Folic acid / lipoic acid modified pH / reduction dual-response mixed polymer cross-linked micelle as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of drug sustained-release and controlled-release materials, and discloses a folic acid / lipoic acid modified pH / reduction dual-response mixed polymer cross-linked micelle as well as a preparation method and application thereof. The mixed polymer cross-linked micelle is formed by mixing, cross-linking and self-assembling a folic acid modified polymer and a lipoic acid modified polymer. The polymer has a drug-loaded hydrophobic core PCL, a pH-sensitive responsive block PDEAEMA, a functional block PHEMA capable of modifying folic acid / lipoic acid and a hydrophilic block PPEGMA for forming a shell, and folic acid and lipoic acid groups endow the polymer with an active targeting effect and a reversible self-crosslinking effect. The mixed polymer cross-linked micelle is used as a drug carrier, has higher stability compared with non-cross-linked drug-loaded micelle, has active targeting and pH / reduction dual responsiveness, can stably exist for a long time in a neutral environment, has less drug leakage, can be actively recognized by tumor cells at a tumor site, can be accumulated and endocytosed, and can smoothly release drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of drug sustained-release and controlled-release materials, and particularly relates to a folic acid / lipoic acid modified pH / reduction dual-responsive mixed polymer cross-linked micelle, and a preparation method and application thereof. Background Art

[0002] Smart responsive nano polymer drug-loaded micelles can flexibly carry anti-tumor drugs to overcome physiological and pathological barriers, complete continuous targeted delivery and controlled release, and achieve the effect of reducing toxicity and enhancing the efficacy of anti-tumor drugs. It is one of the key research and development directions of the current new drug delivery system. It uses amphiphilic polymers to self-assemble in solution to form nanoparticles with core / shell structure. The hydrophobic core can encapsulate hydrophobic drugs and improve drug solubility; the hydrophilic shell stabilizes and protects the micelles, which can reduce the toxic and side effects of drugs in normal human tissues and avoid phagocytosis of the reticuloendothelial system or absorption and clearance by tissues such as the liver, spleen, and kidneys. Functional modification of polymer micelles, such as introducing stimulus-responsive groups or modifying targeting ligands, can be used to give them characteristics such as smart response by utilizing the differences between the normal human cell microenvironment (pH≈7.4, 36-37℃, low GSH) and the tumor tissue microenvironment (pH≈5.0-6.5, 37-42℃, high GSH, etc.).

[0003] Although smart responsive polymer-loaded micelles have made great progress as a new delivery system for anticancer drugs, the stability and controlled release of polymer-loaded micelles, as well as their preparation methods, still need to be further improved. For example: (1) The micelles have poor stability and weak anti-interference ability, which can easily lead to structural damage, resulting in premature release of drugs before reaching tumor tissues, causing toxic side effects on normal tissues; (2) The polymer micelles have poor targeting and can only accumulate at the tumor site through the EPR effect, resulting in a long drug action time; (3) Most multifunctional polymer micelles have long synthesis routes and complicated processes. Summary of the invention

[0004] In order to overcome the shortcomings and deficiencies of the poor stability, weak targeting and low drug controlled release ability of the smart responsive polymer drug-loaded micelles in the above-mentioned prior art, as well as the difficulty in synthesizing multifunctional drug carrier materials, the primary purpose of the present invention is to provide a folic acid / lipoic acid modified pH / reduction dual-responsive mixed polymer cross-linked micelle. The mixed polymer cross-linked micelle has a drug-loaded hydrophobic core (PCL), a pH-sensitive responsive block (PDEAEMA), a functional block (PHEMA) that can modify folic acid / lipoic acid, and a hydrophilic block (PPEGMA) for forming an outer shell, wherein the PDEAEMA, PHEMA and PPEGMA blocks can be prepared by a one-pot method, and the PHEMA block can be introduced by esterification. Folic acid and lipoic acid groups give the polymer active targeting and reversible self-crosslinking.

[0005] Another object of the present invention is to provide a method for preparing the folic acid / lipoic acid modified pH / reduction dual-responsive mixed polymer cross-linked micelles.

[0006] Another object of the present invention is to provide the application of the folic acid / lipoic acid modified pH / reduction dual-responsive mixed polymer cross-linked micelles.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A folic acid / lipoic acid modified pH / reduction dual-responsive mixed polymer cross-linked micelle, wherein the mixed polymer cross-linked micelle is formed by mixed cross-linking self-assembly of a polymer A obtained by modifying folic acid with a polymer P and a polymer B obtained by modifying lipoic acid with a polymer P; wherein the polymer P is PCL-PDEAEMA-PHEMA-PPEGMA, and has a structural formula as shown in formula (1); the polymer A is PCL-PDEAEMA-P(HEMA-g-FA)-PPEGMA, and has a structural formula as shown in formula (2); and the polymer B is PCL-PDEAEMA-P(HEMA-g-LA)-PPEGMA, and has a structural formula as shown in formula (3):

[0009]

[0010]

[0011] Wherein, x is a positive integer of 10 to 20, m and n are positive integers of 0 to 15, and m+n=15.

[0012] The method for preparing the above-mentioned folic acid / lipoic acid modified pH / reduction dual-responsive mixed polymer cross-linked micelles comprises the following steps:

[0013] (1) Under inert gas protection and anhydrous and oxygen-free conditions, ε-caprolactone, a small molecule initiator and a first catalyst are mixed and subjected to a ring-opening polymerization reaction to obtain a polymer PCL;

[0014] (2) Under inert gas protection and anhydrous conditions, the polymer PCL obtained in step (1) is dissolved in a solvent from which water has been removed, an acid binding agent is added, the mixture is cooled to 0° C., a brominating agent is added dropwise and stirred, and after the addition is complete, the mixture is reacted at 0° C. for 1 to 3 hours, and then the reaction is continued at room temperature for 12 to 24 hours. After the reaction, the mixture is filtered, concentrated, washed, and dried to obtain a macromolecular initiator PCL-Br;

