Highly branched poly (beta-amino ester) as well as preparation method and application thereof

Abstract

The invention discloses highly branched poly beta-amino ester as well as a preparation method and application thereof, and belongs to the technical field of chemical synthesis and modification. According to the invention, A < 2 + > B < 3 + > C < 2 > monomers are used for carrying out Michael addition reaction, and the hyperbranched high-molecular polymer with expected size and weight-average molecular weight Mw is obtained by changing the reaction time and the type of a precursor. And an end-capping reagent is introduced, so that the problem of low transfection efficiency caused by A2 type secondary amine formed by A2 type amine reaction is effectively solved. Compared with a carrier in the market, the obtained hyperbranched high-molecular polymer carrier can realize more efficient loading of genes and higher transfection efficiency while ensuring the biological safety. Therefore, a wider market can be provided for high-molecular polymers in nucleic acid drug delivery.

Description

Technical Field

[0001] This invention belongs to the field of chemical synthesis and modification technology, and particularly relates to a highly branched polyβ-amino ester, its preparation method and application. Background Technology

[0002] Urinary tract cancers are among the most common malignant tumors worldwide. Bladder cancer (BCa), in particular, poses a serious threat to human health due to its invasiveness and heterogeneity. Approximately 75% of BCa patients have non-muscle-invasive bladder cancer (NMIBC). Radical cystectomy (TURBT) is the gold standard for bladder cancer treatment, but the 5-year survival rate remains low, at approximately 20% to 40%. In recent years, advancements in gene therapy, immunotherapy, and other treatment modalities have made radical cure of BCa possible. Intravesical instillation therapy maximizes therapeutic efficacy by directly injecting chemotherapy or immunotherapy drugs into the bladder to eradicate residual cancer cells. However, the drug's residence time in the bladder is short, rapidly decreasing during regular urination, often requiring frequent instillation of high concentrations to enhance treatment effectiveness. Furthermore, the toxicity of naked nucleic acid drugs, low cellular uptake, and the effects of enzymes in the body contribute to poor efficacy of direct administration.

[0003] To address these challenges, researchers have developed nanocarrier systems to enhance their accumulation in tumor repositioning. One type is the magnetically responsive carrier, which reacts to external magnetic fields. However, since the bladder is located within the body, an extremely strong external magnetic field is required to retain the magnetic nanoparticles within the bladder. Another type utilizes functional polymers to develop adhesive mucosal systems, thereby prolonging drug retention time in the bladder. For example, positively charged polymers (chitosan-L-lysine) exhibit high binding affinity to the negatively charged bladder mucosa.

[0004] The development of cationic polymers such as linear poly-β-amino esters (LPAE) has shown great potential in the delivery and effective transfection of nucleic acid drugs, and has provided ideas for the development of nucleic acid-carrying polymeric gene vectors. Summary of the Invention

[0005] Considering that three-dimensional (3D) topologies with multiple terminal groups are more conducive to gene transfection, this invention proposes a hyperbranched poly-β-amino ester, its preparation method, and its applications. Hyperbranched poly-β-amino esters (HPAEs) were developed using Michael addition to enhance DNA transfection capabilities. Controlling the ratio of secondary / tertiary amines and DNA in the HPAE component during polymerization and DNA loading is crucial for improving the transfection ability of nucleic acid drugs in bladder cancer cells.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A highly branched polyβ-amino ester, with a structural formula as shown in HPAE-XB or HPAE-XM:

[0008]

[0009] Where n is 10-40.

[0010] The present invention also provides a method for preparing the aforementioned highly branched poly-β-amino ester, comprising the following steps:

[0011] (1) S4 (4-amino-1-butanol), BEDA (bisphenol A ethoxylate diacrylate) and TMPTA (trimethylolpropane triacrylate) were dissolved in DMSO (dimethyl sulfoxide) and heated at a constant temperature of 60-120℃. During the reaction, the Mw of the polymer was determined by GPC (gel permeation chromatography). The reaction was stopped when the Mw of the polymer was close to 3-30 kDa, and the crude product was obtained.

[0012] (2) The crude product is added to DMSO for dilution to obtain the diluted crude product;

[0013] (3) At room temperature, a capping agent was added to the diluted crude product, the polymer Mw = 5-50 kDa was determined, purified and dried to obtain a highly branched poly-β-amino ester with the structure HPAE-XB.

