End-capped zwitterionic functionalized highly branched poly(beta-amino esters) and methods of making and using the same

Abstract

The application discloses a kind of highly branched poly (beta-amino ester) with zwitterionic functionalization at the end and a preparation method and application thereof, and belongs to the technical field of biomedical materials. By selecting different diacrylate monomers, trifunctional branched monomers, small molecule amines, diamine monomers and zwitterionic monomers, highly branched poly (beta-amino ester) containing zwitterions at the end is prepared, and efficient protein delivery in animal cells is realized. Compared with traditional cationic polymers, the polymer is simple to synthesize, has higher cell viability, more effective protein encapsulation and better protein delivery performance. Compared with a representative commercial protein delivery reagent (PULSin), the raw materials are readily available and inexpensive, and have significant clinical application potential in protein drug delivery.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a class of highly branched poly(β-amino esters) with zwitterionic functionalization at the ends, their preparation methods, and applications. Background Technology

[0002] Protein delivery has shown great promise in fields such as inactivated vaccines, oncology, small molecule drug replacement, and gene therapy. Compared to DNA and RNA, protein delivery offers significant advantages, including not requiring entry into the cell nucleus, avoiding transgenic insertion mutations, preventing off-target effects, and eliminating the need for transcription and translation. Proteins act more directly and rapidly on organisms, exhibiting higher efficiency and faster kinetics. Furthermore, many cutting-edge bioengineering techniques require the assistance of proteins; for example, the Cas9 protein can be used for gene editing and disease treatment. However, the current lack of safe and efficient protein delivery vectors severely limits their clinical application.

[0003] Currently, the commonly used commercial reagent PULSin has certain protein delivery capabilities and biocompatibility, but its high price and the need for matching buffer solutions greatly limit its commercial application. Although the potential toxicity of carrier materials can be adjusted by controlling the size and surface modification of metal ions, metal composites, and inorganic nanoparticles, their in vivo accumulation and recalcitrant degradation characteristics also severely limit their biological applications.

[0004] Due to their abundant monomer variety, low cost, easily tunable structure and properties, and biodegradability, poly(β-amino esters) have shown great promise as a highly efficient cationic polymer carrier for drug delivery (such as proteins and nucleic acids). Highly branched poly(β-amino esters) (HPAEs), with their multiple terminal groups and topological structure, have exhibited exceptionally good DNA and RNA delivery performance both in vivo and in vitro. Zwitterions, due to their good biocompatibility and the coexistence of positive and negative charges, are widely used in biomimetic materials and medical devices. However, there are no reports on the use of zwitterion-functionalized poly(β-amino esters) for protein delivery, severely limiting their potential application in protein carrier development and clinical treatment. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a class of highly branched poly(β-amino esters) with zwitterionic functionalization at the ends, their preparation methods and applications, to solve the problem of protein delivery carriers.

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

[0007] This invention discloses a class of highly branched poly(β-amino esters) with zwitterionic functionalization at the ends, the structural formula of which is as follows:

[0008]

[0009] Where n = 5–30; m = 5–30;

[0010] R0 is a diacrylate monomer; R1 is a triacrylate monomer; R2 is a small molecule amine; R3 is a diamine monomer; and R4 is a zwitterionic end-capping agent.

[0011] Preferably, the structural formula of R0 is one of the following:

[0012]

[0013] The structural formula of R1 is one or more of the following:

[0014]

[0015] The structural formula of R2 is one of the following:

[0016]

[0017] The structural formula of R3 is one of the following:

[0018]

[0019] The structural formula of R4 is one of the following:

[0020]

[0021] Preferably, the highly branched poly(β-amino ester) with zwitterionic functionalization at the ends has a molecular weight in the range of 5,000 to 50,000 Da.

[0022] This invention also discloses a method for preparing the above-mentioned type of highly branched poly(β-amino ester) with zwitterionic functionalization at the ends, the steps of which are as follows:

[0023] 1) Add diacrylate monomers, triacrylate monomers, and small molecule amine monomers to dimethyl sulfoxide and mix thoroughly;

[0024] 2) Add the diamine monomer to step 1) and continue the reaction;

[0025] 3) Add a zwitterionic end-capping agent to the polymer after end-capping in step 2) to end-cap, and obtain a highly branched poly(β-amino ester) with zwitterionic functionalization at the end;

[0026] The molar ratio of reactive functional groups of diacrylate monomers, triacrylate monomers and small molecule amine monomers is 1:(0.2-0.6):(0.2-0.8); the molar ratio of diamine monomers to excess acrylate functional groups is (2-5):1.