[0015] (3) placing the macromolecular initiator PCL-Br and the second catalyst obtained in step (2) in a sealed container, repeatedly evacuating and passing argon for three times, and then adding a solvent to fully dissolve; then adding the first monomer and the ligand in sequence, stirring continuously for 10 minutes, and then adding a reducing agent, stirring for 20 minutes to fully react with the complex, and then transferring the container to an oil bath pot and stirring continuously to perform an atom transfer radical polymerization reaction; after reacting for 6 hours, adding the second monomer, continuing the reaction for 9 hours, and then adding the third monomer, and then reacting for 28 hours, cooling to room temperature and passing air, filtering, concentrating, washing, and drying to obtain PCL-PDEAEMA-PHEMA-PPEGMA, which is polymer P;

[0016] (4) Add folic acid and a stirrer into a sealed dry, anhydrous, oxygen-free bottle, repeat vacuuming and argon gas flow three times, then add anhydrous dimethyl sulfoxide (DMSO), and sonicate to fully dissolve the folic acid to obtain a folic acid solution; dissolve the catalyst in anhydrous DMSO, add the solution to the folic acid solution, and react at room temperature for 4-5 hours to activate the carboxyl groups in the folic acid. After the activation is completed, add the polymer P obtained in step (3) that was previously dissolved in anhydrous DMSO, and continue the esterification reaction at room temperature for 18 hours; after the reaction is completed, transfer the reaction solution to a dialysis bag of MWC O3500, the dialysis medium is deionized water, and the deionized water is replaced every 4 hours, and the dialysis is continued for 48 hours. After the dialysis is completed, the dialyzate is filtered with a 0.45 μm water filter, and the obtained filtrate is freeze-dried to obtain PCL-PDEAEMA-P(HEMA-g-FA)-PPEGMA, which is polymer A;

[0017] (5) replacing the folic acid in step (4) with lipoic acid of equal molar mass, and the other conditions are the same as step (4), to obtain PCL-PDEAEMA-P(HEMA-g-LA)-PPEGMA, i.e., polymer B;

[0018] (6) mixing the polymer A obtained in step (4) and the polymer B obtained in step (5) and dissolving them in an organic solvent, and dialyzing to obtain a blank mixed polymer non-cross-linked micelle dialysate;

[0019] (7) The blank mixed polymer non-cross-linked micelle dialysate obtained in step (6) is subjected to self-cross-linking of lipoic acid using a trace amount of a reducing agent, thereby finally obtaining folic acid / lipoic acid modified pH / reduction dual-responsive mixed polymer cross-linked micelles.

[0020] Step (1) The first catalyst is stannous isooctanoate (Sn(Oct)2); the small molecule initiator is n-butanol;

[0021] The solvent in step (2) is tetrahydrofuran; the acid binding agent is triethylamine (TEA); and the brominating agent is 2-bromoisobutyryl bromide;

[0022] Step (3) the second catalyst is copper bromide (CuBr2); the ligand is pentamethyldiethylenetriamine (PMDETA); the reducing agent is stannous isooctanoate (Sn(Oct)2); the solvent is anhydrous tetrahydrofuran; the eluent is tetrahydrofuran; the first monomer is diethylaminoethyl methacrylate (DEAEMA); the second monomer is hydroxyethyl methacrylate (HEMA); the third monomer is polyethylene glycol methyl ether methacrylate (PEGMA);

[0023] The catalyst in step (4) is N,N-dicarbonylimidazole (CDI);

[0024] The reducing agent in step (7) is dithiothreitol (DTT).

[0025] The weight ratio of each substance in step (1) is as follows:

[0026] Small molecule initiator 0.148 parts

[0027] ε-caprolactone 9.13 parts

[0028] 0.027 parts of the first catalyst

[0029] The weight ratio of each substance in step (2) is as follows:

[0030] Polymer PCL 4.56 parts

[0031] 0.81 parts of acid binding agent

[0032] Brominating agent 1.11 parts

[0033] The weight ratio of each substance in step (3) is as follows:

[0034]

[0035] The weight ratio of each substance in step (4) is as follows:

[0036] Folic acid 0.331 parts

[0037] Catalyst 0.122 parts

[0038] Polymer P 0.87 parts

[0039] The weight ratio of each substance in step (5) is as follows:

[0040] Lipoic acid 0.155 parts

[0041] Catalyst 0.122 parts

[0042] Polymer P 0.87 parts

[0043] The weight ratio of the blank mixed polymer non-cross-linked micellar dialysate used in step (7) to the reducing agent is 0.3 parts and 0.0027 parts respectively, based on the weight of the powder obtained by freeze-drying.

[0044] The temperature of the ring-opening polymerization reaction in step (1) is 120-130° C. and the time is 24-48 hours;

[0045] The temperature of the atom transfer radical polymerization reaction in step (3) is 65-70° C.;

[0046] The esterification reaction conditions in step (4) are alkaline environment at room temperature;

[0047] In step (6), the polymer A and polymer B are mixed and dissolved in an organic solvent in a mass ratio of 1 to 3:1 to 3, stirred for 4 to 12 hours, and dialyzed with deionized water for 24 to 72 hours, during which the deionized water is replaced every 3 to 6 hours to obtain a blank mixed polymer non-cross-linked micellar dialysate; the organic solvent is dimethylformamide, dimethyl sulfoxide or acetone;

[0048] The inducing lipoic acid self-crosslinking in step (7) is to add a trace amount of reducing agent to the blank mixed polymer non-crosslinked micelle dialysate, stir the crosslinking reaction at room temperature at pH 8-9 for 6-12 hours, dialyze with deionized water for 12-36 hours after the reaction is completed, replace the deionized water every 3-6 hours, and freeze-dry to obtain folic acid / lipoic acid modified pH / reduction dual-responsive mixed polymer crosslinked micelles.

[0049] The above-mentioned folic acid / lipoic acid modified pH / reduction dual-responsive mixed polymer cross-linked micelles are used in targeted controlled release nano drug delivery systems.

[0050] The targeted controlled-release nano drug delivery system loads and delivers the hydrophobic anticancer drug paclitaxel (PTX).

