Star-shaped poly(β-amino ester) having polyamine as core, and preparation methods therefor and use thereof, and composite nanoparticle based on star-shaped poly(β-amino ester), and preparation methods therefor and use thereof

Abstract

Provided in the present invention are a star-shaped poly(β-amino ester) having a polyamine as a core, and a preparation method therefor and the use thereof, and a composite nanoparticle based on the star-shaped poly(β-amino ester) having a polyamine as a core, and a preparation method therefor and the use thereof. In the present invention, by using linear low-molecular-weight poly(β-amino ester) as arms and diamine and polyamine monomers as core molecules, a class of star-shaped poly(β-amino esters) having a new structure is obtained. The composite nanoparticle formed by the star-shaped poly(β-amino ester) having a polyamine as the core and DNA has uniform distribution, good stability, biodegradability and good biocompatibility. In-vitro transfection results show that the composite nanoparticle formed by the star-shaped poly(β-amino ester) having a polyamine as the core and DNA can efficiently deliver DNA in multiple tissue cell lines. More importantly, the transfection results show that the star-shaped poly(β-amino ester) having a polyamine as the core can also efficiently deliver mRNA and siRNA. These results prove the application potential of the star-shaped poly(β-amino ester) having a polyamine as the core in the delivery of various nucleic acid molecules, which provides an application basis for further gene therapy of genetic diseases and cancers.

Description

Star-shaped poly(β-amino ester) with polyamine as core, composite nanoparticles based thereon, and preparation method and application thereof Technical Field

[0001] The present invention belongs to the field of biomedical materials and relates to a star-shaped poly (β-amino ester) with a polyamine as a core, composite nanoparticles based thereon, and a preparation method and application thereof. Background Art

[0002] Gene therapy, such as DNA and RNA, has shown promising applications in cancer treatment, genetic disease therapy, and vaccine development. However, naked DNA and RNA have difficulty crossing negatively charged and hydrophobic cell membranes and are easily degraded by nucleases and restriction enzymes in the blood or cytoplasm. Therefore, gene therapy requires the use of vectors to encapsulate and protect DNA and RNA. However, the lack of safe and efficient gene vectors has severely limited the clinical application of gene therapy.

[0003] Currently, liposome nanoparticles are one of the most promising carriers for gene delivery, but their clinical application is limited by their high cost, poor serum tolerance, poor stability, complex preparation process, and complex composition. Cationic polymers, including polyethyleneimine (PEI), polydimethylaminoethyl methacrylate (PDMAEMA), and chitosan, have attracted widespread attention due to their many advantages, including a wide range of raw material sources, flexible and diverse chemical compositions, easily adjustable topology, high gene loading efficiency, and good serum tolerance and stability. Although cationic polymer carriers have shown certain application potential, they are prone to high cytotoxicity due to their poor degradation properties and are often composed of a relatively single monomer type.

[0004] Due to their three-dimensional structure, multiple terminal groups, and wider chemical modification space, branched poly(p-amino esters) have high DNA loading efficiency. The resulting composite nanoparticles, formed after DNA compression, have stronger interactions with cells and have shown broad application prospects in DNA delivery. However, current research is often limited to the branched polymers themselves, while the biophysical properties of star-shaped poly(β-amino esters) and their composite nanoparticles with genes and their effects on the transfection efficiency of functional nucleic acid molecules have not been explored, which will greatly limit their application research in gene delivery and gene therapy. 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 star-shaped poly (β-amino ester) with a polyamine as a core, composite nanoparticles based thereon, and a preparation method and application thereof.

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

[0007] A star-shaped poly(β-amino ester) with a polyamine as a core has a structural formula as shown in Formula 1, Formula 2 or Formula 3:

[0008] Wherein, R1 is a group on a diacrylate monomer; the diacrylate monomer is R2 is a group on an organic amine, which is R2-NH2; R3 is a group on a polyamine monomer; R4 is a group on a functionalized end-capping agent monomer, which is R4-NH2; and n=15-55.

[0009] Preferably, the diacrylate monomer is 1,6-hexanediol diacrylate, 1,4-butanediol diacrylate, tetraethylene glycol diacrylate, diethylene glycol diacrylate, 2,2-dithiodiethanol diacrylate, bisphenol A polyoxyethylene ether diacrylate, 1,1′-isopropylidenebis(p-phenyleneoxy)di-2-propanol diacrylate or.

[0010] Preferably, the organic amine is 4-amino-1-butanol, 5-amino-1-pentanol, propylamine, butylamine, hexylamine, octylamine, 1-(2-aminoethyl)piperazine, N-(2-aminoethyl)-morpholine, dodecylamine or octadecylamine.

[0011] Preferably, the polyamine monomer is tris(2-aminoethyl)amine, diethylenetriamine, bis(3-aminopropyl)amine, 1,4-butanediamine, 1,2-ethylenediamine, 1,3-propylenediamine, triethylenetetramine or 1,6-hexanediamine.

[0012] 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, 1,4-bis(3-aminopropyl)piperazine, 1,11-diamino-3,6,9-trioxaundecane or 2-methyl-1,5-pentanediamine.

[0013] The method for preparing the star-shaped poly(β-amino ester) with polyamine as the core comprises the following steps:

[0014] 1) reacting a diacrylate monomer with an organic amine via Michael addition reaction to obtain a linear poly(β-amino ester) with a double bond at the end;

[0015] 2) reacting the linear poly(β-amino ester) with a double bond at the end obtained in step 1) with a polyamine monomer to obtain a star-shaped poly(β-amino ester) with a double bond at the end and a polyamine as a core;

[0016] 3) reacting the star-shaped poly(β-amino ester) with a polyamine core and a double bond at the end obtained in step 2) with a functionalized end-capping agent monomer to perform a functional end-capping reaction to obtain a star-shaped poly(β-amino ester) with a polyamine core.

[0017] Preferably, in step 1), the molar ratio of the diacrylate monomer to the organic amine is (1-5):(0.5-2); in step 2), the molar ratio of the polyamine monomer to the linear poly(β-amino ester) with a double bond at the end is (0.01-1):(1-5).

[0018] The invention discloses a star-shaped poly (beta-amino ester)-based composite nanoparticle with polyamine as core, which is obtained by compounding the star-shaped poly (beta-amino ester) with polyamine as core with DNA.

[0019] The method for preparing the star-shaped poly(β-amino ester)-based composite nanoparticles with polyamine as the core comprises: mixing the solution of the star-shaped poly(β-amino ester) with polyamine as the core with a DNA solution, stirring, and standing to obtain the composite nanoparticles.

[0020] The invention relates to an application of the star-shaped poly(β-amino ester) with polyamine as the core or the star-shaped poly(β-amino ester)-based composite nanoparticle with polyamine as the core in the preparation of a gene carrier for delivering genes to tissues or cells.

