand for 20 minutes to form a composite nanoparticle solution. Then, the composite nanoparticle solution was diluted to 100 μL with serum-free medium, and then 100 μL PicoGreen working solution was added. The excited fluorescence intensity was detected under the conditions of excitation wavelength of 480 nm and emission wavelength of 520 nm. Composite nanoparticles were prepared by a similar method, and the particle size and surface Zeta potential of the composite nanoparticles were tested by dynamic light scattering (DLS), and the morphology of the composite nanoparticles was characterized by TEM.

[0056] Figure 4The results of the affinity test of the hyperbranched poly (β-amino ester) with different molecular weights in Examples 1 to 8 (whose molecular weights are 19.3 kg / mol, 24.3 kg / mol, 24.8 kg / mol, 16.5 kg / mol, 20.1 kg / mol, 13.7 kg / mol, 16.1 kg / mol and 14.2 kg / mol, respectively) for DNA show that when the mass ratio is 60:1, the hyperbranched poly (β-amino ester) with different molecular weights in Examples 1 to 8 (whose molecular weights are 19.3 kg / mol, 24.3 kg / mol, 24.8 kg / mol, 16.5 kg / mol, 20.1 kg / mol, 13.7 kg / mol, 16.1 kg / mol and 14.2 kg / mol, respectively) show higher DNA affinity efficiency of more than 65%, which proves that the prepared hyperbranched poly (β-amino ester) has a wider chemical space and better protection efficiency for DNA.

[0057] Figure 5 The particle size test of the composite nanoparticles of the hyperbranched poly (β-amino ester) with different molecular weights (the molecular weights are 19.3 kg / mol, 24.3 kg / mol, 24.8 kg / mol, 16.5 kg / mol, 20.1 kg / mol, 13.7 kg / mol, 16.1 kg / mol and 14.2 kg / mol) and DNA in Examples 1 to 8. The DLS results confirm that the particle size of the composite nanoparticles formed by the hyperbranched poly (β-amino ester) with different molecular weights and DNA in Examples 1 to 8 is less than 350 nm, which is conducive to the subsequent cell uptake of the composite nanoparticles and gene transfection.

[0058] Figure 6 The zeta potential test of the composite nanoparticles of the hyperbranched poly (β-amino ester) with different molecular weights in Examples 1 to 8 (the molecular weights are 19.3 kg / mol, 24.3 kg / mol, 24.8 kg / mol, 16.5 kg / mol, 20.1 kg / mol, 13.7 kg / mol, 16.1 kg / mol and 14.2 kg / mol, respectively) and DNA. The DLS results confirm that the hyperbranched poly (β-amino ester) with different molecular weights in Examples 1 to 8 can compress the composite nanoparticles formed by DNA, and can effectively shield the negative potential of DNA itself, and the potential on the surface of the composite nanoparticles is greater than +8mV, which further promotes the interaction between the composite nanoparticles and the cell membrane and cell uptake.

[0059] Figure 7This is a microscopic morphology test of the composite nanoparticles of hyperbranched poly (β-amino ester) (whose molecular weight is 20.1 kg / mol) and DNA in Example 5. TEM results confirm that hyperbranched poly (β-amino ester) can compress the composite nanoparticles formed by DNA to form stable spherical particles with relatively uniform distribution, further indicating that the formed composite nanoparticles have excellent stability, providing the necessary conditions for subsequent gene transfection.

[0060] Example 10: HeLa cells and 293T cells were cultured at 2.0×10 4 The cells were inoculated in a 96-well plate at a density of 1 cell / well, and the cells were cultured at 37°C overnight. The sodium acetate buffer solution (specific components are 1.44 μL polymer + 10 μL sodium acetate buffer solution) of hyperbranched poly (β-amino ester) of different molecular weights in Examples 4-7 (whose molecular weights are 16.5 kg / mol, 20.1 kg / mol, 13.7 kg / mol and 16.1 kg / mol, respectively) was mixed with 0.6 μg of sodium acetate buffer solution (specific components are 0.6 μg DNA + 10 μL sodium acetate buffer solution) encoding GFP DNA (Biyuntian D2815), and the mass ratio of 4 hyperbranched poly (β-amino ester) of different molecular weights to DNA was 60:1. After standing for 15 min, it was added to the serum-free culture medium, mixed thoroughly and then slowly added to the cells. Then the new culture medium was replaced every 4 hours, and the cells were cultured for 44 hours, and the cells transfected with GFP DNA were observed under a fluorescence microscope.

