Charge-reversal type amphiphilic star polymer, and preparation method and application thereof

Abstract

This invention relates to the field of antitumor nanomedicine micelles, specifically to a charge-flipping amphiphilic star polymer, its preparation method, and its applications. The structural formula of the charge-flipping amphiphilic star polymer is shown in formula (I). This invention combines pH-responsive charge flipping, long circulation, and efficient cellular uptake with synergistic properties, as well as biodegradability, providing a new strategy to overcome the bottlenecks of traditional anticancer drugs in clinical applications. It is expected to achieve more precise drug release and higher treatment efficiency in tumor therapy.

Description

Technical Field

[0001] This invention relates to the field of antitumor nanodrug-carrying micelles, specifically to a charge-flipping amphiphilic star polymer, its preparation method, and its applications. Background Technology

[0002] Traditional anticancer drugs possess broad-spectrum antitumor activity in cancer treatment, but their clinical application is limited by multiple bottlenecks. For example, the following traditional drug molecules: curcumin enhances chemosensitivity by inhibiting the NF-κB pathway, but its extremely low water solubility (<0.1 mg / mL) results in oral bioavailability of less than 1%; doxorubicin, by intercalating into DNA to inhibit topoisomerase II, is highly effective against breast cancer and lymphoma, but its cardiotoxicity rate is as high as 20%, severely limiting its dosage; paclitaxel, by stabilizing microtubules and inhibiting mitosis, is widely used for ovarian cancer and lung cancer, but neurotoxicity and drug resistance are major challenges to its clinical application. These shortcomings stem from the inherent physicochemical properties (such as hydrophobicity) and mechanisms of action (such as poor passive diffusion targeting) of traditional drug molecules, necessitating novel delivery systems to overcome the bottlenecks in clinical translation.

[0003] Currently, nanodelivery technology can significantly improve drug performance through ingenious carrier design. Among them, charge-flipping nanomicelles have become a research hotspot due to their ability to reverse surface charges in response to specific stimuli. Existing systems mainly rely on external or endogenous stimuli such as light, pH, and enzymes to trigger potential reversal. pH-sensitive charge-flipping nanomicelles utilize the weakly acidic microenvironment of tumor tissue (pH 6.5, compared to 7.4 in normal tissue) to enhance tumor cell adhesion and endocytosis by protonating the surface charge from negative or neutral to positive, which has attracted widespread attention. However, such systems are mostly based on linear polymers and generally suffer from inherent defects such as poor cycling stability and low drug loading efficiency.

[0004] In recent years, amphiphilic star-shaped polymer nanomicelles have been considered an optimization direction for drug delivery systems due to their structural advantages. Star-shaped polymer nanomicelles, with their multi-arm topology providing multiple drug loading sites and their unique three-dimensional configuration inhibiting micelle dissociation, significantly outperform linear systems in terms of drug loading efficiency and cycling stability. However, existing star-shaped drug-loaded nanomicelles mainly rely on passive targeting mechanisms (such as the EPR effect), resulting in insufficient tumor enrichment efficiency. Although the introduction of targeting ligands such as folic acid and RGDS can improve tumor cell uptake, such active targeting strategies often suffer from key drawbacks such as accelerated immune clearance, complex preparation processes, high costs, and potential off-target toxicity, severely hindering their clinical translation. In contrast, charge-flipping nanomicelle technology shows the potential to overcome these limitations. Therefore, developing an amphiphilic star-shaped polymer delivery system with pH-adaptive charge-flipping function is expected to synergistically improve drug delivery efficiency and tumor targeting, possessing outstanding scientific value and broad clinical translation prospects. In view of this, this invention provides a charge-flipping amphiphilic star-shaped polymer, its preparation method, and its applications. Summary of the Invention

[0005] To address the problems of traditional star-shaped polymer nanomicelles in anticancer drug delivery, such as charge staticity (inability to respond to changes in tumor microenvironment pH), the "long circulation-high efficiency uptake" paradox, and the risk of uncontrollable degradation, this invention provides a charge-flipping amphiphilic star-shaped polymer, its preparation method, and its applications. The aim is to combine pH-responsive charge flipping, synergistic properties of long circulation and high efficiency cellular uptake, and biodegradability, providing a new strategy to overcome the bottlenecks of traditional anticancer drugs in clinical applications, and potentially achieving more precise drug release and higher therapeutic efficiency in tumor treatment.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0007] In a first aspect, there is a charge-reversing amphiphilic star polymer, the structural formula of which is shown in formula (I):

[0008] ;

[0009] In formula (Ⅰ), PHC is a polyhydroxy compound; m ranges from 5 to 20; the sum of n1 and n2 ranges from n1+n2=10 to 100; and the ratio of n1 to n2 ranges from n1 / n2=0 to 0.5.

[0010] Based on the above technical solution, the present invention can be further improved as follows.

[0011] Furthermore, the PHC mentioned in formula (Ⅰ) is at least one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, pentaerythritol, xylitol, sorbitol, and inositol.

[0012] Secondly, a method for preparing a charge-reversing amphiphilic star polymer includes the following steps:

[0013] (1) Preparation of star-shaped polycaprolactone (S-PCL-OH) based on polyhydroxy compounds (PHC):

[0014] The polyhydroxy compound, ε-caprolactone, stannous isooctanoate, and a first solvent are mixed, dried to remove moisture, and then reacted. The mixture is then precipitated in an alcohol or petroleum ether to obtain star-shaped polycaprolactone (S-PCL-OH). Molecular sieves can be used to remove moisture during drying.

