Highly branched polymer, preparation method and application thereof, protein delivery nanoparticle and protein delivery method

Abstract

The invention provides a highly branched polymer, a preparation method and application thereof, protein delivery nanoparticles and a protein delivery method, and relates to the technical field of materials. The preparation method of the highly branched polymer provided by the invention comprises the following steps: mixing a monomer, a chain transfer agent and an initiator in a solvent, carrying out reversible addition-fragmentation chain transfer polymerization reaction to prepare a polymer with side group vinyl, and then packaging the side group vinyl of the polymer by adopting PBA or N-(2-aminoethyl) morpholine to obtain the highly branched polymer. Preparing to obtain a highly branched polymer; the monomers are DMAEMA (dimethylaminoethyl methacrylate) and 2, 2 '-dithiobis (ethane-2, 1-diyl). The preparation method is simple, and the prepared highly-branched polymer is good in safety, biodegradable and capable of being used for packaging and efficient delivery of protein.

Description

Technical Field

[0001] The present invention relates to the technical field of materials, and in particular to a hyperbranched polymer, a preparation method and application thereof, a protein delivery nanoparticle, and a protein delivery method. Background Art

[0002] Protein therapy provides a promising approach for treating diseases caused by genetic defects or protein abnormalities, such as inflammation, autoimmune diseases, genetic diseases, cancer, etc. Protein-based drugs, including peptides, hormones, monoclonal antibodies, and vaccines, have been widely recognized and applied due to their high specificity and safety. However, due to their hydrophilicity and large molecular size, the action sites of protein drugs are mainly limited to extracellular targets, which is in sharp contrast to the intracellular targets of many diseases. Therefore, intracellular delivery of proteins has great potential for clinical applications.

[0003] Due to the denaturation and poor stability of proteins in the physiological environment, the direct administration of protein drugs has been severely hindered. A widely used strategy to improve protein delivery efficiency is to package them into nanoparticles. Over the years, various protein delivery carriers have been explored, including lipids, hydrogels, metal-organic frameworks (MOFs), and mesoporous silica nanoparticles (MSNs). However, problems such as poor controllability, complex synthesis, low protein packaging efficiency, and potential toxicity to organisms limit their wide application in protein delivery. Cationic polymers have significant advantages, including simple synthesis, tunable structure, and high stability. Despite these benefits, their efficiency and safety in delivering proteins are still limited by various intracellular and extracellular barriers.

[0004] The positive and negative charges of amino acids are randomly distributed on the protein surface, resulting in uneven charge distribution and different charge densities among different proteins. Therefore, when relying solely on electrostatic interactions, cationic polymers usually exhibit poor protein packaging efficiency. In addition, proteins have a delicate three-dimensional (3D) folded structure, which is biologically affected by minor changes in pH, temperature, and salt concentration. To address these challenges, functional groups such as fluorine, boron, carboxylic acid, guanidine, and heterocycles have been modified onto cationic polymers to improve protein packaging efficiency and nanoparticle stability. These polymers interact with proteins through nitrogen-boric acid (N-B) coordination, cation-π interaction, metal coordination, hydrogen bonding, salt bridges, and hydrophobic interactions. However, the strong interaction between the polymer and the protein may hinder the unpacking efficiency of the protein in the cytoplasm. Therefore, improving substances that promote protein interaction, encapsulation, and those that enable protein unpacking in the cytoplasm is crucial for enhancing protein delivery efficiency. In addition, the topology of the polymer plays a crucial role in its protein delivery performance. The macromolecular structure of cationic polymers can range from linear and branched to star-shaped, cyclic, or spherical dendrimers, determining the spatial distribution and density of functional groups. This, in turn, affects the efficiency of protein delivery.

[0005] In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] The first object of the present invention is to provide a method for preparing a highly branched polymer to solve the problems of low encapsulation efficiency, difficult intracellular release, and poor biocompatibility existing in existing protein delivery carriers.

[0007] The second object of the present invention is to provide a highly branched polymer.

[0008] The third object of the present invention is to provide the application of the above-mentioned highly branched polymer in protein delivery.

[0009] The fourth object of the present invention is to provide a protein delivery nanoparticle.

[0010] The fifth object of the present invention is to provide a method for delivering a protein.

[0011] To achieve the above objects, the following technical solutions are specifically adopted:

[0012] In the first aspect, the present invention provides a method for preparing a highly branched polymer, comprising the following steps:

[0013] The monomer, chain transfer agent and initiator are mixed in a solvent to carry out reversible addition-fragmentation chain transfer polymerization to prepare a polymer with vinyl side groups, and then phenylboronic acid (PBA) or N-(2-aminoethyl)morpholine is used to encapsulate the vinyl side groups of the polymer to prepare a hyperbranched polymer;

[0014] The monomer is dimethylaminoethyl methacrylate (abbreviated as DMAEMA) and 2,2'-dithiobis(ethane-2,1-diyl) (abbreviated as DSDA).