[0014] Alternatively, at room temperature, BEDA is added to the diluted crude product to carry out the reaction, an end-capping agent is added to the product after the reaction, the polymer Mw is measured to be 5-50 kDa, purified, dried, and a highly branched poly-β-amino ester with the structure HPAE-XM is obtained.

[0015] Furthermore, in the process of preparing the crude product, the stoichiometric ratio of S4, BEDA and TMPTA is 1:(0.2-3):(0.3-5).

[0016] Furthermore, in the process of preparing the highly branched polyβ-amino ester with the structure HPAE-XB, 5 equivalents of end-capping agent are added to the diluted crude product.

[0017] In the process of preparing the highly branched polyβ-amino ester with the structure HPAE-XM, 1 equivalent of BEDA is added to the diluted crude product, and 5 equivalents of end-capping agent are added to the product.

[0018] The capping agent is N-(3-aminopropyl)morpholine (MPA).

[0019] Further, the purification step includes: adding excess diethyl ether to the end-capped mixture for precipitation, removing the supernatant, and repeating the operation. The drying refers to drying in a vacuum drying oven at 25°C for 24 hours.

[0020] The present invention also provides a complex with gene transfection capability, which is prepared using the aforementioned highly branched poly-β-amino ester.

[0021] The present invention also provides a method for preparing the aforementioned complex with gene transfection capability, comprising the following steps: dissolving the highly branched poly-β-amino ester in DMSO to obtain a stock solution with a concentration of 50-500 mg / mL; mixing the highly branched poly-β-amino ester with DNA at a mass ratio of (5-40):1; vortexing for 25-30 s; and allowing to stand to obtain the HPAE / DNA complex.

[0022] The present invention also provides the application of the aforementioned highly branched poly-β-amino ester in the preparation of bladder cancer therapeutic drugs.

[0023] The present invention also provides the application of the aforementioned gene transfection-capable complex in the preparation of bladder cancer therapeutic drugs.

[0024] Compared with the prior art, the present invention has the following advantages and technical effects:

[0025] This invention utilizes the Michael addition reaction of "A2+B3+C2" monomers to obtain hyperbranched polymers of the desired size (Mw) by changing the reaction time and the type of precursor. Furthermore, by introducing a capping agent, the low transfection efficiency caused by the formation of secondary amines from the A2-type amine reaction is effectively solved. Compared to commercially available vectors (such as Lipofectamine 3000 and PEI), the obtained hyperbranched polymer vector achieves more efficient gene loading and higher transfection efficiency while ensuring biosafety. This may open up a broader market for polymers in nucleic acid drug delivery. Attached Figure Description

[0026] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings:

[0027] Figure 1 For different HPAE-XB and HPAE-XM 1 HNMR spectrum, x represents 1, 2, 3;

[0028] Figure 2(a) the relative DNA binding affinity of HPAE-XB and HPAE-XM at 5:1; (b) the Zeta potential of HPAE-XB / DNA and HPAE-XM / DNA at 5:1; and (c) the particle size of the HPAE-XB / DNA and HPAE-XM / DNA polymer nanoparticles.

[0029] Figure 3 The images show GFP fluorescence of UM-UC-3 cells with a mass ratio of highly branched poly-β-amino ester to DNA of 20:1 and 40:1, 48 hours after transfection.

[0030] Figure 4 The image shows the GFP fluorescence of UM-UC-3 with a mass ratio of 5:1 and 10:1 of highly branched poly-β-amino ester to DNA 48 hours after transfection. The scale bar is 200 μm.

[0031] Figure 5 The viability of UM-UC-3 cells after transfection with different HPAE / DNA complexes is shown in Figure 1. "*" indicates significantly higher viability than the PEI 25k group and the jetPEI group (p<0.05, Student's test).

[0032] Figure 6 The image shows the GFP fluorescence of SV-HUC-1 with a mass ratio of 5:1 and 10:1 of highly branched poly-β-amino ester to DNA 48 hours after transfection. The scale bar is 200 μm.

[0033] Figure 7 The viability of SV-HUC-1 cells after transfection with different HPAE / DNA complexes is shown in Figure 1. "*" indicates significantly higher viability than the PEI 25k group and the jetPEI group (p<0.05, Student's test).

