A dual stimuli-responsive polymer micelle and a preparation method and application thereof

Abstract

The application discloses a kind of double stimulation response polymer micelles and its preparation method and application, the micelle is formed by the polyethylene glycol monomethyl ether- (alpha-alkynyl propyl-delta-valerolactone-lactide) copolymer of modified phenylboronic acid and the polyethylene glycol monomethyl ether- (alpha-alkynyl propyl-delta-valerolactone-lactide) copolymer of modified catechol is formed together;When the micelle is formed, corresponding borate bond can be formed in the pH, ROS stimulation hydrophobic inner core;The micelle is used to load hydrophobic antitumor drug.The polymer micelle prepared in the application can be stable in the physiological environment of in-vivo circulation, and loose or dissociate in response to tumor microenvironment stimulation, realize the effective delivery of hydrophobic antitumor drug, effectively overcome the problem of micelle instability in vivo, drug pre-leakage, provide theory and practical basis for promoting polymer micelle drug delivery system cancer treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical polymer materials, specifically relating to a dual-stimulus responsive polymer micelle, its preparation method, and its application. Background Technology

[0002] Traditional anti-tumor drugs face multiple challenges in in vivo drug delivery due to poor solubility, high cytotoxicity, and lack of tissue specificity. Drug delivery systems, through specific technologies, improve drug pharmacokinetics and in vivo distribution, increasing effective drug accumulation at the tumor site, thereby enhancing overall therapeutic efficacy and safety.

[0003] Polymer micelles offer advantages such as simple preparation and high drug encapsulation efficiency, making them promising candidates for development. Polymer micelles typically consist of a core-shell structure formed by the self-assembly of amphiphilic polymers, resulting in a hydrophilic surface and a hydrophobic core. Tumor cells exhibit an acidic microenvironment due to abnormal oxidative metabolism and possess abnormal levels of reactive oxygen species (ROS) / glutathione (GSH). By introducing stimuli-responsive groups or linkages onto the amphiphilic polymer structure, polymer micelles with tumor microenvironment stimuli-responsive capabilities can be prepared, enabling controlled targeted release of antitumor drugs. However, traditional polymer micelles usually rely on self-assembly through hydrophilic-hydrophobic interactions, with antitumor drugs loaded within the micelles via hydrophobic / hydrophobic interactions with the hydrophobic polymer fragments. Consequently, the hydrophobic core of traditional drug-loaded polymer micelles is relatively loose, unstable in complex physiological environments, prone to drug pre-leakage, and difficult to achieve effective drug accumulation at the tumor site, leading to significant toxic side effects on normal tissues. These drawbacks significantly limit the clinical application of traditional polymer micelle drug delivery systems.

[0004] Therefore, there is an urgent need to develop a polymer micelle drug delivery system that can circulate stably in complex physiological environments in vivo, has the ability to respond to tumor microenvironment stimuli, and can achieve controlled drug release within tumor tissues or cells. Summary of the Invention

[0005] To address the problems of existing polymer micelle drug delivery systems, this invention aims to provide a dual-stimulus-responsive polymer micelle, its preparation method, and its applications. The dual-stimulus-responsive polymer micelle of this invention is a core-crosslinked polymer micelle with a borate ester structure exhibiting dual pH and ROS-responsive capabilities. The hydrophobic segments of the micelles prepared in this invention are modified by small molecules containing phenylboronic acid and catechol functional groups, which can form a borate ester structure under pH 7.4 conditions. In the specific tumor microenvironment of weak acidity (pH < 7) and high ROS levels, the borate ester bonds break, leading to the dissociation or loosening of the polymer micelle structure, thereby achieving controlled targeted release of hydrophobic antitumor drugs.

[0006] The technical solution adopted in this invention is as follows:

[0007] A dual-stimulation responsive polymer micelle comprises a polyethylene glycol monomethyl ether-(α-propargyl-δ-valerol-lactide) copolymer modified with phenylboronic acid and a polyethylene glycol monomethyl ether-(α-propargyl-δ-valerol-lactide) copolymer modified with catechol.

[0008] Furthermore, the structural formula of the phenylboronic acid-modified polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer is shown in Formula I:

[0009]

[0010] Further, in Formula I, x, y, z, and n represent the degree of polymerization of the repeating unit in the phenylboronic acid-modified polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer, and the ratio of y to z in the hydrophobic segment of the polymer is 1:1 or 1:3. Specifically, when y:z = 1:1, x is 46-227, y is 7-54, z is 7-54, and n / y = 50-72%; when y:z = 1:3, x is 46-227, y is 4-27, z is 12-81, and n / y = 50-72%.

