Preparation method and application of lung organ targeted branched poly (beta-amino ester) group mRNA (messenger ribonucleic acid) compound nanoparticles

Abstract

The invention provides a preparation method and application of lung target organ to branched poly (beta-amino ester) group mRNA (messenger Ribonucleic Acid) compound nanoparticles. Linear low-molecular-weight poly (beta-amino ester) is used as an arm, a diamine monomer is used as a core molecule, and a brand-new-structure lung target organ-branched poly (beta-amino ester) is obtained. The compound nanoparticles formed by the lung organ targeted branched poly (beta-amino ester) and mRNA (messenger ribonucleic acid) are relatively uniform in distribution, good in stability, biodegradable and good in biocompatibility. An in-vitro transfection result shows that the compound nanoparticles formed by the lung organ targeted branched poly (beta-amino ester) and the mRNA can efficiently deliver the mRNA in various tissue cell lines. An in-vivo transfection result shows that the functional branched poly (beta-amino ester) prepared by the invention can safely and efficiently mediate mRNA specific targeting lung tissues, and shows extremely high mRNA transfection efficiency, so that an application basis is provided for lung cancer gene therapy and vaccine development.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials and relates to a preparation method and application of lung organ targeted branched poly (β-amino ester)-based mRNA complex nanoparticles. Background Art

[0002] mRNA therapy can produce various functional genes and has shown broad application prospects in cancer treatment, genetic disease treatment and vaccine development. However, naked mRNA is not easy to cross the cell membrane, which is also negatively charged and hydrophobic, and is easily degraded by nucleases and restriction endonucleases in the blood or cytoplasm. Therefore, mRNA therapy relies heavily on the construction of safe and efficient delivery vectors, which can effectively protect mRNA from serum nuclease degradation, promote target cell uptake, and release mRNA from endosomes into the cytoplasm for protein production, thereby achieving the purpose of disease treatment.

[0003] Currently, liposome nanoparticles (LNPs) have received extensive attention as one of the most promising vectors for gene delivery. Due to the slow blood flow and discontinuous blood vessels in the hepatic sinusoids, LNPs have an affinity for delivering therapeutic RNA payloads to hepatocytes when administered systemically. However, it remains challenging to use LNPs to systemically deliver mRNA to specific cells outside the liver and spleen.

[0004] Due to the three-dimensional structure, multiple terminal groups and wider chemical modification space, the wide source of monomers and the flexible and controllable chemical composition, branched poly (β-amino ester) has excellent MRNA loading efficiency. The complex nanoparticles formed after compressing MRNA have stronger interactions with cells, and it has shown broad application prospects in MRNA delivery. However, current research is often limited to the delivery of MRNA at the cellular level by branched polymers, and the targeted delivery of mRNA to organs outside the liver in vivo by branched poly (β-amino ester) has not been explored, which will greatly limit its 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 lung organ targeted branched poly (β-amino ester), composite nanoparticles based thereon, and a preparation method and application thereof.

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

[0007] A lung organ targeted branched poly (β-amino ester), the structural formula of which is shown in Formula 1, Formula 2 or Formula 3:

[0008]

[0009] 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 diamine monomer; R4 is a group on a functional end-capping agent monomer, which is R4-NH2; n=10-60; m=10-60.

[0010] 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'-isopropyl bis(p-phenyleneoxy)di-2-propanol diacrylate.

[0011] Preferably, the organic amine is 4-amino-1-butanol, 5-amino-1-pentanol, butylamine, pentylamine, hexylamine, octylamine, 1-(2-aminoethyl)piperazine, N-(2-aminoethyl)-morpholine, dodecylamine or octadecylamine.

[0012] Preferably, the diamine monomer is 1,4-butanediamine, 1,2-ethylenediamine, 1,3-propylenediamine, 1,6-hexanediamine, 1,4-bis(3-aminopropyl)piperazine, 1,11-diamino-3,6,9-trioxaundecane or 2-methyl-1,5-pentanediamine.

[0013] Preferably, the functionalized end-capping agent monomer is 1-(3-aminopropyl)-4-methylpiperazine, N-(3-aminopropyl)morpholine, N-(2-aminoethyl)morpholine, 2-methyl-2-morpholin-4-ylpropane-1-amine, 1-(2-aminoethyl)piperazine, 1,4-bis(3-aminopropyl)piperazine, 1,11-diamino-3,6,9-trioxaundecane or 2-methyl-1,5-pentanediamine.

[0014] The method for preparing the lung organ targeted branched poly (β-amino ester) comprises the following steps:

[0015] 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;

[0016] 2) reacting the linear poly(β-amino ester) with a double bond at the end obtained in step 1) with a diamine monomer to obtain a lung organ-targeted branched poly(β-amino ester) with a double bond at the end;

[0017] 3) reacting the lung-targeted branched poly(β-amino ester) containing double bonds at the end obtained in step 2) with a functionalized end-capping agent monomer to perform a functionalized end-capping reaction to obtain the lung-targeted branched poly(β-amino ester).

