ROS-responsive high polymer material, drug-loaded nano-micelle and preparation method of ROS-responsive high polymer material and drug-loaded nano-micelle

Abstract

The invention discloses an ROS response type polymer material, a drug-loaded nano-micelle and a preparation method of the ROS response type polymer material and the drug-loaded nano-micelle, and belongs to the technical field of medical materials. A CDK4 / 6 inhibitor Palbociclib which is approved to be used clinically is used as a model drug load, and the nano-micelle M-Pal loaded with the Palbociclib is prepared by utilizing an autonomously prepared ROS (reactive oxygen species) response type high polymer material MPEG113-b-PCL11-b-PB36 and using a solvent evaporation method. The drug-loaded nano-micelle can be stored for a long time after being freeze-dried; after the water phase is added and re-dispersed, the nano-micelle dispersion liquid is still clear and transparent. The ROS-responsive freeze-dried drug-loaded nano-micelle solves the long-term storage problem of conventional micelle-type drug-loaded nano-materials, provides an effective delivery platform for water-insoluble drugs, has ROS-responsive load release characteristics, and has a good application prospect.

Description

Technical field:

[0001] The present invention belongs to the technical field of medical materials, and in particular relates to a ROS-responsive polymer material, a drug-loaded nano-micelle and a preparation method thereof. Background technology:

[0002] Polymer materials with excellent biocompatibility are widely used in the pharmaceutical field. When used for drug delivery, these polymers can improve drug metabolism and pharmacokinetic properties, reduce toxic side effects, and significantly enhance therapeutic efficacy. Amphiphilic block polymers can self-assemble in aqueous media to form nanomicelles, which can serve as delivery vehicles for poorly water-soluble drugs. With the development of various new amphiphilic block polymers, nanomicelle-based drug delivery systems, as a nanodrug delivery technology with great potential, have been successfully applied in various fields.

[0003] Nanomicelles are ordered, thermodynamically stable colloidal aggregates formed by molecular self-assembly of amphiphilic materials in an aqueous medium. The formation mechanism of nanomicelles is as follows: when the concentration of the amphiphilic material in an aqueous medium exceeds a critical value, the hydrophobic groups aggregate due to hydrophobic interactions to form the core of the nanomicelles, while the hydrophilic groups maintain interaction with water molecules to form the outer shell of the nanomicelles, allowing the nanomicelles to be stably dispersed in the aqueous medium.

[0004] Nanomicelles, as drug delivery materials, have the advantages of small particle size, stable structure, strong drug solubilization ability and low toxicity, and have been widely used for different drug loading. For example, the nanomicelles loaded with paclitaxel developed by Samyang Biopharm, a South Korean biopharmaceutical company PM. The polymer material used in this product is methoxy polyethylene glycol-b-poly D,L-lactide (MPEG-b-PDLLA). PM has been marketed in South Korea for the treatment of metastatic breast cancer, non-small cell lung cancer, and ovarian cancer. In addition, Samyang Biopharm, a South Korean biopharmaceutical company, has developed nano-micelles loaded with docetaxel using methoxypolyethylene glycol-b-poly D,L-lactide (MPEG-b-PDLLA) as a polymer material. M, is currently being used in clinical trials for recurrent or metastatic head and neck squamous cell carcinoma. Clinical trials are also underway for the treatment of metastatic esophageal squamous cell carcinoma with oxaliplatin. Indian pharmaceutical company Sun Pharma has developed cyclosporine A-loaded nanoparticles called Cequa. Cequa promotes tear secretion and has been approved by the US Food and Drug Administration for the treatment of dry eye. Research on drug-loaded nanoparticles has also been reported in China. Paclitaxel-loaded nanoparticles developed by Shanghai Yizhong Pharmaceutical Co., Ltd. have been approved by the China National Medical Products Administration for combination therapy with platinum-based therapies for patients with epidermal growth factor receptor mutation-negative, anaplastic lymphoma kinase-negative, and unresectable locally advanced or metastatic non-small cell lung cancer.