[0011] Furthermore, the structural formula of the catechol-modified polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer is shown in Formula II:

[0012]

[0013] Further, in Formula II, x, y, z, and n represent the degree of polymerization of the repeating unit in the catechol-modified polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer, and the ratio of y to z in the hydrophobic segment of the polymer is 1:1 or 1:3. Specifically, when y:z = 1:1, x is 46-227, y is 7-54, z is 7-54, and n / y = 50-72%; when y:z = 1:3, x is 46-227, y is 4-27, z is 12-81, and n / y = 50-72%.

[0014] Furthermore, the copolymer shown in Formula I is prepared by a divalent copper salt-catalyzed azido-acetylene cycloaddition reaction of polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer and 4-azidomethylphenylboronic acid; the copolymer shown in Formula II is prepared by a divalent copper salt-catalyzed azido-acetylene cycloaddition reaction of polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer and 4-azidoethyl-1,2-catechol; and L-ascorbic acid sodium is also introduced as an additive in the azido-acetylene cycloaddition reaction.

[0015] Furthermore, the main chain molecular weight of the polyethylene glycol monomethyl ether-(α-propargyl-δ-valerol-lactide) copolymer is 4000-25000 Da.

[0016] Furthermore, the polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer has the structure shown in Formula III:

[0017]

[0018] In Formula III, x, y, and z represent the degree of polymerization of the repeating units in the copolymer. The ratio of y to z in the hydrophobic segment of the polymer is 1:1 or 1:3, and the values ​​of the degrees of polymerization x, y, and z are the same as in Formula I or Formula II. The molecular weight of the hydrophilic portion of the polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer is 2000-10000 Da, and the molecular weight of the hydrophobic portion is 2000-15000 Da.

[0019] Furthermore, the polyethylene glycol monomethyl ether-(α-propynyl-δ-valerolactone-lactide) copolymer is obtained by catalytic ring-opening polymerization of lactide and α-propynyl-δ-valerolactone initiated by polyethylene glycol monomethyl ether, and the catalyst used for the catalytic ring-opening polymerization is Sn(Oct)2.

[0020] Furthermore, the molecular weight of the polyethylene glycol monomethyl ether is 2000-10000 Da; more preferably 4000-5000 Da.

[0021] The present invention also provides the application of dual-stimulus responsive polymer micelles in the delivery of hydrophobic antitumor drugs, wherein the hydrophobic antitumor drugs are selected from one or more of paclitaxel, doxorubicin, camptothecin, gemcitabine, mitoxantrone, and derivatives of any of the above drugs.

[0022] Furthermore, the hydrophobic antitumor drug is one of the following:

[0023]

[0024] A hydrophobic antitumor drug micelle delivery system comprises nuclear crosslinked micelles obtained by self-assembly of a phenylboronic acid-modified polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer, a catechol-modified polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer, and a hydrophobic antitumor drug.

[0025] This invention relates to the application of nuclear crosslinked micelles with a borate ester structure that exhibit dual pH and ROS stimuli response in the delivery of hydrophobic antitumor drugs.

[0026] A nuclear crosslinked micelle based on a borate ester structure exhibiting dual pH and ROS stimuli responsiveness, its composition is as follows: Figure 27 As shown.

[0027] The preparation method for the core-crosslinked polymer micelles with dual pH and ROS stimulation response based on borate ester structure is as follows: 5 mg of phenylboronic acid-modified polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer, 5 mg of catechol-modified polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer, and 1 mg of a hydrophobic antitumor drug are added to 1 mL of chloroform and dissolved by sonication. After removing the chloroform to form a thin film using a rotary evaporator, 1 mL of pH 7.4 phosphate buffered saline (PBS) is added, and the solution is rehydrated by sonication at 50 °C. Then, 9 mL of pH 7.4 PBS is added to adjust the total polymer concentration of the solution to 1 mg / mL. Finally, insoluble suspended solids are removed through a syringe filter.

[0028] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0029] 1) The preparation of the basic framework of the micelles in this invention, polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer, is highly controllable; by changing the feed ratio of the hydrophobic monomer α-propargyl-δ-valerolactone to lactide, the proportion of hydrophobic monomers in the polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer can be precisely controlled.

[0030] 2) In this invention, the pendant group of the basic framework of the micelles, polyethylene glycol monomethyl ether-(α-propargyl-δ-valerol-lactide) copolymer, is a hydrophobic alkyne group, which can avoid the influence of unreacted groups on the hydrophobic part after functionalization modification, thereby destroying the stability of the micelles.

