Hyperbranched poly (beta-amino ester)-containing lipid nanoparticle for efficiently delivering mRNA (messenger Ribonucleic Acid) as well as preparation method and application of lipid nanoparticle

Abstract

The invention relates to the technical field of biological medicine. The invention provides highly branched poly (beta-amino ester) and a preparation method thereof. The invention also provides lipid nanoparticles which contain the compound and are used for efficiently delivering mRNA, a preparation method of the lipid nanoparticles, and application of the lipid nanoparticles in preparation of CD5 antibody targeted nucleic acid drug delivery carriers. The highly branched poly (beta-amino ester) prepared by the scheme of the invention has better mRNA binding capacity and in-vivo stability, the obtained lipid nanoparticles have high transfection efficiency, and efficient transfection of specific tissues or cells can be better realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to lipid nanoparticles containing highly branched poly (β-amino ester) for efficient delivery of mRNA, as well as a preparation method and application thereof. Background Art

[0002] In recent years, highly branched poly(β-amino esters) (HPAEs) have attracted widespread attention due to their excellent mRNA binding ability and biocompatibility. However, the transfection efficiency and targeting of HPAEs in existing technologies still need to be further improved.

[0003] Traditional LNP delivery systems usually use ionizable cationic lipid SM102, but they have problems such as insufficient transfection efficiency and poor targeting, especially poor performance in spleen targeted delivery.

[0004] Based on the above, it is very necessary to obtain an efficient targeted modification method to achieve efficient transfection of specific tissues or cells. Summary of the Invention

[0005] The present invention aims to provide lipid nanoparticles containing highly branched poly(β-amino ester) for efficient mRNA delivery, as well as a preparation method and application thereof. The highly branched poly(β-amino ester) prepared using the scheme described in the present invention has good mRNA binding ability and in vivo stability, and the obtained lipid nanoparticles have high transfection efficiency, which can better achieve efficient transfection of specific tissues or cells.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a highly branched poly (β-amino ester), the structural formula of the highly branched poly (β-amino ester) is as follows:

[0008]

[0009] Preferably, in the formula I, m=10-50; 1 The structural formula is as follows:

[0010]

[0011] The R 2 The structural formula is as follows:

[0012]

[0013] The R 3 The structural formula is as follows:

[0014]

[0015] The R 4 Any one of the following structural formulas:

[0016]

[0017] The present invention provides a preparation process of the highly branched poly (β-amino ester), comprising the following steps:

[0018] (1) dissolving bisphenol A glycerate diacrylate, trimethylolpropane triacrylate, 4-(2-aminoethyl)morpholine and dodecylamine in dimethyl sulfoxide and subjecting them to Michael addition reaction to obtain a base polymer;

[0019] (2) reacting the base polymer prepared in step (1) with a capping agent to obtain a highly branched poly(β-amino ester).

[0020] Preferably, the molar ratio of bisphenol A glycerate diacrylate, trimethylolpropane triacrylate, 4-(2-aminoethyl)morpholine and dodecylamine in step (1) is 1.08:(0.06-0.09):(0.6-0.8):0.3.

[0021] Preferably, the end-capping agent in step (2) is 1,11-diamino-3,6,9-trioxoundecane, and the amount of the end-capping agent added is 1.8 times the equivalent of the base polymer.

[0022] The present invention also provides use of the highly branched poly (β-amino ester) in preparing an agent for functional mRNA delivery.

[0023] The present invention also provides a lipid nanoparticle for efficiently delivering mRNA, comprising the following components: the highly branched poly (β-amino ester), auxiliary lipids, DSPE-PEG-Mal-CD5 and EGFP mRNA.

[0024] Preferably, the helper lipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine and / or cholesterol.

[0025] Preferably, the molar ratio of the highly branched poly (β-amino ester), the helper lipid and the DSPE-PEG-Mal-CD5 is (45-55):(47-50):(1-2).

[0026] Preferably, the mass ratio of the highly branched poly (β-amino ester) to EGFP mRNA is (25-35):1.