[0015] (2) Preparation of macromolecular initiators based on star-shaped polycaprolactone:

[0016] The star-shaped polycaprolactone and triethylamine (TEA) are dissolved in a second solvent, and then 2-bromo-2-methylpropionyl bromide (BIBB) is added dropwise under ice bath conditions to carry out the reaction. The mixture is then precipitated in an alcohol or petroleum ether to obtain 2-bromo-2-methylpropionyloxy group-modified star-shaped polycaprolactone (S-PCL-Br), which is a macromolecular initiator based on star-shaped polycaprolactone. After the reaction, the mixture can be washed 3-5 times with saturated NaHCO3 aqueous solution, and then washed 3-5 times with deionized water. The organic phase is dried with anhydrous sodium sulfate, and part of the second solvent is removed using a rotary evaporator. The concentrated organic phase is then precipitated dropwise in an alcohol or petroleum ether. The precipitate is filtered and dried in a vacuum oven to obtain 2-bromo-2-methylpropionyloxy group-modified star-shaped polycaprolactone (S-PCL-Br).

[0017] (3) Preparation of star-shaped polycaprolactone-block polypentafluorophenyl methacrylate (S-PCL-b-PPFPMA):

[0018] The 2-bromo-2-methylpropionyloxy group-modified star-shaped polycaprolactone and pentafluorophenyl methacrylate (PFPMA) were dissolved in a third solvent, and then copper bromide (CuBr2), a nitrogen-containing ligand, and stannous isooctanoate (Sn(EH)2) were added. The reaction was carried out under deoxygenated conditions. The resulting reaction solution was passed through an alumina-packed column and then precipitated in an alcohol or petroleum ether to obtain star-shaped polycaprolactone-block-polypentafluorophenyl methacrylate. The mass fraction of PPFPMA in S-PCL-b-PPFPMA was calculated based on the yield of S-PCL-b-PPFPMA and the amount of S-PCL-Br added.

[0019] (4) Preparation of charge-flipping amphiphilic star polymers:

[0020] The star-shaped polycaprolactone-block-polypentafluorophenyl methacrylate, isopropanolamine (MIPA), and N,N-diisopropylethylenediamine (AEDPA) were dissolved in a fourth solvent and reacted. After the reaction was completed, the mixture was dialyzed and freeze-dried sequentially to obtain the star-shaped polycaprolactone-block-poly(2-hydroxypropyl methacrylamide-copolymer-diisopropylaminoethyl methacrylamide) (S-PCL-bP(HPMA-co-DPAEMA)) (abbreviated as S-PCL-b-PHD), which is a charge-reversing amphiphilic star polymer. In this process, unreacted MIPA, AEDPA, and the pentafluorophenol generated in the reaction were first dialyzed in DMF, and then the mixture was transferred to pure water for dialyzed to remove the DMF used as a solvent.

[0021] Further, in step (1), the molar ratio of the hydroxyl group of the polyhydroxy compound to the ε-caprolactone to the stannous isooctanoate is 1:5~50:0.05~0.5; and the concentration of the ε-caprolactone is 1 mol / L~10 mol / L.

[0022] In step (1), the first solvent includes at least one of toluene, xylene, anisole, cyclohexanone, N-methylpyrrolidone (NMP), dimethylformamide (DMF), and dimethyl sulfoxide (DMSO);

[0023] The alcohols mentioned in step (1) include at least one of methanol and ethanol;

[0024] The reaction conditions after drying and removing moisture in step (1) are: temperature of 110℃~140℃ and time of 12 h~48 h.

[0025] Further, in step (2), the mass ratio of star-shaped polycaprolactone:triethylamine:2-bromo-2-methylpropionyl bromide is 1:0.1~1:0.2~2; the concentration of star-shaped polycaprolactone before the reaction is 20 g / L~200 g / L; the concentration of 2-bromo-2-methylpropionyl bromide before the reaction is 20 g / L~200 g / L.

[0026] In step (2), the second solvent includes at least one of chloroform, dichloromethane (DCM), tetrahydrofuran (THF), and toluene;

[0027] The alcohols mentioned in step (2) include at least one of methanol and ethanol;

[0028] The reaction conditions in step (2) are: react in an ice bath for 1 h to 3 h, and then react at room temperature for 12 h to 24 h.

[0029] Further, in step (3), the molar ratio of copper bromide: nitrogen-containing ligand: stannous isooctanoate: pentafluorophenyl methacrylate is 1:2~20:10~100:5000~20000; the mass ratio of 2-bromo-2-methylpropionyloxy group modified star-shaped polycaprolactone to pentafluorophenyl methacrylate is 1:5~20; and the concentration of 2-bromo-2-methylpropionyloxy group modified star-shaped polycaprolactone (S-PCL-Br) is 0.01g / mL~0.1g / mL;

[0030] The nitrogen-containing ligands mentioned in step (3) include at least one of tris(2-pyridinemethyl)amine, tris[2-(dimethylamino)ethyl]amine, N,N,N′,N′,N′′-pentamethyldiethyltriamine, 1,1,4,7,10,10-hexamethyltriethylenetetramine, and 4,4'-dihexyl-2,2'-bipyridine;

[0031] The third solvent mentioned in step (3) includes at least one of anisole, cyclohexanone, tetrahydrofuran (THF), N-methylpyrrolidone (NMP), toluene, dimethylformamide (DMF), and dimethyl sulfoxide (DMSO);

[0032] The alcohols mentioned in step (3) include at least one of methanol and ethanol;

[0033] The reaction conditions in step (3) under deoxygenation conditions are as follows: high-purity argon gas is bubbled through for 20 min to 30 min to remove oxygen, and then the mixture is transferred to an oil bath at 50℃ to 90℃ and stirred for 24 h to 72 h.