[0034] Figure 8 Let n be the structural formula for HPAE-XB and HPAE-XM, where n is 10-40. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] This invention provides a method for preparing the aforementioned highly branched poly-β-amino ester (highly branched poly-β-amino ester with the structure HPAE-XB), comprising the following steps:

[0041] (1) S4, BEDA and TMPTA are dissolved in DMSO in a stoichiometric ratio of 1:(0.2-3):(0.3-5) and heated at a constant temperature of 60-120℃, preferably at 90℃. During the reaction, the Mw of the polymer is measured by GPC. The reaction is stopped when the Mw of the polymer is close to 5-30kDa, preferably close to 5-20kDa, to obtain the crude product.

[0042] (2) The crude product is added to DMSO for dilution to obtain the diluted crude product;

[0043] (3) The Mw of the polymer was determined using GPC, Mw = 3-30 kDa;

[0044] (4) At room temperature, add 5 times the equivalent of MPA of the crude product to the diluted crude product, add the mixture to excess diethyl ether under vigorous stirring to precipitate, remove the supernatant, repeat the operation 3 times, and purify the product by drying at 25°C for 24 hours in a vacuum oven to obtain a highly branched poly-β-amino ester with the structure HPAE-XB, and determine its GPC to ensure that Mw is 5-50 kDa.

[0045] This invention also provides a method for preparing the aforementioned highly branched poly-β-amino ester (highly branched poly-β-amino ester with the structure HPAE-XM), comprising the following steps:

[0046] (1) S4, BEDA and TMPTA are dissolved in DMSO in a stoichiometric ratio of 1:(0.2-3):(0.3-5) and heated at a constant temperature of 60-120℃, preferably at 90℃. During the reaction, the Mw of the polymer is measured by GPC. The reaction is stopped when the Mw of the polymer is close to 5-30kDa, preferably close to 5-20kDa, to obtain the crude product.

[0047] (2) The crude product is added to DMSO for dilution to obtain the diluted crude product;

[0048] (3) The Mw of the polymer was determined using GPC, Mw = 4-50 kDa;

[0049] (4) At room temperature, 1 equivalent of BEDA was added to the diluted crude product for reaction, and 5 equivalents of MPA were added to the product after reaction. The mixture was added to excess diethyl ether for precipitation under vigorous stirring. The supernatant was removed and the operation was repeated 3 times. The obtained product was dried in a vacuum oven at 25°C for 24 h for purification to obtain a highly branched polyβ-amino ester with the structure HPAE-XM.

[0050] The present invention also provides a complex with gene transfection capability, which is prepared using the aforementioned highly branched poly-β-amino ester.

[0051] The present invention also provides a method for preparing the aforementioned complex with gene transfection capability, comprising the following steps: dissolving the highly branched poly-β-amino ester in DMSO to obtain a stock solution with a concentration of 50-500 mg / mL, preferably 100 mg / mL; mixing the highly branched poly-β-amino ester with DNA at a mass ratio of (5-40):1, vortexing for 25-30 s, and allowing to stand to obtain the HPAE / DNA complex.

[0052] The present invention also provides the application of the aforementioned gene transfection-capable complex in the preparation of bladder cancer drugs.

[0053] Unless otherwise specified, "room temperature" in this invention refers to 25±2℃.

[0054] All raw materials used in the following embodiments of the present invention are commercially available.

[0055] The following embodiments are further illustrations of the technical solution of the present invention.

[0056] Example 1

[0057] (1) S4 (2.492 g, 0.028 mol), BEDA (4.680 g, 0.01 mol) and TMPTA (3.552 g, 0.012 mol) were dissolved in DMSO (25.02 g, 0.320 mol) and reacted under magnetic stirring at 90 °C;

[0058] (2) During the reaction, GPC was used to determine the Mw of the polymer in step (1). When the Mw approached 5 kDa, the temperature was lowered to room temperature to stop the reaction and the crude product was obtained.

[0059] (3) Dilute the crude product in step (2) with DMSO to obtain a diluted crude product with a concentration of 100 mg / mL;

[0060] (4) The product obtained in step (3) was capped with 5 times the equivalent of MPA at room temperature for 48 hours to obtain crude product HPAE-1-B; the Mw of HPAE-1-B was determined by GPC, and Mw = 6.1 kDa.

[0061] (5) Under vigorous stirring, the product obtained in step (4) is slowly added to excess diethyl ether to precipitate and remove the supernatant. This step is repeated 3 times to purify HPAE-1-B.