[0015] As a further technical solution, the molar ratio of dimethylaminoethyl methacrylate to 2,2'-dithiobis(ethane-2,1-diyl) is (1-10):(0.5-5).

[0016] As a further technical solution, the chain transfer agent includes 4-cyano-4-(thiobenzoyl)pentanoic acid (abbreviated as CPADB);

[0017] The initiator includes azobisisobutyronitrile (AIBN);

[0018] The ratio of the monomer, chain transfer agent and initiator is (1.5-11):1:(0.1-0.5).

[0019] As a further technical solution, the solvent includes 2-butanone.

[0020] As a further technical solution, the reversible addition-fragmentation chain transfer polymerization is carried out under a protective atmosphere, and the reaction temperature is 60-85 °C.

[0021] As a further technical solution, the molecular weight of the polymer with vinyl side groups is 5.0-15.0 kDa, and the degree of branching is 30%-70%.

[0022] In a second aspect, the present invention provides a hyperbranched polymer prepared by using the above preparation method.

[0023] In a third aspect, the present invention provides the application of the above hyperbranched polymer in protein delivery.

[0024] In a fourth aspect, the present invention provides a protein delivery nanoparticle, which includes the above hyperbranched polymer and a protein; the protein is loaded on the hyperbranched polymer.

[0025] In a fifth aspect, the present invention provides a protein delivery method, in which the protein is loaded with the above hyperbranched polymer, and then the hyperbranched polymer loaded with the protein is used for the delivery of the protein to cells.

[0026] Compared with the prior art, the hyperbranched polymer provided by the present invention has the following beneficial effects:

[0027] Efficient encapsulation: Multiple interactions between DMAEMA units and PBA parts and proteins significantly improve the encapsulation efficiency.

[0028] Controlled release: The reduction sensitivity of disulfide bonds enables the polymer to degrade intracellularly, realizing the timely release of proteins.

[0029] High delivery efficiency: In SW1353 cells, the delivery efficiency reaches 95.1%, which is 20 times higher than that of the commercial product PULSin.

[0030] Low toxicity: The polymer degrades into low-toxic oligomers intracellularly, and the cell survival rate exceeds 99%. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0032] Figure 1 For the synthesis and characterization of P(DMAEMA-co-DSA)-E; (a) DMAEMA and DSDA are first copolymerized by RAFT polymerization to prepare P(DM-co-DS), and then capped with PBA to obtain P(DM-co-DS)-E; (b) GPC traces of P(DM10-co-DS1) during the polymerization process; (c) GPC traces of P(DM7.5-co-DS2.5) during the polymerization process; (d) GPC traces of P(DM10-co-DS1)-E2 and P(DM7.5-co-DS2.5)-E2; (e) 1H NMR spectra of P(DM10-co-DS1)-E1, P(DM10-co-DS1)-E2, P(DMP5-co-DS2.5)-E1 and P(DM7.5-co-DS2.5)-E2 1 H NMR spectra;

[0033] Figure 2 For the degradation of P(DM-co-DS)-E induced by a reducing agent; (a) DSDA in P(DM7.5-co-DS2.5) can be cleaved, resulting in the degradation of the polymer into oligomers; (b) GPC traces of P(DM7.5-co-DS2.5) before and after degradation; (c) 1H NMR spectra of P(DM7.5-co-DS2.5) before and after degradation;

[0034] Figure 3 Physiological and physicochemical properties of P(DMAEMA-co-DDSA)-E and P(DMAEMA-co-DDA)-E / BSA-FITC nanoparticles; (a) Fluorescence spectra of supernatants of P(DMAEMA10-co-DSDA1)-E1 / BSA-FITC and P(DMAEMA10-co-DSTA1)-E2 / BSA-FITC nanoparticles at mass ratios of 20:1, 40:1, and 60:1; (b) Protein packaging efficiency of P(DMAEMA-co-DDA)-E; (c) Size distribution of nanoparticles at a mass ratio of 60:1; (d) Size and Zeta potential of nanoparticles at a mass ratio of 60:1; (e) Representative TEM image of nanoparticles at a mass ratio of 60:1;