[0031] 3) In this invention, the functionalization modification of the basic framework of micelles, polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer, is prepared by a highly efficient copper-catalyzed azide-alkyne cycloaddition reaction.

[0032] 4) The core crosslinked micelles based on borate ester structure prepared in this invention have better particle size, particle size distribution and drug loading capacity than micelles formed by a single copolymer, and the micelles have better stability.

[0033] 5) The nuclear cross-linked micelles based on borate ester structure prepared in this invention have a high encapsulation efficiency of hydrophobic antitumor drugs with dual pH and ROS stimulation response.

[0034] 6) The borate ester-based nuclear crosslinked micelles prepared in this invention have dual pH and ROS stimulation response capabilities, exhibiting good acidic pH and high-level ROS response capabilities as well as biocompatibility, enabling efficient delivery of hydrophobic antitumor drugs and effective accumulation at tumor sites.

[0035] 7) The nuclear cross-linked micelles based on borate ester structure prepared in this invention have the ability to respond to both pH and ROS stimulation. They can be effectively taken up by cells, achieve good in vivo distribution, and effectively inhibit tumor cell growth.

[0036] The present invention provides a nuclear crosslinked micelle based on a borate ester structure with dual pH and ROS stimulation responsiveness. This mouselle effectively improves the stability of polymer micelles in physiological environments, reduces drug pre-leakage, lowers drug toxicity, and increases drug accumulation at tumor sites. This invention provides theoretical and practical basis for developing novel polymer micelle drug delivery systems for tumor treatment. Attached Figure Description

[0037] Figure 1 Synthesis route diagram of monomer AVL;

[0038] Figure 2 Monomeric AVL 1 H-NMR spectrum;

[0039] Figure 3 Synthesis route diagram of mPPAL;

[0040] Figure 4 mPPAL3 1H-NMR spectrum;

[0041] Figure 5 mPPAL5 1 H-NMR spectrum;

[0042] Figure 6 Synthesis route diagram of N3-PBA;

[0043] Figure 7 N3-PBA 1 H-NMR spectrum;

[0044] Figure 8 Synthetic route diagram of N3-catechol;

[0045] Figure 9 N3-catechol 1 H-NMR spectrum;

[0046] Figure 10 Synthesis roadmaps for mPPAL@PBA and mPPAL@catechol;

[0047] Figure 11 mPPAL3@PBA 1 H-NMR spectrum;

[0048] Figure 12 mPPAL5@PBA 1 H-NMR spectrum;

[0049] Figure 13 mPPAL3@catechol 1 H-NMR spectrum;

[0050] Figure 14 mPPAL5@catechol 1 H-NMR spectrum;

[0051] Figure 15 TEM image of PTX-CLM3;

[0052] Figure 16 TEM image of PTX-CLM5;

[0053] Figure 17 A schematic diagram illustrating the stability of blank micelles;

[0054] Figure 18 Schematic diagram of the stability of drug-loaded micelles;

[0055] Figure 19 Critical micelle concentration results for CLM3;

[0056] Figure 20 Critical micelle concentration results for CLM4;

[0057] Figure 21 Critical micelle concentration results for CLM5;

[0058] Figure 22 Critical micelle concentration results for CLM6;

[0059] Figure 23 Drug release curves of PTX-CLM3 and PTX-CLM5 in different environments;

[0060] Figure 24 Cell viability graphs of HeLa cells treated with different doses of CLM3 and CLM5 and HeLa cells treated with different doses of PTX-CLM3 and PTX-CLM5;

[0061] Figure 25 Fluorescence micrographs of HeLa cells after 2 hours and 6 hours of uptake of different doses of coumarin 6-CLM3 and coumarin 6-CLM5;

[0062] Figure 26 Flow cytometry results of HeLa cells taking up different doses of coumarin 6-CLM3 and coumarin 6-CLM5 for 2 hours and 6 hours.

[0063] Figure 27 This is a compositional diagram of the nuclear cross-linked micelles of hydrophobic antitumor drugs loaded with borate ester structure, which have dual pH and ROS stimulation response capabilities according to the present invention. Detailed Implementation

[0064] The present invention will be further described below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto.