[0005] Although there are many studies on drug-loaded nanomicelles, the number of nanomicelle products that are finally commercialized and used in clinical treatment is still limited. The reasons include the complex preparation process of amphiphilic polymer materials, the high difficulty of quality control, and the difficulty in storing the resulting drug-loaded nanomicelles. Among them, the long-term storage of drug-loaded nanomicelles is one of the major obstacles to the successful commercialization of drug-loaded nanomicelles. Current drug-loaded nanomicelles are mostly stored in the form of aqueous dispersion, which is difficult to store for a long time. Freeze-drying and dehydration of ordinary drug-loaded nanomicelles will destroy their structure, making it impossible to redisperse them evenly in the aqueous medium. Therefore, it is of great significance to develop a type of drug-loaded nanomicelles that can be stored for a long time after freeze-drying and can be redispersed in an aqueous medium for the delivery of drugs that are poorly soluble in water. Summary of the invention:

[0006] The present invention aims to provide a ROS-responsive polymer material, drug-loaded nanomicelles, and methods for their preparation. These drug-loaded nanomicelles have a high drug loading capacity for poorly water-soluble drugs and exhibit reactive oxygen species-responsive drug release properties, making them suitable for the delivery of anti-tumor drugs. Furthermore, these drug-loaded nanomicelles can be stored for long periods after lyophilization and can be redispersed in an aqueous medium, thus resolving the long-term storage challenge of drug-loaded nanomicelles and demonstrating promising application prospects.

[0007] In order to achieve the above objectives, the technical methods adopted in this application are as follows:

[0008] The present invention discloses a ROS-responsive polymer material, specifically MPEG 113 -b-PCL m -b-PB n , using water-soluble monomethoxy polyethylene glycol (MPEG) with a degree of polymerization of 113, first block poly (ε-caprolactone) with different degrees of polymerization m, referred to as PCL m ; then block ROS-responsive poly[4-(acryloyloxymethyl)phenylboronic acid pinacol ester] with different polymerization degrees n, referred to as PB n ; The final preparation is referred to as MPEG 113 -b-PCLm -b-PB n The structural formula is shown in the following general formula I:

[0009]

[0010] Wherein, m and n are selected from 5-100 respectively.

[0011] The ROS-responsive polymer material MPEG 113 -b-PCL m -b-PB n The general preparation method comprises the following steps:

[0012]

[0013] Synthesis of 4-(acryloyloxymethyl)phenylboronic acid pinacol ester. 4-(Hydroxymethyl)phenylboronic acid pinacol ester (CAS No. 302348-51-2) and triethylamine (CAS No. 121-44-8) were dissolved in the organic solvent dichloromethane (CAS No. 75-09-2). After cooling in an ice-water bath and using nitrogen as a blanket, acryloyl chloride (CAS No. 814-68-6) was added dropwise with stirring. After removing the ice bath, the mixture was stirred at room temperature for 12 hours, after which the reaction mixture was extracted with a saturated aqueous solution of sodium chloride (CAS No. 7647-14-5). The dichloromethane solution was separated and concentrated by distillation under reduced pressure. The resulting crude product was purified by silica gel flash column chromatography to obtain 4-(acryloyloxymethyl)phenylboronic acid pinacol ester as a white solid.

[0014] MPEG 113 -b-PCL m Synthesis of such materials. Monomethoxy polyethylene glycol (CAS No. 9004-74-4) with a degree of polymerization of 114 and different equivalents of ε-caprolactone (CAS No. 502-44-3) were dissolved in toluene (CAS No. 108-88-3), and then stannous isooctanoate (CAS No. 301-10-0) was added. The reaction system was kept at 105°C and stirred for 18 hours. The reaction mixture was then added to petroleum ether (CAS No. 64742-49-0) to precipitate MPEG. 113 -b-PCL m .

[0015] MPEG 113 -b-PCL m - Synthesis of BIBB-type materials. 113 -b-PCL mDissolve 2-bromoisobutyryl bromide (CAS No. 20769-85-1) in dichloromethane (CAS No. 75-09-2), add triethylamine (CAS No. 121-44-8), and stir at room temperature for 12 hours. Then add the reaction mixture into petroleum ether (CAS No. 64742-49-0) to precipitate MPEG. 113 -b-PCL m -BIBB.

[0016] MPEG 113 -b-PCL m -b-PB n Synthesis of MPEG-like materials 113 -b-PCL m -BIBB and 4-(acryloyloxymethyl)phenylboronic acid pinacol ester were dissolved in anisole (CAS No. 100-66-3). Cuprous bromide (CAS No. 7787-70-4) and tris[2-(dimethylamino)ethyl]amine (CAS No. 33527-91-2) were added under nitrogen. The reaction mixture was stirred at 60°C for 24 hours, purified by flash column chromatography on neutral alumina, concentrated, and precipitated in petroleum ether (CAS No. 64742-49-0) to obtain MPEG. 113 -b-PCL m -b-PB n .

[0017] For the MPEG 113 -b-PCL m -b-PB n The general formula of the material, the present invention attempts different feed ratios, taking the following different polymer products as examples, the polymerization degrees of each block part are: monomethoxy polyethylene glycol MPEG has a polymerization degree of 113, poly (ε-caprolactone) PCL has a polymerization degree of 11, and poly [4- (acryloyloxymethyl) phenylboronic acid pinacol ester] PB has a polymerization degree of 18, 30, 36, and 40.