[0027] Preferably, the reaction conditions for step 1) are: reacting at 60–120°C for 6–48 h.

[0028] Preferably, in step 2), before adding the diamine monomer, gel permeation chromatography is used to monitor the highly branched poly(β-amino ester) with zwitterionic functionalization at the end. When the molecular weight reaches 4,000 to 20,000 Da, the reaction is terminated.

[0029] Preferably, the reaction conditions for step 2) are: stirring at 20–50°C for 2–6 hours.

[0030] Preferably, the reaction conditions for step 3) are: reacting at 25°C for 4–24 hours.

[0031] This invention also discloses the application of the above-mentioned highly branched poly(β-amino ester) with zwitterionic functionalization at the ends in the preparation of protein drug delivery carriers.

[0032] Preferably, the mass ratio of highly branched poly(β-amino ester) with zwitterionic functionalization at the ends to the protein is (5-100):1.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] This invention provides a class of highly branched poly(β-amino esters) with zwitterionic functionalization at the ends. The polymer is synthesized using a simple and convenient one-pot method. Acrylate monomers and amine small molecules are synthesized into a polymer backbone using a Michael addition strategy. Diamine monomers are used for end-capping, and zwitterionic monomers are used to functionalize the end-capped polymer. After purification, the zwitterionic functionalized polymer encapsulates proteins to form nanoparticles for biological delivery. By using different monomer combinations and controlling the branching structure ratio, and using zwitterionic end-capping monomers, a class of biodegradable highly branched polymers with controllable structural components, structure, and surface charge can be synthesized. Because the polymer backbone contains ester bonds (R0 and R1 monomers contain ester bonds, which can be hydrolyzed under physiological conditions), it exhibits good biodegradability and branching structure control. Ionizable or positively charged groups (tertiary amines generated in the reaction and primary amines obtained from end-capping) can effectively encapsulate proteins, thereby completing delivery. Due to its hyperbranched three-dimensional topology and surface encapsulation, which enhances its ability to encapsulate and protect proteins, it exhibits extremely high protein delivery efficiency and cell activity, showing potential for clinical application. In vitro cell delivery experiments validated the protein delivery potential of highly branched poly(β-amino ester) with zwitterionic functionalization at its ends, demonstrating promising clinical translation prospects.

[0035] This invention provides a method for preparing highly branched poly(β-amino esters) with zwitterionic functionalization at the ends. The reagents used are all commercially available diacrylate monomers, triacrylate monomers, small molecule amines, small molecule diamine monomers, and zwitterionic-terminated monomers. Highly branched poly(β-amino esters) with zwitterionic functionalization at the ends are prepared by a simple Michael addition method. The entire preparation process has advantages such as low cost (the cost of zwitterionic highly branched poly(β-amino esters) is lower than that of the mainstream commercial protein delivery reagent PULSin), simple synthesis route, and easy control of chemical composition and structural properties.

[0036] Furthermore, by monitoring the polymer molecular weight using gel permeation chromatography, the polymer molecular weight was controlled at 4,000–20,000 Da before being capped with diamine monomer, and the molecular weight of the polymer after zwitterionic functionalization was controlled at approximately 5,000–50,000 Da. Attached Figure Description

[0037] Figure 1 This is a schematic diagram illustrating the synthesis of the highly branched poly(β-amino ester) with zwitterionic functionalization at the ends, as described in this invention.

[0038] Figure 2 The image shows the gel permeation chromatography (GPC) curve of the highly branched poly(β-amino ester) diamine monomer with zwitterionic functionalization at the end, which was not purified before being capped in Example 1 of the present invention.

[0039] Figure 3 The diamine monomer with zwitterionic functionalization at the end, prepared in Example 1 of this invention, is capped with a zwitterionic functionalized highly branched poly(β-amino ester) diamine monomer. 1 H NMR spectrum;

[0040] Figure 4 The image shows the gel permeation chromatography (GPC) curves before and after zwitterion capping of a highly branched poly(β-amino ester) with a molecular weight of 18,700 Da and zwitterion-functionalized ends obtained in Example 1 of the present invention.