[0061] Figure 8 Qualitative evaluation of the GFP DNA transfection performance mediated by hyperbranched poly(β-amino ester)s of different molecular weights (the molecular weights are 16.5 kg / mol, 20.1 kg / mol, 13.7 kg / mol and 16.1 kg / mol, respectively) in Examples 4 to 7 of the present invention (fluorescence results). Figure 8 It can be found that in HeLa cells, four hyperbranched poly (β-amino esters) with different molecular weights showed excellent DNA transfection efficiency, which was mainly due to the wider chemical space, multiple terminal groups and high-density amine sites of the polymers, thereby promoting gene transfection.

[0062] Fig. 9The DNA transfection performance mediated by hyperbranched poly(β-amino ester)s of different molecular weights in Examples 4 to 7 of the present invention (whose molecular weights are 16.5 kg / mol, 20.1 kg / mol, 13.7 kg / mol and 16.1 kg / mol, respectively) in HeLa cells was quantitatively evaluated. The flow cytometry results showed that in 293T cells, four hyperbranched poly(β-amino esters) of different molecular weights were able to efficiently mediate DNA transfection, with a transfection efficiency of 78%. These results once again confirmed that the branching strategy can improve the gene transfection efficiency of poly(β-amino ester).

[0063] Fig.10 The cell activity evaluation after DNA transfection mediated by hyperbranched poly(β-amino ester)s of different molecular weights in Examples 4 to 7 of the present invention (the molecular weights are 16.5 kg / mol, 20.1 kg / mol, 13.7 kg / mol and 16.1 kg / mol, respectively) in HeLa cells. The alarmBlue results show that the cell survival rate after transfection with four hyperbranched poly(β-amino ester)-based nanoparticles of different molecular weights exceeds 85%, with the highest reaching 100%, which is mainly due to the excellent biodegradability and biosafety of the composite nanoparticles, further confirming that the composite nanoparticles prepared by the present invention have broad commercial application potential.

[0064] Fig.11 Qualitative evaluation of the GFP DNA transfection performance mediated by hyperbranched poly(β-amino ester)s of different molecular weights (the molecular weights are 16.5 kg / mol, 20.1 kg / mol, 13.7 kg / mol and 16.1 kg / mol, respectively) in Examples 4 to 7 of the present invention (fluorescence results). Fig.11 It can be found that in 293T cells, four hyperbranched poly (β-amino esters) with different molecular weights showed excellent DNA transfection efficiency, which was mainly due to the wider chemical space, multiple terminal groups and high-density amine sites of the polymers, thereby promoting gene transfection.

[0065] Fig.12 The present invention quantitatively evaluates the DNA transfection performance mediated by hyperbranched poly(β-amino ester)s of different molecular weights (the molecular weights are 16.5 kg / mol, 20.1 kg / mol, 13.7 kg / mol and 16.1 kg / mol, respectively) in 293T cells. The flow cytometry results show that in 293T cells, the four hyperbranched poly(β-amino esters) of different molecular weights can efficiently mediate DNA transfection, with a transfection efficiency of 70%. These results once again confirm that the branching strategy can improve the gene transfection efficiency of poly(β-amino ester).

[0066] Fig.13The cell activity after DNA transfection mediated by hyperbranched poly(β-amino ester)s of different molecular weights (molecular weights of 16.5 kg / mol, 20.1 kg / mol, 13.7 kg / mol and 16.1 kg / mol, respectively) in Examples 4 to 7 of the present invention was evaluated in 293T cells. The alarmBlue results showed that the cell viability after transfection with four hyperbranched poly(β-amino ester)-based nanoparticles of different molecular weights exceeded 93%, with the highest reaching 100%, which was mainly due to the excellent biodegradability and biosafety of the composite nanoparticles, further confirming that the composite nanoparticles prepared by the present invention have broad commercial application potential.

[0067] Example 11: SW1353 cells, COS-7 cells and Vero cells were cultured at 2.0 × 10 4 Cells were seeded at a density of 10 cells / well in a 96-well plate and cultured overnight at 37°C. 2 μg of functional highly flexible branched poly (β-amino ester) in sodium acetate buffer was mixed with 0.1 μg of sodium acetate buffer encoding green fluorescent protein mRNA, left to stand for 45 min, then slowly added to the cells, and the culture medium was replaced after 4 hours. The cells were then cultured for 32 hours, and the cells transfected with green fluorescent protein were observed under a fluorescence microscope.

[0068] Fig.14 Qualitative evaluation of the GFP DNA transfection performance mediated by hyperbranched poly(β-amino ester)s of different molecular weights (the molecular weights are 16.5 kg / mol, 20.1 kg / mol, 13.7 kg / mol and 16.1 kg / mol, respectively) in Examples 4 to 7 of the present invention (fluorescence results). Fig.14 It can be found that in SW1353 cells, four hyperbranched poly (β-amino esters) with different molecular weights showed excellent mRNA transfection efficiency, which was mainly due to the wider chemical space, multiple terminal groups and high-density amine sites of the polymers, thereby promoting gene transfection.