[0062] (6) The HPAE-1-B obtained in step (5) was dried in a vacuum oven at 25°C for 24 hours to obtain the final product (polymer highly branched polyβ-amino ester, denoted as HPAE-1-B).

[0063] Example 2

[0064] Same as Example 1, except that in step (4), when Mw approaches 10 kDa, the reaction is stopped at room temperature. The final product is denoted as HPAE-2-B. The Mw of HPAE-1-B is determined using GPC, and Mw = 11.8 kDa.

[0065] Example 3

[0066] Same as Example 1, except that in step (4), when Mw approaches 20 kDa, the reaction is stopped at room temperature. The final product is denoted as HPAE-3-B; the Mw of HPAE-1-B is determined using GPC, and Mw = 22.7 kDa.

[0067] Example 4

[0068] (1) S4 (2.492 g, 0.028 mol), BEDA (4.680 g, 0.01 mol) and TMPTA (3.552 g, 0.012 mol) were dissolved in DMSO (25.02 g, 0.320 mol) and reacted under magnetic stirring at 90 °C;

[0069] (2) During the reaction, GPC was used to determine the Mw of the polymer in step (1). When the Mw approached 5 kDa, the temperature was lowered to room temperature to stop the reaction and the crude product was obtained.

[0070] (3) Dilute the crude product in step (2) with DMSO to obtain a diluted crude product with a concentration of 100 mg / mL;

[0071] (4) React the product obtained in step (3) with 1 equivalent of BEDA at room temperature to obtain the crude product;

[0072] (5) The product obtained in step (4) was capped with 5 times the equivalent of MPA at room temperature for 48 hours to obtain crude product HPAE-1-M; the Mw of HPAE-1-M was determined by GPC, and Mw = 6.5 kDa.

[0073] (6) The product obtained in step (5) was slowly added to an excess of diethyl ether under vigorous stirring to precipitate the product and remove the supernatant. This step was repeated 3 times to purify HPAE-1-M.

[0074] (7) The HPAE-1-M obtained in step (5) is dried in a vacuum oven at 25°C for 24 hours to obtain the final product (polymer highly branched poly-β-amino ester, denoted as HPAE-1-M).

[0075] Example 5

[0076] Same as Example 1, except that in step (4), when Mw approaches 10 kDa, the reaction is stopped by cooling to room temperature. The final product is denoted as HPAE-2-M, and GPC determination shows that the product Mw = 12.5 kDa.

[0077] Example 6

[0078] Same as Example 1, except that in step (4), when Mw approaches 20 kDa, the reaction is stopped by cooling to room temperature. The final product is denoted as HPAE-3-M, and GPC determination shows that the product Mw = 25.8 kDa.

[0079] Performance testing

[0080] 1. Gel permeation chromatography (GPC)

[0081] The number-average molecular weight (Mn), weight-average molecular weight (Mw), and polydispersity index of OPAEs and HPAEs were determined using an Agilent 1260 Infinity II GPC equipped with a refractive index (RI) detector. (Table 1). The sample was dissolved in 1 mL of DMF containing 0.1% lithium bromide, vortexed vigorously for 30 seconds, and then filtered through a 0.22 μm filter. GPC columns (Polarized Gel-MGard, 50 mm × 7.5 mm, Polarized Gel-M, 300 mm × 7.5 mm, two in series) were eluted with DMF (containing 0.1% lithium bromide) at a flow rate of 1 mL / min. The column temperature was maintained at 50 °C. The GPC columns were calibrated using linear poly(methyl methacrylate) (PMMA) standards.

[0082] Table 1. Molecular weight and distribution of different HPAE-XB and HPAE-XM

[0083]

[0084] Table 1 shows the GPC analysis results of the polymers synthesized in Examples 1-6. It can be seen that the Mw values ​​of the obtained polymers are 6100, 6500, 11800, 12500, 22700 and 25800 Da, respectively.

[0085] 2. Nuclear Magnetic Resonance (NMR) Measurement

[0086] Using proton NMR ( 1 H NMR confirmed the chemical composition and structure of HPAEs. Figure 1 The sample was dissolved in CDCl3. 1-hour NMR measurements were performed using a Varian Inova 400MHz spectrometer (Bruker, Switzerland). Chemical changes are reported in parts per million (ppm), involving solvent CDCl3 (7.26 ppm) and TMS (0.00 ppm).

