Dual-stimulus-response polymer micelle as well as preparation method and application thereof

Abstract

The invention discloses a dual stimuli-responsive polymer micelle as well as a preparation method and application thereof. The micelle is jointly formed by a phenylboronic acid modified polyethylene glycol monomethyl ether-(alpha-propargyl-delta-valerolactone-lactide) copolymer and a catechol modified polyethylene glycol monomethyl ether-(alpha-propargyl-delta-valerolactone-lactide) copolymer, wherein the phenylboronic acid modified polyethylene glycol monomethyl ether-(alpha-propargyl-delta-valerolactone-lactide) copolymer is used for modifying the phenylboronic acid; when the micelle is formed, pH and ROS can be formed in a hydrophobic core to stimulate corresponding boric acid ester bonds; the micelle is used for loading hydrophobic antitumor drugs. The polymer micelle prepared by the invention can be stable in an in-vivo circulating physiological environment, and makes stimulation response to a tumor microenvironment to cause structure loosening or dissociation, so that effective delivery of hydrophobic antitumor drugs is realized, the problems of instability of the micelle in vivo and drug pre-leakage are effectively solved, and the drug delivery efficiency is improved. Theoretical and practical basis is provided for promoting cancer treatment of a polymer micelle drug delivery system.

Description

Technical Field

[0001] The invention belongs to the field of biomedical polymer materials, and in particular relates to a dual stimulus-responsive polymer micelle and a preparation method and application thereof. Background Art

[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 use specific technical means to improve the pharmacokinetics and in vivo distribution of drugs, increase the effective accumulation of drugs in tumor sites, and thus improve the overall therapeutic effect and safety.

[0003] Polymer micelles have the advantages of simple preparation and high drug encapsulation efficiency, and have broad development prospects. Polymer micelles are usually formed by amphiphilic polymers through self-assembly to form a core-shell structure with a hydrophilic surface and a hydrophobic core. Due to abnormal oxidative metabolism, tumor cells have an acidic microenvironment and abnormal levels of reactive oxygen species (ROS) / glutathione (GSH). By introducing stimulus-responsive groups or connecting bonds into the amphiphilic polymer structure, polymer micelles with tumor microenvironment stimulus-responsiveness can be prepared, thereby experimentally controlling the targeted release of anti-tumor drugs. However, traditional polymer micelles are usually self-assembled by hydrophilic / hydrophobic interactions, and anti-tumor drugs are loaded into the micelles through hydrophobic / hydrophobic interactions with the hydrophobic segments of the polymer. Therefore, the hydrophobic core of traditional drug-loaded polymer micelles is relatively loose, unstable under complex physiological environments, prone to drug pre-leakage, and difficult to achieve effective drug accumulation at the tumor site, which in turn produces large toxic side effects on normal tissues. The above shortcomings greatly limit the clinical application of traditional polymer micelle drug delivery systems.

[0004] Therefore, it is urgent to develop a polymer micelle drug delivery system that can circulate stably under the complex physiological environment in vivo, has the ability to respond to tumor microenvironment stimulation, and can achieve controlled drug release in tumor tissues or cells. Summary of the invention

[0005] In view of the problems existing in the polymer micelle drug delivery system of the prior art, the purpose of the present invention is to provide a dual stimulus responsive polymer micelle and its preparation method and application. A dual stimulus responsive polymer micelle described in the present invention is a core-crosslinked polymer micelle with dual stimulus responsiveness to pH and ROS based on a boronate structure. The hydrophobic fragments of the micelles prepared in the present invention are modified by small molecules containing phenylboronic acid and catechol functional groups, respectively, and the two can form a boronate structure under pH 7.4. In a specific tumor microenvironment with weak acidity (pH<7) and high levels of ROS, the boronate bond breaks and causes the polymer micelle structure to dissociate or become loose, thereby achieving controllable targeted release of hydrophobic anti-tumor drugs.