[0021] The star-shaped poly(β-amino ester) with a polyamine core or the star-shaped poly(β-amino ester)-based composite nanoparticles with a polyamine core are used in the preparation of nucleic acid molecule carriers. The carriers can be used for mRNA and siRNA delivery, and delivery of nucleic acid molecules to tissues or cells.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention uses linear low molecular weight poly (β-amino ester) as arms and diamine and polyamine monomers as core molecules to obtain a type of star-shaped poly (β-amino ester) with a completely new structure. The composite nanoparticles formed by the star-shaped poly (β-amino ester) with polyamine as the core and DNA are relatively uniformly distributed, have good stability, are biodegradable and have good biocompatibility. The results of in vitro transfection show that the composite nanoparticles formed by the star-shaped poly (β-amino ester) with polyamine as the core and DNA can efficiently deliver DNA in a variety of tissue cell lines, which provides an application basis for the further development of genetic disease and cancer gene therapy. Compared with the commercial cationic polymer transfection reagents mainly used in the current technology in this field, the composite nanoparticles formed by the star-shaped poly (β-amino ester) with polyamine as the core and DNA disclosed in the present invention have higher DNA transfection efficiency and greater clinical application potential.

[0024] The present invention utilizes a star-shaped poly(β-amino ester) with a polyamine core to electrostatically interact with DNA to form composite nanoparticles. By optimizing the chemical composition of the star-shaped poly(β-amino ester) with a polyamine core and the mass ratio of DNA, a series of composite nanoparticles were prepared. These composite nanoparticles exhibit high DNA compression efficiency, small size, high uniformity, high positive surface potential, and a clear spherical microscopic morphology, indicating that the composite nanoparticles have excellent stability. This method for preparing nanoparticles is low-cost, simple in preparation, and requires minimal equipment, making it suitable for industrial production.

[0025] The present invention's star-shaped poly(β-amino ester)-DNA composite nanoparticles, which are based on polyamine cores, are able to efficiently mediate GFP and NOD2 gene transfection in a variety of tissue cells, including hepatocytes (HepG2, HCC-LM3, MHCC-97H, BRL-3A), ovarian cells (A2780), kidney cells (Vero), cervical cells (HeLa), chondrocytes (SW1353), as well as difficult-to-transfect skin cells (HaCaT and NHF), and macrophage RAW264.7 cells. These results demonstrate the highly flexible nature of the polyamine-core star-shaped poly(β-amino ester)-DNA composite nanoparticles, which can be used for simultaneous delivery of two different DNA sizes, further validating the broad applicability of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the GPC curve of the star-shaped poly(β-amino ester) with polyamine as the core in Examples 1 to 8; their weight-average molecular weights are 12.9 kg / mol, 15.5 kg / mol, 11.0 kg / mol, 10.1 kg / mol, 14.9 kg / mol, 19.9 kg / mol, 11.1 kg / mol, and 8.7 kg / mol, respectively.

[0027] Figure 2 shows the weight average molecular weights of star-shaped poly (β-amino ester) with polyamine as the core in Examples 1 to 8, which are 12.9 kg / mol, 15.5 kg / mol, 11.0 kg / mol, 10.1 kg / mol, 14.9 kg / mol, 19.9 kg / mol, 11.1 kg / mol, and 8.7 kg / mol, respectively. 1 HNMR spectrum.

[0028] Figure 3 shows the microscopic morphology of star-shaped poly(β-amino ester) and DNA composite nanoparticles with polyamine as the core in Examples 1 and 2; TEM results show that the two nanoparticles can be evenly and stably distributed under physiological conditions, providing conditions for subsequent gene delivery.

[0029] Figure 4 shows the DNA affinity performance test of the star-shaped poly(β-amino ester) and DNA composite nanoparticles with polyamine as the core in Examples 1 to 8; the PicoGreen test results show that at mass ratios of 20:1, 40:1, and 60:1, the eight star-shaped poly(β-amino ester)s exhibit excellent DNA affinity performance, and confirm that the star-shaped poly(β-amino ester) and DNA composite nanoparticles with polyamine as the core have excellent stability, providing conditions for subsequent gene delivery.

[0030] Figure 5 shows the particle size characterization of star-shaped poly(β-amino ester) and DNA composite nanoparticles with polyamine as the core in Examples 1 to 8; the DLS results show that at mass ratios of 20:1, 40:1, and 60:1, the 24 star-shaped poly(β-amino ester) and DNA composite nanoparticles have smaller sizes, and it is confirmed that the star-shaped poly(β-amino ester) and DNA composite nanoparticles with polyamine as the core have excellent stability, providing conditions for subsequent gene delivery.

[0031] Figure 6 shows the particle size distribution test of the star-shaped poly(β-amino ester) and DNA composite nanoparticles with polyamine as the core prepared in Example 1 and Example 2; the star-shaped poly(β-amino ester) and DNA composite nanoparticles have a narrow particle size distribution, which once again confirms that the star-shaped poly(β-amino ester) and DNA composite nanoparticles with polyamine as the core have excellent stability, providing conditions for subsequent gene delivery.

[0032] Figure 7 shows the zeta test of the potential of the star-shaped poly(β-amino ester) and DNA composite nanoparticles with polyamine as the core in Examples 1 to 8; the DLS results show that at mass ratios of 20:1, 40:1, and 60:1, the 24 star-shaped poly(β-amino ester) and DNA composite nanoparticles have a high positive charge, further promoting the cellular uptake of the nanoparticles.

[0033] Figure 8 shows fluorescence images of cells transfected with DNA encoding green fluorescent protein (GFP) using composite nanoparticles in different cells: (a) Fluorescence images of cells transfected with DNA encoding green fluorescent protein (GFP) using the star-shaped poly(β-amino ester) and DNA composite nanoparticles with polyamine as the core in HeLa cells. The transfection results show that all eight star-shaped poly(β-amino ester) and DNA composite nanoparticles can efficiently mediate GFP gene transfection; (b) Fluorescence images of cells transfected with GFP DNA using the star-shaped poly(β-amino ester) and DNA composite nanoparticles with polyamine as the core in SW1353 cells. The transfection results show that all eight star-shaped poly(β-amino ester) and DNA composite nanoparticles can efficiently mediate GFP gene transfection.

[0034] Figure 9 shows the quantitative transfection results of star-shaped poly(β-amino ester) and DNA composite nanoparticles with polyamine as the core in HeLa cells after transfection of GFP DNA by flow cytometry. The transfection results show that all eight star-shaped poly(β-amino ester) and DNA composite nanoparticles can efficiently mediate GFP gene transfection, among which the transfection efficiency of the nanoparticles in Examples 1 to 8 exceeds 90%.