[0069] Fig.15 The present invention quantitatively evaluates the transfection performance of SW1353 cells mediated by hyperbranched poly(β-amino ester)s of different molecular weights (the molecular weights are 16.5 kg / mol, 20.1 kg / mol, 13.7 kg / mol and 16.1 kg / mol, respectively) in Examples 4 to 7 of the present invention. The flow cytometry results show that in SW1353 cells, four hyperbranched poly(β-amino esters) of different molecular weights can efficiently mediate mRNA transfection, with a transfection efficiency of 57%. These results once again confirm that the branching strategy can improve the gene transfection efficiency of poly(β-amino ester).

[0070] Fig.16 The cell activity evaluation after mRNA transfection mediated by hyperbranched poly(β-amino ester)s of different molecular weights in Examples 4 to 7 of the present invention (the molecular weights are 16.5 kg / mol, 20.1 kg / mol, 13.7 kg / mol and 16.1 kg / mol, respectively) in SW1353 cells was performed. The alarmBlue results showed that the survival rate of cells after transfection with four hyperbranched poly(β-amino ester)-based nanoparticles of different molecular weights exceeded 90%, with the highest reaching 100%, which was mainly due to the excellent biodegradability and biosafety of the composite nanoparticles, further confirming that the composite nanoparticles prepared by the present invention have broad commercial application potential.

[0071] Fig.17 Qualitative evaluation of the GFP DNA transfection performance mediated by hyperbranched poly(β-amino ester)s of different molecular weights (the molecular weights are 16.5 kg / mol, 20.1 kg / mol, 13.7 kg / mol and 16.1 kg / mol, respectively) in Examples 4 to 7 of the present invention (fluorescence results). Fig.17 It can be found that in COS-7 cells, four hyperbranched poly (β-amino esters) with different molecular weights showed excellent mRNA transfection efficiency, which was mainly due to the wider chemical space, multiple terminal groups and high-density amine sites of the polymers, thereby promoting gene transfection.

[0072] Fig.18 The present invention quantitatively evaluates the mRNA transfection performance mediated by hyperbranched poly(β-amino ester)s of different molecular weights (the molecular weights are 16.5 kg / mol, 20.1 kg / mol, 13.7 kg / mol and 16.1 kg / mol, respectively) in COS-7 cells. The flow cytometry results show that in COS-7 cells, the four hyperbranched poly(β-amino esters) of different molecular weights can efficiently mediate mRNA transfection, with a transfection efficiency of 70%. These results once again confirm that the branching strategy can improve the gene transfection efficiency of poly(β-amino ester).

[0073] Fig.19The cell activity evaluation was performed in COS-7 cells after mRNA transfection mediated by hyperbranched poly(β-amino ester)s of different molecular weights (molecular weights of 16.5 kg / mol, 20.1 kg / mol, 13.7 kg / mol and 16.1 kg / mol, respectively) in Examples 4 to 7 of the present invention. The alarmBlue results showed that the cell viability after transfection with the four hyperbranched poly(β-amino ester)-based nanoparticles of different molecular weights exceeded 90%, with the highest reaching 100%, which was mainly due to the excellent biodegradability and biosafety of the composite nanoparticles, further confirming that the composite nanoparticles prepared by the present invention have broad commercial application potential.

[0074] Fig. 20 Qualitative evaluation of the GFP DNA transfection performance mediated by hyperbranched poly(β-amino ester)s of different molecular weights (the molecular weights are 16.5 kg / mol, 20.1 kg / mol, 13.7 kg / mol and 16.1 kg / mol, respectively) in Examples 4 to 7 of the present invention (fluorescence results). Fig. 20 It can be found that in Vero cells, four hyperbranched poly (β-amino esters) with different molecular weights showed excellent mRNA transfection efficiency, which was mainly due to the wider chemical space, multiple terminal groups and high-density amine sites of the polymers, thereby promoting gene transfection.

[0075] Fig.21 The present invention quantitatively evaluates the mRNA transfection performance mediated by hyperbranched poly(β-amino ester)s of different molecular weights (the molecular weights are 16.5 kg / mol, 20.1 kg / mol, 13.7 kg / mol and 16.1 kg / mol, respectively) in Vero cells. The flow cytometry results show that in Vero cells, the four hyperbranched poly(β-amino esters) of different molecular weights can efficiently mediate mRNA transfection, with the highest transfection efficiency reaching 51%. These results once again confirm that the branching strategy can improve the gene transfection efficiency of poly(β-amino ester).