[0006] The technical solution adopted by the present invention is as follows:

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

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

[0009]

[0010] Further, x, y, z, and n in Formula I represent the degree of polymerization of repeating units 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. 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 catechol-modified polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer has a structural formula as shown in Formula II:

[0012]

[0013] Further, x, y, z, and n in Formula II represent the degree of polymerization of the repeating units 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. 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 polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer and 4-azidomethylphenylboronic acid through a divalent copper salt-catalyzed azide-alkyne cycloaddition reaction; the copolymer shown in formula II is prepared by a polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer and 4-azidoethyl-1,2-catechol through a divalent copper salt-catalyzed azide-alkyne cycloaddition reaction; L-sodium ascorbate is also introduced as an additive in the azide-alkyne cycloaddition reaction.

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

[0016] Furthermore, the polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer has a structure as 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 degree of polymerization x, y, and z are the same as those in formula I or II. The molecular weight of the hydrophilic part of the polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer is 2000-10000Da, and the molecular weight of the hydrophobic part is 2000-15000Da.

[0019] Furthermore, the polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer is obtained by catalytic ring-opening polymerization of lactide and α-propargyl-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 2000-10000Da; more preferably 4000-5000Da.

[0021] The present invention also provides an application of a dual stimulus-responsive polymer micelle in the delivery of a hydrophobic anti-tumor drug, wherein the hydrophobic anti-tumor drug is 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, comprising core-crosslinked micelles obtained by self-assembly of phenylboronic acid-modified polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer, catechol-modified polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer and hydrophobic antitumor drugs.

[0025] The present invention discloses an application of a core-crosslinked micelle having a borate structure and having dual stimulation responsiveness to pH and ROS in the delivery of a hydrophobic anti-tumor drug.

[0026] A core-crosslinked micelle based on a boronate structure having dual stimulation responsiveness to pH and ROS, the composition of which is as follows Fig. 27 shown.

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

[0028] Compared with the prior art, the present invention has the following beneficial effects:

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

[0030] 2) The pendant groups of the basic skeleton polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer forming the micelles in the present invention are hydrophobic alkynyl groups, which can avoid the influence of the unreacted groups after functional modification on the hydrophobic part, thereby destroying the stability of the micelles.

[0031] 3) The functional modification of the basic skeleton polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer forming the micelles in the present invention is prepared by an efficient copper-catalyzed azide-alkyne cycloaddition reaction.

[0032] 4) The particle size, particle size distribution and drug loading capacity of the core-crosslinked micelles based on the borate structure prepared in the present invention with dual stimulation responsiveness to pH and ROS are better than those of the micelles formed by a single copolymer, and the micelles have good stability.

[0033] 5) The core-crosslinked micelles based on the borate structure prepared in the present invention with dual stimulation responsiveness to pH and ROS have a high encapsulation rate of hydrophobic anti-tumor drugs.

[0034] 6) The core-crosslinked micelles prepared in the present invention based on a borate structure with dual stimulation responsiveness to pH and ROS have good acidic pH and high-level ROS responsiveness and biosafety, and can achieve efficient delivery of hydrophobic anti-tumor drugs and effective accumulation at the tumor site.

[0035] 7) The core-crosslinked micelles prepared in the present invention based on a borate structure and having dual stimulation responsiveness to pH and ROS can be effectively taken up by cells, achieve good distribution in vivo, and effectively inhibit the growth of tumor cells.