[0065] Specifically, the naming conventions used in this invention are as follows: α-propargyl-δ-valerolactone is abbreviated as AVL; polyethylene glycol monomethyl ether is abbreviated as mPEG; lactide is abbreviated as LA; and the copolymer of polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) is named mPEG. a -bP(AVL-co-LA) bThe abbreviation for mPPAL is as follows: a represents the molecular weight of the hydrophilic portion (mPEG), and b represents the molecular weight of the hydrophobic portion (polyester). Phenylated boric acid is abbreviated as PBA; catechol is abbreviated as Catechol; 4-azidomethylphenylboronic acid is abbreviated as N3-PBA; 4-azidoethyl-1,2-catechol is abbreviated as N3-Catechol; phenylboronic acid-modified polyethylene glycol monomethyl ether-(α-propynyl-δ-valerol-lactide) copolymer is named mPPAL@PBA; catechol-modified polyethylene glycol monomethyl ether-(α-propynyl-δ-valerol-lactide) copolymer is named mPPAL@catechol; core-crosslinked micelles based on borate ester structures are abbreviated as CLM; core-crosslinked micelles of paclitaxel (PTX) supported on borate ester structures are abbreviated as PTX-CLM; core-crosslinked micelles of coumarin 6 supported on borate ester structures are abbreviated as coumarin. 6-CLM; the abbreviation for DiR-CLM is the supported DiR core crosslinked micelles based on borate ester structure.

[0066] The hydrophobic antitumor drug PTX can be replaced by one or more of DOX, CPT, GEM, and MITX.

[0067] In the formula, the encapsulated hydrophobic antitumor drug is:

[0068]

[0069] One of them.

[0070] Example 1: Synthesis of monomer AVL

[0071] 55.2 mL of a 2 mol / L solution of lithium diisopropylamino in tetrahydrofuran / n-hexane (LDA, 110.4 mmol) was added to a 500 mL flask containing 250 mL of anhydrous tetrahydrofuran and stirred at -78 °C. δ-valerolactone (VL, 10.0 g, 99.88 mmol) was thoroughly mixed with 250 mL of anhydrous tetrahydrofuran, and then this mixture was added dropwise over 1 h with stirring. The mixture was stirred for another 0.5 h. A mixture of 3-bromopropyne (14.4 g, 121.0 mmol) and hexamethylphosphoric triamine (HMPA, 20.9 mL, 120.1 mmol) was added dropwise over 2 h with continued stirring at -78 °C. The mixture was stirred for another 2 h at -30 °C. The reactants were quenched by adding 50 mL of saturated NH4Cl solution, and then the temperature was raised to room temperature. After removing tetrahydrofuran by rotary evaporation, the solution was extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed twice with ultrapure water, once with saturated NaCl, dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude product was separated by gradient elution using silica gel column chromatography (n-hexane:ethyl acetate = 9:1-3:1, v / v) to give a pale yellow liquid (6.49 g, 47.0 mmol, yield 47.1%). Further distillation under reduced pressure yielded a pure product as a colorless, viscous liquid AVL (5.45 g, yield 39.4 mmol, 39.4%), the structure of which is shown below. Figure 1 . 1 H-NMR (400MHz, Chloroform-d): δ4.43-4.26(m,2H),2.78-2.60(m,2H),2.54-2.44(m ,1H),2.36-2.23(m,1H),2.01(t,J=2.6Hz,1H),1.99-1.87(m,2H),1.81-1.66(m,1H).

[0072] Example 2: Synthesis of the amphiphilic polymer mPPAL3

[0073] Polyethylene glycol monomethyl ether mPEG with an average molecular weight of 4k is labeled as mPEG. 4k .

[0074] All reaction vessels were thoroughly dried overnight in an oven at 50°C before use. The steps for preparing mPPAL3 are as follows: Under a N2 atmosphere, mPEG... 4k1.00 g (0.25 mmol), AVL (0.49 g, 3.55 mmol), and LA (0.51 g, 3.55 mmol) were added to a 50 mL three-necked flask, along with 25 mL of anhydrous toluene. The mixture was heated to 150 °C to remove residual water by forming an azeotrope with toluene. The temperature was then lowered to 125 °C, and 6-7 drops of Sn(Oct)₂ were added to the reaction mixture. The mixture was stirred at 125 °C for 48 h. After the reaction was complete, the mixture was cooled to room temperature, diluted with dichloromethane, and washed successively with 10% HCl, ultrapure water, and saturated NaCl aqueous solution until the aqueous layer was neutral. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated. The polymer was precipitated at least three times with a mixed solvent (dichloromethane:n-hexane = 1:10, volume ratio), and then dried in a vacuum drying oven. The final product was a yellow solid (1.69 g, 0.21 mmol, yield 84.7%), and its structure is shown below. Figure 4 .

[0075] Example 3: Synthesis of the amphiphilic polymer mPPAL5

[0076] Polyethylene glycol monomethyl ether mPEG with an average molecular weight of 5k is labeled as mPEG. 5k .