[0051] Principle of the present invention:

[0052] The present invention designs and synthesizes a folic acid / lipoic acid modified pH / reduction dual response mixed polymer cross-linked micelle for loading a broad-spectrum anticancer drug, paclitaxel (PTX). The cross-linked micelle uses polycaprolactone (PCL) as a hydrophobic block to provide a hydrophobic core for drug embedding, uses diethylaminoethyl methacrylate (DEAEMA) as a pH response block, uses polyethylene glycol methyl ether methacrylate (PEGMA) with good biocompatibility as a hydrophilic block, and uses hydroxyethyl methacrylate (HEMA) with a hydroxyl group as a functionally modifiable block. The hydroxyl groups of HEMA are modified by folic acid and lipoic acid to give the polymer active targeting and reversible self-crosslinking functions. In order to reduce the difficulty of synthesizing multifunctional materials, the preparation method of the present invention first prepares a polycaprolactone-b-polydiethylaminoethyl methacrylate-b-polyhydroxyethyl methacrylate-b-poly(ethylene glycol) methacrylate) (PCL-PDEAEMA-PHEMA-PPEGMA) polymer P with pH responsiveness; then, the polymer P is subjected to an esterification reaction with folic acid (FA) having an active targeting effect to prepare a folic acid-modified polymer A (PCL-PDEAEMA-P(HEMA-g-FA)-PPEGMA); and the polymer P is subjected to an esterification reaction with lipoic acid (LA) having a reduction responsiveness and a reversible self-crosslinking effect to prepare a lipoic acid-modified polymer B (PCL-PDEAEMA-P(HEMA-g-LA)-PPEGMA); finally, the polymer A and the polymer B are mixed in proportion to prepare a blank mixed polymer non-crosslinked micelle dialysate, and then the mixed polymer crosslinked micelles are formed through self-crosslinking, so that the micelle stability is improved and the mixed polymer has active targeting ability and pH / reduction dual responsiveness. The mixed polymer drug-loaded cross-linked micelles of the present invention are used to load and deliver the hydrophobic anticancer drug paclitaxel, which can improve the stability, targeting and drug controlled release ability of the smart response polymer drug-loaded micelles and reduce the difficulty of synthesizing the multifunctional carrier material.

[0053] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0054] The present invention designs and prepares a folic acid / lipoic acid modified pH / reduction dual responsive mixed polymer drug-loaded cross-linked micelle, which has a uniform particle size distribution and a high drug loading capacity; in order to reduce the difficulty of synthesizing a multifunctional polymer carrier, a mixed micelle strategy is adopted to prepare micelles; after self-crosslinking is initiated by trace dithiothreitol to prepare a mixed polymer cross-linked micelle, the stability is significantly improved compared with non-cross-linked micelles, and it also has active targeting and pH / reduction dual responsiveness. The mixed polymer cross-linked micelle is loaded with anticancer drugs as a drug delivery carrier, and can exist stably for a long time in a neutral environment with little drug leakage; and can be actively recognized, accumulated and internalized by tumor cells at the tumor site, and smoothly release drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 It is the synthetic process route of PCL-PDEAEMA-PHEMA-PPEGMA;

[0056] Figure 2 The synthetic process route of polymer PCL-PDEAEMA-P(HEMA-g-LA / FA)-PPEGMA and the schematic diagram of polymer self-crosslinking;

[0057] Figure 3 PCL obtained in Examples 1 and 2 40 、PCL 40 -Br H NMR spectrum ( 1 H NMR), the solvent was deuterated chloroform (CDCl3);

[0058] Figure 4 is the H NMR spectrum of PCL-PDEAEMA-PHEMA-PPEGMA in Example 3 ( 1 H NMR) spectrum, the solvent is deuterated chloroform (CDCl3);

[0059] Figure 5 The PCL, PCL-Br, and PCL-PDEAEMA-PHEMA-PPEGMA in Example 4 were subjected to gel permeation chromatography (GPC), and the mobile phase was tetrahydrofuran (THF);

[0060] Figure 6 is the critical micelle concentration determination curve of PCL-PDEAEMA-PHEMA-PPEGMA in Example 5;

[0061] Figure 7 is the H NMR spectrum of PCL-PDEAEMA-P(HEMA-g-LA)-PPEGMA in Example 6 ( 1 H NMR) spectrum, the solvent is deuterated dimethyl sulfoxide;

[0062] Figure 8 is the H NMR spectrum of PCL-PDEAEMA-P(HEMA-g-FA)-PPEGMA in Example 6 ( 1 H NMR) spectrum, the solvent is deuterated dimethyl sulfoxide;

[0063] Fig. 9 The graph of the particle size variation over time of the cross-linked blank micelles and the ordinary blank micelles in pH 7.4 phosphate-sodium phosphate buffer in Example 7;

[0064] Fig.10 The DLS particle size distribution and transmission electron microscopy (TEM) image of the mixed drug-loaded micelles in Example 8;

[0065] Fig.11The DLS particle size distribution and transmission electron microscopy (TEM) image of the mixed drug-loaded cross-linked micelles in Example 8;

[0066] Fig.12 is the in vitro drug release curve of the drug-loaded micelles in Example 9;

[0067] Fig.13 The cytotoxicity test results in Example 10; DETAILED DESCRIPTION

[0068] The present invention is further described below with reference to specific embodiments, but they should not be construed as limiting the present invention.

[0069] In the following examples, the hydrophobic anticancer drug paclitaxel is taken as an example, and the names of the substances used in the monomer, acid binding agent, catalyst, brominating agent, initiator, ligand, etc. are as follows:

[0070]

[0071] Example 1

[0072] Synthesis of polymer PCL: ε-caprolactone (13.70 g, 120.0 mmol) and a magnetic stirrer were added to a 100 mL anhydrous and oxygen-free reaction bottle, which was sealed and then vacuumed and argon was passed through for three cycles. A small molecule initiator n-butanol (0.148 g, 2.0 mmol) and a tin-based catalyst Sn(Otc)2 (0.0092 g, 0.02 mmol) were added using a syringe, and the reaction was frozen, vacuumed, and heated for three cycles again. The reaction was then transferred to an oil bath and stirred at 125°C for 24 h. After 24 h, the reaction was removed from the oil bath, quickly frozen with ice water, and passed through the atmosphere to terminate the reaction. 50 mL of tetrahydrofuran was added to the reaction bottle, and the white viscous solid was dissolved by ultrasonication at room temperature for 4 h. The reaction solution was concentrated by rotary evaporation, and the concentrated solution was dripped dropwise into 500 mL of ice methanol / water for precipitation to remove the solvent, unreacted monomers and catalysts. The solution was filtered and the precipitate was vacuum dried at 40°C for 24 h to obtain a white solid polymer PCL. 40 .