[0036] The core-crosslinked micelles prepared by the present invention based on borate structure and having dual stimulation responsiveness to pH and ROS can effectively improve the stability of polymer micelles in physiological environment, reduce drug pre-leakage, reduce the toxic and side effects of drugs on the body, and increase the accumulation of drugs in tumor sites. The present invention provides a theoretical and practical basis for the development of new polymer micelle drug delivery systems for tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Synthesis route of monomer AVL;

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

[0039] Figure 3 Synthesis route of mPPAL;

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

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

[0042] Figure 6 N 3 -Synthesis route of PBA;

[0043] Figure 7 N 3 -PBA 1 H-NMR spectrum;

[0044] Figure 8 N 3 -The synthetic route of catechol;

[0045] Fig. 9 N 3 -catechol 1 H-NMR spectrum;

[0046] Fig.10 Synthesis route of mPPAL@PBA and mPPAL@catechol;

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

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

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

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

[0051] Fig.15 TEM image of PTX-CLM3;

[0052] Fig.16 TEM image of PTX-CLM5;

[0053] Fig.17 Schematic diagram of the stability of blank micelles;

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

[0055] Fig.19 Critical micelle concentration results of CLM3;

[0056] Fig. 20 Critical micelle concentration results of CLM4;

[0057] Fig.21 Critical micelle concentration results of CLM5;

[0058] Fig. 22 Critical micelle concentration results of CLM6;

[0059] Fig.23 Drug release profiles of PTX-CLM3 and PTX-CLM5 in different environments;

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

[0061] Fig.25 Fluorescence microscopy images of HeLa cells taking up different doses of coumarin 6-CLM3 and coumarin 6-CLM5 for 2 hours and 6 hours;

[0062] Fig.26 Flow cytometry quantification results of HeLa cell uptake of different doses of coumarin 6-CLM3 and coumarin 6-CLM5 for 2 hours and 6 hours;

[0063] Fig. 27 This is a composition diagram of the core-crosslinked micelles loaded with hydrophobic anti-tumor drugs based on the borate structure of the present invention and having dual stimulation responsiveness to pH and ROS. DETAILED DESCRIPTION

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

[0065] In particular, the naming method in the present invention is: α-propargyl-δ-valerolactone is abbreviated as AVL; polyethylene glycol monomethyl ether is abbreviated as mPEG; lactide is abbreviated as LA; polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer is named mPEG a -bP(AVL-co-LA) b , which is abbreviated as mPPAL, where a represents the molecular weight of the hydrophilic part mPEG, b represents the molecular weight of the hydrophobic part polyester; phenylboronic acid is abbreviated as PBA; catechol is Catechol; 4-azidomethylphenylboronic acid is abbreviated as N 3 -PBA; 4-azidoethyl-1,2-catechol is abbreviated as N 3-Catechol; phenylboronic acid modified polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer is named mPPAL@PBA; catechol modified polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer is named mPPAL@catechol; the abbreviation of core-crosslinked micelles based on boronate structure is CLM; the abbreviation of paclitaxel (PTX) loaded core-crosslinked micelles based on boronate structure is PTX-CLM; the abbreviation of coumarin 6 (Coumarin 6) loaded core-crosslinked micelles based on boronate structure is coumarin 6-CLM; the abbreviation of DiR loaded core-crosslinked micelles based on boronate structure is DiR-CLM.

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

[0067] Wherein, the entrapped hydrophobic anti-tumor drug is:

[0068]

[0069] One of them.