[0077] All reaction vessels were thoroughly dried overnight in an oven at 50°C before use. The steps for preparing mPPAL5 are as follows: Under a N2 atmosphere, mPEG... 5k 1.00 g (0.20 mmol), AVL (0.78 g, 5.67 mmol), and LA (0.82 g, 5.67 mmol) were added to a 50 mL three-necked flask, along with 25 mL of anhydrous toluene. The mixture was heated to 150 °C to remove residual water by forming an azeotrope with toluene. The temperature was then lowered to 125 °C, and 6-7 drops of Sn(Oct)₂ were added to the reaction mixture. The mixture was stirred at 125 °C for 48 h. After the reaction was complete, the mixture was cooled to room temperature, diluted with dichloromethane, and washed successively with 10% HCl, ultrapure water, and saturated NaCl aqueous solution until the aqueous layer was neutral. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated. The polymer was precipitated at least three times with a mixed solvent (dichloromethane:n-hexane = 1:10, volume ratio), and then dried in a vacuum drying oven. The final product was a yellow solid (1.87 g, 0.14 mmol, yield 71.9%), and its structure is shown below. Figure 5 .

[0078] Using the same stannous isooctanoate catalyst and different molecular weight initiators (mPEG), the ring-opening polymerization method was employed. By controlling the amount of mPEG added and the monomer feed ratio, while keeping other conditions unchanged, mPPAL polymers with different molecular weights and different proportions of hydrophobic monomers were synthesized. The yield of this series of polymers was 66.8%-90.5%. The table below shows the specific molecular weights of hydrophilic and hydrophobic segments and the proportions of hydrophobic monomers of the preferred polymers.

[0079]

[0080] a Molecular weight of the “AVL and LA” copolymer fragments. b The ratio of the number of AVL and LA hydrophobic monomers in mPPAL.

[0081] Example 4: Synthesis of N3-PBA

[0082] 4-(bromomethyl)phenylboronic acid (1.00 g, 4.65 mmol) and sodium azide (0.91 g, 14.0 mmol) were added to a 25 mL round-bottom flask, along with 10 mL of DMF. The reaction mixture was reacted continuously at 50 °C for 48 h. The reactants were then cooled to room temperature and diluted with 100 mL of ultrapure water. The mixture was stirred at room temperature for 10 min, and then extracted with ethyl acetate (100 mL × 3). The organic layers were combined, and the organic phase was washed twice with ultrapure water and once with saturated NaCl aqueous solution. The mixture was dried over anhydrous Na₂SO₄, filtered, and concentrated. The product was further dried in a vacuum drying oven to give a pale yellowish-white solid N₃-PBA (0.78 g, 4.41 mmol, yield 94.8%), the structure of which is shown below. Figure 6 . 1 H-NMR (400MHz, DMSO-d6): δ8.08 (s, 2H), 7.81 (d, J = 7.9 Hz, 2H), 7.32 (d, J = 7.8 Hz, 2H), 4.44 (s, 2H).

[0083] Example 5: Synthesis of N3-catechol

[0084] Sodium azide (1.03 g, 15.8 mmol) and 10 mL of acetonitrile were added to a 25 mL flask and stirred at 0 °C for 10 min. Trifluoromethanesulfonic anhydride (1.06 mL, 6.33 mmol) was added dropwise to the sodium azide suspension over 0.5 h, and the reaction suspension was stirred at 0 °C for 2 h to obtain freshly prepared trifluoromethanesulfonyl azide. In another 100 mL flask, a mixture of dopamine hydrochloride (1.00 g, 5.27 mmol), zinc chloride (71.8 mg, 0.53 mmol), and 25 mL of CH3CN:H2O (volume ratio 7:3) was added, followed by triethylamine (2.21 mL, 15.8 mmol), and then the freshly prepared trifluoromethanesulfonyl azide was rapidly added. The reaction was stirred overnight at room temperature. The mixture was filtered and concentrated by rotary evaporation to remove acetonitrile. The resulting solutions were extracted with ethyl acetate (20 mL × 3). The organic layers were combined, washed twice with ultrapure water and once with saturated NaCl aqueous solution, dried over anhydrous Na₂SO₄, filtered, and concentrated. The product was eluted by silica gel column chromatography (n-hexane:ethyl acetate = 1:1, v / v) to give a brownish-black solid N₃-catechol (0.87 g, 4.86 mmol, yield 92.2%), the structure of which is shown below. Figure 8 . 1 H-NMR(400MHz,Chloroform-d): δ6.81(d,J=8.1Hz,1H),6.74(d,J=2.0Hz,1H),6.6 6(dd,J=8.1,2.1Hz,1H),5.37(s,2H),3.45(t,J=7.2Hz,2H),2.78(t,J=7.2Hz,2H).