[0076] Fig. 22The cell activity evaluation after mRNA transfection mediated by hyperbranched poly(β-amino ester)s of different molecular weights (molecular weights of 16.5 kg / mol, 20.1 kg / mol, 13.7 kg / mol and 16.1 kg / mol, respectively) in Vero cells was performed. The alarmBlue results showed that the cell viability after transfection with four hyperbranched poly(β-amino ester)-based nanoparticles of different molecular weights exceeded 95%, with the highest reaching 99%, which was mainly due to the excellent biodegradability and biosafety of the composite nanoparticles, further confirming that the composite nanoparticles prepared by the present invention have broad commercial application potential.

[0077] Fig.23 For comparative example 1, under normal conditions, trimethylolpropane triacrylate (1 mmol) and 4-amino-1-butanol (2 mmol) were added to dimethyl sulfoxide (DMSO) (2 mL) and reacted at 90°C. The polymerization reaction gelled in a very short time, and the physical state of the polymer in the gel state. These results once again confirm the limitations of the traditional high molar ratio in the preparation of branched poly (β-amino esters), and also illustrate the significance of the present invention in the synthesis of branched poly (β-amino esters).

[0078] Fig.24 For comparative example 2, under normal conditions, trimethylolpropane triacrylate (1 mmol) and 5-amino-1-pentanol (3 mmol) were added to dimethyl sulfoxide (DMSO) (1.5 mL) and reacted at 90°C. The polymerization reaction gelled in a very short time, and the physical state of the polymer in the gel state. These results once again confirm the limitations of the traditional high molar ratio in the preparation of branched poly (β-amino esters), and also illustrate the significance of the present invention in the synthesis of branched poly (β-amino esters).

[0079] 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 hyperbranched poly(β-amino ester), characterized in that Its structural formula is as follows: ； In the formula, R1 is a group on the triacrylate monomer, R2 is a group on the organic amine, R3 is a group on the end-capping agent monomer, and n is the number of repeating units, which ranges from 10 to 45.

2. The hyperbranched poly (β-amino ester) according to claim 1, characterized in that The molecular weight of the hyperbranched poly(β-amino ester) is in the range of 5.0 kg / mol~40.0 kg / mol.

3. The hyperbranched poly (β-amino ester) according to claim 1, characterized in that The organic amine is 1-amino-3-propanol, 4-amino-1-butanol, 5-amino-1-pentanol, 6-amino-1-hexanol, butylamine, pentylamine, 1-(2-aminoethyl)piperazine, N-(2-aminoethyl)-morpholine, dodecylamine or octadecylamine.

4. The hyperbranched poly (β-amino ester) according to claim 1, characterized in that The triacrylate monomers are trimethylolpropane triacrylate and ethoxylated trimethylolpropane triacrylate, and the molecular weights thereof are 428, 692, and 912, respectively.

5. The hyperbranched poly (β-amino ester) according to claim 1, characterized in that The 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, 1,4-bis(3-aminopropyl)piperazine, 1,11-diamino-3,6,9-trioxaundecane, 2-methyl-1,5-pentanediamine, 1,2-ethylenediamine, 1,3-propylenediamine, 1,4-butylenediamine, and 1,6-hexanediamine.

6. The method for preparing a hyperbranched poly(β-amino ester) according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: 1) dissolving triacrylate monomers and organic amines in DMSO, and performing A2+B3 Michael addition reaction to obtain hyperbranched poly(β-amino ester) with a vinyl group at the end; 2) reacting the hyperbranched poly(β-amino ester) having a vinyl group at the end obtained in step 1) with a capping agent to obtain a hyperbranched poly(β-amino ester) having an amine group at the end; in step 1), the reaction molar ratio of the triacrylate monomer to the organic amine is 1:(0.4-1.8), and the reaction is carried out at 60° C.-80° C.; in step 2), the reaction molar ratio of the capping agent to the hyperbranched poly(β-amino ester) having a vinyl group at the end is (1-5):1, and the reaction is carried out at room temperature.

7. The composite nanoparticles are nanoparticles obtained by complexing the hyperbranched poly (β-amino ester) according to any one of claims 1 to 6 with DNA or mRNA.

8. Use of the hyperbranched poly (β-amino ester) according to any one of claims 1 to 6 or the composite nanoparticle according to claim 7 in DNA or mRNA delivery, characterized in that: The hyperbranched poly(β-amino ester) is used to deliver DNA or mRNA to tissues or cells.

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