[0070] Example 1 Synthesis of Monomer AVL

[0071] Add 55.2 mL of 2 mol / L lithium diisopropylamide in tetrahydrofuran / n-hexane solution (LDA, 110.4 mmol) to a 500 mL flask containing 250 mL of anhydrous tetrahydrofuran and stir at -78°C. Mix δ-valerolactone (VL, 10.0 g, 99.88 mmol) with 250 mL of anhydrous tetrahydrofuran and then add the mixed solution dropwise within 1 h under stirring. Stir the mixture for another 0.5 h. Continue stirring at -78°C and add a mixture of 3-bromopropyne (14.4 g, 121.0 mmol) and hexamethylphosphoric acid triamide (HMPA, 20.9 mL, 120.1 mmol) dropwise within 2 h. Stir the mixture at -30°C for another 2 h. Add 50 mL of saturated NH 4 The reaction was quenched with Cl solution and then warmed to room temperature. After removing tetrahydrofuran by rotary evaporation, the resulting solution was extracted with ethyl acetate (100 mL × 3), and the organic phases were combined and washed twice with ultrapure water, once with saturated NaCl, and then with anhydrous Na 2 SO 4Dry, filter and concentrate. The crude product was separated by gradient elution on a silica gel column (n-hexane: ethyl acetate = 9:1-3:1, volume ratio) to obtain a light yellow liquid (6.49 g, 47.0 mmol, yield 47.1%). The product was further distilled under reduced pressure to obtain a pure product as a colorless viscous liquid AVL (5.45 g, yield 39.4 mmol, 39.4%). The structure is shown in 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 amphiphilic polymer mPPAL3

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

[0074] All reaction vessels were thoroughly dried in an oven at 50 °C overnight before use. The steps for preparing mPPAL3 were as follows: 2 Under atmosphere, mPEG 4k (1.00 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, and 25 mL of anhydrous toluene was added. The temperature was raised to 150 ° C, and the residual water in the reaction system was removed by the principle that toluene and water form an azeotrope. Subsequently, the temperature was lowered to 125 ° C, and 6-7 drops of Sn(Oct) were added. 2 The mixture was stirred at 125°C for 48 h. After the reaction was completed, it was cooled to room temperature, diluted with dichloromethane, and washed with 10% HCl, ultrapure water and saturated NaCl aqueous solution in sequence until the water layer was neutral. The organic layer was washed with anhydrous Na 2 SO 4 The polymer was precipitated at least 3 times with a mixed solvent (dichloromethane: n-hexane = 1:10, volume ratio) and then dried in a vacuum oven. The final product was a yellow solid (1.69 g, 0.21 mmol, yield 84.7%). The structure is shown in Figure 4 .

[0075] Example 3 Synthesis of amphiphilic polymer mPPAL5

[0076] Polyethylene glycol monomethyl ether mPEG with an average molecular weight of 5k, labeled as mPEG5k .

[0077] All reaction vessels were thoroughly dried in an oven at 50 °C overnight before use. The steps for preparing mPPAL5 were as follows: 2 Under 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, and 25 mL of anhydrous toluene was added. The temperature was raised to 150 ° C, and the residual water in the reaction system was removed by the principle that toluene and water form an azeotrope. Then, the temperature was lowered to 125 ° C, and 6-7 drops of Sn(Oct) were added. 2 The mixture was stirred at 125°C for 48 h. After the reaction was completed, it was cooled to room temperature, diluted with dichloromethane, and washed with 10% HCl, ultrapure water and saturated NaCl aqueous solution in sequence until the water layer was neutral. The organic layer was washed with anhydrous Na 2 SO 4 The polymer was precipitated at least 3 times with a mixed solvent (dichloromethane: n-hexane = 1:10, volume ratio) and then dried in a vacuum oven. The final product was a yellow solid (1.87 g, 0.14 mmol, yield 71.9%). The structure is shown in Figure 5 .

[0078] The same ring-opening polymerization method using stannous isooctanoate as catalyst and initiator (mPEG) of different molecular weight was used to initiate the polymerization. By controlling the amount of mPEG added and the monomer feed ratio, other conditions remained unchanged to synthesize mPPAL polymers with different molecular weights and different hydrophobic monomer ratios. The yield of this series of polymers was 66.8%-90.5%. The following table shows the specific hydrophilic and hydrophobic segment molecular weights and hydrophobic monomer ratios of the preferred polymers.

[0079]

[0080] a Molecular weight of the "AVL and LA" copolymerized fragment. b The ratio of the designed numbers of two hydrophobic monomers AVL and LA in mPPAL.