[0027] Preferably, when the helper lipids are 1,2-distearoyl-sn-glycero-3-phosphocholine and cholesterol, the molar ratio of 1,2-distearoyl-sn-glycero-3-phosphocholine to cholesterol is 10:(37-40).

[0028] The present invention also provides a method for preparing the lipid nanoparticles, comprising the following steps:

[0029] (1) dissolving highly branched poly(β-amino ester), helper lipid, and DSPE-PEG-Mal-CD5 in an organic solvent to obtain a lipid phase;

[0030] (2) dissolving EGFP mRNA in citric acid buffer to obtain mRNA aqueous phase;

[0031] (3) The lipid phase and the mRNA aqueous phase are mixed, vortexed, and incubated to obtain the lipid nanoparticles.

[0032] Preferably, the volume ratio of the lipid phase to the mRNA aqueous phase is (2-4):1.

[0033] Preferably, the vortex speed in step (3) is 1800-2200 rpm, the incubation time is 13-17 min, and the incubation temperature is 25±2°C.

[0034] The present invention also provides the use of lipid nanoparticles prepared by the preparation method in preparing CD5 antibody-targeted nucleic acid drug delivery carriers.

[0035] By adopting the above technical solution, the present invention has the following beneficial effects:

[0036] 1. The highly branched poly(β-amino ester) prepared using the scheme described in the present invention has good mRNA binding ability and in vivo stability; the lipid nanoparticles obtained using the highly branched poly(β-amino ester) have high transfection efficiency, can better achieve efficient transfection of specific tissues or cells, and are suitable for fields such as gene therapy, vaccine development, and immune regulation.

[0037] 2. The experiments of the present invention also show that the prepared CD5 antibody-targeted lipid nanoparticles containing highly branched poly (β-amino ester) can be used to efficiently deliver EGFP mRNA to specific 293T cells, with good transfection effect and high delivery efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a schematic diagram of the LNP preparation process of the present invention;

[0039] Figure 2 FTIR characterization of highly branched poly (β-amino ester) P441;

[0040] Figure 3 Highly branched poly (β-amino ester) P441 1 H NMR characterization;

[0041] Figure 4 Highly branched poly (β-amino ester) P441 13 C NMR characterization;

[0042] Figure 5 Zeta potential diagram of the LNP of the present invention ( Figure 5 A in the figure represents LNPs modified with EGFP mRNA P441 and CD5, and B represents LNPs modified with EGFP mRNA SM102 and CD5).

[0043] Figure 6 is the standard curve of EGFP mRNA;

[0044] Figure 7 Fluorescence microscopy images of 293T cells transfected with the LNP of the present invention ( Figure 7 A in the figure represents LNPs modified with EGFP mRNA P441 and CD5, and B represents LNPs modified with EGFP mRNA SM102 and CD5). DETAILED DESCRIPTION

[0045] The present invention provides a highly branched poly (β-amino ester), the structural formula of the highly branched poly (β-amino ester) is as follows:

[0046]

[0047] In the present invention, in the formula I, m=10-50; 1 The structural formula is as follows:

[0048]

[0049] The R 2 The structural formula is as follows:

[0050]

[0051] The R 3 The structural formula is as follows:

[0052]

[0053] The R 4 Any one of the following structural formulas:

[0054]

[0055] The present invention provides a preparation process of the highly branched poly (β-amino ester), comprising the following steps:

[0056] (1) dissolving bisphenol A glycerate diacrylate, trimethylolpropane triacrylate, 4-(2-aminoethyl)morpholine and dodecylamine in dimethyl sulfoxide and subjecting them to Michael addition reaction to obtain a base polymer;

[0057] (2) reacting the base polymer prepared in step (1) with a capping agent to obtain a highly branched poly(β-amino ester).

[0058] In the present invention, bisphenol A glycerate diacrylate (DD), trimethylpropane triacrylate (TMPTA), 4-(2-aminoethyl)morpholine (90) and dodecylamine (C12) are dissolved in anhydrous dimethyl sulfoxide (DMSO) and subjected to Michael addition reaction to obtain a base polymer (P4410).