[0035] Figure 4 Protein delivery efficiency mediated by different P(DMAEMA-co-DDA)-E in SW1353 cells; (a) Fluorescence images of SW1353 cells after incubation with different nanoparticles for 24 h; Scale bar represents 100 μm; (b) Fluorescence intensity of SW1353 cells measured by flow cytometry; (c) Protein delivery rate in SW1353 cells; (d) Fluorescence intensity of SW1353 cells; (e) Cell viability after treatment with different P(DMAEMA-co-DDA)-E; Data in the figure are represented as mean ± standard deviation (SD), and statistical comparison was performed using one-way ANOVA with untreated cells as the control ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, ns, not significant;

[0036] Figure 5 For DSDA 1 1H NMR spectrum;

[0037] Figure 6 DMAEMA and DSDA are first copolymerized to form P(DMAEMA-co-DDA), and then capped with MPE;

[0038] Figure 7 For P(DM10-co-DS1) 1 1H NMR spectrum;

[0039] Figure 8 For P(DM7.5-co-DS2.5) 1 1H nuclear magnetic resonance spectrum. Detailed implementation method

[0040] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples. However, those skilled in the art will understand that the following embodiments and examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts belong to the scope of protection of the present invention. Those not specified in specific conditions are carried out under conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified in the manufacturer are all conventional products that can be obtained through commercial purchase.

[0041] In a first aspect, the present invention provides a method for preparing a hyperbranched polymer, comprising the following steps:

[0042] Mixing a monomer, a chain transfer agent, and an initiator in a solvent to carry out reversible addition-fragmentation chain transfer polymerization to prepare a polymer having vinyl side groups, and then encapsulating the vinyl side groups of the polymer with phenylboronic acid (PBA) or N-(2-aminoethyl)morpholine to prepare a hyperbranched polymer;

[0043] The monomer is dimethylaminoethyl methacrylate and 2,2'-dithiobis(ethane-2,1-diyl).

[0044] This preparation method is simple, and the prepared hyperbranched polymer has good safety, is biodegradable, and can be used for efficient delivery of proteins.

[0045] In some alternative embodiments, the molar ratio of dimethylaminoethyl methacrylate to 2,2'-dithiobis(ethane-2,1-diyl) can be, for example, but not limited to, 10:1, 7.5:2.5, 1:5, 5:3, or 10:0.5.

[0046] In some alternative embodiments, the chain transfer agent includes 4-cyano-4-(thiobenzoyl)valeric acid;

[0047] The initiator includes azobisisobutyronitrile;

[0048] The molar ratio of the monomer, the chain transfer agent, and the initiator is 11:1:0.5, 10:1:0.5, 1.5:1:0.1.

[0049] In some alternative embodiments, the solvent includes 2-butanone.

[0050] In some alternative embodiments, the reversible addition-fragmentation chain transfer polymerization is carried out under a protective atmosphere, and the reaction temperature can be, for example, but not limited to, 60°C, 65°C, or 85°C.

[0051] In some alternative embodiments, the molecular weight of the polymer having side vinyl groups can be, for example, but not limited to, 5.0 kDa, 10.0 kDa or 15.0 kDa. Through research by the inventors, it has been found that when the molecular weight of the polymer is in the range of 5.0 kDa - 15.0 kDa, it has better safety and delivery efficiency.

[0052] The degree of branching of the polymer having side vinyl groups can be, for example, but not limited to, 30%, 50% or 70%.

[0053] In a second aspect, the present invention provides a highly branched polymer prepared by the described preparation method.

[0054] This highly branched polymer has good safety, is biodegradable, and can be used for efficient delivery of proteins.

[0055] In a third aspect, the present invention provides the application of the above-mentioned highly branched polymer in protein delivery.

[0056] In a fourth aspect, the present invention provides a protein delivery nanoparticle, comprising the above-mentioned highly branched polymer and a protein; the protein is loaded on the highly branched polymer.

[0057] This protein delivery nanoparticle has high efficiency in delivering proteins.

[0058] In a fifth aspect, the present invention provides a method for delivering a protein, wherein the protein is loaded using the above-mentioned highly branched polymer, and then the highly branched polymer loaded with the protein is used for delivering the protein to cells.

[0059] This delivery method is simple and has high efficiency in delivering proteins.

[0060] The present invention will be further illustrated by specific examples below. However, it should be understood that these examples are only for more detailed illustration and should not be construed as limiting the present invention in any form.