[0018]

[0019] The present invention also discloses a ROS-responsive freeze-dried drug-loaded nano-micelle, which utilizes the ROS-responsive polymer material MPEG 113 -b-PCL m -b-PB n Empty nanomicelles were prepared by nanoprecipitation. Drug-loaded nanomicelles were then prepared by solvent evaporation using the drug load and empty nanomicelles dissolved in an organic solvent. The drug-loaded nanomicelles were then dehydrated using freeze-drying to obtain ROS-responsive, lyophilizable drug-loaded nanomicelles.

[0020] MPEG113 -b-PCL m -b-PB n Select the best matching MPEG from the current screening of similar materials 113 -b-PCL 11 -b-PB 36 Taking the CDK4 / 6 inhibitor Palbociclib (CAS No. 571190-30-2) as an example, ROS-responsive lyophilizable drug-loaded nanomicelles were prepared, including the following steps:

[0021] Preparation of empty nanomicelles: MPEG 113 -b-PCL 11 -b-PB 36 Dissolve in acetone (CAS No. 67-64-1), add twice the volume of ultrapure water while vortexing vigorously, and remove the acetone by vacuum distillation to obtain empty nanomicelles.

[0022] Preparation of drug-loaded nanomicelles: Palbociclib was dissolved in chloroform (CAS 67-66-3) and added dropwise to the empty nanomicelles prepared in the previous step under vigorous stirring (1000 rpm for 4 hours). Stirring was continued for 2 hours. After evaporation of the chloroform, the mixture was freeze-dried to obtain ROS-responsive, lyophilizable, drug-loaded nanomicelles loaded with palbociclib, referred to as M-Pal.

[0023] The beneficial effects of the above technical solution of the present invention are:

[0024] These drug-loaded nanomicelles have a high loading capacity for poorly water-soluble drugs and exhibit reactive oxygen species-responsive drug release, making them suitable for the delivery of anti-tumor drugs. Furthermore, these drug-loaded nanomicelles can be stored for long periods after freeze-drying and can be redispersed in aqueous media, thus resolving the long-term storage challenge of drug-loaded nanomicelles and demonstrating promising application prospects. Description of the drawings:

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 4-(acryloyloxymethyl)phenylboronic acid pinacol ester 1 H-NMR results.

[0027] Figure 24-(acryloyloxymethyl)phenylboronic acid pinacol ester 13 C-NMR results.

[0028] Figure 3 The ESI mass spectrum results of 4-(acryloyloxymethyl)phenylboronic acid pinacol ester are shown in FIG.

[0029] Figure 4 Compound MPEG 113 -b-PCL 11 of 1 H-NMR results.

[0030] Figure 5 Compound MPEG 113 -b-PCL 11 -BIBB's 1 H-NMR results.

[0031] Figure 6 Compound MPEG 113 -b-PCL 11 -b-PB 18 of 1 H-NMR results.

[0032] Figure 7 Compound MPEG 113 -b-PCL 11 -b-PB 30 of 1 H-NMR results.

[0033] Figure 8 Compound MPEG 113 -b-PCL 11 -b-PB 36 of 1 H-NMR results.

[0034] Figure 9 Compound MPEG 113 -b-PCL 11 -b-PB 40 of 1 H-NMR results.

[0035] Figure 10 To prepare MPEG loaded with CDK4 / 6 inhibitor Palbociclib 113 -b-PCL 11 -b-PB 36 Drug-loaded nanomicelles, M-Pal micelles stored for 1 or 30 days after freeze-drying, and the effect after redispersion in ultrapure water.

[0036] Figure 11Graph showing drug loading and encapsulation efficiency of freeze-dried M-Pal.

[0037] Figure 12 Figure 2 shows the ROS-responsive release characteristics of M-Pal after short-term and long-term storage after lyophilization.

[0038] Figure 13 Schematic diagram of the particle size and surface potential of M-Pal after lyophilization for one day (left) and after lyophilization for one month and then redispersed in ultrapure water (right).