[0087] Figure 1 The NMR spectra of the polymers synthesized in Examples 1-6 show that the peak positions of different polymers are close, indicating that the synthesized HPAEs have similar structures.

[0088] 3.1) Determination of HPAEs / DNA binding affinity

[0089] HPAE / DNA binding affinity was assessed using the PicoGreen assay. Highly branched poly-β-amino ester was dissolved in dimethyl sulfoxide to obtain a stock solution with a concentration of 100 mg / mL. 0.5 μg of DNA was used for each sample. Figure 2 A polymer (a gene transfection-capable complex) was formed at a mass ratio of highly branched poly-β-amino ester to DNA of 5:1. Next, an equal volume of PicoGreen solution was added to the polymer, and the mixture was incubated for another 5 minutes. The resulting solution was divided into three aliquots and diluted in 200 μL of serum-free Dulbecco modified Eagle medium (DMEM) in black 96-well plates. Fluorescence measurements were performed using a plate reader at an excitation wavelength of 490 nm and an emission wavelength of 535 nm. Each experiment was repeated at least three times. The negative control consisted of a sample prepared with naked DNA, while the blank was prepared without DNA. The DNA binding affinity of HPAE was calculated using the following formula:

[0090] DNA binding affinity (%) = 1 - (F Sample -F Blank ) / (F DNA -F Blank )

[0091] Among them, F Sample F DNA and F Blank These represent the fluorescence intensity of the sample, control, and blank, respectively.

[0092] 2) Size and zeta potential measurement

[0093] 0.5 μg of DNA was used for each sample. Highly branched poly(β-amino ester) was dissolved in dimethyl sulfoxide to obtain a stock solution with a concentration of 100 mg / mL, as described above. Polymers were prepared at a mass ratio of highly branched poly(β-amino ester) to DNA of 5:1, and then diluted to 1 mL with deionized water. Size and zeta potential were measured using a Morven instrument (Nano ZSE) at a 90° scattering angle. All experiments were repeated at least three times.

[0094] Figure 2 The figures show the determination of HPAE / DNA binding affinity (a), zeta potential (b), and particle size distribution after DNA binding (c). As can be seen from the figures, HPAE-XM exhibits higher DNA affinity compared to HPAE-XB. After binding with DNA, the polymeric nucleic acid drug delivery system shows a higher zeta potential, indicating better stability. Overall, particle size and affinity are negatively correlated.

[0095] 4.1) Cell culture, cell culture products

[0096] Human bladder transitional cell carcinoma (UM-UC-3) and human bladder immortalized epithelial cells (SV-HUC-1) were both derived from MeisenCTCC. UM-UC-3 cells were cultured in DMEM supplemented with 1% penicillin / streptomycin (P / S) and 10% fetal bovine serum (FBS). SV-HUC-1 cells were cultured in Ham's F-12 nutrient medium (F12) supplemented with 1% P / S and 10% FBS. Both cell lines were cultured under standard conditions in a humidified incubator at 37°C and 5% CO2.

[0097] 2) Evaluation of gene transfection efficiency

[0098] UM-UC-3 and SV-HUC-1 cells were grown at 2 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of 1,000 cells / well in 96-well plates and cultured overnight at 37°C. GFP (green fluorescent protein) was used as a reporter gene to qualitatively and quantitatively assess the gene transfection efficiency of HPAEs. 0.5 μg of DNA was used for each transfection well. Highly branched poly(β-amino ester) was dissolved in dimethyl sulfoxide to obtain a stock solution with a concentration of 100 mg / mL. Polymers were prepared at highly branched poly(β-amino ester) to DNA mass ratios of 5:1, 10:1, 20:1, and 40:1. Figures 3-7 Subsequently, 90 μL of DMEM supplemented with 1% P / S and 10% FBS was added to dilute the polyploid solution to a final volume of 100 μL. Cell supernatant was aspirated from the wells using a pipette, and then 100 μL of DMEM containing the prepared peptide was added. Cells transfected with commercial gene transfection reagents PEI25k and jetPEI served as positive controls. Untreated wells served as negative controls. After 48 hours, cells were washed three times with PBS. GFP expression was observed using a fluorescence microscope (OlympuCKX53). All experiments were performed in triplicate.