[0061] Example 1

[0062] 1. Preparation of highly branched poly(dimethylaminoethyl methacrylate - bis(2 - acryloyloxy)ethyl disulfide))

[0063] 1.1 Materials

[0064] Bis-2-hydroxyethyl disulfide (Sigma, 85%), acryloyl chloride (Meyer, 98%), triethylamine (Adamas, water ≤ 50 ppm, 99.5%, SafeDry, with molecular sieve), tetrahydrofuran (Adamas), sodium bicarbonate (Greagent, 99.5% - 100.5%), sodium chloride (Shanghai Macklin, AR), magnesium sulfate (Shanghai Macklin 99.99%), 2-(dimethylamino)ethyl methacrylate (Meyer), 2,2'-azobis(2-methylpropionitrile) (Meyer (99%)), 4-cyano-4-(phenylsulfanylthio)pentanoic acid (Shanghai Macklin, 97%), 2-butanone (Adamlin as, AR), dimethylformamide (DMF, Fisher Scientific, 99.9%), deuterated chloroform (CDCl3, Shanghai Macklin, 99.9%), deuterated dimethyl sulfoxide ((CD3)2SO, Meyer, 99.8%), 4-aminomethylphenylboronic acid hydrochloride (E1, Meyer (97%)), 2-morpholinoethylamine (E2, Shanghai Macklin 99%), tributylphosphine (Bu3P, Macklin, 98%) and fluorescein isothiocyanate isomer (FITC, Shanghai Macklin) were used as received without further purification. Bovine serum albumin (BSA, Aladdin), fetal bovine serum (FBS, Life Technologies), penicillin / streptomycin (P / S, Life Technologies), phosphate buffered saline (PBS, 1X, pH 7.2 - 7.4, Life Technology), sodium acetate (NaAc, pH 5.2, 25 mM, Life Technologies) and PULSin (Polyplus transfection) were used as received or according to the manufacturer's protocol.

[0065] 1.2 Synthesis and purification of 2,2'-dithiobis(ethane-2,1-diyl) (DSDA)

[0066] DSDA was synthesized by reacting bis-2-hydroxyethyl disulfide with an excess of acryloyl chloride in a basic solvent, and the reaction equation is as follows:

[0067]

[0068] Bis-2-hydroxyethyl disulfide (50 mmol) and triethylamine (400 mmol) were dissolved in tetrahydrofuran (150.0 mL). Then acryloyl chloride (200 mmol) was added to the flask, and the mixture was stirred for 50 h. The by-product salt was removed by filtration, and the product was washed three times with water, sodium bicarbonate, and sodium chloride. The organic phase was dried over magnesium sulfate and then passed through a silica column using petroleum ether (PE) and ethyl acetate (EA) as eluents to obtain purified DSDA. The 1 1H NMR spectrum of DSDA is as Figure 5 shown.

[0069] 1.3 Polymer synthesis and purification

[0070] The synthesis process is as Figure 1 shown in a and Figure 6 The monomers, CTA, and initiator were added to two round-bottom flasks in the following ratios:

[0071] 1) P(DM10-co-DS1): DMAEMA (10 equivalents), DSDA (1 equivalent), CPADB (1 equivalent), AIBN (0.5 equivalent), that is, the molar ratio of DMAEMA, DSDA, CPADB, and AIBN is 10:1:1:0.5;

[0072] 2) P(DM7.5-co-DS2.5): DMAEMA (7.5 equivalents), DSDA (2.5 equivalents), CPADB (1 equivalent), AIBN (0.5 equivalent), that is, the molar ratio of DMAEMA, DSDA, CPADB, and AIBN is 7.5:2.5:1:0.5.

[0073] After that, 2-butanone (10 mL) was added to the flasks respectively, and the oxygen was removed by bubbling argon gas into the solution for 30 min. The flasks were immersed in a preheated oil bath at 65 °C, and the growth of the weight-average molecular weight (Mw), number-average molecular weight, and polydispersity index (PDI) was monitored by gel permeation chromatography (GPC). When Mw reached 6.5 kDa and 9.3 kDa, the polymerization was stopped by exposing the solution to air. The polymer was precipitated by adding ether / 2-butanone (10:1, v / v) to remove the unreacted monomers. Then E1 (PBA) and E2 (N-(2-aminoethyl)morpholine) were added in an amount five times that of the vinyl groups and allowed to react for 48 h. Finally, the product was dissolved in DMSO to a concentration of 50 mg / mL.

[0074] 1.4 GPC measurement

[0075] The Mw, Mn, and PDI of the polymers were determined using an Agilent 1260 Infinity II GPC system equipped with a refractive index (RI) detector. 10.0 mg of the sample was dissolved in 1 mL of DMF containing 0.1% lithium bromide and filtered through a 0.22 μm filter. The GPC columns (PolarGel-M Gard, 50 mm × 7.5 mm and PolarGel-M, 300 mm × 7.5 mm, connected in series) were eluted with DMF (containing 0.1% lithium bromide) at a flow rate of 1 mL / min and a temperature of 50 °C. The GPC system was calibrated using linear polymethyl methacrylate (PMMA) standards.