[0085] Example 6: Synthesis of mPPAL3@PBA

[0086] Under light-protected conditions, anhydrous copper acetate (7.17 mg, 0.040 mmol) and sodium L-ascorbate (15.64 mg, 0.079 mmol) were added to 4 mL of anhydrous DMF solution containing mPPAL3 (200.00 mg, 0.026 mmol) and N3-PBA (209.57 mg, 1.18 mmol). The reaction mixture was stirred at 50 °C for 24 h. After cooling to room temperature, the reaction mixture was diluted with 4 mL of ultrapure water and dialyzed against 0.1 mmol of disodium ethylenediaminetetraacetate aqueous solution for 24 h to remove copper, followed by dialyzed against ultrapure water for 48 h. The final product, mPPAL3@PBA, was obtained by freeze-drying, with a click reaction grafting rate of 70.8%. The structure is shown in [Figure number missing]. Figure 10 .

[0087] Example 7: Synthesis of mPPAL3@catechol

[0088] Under light-protected conditions, anhydrous copper acetate (7.17 mg, 0.040 mmol) and sodium L-ascorbate (15.64 mg, 0.079 mmol) were added to 4 mL of anhydrous DMF solution containing mPPAL3 (200.00 mg, 0.026 mmol) and N3-catechol (212.19 mg, 1.18 mmol). The reaction mixture was stirred at 50 °C for 24 h. After cooling to room temperature, the reaction mixture was diluted with 4 mL of ultrapure water and dialyzed against 0.1 mmol of disodium ethylenediaminetetraacetate aqueous solution for 24 h to remove copper, followed by dialyzing against ultrapure water for 48 h. The final product, mPPAL3@catechol, was obtained by freeze-drying, with a click reaction grafting rate of 57.9%. The structure is shown in [Figure number missing]. Figure 10 .

[0089] Example 8: Synthesis of mPPAL5@PBA

[0090] Under light-protected conditions, anhydrous copper acetate (7.33 mg, 0.040 mmol) and sodium L-ascorbate (15.99 mg, 0.081 mmol) were added to 4 mL of anhydrous DMF solution containing mPPAL5 (200.00 mg, 0.018 mmol) and N3-PBA (214.23 mg, 1.21 mmol). The reaction mixture was stirred at 50 °C for 24 h. After cooling to room temperature, the reaction mixture was diluted with 4 mL of ultrapure water and dialyzed against 0.1 mmol of disodium ethylenediaminetetraacetate aqueous solution for 24 h to remove copper, followed by dialyzed against ultrapure water for 48 h. The final product, mPPAL5@PBA, was obtained by freeze-drying, with a click reaction grafting rate of 61.4%. The structure is shown in [Figure number missing]. Figure 10 .

[0091] Example 9: Synthesis of mPPAL5@catechol

[0092] Under light-protected conditions, anhydrous copper acetate (7.33 mg, 0.040 mmol) and sodium L-ascorbate (15.99 mg, 0.081 mmol) were added to 4 mL of anhydrous DMF solution containing mPPAL5 (200.00 mg, 0.018 mmol) and N3-catechol (216.90 mg, 1.21 mmol). The reaction mixture was stirred at 50 °C for 24 h. After cooling to room temperature, the reaction mixture was diluted with 4 mL of ultrapure water and dialyzed against 0.1 mmol of disodium ethylenediaminetetraacetate aqueous solution for 24 h to remove copper, followed by dialyzed against ultrapure water for 48 h. The final product, mPPAL5@catechol, was obtained by freeze-drying, with a click reaction grafting rate of 54.3%. The structure is shown in [Figure number missing]. Figure 10 .

[0093] Example 10: Preparation of nuclear crosslinked micelles

[0094] Blank and drug-loaded micelles were prepared using a thin-film dispersion method. Taking drug-loaded micelle PTX-CLM3 as an example, the polymer materials for PTX-CLM3 were mPPAL3@PBA and mPPAL3@catechol, and the encapsulated hydrophobic antitumor drug was PTX. 5 mg of the polymer materials mPPAL3@PBA, 5 mg of mPPAL3@catechol, and 1 mg of PTX were dissolved in 1 mL of chloroform and sonicated for 10 min to ensure complete dissolution. The chloroform was removed using a rotary evaporator, and the membrane was rehydrated by sonication at 50°C for 0.5 h with 1 mL of PBS solution. Then, 9 mL of PBS solution was added to adjust the polymer concentration to 1 mg / mL, and free PTX was removed using a needle filter to obtain the final drug-loaded micelle PTX-CLM3. Blank micelle CLM3 was prepared using the same method but without the encapsulated hydrophobic antitumor drug. Figure 15 The image shows a TEM image of PTX-CLM3, which is spherical and has a good morphology.