[0099] Figure 3 The images show GFP fluorescence of UM-UC-3 cells with high branched poly-β-amino ester to DNA mass ratios of 20:1 and 40:1 48 hours after transfection. HPAE-3-M showed higher transfection efficiency in UM-UC-3 cells compared to commercially available PEI25k and jetPEI.

[0100] Figure 4 The images show GFP fluorescence of UM-UC-3 cells with a hyperbranched poly(β-amino ester) to DNA mass ratio of 5:1 and 10:1 48 hours after transfection. HPAE-2-M exhibited significantly higher transfection efficiency at a hyperbranched poly(β-amino ester) to DNA mass ratio of 5:1.

[0101] Figure 5 To assess the cell viability of UM-UC-3 cells transfected with different HPAE / DNA complexes, compared to PEI25k and jetPEI, HPAE / DNA exhibited lower cell viability when the mass ratio of highly branched poly-β-amino ester to DNA was 10:1, which may be a potential material for nucleic acid drug delivery.

[0102] Figure 6 The image shows GFP fluorescence in SV-HUC-1 cells 48 h after transfection. In the image, HPAE-2-B and HPAE-2-M showed higher transfection efficiency when the mass ratio of highly branched poly-β-amino ester to DNA was 5:1 and 10:1.

[0103] Figure 7 The figure shows the viability of SV-HUC-1 cells after transfection with different HPAE / DNA complexes. When the mass ratio of highly branched poly-β-amino ester to DNA is 5:1, the biocompatibility of HPAE-3-B and HPAE-3-M is close to that of commercially available PEI25k and jetPEI.

[0104] The above are merely preferred embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A highly branched polyβ-amino ester, characterized in that, The structural formula is shown as HPAE-XB or HPAE-XM: Where n is 10-40.

2. A method for preparing highly branched polyβ-amino ester as described in claim 1, characterized in that, Includes the following steps: S4, BEDA and TMPTA were dissolved in dimethyl sulfoxide, and the reaction was stopped after constant heating until the polymer Mw = 3-30 kDa to obtain the crude product. The crude product was diluted with dimethyl sulfoxide to obtain the diluted crude product. At room temperature, a capping agent was added to the diluted crude product, the polymer Mw was determined to be 5-50 kDa, purified, and dried to obtain a highly branched poly-β-amino ester with the structure HPAE-XB. Alternatively, at room temperature, BEDA is added to the diluted crude product to carry out the reaction, an end-capping agent is added to the product after the reaction, the polymer Mw is measured to be 5-50 kDa, purified, dried, and a highly branched poly-β-amino ester with the structure HPAE-XM is obtained.

3. The method for preparing highly branched polyβ-amino ester according to claim 2, characterized in that, In the process of preparing the crude product, the stoichiometric ratio of S4, BEDA and TMPTA is 1:(0.2-3):(0.3-5); the isothermal heating temperature is 60-120℃.

4. The method for preparing highly branched polyβ-amino ester according to claim 2, characterized in that, In the process of preparing the highly branched polyβ-amino ester with the structure HPAE-XB, 5 times the equivalent of the end-capping agent is added to the diluted crude product. In the process of preparing the highly branched polyβ-amino ester with the structure HPAE-XM, 1 equivalent of BEDA is added to the diluted crude product, and 5 equivalents of end-capping agent are added to the product. The capping agent is 3-marinylamine.

5. The method for preparing highly branched polyβ-amino ester according to claim 2, characterized in that, The purification steps include: adding excess diethyl ether to the end-capped mixture for precipitation, removing the supernatant, and repeating the operation.

6. A complex with gene transfection capability, characterized in that, It is prepared using the highly branched polyβ-amino ester as described in claim 1.

7. A method for preparing a complex with gene transfection capability as described in claim 6, characterized in that, The process includes the following steps: dissolving the highly branched poly-β-amino ester in dimethyl sulfoxide to obtain a stock solution with a concentration of 50-500 mg / mL; mixing the highly branched poly-β-amino ester with DNA at a mass ratio of (5-40):1; vortexing for 25-30 s; and allowing to stand to obtain the HPAE / DNA complex.

8. The use of the highly branched poly-β-amino ester as described in claim 1 in the preparation of a bladder cancer therapeutic agent.

9. The use of the complex with gene transfection capability as described in claim 6 in the preparation of a bladder cancer therapeutic agent.

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