[0059] In the present invention, the molar ratio of bisphenol A glycerate diacrylate (DD), trimethylolpropane triacrylate (TMPTA), 4-(2-aminoethyl)morpholine (90) and dodecylamine (C12) is preferably 1.08:(0.06-0.09):(0.6-0.8):0.3, further preferably 1.08:(0.07-0.085):(0.65-0.75):0.3, and even more preferably 1.08:0.08:0.7:0.3. In the present invention, the total amount of bisphenol A glycerate diacrylate (DD), trimethylpropane triacrylate (TMPTA), 4-(2-aminoethyl)morpholine (90) and dodecylamine (C12) and the amount of anhydrous dimethyl sulfoxide are preferably (2.12-2.38 mol):6 mL, further preferably (2.14-2.19 mol):6 mL, and further preferably 2.16 mol:6 mL.

[0060] In the present invention, the temperature of the Michael addition reaction is preferably 85-95°C, more preferably 88-92°C, and more preferably 90°C; the reaction time is preferably 11-15h, more preferably 12-14h, and more preferably 13h.

[0061] In the present invention, the base polymer (P4410) has Mw=22.0 kDa, PDI=3.9, and the structural formula is as follows:

[0062]

[0063] In the present invention, in the structural formula of the base polymer (P4410), n refers to a repeating unit of 1, and the R1 The structural formula is as follows:

[0064]

[0065] The R 2 The structural formula is as follows:

[0066]

[0067] The R 3 The structural formula is as follows:

[0068]

[0069] In the present invention, the base polymer prepared in step (1) is reacted with a capping agent to obtain a highly branched poly(β-amino ester).

[0070] In the present invention, the end-capping agent is 1,11-diamino-3,6,9-trioxoundecane, and its structural formula is as follows:

[0071]

[0072] In the present invention, the amount of the end-capping agent added is 1.8 times the equivalent weight of the base polymer.

[0073] In the present invention, the base polymer undergoes an aminolysis reaction with a capping agent to simultaneously cap the vinyl group and the amino ester group, and the aminolysis introduces additional amide groups (-CONH-) and amino groups (-NH2). The reaction temperature is preferably 25±1°C, and the reaction time is preferably 45-50 hours, more preferably 47-49 hours, and even more preferably 48 hours.

[0074] In the present invention, the aminolysis reaction is preferably followed by precipitation purification and fractionation.

[0075] In the present invention, the precipitation purification method is preferably: the reaction product is subjected to three ether precipitations to remove unreacted base polymer, vacuum dried, and stored at -20°C. Further preferably, the reaction product is slowly poured into 2 volumes of cold ether, centrifuged at 5000 rpm for 5 minutes, the supernatant is discarded, the precipitate is washed 1-2 times with cold ether, and vacuum dried at -0.09 MPa and 40°C for 12 hours to obtain a purified reaction product.

[0076] In the present invention, the fractionation method is preferably: dissolving the purified reaction product in acetone, gradually adding a diethyl ether / acetone mixture, and centrifuging to separate components of different molecular weights. The volume ratio of diethyl ether / acetone in the diethyl ether / acetone mixture is preferably (2-4):1, more preferably (2.5-3.5):1, and even more preferably 3:1; the centrifugal speed is preferably 7500-8500 rpm, more preferably 7800-8200 rpm, and even more preferably 8000 rpm; and the centrifugal time is preferably 8-12 min, more preferably 9-11 min, and even more preferably 10 min.

[0077] The present invention provides use of the highly branched poly (β-amino ester) in preparing an agent for functional mRNA delivery.

[0078] The present invention also provides a lipid nanoparticle for efficiently delivering mRNA, comprising the following components: the highly branched poly (β-amino ester), auxiliary lipids, DSPE-PEG-Mal-CD5 and EGFP mRNA.

[0079] In the present invention, the helper lipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine and / or cholesterol.

[0080] In the present invention, the concentration of the highly branched poly (β-amino ester) is preferably 3.455 mg / mL, the concentration of the 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) is preferably 0.07901 mg / mL, the concentration of the cholesterol is preferably 0.14886 mg / mL, the concentration of the DSPE-PEG-Mal-CD5 is preferably 0.03764 mg / mL, and the concentration of the EGFP mRNA is preferably 0.1-2 mg / mL.