[0095] The preparation method of drug-loaded micelles PTX-CLM5 is the same as that of drug-loaded micelles PTX-CLM3, except that "mPPAL3@PBA and mPPAL3@catechol are replaced with the same mass of mPPAL5@PBA and mPPAL5@catechol respectively", and the other conditions remain unchanged, and drug-loaded micelles PTX-CLM5 are finally obtained. Figure 16 The image shows a TEM image of PTX-CLM5, which is spherical and has a good morphology.

[0096] Example 11: Investigation of the storage stability of micelle aqueous solutions

[0097] The PBS aqueous solution containing micelles with a polymer concentration of 1 mg / mL, prepared as described in Example 10, was stored at room temperature and in a cool place. Samples were taken at 0, 1, 3, 5, and 7 days after preparation, and the changes in particle size and PDI were detected by DLS. Figure 17 and 18 This indicates that the nuclear cross-linked micelles based on the borate ester structure, which have dual pH and ROS stimulation response capabilities, exhibit good stability over a 7-day period. Among them, both the blank CLM3 and CLM5 and the drug-loaded PTX-CLM3 and PTX-CLM5 show superior stability, suggesting that CLM3 and CLM5 are better hydrophobic antitumor drug delivery carriers.

[0098] Example 12 Determination of Critical Micelle Concentration of Polymer Micelles

[0099] As described in Example 10, a PBS aqueous solution of micelles with a polymer concentration of 1 mg / mL was prepared and then diluted with PBS to obtain polymer micelle aqueous solutions of different concentrations (0.01, 0.05, 0.10, 0.25, 1.00, 5.00, 10.0, 50.0, 100 μg / mL).

[0100] 10 μL of a 0.050 mg / mL Nile Red solution in dichloromethane was transferred to a brown glass bottle. After the dichloromethane evaporated, 1.5 mL of different concentrations (0.01, 0.05, 0.10, 0.25, 1.00, 5.00, 10.0, 50.0, 100 μg / mL) of polymer micelle aqueous solution were prepared and added to the brown glass bottle, and then stirred continuously at room temperature in the dark for 24 h. 200 μL of the polymer micelle aqueous solution was added to the wells of a microplate reader, and the fluorescence intensity was measured at an excitation wavelength of 579 nm and an emission wavelength of 620 nm. A graph was plotted on the fluorescence intensity against the logarithm of the Nile Red concentration. Linear fitting was performed before and after the inflection point of the fitted curve. The logarithm of the concentration corresponding to the x-axis of the intersection of the two fitted lines is the critical micelle concentration (CMC) of the polymer micelles. The results are shown below. Figure 19 , Figure 20 , Figure 21 , Figure 22 The figures shown are the CMC results of polymer micelles formed from the preferred polymers.

[0101] Example 13: In vitro drug release from drug-loaded micelles

[0102] Two mL of a drug-loaded micelle solution (pH 7.4 PBS solution) with a polymer concentration of 1 mg / mL was placed in a dialysis bag (molecular weight cutoff 2000 Da). The dialysis bag was then immersed in centrifuge tubes containing 20 mL of release medium (pH 7.4 PBS solution, pH 7.4 PBS solution + 0.1 mmol H2O2, pH 5.0 PBS solution, pH 5.0 PBS solution + 0.1 mmol H2O2, each release medium containing 0.5% Tweens 80). The centrifuge tubes were placed in a constant temperature water bath at 37°C and 100 rpm. At 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h, 1 mL of release medium was aspirated from the centrifuge tubes, and fresh release medium was added simultaneously. The drug release at each sampling time point was determined by HPLC. All in vitro drug release experiments of drug-loaded micelles were repeated three times, and the drug release was the average of the three parallel experiments. The in vitro drug release of drug-loaded micelles is shown below. Figure 23As shown, the results indicate that drug release from PTX-CLM3 and PTX-CLM5 is inhibited in a simulated physiological environment (pH 7.4 PBS solution); however, in a simulated pathological tumor microenvironment, the drugs can be effectively released from the drug-loaded micelles. This is beneficial for achieving controlled drug release, reducing the side effects caused by drug pre-leakage during administration, and increasing drug accumulation at the tumor site.