[0073] The resulting polymer PCL 40 The results of NMR characterization are as follows Figure 3 As shown, the results all proved that the polymer was successfully synthesized, and the molecular weight obtained by gel chromatography and NMR spectrum integration was close to the expected one.

[0074] Example 2

[0075] Synthesis of macromolecular initiator PCL-Br: Add the polymer PCL obtained in Example 1 into a dry, anhydrous and oxygen-free reaction bottle. 40(7.00g, 1.0mmol) and a stirrer, sealed with a reverse rubber stopper, vacuum-pass argon gas circulation three times for deoxygenation, add 30mL of anhydrous tetrahydrofuran as a solvent, stir for 10min to fully dissolve, then transfer to a low-temperature constant temperature stirring reaction bath, the temperature is set to 0°C, add anhydrous triethylamine (0.51g, 5mmol) and stir for 5min to fully mix, then 2-bromoisobutyryl bromide (1.15g, 5mmol) is slowly dripped into it with a syringe under argon protection, and the speed is controlled at 15 drops / second; then continue to stir and react at 0°C for 3h, and move to room temperature to continue the reaction for 24h. After the reaction is completed, the reaction solution is diluted with tetrahydrofuran, the insoluble triethylamine salt is removed by a neutral alumina chromatography column, and most of the solvent is removed by rotary evaporation of the filtrate. The resulting concentrated solution is dripped into 500mL of ice n-hexane for precipitation and filtered. The precipitate was dissolved with dichloromethane, extracted twice with dilute hydrochloric acid, sodium bicarbonate solution, and water in sequence, concentrated by rotary evaporation, precipitated with ice n-hexane as a precipitant, filtered, and dried in a vacuum drying oven at 40°C overnight to obtain the product macromolecular initiator PCL 40 -Br.

[0076] The obtained macroinitiator PCL 40 -Br was characterized by NMR and the results were as follows Figure 3 As shown, the results all proved that the polymer was successfully synthesized, and the molecular weight obtained by gel chromatography and NMR spectrum integration was close to the expected one.

[0077] Example 3: Synthesis route of PCL-PDEAEMA-PHEMA-PPEGMA Figure 1 Shown

[0078] (1) Synthetic polymer PCL 40 -PDEAEMA 10 -PHEMA 15 -PPEGMA 15 (P1): Add a stirrer, the macroinitiator PCL obtained in Example 2, and 40-Br (4.90 g, 0.7 mmol) and copper bromide (0.019 g, 0.084 mmol) were added to the mixture, sealed with a reverse rubber stopper, vacuumed for 5 min and then purged with argon for 1 min, and the cycle was repeated three times. Then, 20 mL of anhydrous toluene was added with a syringe and stirred for 5 min to fully dissolve. Then, monomer DEAEMA (1.296 g, 7.0 mmol) and ligand pentamethyldiethylenetriamine (PMDETA) (0.146 g, 0.84 mmol) were added in sequence, and stirring was continued for 1 min. 0min; the reducing agent Sn(Otc)2 (0.340g, 0.84mmol) was dissolved in 3mL of anhydrous toluene, injected into the reaction bottle with a syringe, stirred for 20min to make it fully react with the complex, then the reaction bottle was transferred to an oil bath, and stirred and reacted at 70°C; after 6h of reaction, the monomer HEMA (1.282g, 7.0mmol) was added, and after continuing the reaction for 9h, the monomer PEGMA (3.325g, 7.0mmol) was added, and the reaction was continued for 28h. After the reaction was completed, it was quickly cooled to room temperature and vented to the atmosphere. The solution was stirred at room temperature for 20min to oxidize all cuprous ions in the solution to cupric ions, and then excess tetrahydrofuran was added to dilute it, and the residual catalyst copper salt was filtered out through a neutral alumina column. The filtrate was concentrated by rotary evaporation at 20°C, and the concentrate was slowly dripped into n-hexane and filtered. The solid was freeze-vacuum dried to obtain a viscous solid PCL 40 -PDEAEM A 10 -PHEMA 15 -PPEGMA 15 (P1).

[0079] (2) Synthetic polymer PCL 40 -PDEAEMA 15 -PHEMA 15 -PPEGMA 15 (P2): Add a stirrer, the macroinitiator PCL obtained in Example 2, and 40-Br (4.90 g, 0.7 mmol) and copper bromide (0.019 g, 0.084 mmol) were added to the mixture, sealed with a reverse rubber stopper, vacuumed for 5 min and then purged with argon for 1 min, and the cycle was repeated three times. Then, 20 mL of anhydrous toluene was added with a syringe and stirred for 5 min to fully dissolve. Then, monomer DEAEMA (1.296 g, 7.0 mmol) and ligand pentamethyldiethylenetriamine (PMDETA) (0.146 g, 0.84 mmol) were added in sequence, and stirring was continued for 1 min. 0min; dissolve the reducing agent Sn(Otc)2 (0.340g, 0.84mmol) in 3mL of anhydrous toluene, inject it into the reaction bottle with a syringe, stir for 20min to make it fully react with the complex, then transfer the reaction bottle to an oil bath, and continue to stir and react at 70°C; after 6h of reaction, add the monomer HEMA (1.139g, 6.2mmol), continue to react for 9h, add the monomer PEGMA (5.82g, 12.25mmol), and continue to react for 28h. After the reaction is completed, quickly cool to room temperature and ventilate the atmosphere, stir at room temperature for 20min to oxidize all cuprous ions in the solution to cupric ions, then add excess tetrahydrofuran to dilute, pass through a neutral alumina column, filter to remove residual catalyst copper salt, and concentrate the filtrate by rotary evaporation at 20°C. Slowly drip the concentrate into n-hexane and filter it. The solid obtained is freeze-vacuum dried to obtain a viscous solid

[0080] PCL 40 -PDEAEMA 15 -PHEMA 15 -PPEGMA 15 (P2).