[0081] In the present invention, the molar ratio of the highly branched poly (β-amino ester), helper lipid and DSPE-PEG-Mal-CD5 is preferably (45-55):(47-50):(1-2), further preferably (48-52):(48-49):(1.2-1.8), and further preferably 50:48.5:1.5.

[0082] In the present invention, the mass ratio of the highly branched poly (β-amino ester) to EGFP mRNA is preferably (25-35):1, further preferably (28-32):1, and even more preferably 30:1.

[0083] In the present invention, when the auxiliary lipids are 1,2-distearoyl-sn-glycero-3-phosphocholine and cholesterol, the molar ratio of 1,2-distearoyl-sn-glycero-3-phosphocholine and cholesterol is preferably 10:(37-40), further preferably 10:(38-39), and further preferably 10:38.5.

[0084] In the present invention, the DSPE-PEG-Mal-CD5 is a DSPE-PEG-Mal modified with a CD5 antibody, and the modification process is as follows: (1) activating DSPE-PEG-Mal: dissolving DSPE-PEG-Mal in PBS (pH 7.4) and adding EDC / NHS to activate the maleimide group; (2) antibody coupling: reacting a CD5 antibody (containing a free thiol group or reduced by TCEP) with the activated DSPE-PEG-Mal at room temperature for 2-4 hours, with a molar ratio of 1:5-10; (3) purification: removing uncoupled antibodies and small molecule impurities by dialysis (MWCO 10 kDa); and (4) storage: storing at 4°C in the dark for future use.

[0085] In the present invention, the CD5 antibody was purchased from Affinity Biosciences LTD with a catalog number of DF14431; the DSPE-PEG-Mal was purchased from Shanghai Yuanye Biotechnology Co., Ltd. with a CAS number of 474922-22-0.

[0086] The present invention also provides a method for preparing the lipid nanoparticles, comprising the following steps:

[0087] (1) dissolving highly branched poly(β-amino ester), helper lipid, and DSPE-PEG-Mal-CD5 in an organic solvent to obtain a lipid phase;

[0088] (2) dissolving EGFP mRNA in citric acid buffer to obtain mRNA aqueous phase;

[0089] (3) The lipid phase and the mRNA aqueous phase are mixed, vortexed, and incubated to obtain the lipid nanoparticles.

[0090] In the present invention, highly branched poly (β-amino ester), helper lipid and DSPE-PEG-Mal-CD5 are dissolved in an organic solvent and vortex mixed uniformly. The organic solvent is preferably anhydrous ethanol, and the vortex temperature is preferably 25±1°C.

[0091] In the present invention, the pH value of the citric acid buffer is preferably 4.0; the concentration of the mRNA aqueous phase is preferably 1-3 mg / mL, more preferably 1.5-2.5 mg / mL, and even more preferably 2 mg / mL.

[0092] In the present invention, the volume ratio of the lipid phase to the mRNA aqueous phase is preferably (2-4):1, further preferably (2.5-3.5):1, and further preferably 3:1. The vortex speed in the present invention is preferably 1800-2200 rpm, further preferably 1900-2100 rpm, and further preferably 2000 rpm; the vortex time is preferably 50-70 s, further preferably 55-65 s, and further preferably 60 s; the incubation time is preferably 13-17 min, further preferably 14-16 min, and further preferably 15 min; and the incubation temperature is 25±2°C.

[0093] In the present invention, the incubation is preferably followed by purification, which is preferably performed by diluting with PBS, sterilizing through a 0.22 μm filter membrane, and storing at 4°C.

[0094] The present invention also provides the use of lipid nanoparticles prepared by the preparation method in preparing CD5 antibody-targeted nucleic acid drug delivery carriers.