[0041] Figure 5 The physical morphology diagram shows the highly branched poly(β-amino ester) with zwitterionic functionalization at the end obtained in Example 1 of the present invention.

[0042] Figure 6 This is a test diagram of a highly branched poly(β-amino ester) with zwitterionic functionalization at the end, with a molecular weight of 18,700 Da, prepared in Example 1 of the present invention, encapsulating a protein.

[0043] Figure 7 The graph shows the particle size and surface potential test results of the complex formed by the highly branched poly(β-amino ester) with zwitterionic functionalization at the end, which has a molecular weight of 18,700 Da and is prepared in Example 1 of the present invention, and the protein.

[0044] Figure 8 The image shows the microstructure of a complex formed by a highly branched poly(β-amino ester) with zwitterionic functionalization at the ends, which has a molecular weight of 18,700 Da and is prepared in Example 1 of the present invention, and a protein.

[0045] Figure 9 The image shows the fluorescence effect of highly branched poly(β-amino ester) with a molecular weight of 18,700 Da and zwitterionic functionalization at the end, prepared in Example 1 of the present invention, after 24 hours of protein delivery to HeLa cells. From left to right and top to bottom, the images show the fluorescence effect of HeLa cells 24 hours after BSA-FITC delivery, including the commercial reagent PULSin, HPAE-E (mass ratio 5:1), HPAE-ED (mass ratio 5:1), BSA-FITC alone, HPAE-E (mass ratio 10:1), and HPAE-ED (mass ratio 10:1).

[0046] Figure 10 The image shows the fluorescence intensity of HeLa cells 24 hours after delivery of a highly branched poly(β-amino ester) protein with a molecular weight of 18,700 Da and zwitterionic functionalization at the end, prepared in Example 1 of the present invention.

[0047] Figure 11 The image shows the activity results of HeLa cells 24 hours after delivery of a highly branched poly(β-amino ester) protein with a molecular weight of 18,700 Da and zwitterionic functionalization at the end, prepared in Example 1 of the present invention. Detailed Implementation

[0048] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0049] The present invention will now be described in further detail with reference to the accompanying drawings:

[0050] This invention provides a method for synthesizing highly branched poly(β-amino esters) with zwitterionic functionalization at the ends. The synthetic route is described in [reference needed]. Figure 1 This includes the following steps:

[0051] 1) React diacrylate monomers, triacrylate monomers, and small molecule amine monomers at 60–120 °C for 6–48 h;

[0052] The molar ratio of reactive functional groups containing diacrylate monomers, triacrylate monomers, and small molecule amine monomers is 1:(0.2~0.6):(0.2~0.8).

[0053] The diacrylate monomer (R0) is a diacrylate monomer, including one of the following formulas:

[0054]

[0055] The triacrylate monomer (R1) mentioned is one or more of the acrylates in the following formula:

[0056]

[0057] Small molecule amine monomers (R2) include monomers with different numbers of amino groups and molecular structures, and their structures are one of the following formulas:

[0058]

[0059] 2) During the reaction, gel permeation chromatography was used to monitor the highly branched poly(β-amino ester) with zwitterionic functionalization at the end. When the molecular weight of the polymer reached 4,000 to 20,000 Da, the reaction was terminated. A small molecule diamine monomer was added for end capping. The molar ratio of the end capping agent diamine monomer to the excess acrylate functional group was (2 to 5): 1. The end capping conditions were 20 to 50 °C with stirring for 2 to 6 hours.

[0060] The structure of the small molecule diamine monomer (R3) is one of the following formulas:

[0061]

[0062] 3) The polymer after being capped with diamine monomer was purified by diethyl ether precipitation to obtain HPAE-E, which was characterized by NMR to confirm that the carbon-carbon double bond had been completely consumed.

[0063] 4) Add a certain amount of functionalized zwitterionic end-capping agent and dimethyl sulfoxide to the reaction system in step 3) and react at 25°C for 4 h to 24 h.