[0034] Further, in step (4), the molar ratio of N,N-diisopropylethylenediamine (AEDPA) to isopropanolamine (MIPA) is 1:1~20; the molar ratio of pentafluorophenyl methacrylate in the star-shaped polycaprolactone-block-polypentafluorophenyl methacrylate to the total molar ratio of isopropanolamine (MIPA) and N,N-diisopropylethylenediamine (AEDPA) is 1:1~10;

[0035] The fourth solvent in step (4) includes at least one of dimethylformamide (DMF), tetrahydrofuran (THF), N-methylpyrrolidone (NMP), toluene, and dimethyl sulfoxide (DMSO);

[0036] The reaction conditions for step (4) are: reaction in an oil bath at 40℃~60℃ for 20 h~48 h;

[0037] The dialysis bag used in step (4) has a molecular weight cutoff of 1000 Da to 3500 Da; the freeze-drying conditions are: temperature -30±5℃, time 24 h to 72 h.

[0038] Thirdly, the application of a charge-reversing amphiphilic star polymer, wherein the charge-reversing amphiphilic star polymer is used to prepare drug-loaded micelles.

[0039] Fourthly, a method for preparing drug-loaded micelles of a charge-reversing amphiphilic star polymer includes the following steps:

[0040] The charge-reversing amphiphilic star polymer and the anticancer drug are dissolved in a fifth solvent, then added dropwise to pure water while stirring to remove the fifth solvent and unreacted anticancer drug. The solution is then filtered through a microporous membrane (e.g., a 0.22-micron filter) to obtain a drug-loaded micelle aqueous solution.

[0041] Furthermore, the mass ratio of the charge-reversing amphiphilic star polymer to the anticancer drug is 1:(0.5~2); the mass-volume concentration of the charge-reversing amphiphilic star polymer dissolved in the fifth solvent is 1 g / L~10 g / L;

[0042] The volume ratio of the fifth solvent to the water is 1:(3~30).

[0043] The particle size of the drug-loaded micelles is 50 nm to 200 nm;

[0044] The fifth solvent includes at least one of tetrahydrofuran (THF), dimethylformamide (DMF), N-methylpyrrolidone (NMP), and dimethyl sulfoxide (DMSO);

[0045] The anticancer drugs include at least one of curcumin (CUR), doxorubicin, paclitaxel, camptothecin, oxaliplatin, irinotecan, and leucoquinone.

[0046] The present invention achieves breakthroughs in charge-reversing amphiphilic star polymers, their preparation methods, and applications through the following design: (1) using biodegradable star-shaped polycaprolactone (S-PCL) as a hydrophobic core to optimize drug loading efficiency, micelle stability, and biodegradability; (2) using poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA), which has excellent biocompatibility and resistance to non-specific protein adsorption, as a hydrophilic shell to enhance blood circulation performance and biocompatibility; and by introducing tumor pH-sensitive tertiary amine groups into the hydrophilic segments to achieve tumor regeneration. The tumor microenvironment adapts to charge reversal; (4) In the preparation of the hydrophilic shell, the strategy of combining the atom transfer radical polymerization (ATRP) of pentafluorophenyl methacrylate (PFPMA) with the active ester aminolysis reaction avoids the problem of reduced polymerization controllability caused by the competition for coordination between amide monomers (such as N-(2-hydroxypropyl)methacrylamide, HPMA) and Cu catalyst during the polymerization process, and effectively avoids the problem of uncontrollable sequence structure and uneven component distribution caused by the difference in monomer competition rate during copolymerization.

[0047] This invention prepares a charge-flipping amphiphilic star polymer, which, relying on its multi-arm topology providing multiple drug-loading sites and its anti-dissociation three-dimensional configuration, constructs a smart nanodelivery system that combines high drug loading capacity, pH-adaptive charge flipping, and long-term cycling stability. The system carries a negative surface charge at physiological pH, which helps avoid serum protein adsorption and reticuloendothelial system clearance, prolonging blood circulation time. Upon reaching the tumor microenvironment, the tertiary amine groups can trigger a surface potential shift from negative to positive through protonation, enhancing electrostatic adsorption to the negatively charged tumor cell membrane and promoting cell adhesion and endocytosis. Based on the advantages of the star structure's drug loading stability and the precise spatiotemporal control of charge flipping, this system can achieve efficient drug accumulation within tumor cells, overcoming the bottlenecks of drug resistance and off-target toxicity in traditional chemotherapy, and is expected to provide a new pathway for enhanced efficacy and reduced toxicity in precision antitumor therapy.

[0048] The beneficial effects of this invention are:

[0049] (1) The amphiphilic polymer of the present invention is a star polymer. Compared with linear polymers, star polymers have the advantages of being easier to form micelles, having higher drug loading efficiency, more uniform micelle size, and higher stability. They can effectively prevent premature drug release due to micelle rupture during delivery, thus ensuring accurate drug delivery.

[0050] (2) The amphiphilic polymer of the present invention has a pH-adaptive charge-flipping function. By grafting a hydrophilic shell with pH-adaptive charge-flipping function, excellent biocompatibility, and resistance to non-specific protein adsorption onto the outside of the star-shaped polymer, on the one hand, the micelles maintain a negative charge in blood at pH 7.4, thus exhibiting good blood circulation performance; on the other hand, at the weakly acidic tumor site, the micelles acquire a positive charge through charge flipping, thereby improving the micelles' cell entry efficiency. This solves the "long circulation - high efficiency uptake" performance paradox, significantly improving the tumor enrichment efficiency of star-shaped drug-loaded polymer nanomicelles while enhancing blood circulation performance.

[0051] (3) In the construction of the charge-flipped hydrophilic shell, this invention employs a strategy combining atom transfer radical polymerization (ATRP) of pentafluorophenyl methacrylate (PFPMA) with active ester aminolysis. On the one hand, this method avoids the problem of reduced polymerization controllability caused by amide-containing monomers (such as N-(2-hydroxypropyl)methacrylamide, HPMA) competing for coordination with Cu catalyst during polymerization. On the other hand, it effectively avoids the problems of uncontrollable sequence structure and uneven component distribution caused by differences in monomer reactivity during copolymerization, providing a feasible approach for constructing a charge-flipped carrier with precise structure and stable function.