[0081] (3) Synthetic polymer PCL 40 -PDEAEMA 20 -PHEMA 15 -PPEGMA 15 (P3): Add a stirrer, the macroinitiator PCL obtained in Example 2, and 40-Br (4.90 g, 0.7 mmol) and copper bromide (0.019 g, 0.084 mmol) were added to the mixture, sealed with a reverse rubber stopper, vacuumed for 5 min and then purged with argon for 1 min, and the cycle was repeated three times. Then 20 mL of anhydrous toluene was added with a syringe and stirred for 5 min to fully dissolve. Then, monomer DEAEMA (2.592 g, 14.0 mmol) and ligand pentamethyldiethylenetriamine (PMDETA) (0.146 g, 0.84 mmol) were added in sequence, and stirring was continued. 10min; dissolve the reducing agent Sn(Otc)2 (0.340g, 0.84mmol) in 3mL of anhydrous toluene, inject it into the reaction bottle with a syringe, stir for 20min to make it fully react with the complex, then transfer the reaction bottle to an oil bath, and continue to stir and react at 70°C; after 6h of reaction, add the monomer HEMA (1.139g, 6.2mmol), continue to react for 9h, add the monomer PEGMA (3.74g, 7.87mmol), and continue to react for 28h. After the reaction is completed, quickly cool to room temperature and ventilate the atmosphere, stir at room temperature for 20min to oxidize all cuprous ions in the solution to cupric ions, then add excess tetrahydrofuran to dilute, pass through a neutral alumina column, filter to remove residual catalyst copper salt, and concentrate the filtrate by rotary evaporation at 20°C. Slowly drip the concentrate into n-hexane and filter it. The solid obtained is freeze-vacuum dried to obtain a viscous solid

[0082] PCL 40 -PDEAEMA 20 -PHEMA 15 -PPEGMA 15 (P3).

[0083] The polymers P1, P2 and P3 obtained above were characterized by NMR. Figure 4 As shown, it was proved that the polymers were successfully synthesized, and the molecular weights obtained by gel chromatography and nuclear magnetic resonance spectrum integration were close to the expected ones.

[0084] Example 4

[0085] The polymer molecular weight and molecular weight distribution were tested by gel permeation chromatography (GPC). 40 、PCL 40 -Br, polymer P1, polymer P2, polymer P3) were fully dissolved in tetrahydrofuran (HPLC grade) with the concentration controlled at 10 mg / mL, and then filtered with an organic filter head (0.22 μm) and tested at room temperature. The results were as follows Figure 5As shown in the figure, the elution curves of polymers P1, P2 and P3 are all single peaks, proving that the polymers are not doped with other impurities. The increase in polymer molecular weight will affect the peak position of the elution curve. The larger the molecular weight, the earlier the peak time. It can be seen from the figure that the peak positions of the five polymers conform to the peak rule of gel chromatography.

[0086] Example 5

[0087] The critical micelle concentration of the products PCL-PDEAEMA-PHEMA-PPEGMA in Examples 1, 2, and 3 was determined by the fluorescent probe method.

[0088] (1) Preparation of pyrene solution: 6×10 -5 The solution of M.

[0089] (2) Preparation of sample solution: Accurately weigh 10 mg PCL-PDEAEMA-PHEMA-PPEGMA and dissolve it in 5 mL acetone. Add it dropwise into 100 mL deionized water. After evaporating the acetone, a 0.1 mg / mL solution is obtained. Then, dilute it into a series of polymer solutions with different concentrations (0.0001-0.1 mg / ml). Take 20 10 mL brown volumetric flasks, add 0.1 mL pyrene solution to each flask, and then add the above polymer solutions of different concentrations to prepare sample solutions. The concentration of pyrene in the sample solution is 6×10 -7 M.

[0090] (3) Fluorescence spectrum test: Using 373 nm as the emission wavelength, test the excitation spectrum of the sample solution at 300-350 nm, and take I 337 / I 332 The ratio is plotted against the logarithm of the concentration (LogC) Figure 6 ), the turning point of the curve is the critical micelle concentration. 40 -PDEAEMA 10 -PHEMA 15 -PPEGMA 15 、PCL 40 -PD EAEMA 15 -PHEMA 15 -PPEGMA 15 、PCL 40 -PDEAEMA 20 -PHEMA 15 -PPEGMA 15 The critical micelle concentrations were 4.36, 2.25, and 1.09 mg / L, respectively.

[0091] Example 6

[0092] Synthetic polymer PCL-PDEAEMA-P(HEMA-g-LA / FA)-PPEGMA (synthetic process route as shown in Figure 2 As shown): Add lipoic acid (0.75mmol, 0.155g) to a 100ml dry anhydrous oxygen-free bottle, stir the seeds and seal, evacuate and pass argon several times, add 20ml anhydrous dimethyl sulfoxide (DMSO) and ultrasonically dissolve it completely. Dissolve CDI (0.75mmol, 0.122g) in 5ml anhydrous DMSO, add it to the fully dissolved LA after it is completely dissolved, and react at room temperature for 4-5h to activate the carboxyl group in lipoic acid; after activation, add the polymer P3 (0.05mmol, 0.87g) obtained in Example 3 pre-dissolved in anhydrous DMSO, and continue to react at room temperature for 18h. After the reaction is completed, the solution is transferred to a dialysis bag (MWCO3500), the dialysis medium is deionized water, and the deionized water is replaced every 4 hours. The dialysis is continued for 48 hours. After the dialysis is completed, the dialysate is filtered with a 0.45μm water filter head and the solution obtained is freeze-dried to obtain PCL-PDEAEMA-P(HEMA-g-LA)-PPEGMA(LA-P3). Other conditions remain unchanged and lipoic acid is replaced with folic acid of equal molar mass (0.75mmol, 0.331g) to synthesize PCL-PDEAEMA-P(HEMA-g-FA)-PPEGMA(FA-P3).

[0093] The polymers PCL-PDEAEMA-P(HEMA-g-LA)-PPEGMA and PCL-PDEAEMA-P(HEMA-g-FA)-PPEGMA obtained above were characterized by NMR. Figure 7 , 8 As shown, the results all proved that the polymer was successfully synthesized.

[0094] Example 7

[0095] Blank micelles were prepared and micelle stability was tested.

[0096] Preparation of blank micelles: 30 mg of each of polymer P3, polymer LA-P3, and polymer FA-P3 prepared in Example 3 and Example 6 were fully dissolved in 30 ml DMSO, completely dissolved by ultrasonication for 20 min at room temperature, transferred to a dialysis bag (MW3500), dialyzed with deionized water for 48 hours, and the deionized water was replaced every 4 hours. After dialysis, the solution was filtered using a 0.45 μm water filter, and the P3, FA-P3, and LA-P3 solutions were dried in a freeze dryer for 48 hours to obtain solid powders of each blank micelle. A trace amount of DTT was added to the LA-P3 solution and the dialysis was continued in the dark for 24 hours. After freeze drying, the cross-linked blank micelles C-LA-P3 were prepared. The FA-P3 and LA-P3 solutions were mixed in equal amounts, a trace amount of DTT was added, and the dialysis was continued in the dark for 24 hours. After freeze drying, the mixed cross-linked blank micelles C-MIX were prepared.