[0018] Preferably, in step 1), the molar ratio of the diacrylate monomer to the organic amine is (1-4):(0.4-3); in step 2), the molar ratio of the diamine monomer to the linear poly(β-amino ester) with a double bond at the end is (0.02-2):(1-5).

[0019] A lung target organ branched poly (β-amino ester) based composite nanoparticle is obtained by compounding the lung organ targeted branched poly (β-amino ester) with mRNA.

[0020] The method for preparing the branched poly(β-amino ester)-based composite nanoparticles targeting lung target organs comprises: mixing the branched poly(β-amino ester)-based solution targeting lung target organs with mRNA solution, stirring, and standing to obtain composite nanoparticles.

[0021] The lung target organ-directed branched poly (β-amino ester) or the lung target organ-directed branched poly (β-amino ester)-based composite nanoparticles are used in mRNA delivery, thereby specifically delivering it to lung tissue and lung 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 monomers as core molecules to obtain a class of novel branched poly (β-amino ester) for lung target organs. The composite nanoparticles formed by the branched poly (β-amino ester) for lung target organs and mRNA 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 branched poly (β-amino ester) for lung target organs and mRNA can efficiently deliver mRNA in a variety of tissue cell lines. More importantly, the results in vivo show that the composite nanoparticles formed by the multifunctional branched poly (β-amino ester) and mRNA can specifically target lung tissues through tail vein injection, which further provides clinical vector candidates for the development of lung cancer or vaccines. The branched poly (β-amino ester) for lung target organs prepared by the invention has low cost, simple preparation process, low equipment requirements, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are the GPC curves of the lung organ targeted branched poly(β-amino ester) in Examples 1 to 4; their weight average molecular weights are 12.5 kg / mol, 22.1 kg / mol, 18.2 kg / mol, and 14.3 kg / mol, respectively, and their dispersion indices are 3.9, 4.3, 3.7, and 4.5, respectively.

[0025] Figure 2The lung organ targeted branched poly (β-amino ester) weight average molecular weights of Examples 1 to 4 are 12.5 kg / mol, 22.1 kg / mol, 18.2 kg / mol, and 14.3 kg / mol, respectively. 1 HNMR spectrum.

[0026] Figure 3 The mRNA affinity performance of the composite nanoparticles of lung organ-targeted branched poly (β-amino ester) and mRNA in Examples 1 to 4 was tested; the RiboGreen test results showed that at a mass ratio of 40:1 and 60:1 and at different serum concentrations, the four branched poly (β-amino esters) all exhibited excellent mRNA affinity performance of more than 90%, so the composite nanoparticles of lung organ-targeted branched poly (β-amino ester) and mRNA have excellent stability, which provides conditions for subsequent in vitro and in vivo gene delivery.

[0027] Figure 4 The particle size of the composite nanoparticles of branched poly (β-amino ester) and mRNA targeted to lung organs in Examples 1 to 4 is characterized; the results of DLS show that at a mass ratio of 40:1 and 60:1 and at different serum concentrations, the composite nanoparticles of the four branched poly (β-amino ester) and mRNA have a smaller size with a particle size range of 150nm-300nm, and it is confirmed that the composite nanoparticles of branched poly (β-amino ester) and mRNA targeted to lung organs still have excellent stability at a serum concentration of 10%, providing conditions for mRNA delivery in vivo.

[0028] Figure 5 This is a zeta test of the potential of the lung organ-targeted branched poly (β-amino ester) and mRNA composite nanoparticles in Examples 1 to 4; the DLS results show that at a mass ratio of 40:1 and 60:1, the four branched poly (β-amino ester) and mRNA composite nanoparticles have a higher positive charge, further promoting the cellular uptake of the nanoparticles.

[0029] Figure 6 The microscopic morphology of the composite nanoparticles of lung organ-targeted branched poly(β-amino ester) and mRNA in Example 1; the AFM results show that the two nanoparticles can be evenly and stably distributed under physiological conditions.

[0030] Figure 7 The intracellular distribution and uptake performance of the composite nanoparticles of lung organ-targeted branched poly (β-amino ester) and mRNA in Example 1 were evaluated; the results of laser confocal microscopy showed that after incubation of the composite nanoparticles of lung organ-targeted branched poly (β-amino ester) and mRNA with cells for 4 hours, a large number of nanoparticles were able to enter the cells and were distributed around the cell nucleus.

[0031] Figure 8 The endosomal escape ability of the composite nanoparticles of lung organ-targeted branched poly (β-amino ester) and mRNA in Example 1 was evaluated; the results of laser confocal microscopy showed that the composite nanoparticles of lung organ-targeted branched poly (β-amino ester) and mRNA exhibited excellent endosomal escape ability after incubation with cells for 6 hours.