[0095] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0096] Example 1

[0097] A highly branched poly (β-amino ester) P441, prepared as follows:

[0098] (1) 2.16 mol of bisphenol A glycerate diacrylate (DD), trimethylolpropane triacrylate (TMPTA), 4-(2-aminoethyl)morpholine (90), and dodecylamine (C12) in a molar ratio of 1.08:0.08:0.7:0.3 were dissolved in 6 mL of anhydrous dimethyl sulfoxide and subjected to Michael addition reaction at 90°C for 13 h to obtain a base polymer (P4410) with Mw = 22.0 kDa and PDI = 3.9;

[0099] (2) aminolysis reaction of the base polymer (P4410) and 1,11-diamino-3,6,9-trioxonadecane at 25±1°C for 48 hours to obtain a reaction product; the amount of 1,11-diamino-3,6,9-trioxonadecane added is 1.8 times the equivalent of the base polymer (P4410);

[0100] (3) The reaction product was slowly poured into 2 volumes of cold ether, centrifuged at 5000 rpm for 5 min, the supernatant was discarded, the precipitate was washed 1-2 times with cold ether, and vacuum dried at -0.09 MPa and 40°C for 12 h to obtain the purified product;

[0101] (4) The purified product was dissolved in acetone, and a diethyl ether / acetone mixture (diethyl ether / acetone volume ratio of 3:1) was gradually added. The mixture was centrifuged at 8000 rpm for 10 min to separate the components with different molecular weights, and a highly branched poly(β-amino ester) P441 with a molecular weight of 12.4 kDa and a PDI of 2.9 was obtained.

[0102] Example 2

[0103] A lipid nanoparticle for efficient delivery of mRNA (such as Figure 1 ), the preparation method is as follows:

[0104] (1) 1 mM of highly branched poly(β-amino ester) P441, helper lipid, and DSPE-PEG-Mal-CD5 at a molar ratio of 50:48.5:1.5 were dissolved in 1 L of anhydrous ethanol and vortexed to obtain a lipid phase;

[0105] (2) EGFP mRNA was dissolved in a pH 4.0 citric acid buffer at a mass ratio of highly branched poly(β-amino ester) to mRNA of 30:1 to obtain a 2 mg / mL mRNA aqueous phase;

[0106] (3) The lipid phase and the mRNA aqueous phase were mixed at a volume ratio of 3:1, rapidly vortexed at 2000 rpm for 60 seconds, and incubated at 25 ± 2°C for 15 minutes to obtain lipid nanoparticles;

[0107] (4) Dilute the lipid nanoparticles with PBS, sterilize through a 0.22 μm filter, and store at 4°C.

[0108] Comparative Example 1

[0109] The same method as in Example 2 was used, except that the highly branched poly (β-amino ester) P441 was replaced by SM102 (purchased from https: / / www.medchemexpress.cn / sm-102.html).

[0110] Test Example 1

[0111] The highly branched poly (β-amino ester) P441 prepared in Example 1 was characterized using Fourier transform infrared spectroscopy, and two parallel experiments were performed.

[0112] The results showed that the FTIR spectrum was at 1607 cm -1 and 3360cm -1 Absorption peaks appear (such as Figure 2 shown); 1 The H NMR characterization shows that the methylene peak at 3.5-3.9 ppm is enhanced (e.g. Figure 3 shown); 13 The C NMR characterization results are as follows Figure 4 shown.

[0113] Test Example 2

[0114] The lipid nanoparticle sample prepared in Example 2 or Comparative Example 1 was diluted to an appropriate concentration (LNP nanoparticles with a nucleic acid concentration of 200 ng / μL), ultrasonically dispersed for 5 min, injected into the sample cell, equilibrated for 2 min, and the parameters were set (temperature 25°C, refractive index 1.33). The test was run and the zeta potential and particle size distribution were recorded.

[0115] The LNP particle size distribution diagram determined by dynamic light scattering (DLS) (e.g. Figure 5 As shown in the figure, the average particle size of the EGFP mRNAP441-based CD5-modified LNP prepared in Example 2 was 156.1 nm, and the standard deviation was 86.1 nm; the average particle size of the EGFP mRNASM102-based CD5-modified LNP prepared in Comparative Example 1 was 258.6 nm, and the standard deviation was 116.8 nm.