[0035] Figure 10 shows fluorescence images of cells transfected with DNA encoding green fluorescent protein (GFP) using polyamine-based star-shaped poly(β-amino ester) nanoparticles in hepatocytes (HepG2, HCC-LM3, MHCC-97H, BRL-3A), ovarian cells (A2780), kidney cells (Vero), and difficult-to-transfect skin cells (HaCaT and NHF). The transfection results demonstrate that star-shaped poly(β-amino ester) nanoparticles are highly efficient in translating GFP into all eight cell types, preliminarily demonstrating their broad applicability in gene delivery.

[0036] Figure 11 shows the transfection results of composite nanoparticles in different cells after GFP DNA was transfected by flow cytometry; (a) shows the transfection results of composite nanoparticles with polyamine as core and DNA in Example 1 after GFP DNA was transfected by flow cytometry in liver cells (HepG2, HCC-LM3, MHCC-97H, BRL-3A); the transfection results show that the composite nanoparticles with polyamine as core and DNA can efficiently mediate GFP gene transfection in the four types of cells, preliminarily confirming its wide applicability in gene delivery; (b) shows the transfection of composite nanoparticles with polyamine as core and DNA in Example 1 into ovarian cells (A2780), kidney cells (Vero) and skin cells (HaCaT and NHF) that are difficult to transfect. The transfection results were quantitatively analyzed by flow cytometry after DNA addition; the transfection results showed that in four types of cells, the star-shaped poly (β-amino ester) and DNA composite nanoparticles with polyamine as the core were able to efficiently mediate GFP gene transfection, preliminarily confirming its wide applicability in gene delivery.

[0037] Figure 12 shows the protein blotting results of the polyamine-core star-shaped poly(β-amino ester)-DNA composite nanoparticles in Example 1 after NOD2 DNA transfection in RAW264.7 macrophage cells. The transfection results show that the polyamine-core star-shaped poly(β-amino ester)-based nanoparticles can efficiently mediate NOD2 DNA transfection in RAW264.7 cells, preliminarily confirming their application potential in macrophage transfection and cancer immunotherapy.

[0038] Figure 13 is a fluorescence photograph of cells after transfection of green fluorescent protein (GFP) mRNA in COS-7 cells mediated by star-shaped poly(β-amino ester) with polyamine as the core in Examples 1 to 8: The transfection results show that the eight star-shaped poly(β-amino ester) and mRNA composite nanoparticles with polyamine as the core can efficiently mediate GFP mRNA transfection in COS-7 cells, confirming their excellent mRNA delivery performance.

[0039] Figure 14 shows a quantitative evaluation of the performance of the star-shaped poly(β-amino ester)s with polyamine cores in Examples 1 to 8 in mediating transfection of mRNA encoding green fluorescent protein (GFP) in COS-7 cells. Flow cytometry results demonstrate that all eight star-shaped poly(β-amino ester)s with polyamine cores efficiently mediated GFP mRNA transfection in COS-7 cells, with transfection efficiencies reaching 83.8%, demonstrating their excellent mRNA delivery performance.

[0040] Figure 15 shows a qualitative evaluation of the siRNA transfection performance of the star-shaped poly(β-amino ester)s with polyamine cores from Examples 1 to 8 in HeLa cells stably expressing GFP. The transfection results demonstrate that all eight star-shaped poly(β-amino ester)s with polyamine cores are highly efficient in siRNA transfection, with a significant decrease in cell fluorescence intensity following transfection (higher transfection efficiency indicates better siRNA interference, and weaker fluorescence intensity indicates weaker fluorescence intensity), demonstrating their excellent siRNA delivery performance.

[0041] Figure 16 is a quantitative evaluation of the performance of the star-shaped poly(β-amino ester) with a polyamine core in Examples 1 to 8 after mediating siRNA transfection in HeLa cells stably transfected with GFP. Flow cytometry results show that in HeLa cells stably transfected with GFP, the eight star-shaped poly(β-amino ester) with a polyamine core can efficiently mediate siRNA transfection, with a GFP knockdown efficiency of 95% after transfection, confirming their excellent siRNA delivery performance. These results preliminarily confirm the application potential of star-shaped poly(β-amino ester) with a polyamine core in the delivery of various nucleic acid molecules. DETAILED DESCRIPTION

[0042] In order to further understand the present invention, the present invention is described below in conjunction with embodiments. These descriptions are only for further explaining the features and advantages of the present invention and are not intended to limit the claims of the present invention.

[0043] The star-shaped poly(β-amino ester) with a polyamine as a core of the present invention has a structural formula as shown in Formula 1, Formula 2 or Formula 3 below:

[0044] Wherein, R1 is a group on a diacrylate monomer; the diacrylate monomer is R2 is a group on an organic amine, which is R2-NH2; R3 is a group on a polyamine monomer, specifically a methyl group, an ethyl group, a single bond, or an n-butyl group; R4 is a group on a functionalized end-capping agent monomer, which is R4-NH2, and n=15 to 55.

[0045] The diacrylate monomers of the present invention are 1,6-hexanediol diacrylate, 1,4-butanediol diacrylate, tetraethylene glycol diacrylate, diethylene glycol diacrylate, 2,2-dithiodiethanol diacrylate, bisphenol A polyoxyethylene ether diacrylate, and 1,1′-isopropylidene bis(p-phenyleneoxy)di-2-propanol diacrylate, and their structural formulas are shown below:

[0046] The organic amine of the present invention is 4-amino-1-butanol, 5-amino-1-pentanol, propylamine, butylamine, hexylamine, octylamine, 1-(2-aminoethyl)piperazine, N-(2-aminoethyl)-morpholine, dodecylamine or octadecylamine, and their structural formulas are shown below:

[0047] The polyamine monomer of the present invention is tris(2-aminoethyl)amine, diethylenetriamine, bis(3-aminopropyl)amine, 1,4-butanediamine, 1,2-ethylenediamine, 1,3-propylenediamine, triethylenetetramine or 1,6-hexanediamine, and their structural formulas are shown below:

[0048] The functional end-capping agent monomers described in the present invention are 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 or 2-methyl-1,5-pentanediamine, and their structural formulas are shown below:

[0049] The method for preparing a star-shaped poly(β-amino ester) with a polyamine as a core of the present invention comprises the following steps:

[0050] 1) reacting a diacrylate monomer with an organic amine via Michael addition reaction to obtain a linear poly(β-amino ester) with a double bond at the end;

[0051] 2) reacting the linear poly(β-amino ester) with a terminal double bond obtained in step 1) with a polyamine monomer (core molecule) to obtain a star-shaped poly(β-amino ester) with a terminal double bond-containing polyamine as a core;

[0052] 3) reacting the star-shaped poly(β-amino ester) having a double-bond-terminated polyamine as a core obtained in step 2) with a functionalized end-capping agent monomer to perform a functionalized end-capping reaction to obtain a star-shaped poly(β-amino ester) having a high-performance functionalized polyamine as a core;

[0053] Specifically, the method includes the following steps:

[0054] 1) adding a certain amount of diacrylate monomer and organic amine to a solvent (such as dimethyl sulfoxide or tetrahydrofuran) and fully dissolving them by magnetic stirring, wherein the molar ratio of the diacrylate monomer to the organic amine is (1-5):(0.5-2); wherein the reaction is carried out at 30° C. to 90° C. under a nitrogen atmosphere to obtain a linear poly(β-amino ester) with a terminal double bond;

[0055] During the reaction, GPC was used to monitor the polymer molecular weight. When the polymer molecular weight reached a preset range of 2.0 to 5.0 kg / mol, the reaction was terminated.