[0064] The molar ratio of the zwitterionic end-capping agent (R4) to the excess amino functional groups of the polymer is (2-5):1; the end-capping agent is a zwitterionic monomer with one of the following structures:

[0065]

[0066] 4) The product was purified by precipitation and vacuum dried to obtain a highly branched poly(β-amino ester) with zwitterionic functionalization at the ends, namely HPAE-ED, with the following structural formula:

[0067]

[0068] Where n = 5 to 30; m = 5 to 30.

[0069] The highly branched poly(β-amino ester) with zwitterionic functionalization at its ends, prepared according to the present invention, is used for protein delivery, comprising the following steps:

[0070] 1) Cell culture: HeLa cells were cultured under standard culture conditions at a density of 0.5 × 10⁻⁶ cells / mL. 4 ~2.0×10 4 Cells were seeded at a density of 10 cells per well in a 96-well plate.

[0071] 2) When the cell density reaches 60%–90%, a solution of highly branched poly(β-amino ester) with zwitterionic functionalized ends is vortexed at high speed for 15–60 s with a solution of bovine serum albumin (BSA) labeled with FITC (fibrillated isothiocyanate) for 10–30 min, and then transferred to cells for protein delivery. The polymer:protein mass ratio is (5–100):1, and the protein mass per well is 0.1–10 μg.

[0072] 3) After 4 to 48 hours of protein delivery, observe and photograph the cells containing delivered green fluorescent protein under a fluorescence microscope.

[0073] 4) After protein delivery for 4 to 48 hours, remove the culture medium, wash the cells 2 to 3 times with phosphate buffer, and then quickly detect the fluorescence intensity in a microplate reader to test the fluorescence intensity of the cells after protein delivery.

[0074] 5) After protein delivery for 4-48 hours, remove the cell supernatant, add Alamar Blue solution, and incubate in an incubator for 30-120 minutes to perform cell viability testing.

[0075] Example 1

[0076] 1. Synthesis of highly branched poly(β-amino ester) with zwitterionic terminal functionalization

[0077] 1) Bisphenol A ethoxylated diacrylate (EO / phenol = 1.5), trimethylolpropane triacrylate, and 4-amino-1-butanol were added to dimethyl sulfoxide in a molar ratio of 8:2:5, and the Mike addition reaction was carried out in a one-pot manner at 80°C for 12-20 hours.

[0078] 2) During the reaction, gel permeation chromatography (GPC) was used to monitor the highly branched poly(β-amino ester) with zwitterionic functionalization at the ends. The reaction was terminated when the molecular weight of the polymer reached 4,000–20,000 Da. The gel permeation chromatography curve is shown below. Figure 2 As shown;

[0079] 3) Then 1,3-propanediamine and dimethyl sulfoxide were added to the reaction system of step 2) and reacted at 25°C for 4 h to obtain the reaction product; wherein, the molar ratio of 1,3-propanediamine to the excess acrylate functional group of the polymer was 5:1.

[0080] 4) The polymer capped with diamine monomers was purified by ether precipitation to obtain HPAE-E, which was then analyzed by NMR. The results are as follows: Figure 3 As shown, it is confirmed that the carbon-carbon double bond is completely consumed;

[0081] 5) Zwitterionic 2-methacryloyloxyethyl phosphocholine was used as a capping agent, and the polymer purified in step 4) was dissolved in dimethyl sulfoxide. The amino ratio of the capping agent to the polymer was 3:1, and the reaction was carried out at 25°C for 6 hours to obtain the reaction product. The gel permeation chromatogram of the reaction product is shown in Figure 5. Figure 4 As shown;

[0082] 6) The reaction product from step 5) was purified by ether precipitation, vacuum dried, and then lyophilized to obtain a highly branched poly(β-amino ester) with zwitterionic functionalization at the ends, namely HPAE-ED, with a molecular weight of 18,700 Da. The structural formula is as follows:

[0083]

[0084] The physical phase diagram of the branched poly(β-amino ester) obtained above at 25°C is shown below. Figure 5 As shown.

[0085] 2. Protein encapsulation assay was performed on the synthesized highly branched poly(β-amino ester) with zwitterionic terminal functionalization.