[0032] Fig. 9 These are fluorescent photographs of cells after transfection of green fluorescent protein (GFP) mRNA with lung organ-targeted branched poly (β-amino ester) in Examples 1 to 4: The transfection results show that for SW1353 cells, at two different serum concentrations, the four branched poly (β-amino ester) and mRNA composite nanoparticles can all efficiently mediate GFP gene transfection, which is attributed to their excellent serum stability.

[0033] Fig.10 These are fluorescent photographs of cells after transfection of green fluorescent protein (GFP) mRNA with lung organ-targeted branched poly (β-amino ester) in Examples 1 to 4: The transfection results show that for SW1353 cells, at two different serum concentrations, the four branched poly (β-amino ester) and mRNA composite nanoparticles can all efficiently mediate GFP gene transfection, which is attributed to their excellent serum stability.

[0034] Fig.11 Fluorescence photographs of cells after the lung organ-targeted branched poly (β-amino ester) in Examples 1 to 4 mediated mRNA encoding green fluorescent protein (GFP) in 293T, HeLa, and Vero cells: The transfection results showed that the four branched poly (β-amino ester) and mRNA composite nanoparticles were able to efficiently mediate GFP gene transfection in 293T, HeLa, and Vero cells, which confirms their wide applicability.

[0035] Fig.12 The results are a quantitative evaluation of the transfection performance of lung organ-targeted branched poly(β-amino ester) in 293T cells in mediating mRNA encoding green fluorescent protein (GFP). The flow cytometry results show that in 293T cells, four hyperbranched poly(β-amino ester)s with different molecular weights efficiently mediated GFP mRNA transfection, with a transfection efficiency of 90%.

[0036] Fig.13 The results are a quantitative evaluation of the transfection performance of lung organ-targeted branched poly(β-amino esters) in HeLa cells in mediating mRNA encoding green fluorescent protein (GFP). The flow cytometry results show that in HeLa cells, four hyperbranched poly(β-amino esters) with different molecular weights efficiently mediated GFP mRNA transfection, with a transfection efficiency of 72%.

[0037] Fig.14 The results are a quantitative evaluation of the transfection performance of lung organ-targeted branched poly(β-amino esters) in Vero cells in mediating mRNA encoding green fluorescent protein (GFP). The flow cytometry results show that in Vero cells, four hyperbranched poly(β-amino esters) with different molecular weights efficiently mediated GFP mRNA transfection, with a transfection efficiency of 99%.

[0038] Fig.15 The lung organ targeted branched poly (β-amino ester) in Example 1 and Example 2 was used to evaluate the transfection efficiency of Fluc mRNA in mice. The results of in vivo transfection for 6 hours showed that when the mRNA dosage was 0.5 mg, the lung organ targeted branched poly (β-amino ester) in Example 1 and Example 2 were able to efficiently mediate Fluc mRNA transfection in vivo and highly targeted the lung tissue.

[0039] Fig.16 The safety evaluation of lung organ targeted branched poly (β-amino ester) in Example 1 and Example 2 after Fluc mRNA transfection in mice was performed. The results of HE staining 24 hours after in vivo transfection showed that the heart, liver, spleen, lung and kidney of the mice were normal before and after transfection, and no organ damage or obvious toxicity occurred. These results show that the high-performance lung organ targeted branched poly (β-amino ester) of the present invention has the potential for clinical application in the treatment of lung-related diseases. 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 to further explain the features and advantages of the present invention and are not intended to limit the claims of the present invention.

[0043] The lung organ targeted branched poly (β-amino ester) of the present invention has a structural formula as shown in Formula 1, Formula 2 or Formula 3:

[0044]

[0045] 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 diamine monomer, specifically a methyl group, an ethyl group, a single bond or an n-butyl group; R4 is a group on a functional end-capping agent monomer, which is R4-NH2; n=10-60; m=10-60.

[0046] 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, 1,1'-isopropyl bis(p-phenyleneoxy)di-2-propanol diacrylate, and their structural formulas are shown below:

[0047]

[0048] The organic amine of the present invention is 4-amino-1-butanol, 5-amino-1-pentanol, butylamine, pentylamine, hexylamine, octylamine, 1-(2-aminoethyl)piperazine, N-(2-aminoethyl)-morpholine, dodecylamine or octadecylamine, and their structural formulas are shown below:

[0049]

[0050] The diamine monomers of the present invention are 1,4-bis(3-aminopropyl)piperazine, 1,11-diamino-3,6,9-trioxaundecane, 2-methyl-1,5-pentanediamine, 1,4-butanediamine, 1,2-ethylenediamine, 1,3-propylenediamine, and 1,6-hexanediamine, and their structural formulas are shown below:

[0051]

[0052] The functional end-capping agent monomers of 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, and their structural formulas are shown below:

[0053]