[0056] 2) adding a certain amount of low molecular weight linear poly(β-amino ester) with a double bond at the end and a polyamine monomer (core molecule) into a solvent and fully dissolving the monomers by magnetic stirring, wherein the molar ratio of the polyamine monomer to the linear poly(β-amino ester) with a double bond at the end is (0.01-1):(1-5), and reacting at 30° C. to 90° C. to obtain a star-shaped poly(β-amino ester) with a polyamine containing a double bond at the end as the core.

[0057] The polymer molecular weight was monitored using GPC spectroscopy, and the reaction was terminated when the polymer molecular weight approached the set value within the range of 7,000 to 60,000 kg / mol.

[0058] 3) adding a certain amount of functionalized end-capping agent monomer to the reaction system to react with the star-shaped poly(β-amino ester) having a polyamine containing a double bond at the end for end-capping, wherein the molar ratio of the functionalized end-capping agent monomer to the star-shaped poly(β-amino ester) having a polyamine containing a double bond at the end is (1-5):(0.25-2). After the reaction is completed, the product is purified by a precipitation method and freeze-dried for 48h to 72h.

[0059] The method for preparing the star-shaped poly(β-amino ester)-based nanoparticles with polyamine as the core is as follows:

[0060] The star-shaped poly(β-amino ester) prepared by the present invention with a polyamine core was combined with DNA of various sizes to form a series of uniform and stable nanoscale spherical particles through charge interaction. The DNA affinity, the particle size, zeta potential and microscopic morphology of the composite nanoparticles formed by the compressed DNA were tested as follows:

[0061] A certain amount of a polyamine-based star-shaped poly(β-amino ester) solution was quickly added to a DNA solution, vortexed at high speed for 15-60 seconds, and allowed to stand for 5-30 minutes. The mass ratio of the polyamine-based star-shaped poly(β-amino ester) to DNA was (10-100):1. PicoGreen working solution was then added to measure fluorescence intensity, and DNA affinity efficiency was calculated. A similar method was used to prepare composite nanoparticles. After standing for 5-30 minutes, nanoparticles were formed. 1 mL of deionized water was added, and dynamic light scattering (DLS) was used for analysis. Composite nanoparticles were also prepared using the same method. The inorganic salts were washed with deionized water, centrifuged at high speed for 5 minutes, and freeze-dried. The micromorphology of the composite nanoparticles was observed using transmission electron microscopy (TEM).

[0062] The star-shaped poly(β-amino ester) prepared by the present invention with a polyamine core was transfected with DNA encoding green fluorescent protein, and the cytotoxicity was tested as follows:

[0063] 1) Cell culture: HepG2, HCC-LM3, MHCC-97H, BRL-3A, A2780, Vero, HaCaT and NHF cells were cultured at 1.0×10 4 ~2.0×10 4 Cells were seeded at a density of 10 cells / well in 96-well plates and cultured at 37°C overnight.

[0064] 2) A certain amount of star-shaped poly(β-amino ester) with a polyamine core is mixed into a GFP DNA or NOD2 DNA solution, allowed to stand for 5 to 40 minutes, and then added to the cells.

[0065] 3) 12 to 72 hours after transfection, observe and photograph the cells transfected with GFP DNA under a fluorescence microscope.

[0066] 4) 48 h after transfection, the efficiency of GFP DNA transfection was tested using flow cytometry.

[0067] 5) 48 h after transfection, the cell supernatant was removed, and the NOD2 protein expression efficiency was detected by western blotting.

[0068] The star-shaped poly(β-amino ester) prepared by the present invention with a polyamine core is transfected with mRNA or siRNA encoding green fluorescent protein, and the specific method is as follows:

[0069] 1) Cell culture: COS-7 cells or HeLa cells stably transfected with GFP (cells emit green light due to the presence of GFP gene) were cultured at 1.0×10 4 ~2.0×10 4 Cells were seeded at a density of 10 cells / well in 96-well plates and cultured overnight at 37°C.

[0070] 2) A certain amount of star-shaped poly(β-amino ester) with a polyamine core is mixed into a GFP mRNA or siRNA solution, allowed to stand for 5 to 150 minutes, and then added to the cells.

[0071] 3) 48 hours after transfection, the transfected cells were observed and photographed under a fluorescence microscope.

[0072] 4) 48 h after transfection, the efficiency of GFP mRNA or siRNA transfection was tested using flow cytometry.

[0073] Specific embodiment:

[0074] Example 1

[0075] 1,4-Butanediol diacrylate (24 mmol, 4.752 g) and 5-amino-1-pentanol (20 mmol, 2.06 g) were dissolved in dimethyl sulfoxide (DMSO) (4 mL) and reacted at 90°C for 10 h. After the system reached room temperature, DMSO was added to terminate the reaction, synthesizing a linear poly(β-amino ester) with a terminal double bond. Subsequently, the linear poly(β-amino ester) with a terminal double bond and diethylenetriamine were dissolved in the organic solvent DMSO, where the concentration of the linear poly(β-amino ester) with a terminal double bond was 300 mg / mL. Polymerization was carried out at 60°C under magnetic stirring. The molecular weight of the polymer was determined by GPC. An excess of N-(3-aminopropyl)morpholine was then added for end-capping. Finally, the product was precipitated in an excess of diethyl ether and freeze-dried to obtain a star-shaped poly(β-amino ester) with a polyamine core and a weight-average molecular weight of 12.9 kg / mol.

[0076] Figure 1 shows the GPC curve of SPAE-1, a star-shaped poly(β-amino ester) with a molecular weight of 12.9 kg / mol and a polyamine core, prepared in this example, after purification, confirming the successful synthesis of the star-shaped poly(β-amino ester) with a polyamine core. Figure 2 shows the H-NMR spectrum of the star-shaped poly(β-amino ester) with a molecular weight of 12.9 kg / mol and a polyamine core, prepared in this example, after purification, further confirming the successful synthesis of the star-shaped poly(β-amino ester) with a polyamine core.