[0076] 1.5 1 1H nuclear magnetic resonance measurements

[0077] Recorded on a 400 MHz Varian NMR spectrometer (Bruker, Switzerland) 1 1H NMR spectra were used to confirm the chemical structure. The polymer (5 mg) was completely dissolved in CDCl3 (700 μL) and transferred to an NMR tube for measurement. Since P(DM10-co-DS1)-E1 and P(DM7.5-co-DS2.5)-E1 are insoluble in CDCl3 and D2O, these polymers were dissolved in (CD3)2SO. The solvents were CDCl3 (7.26 ppm) and (CD3)2SO (2.49 ppm), and the chemical shifts were expressed in ppm.

[0078] 1.6 Degradation and subsequent analysis of PD7.5-DS2.5-E2

[0079] 0.05 g of purified P(DM7.5-co-DS2.5)-E2 was dissolved in 2 mL of chloroform, and then 30 μL of Bu3P and 100 μL of water were added. The mixture was stirred at room temperature for 24 hours and collected for further analysis.

[0080] 1.7 Synthesis of BSA-FITC

[0081] BSA was dissolved in PBS (pH 7.4) to obtain a 0.2 g / mL solution, while FITC was dissolved in DMSO at a concentration of 0.39 mg / mL. 75 μL of the FITC solution was added to the BSA solution at a molar ratio of 3:1. The solution was stirred at room temperature in the dark for 24 hours, and the unreacted FITC was removed by dialysis in deionized water. Deionized water and DMSO were removed by lyophilization to obtain BSA-FITC.

[0082] 1.8 Preparation of nanoparticles

[0083] Dissolve BSA-FITC in PBS to a concentration of 10 mg / mL. Dilute the polymer solution (50 mg / mL) with NaAc (0.025 M, pH 5.2), and then further dilute the BSA-FITC solution with PBS at a volume ratio of PBS to NaAc of 4:1. Then add the polymer solution to the BSA-FITC solution at different polymer-to-protein mass ratios and stir the mixture at high speed for 25 - 30 seconds. Let the solution stand for 25 minutes to form the polymer.

[0084] 1.9 Size and zeta potential measurement of nanoparticles

[0085] Use 10 μg of BSA-FITC for each sample preparation. Prepare nanoparticles with the polymer and BSA-FITC at a mass ratio of 60:1 according to the above procedure and dilute to 1.0 mL with deionized water. Measure the particle size and zeta potential using a Zetasizer Nano Series-2590 (Malvern) instrument at a scattering angle of 90°. All experiments were repeated at least three times.

[0086] 1.10 Protein encapsulation

[0087] Each sample preparation used 10 μg of BSA-FITC with varying amounts of the polymer. Prepare nanoparticles as described above and dilute to 1.0 mL with PBS. Then centrifuge the nanoparticles and BSA-FITC solution at 15000 rpm for 30 minutes. Collect 100 μL of the supernatant into a 96-well plate. Perform fluorescence measurements using a plate reader (Synergy Hybrid H1, Biotek) with an excitation wavelength of 463 nm and an emission wavelength of 525 nm (λex = 463 nm, λem = 525 nm). All experiments were repeated at least three times. Samples containing BSA-FITC were used as positive controls, and samples without BSA-FITC were used as blanks.

[0088] Protein encapsulation efficiency (%) = 1 - (Fsample - Fblank) / (Fprotein - Fblank);

[0089] where F sample is the fluorescence intensity of the sample group, F protein is the fluorescence intensity of the positive control group, and Fblank is the fluorescence intensity of the blank group.

[0090] 1.11 Cell culture

[0091] Human chondrosarcoma (SW1353) cells obtained from ATCC were cultured in Dulbecco's Modified Eagle Medium (DMEM, GIBCO) containing 10% FBS and 1% penicillin / streptomycin (P / s). Cells were cultured in a humidified incubator at 37 °C and 5% CO2 using standard cell culture techniques.

[0092] 1.12 Evaluation of protein delivery efficiency

[0093] SW1353 cells were seeded into 96-well plates at a density of 2×10 4 cells per well and cultured overnight. Commercial reagents PULSin and BSA-FITC were used as positive and negative controls, respectively. PULSin was used according to the manufacturer's instructions. 1 μg of BSA-FITC was added to each well, and nanoparticles were prepared at different mass ratios as described above. The nanoparticle solution was diluted to 100 μL with complete medium and then added to SW1353 cells, followed by the addition of 100 μL of complete medium. After culturing for 24 hours, the medium was removed, and the cells were washed three times with PBS. The previous medium was removed, and the cells were washed three times with PBS 24 hours later. Fluorescence intensity (λex = 463 nm, λem = 525 nm) was measured qualitatively and quantitatively using a fluorescence microscope, flow cytometer, and microplate reader (Synergy Hybrid H1, Biotek). The fluorescence intensity of cells treated with BSA-FITC was defined as 100%. At least three replicate wells were used for each experiment.