[0054] The method for preparing the lung organ targeted branched poly (β-amino ester) of the present invention comprises the following steps:

[0055] 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;

[0056] 2) reacting the linear poly(β-amino ester) with a double bond at the end obtained in step 1) with a diamine monomer (core molecule) to obtain a branched poly(β-amino ester) with a double bond at the end;

[0057] 3) reacting the branched poly(β-amino ester) containing double bonds at the end obtained in step 2) with a functionalized end-capping agent monomer to perform a functionalized end-capping reaction to obtain a high-performance functionalized lung organ-targeted branched poly(β-amino ester);

[0058] Specifically, the steps include:

[0059] 1) Add a certain amount of diacrylate monomer and organic amine to a solvent (such as dimethyl sulfoxide, tetrahydrofuran), and fully dissolve them by magnetic stirring, wherein the reaction molar ratio of diacrylate monomer to organic amine is (1-5): (0.5-3); 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 double bond at the end. When the molecular weight of the polymer reaches a preset 2.0 to 6.0 kg / mol by GPC monitoring, the reaction is terminated;

[0060] 2) A certain amount of low molecular weight linear poly(β-amino ester) with a double bond at the end and diamine monomer (core molecule) are added to a solvent and the monomer is fully dissolved by magnetic stirring, wherein the molar ratio of the diamine monomer to the linear poly(β-amino ester) with a double bond at the end is (0.04-1):(1-5), and the reaction is carried out at 30°C-90°C to obtain a lung organ-targeted branched poly(β-amino ester) with a double bond at the end. The polymer molecular weight is monitored by GPC, and the reaction is terminated when the set value is within the range of 7.0kg / mol-60.0kg / mol.

[0061] 3) adding a certain amount of functionalized end-capping agent monomer to the reaction system to react with the branched poly(β-amino ester) containing a double bond at the end for end-capping, wherein the molar ratio of the functionalized end-capping agent monomer to the lung organ-targeted branched poly(β-amino ester) 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-72h.

[0062] The preparation method of the lung organ targeted branched poly (β-amino ester) based nanoparticles is as follows:

[0063] The lung organ targeted branched poly (β-amino ester) prepared by the present invention and mRNA of various sizes are combined to form uniform and stable nano-scale spherical particles through charge action, and the mRNA affinity, the particle size of the composite nanoparticles formed by compressing the mRNA, the Zeta potential and the microscopic morphology are tested as follows:

[0064] A certain amount of lung organ targeted branched poly (β-amino ester) solution was quickly added to the mRNA solution, vortexed at high speed for 15 to 60 seconds, and then allowed to stand for 5 to 30 minutes, wherein the mass ratio of lung organ targeted branched poly (β-amino ester) to mRNA was (10 to 100): 1, and then RiboGreen working solution was added to test the fluorescence intensity and calculate the mRNA affinity efficiency. A similar method for preparing composite nanoparticles was used, and nanoparticles were formed after standing for 5 to 30 minutes, 1 mL of deionized water was added, and then tested using dynamic light scattering (DLS). At the same time, composite nanoparticles were prepared using the same method, and inorganic salt ions were washed with deionized water, centrifuged at high speed for 5 minutes, and then freeze-dried. Finally, the microscopic morphology of the composite nanoparticles was observed using an atomic force microscope (AFM).

[0065] The lung organ-targeted branched poly(β-amino ester) prepared by the present invention is transfected with mRNA encoding green fluorescent protein, and the method is as follows:

[0066] 1) Cell culture: SW1353, 293T, HeLa and Vero cells were cultured at 1.0×10 4 ~2.0×10 4 Cells were seeded at a density of 10 cells / well in a 96-well plate and cultured at 37°C overnight.

[0067] 2) A certain amount of lung organ-targeted branched poly (β-amino ester) was mixed into the GFP mRNA solution, allowed to stand for 5 to 150 minutes, and then added to the cells.

[0068] 3) 12 to 72 hours after transfection, the cells transfected with GFP mRNA were observed and photographed under a fluorescence microscope.

[0069] 4) 48 h after transfection, the efficiency of GFP mRNA transfection was tested using flow cytometry.

[0070] The lung organ-targeted branched poly (β-amino ester) prepared by the present invention is transfected with Fluc mRNA in vivo, and the method is as follows:

[0071] 1) Dilute 2.5ug-5ug of Fluc mRNA in 75uL-100uL sodium acetate buffer solution and mix.

[0072] 2) 4uL-8uL of branched poly (β-amino ester) solution was diluted in 11.5uL of ethanol solution, and then 13.5uL of F127 solution was added thereto in sequence and mixed.

[0073] 3) Add the sodium acetate buffer solution of mRNA to the branched poly (β-amino ester) solution, vortex for 25-30 seconds, and incubate at room temperature for 15-30 minutes to form a polyplexes solution.