[0097] Micelle stability test: Each blank micelle was incubated in a phosphate-sodium phosphate buffer at pH 7.4 for 120 h, and the micelle size was measured regularly. The results were repeated three times and the average value was taken. Fig. 9 As shown by Fig. 9 It can be seen that the particle size of the blank micelle FA-P3 increases with the increase of incubation time, and a mutation does not occur until 120 hours. The blank micelle P3 only expands slightly at 24 hours, and the particle size increases significantly after 48 hours, reaching 114.5nm at 72 hours and 133nm at 120 hours. FA-P3 appears to be more stable.

[0098] At the same time, it can be seen that in the environment of phosphate-sodium phosphate buffer with pH = 7.4 simulating normal human tissue fluid, the particle size of the cross-linked blank micelles C-LA-P3 changed very little during the 120h cultivation, and the particle size always remained within 100nm, with basically no change, which indicates that the cross-linking effect of the lipoic acid block significantly improved the stability of the micelles.

[0099] Example 8

[0100] Preparation of polymer drug-loaded micelles P3, FA-P3 and LA-P3: Take 30 mg of each of the polymer P3 obtained in Example 3 and the polymers PCL-PDEAEMA-P(HEMA-g-FA)-PPEGMA and PCL-PDEAEMA-P(HEMA-g-LA)-PPEGMA obtained in Example 6, and fully dissolve them in 30 ml of DMSO with 15 mg of PTX, and completely dissolve them by ultrasonication at room temperature for 20 min, transfer them to a dialysis bag (MW3500), and dialyze them with deionized water for 48 h, and replace the deionized water every 4 h. After dialysis, filter them with a 0.45 μm water filter head, and freeze-dry them in a freeze dryer for 48 h to obtain drug-loaded micelles P3, FA-P3 and LA-P3.

[0101] Preparation of polymer cross-linked drug-loaded micelles C-LA-P3: Take 30 mg of the polymer PCL-PDEAE MA-P(HEMA-g-LA)-PPEGMA obtained in Example 6 and 15 mg of PTX and fully dissolve them in 30 ml of DMSO, ultrasonicate for 20 min at room temperature to completely dissolve, transfer to a dialysis bag (MW3500), dialyze with deionized water for 48 h, and replace the deionized water every 4 h. After dialysis, filter with a 0.45 μm water filter, add a trace amount of DTT and continue to dialyze in the dark for 24 h to prepare polymer cross-linked drug-loaded micelles C-LA-P3.

[0102] Preparation of mixed polymer drug-loaded micelles MIX and mixed polymer cross-linked drug-loaded micelles C-MIX: Take 15 mg of each of the polymers PCL-PDEAEMA-P(HEMA-g-LA)-PPEGMA and PCL-PDE AEMA-P(HEMA-g-FA)-PPEGMA obtained in Example 6 and 15 mg of PTX, fully dissolve them in 30 ml of DMSO, ultrasonicate for 20 min at room temperature to completely dissolve, transfer to a dialysis bag (MW3500), dialyze with deionized water for 48 hours, and replace the deionized water every 4 hours. After dialysis, filter with a 0.45 μm water filter. Part of the solution was dried in a freeze dryer for 48 hours to obtain a solid powder of mixed drug-loaded micelles MIX. Add a trace amount of DTT to part of the mixed micelle solution and continue to dialyze in the dark for 24 hours to prepare mixed polymer drug-loaded cross-linked micelles C-MIX.

[0103] The drug loading capacity (LC) and encapsulation efficiency (EE) of FA-P3 were measured to be 12.46±2.49% and 37.20±5.99%, respectively; the drug loading capacity (LC) and encapsulation efficiency (EE) of LA-P3 were measured to be 14.83±1.35% and 44.00±5.15%, respectively; the drug loading capacity (LC) and encapsulation efficiency (EE) of C-LA-P3 were measured to be 13.82±1.96% and 41.01±7.03%, respectively; the drug loading capacity (LC) and encapsulation efficiency (EE) of MIX were measured to be 15.54±2.48% and 39.20±4.21%, respectively; the drug loading capacity (LC) and encapsulation efficiency (EE) of C-MIX were measured to be 15.31±1.79% and 38.42±5.22%, respectively.

[0104] The particle size test and microscopic morphology characterization results of mixed drug-loaded micelles MIX and mixed drug-loaded cross-linked micelles C-MIX are as follows: Fig.10 , 11 As shown, the obtained particle size is consistent with the microscopic morphology. The particle size of the mixed drug-loaded micelles after cross-linking is reduced, indicating that the cross-linking structure plays a certain role.

[0105] Example 9

[0106] In vitro release experiment:

[0107] A buffer solution of pH = 5.0, 10 mM DTT, was prepared by adding a certain amount of DL-dithiothreitol to an acetic acid-sodium acetate buffer solution of pH = 5.0 to simulate the tumor cell environment, and a buffer solution of pH = 7.4, 0 mM DTT was used to simulate the normal tissue environment of the human body to verify the drug release performance of the drug-loaded micelles. The drug-loaded micelles FA-P3 and C-LA-P3 obtained in Example 8, the mixed drug-loaded micelles MIX and the mixed drug-loaded cross-linked micelles C-MIX were respectively weighed, 5 mg of the drug-loaded micelle solid samples were respectively dissolved in 5 mL of pH = 5.0 acetic acid-sodium acetate buffer and pH = 7.4 phosphate-sodium phosphate buffer, and after being fully dissolved, they were transferred to a dialysis bag (MWCO3500) and dialyzed in a 150 ml beaker, the dialysis medium was 95 mL of the above corresponding buffer solution, and the dialysis temperature was set to 37 ° C. 5 mL of the solution in the beaker was drawn out periodically, and 5 mL of fresh corresponding buffer was added to keep the total volume of the solution in the beaker at 100 mL. The absorbance of the sample at 258 nm was measured, and the cumulative release of paclitaxel in different environments was calculated.