[0094] 1.13 Evaluation of cell viability

[0095] Cell viability after incubation with various nanoparticles was measured using the alamarBlue assay. Briefly, the cell supernatant was removed, and the cells were washed twice with PBS (pH = 7.4). Then, 100 μL of alamarBlue assay solution (diluted to 10% with DMEM without FBS) was added. The cells were incubated for 30 minutes, and the fluorescence intensity (λex = 530 nm, λem = 590 nm) was measured using a microplate reader (Synergy Hybrid H1, Biotek). The cell viability of untreated cells was defined as 100%. At least three replicate wells were used for each experiment.

[0096] 1.14 Statistical analysis

[0097] Fluorescence intensity and cell viability data were analyzed using Student's t-test. Results are expressed as mean ± standard deviation (SD). The mean and SD were calculated based on at least three independent experimental results.

[0098] 2. Experimental results

[0099] Our goal is to achieve effective extracellular protein encapsulation and ensure timely release within the cytoplasm. Considering the influence of topology on the various mechanistic steps involved in protein delivery, two P(DMAEMA-co-DDSA) with different degrees of branching were synthesized at molar ratios of [DMAEMA]:[DSDA] in the feed of 10:1 and 7.5:2.5, respectively. To ensure that the highly branched topology does not cause gelation, the feed ratio of the total monomer to the chain transfer agent 4-cyano-4-(phenylthiobenzoylthio) pentanoic acid (CPADB) was set at 11:1, and the polymerization was carried out at 65 °C. Gel permeation chromatography (GPC) was used to monitor the molecular weight during the polymerization process. In the case of a [DMAEMA]:[DDA] feed ratio of 10:1, due to the relatively high monomer concentration, the GPC trace initially shifted to the left and showed a narrow peak, which facilitated the addition reaction mainly between the monomer and the propagation center. After 1 hour of polymerization, the weight-average molecular weight (Mw) of P(DMAEMA10-co-DSDA1) reached 1.6 kDa, and the polydispersity index (PDI) was 1.2. As the polymerization reaction proceeded, intermolecular binding began to occur. After 7 hours of reaction, a molecular weight of 5.2 kDa and a PDI of 2.6 were observed ( Figure 1 in b of

[0100] Table 1).

[0101]

[0102]

[0103] Previous studies have shown that the molecular weight of polymers with effective protein delivery and safety is usually between 5.0 kDa and 15.0 kDa. Therefore, when the Mw reached 6.5 kDa after 9 hours of polymerization, the reaction was cooled to room temperature. P(DMAEMA10-co-DSDA1) was purified by precipitation with ether, and its chemical structure was confirmed by nuclear magnetic resonance ( 1 1H NMR), showing characteristic signal peaks of carbon-carbon double bonds in the range of 5.5 to 6.5 ppm ( Figure 7 ). The molar ratio of DMAEMA to DSDA was 20:1, slightly higher than the feed ratio. The calculated degree of branching was 67.2%, corresponding to 1.2 branch junctions ( Figure 7and Table 2). Half of P(DMAEMA10-co-DSDA1) was redissolved in dimethyl sulfoxide (DMSO), and an excess of PBA (E1) was added at room temperature to cap the side-chain vinyl groups for 48 h to obtain P(DMAEMA10-co-DSTA1)-E1. The other half of P(DMAEMA10-co-DSDA1) was capped with N-(2-aminoethyl)morpholine (E2), yielding P(DMAEMA10-co-DSDAA1)-E2 with an Mw of 30.0 kDa and a PDI of 7.6, while P(DMAEMA10-co-DSTA1)-E1 was insoluble in the eluent for GPC measurements ( Figure 1 in d). In 1 the 1H NMR spectrum, characteristic signal peaks for the PBA moiety appeared between 7.0 and 8.2 ppm ( Figure 1 in e).

[0104] For a [DMAEMA]:[DSDA] feed ratio of 7.5:2.5, P(DM7.5-co-DS2.5) exhibited a relatively fast polymerization rate ( Figure 1 in c and Table 2), as the DSDA feed ratio was higher compared to P(DMAEMA10-co-DSDA1).

[0105] Table 2 Evolution of molecular weight during the synthesis of P(DM10-co-DS1)

[0106]

[0107]

[0108] After 6 h of polymerization, the degree of branching of P(DMAEMA7.5-co-DSDA2.5) was 39.8%, corresponding to 3.7 branched linkages, and the chemical composition of DMAEMA and DSDA units was 4:1 ( Figure 8 and Table 3).