[0116] The Zeta potential results are shown in Table 1.

[0117] Table 1 Zeta potential of lipid nanoparticles prepared in Example 2 or Comparative Example 1

[0118]

[0119]

[0120] Test Example 3

[0121] A standard curve was prepared using the Invitrogen Ribogreen kit. EGFP mRNA standards were diluted (gradient of 0, 200, 400, 600, 800, and 1000 ng / mL) and an equal volume of RiboGreen reagent (1:200 dilution) was added. After incubation in the dark for 5 minutes, fluorescence was measured using an RNA fluorescence (RiboGreen) quantitative detection kit (purchased from Shanghai Yuduo Biotechnology Co., Ltd.). The linear equation was fitted, F = 33534.38C - 1350000. The standard curve is shown in Figure 2. Figure 6 shown.

[0122] Take 10 μL of the lipid nanoparticle sample prepared in Example 2 or Comparative Example 1, add 90 μL of 0.1% Triton X-100 to lyse it, vortex to mix, and let it stand for 10 minutes. Add 100 μL of RiboGreen working solution, measure fluorescence using the kit, and calculate the total mRNA level based on the standard curve. The total mRNA level is 3684328.

[0123] Take 100 μL of the lipid nanoparticle sample prepared in Example 2 or Comparative Example 1, centrifuge at 10,000 × g for 10 min (molecular weight cutoff 100 kDa), and collect the supernatant (containing free mRNA). Take 50 μL of the supernatant and add 50 μL of RiboGreen working solution. The fluorescence value is measured using the kit, and the amount of free mRNA is calculated according to the above standard curve. The result is 736,866 free mRNA.

[0124] The encapsulation efficiency of each lipid nanoparticle was calculated based on the total mRNA amount and free mRNA amount measured above.

[0125] Encapsulation efficiency (%) = (1-C 游离 / C 总 )×100%

[0126] The results showed that the encapsulation efficiency of the EGFP mRNA P441-based CD5-modified LNP prepared in Example 2 was 95%, and the encapsulation efficiency of the EGFP mRNA SM102-based CD5-modified LNP prepared in Comparative Example 1 was 76%.

[0127] Test Example 4

[0128] Different treatment groups were set up to transfect 293T cells with lipid nanoparticles, and the transfection status of lipid nanoparticles in different treatment groups was measured.

[0129] Example 2 group: EGFPmRNAP441-based CD5-modified LNPs prepared in Example 2

[0130] Comparative Example 1: Comparative Example 1 prepared EGFPmRNA SM102-based CD5 modified LNPs

[0131] Blank control group 1: empty LNP (preparation process is the same as Example 2, only without EGFP mRNA

[0132] Blank control group 2: empty LNP (preparation process is the same as that of comparative example 1, only without EGFP mRNA

[0133] Positive control: commercial transfection reagent Lipofectamine TM 3000 Reagent (purchased from Invitrogen)

[0134] 293T cells (purchased from ATCC CRL-3216) were cultured at 5×10 4 Cells / well were seeded in 24-well plates and cultured overnight at 37°C, 5% CO2 to 70% confluence.

[0135] LNPs from each treatment group were diluted to a final concentration of 50-100 ng mRNA / well and added to serum-free medium, mixing thoroughly to obtain a LNP mixture. The 293T cell culture medium was then aspirated, and 500 μL of the LNP mixture was added to each well. The cells were incubated at 37°C for 4-6 hours, then replaced with complete medium. After an additional 24-48 hours of culture, EGFP expression was assessed using a fluorescence microscope.

[0136] Fluorescence microscopy analysis: Excitation 488 nm, emission 507 nm (EGFP); DAPI channel (358 nm / 461 nm) for nuclear localization. Five fields of view were randomly selected from each well and photographed using a 20× objective lens (maintaining the same exposure parameters).

[0137] ImageJ software was used for analysis, and the channels (EGFP / DAPI) were split and the background was removed by setting a threshold. The fluorescence intensity (Integrated Density) of all target cell areas was measured, and the mean fluorescence intensity (MFI) and the percentage of positive cells were calculated (fluorescence signal > 2 times the SD of the blank control was considered positive). Fluorescence microscopy images were shown in Figure 2. Figure 7 shown.