[0052] (4) The charge-reversing amphiphilic star polymer of the present invention has excellent biocompatibility and in vivo degradability in its constituent materials. The degradation products can be safely metabolized by the body, avoiding the risk of residual carrier materials in vivo. Moreover, the required raw materials are all available from abundant commercial sources, making it easy to scale up production, and showing important clinical application value and industrialization prospects. Attached Figure Description

[0053] Figure 1 The above is the 1H NMR spectrum of the S-PCL-OH of this invention, with CDCl3 as the solvent;

[0054] Figure 2 The above is the 1H NMR spectrum of S-PCL-Br of this invention, with CDCl3 as the solvent;

[0055] Figure 3 The above is the 1H NMR spectrum of the S-PCL-b-PPFPMA of this invention, with deuterated DMF as the solvent;

[0056] Figure 4 The above are the 1H NMR spectra of S-PCL-b-PHD(A) and S-PCL-b-PHPMA(B) of the present invention, with deuterated DMF as the solvent.

[0057] Figure 5 This is a particle size distribution diagram of the S-PCL-b-PHD micelles of the present invention;

[0058] Figure 6 The results of the Zeta potential test of the S-PCL-b-PHD blank micelles of the present invention;

[0059] Figure 7 This is a particle size distribution diagram of the S-PCL-b-PHD@CUR micelles of the present invention;

[0060] Figure 8 The results of Zeta potential tests of S-PCL-b-PHD@CUR micelles at different pH values ​​are shown below.

[0061] Figure 9 This is a morphological feature image of the S-PCL-b-PHD@CUR micelles of the present invention under a transmission electron microscope;

[0062] Figure 10 This is a drug release curve of the S-PCL-b-PHD@CUR micelles of the present invention;

[0063] Figure 11 The cytotoxicity curves of the S-PCL-b-PHD@CUR and S-PCL-b-PHPMA@CUR micelles of the present invention are shown.

[0064] Figure 12 This is a graph showing the H460 flow cytometry quantitative uptake results of the present invention;

[0065] Figure 13 The results are for the cytotoxicity test of blank micelles of S-PCL-b-PHD. Detailed Implementation

[0066] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0067] Materials and reagents source description: β-cyclodextrin (β-CD), ε-caprolactone (CL), stannous isooctanoate (Sn(EH)2), 2-bromo-2-methylpropionyl bromide (BIBB), pentafluorophenyl methacrylate (PFPMA), copper bromide (CuBr2), tris(2-pyridinemethyl)amine (TPMA), isopropanolamine (MIPA), and curcumin (CUR) were all purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; N,N-diisopropylethylenediamine (AEDPA) was purchased from Beijing Innocare Technology Co., Ltd.; human large cell lung cancer cells (H460) were purchased from Wuhan Zishan Biotechnology Co., Ltd.

[0068] Example

[0069] 1. A method for preparing a pH-adaptive charge-flipping amphiphilic star polymer.

[0070] The reaction formula for this preparation method is as follows:

[0071] ;

[0072] The preparation method includes the following steps:

[0073] (1) Preparation of star-shaped polycaprolactone (S-PCL-OH) based on cyclodextrin (CD):

[0074] β-CD (0.11 g), ε-caprolactone (5.35 g) and stannous isooctanoate (0.12 g) were dissolved in 10 mL of toluene, and water was removed by adding 4A molecular sieve for 24 h. The mixture was then stirred in an oil bath at 130 °C for 24 h. After that, the reaction solution was added dropwise to methanol. The precipitate was filtered and dried in a vacuum oven at 45 °C for 24 h to obtain a white powdery star-shaped polycaprolactone (S-PCL-OH). Figure 1 The molecular structure of S-PCL-OH and its proton NMR spectrum are shown; for example... Figure 1 As shown in the spectrum, the proton signal matches the hydrogen atom in the S-PCL-OH molecule structure, indicating that S-PCL-OH was successfully synthesized.

[0075] (2) Preparation of star-shaped polycaprolactone (S-PCL-Br) based on the macromolecular initiator—2-bromo-2-methylpropionyloxy group:

[0076] The S-PCL-OH (3.0 g) prepared in step (1) was dissolved in 30 mL of dichloromethane, and 1.2 mL of triethylamine was added. The mixture was then cooled to 0 °C in an ice bath. 1 mL of 2-bromo-2-methylpropionyl bromide (BIBB) was then dissolved in 25 mL of dichloromethane and added dropwise to the S-PCL-OH solution under nitrogen protection. After the addition was complete, the mixture was reacted in an ice bath for 2 h, and then the reaction continued at room temperature for 24 h. After the reaction was complete, the mixture was washed three times each with 5% NaHCO3 and pure water. The organic phase was dried with anhydrous sodium sulfate for 12 h, and some of the dichloromethane was removed using a rotary evaporator. The concentrated organic phase was added dropwise to methanol, and the precipitate was filtered and dried in a vacuum oven at 45 °C for 24 h to obtain the product S-PCL-Br. Figure 2 The molecular structure of S-PCL-Br and its proton NMR spectrum are shown; such as Figure 2 As shown in the spectrum, the proton signal corresponds to the hydrogen atom in the S-PCL-Br molecular structure, indicating that the macromolecular initiator S-PCL-Br was successfully prepared.