[0109] Table 3 Chemical composition of P(DM-co-DS) copolymers

[0110]

[0111] After capping, P(DMAEMA7.5-co-DSDA2.5)-E1 and P(DMAEMA7.5-co-DSDF2.5)-E2 were obtained ( Figure 1 in e). All these results confirm that the copolymerization of DMAEMA and DSDA can be effectively controlled to produce multifunctional, highly branched polymers with desired molecular weights, customizable degrees of branching, and functionalities.

[0112] The DSDA branched linkage P(DMAEMA-co-DDSA) can be cleaved in the presence of a reducing agent. To verify this, 25 mM of a representative P(DMAEMA7.5-co-DSDA2.5)-E2 was incubated with tributylphosphine (1.5%, v / v). Cleavage of the DSDA branched junction is expected to result in a significant decrease in molecular weight ( Figure 2 In fact, after 24 h of incubation, the GPC trace shifted significantly to the right, and several new peaks appeared in the low molecular weight range ( Figure 2 b). The molecular weight decreased from 10.6 kDa to 1.9 kDa, while the PDI decreased from 3.8 to 1.5. These results confirm that P(DMAEMA7.5-co-DSDA2.5)-E2 has been decomposed into oligomers of different lengths. To further verify the degradation mechanism, 1 The chemical structure of P(DMAEMA7.5-co-DSDA2.5)-E2 before and after degradation was analyzed by H NMR. The spectrum clearly showed that the characteristic peak corresponding to the methylene group adjacent to the disulfide bond shifted downward from 3.0 ppm to 2.9 ppm ( Figure 2 These findings confirm that P(DMAEMA-co-DDA)-E is highly biodegradable via cleavage of disulfide branch linkages under reducing conditions, which not only facilitates protein unpacking but also reduces cytotoxicity.

[0113] We hypothesized that multiple interactions between DMAEMA, PBA moieties, and proteins facilitate protein packaging of P(DMAEMA-co-DDA)-E. Bovine serum albumin (BSA) labeled with fluorescein isothiocyanate (FITC) was used as a model protein to evaluate the protein packaging efficiency at mass ratios of P(DMAEMA-co-DDA)-E1 and BSA-FITC of 20:1, 40:1, and 60:1. The efficient packaging of BSA-FITC by P(DMAEMA-co-DDA)-E1 would lead to co-assembly into nanoparticles, resulting in a significant decrease in the fluorescence intensity in the supernatant. The fluorescence intensity of BSA-FITC solution alone at 1.0 mg / mL was observed to be as high as 24157 absorbance units (AU) at 525 nm ( Figure 3 (a) After incorporation of P(DMAEMA10-co-DSDA1)-E1 at a mass ratio of 20:1, the fluorescence intensity decreased significantly to 11418 AU. Further increasing the mass ratio to 40:1 and 60:1 did not result in a significant additional decrease in fluorescence intensity, and the protein packaging efficiency remained between 40% and 50% ( Figure 3in b). Interestingly, at the same mass ratio, the protein packaging efficiency of P(DMAEMA10-co-DSDA1)-E2 was relatively low, which might be due to the lack of PBA units. Meanwhile, the size of P(DMAEMA-co-DDA)-E1 / BSA-FITC nanoparticles was below 450 nm, the Zeta potential was close to neutral, and they exhibited a uniform spherical morphology ( Figure 3 in c, d, e). In contrast, P(DMAEMA-co-DDSA)-E2 showed a relatively low BSA-FITC encapsulation efficiency, resulting in the formation of P(DMAEMA-co-DDA)-E2 / BSA-FITC nanoparticles with sizes between 450 and 600 nm and an almost neutral Zeta potential. The significant differences in protein packaging efficiency, nanoparticle size, and Zeta potential among different P(DMAEMA-co-DDA)-E / BSA-FITC formulations can be attributed to the different contents of DMAEMA units and PBA moieties, highlighting their importance in promoting N-B coordination and cation-π interactions.