[0108] The drug release test results of the above drug-loaded micelles are as follows Fig.12 As shown in the figure, the cumulative drug release of the mixed drug-loaded micelles is basically between that of the single drug-loaded micelles. Under the conditions of pH = 7.4, 0 mM DTT, the 120 h cumulative drug release of the mixed micelles MIX is 24.9%, and that of the mixed drug-loaded cross-linked micelles C-MIX is 16.8%; under the conditions of pH = 5.0, 10 mM DTT, the 120 h cumulative drug release of MIX reaches 79.48%, and that of C-MIX is slightly lower at 77.1%. This is because the cross-linking structure formed by lipoic acid plays a role. The cumulative drug release of the mixed cross-linked drug-loaded micelles C-MIX increases slowly, but due to the less cross-linking structure, the cumulative drug release in each stage is higher than that of C-LA-P3, and lower than that of FA-P3 and the uncross-linked mixed drug-loaded micelles MIX.

[0109] Example 10

[0110] Cytotoxicity assay

[0111] The CCK-8 (Cell Counting Kit-8) method with good repeatability, strong specificity and low toxicity was selected. The blank micelles, drug-loaded micelles and free paclitaxel were used to conduct cytotoxicity experiments to test the inhibitory effects of samples of different concentrations on liver cancer epithelial cells (HepG2). The specific experimental process is as follows:

[0112] The cultured cell suspension was inoculated in a 96-well plate, and 100 μL / well was ensured, and the cell number concentration was 1×10 4 The culture plate was placed in an incubator (37°C, 5% CO2) for pre-culture for a period of time, and then cultured in serum-free medium for 24 hours. Then, samples of different concentrations and types were added to each well of the culture plate. The types and concentrations of the samples to be tested are as follows:

[0113] (1) Free paclitaxel (0.1, 1, 2, 5, 10, 20 mg / L).

[0114] (2) Blank micelles P3, LA-P3, FA-P3 (1, 5, 10, 100, 200, 300 mg / L);

[0115] (3) Cross-linked blank micelles C-LA-CP3 (1, 5, 10, 100, 200, 300 mg / L);

[0116] (4) mixed cross-linked blank micelles C-MIX (1, ​​5, 10, 100, 200, 300 mg / L);

[0117] (5) drug-loaded micelles P3-PTX (0.625, 6.25, 12.5, 31, 62.5, 125 mg / L);

[0118] (6) drug-loaded micelles LA-P3 (0.667, 6.67, 13.34, 33.35, 66.7, 133.4 mg / L);

[0119] (7) Drug-loaded micelles FA-P3 (0.8, 8, 16, 40, 80, 160 mg / L);

[0120] (8) Cross-linked drug-loaded micelles C-LA-P3-PTX (0.714, 7.14, 14.3, 35.7, 71.4, 143 mg / L);

[0121] (9) Mixed cross-linked drug-loaded micelles C-MIX-PTX (0.653, 6.53, 13.1, 32.7, 65.3, 131 mg / L).

[0122] Each sample was prepared into six concentrations, each concentration of the sample was tested three times, and three control groups were set up: blank control group, DMSO control group and STSP positive control group, and incubated in the dark for 48 hours. Then add CCK-8 solution to each well, and try to control the volume to 10uL. If you are worried about the error being too large, you can directly prepare a culture medium containing 10% CCK-8 and add CCK-8 solution by changing the liquid. Also note that bubbles should be avoided as much as possible during the addition of samples to avoid affecting the OD value reading. After incubation for 4 hours, use an ELISA reader to measure the absorbance of the solution in the well plate at 450nm, and use the following formula to calculate cell viability (Cellviability).

[0123]

[0124] Wherein, Asample is the absorbance at 450 nm, Ablank is the absorbance of the blank well, and Acontrol is the absorbance of the control group.

[0125] The results are as follows Fig.13 As shown in the figure, both blank micelles and blank mixed cross-linked micelles showed low cytotoxicity, and the introduction of lipoic acid (LA) and folic acid (FA) groups had little effect on cell toxicity; free PTX had the strongest inhibitory effect on cells, and the cell survival rate after 48 hours of incubation was only 17.78%, because paclitaxel directly acts on cells and micelles slowly release drugs. Among the drug-loaded micelles, FA-P3-PTX had the lowest cell survival rate, and the cell survival rate after 48 hours of incubation at a concentration of 100μg / mL was only 28.9%, which means that folic acid-modified micelles have stronger inhibitory effects on HepG2; cross-linked micelles C-LA-P3-PTX have slightly weaker inhibitory effects, because the micelle releases drugs through the step of de-crosslinking, so the drug release is slow and the inhibitory effect is low. After LA-P3 and FA-P3 were prepared into active targeted mixed drug-loaded cross-linked micelles C-MIX-PTX, the inhibitory effect on tumor cells was better than that of C-LA-P3-PTX, LA-P3-PTX and P3-PTX.

[0126] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A folic acid / lipoic acid modified pH / reduction dual-responsive mixed polymer cross-linked micelle, characterized in that: The mixed polymer cross-linked micelle is formed by self-assembly of polymer A obtained by modifying folic acid with polymer P and polymer B obtained by modifying lipoic acid with polymer P; wherein polymer P is PCL-PDEAEMA-PHEMA-PPEGMA, and its structural formula is shown in formula (1); polymer A is PCL-PDEAEMA-P(HEMA-g-FA)-PPEGMA, and its structural formula is shown in formula (2); polymer B is PCL-PDEAEMA-P(HEMA-g-LA)-PPEGMA, and its structural formula is shown in formula (3): Wherein, x is a positive integer of 10 to 20, m and n are positive integers of 0 to 15, and m+n=15.