[0077] (3) Preparation of star-shaped polycaprolactone-block polypentafluorophenyl methacrylate (S-PCL-b-PPFPMA):

[0078] S-PCL-Br (0.3 g) and PFPMA (1.8 g) prepared in step (2) were added to a Schlenk reaction tube and dissolved in anisole. Then, copper bromide (CuBr2) (0.00072 mmol), tris(2-pyridinemethyl)amine (TPMA) (0.0072 mmol), and Sn(EH)2 (0.032 mmol) were added. The mixture was bubbled with high-purity argon for 20 min to remove oxygen. The reaction tube was then transferred to a 70℃ oil bath and stirred for 24 h. After the reaction, the solution was passed through an alumina-packed column, and the precipitate was dropped into petroleum ether. After filtration, the precipitate was dried in a 35℃ vacuum oven for 24 h to obtain the product S-PCL-b-PPFPMA. Figure 3 The molecular structure of S-PCL-b-PPFPMA and its 1H NMR spectrum are shown. Compared with S-PCL-Br, the spectrum of S-PCL-b-PPFPMA shows methylene and methyl hydrogen signals at g and h, respectively, indicating that PFPMA was successfully grafted onto star-shaped polycaprolactone.

[0079] (4) Preparation of charge-reversing amphiphilic star polymer—star polycaprolactone-block-poly(2-hydroxypropyl methacrylamide-copolymer-diisopropylaminoethyl methacrylamide) (S-PCL-bP(HPMA-co-DPAEMA)) (abbreviated as S-PCL-b-PHD):

[0080] The S-PCL-b-PPFPMA (50 mg) prepared in step (3) was dissolved in 2 mL of ultra-dry DMF, and 0.042 g (0.56 mmol) of MIPA and 0.02 g (0.14 mmol) of AEDPA were added. The mixture was placed in an oil bath at 50 °C and reacted for 24 h. After the reaction was completed, the reaction liquid was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in 50 mL of DMF for 24 h (the DMF was changed every 4 h). Then, it was transferred to 1 L of pure water and dialyzed for another 48 h (the pure water was changed every 6 h). Finally, it was freeze-dried at -30 °C for 48 h to obtain S-PCL-b-PHD lyophilized powder. Figure 4 Image A shows the molecular structure of S-PCL-b-PHD and its 1H NMR spectrum; as shown in Figure A. Figure 4 As shown in Figure A, the proton signals in the spectrum correspond to the hydrogen atoms in the S-PCL-b-PHD molecular structure, indicating that the pentafluorophenyl ester group in S-PCL-b-PPFPMA reacted successfully with MIPA and AEDPA to obtain S-PCL-b-PHD.

[0081] (5) Preparation of star-shaped polycaprolactone-block-poly(2-hydroxypropylmethacrylamide) (S-PCL-b-PHPMA):

[0082] The S-PCL-b-PPFPMA (50 mg) prepared in step (3) was dissolved in 2 mL of ultra-dry DMF, and 0.047 g (0.63 mmol) of MIPA was added. The mixture was then placed in an oil bath at 50 °C and reacted for 24 h. After the reaction was completed, the reaction liquid was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in 50 mL of DMF for 24 h (the DMF was replaced every 4 h). Then, it was transferred to 1 L of pure water and dialyzed for another 48 h (the pure water was replaced every 6 h). Finally, it was freeze-dried at -30 °C for 48 h to obtain S-PCL-b-PHPMA lyophilized powder. Figure 4 Figure B shows the molecular structure of S-PCL-b-PHPMA and its 1H NMR spectrum; as shown in Figure B. Figure 4 As shown in Figure B, the proton signals in the spectrum correspond to the hydrogen atoms in the S-PCL-b-PHPMA molecular structure, indicating that the pentafluorophenyl ester group in S-PCL-b-PPFPMA reacted successfully with MIPA to obtain S-PCL-b-PHPMA.

[0083] 2. Preparation of charge-flipped amphiphilic star-shaped polymer blank micelles.

[0084] Weigh 5 mg of the prepared S-PCL-b-PHD and dissolve it in 0.5 mL of tetrahydrofuran (THF). Then, add it dropwise to 5 mL of pure water while applying magnetic stirring to assist dispersion. After the addition is complete, continue stirring for 30 min. Remove THF using a rotary evaporator and filter through a 0.22 μm filter to obtain a blank S-PCL-b-PHD micelle aqueous solution.

[0085] 3. Preparation of charge-flipped amphiphilic star-shaped polymer drug-loaded micelles (S-PCL-b-PHD@CUR).

[0086] Weigh 5 mg of the prepared S-PCL-b-PHD and 5 mg of curcumin, dissolve them in 0.5 mL of tetrahydrofuran (THF), mix well, and then add them dropwise to 5 mL of pure water while applying magnetic stirring to assist dispersion. After the addition is complete, continue stirring for 30 min. Remove THF using a rotary evaporator. Centrifuge the remaining aqueous phase at 7200 rpm for 10 min to remove unencapsulated curcumin precipitate. Then filter through a 0.22 μm filter to obtain S-PCL-b-PHD@CUR drug-loaded micelle aqueous solution. Freeze-dry at -30℃ for 48 h to obtain S-PCL-b-PHD@CUR lyophilized powder.

[0087] 4. Preparation of S-PCL-b-PHPMA drug-loaded micelles—S-PCL-b-PHPMA@CUR

[0088] Weigh 5 mg of the prepared S-PCL-b-PHPMA and 5 mg of curcumin, dissolve them in 0.5 mL of tetrahydrofuran (THF), mix well, and then add them dropwise to 5 mL of pure water while applying magnetic stirring to assist dispersion. After the addition is complete, continue stirring for 30 min. Remove THF using a rotary evaporator. Centrifuge the remaining aqueous phase at 7200 rpm for 10 min to remove unencapsulated curcumin precipitate. Then filter through a 0.22 μm filter to obtain S-PCL-b-PHPMA@CUR drug-loaded micelle aqueous solution. Freeze-dry at -30℃ for 48 h to obtain S-PCL-b-PHPMA@CUR lyophilized powder.