[0074] 4) Add 160uL-200uL of PBS to the polyplexes solution, let it stand for 2 minutes, and then inject it into the tail vein.

[0075] 5) Detect the luminescence in vivo and in each organ according to the experiment. Detect the toxicity in vivo by tissue section. Specific embodiment:

[0077] Example 1

[0078] 1,1'-Isopropylidenebis(p-phenyleneoxy)di-2-propanol dipropylene glycol ester (24mmol, 11.63g) and 5-amino-1-pentanol (20mmol, 2.06g) were dissolved in dimethyl sulfoxide (DMSO) (4mL) and reacted at 90℃ for 10h. After the system reached room temperature, DMSO was added to terminate the reaction to synthesize a linear poly(β-amino ester) with a double bond at the end. Then, the linear poly(β-amino ester) with a double bond at the end and ethylenediamine were dissolved in the organic solvent DMSO, wherein the concentration of the linear poly(β-amino ester) with a double bond at the end was 300mg / mL. Under magnetic stirring, the polymerization reaction was carried out at 90℃, and the molecular weight of the polymer was detected by GPC. Then, an excess of N-(3-aminopropyl)morpholine was added for end-capping reaction, and finally precipitated in an excess of ether and freeze-dried to finally obtain a lung organ-targeted branched poly(β-amino ester) with a weight average molecular weight of 12.5kg / mol.

[0079] Figure 1 HPAE-1 in the figure is the GPC curve of the lung-targeted branched poly(β-amino ester) prepared in this example with a molecular weight of 12.5 kg / mol and a dispersion index of 3.9 after purification, which confirms that the lung-targeted branched poly(β-amino ester) was successfully synthesized. Figure 2 The H-NMR spectrum of the lung-targeted branched poly(β-amino ester) with a molecular weight of 12.5 kg / mol prepared in this example after purification once again confirmed that the lung-targeted branched poly(β-amino ester) was successfully synthesized.

[0080] Example 2

[0081] 1,1'-Isopropylidenebis(p-phenyleneoxy)di-2-propanol dipropylene glycol ester (24mmol, 11.63g) and 5-amino-1-pentanol (20mmol, 2.06g) were dissolved in dimethyl sulfoxide (DMSO) (13.06mL), reacted at 60°C for 8h, added dimethyl sulfoxide (DMOS) (concentration in the total reaction system was 10%), reacted at room temperature for 24h, and synthesized a linear poly(β-amino ester) with a double bond at the end. Then, the linear poly(β-amino ester) with a double bond at the end and 1,4-butanediamine were dissolved in an organic solvent THF, wherein the concentration of the linear poly(β-amino ester) with a double bond at the end was 200mg / mL. The polymerization reaction was carried out at 60°C under magnetic stirring, and the molecular weight of the polymer was detected by GPC. Then, an excess of N-(3-aminopropyl)morpholine was added for end-capping reaction. Finally, the product was precipitated in excess ether and freeze-dried to obtain lung-targeted branched poly(β-amino ester) with a molecular weight of 22.1 kg / mol.

[0082] Figure 1 HPAE-2 in the figure is the GPC curve of the lung-targeted branched poly(β-amino ester) prepared in this example with a molecular weight of 22.1 kg / mol and a dispersion index of 4.3, which confirms that the lung-targeted branched poly(β-amino ester) was successfully synthesized. Figure 2 The purified H-NMR spectrum of the lung-targeted branched poly(β-amino ester) with a molecular weight of 22.1 kg / mol prepared in this example, the characteristic peaks of the H-NMR spectrum once again confirmed the successful synthesis of the polymer.

[0083] Example 3

[0084] 1,1'-Isopropylidenebis(p-phenyleneoxy)di-2-propanol dipropylene glycol ester (24mmol, 11.63g) and 4-amino-1-pentanol (20mmol, 1.78g) were dissolved in dimethyl sulfoxide (DMSO) (13.06mL), reacted at 90°C for 5h, added dimethyl sulfoxide (DMOS) (concentration in the total reaction system was 10%), reacted at room temperature for 24h, and synthesized a linear poly(β-amino ester) with a double bond at the end. Then, the linear poly(β-amino ester) with a double bond at the end and propylenediamine were dissolved in the organic solvent DMSO, wherein the concentration of the linear poly(β-amino ester) with a double bond at the end was 200mg / mL. Under magnetic stirring, the polymerization reaction was carried out at 90°C, the molecular weight of the polymer was detected by GPC, and then an excess of 1,11-diamino-3,6,9-trioxaundecane was added for end-capping reaction. Finally, it was precipitated in excess ether and freeze-dried to finally obtain lung organ-targeted branched poly (β-amino ester) with a molecular weight of 18.2 kg / mol.