[0081] Example 4N 3 Synthesis of -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, and 10 mL of DMF was added. After continuous reaction at 50 °C for 48 h, the reactant was cooled to room temperature and diluted with 100 mL of ultrapure water. The mixture was stirred at room temperature for 10 min, and then the resulting solution was extracted with ethyl acetate (100 mL × 3). The organic layers were combined, and the organic phase was washed twice with ultrapure water, once with a saturated aqueous NaCl solution, and anhydrous Na 2 SO 4 Dry, filter and concentrate. The product is further dried in a vacuum oven to obtain a pale yellowish white solid N 3 -PBA (0.78 g, 4.41 mmol, yield 94.8%), structure see Figure 6 . 1 H-NMR (400 MHz, DMSO-d 6 ): δ8.08(s,2H),7.81(d,J=7.9Hz,2H),7.32(d,J=7.8Hz,2H),4.44(s,2H).

[0083] Example 5N 3 Synthesis of -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 within 0.5 h, and the reaction suspension was stirred at 0 °C for 2 h to obtain fresh trifluoromethanesulfonyl azide. Dopamine hydrochloride (1.00 g, 5.27 mmol), zinc chloride (71.8 mg, 0.53 mmol) and 25 mL of CH 3 CN:H 2 O (volume ratio 7:3), then triethylamine (2.21mL, 15.8mmol), and then the freshly prepared trifluoromethanesulfonyl azide prepared above was quickly added. The reaction was stirred overnight at room temperature. The mixture was filtered and concentrated by rotary evaporation to remove acetonitrile, and then the resulting solution was extracted with ethyl acetate (20mL×3) and the organic layer was combined. The organic phase was washed twice with ultrapure water, once with saturated NaCl aqueous solution, and anhydrous Na 2 SO 4 Dry, filter and concentrate. The product was separated by silica gel column chromatography (n-hexane: ethyl acetate = 1:1, volume ratio) to obtain a brown-black solid N 3 -catechol (0.87 g, 4.86 mmol, yield 92.2%), structure see 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-shielded conditions, anhydrous copper acetate (7.17 mg, 0.040 mmol) and sodium L-ascorbate (15.64 mg, 0.079 mmol) were added to mPPAL3 (200.00 mg, 0.026 mmol) and N 3 -PBA (209.57 mg, 1.18 mmol) in 4 mL of anhydrous DMF solution. 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, dialyzed in 0.1 mmol of ethylenediamine-tetraacetic acid disodium salt aqueous solution for 24 h to remove the copper element, and then dialyzed in 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 Fig.10 .

[0087] Example 7 Synthesis of mPPAL3@catechol

[0088] Under light-shielded conditions, anhydrous copper acetate (7.17 mg, 0.040 mmol) and sodium L-ascorbate (15.64 mg, 0.079 mmol) were added to mPPAL3 (200.00 mg, 0.026 mmol) and N 3 -catechol (212.19 mg, 1.18 mmol) in 4 mL of anhydrous DMF solution. 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 in 0.1 mmol of ethylenediamine-tetraacetic acid disodium salt aqueous solution for 24 h to remove the copper element, and then dialyzed in 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 Fig.10 .

[0089] Example 8 Synthesis of mPPAL5@PBA

[0090] Under light-shielded conditions, anhydrous copper acetate (7.33 mg, 0.040 mmol) and sodium L-ascorbate (15.99 mg, 0.081 mmol) were added to mPPAL5 (200.00 mg, 0.018 mmol) and N 3 -PBA (214.23 mg, 1.21 mmol) in 4 mL of anhydrous DMF solution. 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 in 0.1 mmol of ethylenediamine-tetraacetic acid disodium salt aqueous solution for 24 h to remove the copper element, and then dialyzed in 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 Fig.10 .