[0089] 5. Particle size and Zeta potential test of blank micelles of charge-reversed amphiphilic star polymers.

[0090] The prepared S-PCL-b-PHD blank micelle aqueous solution was used to test its particle size distribution and Zeta potential at room temperature using a dynamic light scattering instrument. The particle size distribution is shown in the figure. Figure 5 As shown, the average particle size of the micelles is approximately 155 nm. The Zeta potential is as follows... Figure 6 As shown, the Zeta potential is negative (-20.3 mV) at pH 7.4 and positive (+37.2 mV) at pH 6.5. This indicates that the Zeta potential of S-PCL-b-PHD micelles can respond to the pH of the medium, exhibiting pH-adaptive charge-flipping properties.

[0091] 6. Particle size and Zeta potential testing and morphology observation of charge-flipped amphiphilic star-shaped polymer drug-loaded micelles.

[0092] The prepared S-PCL-b-PHD@CUR micelle aqueous solution was tested for particle size distribution and Zeta potential at room temperature using a dynamic light scattering instrument. The particle size distribution is shown in the figure. Figure 7 As shown, the average particle size of the micelles is approximately 183 nm. The Zeta potential is as follows... Figure 8 As shown, the Zeta potential is negative (-21.1 mV) at pH 7.4 and positive (+37.7 mV) at pH 6.5, indicating that the Zeta potential of S-PCL-b-PHD@CUR micelles can respond to the pH of the medium, exhibiting pH-adaptive charge-flipping characteristics. The morphology of the dried S-PCL-b-PHD@CUR micelles was observed using transmission electron microscopy (TEM). The results are as follows... Figure 9As shown, the dried S-PCL-b-PHD@CUR micelles exhibit a spherical structure with a diameter of approximately 65 nm.

[0093] 7. Drug loading test of charge-flipped amphiphilic star-shaped polymer drug-loaded micelles.

[0094] A certain amount of the prepared S-PCL-b-PHD@CUR lyophilized powder was weighed, dissolved in an appropriate amount of THF, and the absorbance at 423 nm was measured using a UV spectrophotometer. The concentration of CUR in the solution was calculated by the concentration-absorbance standard curve of CUR at 423 nm. The drug loading was calculated by the formula and was found to be 36.2%.

[0095] 8. In vitro drug release behavior of charge-flipped amphiphilic star-shaped polymer drug-loaded micelles.

[0096] A certain amount of the prepared S-PCL-b-PHD@CUR lyophilized powder was weighed and dissolved in an appropriate amount of pure water to obtain an S-PCL-b-PHD@CUR micelle aqueous solution with a CUR concentration of 0.2 mg / mL. 0.2 mL of this solution was transferred into a dialysis bag with a molecular weight cutoff of 3500 Da and immersed in 40 mL of buffer solution containing 0.5% Tween 80 (pH 7.4 and 5.5, respectively). The bag was shaken on a constant temperature shaker (37℃, 120 rpm). At set time points, 3 mL of dialysate was collected and 3 mL of fresh buffer solution (containing 0.5% Tween 80) at the corresponding pH values ​​was added. The absorbance of the collected dialysate was measured at 423 nm. The cumulative drug release was calculated using a concentration-absorbance standard curve of CUR aqueous solution (containing 0.5% Tween 80) at 423 nm, and a drug release curve was plotted. Figure 10 The figure shows the drug release curves of S-PCL-b-PHD@CUR micelles. CUR is released slowly in aqueous solution, and its release rate in pH 5.5 buffer is slightly greater than that in pH 7.4 buffer. At 100 h, the release amounts of CUR in pH 5.5 buffer and pH 7.4 buffer are 72.3% and 70.3%, respectively.

[0097] 9. Cytotoxicity of charge-flipped amphiphilic star-shaped polymer drug-loaded micelles.

[0098] The effect of S-PCL-b-PHD@CUR micelles on inhibiting tumor proliferation in vitro was evaluated using a CCK-8 assay kit. Human large cell lung cancer cells (H460) were cultured at 5 × 10⁶ cells / well. 3Cells were seeded at a density in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours. After incubation, the culture medium was aspirated, and 100 µL of fresh culture medium (pH 6.5) containing different concentrations of S-PCL-b-PHD@CUR micelles was added to each well. As a control, in other wells, after aspirating the culture medium, 100 µL of fresh culture medium (pH 6.5) containing different concentrations of free curcumin (free-CUR) and S-PCL-b-PHPMA@CUR micelles were added, respectively. Incubation continued for another 48 hours. Then, 10 µL of CCK-8 solution was added to each well, and incubation continued for another 3 hours. The absorbance (OD value) at 490 nm was measured using a microplate reader to calculate cell viability. Figure 11 Cytotoxicity curves of S-PCL-b-PHD@CUR and S-PCL-b-PHPMA@CUR micelles; as shown. Figure 11 As shown, compared with S-PCL-b-PHPMA@CUR micelles without charge-flipping function, S-PCL-b-PHD@CUR micelles with charge-flipping function significantly inhibited H460 cell growth. This is because at pH 6.5, the S-PCL-b-PHD@CUR micelles have a positive charge, while the S-PCL-b-PHPMA@CUR micelles have a negative charge. The positively charged micelles exhibit enhanced adhesion to the tumor cell membrane, making them more easily taken up by tumor cells. The cytotoxicity of free curcumin was stronger than that of S-PCL-b-PHD@CUR and S-PCL-b-PHPMA@CUR, possibly due to its extremely low solubility leading to physical precipitation and adhesion to the cell surface, resulting in extremely high local drug concentrations, severe acute physicochemical damage, and rapid drug penetration.