[0085] Figure 1HPAE-3 in the figure is the GPC curve of the lung-targeted branched poly(β-amino ester) prepared in this example with a molecular weight of 18.2 kg / mol and a dispersion index of 3.7, which confirms that the lung-targeted branched poly(β-amino ester) was successfully synthesized. Figure 2 HPAE-3 in the figure is the purified H NMR spectrum of the lung-targeted branched poly(β-amino ester) with a molecular weight of 18.2 kg / mol prepared in this example. The characteristic peaks of the H NMR spectrum once again confirm that the polymer was successfully synthesized.

[0086] Example 4

[0087] 1,1'-Isopropylidenebis(p-phenyleneoxy)di-2-propanol dipropylene glycol ester (24mmol, 11.63g) and 4-amino-1-butanol (20mmol, 2.06g) were dissolved in dimethyl sulfoxide (DMSO) (13.06mL), reacted at 50°C for 10h, added dimethyl sulfoxide (DMOS) (concentration in the total reaction system was 10%), reacted at room temperature for 24h, and synthesized a linear poly(β-amino ester) with a double bond at the end. Then, the linear poly(β-amino ester) with a double bond at the end and hexamethylenediamine were dissolved in the organic solvent DMSO, wherein the concentration of the linear poly(β-amino ester) with a double bond at the end was 400mg / mL. The polymerization reaction was carried out at 60°C under magnetic stirring, and the molecular weight of the polymer was detected by GPC. Then, an excess of N-(2-aminoethyl)morpholine was added for end-capping reaction. Finally, the product was precipitated in excess ether and freeze-dried to obtain lung-targeted branched poly(β-amino ester) with a molecular weight of 14.3 kg / mol.

[0088] Figure 1 HPAE-4 in the figure is the GPC curve of the lung-targeted branched poly(β-amino ester) prepared in this example with a molecular weight of 14.3 kg / mol and a dispersion index of 4.5, which confirms that the lung-targeted branched poly(β-amino ester) was successfully synthesized. Figure 2 HPAE-4 in the figure is the purified H NMR spectrum of the lung-targeted branched poly(β-amino ester) with a molecular weight of 14.3 kg / mol prepared in this example. The characteristic peaks of the H NMR spectrum once again confirm that the polymer was successfully synthesized.

[0089] Example 5

[0090] In Example 1 to Example 4, lung organ targeting branched poly (β-amino ester) (molecular weight is 12.5kg / mol, 22.1kg / mol, 18.2kg / mol, 14.3kg / mol, and dispersion index is 3.9, 4.3, 3.7, 4.5, respectively) and mRNA are used to prepare composite nanoparticles, branched poly (β-amino ester) and mRNA are at a mass ratio of 40: 1, 60: 1 and different serum concentrations, wherein the amount of mRNA is 1.0 μg, and RiboGreen is used to test the mRNA affinity of lung organ targeting branched poly (β-amino ester). First, the star-shaped poly (β-amino ester) is quickly added to the GFP mRNA solution, vortexed at high speed for 30s, and then allowed to stand for 20min to form composite nanoparticles. Then, the composite nanoparticle solution is diluted to 100 μL using serum-free medium, and then 100 μL Ribo Green working solution is added, and the excited fluorescence intensity is detected under the condition of an excitation wavelength of 480nm and an emission wavelength of 520nm. Furthermore, a similar method was used to prepare composite nanoparticles, and dynamic light scattering (DLS) was used to characterize the particle size and particle size distribution of the composite nanoparticles, and AFM was used to characterize their microscopic morphology.

[0091] Figure 3 The mRNA affinity performance of the lung organ targeted branched poly (β-amino ester) and mRNA composite nanoparticles prepared in Examples 1 to 4 was tested, and it was verified that the lung organ targeted branched poly (β-amino ester) and MRNA composite nanoparticles prepared in Examples 1 to 4 showed a binding efficiency of more than 90% mRNA at mass ratios of 40:1 and 60:1 and different serum concentrations, confirming their excellent mRNA affinity performance and excellent serum stability.

[0092] Figure 4 The particle size test of the composite nanoparticles of branched poly (β-amino ester) and mRNA targeted to lung organs prepared in Examples 1 to 4. The results of DLS showed that at a mass ratio of 40:1 and 60:1 and at different serum concentrations, the composite nanoparticles of the four branched poly (β-amino ester) and mRNA had a smaller size, with a particle size range of 150nm-300nm, and confirmed that the composite nanoparticles of branched poly (β-amino ester) and mRNA targeted to lung organs still had excellent stability at a serum concentration of 10%, providing conditions for in vivo mRNA delivery.

[0093] Figure 5Zeta potential test of the composite nanoparticles of branched poly (β-amino ester) and mRNA for lung organ targeting prepared in Examples 1 to 4. The results of DLS showed that when the mass ratio was 40:1 and 60:1, the composite nanoparticles of the four branched poly (β-amino ester) and mRNA had a higher positive charge, further promoting the uptake of nanoparticles by cells.