[0077] Example 2

[0078] 1,6-Hexanediol diacrylate (24 mmol, 5.42 g) and 5-amino-1-pentanol (20 mmol, 2.06 g) were dissolved in dimethyl sulfoxide (DMSO) (13.06 mL) and reacted at 60°C for 8 h. Dimethyl sulfoxide (DMOS) was added (at a concentration of 10% in the total reaction system) and reacted at room temperature for 24 h to synthesize a linear poly(β-amino ester) with a terminal double bond. Subsequently, the linear poly(β-amino ester) with a terminal double bond and 1,4-butanediamine were dissolved in the organic solvent THF to a concentration of 200 mg / mL of the linear poly(β-amino ester) with a terminal double bond. 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. Finally, the product was precipitated in excess ether and freeze-dried to obtain a star-shaped poly(β-amino ester) with a polyamine as the core, whose molecular weight was 15.5 kg / mol.

[0079] Figure 1 shows the GPC curve of SPAE-2, a star-shaped poly(β-amino ester) with a molecular weight of 15.5 kg / mol and a polyamine core, prepared in this example. This curve confirms the successful synthesis of the star-shaped poly(β-amino ester) with a polyamine core. Figure 2 shows the purified H-NMR spectrum of the star-shaped poly(β-amino ester) with a molecular weight of 15.5 kg / mol and a polyamine core, prepared in this example. The characteristic peaks in the H-NMR spectrum further confirm the successful synthesis of the polymer.

[0080] Example 3

[0081] 1,4-Butanediol diacrylate (24 mmol, 4.752 g) and 5-amino-1-pentanol (20 mmol, 2.06 g) were dissolved in dimethyl sulfoxide (DMSO) (13.06 mL) and reacted at 90°C for 5 h. Dimethyl sulfoxide (DMOS) was added (at a concentration of 10% in the total reaction system) and reacted at room temperature for 24 h to synthesize a linear poly(β-amino ester) with a terminal double bond. Subsequently, the linear poly(β-amino ester) with a terminal double bond and tris(2-aminoethyl)amine were dissolved in the organic solvent DMSO to a concentration of 200 mg / mL of the linear poly(β-amino ester) with a terminal double bond. The polymerization reaction was carried out at 90°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-trioxaundecane was added for end-capping reaction. Finally, the product was precipitated in excess ether and freeze-dried to obtain a star-shaped poly(β-amino ester) with a polyamine as the core, whose molecular weight was 11.0 kg / mol.

[0082] Figure 1 shows the GPC curve of SPAE-3, a star-shaped poly(β-amino ester) with a molecular weight of 11.0 kg / mol and a polyamine core, prepared in this example. This curve confirms the successful synthesis of the star-shaped poly(β-amino ester) with a polyamine core. Figure 2 shows the H-NMR spectrum of the purified star-shaped poly(β-amino ester) with a molecular weight of 11.0 kg / mol and a polyamine core, prepared in this example. The characteristic peaks in the H-NMR spectrum further confirm the successful synthesis of the polymer.

[0083] Example 4

[0084] 1,4-Butanediol diacrylate (24 mmol, 4.752 g) and 4-amino-1-butanol (20 mmol, 2.06 g) were dissolved in dimethyl sulfoxide (DMSO) (13.06 mL) and reacted at 50°C for 10 h. Dimethyl sulfoxide (DMOS) was added (at a concentration of 10% in the total reaction system) and reacted at room temperature for 24 h to synthesize a linear poly(β-amino ester) with a terminal double bond. Subsequently, the linear poly(β-amino ester) with a terminal double bond and 1,4-butanediamine were dissolved in the organic solvent DMSO to a concentration of 400 mg / mL of the linear poly(β-amino ester) with a terminal double bond. 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-(2-aminoethyl)morpholine was added for end-capping reaction. Finally, the product was precipitated in excess ether and freeze-dried to obtain a star-shaped poly(β-amino ester) with a polyamine as the core, whose molecular weight was 10.1 kg / mol.

[0085] Figure 1 shows the GPC curve of SPAE-4, a star-shaped poly(β-amino ester) with a molecular weight of 10.1 kg / mol and a polyamine core, prepared in this example. This curve confirms the successful synthesis of the star-shaped poly(β-amino ester) with a polyamine core. Figure 2 shows the H-NMR spectrum of the purified star-shaped poly(β-amino ester) with a molecular weight of 10.1 kg / mol and a polyamine core, prepared in this example. The characteristic peaks in the H-NMR spectrum further confirm the successful synthesis of the polymer.

[0086] Example 5

[0087] 1,4-Butanediol diacrylate (24 mmol, 4.752 g) and 4-amino-1-butanol (20 mmol, 1.78 g) were dissolved in dimethyl sulfoxide (DMSO) (13.06 mL) and reacted at 90°C for 7 h. Dimethyl sulfoxide (DMOS) was added (at a concentration of 10% in the total reaction system) and reacted at room temperature for 24 h to synthesize a linear poly(β-amino ester) with a terminal double bond. Subsequently, the linear poly(β-amino ester) with a terminal double bond and 1,2-butanediamine were dissolved in the organic solvent DMSO, where the concentration of the linear poly(β-amino ester) with a terminal double bond was 200 mg / mL. The polymerization reaction was carried out at 90°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-trioxaundecane was added for end-capping reaction. Finally, the product was precipitated in excess ether and freeze-dried to obtain a star-shaped poly(β-amino ester) with a polyamine as the core, whose molecular weight was 14.9 kg / mol.

[0088] Figure 1 shows the GPC curve of SPAE-5, a star-shaped poly(β-amino ester) with a molecular weight of 14.9 kg / mol and a polyamine core, prepared in this example, confirming the successful synthesis of the star-shaped poly(β-amino ester) with a polyamine core. Figure 2 shows the H-NMR spectrum of SPAE-5, a star-shaped poly(β-amino ester) with a molecular weight of 14.9 kg / mol and a polyamine core, prepared in this example, after purification. The characteristic peaks in the H-NMR spectrum further confirm the successful synthesis of the polymer.

[0089] Example 6

[0090] 1,4-Butanediol diacrylate (24 mmol, 4.752 g) and 4-amino-1-butanol (20 mmol, 1.78 g) were dissolved in dimethyl sulfoxide (DMSO) (13.06 mL) and reacted at 80°C for 6 h. Dimethyl sulfoxide (DMOS) was added (at a concentration of 10% in the total reaction system) and reacted at room temperature for 24 h to synthesize a linear poly(β-amino ester) with a terminal double bond. Subsequently, the linear poly(β-amino ester) with a terminal double bond and tris(2-aminoethyl)amine were dissolved in the organic solvent DMSO, where the concentration of the linear poly(β-amino ester) with a terminal double bond was 300 mg / mL. The polymerization reaction was carried out at 90°C under magnetic stirring, and the molecular weight of the polymer was detected by GPC. Then, an excess of N-(2-aminoethyl)morpholine was added for end-capping reaction. Finally, the product was precipitated in excess ether and freeze-dried to obtain a star-shaped poly(β-amino ester) with a polyamine as the core, whose molecular weight was 19.9 kg / mol.