[0099] 10. Cellular uptake of charge-flipped amphiphilic star-shaped polymer drug-loaded micelles.

[0100] Human large cell lung cancer cells (H460) were cultured at 2.5 × 10⁶ cells per well. 5Cells were seeded at a density in 6-well plates and cultured at 37°C in a 5% CO2 incubator. When the cell density in each well reached approximately 80%, the culture medium was discarded, and 2 mL of fresh culture medium containing free curcumin (CUR), 2 mL of S-PCL-b-PHD@CUR micelles, and 2 mL of S-PCL-b-PHPMA@CUR micelles (pH 6.5, CUR concentration 10 µg / mL) were added to each well, respectively. A blank control group was also included. Cells were incubated at 37°C in a 5% CO2 incubator for 4 h. The drug-containing culture medium was discarded, and the cells were washed three times with sterile PBS. 500 μL of 0.25% trypsin solution was added to each well for digestion. After digestion, 1 mL of fresh culture medium was added, and the cells were gently pipetted to form a single-cell suspension. The suspension was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 200 μL of PBS. The intracellular curcumin fluorescence intensity was detected by flow cytometry. Figure 12 The quantitative uptake results of H460 showed that, compared with the control group, all drug groups exhibited significant CUR fluorescence signals, indicating that free-CUR, S-PCL-b-PHD@CUR, and S-PCL-b-PHPMA@CUR were successfully taken up by H460 cells. Furthermore, the S-PCL-b-PHD@CUR group showed the strongest fluorescence intensity, indicating that S-PCL-b-PHD@CUR micelles are more readily taken up by tumor cells. This is because at pH 6.5, the S-PCL-b-PHD@CUR micelles have a positive charge, resulting in strong adhesion to the tumor cell membrane.

[0101] 11. Biocompatibility of charge-reversed amphiphilic star polymers.

[0102] The prepared S-PCL-b-PHD was dissolved in culture medium to obtain culture media containing different concentrations of S-PCL-b-PHD. Human large cell lung cancer cells (H460) were cultured at a density of 5 × 10⁶ cells / well. 3 Cells were seeded at a density in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours. After incubation, the culture medium was aspirated, and 100 µL of fresh culture medium containing different concentrations of S-PCL-b-PHD was added to each well, followed by incubation for another 48 hours. Then, 10 µL of CCK-8 solution was added to each well, and incubation was continued for another 3 hours. Cell viability was calculated by measuring the absorbance (OD value) at 490 nm using a microplate reader. Figure 13 The cytotoxicity test results for S-PCL-b-PHD, such as... Figure 13 As shown, within the concentration range of 0~500 μg / mL, S-PCL-b-PHD showed almost no cytotoxicity to H460 cells, indicating that S-PCL-b-PHD itself has good biocompatibility.

[0103] In summary, the charge-flipping amphiphilic star polymer prepared in this invention, relying on its multi-arm topology providing multiple drug-loading sites and its anti-dissociation three-dimensional configuration, constructs a smart nanodelivery system that combines high drug loading capacity, pH-adaptive charge flipping, and long-term cycling stability. This system carries a negative surface charge at physiological pH, which helps avoid serum protein adsorption and reticuloendothelial system clearance, prolonging blood circulation time. Upon reaching the tumor microenvironment, the tertiary amine groups can trigger a surface potential shift from negative to positive through protonation, enhancing electrostatic adsorption to the negatively charged tumor cell membrane and promoting cell adhesion and endocytosis. Based on the advantages of drug loading stability of the star structure and the precise spatiotemporal control of charge flipping, this system can achieve efficient drug accumulation in tumor cells, overcoming the bottlenecks of drug resistance and off-target toxicity in traditional chemotherapy, and is expected to provide a new pathway for precision anti-tumor therapy with enhanced efficacy and reduced toxicity.

[0104] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A charge-reversing amphiphilic star polymer, characterized in that, The structural formula of the charge-reversing amphiphilic star polymer is shown in formula (Ⅰ): ； In formula (Ⅰ), PHC is a polyhydroxy compound; the value of m ranges from 5 to 20; the sum of the values ​​of n1 and n2 ranges from n1+n2=10 to 100; and the ratio of n1 to n2 ranges from n1 / n2=0.05 to 1. The PHC mentioned in formula (Ⅰ) is at least one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, xylitol, sorbitol, and inositol.

2. The method for preparing the charge-reversal type amphiphilic star polymer according to claim 1, characterized in that, Includes the following steps: (1) Preparation of star-shaped polycaprolactone based on polyhydroxy compounds: The polyhydroxy compound, ε-caprolactone, stannous isooctanoate, and the first solvent are mixed, dried to remove water, and then reacted. The mixture is then precipitated in an alcohol or petroleum ether to obtain star-shaped polycaprolactone. (2) Preparation of macromolecular initiators based on star-shaped polycaprolactone: The star-shaped polycaprolactone and triethylamine are dissolved in a second solvent, and 2-bromo-2-methylpropionyl bromide is added dropwise under ice bath conditions to carry out the reaction. The mixture is then precipitated in alcohol or petroleum ether to obtain 2-bromo-2-methylpropionyloxy group modified star-shaped polycaprolactone, which is a macromolecular initiator based on star-shaped polycaprolactone. (3) Preparation of star-shaped polycaprolactone-block polypentafluorophenyl methacrylate: The 2-bromo-2-methylpropionyloxy group-modified star-shaped polycaprolactone and pentafluorophenyl methacrylate were dissolved in a third solvent, and then copper bromide, nitrogen-containing ligands, and stannous isooctanoate were added. The reaction was carried out under deoxygenation conditions. The resulting reaction solution was passed through an alumina-packed column and then precipitated in alcohols or petroleum ethers to obtain star-shaped polycaprolactone-block-polypentafluorophenyl methacrylate. (4) Preparation of charge-flipping amphiphilic star polymers: The star-shaped polycaprolactone-block-polypentafluorophenyl methacrylate, isopropanolamine, and N,N-diisopropylethylenediamine were dissolved in a fourth solvent and reacted. After the reaction was completed, the mixture was dialyzed and freeze-dried in sequence to obtain the star-shaped polycaprolactone-block-poly(2-hydroxypropyl methacrylamide-copolymer-diisopropylaminoethyl methacrylamide), which is a charge-reversing amphiphilic star polymer.