[0086] First, highly branched poly(β-amino ester) solutions with zwitterionic functionalization at the ends were dissolved in sodium acetate buffer (pH = 5.2, 0.025 M) at mass ratios of 5:1, 10:1, and 30:1, respectively. The polymer solution was then added to a phosphate buffer solution of protein (BSA-FITC), with 10 μg of protein added. The mixture was vortexed at high speed for 15–60 s, then allowed to stand for 10–30 min. The complex nanoparticle solution was diluted to 1 mL with distilled water. The nanoparticles and BSA-FITC solution were centrifuged at 15000 rpm for 30 min, and 100 μL of the supernatant was collected from each well of a 96-well plate. Fluorescence measurements were performed using a plate reader (Synergy HybridH1, Biotek) at an excitation wavelength of 463 nm and an emission wavelength of 525 nm. All experiments were repeated three times. An equal volume of BSA-FITC (10 μg) served as a negative control, and samples without BSA-FITC were used as blanks. The fluorescence intensity of the excited particles was detected, and the protein was encapsulated by zwitterionic-functionalized, highly branched poly(β-amino ester). Finally, DLS was used to measure the particle size and surface potential, and the composite nanoparticle solution was freeze-dried and characterized by TEM.

[0087] See protein encapsulation test results. Figure 6 , Figure 6It was confirmed that at mass ratios of 5:1, 10:1, and 50:1, the highly branched poly(β-amino ester) with zwitterionic functionalization at the ends, with a molecular weight of 18,700 Da, prepared in Example 1, achieved protein encapsulation efficiencies exceeding 50%, reaching as high as 70%, demonstrating its excellent protein encapsulation properties. DLS analysis results are available in [reference needed]. Figure 7 , Figure 7 The results confirmed that the highly branched poly(β-amino ester) with zwitterionic functionalization at the end and a molecular weight of 18,700 Da prepared in Example 1 could effectively compress proteins, forming 600 nm protein particles into 300–500 nm nanoparticles at a mass ratio of 10:1, further demonstrating that the resulting complex nanoparticles are more conducive to cellular uptake. Meanwhile… Figure 7 The results confirmed that the highly branched poly(β-amino ester) with a molecular weight of 18,700 Da and zwitterionic functionalization at the ends, prepared in Example 1, could effectively shield the negative potential of the protein itself. The higher the zeta potential of the resulting complex nanosurface, the more positively charged the nanoparticles formed. TEM characterization results are shown below. Figure 8 , Figure 8 The results confirmed that the nanoparticles formed by the highly branched poly(β-amino ester) with zwitterionic functionalization at the end and protein with a molecular weight of 18,700 Da prepared in Example 1 had a uniform particle size distribution and a relatively stable structure, demonstrating its good stability.

[0088] 3. Extracellular protein delivery experiments were conducted using the highly branched poly(β-amino ester) with zwitterionic functionalization at its terminals prepared in Example 1.

[0089] HeLa cells were cultured under standard culture conditions at a concentration of 2.0 × 10⁻⁶. 4 Cells were seeded at a density of 50%–80% in 96-well plates. Protein delivery was then performed, with at least three parallel replicates. A corresponding mass of zwitterionic-functionalized highly branched poly(β-amino ester) solution was added to the protein solution, with a zwitterionic-functionalized highly branched poly(β-amino ester) to BSA-FITC mass ratio of 5:1 and 10:1. 1 μg of fluorescent protein was used per well. The mixture was vortexed at high speed for 15–60 s, then allowed to stand for 10–30 min to form composite nanoparticles, which were then transferred to 96-well plates for protein delivery. After culturing for 24 h, the culture medium was discarded, and the cells were washed 2–3 times with phosphate buffer. Cells delivering green fluorescent protein were observed and photographed under a fluorescence microscope. Fluorescence intensity was measured using a microplate reader at an excitation wavelength of 463 nm and an emission wavelength of 525 nm (λex = 463 nm, λem = 525 nm).

[0090] See the protein delivery performance evaluation results. Figure 9 ,Depend on Figure 9 As can be seen, the highly branched poly(β-amino ester) with zwitterionic functionalization at the end prepared in this invention exhibits higher cellular fluorescence than poly(β-amino ester) with amino-terminated end that has not been further zwitterionic functionalized, due to its multiple terminal groups, flexible chemical composition and good biocompatibility.