[0039] Figure 14 Schematic diagram showing the effects of Palbociclib and M-Pal on the viability of pancreatic cancer cells. Specific implementation method:

[0040] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0041] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0042] Example 1: Synthesis of 4-(acryloyloxymethyl)phenylboronic acid pinacol ester

[0043]

[0044] 4-(hydroxymethyl)phenylboronic acid pinacol ester (10g, 0.043mol) and triethylamine (5.2g, 0.051mol) were dissolved in 10mL of the organic solvent dichloromethane. After cooling in an ice-water bath, nitrogen was used as protection, and acryloyl chloride (4.6g, 0.051mol) was added dropwise with stirring. After removing the ice bath, the mixture was stirred at room temperature for 12 hours, and then the reaction mixture was extracted with a saturated aqueous solution of sodium chloride. After separating the dichloromethane solution, the mixture was concentrated by underpressure distillation, and the obtained crude product was purified by silica gel flash column chromatography to obtain 4-(acryloyloxymethyl)phenylboronic acid pinacol ester as a white solid. Figure 1 and Figure 2 4-(acryloyloxymethyl)phenylboronic acid pinacol ester 1 H-NMR and 13 C-NMR results. The peaks and corresponding proton assignments in the spectrum are labeled with lowercase letters. This is calculated by integrating the resonance peaks of the methyl protons in phenylboronic acid (1.34 ppm) and the protons in the acryloyloxy chain (6.45, 6.17, and 5.85 ppm). Figure 3The ESI mass spectrometry results of 4-(acryloyloxymethyl)phenylboronic acid pinacol ester are shown. The molecular weight of 4-(acryloyloxymethyl)phenylboronic acid pinacol ester is 288.153, which is the same as Na + After combining, the adduct ion [M+Na] is formed + m / z311.143, m / z in the mass spectrum

[0045] The peak at 311.142 corresponds to [M+Na] + . 1 H NMR (600MHz, CDCl3) δ7.81(d,J=7.5Hz,2H),7.37(d,J=7.6Hz,2H),6.45(dd,J=17.4,1.3Hz, 1H), 6.17 (dd, J=17.3, 10.4Hz, 1H), 5.85 (dd, J=10.4, 1.3Hz, 1H), 5.21 (s, 2H), 1.34 (s, 12H). 13 C-NMR (150 MHz, CDCl3) δ 165.96, 138.85, 135.02, 131.15, 128.27, 127.30, 83.86, 77.24, 77.03, 76.82, 66.19, 38.89, 37.56, 24.85. ESI-mass spectrometry: [M+Na] calculated for C 16 H 21 BO4:311.143; found 311.142

[0046] Example 2: Compound MPEG 113 -b-PCL 11 Synthesis

[0047]

[0048] Monomethoxypolyethylene glycol (1 g, molecular weight 5000, degree of polymerization 114, 0.0002 mol) and ε-caprolactone (0.25 g, 0.0022 mol) were dissolved in 1 mL of toluene, and stannous isooctanoate (20 mg) was added. The reaction system was stirred at 105°C for 18 hours. The reaction mixture was then added to petroleum ether to precipitate MPEG as a white solid. 113 -b-PCL 11 . Figure 4 Compound MPEG 113 -b-PCL 11 of 1H-NMR results. The peaks and corresponding proton assignments in the spectrum are labeled with lowercase letters. The average degree of polymerization of the polycaprolactone (PCL) block is 11, calculated by comparing the integral of the methylene proton resonance peak in the monomethoxypolyethylene glycol (MPEG) backbone (3.65 ppm, peak b) with the methylene proton resonance peaks in the PCL backbone (peak c: 2.31 ppm, peaks d and f: 1.65 ppm, peak e: 1.39 ppm). 1 H NMR(600MHz, CDCl3)δ4.23(m,2H),4.08–4.04(m,22H),3.76(m,2H),3.65(s,4 54H),3.38(s,3H),2.31(t,J=7.4Hz,22H),1.68–1.62(m,44H),1.39(m,22H).

[0049] Example 3: Compound MPEG 113 -b-PCL 11 Synthesis of -BIBB

[0050] MPEG 113 -b-PCL 11 (5g, 0.00082mol) and 2-bromoisobutyryl bromide (0.14g, 0.00061mol) were dissolved in 5mL of dichloromethane, triethylamine (0.06g, 0.00059mol) was added, and stirred at room temperature for 12 hours. The reaction mixture was then added to petroleum ether to precipitate MPEG. 113 -b-PCL 11 -BIBB. Figure 5 Compound MPEG 113 -b-PCL 11 -BIBB's 1 H-NMR results. The peaks and corresponding protons in the spectrum are assigned using lowercase letters. The successful coupling of 2-bromoisobutyryl bromide was confirmed by comparing the resonance peak of the terminal methyl proton of monomethoxypolyethylene glycol (peak a: 3.38 ppm) with the resonance peak of the terminal methyl proton of 2-bromoisobutyl (peak h: 1.93 ppm). 1 H-NMR (600MHz, CDCl3) δ4.23(m,2H),4.17(m,1H),4.07(m,21H),3.76(m,2H),3.67–3.62(m,453H ),3.38(d,J=2.9Hz,3H),2.31(m,22H),1.93(s,6H),1.65(q,J=6.8Hz,44H),1.42–1.35(m,22H).