[0091] Example 9 Synthesis of mPPAL5@catechol

[0092] Under light-shielded conditions, anhydrous copper acetate (7.33 mg, 0.040 mmol) and sodium L-ascorbate (15.99 mg, 0.081 mmol) were added to mPPAL5 (200.00 mg, 0.018 mmol) and N 3 -catechol (216.90 mg, 1.21 mmol) in 4 mL of anhydrous DMF solution. 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 in 0.1 mmol of ethylenediamine-tetraacetic acid disodium salt aqueous solution for 24 h to remove the copper element, and then dialyzed in 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 Fig.10 .

[0093] Example 10 Preparation of core cross-linked micelles

[0094] Blank and drug-loaded micelles were prepared by thin film dispersion method. Taking drug-loaded micelle PTX-CLM3 as an example, the polymer materials of PTX-CLM3 are mPPAL3@PBA and mPPAL3@catechol, and the encapsulated hydrophobic antitumor drug is PTX. 5mg mPPAL3@PBA, 5mg mPPAL3@catechol, and 1mg PTX were dissolved in 1mL chloroform and ultrasonicated for 10min to ensure complete dissolution. The chloroform was removed by rotary evaporator, and 1mL PBS solution was ultrasonicated at 50℃ for 0.5h to rehydrate the film. Then 9mL PBS solution was added to adjust the polymer concentration to 1mg / mL, and the free PTX was removed by syringe filter to finally obtain drug-loaded micelle PTX-CLM3. Blank micelle CLM3 was prepared by the same method, but without encapsulating hydrophobic antitumor drugs. Fig.15 This is the TEM image of PTX-CLM3. PTX-CLM3 is spherical and has a good morphology.

[0095] The preparation method of drug-loaded micelles PTX-CLM5 repeats the preparation method of drug-loaded micelles PTX-CLM3, with the only difference being that "mPPAL3@PBA and mPPAL3@catechol are replaced with mPPAL5@PBA and mPPAL5@catechol of equal mass, respectively", and the other conditions remain unchanged, finally obtaining drug-loaded micelles PTX-CLM5. Fig.16 This is the TEM image of PTX-CLM5. PTX-CLM5 is spherical and has a good morphology.

[0096] Example 11 Study on the Storage Stability of Micellar Aqueous Solution

[0097] The PBS aqueous solution of micelles with a polymer concentration of 1 mg / mL prepared as described in Example 10 was stored at room temperature in a cool place, and 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. Fig.17 and 18 , indicating that this core-crosslinked micelle based on borate structure with dual stimulation responsiveness to pH and ROS has good stability within 7 days. Among them, both blank CLM3, CLM5 and drug-loaded PTX-CLM3 and PTX-CLM5 have better stability, and CLM3 and CLM5 can be considered as more preferred hydrophobic anti-tumor 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 aqueous solutions of polymer micelles with different concentrations (0.01, 0.05, 0.10, 0.25, 1.00, 5.00, 10.0, 50.0, 100 μg / mL).

[0100] Transfer 10 μL of a 0.050 mg / mL Nile Red dichloromethane solution to a brown glass bottle. After the dichloromethane evaporates, prepare and add 1.5 mL of the above-mentioned polymer micelle aqueous solution of different concentrations (0.01, 0.05, 0.10, 0.25, 1.00, 5.00, 10.0, 50.0, 100 μg / mL) to the brown glass bottle, and then stir continuously for 24 hours at room temperature in the dark. Add 200 μL of the polymer micelle aqueous solution to the well of the microplate reader, and measure the fluorescence intensity at an excitation wavelength of 579 nm and an emission wavelength of 620 nm. Plot the fluorescence intensity value against the logarithm of the Nile Red concentration, and make linear fits before and after the inflection point of the data point fitting curve. The logarithm of the concentration corresponding to the abscissa of the intersection of the two fitting lines is the critical micelle concentration (CMC) of the polymer micelle. The results are shown in Figure 2. Fig.19 , Fig. 20 , Fig.21 , Fig. 22 Shown are the results of measuring CMC for polymer micelles formed by preferred polymers.