[0091] Figure 1 shows the GPC curve of SPAE-6, a star-shaped poly(β-amino ester) with a molecular weight of 19.9 kg / mol and a polyamine core, prepared in this example. This confirms the successful synthesis of the star-shaped poly(β-amino ester) with a polyamine core. Figure 1 shows the H-NMR spectrum of the purified star-shaped poly(β-amino ester) with a molecular weight of 19.9 kg / mol and a polyamine core, prepared in this example. The characteristic peaks in the H-NMR spectrum further confirm the successful synthesis of the polymer.

[0092] Example 7

[0093] 1,4-Butanediol diacrylate (24 mmol, 4.752 g) and dodecylamine (20 mmol, 3.7 g) were dissolved in dimethyl sulfoxide (DMSO) (13.06 mL) and reacted at 60°C for 6 h. Dimethyl sulfoxide (DMOS) was added (at a concentration of 10% in the total reaction system) and reacted at room temperature for 24 h to synthesize a linear poly(β-amino ester) with a terminal double bond. Subsequently, the linear poly(β-amino ester) with a terminal double bond (6.5 g) and tris(2-aminoethyl)amine (0.24 g) were dissolved in the organic solvent DMSO to a concentration of 500 mg / mL of the linear poly(β-amino ester) with a terminal double bond. The polymerization reaction was carried out at 90°C under magnetic stirring, and the molecular weight of the polymer was detected by GPC. Then, an excess of N-(2-aminoethyl)morpholine was added for end-capping reaction. Finally, the product was precipitated in excess ether and freeze-dried to obtain a star-shaped poly(β-amino ester) with a polyamine as the core, whose molecular weight was 11.1 kg / mol.

[0094] Figure 1 shows the GPC curve of SPAE-7, a star-shaped poly(β-amino ester) with a molecular weight of 11.1 kg / mol and a polyamine core, prepared in this example. This confirms the successful synthesis of the star-shaped poly(β-amino ester) with a polyamine core. Figure 2 shows the H-NMR spectrum of SPAE-7, a star-shaped poly(β-amino ester) with a molecular weight of 11.1 kg / mol and a polyamine core, prepared in this example, after purification. The characteristic peaks in the H-NMR spectrum further confirm the successful synthesis of the polymer.

[0095] Example 8

[0096] Diethylene glycol diacrylate (24 mmol, 5.136 g) and 4-amino-1-butanol (20 mmol, 1.78 g) were dissolved in dimethyl sulfoxide (DMSO) (13.06 mL) and reacted at 90°C for 6 h. Dimethyl sulfoxide (DMOS) (10% concentration in the total reaction system) was added and reacted at room temperature for 24 h to synthesize a linear poly(β-amino ester) with a terminal double bond. Subsequently, the linear poly(β-amino ester) with a terminal double bond and 1,2-ethylenediamine were dissolved in the organic solvent DMSO to a concentration of 200 mg / mL. Polymerization was carried out at 90°C under magnetic stirring. The molecular weight of the polymer was determined by GPC. An excess of N-(2-aminoethyl)morpholine was then added for end-capping. Finally, the product was precipitated in an excess of diethyl ether and freeze-dried to obtain a star-shaped poly(β-amino ester) with a polyamine core and a molecular weight of 8.7 kg / mol.

[0097] Figure 1 shows the GPC curve of SPAE-8, a star-shaped poly(β-amino ester) with a molecular weight of 8.7 kg / mol and a polyamine core, prepared in this example. This confirms the successful synthesis of the star-shaped poly(β-amino ester) with a polyamine core. Figure 1 shows the H-NMR spectrum of the purified star-shaped poly(β-amino ester) with a molecular weight of 8.7 kg / mol and a polyamine core, prepared in this example. The characteristic peaks in the H-NMR spectrum further confirm the successful synthesis of the polymer.

[0098] Example 9

[0099] In Examples 1 to 8, composite nanoparticles were prepared from star-shaped poly(β-amino ester)s with polyamine cores (molecular weights of 12.9 kg / mol, 15.5 kg / mol, 11.0 kg / mol, 10.1 kg / mol, 14.9 kg / mol, 19.9 kg / mol, 11.1 kg / mol, and 8.7 kg / mol, respectively) and DNA. The mass ratios of the star-shaped poly(β-amino ester) to DNA were 20:1, 40:1, and 60:1, respectively, with 1.0 μg of DNA used. PicoGreen was used to test the DNA affinity of the composite nanoparticles. First, the star-shaped poly(β-amino ester) was quickly added to the GFP DNA solution, vortexed at high speed for 30 seconds, and then allowed to stand for 20 minutes to form composite nanoparticles. The composite nanoparticle solution was then diluted to 100 μL using serum-free culture medium, and 100 μL of Pico Green working solution was added. The excited fluorescence intensity was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. Furthermore, composite nanoparticles were prepared using a similar method, and their particle size, particle size distribution, and surface zeta potential were measured using dynamic light scattering (DLS). The composite nanoparticles were then washed and freeze-dried, and their micromorphology was characterized using TEM.

[0100] Figure 3 shows the microscopic morphology of the star-shaped poly(β-amino ester)-DNA composite nanoparticles prepared in Examples 1 and 2. TEM results confirm that the star-shaped poly(β-amino ester)-DNA composite nanoparticles prepared in Examples 1 and 2 have a uniform particle size distribution and a relatively stable spherical structure, demonstrating their excellent stability and providing important support for in vitro and in vivo gene delivery.

[0101] Figure 4 shows the DNA affinity test of the star-shaped poly(β-amino ester) and DNA composite nanoparticles with polyamine as the core prepared in Examples 1 to 8, and proves that the star-shaped poly(β-amino ester) and DNA composite nanoparticles with polyamine as the core prepared in Examples 1 to 8 exhibited a DNA binding efficiency of more than 90% when the mass ratio was 20:1, 40:1 and 60:1, respectively. As the mass ratio increased, the binding efficiency also increased, confirming its excellent DNA affinity performance.

[0102] Figure 5 shows the particle size measurements of the star-shaped poly(β-amino ester)-DNA composite nanoparticles with polyamine cores prepared in Examples 1 to 8. DLS results confirm that the particle sizes of the star-shaped poly(β-amino ester)-DNA composite nanoparticles with polyamine cores prepared in Examples 1 to 8 are all less than 350 nm, facilitating cellular uptake.