[0051] Example 4: Compound MPEG 113 -b-PCL 11 -b-PB 18 Synthesis

[0052]

[0053] Compound MPEG 113 -b-PC 11 -BIBB (1 g, 0.00019 mol) and compound 4-(acryloyloxymethyl)phenylboronic acid pinacol ester (1.4 g, 0.0048 mol) were dissolved in 0.56 mL of anisole, an organic reagent, and cuprous bromide (0.056 g, 0.00038 mol) and tris[2-(dimethylamino)ethyl]amine (0.045 g, 0.00038 mol) were added under nitrogen protection. The reaction mixture was stirred at 60°C for 24 hours, purified by flash column chromatography on neutral alumina, and concentrated. MPEG was precipitated in petroleum ether to obtain a light gray solid. 113 -b-PCL 11 -b-PB 18 . Figure 6 Compound MPEG 113 -b-PCL 11 -b-PB 18 of 1 H-NMR results. The peaks and corresponding proton assignments in the spectrum are indicated by lowercase letters. The average degree of polymerization of poly[4-(acryloyloxymethyl)phenylboronic acid pinacol ester] (PB) is 18, calculated by comparing the integral of the proton resonance peak of the terminal methyl group of monomethoxypolyethylene glycol (MPEG) (peak a: 3.38 ppm), the proton resonance peak of the methylene group in the polycaprolactone (PCL) backbone (peak b: 2.35-2.25 ppm), and the proton resonance peak of the benzene ring of poly[4-(acryloyloxymethyl)phenylboronic acid pinacol ester] (PB) (peak c: 7.73 ppm). 1 H-NMR (600MHz, CDCl3) δ7.73(s,36H),7.21(s,36H),4.89(s,36H),4.23(t,J=4.9Hz,2H),4.06(t,J=6.7Hz,18H),3.82–3.49( m,389H),3.38(s,3H),2.35–2.25(m,22H),2.08–1.80(m,30H),1.75–1.52(m,60H),1.49–1.20(m,273H),0.92–0.78(m,43H).

[0054] Example 5: Compound MPEG 113 -b-PCL11 -b-PB 30 Synthesis

[0055]

[0056] Compound MPEG 113 -b-PC 11 -BIBB (1 g, 0.00019 mol) and compound 4-(acryloyloxymethyl)phenylboronic acid pinacol ester (2.8 g, 0.0096 mol) were dissolved in 0.88 mL of anisole, an organic reagent, and cuprous bromide (0.056 g, 0.00038 mol) and tris[2-(dimethylamino)ethyl]amine (0.045 g, 0.00038 mol) were added under nitrogen protection. The reaction mixture was stirred at 60°C for 24 hours, purified by flash column chromatography on neutral alumina, and concentrated. MPEG was precipitated in petroleum ether to obtain a light gray solid. 113 -b-PCL 11 -b-PB 30 . Figure 7 Compound MPEG 113 -b-PCL 11 -b-PB 30 of 1 H-NMR results. The peaks and corresponding proton assignments in the spectrum are indicated by lowercase letters. The average degree of polymerization of poly[4-(acryloyloxymethyl)phenylboronic acid pinacol ester] (PB) is 30. This value was calculated by comparing the integral of the proton resonance peak of the terminal methyl group of monomethoxypolyethylene glycol (MPEG) (peak a: 3.38 ppm), the proton resonance peak of the methylene group in the polycaprolactone (PCL) backbone (peak b: 2.35-2.25 ppm), and the proton resonance peak of the benzene ring of poly[4-(acryloyloxymethyl)phenylboronic acid pinacol ester] (PB) (peak c: 7.73 ppm). 1 H-NMR (600MHz, CDCl3) δ7.73(s,61H),7.21(s,61H),4.89(s,60H),4.23(t,J=4.9Hz,2H),4.06(t,J=6.7Hz,18H),3.82–3.49 (m,397H),3.38(s,3H),2.35–2.25(m,22H),2.08–1.80(m,31H),1.75–1.52(m,61H),1.49–1.20(m,363H),0.92–0.78(m,9H).