3. The method for preparing a charge-reversing amphiphilic star polymer according to claim 2, characterized in that, The molar ratio of the hydroxyl group of the polyhydroxy compound in step (1) to that of ε-caprolactone to that of stannous isooctanoate is 1:5~50:0.05~0.5; the concentration of ε-caprolactone is 1 mol / L~10 mol / L; In step (1), the first solvent includes at least one of toluene, xylene, anisole, cyclohexanone, N-methylpyrrolidone, dimethylformamide, and dimethyl sulfoxide. The alcohols mentioned in step (1) include at least one of methanol and ethanol; The reaction conditions after drying and removing moisture in step (1) are: temperature of 110℃~140℃ and time of 12 h~48 h.

4. The method for preparing a charge-reversing amphiphilic star polymer according to claim 2, characterized in that, The mass ratio of star-shaped polycaprolactone, triethylamine, and 2-bromo-2-methylpropionyl bromide in step (2) is 1:0.1~1:0.2~2; the concentration of star-shaped polycaprolactone before the reaction is 20 g / L~200 g / L; the concentration of 2-bromo-2-methylpropionyl bromide before the reaction is 20 g / L~200 g / L. In step (2), the second solvent includes at least one of chloroform, dichloromethane, tetrahydrofuran, and toluene; The alcohols mentioned in step (2) include at least one of methanol and ethanol; The reaction conditions in step (2) are: react in an ice bath for 1 h to 3 h, and then react at room temperature for 12 h to 24 h.

5. The method for preparing a charge-reversing amphiphilic star polymer according to claim 2, characterized in that, The molar ratio of copper bromide, nitrogen-containing ligand, stannous isooctanoate, and pentafluorophenyl methacrylate in step (3) is 1:2~20:10~100:5000~20000; the mass ratio of 2-bromo-2-methylpropionyloxy group modified star-shaped polycaprolactone to pentafluorophenyl methacrylate is 1:5~20; and the concentration of 2-bromo-2-methylpropionyloxy group modified star-shaped polycaprolactone is 0.01 g / mL~0.1 g / mL. The nitrogen-containing ligands mentioned in step (3) include at least one of tris(2-pyridinemethyl)amine, tris[2-(dimethylamino)ethyl]amine, N,N,N′,N′,N′′-pentamethyldiethyltriamine, 1,1,4,7,10,10-hexamethyltriethylenetetramine, and 4,4'-dihexyl-2,2'-bipyridine; The third solvent mentioned in step (3) includes at least one of anisole, cyclohexanone, tetrahydrofuran, N-methylpyrrolidone, toluene, dimethylformamide, and dimethyl sulfoxide; The alcohols mentioned in step (3) include at least one of methanol and ethanol; The reaction conditions in step (3) under deoxygenation conditions are as follows: high-purity argon gas is bubbled through for 20 min to 30 min to remove oxygen, and then the mixture is transferred to an oil bath at 50℃ to 90℃ and stirred for 24 h to 72 h.

6. The method for preparing a charge-reversing amphiphilic star polymer according to claim 2, characterized in that, The molar ratio of N,N-diisopropylethylenediamine to isopropanolamine in step (4) is 1:1~20; the molar ratio of pentafluorophenyl methacrylate to isopropanolamine and N,N-diisopropylethylenediamine in the star-shaped polycaprolactone-block-polypentafluorophenyl methacrylate is 1:1~10. The fourth solvent in step (4) includes at least one of dimethylformamide, tetrahydrofuran, N-methylpyrrolidone, toluene, and dimethyl sulfoxide; The reaction conditions for step (4) are: reaction in an oil bath at 40℃~60℃ for 20 h~48 h; The dialysis bag used in step (4) has a molecular weight cutoff of 1000 Da to 3500 Da; the freeze-drying conditions are: temperature -30±5℃, time 24 h to 72 h.

7. Use of a charge-flipping amphiphilic star polymer, characterized in that The charge-flipping amphiphilic star polymer of claim 1 is used to prepare drug-loaded micelles.

8. A method for preparing a drug-loaded micelle of a charge-reversal type amphiphilic star polymer, characterized by, Includes the following steps: The charge-reversing amphiphilic star polymer of claim 1 and the anticancer drug are dissolved in a fifth solvent, then added dropwise to pure water while stirring to remove the fifth solvent and unreacted anticancer drug. The solution is then filtered through a microporous membrane to obtain a drug-loaded micelle aqueous solution.

9. The method for preparing drug-loaded micelles of a charge-reversed amphiphilic star polymer according to claim 8, characterized in that, The mass ratio of the charge-reversing amphiphilic star polymer to the anticancer drug is 1:0.5~2; the mass-volume concentration of the charge-reversing amphiphilic star polymer dissolved in the fifth solvent is 1 g / L~10 g / L; The volume ratio of the fifth solvent to the water is 1:3~30; The particle size of the drug-loaded micelles is 50 nm to 200 nm; The fifth solvent includes at least one of tetrahydrofuran, dimethylformamide, N-methylpyrrolidone, and dimethyl sulfoxide; The anticancer drugs include at least one of curcumin, doxorubicin, paclitaxel, camptothecin, oxaliplatin, irinotecan, and leucoquinone.

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