[0103] Example 14 Cytotoxicity assay of micelles / drug-loaded micelles

[0104] This invention uses the MTT assay to assess the biosafety of CLM3 and CLM5, as well as the cytotoxic effects of PTX-CLM3 and PTX-CLM5 on tumor cells (preparation method see Example 10). HeLa cells were seeded in 96-well plates (5000 cells per well) and cultured at 37°C for 24 h. After cell adhesion, 200 μL of DMEM medium was replaced in each well, followed by the addition of a series of gradient concentrations of micelle aqueous solution or free PTX. After 24 h of incubation, 20 μL of 5 mg / mL MTT reagent was added to each well, and incubation continued for another 4 h. Subsequently, the medium was aspirated, 150 μL of DMSO was added, and the absorbance was measured at 490 nm using a microplate reader. Untreated cells served as a positive control, and DMEM served as a negative control. The experimental results are as follows: Figure 24 As shown, CLM3 and CLM5 exhibit good biocompatibility, while PTX-CLM3 and PTX-CLM5 show dose-dependent cytotoxicity.

[0105] Example 15 Cell uptake experiment of fluorescent probe micelles

[0106] This invention uses coumarin 6 as a hydrophobic drug model, encapsulating it in micelles and investigating its uptake by cells in vitro using fluorescence microscopy. HeLa cells were seeded into 6-well plates (100,000 cells per well) and cultured at 37°C for 24 h. 200 μL of DMEM medium was replaced in each well, and cells were cultured with micelle solutions of various concentrations for 2 h and 6 h, respectively. After incubation, the medium was removed, and cells were washed twice with PBS. 1.5 mL of 4% paraformaldehyde was added to each well for fixation at 4°C for 0.5 h, followed by two washes with PBS. Then, 1 mL of 5 μg / mL DAPI was used to localize the cell nuclei. Images were captured using a fluorescence microscope, and the results are shown below. Figure 25 As shown ( Figure 25 The Merge group (referring to the overlapping images of coumarin 6 and DAPI) showed that as the drug concentration increased and the incubation time prolonged, the green fluorescence intensity of coumarin 6 increased, and the degree of overlap with the blue fluorescence of the cell nucleus also increased. CLM3 and CLM5 showed concentration-dependent and time-dependent cellular uptake.

[0107] Cell uptake of HeLa cells by polymer micelle solutions containing 10 μg / mL of coumarin 6 was quantified using flow cytometry. After 2 h and 6 h of culture, HeLa cells were digested, centrifuged, and washed with PBS to remove the culture medium. Fluorescence intensity was measured using flow cytometry, and the results are shown below. Figure 26 As shown, CLM3 and CLM5 can achieve high cell uptake efficiency within 2 hours. As the incubation time is extended to 6 hours, the cell uptake further increases, indicating that polymer micelles can be rapidly taken up by tumor cells.

[0108] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A dual-stimulus responsive polymer micelle, characterized in that... The micelles include polyethylene glycol monomethyl ether-(α-propargyl-δ-valerol-lactide) copolymer modified with phenylboronic acid, and polyethylene glycol monomethyl ether-(α-propargyl-δ-valerol-lactide) copolymer modified with catechol. The phenylboronic acid-modified polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer is prepared by anhydrous copper acetate-catalyzed azido-acetylene cycloaddition reaction of polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer and 4-azidomethylphenylboronic acid; sodium L-ascorbate is also introduced as an additive in the azido-acetylene cycloaddition reaction. The catechol-modified polyethylene glycol monomethyl ether-(α-propargyl-δ-valerol-lactide) copolymer is prepared by a cycloaddition reaction of polyethylene glycol monomethyl ether-(α-propargyl-δ-valerol-lactide) copolymer and 4-azidoethyl-1,2-catechol catalyzed by anhydrous copper acetate; sodium L-ascorbate is also introduced as an additive in the cycloaddition reaction.

2. The dual-stimulus responsive polymer micelles as described in claim 1, characterized in that... The polyethylene glycol monomethyl ether-(α-propynyl-δ-valerolactone-lactide) copolymer is prepared by ring-opening random polymerization of polyethylene glycol monomethyl ether, lactide, and α-propynyl-δ-valerolactone.

3. The application of the dual-stimulation responsive polymer micelles as described in claim 1 in the preparation and delivery of hydrophobic antitumor drugs, characterized in that... The hydrophobic antitumor drug is selected from one or more of paclitaxel, doxorubicin, camptothecin, gemcitabine, and mitoxantrone.

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