[0091] See the results of the pre-fluorescence evaluation of cells. Figure 10 ,Depend on Figure 10 It can be seen that the highly branched poly(β-amino ester) with zwitterionic functionalization at the end prepared by the present invention can significantly improve the fluorescence intensity of poly(β-amino ester) after protein delivery.

[0092] 4. The branched poly(β-amino ester) prepared in Example 1 was tested for its extracellular quantitative protein delivery efficiency and cell viability.

[0093] HeLa cells were cultured under standard culture conditions at a concentration of 2.0 × 10⁻⁶. 4 Cells were seeded at a density of 50%–80% in 96-well plates, and protein delivery was performed, with at least three parallel replicates. A corresponding mass of zwitterionic-functionalized highly branched poly(β-amino ester) solution was added to the protein solution, with a zwitterionic-functionalized highly branched poly(β-amino ester) to BSA-FITC mass ratio of 5:1 and 10:1. 1 μg of fluorescently labeled protein was used per well. The mixture was vortexed at high speed for 15–60 s, then allowed to stand for 10–30 min to form composite nanoparticles, which were then transferred to 96-well plates for protein delivery. After culturing for 24 h, the culture medium was discarded, and the cells were incubated in Alamar Blue solution for 30–120 min for cell viability testing. Fluorescence intensity was measured using a microplate reader at an excitation wavelength of 530 nm and an emission wavelength of 590 nm.

[0094] See cell viability evaluation results. Figure 11 The cell viability results show that, due to the excellent biocompatibility and degradability of highly branched poly(β-amino ester) with zwitterionic functionalization at the ends, cells still maintain high cell viability after protein delivery. This indicates that highly branched poly(β-amino ester) with zwitterionic functionalization at the ends has a significant advantage in protein delivery. See the protein delivery performance evaluation results below. Figure 11 The protein delivery results showed that in HeLa cells, highly branched poly(β-amino ester) with zwitterionic functionalization at the ends can efficiently mediate protein delivery using BSA-FITC as a model due to its multiple terminal groups, flexible chemical composition, good biocompatibility, and high biocompatibility.

[0095] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A class of highly branched poly(β-amino esters) with zwitterionic functionalization at the ends, characterized in that, Its structural formula is as follows: 。 2. The method for preparing a type of highly branched poly(β-amino ester) with zwitterionic functionalization at the end as described in claim 1, characterized in that, The steps are as follows: 1) Add bisphenol A ethoxylated diacrylate, trimethylolpropane triacrylate and 4-amino-1-butanol to dimethyl sulfoxide and mix thoroughly; 2) Add 1,3-propanediamine to step 1) and continue the reaction; 3) Add 2-methacryloyloxyethyl phosphocholine to the polymer after end-capping in step 2) to obtain a highly branched poly(β-amino ester) with zwitterionic functionalization at the end. The molar ratio of the reactive functional groups of bisphenol A ethoxylated diacrylate, trimethylolpropane triacrylate, and 4-amino-1-butanol is 8:2:5; the molar ratio of 1,3-propanediamine to the excess acrylate functional group is 5:

1.

3. The method for preparing a type of highly branched poly(β-amino ester) with zwitterionic functionalization at the end according to claim 2, characterized in that, The reaction conditions for step 1) are: react at 80℃ for 12~20 h.

4. The method for preparing a type of highly branched poly(β-amino ester) with zwitterionic functionalization at the end according to claim 2, characterized in that, In step 2), before adding 1,3-propanediamine, the highly branched poly(β-amino ester) with zwitterionic functionalization at the end is monitored using gel permeation chromatography. The reaction is terminated when the molecular weight reaches 4,000~20,000 Da.

5. The method for preparing a type of highly branched poly(β-amino ester) with zwitterionic functionalization at the end according to claim 2, characterized in that, The reaction conditions for step 2) are: stirring at 20~50 ℃ for 2~6 hours.

6. The method for preparing a type of highly branched poly(β-amino ester) with zwitterionic functionalization at the end according to claim 2, characterized in that, The reaction conditions for step 3) are: react at 25 °C for 4~24 h.

7. The application of the highly branched poly(β-amino ester) with zwitterionic functionalization at the end as described in claim 1 in the preparation of protein drug delivery carriers.

8. The application according to claim 7, characterized in that, The mass ratio of highly branched poly(β-amino ester) with zwitterionic functionalization at the end to protein is (5~100):1.

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