[0094] Figure 6 The microscopic morphology of the composite nanoparticles of lung organ targeted branched poly (β-amino ester) and mRNA in Example 1; The AFM results show that the composite nanoparticles formed by lung organ targeted branched poly (β-amino ester) and mRNA in Example 1 have uniform particle size distribution, confirming their excellent stability. The nanoparticles can be evenly and stably distributed under physiological conditions.

[0095] Example 6

[0096] HeLa cells were expressed at 6.0×10 3 The cells were seeded in a 96-well plate at a density of 10 cells / well, and the cells were cultured overnight at 37°C. When the mass ratio of lung organ targeted branched poly (β-amino ester) to Cy5 fluorescent dye-labeled mRNA in Example 1 was 40:1, the amount of Cy5 fluorescent dye-labeled mRNA was 0.1 μg, and the composite nanoparticles were formed, and then mixed with the culture medium and added to the cells. After incubation for 4h-6h, the culture medium was removed, and the cells were washed three times with PBS, and the endosomes and nuclei of the cells were stained with Lyso-Tracker Green and Hoechst at 37°C for 10min, and then the distribution of DNA in the cells was observed using laser confocal microscopy.

[0097] Figure 7 The distribution and uptake performance of the composite nanoparticles formed by the lung organ targeted branched poly (β-amino ester) and mRNA labeled with Cy5 fluorescent dye in Example 1 were evaluated in cells; the results of laser confocal microscopy showed that after the composite nanoparticles of lung organ targeted branched poly (β-amino ester) and mRNA were incubated with cells for 4 hours, a large number of nanoparticles were able to enter the cells and were distributed around the cell nucleus.

[0098] Figure 8 The endosomal escape ability of the composite nanoparticles of lung organ-targeted branched poly (β-amino ester) and mRNA in Example 1 was evaluated; the results of laser confocal microscopy showed that the composite nanoparticles of lung organ-targeted branched poly (β-amino ester) and mRNA exhibited excellent endosomal escape ability after incubation with cells for 6 hours, which will promote the transfection efficiency of mRNA.

[0099] Example 7

[0100] SW1353, 293T, HeLa and Vero cells were cultured at 1.5×10 4 The density of cells / well was inoculated in a 96-well plate, and the cells were cultured at 37°C overnight. The sodium acetate buffer solution of the lung organ targeted branched poly (β-amino ester) prepared in Example 1 to Example 4 was mixed with 0.5 μg of mRNA encoding green fluorescent protein or sodium acetate buffer solution, the mass ratio of lung organ targeted branched poly (β-amino ester) to mRNA was 40:1, let stand for 15 minutes, added to the serum culture medium, and slowly added to the cells after sufficient mixing. The cells were then cultured for 24 hours, and the cells transfected with GFP mRNA were observed under a fluorescence microscope. The efficiency of transfection of GFP mRNA was tested using a flow cytometer.

[0101] Fig. 9 These are fluorescent photographs of cells after transfection of green fluorescent protein (GFP) mRNA with lung organ-targeted branched poly (β-amino ester) in Examples 1 to 4: The transfection results show that for SW1353 cells, at two different serum concentrations, the four branched poly (β-amino ester) and mRNA composite nanoparticles can all efficiently mediate GFP gene transfection, which is attributed to their excellent serum stability.

[0102] Fig.10 Fluorescence photographs of cells after the lung organ-targeted branched poly (β-amino ester) in Examples 1 to 4 mediated mRNA encoding green fluorescent protein (GFP) in 293T, HeLa, and Vero cells: The transfection results showed that the four branched poly (β-amino ester) and mRNA composite nanoparticles were able to efficiently mediate GFP gene transfection in 293T, HeLa, and Vero cells, which confirms their wide applicability.

[0103] Fig.11 The results are a quantitative evaluation of the transfection performance of lung organ-targeted branched poly(β-amino ester) in 293T cells in mediating mRNA encoding green fluorescent protein (GFP). The flow cytometry results show that in 293T cells, four hyperbranched poly(β-amino ester)s with different molecular weights efficiently mediated GFP mRNA transfection, with a transfection efficiency of 90%.

[0104] Fig.12 The results are a quantitative evaluation of the transfection performance of lung organ-targeted branched poly(β-amino esters) in HeLa cells in mediating mRNA encoding green fluorescent protein (GFP). The flow cytometry results show that in HeLa cells, four hyperbranched poly(β-amino esters) with different molecular weights efficiently mediated GFP mRNA transfection, with a transfection efficiency of 72%.

[0105] Fig.13The results are a quantitative evaluation of the transfection performance of lung organ-targeted branched poly(β-amino ester) in Vero cells encoding green fluorescent protein (GFP). The flow cytometry results show that in Vero cells, four hyperbranched poly(β-amino ester)s with different molecular weights efficiently mediated GFP mRNA transfection, with a transfection efficiency of 99%. These results show that lung organ-targeted branched poly(β-amino ester) has a wide range of application potentials in a variety of tissue cells.