2. The method for preparing a folic acid / lipoic acid modified pH / reduction dual-responsive mixed polymer cross-linked micelle according to claim 1, characterized in that The following steps are involved: (1) Under inert gas protection and anhydrous and oxygen-free conditions, ε-caprolactone, a small molecule initiator and a first catalyst are mixed and subjected to a ring-opening polymerization reaction to obtain a polymer PCL; (2) Under inert gas protection and anhydrous conditions, the polymer PCL obtained in step (1) is dissolved in a solvent from which water has been removed, an acid binding agent is added, the mixture is cooled to 0° C., a brominating agent is added dropwise and stirred, and after the addition is complete, the mixture is reacted at 0° C. for 1 to 3 hours, and then the reaction is continued at room temperature for 12 to 24 hours. After the reaction, the mixture is filtered, concentrated, washed, and dried to obtain a macromolecular initiator PCL-Br; (3) placing the macromolecular initiator PCL-Br and the second catalyst obtained in step (2) in a sealed container, repeatedly evacuating and passing argon for three times, and then adding a solvent to fully dissolve; then adding the first monomer and the ligand in sequence, stirring continuously for 10 minutes, and then adding a reducing agent, stirring for 20 minutes to fully react with the complex, and then transferring the container to an oil bath pot and stirring continuously to perform an atom transfer radical polymerization reaction; after reacting for 6 hours, adding the second monomer, continuing the reaction for 9 hours, and then adding the third monomer, and then reacting for 28 hours, cooling to room temperature and passing air, filtering, concentrating, washing, and drying to obtain PCL-PDEAEMA-PHEMA-PPEGMA, which is polymer P; (4) Add folic acid and a stirrer into a sealed dry anhydrous oxygen-free bottle, repeat vacuuming and argon gas flow three times, then add anhydrous dimethyl sulfoxide, and sonicate to fully dissolve the folic acid to obtain a folic acid solution; dissolve the catalyst in anhydrous dimethyl sulfoxide, add it to the above folic acid solution, and react for 4-5 hours at room temperature to activate the carboxyl groups in the folic acid. After the activation is completed, add the polymer P obtained in step (3) which is pre-dissolved in anhydrous DMSO, and continue the esterification reaction at room temperature for 18 hours; after the reaction is completed, transfer the reaction solution to a dialysis bag with MWCO 3500, the dialysis medium is deionized water, and the deionized water is replaced every 4 hours, and the dialysis is continued for 48 hours. After the dialysis is completed, the dialyzate is filtered with a 0.45 μm water filter head, and the obtained filtrate is freeze-dried to obtain PCL-PDEAEMA-P(HEMA-g-FA)-PPEGMA, which is polymer A; (5) replacing the folic acid in step (4) with lipoic acid of equal molar mass, and the other conditions are the same as step (4), to obtain PCL-PDEAEMA-P(HEMA-g-LA)-PPEGMA, i.e., polymer B; (6) mixing the polymer A obtained in step (4) and the polymer B obtained in step (5) and dissolving them in an organic solvent, and dialyzing to obtain a blank mixed polymer non-cross-linked micelle dialysate; (7) The blank mixed polymer non-cross-linked micelle dialysate obtained in step (6) is subjected to self-cross-linking of lipoic acid using a trace amount of a reducing agent, thereby finally obtaining folic acid / lipoic acid modified pH / reduction dual-responsive mixed polymer cross-linked micelles.

3. The method for preparing a folic acid / lipoic acid modified pH / reduction dual-responsive mixed polymer cross-linked micelle according to claim 2, characterized in that: Step (1) The first catalyst is stannous isooctanoate; the small molecule initiator is n-butanol; The solvent in step (2) is tetrahydrofuran; the acid binding agent is triethylamine; the brominating agent is 2-bromoisobutyryl bromide; Step (3) the second catalyst is copper bromide; the ligand is pentamethyl divinyl triamine; the reducing agent is stannous isooctanoate; the solvent is anhydrous tetrahydrofuran; the eluent is tetrahydrofuran; the first monomer is diethylaminoethyl methacrylate; the second monomer is hydroxyethyl methacrylate; the third monomer is polyethylene glycol methyl ether methacrylate; The catalyst in step (4) is N,N-dicarbonylimidazole; The reducing agent in step (7) is dithiothreitol.

4. The method for preparing a folic acid / lipoic acid modified pH / reduction dual-responsive mixed polymer cross-linked micelle according to claim 2, characterized in that: The weight ratio of each substance in step (1) is as follows: Small molecule initiator 0.148 parts ε-caprolactone 9.13 parts 0.027 parts of the first catalyst The weight ratio of each substance in step (2) is as follows: Polymer PCL 4.56 parts Acid binding agent 0.81 parts Brominating agent 1.11 parts The weight ratio of each substance in step (3) is as follows: The weight ratio of each substance in step (4) is as follows: Folic acid 0.331 parts Catalyst 0.122 parts Polymer P 0.87 parts The weight ratio of each substance in step (5) is as follows: Lipoic acid 0.155 parts Catalyst 0.122 parts Polymer P 0.87 parts The weight ratio of the blank mixed polymer non-cross-linked micellar dialysate used in step (7) to the reducing agent is 0.3 parts and 0.0027 parts respectively, based on the weight of the powder obtained by freeze-drying.

5. The method for preparing a folic acid / lipoic acid modified pH / reduction dual-responsive mixed polymer cross-linked micelle according to claim 2, characterized in that: The temperature of the ring-opening polymerization reaction in step (1) is 120-130° C. and the time is 24-48 hours; The temperature of the atom transfer radical polymerization reaction in step (3) is 65-70° C.; The esterification reaction conditions in step (4) are alkaline environment at room temperature; In step (6), the polymer A and polymer B are mixed and dissolved in an organic solvent in a mass ratio of 1 to 3:1 to 3, stirred for 4 to 12 hours, and dialyzed with deionized water for 24 to 72 hours, during which the deionized water is replaced every 3 to 6 hours to obtain a blank mixed polymer non-cross-linked micellar dialysate; the organic solvent is dimethylformamide, dimethyl sulfoxide or acetone; The inducing lipoic acid self-crosslinking in step (7) is to add a trace amount of reducing agent to the blank mixed polymer non-crosslinked micelle dialysate, stir the crosslinking reaction at room temperature at pH 8-9 for 6-12 hours, dialyze with deionized water for 12-36 hours after the reaction is completed, replace the deionized water every 3-6 hours, and freeze-dry to obtain folic acid / lipoic acid modified pH / reduction dual-responsive mixed polymer crosslinked micelles.

6. Use of the folic acid / lipoic acid modified pH / reduction dual-responsive mixed polymer cross-linked micelles according to claim 1 in a targeted controlled-release nano drug delivery system.

7. The use according to claim 6, characterized in that: The targeted controlled-release nano drug delivery system loads and delivers the hydrophobic anticancer drug paclitaxel.

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