[0057] Example 6: Compound MPEG 113 -b-PCL 11 -b-PB 36 Synthesis

[0058]

[0059] Compound MPEG 113 -b-PC 11 -BIBB (1 g, 0.00019 mol) and compound 4-(acryloyloxymethyl)phenylboronic acid pinacol ester (2.8 g, 0.0096 mol) were reacted without adding solvent. Cuprous bromide (0.056 g, 0.00038 mol) and tris[2-(dimethylamino)ethyl]amine (0.045 g, 0.00038 mol) were added under nitrogen protection. The reaction mixture was stirred at 60°C for 24 hours, purified by flash column chromatography on neutral alumina, and then concentrated. MPEG was precipitated in petroleum ether to obtain a light gray solid. 113 -b-PCL 11 -b-PB 36 . Figure 8 Compound MPEG 113 -b-PCL 11 -b-PB 36 of 1 H-NMR results. The peaks and corresponding proton assignments in the spectrum are indicated by lowercase letters. The average degree of polymerization of poly[4-(acryloyloxymethyl)phenylboronic acid pinacol ester] (PB) is 36. This value was calculated by comparing the integral of the proton resonance peak of the terminal methyl group of monomethoxypolyethylene glycol (MPEG) (peak a: 3.38 ppm), the proton resonance peak of the methylene group in the polycaprolactone (PCL) backbone (peak b: 2.35-2.25 ppm), and the proton resonance peak of the benzene ring of poly[4-(acryloyloxymethyl)phenylboronic acid pinacol ester] (PB) (peak c: 7.73 ppm). 1 H-NMR (600MHz, CDCl3) δ7.73(s,72H),7.21(s,79H),4.89(s,72H),4.23(t,J=4.9Hz,3H),4.06(t,J=6.7Hz,18H),3 .82–3.49(m,393H),3.38(s,3H),2.35–2.25(m,22H),2.08–1.80(m,75H),1.75–1.52(m,23H),1.49–1.20(m,421H).

[0060] Example 7: Compound MPEG 113 -b-PCL 11 -b-PB 40 Synthesis

[0061]

[0062] Compound MPEG 113-b-PC 11 -BIBB (1 g, 0.00019 mol) and compound 4-(acryloyloxymethyl)phenylboronic acid pinacol ester (4.2 g, 0.0144 mol) were dissolved in 1.21 mL of anisole as an organic reagent. Cuprous bromide (0.056 g, 0.00038 mol) and tris[2-(dimethylamino)ethyl]amine (0.045 g, 0.00038 mol) were added under nitrogen protection. The reaction mixture was stirred at 60° C. for 24 hours, purified by flash column chromatography on neutral alumina, and concentrated. MPEG was precipitated in petroleum ether to obtain a dark gray solid. 113 -b-PCL 11 -b-PB 40 . Figure 9 Compound MPEG 113 -b-PCL 11 -b-PB 40 of 1 H-NMR results. The peaks and corresponding proton assignments in the spectrum are indicated by lowercase letters. The average degree of polymerization of poly[4-(acryloyloxymethyl)phenylboronic acid pinacol ester] (PB) is 40. This value was calculated by comparing the integral of the proton resonance peak of the terminal methyl group of monomethoxypolyethylene glycol (MPEG) (peak a: 3.38 ppm), the proton resonance peak of the methylene group in the polycaprolactone (PCL) backbone (peak b: 2.35-2.25 ppm), and the proton resonance peak of the benzene ring of poly[4-(acryloyloxymethyl)phenylboronic acid pinacol ester] (PB) (peak c: 7.73 ppm). 1 H-NMR (600MHz, CDCl3) δ7.73(s,80H),7.21(s,80H),4.89(s,80H),4.23(t,J=4.9Hz,2H),4.06(t,J=6.7Hz,17H),3 .82–3.49(m,389H),3.38(s,3H),2.35–2.25(m,22H),2.08–1.80(m,31H),1.75–1.52(m,60H),1.49–1.20(m,601H).

[0063] Example 8: Preparation of ROS-responsive lyophilizable drug-loaded nanomicelles

[0064] From MPEG 113 -b-PCL m -b-PB n Select the best matching MPEG from the current screening of similar materials 113 -b-PCL 11 -b-PB 36 The preparation method is based on the CDK4 / 6 inhibitor Palbociclib, which includes the following steps:

[0065] (1) Preparation of empty nanomicelles. Weigh 100 mg of MPEG 113 -b-PCL 11 -b-PB 36 Dissolve in 2 ml of acetone, add 4 ml of water while vortexing vigorously, and remove the acetone by vacuum distillation to obtain empty nanomicelles.