[0114] Benchmarked against the leading commercial product PULSin, the protein delivery efficiency of P(DMAEMA-co-DDA)-E was further evaluated in human chondrosarcoma (SW1353) cells. After incubation for 24 h, cells treated with BSA-FITC did not show visible green fluorescence, while the PULSin group showed weak fluorescence ( Figure 4 in a). In contrast, the number of cells with positive green fluorescence increased significantly in cells treated with P(DMAEMA10-co-DSDA1)-E1. Then, the protein delivery ability of P(DMAEMA10-co-DSDA1)-E1 was quantified using a flow cytometer. The histogram distribution of the green fluorescence-positive population in SW1353 cells was observed to shift significantly to the right. When the mass ratios of the polymer to BSA-FITC were 20:1, 40:1, and 60:1, respectively, the protein delivery efficiencies were 39.9%, 78.6%, and 95.1%, which were 8, 16, and 20 times higher than that of PULSin, respectively ( Figure 4 in b and c). Meanwhile, the mean fluorescence intensity (MFI) of cells treated with P(DMAEMA10-co-DSDA1)-E1 / BSA-FITC nanoparticles was 2, 5, and 8 times stronger than that of the PULSin group ( Figure 4d) in []. In sharp contrast, the protein delivery efficiency of all other polymers was lower than 20%. In comparison, when the mass ratio was 40:1, P(DMAEMA7.5-co-DSDA2.5)-E2 had the highest delivery efficiency of 14.6%, although this was still far lower than that of P(DMAEMA10-co-DSDA1)-E1 at the same ratio. Considering that P(DMAEMA10-co-DSDA1)-E2 lacked the PBA moiety and that the DMAEMA content in P(DMAEMA7.5-co-DSDA2.5)-E1 and P(DMAEMA7.5-co-DSDA25.5)-E2 was also lower, we speculated that the terminal PBA and DMAEMA moieties were both crucial for efficient protein delivery. These structural parameters were closely related to the degree of branching of P(DMAEMA-co-DDSA)-E. Encouragingly, regardless of the polymer type, cell viability of over 99% was maintained even at the highest mass ratio of 60:1 ( Figure 4 e) in [], indicating the high safety of P(DMAEMA-co-DDA)-E, which might be due to their biodegradability in the cytoplasm of SW1353 cells.

[0115] In summary, to improve extracellular protein packaging while facilitating its release in the cytoplasm, four P(DMAEMA-co-DDA)-E polymers with different chemical compositions, degrees of branching, and terminal moieties were synthesized by RAFT polymerization without causing gelation. In the presence of a reducing agent, the DSDA branches in P(DMAEMA-co-DDA)-E could be cleaved to form oligomers. Both the DMAEMA and PBA moieties significantly affected the protein packaging efficiency of P(DMAEMA-co-DDA)-E. The optimal P(DMAEMA10-co-DSDA1)-E1, with higher contents of DMAEMA and PBA moieties, showed more efficient protein encapsulation, forming nano-sized, nearly neutral, and uniform nanoparticles. In SW1353 cells, P(DMAEMA10-co-DSDA1)-E1 had the highest protein delivery efficiency of 95.1%, which was 20 times higher than that of PULSin, but did not induce obvious cytotoxicity. P(DMAEMA10-co-DSDA1)-E1 might be a promising candidate for safe and efficient intracellular protein delivery.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for preparing a hyperbranched polymer, characterized in that, Comprising the following steps: Mixing a monomer, a chain transfer agent and an initiator in a solvent to conduct reversible addition-fragmentation chain transfer polymerization reaction to prepare a polymer with side vinyl groups, and then encapsulating the side vinyl groups of the polymer with phenylboronic acid or N-(2-aminoethyl)morpholine to prepare a hyperbranched polymer; The monomer is dimethylaminoethyl methacrylate and 2,2'-dithiobis(ethane-2,1-diyl).

2. The preparation method according to claim 1, characterized in that, The molar ratio of dimethylaminoethyl methacrylate to 2,2'-dithiobis(ethane-2,1-diyl) is (1-10):(0.5-5).

3. The preparation method according to claim 1, characterized in that, The chain transfer agent includes 4-cyano-4-(thiobenzoyl)valeric acid; The initiator includes azobisisobutyronitrile; The molar ratio of the monomer, the chain transfer agent and the initiator is (1.5-11):1:(0.1-0.5).

4. The preparation method according to claim 1, wherein, The solvent includes 2-butanone.

5. The preparation method according to claim 1, characterized in that, The reversible addition-fragmentation chain transfer polymerization reaction is carried out under a protective atmosphere, and the reaction temperature is 60-85 °C.

6. The preparation method according to claim 1, wherein The molecular weight of the polymer with side vinyl groups is 5.0-15.0 kDa, and the degree of branching is 30%-70%.

7. A hyperbranched polymer, characterized in that, Prepared by the preparation method according to any one of claims 1-6.

8. Application of the hyperbranched polymer according to claim 7 in protein delivery.

9. A protein delivery nanoparticle, characterized in that, Comprising the hyperbranched polymer according to claim 7 and a protein; the protein is loaded on the hyperbranched polymer.

10. A method for delivering a protein, characterized in that, Loading the protein with the hyperbranched polymer according to claim 7, and then using the hyperbranched polymer loaded with the protein for the delivery of the protein to cells.

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