[0101] Example 13 Drug release in vitro from drug-loaded micelles

[0102] 2 mL of 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), and the dialysis bag was immersed in 20 mL of release medium (pH 7.4 PBS solution, pH 7.4 PBS solution + 0.1 mmol H 2 O 2 , pH 5.0 PBS solution, pH 5.0 PBS solution + 0.1 mmol H 2 O 2 ) in a centrifuge tube (each release medium contains 0.5% Tweens 80) and placed in a constant temperature water bath shaker at 37°C and 100 rpm. At 1h, 2h, 4h, 8h, 12h, 24h, 36h, and 48h, 1mL of the release medium in the centrifuge tube was aspirated and fresh release medium was added at the same time. The drug release amount at the sampling time point was detected by HPLC. The in vitro drug release experiment of all drug-loaded micelles was repeated 3 times, and the drug release amount was taken as the average of 3 parallel experiments. The in vitro drug release of drug-loaded micelles is shown in Fig.23As shown in the figure, the results show that the drug release of PTX-CLM3 and PTX-CLM5 is inhibited in the simulated physiological environment of pH 7.4 PBS solution; in the pathological environment simulating the tumor microenvironment, the drug can be effectively released from the drug-loaded micelles. This is conducive to achieving controlled release of drugs, reducing the side effects caused by drug pre-leakage during administration, and increasing the accumulation of drugs in the tumor site.

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

[0104] The present invention uses the MTT method to determine and evaluate the biosafety of CLM3 and CLM5, as well as the killing effect of PTX-CLM3 and PTX-CLM5 on tumor cells (see Example 10 for the preparation method). HeLa cells were inoculated in a 96-well plate (5000 cells per well) and cultured at 37°C for 24 hours. After the cells adhered, 200 μL of DMEM culture medium was replaced in each well, and then a series of gradient concentrations of micelle aqueous solution or free PTX were added. After incubation for 24 hours, 20 μL of MTT reagent at a concentration of 5 mg / mL was added to each well and incubated for another 4 hours. The culture medium was then aspirated, 150 μL of DMSO was added, and the absorbance was measured at a wavelength of 490 nm using an enzyme reader. Untreated cells were used as positive controls, and DMEM was used as negative controls. The experimental results are as follows Fig.24 As shown, CLM3 and CLM5 have good biocompatibility, and PTX-CLM3 and PTX-CLM5 exhibit dose-dependent cytotoxicity.

[0105] Example 15 Cellular uptake experiment of fluorescent probe-loaded micelles

[0106] The present invention uses coumarin 6 as a hydrophobic drug model, encapsulates it in micelles, and investigates in vitro cellular uptake by fluorescence microscopy. HeLa cells were seeded into 6-well plates (100,000 cells per well), cultured at 37°C for 24 hours, and 200 μL of DMEM culture medium was replaced in each well, and cultured with a series of micelle solutions of different concentrations for 2 hours and 6 hours, respectively. After incubation, the culture medium was removed, the cells were washed twice with PBS, 1.5 mL of 4% paraformaldehyde was added to each well and fixed at 4°C for 0.5 hours, washed twice with PBS, and then 1 mL of DAPI at a concentration of 5 μg / mL was used to locate the cell nucleus. Images were taken using a fluorescence microscope, and the results are as follows: Fig.25 As shown ( Fig.25 The Merge group refers to the overlapping images of coumarin 6 and DAPI). As the drug concentration increases and the incubation time prolongs, the green fluorescence intensity of coumarin 6 increases, and the overlap with the blue fluorescence of the cell nucleus increases. CLM3 and CLM5 show concentration-dependent and time-dependent cellular uptake.