[0066] (2) Preparation of drug-loaded nanomicelles. Dissolve 10 mg of Palbociclib in 3 ml of chloroform and add dropwise to the empty nanomicelles described in (1) under vigorous stirring. Continue stirring for 2 hours. After the chloroform evaporates, freeze-dry the mixture to obtain ROS-responsive, freeze-dryable drug-loaded nanomicelles loaded with Palbociclib, hereinafter referred to as M-Pal.

[0067] Result analysis: Figure 10 After freeze-drying and storage for 1 day or 30 days, the micellar M-Pal was redispersed in ultrapure water, and the dispersion was clear and transparent without precipitation.

[0068] Example 9: Drug loading and encapsulation efficiency of M-Pal

[0069] High-performance liquid chromatography (HPLC) was used to determine the drug loading and encapsulation efficiency of M-Pal nanomicelles. The mobile phase consisted of 30% acetonitrile and 70% aqueous solution (containing 0.1% trifluoroacetic acid); the detection wavelength was 254 nm; and the column temperature was 30°C. Sample preparation: Palbociclib standards at concentrations of 0, 25, 50, 100, and 200 μg / mL were weighed and prepared for a standard curve. Subsequently, 1 mg of lyophilized micelles was weighed and dissolved in 1 mL of dimethyl sulfoxide for drug content measurement.

[0070] Result analysis: Figure 11 The drug loading capacity of palbociclib in micelle M-Pal was 7.98±0.79% (weight ratio) and the encapsulation efficiency was 79.8±7.85%, which showed high drug loading capacity and encapsulation efficiency.

[0071] Example 10: Active oxygen-responsive drug payload release test of M-Pal

[0072] Figure 12 Cumulative drug release curves of M-Pal in PBS containing hydrogen peroxide (H2O2) and PBS without H2O2 after lyophilization for one day (left) and one month (right), measuring the micellar active oxygen-responsive payload release capacity.

[0073] M-Pal stored for one day and one month after freeze-drying was dispersed in ultrapure water. Phosphate saline (PBS) buffer or PBS containing 100 μM hydrogen peroxide (H2O2) was added and drug release experiments were performed in a 37°C water bath.

[0074] Results: Both short-term and long-term storage of lyophilized M-Pal showed good ROS-responsive release characteristics. There was no significant difference in the drug release characteristics of lyophilized M-Pal after different storage times.

[0075] Example 11: Particle size and surface potential of M-Pal nanomicelles

[0076] Samples were prepared by dispersing 50 mg of lyophilized M-Pal stored for one day and one month in 2 ml of ultrapure water and vortexing. After appropriate dilution, the micelle size and potential were measured using dynamic light scattering (ZetaSizer ZS90, Malvern Instrument, UK).

[0077] Result analysis: Figure 13 The particle size and surface potential of M-Pal after freeze-drying were both smaller after short-term and long-term storage. There was no significant difference in particle size and surface potential between the two groups after freeze-drying and storage for different time periods.

[0078] Example 12: Pancreatic cancer cell viability assay

[0079] Using Palbociclib as a control, M-Pal containing different equivalent concentrations of Palbociclib was co-incubated with human pancreatic cancer cell lines Panc-1 and MIAPaCa-2 and mouse pancreatic cancer primary cells KPC-A548 at 37°C for 72 hours. Cell viability was measured using the CCK-8 assay. The half inhibitory concentration (IC50) was used. 50 ) Characterize the inhibitory ability of Palbociclib and M-Pal on pancreatic cancer cell viability.

[0080] Result analysis: Figure 14 , Palbociclib and M-Pal had similar inhibitory abilities on pancreatic cancer cell viability.

[0081] Definitions and Explanations of Terms:

[0082] Unless otherwise indicated, the definitions of groups and terms in this specification and claims, including definitions used as examples, exemplary definitions, preferred definitions, definitions in tables, and definitions of specific compounds in the Examples, may be arbitrarily combined and coupled with one another. The resulting group definitions and compound structures shall fall within the scope of the specification.

[0083] Unless otherwise indicated, the numerical ranges recited in this specification and claims are equivalent to reciting at least each specific integer value therein. For example, the numerical range "1-10" is equivalent to reciting each integer value in the numerical range "1-10", namely 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. It should be understood that when used herein to describe one, two, or more substituents, "more" should refer to an integer ≥ 3, such as 3, 4, 5, 6, 7, 8, 9, or 10.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A ROS-responsive polymer material, characterized in that: The polymer material is MPEG 113 -b-PCL m -b-PB n , based on water-soluble monomethoxy polyethylene glycol MPEG with a degree of polymerization of 113 113 First, block poly (ε-caprolactone) with different polymerization degrees m, referred to as PCL m ; then block ROS-responsive poly[4-(acryloyloxymethyl)phenylboronic acid pinacol ester] with different polymerization degrees n, referred to as PB n ; The final preparation is referred to as MPEG 113 -b-PCL m -b-PB n , the structural formula is as described in Formula I: Wherein, m and n are selected from 5-100 respectively.