[0106] Example 7

[0107] In vivo organ targeting evaluation. 5ug of Fluc mRNA was diluted in 75uL-100uL sodium acetate buffer solution and mixed. Then 4uL of branched poly (β-amino ester) solution was diluted in 11.5uL of ethanol solution, and 13.5uL of F127 solution was added thereto in sequence and mixed. Finally, the sodium acetate buffer solution of mRNA was added to the branched poly (β-amino ester) solution, vortexed for 25-30s, and incubated at room temperature for 15-30 minutes to form a polyplexes solution. Then 200uL of PBS was added. Let stand for 2 minutes and inject into the tail vein. 6h after injection, the in vivo imaging system was used to illuminate the whole body of the mouse to detect the mRNA transfection efficiency, and then the in vivo toxicity was detected by tissue sections.

[0108] Fig.14 The lung organ targeted branched poly (β-amino ester) in Example 1 and Example 2 was used to evaluate the transfection efficiency of Fluc mRNA in mice. The results of in vivo transfection for 6 hours showed that when the mRNA dosage was 0.5 mg, the lung organ targeted branched poly (β-amino ester) in Example 1 and Example 2 were able to efficiently mediate Fluc mRNA transfection in vivo and highly targeted the lung tissue.

[0109] Fig.15 The lung organ targeted branched poly (β-amino ester) in Example 1 and Example 2 was used to evaluate the transfection efficiency of Fluc mRNA in mice. The results of in vivo transfection for 6 hours showed that when the mRNA dosage was 0.25 mg, the lung organ targeted branched poly (β-amino ester) in Example 1 and Example 2 were able to efficiently mediate Fluc mRNA transfection in vivo and highly targeted the lung tissue.

[0110] Fig.16 The safety evaluation of lung organ targeted branched poly (β-amino ester) in Example 1 and Example 2 after Fluc mRNA transfection in mice was performed. The results of HE staining 24 hours after in vivo transfection showed that the heart, liver, spleen, lung and kidney of mice were normal before and after transfection, and no organ damage or obvious toxicity occurred. These results show that the high-performance lung organ targeted branched poly (β-amino ester) of the present invention has application potential in the clinical treatment of lung-related diseases and the development of viral vaccines.

[0111] 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 lung organ targeted branched poly (β-amino ester), characterized in that Its structural formula is shown in Formula 1 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 diamine monomer; R4 is a group on a functional end-capping agent monomer, which is R4-NH2; n=10-60; m=10-60.

2. The lung organ targeted branched poly (β-amino ester) according to claim 1, characterized in that 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'-isopropyl bis(p-phenyleneoxy)di-2-propanol diacrylate or.

3. The lung organ targeted branched poly (β-amino ester) according to claim 1, characterized in that The organic amine is 4-amino-1-butanol, 5-amino-1-pentanol, butylamine, pentylamine, hexylamine, octylamine, 1-(2-aminoethyl)piperazine, N-(2-aminoethyl)-morpholine, dodecylamine or octadecylamine.

4. The lung organ targeted branched poly (β-amino ester) according to claim 1, characterized in that The diamine monomer is 1,4-butanediamine, 1,2-ethylenediamine, 1,3-propylenediamine, 1,6-hexanediamine, 1,4-bis(3-aminopropyl)piperazine, 1,11-diamino-3,6,9-trioxaundecane or 2-methyl-1,5-pentanediamine.

5. The lung organ targeted branched poly (β-amino ester) 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 the lung organ targeted branched poly (β-amino ester) 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 diamine monomer to obtain a branched poly(β-amino ester) with a double bond at the end; 3) reacting the branched poly(β-amino ester) containing double bonds at the end obtained in step 2) with a functionalized end-capping agent monomer to perform a functionalized end-capping reaction to obtain a lung-targeted branched poly(β-amino ester).

7. The method for preparing a lung organ targeted branched poly (β-amino ester) according to claim 6, characterized in that: In step 1), the molar ratio of the diacrylate monomer to the organic amine is (1-5):(0.5-3); in step 2), the molar ratio of the diamine monomer to the linear poly(β-amino ester) with a double bond at the end is (0.04-1):(1-5).

8. A lung organ targeted branched poly (β-amino ester) based composite nanoparticle, characterized in that: The invention is a composite nanoparticle obtained by compounding the lung organ targeted branched poly (β-amino ester) according to any one of claims 1 to 5 with mRNA.

9. Use of the lung organ targeted branched poly (β-amino ester) according to any one of claims 1 to 5 or the lung organ targeted branched poly (β-amino ester) based composite nanoparticles according to claim 8 in the preparation of gene carriers, characterized in that: The gene vector is used to specifically deliver mRNA to mouse lung tissue.

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