[0107] Flow cytometry was used to quantify the cellular uptake of the polymer micelle solution at a coumarin 6 concentration of 10 μg / mL. After culturing for 2 h and 6 h, the HeLa cells were digested, centrifuged, and the culture medium was removed by rinsing with PBS. The fluorescence intensity was measured using a flow cytometer. The results are shown in Fig.26 As shown, CLM3 and CLM5 can achieve high cellular uptake efficiency in 2 h. As the incubation time is extended to 6 h, the cellular uptake is further improved, indicating that the polymer micelles can be rapidly taken up by tumor cells.

[0108] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A dual stimulus responsive polymer micelle, characterized in that The micelle includes a polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer modified by phenylboronic acid of the structure shown in formula I, and a catechol-modified polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer of the structure shown in formula II; In formula I or formula II, x, y, z, and n represent the degree of polymerization of the repeating units in the copolymer, x is 46-227, and the ratio of y to z in the hydrophobic segment of the polymer is 1:1 or 1:3; when y:z=1:1, y is 7-54, z is 7-54, and the ratio n / y=50-72%; when y:z=1:3, y is 4-27, z is 12-81, and the ratio n / y=50-72%.

2. A dual stimulus responsive polymer micelle as claimed in claim 1, characterized in that The copolymer shown in formula I is prepared from polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer and 4-azidomethylphenylboronic acid through a divalent copper salt-catalyzed azide-alkyne cycloaddition reaction; L-sodium ascorbate is also introduced as an additive in the azide-alkyne cycloaddition reaction.

3. A dual stimulus responsive polymer micelle as claimed in claim 1, characterized in that The copolymer shown in formula II is prepared from polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer and 4-azidoethyl-1,2-catechol through a divalent copper salt-catalyzed azide-alkyne cycloaddition reaction; L-sodium ascorbate is also introduced as an additive in the azide-alkyne cycloaddition reaction.

4. A dual stimulus responsive polymer micelle as claimed in claim 2 or 3, characterized in that The polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer has a structure as shown in Formula III: 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 degree of polymerization x, y, and z are the same as those in formula I or formula II.

5. A dual stimulus responsive polymer micelle as claimed in claim 4, characterized in that The polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer is prepared by ring-opening random polymerization of polyethylene glycol monomethyl ether, lactide and α-propargyl-δ-valerolactone.

6. A dual stimulus responsive polymer micelle as claimed in claim 4, characterized in that The molecular weight of the hydrophilic part of the polyethylene glycol monomethyl ether-(α-propargyl-δ-valerolactone-lactide) copolymer, i.e. the fragment corresponding to the polymerization degree x, is 2000-10000Da, and the molecular weight of the hydrophobic part, i.e. the fragment corresponding to the polymerization degree y+z, is 2000-15000Da.

7. The use of a dual stimulus-responsive polymer micelle as claimed in claim 1 in the delivery of hydrophobic anti-tumor drugs, characterized in that The hydrophobic anti-tumor drug is selected from one or more of paclitaxel, doxorubicin, camptothecin, gemcitabine, mitoxantrone and derivatives of any of the above drugs.

8. A hydrophobic anti-tumor drug micelle delivery system, characterized in that: The invention comprises micelles formed by the copolymers of formula I and II described in claim 1 and loaded with hydrophobic anti-tumor drugs.

9. A hydrophobic anti-tumor drug micelle delivery system according to claim 8, characterized in that: The copolymer represented by formula I, the copolymer represented by formula II, and the hydrophobic antitumor drug are dissolved in chloroform to prepare a solution, the chloroform is removed by a rotary evaporator, and then a PBS solution with a pH of 7.4 is added for ultrasonic rehydration at 50° C. Finally, the insoluble suspended matter is removed by a needle filter to obtain the drug-loaded micelle aqueous solution.

10. A hydrophobic anti-tumor drug micelle delivery system according to claim 9, characterized in that: The mass ratio of the copolymer represented by formula I to the copolymer represented by formula II is 0.5-2:1, and the mass ratio of the total mass of the copolymer represented by formula I and the copolymer represented by formula II to the hydrophobic anti-tumor drug is 8-15:1.

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