[0138] Transfection efficiency = (MFI 实验组 -MFI 空白对照组 ) / (MFI 阳性对照组 -MFI 空白对照组 )×100%

[0139] The results showed that the transfection efficiency of Example 2 group reached 89%, and the transfection efficiency of Comparative Example 1 group reached 76%.

[0140] In summary, it can be concluded that the highly branched poly (β-amino ester) P441 prepared by the scheme described in the present invention has good mRNA binding ability and in vivo stability; the CD5 antibody-targeted lipid nanoparticles prepared using the highly branched poly (β-amino ester) P441 can be used to efficiently deliver EGFP mRNA to 293T cells, with good transfection effect and high delivery efficiency.

[0141] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A highly branched poly (β-amino ester), characterized in that The structural formula of the highly branched poly (β-amino ester) is as follows: Wherein, m = 10 to 50; The R 1 The structural formula is as follows: The R 2 The structural formula is as follows: The R 3 The structural formula is as follows: The R 4 Any one of the following structural formulas:

2. The process for preparing the highly branched poly (β-amino ester) according to claim 1, characterized in that: The steps include: (1) dissolving bisphenol A glycerate diacrylate, trimethylolpropane triacrylate, 4-(2-aminoethyl)morpholine and dodecylamine in dimethyl sulfoxide and subjecting them to Michael addition reaction to obtain a base polymer; (2) reacting the base polymer prepared in step (1) with a capping agent to obtain a highly branched poly(β-amino ester).

3. The preparation process according to claim 2, characterized in that: The molar ratio of bisphenol A glycerate diacrylate, trimethylolpropane triacrylate, 4-(2-aminoethyl)morpholine and dodecylamine in step (1) is 1.08:(0.06-0.09):(0.6-0.8):0.

3.

4. The preparation process according to claim 2, characterized in that: The end-capping agent in step (2) is 1,11-diamino-3,6,9-trioxoundecane, and the amount of the end-capping agent added is 1.8 times the equivalent of the base polymer.

5. Use of the highly branched poly (β-amino ester) prepared by the preparation process of claim 3 or 4 in the preparation of an agent for functional mRNA delivery.

6. A lipid nanoparticle for efficient delivery of mRNA, characterized in that The method comprises the following components: the highly branched poly (β-amino ester) according to claim 1, an auxiliary lipid, DSPE-PEG-Mal-CD5 and EGFP mRNA; The helper lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine and / or cholesterol; The molar ratio of the highly branched poly (β-amino ester), helper lipid and DSPE-PEG-Mal-CD5 is (45-55): (47-50): (1-2); The mass ratio of the highly branched poly (β-amino ester) to EGFP mRNA is preferably (25-35):

1.

7. The lipid nanoparticle according to claim 6, characterized in that When the helper lipids are 1,2-distearoyl-sn-glycero-3-phosphocholine and cholesterol, the molar ratio of 1,2-distearoyl-sn-glycero-3-phosphocholine to cholesterol is 10:(37-40).

8. The method for preparing lipid nanoparticles according to claim 6, characterized in that: The steps include: (1) dissolving highly branched poly(β-amino ester), helper lipid, and DSPE-PEG-Mal-CD5 in an organic solvent to obtain a lipid phase; (2) dissolving EGFP mRNA in citric acid buffer to obtain mRNA aqueous phase; (3) mixing the lipid phase with the mRNA aqueous phase, vortexing, and incubating to obtain the lipid nanoparticles; The volume ratio of the lipid phase to the mRNA aqueous phase is (2-4):

1.

9. The preparation method according to claim 8, characterized in that In step (3), the vortex speed is 1800-2200 rpm, the incubation time is 13-17 min, and the incubation temperature is 25±2°C.

10. Use of lipid nanoparticles prepared by the preparation method according to claim 8 or 9 in preparing CD5 antibody-targeted nucleic acid drug delivery vectors.

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