[0103] Figure 6 shows the particle size distribution of the star-shaped poly(β-amino ester) and DNA composite nanoparticles with polyamine cores prepared in Examples 1 and 2. DLS results confirm that the star-shaped poly(β-amino ester) and DNA composite nanoparticles with polyamine cores prepared in Examples 1 (Figure 6(a)) and 2 (Figure 6(b)) exhibit uniform particle size distribution, which meets the requirements for subsequent precise nanoparticle delivery.

[0104] Figure 7 shows the zeta potential measurements of the star-shaped poly(β-amino ester) / DNA composite nanoparticles with polyamine cores prepared in Examples 1 to 8. DLS results confirm that the star-shaped poly(β-amino ester) / DNA composite nanoparticles with polyamine cores prepared in Examples 1 to 8 effectively shield the negative potential of DNA itself, and that the surface potential of the composite nanoparticles is greater than +10 mV, providing a prerequisite for further DNA delivery.

[0105] Example 10

[0106] Hepatocytes (HepG2, HCC-LM3, MHCC-97H, BRL-3A), ovarian cells (A2780), kidney cells (Vero), cervical cells (HeLa), chondrocytes (SW1353), and skin cells that are difficult to transfect (HaCaT and NHF), macrophage RAW264.7 cells were cultured at 1.5×10 4Cells were seeded at a density of 10 cells / well in a 96-well plate and cultured overnight at 37°C. A sodium acetate buffer solution of the polyamine-based star-shaped poly(β-amino ester) prepared in the example was mixed with a sodium acetate buffer solution of 0.5 μg of DNA encoding green fluorescent protein or NOD2 (at a mass ratio of 40:1). The mixture was allowed to stand for 15 minutes before being added to a serum-containing culture medium, thoroughly mixed, and then slowly added to the cells. The cells were then cultured for an additional 24 hours, and the GFP-transfected cells were observed under a fluorescence microscope.

[0107] Figure 8 shows a qualitative evaluation of the DNA transfection performance of the polyamine-core star-shaped poly(β-amino ester)-DNA composite nanoparticles prepared in Examples 1 to 8. Figure 8(a) shows that in HeLa cells, the polyamine-core star-shaped poly(β-amino ester)-DNA composite nanoparticles exhibit high DNA transfection efficiency due to their excellent DNA affinity, compressibility, and biophysical properties, demonstrating their ability to efficiently deliver DNA into a variety of tissue cells. As shown in Figure 8(b), in SW1353, due to the excellent DNA affinity, compressibility and biophysical properties of the star-shaped poly(β-amino ester) and DNA composite nanoparticles with polyamine as the core, the star-shaped poly(β-amino ester) and DNA composite nanoparticles with polyamine as the core exhibited a higher DNA transfection efficiency, confirming that the multi-component star-shaped poly(β-amino ester) and DNA composite nanoparticles with polyamine as the core can efficiently mediate intracellular DNA delivery.

[0108] Figure 9 shows a quantitative evaluation of the DNA transfection performance of the polyamine-core star-shaped poly(β-amino ester) / DNA composite nanoparticles prepared in Examples 1 to 8. Figure 9 shows that in HeLa cells, the polyamine-core star-shaped poly(β-amino ester) / DNA composite nanoparticles exhibit high DNA transfection efficiency, exceeding 95% for GFP DNA, due to their excellent DNA affinity, compressibility, and biophysical properties.

[0109] Figure 10 shows a quantitative evaluation of the DNA transfection performance of the polyamine-core star-shaped poly(β-amino ester) / DNA composite nanoparticles prepared in Example 1. Figure 10(a) shows that the polyamine-core star-shaped poly(β-amino ester) / DNA composite nanoparticles exhibited high DNA transfection efficiency in HepG2, HCC-LM3, MHCC-97H, and BRL-3A cells due to their excellent DNA affinity, compressibility, and biophysical properties, demonstrating that they can meet the requirements for efficient DNA delivery within liver tissue cells. As shown in Figure 10(b), in A2780, Vero, HaCaT, and NHF cells, the star-shaped poly(β-amino ester) and DNA composite nanoparticles with polyamine as the core exhibited higher DNA transfection efficiency due to their excellent DNA affinity, compressibility, and biophysical properties, proving that they can meet the requirements of efficient DNA delivery of nanoparticles in a variety of tissue cells.

[0110] Figure 11 is a quantitative evaluation of the DNA transfection performance of the polyamine-core star-shaped poly(β-amino ester) and DNA composite nanoparticles prepared in Example 1. Figure 11(a) shows that the polyamine-core star-shaped poly(β-amino ester) and DNA composite nanoparticles exhibited high DNA transfection efficiency in HepG2, HCC-LM3, MHCC-97H, and BRL-3A cells, with the highest transfection efficiency exceeding 80% for GFP DNA. This demonstrates that the nanoparticles can efficiently deliver DNA in a variety of tissue cells. Figure 11(b) shows that the polyamine-core star-shaped poly(β-amino ester) and DNA composite nanoparticles exhibited high DNA transfection efficiency in HepG2, HCC-LM3, MHCC-97H, and BRL-3A cells, with the highest transfection efficiency exceeding 62% for GFP DNA. This demonstrates that the nanoparticles can efficiently deliver DNA in a variety of tissue cells.

[0111] Figure 12 shows a qualitative evaluation of the NOD2 DNA transfection performance mediated by the polyamine-core star-shaped poly(β-amino ester) / DNA composite nanoparticles prepared in Example 1. Western blotting results demonstrate that the polyamine-core star-shaped poly(β-amino ester) / DNA composite nanoparticles can efficiently mediate NOD2 DNA transfection in RAW264.7 cells, providing potential for further cancer treatment.

[0112] This invention uses different diacrylate monomers and polyamine monomers as the core, small amine monomers, and functionalized end-capping agents to prepare star-shaped poly(β-amino ester)s with a functionalized, degradable polyamine core. These nanoparticles, combined with DNA of various sizes, form complex nanoparticles, forming nanosized spherical particles. These nanoparticles achieve safe and efficient transfection of GFP and NOD2 genes in hepatocytes (HepG2, HCC-LM3, MHCC-97H, BRL-3A), ovarian cells (A2780), kidney cells (Vero), cervical cells (HeLa), chondrocytes (SW1353), as well as difficult-to-transfect skin cells (HaCaT and NHF), and macrophage RAW264.7 cells. Compared to mainstream commercial transfection reagents (jetPEI and Lipofectamine 3000), these star-shaped polymer nanoparticles are inexpensive, easy to design and prepare, and offer superior transfection performance. They have significant clinical potential for DNA drug delivery and cancer immunotherapy.