2. A method for preparing a ROS-responsive polymer material according to claim 1, characterized in that: MPEG 113 -b-PCL m -b-PB n The preparation steps of the compound of general formula I described in the material are as follows: Synthesis of 4-(acryloyloxymethyl)phenylboronic acid pinacol ester: 4-(hydroxymethyl)phenylboronic acid pinacol ester and triethylamine were dissolved in an organic solvent, dichloromethane. The mixture was cooled in an ice-water bath and then protected with nitrogen. Acryloyl chloride was added dropwise with stirring. After removing the ice bath, the mixture was stirred at room temperature for 12 hours. The reaction mixture was then extracted with a saturated aqueous solution of sodium chloride. The dichloromethane solution was separated and concentrated by distillation under reduced pressure. The resulting crude product was purified by silica gel flash column chromatography to obtain 4-(acryloyloxymethyl)phenylboronic acid pinacol ester as a white solid. MPEG 113 -b-PCL m Synthesis of similar materials: Monomethoxy polyethylene glycol with a degree of polymerization of 114 and different equivalents of ε-caprolactone were dissolved in toluene, and then stannous isooctanoate was added. The reaction system was stirred at 105°C for 18 hours, and then the reaction mixture was added to petroleum ether to precipitate to obtain MPEG. 113 -b-PCL m ; MPEG 113 -b-PCL m - Synthesis of BIBB-type materials: MPEG 113 -b-PCL m and 2-bromoisobutyryl bromide were dissolved in dichloromethane, triethylamine was added, and the mixture was stirred at room temperature for 12 hours. Then, the reaction mixture was added to petroleum ether to precipitate MPEG. 113 -b-PCL m -BIBB; MPEG 113 -b-PCL m -b-PB n Synthesis of Class Materials: MPEG 113 -b-PCL m -BIBB and 4-(acryloyloxymethyl)phenylboronic acid pinacol ester were dissolved in anisole, and cuprous bromide and tris[2-(dimethylamino)ethyl]amine were added under nitrogen protection. The reaction mixture was stirred at 60°C for 24 hours, purified by neutral alumina flash column chromatography, concentrated, and precipitated in petroleum ether to obtain MPEG. 113 -b-PCL m -b-PB n .

3. A ROS-responsive lyophilizable drug-loaded nanomicelle, characterized in that: First, the ROS-responsive polymer material MPEG according to claim 1 is used 113 -b-PCL m -b-PB n , empty nanomicelles were prepared by nanoprecipitation method, and then drug-loaded nanomicelles and empty nanomicelles dissolved in organic solvent were used to prepare drug-loaded nanomicelles by solvent evaporation method. Finally, the drug-loaded nanomicelles were dehydrated by freeze-drying method to obtain ROS-responsive freeze-dryable drug-loaded nanomicelles.

4. A ROS-responsive lyophilizable drug-loaded nanomicelle as claimed in claim 3, characterized in that: Specifically: from MPEG 113 -b-PCL m -b-PB n Select the best matching MPEG from the current screening of similar materials 113 -b-PCL 11 -b-PB 36 and the CDK4 / 6 inhibitor Palbociclib to prepare ROS-responsive lyophilizable drug-loaded nanomicelles loaded with the CDK4 / 6 inhibitor Palbociclib.

5. A method for preparing ROS-responsive lyophilizable drug-loaded nanomicelles according to claim 4, characterized in that: The following steps are involved: Preparation of empty nanomicelles: MPEG 113 -b-PCL 11 -b-PB 36 Dissolve in acetone, add twice the volume of ultrapure water under vigorous vortexing, and remove acetone by vacuum distillation to obtain empty nanomicelles; Preparation of drug-loaded nanomicelles: Palbociclib was dissolved in chloroform and added dropwise to the prepared empty nanomicelles under vigorous stirring. Stirring was continued for 2 hours. After the chloroform was completely evaporated, the mixture was freeze-dried to obtain ROS-responsive lyophilizable drug-loaded nanomicelles loaded with the CDK4 / 6 inhibitor Palbociclib.

6. The method for preparing ROS-responsive lyophilizable drug-loaded nanomicelles according to claim 5, characterized in that: The palbociclib was dissolved in chloroform and added dropwise to the prepared empty nanomicelles under vigorous stirring at a stirring speed of 1000 r / min for 4 h.

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