[0113] Example 11 COS-7 or HeLa cell lines stably transfected with GFP (cells emit green light because they carry the GFP gene) were seeded in a 96-well plate at a density of 1.0×104 cells / well and cultured overnight at 37°C. A certain amount of star-shaped poly(β-amino ester) with a polyamine core was mixed into 0.1μg GFP mRNA or 100nM GFP siRNA solution, allowed to stand for 5 to 15 minutes, and then added to the cells. The mass ratio of the star-shaped poly(β-amino ester) with a polyamine core to mRNA or siRNA was 30:1, and the concentration of the siRNA mixture was 100nM; the mass ratio of SPAE to siRNA was 20:1. The cells were then cultured for another 24 hours, the transfected cells were observed under a fluorescence microscope, and the transfection efficiency was tested using a flow cytometer.

[0114] Figure 13 is a fluorescence photograph of cells after transfection of green fluorescent protein (GFP) mRNA in COS-7 cells mediated by star-shaped poly(β-amino ester) with polyamine as the core in Examples 1 to 8: The transfection results show that the eight star-shaped poly(β-amino ester) and mRNA composite nanoparticles with polyamine as the core can efficiently mediate GFP mRNA transfection in COS-7 cells, confirming their excellent mRNA delivery performance.

[0115] Figure 14 shows a quantitative evaluation of the performance of the star-shaped poly(β-amino ester)s with polyamine cores in Examples 1 to 8 in mediating transfection of mRNA encoding green fluorescent protein (GFP) in COS-7 cells. Flow cytometry results demonstrate that all eight star-shaped poly(β-amino ester)s with polyamine cores efficiently mediated GFP mRNA transfection in COS-7 cells, with transfection efficiencies reaching 83.8%, demonstrating their excellent mRNA delivery performance.

[0116] Figure 15 shows a qualitative evaluation of the performance of the star-shaped poly(β-amino ester)s with polyamine cores in mediating GFP siRNA transfection in HeLa cells stably transfected with GFP, as described in Examples 1 through 8. The transfection results demonstrate that all eight star-shaped poly(β-amino ester)s with polyamine cores are highly efficient in mediating GFP siRNA transfection, with a significant decrease in cell fluorescence intensity after transfection (higher transfection efficiency indicates better siRNA interference and weaker fluorescence intensity), demonstrating their excellent siRNA delivery performance.

[0117] Figure 16 shows a quantitative evaluation of the performance of the star-shaped poly(β-amino ester)s with polyamine cores in mediating siRNA transfection in HeLa cells stably transfected with GFP, as described in Examples 1 through 8. Flow cytometry results demonstrate that the eight star-shaped poly(β-amino ester)s with polyamine cores are highly efficient in mediating siRNA transfection in HeLa cells stably transfected with GFP, achieving a GFP knockdown efficiency of 95% after transfection, significantly outperforming commercial transfection reagents and demonstrating their excellent siRNA delivery performance.

[0118] The above content is 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 star-shaped poly(β-amino ester) with a polyamine as a core, characterized in that Its structural formula is shown in Formula 1, Formula 2 or Formula 3 below: In the formula, R1 is a group on a diacrylate monomer; the diacrylate monomer is R2 is a group on an organic amine, which is R2-NH2; R3 is a group on a polyamine monomer; R4 is a group on a functional end-capping agent monomer, which is R4-NH2; and n=15-55.

2. The star-shaped poly(β-amino ester) with a polyamine as the core according to claim 1, characterized in that The diacrylate monomers are 1,6-hexanediol diacrylate, 1,4-butanediol diacrylate, tetraethylene glycol diacrylate, diethylene glycol diacrylate, 2,2-dithiodiethanol diacrylate, bisphenol A polyoxyethylene ether diacrylate, and 1,1′-isopropyl bis(p-phenyleneoxy)di-2-propanol diacrylate.

3. The star-shaped poly(β-amino ester) with a polyamine as a core according to claim 1, characterized in that The organic amine is 4-amino-1-butanol, 5-amino-1-pentanol, propylamine, butylamine, hexylamine, octylamine, 1-(2-aminoethyl)piperazine, N-(2-aminoethyl)-morpholine, dodecylamine or octadecylamine.

4. The star-shaped poly(β-amino ester) with a polyamine as a core according to claim 1, characterized in that The polyamine monomer is tris(2-aminoethyl)amine, diethylenetriamine, bis(3-aminopropyl)amine, 1,4-butanediamine, 1,2-ethylenediamine, 1,3-propylenediamine, triethylenetetramine or 1,6-hexanediamine.

5. The star-shaped poly(β-amino ester) with a polyamine as a core according to claim 1, characterized in that The functional 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 or 2-methyl-1,5-pentanediamine.

6. The method for preparing a star-shaped poly(β-amino ester) with a polyamine as a core according to any one of claims 1 to 5, characterized in that: The following steps are involved: 1) reacting a diacrylate monomer with an organic amine through a Michael addition reaction to obtain a linear poly(β-amino ester) having a double bond at the end; 2) reacting the linear poly(β-amino ester) with a double bond at the end obtained in step 1) with a polyamine monomer to obtain a star-shaped poly(β-amino ester) with a double bond at the end and a polyamine as a core; 3) reacting the star-shaped poly(β-amino ester) with a polyamine core and a double bond at the end obtained in step 2) with a functional end-capping agent monomer to perform a functional end-capping reaction to obtain a star-shaped poly(β-amino ester) with a polyamine core.

7. The method for preparing a star-shaped poly(β-amino ester) with a polyamine as a core according to claim 6, characterized in that: In step 1), the molar ratio of the diacrylate monomer to the organic amine is (1-5):(0.5-2); in step 2), the molar ratio of the polyamine monomer to the linear poly(β-amino ester) with a double bond at the end is (0.01-1):(1-5).

8. A star-shaped poly(β-amino ester)-based composite nanoparticle with a polyamine as a core, characterized in that: The invention is obtained by combining the star-shaped poly(β-amino ester) with polyamine as the core as described in any one of claims 1 to 5 with DNA.

9. The method for preparing star-shaped poly(β-amino ester)-based composite nanoparticles with polyamine as core according to claim 8, characterized in that: include: The solution of the star-shaped poly(β-amino ester) with a polyamine as a core according to any one of claims 1 to 5 is mixed with a DNA solution, stirred, and allowed to stand to obtain composite nanoparticles.

10. Use of the star-shaped poly(β-amino ester) with polyamine as the core according to any one of claims 1 to 5 or the star-shaped poly(β-amino ester)-based composite nanoparticle with polyamine as the core according to claim 8 in the preparation of a gene carrier, characterized in that: The gene vector is used to deliver genes to tissues or cells.

11. The star-shaped poly(β-amino ester) with a polyamine as the core according to any one of claims 1 to 5, characterized in that The star-shaped poly(β-amino ester) with polyamine as the core can be used for mRNA and siRNA delivery, and delivery of nucleic acid molecules to tissues or cells.

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